Heterochromatin-associated Protein Research Articles

High copper levels induce premature senescence in 3T3-L1 preadipocytes

Copper (Cu) dyshomeostasis has been linked to obesity and related morbidities and also to aging. Cu levels are higher in older or obese individuals, and adipose tissue (AT) Cu levels correlate with body mass index. Aging and obesity induce similar AT functional and structural changes, including an accumulation of senescent cells. To study the effect of Cu-mediated stress-induced premature senescent (Cu-SIPS) on preadipocytes, 3T3-L1 cell line was exposed to a subcytotoxic concentration of copper sulfate. After Cu treatment, preadipocytes acquired typical senescence characteristics including diminished cell proliferation, cell and nuclei enlargement and increased lysosomal mass (higher Lamp2 expression and a slight increased number of cells positive for β-galactosidase associated with senescence (SA-β-Gal)). Cell cycle arrest was due to upregulation of p16Ink4aInk4a and p21Waf1/Cip1. Accordingly, protein levels of the proliferation marker KI67 were reduced. Cu-SIPS relates with oxidative stress and, in this context, an increase of SOD1 and HO-1 expression was detected in Cu-treated cells. The mRNA expression of senescence-associated secretory phenotype factors, such as Mmp3, Il-6 and Tnf-α, increased in Cu-SIPS 3T3-L1 cells but no effect was observed on the expression of heterochromatin-associated protein 1(HP1).Although the downregulation of Lamin B1 expression is considered a hallmark of senescence, Cu-SIPS cells presented higher levels of Lamin B1. The dysregulation of nuclear lamina was accompanied by an increase of nuclear blebbing, but not of micronuclei number.To conclude, a Cu-SIPS model in 3T3-L1 preadipocytes is here described, which may be an asset to the study of AT dysregulation observed in obesity and aging.

OR11-01 A New Model for Estrogen Receptor-alpha Antagonist Mechanism of Action

Abstract Disclosure: J.C. Nwachukwu: None. J.W. Njeri: None. T. Venables: None. C. Seath: None. M.E. Pipkin: None. Y. Hou: None. B.S. Katzenellenbogen: None. J.A. Katzenellenbogen: None. K.W. Nettles: None. We recently developed Dual Mechanism Estrogen Receptor-α (ERα) Inhibitors (DMERI) with more antagonist efficacy than selective ERα modulators (SERMs) such as tamoxifen, and selective ERα degraders (SERDs) such as fulvestrant in certain breast cancer models. Current models of anti-estrogen action are largely based on genomics studies in growth arrested cells, where the effects primarily reversed estradiol-mediated gene expression. In that context, most ER antagonists, especially the SERDs, show very little gene activation. We were interested in understanding antagonist mechanism of action in asynchronous cells that were growing in intact medium that had not been charcoal-stripped to remove estradiol and growth factors. RNA-seq of asynchronous MCF-7 cells treated with 4-hydroxytamoxifen, fulvestrant, a SERM-like DMERI, and a SERD-like DMERI showed that all four classes of ligands equally up-regulated and down-regulated differentially expressed genes (DEGs), which included a core set of regulatory networks, as well as ligand-selective gene networks. The number of DEGs predicted efficacy, with tamoxifen regulating few genes, and the SERD-like DMERI regulating the most genes. The SERD-like DMERI also inhibited expression of many more genes that encode cell cycle regulators and key cancer drivers, such as ERα, AKT1, HRAS, and several growth factor receptors. Landscape in silico analysis based on chromatin immunoprecipitation (ChIP)-seq data suggests that recruitment of the ERα MegaTrans complex in collaboration with the oxysterol receptor, LXRα, predicts up-regulation, while cooperation with the retinoblastoma-associated proteins, RB1 and RBL1, predicts down-regulation of target genes. APEX2-mediated proximity labeling in asynchronous cells revealed that fulvestrant and the SERD-like DMERI labeled both coactivators and corepressors. Both compounds also increase labeling of STAG1 and other cohesin subunits that coordinate chromatin looping, while also associating with the nuclear matrix protein Lamin B1 and heterochromatin-associated proteins. These data suggest that antagonists have dual roles in genome organization. An shRNAmiR screen targeting over 300 chromatin regulatory factors revealed networks of shared and exclusive coregulators used by antiestrogens, including the heterochromatin protein, CBX1, which was exclusively required for growth inhibition by fulvestrant, and subunits of histone acetylase and methylase complexes such as KMT2A (the catalytic subunit of the MLL1 complex), which were broadly required. Thus, antiestrogen-driven growth suppression involves both common and ligand-selective transcriptional regulatory networks through utilization of both coactivators and corepressors. Supported by NIH R01CA220284 and BCRF-083 and BCRF-084 grants. Presentation Date: Friday, June 16, 2023

Elucidating the mechanism of MBD protein LLPS and its role in heterochromatin formation and transcriptional repression.

Membraneless organelles (MLOs) self-assemble into condensed, biochemically distinct microenvironments through liquid-liquid phase separation (LLPS). Heterochromatin, most recently considered an MLO, assembles through weak, multivalent interactions with its associated proteins that contain intrinsically disordered regions. However, the details of the complex molecular interactions between heterochromatin-associated proteins and methylated DNA that drive LLPS have not been fully explored. It is crucial that we elucidate the molecular mechanisms involved in this process as it regulates vital nuclear processes such as transcriptional repression. Its dysregulation is implicated in neurological disorders and cancer. Here, we focus on two members of the methyl-CpG-binding domain (MBD) family of proteins, MBD2 and MBD3, that interpret methylated residues on heterochromatin's underlying DNA. We utilize biochemical and biophysical techniques to (1) determine the conditions and properties that promote MBD2 and MBD3 LLPS and elucidate the molecular mechanism(s) that underpin this process and (2) identify the role binding partners have on MBD2 and MBD3 LLPS. LLPS droplet formation is monitored using UV-Vis spectroscopy and differential interference contrast (DIC) and fluorescence microscopy. To better understand the molecular basis that drives LLPS, small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS) is used to obtain structural and dynamic details of MBD2 and MBD3. Our results provide details into the mechanism(s) by which MBD2 and MBD3 undergo LLPS individually and how this process is enhanced by binding to each other and methylated DNA. Uncovering the driving forces that assemble MBD protein-based LLPS droplets will give us insight into the higher-order organization of heterochromatin and how it functions within this structure. Additionally, understanding how disease-related mutations lead to aberrant formation of condensates or the inability to form condensates will provide novel therapeutic targets.

Characterization of Hepatoma-Derived Growth Factor-Related Protein 2 Interactions with Heterochromatin.

The expression of genetic information is tightly controlled by chromatin regulatory proteins, including those in the heterochromatin gene repression family. Many of these regulatory proteins work together on the chromatin substrate to precisely regulate gene expression during mammalian development, giving rise to many different tissues in higher organisms from a fixed genomic template. Here we identify and characterize the interactions of two related heterochromatin regulatory proteins, heterochromatin protein 1 alpha (HP1α) and M-phase phosphoprotein 8 (MPP8), with hepatoma-derived growth factor-related protein 2 (HRP2). We find in biochemical experiments that HRP2 copurifies and co-sediments with heterochromatin-associated proteins, including HP1α and MPP8. Using the Chromatin in vivo Assay in multiple cell types, we demonstrate that HP1α-mediated gene repression dynamics are altered by the presence of HRP2. Furthermore, the knockout of HRP2 in MDA-MB-231 cells results in significant changes to chromatin structure and stability, which alter gene expression patterns. Here, we detail a mechanism by which HRP2 contributes to epigenetic transcriptional regulation through engagement with heterochromatin-associated proteins to stabilize the chromatin landscape and influence gene expression.

Destabilizing heterochromatin by APOE mediates senescence.

Apolipoprotein E (APOE) is a component of lipoprotein particles that function in the homeostasis of cholesterol and other lipids. Although APOE is genetically associated with human longevity and Alzheimer's disease, its mechanistic role in aging is largely unknown. Here, we used human genetic, stress-induced and physiological cellular aging models to explore APOE-driven processes in stem cell homeostasis and aging. We report that in aged human mesenchymal progenitor cells (MPCs), APOE accumulation is a driver for cellular senescence. By contrast, CRISPR-Cas9-mediated deletion of APOE endows human MPCs with resistance to cellular senescence. Mechanistically, we discovered that APOE functions as a destabilizer for heterochromatin. Specifically, increased APOE leads to the degradation of nuclear lamina proteins and a heterochromatin-associated protein KRAB-associated protein 1 via the autophagy-lysosomal pathway, thereby disrupting heterochromatin and causing senescence. Altogether, our findings uncover a role of APOE as an epigenetic mediator of senescence and provide potential targets to ameliorate aging-related diseases.

Immunofluorescent Staining for Visualization of Heterochromatin Associated Proteins in Drosophila Salivary Glands.

Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. In this protocol, we describe the immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail.

Diverse heterochromatin-associated proteins repress distinct classes of genes and repetitive elements.

SummaryHeterochromatin, typically marked by histone H3 trimethylation on lysine 9 (H3K9me3) or lysine 27 (H3K27me3), represses different protein-coding genes in different cells, as well as repetitive elements. The basis for locus specificity is unclear. Previously we identified 172 proteins embedded in sonication-resistant heterochromatin (srHC) harboring H3K9me3. Here we investigate in humans how 97 of the H3K9me3-srHC proteins repress heterochromatic genes. We reveal four groups of srHC proteins that each repress many genes and repeat elements in common. Two groups repress H3K9me3-embedded genes with different extents of flanking srHC, one group is specific for srHC genes with H3K9me3 and H3K27me3, and one group is specific for genes with srHC as the primary feature. We find that enhancer of rudimentary homolog (ERH) is conserved from S. pombe in repressing meiotic genes and, in humans, now represses other lineage-specific genes and repeat elements. The study greatly expands our understanding of H3K9me3-based gene repression in vertebrates.

SIRT3 consolidates heterochromatin and counteracts senescence.

Sirtuin 3 (SIRT3) is an NAD+-dependent deacetylase linked to a broad range of physiological and pathological processes, including aging and aging-related diseases. However, the role of SIRT3 in regulating human stem cell homeostasis remains unclear. Here we found that SIRT3 expression was downregulated in senescent human mesenchymal stem cells (hMSCs). CRISPR/Cas9-mediated depletion of SIRT3 led to compromised nuclear integrity, loss of heterochromatin and accelerated senescence in hMSCs. Further analysis indicated that SIRT3 interacted with nuclear envelope proteins and heterochromatin-associated proteins. SIRT3 deficiency resulted in the detachment of genomic lamina-associated domains (LADs) from the nuclear lamina, increased chromatin accessibility and aberrant repetitive sequence transcription. The re-introduction of SIRT3 rescued the disorganized heterochromatin and the senescence phenotypes. Taken together, our study reveals a novel role for SIRT3 in stabilizing heterochromatin and counteracting hMSC senescence, providing new potential therapeutic targets to ameliorate aging-related diseases.

Clinicopathological and Prognostic Significance of CBX3 Expression in Human Cancer: a Systematic Review and Meta-analysis.

Background Chromebox protein homolog 3 (CBX3) as a member of the heterochromatin-associated protein 1 (HP1) family has been reported to be overexpressed in human cancer tissues. Numerous studies have shown the relationship between the CBX3 expression and clinicopathological factor or prognosis in malignant tumors, but their results are inconsistent. To address these results, a meta-analysis was described to investigate the prognostic value and clinicopathological significance of CBX3 expression in human malignant neoplasms. Methods PubMed, Web of Science, Embase, and Chinese National Knowledge Infrastructure (CNKI) were used to search eligible literatures, including publications prior to September 2019. The role of CBX3 in cancer prognosis and clinicopathological characteristics was assessed by pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs). Results Eleven studies with 1682 cancer patients were enrolled in this meta-analysis. This analysis demonstrated that the patients' increased CBX3 expression was significantly associated with poor overall survival (OS) (univariate analysis: HR = 1.81, 95% CI 1.46-2.25; multivariate analysis: HR = 1.95, 95% CI 1.63-2.34). Subgroups analysis by tumor type also indicated that high expression of CBX3 was correlated with poor OS in tongue squamous cell carcinoma (HR = 3.31, 95% CI 2.03-5.39), lung cancer (HR = 1.66, 95% CI 1.21-2.29), genitourinary cancer (HR = 2.03, 95% CI 1.15-3.58), and digestive cancer (HR = 1.48, 95% CI 1.23-1.79). For clinicopathological features, high expression of CBX3 was associated with lymph node metastasis (OR = 2.96, 95% CI 1.42-6.20) and lager tumor size (OR = 1.60, 95% CI 1.12-2.28). Conclusion The results of this meta-analysis indicated that CBX3 expression may be a novel biomarker for predicting patient prognosis and clinicopathological parameters in multiple human cancer.

ZKSCAN3 counteracts cellular senescence by stabilizing heterochromatin.

Zinc finger protein with KRAB and SCAN domains 3 (ZKSCAN3) has long been known as a master transcriptional repressor of autophagy. Here, we identify a novel role for ZKSCAN3 in alleviating senescence that is independent of its autophagy-related activity. Downregulation of ZKSCAN3 is observed in aged human mesenchymal stem cells (hMSCs) and depletion of ZKSCAN3 accelerates senescence of these cells. Mechanistically, ZKSCAN3 maintains heterochromatin stability via interaction with heterochromatin-associated proteins and nuclear lamina proteins. Further study shows that ZKSCAN3 deficiency results in the detachment of genomic lamina-associated domains (LADs) from the nuclear lamina, loss of heterochromatin, a more accessible chromatin status and consequently, aberrant transcription of repetitive sequences. Overexpression of ZKSCAN3 not only rescues premature senescence phenotypes in ZKSCAN3-deficient hMSCs but also rejuvenates physiologically and pathologically senescent hMSCs. Together, these data reveal for the first time that ZKSCAN3 functions as an epigenetic modulator to maintain heterochromatin organization and thereby attenuate cellular senescence. Our findings establish a new functional link among ZKSCAN3, epigenetic regulation, and stem cell aging.

Native Chromatin Proteomics Reveals a Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance.

Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, wedevelop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.

2024 - DEFICIENCY OF THE NOVEL TUMOR SUPPRESSOR C15ORF65 CAUSES MULTIPLE MYELOMA AND PLASMA CELL MALIGNANCY

The previously uncharacterized locus C15orf65 (murine Gm5918) is diminutive, yet highly conserved within and beyond the animal kingdom. We show that it encodes a heterochromatin-associated protein with a hydrophobic pocket and an aromatic cage in its domain of unknown function 4490 (DUF4490) that facilitate its action as a tumor suppressor for multiple myeloma and plasma cell malignancy. Hematopoietic loss of Gm5918 in mice results in the development of transplantable MM at high penetrance between 1-2 years of age. M-spikes developed in the animals as early as 2-3 months before death while Gm5918deficient kidneys showed light chain amyloidosis. 18F PET-CT of animals upon serum M-spike development revealed solitary medullary plasmacytomas or extensive skeletal remodeling, which we found to be accompanied by extramedullary disease on necropsy. Colony forming unit assays and serial competitive transplantation show the defect to be cell intrinsic and cytokine independent after malignant transformation, while IL-7 inducible before disease onset. CD138+ cells from sick animals showed derepression of PRC2 and H3K27me3 targets, and we observed reduced H3K27me3 in tumor-derived cell lines. C15orf65 homodimerizes and co-immunoprecipitates with histone H3 and EED, identifying it as a new class of histone-binding scaffold protein for PRC2-mediated methylation of H3K27. In human MM patients, low C15orf65 correlates with aggressive disease, short time to progression, and increased mortality. Re-expressing C15orf65 in human MM1.R cell lines inhibits growth and establishment post-passaging, while expression of DUF4490-mutant C15orf65 reverses the phenotype. We propose naming C15orf65 SCaffold for HIStone Modification 1 (SCHISM1) and the previously uncharacterized DUF4490 family as a Histone Tail Targeting PRC Scaffold (HTTPS) domain.

Stabilizing heterochromatin by DGCR8 alleviates senescence and osteoarthritis

DiGeorge syndrome critical region 8 (DGCR8) is a critical component of the canonical microprocessor complex for microRNA biogenesis. However, the non-canonical functions of DGCR8 have not been studied. Here, we demonstrate that DGCR8 plays an important role in maintaining heterochromatin organization and attenuating aging. An N-terminal-truncated version of DGCR8 (DR8dex2) accelerated senescence in human mesenchymal stem cells (hMSCs) independent of its microRNA-processing activity. Further studies revealed that DGCR8 maintained heterochromatin organization by interacting with the nuclear envelope protein Lamin B1, and heterochromatin-associated proteins, KAP1 and HP1γ. Overexpression of any of these proteins, including DGCR8, reversed premature senescent phenotypes in DR8dex2 hMSCs. Finally, DGCR8 was downregulated in pathologically and naturally aged hMSCs, whereas DGCR8 overexpression alleviated hMSC aging and mouse osteoarthritis. Taken together, these analyses uncovered a novel, microRNA processing-independent role in maintaining heterochromatin organization and attenuating senescence by DGCR8, thus representing a new therapeutic target for alleviating human aging-related disorders.

Heterochromatin Protein 1 (HP1) inhibits stem cell proliferation induced by ectopic activation of the Jak/STAT pathway in the Drosophila testis

Stem cells can divide asymmetrically with respect to cell fate, producing a copy of themselves (self-renewal), while giving rise to progeny that will differentiate along a specific lineage. Mechanisms that bias the balance towards self-renewal or extend the proliferative capacity of the differentiating progeny can result in tissue overgrowth and, eventually, the formation of tumors. Recent work has explored the role of heterochromatin and heterochromatin-associated proteins in the regulation of stem cell behavior under homeostatic conditions, but less is known about their possible roles in potentiating or suppressing stem cell overproliferation. Here we used ectopic activation of the Jak/STAT pathway in germline and somatic stem cells of the D. melanogaster testis as an in vivo model to probe the function of Heterochromatin Protein 1 (HP1) in stem cell overproliferation. Forced expression of HP1 in either early germ or somatic cells suppressed the overgrowth of testes in response to ectopic Jak/STAT activation. Interestingly, HP1 expression led to distinct phenotypes, depending on whether it was overexpressed in somatic or germ cells, possibly reflecting different cell-autonomous and non-autonomous effects in each cell type. Our results provide a new framework for further in vivo studies aimed at understanding the interactions between heterochromatin and uncontrolled stem cell proliferation, as well as the complex cross-regulatory interactions between the somatic and germline lineages in the Drosophila testis.

FBXO11 Activates Erythroid Gene Transcription By Degrading Heterochromatin-Associated Protein BAHD1

Mature red blood cells (RBC) contain approximately 95% cytosolic hemoglobin for the purpose of blood oxygen transport. This specialized state is achieved during erythropoiesis by regulated gene expression and protein degradation. During late-stage erythropoiesis, ubiquitin ligases eliminate unnecessary proteins and maintain quality control by degrading unstable proteins, including unpaired hemoglobin subunits. However, ubiquitin ligases are expressed at all stages of erythropoiesis and the functions of most are unknown. To study ubiquitin ligases involved in RBC formation, we performed a Cas9/single guide (sg) RNA screen for functional ubiquitin-proteasome components in HUDEP-2 cells, an immortalized human cell line that proliferates as immature erythroblasts and can be induced to undergo terminal maturation. We identified the E3 ubiquitin ligase FBXO11 as a top-ranked candidate. FBXO11 is a member of the F-box protein family that assembles into a SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase complex. Depletion of FBXO11 by 2 different sgRNAs in HUDEP-2 cells inhibited erythroid maturation, as evidenced by reduced hemoglobinization, failure to induce the maturation marker Band3 and persistence of immature cell morphology. In primary human CD34+ cells, suppression of FBXO11 expression by Cas9 + two independent sgRNAs inhibited erythroid maturation, as evidenced by reduced Band3 expression (5.3% vs. 15.6% for non-targeting sgRNA, P < 0.001; n=4). RNA-seq analysis of FBXO11-depleted HUDEP-2 cells revealed 951 decreased transcripts (enriched for erythroid genes) and 339 increased transcripts (enriched for genes expressed in activated T-cells) compared to control cells expressing non-targeting gRNA (P < 0.05). Thus, FBXO11 is required for erythroid maturation and facilitates erythroid gene expression.We sought to establish how FBXO11 modulates erythropoiesis and erythroid gene expression by identifying the relevant ubiquitination substrate(s). Combined quantitative proteome analysis with RNA-seq of FBXO11-depleted HUDEP-2 cells identified several proteins that are upregulated with no change in their corresponding mRNA. We tested whether reduction of these candidate substrates could alleviate the erythroid maturation block conferred by FBXO11 depletion. In FBXO11 gene-disrupted HUDEP-2 cells, suppression of the heterochromatin-associated protein BAHD1 partially rescued hemoglobinization and Band3 expression (4.2% for Cas9 + non-targeting sgRNA vs. 21.7% for Cas9 + BAHD1 sgRNAs, P < 0.01; n=3) . Conversely, stable overexpression of V5-epitope-tagged BAHD1 in WT HUDEP-2 cells reduced Band3 expression from 25.0% to 11.4% (P < 0.001; n=3) and inhibited hemoglobinization. Transcriptome analysis demonstrated a significant inverse correlation between genes deregulated by BAHD1-V5 overexpression and FBXO11 deficiency in HUDEP-2 cells, particularly for erythroid genes that are downregulated (P < 0.0001).BAHD1, named after its bromo-adjacent homology domain that interacts with H3K27me3, is part of a transcriptional repressor complex. We showed that BAHD1 and FBXO11 co-immunoprecipitated in cells and that BAHD1 amino (N)-terminal segments of 188 or 240 amino acids were robustly modified with ubiquitin by SCFFBXO11 complex. Chromatin immunoprecipitation-sequencing analysis of BAHD1-V5-expressing WT HUDEP-2 cells showed strong enrichment for BAHD1 occupancy on erythroid gene promoters that were downregulated by FBXO11-deficiency (P < 0.0001). We next investigated whether a specific set of histone marks distinguish FBXO11-regulated genes in normal erythroblasts. We found that most FBXO11-regulated genes in both HUDEP-2 and primary CD34+ derived erythroblasts harbor histone marks H3K4me3 and H3K27me3, indicating a “bivalent” epigenetic state that supports low level transcription in stem or progenitor cells. Together, these data indicate that FBXO11 activates expression of erythroid genes by ubiquitinating and degrading bivalent promoter-bound BAHD1 repressor complexes with likely resolution to a monovalent transcriptionally active state.Overall, our findings identify FBXO11 as a ubiquitin ligase that utilizes a novel mechanism to activate erythroid genes during RBC formation. This newly identified pathway may contribute to known activities of FBXO11 as a tumor suppressor and developmental regulator in non-erythroid tissues. DisclosuresNo relevant conflicts of interest to declare.

Heterochromatin proteins influence the choice of DNA repair in euchromatin domains

In most eukaryotes including the fission yeast, constitutive heterochromatin in pericentromere and telomere domains is marked by methylation of histone H3K9, which provides a binding platform for specialized proteins including heterochromatin protein 1 (HP1). These proteins contribute to transcriptional silencing, cohesion, and kinetochore recruitment. We showed previously that loss of heterochromatin leads to genome rearrangement in the pericentromere repeats that is exacerbated by replication fork destabilization (e.g., Li et al (2013) Cell Rep. 3:638). This indicates that heterochromatin and fork stability make unique contributions to maintain a repetitive domain and resist recombination.Evidence from mammalian systems has implicated heterochromatin proteins in transient response to induced DNA double strand breaks in euchromatin domains. Unexpectedly, we observed that mutants lacking the histone methyltransferase Clr4, or chromodomain proteins Chp1 or Swi6 show evidence of genome instability in euchromatin even in the absence of exogenous damage. Using molecular and cytological approaches, we have investigated the role of these proteins in maintaining euchromatin genome stability in otherwise unperturbed cells, as well as in response to distinct forms of genome stress. We determined that a subset of heterochromatin‐associated proteins contribute to the choice of repair pathway in euchromatin domains by modulating resection of DNA ends. Thus, H3K9 histone methylation and its associated proteins are not limited to heterochromatin domains and influence chromosome stability broadly throughout the genome.Support or Funding InformationSupport by NIH R35 GM118109This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Carcinogen susceptibility is regulated by genome architecture and predicts cancer mutagenesis.

The development of many sporadic cancers is directly initiated by carcinogen exposure. Carcinogens induce malignancies by creating DNA lesions (i.e., adducts) that can result in mutations if left unrepaired. Despite this knowledge, there has been remarkably little investigation into the regulation of susceptibility to acquire DNA lesions. In this study, we present the first quantitative human genome-wide map of DNA lesions induced by ultraviolet (UV) radiation, the ubiquitous carcinogen in sunlight that causes skin cancer. Remarkably, the pattern of carcinogen susceptibility across the genome of primary cells significantly reflects mutation frequency in malignant melanoma. Surprisingly, DNase-accessible euchromatin is protected from UV, while lamina-associated heterochromatin at the nuclear periphery is vulnerable. Many cancer driver genes have an intrinsic increase in carcinogen susceptibility, including the BRAF oncogene that has the highest mutation frequency in melanoma. These findings provide a genome-wide snapshot of DNA injuries at the earliest stage of carcinogenesis. Furthermore, they identify carcinogen susceptibility as an origin of genome instability that is regulated by nuclear architecture and mirrors mutagenesis in cancer.

Elucidating Protein-DNA Interactions in Human Alphoid Chromatin via Hybridization Capture and Mass Spectrometry.

The centromere is the chromosomal locus where the kinetochore forms and is critical for ensuring proper segregation of sister chromatids during cell division. A substantial amount of effort has been devoted to understanding the characteristic features and roles of the centromere, yet some fundamental aspects of the centromere, such as the complete list of elements that define it, remain obscure. It is well-known that human centromeres include a highly repetitive class of DNA known as alpha satellite, or alphoid, DNA. We present here the first DNA-centric examination of human protein-alpha satellite interactions, employing an approach known as HyCCAPP (hybridization capture of chromatin-associated proteins for proteomics) to identify the protein components of alphoid chromatin in a human cell line. Using HyCCAPP, cross-linked alpha satellite chromatin was isolated from cell lysate, and captured proteins were analyzed via mass spectrometry. After being compared to proteins identified in control pulldown experiments, 90 proteins were identified as enriched at alphoid DNA. This list included many known centromere-binding proteins in addition to multiple novel alpha satellite-binding proteins, such as LRIF1, a heterochromatin-associated protein. The ability of HyCCAPP to reveal both known as well as novel alphoid DNA-interacting proteins highlights the validity and utility of this approach.

EZH2 coupled with HOTAIR to silence MicroRNA‐34a by the induction of heterochromatin formation in human pancreatic ductal adenocarcinoma

MicroRNA-34a (miR-34a) is frequently downregulated in pancreatic ductal adenocarcinoma (PDAC) cells, however, the silencing mechanism remains unclear. Enhancer of zeste homolog 2 (EZH2) is overexpressed in PDAC, and our previous miRNA profiling showed that inhibition of EZH2 in PDAC cells led to the re-expression of a group of tumor suppressor miRNAs including miR-34a. Here, we studied the effect of ectopic EZH2 expression to the silencing of miR-34a, and identified HOTAIR as an interacting partner to induce heterochromatin formation during miR-34a repression. We identified EZH2 as a major player in silencing miR-34a. Inhibition of EZH2 upregulated miR-34a expression in PDAC cells, while EZH2 overexpression in human pancreatic ductal epithelial (HPDE) cells repressed miR-34a expression and decreased the miR-34a promoter activity. We then showed that HOTAIR played a critical role in EZH2-mediated repression of miR-34a, as knockdown of HOTAIR attenuated the miR-34a inhibition effect in EZH2-overexpressing HPDE cells. HOTAIR physically interacted with miR-34a promoter, and the EZH2-interacting region located at 5' HOTAIR RNA was essential in repressing miR-34a and promoting cell proliferation. More importantly, we showed that the interaction between EZH2 and HOTAIR underlay the silencing of miR-34a through induction of heterochromatin formation. We first showed that manipulation of EZH2 level interfered the occupancy of heterochromatin markers H3K9me2, heterochromatin associated protein 1α and 1γ in PDAC cells. In turn, we showed that knockdown of HOTAIR reduced the occupancy of EZH2 at miR-34a promoter. The identification of HOTAIR-guided miR-34a silencing opened a new avenue in miR-34a-oriented therapy against PDAC.

Heterochromatin-Associated Proteins HP1a and Piwi Collaborate to Maintain the Association of Achiasmate Homologs in Drosophila Oocytes.

Accurate segregation of homologous chromosomes during meiosis depends on their ability to remain physically connected throughout prophase I. For homologs that achieve a crossover, sister chromatid cohesion distal to the chiasma keeps them attached until anaphase I. However, in Drosophila melanogaster wild-type oocytes, chromosome 4 never recombines, and the X chromosome fails to cross over in 6-10% of oocytes. Proper segregation of these achiasmate homologs relies on their pericentric heterochromatin-mediated association, but the mechanism(s) underlying this attachment remains poorly understood. Using an inducible RNA interference (RNAi) strategy combined with fluorescence in situ hybridization (FISH) to monitor centromere proximal association of the achiasmate FM7a/X homolog pair, we asked whether specific heterochromatin-associated proteins are required for the association and proper segregation of achiasmate homologs in Drosophila oocytes. When we knock down HP1a, H3K9 methytransferases, or the HP1a binding partner Piwi during mid-prophase, we observe significant disruption of pericentric heterochromatin-mediated association of FM7a/X homologs. Furthermore, for both HP1a and Piwi knockdown oocytes, transgenic coexpression of the corresponding wild-type protein is able to rescue RNAi-induced defects, but expression of a mutant protein with a single amino acid change that disrupts the HP1a-Piwi interaction is unable to do so. We show that Piwi is stably bound to numerous sites along the meiotic chromosomes, including centromere proximal regions. In addition, reduction of HP1a or Piwi during meiotic prophase induces a significant increase in FM7a/X segregation errors. We present a speculative model outlining how HP1a and Piwi could collaborate to keep achiasmate chromosomes associated in a homology-dependent manner.