Mitotic Transcription Installs Sgo1 at Centromeres to Coordinate Chromosome Segregation

Abstract

Human sister chromatids at metaphase are primarily linked by centromeric cohesion, forming the iconic X shape. Premature loss of centromeric cohesion disrupts orderly mitotic progression. Shugoshin (Sgo1) binds to and protects cohesin at inner centromeres. The kinetochore kinase Bub1 phosphorylates histone H2A at T120 (H2A-pT120) and recruits Sgo1 to kinetochores, 0.5 μm from inner centromeres. Here, we show that Sgo1 is a direct reader of the H2A-pT120 mark. Bub1 also recruits RNA polymerase II (Pol II) to unattached kinetochores and promotes active transcription at mitotic kinetochores. Mitosis-specific inactivation of Pol II traps Sgo1 at kinetochores and weakens centromeric cohesion. Sgo1 interacts with Pol II in human cells and with RNA in vitro. We propose that Pol II-dependent transcription enables kinetochore-bound Sgo1 initially recruited by H2A-pT120 to reach cohesin embedded in centromeric chromatin. Our study implicates mitotic transcription in targeting regulatory factors to highly compacted mitotic chromatin.

Insights into centromeric transcription in mitosis

“…Thirdly, several groups, including us, took advantage of the transcription run-on assay and proved that the mitotic chromosomes, especially at centromeres, are able to be transcribed, at least by RNAP II. 10,11 Taken together, I conclude that transcription occurs at centromeres in mitosis.…”

“…Interestingly, I, together with others, found that centromeres are actively transcribed by RNAP II. 10,11 Several lines of evidence support this notion. Firstly, the presence of RNAP II at kinetochores in mitosis can be verified by antibody-based fluorescence microscopy: two antibodies raised against phosphor-CTD of Rpb1, 10,11 the largest subunit of RNAP II, and the GFP antibodies against ectopically expressed GFP-tagged Rpb2, 10 the second largest subunit of RNAP II.…”

“…10,11 Several lines of evidence support this notion. Firstly, the presence of RNAP II at kinetochores in mitosis can be verified by antibody-based fluorescence microscopy: two antibodies raised against phosphor-CTD of Rpb1, 10,11 the largest subunit of RNAP II, and the GFP antibodies against ectopically expressed GFP-tagged Rpb2, 10 the second largest subunit of RNAP II. Secondly, application of the transcriptional inhibitor a-amanitin resulted in significantly weakened centromeric cohesion and decreased CENP-C localization.…”

“…Secondly, application of the transcriptional inhibitor a-amanitin resulted in significantly weakened centromeric cohesion and decreased CENP-C localization. 10,11 Considering the specificity of a-amanitin toward RNAP II, 12,13 it is very likely that the phenotypes were derived from the inhibition of transcriptional activities of RNAP II. Thirdly, several groups, including us, took advantage of the transcription run-on assay and proved that the mitotic chromosomes, especially at centromeres, are able to be transcribed, at least by RNAP II.…”

“…I further demonstrated that the cohesion defects were a result of mislocalization of Sgo1, the cohesin protector, in response to transcription inhibition. 10 Sgo1, together with PP2A, localizes at inner centromeres, referred to the regions between two sister centromeres (Fig. 1), to protect the centromeric cohesion in mitosis, thus preventing chromosome missegregation and chromosome instability.…”