Étude de la régulation transcriptionnelle du gène hoxa5 chez la souris

Les gènes Hox codent pour des facteurs de transcription orchestrant l'identité antéro-postérieure du plan corporel des animaux à symétrie bilatérale. La souris Hoxa5-/- a permis de démontrer que ce gène joue un rôle primordial dans la spécification des squelettes axial et appendiculaire, ainsi que dans l'ontogénie de plusieurs organes. À l'aide d'une approche de transgenèse et de délétions successives de la séquence intergénique Hoxa4-Hoxa5, j'ai identifié deux éléments régulateurs responsables de l'expression du gène Hoxa5 dans les systèmes respiratoire et digestif: un fragment d'ADN NcoI-SacI de 163-pb possédant une activité de type activatrice et dirigeant l'expression au niveau du poumon, de l'estomac et de l'intestin, de même qu'un fragment XbaI- BssHII de 259-pb, nécessaire à une expression complète du gène Hoxa5 au niveau du système digestif. Des expériences de retard sur gel (EMSA) et d'immunoprécipitation de la chromatine (ChIP) m'ont permis de démontrer la liaison du facteur de transcription YY1 à ces deux séquences d'ADN. En mutant ses sites de liaison dans un contexte de transgenèse, j'ai mis en évidence le rôle de YY1 comme activateur transcriptionnel du gène Hoxa5 dans les organes. Il s'agit d'ailleurs d'un des rares exemples où la protéine YY1 ne réprime pas l'expression des gènes Hox. J'ai également appliqué la technique de ChIP pour confirmer que les facteurs de transcription à boîte homéo CDX4 et HOXB9 se lient tous les deux au fragment d'ADN AvrII-Eco47III de 164-pb situé à l'intérieur de l'élément MES. J'ai donc montré que la protéine HOXB9 participe à restreindre caudalement l'expression du gène Hoxa5 au niveau de la prévertèbre 10, supportant ainsi le concept de prévalence postérieure. De plus, j'ai généré deux lignées de souris transgéniques exprimant la recombinase Cre sous le contrôle de deux combinaisons de séquences régulatrices identifiées du gène Hoxa5. Ces lignées ont été caractérisées et fournissent de nouveaux outils utiles pour étudier la fonction de différents gènes dans certains tissus le long de l'axe antéro-postérieur. Enfin, le locus Hoxa5 produit 4 trasncrits de 1.8, 5.0, 9.5 et 11-kb de longueur se chevauchant et pouvant produire une protéine in vitro. Cependant, j'ai démontré que seul le court transcrit de 1.8-kb, correspondant aux deux exons connus du gène Hoxa5, génère une protéine associée à la fonction du gène in vivo. Les différents résultats obtenus seront présentés et discutés.Hox genes encode transcription factors, which orchestrate bilaterian anteroposterior patterning. Using Hoxa5-/- mice as model, we have demonstrated that this gene plays a key role in axial and appendicular skeletal patterning as well as in the formation of several organs such as the respiratory and digestive tracts. Using a transgenesis approach and successive deletions in the Hoxa4-Hoxa5 intergenic region, I have identified two distinct regulatory elements responsible for Hoxa5 expression in respiratory and digestive tracts: a 163-bp NcoI-SacI DNA fragment having enhancer activity that drives expression in lung, stomach and intestine, and a 259-bp XbaI-BssHII fragment necessary for a complete Hoxa5 digestive tract expression. Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays have demonstrated the capacity of the YY1 transcription factor to bind these two DNA sequences. By mutating its binding sites in a transgenesis context, I have highlighted the transcriptional activator role of the YY1 protein in Hoxa5 organ expression, which is very interesting since few examples of Hox gene activation by YY1 are reported in the literature. I have also generated two transgenic mice lines expressing the Cre recombinase under the control of two combinations of identified regulatory sequences. These lines have been charaterized and provide useful genetic tools to study gene function in specific tissues along the anteroposterior axis. I have also applicate ChIP technology to demonstrate the in vivo binding of CDX4 and HOXB9 homeobox transcription factors to the 164-bp AvrII-Eco47III DNA fragment included in the MES regulatory element. Consequently, I have shown that the HOXB9 protein caudally participates to restrict the Hoxa5 gene expression at the level of prevertebra 10, which supports the posterior prevalence concept. Finaly, the Hoxa5 locus encompasses 4 overlapping transcripts of 1.8, 5.0, 9.5 and 11.0-kb that can produce a HOXA5 protein in an in vitro context. However, I have demonstrated that only the short transcript of 1.8-kb corresponding to the two known Hoxa5 gene exons is transcribed into an in vivo HOXA5 protein associated to the gene function. Data will be presented and discussed

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

The 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.We 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.Our 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

YY1 acts as a transcriptional activator of Hoxa5 gene expression in mouse organogenesis.

The Hox gene family encodes homeodomain-containing transcriptional regulators that confer positional information to axial and paraxial tissues in the developing embryo. The dynamic Hox gene expression pattern requires mechanisms that differentially control Hox transcription in a precise spatio-temporal fashion. This implies an integrated regulation of neighbouring Hox genes achieved through the sharing and the selective use of defined enhancer sequences. The Hoxa5 gene plays a crucial role in lung and gut organogenesis. To position Hoxa5 in the regulatory hierarchy that drives organ morphogenesis, we searched for cis-acting regulatory sequences and associated trans-acting factors required for Hoxa5 expression in the developing lung and gut. Using mouse transgenesis, we identified two DNA regions included in a 1.5-kb XbaI-XbaI fragment located in the Hoxa4-Hoxa5 intergenic domain and known to control Hoxa4 organ expression. The multifunctional YY1 transcription factor binds the two regulatory sequences in vitro and in vivo. Moreover, the mesenchymal deletion of the Yy1 gene function in mice results in a Hoxa5-like lung phenotype with decreased Hoxa5 and Hoxa4 gene expression. Thus, YY1 acts as a positive regulator of Hoxa5 expression in the developing lung and gut. Our data also support a role for YY1 in the coordinated expression of Hox genes for correct organogenesis

Characterization of the <i>Hoxa4-Hoxa5</i> intergenic region in E13.5 F0 transgenic mouse embryos.

<p>(A) Schematic representation of the 1.5-kb <i>Xba</i>I-<i>Xba</i>I DNA fragment in the <i>Hoxa4-Hoxa6</i> genomic region. (B) Diagram of the <i>Hoxa5/lacZ</i> constructs used to generate E13.5 F0 transgenic embryos and summary of transgenic expression analyses. A, <i>Acc</i>I; Ap, <i>Apa</i>I; Bh, <i>Bss</i>HII; Bm, <i>Bsm</i>I; RI, <i>EcoR</i>I; H, <i>Hind</i>III; K, <i>Kpn</i>I; Mf, <i>Mfe</i>I; Nc, <i>Nco</i>I; Sc, <i>Sac</i>I; Xb, <i>Xba</i>I; Xh, <i>Xho</i>I. (C–H) Carcass of representative E13.5 transgenic embryos and the associated organs (I–N) stained for β-galactosidase activity showed the effects of the different deletions on the expression pattern. Open arrowheads point the anterior limit of transgene expression in the neural tube. The number in the lower left corner of each panel corresponds to the transgene. h, heart; i, intestine; l, lung; nt, neural tube; pv, prevertebrae; s, stomach; sp, spleen.</p

Characterization of the 1.5-kb <i>Xba</i>I-<i>Xba</i>I DNA fragment in E13.5 F0 transgenic mouse embryos.

<p>(A) Schematic representation of the 1.5-kb <i>Xba</i>I-<i>Xba</i>I DNA fragment in the <i>Hoxa4-Hoxa6</i> genomic region. (B) Diagram of the <i>Hoxa5/lacZ</i> constructs used to generate E13.5 F0 transgenic embryos and summary of transgenic expression analyses. (C–F) Carcass of representative E13.5 transgenic embryos and the associated organs (G–J) stained for β-galactosidase activity showed the effects of the different deletions on the expression pattern. Open arrowhead points the anterior limit of transgene expression in the neural tube. i, intestine; l, lung; nt, neural tube; pv, prevertebrae; s, stomach; sp, spleen.</p

Oligonucleotide sequences.

<p>Oligonucleotide sequences.</p

Characterization of the RARE and YY1 binding sites in E13.5 F0 transgenic mouse embryos.

<p>(A) Schematic representation of the 1.5-kb <i>Xba</i>I-<i>Xba</i>I DNA fragment in the <i>Hoxa4-Hoxa6</i> genomic region. (B) Diagram of the <i>Hoxa5/lacZ</i> constructs used to generate E13.5 F0 transgenic embryos and summary of transgenic expression analyses. The asterisks in constructs 17–18 and the @ symbols in constructs 19–22 correspond to mutations in the RARE and YY1 binding sites, respectively. (C–H) Carcass of representative E13.5 transgenic embryos and the associated organs (I–N) stained for β-galactosidase activity showed the effects of the mutations on the expression pattern. Open arrowhead points the anterior limit of transgene expression in the neural tube. i, intestine; l, lung; nt, neural tube; pv, prevertebrae; s, stomach.</p

Characterization of the 751-bp <i>Bss</i>HII-<i>Sac</i>I DNA fragment in E13.5 F0 transgenic mouse embryos.

<p>(A) Schematic representation of the 1.5-kb <i>Xba</i>I-<i>Xba</i>I DNA fragment in the <i>Hoxa4-Hoxa6</i> genomic region. (B) Diagram of the <i>Hoxa5/lacZ</i> constructs used to generate E13.5 F0 transgenic embryos and summary of transgenic expression analyses. (C–G) Carcass of representative E13.5 transgenic embryos and the associated organs (H–L) stained for β-galactosidase activity showed the effects of the different deletions on the expression pattern. i, intestine; l, lung; nt, neural tube; pv, prevertebrae; s, stomach.</p

Characterization of the 259-bp <i>Xba</i>I-<i>Bss</i>HII DNA fragment by EMSA.

<p>(A) Restriction map of the 1.5-kb <i>Xba</i>I-<i>Xba</i>I sequence extending from +9.3-kb to +10.8-kb in the 3′ half of the <i>Hoxa4</i>-<i>Hoxa5</i> intergenic region. The box denotes the location of the 259-bp <i>Xba</i>I-<i>Bss</i>HII regulatory region. Sequence of the 259-bp <i>Xba</i>I<i>-Bss</i>HII DNA fragment is indicated. Fragments A, B and C used as competitor in EMSA are underlined. Fragment C was further subdivided into Oligos C1, C2, C3 and Oligo RARE and Oligo-18(C3). Boxed nucleotides correspond to RARE-DR5 sequence and YY1 binding sites. Symbols * and @ indicate point mutations into RARE and YY1 binding sites, respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093989#pone-0093989-t001" target="_blank">Table 1B</a> for nucleotide sequences). (B) EMSA with WCE from lung/stomach/intestine of E13.5 embryos and the 259-bp <i>Xba</i>I-<i>Bss</i>HII radiolabelled probe in presence of unlabelled competitors in 100-fold excess showed that protein binding occurred with the Oligo-18(C3) fragment via YY1 binding sites (lanes 9, 12, 18–19). No binding with the RARE site was observed (lanes 4-6). EMSA with <i>in vitro</i>-translated YY1 protein and the 259-bp <i>Xba</i>I-<i>Bss</i>HII probe showed specific binding that was competed by Oligo C3 (lanes 21–24). (C) The binding of WCE with YY1 consensus binding site and the loss of binding when the YY1 antibody was added confirmed the presence of YY1 protein in WCE (lanes 1–5). (D) EMSA with WCE and Oligo C3 radiolabelled probe showed binding that was competed by an excess of cold Oligo C3, Oligo-18(C3) sequence, the YY1 consensus sequence, localized mutations in YY1 sites, and the addition of the YY1-specific antibody (lanes 1–4, 6–9). No competition occurred when a non-specific probe was used (Oligo C2), when several mutations were distributed along the YY1 binding sites in Oligo C3 or when the CDX2 control antibody was used (lanes 5, 10–11). Arrows and brackets indicate the bands corresponding to YY1 binding. COMP, competitor; r. lysate, reticulocyte lysate.</p

Analysis of the lung phenotype in <i>Yy1</i>^flox/flox<i>Dermo1</i>^+/Cre mutants.

<p>(A)Ratios of genotypes of litters obtained from matings between <i>Yy1</i>^flox/+<i>Dermo1</i>^+/Cre and <i>Yy1</i>^flox/flox mice. (B–E) Comparative lung histology of E18.5 <i>Yy1</i>^flox/+<i>Dermo1</i>^+/+, <i>Yy1</i>^flox/+<i>Dermo1</i>^+/Cre, <i>Yy1</i>^flox/flox<i>Dermo1</i>^+/Cre and <i>Hoxa5</i>^-/- embryos. <i>Yy1</i>^flox/+<i>Dermo1</i>^+/+ and <i>Yy1</i>^flox/+<i>Dermo1</i>^+/Cre specimens presented a normal lung structure, whereas lungs from <i>Yy1</i>^flox/flox<i>Dermo1</i>^+/Cre and <i>Hoxa5</i>^-/- embryos were collapsed. (F–M) Characterization of the respiratory epithelium of E18.5 <i>Yy1</i>^flox/flox<i>Dermo1</i>^+/Cre embryos. (F–I) Detection of club cells by CC10 immunostaining showed decreased labelling in lungs from <i>Yy1</i>^flox/flox<i>Dermo1</i>^+/Cre and <i>Hoxa5</i>^-/- specimens. (J–M) Immunostaining with T1α, a marker of type I pneumocytes, was reduced in lungs from <i>Yy1</i>^flox/flox<i>Dermo1</i>^+/Cre and <i>Hoxa5</i>^-/- embryos. (N–P) YY1 immunostaining showed ubiquitous YY1 expression in lung epithelial and mesenchymal compartments in E15.5 control embryos, but an important decreased staining in lung mesenchyme from <i>Yy1</i>^flox/flox<i>Dermo1</i>^+/Cre specimens. e, epithelium; m, mesenchyme. Scale bar: 200 μm. (Q–S) qRT-PCR analysis for <i>Scgb1a1</i>, <i>T1α</i>, <i>Yy1</i>, <i>Hoxa5</i> and <i>Hoxa4</i> expression in lungs from E18.5 <i>Yy1</i>^flox/+<i>Dermo1</i>^+/+ and <i>Yy1</i>^flox/flox<i>Dermo1</i>^+/Cre embryos. Expression levels were significantly diminished for all genes tested in <i>Yy1</i>^flox/flox<i>Dermo1</i>^+/Cre specimens. Values are expressed as means ± SEM. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p