Centromeres consist of specialized centrochromatin containing CENP-A nucleosomes intermingled with H3 nucleosomes carrying transcription-associated modifications. We have designed a novel synthetic biology ‘in situ epistasis' analysis in which H3 dimethylated on lysine 4 (H3K4me2) demethylase LSD2 plus synthetic modules with competing activities are simultaneously targeted to a synthetic alphoidtetO HAC centromere. This allows us to uncouple transcription from histone modifications at the centromere. Here, we report that H3K4me2 loss decreases centromeric transcription, CENP-A assembly and stability and causes spreading of H3K9me3 across the HAC, ultimately inactivating the centromere. Surprisingly, CENP-28/Eaf6-induced transcription of the alphoidtetO array associated with H4K12 acetylation does not rescue the phenotype, whereas p65-induced transcription associated with H3K9 acetylation does rescue. Thus mitotic transcription plus histone modifications including H3K9ac constitute the ‘epigenetic landscape' allowing CENP-A assembly and centrochromatin maintenance. H3K4me2 is required for the transcription and H3K9ac may form a barrier to prevent heterochromatin spreading and kinetochore inactivation at human centromeres.

How eukaryotic chromosomes are compacted during mitosis has been a leading question in cell biology since the nineteenth century. Non-histone proteins such as condensin complexes contribute to chromosome shaping, but appear not to be necessary for mitotic chromatin compaction. Histone modifications are known to affect chromatin structure. As histones undergo major changes in their post-translational modifications during mitotic entry, we speculated that the spectrum of cell-cycle-specific histone modifications might contribute to chromosome compaction during mitosis. To test this hypothesis, we isolated core histones from interphase and mitotic cells and reconstituted chromatin with them. We used mass spectrometry to show that key post-translational modifications remained intact during our isolation procedure. Light, atomic force and transmission electron microscopy analysis showed that chromatin assembled from mitotic histones has a much greater tendency to aggregate than chromatin assembled from interphase histones, even under low magnesium conditions where interphase chromatin remains as separate beads-on-a-string structures. These observations are consistent with the hypothesis that mitotic chromosome formation is a two-stage process with changes in the spectrum of histone post-translational modifications driving mitotic chromatin compaction, while the action of non-histone proteins such as condensin may then shape the condensed chromosomes into their classic mitotic morphology.

We show that Kar9p polarity is instructed by a feedback loop that requires astral microtubules, actin cables, and Myo2p-based transport to enforce Kar9p loading to the bud-ward pole. This novel mechanism also provides the basis for a model unifying Kar9p polarity and the stereotyped pattern of spindle pole inheritance known to occur in yeast.