Plasticity in an embryonic gene regulatory network

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SUMMARY The major objective of the proposed research is to illuminate the molecular basis underlying genetic and epigenetic variation in a major developmental gene regulatory network (GRN). Studies from this and other labs have identified a cascade of “core” zygotically expressed GATA-type transcription factors, and maternal regulatory inputs, that activate the GRN controlling development of the endoderm in C. elegans. The latter include the maternally supplied SKN-1/Nrf2 transcription factor and a triply redundant Wnt, MAPK, and src signaling system that acts through the LIT-1/NLK kinase and the POP-1/Tcf/Lef transcription factor to initiate endoderm development. Removal of any one of these inputs results in an impenetrant phenotype, reflecting a bistable state that shows wide variation between genetically distinct isotypes. Analysis of reciprocal crosses between isotypes with quantitatively different requirements for SKN-1 in this process revealed that endoderm GRN output is also influenced by long-term heritable epigenetic states that differ between natural C. elegans isotypes. This transgenerational epigenetic inheritance (TEI) requires genes involved in piRNA function, the nuclear RNAi pathway, and histone H3K9 methylation. These findings provide a springboard for unveiling the molecular basis for genetic and epigenetic plasticity in the regulation of the endoderm GRN. In Aim 1, we will evaluate hypotheses regarding the mechanisms of action of three genes that differentially alter the requirements for SKN-1 and Wnt signaling. We will assess how expression of the core regulators of endoderm development is influenced by quantitative variation in the requirement for the maternal GRN inputs. We will assess how variation in the requirement for LIT-1 kinase is accommodated in the mechanism that controls asymmetric cell division leading to activation of the endoderm GRN and will test the hypothesis that quantitative variation in the requirement for LIT-1 extends to its global action in many asymmetric cell divisions. In Aim 2, we will develop and implement high-resolution, high-throughput approaches to identifying causal genes underlying variation in the requirement for the major endoderm regulatory inputs. We will test candidate genes for modulation of the SKN-1-dependent activation of the endoderm GRN. In Aim 3, we will analyze the molecular basis for transgenerational inheritance (TEI) of GRN output. We will assess the stages in the endoderm GRN that are modulated by this TEI and test the hypothesis that epigenetic differences in SKN-1 requirement between selected isotypes extends to other regulatory inputs. We will test the hypothesis that TEI results from differences in chromatin states of endoderm genes and that differential expression of non-coding RNAs and endoderm regulatory genes is associated with TEI. Findings from this research will help to illuminate mechanisms of birth defects and can provide a paradigm for understanding relationships between an individual’s genotype and responsiveness to pharmacological agents, of importance to advancing precision medicine. They will also reveal factors that alter the outcome of Wnt signaling, a major regulatory mechanism associated with many cancers.