Germ cells are highly specialized cells with the unique responsibility of producing healthy offspring, thus ensuring the continuity of a species across generations. These cells guard their DNA very carefully to allow the production of sperm and eggs with the right number of chromosomes and no mutations. We wish to understand how germ cells protect their DNA, while turning different genes on and off at the right times to make functional sperm and eggs. To grasp the most important trends, we use global genomic technologies to investigate many genes simultaneously. We are studying germ cell regulation primarily using the model organism C. elegans, a nematode, because of the large number of germ cells it contains, and because of the many experimental advantages it offers. Because the genes in C. elegans are related to those in higher organisms, the results from our studies should help us to understand how germ cells function in humans as well.
The genome carries all the information necessary for the development and function of an organism. This information is embedded at multiple levels - in the regulatory information of individual genes, in the partitioning of that sequence into chromatin domains, and in the spatial segregation of these domains into functionally distinct regions of the nucleus. This linear and spatial organization is essential for effective and precise deployment of genetic information, yet the underlying mechanisms that govern the three-dimensional architecture of the genome are just now being addressed, and fundamental questions remain unanswered: How does genome organization influence epigenetic information, and vice versa? How extensively does genome organization contribute to coordinated expression of functionally related genes? How does genome organization stabilize and instruct tissue-specific gene expression?
We address these questions in vivo with unprecedented cell specificity and comprehensiveness, utilizing innovative methods to investigate how genome structure and organization influences gene expression specifically in the C. elegans germ line. Specific projects are focused on:
Germline-specific expression of piRNA clusters. The C. elegans genome contains a remarkable genomic domain subject to tissue-specific expression. In C. elegans, thousands of individually transcribed loci encoding the piRNA class of small noncoding RNAs are clustered into two sharply demarcated regions on a single chromosome. Amazingly, these piRNAs exhibit synchronized expression in the germ line, despite being interspersed among hundreds of coding genes with diverse expression patterns. piRNA clustering is evolutionarily conserved, indicating that physical proximity is a key feature for coordinated expression, yet how germline-specific expression is implemented is a mystery. We have found that SNPC-4 preferentially binds across both piRNA clusters only in the germ line and promotes piRNA expression. SNPC-4 is a transcription factor known to stimulate the activity of both RNA polymerase II and III, and we have found that RNA polymerase III (pol III) also exhibits increased occupancy in piRNA clusters. Recently, pol III and core components of the pol III complex such as TFIIIC have been demonstrated to establish boundaries between genomic domains, and regulate gene expression within those domains. We are now determining the mechanisms by which SNPC-4 and the core RNA polymerase III machinery coordinately regulate this piRNA-rich genomic domain in a tissue-specific manner, within the native developmental context.
Regulation and function of germline transcription factors. In C. elegans, the germ line maintains a specific gene expression profile largely through the interaction between chromatin state and post-transcriptional RNA regulation. However, certain key transcription factors play a vital role in establishing and maintaining germline identity and separating the germline programs from the soma. In particular, the C. elegans version of the tumor suppressor complex Rb/E2F is vital to distinguishing germline and somatic identities. Gene expression and DNA binding profiles indicate that the complex acts as a repressor in the soma, and an activator in the germ line on completely separate gene targets. How this complex differentially regulates distinct target genes in specific tissues is poorly understood. We are now determining the tissue-specfici chromatin mechanisms by which this complex interacts with target genes. This analysis will have implications not only for germline development but also for human development and tumorigenesis. More broadly, we have defined additional novel regulatory factors that also appear to be expressed specifically in the germline and that have important roles in germline development. We are investigating how these factors are regulated as well as how they function, using a variety of genomic, genetic and biochemical assays.
Genetics; Germ Cells; Stem Cells; Developmental Biology; Caenorhabditis elegans; Genomics; Epigenomics
Genetics, Genomics, Epigenetics; Reproduction