Role Of Chromatin Architecture Research Articles

SMCHD1 has separable roles in chromatin architecture and gene silencing that could be targeted in disease

The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets. Moreover, it also results in enhanced silencing at the facioscapulohumeral muscular dystrophy associated macrosatellite-array, D4Z4, resulting in enhanced repression of DUX4 encoded by this repeat. Heightened SMCHD1 silencing perturbs developmental Hox gene activation, causing a homeotic transformation in mice. Paradoxically, the mutant SMCHD1 appears to enhance insulation against other epigenetic regulators, including PRC2 and CTCF, while depleting long range chromatin interactions akin to what is observed in the absence of SMCHD1. These data suggest that SMCHD1’s role in long range chromatin interactions is not directly linked to gene silencing or insulating the chromatin, refining the model for how the different levels of SMCHD1-mediated chromatin regulation interact to bring about gene silencing in normal development and disease.

Young SINEs in pig genomes impact gene regulation, genetic diversity, and complex traits

Transposable elements (TEs) are a major source of genetic polymorphisms and play a role in chromatin architecture, gene regulatory networks, and genomic evolution. However, their functional role in pigs and contributions to complex traits are largely unknown. We created a catalog of TEs (n = 3,087,929) in pigs and found that young SINEs were predominantly silenced by histone modifications, DNA methylation, and decreased accessibility. However, some transcripts from active young SINEs showed high tissue-specificity, as confirmed by analyzing 3570 RNA-seq samples. We also detected 211,067 dimorphic SINEs in 374 individuals, including 340 population-specific ones associated with local adaptation. Mapping these dimorphic SINEs to genome-wide associations of 97 complex traits in pigs, we found 54 candidate genes (e.g., ANK2 and VRTN) that might be mediated by TEs. Our findings highlight the important roles of young SINEs and provide a supplement for genotype-to-phenotype associations and modern breeding in pigs.

Abstract 751: Transcriptome profiling reveals novel gene fusions in early-onset sporadic rectal cancer

Abstract Implementation of regular screening and adoption of a healthier lifestyle has resulted in a steady decline of colorectal cancer (CRC) incidence in the USA over the past few decades. On the contrary, early-onset sporadic rectal cancer (EOSRC) has exhibited an opposite trend in the more recent past. In addition, EOSRC appears to be the predominant CRC subtype in developing nations where CRC incidence has risen significantly during the past 2 decades. Our previous work revealed the absence of canonical CRC tumorigenesis pathways including Wnt (Wingless, Int-1) activation and mismatch repair inactivation in a significant proportion of EOSRC cases. More importantly, we revealed the enrichment of Ca2+/NFAT signaling leading to chromosomal instability in samples devoid of Wnt activation besides identifying ARID2 as a novel tumor suppressor for EOSRC. In order to improve our understanding of EOSRC driver events, we endeavored to characterize the landscape of gene fusions (GFs); given that the latter provide an attractive druggable target. To this end, we performed paired-end RNA Sequencing on 37 carefully selected and well annotated microsatellite stable EOSRC samples. We employed in silico prediction algorithms viz. FusionCatcher, SOAPFuse and EricScript to identify GFs after removing those predicted (GTEx and ConjoinG databases) to occur in normal tissues. The GFs were further filtered based on read support and identification through at least 2 of the 3 algorithms and validated using reverse transcription PCR and Sanger sequencing. Overall, we identified several known (EIF3E-RSPO2, NCEH1-SPATA16) and novel GFs that arose as inter- or intra-chromosomal events. Computational analyses revealed selective enrichment of Gene Ontologies (GOs) in the 5’- (as compared to 3’-) gene partners which differed from GOs enriched in GFs identified from the TCGA CRC data. More importantly, we analyzed the potential role of chromatin architecture in determining the fusion breakpoints. Analysis of CRC Hi-C data revealed that a majority of EOSRC GFs arose from genes located in open chromatin regions. Interestingly, fusions between 5’ ‘open’ and 3’ ‘closed’ partners often resulted in transcriptional activation of the latter revealing a novel mode of oncogenic activation in cancer. Further analyses to correlate the predicted fusion breakpoints with topologically associating domain (TAD) boundaries as well as with common fragile sites (CFSs) that associate with transcription-replication collision are currently ongoing. Validation of potential oncogenic role of few selected GFs using CRC cell lines and nude mice xenografts is also underway. The study is expected to provide significant insights into the patterns of genomic rearrangements in EOSRC besides highlighting the critical role GFs might play in propagating tumorigenesis in CRC. Citation Format: Asmita Gupta, Vasanth Kumar Mandla, Pratyusha Bala, Murali Dharan Bashyam. Transcriptome profiling reveals novel gene fusions in early-onset sporadic rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 751.

From structure to molecular condensates: emerging mechanisms for Mediator function.

Mediator is a large modular protein assembly whose function as a coactivator of transcription is conserved in all eukaryotes. The Mediator complex can integrate and relay signals from gene-specific activators bound at enhancers to activate the general transcription machinery located at promoters. It has thus been described as a bridge between these elements during initiation of transcription. Here, we review recent studies on Mediator relating to its structure, gene specificity and general requirement, roles in chromatin architecture as well as novel concepts involving phase separation and transcriptional bursting. We revisit the mechanism of action of Mediator and ultimately put forward models for its mode of action in gene activation.

Dynamic regulation of CTCF stability and sub-nuclear localization in response to stress.

The nuclear protein CCCTC-binding factor (CTCF) has diverse roles in chromatin architecture and gene regulation. Functionally, CTCF associates with thousands of genomic sites and interacts with proteins, such as cohesin, or non-coding RNAs to facilitate specific transcriptional programming. In this study, we examined CTCF during the cellular stress response in human primary cells using immune-blotting, quantitative real time-PCR, chromatin immunoprecipitation-sequence (ChIP-seq) analysis, mass spectrometry, RNA immunoprecipitation-sequence analysis (RIP-seq), and Airyscan confocal microscopy. Unexpectedly, we found that CTCF is exquisitely sensitive to diverse forms of stress in normal patient-derived human mammary epithelial cells (HMECs). In HMECs, a subset of CTCF protein forms complexes that localize to Serine/arginine-rich splicing factor (SC-35)-containing nuclear speckles. Upon stress, this species of CTCF protein is rapidly downregulated by changes in protein stability, resulting in loss of CTCF from SC-35 nuclear speckles and changes in CTCF-RNA interactions. Our ChIP-seq analysis indicated that CTCF binding to genomic DNA is largely unchanged. Restoration of the stress-sensitive pool of CTCF protein abundance and re-localization to nuclear speckles can be achieved by inhibition of proteasome-mediated degradation. Surprisingly, we observed the same characteristics of the stress response during neuronal differentiation of human pluripotent stem cells (hPSCs). CTCF forms stress-sensitive complexes that localize to SC-35 nuclear speckles during a specific stage of neuronal commitment/development but not in differentiated neurons. We speculate that these particular CTCF complexes serve a role in RNA processing that may be intimately linked with specific genes in the vicinity of nuclear speckles, potentially to maintain cells in a certain differentiation state, that is dynamically regulated by environmental signals. The stress-regulated activity of CTCF is uncoupled in persistently stressed, epigenetically re-programmed “variant” HMECs and certain cancer cell lines. These results reveal new insights into CTCF function in cell differentiation and the stress-response with implications for oxidative damage-induced cancer initiation and neuro-degenerative diseases.

CRISPR/Cas9 offers a new tool for studying the role of chromatin architecture in disease pathogenesis

A recent study used CRISPR/Cas9 to reveal long-range looping between disease-related genes and their regulatory elements that is mediated by the CCCTC-binding factor (CTCF) in prostate cancer.

Shaping mitotic chromosomes: From classical concepts to molecular mechanisms.

How eukaryotic genomes are packaged into compact cylindrical chromosomes in preparation for cell divisions has remained one of the major unsolved questions of cell biology. Novel approaches to study the topology of DNA helices inside the nuclei of intact cells, paired with computational modeling and precise biomechanical measurements of isolated chromosomes, have advanced our understanding of mitotic chromosome architecture. In this Review Essay, we discuss – in light of these recent insights – the role of chromatin architecture and the functions and possible mechanisms of SMC protein complexes and other molecular machines in the formation of mitotic chromosomes. Based on the information available, we propose a stepwise model of mitotic chromosome condensation that envisions the sequential generation of intra-chromosomal linkages by condensin complexes in the context of cohesin-mediated inter-chromosomal linkages, assisted by topoisomerase II. The described scenario results in rod-shaped metaphase chromosomes ready for their segregation to the cell poles.

Targeting Mycobacterium tuberculosis nucleoid-associated protein HU with structure-based inhibitors

The nucleoid-associated protein HU plays an important role in maintenance of chromosomal architecture and in global regulation of DNA transactions in bacteria. Although HU is essential for growth in Mycobacterium tuberculosis (Mtb), there have been no reported attempts to perturb HU function with small molecules. Here we report the crystal structure of the N-terminal domain of HU from Mtb. We identify a core region within the HU-DNA interface that can be targeted using stilbene derivatives. These small molecules specifically inhibit HU-DNA binding, disrupt nucleoid architecture and reduce Mtb growth. The stilbene inhibitors induce gene expression changes in Mtb that resemble those induced by HU deficiency. Our results indicate that HU is a potential target for the development of therapies against tuberculosis.

HP1a: a structural chromosomal protein regulating transcription

Heterochromatin protein 1 (HP1a in Drosophila) is a conserved eukaryotic chromosomal protein that is prominently associated with pericentric heterochromatin and mediates the concomitant gene silencing. Mechanistic studies implicate HP1 family proteins as 'hub proteins,' able to interact with a variety of chromosomal proteins through the chromo-shadow domain (CSD), as well as to recognize key histone modification sites [primarily histone H3 di/trimethyl Lys9 (H3K9me2/3)] through the chromodomain (CD). Consequently, HP1 has many important roles in chromatin architecture and impacts both gene expression and gene silencing, utilizing a variety of mechanisms. Clearly, HP1 function is altered by context, and potentially by post-translational modifications (PTMs). Here, we report on recent ideas as to how this versatile protein accomplishes its diverse functions.

Distinct isoforms of the Drosophila Brd4 homologue are present at enhancers, promoters and insulator sites

Brd4 is a double bromodomain protein that has been shown to interact with acetylated histones to regulate transcription by recruiting Positive Transcription Elongation Factor b to the promoter region. Brd4 is also involved in gene bookmarking during mitosis and is a therapeutic target for the treatment of acute myeloid leukemia. The Drosophila melanogaster Brd4 homologue is called Fs(1)h and, like its vertebrate counterpart, encodes different isoforms. We have used ChIP-seq to examine the genome-wide distribution of Fs(1)h isoforms. We are able to distinguish the Fs(1)h-L and Fs(1)h-S binding profiles and discriminate between the genomic locations of the two isoforms. Fs(1)h-S is present at enhancers and promoters and its amount parallels transcription levels. Correlations between the distribution of Fs(1)h-S and various forms of acetylated histones H3 and H4 suggest a preference for binding to H3K9acS10ph. Surprisingly, Fs(1)h-L is located at sites in the genome where multiple insulator proteins are also present. The results suggest that Fs(1)h-S may be responsible for the classical role assigned to this protein, whereas Fs(1)h-L may have a new and unexpected role in chromatin architecture by working in conjunction with insulator proteins to mediate intra- or inter-chromosome interactions.

H2B monoubiquitylation is a 5′-enriched active transcription mark and correlates with exon–intron structure in human cells

H2B monoubiquitylation (H2Bub1), which is required for multiple methylations of both H3K4 and H3K79, has been implicated in gene expression in numerous organisms ranging from yeast to human. However, the molecular crosstalk between H2Bub1 and other modifications, especially the methylations of H3K4 and H3K79, remains unclear in vertebrates. To better understand the functional role of H2Bub1, we measured genome-wide histone modification patterns in human cells. Our results suggest that H2Bub1 has dual roles, one that is H3 methylation dependent, and another that is H3 methylation independent. First, H2Bub1 is a 5′-enriched active transcription mark and co-occupies with H3K79 methylations in actively transcribed regions. Second, this study shows for the first time that H2Bub1 plays a histone H3 methylations-independent role in chromatin architecture. Furthermore, the results of this work indicate that H2Bub1 is largely positioned at the exon–intron boundaries of highly expressed exons, and it demonstrates increased occupancy in skipped exons compared with flanking exons in the human and mouse genomes. Our findings collectively suggest that a potentiating mechanism links H2Bub1 to both H3K79 methylations in actively transcribed regions and the exon–intron structure of highly expressed exons via the regulation of nucleosome dynamics during transcription elongation.

Jmjd5, an H3K36me2 histone demethylase, modulates embryonic cell proliferation through the regulation of Cdkn1a expression

Covalent modifications of histones play an important role in chromatin architecture and dynamics. In particular, histone lysine methylation is important for transcriptional control during diverse biological processes. The nuclear protein Jmjd5 (also called Kdm8) is a histone lysine demethylase that contains a JmjC domain in the C-terminal region. In this study, we have generated Jmjd5-deficient mice (Jmjd5(Δ)(/)(Δ)) to investigate the in vivo function of Jmjd5. Jmjd5(Δ)(/)(Δ) embryos showed severe growth retardation, resulting in embryonic lethality at the mid-gestation stage. Mouse embryonic fibroblasts (MEFs) derived from Jmjd5 hypomorphic embryos (Jmjd5(neo/neo)) also showed the growth defect. Quantitative PCR analysis of various cell cycle regulators indicated that only Cdkn1a expression was upregulated in Jmjd5(neo/neo) MEFs and Jmjd5(Δ)(/)(Δ) embryos. A knockdown assay with Cdkn1a-specific small interfering RNAs revealed that the growth defect of Jmjd5(neo/neo) MEFs was significantly rescued. In addition, a genetic study using Jmjd5(Δ)(/)(Δ); Cdkn1a(Δ)(/)(Δ) double-knockout mice showed that the growth retardation of Jmjd5(Δ)(/)(Δ) embryos was partially rescued by Cdkn1a deficiency. Chromatin immunoprecipitation analysis showed that increased di-methylated lysine 36 of histone H3 (H3K36me2) and reduced recruitment of endogenous Jmjd5 were detected in the transcribed regions of Cdkn1a in Jmjd5(neo/neo) MEFs. Taken together, these results suggest that Jmjd5 physiologically moderates embryonic cell proliferation through the epigenetic control of Cdkn1a expression.

Abstract 206: High mobility group A1(HMGA1) proteins localize at the mitochondria and inhibit the p53-mediated intrinsic-apoptotic pathway

Abstract The high mobility group A (HMGA) non-histone chromosomal proteins play key roles in chromatin architecture and orchestrate the assembly of nucleoprotein complexes involved in gene transcription, replication, and chromatin structure. HMGA overexpression and gene rearrangements are frequent events in human cancer, but the molecular basis of HMGA oncogenic activity has not been completely elucidated. Recently, we have defined a new physical and functional interaction between HMGA1 and p53. This interaction modulates the transcription of p53 target genes such as Mdm2, p21waf1, Bax, Bcl-2, and inhibits p53-mediated apoptosis. Here we report that HMGA1 does not act only at transcriptional level but it has also a direct, anti-apoptogenic role at the mitochondria. In fact, we found that in mouse embryonic fibroblast (MEF) and several tumour cell lines overexpressing HMGA1, this protein is present at cytoplasmic level other than in the nucleus. Moreover, we demonstrate that HMGA1 stably localizes at the mitochondria, in which it binds Bcl-2, displacing it from the binding to p53, thus counteracting the release of cytochrome C. In agreement with this observation, the HMGA1-overexpressing cells show a significant reduction of cytochrome C release, after lethal dose of Ultraviolet (UV) light, whereas HMGA1-depleted cells are more susceptibile to UV treatment. These data define a new localization of HMGA1 at the mitochondria level that allows HMGA1 to inhibit by a new mechanism the p53-mediated intrinsic-apoptotic pathway. This mechanism may also have a role in the resistance of malignant cells to chemo- and radio-therapy, in particularly for the tumor cells harboring wild-type form of p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 206. doi:10.1158/1538-7445.AM2011-206

DR396, an apoptotic DNase γ inhibitor, attenuates high mobility group box 1 release from apoptotic cells

High mobility group box1 (HMGB1) is a non-histone chromatin chromosomal protein playing an important role in chromatin architecture and transcriptional regulation. Recently, HMGB1 has been shown to be secreted into extracellular milieu in necrosis and apoptosis, and involved in inflammatory responses. However, the mechanism by which apoptotic cells release HMGB1 is unclear. In this study, to investigate the mechanism of HMGB1 release, we searched inhibitors of HMGB1 release from apoptotic cells. As a result, three compounds, 4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396), Pontacyl Violet 6R (PV6R), and Fmoc-D-Cha-OH (FDCO) in our in-house chemical library were found to inhibit HMGB1 release from staurosporine (STS)-induced apoptotic HeLa S3 cells. Interestingly, these three compounds have been previously categorized into apoptotic DNase γ inhibitors. Therefore, we examined whether apoptotic nucleosomal DNA fragmentation is involved in the release of HMGB1 during apoptosis. Expectedly, DR396, which is the most potent and specific inhibitor of DNase γ, was found to almost completely inhibit both HMGB1 release and internucleosomal DNA cleavage in HeLa S3 cells transfected with DNase γ expression vector and stably expressing DNase γ (HeLa S3/γ cells). These results clearly suggest that nucleosomal DNA fragmentation catalyzed by DNase γ is critical in the release of HMGB1 from apoptotic cells.

Recent advances in MeCP2 structure and functionThis paper is one of a selection of papers published in this Special Issue, entitled 29th Annual International Asilomar Chromatin and Chromosomes Conference, and has undergone the Journal’s usual peer review process.

Mutations in methyl DNA binding protein 2 (MeCP2) cause the neurodevelopmental disorder Rett syndrome (RTT). The mechanism(s) by which the native MeCP2 protein operates in the cell are not well understood. Historically, MeCP2 has been characterized as a proximal gene silencer with 2 functional domains: a methyl DNA binding domain and a transcription repression domain. However, several lines of new data indicate that MeCP2 structure and function relationships are more complex. In this review, we first discuss recent studies that have advanced understanding of the basic structural biochemistry of MeCP2. This is followed by an analysis of cell-based experiments suggesting MeCP2 is a regulator, rather than a strict silencer, of transcription. The new data establish MeCP2 as a multifunctional nuclear protein, with potentially important roles in chromatin architecture, regulation of RNA splicing, and active transcription. We conclude by discussing clinical correlations between domain-specific mutations and RTT pathology to stress that all structural domains of MeCP2 are required to properly mediate cellular function of the intact protein.

Design, Synthesis, and Biological Evaluation of Hybrid Molecules Containing α-Methylene-γ-butyrolactones and Polypyrrole Minor Groove Binders

We have synthesized and evaluated a series of hybrids of polypyrrole minor groove binders structurally related to the natural antitumor agent distamycin A, and alpha-methylene-gamma-butyrolactones with methyl, phenyl, and 4-substituted phenyl groups at the lactone C(gamma) position, denoted 5-17, for in vitro cytotoxic activity against a variety of cancer cell lines. The apoptotic and cytotoxic activities against several tumor cell lines are reported and discussed in terms of their structural differences in relation to both the number of N-methylpyrrole rings and the type of the alkylating unit tethered to the oligopeptidic frame. It may be noted that in general, and especially for 11, 12, and 17, the cytotoxicity of the hybrids was much greater than that of the alpha-methylene-gamma-butyrolactone units 24a-g alone. Using the human leukemia cell line HL-60, we have tested the effects of a selected series of compounds on programmed cell death (apoptosis). The results clearly indicate that 11, 12, and 17, but not 9, are able to induce apoptosis as demonstrated from (i) identification of nuclear changes associated with apoptosis using fluorescence microscopy and (ii) by DNA laddering on agarose gel electrophoresis. Compound 12 was the most potent, especially after a short incubation period. It induced extensive hydrolysis of poly ADP-ribose polymerase (PARP), considered to be a hallmark of apoptosis, which plays a critical role in chromatin architecture and DNA metabolism.

The function of the nuclear matrix attachment region of silkworm rDNA as an autonomously replicating sequence in plasmid and chromosomal replication origin in yeast

Nuclear matrix attachment regions (MARs) play a crucial role in chromatin architecture, gene expression, and DNA replication. Although it is well known that yeast autonomously replicating sequences (ARSs) bind nuclear matrix and MARs also function as ARS elements in yeast, whether a heterologous MAR or ARS element acts as a replication origin in the chromosome has not been elucidated. We previously identified a MAR (rMAR) located in the nontranscribed spacer (NTS) of silkworm Attacus ricini rDNA. We report here that this rMAR contains 10 copies of ARS consensus sequence (ACS) and several DNA unwinding regions. The rMAR employs ARS activity in yeast and a rARS element locates in the 3 ′ region of the rMAR. Furthermore, we have also revealed that either the rMAR or the rARS element functions as a replication origin in the chromosome. Our results provide the first direct evidence to demonstrate that heterologous rMAR and rARS display chromosomal origin activity, suggesting that the chromosome structure and replication origin of rDNA reserve some common features during evolution.

Subcellular Localization of the Human Proto-oncogene Protein DEK

Recent data revealed that DEK associates with splicing complexes through interactions mediated by serine/arginine-repeat proteins. However, the DEK protein has also been shown to change the topology of DNA in chromatin in vitro. This could indicate that the DEK protein resides on cellular chromatin. To investigate the in vivo localization of DEK, we performed cell fractionation studies, immunolabeling, and micrococcal nuclease digestion analysis. Most of the DEK protein was found to be released by DNase treatment of nuclei, and only a small amount by treatment with RNase. Furthermore, micrococcal nuclease digestion of nuclei followed by glycerol gradient sedimentation revealed that DEK co-sedimentates with oligonucleosomes, clearly demonstrating that DEK is associated with chromatin in vivo. Additional chromatin fractionation studies, based on the different accessibilities to micrococcal nuclease, showed that DEK is associated both with extended, genetically active and more densely organized, inactive chromatin. We found no significant change in the amount and localization of DEK in cells that synchronously traversed the cell cycle. In summary these data demonstrate that the major portion of DEK is associated with chromatin in vivo and suggest that it might play a role in chromatin architecture.

A CHD1 gene is Z chromosome linked in the chicken Gallus domesticus

Chromo-helicase-DNA binding 1 (CHD1) is a conserved protein with a putative role in chromatin architecture. Single homologues have been found in mouse, Drosophila and yeast. In birds the situation is different as they possess two homologues. One is known to be W-linked, we show the second, closely related gene is linked to the Z sex chromosome. The basic structure of the Z-linked gene is similar to the homologous genes, however, it does possess an additional, internal 88 amino acid hydrophilic domain, rich in glutamic acid and lysine. Studies on pairs of genes sex-linked in mammals suggests rapid divergence of DNA sequence and function. We suggest the DNA sequences of CHD-W and CHD-Z do not follow this pattern.