Aurora-A mediated histone H3 phosphorylation of threonine 118 controls condensin I and cohesin occupancy in mitosis

Abstract

Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation.DOI: http://dx.doi.org/10.7554/eLife.11402.001

Excess free histone H3 localizes to centrosomes for proteasome-mediated degradation during mitosis in metazoans

“…11 In order to focus on the centrosomal localization of H3 T118ph, we used immunofluorescence analysis of HeLa cells with methanol fixation, which prevents the H3 T118ph antibody from accessing its epitope within chromosomes. We found that H3 T118ph colocalized with the centrosomal protein g-tubulin during prophase through anaphase of mitosis (Fig.…”

“…To do this, we created a panel of stable cell lines using the HEK 293TR Flp-in system (Invitrogen) expressing histone H3:FLAG, H3 T118E:FLAG (to mimic constitutive phosphorylation), H3 T118I:FLAG (to mimic the yeast SIN mutant 24 ), as well as T118A-FLAG (unmodifiable) from the same locus. 11 Structurally, H3 T118I more closely resembles the effect of H3 T118 phosphorylation than H3 T118E because the isoleucine side chain would mimic the rigidity of phosphate group compared to the flexible side chain of glutamic acid. Functionally, H3 T118I caused stronger phenotypes than H3 T118E and that were identical to overexpression of the H3 T118 kinase, Aurora-A.…”

“…Recently, our lab has discerned that two histone H3 modifications, phosphorylation of H3 threonine 118 (H3 T118ph) 11 and phosphorylation of H3 threonine 80 (H3 T80ph), 12 exist at the centrosome during mitosis. The centrosome is the microtubuleorganizing center important for nucleating and orchestrating microtubule spindle dynamics to achieve equal segregation of genomic DNA into daughter cells during mitosis.…”

“…22 Therefore, it was important for us to validate that histone H3 localizes to centrosomes using a method that was independent of H3 T118ph antibodies. Using a stable cell line expressing H3-FLAG generated using 293TR Flp-in technology (Invitrogen), 11 FLAG-tagged H3 was observed at the centrosomes specifically during mitosis, but only upon inhibition of the proteasome by MG132 treatment (Fig. 3C).…”

“…This is likely due to the much higher abundance of H3 on the chromatin and the efficient degradation of free H3, precluding its detection at centrosomes. However, the fact that overexpression of H3-FLAG and H3-YFP (to about 5% the level of endogenous histones 11 ) allows detection of H3 at the centrosomes upon proteasome inhibition (Fig. 3C, D), indicates that excess free H3 localizes to the centrosomes during mitosis for degradation.…”