RESUMO
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.
Assuntos
Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Histonas/metabolismo , Linfócitos/metabolismo , Processamento de Proteína Pós-Traducional , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Cátions Bivalentes , Linhagem Celular Tumoral , Galinhas , Cromatina/efeitos dos fármacos , Cromatina/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Histonas/genética , Humanos , Interfase/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/ultraestrutura , Magnésio/farmacologia , Microscopia de Força Atômica , Mitose/efeitos dos fármacos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Nocodazol/farmacologia , Fosforilação , Moduladores de Tubulina/farmacologiaRESUMO
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.
Assuntos
Epigênese Genética , Código das Histonas , Cinetocoros/metabolismo , Transcrição Gênica , Acetilação , Linhagem Celular Tumoral , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Células HeLa , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Nucleossomos/genética , Nucleossomos/metabolismoRESUMO
In Saccharomyces cerevisiae, Kar9p, one player in spindle alignment, guides the bud-ward spindle pole by linking astral microtubule plus ends to Myo2p-based transport along actin cables generated by the formins Bni1p and Bnr1p and the polarity determinant Bud6p. Initially, Kar9p labels both poles but progressively singles out the bud-ward pole. Here, we show that this polarization requires cell polarity determinants, actin cables, and microtubules. Indeed, in a bud6 Delta bni1 Delta mutant or upon direct depolymerization of actin cables Kar9p symmetry increased. Furthermore, symmetry was selectively induced by myo2 alleles, preventing Kar9p binding to the Myo2p cargo domain. Kar9p polarity was rebuilt after transient disruption of microtubules, dependent on cell polarity and actin cables. Symmetry breaking also occurred after transient depolymerization of actin cables, with Kar9p increasing at the spindle pole engaging in repeated cycles of Kar9p-mediated transport. Kar9p returning to the spindle pole on shrinking astral microtubules may contribute toward this bias. Thus, Myo2p transport along actin cables may support a feedback loop by which delivery of astral microtubule plus ends sustains Kar9p polarized recruitment to the bud-ward spindle pole. Our findings also explain the link between Kar9p polarity and the choice setting aside the old spindle pole for daughter-bound fate.
