GRANT Motif Regulates CENP-A Incorporation and Restricts RNA Polymerase II Accessibility at Centromere

Abstract

Precise chromosome segregation is essential for maintaining genomic stability, and its proper execution centers on the centromere, a chromosomal locus that mounts the kinetochore complex to mediate attachment of chromosomes to the spindle microtubules. The location of the centromere is epigenetically determined by a centromere-specific histone H3 variant, CENP-A. Many human cancers exhibit overexpression of CENP-A, which correlates with occurrence of aneuploidy in these malignancies. Centromeric targeting of CENP-A depends on its histone fold, but recent studies showed that the N-terminal tail domain (NTD) also plays essential roles. Here, we investigated implications of NTD in conferring aneuploidy formation when CENP-A is overexpressed in fission yeast. A series of mutant genes progressively lacking one amino acid of the NTD have been constructed for overexpression in wild-type cells using the intermediate strength nmt41 promoter. Constructs hosting disrupted GRANT (Genomic stability-Regulating site within CENP-A N-Terminus) motif in NTD results in growth retardation, aneuploidy, increased localization to the centromere, upregulated RNA polymerase II accessibility and transcriptional derepression of the repressive centromeric chromatin, suggesting that GRANT residues fine-tune centromeric CENP-A incorporation and restrict RNA polymerase II accessibility. This work highlighted the importance of CENP-A NTD, particularly the GRANT motif, in aneuploidy formation of overexpressed CENP-A in fission yeast.

Set2 regulates Ccp1 and Swc2 to ensure centromeric stability by retargeting CENP-A

“…We noted that shifting to 33°C resulted in a reduction in SpCENP-A cnp1-1 protein levels to a similar extent in both WT and Δset2 (Figure 1F ). To ensure that the decrease centromeric binding of SpCENP-A cnp1-1 was not impacted by such reduction, we increased the protein level of SpCENP-A cnp1-1 via overexpressing from an inducible nmt41 promoter ( 41 , 60 ). Under this condition, we still observed delocalization of overexpressed SpCENP-A cnp1-1 from the centromere upon shifting the cells to the restrictive temperature ( Supplementary Figure S1A and B ).…”

“…To further ascertain whether Swc2 may mediate the cnp1-1 suppression by Δset2 , we overexpressed swc2 + in cnp1-1 and Δset2cnp1-1 , using the inducible nmt41 promoter ( 41 , 62 ) ( Supplementary Figure S8 ). Overexpression of swc2 + resulted in approximately 25-fold suppression of cnp1-1 at the restrictive temperature of 33°C relative to the vector control, as observed previously (Figure 4A ).…”

“…The detection of transcripts arising from centromeric sequences is correlated with the disruption of centromeric chromatin ( 24 , 39–42 ), and is probably associated with the uninhibited access of RNAPII, which disrupts the compacted chromatin of the centromere and facilitates the delocalization of centromere-associated factors including CENP-A ( 41 ). CENP-A can be delocalized from the centromere under several scenarios that may include abnormal disruption of centromere chromatin, or overexpression of CENP-A.…”

Resistance to Chemotherapeutic 5-Fluorouracil Conferred by Modulation of Heterochromatic Integrity through Ino80 Function in Fission Yeast

“…Standard microscopy procedure was carried out as previously described [ 82 , 84 , 85 , 86 ]. Log-phase cells were treated with the appropriate concentrations of drug for 4 h at 30 °C before fixation in 1/10 volume glutaraldehyde on ice for 15 min, washed three times with cold 1x phosphate-buffered saline (PBS).…”