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CENP-B promotes the centromeric localization of ZFAT to control transcription of noncoding RNA.

ABSTRACT: The centromere is a chromosomal locus that is essential for the accurate segregation of chromosomes during cell division. Transcription of noncoding RNA (ncRNA) at the centromere plays a crucial role in centromere function. The zinc-finger transcriptional regulator ZFAT binds to a specific 8-bp DNA sequence at the centromere, named the ZFAT box, to control ncRNA transcription. However, the precise molecular mechanisms by which ZFAT localizes to the centromere remain elusive. Here we show that the centromeric protein CENP-B is required for the centromeric localization of ZFAT to regulate ncRNA transcription. The ectopic expression of CENP-B induces the accumulation of both endogenous and ectopically expressed ZFAT protein at the centromere in human cells, suggesting that the centromeric localization of ZFAT requires the presence of CENP-B. Coimmunoprecipitation analysis reveals that ZFAT interacts with the acidic domain of CENP-B, and depletion of endogenous CENP-B reduces the centromeric levels of ZFAT protein, further supporting that CENP-B is required for the centromeric localization of ZFAT. In addition, knockdown of CENP-B significantly decreased the expression levels of ncRNA at the centromere where ZFAT regulates the transcription, suggesting that CENP-B is involved in the ZFAT-regulated centromeric ncRNA transcription. Thus, we concluded that CENP-B contributes to the establishment of the centromeric localization of ZFAT to regulate ncRNA transcription.

SUBMITTER: Ishikura S

PROVIDER: S-EPMC8496178 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Functional centromeres are essential for proper cell division. Centromeres are established largely by epigenetic processes resulting in incorporation of the histone H3 variant CENP-A. Here, we demonstrate the direct involvement of H2B monoubiquitination, mediated by RNF20 in humans or Brl1 in Schizosaccharomyces pombe, in centromeric chromatin maintenance. Monoubiquinated H2B (H2Bub1) is needed for this maintenance, promoting noncoding transcription, centromere integrity and accurate chromosomal segregation. A transient pulse of centromeric H2Bub1 leads to RNA polymerase IIM-bM-^@M-^Smediated transcription of the centromereM-bM-^@M-^Ys central domain, coupled to decreased H3 stability. H2Bub1-deficient cells have centromere cores that, despite their intact centromeric heterochromatin barriers, exhibit characteristics of heterochromatin, such as silencing histone modifications, reduced nucleosome turnover and reduced levels of transcription. In the H2Bub1-deficient cells, centromere functionality is hampered, thus resulting in unequal chromosome segregation. Therefore, centromeric H2Bub1 is essential for maintaining active centromeric chromatin. ChIP: In total 17 samples; 15 ChIP DNA files (7 different conditions) 2 Input files. Cnp1_WT_input.bar (antibody against Cnp1 in strains Hu303, Hu29 (WT) (Cnp1_Hu303.CEL, AS_10.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); Cnp1_Hu2640_WT.bar (antibody against Cnp1 in strain Hu2640 (htb1K119R) (Cnp1_Hu2640_A.CEL, Cnp1_Hu2640_B.CEL) vs strains Hu303, Hu29 (WT) (Cnp1_Hu303.CEL, AS_10.CEL)); Cnp1_Hu2640_input.bar (antibody against Cnp1 in strain Hu2640 (htb1K119R) (Cnp1_Hu2640_A.CEL, Cnp1_Hu2640_B.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); H2Bub1_Hu303_input.bar (antibody against H2Bub1 in strain Hu303 (WT) (H2Bub1_Hu303_A.CEL, H2Bub1_Hu303_B.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); H3_WT_input.bar (antibody against H3 in strains Hu303, Hu29 (WT) (H3_Hu303.CEL, As_12.CEL) vs input (JW_1.CEL, JW_2_2.CEL); H3_Hu2640_WT.bar (antibody against H3 in strain Hu2640 (htb1K119R) (H3_Hu2640_A.CEL, H3_Hu2640_B.CEL, H3_Hu2640_C.CEL) vs strains Hu303, Hu29 (WT) (H3_Hu303.CEL, AS_12.CEL)); H3_Hu2640_input.bar (antibody against H3 in strain Hu2640 (htb1K119R) (H3_Hu2640_A.CEL, H3_Hu2640_B.CEL, H3_Hu2640_C.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); H3K9me2_Hu303_input.bar (antibody against H3K9me2 in strains Hu303, Hu29 (WT) (H3K9me2_Hu303.CEL, AS_6.CEL) vs input (JW_1.CEL, JW_2_2.CEL)); H3k9me2_Hu2640_Hu303.bar (antibody against H3K9me2 in strain Hu2640 (htb1K119R) (H3K9me2_Hu2640_A.CEL, H3K9me2_Hu2640_B.CEL) vs strain Hu303, Hu29 (WT) (H3K9me2_Hu303.CEL, AS_6.CEL)); H3K9me2_Hu2640_input.bar (antibody against H3K9me2 in strain Hu2640 (htb1K119R) (H3K9me2_Hu2640_A.CEL, H3K9me2_Hu2640_B.CEL) vs input (JW_1.CEL, JW_2_2.CEL)). RNA: 4 RNA samples: 2 replicates of WT RNA (RNA_Hu303_A.CEL, RNA_Hu303_B.CEL) and 2 replicates of htb1-K119R RNA (RNA_Hu2640_A.CEL, RNA_Hu2640_B.CEL).

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CENP-B promotes the centromeric localization of ZFAT to control transcription of noncoding RNA.

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