Functions of the histone chaperones FACT and bromodomain AAA+ ATPases in heterochromatin spreading and centromere biology
Final Report Abstract
The eukaryotic genome consists of transcriptionally active euchromatin and heterochromatin, which is transcriptionally silent and plays a crucial role in genome stability, chromosome segregation, and maintaining cell identity. Heterochromatin formation is a stepwise process involving: nucleation, spreading along the chromosome arms, and stable maintenance after DNA replication. Disruption of any of these steps leads to heterochromatin defects and gene misexpression. The histone chaperone FACT (Facilitates Chromatin Transcription) has been implicated in heterochromatin silencing, but its exact role remained unclear. In the first project, we identified FACT's specific function in heterochromatin spreading in Schizosaccharomyces pombe. In this model organism, heterochromatin assembly begins at nucleation sites, where various mechanisms recruit the sole H3K9 methyltransferase, Clr4. Once recruited, Clr4 initiates spreading through a "write" and "read" mechanism. As the "writer," Clr4 not only adds the H3K9 methylation mark but also recognizes it via its chromodomain. Additionally, spreading requires the involvement the "reader," Heterochromatin Protein 1 (Swi6 in S. pombe), which binds to the H3K9me2/3 marks through its chromodomain. Using genomics, yeast genetics, and single-cell fluorescence reporter assays, we showed that FACT does not regulate nucleation but facilitates heterochromatin spreading along chromosomal arms. FACT mutants exhibited reduced accumulation of H3K9me3, and Swi6 at subtelomeres and derepression of genes near heterochromatin boundaries. A heterochromatin spreading assay further revealed impaired spreading at the mating-type locus in the chaperone mutants. Clr4 recruitment to nucleation sites remained unaffected. We demonstrated that the spreading defect in FACT mutants is due to impaired transition from H3K9me2 to H3K9me3, a key step in the spreading process. H3K9me3 methylation by Clr4 is slower than mono- and dimethylation and requires longer residency time on the nucleosome. By reducing heterochromatic nucleosome turnover, FACT facilitates H3K9 tri-methylation and efficient heterochromatin spreading. Together, our findings provide crucial insights into heterochromatin spreading mechanism and highlight the role of the conserved histone chaperone in this process. In the second, unpublished project, we studied the novel bromodomain AAA+ ATPase, Abo2, in centromere functions in S. pombe. We found that Abo2, but not its paralog Abo1, specifically localizes to centromeric chromatin. Abo2 mutant, abo2∆, displays mitotic phenotypes, including TBZ sensitivity, lagging chromosomes, and unequal chromosome segregation, linking Abo2 to chromosome segregation functions. Although assembly of the centromeric histone variant CENP-A (Cnp1 in S. pombe) was unaffected in the abo2∆ strain, Cnp1 levels were reduced at the boundaries between centromere and pericentromeric heterochromatin, suggesting Abo2 may function as a boundary factor at centromere. Additionally, FACT accumulates at centromere in the absence of Abo2, indicating that Abo2 regulates the chaperone dynamics at centromeric chromatin. Abo2 mutant shows synthetic genetic interactions with several centromere components. Future studies will aim to investigate the mechanism of Abo2 in chromosome segregation in S. pombe and its conservation in mammalian cells.
Publications
-
The histone chaperone FACT facilitates heterochromatin spreading by regulating histone turnover and H3K9 methylation states. Cell Reports, 37(5), 109944.
Murawska, Magdalena; Greenstein, R.A.; Schauer, Tamas; Olsen, Karl C.F.; Ng, Henry; Ladurner, Andreas G.; Al-Sady, Bassem & Braun, Sigurd
-
Chaperoning heterochromatin: new roles of FACT in chromatin silencing. Trends in Genetics, 38(7), 646-649.
Murawska, Magdalena & Braun, Sigurd
-
Pioneers conquer core histones at the chromatin frontier. Nature Structural & Molecular Biology, 30(8), 1050-1053.
Murawska, Magdalena; Ladurner, Andreas G. & Margulies, Carla E.
