Specific Chromatin Structure Research Articles

The Epigenome of Evolving Drosophila Neo-Sex Chromosomes: Dosage Compensation and Heterochromatin Formation

Author SummarySex chromosomes differ from non-sex chromosomes (“autosomes”) at the genomic, transcriptomic, and epigenomic level, yet the X and Y share a common evolutionary origin. The Drosophila Y chromosome is gene-poor and associated with a compact and transcriptionally inactive form of genetic material called heterochromatin. The X, in contrast, is enriched for activating chromatin marks and is consequently hyper-transcribed, a process thought to be an adaptation to decay and silencing of genes on the Y, resulting in “dosage compensation.” How sex chromosomes have altered their chromatin structure, and what genomic changes led to this dramatically different epigenetic makeup, however, has remained a mystery. By studying the genome, epigenome, and transcriptome of a species with a very recently evolved pair of sex chromosomes (the neo-X and neo-Y of a fruit fly, Drosophila miranda), we here recapitulate how both dosage compensation and heterochromatin formation evolve in Drosophila and establish several novel and important principles governing the evolution of chromatin structure. We dissect the evolutionary history of over 60 novel binding sites for the dosage compensation complex that evolved by natural selection on the neo-X within the last one million years. We show that the 21-bp consensus motifs for recruiting the dosage compensation complex were acquired by diverse molecular mechanisms along the neo-X, while the onset of heterochromatin formation is triggered by the accumulation of transposable elements, leading to silencing of adjacent neo-Y genes. We find that spreading of these chromatin modifications results in massive mis-expression of neo-sex linked genes, and that little correspondence exists between functional activity of genes on the neo-Y and whether they are dosage-compensated on the neo-X. Intriguingly, the genomic regions being targeted by the dosage compensation complex on the neo-X and those that are heterochromatic on the neo-Y show little overlap, possibly reflecting different propensities of the ancestral chromosome that formed the sex chromosome to evolve active versus repressive chromatin configurations. These findings have broad implications for current models of sex chromosome evolution.

Nucleosome Distribution near the 3′ Ends of Genes in the Human Genome

By systematic analysis of high-throughput sequencing datasets from the human genome, we found that protein-coding genes have a specific chromatin structure near transcription termination sites relative to non-coding genes, one related to polyadenylation. Nucleosome was depleted near the site of cleavage/polyadenylation (polyA site) regardless of its relative position in the gene. DNA sequence plays an improtant role in nucleosome distribution, and conservative sequence elements and the protein binding to them are major determinants in causing nucleosome depletion near polyA sites. Furthermore, nucleosome occupancy was regulated by gene transcription and RNA polymerase II (RNAPII) occupancy. Our results reveal influences on nucleosome occupancy near polyadenylation sites and constitute evidence indicating that nucleosome distribution regulates 3' end processing of protein-coding genes.

UV light-induced DNA lesions cause dissociation of yeast RNA polymerases-I and establishment of a specialized chromatin structure at rRNA genes

The cytotoxicity of UV light-induced DNA lesions results from their interference with transcription and replication. DNA lesions arrest elongating RNA polymerases, an event that triggers transcription-coupled nucleotide excision repair. Since arrested RNA polymerases reduce the accessibility of repair factors to DNA lesions, they might be displaced. The fate of arrested RNA polymerases-II at DNA lesions has been extensively studied, yielding partially contradictory results. Considerably less is known about RNA polymerases-I that transcribe nucleosomes-depleted rRNA genes at very high rate. To investigate the fate of arrested RNA polymerases-I at DNA lesions, chromatin-immunoprecipitation, electron microscopy, transcription run-on, psoralen-cross-linking and chromatin-endogenous cleavage were employed. We found that RNA polymerases-I density increased at the 5′-end of the gene, likely due to continued transcription initiation followed by elongation and pausing/release at the first DNA lesion. Most RNA polymerases-I dissociated downstream of the first DNA lesion, concomitant with chromatin closing that resulted from deposition of nucleosomes. Although nucleosomes were deposited, the high mobility group-box Hmo1 (component of actively transcribed rRNA genes) remained associated. After repair of DNA lesions, Hmo1 containing chromatin might help to restore transcription elongation and reopening of rRNA genes chromatin.

Abstract 4857: Short overall telomere length and chromosome 17p-specific telomere length in peripheral blood leukocytes are associated with increased risk of esophageal adenocarcinoma.

Abstract Esophageal adenocarcinoma (EAC) is a deadly cancer. The incidence of EAC has increased dramatically in the United States in the past three decades. The biological mechanisms of EAC development are not fully understood. Telomeres are specialized chromatin structures essential for the maintenance of chromosomal integrity. Telomere shortening in somatic tissues has been shown as an early event during carcinogenesis. Previous studies also suggested that constitutive short telomeres in peripheral blood leukocytes (PBLs) are associated with increased risks of several cancers, including esophageal cancer. In this study, we measured overall telomere length in PBLs using real-time PCR in a large case control study of 805 EAC cases and 805 age and gender-matched controls. Furthermore, we developed a fluorescence in site hybridization (FISH) based method to evaluate several chromosome-specific telomere length with the risk of EAC in a subset of subjects. The mean overall telomere length in cases was significantly shorter than that in controls (p<0.001). Dichotomized at the median telomere length in controls, individuals with short PBL telomere length was associated with a 1.53-fold (95% CI, 1.26-1.87) increased risk of EAC. There was a significant dose-effect in quartile analysis: Individuals in quartile with the shortest telomeres showed a nearly 2-fold (95%CI: 1.47-2.57) elevated EAC risk compared to the reference group of the longest quartile of telomere length. In chromosome-specific telomere length analysis, we found that short chromosome 17p-specific telomere length (defined as the ratio of chromosome 17p-specific telomere length to overall telomere length), but not 2p- and 7q-specific telomere length, was associated with a significantly increased risk of EAC (OR=2.02, 95% CI, 1.00-4.08). This study validates that short overall telomere length in PBLs predisposes to EAC with a much larger sample size and supports the role of chromosome 17p aberrations in EAC development. Citation Format: Meng Chen, Jian Gu, Yilei Gong, Jaffer A. Ajani, Xifeng Wu. Short overall telomere length and chromosome 17p-specific telomere length in peripheral blood leukocytes are associated with increased risk of esophageal adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4857. doi:10.1158/1538-7445.AM2013-4857

MeCP2's Role in Chromatin Structure and Stability

Autism spectrum disorders (ASDs) affect 1 in 110 children. Rett Syndrome (RTT) is one of the few ASDs that have a direct genetic link, which make it great model to understand some underlying causes of ASD. RTT is caused by mutation in the X‐linked Methyl CpG Binding Protein 2 (MeCP2). MeCP2 is associated with transcriptional repression and can act on naked DNA or, if found in high enough quantities, alters chromatin structure. Therefore understanding the functional implications on both specific genes and chromatin structure is critical in developing therapeutic strategies. After neural development MeCP2 is found in levels equivalent to H1 in neuronal cells and is believed to compete with H1. Like H1, MeCP2 binds at the entry/exit site of nucleosomal DNA, and could regulate the rate of wrapping and unwrapping around the nucleosome, thus being critical in target gene accessibility. Our goal is to understand how MeCP2 alters gene accessibility through dynamic changes to chromatin structure. We will use FRET measurements to determine the influence of MeCP2 on nucleosome stability, site accessibility, and higher order compaction; providing insight to the mechanistic functions of MeCP2 on gene expression.

Association of Leukocyte Telomere Length With Breast Cancer Risk: Nested Case-Control Findings From the Shanghai Women's Health Study

Telomeres are specialized chromatin structures essential for the maintenance of chromosomal integrity and stability. Telomere shortening has been linked to multiple aging-related diseases, including cancer. Evidence associating telomere length with breast cancer risk-most of which has been from retrospective case-control studies-is conflicting. We conducted a nested case-control study based on the Shanghai Women's Health Study (1997-2009) in which we evaluated the association of telomere length and breast cancer risk using peripheral blood samples collected before cancer diagnosis (601 cases and 695 controls). We used monochrome multiplex quantitative polymerase chain reaction to measure relative telomere length. Multiple logistic regressions were used to derive adjusted odds ratios with 95% confidence intervals as the measure of association. Telomere length was inversely correlated with age (r = -0.22). Women with moderately long telomeres (those in the fourth quintile) had the lowest breast cancer risk. Risk increased in a dose-response manner with decreasing quintile of telomere length; odds ratios were 1.39 (95% confidence interval (CI): 0.95, 2.04), 1.79 (95% CI: 1.17, 2.75), and 2.39 (95% CI: 1.45, 3.92), respectively, for the third, second, and first quintiles compared with the fourth quintile. A slightly elevated risk of breast cancer (odds ratio = 1.35, 95% CI: 0.90, 2.04), although one that was not statistically significant, was found in the top quintile (longest telomeres). Our results support the hypothesis that telomere shortening is associated with increased risk of breast cancer and suggest a possible elevated risk associated with long telomeres.

Evolutionary Mobility of the Ribosomal DNA Array in Yeasts

The ribosomal DNA (rDNA) of eukaryotes is organized as large tandem arrays. Here, we compare the genomic locations of rDNA among yeast species and show that, despite its huge size (>1 Mb), the rDNA array has moved around the genome several times within the family Saccharomycetaceae. We identify an ancestral, nontelomeric, rDNA site that is conserved across many species including Saccharomyces cerevisiae. Within the genus Lachancea, however, the rDNA apparently transposed from the ancestral site to a new site internal to a different chromosome, becoming inserted into a short intergenic region beside a tRNA gene. In at least four other yeast lineages, the rDNA moved from the ancestral site to telomeric locations. Remarkably, both the ancestral rDNA site and the new site in Lachancea are adjacent to protein-coding genes whose products maintain the specialized chromatin structure of rDNA (HMO1 and CDC14, respectively). In almost every case where the rDNA was lost from the ancestral site, the entire array disappeared without any other rearrangements in the region, leaving just an intergenic spacer of less than 2 kb. The mechanism by which this large and complex locus moves around the genome is unknown, but we speculate that it may involve the formation of double-strand DNA breaks by Fob1 protein or the formation of extrachromosomal rDNA circles.

Heterochromatinization associated with cell differentiation as a model to study DNA double strand break induction and repair in the context of higher-order chromatin structure

Cell differentiation is associated with extensive gene silencing, heterochromatinization and potentially decreasing need for repairing DNA double-strand breaks (DSBs). Differentiation stages of blood cells thus represent an excellent model to study DSB induction, repair and misrepair in the context of changing higher-order chromatin structure. We show that immature granulocytes form γH2AX and 53BP1 foci, contrary to the mature cells; however, these foci colocalize only rarely and DSB repair is inefficient. Moreover, specific chromatin structure of granulocytes probably influences DSB induction.

Association of Leukocyte Telomere Length with Colorectal Cancer Risk: Nested Case–Control Findings from the Shanghai Women's Health Study

Telomeres are specialized chromatin structures essential for maintenance of chromosomal integrity and stability. Abnormal alteration of telomere length has been linked to several cancers; however, epidemiologic evidence about the association of telomere length with colorectal cancer risk has been conflicting. We conducted a nested case-control study to evaluate the association between telomere length and colorectal cancer risk using peripheral blood samples collected before cancer diagnosis. The study included 441 women with incident colorectal cancer and 549 matched controls. Monochrome multiplex quantitative PCR was applied to measure relative telomere length. Multiple logistic regressions were used to derive adjusted OR with 95% confidence intervals (CI) as the measure of association between telomere length and subsequent colorectal cancer risk. A U-shaped association was observed between telomere length and colorectal cancer risk (test for nonlinearity P = 0.0112). Women with telomere length in the third quintile (40th-60th percentiles) had the lowest risk of colorectal cancer, and the risks were elevated with a shorter or longer telomere length. This U-shaped association did not statistically differ for colon cancer and rectum cancer. Our prospective study revealed a U-shaped association between telomere length in peripheral blood cells and colorectal cancer risk. Our findings provide strong evidence that both very short and very long telomeres are associated with increased risk of colorectal cancer.

Histone Variant H2A.Bbd Is Associated with Active Transcription and mRNA Processing in Human Cells

Variation in chromatin composition and organization often reflects differences in genome function. Histone variants, for example, replace canonical histones to contribute to regulation of numerous nuclear processes including transcription, DNA repair, and chromosome segregation. Here we focus on H2A.Bbd, a rapidly evolving variant found in mammals but not in invertebrates. We report that in human cells, nucleosomes bearing H2A.Bbd form unconventional chromatin structures enriched within actively transcribed genes and characterized by shorter DNA protection and nucleosome spacing. Analysis of transcriptional profiles from cells depleted for H2A.Bbd demonstrated widespread changes in gene expression with a net downregulation of transcription and disruption of normal mRNA splicing patterns. In particular, we observed changes in exon inclusion rates and increased presence of intronic sequences in mRNA products upon H2A.Bbd depletion. Taken together, our results indicate that H2A.Bbd is involved in formation of a specific chromatin structure that facilitates both transcription and initial mRNA processing.

The chromatin remodeling complex NuRD establishes the poised state of rRNA genes characterized by bivalent histone modifications and altered nucleosome positions

rRNA genes (rDNA) exist in two distinct epigenetic states, active promoters being unmethylated and marked by euchromatic histone modifications, whereas silent ones are methylated and exhibit heterochromatic features. Here we show that the nucleosome remodeling and deacetylation (NuRD) complex establishes a specific chromatin structure at rRNA genes that are poised for transcription activation. The promoter of poised rRNA genes is unmethylated, associated with components of the preinitiation complex, marked by bivalent histone modifications and covered by a nucleosome in the "off" position, which is refractory to transcription initiation. Repression of rDNA transcription in growth-arrested and differentiated cells correlates with elevated association of NuRD and increased levels of poised rRNA genes. Reactivation of transcription requires resetting the promoter-bound nucleosome into the "on" position by the DNA-dependent ATPase CSB (Cockayne syndrome protein B). The results uncover a unique mechanism by which ATP-dependent chromatin remodeling complexes with opposing activities establish a specific chromatin state and regulate transcription.

Chromatin structure of the LCR in the human β-globin locus transcribing the adult δ- and β-globin genes

The β-like globin genes are transcribed in a developmental stage specific fashion in erythroid cells. The specific transcription of globin genes is conferred by the locus control region (LCR), but the chromatin structure of the LCR in the human adult β-globin locus transcribing the δ- and β-globin genes is not clear. Here, we employed hybrid MEL cells that contain a human chromosome 11. The δ- and β-globin genes were highly transcribed in hybrid MEL/ch11 cells after transcriptional induction. LCR HS3 and HS2 were strongly occupied by erythroid specific transcriptional activators and co-factors in the induced locus. These HSs, but not HS4 and HS1, were in close proximity with the active globin genes as revealed by high resolution 3C experiments. The active features at HS3 were markedly established after transcriptional induction, while HS2 was in a relatively active conformation before the induction. Unexpectedly, HS1 did not show notable active features except histone hyperacetylation. Taken together, the LCR of the human β-globin locus transcribing the adult δ- and β-globin genes has HS specific chromatin structure. The structure at each HS, which is different from the locus transcribing the fetal globin genes, might relate to its role in transcribing the adult genes.

Mapping personal functional data to personal genomes

The sequencing of personal genomes enabled analysis of variation in transcription factor (TF) binding, chromatin structure and gene expression and indicated how they contribute to phenotypic variation. It is hypothesized that using the reference genome for mapping ChIP-seq or RNA-seq reads may introduce errors, especially at polymorphic genomic regions. We developed a Personal Genome Editor (perEditor) that changes the reference human genome (NCBI36/hg18) into an individual genome, taking into account single nucleotide polymorphisms (SNPs), insertions and deletions, copy number variation, and chromosomal rearrangements. perEditor outputs two alleles (maternal, paternal) of the individual genome that is ready for mapping ChIP-seq and RNA-seq reads, and enabling the analyses of allele specific binding, chromatin structure and gene expression. perEditor is available at http://biocomp.bioen.uiuc.edu/perEditor. szhong@illinois.edu.

Endogenous Transcription at the Centromere Facilitates Centromere Activity in Budding Yeast

The centromere (CEN) DNA-kinetochore complex is the specialized chromatin structure that mediates chromosome attachment to the spindle and is required for high-fidelity chromosome segregation. Although kinetochore function is conserved from budding yeast to humans, it was thought that transcription had no role in centromere function in budding yeast, in contrast to other eukaryotes including fission yeast. We report here that transcription at the centromere facilitates centromere activity in the budding yeast Saccharomyces cerevisiae. We identified transcripts at CEN DNA and found that Cbf1, which is a transcription factor that binds to CEN DNA, is required for transcription at CEN DNA. Chromosome instability of cbf1Δ cells is suppressed by transcription driven from an artificial promoter. Furthermore, we have identified Ste12, which is a transcription factor, and Dig1, a Ste12 inhibitor, as a novel CEN-associated protein complex by an in vitro kinetochore assembly system. Dig1 represses Ste12-dependent transcription at the centromere. Our studies reveal that transcription at the centromere plays an important role in centromere function in budding yeast.

Conference Scene: Chromatin, replication and chromosomal stability

The Chromatin, Replication and Chromosomal Stability Conference took place on June 20-21 in Stockholm, Sweden. In this article, I outline the broad scientific program of the meeting which reflected the wide diversity in epigenetics research. Distinct histone modifications are linked with specific chromatin structures and intranuclear positioning, thereby impacting replication timing and replication initiation, which in turn are related to gene expression and cell differentiation. Interference in any of these interconnected mechanisms can result in DNA breakage and lead to the activation of repair pathways. The DNA repair mechanisms again are influenced by the chromatin structure. In summary, the conference highlighted the functional implication of epigenetics in chromatin compaction, transcription regulation, replication control and DNA repair. The tight control of all these mechanisms defines the final cellular character.

The Telomere Binding Protein TRF2 Induces Chromatin Compaction

Mammalian telomeres are specialized chromatin structures that require the telomere binding protein, TRF2, for maintaining chromosome stability. In addition to its ability to modulate DNA repair activities, TRF2 also has direct effects on DNA structure and topology. Given that mammalian telomeric chromatin includes nucleosomes, we investigated the effect of this protein on chromatin structure. TRF2 bound to reconstituted telomeric nucleosomal fibers through both its basic N-terminus and its C-terminal DNA binding domain. Analytical agarose gel electrophoresis (AAGE) studies showed that TRF2 promoted the folding of nucleosomal arrays into more compact structures by neutralizing negative surface charge. A construct containing the N-terminal and TRFH domains together altered the charge and radius of nucleosomal arrays similarly to full-length TRF2 suggesting that TRF2-driven changes in global chromatin structure were largely due to these regions. However, the most compact chromatin structures were induced by the isolated basic N-terminal region, as judged by both AAGE and atomic force microscopy. Although the N-terminal region condensed nucleosomal array fibers, the TRFH domain, known to alter DNA topology, was required for stimulation of a strand invasion-like reaction with nucleosomal arrays. Optimal strand invasion also required the C-terminal DNA binding domain. Furthermore, the reaction was not stimulated on linear histone-free DNA. Our data suggest that nucleosomal chromatin has the ability to facilitate this activity of TRF2 which is thought to be involved in stabilizing looped telomere structures.

Zebrafish as a Model System to Study the Physiological Function of Telomeric Protein TPP1

Telomeres are specialized chromatin structures at the end of chromosomes. Telomere dysfunction can lead to chromosomal abnormalities, DNA damage responses, and even cancer. In mammalian cells, a six-protein complex (telosome/shelterin) is assembled on the telomeres through the interactions between various domain structures of the six telomere proteins (POT1, TPP1, TIN2, TRF1, TRF2 and RAP1), and functions in telomere maintenance and protection. Within the telosome, TPP1 interacts directly with POT1 and TIN2 and help to mediate telosome assembly. Mechanisms of telomere regulation have been extensively studied in a variety of model organisms. For example, the physiological roles of telomere-targeted proteins have been assessed in mice through homozygous inactivation. In these cases, early embryonic lethality has prevented further studies of these proteins in embryogenesis and development. As a model system, zebrafish offers unique advantages such as genetic similarities with human, rapid developmental cycles, and ease of manipulation of its embryos. In this report, we detailed the identification of zebrafish homologues of TPP1, POT1, and TIN2, and showed that the domain structures and interactions of these telosome components appeared intact in zebrafish. Importantly, knocking down TPP1 led to multiple abnormalities in zebrafish embryogenesis, including neural death, heart malformation, and caudal defect. And these embryos displayed extensive apoptosis. These results underline the importance of TPP1 in zebrafish embryogenesis, and highlight the feasibility and advantages of investigating the signaling pathways and physiological function of telomere proteins in zebrafish.

CTCF Prevents the Epigenetic Drift of EBV Latency Promoter Qp

The establishment and maintenance of Epstein-Barr Virus (EBV) latent infection requires distinct viral gene expression programs. These gene expression programs, termed latency types, are determined largely by promoter selection, and controlled through the interplay between cell-type specific transcription factors, chromatin structure, and epigenetic modifications. We used a genome-wide chromatin-immunoprecipitation (ChIP) assay to identify epigenetic modifications that correlate with different latency types. We found that the chromatin insulator protein CTCF binds at several key regulatory nodes in the EBV genome and may compartmentalize epigenetic modifications across the viral genome. Highly enriched CTCF binding sites were identified at the promoter regions upstream of Cp, Wp, EBERs, and Qp. Since Qp is essential for long-term maintenance of viral genomes in type I latency and epithelial cell infections, we focused on the role of CTCF in regulating Qp. Purified CTCF bound ∼40 bp upstream of the EBNA1 binding sites located at +10 bp relative to the transcriptional initiation site at Qp. Mutagenesis of the CTCF binding site in EBV bacmids resulted in a decrease in the recovery of stable hygromycin-resistant episomes in 293 cells. EBV lacking the Qp CTCF site showed a decrease in Qp transcription initiation and a corresponding increase in Cp and Fp promoter utilization at 8 weeks post-transfection. However, by 16 weeks post-transfection, bacmids lacking CTCF sites had no detectable Qp transcription and showed high levels of histone H3 K9 methylation and CpG DNA methylation at the Qp initiation site. These findings provide direct genetic evidence that CTCF functions as a chromatin insulator that prevents the promiscuous transcription of surrounding genes and blocks the epigenetic silencing of an essential promoter, Qp, during EBV latent infection.

Chromatin Conformation Signatures: Ideal Human Disease Biomarkers?

Human health is related to information stored in our genetic code, which is highly variable even amongst healthy individuals. Gene expression is orchestrated by numerous control elements that may be located anywhere in the genome, and can regulate distal genes by physically interacting with them. These DNA contacts can be mapped with the chromosome conformation capture and related technologies. Several studies now demonstrate that gene expression patterns are associated with specific chromatin structures, and may therefore correlate with chromatin conformation signatures. Here, we present an overview of genome organization and its relationship with gene expression. We also summarize how chromatin conformation signatures can be identified and discuss why they might represent ideal biomarkers of human disease in such genetically diverse populations.

Daxx is an H3.3-specific histone chaperone and cooperates with ATRX in replication-independent chromatin assembly at telomeres

The histone variant H3.3 is implicated in the formation and maintenance of specialized chromatin structure in metazoan cells. H3.3-containing nucleosomes are assembled in a replication-independent manner by means of dedicated chaperone proteins. We previously identified the death domain associated protein (Daxx) and the alpha-thalassemia X-linked mental retardation protein (ATRX) as H3.3-associated proteins. Here, we report that the highly conserved N terminus of Daxx interacts directly with variant-specific residues in the H3.3 core. Recombinant Daxx assembles H3.3/H4 tetramers on DNA templates, and the ATRX-Daxx complex catalyzes the deposition and remodeling of H3.3-containing nucleosomes. We find that the ATRX-Daxx complex is bound to telomeric chromatin, and that both components of this complex are required for H3.3 deposition at telomeres in murine embryonic stem cells (ESCs). These data demonstrate that Daxx functions as an H3.3-specific chaperone and facilitates the deposition of H3.3 at heterochromatin loci in the context of the ATRX-Daxx complex.