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Christopher L Woodcock

Publications and source records attributed to Christopher L Woodcock.

13 recordsLinked to original sources
PubMed2006

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

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.

Chromatin↗
PubMed2006

Chromatin architecture.

A complete understanding of the structure-function relationships of chromatin requires extending primarily one dimensional information, obtained from molecular genetic techniques and based on the underlying linear DNA sequence, to the three dimensional conformation. Recent progress in this endeavor has included the examination of fully defined nucleosomes and nucleosomal arrays assembled in vitro using X-ray diffraction, NMR spectroscopy, electron microscopy and atomic force microscopy. These studies have provided valuable insights into the structural roles of histone variants, the impact of histone mutations and the compaction of nucleosomal arrays. In addition, the diverse structural consequences of the binding of specific chromatin 'architectural' proteins are becoming apparent. These approaches provide an essential basis for understanding the conformation of the 'epigenome'.

Animals↗
PubMed2006

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.

Animals↗
PubMed2005

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.

Animals↗
PubMed2005

Organization of interphase chromatin.

The organization of interphase chromatin spans many topics, ranging in scale from the molecular level to the whole nucleus, and its study requires a concomitant range of experimental approaches. In this review, we examine these approaches, the results they have generated, and the interfaces between them. The greatest challenge appears to be the integration of information on whole nuclei obtained by light microscopy with data on nucleosome-nucleosome interactions and chromatin higher-order structures, obtained in vitro using biophysical characterization, atomic force microscopy, and electron microscopy. We consider strategies that may assist in the integration process, and we review emerging technologies that promise to reduce the "resolution gap."

Animals↗
PubMed2004

Dynamic relocation of epigenetic chromatin markers reveals an active role of constitutive heterochromatin in the transition from proliferation to quiescence.

Quiescent lymphocytes have small nuclei, filled with masses of facultative heterochromatin. Upon receiving mitogenic signals, these cells undergo nuclear enlargement, chromatin decondensation, the reactivation of cell proliferation, and changes in the intranuclear positioning of key genes. We examined the levels and intranuclear localization of major histone modifications and non-histone heterochromatin proteins in quiescent and reactivated mouse spleen lymphocytes. Dramatic and selective changes in localization of two heterochromatin-associated proteins, the histone variant macroH2A and HP1alpha occurred during lymphocyte reactivation. Reciprocal changes in the locations of these two proteins were observed in activated lymphocytes and cultured mouse fibroblasts induced into quiescence. We also describe a new apocentric nuclear compartment with a unique set of histone modifications that occurs as a zone of chromatin surrounding centromeric heterochromatin in differentiated lymphocytes. It is within this zone that the most significant changes occur in the transition from proliferation to quiescence. Our results suggest that constitutive centromeric heterochromatin plays an active role in cell differentiation and reactivation.

3T3 Cells↗
PubMed2004

Chromatin compaction by a polycomb group protein complex.

Polycomb group proteins preserve body patterning through development by maintaining transcriptional silencing of homeotic genes. A long-standing hypothesis is that silencing involves creating chromatin structure that is repressive to gene transcription. We demonstrate by electron microscopy that core components of Polycomb Repressive Complex 1 induce compaction of defined nucleosomal arrays. Compaction by Polycomb proteins requires nucleosomes but not histone tails. Each Polycomb complex can compact about three nucleosomes. A region of Posterior Sex Combs that is important for gene silencing in vivo is also important for chromatin compaction, linking the two activities. This mechanism of chromatin compaction might be central to stable gene silencing by the Polycomb group.

Chromatin↗
PubMed2004

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.

Animals↗
PubMed2003

Chromatin compaction by human MeCP2. Assembly of novel secondary chromatin structures in the absence of DNA methylation.

MeCP2 is a transcriptional repressor that contains an N-terminal methylated DNA-binding domain, a central transcription regulation domain, and a C-terminal domain of unknown function. Whereas current models of MeCP2 function evoke localized recruitment of histone deacetylases to specific methylated regions of the genome, it is unclear whether MeCP2 requires DNA methylation to bind to chromatin or whether MeCP2 binding influences chromatin structure in the absence of other proteins. To address these issues, we have characterized the complexes formed between MeCP2 and biochemically defined nucleosomal arrays. At molar ratios near 1 MeCP2/nucleosome, unmethylated nucleosomal arrays formed both extensively condensed ellipsoidal particles and oligomeric suprastructures. Furthermore, MeCP2-mediated chromatin compaction occurred in the absence of monovalent or divalent cations, in distinct contrast to all other known chromatin-condensing proteins. Analysis of specific missense and nonsense MeCP2 mutants indicated that the ability to condense chromatin resides in region(s) of the protein other than the methylated DNA-binding domain. These data demonstrate that MeCP2 assembles novel secondary chromatin structures independent of DNA modification and suggest that the ability of MeCP2 to silence chromatin may be related in part to its effects on large-scale chromatin organization.

Chromatin↗
PubMed2003

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.

Adenosine Triphosphatases↗
PubMed2003

H1 linker histones are essential for mouse development and affect nucleosome spacing in vivo.

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.

Alleles↗