An ultraconserved Hox–Pbx responsive element resides in the coding sequence of Hoxa2 and is active in rhombomere 4

Abstract

The Hoxa2 gene has a fundamental role in vertebrate craniofacial and hindbrain patterning. Segmental control of Hoxa2 expression is crucial to its function and several studies have highlighted transcriptional regulatory elements governing its activity in distinct rhombomeres. Here, we identify a putative Hox–Pbx responsive cis-regulatory sequence, which resides in the coding sequence of Hoxa2 and is an important component of Hoxa2 regulation in rhombomere (r) 4. By using cell transfection and chromatin immunoprecipitation (ChIP) assays, we show that this regulatory sequence is responsive to paralogue group 1 and 2 Hox proteins and to their Pbx co-factors. Importantly, we also show that the Hox–Pbx element cooperates with a previously reported Hoxa2 r4 intronic enhancer and that its integrity is required to drive specific reporter gene expression in r4 upon electroporation in the chick embryo hindbrain. Thus, both intronic as well as exonic regulatory sequences are involved in Hoxa2 segmental regulation in the developing r4. Finally, we found that the Hox–Pbx exonic element is embedded in a larger 205-bp long ultraconserved genomic element (UCE) shared by all vertebrate genomes. In this respect, our data further support the idea that extreme conservation of UCE sequences may be the result of multiple superposed functional and evolutionary constraints.

Coding exons function as tissue-specific enhancers of nearby genes

“…Given that several eExons were previously discovered to regulate the gene they reside in (Neznanov et al 1997;Lampe et al 2008;Tumpel et al 2008;Ritter et al 2012), we explicitly set out to search for coding eExons that do not autoregulate but rather could regulate their nearby genes. This was of interest to us due to the phenotypic consequences that coding mutations could have on their nearby genes.…”

“…In addition, a recent study scanning for synonymous constraint in protein coding regions (Lin et al 2011) found an overlap between two of these eExons (Lampe et al 2008;Tumpel et al 2008) and synonymous constraint elements. Here, we analyzed 25 available ChIP-seq data sets of enhancer marks (H3K4me1, H3K27ac, and EP300, also known as p300) for their overlap with coding exons.…”

“…Previous exonic enhancers (eExons) have been reported in vertebrates (Neznanov et al 1997;Lampe et al 2008;Tumpel et al 2008;Dong et al 2010;Eichenlaub and Ettwiller 2011;Ritter et al 2012). In addition, a recent study scanning for synonymous constraint in protein coding regions (Lin et al 2011) found an overlap between two of these eExons (Lampe et al 2008;Tumpel et al 2008) and synonymous constraint elements.…”

Cellular and molecular insights into Hox protein action

“…They share the ability to bind and regulate certain target genes and enhancers in the hindbrain (Alexander et al, 2009;Lampe et al, 2008;Matis et al, 2007), but they display opposite activities in regulating oligodendrocyte precursor cells in this territory. Indeed, Hoxa2 has been shown to inhibit whereas Hoxb2 promotes oligodendrogenesis at its early steps.…”

KPC2 relocalizes HOXA2 to the cytoplasm and decreases its transcriptional activity

“…Gateway® expression vectors for AD-Hoxa2, DB-Hoxa2 and FLAG-Hoxa2 [26], construction of expression vectors for Hoxa2 [31], pCMVlacZ [32], pCMV-PBX1a [33], pCS2-Prep1 [34], Hoxa2 r4 HRE enhancer [35] and the pKS-Hoxa2 [36] plasmids have been described elsewhere. Gateway® entry vectors (pEnt) for human KPC2 (refers as UBAC1) was obtained from the hORFeome v3.1 (http://horfdb.dfci.harvard.edu) [37] and HOXA1, HOXA2, HOXA3, HOXC4, HOXB5, HOXD10 and HOXC11 genes were obtained from the hORFeome v7.1 (http://horfdb.dfci.harvard.edu/hv7/).…”