The evolutionary differentiation of two histone H2A.Z variants in chordates (H2A.Z-1 and H2A.Z-2) is mediated by a stepwise mutation process that affects three amino acid residues

Background  

The histone H2A family encompasses the greatest number of core histone variants of which the replacement variant H2A.Z is currently one of the most heavily studied. No clear mechanism for the functional variability that H2A.Z imparts to chromatin has yet been proposed. While most of the past studies have referred to H2A.Z generically as a single protein, in vertebrates it is a mixture of two protein forms H2A.Z-1 (previously H2A.Z) and H2A.Z-2 (previously H2A.F/Z or H2A.V) that differ by three amino acids.     

Distinct Distribution of Ectopically Expressed Histone Variants H2A.Bbd and MacroH2A in Open and Closed Chromatin Domains

Background

It becomes increasingly evident that nuclesomes are far from being identical to each other. This nucleosome diversity is due partially to the existence of histone variants encoded by separate genes. Among the known histone variants the less characterized are H2A.Bbd and different forms of macroH2A. This is especially true in the case of H2A.Bbd as there are still no commercially available antibodies specific to H2A.Bbd that can be used for chromatin immunoprecipitation (ChIP).

Methods

We have generated HeLa S3 cell lines stably expressing epitope-tagged versions of macroH2A1.1, H2A.Bbd or canonical H2A and analyzed genomic distribution of the tagged histones using ChIP-on-chip technique.

Results

The presence of histone H2A variants macroH2A1.1 and H2A.Bbd has been analyzed in the chromatin of several segments of human chromosomes 11, 16 and X that have been chosen for their different gene densities and chromatin status. Chromatin immunoprecipitation (ChIP) followed by hybridization with custom NimbleGene genomic microarrays demonstrated that in open chromatin domains containing tissue-specific along with housekeeping genes, the H2A.Bbd variant was preferentially associated with the body of a subset of transcribed genes. The macroH2A1.1 variant was virtually absent from some genes and underrepresented in others. In contrast, in closed chromatin domains which contain only tissue-specific genes inactive in HeLa S3 cells, both macroH2A1.1 and H2A.Bbd histone variants were present and often colocalized.

Conclusions

Genomic distribution of macro H2A and H2A.Bbd does not follow any simple rule and is drastically different in open and closed genomic domains.     

A lesson learned from the H3.3K27M mutation found in pediatric glioma

Glioblastoma (GBM) is the most aggressive primary brain tumor in human. Recent studies on high-grade pediatric GBM have identified two recurrent mutations (K27M and G34R/V) in genes encoding histone H3 (H3F3A for H3.3 and HIST1H3B for H3.1).^1,2 The two histone H3 mutations are mutually exclusive and give rise to tumors in different brain compartments.³ Recently, we⁴ and others⁵ have shown that the histone H3 K27M mutation specifically altered the di- and tri-methylation of endogenous histone H3 at Lys27. Genome-wide studies using ChIP-seq on H3.3K27M patient samples indicate a global reduction of H3K27me3 on chromatin. Remarkably, we also found a dramatic enrichment of H3K27me3 and EZH2 (the catalytic subunit H3K27 methyltransferase) at hundreds of gene loci in H3.3K27M patient cells. Here, we discuss potential mechanisms whereby H3K27me3 is enriched at chromatin loci in cells expressing the H3.3K27M mutation and report effects of Lys-to-Met mutations of other well-studied lysine residues of histone H3.1/H3.3 and H4 on the corresponding endogenous lysine methylation. We suggest that mutation(s) on histones may be found in a variety of human diseases, and the expression of mutant histones may help to address the function of histone lysine methylation and possibly other modifications in mammalian cells.     

Histone H2A and H2B Are Monoubiquitinated at AID-Targeted Loci

Background

Somatic hypermutation introduces base substitutions into the rearranged and expressed immunoglobulin (Ig) variable regions to promote immunity. This pathway requires and is initiated by the Activation Induced Deaminase (AID) protein, which deaminates cytidine to produce uracils and UG mismatches at the Ig genes. Subsequent processing of uracil by mismatch repair and base excision repair factors contributes to mutagenesis. While selective for certain genomic targets, the chromatin modifications which distinguish hypermutating from non-hypermutating loci are not defined.

Methodology/Principal Findings

Here, we show that AID-targeted loci in mammalian B cells contain ubiquitinated chromatin. Chromatin immunoprecipitation (ChIP) analysis of a constitutively hypermutating Burkitt''s B cell line, Ramos, revealed the presence of monoubiquitinated forms of both histone H2A and H2B at two AID-associated loci, but not at control loci which are expressed but not hypermutated. Similar analysis using LPS activated primary murine splenocytes showed enrichment of the expressed V_H and Sγ3 switch regions upon ChIP with antibody specific to AID and to monoubiquitinated H2A and H2B. In the mechanism of mammalian hypermutation, AID may interact with ubiquitinated chromatin because confocal immunofluorescence microscopy visualized AID colocalized with monoubiquitinated H2B within discrete nuclear foci.

Conclusions/Significance

Our results indicate that monoubiquitinated histones accompany active somatic hypermutation, revealing part of the histone code marking AID-targeted loci. This expands the current view of the chromatin state during hypermutation by identifying a specific nucleosome architecture associated with somatic hypermutation.     

The Schizosaccharomyces pombe JmjC-Protein,Msc1, Prevents H2A.Z Localization in Centromeric and Subtelomeric Chromatin Domains

Eukaryotic genomes are repetitively packaged into chromatin by nucleosomes, however they are regulated by the differences between nucleosomes, which establish various chromatin states. Local chromatin cues direct the inheritance and propagation of chromatin status via self-reinforcing epigenetic mechanisms. Replication-independent histone exchange could potentially perturb chromatin status if histone exchange chaperones, such as Swr1C, loaded histone variants into wrong sites. Here we show that in Schizosaccharomyces pombe, like Saccharomyces cerevisiae, Swr1C is required for loading H2A.Z into specific sites, including the promoters of lowly expressed genes. However S. pombe Swr1C has an extra subunit, Msc1, which is a JumonjiC-domain protein of the Lid/Jarid1 family. Deletion of Msc1 did not disrupt the S. pombe Swr1C or its ability to bind and load H2A.Z into euchromatin, however H2A.Z was ectopically found in the inner centromere and in subtelomeric chromatin. Normally this subtelomeric region not only lacks H2A.Z but also shows uniformly lower levels of H3K4me2, H4K5, and K12 acetylation than euchromatin and disproportionately contains the most lowly expressed genes during vegetative growth, including many meiotic-specific genes. Genes within and adjacent to subtelomeric chromatin become overexpressed in the absence of either Msc1, Swr1, or paradoxically H2A.Z itself. We also show that H2A.Z is N-terminally acetylated before, and lysine acetylated after, loading into chromatin and that it physically associates with the Nap1 histone chaperone. However, we find a negative correlation between the genomic distributions of H2A.Z and Nap1/Hrp1/Hrp3, suggesting that the Nap1 chaperones remove H2A.Z from chromatin. These data describe H2A.Z action in S. pombe and identify a new mode of chromatin surveillance and maintenance based on negative regulation of histone variant misincorporation.     

Evolution and diversity of Rickettsia bacteria

Background

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant neuromuscular disorder associated with the partial deletion of integral numbers of 3.3 kb D4Z4 DNA repeats within the subtelomere of chromosome 4q. A number of candidate FSHD genes, adenine nucleotide translocator 1 gene (ANT1), FSHD-related gene 1 (FRG1), FRG2 and DUX4c, upstream of the D4Z4 array (FSHD locus), and double homeobox chromosome 4 (DUX4) within the repeat itself, are upregulated in some patients, thus suggesting an underlying perturbation of the chromatin structure. Furthermore, a mouse model overexpressing FRG1 has been generated, displaying skeletal muscle defects.

Results

In the context of myogenic differentiation, we compared the chromatin structure and tridimensional interaction of the D4Z4 array and FRG1 gene promoter, and FRG1 expression, in control and FSHD cells. The FRG1 gene was prematurely expressed during FSHD myoblast differentiation, thus suggesting that the number of D4Z4 repeats in the array may affect the correct timing of FRG1 expression. Using chromosome conformation capture (3C) technology, we revealed that the FRG1 promoter and D4Z4 array physically interacted. Furthermore, this chromatin structure underwent dynamic changes during myogenic differentiation that led to the loosening of the FRG1/4q-D4Z4 array loop in myotubes. The FRG1 promoter in both normal and FSHD myoblasts was characterized by H3K27 trimethylation and Polycomb repressor complex binding, but these repression signs were replaced by H3K4 trimethylation during differentiation. The D4Z4 sequences behaved similarly, with H3K27 trimethylation and Polycomb binding being lost upon myogenic differentiation.

Conclusion

We propose a model in which the D4Z4 array may play a critical chromatin function as an orchestrator of in cis chromatin loops, thus suggesting that this repeat may play a role in coordinating gene expression.     

A conserved function for the H2A.Z C terminus

Histone H2A variants generate diversity in chromatin structure and functions, as nucleosomes containing variant H2A histones have altered physical, chemical, and biological properties. H2A.Z is an evolutionarily ancient and highly conserved H2A variant that regulates processes ranging from gene expression to the DNA damage response. Here we find that the unstructured portion of the C-terminal tail of H2A.Z is required for the normal functions of this histone variant in budding yeast. We have also identified a novel splice isoform of the human H2A.Z-2 gene that encodes a C-terminally truncated H2A.Z protein that is similar to the truncation mutants we identified in yeast. The short forms of H2A.Z in both yeast and human cells are more loosely associated with chromatin than the full-length proteins, indicating a conserved function for the H2A.Z C-terminal tail in regulating the association of H2A.Z with nucleosomes.