MLL and KDM5A activity during cell cycle progression depend on Ras signalling

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Kirtana, Soumen Manna This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4332860/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cell cycle progression is regulated by many extracellular stimuli and intracellular signaling. Interaction between different epigenetic modifiers and transcription factors regulate the expression of genes encoding proteins involved in cell cycle control. Along with the cyclin-CDK complexes and phosphatases, RAS- signaling play crucial role to direct the cell passage through different stages of cell cycle. In this scenario, chromatin configuration is important for the progression of cell division and chromatin modifications (DNA methylation and histone modifications) helps to attain correct chromatin folds. Here, in this study we analyzed how modulation of H3K4me3 by MLL1 and KDM5A affect cell cycle progression. As slow and fast cycling cell lines exhibited differences in mechanisms of regulation, from in-silico screening and experimental demonstration we deciphered that the expression of the MAPK effector, RAS is involved to controlling the expression and activity of KDM5A and MLL proteins to balance H3K4me3 oscillation throughout cell cycle. Cell Cycle & Proliferation Cell cycle KDM5A MLL1 H3K4me3 RAS signalling Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The mammalian cell cycle includes four successive phases: G1 (the post mitotic interphase), S (the DNA synthetic phase), G2 (the post synthetic phase) and M (mitotic phase) and progression through these phases is an outcome of coordinated regulation of multiple protein complexes and inhibitory signals (i.e., checkpoints). The epigenetic landscape of a cell also dictates progression through a certain phase. Although most of the chromatin modifications were reported to change during cell cycle, but global H3K4me3 level were shown to be constant during cell cycle [ 1 , 2 ] along with the respective writers (MLLs) and erasers (KDM5A) [ 3 , 4 ]. Though functional characterization of the exact roles of MLL and KDM5 family proteins is lacking, altering the expression of these proteins led to cell cycle arrest by different mechanisms. Some studies have shown that siMLL1 treatment led to G2/M arrest associated with downregulation of cyclin A, cyclin B and p57 [ 5 ], and overexpression of MLL1 led to S-phase arrest [ 6 ], others suggested a G0/G1 arrest when amino-terminal MLL mutants were expressed in U937 cells whereas overexpression of carboxy terminal sequences lead to accumulation of population in S-phase [ 7 ]. Some of the earlier investigations implicated that reduction in the histone demethylase, KDM5A, mostly led to G1 arrest associated with reduced cyclin D/E expression and enhanced CDK inhibitors (p16, p21, p27, 57) in breast, gastric, lung, osteosarcoma, renal and neuroendocrine cancers [ 8 – 12 ]. During mouse MEF and ES differentiation, KDM5A was reported to coordinate with pRB protein and E2F4 transcription factors and repress mitochondrial and cell cycle related genes respectively [ 13 – 14 ]. In contrast to the above results, a study on adipocyte differentiation found that cells with KDM5A-KD could not induce cell cycle genes during early differentiation and displayed significant inhibition of DNA synthesis and mitotic expansion proving that KDM5A is required for the induction of cell cycle genes [ 15 ]. Knockdown of KDM5B (a member of KDM5 family) in gastric and hepatic cancers also led to G1/S arrest with enhanced p15 and p27 expression [ 16 – 17 ]. In studies with KDM5B-KD treated melanoma cells, when cells were stimulated with different concentrations of FCS (i.e., 2%, 5%, and 10%) over different time points, a G2/M arrest was detected but KD in serum starvation conditions (i.e., 0% FCS) led to increase in S-phase population [ 18 ]. The process of cell cycle is monitored by chromatin architecture, but certain epigenetic modifiers are regulated by cell cycle machinery as well [ 1 , 2 ]. Modulation of gene expression following activation by CDK2 mediated phosphorylation of MLL2 – at thr5099 was reported to enhance G1-specific transcription [ 19 ]. Further, the inactive precursor MLL2 could not induce cyclins (E, A and B) [ 20 ]. Histone demethylase KDM5B was reported to be phosphorylated by CDK1 thereby reducing its potential to be recruited to SOX2 and NANOG promoters [ 21 ]. ]. RAS-signaling exerts oncogenic signal [ 22 ], and imparts in cancer progression by regulation of DNA methylation [ 23 , 24 ]. With all these diverse reports on how H3K4me3 writers and erasers regulate cell cycle, it is unclear whether changes in KDM5A and MLLs affects cell cycle progression by altering global H3K4me3 or regulate cell cycle related gene expression (i.e., cyclins, CDKs or CKIs). Through rigorous investigations, we report how KDM5A impart cell cycle progression. We discovered that KDM5A exhibits diverse functionality in different cell lines, depending on other signalling events that might enhance its function. Further, we demonstrated that RAS proteins regulate the activity of KDM5A affecting global levels of H3K4me3. Materials and methods Cell culture conditions HeLa and HaCaT cells were cultured in MEM and DMEM media respectively, with 10% FBS (Gibco − 10270106) and 1% anti-anti. PC3 was grown in F12 media supplemented with 15% FBS, 1% anti-anti and (Gibco − 15240-062) L-glutamine. During treatments, depending on cell doubling size, seeding density was calculated to attain 70% confluency within 24hrs, following transfections. For microscopy experiments, seeding density was reduced to avoid overcrowding. siRNA and pcDNA transfection In HeLa and HaCaT cell lines, both knockdown and overexpression was performed for 48hrs, but in PC3 the treatment duration was 24hrs. Transfection was attained using lipofectamine 3000 (Invitrogen L3000-15) following manufacturer’s instructions. Plasmid concentration varied from 6 well to 60mm plate and we used 5 or 10ug respectively to induce KDM5A overexpression. The pcDNA-triple epitope SFB-tagged-RBP2 (KDM5A) overexpression construct was borrowed from Dr. Shweta Tyagi (CDFD). The YFP-KRAS pcDNA3.1 was acquired from Addgene. Cell cycle distribution analysis by flow cytometry: Cell cycle distribution was assessed by plating 1* 10 5 in a 6 well plate, and the following day, the cells were treated with siRNA or pcDNA constructs for 48hrs, followed by trypsinization, washed with PBS (twice), fixed with 100% ethanol for an hour at -20 o C, followed by treatment with 10ug/ml of RNase-A (Himedia – MB087) at 37 o C in PBS for an hour and addition of 1ug/µl propidium iodide (Sigma − 81845). Cellular DNA content was measured using BD accuriC6 FL2 filter and cell cycle distribution was analysed with BD software. While analysing cell cycle in serum starved cells, HeLa and HaCaT cells were maintained in media with antibiotics for three days, following treatments in Opti-MEM. Gene expression profiling: UALCAN database was used to assess GRB2, SOS1 and different RAS transcript profile in cervical and prostate tumor tissues relative to its expression in normal tissue. The expression profile of the above-mentioned genes across multiple cell lines was extracted from human protein atlas. Western blotting Whole cell lysate prepared by RIPA lysis buffer (sigma –R0278-50ML) were electrophoresed on 8–12% SDS-PAGE gels depending on target proteins analysed as per previous protocol [ 25 ], resolved and transferred to nitrocellulose membrane (Axiva – 160300RI), blocked with 5% skim milk for an hour. All primary antibodies were prepared in 1% BSA (Himedia-MB083) in PBST and the blots were probed with primary antibodies at 4 o C overnight. Following three washes with PBST, the blots were incubated with either anti-rabbit (Invitrogen-65-6120) or anti-mouse (Santa Cruz – SC516102) secondary antibody for an hour. The blots were then washed thrice using PBST, visualized using ECL chemiluminescence detection system (Thermo- 34580). Antibodies used: KDM5A (SC-365993) and β-actin (SC-47778) were from Santa Cruz. Immunofluorescence HeLa, HaCaT and PC3 cells were seeded on a coverslip in a 6-well plate, transfected with YFP-RAS construct for 48hrs followed by fixation using 100% methanol for 5 minutes, permeabilized with PBST on ice for 10 minutes and blocked using 1% BSA in PBST for 1hr. The cells were incubated with anti-H3K4me3 (Invitrogen MA5-11199) and MLL2 (Abcam - ab56770) antibody overnight at 4 o C in a humid chamber. Following 3 washes with PBS, the cells were incubated in Alexa488 (anti-rabbit ab150077) and Alexa 647 (anti-mouse – ab150119) washed thrice with PBS and images were acquired using Leica microsystems. Statistical analysis Either student’s ‘t’ test or two-way ANOVA followed by Bonferroni correction were employed to test the significance, and the test employed along with the p-values and their inference is mentioned in Figure legends. Results To understand the cell cycle distribution under KDM5A knockdown, overexpression and MLL1 knockdown conditions, we choose cervical (HeLa), Prostate (PC3) and Keratinocyte (HaCaT) cell lines. Following transfections and PI staining, we observed that in HeLa ( Fig. 1 a-b ) and HaCaT ( Fig. 1 c-d ) cell lines, KDM5A knockdown led to an increase in G2/M population compared to siControl, and KDM5A overexpression (KDM5A-OE) led to S-phase arrest. MLL1-knockdown (MLL1-KD) also led to S-phase arrest. From the cell cycle distributions, we understood that these observations are a consequence of global changes in H3K4me3 as siKDM5A would increase the active mark and render the chromatin in open configuration thereby influencing the G2/M phase transition, as normally chromatin compaction should commence at this stage and enhanced H3K4me3 hinders with this process. In KDM5A-OE and ML1-KD, as both these conditions reduce H3K4me3, an S-phase arrest was noted due to highly packaged DNA being rendered inaccessible for replication process in S-phase. In PC3 cell line, although the effects were subtle but a reverse trend was observed, i.e., siKDM5A led to S-phase arrest and KDM5A-OE led to G2/M arrest ( Fig. 1 e-f ) . One difference between PC3 and the other two cell lines used is that PC3 is a slow growing cell line. So, to test whether a slow culturing HeLa cell would also exhibit similar cell cycle distribution, we serum starved HeLa cells to reduce their high proliferative ability and performed similar knockdown and overexpression experiments as mentioned above. To our surprise, serum starved HeLa cell cycle distribution ( Fig. 2 a-b ) was similar to the PC3 distribution rather than normally cultured HeLa (10% FBS) cells. Serum starvation and siKDM5A treatment led to S-phase arrest instead of G2/M; whereas KDM5A overexpression and siMLL1 led to an increase in G2/M population instead of S-phase as observed in Fig. 1 a-b. Further, from earlier reports on KDM5B (a member of KDM family of demethylases) in melanoma cells, when the cells were serum starved, knockdown of KDM5B led to S- phase increase but when stimulated with different concentrations of FCS, a rise in G2/M population occurred as seen in our experiments. In line with this, when we assessed the expression of KDM5A at different serum concentrations, serum starvation (0% FBS) enhanced KDM5A but high percentage of FBS (20%) reduced KDM5A levels ( Fig. 2 c ). From our observations so far and other literature, we hypothesized that an interplay exists between KDM5A and some factors of FBS. As growth factors are the major signaling molecules of FBS and can trigger the RTK signaling during cell cycle according to KEGG identifier 04110N pathway, we sought to assess the differences among growth factor signaling molecules. As we used different cell lines in the study which could be triggered by different growth factors and possess diverse receptors, we assessed the initial cytosolic effector molecules of RTK signaling and analyzed GRB1, SOS1 and RAS expression among cervical and prostate cancers using TGCA datasets. We observed that GRB2 expression was marginally enhanced in cervical cancer patients but not in prostate cancer ( Fig. 3 ) , and SOS1 was downregulated in both ( Fig. 3 ) . As RAS proteins are most mutated among cancers, we assessed KRAS, NRAS and HRAS, and found that KRAS and NRAS were highly expressed in cervical cancer patients and in HeLa and HaCaT cell lines when compared to PC3 in which they were unaffected and less expressed ( Fig. 4 , a &b). Unlike KRAS and NRAS, HRAS was more expressed in PC3 cell line, and enhanced in both prostate and cervical cancer patients, but its expression was twice that of normal in cervical patients compared to PC3 where only marginal changed were noted ( Fig. 4 c ). Following the in-silico gene expression analysis, we speculated that although GRB2 and SOS1 were slightly upregulated (in cervical) or remained unregulated, but RAS proteins were highly expressed in HeLa and HaCaT compared to PC3, and might affect KDM5A expression or activity. From literature survey we noticed that many epigenetic markers are target to CDKs, and from our results we see that cells grown in serum starved conditions exhibited altered KDM5A or MLL1 functioning when compared to cells grown in 10% FBS, we hypothesized that RAS proteins might activate/enhance KDM5A enzymatic activity or reduce MLL1 methyltransferase activity downstream of MAPK signalling. To test our hypothesis, we overexpressed KRAS ( Fig. 5 a ) and analyzed if RAS overexpression affects KDM5A expression. As shown in Fig. 5 b, we traced that KRAS overexpression led to reduction in KDM5A protein. KDM5A protein content is reduced in enhanced RAS conditions, however, RAS might enhance the demethylase activity of the residual KDM5A. To test whether RAS enhanced KDM5A demethylase activity, we overexpressed RAS in HaCaT cells and assessed the H3K4me3 levels as an indirect estimate of the demethylase function of KDM5A. We noted that RAS overexpression reduced H3K4me3 ( Fig. 5 c ) , supporting that RAS might activate/enhance KDM5A’s enzymatic function. Along this line, we also tested if inhibiting MAPK signalling enhanced H3K4me3 levels. Using U0126 (MEK inhibitor) in HaCaT cells, we analysed the levels of the writer (MLL2) and the modification and observed that both were increased (Fig. 5 d). From this, we concluded that reduced MAPK signalling activates MLLs function thereby increasing H3K4me3, and increased MAPK activity increases KDM5A’s demethylase function thereby reducing H3K4me3. As we have observed that RAS overexpression reduced H3K4me3, we tested whether transfection of both RAS and KDM5A together in serum starved HeLa and HaCaT cells would enhance KDM5A’s demethylase activity and enhance cell population in S-phase instead of G2/M as observed in Fig. 2 . So, when this double transfection was performed, an increase in S-phase population was noted when compared to KDM5A and RAS transfection alone ( Fig. 6 , a-d ) . Further, in PC3 cells which exhibit low RAS proteins and KDM5A expression in normal conditions (10% FBS) led to G2/M arrest, RAS and KDM5A double transfection led to increase in S-phase cells ( Fig. 6 e &f) , proving that KDM5A activity was monitored by RAS or the downstream signalling molecules and global H3K4me3 status dictated by KDM5/MLL ratio was responsible for the changes observed in this study. Discussion Transition through different phases of cell cycle is majorly governed by three classes of proteins; CDKs, cyclins, and phosphatases; while precision is maintained by multiple proteins at restriction check points. Apart from the proteins that ensure faithful replication and segregation, chromatin configuration was described to be the heart of cell cycle progression [ 26 ] and many histone modifications are said to play a role during replicative licencing, act as barrier to prevent re-replication and aid in compaction of chromatin into chromosomes. Chromatin structure is dictated by the epigenetic modifications and how changes in individual modification effects cell cycle is yet be elucidated. Changes in the amount of writer and eraser proteins alter global and gene specific occupancy of different modifications. When a certain epigenetic modifier expression is targeted by knockdown or overexpression, whether the global changes or gene-specific expression pattern lay influence over cell cycle is a question that need addressing. Recently, we have deciphered that KDM5A in association with MLL1/2 regulates expression of genes associated with EMT; where, as repressor of genes KDM5A demethylates H3K4me3 to H3K4me, and activator of gene it does not demethylate H3K4me3 but in association with MLL inhibits histone deacetylase activity [ 27 ]. Here, through this study, we elucidated how changes in global H3K4me3 effect cell cycle distribution. We targeted the writer MLL1 and eraser KDM5A to reveal the changes that emerge following their knockdown and overexpression in HeLa, HaCaT and PC3 cell lines. When KDM5A knockdown was performed in HeLa and HaCaT cell lines, G2/M arrest was observed as increased H3K4me3 obstruct chromatin compaction, while KDM5A-OE and MLL1-KD led to an increase in S-phase population as reduced H3K4me3 could not allow DNA synthesis due to compacted structure. But a reverse trend was observed in PC3 cell line. In our culture conditions, PC3 was slow growing compared to HeLa and HaCaT, and from literature review, we understood that KDM5B-KD in low serum conditions led to S-phase arrest and when knockdown was performed in cells cultured with serum - G2/M arrest was seen [ 20 ]. We thus performed in-silico screening of effector molecules of growth factor signalling – i.e., MAPK pathway. From gene expression data obtained through TCGA datasets and transcript profile of cell lines acquired from human protein atlas, we understood that RAS expression is reduced in PC3 cell line compared to HeLa and HaCaT. As many reports present that cell cycle regulators can activate or suppress epigenetic modifier activity, we tested whether RAS could have any effect of the demethylase activity of KDM5A. As HeLa and HaCaT had more RAS expression, we speculated that KDM5A overexpression in these cell lines at normal FBS conditions (10%) led to S-phase arrest as KDM5A demethylase was active, but in PC3 where RAS expression is inherently low, KDM5A overexpression did not arrest cells in S-phase but instead in G2/M probably because KDM5A could not be enzymatically activated. When such slow culturing conditions were replicated in HeLa cells, cell cycle distribution similar to PC3 was observed. As lack of RAS mediated signalling led to reduced demethylase activity in PC3 and serum starved HeLa cells – which led to G2/M arrest instead of S-phase, we overexpressed RAS in serum starved HeLa and HaCaT cells to see if supplementing RAS overexpression in place of serum would enhance KDM5A activity. As expected, when RAS and KDM5A were overexpressed together in serum starved cells, and increase in S-phase population was noted when compared to KDM5A or RAS overexpression alone. Similar results were observed in PC3, proving that RAS somehow enhanced the demethylase activity of KDM5A. Concluding our study, we depicted KDM5A and MLL1 enzyme activity is cell line or tissue dependent as many proteins of different signalling pathways activate these proteins, MAPK being one among them. It was not just KDM5A, we unveiled that MAPK pathway regulate both writers and erasers of H3K4me3 to balance the distribution of this active mark as inhibiting MEK activity enhanced MLL2 and H3K4me3 levels; earlier work from our laboratory has shown MAPK pathway inhibition using U0126 [ 28 ]. Although many reports claim that the expression of KDM5A [ 4 ], and MLLs [ 3 ] do not change through cell cycle, and so is the H3K4me3 modification, recent reports claim that MLLs localization during cell cycle varies and H3K4me3 at gene promoters is modified in a phase dependent manner. Here we show that global changes in H3K4me3 affects cell cycle distribution and a more detailed investigation on the mechanism of regulation is needed. Declarations Acknowledgements: R. Kirtana received fellowship from CSIR, Govt. of India (CSIR-09/983(0018)/2017-EMRI). S. Manna is thankful to NIT-Rourkela for fellowships under the Institute Research Scheme, NIT-Rourkela. We thank Dr. Shweta Tyagi (CDFD Hyderabad) for SFB-RBP2 and GST-RBP2 deletion (D1-D5) constructs. Author Contributions: Samir Kumar Patra: Conceptualization, Supervision, and Editing. Kirtana R: Formulation, Methodology, Data curation, Analysis, Original draft preparation, and Review. Soumen Manna: Data curation and analysis. Conflict of Interest: None Funding: This work is supported in parts by the Department of Biotechnology (Government of India) project No.: BT/PR21318/MED/12/742/2016 to SKP. References Epigenetic dynamics across the cell cycle. Tony Bou Kheir and Anders H. Lund1. Essays Biochem. (2010) 48, 107–120; doi:10.1042/BSE0480107 Epigenetics and cell cycle regulation in cystogenesis. Xiaogang Lia,b,⁎. Cellular Signalling 68 (2020) 109509. Dynamic association of MLL1, H3K4 trimethylation with chromatin and Hox gene expression during the cell cycle. Bibhu P. Mishra, Khairul I. Ansari and Subhrangsu S. Mandal. FEBS Journal 276 (2009) 1629–1640. 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Mallory, David Latrasse, Teddy Jégu, Quentin Bruggeman, Marianne Delarue, Catherine Bergounioux, Moussa Benhamed, Journal of Experimental Botany , (2014) 65:2677–2689. KDM5A noncanonically binds antagonists MLL1/2 to mediate gene regulation and promotes epithelial to mesenchymal transition. Kirtana R, Manna S, Patra SK. Biochim Biophys Acta Gene Regul Mech. 2023 Dec;1866(4):194986. doi: 10.1016/j.bbagrm.2023.194986. Epigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer. Pradhan N, Parbin S, Kar S, Das L, Kirtana R, Suma Seshadri G, Sengupta D, Deb M, Kausar C, Patra SK. Biochim Biophys Acta Mol Basis Dis. (2019) 1865(6):1651-1665. Additional Declarations The authors declare no competing interests. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4332860","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":296073484,"identity":"ae37e1a9-0223-4444-abc2-734f296c6227","order_by":0,"name":"Samir Patra","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAz0lEQVRIiWNgGAWjYJCCAwwMNgxsDAzMDA9AXGbitKQxsLEB1SYQqwUIDjMwwLUQAvLtpxMPV+44n8cn3/vYIIHBTp6BnfcAXi0GZ3I3HDx75nYxGxu7cUICQ7JhAzMffssMGIBaGttuJ7axsTEfSGBgBiIeA/wO638L0nIOpqWesBaGG2BbDoC1AB12mLAWgxsgW84kA7WkMRskGBw3bCPssNzNHxt32CXObz7GLPGholqen/8MAYeBAGMD3FJQ/BADEFpGwSgYBaNgFGABAE1gP4Mw9DqAAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5641-1835","institution":"National Institute of Technology Rourkela","correspondingAuthor":true,"prefix":"","firstName":"Samir","middleName":"","lastName":"Patra","suffix":""},{"id":296074400,"identity":"1044c504-5cf5-4ccf-ac10-94baff4986bc","order_by":1,"name":"R. Kirtana","email":"","orcid":"","institution":"National Institute of Technology Rourkela","correspondingAuthor":false,"prefix":"","firstName":"R.","middleName":"","lastName":"Kirtana","suffix":""},{"id":296074401,"identity":"65f5eb07-3ca3-4389-9b65-bd48694766ad","order_by":2,"name":"Soumen Manna","email":"","orcid":"","institution":"National Institute of Technology Rourkela","correspondingAuthor":false,"prefix":"","firstName":"Soumen","middleName":"","lastName":"Manna","suffix":""}],"badges":[],"createdAt":"2024-04-27 07:35:24","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4332860/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4332860/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55569598,"identity":"8fab7147-e0a1-4198-bcc7-6d574509bbe4","added_by":"auto","created_at":"2024-04-30 05:01:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":637744,"visible":true,"origin":"","legend":"\u003cp\u003eCell cycle distribution assessed by using PI stain followed by flow cytometry\u003cstrong\u003e. \u003c/strong\u003eHeLa \u003cstrong\u003e(a, b)\u003c/strong\u003e, HaCaT \u003cstrong\u003e(c, d)\u003c/strong\u003e and PC3 \u003cstrong\u003e(e, f) \u003c/strong\u003ecells were transfected with siKDM5A, KDM5A overexpression (OE) construct (i.e., pcDNA-SFB-RBP2) and siMLL1 for 48hrs followed by PI staining and flow cytometric analysis to assess cell cycle distribution. The experiments were performed in triplicate and the bar graphs in are representative of three independent replicates shown as mean ± SD, and p≤0.05 as calculated by Prism5.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4332860/v1/d8e0def9184b0eef64c5f61f.png"},{"id":55569599,"identity":"9b794628-2c73-41cd-955b-472ff33b588f","added_by":"auto","created_at":"2024-04-30 05:01:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":509263,"visible":true,"origin":"","legend":"\u003cp\u003eFlow cytometric distribution of serum starved HeLa cells following siKDM5A, KDM5A-OE and siMLL1 transfections for 48hrs followed by PI staining. The percentage of cells in different phases were quantified using BD-accuri C6 software and the treated samples were compared to either siControl or normal cells \u003cstrong\u003e(a)\u003c/strong\u003e. Triplicate samples were assessed and p\u0026lt;0.05 as measured by Prism5, data is presented as mean ± SD \u003cstrong\u003e(b)\u003c/strong\u003e. \u003cstrong\u003e(c). \u003c/strong\u003eImmunoblots representing KDM5A expression following serum starvation and excess FBS supplementation (i.e., 20%)\u003cstrong\u003e. \u003c/strong\u003eThe densitometric estimation of immunoblots by ImageJ software and presented as mean ± SD, followed by analyzing statistical significance where **P≤0.01, ***P≤0.001 and ns is non-significant when compared to 10% FBS treated cells.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4332860/v1/85445e4b9bb43b4bd2c81e1a.png"},{"id":55569600,"identity":"7492c1f1-5b8c-4354-b888-0b1f2e20249b","added_by":"auto","created_at":"2024-04-30 05:01:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":128022,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a). \u003c/strong\u003eBox plots indicating the expression of GRB2 in cervical (CESC) and prostate (PRAD) tumors compared to their respective normal tissues was obtained using UALCAN database. GRB2 was observed to be marginally enhanced in cervical cancer patients (n=305) compared to normal (n=3), but reduced in prostate cancer patients (n=497) vs normal (n=52)\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003e(b). \u003c/strong\u003eBox plots indicating the expression of SOS1 in cervical (CESC) and prostate (PRAD) tumors compared to their respective normal tissues was obtained using UALCAN database. SOS1 was observed to be reduced in both cervical cancer (n=305) and prostate cancer patients\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4332860/v1/a8e8de52a5028b482d9a182a.png"},{"id":55569601,"identity":"668caa04-8cd2-432a-b091-1e3d062a3306","added_by":"auto","created_at":"2024-04-30 05:01:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1166813,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a). \u003c/strong\u003eKRAS expression was observed to be higher in HeLa and HaCaT cell lines compared to PC3, and the while it remained unaltered in prostate cancer patient samples, KRAS expression doubled in cervical cancer patient samples when compared to normal. \u003cstrong\u003e(b).\u003c/strong\u003e NRAS expression was observed to be three times higher in cervical patient samples vs normal and reduced in prostate cancer patients as observed from TGCA datasets. Comparatively, HeLa and HaCaT exhibited higher transcript expression than PC3 cell line as seen in Human protein atlas data. \u003cstrong\u003e(c). \u003c/strong\u003eA marginal increase in HRAS expression was noticed in prostate cancer patient samples when compared to normal, and in cervical cancer patients, the expression almost doubled (median TPM = 97.165) when compared to normal (median TPM = 43.194). PC3 cell line exhibited higher HRAS expression than HeLa and HaCaT.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4332860/v1/a4dd6fafb3a6a5097db5d9db.png"},{"id":55569604,"identity":"12686fcd-1915-4d2d-9ba7-fb6760d55c87","added_by":"auto","created_at":"2024-04-30 05:01:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2633831,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a). \u003c/strong\u003eConfirmation of YFP-tagged RAS overexpression by microscopy following transient transfection of YFP-KRAS pcDNA3.1 in HeLa and PC3 cells. \u003cstrong\u003e(b). \u003c/strong\u003eAnalyzing KDM5A expression following YFP-RAS overexpression. Hela, HaCaT and PC3 cells were transfected with RAS overexpression construct and the whole cell protein lysate was subjected to SDS-PAGE followed by immunoblotting using anti-KDM5A antibody. Blots presented are one of the triplicates, and statistical significance is represented as mean ± SD with P\u0026lt;0.001 when compared to untreated control. \u003cstrong\u003e(c). \u003c/strong\u003eImmunofluorescence images showing reduced H3K4me3 on RAS overexpression in HaCaT cells, the experiments were performed in triplicates the mean fluorescence intensity with SD\u003cstrong\u003e, \u003c/strong\u003eand the statistical significance was calculated using Prism5 software where ***P≤0.001 when compared to control. \u003cstrong\u003e(d). \u003c/strong\u003eImmunofluorescence images showing enhanced H3K4me3 associated with increased MLL2 expression in HaCaT cells; the experiments were performed in triplicates, the mean fluorescence intensity with SD is presented as bar graph\u003cstrong\u003e, \u003c/strong\u003eand the statistical significance was calculated using Prism5 software where *P≤0.05 when compared to control.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4332860/v1/641fcdcdd9a0f5ca345d93ce.png"},{"id":55569603,"identity":"419ddcd8-510b-449d-bb39-5d4062b62b7d","added_by":"auto","created_at":"2024-04-30 05:01:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":401007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a-b). \u003c/strong\u003ePercentage of cells distributed into different phases of cell cycle as analyzed by flow cytometric histograms following overexpression treatments in serum starved HeLa cells, triplicate experiments were performed and data is presented as mean ± SD and p\u0026lt;0.05 as calculated by Prism5. \u003cstrong\u003e\u0026nbsp;(c, d). \u003c/strong\u003eFlow cytometry of serum starved HaCaT cells showing distribution of cell population after overexpression treatments\u003cstrong\u003e, \u003c/strong\u003equantitative analysis of percentage of cells in different phases of cell cycle represented as mean ± SD with a significant difference of p\u0026lt;0.05 when compared with control groups. \u003cstrong\u003e(e, f). \u003c/strong\u003eEffects of KDM5A+ RAS overexpression treatment on the cell cycle distribution of PC3 cells as assessed by flow cytometry\u003cstrong\u003e, \u003c/strong\u003ethe data is shown as mean\u003cstrong\u003e \u003c/strong\u003e± SD from three independent experiments\u003cstrong\u003e, \u003c/strong\u003eand p\u0026lt;0.05 when compared to untreated control.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4332860/v1/11036f580de550752b77a434.png"},{"id":55570037,"identity":"b5baa815-fa45-47b0-89fc-1f51c9700030","added_by":"auto","created_at":"2024-04-30 05:09:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2238984,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4332860/v1/e6f0dba4-0ba5-4fa5-8978-f5a180c26d59.pdf"},{"id":55569597,"identity":"4925f6e8-72e2-425a-bba3-9b0c560715c3","added_by":"auto","created_at":"2024-04-30 05:01:43","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":177247,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.png","url":"https://assets-eu.researchsquare.com/files/rs-4332860/v1/e6de7d7935714b3139b15fa6.png"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eMLL and KDM5A activity during cell cycle progression depend on Ras signalling\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe mammalian cell cycle includes four successive phases: G1 (the post mitotic interphase), S (the DNA synthetic phase), G2 (the post synthetic phase) and M (mitotic phase) and progression through these phases is an outcome of coordinated regulation of multiple protein complexes and inhibitory signals (i.e., checkpoints). The epigenetic landscape of a cell also dictates progression through a certain phase. Although most of the chromatin modifications were reported to change during cell cycle, but global H3K4me3 level were shown to be constant during cell cycle [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] along with the respective writers (MLLs) and erasers (KDM5A) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThough functional characterization of the exact roles of MLL and KDM5 family proteins is lacking, altering the expression of these proteins led to cell cycle arrest by different mechanisms. Some studies have shown that siMLL1 treatment led to G2/M arrest associated with downregulation of cyclin A, cyclin B and p57 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], and overexpression of MLL1 led to S-phase arrest [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], others suggested a G0/G1 arrest when amino-terminal MLL mutants were expressed in U937 cells whereas overexpression of carboxy terminal sequences lead to accumulation of population in S-phase [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSome of the earlier investigations implicated that reduction in the histone demethylase, KDM5A, mostly led to G1 arrest associated with reduced cyclin D/E expression and enhanced CDK inhibitors (p16, p21, p27, 57) in breast, gastric, lung, osteosarcoma, renal and neuroendocrine cancers [\u003cspan additionalcitationids=\"CR9 CR10 CR11\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. During mouse MEF and ES differentiation, KDM5A was reported to coordinate with pRB protein and E2F4 transcription factors and repress mitochondrial and cell cycle related genes respectively [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In contrast to the above results, a study on adipocyte differentiation found that cells with KDM5A-KD could not induce cell cycle genes during early differentiation and displayed significant inhibition of DNA synthesis and mitotic expansion proving that KDM5A is required for the induction of cell cycle genes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Knockdown of KDM5B (a member of KDM5 family) in gastric and hepatic cancers also led to G1/S arrest with enhanced p15 and p27 expression [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In studies with KDM5B-KD treated melanoma cells, when cells were stimulated with different concentrations of FCS (i.e., 2%, 5%, and 10%) over different time points, a G2/M arrest was detected but KD in serum starvation conditions (i.e., 0% FCS) led to increase in S-phase population [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe process of cell cycle is monitored by chromatin architecture, but certain epigenetic modifiers are regulated by cell cycle machinery as well [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Modulation of gene expression following activation by CDK2 mediated phosphorylation of MLL2 \u0026ndash; at thr5099 was reported to enhance G1-specific transcription [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Further, the inactive precursor MLL2 could not induce cyclins (E, A and B) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Histone demethylase KDM5B was reported to be phosphorylated by CDK1 thereby reducing its potential to be recruited to SOX2 and NANOG promoters [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. ]. RAS-signaling exerts oncogenic signal [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and imparts in cancer progression by regulation of DNA methylation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith all these diverse reports on how H3K4me3 writers and erasers regulate cell cycle, it is unclear whether changes in KDM5A and MLLs affects cell cycle progression by altering global H3K4me3 or regulate cell cycle related gene expression (i.e., cyclins, CDKs or CKIs). Through rigorous investigations, we report how KDM5A impart cell cycle progression. We discovered that KDM5A exhibits diverse functionality in different cell lines, depending on other signalling events that might enhance its function. Further, we demonstrated that RAS proteins regulate the activity of KDM5A affecting global levels of H3K4me3.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eCell culture conditions\u003c/h2\u003e\n\u003cp\u003eHeLa and HaCaT cells were cultured in MEM and DMEM media respectively, with 10% FBS (Gibco \u0026minus;\u0026thinsp;10270106) and 1% anti-anti. PC3 was grown in F12 media supplemented with 15% FBS, 1% anti-anti and (Gibco \u0026minus;\u0026thinsp;15240-062) L-glutamine. During treatments, depending on cell doubling size, seeding density was calculated to attain 70% confluency within 24hrs, following transfections. For microscopy experiments, seeding density was reduced to avoid overcrowding.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003esiRNA and pcDNA transfection\u003c/h2\u003e\n\u003cp\u003eIn HeLa and HaCaT cell lines, both knockdown and overexpression was performed for 48hrs, but in PC3 the treatment duration was 24hrs. Transfection was attained using lipofectamine 3000 (Invitrogen L3000-15) following manufacturer\u0026rsquo;s instructions. Plasmid concentration varied from 6 well to 60mm plate and we used 5 or 10ug respectively to induce KDM5A overexpression. The pcDNA-triple epitope SFB-tagged-RBP2 (KDM5A) overexpression construct was borrowed from Dr. Shweta Tyagi (CDFD). The YFP-KRAS pcDNA3.1 was acquired from Addgene.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003eCell cycle distribution analysis by flow cytometry:\u003c/h2\u003e\n\u003cp\u003eCell cycle distribution was assessed by plating 1* 10\u003csup\u003e5\u003c/sup\u003e in a 6 well plate, and the following day, the cells were treated with siRNA or pcDNA constructs for 48hrs, followed by trypsinization, washed with PBS (twice), fixed with 100% ethanol for an hour at -20 \u003csup\u003eo\u003c/sup\u003eC, followed by treatment with 10ug/ml of RNase-A (Himedia \u0026ndash; MB087) at 37 \u003csup\u003eo\u003c/sup\u003eC in PBS for an hour and addition of 1ug/\u0026micro;l propidium iodide (Sigma \u0026minus;\u0026thinsp;81845). Cellular DNA content was measured using BD accuriC6 FL2 filter and cell cycle distribution was analysed with BD software. While analysing cell cycle in serum starved cells, HeLa and HaCaT cells were maintained in media with antibiotics for three days, following treatments in Opti-MEM.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003eGene expression profiling:\u003c/h2\u003e\n\u003cp\u003eUALCAN database was used to assess GRB2, SOS1 and different RAS transcript profile in cervical and prostate tumor tissues relative to its expression in normal tissue. The expression profile of the above-mentioned genes across multiple cell lines was extracted from human protein atlas.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n\u003ch2\u003eWestern blotting\u003c/h2\u003e\n\u003cp\u003eWhole cell lysate prepared by RIPA lysis buffer (sigma \u0026ndash;R0278-50ML) were electrophoresed on 8\u0026ndash;12% SDS-PAGE gels depending on target proteins analysed as per previous protocol [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e], resolved and transferred to nitrocellulose membrane (Axiva \u0026ndash; 160300RI), blocked with 5% skim milk for an hour. All primary antibodies were prepared in 1% BSA (Himedia-MB083) in PBST and the blots were probed with primary antibodies at 4\u003csup\u003eo\u003c/sup\u003eC overnight. Following three washes with PBST, the blots were incubated with either anti-rabbit (Invitrogen-65-6120) or anti-mouse (Santa Cruz \u0026ndash; SC516102) secondary antibody for an hour. The blots were then washed thrice using PBST, visualized using ECL chemiluminescence detection system (Thermo- 34580).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibodies used:\u0026nbsp;\u003c/strong\u003eKDM5A (SC-365993) and \u0026beta;-actin (SC-47778) were from Santa Cruz.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n\u003ch2\u003eImmunofluorescence\u003c/h2\u003e\n\u003cp\u003eHeLa, HaCaT and PC3 cells were seeded on a coverslip in a 6-well plate, transfected with YFP-RAS construct for 48hrs followed by fixation using 100% methanol for 5 minutes, permeabilized with PBST on ice for 10 minutes and blocked using 1% BSA in PBST for 1hr. The cells were incubated with anti-H3K4me3 (Invitrogen MA5-11199) and MLL2 (Abcam - ab56770) antibody overnight at 4\u003csup\u003eo\u003c/sup\u003eC in a humid chamber. Following 3 washes with PBS, the cells were incubated in Alexa488 (anti-rabbit ab150077) and Alexa 647 (anti-mouse \u0026ndash; ab150119) washed thrice with PBS and images were acquired using Leica microsystems.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eEither student\u0026rsquo;s \u0026lsquo;t\u0026rsquo; test or two-way ANOVA followed by Bonferroni correction were employed to test the significance, and the test employed along with the p-values and their inference is mentioned in Figure legends.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eTo understand the cell cycle distribution under KDM5A knockdown, overexpression and MLL1 knockdown conditions, we choose cervical (HeLa), Prostate (PC3) and Keratinocyte (HaCaT) cell lines. Following transfections and PI staining, we observed that in HeLa \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b\u003cb\u003e)\u003c/b\u003e and HaCaT \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ec-d\u003cb\u003e)\u003c/b\u003e cell lines, KDM5A knockdown led to an increase in G2/M population compared to siControl, and KDM5A overexpression (KDM5A-OE) led to S-phase arrest. MLL1-knockdown (MLL1-KD) also led to S-phase arrest. From the cell cycle distributions, we understood that these observations are a consequence of global changes in H3K4me3 as siKDM5A would increase the active mark and render the chromatin in open configuration thereby influencing the G2/M phase transition, as normally chromatin compaction should commence at this stage and enhanced H3K4me3 hinders with this process. In KDM5A-OE and ML1-KD, as both these conditions reduce H3K4me3, an S-phase arrest was noted due to highly packaged DNA being rendered inaccessible for replication process in S-phase.\u003c/p\u003e \u003cp\u003eIn PC3 cell line, although the effects were subtle but a reverse trend was observed, i.e., siKDM5A led to S-phase arrest and KDM5A-OE led to G2/M arrest \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ee-f\u003cb\u003e)\u003c/b\u003e. One difference between PC3 and the other two cell lines used is that PC3 is a slow growing cell line. So, to test whether a slow culturing HeLa cell would also exhibit similar cell cycle distribution, we serum starved HeLa cells to reduce their high proliferative ability and performed similar knockdown and overexpression experiments as mentioned above. To our surprise, serum starved HeLa cell cycle distribution \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-b\u003cb\u003e)\u003c/b\u003e was similar to the PC3 distribution rather than normally cultured HeLa (10% FBS) cells. Serum starvation and siKDM5A treatment led to S-phase arrest instead of G2/M; whereas KDM5A overexpression and siMLL1 led to an increase in G2/M population instead of S-phase as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003ea-b.\u003c/p\u003e \u003cp\u003eFurther, from earlier reports on KDM5B (a member of KDM family of demethylases) in melanoma cells, when the cells were serum starved, knockdown of KDM5B led to S- phase increase but when stimulated with different concentrations of FCS, a rise in G2/M population occurred as seen in our experiments. In line with this, when we assessed the expression of KDM5A at different serum concentrations, serum starvation (0% FBS) enhanced KDM5A but high percentage of FBS (20%) reduced KDM5A levels \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003eFrom our observations so far and other literature, we hypothesized that an interplay exists between KDM5A and some factors of FBS. As growth factors are the major signaling molecules of FBS and can trigger the RTK signaling during cell cycle according to KEGG identifier 04110N pathway, we sought to assess the differences among growth factor signaling molecules. As we used different cell lines in the study which could be triggered by different growth factors and possess diverse receptors, we assessed the initial cytosolic effector molecules of RTK signaling and analyzed GRB1, SOS1 and RAS expression among cervical and prostate cancers using TGCA datasets.\u003c/p\u003e \u003cp\u003eWe observed that GRB2 expression was marginally enhanced in cervical cancer patients but not in prostate cancer \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e, and SOS1 was downregulated in both \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e. As RAS proteins are most mutated among cancers, we assessed KRAS, NRAS and HRAS, and found that KRAS and NRAS were highly expressed in cervical cancer patients and in HeLa and HaCaT cell lines when compared to PC3 in which they were unaffected and less expressed \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e, a\u003cb\u003e\u0026amp;b).\u003c/b\u003e Unlike KRAS and NRAS, HRAS was more expressed in PC3 cell line, and enhanced in both prostate and cervical cancer patients, but its expression was twice that of normal in cervical patients compared to PC3 where only marginal changed were noted \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFollowing the in-silico gene expression analysis, we speculated that although GRB2 and SOS1 were slightly upregulated (in cervical) or remained unregulated, but RAS proteins were highly expressed in HeLa and HaCaT compared to PC3, and might affect KDM5A expression or activity. From literature survey we noticed that many epigenetic markers are target to CDKs, and from our results we see that cells grown in serum starved conditions exhibited altered KDM5A or MLL1 functioning when compared to cells grown in 10% FBS, we hypothesized that RAS proteins might activate/enhance KDM5A enzymatic activity or reduce MLL1 methyltransferase activity downstream of MAPK signalling. To test our hypothesis, we overexpressed KRAS \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ea\u003cb\u003e)\u003c/b\u003e and analyzed if RAS overexpression affects KDM5A expression. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, we traced that KRAS overexpression led to reduction in KDM5A protein. KDM5A protein content is reduced in enhanced RAS conditions, however, RAS might enhance the demethylase activity of the residual KDM5A.\u003c/p\u003e \u003cp\u003eTo test whether RAS enhanced KDM5A demethylase activity, we overexpressed RAS in HaCaT cells and assessed the H3K4me3 levels as an indirect estimate of the demethylase function of KDM5A. We noted that RAS overexpression reduced H3K4me3 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e, supporting that RAS might activate/enhance KDM5A\u0026rsquo;s enzymatic function. Along this line, we also tested if inhibiting MAPK signalling enhanced H3K4me3 levels. Using U0126 (MEK inhibitor) in HaCaT cells, we analysed the levels of the writer (MLL2) and the modification and observed that both were increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). From this, we concluded that reduced MAPK signalling activates MLLs function thereby increasing H3K4me3, and increased MAPK activity increases KDM5A\u0026rsquo;s demethylase function thereby reducing H3K4me3.\u003c/p\u003e \u003cp\u003eAs we have observed that RAS overexpression reduced H3K4me3, we tested whether transfection of both RAS and KDM5A together in serum starved HeLa and HaCaT cells would enhance KDM5A\u0026rsquo;s demethylase activity and enhance cell population in S-phase instead of G2/M as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. So, when this double transfection was performed, an increase in S-phase population was noted when compared to KDM5A and RAS transfection alone \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003e, a-d\u003cb\u003e)\u003c/b\u003e. Further, in PC3 cells which exhibit low RAS proteins and KDM5A expression in normal conditions (10% FBS) led to G2/M arrest, RAS and KDM5A double transfection led to increase in S-phase cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e6\u003c/span\u003ee\u003cb\u003e\u0026amp;f)\u003c/b\u003e, proving that KDM5A activity was monitored by RAS or the downstream signalling molecules and global H3K4me3 status dictated by KDM5/MLL ratio was responsible for the changes observed in this study.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTransition through different phases of cell cycle is majorly governed by three classes of proteins; CDKs, cyclins, and phosphatases; while precision is maintained by multiple proteins at restriction check points. Apart from the proteins that ensure faithful replication and segregation, chromatin configuration was described to be the heart of cell cycle progression [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and many histone modifications are said to play a role during replicative licencing, act as barrier to prevent re-replication and aid in compaction of chromatin into chromosomes. Chromatin structure is dictated by the epigenetic modifications and how changes in individual modification effects cell cycle is yet be elucidated. Changes in the amount of writer and eraser proteins alter global and gene specific occupancy of different modifications. When a certain epigenetic modifier expression is targeted by knockdown or overexpression, whether the global changes or gene-specific expression pattern lay influence over cell cycle is a question that need addressing. Recently, we have deciphered that KDM5A in association with MLL1/2 regulates expression of genes associated with EMT; where, as repressor of genes KDM5A demethylates H3K4me3 to H3K4me, and activator of gene it does not demethylate H3K4me3 but in association with MLL inhibits histone deacetylase activity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere, through this study, we elucidated how changes in global H3K4me3 effect cell cycle distribution. We targeted the writer MLL1 and eraser KDM5A to reveal the changes that emerge following their knockdown and overexpression in HeLa, HaCaT and PC3 cell lines. When KDM5A knockdown was performed in HeLa and HaCaT cell lines, G2/M arrest was observed as increased H3K4me3 obstruct chromatin compaction, while KDM5A-OE and MLL1-KD led to an increase in S-phase population as reduced H3K4me3 could not allow DNA synthesis due to compacted structure. But a reverse trend was observed in PC3 cell line. In our culture conditions, PC3 was slow growing compared to HeLa and HaCaT, and from literature review, we understood that KDM5B-KD in low serum conditions led to S-phase arrest and when knockdown was performed in cells cultured with serum - G2/M arrest was seen [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. We thus performed in-silico screening of effector molecules of growth factor signalling \u0026ndash; i.e., MAPK pathway.\u003c/p\u003e \u003cp\u003eFrom gene expression data obtained through TCGA datasets and transcript profile of cell lines acquired from human protein atlas, we understood that RAS expression is reduced in PC3 cell line compared to HeLa and HaCaT. As many reports present that cell cycle regulators can activate or suppress epigenetic modifier activity, we tested whether RAS could have any effect of the demethylase activity of KDM5A.\u003c/p\u003e \u003cp\u003eAs HeLa and HaCaT had more RAS expression, we speculated that KDM5A overexpression in these cell lines at normal FBS conditions (10%) led to S-phase arrest as KDM5A demethylase was active, but in PC3 where RAS expression is inherently low, KDM5A overexpression did not arrest cells in S-phase but instead in G2/M probably because KDM5A could not be enzymatically activated. When such slow culturing conditions were replicated in HeLa cells, cell cycle distribution similar to PC3 was observed.\u003c/p\u003e \u003cp\u003eAs lack of RAS mediated signalling led to reduced demethylase activity in PC3 and serum starved HeLa cells \u0026ndash; which led to G2/M arrest instead of S-phase, we overexpressed RAS in serum starved HeLa and HaCaT cells to see if supplementing RAS overexpression in place of serum would enhance KDM5A activity. As expected, when RAS and KDM5A were overexpressed together in serum starved cells, and increase in S-phase population was noted when compared to KDM5A or RAS overexpression alone. Similar results were observed in PC3, proving that RAS somehow enhanced the demethylase activity of KDM5A.\u003c/p\u003e \u003cp\u003eConcluding our study, we depicted KDM5A and MLL1 enzyme activity is cell line or tissue dependent as many proteins of different signalling pathways activate these proteins, MAPK being one among them. It was not just KDM5A, we unveiled that MAPK pathway regulate both writers and erasers of H3K4me3 to balance the distribution of this active mark as inhibiting MEK activity enhanced MLL2 and H3K4me3 levels; earlier work from our laboratory has shown MAPK pathway inhibition using U0126 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Although many reports claim that the expression of KDM5A [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and MLLs [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] do not change through cell cycle, and so is the H3K4me3 modification, recent reports claim that MLLs localization during cell cycle varies and H3K4me3 at gene promoters is modified in a phase dependent manner. Here we show that global changes in H3K4me3 affects cell cycle distribution and a more detailed investigation on the mechanism of regulation is needed.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements:\u003c/h2\u003e\n\u003cp\u003eR. Kirtana received fellowship from CSIR, Govt. of India (CSIR-09/983(0018)/2017-EMRI). S. Manna is thankful to NIT-Rourkela for fellowships under the Institute Research Scheme, NIT-Rourkela. We thank Dr. Shweta Tyagi (CDFD Hyderabad) for SFB-RBP2 and GST-RBP2 deletion (D1-D5) constructs.\u003c/p\u003e\n\u003ch2\u003eAuthor Contributions:\u003c/h2\u003e\n\u003cp\u003eSamir Kumar Patra: Conceptualization, Supervision, and Editing. Kirtana R: Formulation, Methodology, Data curation, Analysis, Original draft preparation, and Review. Soumen Manna: Data curation and analysis.\u003c/p\u003e\n\u003ch2\u003eConflict of Interest:\u003c/h2\u003e\n\u003cp\u003eNone\u003c/p\u003e\n\u003ch2\u003eFunding:\u003c/h2\u003e\n\u003cp\u003eThis work is supported in parts by the Department of Biotechnology (Government of India) project No.: BT/PR21318/MED/12/742/2016 to SKP.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eEpigenetic dynamics across the cell cycle. Tony Bou Kheir and Anders H. Lund1. Essays Biochem. (2010) 48, 107\u0026ndash;120; doi:10.1042/BSE0480107\u003c/li\u003e\n\u003cli\u003eEpigenetics and cell cycle regulation in cystogenesis. Xiaogang Lia,b,⁎. Cellular Signalling 68 (2020) 109509.\u003c/li\u003e\n\u003cli\u003eDynamic association of MLL1, H3K4 trimethylation with chromatin and Hox gene expression during the cell cycle. Bibhu P. Mishra, Khairul I. Ansari and Subhrangsu S. Mandal. 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Mallory, David Latrasse, Teddy J\u0026eacute;gu, Quentin Bruggeman, Marianne Delarue, Catherine Bergounioux, Moussa Benhamed, \u003cem\u003eJournal of Experimental Botany\u003c/em\u003e, (2014) 65:2677\u0026ndash;2689.\u003c/li\u003e\n\u003cli\u003eKDM5A noncanonically binds antagonists MLL1/2 to mediate gene regulation and promotes epithelial to mesenchymal transition. Kirtana R, Manna S, Patra SK. Biochim Biophys Acta Gene Regul Mech. 2023 Dec;1866(4):194986. doi: 10.1016/j.bbagrm.2023.194986.\u003c/li\u003e\n\u003cli\u003eEpigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer. Pradhan N, Parbin S, Kar S, Das L, Kirtana R, Suma Seshadri G, Sengupta D, Deb M, Kausar C, Patra SK. Biochim Biophys Acta Mol Basis Dis. (2019) 1865(6):1651-1665.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cell cycle, KDM5A, MLL1, H3K4me3, RAS signalling","lastPublishedDoi":"10.21203/rs.3.rs-4332860/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4332860/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCell cycle progression is regulated by many extracellular stimuli and intracellular signaling. Interaction between different epigenetic modifiers and transcription factors regulate the expression of genes encoding proteins involved in cell cycle control. Along with the cyclin-CDK complexes and phosphatases, RAS- signaling play crucial role to direct the cell passage through different stages of cell cycle. In this scenario, chromatin configuration is important for the progression of cell division and chromatin modifications (DNA methylation and histone modifications) helps to attain correct chromatin folds. Here, in this study we analyzed how modulation of H3K4me3 by MLL1 and KDM5A affect cell cycle progression. As slow and fast cycling cell lines exhibited differences in mechanisms of regulation, from in-silico screening and experimental demonstration we deciphered that the expression of the MAPK effector, RAS is involved to controlling the expression and activity of KDM5A and MLL proteins to balance H3K4me3 oscillation throughout cell cycle.\u003c/p\u003e","manuscriptTitle":"MLL and KDM5A activity during cell cycle progression depend on Ras signalling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-30 05:01:38","doi":"10.21203/rs.3.rs-4332860/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d929eb83-4f15-4241-a429-930973e26c14","owner":[],"postedDate":"April 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":31228113,"name":"Cell Cycle \u0026 Proliferation"}],"tags":[],"updatedAt":"2024-04-30T05:01:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-30 05:01:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4332860","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4332860","identity":"rs-4332860","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}