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Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1β, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent phosphorylation of HP1γ may participate in the regulation of dynamic interaction between HP1γ and histone H3K9me3 to promote DNA damage repair. This HIPK2/HP1γ pathway may uncover a new functional aspect of HIPK2 as a tumor suppressor. Show less

We measured the dynamics of an essential epigenetic modifier, HP1β, in human cells at different stages of differentiation using Fluorescence Recovery After Photobleaching (FRAP). We found that HP1β mobility is similar in human embryonic stem cells (hES) and iPS cells where it is more mobile compared to fibroblasts; HP1β is less mobile in senescent fibroblasts than in young (dividing) fibroblasts. Introduction of "reprogramming factors", Oct4, Sox2, Klf4, cMyc and Lin28, into senescent fibroblasts and measuring the changes in HP1β mobility as reprogramming proceeds shows that the mobility of HP1β in senescent cells increases and by day 9 is the same as that found in young fibroblasts. Thus the dynamics of a key epigenetic modifier can be rejuvenated without de-differentiation through an embryonic stage. Future work will test whether other aspects of cellular physiology that age can be so rejuvenated without de-differentiation. Show less

We report here a peptide-driven approach to create first inhibitors of the chromobox homolog 7 (CBX7), a methyllysine reader protein. CBX7 uses its chromodomain to bind histone 3, lysine 27 trimethylated (H3K27me3), and this recognition event is implicated in silencing multiple tumor suppressors. Small trimethyllysine containing peptides were used as the basic scaffold from which potent ligands for disruption of CBX7-H3K27me3 complex were developed. Potency of ligands was determined by fluorescence polarization and/or isothermal titration calorimetry. Binding of one ligand was characterized in detail using 2D NMR and X-ray crystallography, revealing a structural motif unique among human CBX proteins. Inhibitors with a ∼200 nM potency for CBX7 binding and 10-fold/400-fold selectivity over related CBX8/CBX1 proteins were identified. These are the first reported inhibitors of any chromodomain. Show less

The human genome consists of roughly 30,000 genes coding for over 500,000 different proteins, of which more than 10,000 proteins can be produced by the cell at any given time (the cellular "proteome"). It has been estimated that over 80% of proteins do not operate alone, but in complexes. These protein-protein interactions (PPI) are regulated by several mechanisms. For example, post-translational modifications (methylation, acetylation, phosphorylation, or ubiquitination) or metal-binding can lead to conformational changes that alter the affinity and kinetic parameters of the interaction. Many PPIs are part of larger cellular networks of interactions or interactomes. Indeed, these interactions are at the core of the entire interactomics system of any living cell, and so, aberrant PPIs are the basis of multiple diseases, such as neurodegenerative diseases and cancer. The objective of this study was to develop a method of monitoring protein-protein interactions and proximity dependence in vivo. The biotin ligase BirA was fused to the protein of interest, and the Biotin Acceptor Peptide (BAP) was fused to an interacting partner to make the detection of its biotinylation possible by western blot or mass spectrometry. Using several experimental systems (BirA.A + BAP.B), we showed that the biotinylation is interaction/proximity dependent. Here, A and B are the next nuclear proteins used in the experiments - 3 paralogues of heterochromatin protein HP1α (CBX5), HP1β (CBX1), HP1γ (CBX3), wild type and transcription mutant factor Kap1, translesion DNA polymerase PolH and E3, ubiquitin ligase RAD18, Proliferative Cell Nuclear Antigen (PCNA), ubiquitin Ub, SUMO-2/3, different types and isoforms of histones H2A, H2Az, H3.1, H3.3, CenpA, H2A.BBD, and macroH2A. The variant of this approach is termed PUB-NChIP (Proximity Utilizing Biotinylation with Native Chromatin Immuno-precipitation) and is designed to purify and study the protein composition of chromatin in proximity to the nuclear protein of interest. Using the RAD18 protein as a model, we demonstrated that the RAD18-proximal chromatin is enriched in some H4 acetylated species. Moreover, the RAD18-proximal chromatin containing a replacement histone H2Az has a different pattern of H4 acetylation. Progress in the last decade in cancer drug therapy has led us to the conclusion that the nucleus of eukaryotic cells is an active site for many cellular processes important to the development of cancer. These processes include changes in genetic and epigenetic landscape (e. g. methylation of DNA, modification of histones) and the expression levels of transcription factors, which regulates gene products (e.g. hypoxia-inducible factor 1α (HIF-1α) in chronic anemia, etc.) where protein-protein interactions play important role. Understanding the nature of protein-protein interactions may improve design strategies for small-molecule PPI modulators. PPI assay technologies that closely reflect physiological conditions hold the key to developing specific anti-cancer drugs. Show less

Ribosomes are essential intracellular machines composed of proteins and RNA molecules. The DNA sequences (rDNA) encoding ribosomal RNAs (rRNAs) are tandemly repeated and give origin to the nucleolus. Here we develop a computational method for estimating rDNA dosage (copy number) and mitochondrial DNA abundance using whole-genome short-read DNA sequencing. We estimate these attributes across hundreds of human genomes and their association with global gene expression. The analyses uncover abundant variation in rDNA dosage that is coupled with the expression of hundreds of functionally coherent gene sets. These include associations with genes coding for chromatin components that target the nucleolus, including CTCF and HP1β. Finally, the data show an inverse association between rDNA dosage and mitochondrial DNA abundance that is manifested across genotypes. Our findings uncover a novel and cryptic source of hypervariable genomic diversity with global regulatory consequences (ribosomal eQTL) in humans. The variation provides a mechanism for cellular homeostasis and for rapid and reversible adaptation. Show less

Heterochromatin protein 1 (HP1) is an epigenetic modifier of gene regulation and chromatin packing via binding to trimethylated histone H3 lysine 9 (H3K9). HP1 plays an important role in gene activation as well as gene repression in heterochromatin and euchromatin. However, the role of individual HP1 proteins in human diseases remains elusive. Here, we show that HP1β negatively regulates the expression and activation of matrix metallopeptidase (MMP)2, which mediates cancer metastasis by destructing type Ⅳ collagen. Reduced HP1β expression correlates with the increased level of pro- and active-MMP2 in colon cancer cells. Consistently, HP1β knockdown (KD) increased and HP1β overexpression decreased the mRNA level of MMP2 and membrane type 1 metallopeptidase (MT1-MMP). Furthermore, cancer cells overexpressing HP1β showed impaired migratory ability, whereas HP1β‑deleted cancer cells had increased migration. HP1β negatively regulates MMP2 expression in a transcriptional level and prevents MMP2 activation through reducing the expression of MT1‑MMP. These findings shed new light on HP1β as a molecular regulator and an efficient therapeutic target of metastatic cancer. Show less

The brains of teleost fish show extensive adult neurogenesis and neuronal regeneration. The patterns of gene regulation during fish brain aging are unknown. The short-lived teleost fish Nothobranchius furzeri shows markers of brain aging including reduced learning performances, gliosis, and reduced adult neurogenesis. We used RNA-seq to quantify genome-wide transcript regulation and sampled five different time points to characterize whole-genome transcript regulation during brain aging of N. furzeri. Comparison with human datasets revealed conserved up-regulation of ribosome, lysosome, and complement activation and conserved down-regulation of synapse, mitochondrion, proteasome, and spliceosome. Down-regulated genes differ in their temporal profiles: neurogenesis and extracellular matrix genes showed rapid decay, synaptic and axonal genes a progressive decay. A substantial proportion of differentially expressed genes (~40%) showed inversion of their temporal profiles in the last time point: spliceosome and proteasome showed initial down-regulation and stress-response genes initial up-regulation. Extensive regulation was detected for chromatin remodelers of the DNMT and CBX families as well as members of the polycomb complex and was mirrored by an up-regulation of the H3K27me3 epigenetic mark. Network analysis showed extensive coregulation of cell cycle/DNA synthesis genes with the uncharacterized zinc-finger protein ZNF367 as central hub. In situ hybridization showed that ZNF367 is expressed in neuronal stem cell niches of both embryonic zebrafish and adult N. furzeri. Other genes down-regulated with age, not previously associated with adult neurogenesis and with similar patterns of expression are AGR2, DNMT3A, KRCP, MEX3A, SCML4, and CBX1. CBX7, on the other hand, was up-regulated with age. Show less

Owing to the loss of heterochromatin integrity that occurs during thyroid tumorigenesis, the expression of Heterochromatin Protein 1 isoforms HP1α and HP1β was assessed by immunohistochemistry in 189 thyroid tumors and non-neoplastic tissues. Expression of HP1β was significantly decreased in all thyroid lesions, except in follicular adenomas, when compared with matched adjacent normal tissue. This loss of HP1β expression may in part be caused by microRNA dysregulation. An example is miR-205, a microRNA that is abundantly upregulated in thyroid carcinomas and shown to reduce the expression of HP1β. In contrast to HP1β, HP1α expression was only reduced in metastatic carcinomas and poorly differentiated lesions. These results suggest the reduction of HP1β followed by a decrease in HP1α contributes to the pathogenesis of thyroid carcinomas, and their loss is a potential marker of thyroid malignancy and metastatic potential, respectively. Show less

Heterochromatin protein 1 (HP1) proteins are chromatin-bound transcriptional regulators. While their chromodomain binds histone H3 methylated on lysine 9, their chromoshadow domain associates with the H3 histone fold in a region involved in chromatin remodeling. Here, we show that phosphorylation at histone H3 threonine 45 and serine 57 within this latter region differentially affects binding of the three mammalian HP1 isoforms HP1α, HP1β and HP1γ. Both phosphorylation events are dependent on the activity of the DYRK1A kinase that antagonizes HP1-mediated transcriptional repression and participates in abnormal activation of cytokine genes in Down's syndrome-associated megakaryoblastic leukemia. Show less

The optimal repair of DNA lesions is fundamental for physiological processes. We asked whether the recruitment of HP1β, 53BP1 and BMI1 proteins to ultraviolet (UVA)-induced DNA lesions requires functional A-type lamins. We found that UVA irradiation of nuclear lamina abolished the fluorescence of mCherry-tagged A-type lamins and destroyed the nuclear lamina as also observed by electron microscopy studies. Similarly, an absence of endogenous A- and B-type lamins was found in irradiated regions by UVA. However, irradiation did not affect the recruitment of HP1β, 53BP1 and BMI1 to DNA lesions. The UVA-induced shrinkage of the nuclear lamina, which anchors chromatin, explains why UVA-micro-irradiated chromatin is relaxed. Conversely, additional experiments with γ-irradiation showed that the nuclear lamina remained intact and the genome-wide level of HP1β was stable. Fluorescence intensity of HP1β and BMI1 in UVA-induced DNA lesions and level of HP1β after γ-irradiation were unaffected by deficiency in A-type lamins, whereas those parameters of 53BP1 were changed. We conclude that only the 53BP1 status in DNA lesions, induced by UVA or γ-rays, is affected by A-type lamin deficiency, which was not observed for heterochromatin-related proteins HP1β and BMI1. Show less

Proteins of the Heterochromatin Protein 1 (HP1) family are regulators of chromatin structure and genome function in eukaryotes. Post-translational modifications expand the repertoire of the chemical diversity of HP1 proteins and regulate their activity. Here, we investigated the effect of phosphorylation by Casein kinase 2 (CK2) on the structure, dynamics and binding activity of human HP1β. We show that Ser89 in the hinge region is the most effective substrate, followed by Ser175 at the C-terminal tail. Phosphorylation at these sites results in localized conformational changes in HP1β that do not compromise the ability of the protein to bind chromatin. Show less

New protein lysine methyltransferase (PKMT) assays are needed to facilitate screening for improved PKMT inhibitors, because PKMTs are mutated or overexpressed in several cancers. In cells, methylated lysine residues are recognized by reading domains such as the chromodomain of HP1β, which bind to target proteins in a lysine-methylation-specific manner. Herein we describe a sensitive, robust, and non-radioactive high-throughput PKMT assay that employs the HP1β chromodomain to detect the methylation of peptide substrates by the human SUV39H1 and SUV39H2 PKMTs. The assay has a very good dynamic range and high signal-to-noise ratio. It can be used to screen for PKMT inhibitors, as illustrated by analyzing the inhibition of SUV39H1 by chaetocin. The IC50 value of this inhibition was found to be 480 nM, which is close to its published value. Our data indicate that natural reading domains can be used as alternates to methyl-specific antibodies in PKMT assays. Reading domains can be produced recombinantly in E. coli at low cost and consistent quality, and they are accessible to protein design. Show less

Biobed substrates commonly exhibit high degradation capacity. However, degradation does not always lead to detoxification and information on the metabolic pathways of pesticides in biobeds is scarce. We studied the degradation and metabolism of three pesticides in selected biomixtures and soil. Biomixtures stimulated degradation of terbuthylazine and metribuzin, whereas chlorpyrifos degraded faster in soil. The latter was attributed to the lipophilicity of chlorpyrifos which increased adsorption and limited biodegradation in organic-rich biomixtures. Although the same metabolites were detected in all substrates, qualitative and quantitative differences in the metabolic routes of pesticides in the various substrates were observed. Chlorpyrifos was hydrolyzed to 3,5,6-tricholorpyridinol (TCP) which was further degraded only in compost-biomixture CBX1. Metabolism of terbuthylazine in compost biomixtures (BX) and soil resulted in the formation of desethyl-terbuthylazine (DES) which was fully degraded only in the compost-biomixture CBX2, whereas peat-based biomixture (OBX) promoted the hydroxylation of terbuthylazine. Desamino- (DA) (dominant) and diketo- (DK) metribuzin appear as intermediate metabolites in all substrates and were further transformed to desamino-diketo-metribuzin (DADK) which was fully degraded only in compost-biomixture GSBX. Overall, lower amounts of metabolites were accumulated in biomixtures compared to soil stressing the higher depuration efficiency of biobeds. Show less

Mouse embryonic fibroblasts (MEFs) differentiate into fully functional chondrocytes in response to bone morphogenetic protein-2 (BMP-2). However, the comprehensive proteomic aspect of BMP-2-induced chondrogenesis remains unknown. We took advantage of quantitative proteomic analysis based on isobaric tag for relative and absolute quantitation (iTRAQ) and on-line 2D nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS) to identify proteins differentially expressed during BMP-2-induced chondrogenic differentiation of MEFs. We found 85 downregulated proteins, and ingenuity pathways analysis (IPA) revealed a protein-protein network with chromodomain-helicase-DNA-binding protein 4 (Chd4) in the center. Chromatin immunoprecipitation (ChIP) and nuclease hypersensitivity assays showed that Chd4, interacting with Hdac1/2, cooperates with its related proteins Kap1 and Cbx1 to bind at -207/-148 of the Sox9 promoter. We also provided evidence that let-7a targets the 3'UTR of Chd4 to promote chondrogenesis of MEFs. Together, our findings indicate that BMP-2 induced the upregulation of let-7a, targeting Chd4 and positively controlling the chondrogenic differentiation of MEFs. These findings illustrate epigenetic regulation of the chondrogenic differentiation process and also expand the understanding of the involved intracellular mechanisms. Show less

Retrovirus-based vectors are commonly used as delivery vehicles to correct genetic diseases because of their ability to integrate new sequences stably. However, adverse events in which vector integration activates proto-oncogenes, leading to clonal expansion and leukemogenesis hamper their application. The host cell-encoded lens epithelium-derived growth factor (LEDGF/p75) binds lentiviral integrase and targets integration to active transcription units. We demonstrated earlier that replacing the LEDGF/p75 chromatin interaction domain with an alternative DNA-binding protein could retarget integration. Here, we show that transient expression of the chimeric protein using mRNA electroporation efficiently redirects lentiviral vector (LV) integration in wild-type (WT) cells. We then employed this technology in a model for X-linked chronic granulomatous disease (X-CGD) using myelomonocytic PLB-985 gp91(-/-) cells. Following electroporation with mRNA encoding the LEDGF-chimera, the cells were treated with a therapeutic lentivector encoding gp91(phox). Integration site analysis revealed retargeted integration away from genes and towards heterochromatin-binding protein 1β (CBX1)-binding sites, in regions enriched in marks associated with gene silencing. Nevertheless, gp91(phox) expression was stable for at least 6 months after electroporation and NADPH-oxidase activity was restored to normal levels as determined by superoxide production. Together, these data provide proof-of-principle that transient expression of engineered LEDGF-chimera can retarget lentivector integration and rescues the disease phenotype in a cell model, opening perspectives for safer gene therapy.Molecular Therapy - Nucleic Acids (2013) 2, e77; doi:10.1038/mtna.2013.4; published online 5 March 2013. Show less

Androgen receptor (AR) signaling is critical for the tumorigenesis and development of prostate cancer, as well as the progression to castration-resistant prostate cancer. We previously showed that the heterochromatin protein 1 (HP1) β isoform plays a critical role in transactivation of AR signaling as an AR coactivator that promotes prostate cancer cell proliferation. However, the roles of other HP1 isoforms, HP1α and HP1γ, in AR expression and prostate cancer remain unclear. Here, we found that knockdown of HP1γ, but not HP1α, reduced AR expression and cell proliferation by inducing cell cycle arrest at G1 phase in LNCaP cells. Conversely, overexpression of full-length HP1α and its C-terminal deletion mutant increased AR expression and cell growth, whereas overexpression of HP1γ had no effect. Similarly, HP1α overexpression promoted 22Rv1 cell growth, whereas HP1γ knockdown reduced the proliferation of CxR cells, a castration-resistant LNCaP derivative. Taken together, HP1 isoforms distinctly augment AR signaling and cell growth in prostate cancer. Therefore, silencing of HP1β and HP1γ may be a promising therapeutic strategy for treatment of prostate cancer. Show less

As essential components of the molecular machine assembling heterochromatin in eukaryotes, HP1 (Heterochromatin Protein 1) proteins are key regulators of genome function. While several high-resolution structures of the two globular regions of HP1, chromo and chromoshadow domains, in their free form or in complex with recognition-motif peptides are available, less is known about the conformational behavior of the full-length protein. Here, we used NMR spectroscopy in combination with small angle X-ray scattering and dynamic light scattering to characterize the dynamic and structural properties of full-length human HP1β (hHP1β) in solution. We show that the hinge region is highly flexible and enables a largely unrestricted spatial search by the two globular domains for their binding partners. In addition, the binding pockets within the chromo and chromoshadow domains experience internal dynamics that can be useful for the versatile recognition of different binding partners. In particular, we provide evidence for the presence of a distinct structural propensity in free hHP1β that prepares a binding-competent interface for the formation of the intermolecular β-sheet with methylated histone H3. The structural plasticity of hHP1β supports its ability to bind and connect a wide variety of binding partners in epigenetic processes. Show less

Apoptotic bodies are the most condensed form of chromatin. In general, chromatin structure and function are mostly dictated by histone post-translational modifications. Thus, we have analyzed the histone signature in apoptotic cells, characterized by pronounced chromatin condensation. Here, H2B mono-acetylation, and H3K9 and H4 acetylation was significantly decreased in apoptotic cells, which maintained a high level of H3K9 methylation. This phenotype was independent of p53 function and distinct levels of anti-apoptotic Bcl2 protein. Interestingly, after etoposide treatment of leukemia and multiple myeloma cells, H3K9 and H4 hypoacetylation was accompanied by increased H3K9me2, but not H3K9me1 or H3K9me3. In adherent mouse fibroblasts, a high level of H3K9me3 and histone deacetylation in apoptotic bodies was likely responsible for the pronounced (∼40%) recovery of GFP-HP1α and GFP-HP1β after photobleaching. HP1 mobility in apoptotic cells appeared to be unique because limited exchange after photobleaching was observed for other epigenetically important proteins, including GFP-JMJD2b histone demethylase (∼10% fluorescence recovery) or Polycomb group-related GFP-BMI1 protein (∼20% fluorescence recovery). These findings imply a novel fact that only certain subset of proteins in apoptotic bodies is dynamic. Show less

Terminally-differentiated cells cease to proliferate and acquire specific sets of expressed genes and functions distinguishing them from less differentiated and cancer cells. Mature granulocytes show lobular structure of cell nuclei with highly condensed chromatin in which HP1 proteins are replaced by MNEI. These structural features of chromatin correspond to low level of gene expression and the loss of some important functions as DNA damage repair, shown in this work and, on the other hand, acquisition of a new specific function consisting in the release of chromatin extracellular traps in response to infection by pathogenic microbes. Granulocytic differentiation is incomplete in myeloid leukemia and is manifested by persistence of lower levels of HP1γ and HP1β isoforms. This immaturity is accompanied by acquisition of DDR capacity allowing to these incompletely differentiated multi-lobed neutrophils of AML patients to respond to induction of DSB by γ-irradiation. Immature granulocytes persist frequently in blood of treated AML patients in remission. These granulocytes contrary to mature ones do not release chromatin for NETs after activation with phorbol myristate-12 acetate-13 and do not exert the neutrophil function in immune defence. We suggest therefore the detection of HP1 expression in granulocytes of AML patients as a very sensitive indicator of their maturation and functionality after the treatment. Our results show that the changes in chromatin structure underlie a major transition in functioning of the genome in immature granulocytes. They show further that leukemia stem cells can differentiate ex vivo to mature granulocytes despite carrying the translocation BCR/ABL. Show less

DNA repair events have functional significance especially for genome stability. Although the DNA damage response within the whole genome has been extensively studied, the region-specific characteristics of nuclear sub-compartments such as the nucleolus or fragile sites have not been fully elucidated. Here, we show that the heterochromatin protein HP1 and PML protein recognize spontaneously occurring 53BP1- or γ-H2AX-positive DNA lesions throughout the genome. Moreover, 53BP1 nuclear bodies, which co-localize with PML bodies, also occur within the nucleoli compartments. Irradiation of the human osteosarcoma cell line U2OS with γ-rays increases the degree of co-localization between 53BP1 and PML bodies throughout the genome; however, the 53BP1 protein is less abundant in chromatin of ribosomal genes and fragile sites (FRA3B and FRA16D) in γ-irradiated cells. Most epigenomic marks on ribosomal genes and fragile sites are relatively stable in both non-irradiated and γ-irradiated cells. However, H3K4me2, H3K9me3, H3K27me3 and H3K79me1 were significantly changed in promoter and coding regions of ribosomal genes after exposure of cells to γ-rays. In fragile sites, γ-irradiation induces a decrease in H3K4me3, changes the levels of HP1β, and modifies the levels of H3K9 acetylation, while the level of H3K9me3 was relatively stable. In these studies, we confirm a specific DNA-damage response that differs between the ribosomal genes and fragile sites, which indicates the region-specificity of DNA repair. Show less

Methyl-CpG-binding protein 2 (MeCP2) is generally considered to act as a transcriptional repressor, whereas recent studies suggest that MeCP2 is also involved in transcription activation. To gain insight into this dual function of MeCP2, we assessed the impact of MeCP2 on higher-order chromatin structure in living cells using mammalian cell systems harbouring a lactose operator and reporter gene-containing chromosomal domain to assess the effect of lactose repressor-tagged MeCP2 (and separate MeCP2 domains) binding in living cells. Our data reveal that targeted binding of MeCP2 elicits extensive chromatin unfolding. MeCP2-induced chromatin unfolding is triggered independently of the methyl-cytosine-binding domain. Interestingly, MeCP2 binding triggers the loss of HP1γ at the chromosomal domain and an increased HP1γ mobility, which is not observed for HP1α and HP1β. Surprisingly, MeCP2-induced chromatin unfolding is not associated with transcriptional activation. Our study suggests a novel role for MeCP2 in reorganizing chromatin to facilitate a switch in gene activity. Show less

Heterochromatin protein 1 (HP1), a small non-histone chromosomal protein, was recently shown to form a complex in vivo with Proliferating Cell Nuclear Antigen (PCNA), a key factor in DNA replication. The complex, which requires HP1β in a form of a dimer, is engaged in DNA repair and replication. We now provide further evidence based on FRET-FLIM live cell studies confirming the association and close proximity between HP1β and PCNA in the complex. We also demonstrate using FRAP, that although HP1β-PCNA complexes are highly mobile in nonreplicating nuclei, when engaged in DNA replication, they become bound and do not exchange with the mobile pool. These observations are in agreement with a notion that a subpopulation of HP1 molecules interact with PCNA in vivo during DNA replication. Similarly, HP1β which is associated with PCNA in regions of DNA repair, is bound and does not exchange with the mobile pool, suggesting that HP1β in association with PCNA may be a component of a DNA repair complex. Show less

Lysine methylation occurs on both histone and nonhistone proteins. However, our knowledge on the prevalence and function of nonhistone protein methylation is poor. We describe an approach that combines peptide array, bioinformatics, and mass spectrometry to systematically identify lysine methylation sites and map methyllysine-driven protein-protein interactions. Using this approach, we identified a high-confidence and high-resolution interactome of the heterochromatin protein 1β (HP1β) and uncovered, simultaneously, numerous methyllysine sites on nonhistone proteins. We found that HP1β binds to DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and regulates its localization to double-strand breaks (DSBs) during DNA damage response (DDR). Mutation of the methylation sites in DNA-PKcs or depletion of HP1β in cells caused defects in DDR. Furthermore, we showed that the methylation of DNA-PKcs and many other proteins in the HP1β interactome undergoes large changes in response to DNA damage, indicating that Lys methylation is a highly dynamic posttranslational modification. Show less

Sex chromosome inactivation is essential epigenetic programming in male germ cells. However, it remains largely unclear how epigenetic silencing of sex chromosomes impacts the evolution of the mammalian genome. Here we demonstrate that male sex chromosome inactivation is highly conserved between humans and mice and has an impact on the genetic evolution of human sex chromosomes. We show that, in humans, sex chromosome inactivation established during meiosis is maintained into spermatids with the silent compartment postmeiotic sex chromatin (PMSC). Human PMSC is illuminated with epigenetic modifications such as trimethylated lysine 9 of histone H3 and heterochromatin proteins CBX1 and CBX3, which implicate a conserved mechanism underlying the maintenance of sex chromosome inactivation in mammals. Furthermore, our analyses suggest that male sex chromosome inactivation has impacted multiple aspects of the evolutionary history of mammalian sex chromosomes: amplification of copy number, retrotranspositions, acquisition of de novo genes, and acquisition of different expression profiles. Most strikingly, profiles of escape genes from postmeiotic silencing diverge significantly between humans and mice. Escape genes exhibit higher rates of amino acid changes compared with non-escape genes, suggesting that they are beneficial for reproductive fitness and may allow mammals to cope with conserved postmeiotic silencing during the evolutionary past. Taken together, we propose that the epigenetic silencing mechanism impacts the genetic evolution of sex chromosomes and contributed to speciation and reproductive diversity in mammals. Show less

Gene positioning and regulation of nuclear architecture are thought to influence gene expression. Here, we show that, in mouse olfactory neurons, silent olfactory receptor (OR) genes from different chromosomes converge in a small number of heterochromatic foci. These foci are OR exclusive and form in a cell-type-specific and differentiation-dependent manner. The aggregation of OR genes is developmentally synchronous with the downregulation of lamin b receptor (LBR) and can be reversed by ectopic expression of LBR in mature olfactory neurons. LBR-induced reorganization of nuclear architecture and disruption of OR aggregates perturbs the singularity of OR transcription and disrupts the targeting specificity of the olfactory neurons. Our observations propose spatial sequestering of heterochromatinized OR family members as a basis of monogenic and monoallelic gene expression. Show less

The nuclear lamina is a key determinant of nuclear architecture, integrity and functionality in metazoan nuclei. Mutations in the human lamin A gene lead to highly debilitating genetic diseases termed as laminopathies. Expression of lamin A mutations or reduction in levels of endogenous A-type lamins leads to nuclear defects such as abnormal nuclear morphology and disorganization of heterochromatin. This is accompanied by increased proteasomal degradation of certain nuclear proteins such as emerin, nesprin-1α, retinoblastoma protein and heterochromatin protein 1 (HP1). However, the pathways of proteasomal degradation have not been well characterized. To investigate the mechanisms underlying the degradation of HP1 proteins upon lamin misexpression, we analyzed the effects of shRNA-mediated knock-down of lamins A and C in HeLa cells. Cells with reduced levels of expression of lamins A and C exhibited proteasomal degradation of HP1α and HP1β but not HP1γ. Since specific ubiquitin ligases are upregulated in lamin A/C knock-down cells, further studies were carried out with one of these ligases, RNF123, which has a putative HP1-binding motif. Ectopic expression of GFP-tagged RNF123 directly resulted in degradation of HP1α and HP1β. Mutational analysis showed that the canonical HP1-binding pentapeptide motif PXVXL in the N-terminus of RNF123 was required for binding to HP1 proteins and targeting them for degradation. The role of endogenous RNF123 in the degradation of HP1 isoforms was confirmed by RNF123 RNAi experiments. Furthermore, FRAP analysis suggested that HP1β was displaced from chromatin in laminopathic cells. Our data support a role for RNF123 ubiquitin ligase in the degradation of HP1α and HP1β upon lamin A/C knock-down. Hence lamin misexpression can cause degradation of mislocalized proteins involved in key nuclear processes by induction of specific components of the ubiquitin-proteasome system. Show less

Genome-wide association studies (GWAS) have served crucial roles in investigating disease susceptible loci for single traits. On the other hand, GWAS have been limited in measuring genetic risk factors for multivariate phenotypes from pleiotropic genetic effects of genetic loci. This work reports a data mining approach to discover patterns of multivariate phenotypes expressed as association rules, and presents an analytical scheme for GWAS of those newly defined multivariate phenotypes. We identified 13 SNPs for four genes (CSMD1, NFE2L1, CBX1, and SKAP1) associated with a new multivariate phenotype defined as low levels of low density lipoprotein cholesterol (LDL-C < or = 100 mg/dl) and high levels of triglycerides (TG > or = 180 mg/dl). Compared with a traditional approach to GWAS, the use of discovered multivariate phenotypes can be advantageous in identifying pleiotropic genetic risk factors, which may have a common etiological role for the multivariate phenotypes. Show less

Histone post-translational modifications (PTMs) constitute a branch of epigenetic mechanisms that can control the expression of eukaryotic genes in a heritable manner. Recent studies have identified several PTM-binding proteins containing diverse specialized domains whose recognition of specific PTM sites leads to gene activation or repression. Here, we present a high-throughput proteogenomic platform designed to characterize the nucleosomal make-up of chromatin enriched with a set of histone PTM binding proteins known as histone PTM readers. We support our findings with gene expression data correlating to PTM distribution. We isolated human mononucleosomes bound by the bromodomain-containing proteins Brd2, Brd3 and Brd4, and by the chromodomain-containing heterochromatin proteins HP1β and HP1α. Histone PTMs were quantified by mass spectrometry (ChIP-qMS), and their associated DNAs were mapped using deep sequencing. Our results reveal that Brd- and HP1-bound nucleosomes are enriched in histone PTMs consistent with actively transcribed euchromatin and silent heterochromatin, respectively. Data collected using RNA-Seq show that Brd-bound sites correlate with highly expressed genes. In particular, Brd3 and Brd4 are most enriched on nucleosomes located within HOX gene clusters, whose expression is reduced upon Brd4 depletion by short hairpin RNA. Proteogenomic mapping of histone PTM readers, alongside the characterization of their local chromatin environments and transcriptional information, should prove useful for determining how histone PTMs are bound by these readers and how they contribute to distinct transcriptional states. Show less

Developmental regulation of gene expression is controlled by distinct epigenetic signatures catalyzed by various epigenetic modifiers. Little is known about the ontogeny and tissue distribution of these epigenetic modifiers. In the present study, we used a novel approach of RNA-sequencing to elucidate hepatic ontogeny and tissue distribution of mRNA expression of 142 epigenetic modifiers, including enzymes involved in DNA methylation/demethylation, histone acetylation/deacetylation, histone methylation/demethylation, histone phosphorylation and chromosome remodeling factors in male C57BL/6 mice. Livers from male C57BL/6 mice were collected at 12 ages from prenatal to adulthood. Many of these epigenetic modifiers were expressed at much higher levels in perinatal livers than adult livers, such as Dnmt1, Dnmt3a, Dnmt3b, Apobec3, Kat1, Ncoa4, Setd8, Ash2l, Dot1l, Cbx1, Cbx3, Cbx5, Cbx6, Ezh2, Suz12, Eed, Suv39h1, Suv420h2, Dek, Hdac1, Hdac2, Hdac7, Kdm2b, Kdm5c, Kdm7, Prmt1-5, Prmt7, Smarca4, Smarcb1, Chd4 and Ino80e. In contrast, hepatic mRNA expression of a few epigenetic modifiers increased during postnatal liver development, such as Smarca2, Kdm1b, Cbx7 and Chd3. In adult mice (60 d of age), most epigenetic modifiers were expressed at moderately (1-3-fold) higher levels in kidney and/or small intestine than liver. In conclusion, this study, for the first time, unveils developmental changes in mRNA abundance of all major known epigenetic modifiers in mouse liver. These data suggest that ontogenic changes in mRNA expression of epigenetic modifiers may play important roles in determining the addition and/or removal of corresponding epigenetic signatures during liver development. Show less

CBX5, CBX1, and CBX3 (HP1α, β, and γ, respectively) play an evolutionarily conserved role in the formation and maintenance of heterochromatin. In addition, CBX5, CBX1, and CBX3 may also participate in transcriptional regulation of genes. Recently, CBX3 binding to the bodies of a subset of genes has been observed in human and murine cells. However, the generality of this phenomenon and the role CBX3 may play in this context are unknown. Genome-wide localization analysis reveals CBX3 binding at genic regions, which strongly correlates with gene activity across multiple cell types. Depletion of CBX3 resulted in down-regulation of a subset of target genes. Loss of CBX3 binding leads to a more dramatic accumulation of unspliced nascent transcripts. In addition, we observed defective recruitment of splicing factors, including SNRNP70, to CBX3 target genes. Collectively, our data suggest a role for CBX3 in aiding in efficient cotranscriptional RNA processing. Show less