Abstract Changes that can persist for many generations without altering DNA sequence have been reported in many organisms. The existence of such heritable epigenetic changes has also been claimed in humans in response to some stressful experiences such as starvation and psychological trauma. However, there is poor understanding of the mechanistic basis for the persistence of epigenetic changes across generations in any organism. Although molecules mediating such changes have been identified in multiple systems (e.g., DNA methylation, chromatin modifications, small RNAs, etc.), the mere presence of these molecules and chemical modifications do not predict the persistence of any newly introduced epigenetic change. Recent conceptual advances that provide a system-level understanding of heredity suggest that changes in regulatory architectures drive and maintain heritable epigenetic changes. Therefore, focusing on the regulatory architectures can reveal the important interactions required for explaining any change that can last for multiple generations regardless of the particular molecules that mediate these interactions. In the nematode C. elegans, multiple phenomena have been reported whereby RNA-mediated regulation is used to sustain the silencing of gene expression for hundreds of generations. These phenomena thus provide opportunities for analyzing heritable epigenetic changes at single-gene resolution to gain insights. In addition to the expected broad applicability of insights into regulatory architectures, even some of the specific molecules used to regulate heritable epigenetic changes in C. elegans such as piRNAs, phase-separated RNA granules, the double-stranded RNA importer SID-1, and small RNA-bound Argonaute proteins are conserved in many organisms, including humans. Our preliminary results have revealed insights into a regulatory architecture that promotes stable RNA silencing of a model transgene encoding a fluorescent protein and endogenous genes that are similarly susceptible to heritable epigenetic changes in gene expression. The aims of this proposal are: (1) to analyze model genes to discover the regulatory sequences and perturbations that promote transgenerational gene silencing; and (2) to analyze regulation of endogenous genes to elucidate the regulatory architectures that have evolved to control heritable epigenetic changes in gene expression within the germline. Completion of these goals will provide both a general view of heritable epigenetic changes at any gene and reveal the regulatory architecture of evolved mechanisms. These results will provide a better understanding of hereditary diseases, particularly diseases that could have an epigenetic origin in earlier generations.