N-Terminus Does Not Govern Protein Turnover of Schizosaccharomyces pombe CENP-A

Abstract

Centromere integrity underlies an essential framework for precise chromosome segregation and epigenetic inheritance. Although centromeric DNA sequences vary among different organisms, all eukaryotic centromeres comprise a centromere-specific histone H3 variant, centromeric protein A (CENP-A), on which other centromeric proteins assemble into the kinetochore complex. This complex connects chromosomes to mitotic spindle microtubules to ensure accurate partitioning of the genome into daughter cells. Overexpression of CENP-A is associated with many cancers and is correlated with its mistargeting, forming extra-centromeric kinetochore structures. The mislocalization of CENP-A can be counteracted by proteolysis. The amino (N)-terminal domain (NTD) of CENP-A has been implicated in this regulation and shown to be dependent on the proline residues within this domain in Saccharomyces cerevisiae CENP-A, Cse4. We recently identified a proline-rich GRANT motif in the NTD of Schizosaccharomyces pombe CENP-A (SpCENP-A) that regulates the centromeric targeting of CENP-A via binding to the CENP-A chaperone Sim3. Here, we investigated whether the NTD is required to confer SpCENP-A turnover (i.e., counter stability) using various truncation mutants of SpCENP-A. We show that sequential truncation of the NTD did not improve the stability of the protein, indicating that the NTD of SpCENP-A does not drive turnover of the protein. Instead, we reproduced previous observations that heterochromatin integrity is important for SpCENP-A stability, and showed that this occurs in an NTD-independent manner. Cells bearing the null mutant of the histone H3 lysine 9 methyltransferase Clr4 (Δclr4), which have compromised constitutive heterochromatin integrity, showed reductions in the proportion of SpCENP-A in the chromatin-containing insoluble fraction of the cell extract, suggesting that heterochromatin may promote SpCENP-A chromatin incorporation. Thus, a disruption in heterochromatin may result in the delocalization of SpCENP-A from chromatin, thus exposing it to protein turnover. Taken together, we show that the NTD is not required to confer SpCENP-A protein turnover.

Low-Dose Radiation Can Cause Epigenetic Alterations Associated With Impairments in Both Male and Female Reproductive Cells

“…In addition, histone modifications including activation markers (H3K4me1 and H3K4me3) and silencing marks (H3K27me3) at distal promoters were telomere length dependent, suggesting that the epigenetic state of telomere-distal promoters could be influenced by telomere length ( Mukherjee et al, 2018 ). Moreover, meiotic chromosome segregation is essential for the maintenance of genomic integrity of gametes and requires functional centromeres that are required for a precise epigenetic inheritance ( Mahlke and Nechemia-Arbely, 2020 ; Tan et al, 2020 ).…”

E3 Ligase for CENP-A (Part 2)

“…Meanwhile, the functions of the N-terminal CENP-A are also reported for some species [16][17][18][19][20][21][22][23][24][25][26][27] (see also previous chapter, Sections 2.1, 2.2.2, 2.4.1, 2.9 and this chapter, Sections 2.1, 2.3, 2.4, 3.1, 4.1, 4.6, and 5.1). Loading of CENP-A at centromeres and its incorporation/deposition and maintenance in centromeric chromatin is cell cycle-regulated.…”

“…Domain-specific function, such as the N-terminal function, of fission yeast Cnp1/ CENP-A is also reported as budding yeast Cse4 [24,25] (see also previous chapter, Section 2.4). Folco et al demonstrated that alteration of the Cnp1 N-tail does not affect Cnp1 loading at centromeres, outer kinetochore recruitment, or spindle checkpoint signaling but significantly increases chromosome loss [17].…”

“…However, they suggest that the GRANT-prolines of Cnp1 do not coordinate proteolysis of the SpCENP-A protein as do proline residues in the budding yeast Cse4 NTD. In addition, Tan et al showed that sequential truncation of the NTD did not improve the stability of the protein, suggesting that the NTD of Cnp1 does not regulate the turnover of the protein [25]. Instead, they proposed that heterochromatin integrity may contribute to Cnp1 stability and promote its chromatin incorporation.…”

E3 Ligase for CENP-A (Part 1)

“…Their results using cse4KR mutants suggest that Psh1 is not the sole regulator of Cse4 proteolysis and that Doa1 facilitates Cse4 N-terminus-dependent proteolysis. We also note that N-terminal functions of CENP-A were described for some species [12,28,45,[47][48][49][50][51][52][53][54][55] (see also Sections 1.1, 1.4.1, 1.9 and next chapter, sections 2.1, 2.3, 2.4, 3.1, 4.1, 4.6, and 5.1).…”