The switch from proliferation to differentiation is a key regulatory point in the adult stem cell lineages that underlie tissue maintenance and repair, and failure to cleanly switch may contribute to genesis of cancer. My laboratory has long used the Drosophila male germ line as a model to investigate how self-renewal, proliferation and differentiation are regulated in adult stem cell lineages. Several lines of our inquiry have recently converged on the molecular mechanisms underlying the developmentally programmed transition from mitotic proliferation to onset of meiosis and differentiation, implicating a number of molecular and cellular mechanisms in regulating this critical switch. We find that RNA binding proteins involved in translational control and alternative splicing act cell autonomously to regulate the cessation of proliferation and that progression of differentiation requires communication from associated somatic support cells. We discovered that a developmentally regulated alternate choice of site at which certain nascent transcripts are cut to form 3' ends, leading to production of novel mRNA isoforms with shortened 3'UTRs, controls dramatic changes in the suite of proteins expressed in differentiating spermatocytes compared to proliferating spermatogonia. We found that dramatic changes in chromatin open over 2000 new promoters with novel core sequence structure to turn on the new cell type specific transcription program when cells initiate spermatocyte differentiation. Some of the earliest genes turned on in this differentiation program encode chromatin associated proteins that prevent spurious opening of normally cryptic promoters, thus preventing massive misexpression of genes associated with the wrong cell type. Other transcripts upregulated with differentiation onset encode cell type-specific translational regulators that delay production of core G2/M cell cycle machinery to program the extended G2 phase of meiotic prophase. Over the next 5 years, we propose to map how these processes collaborate to form the regulatory circuitry that initiates then executes the switch from proliferation to differentiation. We will investigate how the RNA binding proteins Bam and Bgcn trigger the switch from mitosis to meiosis by repressing expression of the alternative splice factor HOW, identify candidate substrates of HOW by immunoprecipitation followed by RNA-Seq, and assess their function in vivo, including whether they communicate with adjacent somatic support cells. We will investigate how the switch in proteins expressed due to alternative 3' end cut site selection on nascent transcripts is regulated and influences differentiation. We will investigate how the differentiation program is kept off in precursor cells and how cell-type specific chromatin regulators and proteins that recruit them to target loci set up the new transcription program for differentiation. To elucidate how the developmental program remodels the cell cycle, we will investigate how cell-type specific RNA binding proteins first repress, then activate translation of cyclin B during meiotic prophase and how the DAZ homolog Boule regulates progression into the meiotic divisions. .
The proposed studies seek an integrated, systems level understanding of how multiple molecular mechanisms collaborate to control the critical biological switch from proliferation to differentiation, a key regulatory point in the adult stem cell lineages that underlie tissue maintenance and repair. Failure to cleanly execute this switch may contribute to genesis of cancer. Our results will reveal how cell type and stage specific alternative mRNA processing, translational control, changes in chromatin architecture, cell type specific transcriptional regulators, and cell-cell communication act together to ensure that proliferation of precursor cells ceases and the correct gene expression program for differentiation of the right specialized cell type is turned on.
