Project description:We report the proteomic characterization of gonads from wild P. lividus collected along coastal Sardinia, and describe the changes occurring in gonads according to sex and developmental stage. Gonads in the recovery, pre-mature, mature, and spent stages were analyzed using a shotgun proteomics approach based on filter-aided sample preparation followed by tandem mass spectrometry and label-free differential analysis. A detailed characterization of the proteome changes occurring in gonads of both sexes along maturation was achieved. Significant changes were seen in numerous proteins involved in nutrient accumulation and in gamete biology and maturation. Adding to an improved understanding of the P. lividus reproductive cycle in its natural environment, the results described in this work form the basis for defining novel protein markers and procedures for an easier sexing and staging, and for monitoring sea urchin gonad maturation in aquaculture plants.
Project description:The eukaryotic nucleosome is the fundamental subunit of chromatin and plays functional roles in DNA templated processes including replication and transcription. In eukaryotic promoters, nucleosome organization is generally thought to be highly structured, with nucleosomes occupying canonical positions flanking the transcription start site (TSS), thereby regulating access of the transcriptional machinery to the underlying DNA. We sought to determine whether this canonical distribution is present in the purple sea urchin, Strongylocentrotus purpuratus, an important developmental model organism. We used titrations of micrococcal nuclease to produce high throughput maps of nucleosome distribution, sensitivity to digestion, and subnucleosomal footprints from female urchin gonad tissue. Unlike yeast, flies, zebrafish, maize, or humans, urchins lack a nucleosome depleted region over TSSs. Urchin promoters are dominated by strongly positioned and highly occupied +1 and +2 nucleosomes which are most prominent in highly expressed genes. Additionally, urchin promoters exhibit distinct patterns of sensitivity to nuclease digestion, with heightened sensitivity upstream of the TSS and limited resistance to nuclease digestion. Discretely positioned sensitive nucleosomes were enriched in promoters of highly expressed genes, suggesting a relationship between nucleosome sensitivity and transcriptional regulation. Moreover, urchin promoters were enriched for small fragment-protected footprints associated with known regulatory motifs. Collectively, we present a comprehensive overview of the unique interplay between nucleosome positioning, chromatin sensitivity, cis-regulatory interactions, and gene expression in sea urchins. Our study not only provides a reproducible methodology applicable to other organisms, but also advances our understanding of functional chromatin organization and gene regulation in urchins.
Project description:We use multi-omics (ATAC-seq, single cell RNA-seq and differential bulk RNA-seq) to increase resolution of the sea urchin posterior gut GRN for the cells expressing the ParaHox gene Sp-Pdx1.
Project description:Purpose: The Tbrain transcription factor has demonstrated an evolved preference for low-affinity, secondary site binding motifs between the sea star and sea urchin orthologs. We sought to identify targets of sea urchin and sea star orthologs of Tbr. Because less is known about the function of Tbr during sea star development, we used RNA-seq in conjuction with ChIP-seq studies (GEO:xxxx) to determine the targets of sea star Tbr in early development. Methods: Sea star (Patiria miniata) embryos were injected with translation-blocking morpholino antisense oligonucleotides to knock-down PmTbr expression, as described previously. Control morpholinos were injected into sibling embryos. Embryos were allowed to develop until hatching (30-36 hpf) at which point injected embryos were collected and RNA was extracted. RNA-seq libraries were prepared, sequenced, and analyzed using standard protocols. Results: There are 2,562 genes that are significantly differentially expressed relative to control morpholino inected embryos (FDR < 0.05). There are roughly equivalent numbers of genes down-regulated (1,041) and up-regulated (1,521) by Pm-tbr knockdown, suggesting that PmTbr may act as both a transcriptional activator and repressor. 1,165 differentially expressed genes are located within 75 kb of a PmTbr binding site determined using ChIP-seq, and this set is used as a basis for comparison between sea star and sea urchin binding sites. Conclusions: 1,165 targets of the PmTbr transcription factor were identified based on differential expression following knockdown and the presence of transcription factor binding sites proximal to differentially expressed genes. There are an equal number of up- and down-regulated targets, suggesting Tbr may function as a transcriptional activator and repressor, depending on context and target gene. There was no clear association of motif utilization with either the direction of differential expression or ontological category of the target gene. There are only a small fraction of target genes (approximately 10%) that are in common between the sea star and sea urchin sets.