Altered Nucleosome Positioning Research Articles

The Role of Histone Variant H3.3 Regulation By Hira in Hematopoiesis

A large proportion of adult hematopoietic stem cells (HSCs) remain quiescent throughout life, rarely undergoing cell division. These quiescent HSCs must undergo global changes in gene expression patterns in order to leave their dormant state and differentiate. Chromatin modifiers that act independent of cell division may therefore be of particular importance in regulating the behavior of quiescent HSCs through alterations in the chromatin landscape.One such chromatin modifier is the histone variant H3.3 chaperone, Hira, which is responsible for incorporating H3.3 independent of DNA replication at poised and transcriptionally active promoters. Despite the fact that this histone variant only differs from the canonical H3 histones by four or five amino acids, its gene-specific association is required for the proper establishment of gene expression and lineage specification in murine embryonic stem cells (mESCs), post-mitotic neurons, and oocytes. Since Hira deposits H3.3 outside of S phase, we hypothesize that Hira plays a critical role in the maintenance of quiescent HSCs through the regulation of H3.3 independent of cell division.HSCs divide rapidly in the fetal liver but become quiescent in adulthood, providing an interesting opportunity to study HSCs that are vastly different in terms of cell cycle properties. In order to study the role of Hira in HSCs, we used a conditional knockout (cKO) mouse model to delete Hira in hematopoietic cells in early development (Vav-iCre; Hirafl/fl). Importantly, we observed severe depletion of adult, but not fetal, HSCs upon loss of Hira. This suggests that Hira is important for the maintenance of quiescent adult HSCs, but not rapidly dividing fetal HSCs, and supports our model of Hira as a regulator of HSC quiescence through the DNA replication-independent incorporation of H3.3. We additionally observed altered differentiation potential, reduced reconstitution capacity, and increased cycling in adult HSCs upon Hira cKO. These results demonstrate that Hira is an essential regulatory factor for the control of adult HSC function.To obtain adult Hira cKO HSCs for further analysis, we also used an inducible model of Hira deletion in the hematopoietic system upon polyinosinic-polycytidylic (pIpC) treatment (Mx1-Cre; Hirafl/fl). We performed RNA-seq analysis on adult Hira cKO HSCs, which showed widespread alterations in gene expression patterns and upregulation of PRC2 target genes. Since Hira is known to recruit polycomb repressive complex 2 (PRC2) to bivalent promoters in mESCs in an H3.3-dependent manner (Banaszynski et. al. Cell 2013), chromatin-immunoprecipitation and sequencing (ChIP-seq) for H3K27me3 will be used to show how loss of Hira affects PRC2 activity in HSCs. We have additionally performed assay for transposase-accessible chromatin and sequencing (ATAC-seq) in order to determine how patterns of DNA accessibility in HSCs are affected by Hira cKO. Altered nucleosome positioning and PRC2 activity will be discussed as potential mechanisms for the role of Hira in regulating the behavior of quiescent HSCs.Our findings reveal a novel paradigm for epigenetic state regulation in adult HSCs. Knowledge gained from this study may inform future research for clinical applications, such as for improved methods of in vitro HSC expansion. Characterizing the importance of H3.3 regulation by Hira in normal HSC activity will therefore provide invaluable information regarding the epigenetic mechanisms underlying hematopoietic homeostasis. DisclosuresNo relevant conflicts of interest to declare.

Topoisomerase activity is linked to altered nucleosome positioning and transcriptional regulation in the fission yeast fbp1 gene.

Chromatin structure, including nucleosome positioning, has a fundamental role in transcriptional regulation through influencing protein-DNA interactions. DNA topology is known to influence chromatin structure, and in doing so, can also alter transcription. However, detailed mechanism(s) linking transcriptional regulation events to chromatin structure that is regulated by changes in DNA topology remain to be well defined. Here we demonstrate that nucleosome positioning and transcriptional output from the fission yeast fbp1 and prp3 genes are altered by excess topoisomerase activity. Given that lncRNAs (long noncoding RNAs) are transcribed from the fbp1 upstream region and are important for fbp1 gene expression, we hypothesized that local changes in DNA topological state caused by topoisomerase activity could alter lncRNA and fbp1 transcription. In support of this, we found that topoisomerase overexpression caused destabilization of positioned nucleosomes within the fbp1 promoter region, which was accompanied by aberrant fbp1 transcription. Similarly, the direct recruitment of topoisomerase, but not a catalytically inactive form, to the promoter region of fbp1 caused local changes in nucleosome positioning that was also accompanied by altered fbp1 transcription. These data indicate that changes in DNA topological state induced by topoisomerase activity could lead to altered fbp1 transcription through modulating nucleosome positioning.

General Regulatory Factors Control the Fidelity of Transcription by Restricting Non-coding and Ectopic Initiation

The fidelity of transcription initiation is essential for accurate gene expression, but the determinants of start site selection are not fully understood. Rap1 and other general regulatory factors (GRFs) control the expression of many genes in yeast. We show that depletion of these factors induces widespread ectopic transcription initiation within promoters. This generates many novel non-coding RNAs and transcript isoforms with diverse stability, drastically altering the coding potential of the transcriptome. Ectopic transcription initiation strongly correlates with altered nucleosome positioning. We provide evidence that Rap1 can suppress ectopic initiation by a "place-holder" mechanism whereby it physically occludes inappropriate sites for pre-initiation complex formation. These results reveal an essential role for GRFs in the fidelity of transcription initiation and in the suppression of pervasive transcription, profoundly redefining current models for their function. They have important implications for the mechanism of transcription initiation and the control of gene expression.

Divergent Residues Within Histone H3 Dictate a Unique Chromatin Structure in Saccharomyces cerevisiae.

Histones are among the most conserved proteins known, but organismal differences do exist. In this study, we examined the contribution that divergent amino acids within histone H3 make to cell growth and chromatin structure in Saccharomyces cerevisiae. We show that, while amino acids that define histone H3.3 are dispensable for yeast growth, substitution of residues within the histone H3 α3 helix with human counterparts results in a severe growth defect. Mutations within this domain also result in altered nucleosome positioning, both in vivo and in vitro, which is accompanied by increased preference for nucleosome-favoring sequences. These results suggest that divergent amino acids within the histone H3 α3 helix play organismal roles in defining chromatin structure.

ACF chromatin-remodeling complex mediates stress-induced depressive-like behavior.

Improved treatment for major depressive disorder (MDD) remains elusive due to limited understanding of its underlying biological mechanisms. Stress–induced maladaptive transcriptional regulation within limbic neural circuits likely contributes to the development of MDD, possibly through epigenetic factors that regulate chromatin structure. We establish that persistent upregulation of the ACF ATP–dependent chromatin remodeling complex, occurring in the nucleus accumbens of stress–susceptible mice and depressed humans, is necessary for stress–induced depressive–like behaviors. Altered ACF binding after chronic stress is correlated with altered nucleosome positioning, particularly around the transcription start sites of affected genes. These alterations in ACF binding and nucleosome positioning are associated with repressed expression of genes implicated in susceptibility to stress. Together, we identify the ACF chromatin remodeling complex as a critical component in the development of susceptibility to depression and in regulating stress–related behaviors.

Altered Nucleosome Positioning at the Transcription Start Site and Deficient Transcriptional Initiation in Friedreich Ataxia

Most individuals with Friedreich ataxia (FRDA) are homozygous for an expanded GAA triplet repeat (GAA-TR) mutation in intron 1 of the FXN gene, which results in deficiency of FXN transcript. Consistent with the expanded GAA-TR sequence as a cause of variegated gene silencing, evidence for heterochromatin has been detected in intron 1 in the immediate vicinity of the expanded GAA-TR mutation in FRDA. Transcriptional deficiency in FRDA is thought to result from deficient elongation through the expanded GAA-TR sequence because of repeat-proximal heterochromatin and abnormal DNA structures adopted by the expanded repeat. There is also evidence for deficient transcriptional initiation in FRDA, but its relationship to the expanded GAA-TR mutation remains unclear. We show that repressive chromatin extends from the expanded GAA-TR in intron 1 to the upstream regions of the FXN gene, involving the FXN transcriptional start site. Using a chromatin accessibility assay and a high-resolution nucleosome occupancy assay, we found that the major FXN transcriptional start site, which is normally in a nucleosome-depleted region, is rendered inaccessible by altered nucleosome positioning in FRDA. Consistent with the altered epigenetic landscape the FXN gene promoter, a typical CpG island promoter, was found to be in a transcriptionally non-permissive state in FRDA. Both metabolic labeling of nascent transcripts and an unbiased whole transcriptome analysis revealed a severe deficiency of transcriptional initiation in FRDA. Deficient transcriptional initiation, and not elongation, is the major cause of FXN transcriptional deficiency in FRDA, and it is related to the spread of repressive chromatin from the expanded GAA-TR mutation.

CpG domains downstream of TSSs promote high levels of gene expression

CpG dinucleotides are known to play a crucial role in regulatory domains, affecting gene expression in their natural context. Here, we demonstrate that intragenic CpG frequency and distribution impacts transgene and genomic gene expression levels in mammalian cells. As shown for the Macrophage Inflammatory Protein 1α, de novo RNA synthesis correlates with the number of CpG dinucleotides, whereas RNA splicing, stability, nuclear export and translation are not affected by the sequence modification. Differences in chromatin accessibility in vivo and altered nucleosome positioning in vitro suggest that increased CpG levels destabilize the chromatin structure. Moreover, enriched CpG levels correlate with increased RNA polymerase II elongation rates in vivo. Interestingly, elevated CpG levels particularly at the 5′ end of the gene promote efficient transcription. We show that this is a genome-wide feature of highly expressed genes, by identifying a domain of ∼700 bp with high CpG content downstream of the transcription start site, correlating with high levels of transcription. We suggest that these 5′ CpG domains are required to distort the chromatin structure and to increase gene activity.

A Compendium of Nucleosome and Transcript Profiles Reveals Determinants of Chromatin Architecture and Transcription

Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies.

Dissecting Epigenetic Silencing Complexity in the Mouse Lung Cancer Suppressor Gene Cadm1

Disease-oriented functional analysis of epigenetic factors and their regulatory mechanisms in aberrant silencing is a prerequisite for better diagnostics and therapy. Yet, the precise mechanisms are still unclear and complex, involving the interplay of several effectors including nucleosome positioning, DNA methylation, histone variants and histone modifications. We investigated the epigenetic silencing complexity in the tumor suppressor gene Cadm1 in mouse lung cancer progenitor cell lines, exhibiting promoter hypermethylation associated with transcriptional repression, but mostly unresponsive to demethylating drug treatments. After predicting nucleosome positions and transcription factor binding sites along the Cadm1 promoter, we carried out single-molecule mapping with DNA methyltransferase M.SssI, which revealed in silent promoters high nucleosome occupancy and occlusion of transcription factor binding sites. Furthermore, M.SssI maps of promoters varied within and among the different lung cancer cell lines. Chromatin analysis with micrococcal nuclease also indicated variations in nucleosome positioning to have implications in the binding of transcription factors near nucleosome borders. Chromatin immunoprecipitation showed that histone variants (H2A.Z and H3.3), and opposing histone modification marks (H3K4me3 and H3K27me3) all colocalized in the same nucleosome positions that is reminiscent of epigenetic plasticity in embryonic stem cells. Altogether, epigenetic silencing complexity in the promoter region of Cadm1 is not only defined by DNA hypermethylation, but high nucleosome occupancy, altered nucleosome positioning, and ‘bivalent’ histone modifications, also likely contributed in the transcriptional repression of this gene in the lung cancer cells. Our results will help define therapeutic intervention strategies using epigenetic drugs in lung cancer.

Hepatic eEF2K downregulation is associated with altered nucleosome positioning in neonatal IUGR rats

Uteroplacental insufficiency (UPI) alters protein synthesis in intrauterine growth restriction (IUGR). Protein synthesis is regulated in part, by eukaryotic elongation factor 2 kinase (eEF2K). eEF2K, a CpG‐rich gene, is down regulated in IUGR rat liver. Our group has shown that UPI alters epigenetic events on genes that have multiple promoters and splice variants. What is not known is how UPI influences epigenetic regulation in CpG island genes. We therefore analyzed the effects of UPI on eEF2K in terms of DNA methylation, histone covalent modifications, and nucleosome positioning around the transcription start in IUGR rat liver. Bisulfite sequencing, ChIP and SssI MTase footprining were performed. The eEF2K promoter had significantly increased DNA methylation, decreased H3acK9, K14, me1K4, me3K4, and me3K36. In addition, the +1 nucleosome accessibility was significantly restricted, together with increased H2A occupancy and decreased RNA polII loading in IUGR relative to the control. In conclusion, UPI induced changes in DNA and histone covalent modifications, nucleosome position and polII loading. We speculate that these changes contribute to eEF2K downregulation.