Proteomics analysis of the centromere complex from HeLa interphase cells: UV-damaged DNA binding protein 1 (DDB-1) is a component of the CEN-complex, while BMI-1 is transiently co-localized with the centromeric region in interphase.
CENP-A, a centromere-specific histone H3, is conserved throughout eukaryotes, and formation of CENP-A chromatin defines the active centromere region. Here, we report the isolation of CENP-A chromatin from HeLa interphase nuclei by chromatin immunoprecipitation using anti-CENP-A monoclonal antibody, and systematic identification of its components by mass spectrometric analyses. The isolated chromatin contained CENP-B, CENP-C, CENP-H, CENP-I/hMis 6 and hMis 12 as well as CENP-A, suggesting that the isolated chromatin may represent the centromere complex (CEN-complex). Mass spectrometric analyses of the CEN-complex identified approximately 40 proteins, including the previously reported centromere proteins and the proteins of unknown function. In addition, we unexpectedly identified a series of proteins previously reported to be related to functions other than chromosome segregation, such as uvDDB-1, XAP8, hSNF2H, FACTp180, FACTp80/SSRP1, polycomb group proteins (BMI-1, RING1, RNF2, HPC3 and PHP2), KNL5 and racGAP. We found that uvDDB-1 was actually localized to the centromeric region throughout cell cycle, while BMI-1 was transiently co-localized with the centromeres in interphase. These results give us new insights into the architecture, dynamics and function of centromeric chromatin in interphase nuclei, which might reflect regulation of cell proliferation and differentiation.
Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan.
Chikashi
C
Obuse
Hua
H
Yang
Naohito
N
Nozaki
Shouhei
S
Goto
Tuneko
T
Okazaki
Kinya
K
Yoda
D001324:Autoantigens / Q000032:analysis* / Q000302:isolation & purification; D002503:Centromere / Q000737:chemistry*; D000076247:Centromere Protein A; D002843:Chromatin / Q000737:chemistry / Q000302:isolation & purification; D002868:Chromosomal Proteins, Non-Histone / Q000032:analysis* / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000032:analysis* / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry; D006801:Humans; D007399:Interphase; D013058:Mass Spectrometry; D009687:Nuclear Proteins / Q000032:analysis*; D063150:Polycomb Repressive Complex 1; D011233:Precipitin Tests; D040901:Proteomics / Q000379:methods; D011518:Proto-Oncogene Proteins / Q000032:analysis*; D034741:RNA, Small Interfering / Q000494:pharmacology; D012097:Repressor Proteins / Q000032:analysis*
D001324:Autoantigens; C069077:BMI1 protein, human; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; C091435:DDB1 protein, human; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D063150:Polycomb Repressive Complex 1