Yuhong Fan

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.

Ring1B Compacts Chromatin Structure and Represses Gene Expression Independent of Histone Ubiquitination

How polycomb group proteins repress gene expression in vivo is not known. While histone-modifying activities of the polycomb repressive complexes (PRCs) have been studied extensively, in vitro data have suggested a direct activity of the PRC1 complex in compacting chromatin. Here, we investigate higher-order chromatin compaction of polycomb targets in vivo. We show that PRCs are required to maintain a compact chromatin state at Hox loci in embryonic stem cells (ESCs). There is specific decompaction in the absence of PRC2 or PRC1. This is due to a PRC1-like complex, since decompaction occurs in Ring1B null cells that still have PRC2-mediated H3K27 methylation. Moreover, we show that the ability of Ring1B to restore a compact chromatin state and to repress Hox gene expression is not dependent on its histone ubiquitination activity. We suggest that Ring1B-mediated chromatin compaction acts to directly limit transcription in vivo.

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.

Involvement of Histone H1.2 in Apoptosis Induced by DNA Double-Strand Breaks

It is poorly understood how apoptotic signals arising from DNA damage are transmitted to mitochondria, which release apoptogenic factors into the cytoplasm that activate downstream destruction programs. Here, we identify histone H1.2 as a cytochrome c-releasing factor that appears in the cytoplasm after exposure to X-ray irradiation. While all nuclear histone H1 forms are released into the cytoplasm in a p53-dependent manner after irradiation, only H1.2, but not other H1 forms, induced cytochrome c release from isolated mitochondria in a Bak-dependent manner. Reducing H1.2 expression enhanced cellular resistance to apoptosis induced by X-ray irradiation or etoposide, but not that induced by other stimuli including TNF-alpha and UV irradiation. H1.2-deficient mice exhibited increased cellular resistance in thymocytes and the small intestine to X-ray-induced apoptosis. These results indicate that histone H1.2 plays an important role in transmitting apoptotic signals from the nucleus to the mitochondria following DNA double-strand breaks.

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.

Global chromatin compaction limits the strength of the DNA damage response

In response to DNA damage, chromatin undergoes a global decondensation process that has been proposed to facilitate genome surveillance. However, the impact that chromatin compaction has on the DNA damage response (DDR) has not directly been tested and thus remains speculative. We apply two independent approaches (one based on murine embryonic stem cells with reduced amounts of the linker histone H1 and the second making use of histone deacetylase inhibitors) to show that the strength of the DDR is amplified in the context of “open” chromatin. H1-depleted cells are hyperresistant to DNA damage and present hypersensitive checkpoints, phenotypes that we show are explained by an increase in the amount of signaling generated at each DNA break. Furthermore, the decrease in H1 leads to a general increase in telomere length, an as of yet unrecognized role for H1 in the regulation of chromosome structure. We propose that slight differences in the epigenetic configuration might account for the cell-to-cell variation in the strength of the DDR observed when groups of cells are challenged with DNA breaks.

Individual Somatic H1 Subtypes Are Dispensable for Mouse Development Even in Mice Lacking the H10 Replacement Subtype

ABSTRACT H1 linker histones are involved in facilitating the folding of chromatin into a 30-nm fiber. Mice contain eight H1 subtypes that differ in amino acid sequence and expression during development. Previous work showed that mice lacking H10, the most divergent subtype, develop normally. Examination of chromatin in H10−/− mice showed that other H1s, especially H1c, H1d, and H1e, compensate for the loss of H10 to maintain a normal H1-to-nucleosome stoichiometry, even in tissues that normally contain abundant amounts of H10 (A. M. Sirotkin et al., Proc. Natl. Acad. Sci. USA 92:6434–6438, 1995). To further investigate the in vivo role of individual mammalian H1s in development, we generated mice lacking H1c, H1d, or H1e by homologous recombination in mouse embryonic stem cells. Mice lacking any one of these H1 subtypes grew and reproduced normally and did not exhibit any obvious phenotype. To determine whether one of these H1s, in particular, was responsible for the compensation present in H10−/− mice, each of the three H1 knockout mouse lines was bred with H10 knockout mice to generate H1c/H10, H1d/H10, or H1e/H10double-knockout mice. Each of these doubly H1-deficient mice also was fertile and exhibited no anatomic or histological abnormalities. Chromatin from the three double-knockout strains showed no significant change in the ratio of total H1 to nucleosomes. These results suggest that any individual H1 subtype is dispensable for mouse development and that loss of even two subtypes is tolerated if a normal H1-to-nucleosome stoichiometry is maintained. Multiple compound H1 knockouts will probably be needed to disrupt the compensation within this multigene family.

Mammalian linker-histone subtypes differentially affect gene expression in vivo

Posttranslational modifications and remodeling of nucleosomes are critical factors in the regulation of transcription. Higher-order folding of chromatin also is likely to contribute to the control of gene expression, but the absence of a detailed description of the structure of the chromatin fiber has impaired progress in this area. Mammalian somatic cells contain a set of H1 linker-histone subtypes, H1 (0) and H1a to H1e, that bind to nucleosome core particles and to the linker DNA between nucleosomes. To determine whether the H1 histone subtypes play differential roles in the regulation of gene expression, we combined mice lacking specific H1 histone subtypes with mice carrying transgenes subject to position effects. Because position effects result from the unique chromatin structure created by the juxtaposition of regulatory elements in the transgene and at the site of integration, transgenes can serve as exquisitely sensitive indicators of chromatin structure. We report that some, but not all, linker histones can attenuate or accentuate position effects. The results suggest that the linker-histone subtypes play differential roles in the control of gene expression and that the sequential arrangement of the linker histones on the chromatin fiber might regulate higher-order chromatin structure and fine-tune expression levels.

CHD8 suppresses p53-mediated apoptosis through histone H1 recruitment during early embryogenesis.

The chromodomain helicase DNA-binding (CHD) family of enzymes is thought to regulate gene expression, but their role in the regulation of specific genes has been unclear. Here we show that CHD8 is expressed at a high level during early embryogenesis and prevents apoptosis mediated by the tumour suppressor protein p53. CHD8 was found to bind to p53 and to suppress its transactivation activity. CHD8 promoted the association of p53 and histone H1, forming a trimeric complex on chromatin that was required for inhibition of p53-dependent transactivation and apoptosis. Depletion of CHD8 or histone H1 resulted in p53 activation and apoptosis. Furthermore, Chd8−/− mice died early during embryogenesis, manifesting widespread apoptosis, whereas deletion of p53 ameliorated this developmental arrest. These observations reveal a mode of p53 regulation mediated by CHD8, which may set a threshold for induction of apoptosis during early embryogenesis by counteracting p53 function through recruitment of histone H1.

H1° histone and differentiation of dendritic cells. A molecular target for tumor‐derived factors

Dendritic cells (DC) play a central role in antitumor immune responses. Abnormal differentiation of DC and their inability to stimulate T cells are important factors in tumor escape from immune‐system control. However, the mechanisms of this process remain elusive. Here, we have described one possible molecular mechanism that involves replacement linker histone H1°. A close association between expression of H1° and DC differentiation in vitro has been found. DC production in H1°‐deficient mice was decreased significantly, whereas generation and function of macrophages, granulocytes, and lymphocytes appear to be normal. However, these mice had a significantly reduced response to vaccination with antigens. Tumor‐derived factors considerably reduced h1° expression in hematopoietic progenitor cells. We have demonstrated that transcription factor NF‐κB is involved actively in regulation of h1°. Thus, H1° histone may be an important factor in normal DC differentiation. Tumor‐derived factors may inhibit DC differentiation by affecting H1° expression.