Chromatin and Epigenetics

Poster

F-2072 - SIMPLE SEQUENCE REPEATS (SSRs) - GUARDIANS OF THE CHROMATIN GALAXY

6/22/2018
19:00 - 20:00

Establishment, maintenance and dynamic rearrangement of euchromatin and heterochromatin domains in the genome guide the processes of development and differentiation in organisms. Simple Sequence Repeats (SSRs) or Microsatellites are short tandem repeats of 1-6 nucleotide motifs, with the repetitive unit occurring between 10-20 times. SSRs occur in most organisms, in coding and non-coding DNA. In the human genome, they are twice as abundant as the protein-coding DNA, but their functions are unknown. They are used widely in genetic linkage mapping analysis and the triplet repeat disorders caused by aberrant expansions in SSRs are well known. Studies suggest that SSRs may have essential roles in gene regulation and genome organisation. Chromatin domains in the genome are defined by DNA elements called Insulators/boundary elements. They function as enhancer-blockers, which prevent communication between a promoter and enhancer when placed between them and/or barriers, which protect a flanked transgene from position-dependent silencing. The globin insulator, the Drosophila Gypsy insulator and the boundaries of Drosophila Hox loci are well-known insulators. Though many insulator proteins in Drosophila are known, the CCCTC-repeat binding factor, CTCF is the only insulator protein identified in vertebrates. It functions as a transcription factor and chromatin organising protein and is shown to be critical for stem cell proliferation. We tested whether SSRs are capable of enhancer-blocker and barrier activities. We selected 23 human SSRs based on their ‘length preference’ (Ramamoorthy S. et al. 2014) and cloned the oligos into vectors for cell-based functional assays to study their cis-regulatory potential. The 23 SSRs which are mostly intergenic or intronic modulated promoter activity in transient luciferase assays in six different cell lines, in a cell-type-dependent manner. 15 of 23 SSRs showed insulator activity in K562 cell line. Electrophoretic Mobility Shift Assay (EMSA) using nuclear extracts from Drosophila embryos and human cell lines revealed that these SSRs bind specific proteins. Using biotin-affinity purification and proteomics approach, we aim to identify novel SSR-binding proteins, which may have important roles in organisation and regulation of the genome.




 

Fathima Athar

CSIR-CENTRE FOR CELLULAR AND MOLECULAR BIOLOGY (CCMB), Telangana, India

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