Dissecting histone regulation of the DNA-damage checkpoint in proliferating cells

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Cell sizes vary across several orders of magnitude both within and between species. Yet, nuclear volumes and genomic content typically scale with cell size leading to a robustly consistent ratio of nucleus to cytoplasm (N/C ratio). One exception to this N/C ratio rule is the early embryo of many externally developing species which contains all the material to produce the resultant offspring but starts with only a single diploid nucleus. These embryos immediately undertake a series of reductive cleavage divisions to restore more typical N/C ratios. These cleavage divisions halt and transcription starts at the Mid-Blastula Transition (MBT). A longstanding hypothesis has been that the embryo measures the N/C ratio to time the MBT through titration of some maternally provided factor. We and others have proposed that histones, which are maternally provided in vast excess of DNA in the early embryo are one such factor. However, how the molecular mechanism by which histone availability is translated into a cell cycle signal has remained a mystery. Recently, we discovered that histone H3 promote cell cycle progression and inhibit the DNA damage kinase, Chk1. Chk1, in turn is required to stop the cell cycle at the MBT. Here we propose to test the hypothesis that histone H3 is a direct Chk1 competitive inhibitor in the early embryo and extend this hypothesis to other contexts of DNA damage. In Specific Aim 1 we will directly measure the in vitro affinities and in vivo concentrations of the relevant species to construct a kinetic model of H3 dependent Chk1 activity at the MBT. We will then test this model by measuring Chk1 activity and cell cycle behavior in response to a variety of H3 perturbations using fluorescent biosensors. In Specific Aim 2 we will extend these findings to Chk1 mediated DNA damage response triggered by irradiation, chemical damaging agents, and oncogenic replication stress in both the embryo and wing imaginal disc. Successful completion of this work will answer the longstanding question of how cells in the early embryo senses its N/C ratio and provide a potential new regulatory arm to the DNA- damage response pathway that may have implications for carcinogenesis. The BioMT COBRE will benefit the project by providing essential core infostructure, strong mentoring support, and providing a community of other like-minded junior investigators for scientific discussion and collaboration. In addition, the BioMT COBRE will support the acquisition of the data necessary to extend my work beyond the early embryo and into the wing disc and provide sufficient preliminary data to be competitive for extramural funding. Project funding will also help to expand the community of biologists interested in molecular targeting at Dartmouth to help nucleate a mutually supportive research cluster. .