Nuclear mobility and mitotic chromosome binding: similarities between pioneer transcription factor FoxA and linker histone H1.
ABSTRACT There exists a hierarchy by which transcription factors can engage their target sites in chromatin, in that a subset of factors can bind transcriptionally silent, nucleosomal DNA, whereas most factors cannot, and this hierarchy is reflected, at least in part, in the developmental function of the factors. For example, transcription factors possessing the Forkhead box (Fox) DNA-binding domain contain an overall fold resembling that of linker histone and thus are structured to bind DNA, site specifically, in a nucleosomal context. Where tested, Fox factors bind early in the developmental or physiological activation of target genes, thereby enabling the binding of other factors that cannot engage chromatin on their own. To investigate the basis for early chromatin binding, we have used fluorescence recovery after photobleaching (FRAP) to analyze the mobility, in the live cell nucleus, of FoxA factors in comparison to linker histone and other transcription factors. We have further analyzed the factors for their ability to bind to chromatin in mitosis and thereby serve as epigenetic marks. The results indicate that the "pioneer" features of FoxA factors involve various chromatin-binding parameters seen in linker histones and that distinguish the factors with respect to their regulatory and mechanistic functions.
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ABSTRACT: Nucleosomes are present throughout the genome and must be dynamically regulated to accommodate binding of transcription factors and RNA polymerase machineries by various mechanisms. Despite the development of protocols and techniques that have enabled us to map nucleosome occupancy genome-wide, the dynamic properties of nucleosomes remain poorly understood, particularly in mammalian cells. The histone variant H3.3 is incorporated into chromatin independently of DNA replication and requires displacement of existing nucleosomes for its deposition. Here, we measure H3.3 turnover at high resolution in the mammalian genome in order to present a genome-wide characterization of replication-independent H3.3-nucleosome dynamics. We developed a system to study the DNA replication-independent turnover of nucleosomes containing the histone variant H3.3 in mammalian cells. By measuring the genome-wide incorporation of H3.3 at different time points following epitope-tagged H3.3 expression, we find three categories of H3.3-nucleosome turnover in vivo: rapid turnover, intermediate turnover and, specifically at telomeres, slow turnover. Our data indicate that H3.3-containing nucleosomes at enhancers and promoters undergo rapid turnover that is associated with active histone modification marks including H3K4me1, H3K4me3, H3K9ac, H3K27ac and the histone variant H2A.Z. The rate of turnover is negatively correlated with H3K27me3 at regulatory regions and with H3K36me3 at gene bodies. We have established a reliable approach to measure turnover rates of H3.3-containing nucleosomes on a genome-wide level in mammalian cells. Our results suggest that distinct mechanisms control the dynamics of H3.3 incorporation at functionally different genomic regions.Genome biology 10/2013; 14(10):R121. DOI:10.1186/gb-2013-14-10-r121 · 10.47 Impact Factor
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ABSTRACT: The histone variant H3.3 plays a critical role in maintaining the pluripotency of embryonic stem cells (ESCs) by regulating gene expression programs important for lineage specification. H3.3 is deposited by various chaperones at regulatory sites, gene bodies, and certain heterochromatic sites such as telomeres and centromeres. Using Tet-inhibited expression of epitope-tagged H3.3 combined with ChIP-Seq we undertook genome-wide measurements of H3.3 dissociation rates across the ESC genome and examined the relationship between H3.3-nucleosome turnover and ESC-specific transcription factors, chromatin modifiers, and epigenetic marks. Our comprehensive analysis of H3.3 dissociation rates revealed distinct H3.3 dissociation dynamics at various functional chromatin domains. At transcription start sites, H3.3 dissociates rapidly with the highest rate at nucleosome-depleted regions (NDRs) just upstream of Pol II binding, followed by low H3.3 dissociation rates across gene bodies. H3.3 turnover at transcription start sites, gene bodies, and transcription end sites was positively correlated with transcriptional activity. H3.3 is found decorated with various histone modifications that regulate transcription and maintain chromatin integrity. We find greatly varying H3.3 dissociation rates across various histone modification domains: high dissociation rates at active histone marks and low dissociation rates at heterochromatic marks. Well- defined zones of high H3.3-nucleosome turnover were detected at binding sites of ESC-specific pluripotency factors and chromatin remodelers, suggesting an important role for H3.3 in facilitating protein binding. Among transcription factor binding sites we detected higher H3.3 turnover at distal cis-acting sites compared to proximal genic transcription factor binding sites. Our results imply that fast H3.3 dissociation is a hallmark of interactions between DNA and transcriptional regulators. Our study demonstrates that H3.3 turnover and nucleosome stability vary greatly across the chromatin landscape of embryonic stem cells. The presence of high H3.3 turnover at RNA Pol II binding sites at extragenic regions as well as at transcription start and end sites of genes, suggests a specific role for H3.3 in transcriptional initiation and termination. On the other hand, the presence of well-defined zones of high H3.3 dissociation at transcription factor and chromatin remodeler binding sites point to a broader role in facilitating accessibility.Epigenetics & Chromatin 12/2014; 7(1):38. DOI:10.1186/1756-8935-7-38 · 4.46 Impact Factor
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ABSTRACT: Compaction of the eukaryotic genome into the confined space of the cell nucleus must occur faithfully throughout each cell cycle to retain gene expression fidelity. For decades, experimental limitations to study the structural organization of the interphase nucleus restricted our understanding of its contributions towards gene regulation and disease. However, within the past few years, our capability to visualize chromosomes in vivo with sophisticated fluorescence microscopy, and to characterize chromosomal regulatory environments via massively-parallel sequencing methodologies have drastically changed how we currently understand epigenetic gene control within the context of three-dimensional nuclear structure. The rapid rate at which information on nuclear structure is unfolding brings challenges to compare and contrast recent observations with historic findings. In this review, we discuss experimental breakthroughs that have influenced how we understand and explore the dynamic structure and function of the nucleus, and how we can incorporate historical perspectives with insights acquired from the ever-evolving advances in molecular biology and pathology. J. Cell. Physiol. © 2013 Wiley Periodicals, Inc.Journal of Cellular Physiology 06/2014; 229(6). DOI:10.1002/jcp.24508 · 3.87 Impact Factor