Formation of facultative heterochromatin in the absence of HP1

Facultative heterochromatin is a cytological manifestation of epigenetic mechanisms that regulate gene expression. Constitutive heterochromatin is marked by distinctive histone H3 methylation and the presence of HP1 proteins, but the chromatin modifications of facultative heterochromatin are less clear. We have examined histone modifications and HP1 in the facultative heterochromatin of nucleated erythrocytes and show that mouse and chicken erythrocytes have different mechanisms of heterochromatin formation. Mouse embryonic erythrocytes have abundant HP1, increased tri-methylation of H3 at K9 and loss of H3 tri-methylation at K27. In contrast, we show that HP1 proteins are lost during the differentiation of chicken erythrocytes, and that H3 tri-methylation at both K9 and K27 is reduced. This coincides with the appearance of the variant linker histone H5. HP1s are also absent from erythrocytes of Xenopus and zebrafish. Our data show that in the same cell lineage there are different mechanisms for forming facultative heterochromatin in vertebrates. To our knowledge, this is the first report of cell types that lack HP1s and that have gross changes in the levels of histone modifications.     

Three dimensional analysis of histone methylation patterns in normal and tumor cell nuclei

Histone modifications represent an important epigenetic mechanism for the organization of higher order chromatin structure and gene regulation. Methylation of position-specific lysine residues in the histone H3 and H4 amino termini has linked with the formation of constitutive and facultative heterochromatin as well as with specifically repressed single gene loci. Using an antibody, directed against dimethylated lysine 9 of histone H3 and several other lysine methylation sites, we visualized the nuclear distribution pattern of chromatin flagged by these methylated lysines in 3D preserved nuclei of normal and malignant cell types. Optical confocal serial sections were used for a quantitative evaluation. We demonstrate distinct differences of these histone methylation patterns among nuclei of different cell types after exit of the cell cycle. Changes in the pattern formation were also observed during the cell cycle. Our data suggest an important role of methylated histones in the reestablishment of higher order chromatin arrangements during telophase/early G1. Cell type specific histone methylation patterns are possibly casually involved in the formation of cell type specific heterochromatin compartments, composed of (peri)centromeric regions and chromosomal subregions from neighboring chromosomes territories, which contain silent genes.     

Histone modifications and nuclear architecture: a review.

Epigenetic modifications, such as acetylation, phosphorylation, methylation, ubiquitination, and ADP ribosylation, of the highly conserved core histones, H2A, H2B, H3, and H4, influence the genetic potential of DNA. The enormous regulatory potential of histone modification is illustrated in the vast array of epigenetic markers found throughout the genome. More than the other types of histone modification, acetylation and methylation of specific lysine residues on N-terminal histone tails are fundamental for the formation of chromatin domains, such as euchromatin, and facultative and constitutive heterochromatin. In addition, the modification of histones can cause a region of chromatin to undergo nuclear compartmentalization and, as such, specific epigenetic markers are non-randomly distributed within interphase nuclei. In this review, we summarize the principles behind epigenetic compartmentalization and the functional consequences of chromatin arrangement within interphase nuclei.     

Chromatin decondensation and nuclear reprogramming by nucleoplasmin

Somatic cell nuclear cloning has repeatedly demonstrated striking reversibility of epigenetic regulation of cell differentiation. Upon injection into eggs, the donor nuclei exhibit global chromatin decondensation, which might contribute to reprogramming the nuclei by derepressing dormant genes. Decondensation of sperm chromatin in eggs is explained by the replacement of sperm-specific histone variants with egg-type histones by the egg protein nucleoplasmin (Npm). However, little is known about the mechanisms of chromatin decondensation in somatic nuclei that do not contain condensation-specific histone variants. Here we found that Npm could widely decondense chromatin in undifferentiated mouse cells without overt histone exchanges but with specific epigenetic modifications that are relevant to open chromatin structure. These modifications included nucleus-wide multiple histone H3 phosphorylation, acetylation of Lys 14 in histone H3, and release of heterochromatin proteins HP1beta and TIF1beta from the nuclei. The protein kinase inhibitor staurosporine inhibited chromatin decondensation and these epigenetic modifications with the exception of H3 acetylation, potentially linking these chromatin events. At the functional level, Npm pretreatment of mouse nuclei facilitated activation of four oocyte-specific genes from the nuclei injected into Xenopus laevis oocytes. Future molecular elucidation of chromatin decondensation by Npm will significantly contribute to our understanding of the plasticity of cell differentiation.     

Interplay between histone deacetylase SIR-2, linker histone H1 and histone methyltransferases in heterochromatin formation

Maintenance of intact heterochromatin structure through epigenetic mechanisms is essential for cell survival. Defects in heterochromatin formation caused by loss of chromatin-modifying enzymes lead to genomic instability and cellular senescence. The NAD+-dependent histone deacetylase SIR-2 and the H1 linker histone are intriguing chromatin elements that are connected to chromatin regulation and cell viability in the single cellular eukaryotic organism yeast. However, it remains an open question how SIR-2 and H1 mediate heterochromatin formation in simple multi-cellular organisms such as C. elegans and in even more complex organisms such as mammals. Recently we have identified SIR-2.1 and the H1 histone subtype, HIS-24 as factors involved in heterochromatin regulation at subtelomeric regions in C. elegans. In addition we show that SIR-2.1, HIS-24, and MES-2, a ortholog to Enhancer of zeste E(Z) are functionally related in heterochromatin formation contributing to fertility and embryogenesis. Here we discuss the interplay between SIR-2, H1 histone and histone methyltransferases in modulation of chromatin structure in further detail.     

Novel nucleosomal particles containing core histones and linker DNA but no histone H1

Eukaryotic chromosomal DNA is assembled into regularly spaced nucleosomes, which play a central role in gene regulation by determining accessibility of control regions. The nucleosome contains ∼147 bp of DNA wrapped ∼1.7 times around a central core histone octamer. The linker histone, H1, binds both to the nucleosome, sealing the DNA coils, and to the linker DNA between nucleosomes, directing chromatin folding. Micrococcal nuclease (MNase) digests the linker to yield the chromatosome, containing H1 and ∼160 bp, and then converts it to a core particle, containing ∼147 bp and no H1. Sequencing of nucleosomal DNA obtained after MNase digestion (MNase-seq) generates genome-wide nucleosome maps that are important for understanding gene regulation. We present an improved MNase-seq method involving simultaneous digestion with exonuclease III, which removes linker DNA. Remarkably, we discovered two novel intermediate particles containing 154 or 161 bp, corresponding to 7 bp protruding from one or both sides of the nucleosome core. These particles are detected in yeast lacking H1 and in H1-depleted mouse chromatin. They can be reconstituted in vitro using purified core histones and DNA. We propose that these ‘proto-chromatosomes’ are fundamental chromatin subunits, which include the H1 binding site and influence nucleosome spacing independently of H1.     

Histone H1

Linker histones of which histone H1 is a representative are a diverse family of architectural proteins within the eukaryotic nucleus. These proteins have a variety of structures, but invariably contain a region enriched in lysine, serine, alanine and profine. All metazoan histone His also include a structured domain that binds to DNA through a helix-turn-helix motif. By binding to the linker DNA flanking the nucleosome core they contribute to the assembly of higher-order chromatin structures. Surprisingly, the use of “knockout” technology to eliminate histone H1 in isolated cells and Xenopus does not prevent the assembly of chromosomes or nuclei, however specific genes are activated or repressed indicative of targeted regulatory functions. A dual role for histone HI in chromatin structure and gene regulation might contribute to epigenetic phenomena in which heritable states of gene activity are maintained through mechanisms independent of gene sequence. This may have important implications for biotechnological and medical research.     

The basic linker of macroH2A stabilizes DNA at the entry/exit site of the nucleosome

MacroH2A is a histone H2A variant that is typically found in heterochromatic regions of the genome. A positively charged linker that connects the histone-fold with the macro-domain was suggested to have DNA-binding properties, and has been shown to promote oligomerization of chromatin fibers. Here we examine the influence of this basic linker on DNA of mononucleosomes. We find that the macro-linker reduces accessibility to extranucleosomal DNA, and appears to increase compaction of the nucleosome. These properties arise from interactions between the H1-like basic linker region and DNA around the entry/exit site, which increases protection of nucleosomal DNA from exonuclease III digestion by ∼10 bp. By stabilizing the wrapping of DNA around the histone core, this basic linker of macroH2A may alter the distribution of nucleosome-associated factors, and potentially contribute to the more compacted nature of heterochromatin.     

Heterochromatin and its Relationship to Cell Senescence and Cancer Therapy

Our goal is to understand the impact of chromatin structure on cell proliferation, cell and tissue aging, cancer and cancer therapies. To this end, we have investigated the formation of specialized domains of facultative heterochromatin, called Senescence Associated Heterochromatin Foci (SAHF), in senescent human cells. A complex of histone chaperones, HIRA and ASF1a, drives formation of SAHF. Remarkably, although SAHF are highly compacted domains of heterochromatin, these domains of facultative heterochromatin largely exclude other domains of chromatin at telomeres and pericentromeres, which are themselves thought to be constitutively heterochromatic. The relationship between SAHF formation and these other domains of heterochromatin is discussed. Also, in the course of our studies, we have obtained evidence that points to a novel function for the widely-studied but poorly-understood family of heterochromatin proteins, HP1 proteins. We propose that HP1 proteins are essential components of a dynamic nuclear response that senses and rectifies defects in epigenetic information, encoded in chromatin through histone modifications and DNA methylation. We further propose that defects in this essential "chromatin repair" response in transformed human cells contributes to the preferential killing of cancer cells by the epigenetic cancer therapies that are currently in clinical development.     

Linker DNA destabilizes condensed chromatin.

The contribution of the linker region to maintenance of condensed chromatin was examined in two model systems, namely sea urchin sperm nuclei and chicken red blood cell nuclei. Linkerless nuclei, prepared by extensive digestion with micrococcal nuclease, were compared with Native nuclei using several assays, including microscopic appearance, nuclear turbidity, salt stability, and trypsin resistance. Chromatin in the Linkerless nuclei was highly condensed, resembling pyknotic chromatin in apoptotic cells. Linkerless nuclei were more stable in low ionic strength buffers and more resistant to trypsin than Native nuclei. Analysis of histones from the trypsinized nuclei by polyacrylamide gel electrophoresis showed that specific histone H1, H2B, and H3 tail regions stabilized linker DNA in condensed nuclei. Thermal denaturation of soluble chromatin preparations from differentially trypsinized sperm nuclei demonstrated that the N-terminal regions of histones Sp H1, Sp H2B, and H3 bind tightly to linker DNA, causing it to denature at a high temperature. We conclude that linker DNA exerts a disruptive force on condensed chromatin structure which is counteracted by binding of specific histone tail regions to the linker DNA. The inherent instability of the linker region may be significant in all eukaryotic chromatins and may promote gene activation in living cells.     

Mouse oocytes and early embryos express multiple histone H1 subtypes

Oocytes and embryos of many species, including mammals, contain a unique linker (H1) histone, termed H1oo in mammals. It is uncertain, however, whether other H1 histones also contribute to the linker histone complement of these cells. Using immunofluorescence and radiolabeling, we have examined whether histone H10, which frequently accumulates in the chromatin of nondividing cells, and the somatic subtypes of H1 are present in mouse oocytes and early embryos. We report that oocytes and embryos contain mRNA encoding H10. A polymerase chain reaction-based test indicated that the poly(A) tail did not lengthen during meiotic maturation, although it did so beginning at the four-cell stage. Antibodies raised against histone H10 stained the nucleus of wild-type prophase-arrested oocytes but not of mice lacking the H10 gene. Following fertilization, H10 was detected in the nuclei of two-cell embryos and less strongly at the four-cell stage. No signal was detected in H10 -/- embryos. Radiolabeling revealed that species comigrating with the somatic H1 subtypes H1a and H1c were synthesized in maturing oocytes and in one- and two-cell embryos. Beginning at the four-cell stage in both wild-type and H10 -/- embryos, species comigrating with subtypes H1b, H1d, and H1e were additionally synthesized. These results establish that histone H10 constitutes a portion of the linker histone complement in oocytes and early embryos and that changes in the pattern of somatic H1 synthesis occur during early embryonic development. Taken together with previous results, these findings suggest that multiple H1 subtypes are present on oocyte chromatin and that following fertilization changes in the histone H1 complement accompany the establishment of regulated embryonic gene expression.     

RNAi-directed assembly of heterochromatin in fission yeast

Heterochromatin is an epigenetically heritable and conserved feature of eukaryotic chromosomes with important roles in chromosome segregation, genome stability, and gene regulation. The formation of heterochromatin involves an ordered array of chromatin changes, including histone deacetylation, histone H3-lysine 9 methylation, and recruitment of histone binding proteins such as Swi6/HP1. Recent discoveries have uncovered a role for the RNA interference (RNAi) pathway in heterochromatin assembly in the fission yeast Schizosaccharomyces pombe and other eukaryotes. Purification of two RNAi complexes, RITS and RDRC, from fission yeast has provided further insight into the mechanism of RNAi-mediated heterochromatin assembly. These discoveries have given rise to a model in which small interfering RNA molecules act as specificity factors that initiate epigenetic chromatin modifications and double strand RNA synthesis at specific chromosome regions.