bIn response to genotoxic stress, ATR and ATM kinases phosphorylate H2A in fungi and H2AX in animals on a C-terminal serine. The resulting modified histone, called ␥H2A, recruits chromatin-binding proteins that stabilize stalled replication forks or promote DNA double-strand-break repair. To identify genomic loci that might be prone to replication fork stalling or DNA breakage in Neurospora crassa, we performed chromatin immunoprecipitation (ChIP) of ␥H2A followed by next-generation sequencing (ChIP-seq). ␥H2A-containing nucleosomes are enriched in Neurospora heterochromatin domains. These domains are comprised of A·T-rich repetitive DNA sequences associated with histone H3 methylated at lysine-9 (H3K9me), the H3K9me-binding protein heterochromatin protein 1 (HP1), and DNA cytosine methylation. H3K9 methylation, catalyzed by DIM-5, is required for normal ␥H2A localization. In contrast, ␥H2A is not required for H3K9 methylation or DNA methylation. Normal ␥H2A localization also depends on HP1 and a histone deacetylase, HDA-1, but is independent of the DNA methyltransferase DIM-2. ␥H2A is globally induced in dim-5 mutants under normal growth conditions, suggesting that the DNA damage response is activated in these mutants in the absence of exogenous DNA damage. Together, these data suggest that heterochromatin formation is essential for normal DNA replication or repair.

Chromosome replication in Saccharomyces cerevisiae is initiated from ~300 origins that are regulated by DNA sequence and by the limited abundance of six trans-acting initiation proteins (Sld2, Sld3, Dpb11, Dbf4, Sld7 and Cdc45). We set out to determine how the levels of individual factors contribute to time of origin activation and/or origin efficiency using induced depletion of single factors and overexpression of sets of multiple factors. Depletion of Sld2 or Sld3 slows growth and S phase progression, decreases origin efficiency across the genome and impairs viability as a result of incomplete replication of the rDNA. We find that the most efficient early origins are relatively unaffected by depletion of either Sld2 or Sld3. However, Sld3 levels, and to a lesser extent Sld2 levels, are critical for firing of the less efficient early origins. Overexpression of Sld3 simultaneously with Sld2, Dpb11 and Dbf4 preserves the relative efficiency of origins. Only when Cdc45 and Sld7 are also overexpressed is origin efficiency equalized between early- and late-firing origins. Our data support a model in which Sld3 together with Cdc45 (and/or Sld7) is responsible for the differential efficiencies of origins across the yeast genome.

Edited by Urs GreberAbbreviations ADP, adenovirus death protein; Ad5 or Ad 12, human adenovirus type 5 or 12, or other type referenced; BHK, baby hamster kidney; BrdU, bromodeoxyuridine; CTCF, CCCTC binding factor; DBS, double stranded break; DDR, DNA damage response; E1A, early 1 A; E4orf3 or E4orf6, early 4 open reading frame 3 or 6; HAdV-A12, human adenovirus subgroup A type 12, or different subgroup or type referenced; HMGB, high-mobility group box; hPaf1, human RNA polymerase II-associated factor 1; ISGs, interferon-stimulated genes; PML, promyelocytic leukemia; snRNP, small nuclear ribonuclear protein; SET or TAF-1, SE translocation or template activating factor; VRCs, viral replication centers.

Virus infection necessarily requires redirecting cellular resources toward viral progeny production. Adenovirus encodes the histone-like protein VII, which causes catastrophic global reorganization of host chromatin to promote virus infection. Protein VII recruits the family of high mobility group box (HMGB) proteins to chromatin along with the histone chaperone SET. As a consequence of this recruitment, we find that protein VII causes chromatin depletion of several linker histone H1 isoforms. The relationship between linker histone H1 and the functionally opposite HMGB proteins is critical for higher-order chromatin structure. However, the physiological consequences of perturbing this relationship are largely unknown. Here, we employ complementary systems in Saccharomyces cerevisiae and human cells to demonstrate that adenovirus protein VII disrupts the H1-HMGB balance to obstruct the cell cycle. We find that protein VII causes an accumulation of G2/M cells both in yeast and human systems, underscoring the high conservation of this chromatin vulnerability. In contrast, adenovirus E1A and E1B proteins are well established to override cell cycle regulation and promote transformation of human cells. Strikingly, we find that protein VII obstructs the cell cycle, even in the presence of E1A and E1B. We further show that, in a protein-VII-deleted infection, several cell cycle markers are regulated differently compared to wild-type infection, supporting our model that protein VII plays an integral role in hijacking cell cycle regulation during infection. Together, our results demonstrate that protein VII targets H1-HMGB1 antagonism to obstruct cell cycle progression, revealing an unexpected chromatin vulnerability exploited for viral benefit.

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