Project Summary / Abstract The pairing of homologous chromosomes is a fundamental process for meiotic recombination but also occurs in non-meiotic cells in a broad range of organisms. Recent studies have revealed that non-meiotic homolog pairing is an actively regulated process and provides an opportunity for interchromosomal interaction. The local pairing status of a particular gene locus differs in a cell type-specific manner and correlates with local chromatin states, suggesting the possibility that non-meiotic homolog pairing may take part in gene regulation. Drosophila male germline stem cells (GSCs) constantly divide asymmetrically to produce one GSC and one differentiating gonialblast (GB). In this system, stem cell-specific genes, including Signal transducer and activator of transcription 92E (Stat92E), are quickly downregulated, providing an excellent model to study preprogrammed changes of gene expression states in vivo. Results from the previous funding periods showed that the homologous regions of Stat92E always closely associate with each other in GSCs (paired) but separate immediately in GBs (unpaired), and the change in pairing states is required for prompt downregulation of Stat92E transcription. It has been shown that GSCs tend to retain old histones H3 and H4, while GBs tend to inherit newly-synthesized histones H3 and H4. When this histone inheritance was compromised by expressing non- phosphorylatable histone H3T3A, the change in Stat92E pairing upon differentiation did not occur, raising the exciting possibility that the pairing states of key genes are epigenetically programmed through the inheritance of old vs. new histones. These observations suggested that the non-meiotic homologous pairing may be a process to reprogram gene activity, and that the alteration of pairing states is an important mechanism that regulates key genes during stem-cell differentiation. In this proposal, we aim to define the role of interchromosomal interaction in non-meiotic stages of germline on cell fate determination and to elucidate the underlying mechanisms. First, we will identify a list of genes differently paired in GSC and GB. Next, we will characterize genome-wide homolog interaction in GSCs and differentiating cells. Lastly, we will investigate mechanisms by which changes in pairing state influence transcription using Stat92E and other identified genes. These experiments will allow us to understand how pairing is developmentally regulated and how it impacts cell fate determination. Homologous allelic pairing also occurs in mammalian stem cells, where alleles of Oct4 transiently pair to share repressive chromatin marks during the transition from pluripotency to lineage commitment. Thus these proposed studies of pairing regulation may elucidate common mechanisms of stem cell differentiation.