Post Date
Sep 19 2021

Kinome-wide RNAi screen uncovers the role of Drosophila Ballchen in maintenance of epigenetic cell memory

Year
2020
Supervisor:
Dr. Muhammad Tariq
Students:
Muhammad Haider Farooq Khan
MS/PhD
PhD
Reference / Filters
Biology
Abstract:
The capacity of cells to diversify in different cell lineages and the ability to remember their identity is central to the development of multicellular organisms. Once the cell fate is determined during early development, the identity of different cell types is maintained during subsequent development which involves maintenance of cell type specific gene expression patterns through successive cell divisions. Polycomb group (PcG) and trithorax group (trxG) proteins are evolutionarily conserved factors that maintain cellular identity after the establishment of cell fates. PcG proteins behave as repressors to maintain heritable patterns of gene silencing and trxG proteins act as anti-silencers by maintaining active gene expression profile linked to specific cell fate. Genetic and molecular analysis has revealed extensive details about how PcG and trxG antagonize to maintain cell fate, but the cellular signaling components that contribute to trxG mediated gene activation or PcG mediated repression have remained elusive. The aim of this thesis is to discover novel contributions of cellular signaling components, specifically protein kinases in facilitating trxG to counteract PcG mediated gene repression. To this end, an RNAi based reverse genetics approach is employed to determine the novel role of kinases and cell signaling proteins in maintenance of gene activation by trxG proteins in Drosophila. The ex vivo kinome-wide RNAi screen led to identification of twenty-eight genes shortlisted as potential regulators of trxG. Serine-threonine protein kinases from the primary list of candidates were validated by performing a secondary screen. Drosophila Ballchen (Ball), a histone kinase among the candidates, was further characterized as a novel trxG regulator. The ball (ball2) mutant strongly suppressed the extra sex comb phenotype of Pc mutants and enhanced the loss of abdominal pigmentation phenotype of trx mutants. In addition, depletion of Ball in homozygous ball2 embryos and mitotic clones resulted in downregulation of trxG target genes. Interestingly, diminished amounts of H3K4 trimethylation and H3K27 acetylation, two histone marks associated with anti-silencing activity of trxG, were also observed in ball2 mitotic clones. Moreover, Ball co-localizes with Trx on chromatin and inhibits H2AK118 ubiquitination, which is a histone mark central to PcG mediated gene silencing. Together, this data suggests that Ball mediated phosphorylation contributes to a binary switch at H2A which facilitates trxG to counteract repression by PcG. Thus , a novel role of a protein kinase, Ball, is discovered in controlling PcG/trxG mediated cell fate maintenance. Further molecular and biochemical characterization of hitherto unknown link between trxG and Ball will reveal effect of cell signaling in maintaining dynamic state of gene expression patterns involved in epigenetic cell memory.

Publication:

Umer, Z*., Akhtar, J*., Khan, MHF*., Shaheen, N., Haseeb, MA., Mazhar, K., Mithani, A., Anwar, S and Tariq, M. (2019). Genome-wide RNAi screen in Drosophila reveals Enok as a novel Trithorax group regulator. Epigenetics and Chromatin 12, 55 (2019) doi:10.1186/s13072-019-0301-x