Regulatory mechanisms governing imprinted domains during early development - ABSTRACT Genomic imprinting is an epigenetic regulatory process that restricts expression of specific genes to one parental allele. Understanding genomic imprint regulation during early development has significant medical and societal implications, including for medically assisted reproduction, which is associated with rare imprinting disorders. However, significant knowledge gaps exist for the regulation of genomic imprinting during preimplantation development. While DNA methylation at imprinting control regions (ICRs) plays a critical role in embryos as an inherited silencing mark, the specific mechanisms that establish and/or maintain an active transcription compartment at the unmethylated ICR, permitting long noncoding RNA (lncRNA) transcription and in turn, repressive function across an imprinted domain, remain unclear. To address these gaps, we performed an innovative, RNA interference screen for epigenetic regulators of the Kcnq1ot1 imprinted domain (as a model domain) in mouse extraembryonic endoderm (XEN) stem cells. We identified 41 candidates, including chromatin remodelers, activators and repressors. Our investigation of one candidate, nucleoporin (NUP) 107, led to the discovery of a novel NUP107 and NUP153 mechanism of imprinted domain regulation. Here, we hypothesize that NUP107 and NUP153 function as a nuclear scaffold for promoting an active transcription compartment at ICRs in zygotes by recruiting chromatin remodelers and activators for lncRNA transcription, which in turn, leads to allelic silencing of neighboring genes. In Aim 1, we will examine the postfertilization role of NUP107 and NUP153 at the Kcnq1ot1 ICR by assessing physical interactions between these proteins, the Kcnq1ot1 domain and its lncRNA in late 1-cell to blastocyst-stage embryos. Wildtype embryos will be compared to embryos with NUP107 or NUP153 degradation, 2 maternal genomes (parthenotes) or 2 paternal- like genomes (maternal Dnmt3a/3b null). Next, we will assess genomic localization of NUP107 and NUP153, as well as chromatin state, using ultralow-input CUT&RUN in these embryos. To further define their function at imprinted domains, ChIP-seq with NUP107 and NUP153 antibodies and RNA-seq will be conducted on control, Nup107- and Nup153-depleted XEN cells. In Aim 2, we will determine the function of chromatin remodelers, SMARCA5 and SMARCC2, and activators, KAT2A and TAF6L, in generating active transcription compartments at ICRs. Using candidate depletion in XEN cells, we will ascertain the role of these proteins on Kcnq1ot1 lncRNA expression and its localization at the domain, open/active chromatin formation, allelic repression of neighboring genes in the Kcnq1ot1 domain, as well as their function at other imprinted domains. Next, we will analyze NUP107 and NUP153’s ability to recruit these chromatin remodelers and activators to carry out their function at ICRs. Finally, we will assess the role of SMARCA5, SMARCC2, TAF6L and KAT2A in establishing active transcription at ICRs in preimplantation embryos. This work will lead to the discovery of novel mechanisms of imprinted domain regulation during early development.
