Changes in the histone H2A variant H2A.Z and polyubiquitinated histone species in developing trout testis

The trout histone H2A variant H2A.Z has been identified by its electrophoretic mobility on two-dimensional polyacrylamide gels and its N-terminal amino acid sequence. Similar to bovine H2A.Z and chicken H2A.F (also called H2A.Z and M1), the trout H2A.Z had a two-residue extension when aligned with trout H2A and a 67% sequence homology with the N-terminal portion of trout H2A. The first 29 amino acids of trout H2A.Z were identical with those of chicken H2A.F and differed from those of bovine H2A.Z at only one position. Thus, the N-terminal part of histone H2A.Z appears to be highly conserved. The levels of histone H2A.Z and ubiquitinated species of the histones H2A, H2A.Z, and H2B, which were detected with an anti-ubiquitin antibody, were studied at various stages of trout testis development. At the final stages of spermatogenesis in trout, histones are replaced by protamines. Ubiquitinated and diubiquitinated histone H2A remained at similar levels in early and late stage testis nucleohistone. In the late stage testis chromatin (nucleohistone), ubiquitinated histone H2A.Z was not detected, the level of ubiquitinated histone H2B was reduced, and the amount of diubiquitinated histone H2B increased. There was also a marked reduction in the level of histone H2A.Z. This observation suggests nucleosomes with this histone variant were selectively disassembled during the transition from nucleohistone to nucleoprotamine, indicating that protamine deposition is not a random process in rainbow trout.     

基于组蛋白甲基化信息的H2A.Z和H2A核小体识别

组蛋白H2A的变体H2A.Z在基因的表达过程中发挥着重要的作用。根据H2A.Z和H2A核小体中组蛋白甲基化修饰方式的不同,作者应用多样性增量二次判别方法(increment of diversity with quadratic discriminant,IDQD)成功地对H2A.Z和H2A核小体进行了识别,说明了以组蛋白甲基化信息作为特征参数的IDQD模型对H2A.Z和H2A核小体识别的有效性。通过计算DNA序列的柔性,发现H2A.Z核小体对应的DNA序列的平均柔性比常规H2A核小体对应的DNA序列的平均柔性弱。     

H2A.Z.2.2 is an alternatively spliced histone H2A.Z variant that causes severe nucleosome destabilization

The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.     

Key functional regions in the histone variant H2A.Z C-terminal docking domain

The incorporation of histone variants into nucleosomes represents one way of altering the chromatin structure to accommodate diverse functions. Histone variant H2A.Z has specific roles in gene regulation, heterochromatin boundary formation, and genomic integrity. The precise features required for H2A.Z to function and specify an identity different from canonical H2A remain to be fully explored. Analysis of the C-terminal docking domain of H2A.Z in Saccharomyces cerevisiae using epistatic miniarray profile (E-MAP) uncovered nuanced requirements of the H2A.Z C-terminal region for cell growth when additional genes were compromised. Moreover, the H2A.Z(1-114) truncation, lacking the last 20 amino acids of the protein, did not support regular H2A.Z functions, such as resistance to genotoxic stress, restriction of heterochromatin in its native context, GAL1 gene activation, and chromatin anchoring. The corresponding region of H2A could fully rescue the strong defects caused by loss of this functionally essential region in the C terminus of H2A.Z. Despite the dramatic reduction in function, the H2A.Z(1-114) truncation still bound the H2A.Z deposition complex SWR1-C, the histone chaperone Chz1, and histone H2B. These data are consistent with a model in which retaining the variant in chromatin after its deposition by SWR1-C is a crucial determinant of its function.