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

SummaryLinker 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

BEN domain protein Elba2 can functionally substitute for linker histone H1 in Drosophila in vivo

Metazoan linker histones are essential for development and play crucial roles in organization of chromatin, modification of epigenetic states and regulation of genetic activity. Vertebrates express multiple linker histone H1 isoforms, which may function redundantly. In contrast, H1 isoforms are not present in Dipterans, including D. melanogaster, except for an embryo-specific, distantly related dBigH1. Here we show that Drosophila BEN domain protein Elba2, which is expressed in early embryos and was hypothesized to have insulator-specific functions, can compensate for the loss of H1 in vivo. Although the Elba2 gene is not essential, its mutation causes a disruption of normal internucleosomal spacing of chromatin and reduced nuclear compaction in syncytial embryos. Elba2 protein is distributed ubiquitously in polytene chromosomes and strongly colocalizes with H1. In H1-depleted animals, ectopic expression of Elba2 rescues the increased lethality and ameliorates abnormalities of chromosome architecture and heterochromatin functions. We also demonstrate that ectopic expression of BigH1 similarly complements the deficiency of H1 protein. Thus, in organisms that do not express redundant H1 isoforms, the structural and biological functions performed by canonical linker histones in later development, may be shared in early embryos by weakly homologous proteins, such as BigH1, or even unrelated, non-homologous proteins, such as Elba2

Drosophila SUMM4 complex couples insulator function and DNA replication control

Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underrelicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins

A Large Gene Network in Immature Erythroid Cells Is Controlled by the Myeloid and B Cell Transcriptional Regulator PU.1

PU.1 is a hematopoietic transcription factor that is required for the development of myeloid and B cells. PU.1 is also expressed in erythroid progenitors, where it blocks erythroid differentiation by binding to and inhibiting the main erythroid promoting factor, GATA-1. However, other mechanisms by which PU.1 affects the fate of erythroid progenitors have not been thoroughly explored. Here, we used ChIP-Seq analysis for PU.1 and gene expression profiling in erythroid cells to show that PU.1 regulates an extensive network of genes that constitute major pathways for controlling growth and survival of immature erythroid cells. By analyzing fetal liver erythroid progenitors from mice with low PU.1 expression, we also show that the earliest erythroid committed cells are dramatically reduced in vivo. Furthermore, we find that PU.1 also regulates many of the same genes and pathways in other blood cells, leading us to propose that PU.1 is a multifaceted factor with overlapping, as well as distinct, functions in several hematopoietic lineages

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