Functional analysis of the Vsx2 super-enhancer uncovers distinct cis-regulatory circuits controlling Vsx2 expression during retinogenesis

Abstract

Vsx2 is a transcription factor essential for retinal proliferation and bipolar cell differentiation but the molecular mechanisms underlying its developmental roles are unclear. Here, we profiled VSX2 genomic occupancy during mouse retinogenesis, revealing extensive retinal genetic programs associated with VSX2 during development. VSX2 binds and transactivates its enhancer in association with the transcription factor PAX6. Mice harboring deletions in the Vsx2 regulatory landscape exhibit specific abnormalities in retinal proliferation and bipolar cell differentiation. In one of those deletions, a complete loss of bipolar cells is associated with a bias toward photoreceptor production. VSX2 occupies cis-regulatory elements nearby genes associated with photoreceptor differentiation and homeostasis in the adult mouse and human retina, including a conserved region nearby Prdm1, a factor implicated in the specification of rod photoreceptors and suppression of bipolar cell fate. VSX2 interacts with the transcription factor OTX2 and can act to suppress OTX2-dependent enhancer transactivation of Prdm1 enhancer. Taken together, our analyses indicate that Vsx2 expression can be temporally and spatially uncoupled at the enhancer level and illuminate important mechanistic insights on how VSX2 is engaged with gene regulatory networks essential for retinal proliferation and cell fate acquisition.

Aberrant homeodomain-DNA cooperative dimerization underlies distinct developmental defects in two dominantCRXretinopathy models

“…The CRX K88N’s DNA binding preference at Q 50 HD motifs resembles that of other important retinal HD TFs belonged to the native paired-class Q 50 HD family (11,40). In the developing mouse retinas, these HD TFs are highly expressed in retinal progenitor cells, coordinate the dynamic chromatin landscape changes during retinal neurogenesis, and are down-regulated in differentiating photoreceptors (41,42). We thus predicted that CRX K88N ectopic activities affect the chromatin landscape changes at subset of progenitor CREs that are usually bound by progenitor Q 50 homeoproteins.…”

“…Using a previously published ATAC-seq dataset of normal mouse retinal development (28), we found that Crx K88N -increased ATAC-seq peaks showed the strongest accessibility at neonatal ages P0 and P3, followed by a gradual decrease in accessibility as photoreceptors undergo differentiation (Figure 4C). Re-analysis of a published VSX2 (Q50 HD TF) retinal ChIP-seq data (42) showed that embryonic day 14.5 (E14.5) but not adult VSX2 binding is enriched at the Crx K88N -increased ATAC-seq peaks (Figure 4D). In the embryonic retina, VSX2 is expressed in retinal progenitor cells; in the adult retina, VSX2 expression is maintained in bipolar cells and Müller glia but lost in mature photoreceptors (43)(44)(45).…”

“…The VSX2 ChIP-seq data from the Bian et al study (29) was obtained from GEO under accession number GSE196106. The reads were processed similarly as the CRX ChIP-seq data described in our previous study (17).…”

A framework to identify functional interactors that contribute to disrupted early retinal development in Vsx2 ocular retardation J mice

“…2,8,17 However, the retinal domain is still specified, optic cup morphogenesis occurs, and Vsx2 mRNA (but not protein) remains expressed. 2,6,7,18,19 Genetic analysis of the neomorphic DNA-binding defective mutant Vsx2 R227W , which exhibits a more severe phenotype than the orJ mutant, suggests that Vsx2 ensures lineage fidelity, allowing the retinal gene expression program to occur in the absence of extra-lineage or cryptic gene expression programs. 6 RPC proliferation is greatly reduced in all existing Vsx2 mouse mutants but nevertheless persists through retinal development and at least for the orJ retina, into adult stages.…”

“…Best studied with the recessive loss‐of‐function ocular retardation J allele (homozygotes are referred to as orJ ), which encodes a premature stop codon in the Vsx2 homeodomain, retinal identity is compromised as revealed by ectopic expression of genes normally restricted to RPE or ciliary epithelium 2,8,17 . However, the retinal domain is still specified, optic cup morphogenesis occurs, and Vsx2 mRNA (but not protein) remains expressed 2,6,7,18,19 . Genetic analysis of the neomorphic DNA‐binding defective mutant Vsx2 R227W , which exhibits a more severe phenotype than the orJ mutant, suggests that Vsx2 ensures lineage fidelity, allowing the retinal gene expression program to occur in the absence of extra‐lineage or cryptic gene expression programs 6 .…”