This project seeks to identify and test mechanisms that establish the transcriptional regulatory potency of early embryos. The relationships among DNA accessibility, nucleosome lability, transcription, histone variants and DNA sequences interacting with nucleosomes and transcription factors in early embryos and differentiated cell types will be identified with genomic methods. It is proposed that regulatory potential may be primed at some transcriptional regulatory elements (TREs, inclusive of promoters and enhancers/silencers) by DNA sequences promoting weak histone interactions. Such sequence-based priming may synergize with the embryonic abundance of histone variants known to promote TRE accessibility. In particular, H2A.Z/H3.3 histone variant nucleosomes associate with low occupancy TREs. Furthermore, H2A.Z mRNA expression is high in blastula embryos and multipotent adult cell precursors of indirectly developing sea urchins and polychaetes but low in differentiated cells, therefore supporting a general H2A.Z transcriptional multipotency role. PRO-seq (Precision Run-On Sequencing, a method to detect actively transcribing TREs) followed by dREG (Discriminative Regulatory Element Detection, a vector machine TRE prediction tool) will identify transcriptionally active TREs in early embryos and differentiated adult cell types. OmniATAC experiments will test if in early embryo and differentiated cell TREs have incipient DNA accessibility. Quantitative micrococcal nuclease and sequencing (qMNase-seq) will test if TREs with incipient DNA accessibility in early embryos are occupied by labile nucleosomes. H2A.Z and H3.3 CUT&RUN (Cleavage Under Targets & Release Using Nuclease) will test the prevalence of these histone variants at labile nucleosomes. The new data sets will synergize with existing ATAC-seq and PRO-seq characterizations during sea urchin embryogenesis and differentiation. Comparison among genomic profiles will test if transcriptionally disengaged but accessible TREs in early embryos associate with labile nucleosomes, H2A.Z, H3.3, A/T-GC sequence periodicities and/or pioneeer transcription factor binding sites (TFBSs). Similar experiments in terminally differentiated cell types with low H2A.Z expression will test if incipient accessibility is an inherent property of TREs entirely based on A/T-GC sequence periodicities. Accessibility sequences setting incipient TRE accessibility during embryogenesis will be experimentally tested by DNase-I-seq of zygotically microinjected TREs harboring mutations that alter A/T-GC sequence periodicities or pioneer TFBSs. The project will be integrated with parallel efforts to implement “research in the classroom” in existing genomic courses that will recruit, train and motivate diverse undergraduate and graduate students at CUNY. The understanding of basic transcriptional multipotency mechanisms in sea urchin embryos is relevant to understand the evolution of developmental gene regulation and to advance futu...