I. Chromosome organization and function:
The structural organization of chromosomes
influences various important chromosomal processes such as transcription,
recombination and repair, maintenance of genomic integrity and the accurate
inheritance of chromosomes during cell division. Perturbation of chromosome
organization can result in defects in these processes, which can manifest
as defects in normal growth, development and accumulation of chromosome
aberrations, observed in case of disease conditions such as cancer and
some developmental disorders.
The focus of research of my group is to understand the molecular basis of chromosome organization and explore its relation with these chromosomal processes. We utilize a multifaceted approach including molecular, genetic, cytological and biochemical tools to investigate these processes in the budding yeast Saccharomyces cerevisiae, an excellent model system for such studies. Since several aspects of chromosome organization and its regulation are conserved, we plan to extend our findings in yeast to human cell lines, as a step towards exploring the relevance of these findings in understanding human diseases involving chromosomal abnormalities.
A. Determinants of chromosome architecture and genomic integrity.
Eukaryotic chromosomes undergo extensive
structural reorganization in order to facilitate accurate segregation
during mitosis. Newly replicated sister chromatids are paired intimately
at centromeres and chromosome arms. Chromosomes are also condensed prior
to metaphase of mitosis. These structural attributes (cohesion and condensation)
are dependent on Smc (Structural maintenance of chromosomes) protein
complexes, which are evolutionarily conserved, essential for viability
and for maintenance of chromosome organization. Cohesin, Condensin and
the Smc5/6 complex are three well-known Smc complexes. Dysfunction of
cohesion proteins results in disorders in humans such as Cornelia de
Lange syndrome, Roberts syndrome and SC phocomelia, characterized by
severe developmental abnormalities, mental retardation, and neurodevelopmental
abnormalities, highlighting the biomedical relevance of functional studies
of Smc proteins.
As a step towards understanding the molecular basis of cohesion, I had earlier identified the chromosomal addresses of Mcd1p, a component of cohesin (4). Mcd1p is required for both cohesion and condensation. By using a combination of chromatin immunoprecipitation and quantitative PCR, we identified numerous cohesion associated regions (CARs) on single-copy and repetitive regions on chromosome arms. Our study also included the tandemly repeated ribosomal-DNA locus, which has been shown to be a target site for both cohesin and condensin action in vivo, and has one cohesin binding site (CARL3) per repeat. Our study revealed conserved spatial relationships of CARs with transcriptional units and some silenced chromatin domains; CARs are preferentially located in intergenic regions and some CARs overlap with known or putative boundaries of silenced regions. Recently, cohesin and the Smc5/6 complex have been implicated in the repair of DNA double strand breaks. Our current interest is directed toward understanding the regulation of chromatin association of Smc protein complexes and its relevance in maintaining chromosomal functions and preserving genomic integrity.
B. Determinants of Euchromatin.
In eukaryotic cells, genomic DNA exists as chromatin in association with histone octamers called nucleosomes, and various other chromatin proteins. Chromatin structure varies along the chromosome and this influences the state of gene expression. Based on such variations in structure and gene expression, chromatin can be broadly classified into euchromatin (transcriptionally active) and heterochromatin (transcriptionally repressed). Sequences termed silencing barriers or boundary elements can limit the spread of heterochromatin, thereby protecting nearby euchromatin from its repressive influence. In budding yeast , such sequences have been identified flanking the silent HMR mating locus on chromosome III. We have recently identified new silencing barrier sequences in budding yeast (3). We are interested in investigating the mechanism and requirements for the barrier activity of these sequences.
II. Ubiquitin E3 ligases.
Ubiquitin is a short polypeptide, which when conjugated to cellular proteins tags them for degradation by the proteasome or occasionally modifies their intracellular function or localization. The attachment of ubiquitin to target proteins is brought about by a multi-step enzymatic reaction usually involving a ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and a ubiquitin-ligase (E3), which catalyzes the conjugation of ubiquitin to appropriate target proteins. E3-ligases are particularly interesting because in addition to their catalytic activity, they determine substrate selectivity and specificity, which is critical for correct regulation of cellular processes. We are interested in identifying and characterizing functional domains and cellular targets of E3 ligase enzymes, with emphasis on those involved in cell cycle regulation and chromosome organization and function.
Kothiwal D, and Laloraya S. A SIR-independent role for cohesin in subtelomeric silencing and organization. Proc Natl Acad Sci U S A. 2019 Mar 19;116(12):5659-5664. doi: 10.1073/pnas.1816582116. Epub 2019 Mar 6.
Laloraya S. Asymmetric tyrosination of spindle microtubules facilitates selfish inheritance. Trends in Cell Biology. 2018 Jun;28(6):417-419. J doi: 10.1016/j.tcb.2018.03.005. Epub 2018 Apr 10.
Wani S, Maharshi N, Kothiwal D, Mahendrawada L, Kalaivani R, Laloraya S. Interaction of the Saccharomyces cerevisiae RING-domain protein Nse1 with Nse3 and the Smc5/6 complex is required for chromosome replication and stability. Curr Genet. 2018 Jun;64(3):599-617. doi: 10.1007/s00294-017-0776-6. Epub 2017 Nov 8.
Rai R., Varma S. P., Shinde N., Ghosh S., Kumaran S. P., Skariah G., and Laloraya S. Small Ubiquitin-related Modifier Ligase activity of Mms21 is required for maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in Saccharomyces cerevisiae. J Biol Chem. 2011 Apr 22;286(16):14516-30. Epub 2011 Feb 15.
Biswas M., Maqani N., Rai R., Kumaran S. P., Iyer K. R., Sendinc E., Smith J. S. and Laloraya S. Limiting the extent of the RDN1 heterochromatin domain by a silencing barrier and Sir2 protein levels in Saccharomyces cerevisiae. Mol Cell Biol. 2009 May; 29, 2889-2898
Laloraya, S., Guacci, V., and Koshland, D. Chromosomal addresses of the cohesin component, Mcd1p. J. Cell Biol. 2000 Nov 27; 151, 1047-1056.
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