Spt2 is a chromatin component with roles in transcription and posttranscriptional regulation. Recently, we found that Spt2 travels with RNA polymerase II (RNAP II), is involved in elongation, and plays important roles in chromatin modulations associated with this process. In this work, we dissect the function of Spt2 in the repression of SER3. This gene is repressed by a transcription interference mechanism involving the transcription of an adjacent intergenic region, SRG1, that leads to the production of a noncoding RNA (ncRNA). We find that Spt2 and Spt6 are required for the repression of SER3 by SRG1 transcription. Intriguingly, we demonstrate that these effects are not mediated through modulations of the SRG1 transcription rate. Instead, we show that the SRG1 region overlapping the SER3 promoter is occluded by randomly positioned nucleosomes that are deposited behind RNAP II transcribing SRG1 and that their deposition is dependent on the presence of Spt2. Our data indicate that Spt2 is required for the major chromatin deposition pathway that uses old histones to refold nucleosomes in the wake of RNAP II at the SRG1-SER3 locus. Altogether, these observations suggest a new mechanism of repression by ncRNA transcription involving a repressive nucleosomal structure produced by an Spt2-dependent pathway following RNAP II passage.

During mitosis, Bub1 kinase phosphorylates histone H2A-T120 to promote centromere sister chromatid cohesion through recruitment of shugoshin (Sgo) proteins. The regulation and dynamics of H2A-T120 phosphorylation are poorly understood. Using quantitative phosphoproteomics we show that Bub1 is autophosphorylated at numerous sites. We confirm mitosis-specific autophosphorylation of a several residues and show that Bub1 activation is primed in interphase but fully achieved only in mitosis. Mutation of a single autophosphorylation site T589 alters kinetochore turnover of Bub1 and results in uniform H2A-T120 phosphorylation and Sgo recruitment along chromosome arms. Consequently, improper sister chromatid resolution and chromosome segregation errors are observed. Kinetochore tethering of Bub1-T589A refocuses H2A-T120 phosphorylation and Sgo1 to centromeres. Recruitment of the Bub1-Bub3-BubR1 axis to kinetochores has recently been extensively studied. Our data provide novel insight into the regulation and kinetochore residency of Bub1 and indicate that its localization is dynamic and tightly controlled through feedback autophosphorylation.

Background: The N terminus is required for localization and functions of Mps1, Bub1, and BubR1 kinases.Results: A novel Bub1/BubR1-related TPR motif is identified in Mps1 and is required for kinase activity.Conclusion: TPR domain of Mps1 regulates kinase activity, Mps1 chromosome alignment, and checkpoint functions.Significance: Identification of a novel domain in Mps1 enhances our understanding of its contribution to maintaining genome integrity.

Tyrosine phosphorylation is closely associated with cell proliferation. During the cell cycle, serine and threonine phosphorylation plays the leading role, and such phosphorylation events are most dynamic during the mitotic phase of the cell cycle. However, mitotic phosphotyrosine is not well characterized. Although a few functionally-relevant mitotic phosphotyrosine sites have been characterized, evidence suggests that this modification may be more prevalent than previously appreciated. Here, we examined tyrosine phosphorylation in mitotic human cells including those on spindle-associated proteins.? Database mining confirmed ~2000 mitotic phosphotyrosine sites, and network analysis revealed a number of subnetworks that were enriched in tyrosine-phosphorylated proteins, including components of the kinetochore or spindle and SRC family kinases. We identified Polo-like kinase 1 (PLK1), a major signaling hub in the spindle subnetwork, as phosphorylated at the conserved Tyr in the kinase domain. Substitution of Tyr with a phosphomimetic residue eliminated PLK1 activity in vitro and in cells. Further analysis showed that Tyr phosphorylation reduced the phosphorylation of Thr in the activation loop, a phosphorylation event necessary for PLK1 activity. Our data indicate that mitotic tyrosine phosphorylation regulated a key serine/threonine kinase hub in mitotic cells and suggested that spatially separating tyrosine phosphorylation events can reveal previously unrecognized regulatory events and complexes associated with specific structures of the cell cycle.