Genome-wide chromatin mapping with size resolution reveals a dynamic sub-nucleosomal landscape in Arabidopsis.

All eukaryotic genomes are packaged as chromatin, with DNA interlaced with both regularly patterned nucleosomes and sub-nucleosomal-sized protein structures such as mobile and labile transcription factors (TF) and initiation complexes, together forming a dynamic chromatin landscape. Whilst details of nucleosome position in Arabidopsis have been previously analysed, there is less understanding of their relationship to more dynamic sub-nucleosomal particles (subNSPs) defined as protected regions shorter than the ~150bp typical of nucleosomes. The genome-wide profile of these subNSPs has not been previously analysed in plants and this study investigates the relationship of dynamic bound particles with transcriptional control. Here we combine differential micrococcal nuclease (MNase) digestion and a modified paired-end sequencing protocol to reveal the chromatin structure landscape of Arabidopsis cells across a wide particle size range. Linking this data to RNAseq expression analysis provides detailed insight into the relationship of identified DNA-bound particles with transcriptional activity. The use of differential digestion reveals sensitive positions, including a labile -1 nucleosome positioned upstream of the transcription start site (TSS) of active genes. We investigated the response of the chromatin landscape to changes in environmental conditions using light and dark growth, given the large transcriptional changes resulting from this simple alteration. The resulting shifts in the suites of expressed and repressed genes show little correspondence to changes in nucleosome positioning, but led to significant alterations in the profile of subNSPs upstream of TSS both globally and locally. We examined previously mapped positions for the TFs PIF3, PIF4 and CCA1, which regulate light responses, and found that changes in subNSPs co-localized with these binding sites. This small particle structure is detected only under low levels of MNase digestion and is lost on more complete digestion of chromatin to nucleosomes. We conclude that wide-spectrum analysis of the Arabidopsis genome by differential MNase digestion allows detection of sensitive features hereto obscured, and the comparisons between genome-wide subNSP profiles reveals dynamic changes in their distribution, particularly at distinct genomic locations (i.e. 5'UTRs). The method here employed allows insight into the complex influence of genetic and extrinsic factors in modifying the sub-nucleosomal landscape in association with transcriptional changes.

SWI/SNF-like chromatin remodeling factor Fun30 supports point centromere function in S. cerevisiae.

Budding yeast centromeres are sequence-defined point centromeres and are, unlike in many other organisms, not embedded in heterochromatin. Here we show that Fun30, a poorly understood SWI/SNF-like chromatin remodeling factor conserved in humans, promotes point centromere function through the formation of correct chromatin architecture at centromeres. Our determination of the genome-wide binding and nucleosome positioning properties of Fun30 shows that this enzyme is consistently enriched over centromeres and that a majority of CENs show Fun30-dependent changes in flanking nucleosome position and/or CEN core micrococcal nuclease accessibility. Fun30 deletion leads to defects in histone variant Htz1 occupancy genome-wide, including at and around most centromeres. FUN30 genetically interacts with CSE4, coding for the centromere-specific variant of histone H3, and counteracts the detrimental effect of transcription through centromeres on chromosome segregation and suppresses transcriptional noise over centromere CEN3. Previous work has shown a requirement for fission yeast and mammalian homologs of Fun30 in heterochromatin assembly. As centromeres in budding yeast are not embedded in heterochromatin, our findings indicate a direct role of Fun30 in centromere chromatin by promoting correct chromatin architecture.