Enhancer timing of Hox gene expression: deletion of the endogenousHoxc8early enhancer

Abstract

The proper expression of Hox genes is necessary for the accurate patterning of the body plan. The elucidation of the developmental genetic basis of transcriptional regulation of Hox genes by the study of their cis-regulatory elements provides crucial information regarding the establishment of axial specification. In this report, we investigate the role of the early enhancer(EE) of the murine Hoxc8 gene to better understand its role in pattern formation. Previous reports show that knockouts of the endogenous Hoxc8 coding region result in a combination of neural, behavioral and skeletal phenotypes. In this report, we limit ourselves to a consideration of the skeletal abnormalities. Early reports from our laboratory based on exogenous transgenic reporter constructs implicate a 200 bp non-coding element 3 kb upstream of the Hoxc8 promoter as a crucial enhancer that regulates the transcription of Hoxc8. In the present work, we have deleted this regulatory region from the endogenous genome using embryonic stem cell technology. Our results show that the deletion of the EE results in a significant delay in the temporal expression of Hoxc8. We also show that the deletion of the EE does not eliminate the expression of the Hoxc8 protein, but delays the attainment of control levels of expression and anterior and posterior boundaries of expression on the AP axis. The temporal delay in Hoxc8 expression is sufficient to produce phenocopies of many of the axial skeletal defects associated with the complete absence of Hoxc8 gene product as previously reported for the Hoxc8-null mutation. Our results are consistent with emerging evidence that the precise temporal expression of Hox genes is crucial for the establishment of regional identities. The fact that the EE deletion does not eliminate Hoxc8expression indicates the existence of a Hoxc8 transcriptional regulatory apparatus independent to some degree of the Hoxc8 EE. In a comparison of our results with those reported previously by others investigating temporal control of Hox gene expression, we have discovered a structural similarity between the Hoxc8 EE reported here and a transcriptional control element located in the Hoxd11 region. We speculate that a distributed system of expression timing control may exist that is similar the one we propose for Hoxc8. Last, our data is consistent with the position that disparate regulatory pathways are responsible for the expression of Hoxc8 in the organogenesis of somites, neural tube and limb bud.

GTF2IRD1 regulates transcription by binding an evolutionarily conserved DNA motif ‘GUCE’

“…The importance of the Hoxc8 EE for transcriptional control was demonstrated in mutant mouse models deleted for the 200 bp Hoxc8 EE (encompassing GUCE) [32] which showed significant delay in the temporal expression of Hoxc8 and also shares aspects of its phenotype with our Gtf2ird1 null mouse [2]; both display an abnormal clasping of the limbs when suspended by the tail. This leads us to speculate that aspects of the neurological phenotypes observed in WBS could be influenced by GTF2IRD1 induced misregulation of HOXC8.…”

“…DNA duplexes (50 pmol) were end-labelled with [c- 32 P] ATP and purified on Sephadex G50 columns (Amersham Biosciences). Following ethanol precipitation, the probes were resuspended in 10 mM TrisHCl (pH 7.0), 50 mM NaCl.…”

The significance of Hox gene collinearity

“…In vertebrates, these include retinoic acid, FGF, Gdf11 and Cdx proteins. We know that Hox/lacZ reporters are not just responding to auto-regulatory cues from endogenous Hox genes, as has been suggested (Gould et al, 1997), because for example, multiple Cdx binding sites within the transgenes (Charite et al, 1998, Gaunt et al, 2004, or increased Cdx dosage (Juan andRuddle, 2003, Schyr et al, 2012), can drive lacZ expression both earlier and anterior to expression of the endogenous gene. While results for Hox/lacZ transgenes argue against the instructive form of the chromatin opening model they may still be accommodated by the restrictive form.…”

The regulation of Hox gene expression during animal development

“…This latter and belated regulatory phase seems to rely on the activity of a second tier of control elements that produce specific Hox expression patterns, which are later maintained by the PcG and trxG systems (Alexander et al, 2009;Deschamps and van Nes, 2005). The tempo of Hox gene activation is functionally important because experimental conditions resulting in premature or delayed Hox gene activation have been shown to produce phenotypic alterations, even in cases when the final Hox expression patterns are preserved Juan and Ruddle, 2003;Gérard et al, 1997;Kondo and Duboule, 1999). This is consistent with the existence of distinct functional activities associated with early and late phases of vertebrate Hox gene expression (Carapuço et al, 2005).…”