Investigating connections between variability in DNA damage responses and global genome organization in regulation of tissue-specific differentiation and cancer development.

DNA damage is ubiquitous in nature, and cellular response to it as diverse. A vast body of literature has established an intimate link between DNA damage responses (DDR) and the emergence of cancer, neurodegeneration and premature aging. Perturbations of specific DNA repair pathways are often associated with cancers in specific tissues. The very broad aims of my research are to elucidate the tissue-specific emergence of cancer with mutations in specific DNA repair pathways, and investigate the roles of DDR in tissue-specific differentiation.

Years of concerted efforts by several groups have identified most of the key players in the different DNA repair pathways. But despite this wealth of biochemical knowledge, the connections between DDR and the local and global physical structure of the chromatin are only beginning to be discovered. Any corrective responses to DNA damage has to take place in the context of the highly non-random spatial organization within the eukaryotic cell nucleus, which in turn may regulate the processes of transcription and repair in a tissue-specific manner. Indeed there have been suggestions that the tissue-specificity of gene expression is coded at a broad level in the spatial organization of the chromatin. Efforts in my group are focused towards elucidating the connections between physical chromatin structure and DDR in tissues with genotoxic stress. Further connecting the themes of tissue-specific gene expression and DDR, we aim to explore the roles of DDR in processes of differentiation and development. With the progress of single-cell sequencing techniques, it is increasingly becoming clear that fairly large copy number variations of genetic material can exist among cells in differentiated tissue. While this is thought to be due to regulated genomic instability, it is not clear if this CNV serves a functional consequence. It is also known DNA damage pushes undifferentiated embryonic stem cells towards differentiation by repressing pluripotency programs, but again it is not well understood if this differentiation is erratic or lineage-specific. In terms of epigenetic changes too, enzymes evolved for DNA repair can be exploited. In all these contexts, single cell microscopy experiments in conjunction with current single cell sequencing efforts could provide unprecedented insight into the regulation of differentiation processes by DNA damage and repair. Thus in addition to seeking the roots of tissue-specificity of cancers, we aim to explore if Nature generally exploits DDR to regulate processes of differentiation.

It is only recently that the mechanical aspects of DDR are getting to be appreciated, and it is an exciting avenue of research, ripe for applications of both physical techniques and rigorous mathematical modeling through collaborative efforts. Further we mean to develop means to take these experiments to a higher throughput context to gain a systems level understanding of DDR. In the coming days interdisciplinary approaches will be crucial for furthering our knowledge of stress responses like DDR, and elucidating the emergence of disease states.

Single transcript imaging in a Mouse Embryonic Fibroblast cell. Nucleus (blue), and individual transcripts (red) of an integrated transgene for a DNA repair glycosylase are shown. The bright spot in the nucleus is the site of the transgene integration. Single mRNA counting assays such as these facilitate the measurement of transcription on a cell by cell basis, and thus beyond mean transcription, allow us to get at the heterogeneity of DNA damage responses.