Munira A. Basrai, Ph.D.
Building 41, Room B629B
Bethesda, MD 20892-5055
Molecular Determinants of Faithful Chromosome Transmission and Cell Cycle Checkpoint Regulation
A fundamental requirement of the cell division cycle is the maintenance, replication, and segregation of chromosomal DNA. Failure of complex mechanisms involved in maintaining genome integrity has been implicated in cancer, aging, and congenital birth defects. Research in our laboratory focuses on the molecular mechanisms of high fidelity chromosome transmission, the organization of chromatin structure, and the checkpoint regulatory mechanisms that ensure the proper execution of the cell cycle in S. cerevisiae and the study of its human homologs. The high degree of conservation between S. cerevisiae and human genes makes budding yeast an excellent model system to molecularly dissect the interplay between centromeric chromatin, cell cycle regulation, gene dosage, and genome stability. Our research is focused on understanding the assembly and regulation of centromeric chromatin for faithful chromosome segregation.
We have pioneered studies in these areas and have shown that a balanced stoichiometry of centromere binding kinetochore proteins, topology of centromeric chromatin, post-translational modifications of centromeric histones, and haploinsufficiency (HI) of the conserved γ-tubulin complex affect faithful chromosome segregation. Our studies have also led to the identification of about 300 new genes in budding yeast. These results highlight the evolutionary conservation of pathways required for genome stability in yeast and humans and demonstrate that the yeast model system can be used to study the fundamental process of chromosome segregation.
A) The assembly and regulation of centromeric chromatin for faithful chromosome segregation. Evolutionarily conserved Cse4, the centromeric histone H3 variant (CENP-A in humans) and its chaperone Scm3 (HJURP in humans), both of which are essential for chromosome segregation, are overexpressed and mis-localized in many cancers. Patients with elevated HJURP expression show a reduced survival rate. The role of HJURP overexpression in tumorigenesis is not yet understood. We are investigating the molecular mechanisms that regulate expression and localization of Cse4/CENP-A and its interacting proteins Scm3/HJURP and Pat1 for faithful chromosome segregation. We have shown that the imbalanced stoichiometry of HJURP and Scm3 lead to chromosome mis-segregation in both human and yeast cells thereby providing a link between HJURP overexpression and mitotic defects in cancers (Mishra et al., 2011). Future studies will utilize genome-wide screens to identify genes/pathways that show lethality with overexpression of HJURP for possible treatment of cancers with deregulated HJURP expression. Scm3 interacts with Pat1 (Protein associated with topoisomerase II) and we have uncovered a role for Pat1 in the topology of centromeric chromatin and chromosome segregation (Mishra et al., 2013). We used a pat1 deletion strain to define the number of Cse4 molecules at the yeast kinetochore (Hasse, Mishra 2013, Mishra et al., 2015). Our results show that Pat1 regulates the structural integrity of centromeric chromatin and localization of Cse4 for faithful chromosome segregation. Ongoing research is aimed at understanding how topology of centromeric chromatin affects chromosome segregation an area of research that is largely unexplored at the present time. In addition to kinetochore proteins, association of cohesins with centromeres and along the length of the chromosomes ensures faithful segregation of sister chromatids during mitosis. Our studies have shown that evolutionarily conserved polo kinase, Cdc5 associates with centromeric chromatin to facilitate the removal of centromeric cohesins (Mishra et al., 2016). Future studies will allow us to understand the mechanism by which Cdc5 regulates kinetochore structure.
B) Post-translational modifications (PTMs) of centromeric histones affect chromosome segregation. We investigated the nature and role of PTMs of Cse4 and centromeric histone H4 in chromosome segregation. Distinctive acetylation pattern of centromeric histone H4 has been previously reported in other systems, however, the physiological role for this pattern is not fully understood. Using budding yeast as a model, we determined that the acetylation pattern of centromeric histone H4 affects chromosome segregation. We provide the first evidence that yeast centromeres contain hypoacetylated histone H4 and that increased acetylation of histone H4 on lysine 16 (H4K16) leads to chromosome mis-segregation (Choy et al., 2011). A balance in H4K16 acetyltransferase, Sas2, and H4K16 deacetylase, Sir2, is required for faithful chromosome segregation. Notably, both Sas2 and Sir2 have human homologs. Even though HDAC inhibitors (HDACi) are used in clinical trials we do not fully understand their mode of action. Hence, we performed a genome-wide screen with an HDACi to identify pathways that are vulnerable to altered histone acetylation. Our results showed that chromosome segregation mutants are more sensitive to HDACi. Future studies will examine if combining HDACi with drugs that affect chromosome segregation are more effective for cancer treatment with a minimal effect on normal cells.
Our studies for acetylation of centromeric H4 prompted us to investigate the role of PTM of Cse4. We determined that phosphorylation and methylation of Cse4 regulate chromosome segregation. An innovative approach for the biochemical purification of Cse4, allowed us to provide the first comprehensive analysis of PTMs of Cse4 (Boeckmann et al., 2013). Conserved sites for acetylation, methylation, and phosphorylation in Cse4 were identified. We generated a phospho-specific antibody and showed the association of phosphorylated Cse4 with centromeres and determined that evolutionarily conserved Aurora B/Ipl1 kinase phosphorylates Cse4 in vivo and in vitro for faithful chromosome segregation. Future studies will allow us to understand the molecular role of Cse4 phosphorylation and methylation in chromosome segregation and determine if these PTMs are conserved in human CENP-A.
Overexpression and mislocalization of CENP-A to euchromatin are observed in colorectal cancers and lead to aneuploidy in flies. Our research is aimed at identifying pathways that, when disrupted, will lead to selective killing of CENP-A overexpressing cancer cells. We showed previously that S. cerevisiae spt4 mutants show mislocalization of Cse4 and chromosome segregation defects that are complemented by human SPT4 (Crotti and Basrai 2004). We have now established the cause and effect of Cse4 mislocalization by showing that altered histone dosage and mislocalization of Cse4 to euchromatin correlate with chromosome loss (Au et al., 2008). One mechanism that prevents mislocalization of Cse4 is ubiquitin-mediated proteolysis of Cse4 by E3 ligase Psh1. We identified a novel role for the N terminus of Cse4 in ubiquitin (Ub)-mediated proteolysis for faithful chromosome segregation (Au et al., 2013). We recently reported that Cse4 is sumoylated and ubiquitination of sumoylated Cse4 by Slx5 regulates its proteolysis to prevent mislocalization to euchromatin (Ohkuni et al., 2016). We have undertaken genome-wide approaches to identify regulators that prevent mislocalization of Cse4 to euchromatin. Our studies have revealed a role for histone chaperones and other E3 Ub ligases in Cse4 proteolysis. We have now established a cell biology based reporter system to investigate the causes and consequences of CENP-A mislocalization in human cells. These studies have allowed us to conclude that misllocalization of CENP-A contiibutes to aneuploidy and the function of human homologs of the yeast genes we have identified is evolutionraily conserved. Our long-term objective is to identify pathways that, when disrupted, will lead to selective killing of cancer cells overexpressing CENP-A.
C) Functional genomics of genes with small open reading frames (sORFs). Genes with sORFs (<100 amino acids) represent an untapped source of important biology. sORFs largely escaped analysis since they were difficult to predict computationally and less likely to be targeted by genetic screens. Thus, the substantial number of sORFs and their potential importance has only recently become clear. To investigate sORF function, we undertook the first functional studies of sORFs in any system, using the model eukaryote Saccharomyces cerevisiae (Velculescu et al., 1997, Kastenmayer et al., 2006). Based on independent experimental approaches and computational analyses, evidence now exists for 299 sORFs in the S. cerevisiae genome representing about 5% of annotated S. cerevisiae ORFs. We determined that a similar percentage sORFs are annotated in other eukaryotes, including humans, and 184 of the S. cerevisiae sORFs exhibit significant similarity with ORFs in other organisms. To investigate sORF function, we constructed a collection of gene-deletion mutants of 141 newly identified sORFs, each of which contains a strain-specific 'molecular bar code', bringing the total number of sORF deletion strains to 248. Phenotypic analyses identified 20 sORFs required for haploid growth, growth at high temperature, growth in the presence of a non-fermentable carbon source or growth in the presence of DNA damage and replication arrest agents. We provide a collection of sORF deletion strains that can be integrated into the existing deletion collection as a resource for the yeast community for elucidating gene function. Further studies with one of the sORFs have led to the annotation of HUG1 (HU, UV, Gamma) induced gene (Basrai et al., 1999). We determined that Hug1 is a critical downstream effector of the evolutionarily conserved MEC1/ATM pathway that regulates recovery from checkpoint arrest (Ainsworth et al., 2014). Our analyses of S. cerevisiae sORFs establish that sORFs are conserved across eukaryotes and have important biological functions.
Dr. Basrai received her Ph.D. from the University of Tennessee at Knoxville and did her postdoctoral research with Dr. Philip Hieter in the jDepartment of Molecular Biology and Genetics at Johns Hopkins University School of Medicine in Maryland. Dr. Basrai joined the Genetics Branch at the NCI in 1998 and was promoted to tenure in 2006. Her research interests are genome stability and cell cycle regulation in yeast and human cancers. Dr. Basrai co-chairs the Cell Cycle Interest Group since 2005 and serves on the steering committee for the Center of Excellence of Chromosome Biology (CECB) since 2007. Dr. Basrai served on the Cancer Advisory Board for three years (2008-2011). Dr. Basrai has received the NIH Director’s Award and the NIH Award for Outstanding Mentorship towards the education of post-baccalaureate trainees. Her trainees have recieved numerous awards such as NIH Fellows Award for Excellence in Research and exceptional pay increase.
Shrestha RL, Ahn GS, Staples MI, Sathyan KM, Karpova TS, Foltz DR, Basrai MA. Mislocalization of centromeric histone H3 variant CENP-A contributes to chromosomal instability (CIN) in human cells. Oncotarget. 2017;8(29):46781-46800.
Ohkuni K, Takahashi Y, Fulp A, Lawrimore J, Au WC, Pasupala N, Levy-Myers R, Warren J, Strunnikov A, Baker RE, Kerscher O, Bloom K, Basrai MA. SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin. Mol Biol Cell. 2016.
Mishra PK, Ciftci-Yilmaz S, Reynolds D, Au WC, Boeckmann L, Dittman LE, Jowhar Z, Pachpor T, Yeh E, Baker RE, Hoyt MA, D'Amours D, Bloom K, Basrai MA. Polo kinase Cdc5 associates with centromeres to facilitate the removal of centromeric cohesin during mitosis. Mol Biol Cell. 2016;27(14):2286-300.
Mishra PK, Guo J, Dittman LE, Haase J, Yeh E, Bloom K, Basrai MA. Pat1 protects centromere-specific histone H3 variant Cse4 from Psh1-mediated ubiquitination. Mol Biol Cell. 2015;26(11):2067-79.
Choy JS, O'Toole E, Schuster BM, Crisp MJ, Karpova TS, McNally JG, Winey M, Gardner MK, Basrai MA. Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability. Mol Biol Cell. 2013;24(17):2753-63.
Related Scientific Focus Areas
Genetics and Genomics
Molecular Biology and Biochemistry
This page was last updated on July 23rd, 2017