Transcriptional potency in early embryos

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 future therapeutics of tissue repair and regeneration.

Grant Number: 1R15HD115134-01
NIH Institute/Center: NIH
Principal Investigator: CESAR ARENAS-MENA