Shared retinoic acid responsive enhancers coordinately regulate nascent transcription of Hoxb coding and non-coding RNAs in the developing mouse neural tube

Abstract

Signaling pathways regulate the patterns of Hox gene expression that underlie their functions in specification of axial identity. Little is known about the properties of cis-regulatory elements and underlying transcriptional mechanisms that integrate graded signaling inputs to coordinately control Hox expression. Here we optimized single molecule fluorescent in situ hybridization (smFISH) technique with probes spanning introns to evaluate how three shared retinoic acid response element (RARE)-dependent enhancers in the Hoxb cluster regulate patterns of nascent transcription in vivo at the level of single cells in wild type and mutant embryos. We predominately detect nascent transcription of only a single Hoxb gene in each cell, with no evidence for simultaneous co-transcriptional coupling of all or specific subsets of genes. Single and/or compound RARE mutations indicate each enhancer differentially impacts global and local patterns of nascent transcription, suggesting that selectivity and competitive interactions between these enhancers is important to robustly maintain the proper levels and patterns of nascent Hoxb transcription. This implies rapid and dynamic regulatory interactions potentiate transcription of genes through combined inputs from these enhancers in coordinating the RA response.

Direct comparison of Hoxb8-driven reporter distribution in the brains of four transgenic mouse lines: towards a spinofugal projection atlas

“…transgene insertions to report transcriptional activity of a given gene is affected (either positively or negatively, depending on the question being asked) by the absence of regulatory elements controlling endogenous expression. In the case of the construct used to generate Hoxb8-Cre and Hoxb8-FlpO mice, the transgenes are no longer in the vicinity of an important retinoic acid response element (RARE) called the distal element (DE), positioned nearby between the Hoxb4 and Hoxb5 genes (Oosterveen et al, 2003;Valarché et al, 2003;Afzal et al, 2023), which is responsible for extension Hoxb8 expression from C4 to the obex. Ectopic recombination, on the other hand, could be the result of the absence of suppressive regulatory sequences, insertion of the transgene in the vicinity of non-canonical promoters or enhancers, or temporally-altered chromatin accessibility.…”

“…Whichever mechanism (or combination of mechanisms) is responsible, it still requires a stimulus for the activation of Hoxb8 expression. The dependence of Hoxb8 transcription on retinoic acid, elegantly dissected by the Deschamps and Krumlauf groups (Charité et al, 1995;Bel-Vialar et al, 2002;Forlani et al, 2003;Afzal et al, 2023), makes developmental exposure to this diffusible signaling molecule a likely cause. Indeed, ectopic recombination occurred close to tissues expressing retinoic synthesizing enzymes (RALDH1 and RALDH2) (olfactory epithelium, retina, craniofacial mesenchyme below the cephalic flexure), choroid plexus [Ruberte et al, 1993;Yamamoto et al, 1998;Luo et al, 2004 and atlas.brain-map.org], and/or in brain regions rendered sensitive to endogenous retinoic acid using a RARE-driven LacZ reporter (anterior thalamic nuclei, dentate gyrus) (Luo et al, 2004).…”

“…Retinoic acid-dependent Hox gene regulation has many layers, and relief from Hoxb8 suppression must contribute to ectopic expression. Recently, Afzal et al (2023), used CRISPR/Cas9 to generate mutations in the DE and two other RAREs (B4U and ENE) flanking the Hoxb4 coding sequence to determine their role in Hoxb regulation. With DE and ENE mutations, there was increased transcription of both Hoxb9 and Hoxb1, with a greater increase in the ENE mutant embryos (Afzal et al, 2023).…”

“…Recently, Afzal et al (2023), used CRISPR/Cas9 to generate mutations in the DE and two other RAREs (B4U and ENE) flanking the Hoxb4 coding sequence to determine their role in Hoxb regulation. With DE and ENE mutations, there was increased transcription of both Hoxb9 and Hoxb1, with a greater increase in the ENE mutant embryos (Afzal et al, 2023). The B4U mutation did not result in changes from wild-type mice with respect to Hoxb9 or Hoxb1 transcription, but Hoxb4 transcription was increased.…”

“…The B4U mutation did not result in changes from wild-type mice with respect to Hoxb9 or Hoxb1 transcription, but Hoxb4 transcription was increased. This suggests that the DE and ENE are important in the regulation of all Hoxb genes, while B4U acts on more closely adjacent sequences in the Hoxb cluster (Afzal et al, 2023). Given that loss of control by ENE led to an increase in Hoxb9 transcription, its absence may similarly affect the Hoxb8 transgene leading to its ectopic expression.…”