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

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.

Changing homeodomain residues 2 and 3 of Hoxa1 alters its activity in a cell-type and enhancer dependent manner.

The second and third amino acid residues of the N-terminal arm of most Hox protein homeodomains are basic (lysine or arginine), whereas they are asparagine and alanine, respectively, in the Hoxa1 homeodomain. Previous reports pinpointed these residues as specificity determinants in the function of Hoxa1 when it is acting as a monomer. However, in vitro data supported that these residues do not influence the target specificity of Hoxa1 in Pbx1a-Hoxa1 heterodimers. Here, we have analysed the transcriptional activity of a Hoxa1(NA-KR) mutant for which the asparagine and alanine residues of the homeodomain have been replaced by lysine and arginine, respectively. Comparison between the wild-type and mutant Hoxa1 reveals that they show distinct activity on the TSEII enhancer of the somatostatin gene, but that they are equally active in the presence of Pbx and Prep cofactors. This therefore corroborates the biochemical evidence having shown that the second and third residues of the homeodomain do not contribute to the DNA binding of Hoxa1-Pbx dimers. However, on the hoxb1 autoregulatory enhancer, Hoxa1 and Hoxa1(NA-KR) may display distinct activity despite the presence of Pbx, in a cell-type dependent manner. Therefore, our data suggest that, depending on the enhancer, these residues may contribute to the functional specificity of Hoxa1 and that this contribution may not be abrogated by the interaction with Pbx.

Aberrant promoter methylation and expression of UTF1 during cervical carcinogenesis.

Promoter methylation profiles are proposed as potential prognosis and/or diagnosis biomarkers in cervical cancer. Up to now, little is known about the promoter methylation profile and expression pattern of stem cell (SC) markers during tumor development. In this study, we were interested to identify SC genes methylation profiles during cervical carcinogenesis. A genome-wide promoter methylation screening revealed a strong hypermethylation of Undifferentiated cell Transcription Factor 1 (UTF1) promoter in cervical cancer in comparison with normal ectocervix. By direct bisulfite pyrosequencing of DNA isolated from liquid-based cytological samples, we showed that UTF1 promoter methylation increases with lesion severity, the highest level of methylation being found in carcinoma. This hypermethylation was associated with increased UTF1 mRNA and protein expression. By using quantitative RT-PCR and Western Blot, we showed that both UTF1 mRNA and protein are present in epithelial cancer cell lines, even in the absence of its two main described regulators Oct4A and Sox2. Moreover, by immunofluorescence, we confirmed the nuclear localisation of UTF1 in cell lines. Surprisingly, direct bisulfite pyrosequencing revealed that the inhibition of DNA methyltransferase by 5-aza-2'-deoxycytidine was associated with decreased UTF1 gene methylation and expression in two cervical cancer cell lines of the four tested. These findings strongly suggest that UTF1 promoter methylation profile might be a useful biomarker for cervical cancer diagnosis and raise the questions of its role during epithelial carcinogenesis and of the mechanisms regulating its expression.

Hox and Pbx factors control retinoic acid synthesis during hindbrain segmentation.

In vertebrate embryos, retinoic acid (RA) synthesized in the mesoderm by Raldh2 emanates to the hindbrain neuroepithelium, where it induces anteroposterior (AP)-restricted Hox expression patterns and rhombomere segmentation. However, how appropriate spatiotemporal RA activity is generated in the hindbrain is poorly understood. By analyzing Pbx1/Pbx2 and Hoxa1/Pbx1 null mice, we found that Raldh2 is itself under the transcriptional control of these factors and that the resulting RA-deficient phenotypes can be partially rescued by exogenous RA. Hoxa1-Pbx1/2-Meis2 directly binds a specific regulatory element that is required to maintain normal Raldh2 expression levels in vivo. Mesoderm-specific Xhoxa1 and Xpbx1b knockdowns in Xenopus embryos also result in Xraldh2 downregulation and hindbrain defects similar to mouse mutants, demonstrating conservation of this Hox-Pbx-dependent regulatory pathway. These findings reveal a feed-forward mechanism linking Hox-Pbx-dependent RA synthesis during early axial patterning with the establishment of spatially restricted Hox-Pbx activity in the developing hindbrain.

Identification of Lmo1 as part of a Hox-dependent regulatory network for hindbrain patterning.

The embryonic functions of Hox proteins have been extensively investigated in several animal phyla. These transcription factors act as selectors of developmental programmes, to govern the morphogenesis of multiple structures and organs. However, despite the variety of morphogenetic processes Hox proteins are involved in, only a limited set of their target genes has been identified so far. To find additional targets, we used a strategy based upon the simultaneous overexpression of Hoxa2 and its cofactors Pbx1 and Prep in a cellular model. Among genes whose expression was upregulated, we identified LMO1, which codes for an intertwining LIM-only factor involved in protein-DNA oligomeric complexes. By analysing its expression in Hox knockout mice, we show that Lmo1 is differentially regulated by Hoxa2 and Hoxb2, in specific columns of hindbrain neuronal progenitors. These results suggest that Lmo1 takes part in a Hox paralogue 2-dependent network regulating anteroposterior and dorsoventral hindbrain patterning.

The Hoxa2 enhancer 2 contains a critical Hoxa2 responsive regulatory element.

Rhombomeres are embryonic territories arising from the transient segmentation of the hindbrain. Their identity is specified by Hox genes from paralogous groups 1-4. Hoxa2 is the only Hox gene to be expressed in the second rhombomere and the regulatory cues leading to this region-specific expression have been poorly investigated. A 2.5-kb DNA fragment overlapping with the 3' end of Hoxa2 was previously shown to specifically direct the expression of a reporter gene in the second rhombomere and the rostral somites of mouse embryos. Here, we report that this enhancer region is activated in vitro by Hoxa2 and that this activation is strictly dependent on a short 10-bp sequence matching the consensus for Hox-Pbx recognition sites.