The nucleosome is the primary unit of chromatin structure and commonly imputed as a regulator of nuclear events, although the exact mechanisms remain unclear. Recent studies have shown that certain nucleosomes can have different sensitivities to micrococcal nuclease (MNase) digestion, resulting in the release of populations of nucleosomes dependent on the concentration of MNase. Mapping MNase sensitivity of nucleosomes at transcription start sites genome-wide reveals an important functional nucleosome organization that correlates with gene expression levels and transcription factor binding. In order to understand nucleosome distribution and sensitivity dynamics during a robust genome response, we mapped nucleosome position and sensitivity using multiple concentrations of MNase. We used the innate immune response as a model system to understand chromatin-mediated regulation. Herein we demonstrate that stimulation of a human lymphoblastoid cell line (GM12878) with heat-killed Salmonella typhimurium (HKST) results in changes in nucleosome sensitivity to MNase. We show that the HKST response alters the sensitivity of -1 nucleosomes at highly expressed promoters. Finally, we correlate the increased sensitivity with response-specific transcription factor binding. These results indicate that nucleosome sensitivity dynamics reflect the cellular response to HKST and pave the way for further studies that will deepen our understanding of the specificity of genome response.

The highly conserved FACT (Facilitates Chromatin Transactions) complex performs essential functions in eukaryotic cells through the reorganization of nucleosomes. During transcription, FACT reorganizes nucleosomes to allow passage of RNA Polymerase II and then assists in restoring these nucleosomes after RNA Polymerase II has passed. We have previously shown, consistent with this function, that Spt16 facilitates repression of the Saccharomyces cerevisiae SER3 gene by maintaining nucleosome occupancy over the promoter of this gene as a consequence of intergenic transcription of SRG1 noncoding DNA. In this study, we report the results of a genetic screen to identify mutations in SPT16 that derepress SER3. Twenty-five spt16 mutant alleles were found to derepress SER3 without causing significant reductions in either SRG1 RNA levels or Spt16 protein levels. Additional phenotypic assays indicate that these mutants have general transcription defects related to altered chromatin structure. Our analyses of a subset of these spt16 mutants reveal defects in SRG1 transcription-coupled nucleosome occupancy over the SER3 promoter. We provide evidence that these mutants broadly impair transcription-coupled nucleosome occupancy at highly transcribed genes but not at lowly transcribed genes. Finally, we show that one consequence shared by these mutations is the reduced binding of mutant Spt16 proteins across SRG1 and other highly transcribed genes. Taken together, our results highlight an important role for Spt16 in orchestrating transcription-coupled nucleosome assembly at highly transcribed regions of the genome, possibly by facilitating the association of Spt16 during this process.

Kinetochores assemble on centromeres to drive chromosome segregation in eukaryotic cells. Humans and budding yeast share most of the structural subunits of the kinetochore, whereas protein sequences have diverged considerably. The conserved centromeric histone H3 variant, CenH3 (CENP-A in humans and Cse4 in budding yeast), marks the site for kinetochore assembly in most species. A previous effort to complement Cse4 in yeast with human CENP-A was unsuccessful; however, co-complementation with the human core nucleosome was not attempted. Previously, our lab successfully humanized the core nucleosome in yeast; however, this severely affected cellular growth. We hypothesized that yeast Cse4 is incompatible with humanized nucleosomes and that the kinetochore represented a limiting factor for efficient histone humanization. Thus, we argued that including the human CENP-A or a Cse4-CENP-A chimera might improve histone humanization and facilitate kinetochore function in humanized yeast. The opposite was true: CENP-A expression reduced histone humanization efficiency, was toxic to yeast, and disrupted cell cycle progression and kinetochore function in wild-type (WT) cells. Suppressors of CENP-A toxicity included gene deletions of subunits of 3 conserved chromatin remodeling complexes, highlighting their role in CenH3 chromatin positioning. Finally, we attempted to complement the subunits of the NDC80 kinetochore complex, individually and in combination, without success, in contrast to a previous study indicating complementation by the human NDC80/HEC1 gene. Our results suggest that limited protein sequence similarity between yeast and human components in this very complex structure leads to failure of complementation.

Extremophilic organisms demonstrate the flexibility and adaptability of basic biological processes by highlighting how cell physiology adapts to environmental extremes. Few eukaryotic extremophiles have been well studied and only a small number are amenable to laboratory cultivation and manipulation. A detailed characterization of the genome architecture of such organisms is important to illuminate how they adapt to environmental stresses. One excellent example of a fungal extremophile is the halophile Hortaea werneckii (Pezizomycotina, Dothideomycetes, Capnodiales), a yeast-like fungus able to thrive at near-saturating concentrations of sodium chloride and which is also tolerant to both UV irradiation and desiccation. Given its unique lifestyle and its remarkably recent whole genome duplication, H. werneckii provides opportunities for testing the role of genome duplications and adaptability to extreme environments. We previously assembled the genome of H. werneckii using short-read sequencing technology and found a remarkable degree of gene duplication. Technology limitations, however, precluded high-confidence annotation of the entire genome. We therefore revisited the H. wernickii genome using long-read, single-molecule sequencing and provide an improved genome assembly which, combined with transcriptome and nucleosome analysis, provides a useful resource for fungal halophile genomics. Remarkably, the ∼50 Mb H. wernickii genome contains 15,974 genes of which 95% (7608) are duplicates formed by a recent whole genome duplication (WGD), with an average of 5% protein sequence divergence between them. We found that the WGD is extraordinarily recent, and compared to Saccharomyces cerevisiae, the majority of the genome's ohnologs have not diverged at the level of gene expression of chromatin structure.