Histone H3.3 regulates mitotic progression in mouse embryonic fibroblasts

H3.3 is a histone variant that marks transcription start sites as well as telomeres and heterochromatic sites on the genome. The presence of H3.3 is thought to positively correlate with the transcriptional status of its target genes. Using a conditional genetic strategy against H3.3B, combined with short hairpin RNAs against H3.3A, we essentially depleted all H3.3 gene expression in mouse embryonic fibroblasts. Following nearly complete loss of H3.3 in the cells, our transcriptomic analyses show very little impact on global gene expression or on the localization of histone variant H2A.Z. Instead, fibroblasts displayed slower cell growth and an increase in cell death, coincident with large-scale chromosome misalignment in mitosis and large polylobed or micronuclei in interphase cells. Thus, we conclude that H3.3 may have an important under-explored additional role in chromosome segregation, nuclear structure, and the maintenance of genome integrity. © 2017 Published by NRC Research Press

A Novel Pzg-NURF Complex Regulates Notch Target Gene Activity

The Putzig (Pzg) protein is associated with the NURF nucleosome remodeling complex, thereby promoting Notch target gene expression. Our findings suggest a novel Pzg-NURF complex that is responsible for the epigenetic regulation of Notch target genes

An All-Atom Model of the Chromatin Fiber Containing Linker Histones Reveals a Versatile Structure Tuned by the Nucleosomal Repeat Length

In the nucleus of eukaryotic cells, histone proteins organize the linear genome into a functional and hierarchical architecture. In this paper, we use the crystal structures of the nucleosome core particle, B-DNA and the globular domain of H5 linker histone to build the first all-atom model of compact chromatin fibers. In this 3D jigsaw puzzle, DNA bending is achieved by solving an inverse kinematics problem. Our model is based on recent electron microscopy measurements of reconstituted fiber dimensions. Strikingly, we find that the chromatin fiber containing linker histones is a polymorphic structure. We show that different fiber conformations are obtained by tuning the linker histone orientation at the nucleosomes entry/exit according to the nucleosomal repeat length. We propose that the observed in vivo quantization of nucleosomal repeat length could reflect nature's ability to use the DNA molecule's helical geometry in order to give chromatin versatile topological and mechanical properties

Histone H1 Subtypes Differentially Modulate Chromatin Condensation without Preventing ATP-Dependent Remodeling by SWI/SNF or NURF

Although ubiquitously present in chromatin, the function of the linker histone subtypes is partly unknown and contradictory studies on their properties have been published. To explore whether the various H1 subtypes have a differential role in the organization and dynamics of chromatin we have incorporated all of the somatic human H1 subtypes into minichromosomes and compared their influence on nucleosome spacing, chromatin compaction and ATP-dependent remodeling. H1 subtypes exhibit different affinities for chromatin and different abilities to promote chromatin condensation, as studied with the Atomic Force Microscope. According to this criterion, H1 subtypes can be classified as weak condensers (H1.1 and H1.2), intermediate condensers (H1.3) and strong condensers (H1.0, H1.4, H1.5 and H1x). The variable C-terminal domain is required for nucleosome spacing by H1.4 and is likely responsible for the chromatin condensation properties of the various subtypes, as shown using chimeras between H1.4 and H1.2. In contrast to previous reports with isolated nucleosomes or linear nucleosomal arrays, linker histones at a ratio of one per nucleosome do not preclude remodeling of minichromosomes by yeast SWI/SNF or Drosophila NURF. We hypothesize that the linker histone subtypes are differential organizers of chromatin, rather than general repressors

In Vivo Chromatin Organization of Mouse Rod Photoreceptors Correlates with Histone Modifications

BACKGROUND: The folding of genetic information into chromatin plays important regulatory roles in many nuclear processes and particularly in gene transcription. Post translational histone modifications are associated with specific chromatin condensation states and with distinct transcriptional activities. The peculiar chromatin organization of rod photoreceptor nuclei, with a large central domain of condensed chromatin surrounded by a thin border of extended chromatin was used as a model to correlate in vivo chromatin structure, histone modifications and transcriptional activity. METHODOLOGY: We investigated the functional relationships between chromatin compaction, distribution of histone modifications and location of RNA polymerase II in intact murine rod photoreceptors using cryo-preparation methods, electron tomography and immunogold labeling. Our results show that the characteristic central heterochromatin of rod nuclei is organized into concentric domains characterized by a progressive loosening of the chromatin architecture from inside towards outside and by specific combinations of silencing histone marks. The peripheral heterochromatin is formed by closely packed 30 nm fibers as revealed by a characteristic optical diffraction signal. Unexpectedly, the still highly condensed most external heterochromatin domain contains acetylated histones, which are usually associated with active transcription and decondensed chromatin. Histone acetylation is thus not sufficient in vivo for complete chromatin decondensation. The euchromatin domain contains several degrees of chromatin compaction and the histone tails are hyperacetylated, enriched in H3K4 monomethylation and hypo trimethylated on H3K9, H3K27 and H4K20. The transcriptionally active RNA polymerases II molecules are confined in the euchromatin domain and are preferentially located at the vicinity of the interface with heterochromatin. CONCLUSIONS: Our results show that transcription is located in the most decondensed and highly acetylated chromatin regions, but since acetylation is found associated with compact chromatin it is not sufficient to decondense chromatin in vivo. We also show that a combination of histone marks defines distinct concentric heterochromatin domains

Chromatin compaction in terminally differentiated avian blood cells: the role of linker histone H5 and non-histone protein MENT

Chromatin has a tendency to shift from a relatively decondensed (active) to condensed (inactive) state during cell differentiation due to interactions of specific architectural and/or regulatory proteins with DNA. A promotion of chromatin folding in terminally differentiated avian blood cells requires the presence of either histone H5 in erythrocytes or non-histone protein, myeloid and erythroid nuclear termination stage-specific protein (MENT), in white blood cells (lymphocytes and granulocytes). These highly abundant proteins assist in folding of nucleosome arrays and self-association of chromatin fibers into compacted chromatin structures. Here, we briefly review structural aspects and molecular mode of action by which these unrelated proteins can spread condensed chromatin to form inactivated regions in the genome

RSF Governs Silent Chromatin Formation via Histone H2Av Replacement

Human remodeling and spacing factor (RSF) consists of a heterodimer of Rsf-1 and hSNF2H, a counterpart of Drosophila ISWI. RSF possesses not only chromatin remodeling activity but also chromatin assembly activity in vitro. While no other single factor can execute the same activities as RSF, the biological significance of RSF remained unknown. To investigate the in vivo function of RSF, we generated a mutant allele of Drosophila Rsf-1 (dRsf-1). The dRsf-1 mutant behaved as a dominant suppressor of position effect variegation. In dRsf-1 mutant, the levels of histone H3K9 dimethylation and histone H2A variant H2Av were significantly reduced in an euchromatic region juxtaposed with heterochromatin. Furthermore, using both genetic and biochemical approaches, we demonstrate that dRsf-1 interacts with H2Av and the H2Av-exchanging machinery Tip60 complex. These results suggest that RSF contributes to histone H2Av replacement in the pathway of silent chromatin formation

Plasmodium falciparum Nucleosomes Exhibit Reduced Stability and Lost Sequence Dependent Nucleosome Positioning

The packaging and organization of genomic DNA into chromatin represents an additional regulatory layer of gene expression, with specific nucleosome positions that restrict the accessibility of regulatory DNA elements. The mechanisms that position nucleosomes in vivo are thought to depend on the biophysical properties of the histones, sequence patterns, like phased di-nucleotide repeats and the architecture of the histone octamer that folds DNA in 1.65 tight turns. Comparative studies of human and P. falciparum histones reveal that the latter have a strongly reduced ability to recognize internal sequence dependent nucleosome positioning signals. In contrast, the nucleosomes are positioned by AT-repeat sequences flanking nucleosomes in vivo and in vitro. Further, the strong sequence variations in the plasmodium histones, compared to other mammalian histones, do not present adaptations to its AT-rich genome. Human and parasite histones bind with higher affinity to GC-rich DNA and with lower affinity to AT-rich DNA. However, the plasmodium nucleosomes are overall less stable, with increased temperature induced mobility, decreased salt stability of the histones H2A and H2B and considerable reduced binding affinity to GC-rich DNA, as compared with the human nucleosomes. In addition, we show that plasmodium histone octamers form the shortest known nucleosome repeat length (155bp) in vitro and in vivo. Our data suggest that the biochemical properties of the parasite histones are distinct from the typical characteristics of other eukaryotic histones and these properties reflect the increased accessibility of the P. falciparum genome