Mutation of histone H3 serine 86 disrupts GATA factor Ams2 expression and precise chromosome segregation in fission yeast

Abstract

Eukaryotic genomes are packed into discrete units, referred to as nucleosomes, by organizing around scaffolding histone proteins. The interplay between these histones and the DNA can dynamically regulate the function of the chromosomal domain. Here, we interrogated the function of a pair of juxtaposing serine residues (S86 and S87) that reside within the histone fold of histone H3. We show that fission yeast cells expressing a mutant histone H3 disrupted at S86 and S87 (hht2-S86AS87A) exhibited unequal chromosome segregation, disrupted transcriptional silencing of centromeric chromatin, and reduced expression of Ams2, a GATA-factor that regulates localization of the centromere-specific histone H3 variant CENP-A. We found that overexpression of ams2+ could suppress the chromosome missegregation phenotype that arose in the hht2-S86AS87A mutant. We further demonstrate that centromeric localization of SpCENP-Acnp1-1 was significantly compromised in hht2-S86AS87A, suggesting synergism between histone H3 and the centromere-targeting domain of SpCENP-A. Taken together, our work presents evidence for an uncharacterized serine residue in fission yeast histone H3 that affects centromeric integrity via regulating the expression of the SpCENP-A-localizing Ams2 protein. [173/200 words]

Histone H3 lysine 36 methyltransferase mobilizes NER factors to regulate tolerance against alkylation damage in fission yeast

“…Asynchronous cells growing at 30°C were incubated with 0.01% MMS. Cells were collected at different time points up to 4 h. Total protein was extracted using trichloroacetic acid ( 46 , 47 ), separated on polyacrylamide gels, and transferred to Hybond ECL nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). Membranes were incubated with primary antibodies for 1 h at room temperature, washed, and then incubated with secondary antibodies for 1 h. Chemiluminescence was performed with Amersham ECL Prime (GE Healthcare) and chemiluminescence detection and band intensity quantification were achieved using an ImageQuant LAS 4000 imager (GE Healthcare).…”

Prolyl isomerization of the CENP-A N-terminus regulates centromeric integrity in fission yeast

“…To determine whether Ani1 and Ani2 are required for chromosome segregation, we disrupted the genes encoding these two prolyl isomerases. Null mutants of ani1 ( Δani1 ) and ani2 ( Δani2 ) resulted in a similar chromosome missegregation phenotype as cnp1–4PA (Figure 4A ); the mutants also caused a de-repression of the inner centromeric DNA sequence that is associated with a disruption to centromeric chromatin architecture ( 47 ) (Figure 4B ).…”

Regulation of transcriptional silencing and chromodomain protein localization at centromeric heterochromatin by histone H3 tyrosine 41 phosphorylation in fission yeast

“…A previously published western blotting procedure was followed ( 39 ). Antibodies used for this study were H3Y41p (Merck Millipore, Billerica, MA, 1:100), Cdc2 (Santa Cruz Biotechnology, Dallas, TX, 1:500), H2A (Merck Millipore, 1:200), H2B (Merck Millipore, 1:200), H3 (Merck Millipore, 1:500), H4 (Merck Millipore, 1:500), HA (Roche Applied Science, 1:500), Swi6 (Abcam, UK, 1:1000), Chp1 (Abcam, 1:100), GFP (Roche Applied Science, 1:500), FLAG (Wako, Japan, 1:500).…”

“…Cells were then washed with PBS thrice. ChIP was conducted as previously described ( 33 , 39 ). Relative fold enrichment was calculated by the ΔΔCt method, whereby NDR or act1 was used as internal control and further titrated from the whole cell extract (WCE) or histone H3 immunoprecipitant as backgrounds.…”