High nucleosome occupancy is encoded at X-linked gene promoters in C. elegans.
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ABSTRACT:
SUBMITTER: Ercan S
PROVIDER: S-EPMC3032927 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The packaging of DNA into nucleosomes influences the accessibility of underlying regulatory information. Nucleosome occupancy and positioning are best characterized in the budding yeast Saccharomyces cerevisiae, albeit in asynchronous cell populations or on individual promoters such as PHO5 and GAL1-10. Using FAIRE (formaldehyde-assisted isolation of regulatory elements) and whole-genome microarrays, we examined changes in nucleosome occupancy throughout the mitotic cell cycle in synchronized populations of S. cerevisiae. Perhaps surprisingly, nucleosome occupancy did not exhibit large, global variation between cell cycle phases. However, nucleosome occupancy at the promoters of cell cycle-regulated genes was reduced specifically at the cell cycle phase in which that gene exhibited peak expression, with the notable exception of S-phase genes. We present data that establish FAIRE as a high-throughput method for assaying nucleosome occupancy. For the first time in any system, nucleosome occupancy was mapped genome-wide throughout the cell cycle. Fluctuation of nucleosome occupancy at promoters of most cell cycle-regulated genes provides independent evidence that periodic expression of these genes is controlled mainly at the level of transcription. The promoters of G2/M genes are distinguished from other cell cycle promoters by an unusually low baseline nucleosome occupancy throughout the cell cycle. This observation, coupled with the maintenance throughout the cell cycle of the stereotypic nucleosome occupancy states between coding and non-coding loci, suggests that the largest component of variation in nucleosome occupancy is "hard wired," perhaps at the level of DNA sequence.
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Project description:The human transcription factor TP53 is a pivotal roadblock against cancer. A key unresolved question is how the p53 protein selects its genomic binding sites in vivo out of a large pool of potential consensus sites. We hypothesized that chromatin may play a significant role in this site-selection process. To test this, we used a custom DNA microarray to measure p53 binding at approximately 2000 sites predicted to possess high-sequence specificity, and identified both strongly bound and weakly bound sites. When placed within a plasmid, weakly bound sites become p53 responsive and regain p53 binding when stably integrated into random genomic locations. Notably, strongly bound sites reside preferentially within genomic regions whose DNA sequence is predicted to encode relatively high intrinsic nucleosome occupancy. Using in vivo nucleosome occupancy measurements under conditions where p53 is inactive, we experimentally confirmed this prediction. Furthermore, upon p53 activation, nucleosomes are partially displaced from a relatively broad region surrounding the bound p53 sites, and this displacement is rapidly reversed upon inactivation of p53. Thus, in contrast to the general assumption that transcription-factor binding is preferred in sites that have low nucleosome occupancy prior to factor activation, we find that p53 binding occurs preferentially within a chromatin context of high intrinsic nucleosome occupancy.
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Project description:Nucleosomes can block access to transcription factors. Thus the precise localization of nucleosomes relative to transcription start sites and other factor binding sites is expected to be a critical component of transcriptional regulation. Recently developed microarray approaches have allowed the rapid mapping of nucleosome positions over hundreds of kilobases (kb) of human genomic DNA, although these approaches have not yet been widely used to measure chromatin changes associated with changes in transcription. Here, we use custom tiling microarrays to reveal changes in nucleosome positions and abundance that occur when hormone-bound glucocorticoid receptor (GR) binds to sites near target gene promoters in human osteosarcoma cells. The most striking change is an increase in measured nucleosome occupancy at sites spanning ∼1 kb upstream and downstream of transcription start sites, which occurs one hour after addition of hormone, but is lost at 4 hours. Unexpectedly, this increase was seen both on GR-regulated and GR-non-regulated genes. In addition, the human SWI/SNF chromatin remodeling factor (a GR co-activator) was found to be important for increased occupancy upon hormone treatment and also for low nucleosome occupancy without hormone. Most surprisingly, similar increases in nucleosome occupancy were also seen on both regulated and non-regulated promoters during differentiation of human myeloid leukemia cells and upon activation of human CD4+ T-cells. These results indicate that dramatic changes in chromatin structure over ∼2 kb of human promoters may occur genomewide and in response to a variety of stimuli, and suggest novel models for transcriptional regulation.
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Project description:Precise nucleosome-positioning patterns at promoters are thought to be crucial for faithful transcriptional regulation. However, the mechanisms by which these patterns are established, are dynamically maintained, and subsequently contribute to transcriptional control are poorly understood. The switch/sucrose non-fermentable chromatin remodeling complex, also known as the Brg1 associated factors complex, is a master developmental regulator and tumor suppressor capable of mobilizing nucleosomes in biochemical assays. However, its role in establishing the nucleosome landscape in vivo is unclear. Here we have inactivated Snf5 and Brg1, core subunits of the mammalian Swi/Snf complex, to evaluate their effects on chromatin structure and transcription levels genomewide. We find that inactivation of either subunit leads to disruptions of specific nucleosome patterning combined with a loss of overall nucleosome occupancy at a large number of promoters, regardless of their association with CpG islands. These rearrangements are accompanied by gene expression changes that promote cell proliferation. Collectively, these findings define a direct relationship between chromatin-remodeling complexes, chromatin structure, and transcriptional regulation.
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Project description:We have characterized two post-translational histone modifications in Caenorhabditis elegans on a genomic scale. Micrococcal nuclease digestion and immunoprecipitation were used to obtain distinct populations of single nucleosome cores, which were analyzed using massively parallel DNA sequencing to obtain positional and coverage maps. Two methylated histone H3 populations were chosen for comparison: H3K4 histone methylation (associated with active chromosomal regions) and H3K9 histone methylation (associated with inactivity). From analysis of the sequence data, we found nucleosome cores with these modifications to be enriched in two distinct partitions of the genome; H3K4 methylation was particularly prevalent in promoter regions of widely expressed genes, while H3K9 methylation was enriched on specific chromosomal arms. For each of the six chromosomes, the highest level of H3K9 methylation corresponds to the pairing center responsible for chromosome alignment during meiosis. Enrichment of H3K9 methylation at pairing centers appears to be an early mark in meiotic chromosome sorting, occurring in the absence of components required for proper pairing of homologous chromosomes. H3K9 methylation shows an intricate pattern within the chromosome arms with a particular anticorrelation to regions that display a strong approximately 10.5 bp periodicity of AA/TT dinucleotides that is known to associate with germline transcription. By contrast to the global features observed with H3K9 methylation, H3K4 methylation profiles were most striking in their local characteristics around promoters, providing a unique promoter-central landmark for 3,903 C. elegans genes and allowing a precise analysis of nucleosome positioning in the context of transcriptional initiation.
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Project description:Spt6 is a transcriptional elongation factor and histone chaperone that reassembles transcribed chromatin. Genome-wide H3 mapping showed that Spt6 preferentially maintains nucleosomes within the first 500 bases of genes and helps define nucleosome-depleted regions in 5' and 3' flanking sequences. In Spt6-depleted cells, H3 loss at 5' ends correlates with reduced pol II density suggesting enhanced transcription elongation. Consistent with its 'Suppressor of Ty' (Spt) phenotype, Spt6 inactivation caused localized H3 eviction over 1-2 nucleosomes at 5' ends of Ty elements. H3 displacement differed between genes driven by promoters with 'open'/DPN and 'closed'/OPN chromatin conformations with similar pol II densities. More eviction occurred on genes with 'closed' promoters, associated with 'noisy' transcription. Moreover, swapping of 'open' and 'closed' promoters showed that they can specify distinct downstream patterns of histone eviction/deposition. These observations suggest a novel function for promoters in dictating histone dynamics within genes possibly through effects on transcriptional bursting or elongation rate.
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Project description:Chromatin structure plays a key role in regulating gene expression and embryonic differentiation; however, the factors that determine the organization of chromatin around regulatory sites are not fully known. Here we show that HMGN1, a nucleosome-binding protein ubiquitously expressed in vertebrate cells, preferentially binds to CpG island-containing promoters and affects the organization of nucleosomes, DNase I hypersensitivity, and the transcriptional profile of mouse embryonic stem cells and neural progenitors. Loss of HMGN1 alters the organization of an unstable nucleosome at transcription start sites, reduces the number of DNase I-hypersensitive sites genome wide, and decreases the number of nestin-positive neural progenitors in the subventricular zone (SVZ) region of mouse brain. Thus, architectural chromatin-binding proteins affect the transcription profile and chromatin structure during embryonic stem cell differentiation.
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Project description:In eukaryotic organisms, packaging of DNA into nucleosomes controls gene expression by regulating access of the promoter to transcription factors. The human malaria parasite Plasmodium falciparum encodes relatively few transcription factors, while extensive nucleosome remodeling occurs during its replicative cycle in red blood cells. These observations point towards an important role of the nucleosome landscape in regulating gene expression. However, the relation between nucleosome positioning and transcriptional activity has thus far not been explored in detail in the parasite.Here, we analyzed nucleosome positioning in the asexual and sexual stages of the parasite's erythrocytic cycle using chromatin immunoprecipitation of MNase-digested chromatin, followed by next-generation sequencing. We observed a relatively open chromatin structure at the trophozoite and gametocyte stages, consistent with high levels of transcriptional activity in these stages. Nucleosome occupancy of genes and promoter regions were subsequently compared to steady-state mRNA expression levels. Transcript abundance showed a strong inverse correlation with nucleosome occupancy levels in promoter regions. In addition, AT-repeat sequences were strongly unfavorable for nucleosome binding in P. falciparum, and were overrepresented in promoters of highly expressed genes.The connection between chromatin structure and gene expression in P. falciparum shares similarities with other eukaryotes. However, the remarkable nucleosome dynamics during the erythrocytic stages and the absence of a large variety of transcription factors may indicate that nucleosome binding and remodeling are critical regulators of transcript levels. Moreover, the strong dependency between chromatin structure and DNA sequence suggests that the P. falciparum genome may have been shaped by nucleosome binding preferences. Nucleosome remodeling mechanisms in this deadly parasite could thus provide potent novel anti-malarial targets.
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Project description:The response to stressful stimuli requires rapid, precise, and dynamic gene expression changes that must be coordinated across the genome. To gain insight into the temporal ordering of genome reorganization, we investigated dynamic relationships between changing nucleosome occupancy, transcription factor binding, and gene expression in Saccharomyces cerevisiae yeast responding to oxidative stress. We applied deep sequencing to nucleosomal DNA at six time points before and after hydrogen peroxide treatment and revealed many distinct dynamic patterns of nucleosome gain and loss. The timing of nucleosome repositioning was not predictive of the dynamics of downstream gene expression change but instead was linked to nucleosome position relative to transcription start sites and specific cis-regulatory elements. We measured genome-wide binding of the stress-activated transcription factor Msn2p over time and found that Msn2p binds different loci with different dynamics. Nucleosome eviction from Msn2p binding sites was common across the genome; however, we show that, contrary to expectation, nucleosome loss occurred after Msn2p binding and in fact required Msn2p. This negates the prevailing model that nucleosomes obscuring Msn2p sites regulate DNA access and must be lost before Msn2p can bind DNA. Together, these results highlight the complexities of stress-dependent chromatin changes and their effects on gene expression.
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Project description:MAGEA11 is a cancer germline (CG) antigen and androgen receptor co-activator. Its expression in cancers other than prostate, and its mechanism of activation, has not been reported. In silico analyses reveal that MAGEA11 is frequently expressed in human cancers, is increased during tumor progression, and correlates with poor prognosis and survival. In prostate and epithelial ovarian cancers (EOC), MAGEA11 expression was associated with promoter and global DNA hypomethylation, and with activation of other CG genes. Pharmacological or genetic inhibition of DNA methyltransferases (DNMTs) and/or histone deacetylases (HDACs) activated MAGEA11 in a cell line specific manner. MAGEA11 promoter activity was directly repressed by DNA methylation, and partially depended on Sp1, as pharmacological or genetic targeting of Sp1 reduced MAGEA11 promoter activity and endogenous gene expression. Importantly, DNA methylation regulated nucleosome occupancy specifically at the -1 positioned nucleosome of MAGEA11. Methylation of a single Ets site near the transcriptional start site (TSS) correlated with -1 nucleosome occupancy and, by itself, strongly repressed MAGEA11 promoter activity. Thus, DNA methylation regulates nucleosome occupancy at MAGEA11, and this appears to function cooperatively with sequence-specific transcription factors to regulate gene expression. MAGEA11 regulation is highly instructive for understanding mechanisms regulating CG antigen genes in human cancer.
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