SIRT6-dependent functional switch via K494 modifications of RE-1 Silencing Transcription factor

SIRT6-dependent functional switch via K494 modifications of RE-1 Silencing Transcription factor | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SIRT6-dependent functional switch via K494 modifications of RE-1 Silencing Transcription factor Debra Toiber, Adam Zaretsky, Alfredo Garcia Venzor, Ekaterina Eremenko, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4371623/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Nov, 2024 Read the published version in Cell Death & Disease → Version 1 posted 8 You are reading this latest preprint version Abstract RE-1 Silencing Transcription factor (REST) is a key repressor of neural genes. REST is upregulated under stress signals, aging and neurodegenerative diseases, but although it is upregulated, it loses its function in Alzheimer's Disease. However, why it becomes inactive remains unclear. Here, we show that the NAD-dependent deacetylase SIRT6 regulates REST expression, location and activity. In SIRT6 absence, REST is overexpressed but mislocalized, and loses part of its activity, becoming toxic. SIRT6 deficiency abrogates REST and EZH2 interaction, perturbs its location to heterochromatin Lamin B ring, and leads to REST target gene overexpression. SIRT6 reintroduction or REST methyl-mimic K494M expression rescues this phenotype, while an acetyl-mimic mutant loses its function even in WT cells. Our studies define a novel regulatory switch, where the function of a critical repressor is regulated by post-translational modifications on K494, depending on SIRT6 existence and, in turn, modulating neuronal gene expression. Biological sciences/Molecular biology/Epigenetics Biological sciences/Molecular biology/Transcription Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction The RE-1 silencing transcription factor (REST) is a zinc finger DNA binding transcription factor (TF). REST is a master regulator of neural genes 1 which predominantly acts as a repressor 2 . During development, REST is a key factor for proper cell differentiation and a repressor of neural phenotype in non-neural somatic tissues 3 . REST is silenced in differentiated neurons during adulthood but upregulated under neuronal intrinsic or extrinsic insults 4 . Interestingly, REST expression is elevated in aging brains, serving as a neuroprotective factor 5 . However, in the brain of AD patients, REST fails to localize to the nucleus, therefore failing in its neuroprotective role as a gene expression regulator 5 . Using differentiated induced pluripotent stem cells (iPSC) derived from AD patient fibroblasts, it was shown that these patients have a distinct gene expression profile, presenting upregulation in neurodevelopment and neural activity genes. Interestingly, most of these genes are REST targets, suggesting that in neurodegeneration, REST target genes are misregulated; however, the impaired function of REST as a repressor remains unclear 6 . Sirtuin 6 (SIRT6) is a histone deacetylase and ADP-ribosyl transferase with important roles in various cellular functions such as gene expression 7 and DNA repair 8 , both involved in organismal aging and neurodegeneration 7,9–11 . “SIRT6-KO monkeys”, die right after birth, presenting severe physiological abnormalities - specifically, impaired brain development. Moreover, in humans with mutant SIRT6, rendering it inactive, embryonic lethality and neurodevelopmental disorders occur. On the other hand, during aging, SIRT6 diminishes, and an even more pronounced reduction is observed in AD patients. Brain-specific SIRT6 deficient mice develop a neurodegenerative-like phenotype with hyperphosphorylated and hyper-acetylated Tau, learning impairments, and increased cell death 10,12 . Therefore, REST and SIRT6 both work in brain development and maintenance, preventing neurodegeneration. In this study, we find that SIRT6-KO brains show that a significant part of the upregulated genes are REST targets. We therefore expected REST to be downregulated. Instead, we find that in the absence of SIRT6, REST is overexpressed, as in AD patients. However, its repressive function is impaired, leading to an upregulation of its target genes. Although REST itself is upregulated, it is mislocalized inside the nucleus and in the cytoplasm, where it forms aggregate-like structures due to changes in a post-translational modification in residue K494. In the nucleus, it cannot localize properly to the Lamin B domain, and the recruitment EZH2 and binding to H3K27me3 is impaired. This suggests that, in the absence of SIRT6, the upregulation of REST cannot protect the brains due to its loss of function. Results SIRT6-deficient brains show impaired expression of neuronal activity genes. To characterize the transcriptional profile of the brS6KO brain tissue, we performed whole-brain RNA sequencing on brS6KO and WT mice (Fig. S1 A). Gene enrichment against several datasets of the differentially overexpressed genes showed enrichment of categories in Gene Ontology (GO) related to neural activity and development (Fig. 1 A, Table S1 ). These results suggest that SIRT6 is involved in regulating brain function and neuronal activity. To better understand the changes in cortical neurons specifically, we performed ATAC sequencing (ATAC-seq) on WT and brS6KO cortical neurons isolated from 10 month old mice, allowing us to recognize genomic regions accessible to transcription mechanisms (Fig. S1 B, S1C). Enrichment of regions differentially accessed in brS6KO neurons reveals that GO categories associated with neural activity are significantly enriched (Fig. 1 B, Table S2 ). Genes represented in both RNA-seq and ATAC-seq were mainly related to neural activity, morphology, and development (Fig. 1 A, 1 B). SIRT6-KO differentially expressed genes are enriched for REST regulation. Next, we asked whether these genes might be regulated by a common transcription factor (TF). To find potential TF candidates, we performed an enrichment analysis of the brS6KO cortical neuron ATAC-seq and the brS6KO RNA-seq against the ENCODE, and ChEA consensus TFs from a ChIPX database. We then performed a hypergeometric meta-analysis encompassing the enriched TFs to find the candidates overlapping both neuron chromatin accessibility and brain expression. Our results filtered three potential TFs: SUZ12, SMAD4, and REST (Fig. 2 A). To narrow our focus, we tested their relevance to SIRT6 expression in the human brain by comparing the transcription profile of SIRT6 from six human brains (Allen Brain Atlas) to the profile of each TF 13 . Interestingly, the RE-1 Silencing Transcription factor (REST) correlates negatively with SIRT6 and, most significantly, in the brains of older individuals. The transcription factors SMAD4 and SUZ12 showed significant, but less prominent correlation with SIRT6 in most individuals. (Fig. 2 B, Fig. S1 A). REST is considered a repressive factor crucial for mediating the expression of neural development and activity genes containing the RE-1 element in their promoter 14 . Therefore, these results suggest that upregulated genes in brS6KO might be mediated through a lack of REST repression. Next, we examined the proportion of REST targets among the upregulated genes in brS6KO. To do this, we overlapped the SIRT6 RNA-seq results with publicly available REST ChIP-seq data of SHSY-5Y cells 5 , and H1 cell line ChIP seq (GSM803365) (Fig. 2 C, Fig. 2 E). Over 100 genes upregulated in brS6KO are REST targets in the SHSY-5Y cell line, enriched in GO categories related to neural activity and development (Fig. 2 D, Table S3 ). In H1 cells, more than 500 REST targets are represented in brS6KO upregulated genes and are associated with neural morphology and activity (Fig. 2 F, Table S4 ). Altogether, our results suggest that the transcriptional alteration in brS6KO, specifically the upregulated neural genes, is mediated through REST. REST is overexpressed in SIRT6-KO brains and cells. REST is known as a neural gene repressor, and since its targets were upregulated, we expected to observe reduced levels of REST in brS6KO. Surprisingly, REST levels were higher in the brS6KO RNA-seq data (Fig. 3 A). To validate these results in cells, we measured mRNA and protein levels of REST in the SHSY-5Y cell line with CRISPR deleted SIRT6 (SIRT6-KO). In these cells, REST was overexpressed in both mRNA and protein levels of chromatin extractions (Fig. 3 B, 3 C). In brS6KO mice, REST protein levels were increased in chromatin extracts (Fig. 3 D). To confirm that this overexpression is a product of SIRT6 absence, we introduced recombinant SIRT6 to the SIRT6-KO cells. mRNA measurement by qPCR shows rescue of REST levels with the introduction of SIRT6 (Fig. 3 E), suggesting that the expression of this factor is SIRT6 dependent. To test if this is a conserved phenomenon and if we could use tissue culture to understand REST-related changes, we selected REST target genes from the RNA-seq results to measure in the SIRT6-KO cells (Fig. S3 A). We measured mRNA levels for two representative REST target genes, SYN1 and GRIK2, and found that in both genes, mRNA levels were higher in SIRT6-KO cells, and reintroduction of SIRT6 to these cells rescued their expression (Fig. 3 F). In the context of AD, SIRT6 expression declines with Braak stage progression 10 . To assess the levels of REST expression in AD, we used microarray data from AD and non-AD patients from GSE48350 database. By dividing the samples according to their Braak stage, we concluded that REST expression is higher in later Braak stages than earlier ones, suggesting loss of expression regulation (Fig S3 B). REST has several isoforms, some of which lack repressive capabilities and thus quench the function of the main isoform 15,16 . To test for the possibility of alternative splicing, we constructed probes that recognize the exon 2 and exon 3 (E2 + E3) exon junction (present in most of the transcripts) and the exon 3 and exon 4 (E3 + E4) exon junction (present in the full isoform), and we measured the abundance of the inactive splice variant relative to the whole isoform population (Fig. S3 C). According to our measurements, there was no significant difference in the main REST isoform population between WT and SIRT6-KO cells (Fig. S3 D), indicating that REST loss of function does not correlate with REST alternative splicing, and the lack of repressive capacity is not from the generation of different alternative spliced variants. REST sub-cellular localization. To address the hypothesis of REST loss of function in SIRT6 KO cells, we first tested changes in cellular localization. Previous studies showed that REST could be nuclear and cytoplasmic 17 . We hypothesized that changes in REST localization might hint at the causality of its impairment. To observe nuclear morphology and different nuclear regions, we performed immunofluorescence (IF) against the endogenous REST in SIRT6-KO cells (Fig. 3 G). When measuring total cell intensity, REST intensity significantly increased in KO cells (Fig. 3 G-H). Dividing the cellular area to its nuclear and cytoplasmic regions, REST intensity was higher in both compartments (Fig. 3 H and S3E). Additionally, in the SIRT6-KO cells, REST was observed as condensates in the cytoplasm and closer to the cellular membrane, in addition to its nuclear location (Fig. 3 G). This phenotype was not observed in WT cells. Quantification of these bodies shows that REST condensates accumulate in the cytoplasm of the SIRT6-KO cells (Fig. 3 I). To test whether this phenomenon is reversible, we used an inducible shSIRT6 system that silences SIRT6 for 21 days using doxycycline, followed by 9 more days of recovery of SIRT6 levels (Fig. 3 J and S3F). After the tenth day of silencing, REST cellular intensity increased significantly, in agreement with our previous results from SIRT6-KO cells. Interestingly, the recovery period did not reduce the mean nuclear intensity of REST, in addition to whole cell and cytoplasmic region measurements (Fig. 3 J-M). Our previous results show rescue of mRNA levels of REST after 48 hours of SIRT6 reintroduction (Fig. 3 E). However, in the inducible experiment, the intensity of REST does not decrease after SIRT6 recovery, suggesting that the phenotype is irreversible at the protein level, at least not within 10 days after SIRT6 re-expression. To test whether this was due to high levels of REST leading to its aggregation, we overexpressed REST with Flag-REST plasmid under the CMV promoter, followed by immunofluorescence using Flag antibodies, which recognizes only the exogenous protein. In this experiment, we failed to see cytoplasmic REST bodies. To understand the discrepancy between exogenous REST and endogenous staining, we cloned a Flag-REST-GFP plasmid (Fig. S4 A-B), since the antibodies are targeted for the C-terminus and Flag was in the N-terminus. Overexpression of this construct showed cytoplasmic aggregates (Fig S3 G). It was recently published that REST accumulated in cytoplasmic autophagosomes 5 ; therefore, we measured co-localization of REST with LC3. A fraction of REST co-localized with LC3 in both WT and SIRT6-KO cells, but there was a significant increase in SIRT6-KO cells with both Flag and GFP (Fig. 3 N-O). Last, we collected brains from mice at different ages, and separated proteins into chromatin bound and cytoplasmic fractions. With age, REST tends to localize more to the cytoplasmic fractions almost two-fold, while being depleted from the chromatin bound fraction. This suggests that the overexpression of REST first leads to its increase in all fractions, while slowly accumulating in the cytoplasm with age (Fig. 3SH-I). Note that in this method we are missing the nuclear fraction, unlike that for IF. SIRT6 has a declining trend with age but presents some variability (Fig. 3SJ). REST is present at the nuclear Lamina. Interestingly, we detected REST accumulating closer to the nuclear lamina, forming a ring almost co-localizing with Lamin B in WT cells (Fig. 4 A-B). This was not previously seen for REST. In contrast, SIRT6-KO cells exhibited less colocalization of REST with the nuclear lamina (70% less) (Fig. 4 C). When a ring was detectable, it was more homogenous and thicker inside the nucleus, measured by the co-localization of REST to the nuclear lamina and the thickness of the REST peak near the lamina (Fig. 4 B and D-F), suggesting a loss of heterochromatin binding, or loss of chromatin condensation capability. Moreover, Flag and GFP staining co-localize in this ring, but not in all the cells, with GFP having a more homogenous nuclear distribution, and higher appearance in the cytoplasmic fraction (Fig. S4 B). These results suggested that REST could be cleaved, separating the C and N terminus. To test that, we transfected Flag-REST-GFP and measured total protein extracts as well as cytoplasmic and chromatin bound fraction with Flag, GFP and REST antibodies. We can clearly see REST fragmentation overall (also with a higher band, only in total extraction). While Flag shows mainly a chromatin bound presence, GFP could be seen in the cytoplasm, as well as REST (which combined both exogenous and endogenous forms). This strengthens the point at which N-terminal REST is mainly nuclear and chromatin bound, while the C-terminus is cleaved and appears more in the nucleus as cytoplasm (Fig. S4 F-H). Next, we performed ChIP-seq to better understand the changes in REST targets in SIRT6-WT and KO cells. We confirmed a general enrichment for neuronal related genes (Fig. 4 G). SIRT6 deficient cells have almost double the amount of peaks, probably due to higher levels in the cells (Fig. 4 H), but with no difference in the distribution to promoter, intron or exon distribution (Fig. 4 I). Importantly, in both WT and KO cells, a significant part of REST localizes to Lamin Associated Domains (LADs) confirming our IF results. However, the KO cells show a slightly higher proportion in the LADs (Fig. 4 J). A small but significant fraction of REST co-localized with heterochromatic mark H3K27me3 in both cells (Fig. 4 K),but had zero overlap with H3K9me3 mark. REST gained categories such as neurotransmitter transport and other ion activities, but lost monoatomic ion channel activity, possibly affecting neuronal activity (Fig. 4 L). REST interaction with EZH2 is abrogated in SIRT6-KO cells. Since REST binding was not diminished in the SIRT6 deficient cells, we hypothesized that REST function as a scaffold protein 15 , recruiting various chromatin remodelers to its RE-1 element in neural promoters could be impaired, failing to repress its target genes. EZH2 is an H3K27 methyl transferase, part of the PRC2 silencing complex 18 . EZH2 is a known REST interactor and an important chromatin silencer in the brain 18,19 . Previous works showed that the interaction of REST and EZH2 is important for repressing REST target genes 20 . Interestingly, 190 genes upregulated in brS6KO are EZH2 targets (Fig. 5 A) (Using publicly available hESC ChIP-seq data). To better understand REST and EZH2 gene co-regulation in SIRT6 KO cells, we analyzed the EZH2 targets together with the REST consensus peaks from our ChIP-seq data (Fig. S5 A). Functional enrichment analysis of REST and EZH2 targets revealed that overlapping pathways are neuron related (Fig. S5 B-C). We then checked if upregulated REST targets in brS6KO are also targeted by EZH2. A permutation test showed that REST and EZH2 regulate 16 genes in WT cells that are upregulated in brS6KO (Fig. 5 B), and 21 genes in SIRT6 KO cells that are upregulated in brS6KO (Fig. 5 C). Functional similarity between these genes allows significant enrichment in GO categories associated with neural pathways (Fig. 5 E). Due to the small coverage of our ChIP-seq, we analyzed data from hESC for EZH2 and REST, and found that they overlap in about 500 genes, with 51 being also overexpressed in SIRT6-KO brains (Fig. 5 D). Genes were enriched for synaptic and brain functioning, similarly to the ATAC-seq and RNA-seq results (Fig. 1 A-B). To confirm these bioinformatic results, we tested the levels of EZH2 in SIRT6 deficient cells. Although total levels of EZH2 were not altered in the total cell extract (Fig. 5 F and S5G), the chromatin bound fractions were reduced (Fig. 5 G and S5E). To determine if the interaction between REST and EZH2 is affected in the absence of SIRT6, we performed Co-Immunoprecipitation (Co-IP) of the recombinant Flag-REST expressed in WT vs. SIRT6 KO SHSY cells. We found a significant reduction in the interaction between REST and EZH2 in SIRT6-KO cells (Fig. 5 H). Although REST did not show differential binding between WT and SIRT6 KO cells in our ChIP-seq (Fig. 4 K-I), the impaired interaction with EZH2 led us to hypothesize that there are less H3K27me3 in regions bound by REST. To test this, we performed Co-IP against H3K27me3 from chromatin preps in WT and SIRT6 KO SHSY-5Y cells, and found that REST is less enriched in H3K27me3 in SIRT6 KO cells (Fig. 5 I). These results suggest that, although REST levels are increased and its DNA binding capability not impaired, interaction with key chromatin remodelers and epigenetic markers such as EZH2 and H3K27me3 are disrupted, leading to less chromatin condensation in REST bound loci. To assess the loss and gain of REST interaction depending on SIRT6 genotype, we overexpressed recombinant REST in WT and SIRT6KO HEK293T cells and sent the triplicate samples to mass spectrometry. We obtained 141 proteins that pass the filter (excluding non-specific protein reads that were present in the empty vector sample threshold and in 3 independent IPs). Our results show one unique interaction for REST in WT and nine novel interactions in SIRT6-KO cells. The unique interactions were defined as proteins present specifically in the WT or KO samples (Fig. S6 A). In addition, we performed a differential interaction analysis. We discovered a small but significant number of proteins ( 9 ) that are gained in SIRT6-KO cells, mainly related to translation, while the lost interactions ( 5 ) were related to DNA repair and remodeling, potentially affecting chromatin condensation function (Fig. S6 B-C). STRING analysis revealed that REST interacts with proteins involved in histone and chromatin remodeling (Fig. S6 D-E). For our lost and gained interactions, we performed STRING analysis and allowed the addition of a second layer of interactors to better define the functionality of these proteins (Fig S6 F-G). Enrichment analysis of the cellular components of these networks revealed that, in SIRT6 KO cells, REST loses interaction with proteins involved in DNA repair, while gaining interaction with translation related complexes - which are chiefly cytoplasmatic (Fig S6 H). These results support the fact that, in SIRT6 KO, REST exists more prominently in the cytoplasm and loses its function in the nucleus by losing key interactors. However, we cannot discard the possibility that these changes could be the result of different protein content in a SIRT6-deficient background. Acetylation of REST on Lysine 494 results in loss of association with the heterochromatin. It was shown that EZH2 methylates REST on Lysine 494 (K494), increasing the interaction between these two proteins, and this modification is critical for the repression of REST target genes 20 . Since the interaction with EZH2 is diminished, we hypothesized that methylation could be impaired by the presence of different PTMs, such as acetylation. Since there are no commercially available antibodies for K494Ac, we performed immunoprecipitation (IP) of Flag-REST in WT and KO cells, and measured pan-acetyl-Lysine antibody (Fig. 6 A). We discovered that, not only is REST acetylated, but is even more so in the absence of SIRT6. The reduced interaction with EZH2 and the existence of acetylated REST led us to hypothesize that the K494 residue might be responsible for these alterations. To check this, we generated a K494A REST mutant. Overexpression of REST variants shows that WT-REST is hyperacetylated in KO cells, while the non-modifiable K494A is generally less acetylated in WT cells and shows no change in KO cells, indicating that this is the main change occurring in SIRT6-KO cells. A similar trend is observed in the mutated K494R (Fig. 6 B). These results suggest that SIRT6 mediates the balance between acetylated or methylated REST. To test the proportion of acetylated or methylated REST in SIRT6 KO cells, we performed immunoprecipitation using pan-methyl lysine (MeK) and pan-acetyl lysine (AcK) antibodies. SIRT6 KO cells transfected with WT REST showed more REST enrichment when using AcK antibody (Fig. 6 C), and less when using MeK (Fig. 6 D), suggesting that REST is more likely to get acetylated than methylated in SIRT6 KO cells. In addition, cells transfected with the non-modifiable REST K494A mutant using MeK antibody showed less enrichment of REST, regardless of SIRT6 genotype - while in AcK, REST enriched similarly to the control. These results suggest that, in addition to the differential proportion of Me-REST and Ac-REST in SIRT6 KO cells, lysine 494 has a critical role in influencing REST PTM. To understand if REST can become toxic under the different PTMs, we transfected the cells with the different REST variants and Apo-Alert Active caspase 3 detector. Interestingly, WT-REST was no different between SIRT6-WT and KO cells, but the different REST variants, particularly 494M and a lesser extent 494Q could rescue the WT cells but became more toxic in the KO cells (Fig. 6 E). Suggesting that in the presence of SIRT6, REST can be neuroprotective, but in its absence, as it happens during AD, they become toxic. REST K494 PTMs affect its nuclear location. To assess if K494 affects REST nuclear localization, we mutated WT REST in K494 to alanine (K494A), methionine (K494M) and glutamine (K494Q). K494M and K494Q serve as PTM mimics for methylation and acetylation in this lysine residue, respectively, while alanine is a non-modifiable mutation. REST WT and the point mutants were transfected to WT and SIRT6 KO SHSY-5Y cells (Fig. 7 A-D, S7A). Both WT, K494A, K494M - and K494Q REST - appear in the nucleus 48 hours after transfection. As previously shown, WT REST co-localizes closely to the nuclear lamina in WT cells, while a more diffuse pattern is seen in SIRT6-KO cells, losing the ring feature (Fig. 7 A). Interestingly, K494A REST fails to localize to the nuclear lamina in both WT and SIRTKO cells (Fig. 7 B). The methyl mimic K494M showed higher association with the nuclear periphery even in KO cells (Fig. 7 C), while the acetyl mimic showed lower association even in SIRT6-WT cells (Fig. 7 D). This indicates that K494 methylation induces its Lamin-associated location, while non modifiable or acetylated REST shows impaired recruitment to the nuclear envelope. In addition, we measured the width of the peaks that the REST mutants generate. SIRT6 genotype did not affect the width of peaks of the K494A and K494Q mutants, probably because of the decreased accumulation of these mutants to the nuclear lamina (Fig. S7 C-D). In the K494M mutant, the width of the REST peak was wider in KO than in WT cells (Fig. 7 B), although the laminar colocalization value improved in the KO cells. This might suggest that although REST is more localized to the nuclear lamina when methylated, it still depends on the presence of SIRT6 to have a sharper localization, probably through other effects of SIRT6 on nuclear lamina function. To understand the changes in binding activity, we performed ChIP-seq using anti-Flag antibodies expressed REST WT, and the K494A, M and Q mutants in SHSY cells. Enrichment analysis of consensus peaks between all the mutants showed high enrichment of processes and pathways associated with neural activity (Fig. 7 E-F). Once again, the different REST mutants did not affect the distribution of REST to the different regions such as promoter, introns, exon etc., similarly to what we saw with endogenous REST in SIRT6 WT and KO cells (Fig. 7 E). We performed an analysis with 2 out of 3 ChIPs presenting the peak and a more stringent test with the 3 peaks present. Both analyses show the same trend (Fig. S7 D-F). Interestingly, the K494M mutation bound ~ 4 times more unique targets than K494Q, and ~ 22 times more than K494A. Additionally, K494M shares more targets with REST WT than with K494Q (~ 3 times) and K494A (~ 8 times) (Fig S7 D). These results suggest that under normal conditions REST is methylated in the cells, allowing improved binding, while non modifiable or acetyl mimic shown an impairment. The difference between the point mutants is mainly in the amount of sites they can bind, but enrichment the for different categories is also affected. For example, WT binds the monoatomic channels, neurotransmitter and synaptic activity. Interestingly, only WT and acetyl mimic bind glutamate receptor activity, one of the enriched categories in the DE expressed genes in brS6KO - particularly the alanine mutant loss and monoatomic ion gated channel activity. Methyl mimic lost some categories related to glutamate receptor, symporter and general transmembrane transport, but gained guanyl nucleotide exchange factor activities. One unique acetyl mimic category was syntaxin binding, also related to SNARE binding enriched in the Alanine mutant, suggesting an overall important role for REST in membrane constitution - from ion channels to membrane composition and endocytosis (Fig. 7 G). Moreover, we see that the proportion of REST in LADs is conserved among the mutants, strengthening the notion that the plasmids are indeed binding the correct sequences as the endogenous REST, and that it is not an artifact of the Flag (Fig. 7 H). Rockowitz et. al 21 showed that REST associates mainly with the transient heterochromatin mark H3K27me3 in neurons and less with the more permanent H3K9me3 mark. We see the same results here; although with smaller numbers, it is significantly enriched (Fig. 7 I). The association of REST K494M with the nuclear periphery led us to hypothesize that this mutant might help to repress REST target genes, even in the absence of SIRT6. To check this, we introduced WT or K494M REST to SIRT6 normal or deficient cells and measured the mRNA levels of REST targets SYN1 and GRIK2 (Fig. S7 G). As shown previously, SIRT6 KO influences the expression of REST targets even when overexpressed in its WT form. However, REST K494M rescues the level of these target genes, even in the context of SIRT6 deficiency. These results not only suggest that the methylation of REST is important for its repressive function, but also that SIRT6 regulates REST function through the K494 residue acetylation/methylation switch. Discussion Our results indicate that SIRT6 regulates REST in multiple layers, from mRNA levels to the nucleus and cytoplasm, and repressive activity at target genes. We show that REST loses its capacity to regulate gene expression through a lack of interaction with the repressor EZH2 depending on modifications in residue 494, impairing not only gene expression, but nuclear and cytoplasmic distribution. Moreover, REST accumulates and forms cytoplasmic bodies that could become irreversibly toxic. SIRT6-REST neuronal targets overlap. The upregulation of neural genes in brS6KO led us to suspect misregulation through key TFs. A considerable number of upregulated genes in SIRT6-KO brains were found as targets of REST, in addition to other components of the PRC2 complex, like SUZ12 and EZH2. In our results, REST and SUZ12 were both enriched factors targeting upregulated genes in brS6KO. In cortical neurons, particularly, we could detect changes in chromatin accessibility to REST targets in SIRT6-KO brains. Absence of SIRT6 results in a dramatic transcriptomic alteration that pushes cells toward neural differentiation and unregulated neural activity, as in the phenotype of loss of REST 22 . This is similar to iPSC from AD patients, which experiences faster differentiation to neuronal progenitor cells and later to neurons 6 . In aging brain tissue, REST represses neural activity and development to reduce neurotoxicity and maintain remaining progenitor cells. Unregulated REST can lead to increased neural activity and diminish the progenitor cell population, leading to higher tissue vulnerability 6,23 (Pereira et al., 2010; Meyer et al., 2019). We suggest that SIRT6 reduction during aging can mis regulate REST targets in the brain, preventing its protection. Excessive REST in SIRT6-KO and aging Our initial hypothesis was that REST levels were low in SIRT6-KO in contrast to those seen in healthy aging, where REST was found to be overexpressed, serving as a neuroprotective factor 5 . Loss of REST could explain the deficit in the repression of its target genes. Surprisingly, REST is overexpressed in AD, brS6KO and SIRT6-KO cells (qPCR, RNA-seq and protein levels), but so are its target genes. To understand REST relevance in the brains, we analyzed REST and SIRT6 expression in human brains (Allen brain Atlas) and found a significant negative correlation between the levels of SIRT6 and REST, particularly in older samples. The decreased levels of SIRT6 in aging brains, even more pronounced in AD patients 10 , could influence the levels of REST in aging brains. In the absence of SIRT6, REST fails to protect the brains. REST- “ full gas in neutral” In normal aging brains, REST overexpression is protective, while in AD 5 and sporadic AD cells 6 , REST target genes were overexpressed due to lack of REST localization to the nucleus. This impairment was also relevant in Parkinson’s disease 5,6,24 . We showed that REST was overexpressed in SIRT6-KO brains and cells; however, it was inactive, in accordance to what was observed in AD and Parkinson’s patients. Since SIRT6 is reduced in aging brains and AD, this could be a causal link. In some isoforms, REST loses its repressive domains but retains the DNA binding domain 16 . This can lead to a dominant negative phenotype, where REST isoform (REST4) competes with the full-length REST on target promoter binding. In our results, there was no difference between the main REST isoform and the total population of possible isoforms, leading us to the conclusion that regulation at the protein level was required. REST is mislocalized in the cytoplasm. To better understand REST loss of function we tested whether it could be mislocalized, as it was observed in AD and PD patients. First, we observed endogenous REST protein localization in the SIRT6-KO cells. In the absence of SIRT6, REST was nuclear but also showed an increase in the cytoplasm, in polarized and “ condensate-like” forms. Interestingly, these bodies are accumulated irreversibly and partially co-localized in autophagosomes. While re-introduction of SIRT6 can reduce REST mRNA levels in 48 hours (Fig. 3F), re-expression of SIRT6 for 10 days could not reverse this phenotype, suggesting that even the loss of SIRT6 for a short period could lead to irreversible phenotype regarding REST protein. REST is mislocalized in the nucleus. In SIRT6 deficient cells, REST is highly enriched in the nucleus, with a more homogeneous location, and in 70% of the cases, fails to localize in a LaminB region. Our ChIP-seq results suggest that REST binding is not reduced, nor the location impaired. Therefore, its lack of function could not be explained by lack of binding. REST repressive function results from recruiting chromatin remodeling enzymes that condense histones around REST targets such as EZH2 2 . EZH2 is a H3K27 methyl transferase and a component of the PRC2 complex 18,20 . In mouse cortex, loss of EZH2 leads to cortical progenitor cell depletion from the Sub Ventricular Zone and enrichment of genes related to neural development 25 . In SIRT6-KO cells, EZH2 is less abundant in chromatin. In addition, interaction between EZH2 and REST is impaired in SIRT6-KO cells, suggesting why it fails to repress its target genes. Moreover, REST usually interacts with proteins that help condensed chromatin. Our Co-IP of REST, and the loss of interaction found in SIRT6c cells by mass spec, suggest loss of chromatin remodelers and heterochromatin formation. These results might explain the appearance of a thicker REST ‘ring’ in KO cells (Fig. 4H, 4I), since REST still arrives to chromatin (Fig. 3C, 5A). Interestingly, during aging, the nuclear envelope becomes damaged, and loss of heterochromatin is observed, altering gene expression 26,27 . Loss of REST function at LADs could contribute to this phenomenon. REST location and function can be controlled by modifications at residue K494. EZH2 can methylate REST in a specific lysine residue (K494), which increases its interaction with EZH2. We hypothesized that this residue is also acetylated. Our results show that REST can indeed be acetylated and, interestingly, more so in SIRT6-KO cells. This might suggest that there is a PTM balance that can influence REST function. If methylation of K494 is beneficial for REST function and interaction with EZH2, an acetylation event can keep this residue from being methylated. When the K494 residue is mutated to alanine, REST acetylation is reduced; moreover, the acetylation increase seen in KO cells is lost, suggesting SIRT6 could be the deacetylase. However, we failed to see direct interaction between SIRT6 and REST by Co-IP or mass spec (although substrate-enzyme interactions are fast and maybe undetected in our system). Because of the lack of specific antibodies, we could not effectively measure deacetylation in vitro, and this question remains open. REST nuclear distribution is lost when K494 is changed to alanine as a preventive for charged modifications and Q (mimicking acetylation) in both WT and SIRT6 deficient cells. Moreover, the M mutant, which mimics methylation, pushes REST to its “ functional” phenotypes even in the SIRT6-KO cells. Moreover, REST-494M can restore the gene expression of REST target genes in SIRT6 deficient cells, while WT-REST cannot, indicating that location and function can be interconnected. All the REST PTMs mutants have a similar distribution in the genome (regarding regions they prefer to bind). Still, methyl-mimic can bind many more target genes that the acetyl or the non-modifiable mutants, strengthening the point that methylation helps REST in its repressive function. Overall, we found that SIRT6 regulates REST expression and activity in the cell, allowing it to repress neuronal genes. This is done by a molecular switch in the acetylation/methylation status of REST, allowing it to interact with EZH2 and localize to nuclear lamina. The functional relevance of SIRT6 depletion in the aging brain and neurodegenerative diseases could influence the capacity of REST to protect the brain from neurodegeneration and could be the cause of REST loss of function, even when expressed at high levels, as observed in AD and PD; therefore, the SIRT6-REST-EZH2 pathway could be relevant to target for future research. Declarations Author contributions AZ planned, performed the experiments and wrote the paper. AGV, EE, DS, YR, RD and ME performed experiments, DS, DK and EK analyzed data and DT planned and wrote the paper Funding The study was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 849029), the David and Inez Myers foundation, the Israeli Ministry of Science and Technology (MOST), the High-tech, Bio-tech and Negev fellowships of Kreitman School of Advanced Research of Ben Gurion University. 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Additional Declarations (Not answered) Supplementary Files keyresourcetableandmethods.docx TableS1.csv TableS2.csv TableS3.csv TableS4.csv TableS5.xlsx TableS6.xlsx TableS7.xlsx TableS8.xlsx S1.jpg S2.jpg S3.jpg S4.jpg S5.jpg S5contin.jpg S7.jpg GraphicalAbstract.jpg Cite Share Download PDF Status: Published Journal Publication published 07 Nov, 2024 Read the published version in Cell Death & Disease → Version 1 posted Editorial decision: revise 12 Sep, 2024 Review # 1 received at journal 22 Jul, 2024 Reviewer # 2 agreed at journal 12 Jul, 2024 Reviewer # 1 agreed at journal 10 Jul, 2024 Reviewers invited by journal 21 Jun, 2024 Submission checks completed at journal 15 May, 2024 Editor assigned by journal 14 May, 2024 First submitted to journal 14 May, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Negev","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"Dryer","suffix":""},{"id":317372473,"identity":"026e1171-bd8f-43a9-a2a4-170744c80747","order_by":8,"name":"Monica Einav","email":"","orcid":"","institution":"Ben Gurion University of the Negev","correspondingAuthor":false,"prefix":"","firstName":"Monica","middleName":"","lastName":"Einav","suffix":""},{"id":317372474,"identity":"3c889d99-4657-4044-9232-7005e473bdd7","order_by":9,"name":"Dmitrii Kriukov","email":"","orcid":"","institution":"Skolkovo Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Dmitrii","middleName":"","lastName":"Kriukov","suffix":""},{"id":317372475,"identity":"a41a9517-c673-4637-b50e-00831b91dc6a","order_by":10,"name":"Ekaterina Khrameeva","email":"","orcid":"","institution":"Skolkovo Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ekaterina","middleName":"","lastName":"Khrameeva","suffix":""}],"badges":[],"createdAt":"2024-05-05 12:25:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4371623/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4371623/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-024-07160-0","type":"published","date":"2024-11-07T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60521040,"identity":"e0522e19-a850-4130-a727-59c90685913e","added_by":"auto","created_at":"2024-07-17 16:31:11","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1275922,"visible":true,"origin":"","legend":"\u003cp\u003eNeural genes are upregulated in brS6KO brain\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eEnrichment analysis of upregulated genes in brS6KO mice. \u003cstrong\u003e(B) \u003c/strong\u003eEnrichment analysis of differentially accessed genes in SIRT6-KO cortical neurons. BP = Biological Processes, CC = Cellular Component, MF = Molecular Function. Dashed line represents Adjusted P-value=0.05.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4371623/v1/ae105d5a56d49e4f7acba14c.jpg"},{"id":60520451,"identity":"a0d1a38b-4d3e-49d4-88ac-b231f9183e95","added_by":"auto","created_at":"2024-07-17 16:23:11","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1185540,"visible":true,"origin":"","legend":"\u003cp\u003eREST regulates upregulated genes in SIRT6 KO.\u003c/p\u003e\n\u003cp\u003e(A) Hypergeometric test of TFs targeting differentially accessed genes in SIRTKO cortical neurons ATAC-seq, with differentially expressed genes in brS6KO RNA-seq. In both data sets SUZ12, SMAD4 and REST are common between these two enrichment analyses. (B) Hierarchical clustering of expression profile correlation between SIRT6 and each of the candidate transcription factors. n(brains)=6. Value in each cell represents Pearson R. (C) Hypergeometric test of genes upregulated in brS6KO, and genes significantly targeted by REST SHSY-5Y ChIP-seq performed by Lu et al., 2014. (D) Genes that were common in both datasets in C were enriched against GO categories. (E) Hypergeometric test of genes upregulated in brS6KO, and genes significantly targeted by REST in human embryonic stem cells (H1) ChIP-seq (GSM803365). (F) Genes that were common in both datasets in E were enriched against GO categories. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001. BP = Biological Processes, CC = Cellular Component, MF = Molecular Function. Dashed line represents Adjusted P-value=0.05.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4371623/v1/66d73e7f40c5218b9bd69440.jpg"},{"id":60521041,"identity":"0b7f9a72-3fcd-45ce-850d-32e7761f9d83","added_by":"auto","created_at":"2024-07-17 16:31:11","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2766728,"visible":true,"origin":"","legend":"\u003cp\u003eREST expression in is upregulated in SIRT6 KO\u003c/p\u003e\n\u003cp\u003e(A) Normalized counts from brS6KO RNA-seq of REST, n(mice)=5 (B) mRNA fold change via qPCR of REST in SHSY-5Y cell lines. n=3. (C) Chromatin protein extraction from SHSY-5Y cell line. n=3. (D) REST protein levels in chromatin extraction from WT and SIRT6 KO mice brains, n (mice per group)=8. (E) mRNA fold change via qPCR of REST in WT vs KO SHSY-5Y cells transfected SIRT6 or Control vector. n=3. (F) mRNA fold change via qPCR of SYN1 n=4 and GRIK2 n=3 in SHSY-5Y cell line. (G) Immunofluorescence of REST (Alexa flour 488) in WT and KO SHSY-5Y cells. (H) Quantification of nuclear REST intensity in WT and KO SHSY-5Y cells. n(cells)=294[WT],369[KO]. (I) Quantification of REST condensations via ImageJ tool ”AggreCount”. n(cells)=256. (J) Immunofluorescence of endogenous REST in SHSY-5Y cells with inducible SIRT6 silencing. shSIRT6 (shS6) Day 0 (d0) dox represent cells with shSRIT6 without silencing. shS6 d21 dox represent cells with shSIRT6 treated 21 days with dox (21 days of silencing). d30 recovery are cells that did not receive Dox since d21. shCtrl are cells induced to express scramble shRNA. DMSO is vehicle control instead of doxycycline. (K-M) Quantification of REST intensity in whole cell, nuclear and cytoplasm in shSIRT6 inducible system, 21 days of shSIRT6 induction, followed by recovery (until day 30). n(cells)=72[Day 0 dox],66[Day 5 dox], 130[Day 10 dox], 99[Day 21 dox], 93[Day 30 rec], 179[Day 21 DMSO], 149[Day 21 shCtrl]. (N) Immunofluorescence of Flag-REST-GFP transfected WT and SIRT6 KO SHSY-5Y cells. The Flag channel is in red, the GFP channel is in green, the LC3 channel is in magenta, DAPI channel is in blue. (O) Quantification of Flag/GFP positive phagosomes detected by cytoplasmic LC3. Each dot represents a cell. n(cells)=20[WT],22[KO]. Bars and error bars represent Mean±SEM. A-D, H, O Unpaired t-test. E-F 2-Way ANOVA followed by Sidak’s multiple comparison. F(SYN1) Multiple t-test. I - M 2-way ANOVAova followed by Sidak’s/Dunnett’s multiple comparison. Two tailed *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ****p\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4371623/v1/75174be9410537938769068c.jpg"},{"id":60521043,"identity":"c65d9eff-a756-4519-a98f-c874af981a82","added_by":"auto","created_at":"2024-07-17 16:31:11","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2079738,"visible":true,"origin":"","legend":"\u003cp\u003eREST localization alters in SIRT6 KO.\u003c/p\u003e\n\u003cp\u003e(A) Nuclear distribution of Flag-REST in SHSY-5Y WT and KO cells. Cells were immuno-stained for recombinant REST (Anti-Flag) and LAMINB as nuclear envelope reference. The dashed frame marks representative cells for quantification. (B) Representing spectra of DAPI, LAMINB, and Flag channels across the dashed line in A. Distance and intensity are scaled according to the maximum value. (C) Percentage of “REST ring” positive cells out of total transfection positive cells. n=3. (D) Quantification of Nuclear REST distribution. The swarm plot represents the REST nuclear distribution value per cell. n(cells)=52[WT],50[KO]. (E) Swarm plot representing the thickness of the REST peak close to the nuclear lamina, among cells that present REST. N(cells)=33[WT],36[KO]. (F) Immunofluorescence of Flag-REST-GFP transfected WT and SIRT6 KO SHSY-5Y cells. Flag channel in magenta. GFP channel in green. (G) Functional enrichment analysis of consensus REST peaks from 3 independent replicates. (H) Venn diagram representing the number of consensus peaks in WT and SIRT6 KO cells. (J) Fraction of REST peaks overlapping with laminin-associated domains (LADs) in WT vs SIRT6 KO cells. (K) Fraction of REST peaks overlapping with H3K27me3 domains. (I) Feature distribution of REST peaks in WT vs SIRT6 KO cells. (L) Go analysis of WT and SIRT6 KO REST peaks and Enrichment analysis of REST peaks specific to SIRT6 KO cells. All data represents Mean±SEM. C-E Unpaired t-test. Two tailed *p\u0026lt;0.05, **p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4371623/v1/5e691ab9f8c106f88ede5f78.jpg"},{"id":60521045,"identity":"1c2864d0-9289-419e-bbf2-946da1393e17","added_by":"auto","created_at":"2024-07-17 16:31:11","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1525568,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eREST loses interaction with EZH2 in SIRT6 KO.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Venn diagram of upregulated genes in brS6KO and EZH2 hESC targets. P represents hypergeometric test p value. (B-C) . Overlapping genes between REST peaks in WT (B) or SIRT6 KO (B) cells with EZH2 targets form hESC and genes upregulated in brS6KO. Calculated P resembles permutation test on the background of all available human protein-coding genes (HGNC). (D) Overlap between REST targets, EZH2 targets and brS6KO upregulated genes. Calculated P resembles permutation test on the background of all available human protein coding genes (HGNC). (E) Enrichment analysis for overlapping genes in Fig. 5B. (F) Total EZH2 from total protein extraction. n=3. (G) Chromatin extraction of SHSY-5Y cells. n=3. (H) Co-immunoprecipitation of Flag-REST. Loading normalized to equal levels of REST in the IP. n(replicates)=3. (I) Co-immunoprecipitation of H3K27me3 in WT vs SIRT6 KO SHSY-5Y cells. Quantification represents intensities of REST/H3K27me3. n=3. Bars and error bars represent Mean±SEM. A P value represents hypergeometric test. B-D Permutation test P value. F-H Unpaired t-test. H-I Paired t-test. *p\u0026lt;0.05,**p\u0026lt;0.01. ns=non-significant. BP = Biological Processes, CC = Cellular Component, MF = Molecular Function. Dashed line represents Adjusted P-value=0.05.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4371623/v1/53e5e278b0ef54b28eb973ee.jpg"},{"id":60520461,"identity":"0fda9f0f-ab22-4ea8-b32a-88d0237ab2a1","added_by":"auto","created_at":"2024-07-17 16:23:11","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1237077,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eREST can be acetylated.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eImmunoprecipitation of REST in SYSY-5Y cells transfected with Flag-REST. n(replicates)=3 \u003cstrong\u003e(B) \u003c/strong\u003eTotal protein extraction of SHSY-5Y cells transfected with WT REST and Arginine (K494R) or Alanine mutant (K494A). \u003cstrong\u003e(C) \u003c/strong\u003eImmunoprecipitation using AcK antibody. n(replicates)=3. \u003cstrong\u003e(D) \u003c/strong\u003eImmunoprecipitation using Me-K antibody. n(replicates)=3. \u003cstrong\u003e(E) \u003c/strong\u003eApoAlert caspase 3 detector assay: SHSY5Y cells were co-transfected with ApoAlert and the different REST variants. The percentage of apoptotic cells was obtained per treatment and ANOVA used for analysis.Bars and error bars represent Mean±SEM. A, Unpaired t-test. C, 2-way ANOVA followed by Tukey's multiple comparisons test\u003cstrong\u003e. \u003c/strong\u003eD, Unpaired t-test. *p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4371623/v1/c0502f818cbd94d3cab33c8c.jpg"},{"id":60521046,"identity":"0f2c23b1-a065-4aac-9a93-ade1c2e8c2a0","added_by":"auto","created_at":"2024-07-17 16:31:11","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2497501,"visible":true,"origin":"","legend":"\u003cp\u003eREST K494 residue influences nucellar distribution regardless of SIRT6 existence.\u003c/p\u003e\n\u003cp\u003e(A-D) SHSY-5Y WT and SIRT6KO cells transfected with REST WT, REST K49A, REST K494M, REST K49Q, respectively. LUT editing allows to observe a heat map accumulation of REST in the nucleus.(E) Feature frequency for each mutant REST in 3 replicates. (F) Venn diagram of all peaks for 3 replicates for each REST mutant (G) GO enrichment analysis for 2 out of 3 replicates for peaks of REST WT, K494A, K494M and K494Q. (H)Fraction of REST peaks overlapping with H3K27me3 domains. (I) Fraction of REST peaks overlapping with laminin-associated domains (LADs).\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4371623/v1/d230e3cf5e0956396ffc49c7.jpg"},{"id":68524275,"identity":"65696f35-96ee-4459-9f4e-907568acec33","added_by":"auto","created_at":"2024-11-08 08:10:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13219598,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4371623/v1/750cb89b-9ec4-4fc2-af6b-e41872f84328.pdf"},{"id":60521279,"identity":"fd58f385-9930-4420-a862-e1b70854705c","added_by":"auto","created_at":"2024-07-17 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REST is a master regulator of neural genes\u003csup\u003e1\u003c/sup\u003e which predominantly acts as a repressor \u003csup\u003e2\u003c/sup\u003e. During development, REST is a key factor for proper cell differentiation and a repressor of neural phenotype in non-neural somatic tissues\u003csup\u003e3\u003c/sup\u003e. REST is silenced in differentiated neurons during adulthood but upregulated under neuronal intrinsic or extrinsic insults\u003csup\u003e4\u003c/sup\u003e. Interestingly, REST expression is elevated in aging brains, serving as a neuroprotective factor\u003csup\u003e5\u003c/sup\u003e. However, in the brain of AD patients, REST fails to localize to the nucleus, therefore failing in its neuroprotective role as a gene expression regulator\u003csup\u003e5\u003c/sup\u003e. Using differentiated induced pluripotent stem cells (iPSC) derived from AD patient fibroblasts, it was shown that these patients have a distinct gene expression profile, presenting upregulation in neurodevelopment and neural activity genes. Interestingly, most of these genes are REST targets, suggesting that in neurodegeneration, REST target genes are misregulated; however, the impaired function of REST as a repressor remains unclear\u003csup\u003e6\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSirtuin 6 (SIRT6) is a histone deacetylase and ADP-ribosyl transferase with important roles in various cellular functions such as gene expression\u003csup\u003e7\u003c/sup\u003e and DNA repair\u003csup\u003e8\u003c/sup\u003e, both involved in organismal aging and neurodegeneration\u003csup\u003e7,9\u0026ndash;11\u003c/sup\u003e. \u0026ldquo;SIRT6-KO monkeys\u0026rdquo;, die right after birth, presenting severe physiological abnormalities - specifically, impaired brain development. Moreover, in humans with mutant SIRT6, rendering it inactive, embryonic lethality and neurodevelopmental disorders occur. On the other hand, during aging, SIRT6 diminishes, and an even more pronounced reduction is observed in AD patients. Brain-specific SIRT6 deficient mice develop a neurodegenerative-like phenotype with hyperphosphorylated and hyper-acetylated Tau, learning impairments, and increased cell death\u003csup\u003e10,12\u003c/sup\u003e. Therefore, REST and SIRT6 both work in brain development and maintenance, preventing neurodegeneration.\u003c/p\u003e \u003cp\u003eIn this study, we find that SIRT6-KO brains show that a significant part of the upregulated genes are REST targets. We therefore expected REST to be downregulated. Instead, we find that in the absence of SIRT6, REST is overexpressed, as in AD patients. However, its repressive function is impaired, leading to an upregulation of its target genes. Although REST itself is upregulated, it is mislocalized inside the nucleus and in the cytoplasm, where it forms aggregate-like structures due to changes in a post-translational modification in residue K494. In the nucleus, it cannot localize properly to the Lamin B domain, and the recruitment EZH2 and binding to H3K27me3 is impaired. This suggests that, in the absence of SIRT6, the upregulation of REST cannot protect the brains due to its loss of function.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eSIRT6-deficient brains show impaired expression of neuronal activity genes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo characterize the transcriptional profile of the brS6KO brain tissue, we performed whole-brain RNA sequencing on brS6KO and WT mice (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Gene enrichment against several datasets of the differentially overexpressed genes showed enrichment of categories in Gene Ontology (GO) related to neural activity and development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These results suggest that SIRT6 is involved in regulating brain function and neuronal activity. To better understand the changes in cortical neurons specifically, we performed ATAC sequencing (ATAC-seq) on WT and brS6KO cortical neurons isolated from 10 month old mice, allowing us to recognize genomic regions accessible to transcription mechanisms (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB, S1C). Enrichment of regions differentially accessed in brS6KO neurons reveals that GO categories associated with neural activity are significantly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Genes represented in both RNA-seq and ATAC-seq were mainly related to neural activity, morphology, and development (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eSIRT6-KO differentially expressed genes are enriched for REST regulation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eNext, we asked whether these genes might be regulated by a common transcription factor (TF). To find potential TF candidates, we performed an enrichment analysis of the brS6KO cortical neuron ATAC-seq and the brS6KO RNA-seq against the ENCODE, and ChEA consensus TFs from a ChIPX database. We then performed a hypergeometric meta-analysis encompassing the enriched TFs to find the candidates overlapping both neuron chromatin accessibility and brain expression. Our results filtered three potential TFs: SUZ12, SMAD4, and REST (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To narrow our focus, we tested their relevance to SIRT6 expression in the human brain by comparing the transcription profile of SIRT6 from six human brains (Allen Brain Atlas) to the profile of each TF\u003csup\u003e13\u003c/sup\u003e. Interestingly, the RE-1 Silencing Transcription factor (REST) correlates negatively with SIRT6 and, most significantly, in the brains of older individuals. The transcription factors SMAD4 and SUZ12 showed significant, but less prominent correlation with SIRT6 in most individuals. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). REST is considered a repressive factor crucial for mediating the expression of neural development and activity genes containing the RE-1 element in their promoter\u003csup\u003e14\u003c/sup\u003e. Therefore, these results suggest that upregulated genes in brS6KO might be mediated through a lack of REST repression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we examined the proportion of REST targets among the upregulated genes in brS6KO. To do this, we overlapped the SIRT6 RNA-seq results with publicly available REST ChIP-seq data of SHSY-5Y cells\u003csup\u003e5\u003c/sup\u003e, and H1 cell line ChIP seq (GSM803365) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Over 100 genes upregulated in brS6KO are REST targets in the SHSY-5Y cell line, enriched in GO categories related to neural activity and development (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). In H1 cells, more than 500 REST targets are represented in brS6KO upregulated genes and are associated with neural morphology and activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Altogether, our results suggest that the transcriptional alteration in brS6KO, specifically the upregulated neural genes, is mediated through REST.\u003c/p\u003e \u003cp\u003e \u003cb\u003eREST is overexpressed in SIRT6-KO brains and cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eREST is known as a neural gene repressor, and since its targets were upregulated, we expected to observe reduced levels of REST in brS6KO. Surprisingly, REST levels were higher in the brS6KO RNA-seq data (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). To validate these results in cells, we measured mRNA and protein levels of REST in the SHSY-5Y cell line with CRISPR deleted SIRT6 (SIRT6-KO). In these cells, REST was overexpressed in both mRNA and protein levels of chromatin extractions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In brS6KO mice, REST protein levels were increased in chromatin extracts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo confirm that this overexpression is a product of SIRT6 absence, we introduced recombinant SIRT6 to the SIRT6-KO cells. mRNA measurement by qPCR shows rescue of REST levels with the introduction of SIRT6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE), suggesting that the expression of this factor is SIRT6 dependent. To test if this is a conserved phenomenon and if we could use tissue culture to understand REST-related changes, we selected REST target genes from the RNA-seq results to measure in the SIRT6-KO cells (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA). We measured mRNA levels for two representative REST target genes, SYN1 and GRIK2, and found that in both genes, mRNA levels were higher in SIRT6-KO cells, and reintroduction of SIRT6 to these cells rescued their expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). In the context of AD, SIRT6 expression declines with Braak stage progression\u003csup\u003e10\u003c/sup\u003e. To assess the levels of REST expression in AD, we used microarray data from AD and non-AD patients from GSE48350 database. By dividing the samples according to their Braak stage, we concluded that REST expression is higher in later Braak stages than earlier ones, suggesting loss of expression regulation (Fig \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eREST has several isoforms, some of which lack repressive capabilities and thus quench the function of the main isoform\u003csup\u003e15,16\u003c/sup\u003e. To test for the possibility of alternative splicing, we constructed probes that recognize the exon 2 and exon 3 (E2\u0026thinsp;+\u0026thinsp;E3) exon junction (present in most of the transcripts) and the exon 3 and exon 4 (E3\u0026thinsp;+\u0026thinsp;E4) exon junction (present in the full isoform), and we measured the abundance of the inactive splice variant relative to the whole isoform population (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC). According to our measurements, there was no significant difference in the main REST isoform population between WT and SIRT6-KO cells (Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD), indicating that REST loss of function does not correlate with REST alternative splicing, and the lack of repressive capacity is not from the generation of different alternative spliced variants.\u003c/p\u003e \u003cp\u003e \u003cb\u003eREST sub-cellular localization.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo address the hypothesis of REST loss of function in SIRT6 KO cells, we first tested changes in cellular localization. Previous studies showed that REST could be nuclear and cytoplasmic\u003csup\u003e17\u003c/sup\u003e. We hypothesized that changes in REST localization might hint at the causality of its impairment. To observe nuclear morphology and different nuclear regions, we performed immunofluorescence (IF) against the endogenous REST in SIRT6-KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). When measuring total cell intensity, REST intensity significantly increased in KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H). Dividing the cellular area to its nuclear and cytoplasmic regions, REST intensity was higher in both compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH and S3E). Additionally, in the SIRT6-KO cells, REST was observed as condensates in the cytoplasm and closer to the cellular membrane, in addition to its nuclear location (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). This phenotype was not observed in WT cells. Quantification of these bodies shows that REST condensates accumulate in the cytoplasm of the SIRT6-KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). To test whether this phenomenon is reversible, we used an inducible shSIRT6 system that silences SIRT6 for 21 days using doxycycline, followed by 9 more days of recovery of SIRT6 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ and S3F). After the tenth day of silencing, REST cellular intensity increased significantly, in agreement with our previous results from SIRT6-KO cells. Interestingly, the recovery period did not reduce the mean nuclear intensity of REST, in addition to whole cell and cytoplasmic region measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ-M). Our previous results show rescue of mRNA levels of REST after 48 hours of SIRT6 reintroduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). However, in the inducible experiment, the intensity of REST does not decrease after SIRT6 recovery, suggesting that the phenotype is irreversible at the protein level, at least not within 10 days after SIRT6 re-expression.\u003c/p\u003e \u003cp\u003eTo test whether this was due to high levels of REST leading to its aggregation, we overexpressed REST with Flag-REST plasmid under the CMV promoter, followed by immunofluorescence using Flag antibodies, which recognizes only the exogenous protein. In this experiment, we failed to see cytoplasmic REST bodies. To understand the discrepancy between exogenous REST and endogenous staining, we cloned a Flag-REST-GFP plasmid (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA-B), since the antibodies are targeted for the C-terminus and Flag was in the N-terminus. Overexpression of this construct showed cytoplasmic aggregates (Fig \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eG). It was recently published that REST accumulated in cytoplasmic autophagosomes\u003csup\u003e5\u003c/sup\u003e; therefore, we measured co-localization of REST with LC3. A fraction of REST co-localized with LC3 in both WT and SIRT6-KO cells, but there was a significant increase in SIRT6-KO cells with both Flag and GFP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN-O).\u003c/p\u003e \u003cp\u003eLast, we collected brains from mice at different ages, and separated proteins into chromatin bound and cytoplasmic fractions. With age, REST tends to localize more to the cytoplasmic fractions almost two-fold, while being depleted from the chromatin bound fraction. This suggests that the overexpression of REST first leads to its increase in all fractions, while slowly accumulating in the cytoplasm with age (Fig.\u0026nbsp;3SH-I). Note that in this method we are missing the nuclear fraction, unlike that for IF. SIRT6 has a declining trend with age but presents some variability (Fig.\u0026nbsp;3SJ).\u003c/p\u003e \u003cp\u003e \u003cb\u003eREST is present at the nuclear Lamina.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eInterestingly, we detected REST accumulating closer to the nuclear lamina, forming a ring almost co-localizing with Lamin B in WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). This was not previously seen for REST. In contrast, SIRT6-KO cells exhibited less colocalization of REST with the nuclear lamina (70% less) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). When a ring was detectable, it was more homogenous and thicker inside the nucleus, measured by the co-localization of REST to the nuclear lamina and the thickness of the REST peak near the lamina (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and D-F), suggesting a loss of heterochromatin binding, or loss of chromatin condensation capability. Moreover, Flag and GFP staining co-localize in this ring, but not in all the cells, with GFP having a more homogenous nuclear distribution, and higher appearance in the cytoplasmic fraction (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB). These results suggested that REST could be cleaved, separating the C and N terminus. To test that, we transfected Flag-REST-GFP and measured total protein extracts as well as cytoplasmic and chromatin bound fraction with Flag, GFP and REST antibodies. We can clearly see REST fragmentation overall (also with a higher band, only in total extraction). While Flag shows mainly a chromatin bound presence, GFP could be seen in the cytoplasm, as well as REST (which combined both exogenous and endogenous forms). This strengthens the point at which N-terminal REST is mainly nuclear and chromatin bound, while the C-terminus is cleaved and appears more in the nucleus as cytoplasm (Fig. \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eF-H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we performed ChIP-seq to better understand the changes in REST targets in SIRT6-WT and KO cells. We confirmed a general enrichment for neuronal related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). SIRT6 deficient cells have almost double the amount of peaks, probably due to higher levels in the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), but with no difference in the distribution to promoter, intron or exon distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Importantly, in both WT and KO cells, a significant part of REST localizes to Lamin Associated Domains (LADs) confirming our IF results. However, the KO cells show a slightly higher proportion in the LADs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). A small but significant fraction of REST co-localized with heterochromatic mark H3K27me3 in both cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK),but had zero overlap with H3K9me3 mark. REST gained categories such as neurotransmitter transport and other ion activities, but lost monoatomic ion channel activity, possibly affecting neuronal activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003e \u003cb\u003eREST interaction with EZH2 is abrogated in SIRT6-KO cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eSince REST binding was not diminished in the SIRT6 deficient cells, we hypothesized that REST function as a scaffold protein\u003csup\u003e15\u003c/sup\u003e, recruiting various chromatin remodelers to its RE-1 element in neural promoters could be impaired, failing to repress its target genes. EZH2 is an H3K27 methyl transferase, part of the PRC2 silencing complex\u003csup\u003e18\u003c/sup\u003e. EZH2 is a known REST interactor and an important chromatin silencer in the brain\u003csup\u003e18,19\u003c/sup\u003e. Previous works showed that the interaction of REST and EZH2 is important for repressing REST target genes\u003csup\u003e20\u003c/sup\u003e. Interestingly, 190 genes upregulated in brS6KO are EZH2 targets (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) (Using publicly available hESC ChIP-seq data). To better understand REST and EZH2 gene co-regulation in SIRT6 KO cells, we analyzed the EZH2 targets together with the REST consensus peaks from our ChIP-seq data (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA). Functional enrichment analysis of REST and EZH2 targets revealed that overlapping pathways are neuron related (Fig. \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eB-C). We then checked if upregulated REST targets in brS6KO are also targeted by EZH2. A permutation test showed that REST and EZH2 regulate 16 genes in WT cells that are upregulated in brS6KO (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), and 21 genes in SIRT6 KO cells that are upregulated in brS6KO (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Functional similarity between these genes allows significant enrichment in GO categories associated with neural pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Due to the small coverage of our ChIP-seq, we analyzed data from hESC for EZH2 and REST, and found that they overlap in about 500 genes, with 51 being also overexpressed in SIRT6-KO brains (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Genes were enriched for synaptic and brain functioning, similarly to the ATAC-seq and RNA-seq results (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). To confirm these bioinformatic results, we tested the levels of EZH2 in SIRT6 deficient cells. Although total levels of EZH2 were not altered in the total cell extract (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and S5G), the chromatin bound fractions were reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG and S5E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine if the interaction between REST and EZH2 is affected in the absence of SIRT6, we performed Co-Immunoprecipitation (Co-IP) of the recombinant Flag-REST expressed in WT vs. SIRT6 KO SHSY cells. We found a significant reduction in the interaction between REST and EZH2 in SIRT6-KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Although REST did not show differential binding between WT and SIRT6 KO cells in our ChIP-seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK-I), the impaired interaction with EZH2 led us to hypothesize that there are less H3K27me3 in regions bound by REST. To test this, we performed Co-IP against H3K27me3 from chromatin preps in WT and SIRT6 KO SHSY-5Y cells, and found that REST is less enriched in H3K27me3 in SIRT6 KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). These results suggest that, although REST levels are increased and its DNA binding capability not impaired, interaction with key chromatin remodelers and epigenetic markers such as EZH2 and H3K27me3 are disrupted, leading to less chromatin condensation in REST bound loci.\u003c/p\u003e \u003cp\u003eTo assess the loss and gain of REST interaction depending on SIRT6 genotype, we overexpressed recombinant REST in WT and SIRT6KO HEK293T cells and sent the triplicate samples to mass spectrometry. We obtained 141 proteins that pass the filter (excluding non-specific protein reads that were present in the empty vector sample threshold and in 3 independent IPs). Our results show one unique interaction for REST in WT and nine novel interactions in SIRT6-KO cells. The unique interactions were defined as proteins present specifically in the WT or KO samples (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eA). In addition, we performed a differential interaction analysis. We discovered a small but significant number of proteins (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) that are gained in SIRT6-KO cells, mainly related to translation, while the lost interactions (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) were related to DNA repair and remodeling, potentially affecting chromatin condensation function (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eB-C). STRING analysis revealed that REST interacts with proteins involved in histone and chromatin remodeling (Fig. \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eD-E). For our lost and gained interactions, we performed STRING analysis and allowed the addition of a second layer of interactors to better define the functionality of these proteins (Fig \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eF-G). Enrichment analysis of the cellular components of these networks revealed that, in SIRT6 KO cells, REST loses interaction with proteins involved in DNA repair, while gaining interaction with translation related complexes - which are chiefly cytoplasmatic (Fig \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eH). These results support the fact that, in SIRT6 KO, REST exists more prominently in the cytoplasm and loses its function in the nucleus by losing key interactors. However, we cannot discard the possibility that these changes could be the result of different protein content in a SIRT6-deficient background.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAcetylation of REST on Lysine 494 results in loss of association with the heterochromatin.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIt was shown that EZH2 methylates REST on Lysine 494 (K494), increasing the interaction between these two proteins, and this modification is critical for the repression of REST target genes\u003csup\u003e20\u003c/sup\u003e. Since the interaction with EZH2 is diminished, we hypothesized that methylation could be impaired by the presence of different PTMs, such as acetylation. Since there are no commercially available antibodies for K494Ac, we performed immunoprecipitation (IP) of Flag-REST in WT and KO cells, and measured pan-acetyl-Lysine antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We discovered that, not only is REST acetylated, but is even more so in the absence of SIRT6. The reduced interaction with EZH2 and the existence of acetylated REST led us to hypothesize that the K494 residue might be responsible for these alterations. To check this, we generated a K494A REST mutant. Overexpression of REST variants shows that WT-REST is hyperacetylated in KO cells, while the non-modifiable K494A is generally less acetylated in WT cells and shows no change in KO cells, indicating that this is the main change occurring in SIRT6-KO cells. A similar trend is observed in the mutated K494R (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These results suggest that SIRT6 mediates the balance between acetylated or methylated REST. To test the proportion of acetylated or methylated REST in SIRT6 KO cells, we performed immunoprecipitation using pan-methyl lysine (MeK) and pan-acetyl lysine (AcK) antibodies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSIRT6 KO cells transfected with WT REST showed more REST enrichment when using AcK antibody (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), and less when using MeK (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD), suggesting that REST is more likely to get acetylated than methylated in SIRT6 KO cells. In addition, cells transfected with the non-modifiable REST K494A mutant using MeK antibody showed less enrichment of REST, regardless of SIRT6 genotype - while in AcK, REST enriched similarly to the control. These results suggest that, in addition to the differential proportion of Me-REST and Ac-REST in SIRT6 KO cells, lysine 494 has a critical role in influencing REST PTM.\u003c/p\u003e \u003cp\u003eTo understand if REST can become toxic under the different PTMs, we transfected the cells with the different REST variants and Apo-Alert Active caspase 3 detector. Interestingly, WT-REST was no different between SIRT6-WT and KO cells, but the different REST variants, particularly 494M and a lesser extent 494Q could rescue the WT cells but became more toxic in the KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Suggesting that in the presence of SIRT6, REST can be neuroprotective, but in its absence, as it happens during AD, they become toxic.\u003c/p\u003e \u003cp\u003e \u003cb\u003eREST K494 PTMs affect its nuclear location.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess if K494 affects REST nuclear localization, we mutated WT REST in K494 to alanine (K494A), methionine (K494M) and glutamine (K494Q). K494M and K494Q serve as PTM mimics for methylation and acetylation in this lysine residue, respectively, while alanine is a non-modifiable mutation. REST WT and the point mutants were transfected to WT and SIRT6 KO SHSY-5Y cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D, S7A). Both WT, K494A, K494M - and K494Q REST - appear in the nucleus 48 hours after transfection. As previously shown, WT REST co-localizes closely to the nuclear lamina in WT cells, while a more diffuse pattern is seen in SIRT6-KO cells, losing the ring feature (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Interestingly, K494A REST fails to localize to the nuclear lamina in both WT and SIRTKO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The methyl mimic K494M showed higher association with the nuclear periphery even in KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC), while the acetyl mimic showed lower association even in SIRT6-WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). This indicates that K494 methylation induces its Lamin-associated location, while non modifiable or acetylated REST shows impaired recruitment to the nuclear envelope. In addition, we measured the width of the peaks that the REST mutants generate. SIRT6 genotype did not affect the width of peaks of the K494A and K494Q mutants, probably because of the decreased accumulation of these mutants to the nuclear lamina (Fig. \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eC-D). In the K494M mutant, the width of the REST peak was wider in KO than in WT cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), although the laminar colocalization value improved in the KO cells. This might suggest that although REST is more localized to the nuclear lamina when methylated, it still depends on the presence of SIRT6 to have a sharper localization, probably through other effects of SIRT6 on nuclear lamina function.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo understand the changes in binding activity, we performed ChIP-seq using anti-Flag antibodies expressed REST WT, and the K494A, M and Q mutants in SHSY cells. Enrichment analysis of consensus peaks between all the mutants showed high enrichment of processes and pathways associated with neural activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-F). Once again, the different REST mutants did not affect the distribution of REST to the different regions such as promoter, introns, exon etc., similarly to what we saw with endogenous REST in SIRT6 WT and KO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). We performed an analysis with 2 out of 3 ChIPs presenting the peak and a more stringent test with the 3 peaks present. Both analyses show the same trend (Fig. \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eD-F). Interestingly, the K494M mutation bound\u0026thinsp;~\u0026thinsp;4 times more unique targets than K494Q, and ~\u0026thinsp;22 times more than K494A. Additionally, K494M shares more targets with REST WT than with K494Q (~\u0026thinsp;3 times) and K494A (~\u0026thinsp;8 times) (Fig \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eD). These results suggest that under normal conditions REST is methylated in the cells, allowing improved binding, while non modifiable or acetyl mimic shown an impairment.\u003c/p\u003e \u003cp\u003eThe difference between the point mutants is mainly in the amount of sites they can bind, but enrichment the for different categories is also affected. For example, WT binds the monoatomic channels, neurotransmitter and synaptic activity. Interestingly, only WT and acetyl mimic bind glutamate receptor activity, one of the enriched categories in the DE expressed genes in brS6KO - particularly the alanine mutant loss and monoatomic ion gated channel activity. Methyl mimic lost some categories related to glutamate receptor, symporter and general transmembrane transport, but gained guanyl nucleotide exchange factor activities. One unique acetyl mimic category was syntaxin binding, also related to SNARE binding enriched in the Alanine mutant, suggesting an overall important role for REST in membrane constitution - from ion channels to membrane composition and endocytosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003eMoreover, we see that the proportion of REST in LADs is conserved among the mutants, strengthening the notion that the plasmids are indeed binding the correct sequences as the endogenous REST, and that it is not an artifact of the Flag (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eRockowitz et. al\u003csup\u003e21\u003c/sup\u003e showed that REST associates mainly with the transient heterochromatin mark H3K27me3 in neurons and less with the more permanent H3K9me3 mark. We see the same results here; although with smaller numbers, it is significantly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003eThe association of REST K494M with the nuclear periphery led us to hypothesize that this mutant might help to repress REST target genes, even in the absence of SIRT6. To check this, we introduced WT or K494M REST to SIRT6 normal or deficient cells and measured the mRNA levels of REST targets SYN1 and GRIK2 (Fig. \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003eG). As shown previously, SIRT6 KO influences the expression of REST targets even when overexpressed in its WT form. However, REST K494M rescues the level of these target genes, even in the context of SIRT6 deficiency. These results not only suggest that the methylation of REST is important for its repressive function, but also that SIRT6 regulates REST function through the K494 residue acetylation/methylation switch.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results indicate that SIRT6 regulates REST in multiple layers, from mRNA levels to the nucleus and cytoplasm, and repressive activity at target genes. We show that REST loses its capacity to regulate gene expression through a lack of interaction with the repressor EZH2 depending on modifications in residue 494, impairing not only gene expression, but nuclear and cytoplasmic distribution. Moreover, REST accumulates and forms cytoplasmic bodies that could become irreversibly toxic.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSIRT6-REST neuronal targets overlap.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe upregulation of neural genes in brS6KO led us to suspect misregulation through key TFs. A considerable number of upregulated genes in SIRT6-KO brains were found as targets of REST, in addition to other components of the PRC2 complex, like SUZ12 and EZH2. In our results, REST and SUZ12 were both enriched factors targeting upregulated genes in brS6KO. In cortical neurons, particularly, we could detect changes in chromatin accessibility to REST targets in SIRT6-KO brains. Absence of SIRT6 results in a dramatic transcriptomic alteration that pushes cells toward neural differentiation and unregulated neural activity, as in the phenotype of loss of REST \u003csup\u003e22\u003c/sup\u003e. This is similar to iPSC from AD patients, which experiences faster differentiation to neuronal progenitor cells and later to neurons\u003csup\u003e6\u003c/sup\u003e. In aging brain tissue, REST represses neural activity and development to reduce neurotoxicity and maintain remaining progenitor cells. Unregulated REST can lead to increased neural activity and diminish the progenitor cell population, leading to higher tissue vulnerability\u003csup\u003e6,23\u003c/sup\u003e (Pereira et al., 2010; Meyer et al., 2019). We suggest that SIRT6 reduction during aging can mis regulate REST targets in the brain, preventing its protection.\u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExcessive REST in SIRT6-KO and aging\u003c/h2\u003e \u003cp\u003eOur initial hypothesis was that REST levels were low in SIRT6-KO in contrast to those seen in healthy aging, where REST was found to be overexpressed, serving as a neuroprotective factor \u003csup\u003e5\u003c/sup\u003e. Loss of REST could explain the deficit in the repression of its target genes. Surprisingly, REST is overexpressed in AD, brS6KO and SIRT6-KO cells (qPCR, RNA-seq and protein levels), but so are its target genes. To understand REST relevance in the brains, we analyzed REST and SIRT6 expression in human brains (Allen brain Atlas) and found a significant negative correlation between the levels of SIRT6 and REST, particularly in older samples. The decreased levels of SIRT6 in aging brains, even more pronounced in AD patients\u003csup\u003e10\u003c/sup\u003e, could influence the levels of REST in aging brains.\u003c/p\u003e \u003cp\u003eIn the absence of SIRT6, REST fails to protect the brains.\u003c/p\u003e \u003c/div\u003e\u003cp\u003e\u003cstrong\u003eREST-\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cspan dir=\"RTL\"\u003e\u0026ldquo;\u003c/span\u003e\u003c/strong\u003e\u003cstrong\u003efull gas in neutral\u0026rdquo;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn normal aging brains, REST overexpression is protective, while in AD\u003csup\u003e5\u003c/sup\u003e and sporadic AD cells\u003csup\u003e6\u003c/sup\u003e, REST target genes were overexpressed due to lack of REST localization to the nucleus. This impairment was also relevant in Parkinson\u0026rsquo;s disease\u003csup\u003e5,6,24\u003c/sup\u003e. We showed that REST was overexpressed in SIRT6-KO brains and cells; however, it was inactive, in accordance to what was observed in AD and Parkinson\u0026rsquo;s patients. Since SIRT6 is reduced in aging brains and AD, this could be a causal link. In some isoforms, REST loses its repressive domains but retains the DNA binding domain\u0026nbsp;\u003csup\u003e16\u003c/sup\u003e. This can lead to a dominant negative phenotype, where REST isoform (REST4) competes with the full-length REST on target promoter binding. In our results, there was no difference between the main REST isoform and the total population of possible isoforms, leading us to the conclusion that regulation at the protein level was required.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eREST is mislocalized in the cytoplasm.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo better understand REST loss of function we tested whether it could be mislocalized, as it was observed in AD and PD patients. First, we observed endogenous REST protein localization in the SIRT6-KO cells. In the absence of SIRT6, REST was nuclear but also showed an increase in the cytoplasm, in polarized and \u003cspan dir=\"RTL\"\u003e\u0026ldquo;\u003c/span\u003econdensate-like\u0026rdquo; forms. Interestingly, these bodies are accumulated irreversibly and partially co-localized in autophagosomes. While re-introduction of SIRT6 can reduce REST mRNA levels in 48 hours (Fig. 3F), re-expression of SIRT6 for 10 days could not reverse this phenotype, suggesting that even the loss of SIRT6 for a short period could lead to irreversible phenotype regarding REST protein.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eREST is mislocalized in the nucleus.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn SIRT6 deficient cells, REST is highly enriched in the nucleus, with a more homogeneous location, and in 70% of the cases, fails to localize in a LaminB region. Our ChIP-seq results suggest that REST binding is not reduced, nor the location impaired. Therefore, its lack of function could not be explained by lack of binding. REST repressive function results from recruiting chromatin remodeling enzymes that condense histones around REST targets such as EZH2 \u003csup\u003e2\u003c/sup\u003e. EZH2 is a H3K27 methyl transferase and a component of the PRC2 complex \u003csup\u003e18,20\u003c/sup\u003e. In mouse cortex, loss of EZH2 leads to cortical progenitor cell depletion from the Sub Ventricular Zone and enrichment of genes related to neural development \u003csup\u003e25\u003c/sup\u003e. In SIRT6-KO cells, EZH2 is less abundant in chromatin. In addition, interaction between EZH2 and REST is impaired in SIRT6-KO cells, suggesting why it fails to repress its target genes. Moreover, REST usually interacts with proteins that help condensed chromatin. Our Co-IP of REST, and the loss of interaction found \u0026nbsp;in SIRT6c cells by mass spec, suggest loss of chromatin remodelers and heterochromatin formation. These results might explain the appearance of a thicker REST \u0026lsquo;ring\u0026rsquo; in KO cells (Fig. 4H, 4I), since REST still arrives to chromatin (Fig. 3C, 5A). Interestingly, during aging, the nuclear envelope becomes damaged, and loss of heterochromatin is observed, altering gene expression \u003csup\u003e26,27\u003c/sup\u003e. Loss of REST function at LADs could contribute to this phenomenon.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eREST location and function can be controlled by modifications at residue K494.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEZH2 can methylate REST in a specific lysine residue (K494), which increases its interaction with EZH2. We hypothesized that this residue is also acetylated. Our results show that REST can indeed be acetylated and, interestingly, more so in SIRT6-KO cells. This might suggest that there is a PTM balance that can influence REST function. If methylation of K494 is beneficial for REST function and interaction with EZH2, an acetylation event can keep this residue from being methylated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen the K494 residue is mutated to alanine, REST acetylation is reduced; moreover, the acetylation increase seen in KO cells is lost, suggesting SIRT6 could be the deacetylase. However, we failed to see direct interaction between SIRT6 and REST by Co-IP or mass spec (although substrate-enzyme interactions are fast and maybe undetected in our system). Because of the lack of specific antibodies, we could not effectively measure deacetylation in vitro, and this question remains open.\u003c/p\u003e\n\u003cp\u003eREST nuclear distribution is lost when K494 is changed to alanine as a preventive for charged modifications and Q (mimicking acetylation) in both WT and SIRT6 deficient cells. Moreover, the M mutant, which mimics methylation, pushes REST to its\u0026nbsp;\u003cspan dir=\"RTL\"\u003e\u0026ldquo;\u003c/span\u003efunctional\u0026rdquo; phenotypes even in the SIRT6-KO cells. Moreover, REST-494M can restore the gene expression of REST target genes in SIRT6 deficient cells, while WT-REST cannot, indicating that location and function can be interconnected.\u003c/p\u003e\n\u003cp\u003eAll the REST PTMs mutants have a similar distribution in the genome (regarding regions they prefer to bind). Still, methyl-mimic can bind many more target genes that the acetyl or the non-modifiable mutants, strengthening the point that methylation helps REST in its repressive function.\u003c/p\u003e\n\u003cp\u003eOverall, we found that SIRT6 regulates REST expression and activity in the cell, allowing it to repress neuronal genes. This is done by a molecular switch in the acetylation/methylation status of REST, allowing it to interact with EZH2 and localize to nuclear lamina. The functional relevance of SIRT6 depletion in the aging brain and neurodegenerative diseases could influence the capacity of REST to protect the brain from neurodegeneration and could be the cause of REST loss of function, even when expressed at high levels, as observed in AD and PD; therefore, the SIRT6-REST-EZH2 pathway could be relevant to target for future research.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAZ planned, performed the experiments and wrote the paper. AGV, EE, DS, YR, RD and ME performed experiments, DS, DK and EK analyzed data and DT planned and wrote the paper\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was funded by the European Research Council (ERC) under the European Union\u0026rsquo;s Horizon 2020 research and innovation program (grant agreement No 849029), the David and Inez Myers foundation, the Israeli Ministry of Science and Technology (MOST), the High-tech, Bio-tech and Negev fellowships of Kreitman School of Advanced Research of Ben Gurion University.\u003c/p\u003e\u003ch2\u003eData and code availability\u003c/h2\u003e \u003cp\u003e ATAC-seq and ChIP-seq data have been deposited at GEO and are publicly available as of the date of publication. Accession numbers are listed in the key resources table.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChong, J.A., Tapia-Ramirez, J., Kim, S., Toledo-Aral, J.J., Zheng, Y., Boutros, M.C., Altshuller, Y.M., Frohman, M.A., Kraner, S.D., and Mandel, G. (1995). REST: A mammalian silencer protein that restricts sodium channel gene expression to neurons. Cell \u003cem\u003e80\u003c/em\u003e, 949\u0026ndash;957. 10.1016/0092-8674(95)90298-8.\u003c/li\u003e\n\u003cli\u003eThiel, G., Ekici, M., and R\u0026ouml;ssler, O.G. (2015). RE-1 silencing transcription factor (REST): a regulator of neuronal development and neuronal/endocrine function. Cell Tissue Res \u003cem\u003e359\u003c/em\u003e, 99\u0026ndash;109. 10.1007/s00441-014-1963-0.\u003c/li\u003e\n\u003cli\u003eJones, F.S., and Meech, R. (1999). Knockout of REST/NRSF shows that the protein is a potent repressor of neuronally expressed genes in non-neural tissues. 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Nucleic Acids Research \u003cem\u003e51\u003c/em\u003e, D1003\u0026ndash;D1009. 10.1093/nar/gkac888.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4371623/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4371623/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eRE-1 Silencing Transcription factor (REST)\u003c/em\u003e \u003cem\u003eis a key repressor of neural genes. REST is upregulated under stress signals, aging and neurodegenerative diseases, but although it is upregulated, it loses its function in Alzheimer's Disease. However, why it becomes inactive remains unclear. Here, we show that the NAD-dependent deacetylase SIRT6 regulates REST expression, location and activity. In SIRT6 absence, REST is overexpressed but mislocalized, and loses part of its activity, becoming toxic. SIRT6 deficiency abrogates REST and EZH2 interaction, perturbs its location to heterochromatin Lamin B ring, and leads to REST target gene overexpression. SIRT6 reintroduction or REST methyl-mimic K494M expression rescues this phenotype, while an acetyl-mimic mutant loses its function even in WT cells. Our studies define a novel regulatory switch, where the function of a critical repressor is regulated by post-translational modifications on K494, depending on SIRT6 existence and, in turn, modulating neuronal gene expression.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"SIRT6-dependent functional switch via K494 modifications of RE-1 Silencing Transcription factor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-17 16:23:06","doi":"10.21203/rs.3.rs-4371623/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-09-12T10:13:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-07-22T12:34:35+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-12T15:49:53+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-07-10T16:39:32+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-06-21T14:13:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-05-15T10:48:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-14T17:45:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2024-05-14T17:45:52+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b92046ce-de34-4b64-b20f-5e73e1cb599d","owner":[],"postedDate":"July 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":33562933,"name":"Biological sciences/Molecular biology/Epigenetics"},{"id":33562934,"name":"Biological sciences/Molecular biology/Transcription"}],"tags":[],"updatedAt":"2024-11-08T08:10:24+00:00","versionOfRecord":{"articleIdentity":"rs-4371623","link":"https://doi.org/10.1038/s41419-024-07160-0","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2024-11-07 05:00:00","publishedOnDateReadable":"November 7th, 2024"},"versionCreatedAt":"2024-07-17 16:23:06","video":"","vorDoi":"10.1038/s41419-024-07160-0","vorDoiUrl":"https://doi.org/10.1038/s41419-024-07160-0","workflowStages":[]},"version":"v1","identity":"rs-4371623","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4371623","identity":"rs-4371623","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}