Transcription factor interactions explain the context-dependent activity of CRX binding sites
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus synthetic construct
PROVIDER: GSE225867 | GEO | 2023/03/05
REPOSITORIES: GEO
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Project description:Death of photoreceptors and/or Retinal Pigment Epithelium (RPE) cells is a common cause of age related and inherited retinal dystrophies, thus their replenishment from renewable stem cell sources is a well sought therapeutic goal. Human pluripotent stem cells provide a useful cell source in view of their limitless self-renewal capacity and potential to differentiate into all key retinal cell types either in isolation or as part of three dimensional retinal organoids. Photoreceptor precursors have been isolated from differentiating human pluripotent stem cells either through application of cell surface markers or fluorescent reporter approaches and shown to share a transcriptional profile akin to foetal photoreceptors. In this study we investigated the transcriptional profile of CRX+ photoreceptor precursors derived from human embryonic stem cells (hESC) using single cell RNA sequencing and their engraftment capacity in an animal model of retinitis pigmentosa (C3H/rd1). Single cell RNA seq analysis revealed the presence of dominant cell cluster which displayed the hallmarks of early cone photoreceptor expression. When transplanted subretinally into the C3H/rd1 mice, the Crx positive cells settled next to the inner nuclear layer of host retina, matured into cone photoreceptors and made connections with the inner neurones of the host retina. Cellular transfer between the host retina and donor photoreceptors was investigated and shown to be minimal. Together our data provide valuable molecular insights into the transcriptional profile of human pluripotent stem cells derived CRX+ photoreceptor precursors and indicate their usefulness as a source of transplantable cone photoreceptors.
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Project description:Cone-rod homeobox (CRX) is a paired-like homeodomain transcription factor (TF) and a master regulator of photoreceptor development in vertebrates. The in vitro DNA binding preferences of CRX have been described in detail, but the degree to which in vitro binding affinity is correlated with in vivo enhancer activity is not known. In addition, paired-class homeodomain TFs can bind DNA cooperatively as both homodimers and heterodimers at inverted TAAT half-sites separated by two or three nucleotides. This dimeric configuration is thought to mediate target specificity, but whether monomeric and dimeric sites encode distinct levels of activity is not known. Here, we use a massively parallel reporter assay, CRE-seq, to determine how local sequence context shapes the regulatory activity of CRX binding sites in mouse photoreceptors. We assay inactivating mutations in >1700 TFBSs, and we find that dimeric CRX binding sites act as stronger enhancers than monomeric CRX binding sites. Furthermore, the activity of dimeric half-sites is cooperative, dependent on a strict three-base-pair spacing, and tuned by the identity of the spacer nucleotides. Saturating single-nucleotide mutagenesis of 195 CRX binding sites shows that, on average, changes in TFBS affinity are correlated with changes in regulatory activity, but this relationship is obscured when considering mutations across multiple CREs. Taken together, these results demonstrate that the activity of CRX binding sites is highly dependent on sequence context, providing insight into photoreceptor gene regulation and illustrating functional principles of homeodomain binding sites that may be conserved in other cell types.
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Project description:We developed a single-cell massively parallel reporter assay (scMPRA) to measure the activity of libraries of cis-regulatory sequences (CRSs) across multiple cell-types simultaneously. As a proof of concept, we assayed a library of core promoters in a mixture of HEK293 and K562 cells and showed that scMPRA is a reproducible, highly parallel, single-cell reporter gene assay. Our results show that housekeeping promoters and CpG island promoters have lower activity in K562 cells relative to HEK293, which likely reflects developmental differences between the cell lines. Within K562 cells, scMPRA identified a subset of developmental promoters that are upregulated in the CD34+/CD38- sub-state, confirming this state as more “stem-like.” Finally, we deconvolved the intrinsic and extrinsic components of cell-to-cell variability and found that developmental promoters have a higher proportion of extrinsic noise compared to housekeeping promoters. We anticipate scMPRA will be widely applicable for studying the role of CRSs across diverse cell types.
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Project description:RNA sequencing (RNAseq) is being used to study inflammatory pathways in chronic rhinosinusitis (CRS). Our goal was to probe validity of tissue eosinophilia as a metric to study RNAseq in CRS. The study was conducted on prospectively enrolled subjects undergoing sinonasal surgery. Subjects were categorized as control, CRS, CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). CRS was also categorized by tissue eosinophil levels per high power field (EOS/HPF) as <10 EOS/HPF or ≥10 EOS/HPF. Ethmoidal tissue samples were processed, differentially expressed (DE) genes were calculated and Ingenuity pathway analysis (IPA) performed.Controls separated clearly from CRS by both study criteria (polyp status, EOS/HPF). In both analyses, CRS differentiated into two distinct CRS subgroups. However, heatmaps showed greater homogeneity within each CRS subtype when studied by eosinophilia versus polyp status. Overall, high differential gene expression was found in CRS versus controls, with 736 statistically significant differentially expressed (DE) genes. On comparison between between CRSwNP and CRSsNP, 60 DE genes were found and on analyzing by tissue EOS/HPF, 110DE genes were statistically significant between CRS <10 EOS/HPF and CRS ≥10 EOS/HPF analyses. Tissue eosinophilia as a metric was further validated by finding of IL 17 signalling pathway between <10EOS/HPF (increased) versus ≥10 EOS/HPF samples on pathway analysis. As a metric, tissue eosinophilia is at least as effective as analysis by polyp status for RNA sequencing and may potentially offer superior insights into mechanistic pathways.
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