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A Conserved Function for the H2A.Z C Terminus

Time:2018-02-11 18:22Turbochargers information Click:

Function Conserved H2A.Z Termi

Variant forms of H2A have heterogeneous C termini that appear to be central to the functional identity of these proteins. Post-translational modifications of the H2A.Z C terminus have previously been implicated in the DNA damage response in yeast and in heterochromatic silencing in mammals (, ). The experiments presented here demonstrate that in addition to modification at specific residues, the length of the H2A.Z C-terminal tail is also important for the normal functions of the protein.

Truncated Htz1 proteins are unable to perform the functions of full-length Htz1 in yeast, resulting in synthetic genetic interactions with asf1Δ and sensitivity of cells to MMS. While the shorter truncations Δ128–133 and Δ125–133 are not MMS sensitive, they were isolated as having growth defects in our original screen. They may therefore have subtle phenotypes that are not as severe as larger deletions and that do not affect the response to DNA damage. Known sites of sumoylation in the Htz1 C terminus that regulate the response to an irreparable DNA double-strand break () are not required for MMS resistance, indicating that the function of this region is not dependent on modification of these residues. Indeed, portions of the C-terminal tail of major H2A with markedly different amino acid sequences can fully compensate for the Htz1 C terminus in mediating resistance to MMS.

Removal of amino acids 111 onwards reduces the occupancy of H2A.Z in yeast chromatin, explaining the loss of function phenotypes observed for this mutant. For this reason, we have not characterized this mutant further, as chromatin localization is upstream of most H2A.Z functions. It is interesting to note that this mutant should have normal chromatin occupancy as it contains the M6 region that is required for association with the Htz1 deposition complex SWR-C (, ). This finding agrees with the work of Wang et al. (), who recently showed that H2A.Z lacking the last 20 amino acids (Δ114–133) is not functional in yeast cells despite interacting with SWR-C components. As Htz1 can be enzymatically removed from nucleosomes by the INO80-C (), one possibility is that the C-terminal tail regulates this process. Alternatively, the removal of amino acids may simply reduce the contact surfaces between Htz1 and other nucleosomal components, making the protein less able to stably associate. The C-terminal tail of major H2A is also required for stable association with nucleosomes and chromatin (, ), indicating that regulation of association with nucleosomes may be a general function of the C termini of H2A family proteins.

The mutant that we have focused on is the Δ120–133 truncation, which is not functional despite being associated with the chromatin fraction and having a normal localization pattern across the yeast genome. The slight but reproducible destabilization of this mutant relative to full-length Htz1, as measured by salt washes and ChIP, is the only detectable difference between the functional full-length protein and the non-functional Δ120–133 mutant. These data link the dynamics of the nucleosomal association of Htz1 to function in yeast cells.

Although the yeast C-terminal truncation mutants reveal a function for the H2A.Z C terminus and will be useful in further studies of the effects of modulating H2A.Z nucleosomal stability they do not, to our knowledge, represent physiological proteins. The novel splice isoform of human H2A.Z-2 containing exon 6, on the other hand, is a naturally occurring example of truncated H2A.Z. This splice isoform is expressed predominantly in human brain, skeletal muscle and liver, suggesting that this transcript may have tissue-specific roles. Although other transcripts derived from the H2A.Z-2 gene may exist in vivo, they encode proteins lacking sequences within the histone fold domain, and they do not produce stable protein in human cells.

A direct comparison of the characteristics of H2A.Z-2.1 and H2A.Z-2.2 in human cells showed that H2A.Z-2.2 is markedly less stably associated with chromatin than H2A.Z-2.1, as revealed by washing with buffers containing various salt concentrations. Additionally, isoform 1 is almost entirely chromatin bound while there is a large pool of soluble isoform 2. It is possible that the soluble pool of H2A.Z-2.2 is a result of the protein level in our system being above normal physiological levels, as isoforms 1 and 2 were induced to the same levels to allow direct comparison between them while RT-Q-PCR analysis indicates that the endogenous transcript encoding isoform 2 is less abundant than that encoding isoform 1. Nonetheless, the different behaviors of the proteins can only be attributed to the differences between their C termini. It will be interesting to discover whether the novel sequence in H2A.Z-2.2 provides a unique docking site for proteins regulating H2A.Z chromatin occupancy or if the shortening of the C terminus directly regulates stability.

Why might the nucleosomal stability of H2A.Z be important for its functions? H2A.Z is found in nucleosomes close to the position of transcription initiation in all organisms studied to date (,,). These nucleosomes represent key control points as they can sterically occlude transcription factors and other proteins of the transcription machinery but may need to be easily disrupted in response to induction signals. Correspondingly, H2A.Z is required for normal kinetics of gene activation, and its chromatin occupancy is dynamic during gene activation and repression in yeast and mammalian cells (,,). Yeast H2A.Z is dissociated from nucleosomes at lower salt concentrations than H2A () and nucleosomes containing H2A.Z are more susceptible to nuclease digestion than their canonical counterparts (), indicating that H2A.Z nucleosomes in yeast are less stable than those containing H2A. In mammalian cells, nucleosomes containing H2A.Z at promoters and other regulatory regions are also highly salt-labile (). These characteristics have been interpreted as important for H2A.Z functions but the effects of altering the stability of H2A.Z have not previously been tested. Our findings that a C-terminal truncation mutant with phenotypic defects is present with a normal distribution in chromatin but is nonetheless unable to function like wild-type Htz1 indicates that the altered stability of this mutant in nucleosomes interferes with its ability to function normally in yeast. In human cells, the naturally occurring shorter H2A.Z-2 isoform also displays altered affinity for chromatin relative to the full-length protein. The tissue-specific distribution of this isoform indicates that the relative instability of H2A.Z.2–2 may be harnessed as a novel regulatory mechanism in certain cell types. As the importance of the dynamic behavior of nucleosomes becomes clearer, the contribution of the H2A.Z C-terminal tail to nucleosome stability will help to reconcile the complex and diverse functional roles of H2A.Z.

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