Christopher L. Woodcock

Ezh1 and Ezh2 maintain repressive chromatin through different mechanisms

Polycomb group proteins are critical to maintaining gene repression established during Drosophila development. Part of this group forms the PRC2 complex containing Ez that catalyzes di- and trimethylation of histone H3 lysine 27 (H3K37me2/3), marks repressive to transcription. We report that the mammalian homologs Ezh1 and Ezh2 form similar PRC2 complexes but exhibit contrasting repressive roles. While PRC2-Ezh2 catalyzes H3K27me2/3 and its knockdown affects global H3K27me2/3 levels, PRC2-Ezh1 performs this function weakly. In accordance, Ezh1 knockdown was ineffectual on global H3K27me2/3 levels. Instead, PRC2-Ezh1 directly and robustly represses transcription from chromatinized templates and compacts chromatin in the absence of the methyltransferase cofactor SAM, as evidenced by electron microscopy. Ezh1 targets a subset of Ezh2 genes, yet Ezh1 is more abundant in nonproliferative adult organs while Ezh2 expression is tightly associated with proliferation, as evidenced when analyzing aging mouse kidney. These results might reflect subfunctionalization of a PcG protein during evolution.

Histone H1 Depletion in Mammals Alters Global Chromatin Structure but Causes Specific Changes in Gene Regulation

Linker histone H1 plays an important role in chromatin folding in vitro. To study the role of H1 in vivo, mouse embryonic stem cells null for three H1 genes were derived and were found to have 50% of the normal level of H1. H1 depletion caused dramatic chromatin structure changes, including decreased global nucleosome spacing, reduced local chromatin compaction, and decreases in certain core histone modifications. Surprisingly, however, microarray analysis revealed that expression of only a small number of genes is affected. Many of the affected genes are imprinted or are on the X chromosome and are therefore normally regulated by DNA methylation. Although global DNA methylation is not changed, methylation of specific CpGs within the regulatory regions of some of the H1 regulated genes is reduced. These results indicate that linker histones can participate in epigenetic regulation of gene expression by contributing to the maintenance or establishment of specific DNA methylation patterns.

Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length

Despite a great deal of attention over many years, the structural and functional roles of the linker histone H1 remain enigmatic. The earlier concepts of H1 as a general transcriptional inhibitor have had to be reconsidered in the light of experiments demonstrating a minor effect of H1 deletion in unicellular organisms. More recent work analysing the results of depleting H1 in mammals through genetic knockouts of selected H1 subtypes in the mouse has shown that cells and tissues can tolerate a surprisingly low H1 content. One common feature of H1-depleted nuclei is a reduction in nucleosome repeat length (NRL). Moreover, there is a robust linear relationship between H1 stoichiometry and NRL, suggesting an inherent homeostatic mechanism that maintains intranuclear electrostatic balance. It is also clear that the 1 H1 per nucleosome paradigm for higher eukaryotes is the exception rather than the rule. This, together with the high mobility of H1 within the nucleus, prompts a reappraisal of the role of linker histone as an obligatory chromatin architectural protein.

H1 Linker Histones Are Essential for Mouse Development and Affect Nucleosome Spacing In Vivo

ABSTRACT Most eukaryotic cells contain nearly equimolar amounts of nucleosomes and H1 linker histones. Despite their abundance and the potential functional specialization of H1 subtypes in multicellular organisms, gene inactivation studies have failed to reveal essential functions for linker histones in vivo. Moreover, in vitro studies suggest that H1 subtypes may not be absolutely required for assembly of chromosomes or nuclei. By sequentially inactivating the genes for three mouse H1 subtypes (H1c, H1d, and H1e), we showed that linker histones are essential for mammalian development. Embryos lacking the three H1 subtypes die by mid-gestation with a broad range of defects. Triple-H1-null embryos have about 50% of the normal ratio of H1 to nucleosomes. Mice null for five of these six H1 alleles are viable but are underrepresented in litters and are much smaller than their littermates. Marked reductions in H1 content were found in certain tissues of these mice and in another compound H1 mutant. These results demonstrate that the total amount of H1 is crucial for proper embryonic development. Extensive reduction of H1 in certain tissues did not lead to changes in nuclear size, but it did result in global shortening of the spacing between nucleosomes.

A Histone-Fold Complex and FANCM Form a Conserved DNA-Remodeling Complex to Maintain Genome Stability

FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.

Structural repeating units in chromatin. I. Evidence for their general occurrence.

Abstract When chromatin from a diverse range of eukaryotes is prepared for the electron microscope by aldehyde fixation followed by centrifugation onto the grid, the fibers are composed of interconnected spherical particles, about 70 A in diameter. Evidence is presented which suggests that the particles are an in vivo structural repeating unit of deoxyribonucleoprotein, and not a preparation-induced artefact.

Evidence for heteromorphic chromatin fibers from analysis of nucleosome interactions

The architecture of the chromatin fiber, which determines DNA accessibility for transcription and other template-directed biological processes, remains unknown. Here we investigate the internal organization of the 30-nm chromatin fiber, combining Monte Carlo simulations of nucleosome chain folding with EM-assisted nucleosome interaction capture (EMANIC). We show that at physiological concentrations of monovalent ions, linker histones lead to a tight 2-start zigzag dominated by interactions between alternate nucleosomes (i ± 2) and sealed by histone N-tails. Divalent ions further compact the fiber by promoting bending in some linker DNAs and hence raising sequential nucleosome interactions (i ± 1). Remarkably, both straight and bent linker DNA conformations are retained in the fully compact chromatin fiber as inferred from both EMANIC and modeling. This conformational variability is energetically favorable as it helps accommodate DNA crossings within the fiber axis. Our results thus show that the 2-start zigzag topology and the type of linker DNA bending that defines solenoid models may be simultaneously present in a structurally heteromorphic chromatin fiber with uniform 30 nm diameter. Our data also suggest that dynamic linker DNA bending by linker histones and divalent cations in vivo may mediate the transition between tight nucleosome packing within discrete 30-nm fibers and self-associated higher-order chromosomal forms.

Structural analysis of the yeast SWI/SNF chromatin remodeling complex

Elucidating the mechanism of ATP-dependent chromatin remodeling is one of the largest challenges in the field of gene regulation. One of the missing pieces in understanding this process is detailed structural information on the enzymes that catalyze the remodeling reactions. Here we use a combination of subunit radio-iodination and scanning transmission electron microscopy to determine the subunit stoichiometry and native molecular weight of the yeast SWI/SNF complex. We also report a three-dimensional reconstruction of yeast SWI/SNF derived from electron micrographs.

Multiple modes of interaction between the methylated DNA binding protein MeCP2 and chromatin.

ABSTRACT Mutations of the methylated DNA binding protein MeCP2, a multifunctional protein that is thought to transmit epigenetic information encoded as methylated CpG dinucleotides to the transcriptional machinery, give rise to the debilitating neurodevelopmental disease Rett syndrome (RTT). In this in vitro study, the methylation-dependent and -independent interactions of wild-type and mutant human MeCP2 with defined DNA and chromatin substrates were investigated. A combination of electrophoretic mobility shift assays and visualization by electron microscopy made it possible to understand the different conformational changes underlying the gel shifts. MeCP2 is shown to have, in addition to its well-established methylated DNA binding domain, a methylation-independent DNA binding site (or sites) in the first 294 residues, while the C-terminal portion of MeCP2 (residues 295 to 486) contains one or more essential chromatin interaction regions. All of the RTT-inducing mutants tested were quantitatively bound to chromatin under our conditions, but those that tend to be associated with the more severe RTT symptoms failed to induce the extensive compaction observed with wild-type MeCP2. Two modes of MeCP2-driven compaction were observed, one promoting nucleosome clustering and the other forming DNA-MeCP2-DNA complexes. MeCP2 binding to DNA and chromatin involves a number of different molecular interactions, some of which result in compaction and oligomerization. The multifunctional roles of MeCP2 may be reflected in these different interactions.

Closed chromatin loops at the ends of chromosomes

The termini of eukaryotic chromosomes contain specialized protective structures, the telomeres, composed of TTAGGG repeats and associated proteins which, together with telomerase, control telomere length. Telomere shortening is associated with senescence and inappropriate telomerase activity may lead to cancer. Little is known about the chromatin context of telomeres, because, in most cells, telomere chromatin is tightly anchored within the nucleus. We now report the successful release of telomere chromatin from chicken erythrocyte and mouse lymphocyte nuclei, both of which have a reduced karyoskeleton. Electron microscopy reveals telomere chromatin fibers in the form of closed terminal loops, which correspond to the “t-loop” structures adopted by telomere DNA. The ability to recognize isolated telomeres in their native chromatin conformation opens the way for detailed structural and compositional studies.