The overall goal of this proposal is to understand mechanisms of gene expression that occur in the meiotic cell. In particular, we wish to define the molecular order of events that occurs when an inactive gene undergoes a developmentally-controlled transition to a state in which it is expressed. The meiotic differentiation program is an attractive biological system to study such events because changes occur on a relatively slow time-scale. In addition, the appearance of different types of male germ cells is synchronized with age, allowing one to examine the progression of events. Gene expression during meiotic prophase is also unique in that new, non-somatic, forms of chromatin packaging and general transcription factors are expressed. The proposal has three main objectives, and will use the germ cell-specific gene that encodes the transcription factor ALF as the model. The first is to define the sequence of events leading up to meiotic expression of the ALF gene. Recent studies of gene activation in somatic cells highlight the importance of DNA methylation, histone modifications, changes in chromatin accessibility, and occupancy by TFIlD. We intend to bring methods used in these studies to bear on our studies of ALF. This is especially interesting because expression from meiotic chromosomes may be influenced by changes in DNA packaging and organization that do not occur in somatic cells. The second objective will be to identify the minimal sequences required for proper expression using a transgenic mouse model and to identify factors that bind to those sequences. The third objective will be to address the hypothesis that the variations in the patterns of methylation at the ALF promoter reflect variations in the positioning of nucleosomes. Overall, we expect the work will contribute to our understanding of the basic mechanisms of gene expression, and will provide a foundation for understanding mechanisms of infertility and other disorders related to human reproductive health. ? ?
