Multiple Promoters and Alternative Splicing: Hoxa5 Transcriptional Complexity in the Mouse Embryo

Abstract

BackgroundThe genomic organization of Hox clusters is fundamental for the precise spatio-temporal regulation and the function of each Hox gene, and hence for correct embryo patterning. Multiple overlapping transcriptional units exist at the Hoxa5 locus reflecting the complexity of Hox clustering: a major form of 1.8 kb corresponding to the two characterized exons of the gene and polyadenylated RNA species of 5.0, 9.5 and 11.0 kb. This transcriptional intricacy raises the question of the involvement of the larger transcripts in Hox function and regulation.Methodology/Principal FindingsWe have undertaken the molecular characterization of the Hoxa5 larger transcripts. They initiate from two highly conserved distal promoters, one corresponding to the putative Hoxa6 promoter, and a second located nearby Hoxa7. Alternative splicing is also involved in the generation of the different transcripts. No functional polyadenylation sequence was found at the Hoxa6 locus and all larger transcripts use the polyadenylation site of the Hoxa5 gene. Some larger transcripts are potential Hoxa6/Hoxa5 bicistronic units. However, even though all transcripts could produce the genuine 270 a.a. HOXA5 protein, only the 1.8 kb form is translated into the protein, indicative of its essential role in Hoxa5 gene function. The Hoxa6 mutation disrupts the larger transcripts without major phenotypic impact on axial specification in their expression domain. However, Hoxa5-like skeletal anomalies are observed in Hoxa6 mutants and these defects can be explained by the loss of expression of the 1.8 kb transcript. Our data raise the possibility that the larger transcripts may be involved in Hoxa5 gene regulation.SignificanceOur observation that the Hoxa5 larger transcripts possess a developmentally-regulated expression combined to the increasing sum of data on the role of long noncoding RNAs in transcriptional regulation suggest that the Hoxa5 larger transcripts may participate in the control of Hox gene expression.

Partial functional redundancy betweenHoxa5andHoxb5paralog genes during lung morphogenesis

“…1, F and H). In contrast, Hoxb5 expression remained unchanged in lungs from Even though mutant transcripts are produced from the Hoxa5 and Hoxb5 mutant alleles, both Hoxa5 and Hoxb5 mutations generate a null allele with no production of protein (11,43). We observed a loss of Hoxa5 expression in lungs from Hoxa5 Ϫ/Ϫ embryos and a reduced Hoxb5 expression in Hoxb5 Ϫ/Ϫ specimens that might reflect the interference of the neo selection cassette on Hox transcription as reported (Fig.…”

Bimodal control of Hoxd gene transcription in the spinal cord defines two regulatory subclusters

“…Promoter competition and enhancer titration Hox clusters display very dense transcriptional activities, owing to the presence of numerous alternative transcription start sites and enhancers intermingled within the coding regions (Mainguy et al, 2007;Rinn et al, 2007;Coulombe et al, 2010). Accordingly, we find extensive transcription outside the annotated coding sequences of the HoxD complex, which contribute to delineate the two distinct transcription blocks.…”

The regulation of Hox gene expression during animal development

“…The role of such a molecule is still unclear, as functional analysis in transgenic mice indicate that it is not able to activate the typical Hoxa10 patterning programme in the axial skeleton or interfere with it (Guerreiro et al, 2012). Hoxb3 and Hoxa5 also produce different mRNA isoforms through a combination of alternative splicing and alternative promoter usage; interestingly, the resulting isoforms are expressed in different domains during embryonic development (Chan et al, 2010;Coulombe et al, 2010;Sham et al, 1992), once again suggesting that the resulting isoforms might carry out specific functions. Finally, a recent bioinformatic study (P. Patraquim and C.R.A., unpublished) focused on mouse Hox genes revealed that approximately a third of them produce a spectrum of mRNA isoforms generated by different types of RNA processing, many of which are evolutionarily conserved between mouse and human.…”