Three-dimensional genome organization, in which the linear DNA sequence is partitioned into functionally distinct domains, is essential for the precise deployment of genetic information in every cell type. An increasing number of devastating disorders and diseases have recently been attributed to disruption of genome organization, yet the mechanistic underpinnings remain poorly understood, especially in vivo. In particular, how the activity of binding factors and sequence elements is translated to epigenetic states and three-dimensional genomic domains to govern gene expression remains mysterious. The germ line of C. elegans provides a relevant and experimentally advantageous in vivo model to dissect the mechanisms governing proper establishment of genomic domains with coordinated regulation. In this system, we investigate diverse large-scale gene regulatory mechanisms controlling piRNA biogenesis, X chromosome regulation, and transformation from the germline to the somatic fate. We have recently developed the ability to reliably purify germ nuclei at quantities sufficient for genomic analyses, including ChIP-seq, RNA-seq, and chromatin capture conformation assays. This achievement removes a major barrier in the field to dissect gene expression mechanisms in the germ line. With this new capacity, we will now characterize fundamental transcriptional and epigenetic mechanisms in wild type germ nuclei with unprecedented specificity. Using these data, we will now define key mechanisms governing the germline-to-soma transformation, the coordinated, germline-specific regulation of genomic domains containing thousands of piRNA genes, and the temporal and global regulation of X chromosome gene expression in the germ line. The successful completion of the proposed experiments will illuminate the poorly understood yet vitally important in vivo mechanisms by which large-scale genome organization influences gene expression, and vice versa.