Plc1p is required for proper chromatin structure and activity of the kinetochore in Saccharomyces cerevisiae by facilitating recruitment of the RSC complex

Abstract

High-fidelity chromosome segregation during mitosis requires kinetochores, protein complexes that assemble on centromeric DNA and mediate chromosome attachment to spindle microtubules. In budding yeast, phosphoinositide-specific phospholipase C (Plc1p encoded by PLC1 gene) is important for function of kinetochores. Deletion of PLC1 results in alterations in chromatin structure of centromeres, reduced binding of microtubules to minichromosomes, and a higher frequency of chromosome loss. The mechanism of Plc1p's involvement in kinetochore activity was not initially obvious; however, a testable hypothesis emerged with the discovery of the role of inositol polyphosphates (InsPs), produced by a Plc1p-dependent pathway, in the regulation of chromatin-remodeling complexes. In addition, the remodels structure of chromatin (RSC) chromatin-remodeling complex was found to associate with kinetochores and to affect centromeric chromatin structure. We report here that Plc1p and InsPs are required for recruitment of the RSC complex to kinetochores, which is important for establishing proper chromatin structure of centromeres and centromere proximal regions. Mutations in PLC1 and components of the RSC complex exhibit strong genetic interactions and display synthetic growth defect, altered nuclear morphology, and higher frequency of minichromosome loss. The results thus provide a mechanistic explanation for the previously elusive role of Plc1p and InsPs in kinetochore function.

Facilitated Assembly of the Preinitiation Complex by Separated Tail and Head/Middle Modules of the Mediator

“…In vivo chromatin crosslinking and immunoprecipitation were performed as described previously 59,61,64 with the following antibodies: anti-myc polyclonal antibody A-14, anti-HA monoclonal antibody F7, anti-Yap1p polyclonal antibody (all Santa Cruz Biotechnology), and anti-RNA polymerase II monoclonal antibody 8WG16 (Covance). Primers used for real-time PCR analysis are as follows: POL1 (5’-TCCTGACAAAGAAGGCAATAGAAG-3’and 5’-TAAAACACCC TGATCCACCTCTG -3’), FLR1 promoter (5’-AATGGGCGGGATAATTAGTCAG-3’ and 5’-GTGTGTCTGTACGTT GAAGTGTATACC-3’), FLR1 , middle of the coding region (5’-GTTGGTTGTGCTACTGT GCATAAC-3’ and 5’-AAACGAGAGGAACCATTTCTGG-3’), FLR1 , 3’ end of the coding region (5’-TACCCAAAGTATGTTGCATCCG-3’ and 5’-CCATGCCACAGGATAGTTCTT AGT-3’), PHO5 (5’-CCATTTGGGATAAGGGTAAACATC-3’ and 5’-AGAGATGAAGCCATACTAACCTCG -3’), GAL1 (5’-CGCTTAACTGCTCATTGCTATATTG-3’ and 5’-TTGTTCGGAGCAGTGCGG-3’), TRX2 (5’-ATACGACAGTGCTTTAGCATCTGG-3’ and 5’-GTTCTGCAAACTTTTCAATCATTG-3’), BNA1 (5’-GCGTCAGGATAAATTGTGAGTCAC-3’ and CCTTCGTTCTCCTTCAACCATTTG-3’), HSP12 (5’-AAGCACTCTAGACGGAGAGTAACTAG-3’ and 5’-GAATCCTTTTCTACCTGCGTCAG-3’), HSP26 (5’-CGGATCTATGCACGTTCTTGAGTG-3’ and 5’-CCTTCACCCAGCAATCTGTTAAAG-3’).…”

Roles of inositol phosphates and inositol pyrophosphates in development, cell signaling and nuclear processes

“…Recently, Vancura’s group showed that Plc1 and IPs are involved in regulation of activity of the kinetochore by facilitating recruitment of another chromatin remodeling complex, RSC, which stands for Remodels Structure of Chromatin. RSC has been shown to be required for maintaining the structure of the centromere and for proper kinetochore function (Cairns et al, 1996; Desai et al, 2009). …”

Tension-dependent nucleosome remodeling at the pericentromere in yeast

“…Loss of Sth1p/Nps1p function results in reduced histone occupancy around the centromere by nucleosome-scanning assay and chromatin immunoprecipitation (Desai et al , 2009) and leads to cell cycle arrest at G2/M. Nucleosomes flanking the centromere are subject to disruptive tension from the spindle, which would require removing mislocalized nucleosomes and reloading them in the proper position.…”