Therefore, epigenetic regulation is at present focused on in the field of autoimmune diseases

s PBTZ 169 chemical information Aurora B. Adding nocodazole along with reversine and HI resulted in a significant drop in Aurora B levels compared with reversine and HI. Inhibition EB1 and microtubules control Aurora B recruitment Banerjee et al. 951 952 JCB VOLUME 204 NUMBER 6 2014 of MPS1 by 1 M reversine has been previously shown to not affect Aurora B levels. However, we note that these experiments were performed in nocodazole, in which we observe reduced levels of Aurora B. In agreement with the previous study, we find Aurora B levels in nocodazole were not substantially reduced upon addition of reversine. We conclude that microtubules can recruit Aurora B to inner centromeres independent of the histone phosphorylation pathways, but the histone phosphorylation pathways are required to obtain the full enrichment of centromeric Aurora B. These data suggest that EB1 and microtubules are either upstream or work in combination with histone phosphorylation pathways that recruit CPC. We asked whether targeting Aurora B to centromeres suppressed the reduction of the histone phosphorylation pathways observed after EB1 depletion. We depleted EB1 from U2OS-TR cells containing an integrated transgene encoding CENPB1158 fused to INCENP47920 driven by a doxycycline-inducible promoter. CENPB1158 binds -satellite DNA sequences at the centromere so that Aurora B is targeted independent of the normal pathways. INCENP47920 cannot bind Survivin and Borealin but does bind Aurora B. EB1 depletion in U2OS-TR cells also reduced phosphoKNL1 and Bub1 at kinetochores and phosphohistone H3Thr3 levels when the CENPB-INCENP was not expressed. However, expressing CENPB-INCENP in EB1-depleted cells rescued both Bub1 and phospho-KNL1 levels PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19834545 at the kinetochore bypassing the requirement of EB1. We also observed recovery of phospho-H3Thr3 levels on CENPB-INCENP expression. We conclude that the EB1microtubule pathway works either upstream or is interdependent with the histone H2A and H3 phosphorylation pathways to recruit Aurora B to the centromeres. EB1 interacts with Aurora B at the centromeres in prometaphase immunofluorescence staining with anti-EB1 and anti-Borealin antibodies showed spindle and inner centromere localization as previously shown. However, most EB1Aurora B interactions identified by PLA were only found near inner centromeres in prometaphase cells. This was confirmed by costaining for Borealin in these cells. Similarly, EB1Aurora B interactions were proximal to microtubuleinner centromere junctions in metaphasealigned chromosomes. Consistent with the fact that there are no microtubules within the prophase nuclei, we don’t see any EB1Aurora B interactions in prophase cells. To verify the specificity of the interaction, we performed PLA reactions lacking either the EB1 or the Aurora B primary antibodies, and very little PLA signal was produced. We conclude that subsets of EB1 and Aurora B interact near or at inner centromeres, which is consistent with the regulation of Aurora B localization by EB1. Aurora B interacts with K-fibers, preKfibers, and astral microtubules in mitosis Recombinant Xenopus Aurora B bound to a C-terminal fragment of Xenopus INCENP that includes the IN-box purified from Escherichia coli has a basal amount of activity. The addition of microtubules stimulated Aurora B kinase activity in vitro between four- to sixfold on a myelin basic protein substrate over a range of kinase concentrations. However, the addition of EB1 did not further stimulate kinase activity