Browsing by Subject "Chromatin"

Now showing 1 - 12 of 12

The Analysis of the MCF7 Cancer Model System and the Effects of 5-AZA-2'-Deoxycytidine Treatment on the Chromantin State Using a Novel Microarray-Based Technology for High Resolution Global Chromatin State Measurement
(2006-07-10) Weil, Michael Ryan; Garner, Harold R.
A microarray method to measure the global chromatin state of the human genome was developed in order to provide a novel view of gene regulation. The 'chromatin array' employs traditional methods of chromatin isolation, microarray technology, and advanced data analysis, and was applied to a cancer model system. Chromatin is first separated by its condensation state using chromatin fractionation. By probing with a comparative genomic hybridization-style microarray, the chromatin condensation state of thousands of individual loci in an MCF7 tumor model cell line was determined and correlated with transcriptional activity. The chromatin array showed a significant portion (>3,000) of the genes were in a condensation state that was neither condensed or relaxed as a result of heterogeneity in the condensation states in the population. The utility of the chromatin array in deciphering gene regulation was demonstrated in a MCF7 cell line treated with 5 Aza dC, which disrupts genome methylation, and as a result causes global relaxation of chromatin structure. 5 Aza dC treatment results in strong changes in expression, and a normalized global chromatin relaxation of two-fold. A significant subset of 378 genes was condensed by 5 Aza dC treatment, indicating that a mechanism of chromatin regulation exists that can resist the effects of 5 Aza dC treatment. The genes with the largest changes in response to 5 Aza dC treatment showed a strong correlation with CpG island-based regulation (p < 0.0001), and a restoration of transcription patterns associated with normal mammary tissue. Analysis using splice-form specific microarray probes demonstrated that the chromatin state was not uniform across a gene. These findings indicate that certain gene regions exhibit differential sensitivity to 5 Aza dC treatment, and therefore may be regulated independently. Using functional annotation, expression microarray, and comparative genomic hybridization data, this work should provide a framework through which the biological implications of the relationship between chromatin accessibility and expression may be deciphered.
Control of Regulatory Element Function by Histone H3.3
(2022-05) Tafessu, Amanuel Melesse; Yu, Hongtao; Banaszynski, Laura; Chiang, Cheng-Ming; Xu, Jian
In eukaryotic cells, DNA is wrapped around histone proteins to form nucleosomes, the fundamental repeating unit of chromatin. While chromatin functions in part to organize a large amount of genomic material within the confines of the nucleus, regulatory DNA sequences consequently become masked to transcription machinery. Such regulatory sequences are enriched in specific histone variants and post-translational modifications (PTMs). The histone variant H3.3 is enriched at transcriptionally active regulatory elements such as promoters and enhancers. While recent studies have revealed a role for H3.3 in silencing repetitive elements and repressing developmentally regulated promoters, it is unclear how H3.3 contributes to chromatin states at active promoters and enhancers. In this study, we performed genomic analyses of chromatin features associated with active regulatory elements in mouse embryonic stem cells (ESCs) and found evidence of subtle yet widespread dysregulation in the absence of H3.3. Loss of H3.3 or HIRA- the chaperone responsible for H3.3 deposition to transcriptionally active regions- reduces chromatin accessibility and transcription factor (TF) footprinting at promoters. Further, H3.3 KO ESCs show reduced promoter enrichment of p300- a transcriptional coactivator responsible for H3 acetylation at lysine 27 (H3K27ac). Consequently, H3.3 KO ESCs show reduced H3K27ac at promoters, along with reduced enrichment of the acetyllysine reader BRD4. Despite the enrichment of H3.3 at both promoters and enhancers, it appears to play distinct roles at these regions. ESCs lacking H3.3 or HIRA are able to maintain both accessibility and TF footprinting at enhancers, but still show reduced H3K27ac. Unlike promoters, enhancers show no deficit of p300 enrichment in the absence of H3.3. The loss of H3K27ac observed at enhancers of H3.3 KO ESCs can be attributed to reduced catalytic activity of p300. In particular, phosphorylation of Ser31, the only residue unique to the N-terminal tail of H3.3, facilitates p300 activity and H3K27ac enrichment. In spite of extensive chromatin dysregulation and reduced active RNA polymerase II (RNAPII) engagement, ESCs maintain transcription from ESC-specific genes in the absence of H3.3. However, H3.3 KO ESCs are unable to initiate lineage-specific transcription upon undirected differentiation. In line with their differentiation defect, H3.3 KO ESCs retain footprinting of ESC-specific TFs and fail to generate footprints of lineage-specific TFs. Further, H3.3 KO ESCs fail to "open" and acetylate developmentally regulated enhancers. Overall, our study shows that H3.3 facilitates the establishment of transcriptionally permissive chromatin at regulatory elements, with context-dependent outcomes for transcriptional output. While H3.3 is not required for maintaining transcription in ESCs, it plays a key role in activating promoters and enhancers during differentiation.
Epigenetic Mechanisms in Drug Addiction
(2010-11-02) Renthal, William Russell; Nestler, Eric J.
Changes in gene expression in brain reward regions are thought to contribute to the pathogenesis and persistence of drug addiction. Recent studies have begun to focus on the molecular mechanisms by which drugs of abuse, and related environmental stimuli, such as drug-associated associated cues or stress, converge on the genome to alter specific geneprograms. Increasing evidence suggests that these stable gene expression changes in neurons are mediatedin part by epigenetic mechanisms that alter chromatin structure on specific gene promoters. Indeed, genome-wide analysis using chromatin immunoprecipitation coupled with promoter microarrays in vivo, identified on which genes chronic cocaine exposure alters histone acetylation and methylation in the nucleus accumbens, a key brain reward region. In addition to providing novel insight into basic transcriptional mechanisms co-opted by cocaine, these data revealed a new class of cocaine-regulated genes, the sirtuins, which potently regulate reward behavior. In order to further understand the mechanisms by which cocaine regulates chromatin structure, I investigated enzymes which control levels of histone acetylation, histone deacetylases (HDACs). Chronic, but not acute, exposure to cocaine decreased the function of a class II HDAC, HDAC5, in the NAc, which allows for increased histone acetylation and transcription of HDAC5 target genes. This regulation is behaviorally important, as loss of HDAC5 causes hypersensitive responses to chronic, but not acute, cocaine. I have also identified a key roleof the class I HDAC, HDAC1, which interacts with the drug-induced transcription factor, ΔFosB, to repress c-fos gene induction in striatum after chronic psychostimulant exposure. Taken together, these findings suggest that proper balance of histone acetylation in the NAc is a crucial factor in the saliency of cocaine action, and that disruption of this balance may be involved in the transition from acuteadaptive responses to chronic psychiatric illness.
HP1BP3, A Chromatin Retention Factor for Co-Transcriptional MicroRNA Processing
(2016-06-27) Liu, Haoming; Tu, Benjamin; Liu, Qinghua; Roth, Michael G.; Orth, Kim
RNA interference (RNAi) is a post-transcriptional gene silencing mechanism found in all eukaryotic organisms. It is characterized by a family of small non-coding RNAs, either endogenous (in the case of microRNAs) or exogenous (in the case of siRNAs), that inhibits gene expression post-transcriptionally. MicroRNAs (miRNAs) are a family of ~21-nt cellular RNAs that govern numerous pathological and physiological processes by mediating translational repression and deadenylation/decay of cognate mRNA. Dysregulation of miRNA expression have been associated with various types of cancer and developmental diseases. Typically, primary (pri-)miRNA transcripts are processed by Drosha complex into precursor (pre-)miRNAs, and then by cytoplasmic Dicer complex into mature miRNAs. The processing of pri-miRNAs is the most highly regulated step in the miRNA biogenesis pathway. Therefore, understanding the molecular mechanisms of pri-miRNA processing and its regulation represents a very important objective in the miRNA filed. Recent studies suggest that the Drosha-DGCR8 complex can be recruited to chromatin to catalyze co-transcriptional processing of primary microRNAs (pri-miRNAs) in mammalian cells. However, the molecular mechanism of co-transcriptional miRNA processing is poorly understood. Here, we find that HP1BP3, a histone H1-like chromatin protein, specifically associates with the Microprocessor and promotes global miRNA biogenesis in HeLa cells. Accordingly, chromatin immunoprecipitation (ChIP) studies reveal genome-wide co-localization of HP1BP3 & Drosha and HP1BP3-dependent Drosha binding to actively transcribed miRNA loci. Moreover, HP1BP3 exhibits a novel pri-miRNA binding activity and promotes the Drosha-pri-miRNA association in vivo. Knockdown of HP1BP3 compromises pri-miRNA processing by resulting in premature release of pri-miRNA transcripts from the chromatin. Taken together, these studies suggest that HP1BP3 promotes co-transcriptional miRNA processing via chromatin retention of nascent pri-miRNA transcripts. This work expands the functional repertoire of the H1 family of proteins and suggests a new concept of chromatin retention factor for widespread co-transcriptional miRNA processing.
Investigations into the Role of Paf1 Complex Proteins in Drosophila Ovaries
(2014-10-23) Chaturvedi, Dhananjay; Li, Bing; Olson, Eric N.; Jiang, Jin; Buszczak, Michael
Over the past decade considerable interest has grown in epigenetics and chromatin modifications. The ability of two cells with identical genomes to have entirely different transcriptomes and therefore cellular behaviors has piqued the curiosity of many researchers creating the whole field of Chromatin Biology. The difference in behavior of otherwise identical cells comes from an array of covalent modifications to protein spools wrapped by DNA in each cell. The complex of DNA and proteins is referred to as chromatin. Chromatin modifications influence the expression of regulatory proteins that control effectors of cellular physiology and metabolism. The outcome of protein function within a cell decides its fate from size, shape, function, the ability to divide or the lack thereof. The behavior of cells that can divide to give rise to themselves or progeny with a distinct specified function in an organism is of interest the biomedical community at large. These cells, called stem cells, hold promise in regenerative medicine to treat dystrophic diseases and injury. Work in cell culture systems shows that proteins modifying stem cell chromatin control their fate to a large extent. In this thesis I present to you, my efforts at understanding the role of a chromatin modifying complex: the Paf1 complex in the maintenance and differentiation of in vivo stem cell populations that control the maintenance of Drosophila ovaries.
KAP1 Is a Chromatin Reader that Couples Steps of RNA Polymerase II Transcription to Sustain Oncogenic Programs
(2020-05-01T05:00:00.000Z) Bacon, Curtis Wayne; Conrad, Nicholas; Kraus, W. Lee; Corey, David; D'Orso, Iván
Precise control of the RNA polymerase II (Pol II) cycle, including pausing and pause release, maintains transcriptional homeostasis and organismal functions. Much previous work to understand individual transcription steps, but insight into how these steps might be integrated is lacking. Here we reveal a mechanism that integrates Pol II cycle transitions. Surprisingly, the transcription factor KAP1/TRIM28 uses a previously uncharacterized chromatin reader cassette to bind hypo-acetylated histone 4 tails at promoters thereby guaranteeing a continuous progression of Pol II entry to, and exit from, the pause state. Upon chromatin docking, KAP1 first associates with Pol II and then recruits a pathway-specific transcription factor (SMAD2) in response to cognate ligands thereby enabling gene-selective CDK9-dependent pause release. This coupling mechanism is exploited by colorectal cancer cells to aberrantly sustain transcriptional programs commonly dysregulated in cancer patients. The discovery of a factor integrating transcription steps expands the functional repertoire by which chromatin readers operate and provides mechanistic understanding of transcription regulation, offering alternative therapeutic opportunities to target transcriptional dysregulation.
Molecular Dissection of Hand2 During the Formation of Pacemaker-Like Myocytes During Direct Reprogramming
(2019-03-06) Fernandez-Perez, Antonio; Zhang, Chun-Li; Cleaver, Ondine; Olson, Eric N.; Munshi, Nikhil
Direct reprogramming of one cell type into another has great promise for regenerative medicine, disease modeling, and lineage specification. Currently, the conversion of fibroblasts into induced cardiomyocytes (iCM) by Gata4, Mef2c, and Tbx5 (GMT) represents an important avenue for generating de novo cardiac myocytes. Recent evidence has shown that iCM formation and diversity can be enhanced by the addition of Hand2 to GMT (GHMT). These four transcription factors give rise to a heterogenous CM population, consisting of atrial (iAM), ventricular (iVM), and pacemaker myocytes (iPM). However, the molecular mechanisms that drive this plastic fate conversion remain poorly understood. Although chromatin and single-cell studies in GMT-iCM have shown the existence of a set of temporal steps that orchestrate iCM formation, little is known about how Hand2 enhances this process. In the present study, we seek to characterize these Hand2-dependent mechanisms. We hypothesize that Hand2 regulates a discrete pacemaker regulatory network that becomes active during GHMT-iCM reprogramming. To test this, we compared the transcriptional and genomic profiles of fibroblasts, GMT, GHMT, and endogenous mouse Pacemaker cells. We observe similar chromatin landscape and gene expression profiles between Hand2-iPM and endogenous sinoatrial node (SAN), however several known key PM pathways are not active. Activation of these networks further enhances iCM-iPM fo Moreover, we show that Hand2 enhances chromatin accessibility in regions related to sarcomere function and electrical coupling, as well as promoting the closing of regions related to alternative fates. Utilizing integrative genomics between ATAC-seq and RNA-seq datasets, we identify the desmosome machinery as an important feature of iPM formation. In parallel, we define a novel Hand2 domain region that regulates cardiac subtype diversity. Taken together, our results showcase Hand2-dependent mechanisms for iPM formation and gives insight into the improvement of future iPM engineering.
Regulation of Regeneration and Clonal Fitness in Normal and Diseased Liver
(2022-08) Jia, Yuemeng; Mendell, Joshua T.; Buszczak, Michael; Hon, Gary C.; Zhu, Hao
The potent regenerative capabilities of planaria, newts, and zebrafish are largely absent in mammals. In humans, impaired regeneration contributes to poor outcomes after acute trauma, tissue damage, or organ transplant, as well as in chronic diseases such as liver cirrhosis, inflammatory bowel disease, and diabetes. A central goal of my thesis is to identify unexpected pathways in tissue repair by establishing and exploiting in vivo forward genetic screening approaches, and two key questions were tackled with these platforms. The first goal was to identify druggable chromatin regulators of regeneration. To facilitate the discovery process, direct in vivo CRISPR knockout and CRISPR activation screening platforms in mouse livers were developed. Among 164 epigenetic regulators, both gain- and loss-of-function screens identified imitation-SWI (ISWI) chromatin remodeling components BAZ2A and BAZ2B. In vivo sgRNA, siRNA, and knockout mouse experiments against either paralog confirmed increased liver regeneration. Two distinct BAZ2 bromodomain inhibitors GSK2801 and BAZ2-ICR also resulted in accelerated liver healing after diverse injuries. Mechanistically, BAZ2 inhibition resulted in an increase in ribosomal biogenesis and protein synthesis by directly regulating the expressions of ribosomal proteins, resulting in an expanded reservoir of ribosomes, which allowed a more rapid cell cycle entry. The second part of the thesis was to understand the functional impact of somatic mutations on tissue regeneration in the context of tissue damage. Through exome and ultra-deep sequencing of 82 human cirrhotic liver samples, functional mutations in PKD1, KMT2D, and ARID1A were identified. Heterozygous deletion of these genes in mice promoted regeneration after liver injuries. The key concept from this work is that chronic injury, and perhaps aging, can select for mutations that promote adaptive or regenerative phenotypes rather than carcinogenesis. Together, these discoveries have established new methods for gene discovery in mammalian tissues, identified new targets for regenerative medicine, and uncovered adaptive mechanisms in chronic tissue injury that do not necessarily drive cancer.
Regulatory Mechanisms for the Pol II Associated Histone Methyltransferase Set2
(2014-11-20) Wang, Yi; Roth, Michael G.; Kraus, W. Lee; Liu, Yi; Li, Bing
Nucleosomes are building blocks of the eukaryotic chromatin which package genomic DNA with histones. The modification patterns of histones constitute an important signaling pathway for various nuclear processes. H3K36 methylation is catalyzed by the histone methyltranserase Set2 during transcription elongation. This important histone mark is ubiquitously presented in all organisms from yeast to mammals. In this study, we set out to investigate the molecular mechanisms by which the Set2 activity is precisely regulated during dynamic transcription cycle. In the first part of the study we discovered a novel role of the Set2 SRI domain, which is responsible for the binding of Set2 to elongating RNA polymerase II. We show that SRI also binds to DNA which determines the substrate specificity of Set2. In addition, we identified a novel auto-inhibitory role for the middle region of Set2 in regulating catalytic activity of Set2. Remarkably, mutations at this region cause hyperactivities, which in turn lead to synthetic phenotype with an essential histone chaperone FACT. Our data suggests that a temporal control for dynamic chromatin regulation is needed during transcription elongation process. In the second part, we investigated the molecular mechanism by which elongating Pol II regulated the Set2 activity beyond its initial recruitment. Surprisingly, we found the excessive amount of phosphorylated serine residues on Pol II CTD inhibited Set2 activity in vitro. Subsequent biophysical examination revealed that the additional phosphorylated CTD repeats collaterally occupied the surface of SRI where SRI contacts DNA. Finally, we determined that the minimal recognition unit of Set2 on fully phosphorylated CTD tail is three heptad repeats, and demonstrated that this minimal unit is sufficient for the Set2 recruitment without disrupting its catalytic activity. Since Pol II CTD utilizes repeating sequence as a scaffold for multiple factors, our results implicate that an organized spatial arrangement of these factors along CTD is necessary for accommodating their individual functions. In the last part of this work, we examined the state-specific functions of H3K36 methylation. By manipulating the catalytic domain of Set2, we obtained two mutants that can catalyze specific methyl-states of H3K36 both in vitro and in vivo. Genetic studies showed that cells carrying these two mutants displayed distinct phenotypes in several functional pathways, including histone chaperone, CTD proline isomerization and double-strand DNA repair. Our results suggest individual methyl-state of H3K36 plays non-redundant biological roles in cells. In summary, we discovered multiple mechanisms by which Set2 is dynamically regulated by elongating Pol II. Setd2, the human homolog of yeast Set2, has been shown recently to be one of the most important tumor suppressors among chromatin regulators. Given the highly conserved nature of this histone methyltransferase family, we believe that our mechanistic studies here may shed lights on the roles of Setd2 in tumorigenesis.
The Role of Chromatin Remodeling in Hippocampus in Depression and Antidepressant Action
(2008-05-13) Tsankova, Nadejda Mincheva; Nestler, Eric J.
This thesis presents a novel level by which neuroplastic changes in the brain may be disrupted with depression and reversed by treatment with antidepressants: regulation at the level of chromatin remodeling. The technique of brain chromatin immunoprecipitation was pioneered to directly measure the in vivo modifications of histones, a form of chromatin remodeling, at gene promoter regions in the hippocampus after chronic defeat stress, a model of depression, and chronic treatment with the antidepressants imipramine and electroconvulsive seizure (ECS). Chromatin modifications and transcriptional changes were assayed in one gene in particular, the brain-derived neurotrophic factor (BDNF). BDNF is alternatively spliced to generate several mRNA transcripts, driven by unique promoters. I measured the expression levels of each BDNF transcript (I-IV) in rat after ECS, as well as each BDNF transcript (I-V) in mice after chronic stress and imipramine treatments, and found that these chronic treatments induce lasting changes in the expression of specific BDNF splice variants. These changes correlated with sustained modifications in histones at the exact promoter regions, driving the differential changes in BDNF expression. Chronic defeat stress induced robust enrichment of H3-K27 methylation at BDNF P3 and P4 promoters (modifications expected to repress promoter activity), while chronic imipramine in defeated animals lead to lasting upregulation in the levels of H3 acetylation and H3-K4 methylation at P3 and P4 (modifications expected to stimulate promoter activity). Finally, I discovered a novel role for the histone deacetylase HDAC5 in the therapeutic efficacy of chronic imipramine after defeat stress. I found that chronic imipramine downregulates HDAC5 after stress, that HDAC5 overexpression in the hippocampus blocks the behavioral effects of imipramine in defeated mice, that HDAC5 inhibition exerts a subtle antidepressant-like effect, and that HDAC5 deficiency reduces the pathological response to stress. This unexpected role for HDAC5 provides an important mechanistic link between the adaptive chromatin remodeling changes at genes and the ability of chronic antidepressants to exert therapeutic efficacy after chronic stress. These experiments provide one of the first endeavors to understand the role of chromatin remodeling in modulating long-term adaptive changes in brain associated with complex psychiatric conditions, such as depression.
Transcription Factor Dynamics Investigated through Single-Molecule Imaging, High-Throughput Sequencing, and Neural Networks
(2020-08-01T05:00:00.000Z) Stevens, Evan McAllister; Brekken, Rolf A.; D'Orso, Iván; Le, Lu Q.
Recent chromatin characterization and sequencing technologies, paired with growing power in computational and bioinformatic analysis, have enabled a deeper understanding of the highly sequence-dependent nature of protein-DNA interactions. Further, these tools have brought to the forefront gaps in our understanding of how changing chromatin landscapes shape cell and tissue identity, and particularly how proteins with stable and transient DNA associations provide feedback to this process. Chromatin remodeling and reorganization serve as umbrella terms to describe diverse mechanisms altering cell epigenetic identity. Transcription factors interacting with chromatin can be influenced by chromatin remodeling processes specifically through cognate sites modifications or generally through a variety of mechanisms, but the degree to which chromatin remodeling alters transcription factor dynamics and activity through general or specific mechanisms is poorly understood. We applied the techniques of Single-Molecule Tracking (SMT) to study the changing dynamics of transcription factors through a B cell activation process marked by widespread chromatin reorganization. First, we identified that during B cell activation, and specifically by the process of nanodomain decompaction, residence time for transcription factors is decreased, suggesting an increased efficiency in transcription. Further studies will be needed to determine if this association between transcription factor residence time and gene transcription is reproducible, and the mechanism underlying it. Second, we identified that the process by which transcription factors scan DNA to identify cognate binding sites, measured by transcription factor random collisions and search time, occurred more rapidly in activated B cells. Given that our work gave additional evidence of the effect of chromatin organization on transcription factor residence time and transcription, we aimed to systematically identify proteins that work upstream to influence the accessibility of chromatin. We generated datasets measuring chromatin accessibility in a variety of mouse tissues and cells, with significant contribution of immune cell subsets. Our accessibility data showed patterns for regulatory elements that fall in line with literature describing significant regions of the genome dedicated to cell-specific regulation, rather than universal regulation. Using a neural network tool known as DeepLIFT with motif identification tools TF-MoDISco and HOMER, we tracked patterns of transcription factor contributions to accessibility across these cell and tissue types, and especially through cell lineages. We identified orphan motifs with no assigned transcription factor, and further identified pleiotropic transcription factors predicting overlooked immune cell functions. Our work stands as a valuable resource for connecting chromatin reorganization and transcription factor dynamics, as well as for testing limits for systematic approaches to predicting contributions of transcription factors to chromatin accessibility.
Transcription of the Long Noncoding RNA, UpperHand, Is Required for Heart Development
(2016-10-03) Anderson, Kelly Marie; Mendell, Joshua T.; Olson, Eric N.; Graff, Jonathan M.; Conrad, Nicholas; Stull, James
The basic helix-loop-helix transcription factor HAND2 is an ancestral regulator of heart development and one of four cardiac transcription factors that controls the reprogramming of fibroblasts to cardiomyocytes. Genetic deletion of Hand2 in mice results in hypoplasia of the right ventricle and embryonic lethality. The embryonic expression of Hand2 is tightly regulated by well-characterized upstream enhancer elements, which reside within a super-enhancer delineated by H3K27ac histone modifications. Here, I show that transcription of a long noncoding RNA upstream of Hand2, which I named UpperHand (UPH), is required to maintain the H3K27ac super-enhancer signature and elongation of RNA polymerase II through the Hand2 locus. Blockade of UPH transcription by insertion of a transcription termination cassette, but not knockdown of the mature transcript, abolished Hand2 expression, causing right ventricular hypoplasia and embryonic lethality in mice. Given the substantial number of uncharacterized promoter-associated lncRNAs encoded by the mammalian genome, the UPH-Hand2 regulatory partnership offers a mechanism by which noncoding transcription can establish a permissive chromatin environment necessary for the expression of a neighboring protein-coding gene.