Hox proteins display a common and ancestral ability to diversify their interaction mode with the PBC class cofactors.

Hox transcription factors control a number of developmental processes with the help of the PBC class proteins. In vitro analyses have established that the formation of Hox/PBC complexes relies on a short conserved Hox protein motif called the hexapeptide (HX). This paradigm is at the basis of the vast majority of experimental approaches dedicated to the study of Hox protein function. Here we questioned the unique and general use of the HX for PBC recruitment by using the Bimolecular Fluorescence Complementation (BiFC) assay. This method allows analyzing Hox-PBC interactions in vivo and at a genome-wide scale. We found that the HX is dispensable for PBC recruitment in the majority of investigated Drosophila and mouse Hox proteins. We showed that HX-independent interaction modes are uncovered by the presence of Meis class cofactors, a property which was also observed with Hox proteins of the cnidarian sea anemone Nematostella vectensis. Finally, we revealed that paralog-specific motifs convey major PBC-recruiting functions in Drosophila Hox proteins. Altogether, our results highlight that flexibility in Hox-PBC interactions is an ancestral and evolutionary conserved character, which has strong implications for the understanding of Hox protein functions during normal development and pathologic processes.

Protein interactions of the transcription factor Hoxa1.

BACKGROUND: Hox proteins are transcription factors involved in crucial processes during animal development. Their mode of action remains scantily documented. While other families of transcription factors, like Smad or Stat, are known cell signaling transducers, such a function has never been squarely addressed for Hox proteins. RESULTS: To investigate the mode of action of mammalian Hoxa1, we characterized its interactome by a systematic yeast two-hybrid screening against ~12,200 ORF-derived polypeptides. Fifty nine interactors were identified of which 45 could be confirmed by affinity co-purification in animal cell lines. Many Hoxa1 interactors are proteins involved in cell-signaling transduction, cell adhesion and vesicular trafficking. Forty-one interactions were detectable in live cells by Bimolecular Fluorescence Complementation which revealed distinctive intracellular patterns for these interactions consistent with the selective recruitment of Hoxa1 by subgroups of partner proteins at vesicular, cytoplasmic or nuclear compartments. CONCLUSIONS: The characterization of the Hoxa1 interactome presented here suggests unexplored roles for Hox proteins in cell-to-cell communication and cell physiology.

Comparison of a Barcode-Based Smartphone Application to a Questionnaire to Assess the Use of Cleaning Products at Home and Their Association with Asthma Symptoms.

Household disinfectant and cleaning products (HDCPs) assessment is challenging in epidemiological research. We hypothesized that a newly-developed smartphone application was more objective than questionnaires in assessing HDCPs. Therefore, we aimed to compare both methods, in terms of exposure assessments and respiratory health effects estimates. The women of the SEPAGES birth cohort completed repeated validated questionnaires on HDCPs and respiratory health and used an application to report HDCPs and scan products barcodes, subsequently linked with an ingredients database. Agreements between the two methods were assessed by Kappa coefficients. Logistic regression models estimated associations of HDCP with asthma symptom score. The 101 participants (18 with asthma symptom score ≥1) scanned 617 different products (580 with available ingredients list). Slight to fair agreements for sprays, bleach and scented HDCP were observed (Kappa: 0.35, 0.25, 0.11, respectively). Strength of the associations between HDCP and asthma symptom score varied between both methods but all odds ratios (OR) were greater than one. The number of scanned products used weekly was significantly associated with the asthma symptom score (adjusted-OR [CI 95%]: 1.15 [1.00-1.32]). This study shows the importance of using novel tools in epidemiological research to objectively assess HDCP and therefore reduce exposure measurement errors.

Pentapeptide insertion mutagenesis of the Hoxa1 protein: mapping of transcription activation and DNA-binding regulatory domains.

The mode of action of Hoxa1, like that of most Hox proteins, remains poorly characterized. In an effort to identify functional determinants contributing to the activity of Hoxa1 as a transcription factor, we generated 18 pentapeptide insertion mutants of the Hoxa1 protein and we assayed them in transfected cells for their activity on target enhancers from the EphA2 and Hoxb1 genes known to respond to Hoxa1 in the developing hindbrain. Only four mutants displayed a complete loss-of-function. Three of them contained an insertion in the homeodomain of Hoxa1, whereas the fourth loss-of-function mutant harbored an insertion in the very N-terminal end of the protein. Transcription activation assays in yeast further revealed that the integrity of both the N-terminal end and homeodomain is required for Hoxa1-mediated transcriptional activation. Furthermore, an insertion in the serine-threonine-proline rich C-terminal extremity of Hoxa1 induced an increase in activity in mammalian cells as well as in the yeast assay. The C-terminal extremity thus modulates the transcriptional activation capacity of the protein. Finally, electrophoretic mobility shift assays revealed that the N-terminal extremity of the protein also exerts a modulatory influence on DNA binding by Hoxa1-Pbx1a heterodimers.

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.

Loss of function but no gain of function caused by amino acid substitutions in the hexapeptide of Hoxa1 in vivo.

Homeodomain containing transcription factors of the Hox family play critical roles in patterning the anteroposterior embryonic body axis, as well as in controlling several steps of organogenesis. Several Hox proteins have been shown to cooperate with members of the Pbx family for the recognition and activation of identified target enhancers. Hox proteins contact Pbx via a conserved hexapeptide motif. Previous biochemical studies provided evidence that critical amino acid substitutions in the hexapeptide sequence of Hoxa1 abolish its interaction with Pbx. As a result, these substitutions also abolish Hoxa1 activity on known target enhancers in cellular models, suggesting that Hoxa1 activity relies on its capacity to interact with Pbx. Here, we show that mice with mutations in the Hoxa1 hexapeptide display hindbrain, cranial nerve, and skeletal defects highly reminiscent of those reported for the Hoxa1 loss of function. Since similar hexapeptide mutations in the mouse Hoxb8 and the Drosophila AbdA proteins result in activity modulation and gain of function, our data demonstrate that the functional importance of the hexapeptide in vivo differs according to the Hox proteins.

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.

Mutation analysis of the HOX paralogous 4-13 genes in children with acute lymphoid malignancies: identification of a novel germline mutation of HOXD4 leading to a partial loss-of-function.

The molecular basis of susceptibility to childhood malignant hemopathy remains largely unknown. An excess of skeletal congenital anomalies has been reported among children with hematological malignancy and points towards involvement of developmental genes, like those belonging to the HOX gene family. In addition to their role in embryogenesis, HOX transcription factors are known to be regulators of proliferation and differentiation of hematopoietic cells. We aimed to explore the possibility that germline alterations of HOX genes might be involved in childhood acute lymphoid malignancies. A cohort of 86 children diagnosed with acute lymphoid malignancy was studied, 20 of them concurrently presenting a congenital anomaly of the skeleton. First, we screened for nucleotide changes throughout the HOX genes of paralogous groups 4 to 13 in the 20 patients with skeletal defects, following a skeletal phenotype-based strategy. Subsequently, we extended the HOX mutation screening to the other 66 children having a malignant lymphoproliferative disorder, but without skeletal defects. In total, 16 germline mutations were identified. While 13 changes were also observed in healthy controls, three variants were exclusively found in acute lymphoid malignancy cases. These comprised the germline c.242A>T (p.Glu81Val) missense mutation of HOXD4, detected in two children diagnosed with acute lymphoblastic leukemia (ALL). Furthermore, this mutation was found in association with other specific HOX variants of cluster D (2q31-q37), defining a unique haplotype. Functional analysis of the murine Hoxd4 homolog revealed that mutant Hoxd4 protein had lower transcriptional activity than wild-type protein in vitro. The p.Glu81Val mutation of HOXD4 thus results in a partial loss-of-function, which might be involved in childhood ALL.

A retinoic acid responsive Hoxa3 transgene expressed in embryonic pharyngeal endoderm, cardiac neural crest and a subdomain of the second heart field.

A transgenic mouse line harbouring a ß-galacdosidase reporter gene controlled by the proximal 2 kb promoter of Hoxa3 was previously generated to investigate the regulatory cues governing Hoxa3 expression in the mouse. Examination of transgenic embryos from embryonic day (E) 8.0 to E15.5 revealed regionally restricted reporter activity in the developing heart. Indeed, transgene expression specifically delineated cells from three distinct lineages: a subpopulation of the second heart field contributing to outflow tract myocardium, the cardiac neural crest cells and the pharyngeal endoderm. Manipulation of the Retinoic Acid (RA) signaling pathway showed that RA is required for correct expression of the transgene. Therefore, this transgenic line may serve as a cardiosensor line of particular interest for further analysis of outflow tract development.

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 nuclear factor kappaB-activator gene PLEKHG5 is mutated in a form of autosomal recessive lower motor neuron disease with childhood onset.

Lower motor neuron diseases (LMNDs) include a large spectrum of clinically and genetically heterogeneous disorders. Studying a large inbred African family, we recently described a novel autosomal recessive LMND variant characterized by childhood onset, generalized muscle involvement, and severe outcome, and we mapped the disease gene to a 3.9-cM interval on chromosome 1p36. We identified a homozygous missense mutation (c.1940 T-->C [p.647 Phe-->Ser]) of the Pleckstrin homology domain-containing, family G member 5 gene, PLEKHG5. In transiently transfected HEK293 and MCF10A cell lines, we found that wild-type PLEKHG5 activated the nuclear factor kappa B (NF kappa B) signaling pathway and that both the stability and the intracellular location of mutant PLEKHG5 protein were altered, severely impairing the NF kappa B transduction pathway. Moreover, aggregates were observed in transiently transfected NSC34 murine motor neurons overexpressing the mutant PLEKHG5 protein. Both loss of PLEKHG5 function and aggregate formation may contribute to neurotoxicity in this novel form of LMND.