Zbtb20 zygosity affects the immunological profile of experimental animals  

Zbtb20 zygosity affects the immunological profile of experimental animals | 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 Research Article Zbtb20 zygosity affects the immunological profile of experimental animals Lidiya Kechidzhieva, Katerina Ilieva, Viktoria Hranova, Valentina Ivanova, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6876906/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 Members of the Zbtb family of transcription factors is vital for the development and function of many cells and organs. One member in particular, Zbtb20, is critical for the maturation of the nervous system, the pituitary gland and the liver. In the present research we investigate the effect that the Zbtb20 TF has on the immune system of animals that lack one allele of the Zbtb20 gene. We report that greater number of hematopoietic cells, but lower levels of B-1 cells, is found in the bone marrow of Zbtb20 -heterozygous mutants. More CD19 + cells, than CD3 + cells are present in the spleen, with CD4 + cells being less abundant than the CD8 + cells of these animals, as well. The ratio between natural IgM and IgG antibodies differs in the two genotypes, with more IgM antibodies being secreted from less B-1 cells in the heterozygous mice. Together, our results shed additional light on the Zbtb20 -mediated regulation of the immune system. Zbtb20 Immunogenetics Heterozygous animals Hematopoietic Stem Cells Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The genetic impact on tissue development is well established, with transcription factors (TFs) playing key role in all organs. For example, Oct4 and Sox2 were proven as vital participants in keeping the pluripotency of the embryonic stem cells, with deficiencies leading to failure in forming the inner cell mass. (Rodda et al., 2005 ) TP53 deficiency leads to aggressive cancer development. (Ignatius et al., 2018 ) This makes the discovery of new regulatory functions, of known TF families, indispensable for the understanding of the development of the organism and its systems. The immune system has many factors that are involved in its development and functioning. Mutations in the gene GATA-2 lead to unfunctional proteins and facilitates the ongoing of opportunistic infections. (Crispino & Horwitz, 2017 ) AIRE, and its related protein, is responsible for the regulation of the autoimmunity reactions in the body (Zhao, Chang, Fu, Sun, & Yang, 2018 ) NAIP is important for the formation of the inflammasome and its deficiency leads to a lack of protection. (Mitchell et al., 2020 ) The ZBTB family of TFs is another example of developmentally-important family of proteins first identified in Drosophilas and, named after three Drosophila genes. (Zollman, Godt, Privé, Couderc, & Laski, 1994 ) They possess a N-terminal BTB/POZ ( b road complex t ramtrack b ric-a-brac/ po xvirus) domain involved in protein-protein interactions, and a z inc finger domain, importnat for the recognition and DNA interaction. (Lee & Maeda, 2012 ) The family became even more important after the discovery that a protein, named DPZF (W. Zhang et al., 2001 ) or HOF (Mitchelmore et al., 2002 ), can be found in many developmental processess and cancerous conditions. (Kelly & Daniel, 2006 ) Now, it is becoming more and more evident that many of the Zbtb TFs have multiple functions. Zbtb1 acts as a trasncriptional repressor and is vital for the T cell development. (Matic et al., 2010 ; J. Wang et al., 2022 ) Zbtb17 (MIZ-1) is a TF equally important for the proper gastrulation and the “β-selection” checkpoint of the pre-TCR. (Adhikary et al., 2003 ; Rashkovan et al., 2014 ) Breast cancer prognosys and myeloid lineage development are associated with Zbtb7 (THPOK, LRF) (Basu et al., 2023 ; H. Qu et al., 2010) The well studied Zbtb27 (BCL-6) has a great clinical value for the B cell lymphomas diagnotics and the regulation of T cells. (J. Choi & Crotty, 2021 ; Zhu, Chen, Zhao, Gao, & Wang, 2018 ) This multiple activity is valid as well for ZBTB35 (ZNF131), ZBTB19 (PATZ1, MAZR), ZBTB29 (HIC1) and many more (Z.-Y. Cheng, He, Gao, Zhao, & Wang, 2021 ) An important member of the family, the TF Zbtb20 was discovered during brain development (Mitchelmore et al., 2002 ), and during the development of the astrocytes and chondrocytes. (Z. Qu et al., 2016 ) During gliogenesis, Zbtb20 acts in concert with Sox9 and NFIA to regulate astrogliogenesis, and a similar molecular mechanisms apparently exists during chondrocyte final differentiatin. (Zhou et al., 2015 ) The Pituitary gland development and the functioning of the liver are highly dependable on this gene (D. Cao et al., 2016 ; Xie et al., 2008 ) ZBTB20 loss-of-function in humans causes Primrose syndrome. (Cordeddu et al., 2014 ) Throughout the years, several groups have discovered a role of Zbtb20 in immunity. The transcriptional activity of the protein is elevated when long-lived plasma cells are being formed and differentiated in the germinal centers. (Chevrier et al., 2014 ) It helps the long term antibody response and it plays a significant role in the development of the B-cells from progenitor cells in the bone marrow (BM). (Lee & Maeda, 2012 ; Y. Wang & Bhattacharya, 2014 ) Zbtb20 has a role in the T cell activation, specifically the decision to generate CD8 + cells or T regs, and this was shown in multiple mouse experiments. Further, Zbtb20 is able to modulate the tumor-specific T-cell response, the NF-kB signal pathway and the intestine immunological homeostasis. (Krzyzanowska et al., 2022 ; X. Liu et al., 2013 ; Sun et al., 2020 ) Yet, more is to be discovered about the role of ZBTB20 on the immune system. In the present research we studied wether the loss of one allele of Zbtb20 will affect the phenotype and antibody secretion properties of BM and spleen cells. Materials and methods Animal experiments All animals included in the present study were handled in accordance with the Bulgarian national regulations. The experiments were carried out in strict accordance with the Guidelines for the Care and Use of Laboratory Animals of the European Union (EU Directive 2010/63/EU), and the manipulations were approved by the animal care commission at the Bulgarian Food Safety Agency, approval protocol Nr. 392/21.04.2024. The mice were bread and kept under specific pathogen free (SPF) conditions at the Institute of Microbiology, Bulgarian Academy of Sciences and housed at 20–22 o C with a light/dark cycle of 12/12 hrs. We used 3–6 months old male and female 129s mice in all experiments. Heterozygous Zbtb20 mice generation The Zbtb20 mutant mice generation was previously described. (Rosenthal, Tonchev, Stoykova, & Chowdhury, 2012 ; Tonchev, Tuoc, Rosenthal, Studer, & Stoykova, 2016 ) Briefly, the BTB/POZ protein domain and the first zinc fingers were replaced by a lacZ-neomycin cassette. Animals carrying the mutation in a homozygous state Zbtb20 LacZ/LacZ were not vital for more than a month, with many physiological alterations being observed. In order to preserve the mutation, heterozygous animals Zbtb20 LacZ/+ were intecrossed and based on the Mendelian law of segregation, a genotypic ratio of 1:2:1 (WT: Zbtb20 LacZ/+ : Zbtb20 LacZ/LacZ ) was expected. DNA isolation and genotyping On day 5 after birth, mice of the Zbtb20 LacZ/+ intercross progeny were subjected to toe clipping. Briefly, the pad of each animal was cleaned with alcohol prior the procedure. Sterilized sharp scissors were used and were disinfected between animals. A clean gauze sponge was applied over the site with gentle pressure to stop the bleeding. The isolated distal phalangeal bone was put in a sterile Eppendorf tube and kept on ice until all samples were collected. The so obtained tissues were subjected to “dirty” DNA isolation. (Truett et al., 2000 ) Alcaline lysing solution containing 25mM NaOH and 0.2mM EDTA was added to each tube prior of putting it to a thermocycler at 98 o C for 1 hour. The solution was afterwards neutralized with the same volume of 40 mM Tris HCl (pH 5.5). After a brief centrifuge at 1000 RCF for 3 minutes the isolated DNA was subjected to a standard PCR amplification in order to verify the zigoucity of the animals in the progeny. The primers used in the reaction are as follows: ER_Zbtb_F, 50-TCACAGCCAAACAGAACTACG-30; ER_Neo_F, 50-TCTTCTGAGGGGATCAATTCTC-30; ER_Zbtb_R, 50-CAAGCTTTGGACCCACACTA-30. After the genotyping, all mice were divided into one of the following groups: WT, Zbtb20 LacZ/+ and Zbtb20 LacZ/LacZ , as only WT and Zbtb20 LacZ/+ were part of the study. Antibodies for flow cytometry The following antibodies for flow cytometry were used in the study. For the BM flow cytometry: FITC anti-mouse Lineage Cocktail with Isotype Ctrl (Biolegend, 133302), PE anti-mouse CD117 (c-kit) Antibody (Biolegend, 135106), PE/Cyanine7 anti-mouse Ly-6A/E (Sca-1) Antibody (Biolegend, 108114), Pacific Blue™ anti-mouse CD19 Antibody (Biolegend, 115523). For the flow cytometry of splenocytes: FITC anti-mouse CD45 Antibody (Biolegend, 103107), PE anti-mouse CD19 Antibody (Biolegend, 152408), PE/Cyanine7 anti-mouse CD3ε Antibody (Biolegend, 100320), Pacific Blue™ anti-mouse CD4 Antibody (Biolegend, 100428), APC anti-mouse CD8a Antibody (Biolegend, 100712) Flow cytometry of the BM cells Analysis of the hematopoietic stem cells (HSCs) of the animals was performed using flow cytometry. 7 WT and 7 Zbtb20 LacZ/+ mice were euthanized by cervical dislocation and BM was isolated from their femurs as described. (Amend, Valkenburg, & Pienta, 2016 ) The long bones were put into 0.5ml tubes, nested in 1.5ml centrifuge tubes. They were spun at 10000 RCF for 15sec. The erythrocytes from the isolated cellular fraction were lysed for a minute with a lysis buffer containting NH 4 Cl, KHCO 3 and EDTA and the suspension was washed 2 times with PBS at 150 RCF / 4 o C / 10min to remove the debris. 2×10 5 cells per FACS tube were distributed in ice-cold PBS (containing 2.5% FCS and 0.05% sodium azide) and incubated with the Lineage cocktail, CD1117 (c-kit), Ly-6A/E (Sca-1) and CD19 antibodies for 20min/ 4oC, followed by double wash with PBS/FCS to remove the excess of antibodies. The pellet was finally resuspended in PBS/FCS and analyzed. Cells populations were defined as L-S + K+ (Lin-Sca-1 + c-Kit+), L-S + CD19+ (Lin-Sca-1 + CD19+) and CD19 + B220 + S+ (CD19 + B220 + Sca-1+). At least thirty thousand cells were analyzed from each sample with a BD LSR II flow cytometer (BD Biosciences, San Jose, CA) using Diva 6.1.1. software (BD Biosciences, Mountain View, CA, USA). Flow cytometry of spleen cells Analysis of the spleen lymphocyte populations was performed using flow cytometry. Spleens from the euthanized mice were taken and subjected to splenocytes isolation by disruption with cell stainer, lysing and washing as previously described. 2×10 5 cells per FACS tube were distributed in ice-cold PBS / FBS and an antibody mix containing the CD45, CD19, CD3ε, CD4, CD8a antibodies was added for for 20min/ 4 o C. After a double PBS/ FBS wash, the cells were analyzed by flow cytometry as at least thirty thousand cells were counted. The screened populations were defined as: CD45 + CD19+, CD45 + CD3+, CD45 + CD3 + CD4+, CD45 + CD3 + CD8+. Sandwich ELISA of sera Sandwich ELISA assay was performed to screen the sera from different animals, for the major antibodies isotypes – IgG, IgM and IgA. Blood samples were collected from the retro-orbital plexuses of the experimental animals and left for 2h at 4 o C for clot formation. The clotted blood was consecutively centrifuged first at 200 RCF, next at 300 RCF and the obtained sera were kept at -80 o C for further analysis. When enough samples were collected, sandwich ELISA was performed as previously described. (Kohl & Ascoli, 2017 ) Briefly, 96-well Maxisorp immunoplates (Nunc, Roskilde, Denmark) were coated with Goat Anti-Mouse IgG + IgM + IgA H&L (ab102445, Abcam), 5ug/ml in PBS overnight at 4 o C. The plates were then blocked with 1.0% Tween-20, incubated with 100x diluted sera and later detected with biotin anti-mouse IgA Antibody (407004, Biolegend), biotin anti-mouse IgM Antibody (406504, Biolegend) and biotin goat anti-mouse IgG (minimal x-reactivity) antibody (405303, Biolegend). The samples were developed using ABTS (2,20-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) solution (11684302001, Sigma-Aldrich) and read at 405nm. Results Zbtb20 LacZ/+ animals show significant changes in the Lin − BM populations Flow cytometry analysis of BM cells was used to identify the L-S + K+, L-CD19 + and L-CD19 + S + cell populations. After the cells were gated through the Lin–/Lin + populations, major mouse hematopoetic markers Sca-1 and c-Kit were used to segregate the 2 groups of animals. Gating through the double positive cells was performed for the L-S + K+. As Zbtb20 is associated with the development, a change was expected in the hematopoietic cellular department. As depicted, the percent of the L-S + K + population was significantly lower in the WT animals, compared to the Zbtb20 LacZ/+ (Fig. 1 , pannel a). This result led to the expectation that all populations will be less in the BM of the WT animals. Interestingly, the L-CD19 + cells which here, we consider as B-1 cells, were less in the heterozygous animals (Fig. 1 , pannel b), leading to the conclusion that this side of the innate immunity will be underdevelopped in these animas. The hematopietic capacity of these B-1 cells, was assed with both c-Kit and Sca-1 markers, and while any Lin-CD19 + c-Kit + positive cells were not observed (data not shown), the levels of L-CD19 + S + cells were different in both genotypes, showing a role of Zbtb20 in the development of this particular population (Fig. 1 , pannel c) Development of the B cells in the BM is augmented in heterozygous animals As regular B cell formation is undergone in the BM, a screening for the CD19 + B220 + cells was perfomed. Here we consider these cells as developping conventional B cells, that include the Pro-B, Pre-B, Immature B and the Transitional B cells as both markers are observed during all the stages. What we observed was a higher percent of the CD19 + B220 + cells in the Zbtb20 LacZ/+ animals (Fig. 2 , pannel a), leading to the assumption that these mice will have a higher antibody levels in the sera. The hematopoietic potential of these cells (expressed as CD19 + B220 + S+) in the heterozygous animals is visible, which can be related to the overall HSC activity in this genotype (Fig. 2 , pannel b). General splenocytes populations are affected in heterozygous animals Based on the high HSC levels in the BM as a primary lymphoid organ, a change in the immune cells populations in the systemic lymphoid organs was expected in the heterozygous animals. A screening of the spleen cells was performed for the major immune cells populations, like CD45, CD19, CD3, CD4 and CD8. The comparison between the CD45 + CD19 + and the CD45 + CD3 + cells (generally considered as B and T cells) presented more B cells and less T cells in the Zbtb20 LacZ/+ splenocytes. The screening of the WT spleen cells, showed the opposite trend (Fig. 3 , pannel a and pannel b). This was not a surprise, as the levels of the developping B cells in the BM were more in the animals bearing the heterozygous genotype. Next, we analyzed two T cell subsets - CD45 + CD3 + CD4+ (regarded as helper T cells) and CD45 + CD3 + CD8+ (regarded as cytotoxic T cells). A noticeable difference is observed between the homo- and the heterozygous animals regarding the helper T cells. The Zbtb20 LacZ/+ mice had significantly lower numbers of CD45 + CD3 + CD4 + cells suggesting a weakened adaptive immunity response and overall activation. This data supports previous research results on the role of other Zbtb factors on the development of helper T cells. (He et al., 2005 ) Although visible, the contrast in the levels of CD45 + CD3 + CD8 + cells between the two genotypes can not be considered as a significant one. (Fig. 3 c and 3 d) Altered circulating natural antibodies in Zbtb20 heterozygous mice The higher levels of B cells in the BM and the spleen of the heterozygous animals could lead to more natural antibodies in the blood of these mice. We probed this possibility by a sandwich ELISA of the sera of +/+ and +/- animals. We screened the natural IgG, IgM and IgA antibodies, obtained from peripheral blood. The IgA antibodies were not expected to vary, as IgA is predominantly found in the mucosal tissues (Fig. 4 , right pannel), although there is a slight difference. On the other hand, IgG and IgM antibodies exhibited different trends. The natural IgG antibodies were slighly diminished in the Zbtb20 LacZ/+ animals (Fig. 4 , left pannel), while the levels of natural IgM antibodies were elevated, similarly to the B cells in the BM and the spleen of the WT animals (Fig. 4 , center pannel), opposing the lower levels of the B-1 cells in the BM, but suggesting a higher secreting capacity of this particular population. Discussion The present paper confirms the importance of Zbtb20 in the regulation of the immune system homeostasis. Although there was no obvious contrast in the physical appearance of the WT and Zbtb20 LacZ/+ animals in our study, there was a dramatic difference in the level of L-S + K + cells. This came to a surprise, given the effect of a similary designed heterozygous condition regarding the Zbtb11 TF. (H. Cao et al., 2023 ) Usually overexpression of Zbtb genes leads to diminishing of cell numbers. (Satpathy et al., 2012 ; Tang et al., 2025 ) On the other hand HSC activity is affected by many checkpoint regulators and is dependent on DNA methylation. (T. Cheng et al., 2000 ; Yanai et al., 2024 ) While we can not exclude a role of Zbtb20 in all these processes, more research is needed to address this issue. (Maeda, 2016 ) There is also a possibility for a genetic compensation by other Zbtb family member – a hypothesys that is being investigated in the last years in different models. (El-Brolosy & Stainier, 2017 ; Rouf et al., 2023 ) Surprisingly to the high levels of HSC cells in the BM of the Zbtb20 heterozygous mice, their B-1 cells were decreased. We expected to detect higher numbers of B-1 cells in the mutants, but the observed opposite result confirms the critical importance of Zbtb20 on B-1 cells. (J. Liu & Zhang, 2024 ) Development of the two substypes of B cells (B-1 cells and B-2 cells) takes place in 3 distinct stages: the first one occurs in the yolk sac, the second one - in the fetal liver, and the third one - in the BM. (Mattos, Vandendriessche, Waisman, & Marques, 2024) The two B cell subsets have different roles in immunity. While the B-2 cells are considered as “classical” ones, the B-1 cells act more like innate-like cells, found in the cavities and being able to go through a T cell independent differentiation to IgM secreting plasma cells. (Y. S. Choi, Dieter, Rothaeusler, Luo, & Baumgarth, 2012 ; Montecino-Rodriguez et al., 2016 ) Having lower numbers of B-1 cells in the BM of the Zbtb20 mutants, we expected, that the levels of natural IgM antibodies would be lower as well. To our surprise, 3 of the animals showed higher levels of IgM antibody secretion. The number of B-1 cells in these animals was not above the average in the cohort. Furthermore, the levels of Sca-1 on these cells were also at the average for the study group. While Sca-1 is known to affect the number of B-1 cells, its role on the secretion of IgM antibodies is less clear. (Long, Pavlath, & Montano, 2011 ) This phenomenon suggests a possible involvement of another member of the Zbtb TF family, possibly Zbtb32, in the IgM production. (Chevrier et al., 2014 ) Another possibility could be that the higher levels of IgM in these 3 animals is related to a loss of heterozygosity, and the subsequent over activation of the remaining allele on one of the chromosomes. (X. Zhang & Sjöblom, 2021 ). The function of Zbtb20 in B cell development and differentiation outside the BM was already defined. (Chevrier et al., 2014 ) Nevertheless, it is intriguing how and why the Zbtb20 LacZ/+ mice get more developing conventional B cells in the BM and in the spleen. There is a probability, that the heterozygous mice fail to activate the controlling mechanisms in the B cell compartment, thus leading to a higher percent of B cells. (Nemazee, 2017 ) This may be a consequence of an interaction between Zbtb20 and miRNA factors – a dual system, found in some malignancies. (J. Liu et al., 2018 ) Still, more analyses should be done to prove this theory, as Zbtb20 heterozygous mice are expected to have lower Zbtb20 activity. While screening the regular CD3 cells, we found a lower number of CD3 + cells and CD4 + cells in the Zbtb20 LacZ/+ . Zbtb7a (ThPOK) is a well known regulator of the T cell activity, as it is able to supress the CD8 + T cells. (He, Park, & Kappes, 2010 ; L. Wang et al., 2008 ) Recent data demonstrate that Zbtb20 is also able to coordinate the development of the T cells. And what we see in our research confirms the results that define Zbtb20 as a supressor of the CD8 T cell activity (although our data is not statistically confirmable). (Preiss et al., 2023 ; Sun et al., 2020 )We obsercve a lower number of CD4 T cells in the Zbtb20 LacZ/+ mice, compared to the WT, a fact that sheds light on the effect ot this particular gene on the development of Th cells. In summary, the present study provides new information on the BM HSCs that are affected by the loss of allele of Zbtb20. The ratio between the T cell populations was also affected in the mutants, as were the levels IgM secretion, that were higher in some of the heterozygous animals. Future studies will help investigate more deeply the role that Zbtb20 has on the immune system, but together these results present a good platform to continue the work in several directions. Declarations Funding This research was supported by The National Science Fund of Bulgaria, project number KP-06-H61/5 (IM), and NextGenerationEU via Bulgarian National Recovery and Resilience Plan, Project #BG-RRP-2.004-0009-C03 (DS, ABT). Author Contribution L.K., K.I., V.H. and V.I. researched the data, I.M. wrote the manuscript and prepared the figures. D.S., A.T. and A.T. helped with the discussion of the results. All authors reviewed the manuscript. Acknowledgement This research was supported by The National Science Fund of Bulgaria, project number KP-06-H61/5 (IM), and NextGenerationEU via Bulgarian National Recovery and Resilience Plan, Project #BG-RRP-2.004-0009-C03 (DS, ABT). Data Availability The data that support the findings of this study are available from the corresponding author. References Adhikary S, Peukert K, Karsunky H, Beuger V, Lutz W, Elsässer HP, Eilers M (2003) Miz1 is required for early embryonic development during gastrulation. Mol Cell Biol 23(21):7648–7657. 10.1128/mcb.23.21.7648-7657.2003 Amend SR, Valkenburg KC, Pienta KJ (2016) Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation. J Vis Exp 11010.3791/53936 Basu J, Olsson A, Ferchen, Kyle, Titerina EK, Chetal K, Nicolas, Emmanuelle,.. Kappes, Dietmar J (2023) ThPOK is a critical multifaceted regulator of myeloid lineage development. Nat Immunol 24(8):1295–1307. 10.1038/s41590-023-01549-3 Cao D, Ma X, Cai J, Luan J, Liu AJ, Yang R, Zhang WJ (2016) ZBTB20 is required for anterior pituitary development and lactotrope specification. Nat Commun 7:11121. 10.1038/ncomms11121 Cao H, Naik SH, Amann-Zalcenstein D, Hickey P, Salim A, Cao B, Lieschke GJ (2023) Late fetal hematopoietic failure results from ZBTB11 deficiency despite abundant HSC specification. Blood Adv 7(21):6506–6519. 10.1182/bloodadvances.2022009580 Cheng T, Rodrigues N, Shen H, Yang Y, Dombkowski D, Sykes M, Scadden DT (2000) Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 287(5459):1804–1808. 10.1126/science.287.5459.1804 Cheng Z-Y, He T-T, Gao X-M, Zhao Y, Wang J (2021) ZBTB Transcription Factors: Key Regulators of the Development, Differentiation and Effector Function of T Cells. 12 . 10.3389/fimmu.2021.713294 Chevrier S, Emslie D, Shi W, Kratina T, Wellard C, Karnowski A, Corcoran LM (2014) The BTB-ZF transcription factor Zbtb20 is driven by Irf4 to promote plasma cell differentiation and longevity. J Exp Med 211(5):827–840. 10.1084/jem.20131831 Choi J, Crotty S (2021) Bcl6-Mediated Transcriptional Regulation of Follicular Helper T cells (T(FH)). Trends Immunol 42(4):336–349. 10.1016/j.it.2021.02.002 Choi YS, Dieter JA, Rothaeusler K, Luo Z, Baumgarth N (2012) B-1 cells in the bone marrow are a significant source of natural IgM. Eur J Immunol 42(1):120–129. 10.1002/eji.201141890 Cordeddu V, Redeker B, Stellacci E, Jongejan A, Fragale A, Bradley TE, Hennekam RC (2014) Mutations in ZBTB20 cause Primrose syndrome. Nat Genet 46(8):815–817. 10.1038/ng.3035 Crispino JD, Horwitz MS (2017) GATA factor mutations in hematologic disease. Blood 129(15):2103–2110. 10.1182/blood-2016-09-687889 El-Brolosy MA, Stainier DYR (2017) Genetic compensation: A phenomenon in search of mechanisms. PLoS Genet 13(7):e1006780. 10.1371/journal.pgen.1006780 He X, He X, Dave VP, Zhang Y, Hua X, Nicolas E, Kappes DJ (2005) The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 433(7028):826–833. 10.1038/nature03338 He X, Park K, Kappes DJ (2010) The role of ThPOK in control of CD4/CD8 lineage commitment. Annu Rev Immunol 28:295–320. 10.1146/annurev.immunol.25.022106.141715 Ignatius MS, Hayes MN, Moore FE, Tang Q, Garcia SP, Blackburn PR, Langenau DM (2018) tp53 deficiency causes a wide tumor spectrum and increases embryonal rhabdomyosarcoma metastasis in zebrafish. Elife 7. 10.7554/eLife.37202 Kelly KF, Daniel JM (2006) POZ for effect–POZ-ZF transcription factors in cancer and development. Trends Cell Biol 16(11):578–587. 10.1016/j.tcb.2006.09.003 Kohl TO, Ascoli CA (2017) Immunometric Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay. Cold Spring Harb Protoc 2017(6). pdb.prot093724 Krzyzanowska AK, Ii H, Kovalovsky RAH, Lin D, Osorio HC, Edelblum L, Sant'Angelo KL, D. B (2022) Zbtb20 identifies and controls a thymus-derived population of regulatory T cells that play a role in intestinal homeostasis. Sci Immunol 7(71):eabf3717. 10.1126/sciimmunol.abf3717 Lee SU, Maeda T (2012) POK/ZBTB proteins: an emerging family of proteins that regulate lymphoid development and function. Immunol Rev 247(1):107–119. 10.1111/j.1600-065X.2012.01116.x Liu J, Jiang J, Hui X, Wang W, Fang D, Ding L (2018) Mir-758-5p Suppresses Glioblastoma Proliferation, Migration and Invasion by Targeting ZBTB20. Cell Physiol Biochem 48(5):2074–2083. 10.1159/000492545 Liu J, Zhang H (2024) Zinc Finger and BTB Domain-Containing 20: A Newly Emerging Player in Pathogenesis and Development of Human Cancers. Biomolecules 14(2). 10.3390/biom14020192 Liu X, Zhang P, Bao Y, Han Y, Wang Y, Zhang Q, Cao X (2013) Zinc finger protein ZBTB20 promotes Toll-like receptor-triggered innate immune responses by repressing IκBα gene transcription. Proc Natl Acad Sci U S A 110(27):11097–11102. 10.1073/pnas.1301257110 Long KK, Pavlath GK, Montano M (2011) Sca-1 influences the innate immune response during skeletal muscle regeneration. Am J Physiol Cell Physiol 300(2):C287–294. 10.1152/ajpcell.00319.2010 Maeda T (2016) Regulation of hematopoietic development by ZBTB transcription factors. Int J Hematol 104(3):310–323. 10.1007/s12185-016-2035-x Matic I, Schimmel J, Hendriks IA, van Santen MA, van de Rijke F, van Dam H, Vertegaal AC (2010) Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif. Mol Cell 39(4):641–652. 10.1016/j.molcel.2010.07.026 Mattos M, Silvério, Vandendriessche, Sofie, Waisman A, Marques PE (2024) The immunology of B-1 cells: from development to aging. Immun Ageing 21(1):54. 10.1186/s12979-024-00455-y Mitchell PS, Roncaioli JL, Turcotte EA, Goers L, Chavez RA, Lee AY, Vance RE (2020) NAIP-NLRC4-deficient mice are susceptible to shigellosis. Elife 9. 10.7554/eLife.59022 Mitchelmore C, Kjaerulff KM, Pedersen HC, Nielsen JV, Rasmussen TE, Fisker MF, Jensen NA (2002) Characterization of two novel nuclear BTB/POZ domain zinc finger isoforms. Association with differentiation of hippocampal neurons, cerebellar granule cells, and macroglia. J Biol Chem 277(9):7598–7609. 10.1074/jbc.M110023200 Montecino-Rodriguez E, Fice M, Casero D, Berent-Maoz B, Barber CL, Dorshkind K (2016) Distinct Genetic Networks Orchestrate the Emergence of Specific Waves of Fetal and Adult B-1 and B-2 Development. Immunity 45(3):527–539. 10.1016/j.immuni.2016.07.012 Nemazee D (2017) Mechanisms of central tolerance for B cells. Nat Rev Immunol 17(5):281–294. 10.1038/nri.2017.19 Preiss NK, Kamal Y, Wilkins OM, Li C, Kolling FW th, Trask HW, Usherwood EJ (2023) Characterizing control of memory CD8 T cell differentiation by BTB-ZF transcription factor Zbtb20. Life Sci Alliance 6(9). 10.26508/lsa.202201683 Qu H Danni, Qu, Fuhui, Chen, Zhenyu, Zhang, Baogang, Liu, & and Liu, Haifeng. (2010). ZBTB7 Overexpression Contributes to Malignancy in Breast Cancer. Cancer Invest, 28 (6), 672–678. 10.3109/07357901003631007 Qu Z, Zhang H, Huang M, Shi G, Liu Z, Xie P, Xu Y (2016) Loss of ZBTB20 impairs circadian output and leads to unimodal behavioral rhythms. Elife 5. 10.7554/eLife.17171 Rashkovan M, Vadnais C, Ross J, Gigoux M, Suh WK, Gu W, Möröy T (2014) Miz-1 regulates translation of Trp53 via ribosomal protein L22 in cells undergoing V(D)J recombination. Proc Natl Acad Sci U S A 111(50):E5411–5419. 10.1073/pnas.1412107111 Rodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, Robson P (2005) Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem 280(26):24731–24737. 10.1074/jbc.M502573200 Rosenthal EH, Tonchev AB, Stoykova A, Chowdhury K (2012) Regulation of archicortical arealization by the transcription factor Zbtb20. Hippocampus 22(11):2144–2156. 10.1002/hipo.22035 Rouf MA, Wen L, Mahendra Y, Wang J, Zhang K, Liang S, Wang G (2023) The recent advances and future perspectives of genetic compensation studies in the zebrafish model. Genes Dis 10(2):468–479. 10.1016/j.gendis.2021.12.003 Satpathy AT, Kc W, Albring JC, Edelson BT, Kretzer NM, Bhattacharya D, Murphy KM (2012) Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages. J Exp Med 209(6):1135–1152. 10.1084/jem.20120030 Sun Y, Preiss NK, Valenteros KB, Kamal Y, Usherwood YK, Frost HR, Usherwood EJ (2020) Zbtb20 Restrains CD8 T Cell Immunometabolism and Restricts Memory Differentiation and Antitumor Immunity. J Immunol 205(10):2649–2666. 10.4049/jimmunol.2000459 Tang X, Deng C, Liu, Yang, Pu, Shengyu, Zheng, Qi, Zhou Y, Hao N (2025) ZBTB6 promotes breast cancer progression by inhibiting ARHGAP6 transcription and modulating the STAT3 signaling pathway. J Translational Med 23(1):370. 10.1186/s12967-025-06364-y Tonchev AB, Tuoc TC, Rosenthal EH, Studer M, Stoykova A (2016) Zbtb20 modulates the sequential generation of neuronal layers in developing cortex. Mol Brain 9(1):65. 10.1186/s13041-016-0242-2 Truett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML (2000) Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques 29(1):52, 54. 10.2144/00291bm09 Wang J, Shi, Chunwei, Cheng M, Lu Y, Zhang X, Li, Fengdi, Cao (2022) Xin. Effects of the Zbtb1 Gene on Chromatin Spatial Structure and Lymphatic Development: Combined Analysis of Hi-C, ATAC-Seq and RNA-Seq. 10 . 10.3389/fcell.2022.874525 Wang L, Wildt KF, Castro E, Xiong Y, Feigenbaum L, Tessarollo L, Bosselut R (2008) The zinc finger transcription factor Zbtb7b represses CD8-lineage gene expression in peripheral CD4 + T cells. Immunity 29(6):876–887. 10.1016/j.immuni.2008.09.019 Wang Y, Bhattacharya D (2014) Adjuvant-specific regulation of long-term antibody responses by ZBTB20. J Exp Med 211(5):841–856. 10.1084/jem.20131821 Xie Z, Zhang H, Tsai W, Zhang Y, Du Y, Zhong J, Zhang WJ (2008) Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver. Proc Natl Acad Sci U S A 105(31):10859–10864. 10.1073/pnas.0800647105 Yanai H, McNeely T, Ayyar S, Leone M, Zong L, Park B, Beerman I (2024) DNA methylation drives hematopoietic stem cell aging phenotypes after proliferative stress. Geroscience. 10.1007/s11357-024-01360-4 Zhang W, Mi J, Li N, Sui L, Wan T, Zhang J, Cao X (2001) Identification and characterization of DPZF, a novel human BTB/POZ zinc finger protein sharing homology to BCL-6. Biochem Biophys Res Commun 282(4):1067–1073. 10.1006/bbrc.2001.4689 Zhang X, Sjöblom T (2021) Targeting Loss of Heterozygosity: A Novel Paradigm for Cancer Therapy. Pharmaceuticals (Basel) 14(1). 10.3390/ph14010057 Zhao B, Chang L, Fu H, Sun G, Yang W (2018) The Role of Autoimmune Regulator (AIRE) in Peripheral Tolerance. J Immunol Res, 2018 , 3930750. 10.1155/2018/3930750 Zhou G, Jiang X, Zhang H, Lu Y, Liu A, Ma X, Xie Z (2015) Zbtb20 regulates the terminal differentiation of hypertrophic chondrocytes via repression of Sox9. Development 142(2):385–393. 10.1242/dev.108530 Zhu C, Chen G, Zhao Y, Gao XM, Wang J (2018) Regulation of the Development and Function of B Cells by ZBTB Transcription Factors. Front Immunol 9:580. 10.3389/fimmu.2018.00580 Zollman S, Godt D, Privé GG, Couderc JL, Laski FA (1994) The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc Natl Acad Sci U S A 91(22):10717–10721. 10.1073/pnas.91.22.10717 Additional Declarations No competing interests reported. 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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-6876906","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":479692393,"identity":"821dabbc-911f-41e4-a6ab-f8ba123cb303","order_by":0,"name":"Lidiya Kechidzhieva","email":"","orcid":"","institution":"Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lidiya","middleName":"","lastName":"Kechidzhieva","suffix":""},{"id":479692394,"identity":"70987528-c98a-4898-8bf8-1f70b9ab9334","order_by":1,"name":"Katerina Ilieva","email":"","orcid":"","institution":"Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Katerina","middleName":"","lastName":"Ilieva","suffix":""},{"id":479692396,"identity":"e4a5b913-be70-4a6f-91d4-46789b345c43","order_by":2,"name":"Viktoria Hranova","email":"","orcid":"","institution":"Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Viktoria","middleName":"","lastName":"Hranova","suffix":""},{"id":479692398,"identity":"9c9a35da-bfb9-417b-ba2d-fcb8ee3da58e","order_by":3,"name":"Valentina Ivanova","email":"","orcid":"","institution":"Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Valentina","middleName":"","lastName":"Ivanova","suffix":""},{"id":479692400,"identity":"4d0afb49-fd8d-4ff1-b721-eedf79007db8","order_by":4,"name":"Dimo Stoyanov","email":"","orcid":"","institution":"Medical University of Varna","correspondingAuthor":false,"prefix":"","firstName":"Dimo","middleName":"","lastName":"Stoyanov","suffix":""},{"id":479692403,"identity":"fe0166e6-7086-4ba1-83a9-56e616a71b2d","order_by":5,"name":"Anton Tonchev","email":"","orcid":"","institution":"Medical University of Varna","correspondingAuthor":false,"prefix":"","firstName":"Anton","middleName":"","lastName":"Tonchev","suffix":""},{"id":479692404,"identity":"e1c9f243-b93f-4543-bda2-e560d2f798b2","order_by":6,"name":"Andrey Tchorbanov","email":"","orcid":"","institution":"Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Andrey","middleName":"","lastName":"Tchorbanov","suffix":""},{"id":479692405,"identity":"85b96924-49d1-4a77-98de-9a723fa00f76","order_by":7,"name":"Iliyan Manoylov","email":"data:image/png;base64,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","orcid":"","institution":"Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Iliyan","middleName":"","lastName":"Manoylov","suffix":""}],"badges":[],"createdAt":"2025-06-12 06:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6876906/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6876906/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86016954,"identity":"d95e856a-8a30-4ff1-9790-8fdd90852fa3","added_by":"auto","created_at":"2025-07-04 10:58:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":449800,"visible":true,"origin":"","legend":"\u003cp\u003eZbtb20 affects the HSCs and L-CD19+ (B-1) cells in \u0026nbsp;the BM of the Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e mice. \u0026nbsp;The Lin-Sca-1+c-Kit+ (L-S+K+) (pannel a),\u0026nbsp; Lin- CD19+ (L-CD19+) (pannel b) and\u0026nbsp; Lin-CD19+Sca-1+ (L-CD19+S+) (pannel c) populations were analysed in BMs of experimental WT and Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e mice. 7 animals per group were used in the analysis. Data is represented as mean ± standard deviation (SD) values; P-values were calculated by unparired t-test (n = 7) \u0026nbsp;(*P\u0026lt;0.1, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P\u0026lt;0.0001)\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6876906/v1/d6512ce79792a6ccf94cb2e0.jpg"},{"id":86015837,"identity":"3fc33047-ab43-4ac4-9166-dd8a92ac8fb6","added_by":"auto","created_at":"2025-07-04 10:50:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":321915,"visible":true,"origin":"","legend":"\u003cp\u003eRegular B cells development is affected in the BM of the Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e mice. \u0026nbsp;The CD19+B220+ population from homo- and heterozygous animals was analysed. 7 animals per group were used in the analysis. Data is represented as mean ± standard deviation (SD) values; P-values were calcualted by unparired t-test (n = 7) \u0026nbsp;(*P\u0026lt;0.1, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P\u0026lt;0.0001)\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6876906/v1/253bf657ceff4b01ccd885ea.jpg"},{"id":86016955,"identity":"0b2ba5b1-e75f-4d61-a83d-a0caf409b8da","added_by":"auto","created_at":"2025-07-04 10:58:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":692000,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of zygosity for Zbtb20 on the CD19+, CD4+ and CD8+ populations. Screening of the C45+CD19+ (pannel a), CD45+CD3+ (pannel b), CD45+CD3+CD4+ (pannel c) an CD45+CD3+CD8+ (pannel d) cells isolated from splenocytes of homo- and heterozygous animals. 7 animals per group were used in the analysis. Data is represented as mean ± standard deviation (SD) values; P-values were calcualted by unparired t-test (n = 7) \u0026nbsp;(*P\u0026lt;0.1, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P\u0026lt;0.0001)\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6876906/v1/d299fd7288b643431be5406a.jpg"},{"id":86016958,"identity":"f1510b46-d8b7-44ab-ab26-c57251cf1999","added_by":"auto","created_at":"2025-07-04 10:58:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":954357,"visible":true,"origin":"","legend":"\u003cp\u003eELISA assay for natural IgG, IgM and IgA antibodies in sera of WT and Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e mice. 7 animals per group were used in the analysis. All samples were triplicated. Data is represented as mean ± standard deviation (SD) values; P-values were calcualted by unparired t-test (n = 7) \u0026nbsp;(*P\u0026lt;0.1, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P\u0026lt;0.0001)\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6876906/v1/8e1ac50769cfd3e9931990dd.jpg"},{"id":92929503,"identity":"3f47bd4a-60b5-4092-971a-c6851219bb88","added_by":"auto","created_at":"2025-10-07 08:47:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3055626,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6876906/v1/92a3409b-7fec-4494-a558-8623cc58b419.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Zbtb20 zygosity affects the immunological profile of experimental animals ","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe genetic impact on tissue development is well established, with transcription factors (TFs) playing key role in all organs. For example, Oct4 and Sox2 were proven as vital participants in keeping the pluripotency of the embryonic stem cells, with deficiencies leading to failure in forming the inner cell mass. (Rodda et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) TP53 deficiency leads to aggressive cancer development. (Ignatius et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) This makes the discovery of new regulatory functions, of known TF families, indispensable for the understanding of the development of the organism and its systems.\u003c/p\u003e \u003cp\u003eThe immune system has many factors that are involved in its development and functioning. Mutations in the gene GATA-2 lead to unfunctional proteins and facilitates the ongoing of opportunistic infections. (Crispino \u0026amp; Horwitz, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) AIRE, and its related protein, is responsible for the regulation of the autoimmunity reactions in the body (Zhao, Chang, Fu, Sun, \u0026amp; Yang, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) NAIP is important for the formation of the inflammasome and its deficiency leads to a lack of protection. (Mitchell et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe ZBTB family of TFs is another example of developmentally-important family of proteins first identified in \u003cem\u003eDrosophilas\u003c/em\u003e and, named after three \u003cem\u003eDrosophila\u003c/em\u003e genes. (Zollman, Godt, Priv\u0026eacute;, Couderc, \u0026amp; Laski, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1994\u003c/span\u003e) They possess a N-terminal BTB/POZ (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eb\u003c/span\u003eroad complex\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003et\u003c/span\u003e ramtrack \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eb\u003c/span\u003eric-a-brac/\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003epo\u003c/span\u003exvirus) domain involved in protein-protein interactions, and a \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ez\u003c/span\u003einc finger domain, importnat for the recognition and DNA interaction. (Lee \u0026amp; Maeda, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) The family became even more important after the discovery that a protein, named DPZF (W. Zhang et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) or HOF (Mitchelmore et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), can be found in many developmental processess and cancerous conditions. (Kelly \u0026amp; Daniel, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) Now, it is becoming more and more evident that many of the Zbtb TFs have multiple functions.\u003c/p\u003e \u003cp\u003eZbtb1 acts as a trasncriptional repressor and is vital for the T cell development. (Matic et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; J. Wang et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) Zbtb17 (MIZ-1) is a TF equally important for the proper gastrulation and the \u0026ldquo;β-selection\u0026rdquo; checkpoint of the pre-TCR. (Adhikary et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Rashkovan et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) Breast cancer prognosys and myeloid lineage development are associated with Zbtb7 (THPOK, LRF) (Basu et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; H. Qu et al., 2010) The well studied Zbtb27 (BCL-6) has a great clinical value for the B cell lymphomas diagnotics and the regulation of T cells. (J. Choi \u0026amp; Crotty, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Zhu, Chen, Zhao, Gao, \u0026amp; Wang, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) This multiple activity is valid as well for ZBTB35 (ZNF131), ZBTB19 (PATZ1, MAZR), ZBTB29 (HIC1) and many more (Z.-Y. Cheng, He, Gao, Zhao, \u0026amp; Wang, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eAn important member of the family, the TF Zbtb20 was discovered during brain development (Mitchelmore et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), and during the development of the astrocytes and chondrocytes. (Z. Qu et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) During gliogenesis, Zbtb20 acts in concert with Sox9 and NFIA to regulate astrogliogenesis, and a similar molecular mechanisms apparently exists during chondrocyte final differentiatin. (Zhou et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) The Pituitary gland development and the functioning of the liver are highly dependable on this gene (D. Cao et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) \u003cem\u003eZBTB20\u003c/em\u003e loss-of-function in humans causes Primrose syndrome. (Cordeddu et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) Throughout the years, several groups have discovered a role of Zbtb20 in immunity. The transcriptional activity of the protein is elevated when long-lived plasma cells are being formed and differentiated in the germinal centers. (Chevrier et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) It helps the long term antibody response and it plays a significant role in the development of the B-cells from progenitor cells in the bone marrow (BM). (Lee \u0026amp; Maeda, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Y. Wang \u0026amp; Bhattacharya, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) \u003cem\u003eZbtb20\u003c/em\u003e has a role in the T cell activation, specifically the decision to generate CD8\u0026thinsp;+\u0026thinsp;cells or T regs, and this was shown in multiple mouse experiments. Further, Zbtb20 is able to modulate the tumor-specific T-cell response, the NF-kB signal pathway and the intestine immunological homeostasis. (Krzyzanowska et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; X. Liu et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) Yet, more is to be discovered about the role of ZBTB20 on the immune system.\u003c/p\u003e \u003cp\u003eIn the present research we studied wether the loss of one allele of \u003cem\u003eZbtb20\u003c/em\u003e will affect the phenotype and antibody secretion properties of BM and spleen cells.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003e All animals included in the present study were handled in accordance with the Bulgarian national regulations. The experiments were carried out in strict accordance with the Guidelines for the Care and Use of Laboratory Animals of the European Union (EU Directive 2010/63/EU), and the manipulations were approved by the animal care commission at the Bulgarian Food Safety Agency, approval protocol Nr. 392/21.04.2024. The mice were bread and kept under specific pathogen free (SPF) conditions at the Institute of Microbiology, Bulgarian Academy of Sciences and housed at 20\u0026ndash;22\u003csup\u003eo\u003c/sup\u003eC with a light/dark cycle of 12/12 hrs. We used 3\u0026ndash;6 months old male and female 129s mice in all experiments.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHeterozygous Zbtb20 mice generation\u003c/h3\u003e\n\u003cp\u003eThe Zbtb20 mutant mice generation was previously described. (Rosenthal, Tonchev, Stoykova, \u0026amp; Chowdhury, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Tonchev, Tuoc, Rosenthal, Studer, \u0026amp; Stoykova, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) Briefly, the BTB/POZ protein domain and the first zinc fingers were replaced by a lacZ-neomycin cassette. Animals carrying the mutation in a homozygous state Zbtb20\u003csup\u003eLacZ/LacZ\u003c/sup\u003e were not vital for more than a month, with many physiological alterations being observed. In order to preserve the mutation, heterozygous animals Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e were intecrossed and based on the Mendelian law of segregation, a genotypic ratio of 1:2:1 (WT: Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e: Zbtb20\u003csup\u003eLacZ/LacZ\u003c/sup\u003e) was expected.\u003c/p\u003e\n\u003ch3\u003eDNA isolation and genotyping\u003c/h3\u003e\n\u003cp\u003eOn day 5 after birth, mice of the Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e intercross progeny were subjected to toe clipping. Briefly, the pad of each animal was cleaned with alcohol prior the procedure. Sterilized sharp scissors were used and were disinfected between animals. A clean gauze sponge was applied over the site with gentle pressure to stop the bleeding. The isolated distal phalangeal bone was put in a sterile Eppendorf tube and kept on ice until all samples were collected.\u003c/p\u003e \u003cp\u003eThe so obtained tissues were subjected to \u0026ldquo;dirty\u0026rdquo; DNA isolation. (Truett et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) Alcaline lysing solution containing 25mM NaOH and 0.2mM EDTA was added to each tube prior of putting it to a thermocycler at 98\u003csup\u003eo\u003c/sup\u003eC for 1 hour. The solution was afterwards neutralized with the same volume of 40 mM Tris HCl (pH 5.5). After a brief centrifuge at 1000 RCF for 3 minutes the isolated DNA was subjected to a standard PCR amplification in order to verify the zigoucity of the animals in the progeny. The primers used in the reaction are as follows: ER_Zbtb_F, 50-TCACAGCCAAACAGAACTACG-30; ER_Neo_F, 50-TCTTCTGAGGGGATCAATTCTC-30; ER_Zbtb_R, 50-CAAGCTTTGGACCCACACTA-30.\u003c/p\u003e \u003cp\u003eAfter the genotyping, all mice were divided into one of the following groups: WT, Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e and Zbtb20\u003csup\u003eLacZ/LacZ\u003c/sup\u003e, as only WT and Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e were part of the study.\u003c/p\u003e\n\u003ch3\u003eAntibodies for flow cytometry\u003c/h3\u003e\n\u003cp\u003eThe following antibodies for flow cytometry were used in the study. For the BM flow cytometry: FITC anti-mouse Lineage Cocktail with Isotype Ctrl (Biolegend, 133302), PE anti-mouse CD117 (c-kit) Antibody (Biolegend, 135106), PE/Cyanine7 anti-mouse Ly-6A/E (Sca-1) Antibody (Biolegend, 108114), Pacific Blue\u0026trade; anti-mouse CD19 Antibody (Biolegend, 115523). For the flow cytometry of splenocytes: FITC anti-mouse CD45 Antibody (Biolegend, 103107), PE anti-mouse CD19 Antibody (Biolegend, 152408), PE/Cyanine7 anti-mouse CD3ε Antibody (Biolegend, 100320), Pacific Blue\u0026trade; anti-mouse CD4 Antibody (Biolegend, 100428), APC anti-mouse CD8a Antibody (Biolegend, 100712)\u003c/p\u003e\n\u003ch3\u003eFlow cytometry of the BM cells\u003c/h3\u003e\n\u003cp\u003eAnalysis of the hematopoietic stem cells (HSCs) of the animals was performed using flow cytometry. 7 WT and 7 Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e mice were euthanized by cervical dislocation and BM was isolated from their femurs as described. (Amend, Valkenburg, \u0026amp; Pienta, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) The long bones were put into 0.5ml tubes, nested in 1.5ml centrifuge tubes. They were spun at 10000 RCF for 15sec. The erythrocytes from the isolated cellular fraction were lysed for a minute with a lysis buffer containting NH\u003csub\u003e4\u003c/sub\u003eCl, KHCO\u003csub\u003e3\u003c/sub\u003e and EDTA and the suspension was washed 2 times with PBS at 150 RCF / 4\u003csup\u003eo\u003c/sup\u003eC / 10min to remove the debris. 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per FACS tube were distributed in ice-cold PBS (containing 2.5% FCS and 0.05% sodium azide) and incubated with the Lineage cocktail, CD1117 (c-kit), Ly-6A/E (Sca-1) and CD19 antibodies for 20min/ 4oC, followed by double wash with PBS/FCS to remove the excess of antibodies. The pellet was finally resuspended in PBS/FCS and analyzed. Cells populations were defined as L-S\u0026thinsp;+\u0026thinsp;K+ (Lin-Sca-1\u0026thinsp;+\u0026thinsp;c-Kit+), L-S\u0026thinsp;+\u0026thinsp;CD19+ (Lin-Sca-1\u0026thinsp;+\u0026thinsp;CD19+) and CD19\u0026thinsp;+\u0026thinsp;B220\u0026thinsp;+\u0026thinsp;S+ (CD19\u0026thinsp;+\u0026thinsp;B220\u0026thinsp;+\u0026thinsp;Sca-1+). At least thirty thousand cells were analyzed from each sample with a BD LSR II flow cytometer (BD Biosciences, San Jose, CA) using Diva 6.1.1. software (BD Biosciences, Mountain View, CA, USA).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry of spleen cells\u003c/h2\u003e \u003cp\u003eAnalysis of the spleen lymphocyte populations was performed using flow cytometry. Spleens from the euthanized mice were taken and subjected to splenocytes isolation by disruption with cell stainer, lysing and washing as previously described. 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per FACS tube were distributed in ice-cold PBS / FBS and an antibody mix containing the CD45, CD19, CD3ε, CD4, CD8a antibodies was added for for 20min/ 4\u003csup\u003eo\u003c/sup\u003eC. After a double PBS/ FBS wash, the cells were analyzed by flow cytometry as at least thirty thousand cells were counted. The screened populations were defined as: CD45\u0026thinsp;+\u0026thinsp;CD19+, CD45\u0026thinsp;+\u0026thinsp;CD3+, CD45\u0026thinsp;+\u0026thinsp;CD3\u0026thinsp;+\u0026thinsp;CD4+, CD45\u0026thinsp;+\u0026thinsp;CD3\u0026thinsp;+\u0026thinsp;CD8+.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSandwich ELISA of sera\u003c/h3\u003e\n\u003cp\u003eSandwich ELISA assay was performed to screen the sera from different animals, for the major antibodies isotypes \u0026ndash; IgG, IgM and IgA. Blood samples were collected from the retro-orbital plexuses of the experimental animals and left for 2h at 4\u003csup\u003eo\u003c/sup\u003eC for clot formation. The clotted blood was consecutively centrifuged first at 200 RCF, next at 300 RCF and the obtained sera were kept at -80\u003csup\u003eo\u003c/sup\u003eC for further analysis. When enough samples were collected, sandwich ELISA was performed as previously described. (Kohl \u0026amp; Ascoli, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) Briefly, 96-well Maxisorp immunoplates (Nunc, Roskilde, Denmark) were coated with Goat Anti-Mouse IgG\u0026thinsp;+\u0026thinsp;IgM\u0026thinsp;+\u0026thinsp;IgA H\u0026amp;L (ab102445, Abcam), 5ug/ml in PBS overnight at 4\u003csup\u003eo\u003c/sup\u003eC. The plates were then blocked with 1.0% Tween-20, incubated with 100x diluted sera and later detected with biotin anti-mouse IgA Antibody (407004, Biolegend), biotin anti-mouse IgM Antibody (406504, Biolegend) and biotin goat anti-mouse IgG (minimal x-reactivity) antibody (405303, Biolegend). The samples were developed using ABTS (2,20-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) solution (11684302001, Sigma-Aldrich) and read at 405nm.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003eZbtb20\u003c/b\u003e\u003csup\u003e\u003cb\u003eLacZ/+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eanimals show significant changes in the Lin\u003c/b\u003e\u003csup\u003e\u003cb\u003e\u0026minus;\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eBM populations\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eFlow cytometry analysis of BM cells was used to identify the L-S\u0026thinsp;+\u0026thinsp;K+, L-CD19\u0026thinsp;+\u0026thinsp;and L-CD19\u0026thinsp;+\u0026thinsp;S\u0026thinsp;+\u0026thinsp;cell populations. After the cells were gated through the Lin\u0026ndash;/Lin\u0026thinsp;+\u0026thinsp;populations, major mouse hematopoetic markers Sca-1 and c-Kit were used to segregate the 2 groups of animals. Gating through the double positive cells was performed for the L-S\u0026thinsp;+\u0026thinsp;K+. As Zbtb20 is associated with the development, a change was expected in the hematopoietic cellular department. As depicted, the percent of the L-S\u0026thinsp;+\u0026thinsp;K\u0026thinsp;+\u0026thinsp;population was significantly lower in the WT animals, compared to the Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, pannel a). This result led to the expectation that all populations will be less in the BM of the WT animals. Interestingly, the L-CD19\u0026thinsp;+\u0026thinsp;cells which here, we consider as B-1 cells, were less in the heterozygous animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, pannel b), leading to the conclusion that this side of the innate immunity will be underdevelopped in these animas. The hematopietic capacity of these B-1 cells, was assed with both c-Kit and Sca-1 markers, and while any Lin-CD19\u0026thinsp;+\u0026thinsp;c-Kit\u0026thinsp;+\u0026thinsp;positive cells were not observed (data not shown), the levels of L-CD19\u0026thinsp;+\u0026thinsp;S\u0026thinsp;+\u0026thinsp;cells were different in both genotypes, showing a role of Zbtb20 in the development of this particular population (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, pannel c)\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDevelopment of the B cells in the BM is augmented in heterozygous animals\u003c/h2\u003e \u003cp\u003eAs regular B cell formation is undergone in the BM, a screening for the CD19\u0026thinsp;+\u0026thinsp;B220\u0026thinsp;+\u0026thinsp;cells was perfomed. Here we consider these cells as developping conventional B cells, that include the Pro-B, Pre-B, Immature B and the Transitional B cells as both markers are observed during all the stages. What we observed was a higher percent of the CD19\u0026thinsp;+\u0026thinsp;B220\u0026thinsp;+\u0026thinsp;cells in the Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, pannel a), leading to the assumption that these mice will have a higher antibody levels in the sera. The hematopoietic potential of these cells (expressed as CD19\u0026thinsp;+\u0026thinsp;B220\u0026thinsp;+\u0026thinsp;S+) in the heterozygous animals is visible, which can be related to the overall HSC activity in this genotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, pannel b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGeneral splenocytes populations are affected in heterozygous animals\u003c/h2\u003e \u003cp\u003eBased on the high HSC levels in the BM as a primary lymphoid organ, a change in the immune cells populations in the systemic lymphoid organs was expected in the heterozygous animals. A screening of the spleen cells was performed for the major immune cells populations, like CD45, CD19, CD3, CD4 and CD8. The comparison between the CD45\u0026thinsp;+\u0026thinsp;CD19\u0026thinsp;+\u0026thinsp;and the CD45\u0026thinsp;+\u0026thinsp;CD3\u0026thinsp;+\u0026thinsp;cells (generally considered as B and T cells) presented more B cells and less T cells in the Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e splenocytes. The screening of the WT spleen cells, showed the opposite trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, pannel a and pannel b). This was not a surprise, as the levels of the developping B cells in the BM were more in the animals bearing the heterozygous genotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, we analyzed two T cell subsets - CD45\u0026thinsp;+\u0026thinsp;CD3\u0026thinsp;+\u0026thinsp;CD4+ (regarded as helper T cells) and CD45\u0026thinsp;+\u0026thinsp;CD3\u0026thinsp;+\u0026thinsp;CD8+ (regarded as cytotoxic T cells). A noticeable difference is observed between the homo- and the heterozygous animals regarding the helper T cells. The Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e mice had significantly lower numbers of CD45\u0026thinsp;+\u0026thinsp;CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;cells suggesting a weakened adaptive immunity response and overall activation. This data supports previous research results on the role of other Zbtb factors on the development of helper T cells. (He et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) Although visible, the contrast in the levels of CD45\u0026thinsp;+\u0026thinsp;CD3\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;cells between the two genotypes can not be considered as a significant one. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAltered circulating natural antibodies in Zbtb20 heterozygous mice\u003c/h2\u003e \u003cp\u003eThe higher levels of B cells in the BM and the spleen of the heterozygous animals could lead to more natural antibodies in the blood of these mice. We probed this possibility by a sandwich ELISA of the sera of +/+ and +/- animals. We screened the natural IgG, IgM and IgA antibodies, obtained from peripheral blood. The IgA antibodies were not expected to vary, as IgA is predominantly found in the mucosal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, right pannel), although there is a slight difference. On the other hand, IgG and IgM antibodies exhibited different trends. The natural IgG antibodies were slighly diminished in the Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, left pannel), while the levels of natural IgM antibodies were elevated, similarly to the B cells in the BM and the spleen of the WT animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, center pannel), opposing the lower levels of the B-1 cells in the BM, but suggesting a higher secreting capacity of this particular population.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present paper confirms the importance of Zbtb20 in the regulation of the immune system homeostasis. Although there was no obvious contrast in the physical appearance of the WT and Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e animals in our study, there was a dramatic difference in the level of L-S\u0026thinsp;+\u0026thinsp;K\u0026thinsp;+\u0026thinsp;cells. This came to a surprise, given the effect of a similary designed heterozygous condition regarding the Zbtb11 TF. (H. Cao et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) Usually overexpression of Zbtb genes leads to diminishing of cell numbers. (Satpathy et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Tang et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) On the other hand HSC activity is affected by many checkpoint regulators and is dependent on DNA methylation. (T. Cheng et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Yanai et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) While we can not exclude a role of Zbtb20 in all these processes, more research is needed to address this issue. (Maeda, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) There is also a possibility for a genetic compensation by other Zbtb family member \u0026ndash; a hypothesys that is being investigated in the last years in different models. (El-Brolosy \u0026amp; Stainier, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Rouf et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eSurprisingly to the high levels of HSC cells in the BM of the \u003cem\u003eZbtb20\u003c/em\u003e heterozygous mice, their B-1 cells were decreased. We expected to detect higher numbers of B-1 cells in the mutants, but the observed opposite result confirms the critical importance of Zbtb20 on B-1 cells. (J. Liu \u0026amp; Zhang, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Development of the two substypes of B cells (B-1 cells and B-2 cells) takes place in 3 distinct stages: the first one occurs in the yolk sac, the second one - in the fetal liver, and the third one - in the BM. (Mattos, Vandendriessche, Waisman, \u0026amp; Marques, 2024) The two B cell subsets have different roles in immunity. While the B-2 cells are considered as \u0026ldquo;classical\u0026rdquo; ones, the B-1 cells act more like innate-like cells, found in the cavities and being able to go through a T cell independent differentiation to IgM secreting plasma cells. (Y. S. Choi, Dieter, Rothaeusler, Luo, \u0026amp; Baumgarth, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Montecino-Rodriguez et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) Having lower numbers of B-1 cells in the BM of the Zbtb20 mutants, we expected, that the levels of natural IgM antibodies would be lower as well. To our surprise, 3 of the animals showed higher levels of IgM antibody secretion. The number of B-1 cells in these animals was not above the average in the cohort. Furthermore, the levels of Sca-1 on these cells were also at the average for the study group. While Sca-1 is known to affect the number of B-1 cells, its role on the secretion of IgM antibodies is less clear. (Long, Pavlath, \u0026amp; Montano, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) This phenomenon suggests a possible involvement of another member of the Zbtb TF family, possibly Zbtb32, in the IgM production. (Chevrier et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) Another possibility could be that the higher levels of IgM in these 3 animals is related to a loss of heterozygosity, and the subsequent over activation of the remaining allele on one of the chromosomes. (X. Zhang \u0026amp; Sj\u0026ouml;blom, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe function of Zbtb20 in B cell development and differentiation outside the BM was already defined. (Chevrier et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) Nevertheless, it is intriguing how and why the Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e mice get more developing conventional B cells in the BM and in the spleen. There is a probability, that the heterozygous mice fail to activate the controlling mechanisms in the B cell compartment, thus leading to a higher percent of B cells. (Nemazee, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) This may be a consequence of an interaction between Zbtb20 and miRNA factors \u0026ndash; a dual system, found in some malignancies. (J. Liu et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) Still, more analyses should be done to prove this theory, as Zbtb20 heterozygous mice are expected to have lower Zbtb20 activity. While screening the regular CD3 cells, we found a lower number of CD3\u0026thinsp;+\u0026thinsp;cells and CD4\u0026thinsp;+\u0026thinsp;cells in the Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e. Zbtb7a (ThPOK) is a well known regulator of the T cell activity, as it is able to supress the CD8\u0026thinsp;+\u0026thinsp;T cells. (He, Park, \u0026amp; Kappes, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; L. Wang et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) Recent data demonstrate that Zbtb20 is also able to coordinate the development of the T cells. And what we see in our research confirms the results that define Zbtb20 as a supressor of the CD8 T cell activity (although our data is not statistically confirmable). (Preiss et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e)We obsercve a lower number of CD4 T cells in the Zbtb20\u003csup\u003eLacZ/+\u003c/sup\u003e mice, compared to the WT, a fact that sheds light on the effect ot this particular gene on the development of Th cells.\u003c/p\u003e \u003cp\u003eIn summary, the present study provides new information on the BM HSCs that are affected by the loss of allele of Zbtb20. The ratio between the T cell populations was also affected in the mutants, as were the levels IgM secretion, that were higher in some of the heterozygous animals. Future studies will help investigate more deeply the role that Zbtb20 has on the immune system, but together these results present a good platform to continue the work in several directions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by The National Science Fund of Bulgaria, project number KP-06-H61/5 (IM), and NextGenerationEU via Bulgarian National Recovery and Resilience Plan, Project #BG-RRP-2.004-0009-C03 (DS, ABT).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eL.K., K.I., V.H. and V.I. researched the data, I.M. wrote the manuscript and prepared the figures. D.S., A.T. and A.T. helped with the discussion of the results. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was supported by The National Science Fund of Bulgaria, project number KP-06-H61/5 (IM), and NextGenerationEU via Bulgarian National Recovery and Resilience Plan, Project #BG-RRP-2.004-0009-C03 (DS, ABT).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdhikary S, Peukert K, Karsunky H, Beuger V, Lutz W, Els\u0026auml;sser HP, Eilers M (2003) Miz1 is required for early embryonic development during gastrulation. Mol Cell Biol 23(21):7648\u0026ndash;7657. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1128/mcb.23.21.7648-7657.2003\u003c/span\u003e\u003cspan address=\"10.1128/mcb.23.21.7648-7657.2003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmend SR, Valkenburg KC, Pienta KJ (2016) Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation. J Vis Exp 11010.3791/53936\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBasu J, Olsson A, Ferchen, Kyle, Titerina EK, Chetal K, Nicolas, Emmanuelle,.. Kappes, Dietmar J (2023) ThPOK is a critical multifaceted regulator of myeloid lineage development. Nat Immunol 24(8):1295\u0026ndash;1307. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41590-023-01549-3\u003c/span\u003e\u003cspan address=\"10.1038/s41590-023-01549-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao D, Ma X, Cai J, Luan J, Liu AJ, Yang R, Zhang WJ (2016) ZBTB20 is required for anterior pituitary development and lactotrope specification. Nat Commun 7:11121. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ncomms11121\u003c/span\u003e\u003cspan address=\"10.1038/ncomms11121\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCao H, Naik SH, Amann-Zalcenstein D, Hickey P, Salim A, Cao B, Lieschke GJ (2023) Late fetal hematopoietic failure results from ZBTB11 deficiency despite abundant HSC specification. Blood Adv 7(21):6506\u0026ndash;6519. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/bloodadvances.2022009580\u003c/span\u003e\u003cspan address=\"10.1182/bloodadvances.2022009580\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng T, Rodrigues N, Shen H, Yang Y, Dombkowski D, Sykes M, Scadden DT (2000) Hematopoietic stem cell quiescence maintained by p21cip1/waf1. Science 287(5459):1804\u0026ndash;1808. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/science.287.5459.1804\u003c/span\u003e\u003cspan address=\"10.1126/science.287.5459.1804\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng Z-Y, He T-T, Gao X-M, Zhao Y, Wang J (2021) ZBTB Transcription Factors: Key Regulators of the Development, Differentiation and Effector Function of T Cells. \u003cem\u003e12\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2021.713294\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2021.713294\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChevrier S, Emslie D, Shi W, Kratina T, Wellard C, Karnowski A, Corcoran LM (2014) The BTB-ZF transcription factor Zbtb20 is driven by Irf4 to promote plasma cell differentiation and longevity. J Exp Med 211(5):827\u0026ndash;840. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1084/jem.20131831\u003c/span\u003e\u003cspan address=\"10.1084/jem.20131831\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi J, Crotty S (2021) Bcl6-Mediated Transcriptional Regulation of Follicular Helper T cells (T(FH)). Trends Immunol 42(4):336\u0026ndash;349. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.it.2021.02.002\u003c/span\u003e\u003cspan address=\"10.1016/j.it.2021.02.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChoi YS, Dieter JA, Rothaeusler K, Luo Z, Baumgarth N (2012) B-1 cells in the bone marrow are a significant source of natural IgM. Eur J Immunol 42(1):120\u0026ndash;129. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/eji.201141890\u003c/span\u003e\u003cspan address=\"10.1002/eji.201141890\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCordeddu V, Redeker B, Stellacci E, Jongejan A, Fragale A, Bradley TE, Hennekam RC (2014) Mutations in ZBTB20 cause Primrose syndrome. Nat Genet 46(8):815\u0026ndash;817. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/ng.3035\u003c/span\u003e\u003cspan address=\"10.1038/ng.3035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrispino JD, Horwitz MS (2017) GATA factor mutations in hematologic disease. Blood 129(15):2103\u0026ndash;2110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1182/blood-2016-09-687889\u003c/span\u003e\u003cspan address=\"10.1182/blood-2016-09-687889\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Brolosy MA, Stainier DYR (2017) Genetic compensation: A phenomenon in search of mechanisms. PLoS Genet 13(7):e1006780. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1371/journal.pgen.1006780\u003c/span\u003e\u003cspan address=\"10.1371/journal.pgen.1006780\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe X, He X, Dave VP, Zhang Y, Hua X, Nicolas E, Kappes DJ (2005) The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 433(7028):826\u0026ndash;833. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nature03338\u003c/span\u003e\u003cspan address=\"10.1038/nature03338\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHe X, Park K, Kappes DJ (2010) The role of ThPOK in control of CD4/CD8 lineage commitment. Annu Rev Immunol 28:295\u0026ndash;320. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1146/annurev.immunol.25.022106.141715\u003c/span\u003e\u003cspan address=\"10.1146/annurev.immunol.25.022106.141715\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIgnatius MS, Hayes MN, Moore FE, Tang Q, Garcia SP, Blackburn PR, Langenau DM (2018) tp53 deficiency causes a wide tumor spectrum and increases embryonal rhabdomyosarcoma metastasis in zebrafish. Elife 7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7554/eLife.37202\u003c/span\u003e\u003cspan address=\"10.7554/eLife.37202\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKelly KF, Daniel JM (2006) POZ for effect\u0026ndash;POZ-ZF transcription factors in cancer and development. Trends Cell Biol 16(11):578\u0026ndash;587. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tcb.2006.09.003\u003c/span\u003e\u003cspan address=\"10.1016/j.tcb.2006.09.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohl TO, Ascoli CA (2017) Immunometric Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay. Cold Spring Harb Protoc 2017(6). pdb.prot093724\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKrzyzanowska AK, Ii H, Kovalovsky RAH, Lin D, Osorio HC, Edelblum L, Sant'Angelo KL, D. B (2022) Zbtb20 identifies and controls a thymus-derived population of regulatory T cells that play a role in intestinal homeostasis. Sci Immunol 7(71):eabf3717. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1126/sciimmunol.abf3717\u003c/span\u003e\u003cspan address=\"10.1126/sciimmunol.abf3717\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee SU, Maeda T (2012) POK/ZBTB proteins: an emerging family of proteins that regulate lymphoid development and function. Immunol Rev 247(1):107\u0026ndash;119. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1600-065X.2012.01116.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1600-065X.2012.01116.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Jiang J, Hui X, Wang W, Fang D, Ding L (2018) Mir-758-5p Suppresses Glioblastoma Proliferation, Migration and Invasion by Targeting ZBTB20. Cell Physiol Biochem 48(5):2074\u0026ndash;2083. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1159/000492545\u003c/span\u003e\u003cspan address=\"10.1159/000492545\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Zhang H (2024) Zinc Finger and BTB Domain-Containing 20: A Newly Emerging Player in Pathogenesis and Development of Human Cancers. Biomolecules 14(2). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/biom14020192\u003c/span\u003e\u003cspan address=\"10.3390/biom14020192\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu X, Zhang P, Bao Y, Han Y, Wang Y, Zhang Q, Cao X (2013) Zinc finger protein ZBTB20 promotes Toll-like receptor-triggered innate immune responses by repressing IκBα gene transcription. Proc Natl Acad Sci U S A 110(27):11097\u0026ndash;11102. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1301257110\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1301257110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLong KK, Pavlath GK, Montano M (2011) Sca-1 influences the innate immune response during skeletal muscle regeneration. Am J Physiol Cell Physiol 300(2):C287\u0026ndash;294. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/ajpcell.00319.2010\u003c/span\u003e\u003cspan address=\"10.1152/ajpcell.00319.2010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaeda T (2016) Regulation of hematopoietic development by ZBTB transcription factors. Int J Hematol 104(3):310\u0026ndash;323. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s12185-016-2035-x\u003c/span\u003e\u003cspan address=\"10.1007/s12185-016-2035-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatic I, Schimmel J, Hendriks IA, van Santen MA, van de Rijke F, van Dam H, Vertegaal AC (2010) Site-specific identification of SUMO-2 targets in cells reveals an inverted SUMOylation motif and a hydrophobic cluster SUMOylation motif. Mol Cell 39(4):641\u0026ndash;652. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.molcel.2010.07.026\u003c/span\u003e\u003cspan address=\"10.1016/j.molcel.2010.07.026\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMattos M, Silv\u0026eacute;rio, Vandendriessche, Sofie, Waisman A, Marques PE (2024) The immunology of B-1 cells: from development to aging. Immun Ageing 21(1):54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12979-024-00455-y\u003c/span\u003e\u003cspan address=\"10.1186/s12979-024-00455-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitchell PS, Roncaioli JL, Turcotte EA, Goers L, Chavez RA, Lee AY, Vance RE (2020) NAIP-NLRC4-deficient mice are susceptible to shigellosis. Elife 9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7554/eLife.59022\u003c/span\u003e\u003cspan address=\"10.7554/eLife.59022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMitchelmore C, Kjaerulff KM, Pedersen HC, Nielsen JV, Rasmussen TE, Fisker MF, Jensen NA (2002) Characterization of two novel nuclear BTB/POZ domain zinc finger isoforms. Association with differentiation of hippocampal neurons, cerebellar granule cells, and macroglia. J Biol Chem 277(9):7598\u0026ndash;7609. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1074/jbc.M110023200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M110023200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontecino-Rodriguez E, Fice M, Casero D, Berent-Maoz B, Barber CL, Dorshkind K (2016) Distinct Genetic Networks Orchestrate the Emergence of Specific Waves of Fetal and Adult B-1 and B-2 Development. Immunity 45(3):527\u0026ndash;539. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.immuni.2016.07.012\u003c/span\u003e\u003cspan address=\"10.1016/j.immuni.2016.07.012\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNemazee D (2017) Mechanisms of central tolerance for B cells. Nat Rev Immunol 17(5):281\u0026ndash;294. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/nri.2017.19\u003c/span\u003e\u003cspan address=\"10.1038/nri.2017.19\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePreiss NK, Kamal Y, Wilkins OM, Li C, Kolling FW th, Trask HW, Usherwood EJ (2023) Characterizing control of memory CD8 T cell differentiation by BTB-ZF transcription factor Zbtb20. Life Sci Alliance 6(9). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.26508/lsa.202201683\u003c/span\u003e\u003cspan address=\"10.26508/lsa.202201683\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQu H Danni, Qu, Fuhui, Chen, Zhenyu, Zhang, Baogang, Liu, \u0026amp; and Liu, Haifeng. (2010). ZBTB7 Overexpression Contributes to Malignancy in Breast Cancer. Cancer Invest, \u003cem\u003e28\u003c/em\u003e(6), 672\u0026ndash;678. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3109/07357901003631007\u003c/span\u003e\u003cspan address=\"10.3109/07357901003631007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQu Z, Zhang H, Huang M, Shi G, Liu Z, Xie P, Xu Y (2016) Loss of ZBTB20 impairs circadian output and leads to unimodal behavioral rhythms. Elife 5. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.7554/eLife.17171\u003c/span\u003e\u003cspan address=\"10.7554/eLife.17171\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRashkovan M, Vadnais C, Ross J, Gigoux M, Suh WK, Gu W, M\u0026ouml;r\u0026ouml;y T (2014) Miz-1 regulates translation of Trp53 via ribosomal protein L22 in cells undergoing V(D)J recombination. Proc Natl Acad Sci U S A 111(50):E5411\u0026ndash;5419. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.1412107111\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1412107111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodda DJ, Chew JL, Lim LH, Loh YH, Wang B, Ng HH, Robson P (2005) Transcriptional regulation of nanog by OCT4 and SOX2. J Biol Chem 280(26):24731\u0026ndash;24737. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1074/jbc.M502573200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M502573200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenthal EH, Tonchev AB, Stoykova A, Chowdhury K (2012) Regulation of archicortical arealization by the transcription factor Zbtb20. Hippocampus 22(11):2144\u0026ndash;2156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1002/hipo.22035\u003c/span\u003e\u003cspan address=\"10.1002/hipo.22035\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRouf MA, Wen L, Mahendra Y, Wang J, Zhang K, Liang S, Wang G (2023) The recent advances and future perspectives of genetic compensation studies in the zebrafish model. Genes Dis 10(2):468\u0026ndash;479. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.gendis.2021.12.003\u003c/span\u003e\u003cspan address=\"10.1016/j.gendis.2021.12.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSatpathy AT, Kc W, Albring JC, Edelson BT, Kretzer NM, Bhattacharya D, Murphy KM (2012) Zbtb46 expression distinguishes classical dendritic cells and their committed progenitors from other immune lineages. J Exp Med 209(6):1135\u0026ndash;1152. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1084/jem.20120030\u003c/span\u003e\u003cspan address=\"10.1084/jem.20120030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Preiss NK, Valenteros KB, Kamal Y, Usherwood YK, Frost HR, Usherwood EJ (2020) Zbtb20 Restrains CD8 T Cell Immunometabolism and Restricts Memory Differentiation and Antitumor Immunity. J Immunol 205(10):2649\u0026ndash;2666. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4049/jimmunol.2000459\u003c/span\u003e\u003cspan address=\"10.4049/jimmunol.2000459\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang X, Deng C, Liu, Yang, Pu, Shengyu, Zheng, Qi, Zhou Y, Hao N (2025) ZBTB6 promotes breast cancer progression by inhibiting ARHGAP6 transcription and modulating the STAT3 signaling pathway. J Translational Med 23(1):370. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12967-025-06364-y\u003c/span\u003e\u003cspan address=\"10.1186/s12967-025-06364-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTonchev AB, Tuoc TC, Rosenthal EH, Studer M, Stoykova A (2016) Zbtb20 modulates the sequential generation of neuronal layers in developing cortex. Mol Brain 9(1):65. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s13041-016-0242-2\u003c/span\u003e\u003cspan address=\"10.1186/s13041-016-0242-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTruett GE, Heeger P, Mynatt RL, Truett AA, Walker JA, Warman ML (2000) Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and tris (HotSHOT). Biotechniques 29(1):52, 54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.2144/00291bm09\u003c/span\u003e\u003cspan address=\"10.2144/00291bm09\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Shi, Chunwei, Cheng M, Lu Y, Zhang X, Li, Fengdi, Cao (2022) Xin. Effects of the Zbtb1 Gene on Chromatin Spatial Structure and Lymphatic Development: Combined Analysis of Hi-C, ATAC-Seq and RNA-Seq. \u003cem\u003e10\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fcell.2022.874525\u003c/span\u003e\u003cspan address=\"10.3389/fcell.2022.874525\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang L, Wildt KF, Castro E, Xiong Y, Feigenbaum L, Tessarollo L, Bosselut R (2008) The zinc finger transcription factor Zbtb7b represses CD8-lineage gene expression in peripheral CD4\u0026thinsp;+\u0026thinsp;T cells. Immunity 29(6):876\u0026ndash;887. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.immuni.2008.09.019\u003c/span\u003e\u003cspan address=\"10.1016/j.immuni.2008.09.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Bhattacharya D (2014) Adjuvant-specific regulation of long-term antibody responses by ZBTB20. J Exp Med 211(5):841\u0026ndash;856. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1084/jem.20131821\u003c/span\u003e\u003cspan address=\"10.1084/jem.20131821\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Z, Zhang H, Tsai W, Zhang Y, Du Y, Zhong J, Zhang WJ (2008) Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver. Proc Natl Acad Sci U S A 105(31):10859\u0026ndash;10864. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.0800647105\u003c/span\u003e\u003cspan address=\"10.1073/pnas.0800647105\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYanai H, McNeely T, Ayyar S, Leone M, Zong L, Park B, Beerman I (2024) DNA methylation drives hematopoietic stem cell aging phenotypes after proliferative stress. Geroscience. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s11357-024-01360-4\u003c/span\u003e\u003cspan address=\"10.1007/s11357-024-01360-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang W, Mi J, Li N, Sui L, Wan T, Zhang J, Cao X (2001) Identification and characterization of DPZF, a novel human BTB/POZ zinc finger protein sharing homology to BCL-6. Biochem Biophys Res Commun 282(4):1067\u0026ndash;1073. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1006/bbrc.2001.4689\u003c/span\u003e\u003cspan address=\"10.1006/bbrc.2001.4689\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Sj\u0026ouml;blom T (2021) Targeting Loss of Heterozygosity: A Novel Paradigm for Cancer Therapy. Pharmaceuticals (Basel) 14(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/ph14010057\u003c/span\u003e\u003cspan address=\"10.3390/ph14010057\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao B, Chang L, Fu H, Sun G, Yang W (2018) The Role of Autoimmune Regulator (AIRE) in Peripheral Tolerance. \u003cem\u003eJ Immunol Res, 2018\u003c/em\u003e, 3930750. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1155/2018/3930750\u003c/span\u003e\u003cspan address=\"10.1155/2018/3930750\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou G, Jiang X, Zhang H, Lu Y, Liu A, Ma X, Xie Z (2015) Zbtb20 regulates the terminal differentiation of hypertrophic chondrocytes via repression of Sox9. Development 142(2):385\u0026ndash;393. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1242/dev.108530\u003c/span\u003e\u003cspan address=\"10.1242/dev.108530\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu C, Chen G, Zhao Y, Gao XM, Wang J (2018) Regulation of the Development and Function of B Cells by ZBTB Transcription Factors. Front Immunol 9:580. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fimmu.2018.00580\u003c/span\u003e\u003cspan address=\"10.3389/fimmu.2018.00580\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZollman S, Godt D, Priv\u0026eacute; GG, Couderc JL, Laski FA (1994) The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila. Proc Natl Acad Sci U S A 91(22):10717\u0026ndash;10721. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1073/pnas.91.22.10717\u003c/span\u003e\u003cspan address=\"10.1073/pnas.91.22.10717\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Zbtb20, Immunogenetics, Heterozygous animals, Hematopoietic Stem Cells","lastPublishedDoi":"10.21203/rs.3.rs-6876906/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6876906/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMembers of the Zbtb family of transcription factors is vital for the development and function of many cells and organs. One member in particular, Zbtb20, is critical for the maturation of the nervous system, the pituitary gland and the liver. In the present research we investigate the effect that the Zbtb20 TF has on the immune system of animals that lack one allele of the \u003cem\u003eZbtb20\u003c/em\u003e gene. We report that greater number of hematopoietic cells, but lower levels of B-1 cells, is found in the bone marrow of \u003cem\u003eZbtb20\u003c/em\u003e-heterozygous mutants. More CD19\u0026thinsp;+\u0026thinsp;cells, than CD3\u0026thinsp;+\u0026thinsp;cells are present in the spleen, with CD4\u0026thinsp;+\u0026thinsp;cells being less abundant than the CD8\u0026thinsp;+\u0026thinsp;cells of these animals, as well. The ratio between natural IgM and IgG antibodies differs in the two genotypes, with more IgM antibodies being secreted from less B-1 cells in the heterozygous mice. Together, our results shed additional light on the \u003cem\u003eZbtb20\u003c/em\u003e-mediated regulation of the immune system.\u003c/p\u003e","manuscriptTitle":"Zbtb20 zygosity affects the immunological profile of experimental animals ","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-04 10:50:26","doi":"10.21203/rs.3.rs-6876906/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":"4056a147-074e-4a04-8fbc-90b271b1d25e","owner":[],"postedDate":"July 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-07T08:39:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-04 10:50:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6876906","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6876906","identity":"rs-6876906","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}