MBI PhD Qualifying Exam 

Time: 11am
Date: Thursday, 22 March 2018
Venue: MBI, level 5 meeting rooms

Supervisor:  Prof G. V. Shivashankar

Role of cell geometry and chromatin architecture in modulating transcriptional response to environmental stimuli

by Saradha VENKATACHALAPATHY, Shivashankar Group

 Cells in tissues integrate a multitude of mechanical and chemical stimuli from their microenvironment to modulate gene expression patterns and thereby, cell behavior. Most studies have been done on a population cells with heterogeneous cell geometries with the assumption that population means are descriptive of cellular phenomena. Recent studies have challenged this by demonstrating that cells of different geometries differentially modulate nuclear morphology, chromosome organization and importantly, transcription regulation (Versaeval Nat.Comm, Wang NAR, Jain PNAS). However, the importance of the cell geometry in determining cellular response to an environmental stimuli has not been studied. In our study, we used NIH3T3 mouse fibroblast cells grown on rectangular (1800μm2 AR 1:5) and circular (500μm2) fibronectin coated micropatterns and exposed them to the inflammatory cytokine TNFα and compressive force (CF). When we measured the transcriptional response of cells in these conditions we found that there is geometry dependent expression of genes in response to TNFalpha and compressive load. In addition, we show that such geometry specificity is exhibited by target genes of relevant transcription factors. Collectively, these results suggest the presence of an additional layer of transcription regulation that is driven by cell geometry. Since cell geometry was shown to modulate inter-chromosomal interactions, we went on the characterize the transcriptional significance of such regions(Wang NAR). Based on an integrative approach that leverages 1D functional genomic features (e.g., epigenetic marks) with 3D chromatin interactions from Hi-C data, we demonstrate using 3D fluorescence in situ hybridization (FISH) experiments, that active RNAPII is enriched in active chromosome intermingling clusters in fibroblasts. We therefore hypothesize that the chromosome intermingling domains to be a part of the cell geometry driven differential transcriptional response to stimuli. In order to test our hypothesis, we have established a cancer spheroidfibroblast co-culture model system in 3D collagen matrix. Our preliminary experiments show that protein levels of aSMA, Vimentin and FAP-A as well as the nuclear abundance of the transcription factor MKL in the nucleus is higher in fibroblasts co-cultured with cancer spheroids. In addition, our results show that the nuclear enrichment of MKL is dependent on nuclear morphometrics, which is regulated by cell geometry. Going forward, we will be using this system to characterize the importance of cell geometric heterogeneity and 3D chromatin structure in integrating and regulating the transcriptional responses to environmental signals.

**Please note the examination following the seminar is closed-door**