Project Summary Parental environmental influences such as diet affect energy metabolism in the next generation but the underlying mechanisms for such transgenerational epigenetic inheritance are not well- understood. While small RNAs, histone modifications and DNA methylation in germ cells can function as signals mediating transgenerational epigenetic inheritance, very little is known on the mechanisms how these signals are regulated. This in part due to a lack of genetic model systems, suitable for carrying out detailed investigations. The X-linked gene Rlim encodes a RING finger E3 ligase that functions as a major epigenetic regulator in female mice, as it is crucial for imprinted X chromosome inactivation (iXCI), the epigenetic silencing of the paternally transmitted X chromosome in female preimplantation embryos to achieve X dosage compensation. Indeed, a maternally transmitted Rlim KO allele results in early embryonic lethality in females, while males lacking Rlim grow into adulthood. We recently showed that in testes of males Rlim is highly expressed both in Sertoli cells (SCs) and in spermatogenic cells at the round spermatid stage. Despite being fertile, mice with systemic ablation of Rlim produce less sperm which additionally is dysfunctional. Targeting a conditional KO (cKO) to testicular cell types, our published results demonstrate that Rlim activity in spermatogenic cells but not SCs is required for normal spermatogenesis. We found that offspring sired by fathers systemically lacking Rlim is protected from high fat diet (HFD)-induced obesity in a transgenerational epigenetic effect. Targeting the cKO of Rlim in fathers, we demonstrate that lack of Rlim in SCs recapitulates this effect. As paternal signals transmitting transgenerational effects are transmitted in sperm, our unpublished results provide genetic evidence for a novel SCspermHFD-response in offspring pathway. Our future research research will exploit our unique Rlim cKO mice to investigate this pathway to A) elucidate molecular mechanisms of Rlim action in SCs, B) identify the epigenetic signal in sperm and how it is generated, and C) uncover downstream effects in offspring ultimately leading to changes in HFD-response. The overarching goal of the future research is to establish the Rlim mouse model as a novel paradigm for transgenerational epigenetic regulation. This research will define the SCsperm offspring pathway in a larger biological context and, using cutting edge technologies, will identify new and fundamental epigenetic mechanisms.