Accessibility and activity of transcriptional regulatory elements during sea urchin embryogenesis and differentiation [PRO-seq]
Transcriptional regulatory elements (TREs) are the primary nodes of the gene regulatory networks that control development.   TREs are identified by PRO-seq and their accessibility by ATAC-seq during sea urchin embryonic development and differentiation.  Our analysis identifies surprisingly early accessibility in 4-cell cleavage embryo TREs that is not necessarily followed by subsequent transcription, and an excess of ATAC-seq peaks transcriptionally disengaged during the stages analyzed.  Embryonic accessibility shifts are driven by transcriptionally engaged TREs, and PRO-seq transcriptional differences at TREs provide more contrast among embryonic stages than ATAC-seq accessibility differences.  TRE accessibility reaches a maximum around the 20-hour late blastula, which coincides with major embryo regionalizations. At the same time, a large number of distal TREs become transcriptionally disengaged, in support of an early Pol II primed model for developmental gene regulation that eventually resolves in transcriptional activation or silencing.  A transcriptional potency model based on labile nucleosome TRE occupancy driven by DNA sequences and the prevalence of histone variants is proposed in order to explain the basal accessibility of transcriptionally inactive TREs during early embryogenesis. Overall design: PRO-seq in embryos and larvae followed previously stablished protocols (Arenas-Mena et al., 2021), except that for red spherule cells the lysis buffer during the nuclear prep was supplemented with 0.1 % NP40, 0.1% Tween-20, and 1% BSA, and the density gradient solutions were supplemented with 1% BSA to diminish nuclear shearing. Red spherule cells were purified from the coelomic fluid of adult sea urchins following a previously stablished method (Smith et al., 2019).  OmniATAC in 72 hour embryos raised at 15 degrees C and red spherule cells was performed as previously described (Corces et al., 2017).    Quick PRO-seq in red spherule cells was performed according to a previously stablished method (Judd et al., 2019) with minor modifications.  Permeabilization and wash buffers were supplemented with 1% BSA, cells were collected in a fixed angle centrifuge at 0 oC and 1,000 G for 10 minutes.  The run-on reaction was done at 15 oC for 15 minutes.  Dynabeads Myone ® Streptoavidin T1 beads were used to pull down the biotinylated RNAs. Free biotinylated nucleotides were removed by filtration with SigmaspinTM post-reaction clean-up columns.  On-Bead 5' Decapping reaction was done using NEBuffer 2 buffer (NEB, B7002S ®). Off-Bead Reverse Transcription followed the instructions in SuperScript ® IV Reverse transcriptase kit. The Quick PRO-seq PRO-seq libraries were purified from 5% acrylamide gel to remove adaptor dimers.