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The Mouse HP1 Proteins Are Not Required for Cell Viability but Are Essential For

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bioRxiv preprint doi: https://doi.org/10.1101/441279; this version posted October 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 The mouse HP1 proteins are not required for cell viability but are essential for 2 preventing liver tumorigenesis 3 4 5 Nehmé Saksouk1-4*, Shefqet Hajdari1-4*, Marine Pratlong5, Célia Barrachina5, Aliki Zavoriti1-4, 6 Amélie Sarrazin6, Nelly Pirot1-4, Jean-Yohan Noël1-4, Lakhdar Khellaf4, Eric Fabbrizio1-4, 7, 7 Florence M. Cammas1-4, 7 8 9 1 IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier, F-34298, France. 10 2 INSERM, U1194, Montpellier, F-34298, France. 11 3 Université de Montpellier, Montpellier, F-34090, France. 12 4 Institut régional du Cancer de Montpellier, Montpellier, F-34298, France. 13 5 MGX, Biocampus Montpellier, CNRS, INSERM, Univ Montpellier, Montpellier, France 14 6 MRI, BioCampus Montpellier, CNRS, INSERM, Univ Montpellier, Montpellier, France 15 7 CNRS, Route de Mende, Montpellier, France 16 17 Corresponding author: Florence Cammas, [email protected] 18 19 * Contributed equally to the work 1 bioRxiv preprint doi: https://doi.org/10.1101/441279; this version posted October 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 20 Abstract 21 Chromatin organization plays essential roles in cell identity and functions. Here, we 22 demonstrated by hepatocyte-specific inactivation of the genes encoding the three 23 Heterochromatin Protein 1 (HP1α, β and γ) in mice (HP1-TKO) that these proteins are 24 dispensable for hepatocyte activity and survival. Conversely, the chronic absence of these 25 proteins led to a drastic increased incidence of liver tumor development. Molecular 26 characterization of HP1-TKO hepatocytes revealed that HP1 proteins are required for 27 maintenance of histone marks associated with heterochromatin, and for the appropriate 28 expression of large number of genes involved in liver-specific functions as well as in genes 29 encoding for transcriptional repressors of the KRAB-ZFP family. Moreover, several specific 30 endogenous retrovirus families were upregulated in HP1-TKO hepatocytes, leading to the 31 deregulated expression of genes in their vicinity. Our findings indicate that HP1 proteins act 32 as guardians of liver homeostasis to prevent tumor development through the modulation of 33 multiple chromatin-associated events. 34 Keywords: chromatin; HP1; cancer; liver; transcriptional silencing; endogenous retrovirus 35 36 2 bioRxiv preprint doi: https://doi.org/10.1101/441279; this version posted October 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 37 Introduction 38 Chromatin is a large structure that is made of DNA fibers associated with histone 39 proteins, and that is essential for the interpretation of genetic information in a cell-type and 40 tissue-specific manner (Jenuwein and Allis 2001). Its alterations can have devastating 41 consequences, as evidenced by the large number of diseases induced by mutations in 42 chromatin-associated proteins (Koschmann et al. 2017; Mirabella et al. 2016), as well as by 43 the dramatic changes in chromatin organization observed in cancer cells (Stein et al. 2000). 44 Although extensively studied in the past three decades, it is still largely unknown how 45 chromatin organization is regulated and involved in whole organism homeostasis. 46 Chromatin can be divided, according to its structural and functional features, in 47 euchromatin and heterochromatin. Euchromatin displays low compaction level, is highly 48 enriched in genes, and is transcriptionally competent. Conversely heterochromatin is highly 49 compacted, enriched in repetitive DNA sequences, and mostly silent. Although, 50 heterochromatin was considered for a long time as an inert compartment with the single 51 function of silencing parasite DNA sequences, it is now recognized as a dynamic and 52 functional compartment (Janssen et al. 2018a). Heterochromatin Protein 1 (HP1) proteins 53 were first isolated as major heterochromatin components in Drosophila (James and Elgin 54 1986), and they are highly conserved from yeast to mammals which express three isoforms 55 (HP1α, HP1β and HP1γ). These proteins are characterized by an N-terminal chromodomain 56 (CD), which is involved in the recognition of the heterochromatin-associated histone mark H3 57 lysine-9 di- or trimethylated (H3K9me2/3), and a C-terminal chromoshadow domain (CSD), 58 which constitutes a platform for interaction with many protein partners. These two domains 59 are separated by the hinge domain that is crucial for HP1 association with RNA and 60 recruitment to heterochromatin (Eissenberg and Elgin 2014; Lomberk et al. 2006). Thus, HP1 61 proteins are at the crossroads of the structural and functional organization of chromatin. 62 Accordingly, HP1 are important for heterochromatin silencing, chromosome segregation, 63 regulation of gene expression, DNA repair and DNA replication (Dinant and Luijsterburg 3 bioRxiv preprint doi: https://doi.org/10.1101/441279; this version posted October 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 64 2009; Fanti and Pimpinelli 2008; Nishibuchi and Nakayama 2014). Moreover, HP1 proteins 65 are essential for embryonic development in several organisms, including Drosophila 66 (Eissenberg et al. 1992), C. elegans (Schott et al. 2006) and the mouse (our unpublished 67 data). HP1α is essential for the plasticity of T helper (Th2) lymphocytes (Allan et al. 2012), 68 HP1β for neuro-muscular junctions (Aucott et al. 2008) and HP1γ for spermatogenesis (Abe 69 et al. 2011; Brown et al. 2010). Several studies also suggested a correlation between HP1 70 expression level and cancer development and/or metastasis formation; however, HP1 role in 71 tumorigenesis remains to be clarified (Dialynas et al. 2008; Vad-Nielsen and Nielsen 2015). 72 To assess the functions of this family of proteins in homeostasis and to bypass the 73 embryonic lethality of their genetic ablation, we inactivated all HP1 encoding genes in mouse 74 hepatocytes (HP1-TKO mice). Liver chromatin organization has been well characterized in 75 several physio-pathological conditions (Janssen et al. 2018b). In addition, several known 76 HP1 partners, including the transcription cofactors TRIM24 and TRIM28, and the histone- 77 lysine N-methyltransferase SUV39H1, play key roles in hepatocytes. Therefore, hepatocytes 78 constitute an excellent model to investigate HP1 roles in chromatin functions ((Herquel et al. 79 2011; Fan et al. 2013; Bojkowska et al. 2012a; Khetchoumian et al. 2007; Hardy and Mann 80 2016)). Unexpectedly, we found that in HP1-TKO mice, hepatocytes can differentiate and 81 contribute to liver functions throughout life. Conversely, HP1 proteins are critical for 82 preventing liver tumorigenesis. Finally, molecular characterization of HP1-TKO mice revealed 83 that HP1 are involved in the regulation of heterochromatin organization and of the expression 84 of genes and Endogenous Retroviruses (ERV). Altogether, these data highlight a new 85 function of HP1 proteins as guardians of liver homeostasis through the modulation of various 86 chromatin-associated events. 87 88 4 bioRxiv preprint doi: https://doi.org/10.1101/441279; this version posted October 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 89 RESULTS 90 91 HP1 proteins are not required for hepatocyte proliferation and survival 92 To unravel HP1 functions in homeostasis, the HP1β and HP1γ encoding-genes (Cbx1 and 93 Cbx3, respectively) were inactivated in the liver of HP1αKO mice (Allan et al. 2012) using the 94 Cre recombinase expressed under the control of the hepatocyte-specific albumin promoter 95 (Postic et al. 1999) (Fig. 1A). These animals were called HP1-TKO. Liver-specific excision of 96 the Cbx1 and Cbx3 alleles was confirmed by PCR (Fig. 1B), and the absence of HP1β and 97 HP1γ protein expression was checked by western blotting. At 7 weeks post-partum as well 98 as at middle-aged, the overall level of HP1β and HP1γ was decreased by about 60% in HP1- 99 TKO liver (Fig. 1C). Furthermore, immuno-fluorescence analysis of liver cryosections using 100 anti-HP1β and -HP1γ antibodies showed that their expression was lost in about 60% of liver 101 cells in HP1-TKO mice compared with controls (wild type littermates or age-matched wild 102 type animals) (Fig. 1D). As this percentage corresponds to the hepatocyte fraction within the 103 liver, these findings indicate that both proteins were concomitantly depleted in most 104 hepatocytes. As expected since Cbx5 (the HP1α-encoding gene) was knocked out in all body 105 cells HP1α could not be detected HP1-TKO liver (Fig. 1B-C). Histological analysis of paraffin- 106 embedded liver tissue sections of 7-week-old (young) and 3-6-month-old (middle-aged) 107 control and HP1-TKO animals after hematoxylin/eosin/Safran staining did not reveal any 108 significant difference in the structural organization/morphology of hepatocytes and of the 109 parenchyma (Supplementary Figure 1). In agreement, analysis by immunohistochemistry 110 (IHC) of Tissue Micro Arrays (TMA) made of liver samples using anti-Ki67 and anti-activated 111 caspase 3 antibodies did not reveal any significant difference in cell proliferation and 112 apoptosis in young and middle-aged HP1-TKO and control mice (Fig.
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  • Mice Lacking Methyl-Cpg Binding Protein 1 Have Deficits in Adult Neurogenesis and Hippocampal Function

    Mice Lacking Methyl-Cpg Binding Protein 1 Have Deficits in Adult Neurogenesis and Hippocampal Function

    Mice lacking methyl-CpG binding protein 1 have deficits in adult neurogenesis and hippocampal function Xinyu Zhao*, Tetsuya Ueba*†, Brian R. Christie‡, Basam Barkho*, Michael J. McConnell§, Kinichi Nakashima*, Edward S. Lein*, Brennan D. Eadie‡, Andrew R. Willhoite*, Alysson R. Muotri*, Robert G. Summers*, Jerold Chun§, Kuo-Fen Lee¶, and Fred H. Gage*ʈ *Laboratory of Genetics and ¶Peptide Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; §Department of Pharmacology, University of California, 9500 Gilman Drive, La Jolla, CA 92093; and ‡Department of Psychology, University of British Columbia, 2136 West Mall, Vancouver, BC, Canada V6T 1Z4 Communicated by Inder M. Verma, The Salk Institute for Biological Studies, San Diego, CA, April 2, 2003 (received for review March 7, 2003) DNA methylation-mediated epigenetic regulation plays critical roles in vivo function of MBD1 is still unclear. Because MBD1 has no in regulating mammalian gene expression, but its role in normal brain sequence specificity, except for methylated CpG, its in vivo target function is not clear. Methyl-CpG binding protein 1 (MBD1), a member genes are not known. of the methylated DNA-binding protein family, has been shown to Maintaining normal DNA methylation levels is critical during bind methylated gene promoters and facilitate transcriptional repres- mammalian development, as demonstrated by the embryonic Ϫ Ϫ sion in vitro. Here we report the generation and analysis of MBD1؊/؊ lethality of Dnmt1 / mice (17). Mutation of the de novo DNA -mice. MBD1؊/؊ mice had no detectable developmental defects and methyltransferase 1-36 (Dnmt3b) causes immunodeficiency, cen -appeared healthy throughout life. However, we found that MBD1؊/؊ tromeric instability, and facial anomalies (ICF) syndrome in hu neural stem cells exhibited reduced neuronal differentiation and mans, with characteristics of genomic instability (18).