Lucie Jeannotte

Structure of the rat pro-opiomelanocortin (POMC) gene

The gene encoding pro‐opiomelanocortin (POMC) presents unique regulatory features. In particular, glucocorticoids inhibit transcription of the POMC gene in the anterior pituitary, but not in the intermediate pituitary. In order to study the mechanism leading to transcriptional inhibition of POMC by glucocorticoid and the interaction of the glucocorticoid receptor complex with specific DNA sequences along the POMC gene, we have cloned the rat POMC gene and determined its structure. The gene is composed of three exons and appears to be present at a single copy per haploid genome. Besides the usual regulatory signals like ‘TATA’ and ‘CCAAT’ boxes, the upstream region contains sequences homologous to known enhancer sequences and to the glucocorticoid receptor binding site observed in glucocorticoid‐responsive genes.

Leukaemic transformation by CALM¿AF10 involves upregulation of Hoxa5 by hDOT1L

Chromosomal translocation is a common cause of leukaemia and the most common chromosome translocations found in leukaemia patients involve the mixed lineage leukaemia (MLL) gene. AF10 is one of more than 30 MLL fusion partners in leukaemia. We have recently demonstrated that the H3K79 methyltransferase hDOT1L contributes to MLL–AF10-mediated leukaemogenesis through its interaction with AF10 (ref. 5). In addition to MLL, AF10 has also been reported to fuse to CALM (clathrin-assembly protein-like lymphoid–myeloid) in patients with T-cell acute lymphoblastic leukaemia (T-ALL) and acute myeloid leukaemia (AML). Here, we analysed the molecular mechanism of leukaemogenesis by CALM–AF10. We demonstrate that CALM–AF10 fusion is both necessary and sufficient for leukaemic transformation. Additionally, we provide evidence that hDOT1L has an important role in the transformation process. hDOT1L contributes to CALM–AF10-mediated leukaemic transformation by preventing nuclear export of CALM–AF10 and by upregulating the Hoxa5 gene through H3K79 methylation. Thus, our study establishes CALM–AF10 fusion as a cause of leukaemia and reveals that mistargeting of hDOT1L and upregulation of Hoxa5 through H3K79 methylation is the underlying mechanism behind leukaemia caused by CALM–AF10 fusion.

Sustained Hox5 Gene Activity is Required for Respiratory Motor Neuron Development

Respiration in mammals relies on the rhythmic firing of neurons in the phrenic motor column (PMC), a motor neuron group that provides the sole source of diaphragm innervation. Despite their essential role in breathing, the specific determinants of PMC identity and patterns of connectivity are largely unknown. We show that two Hox genes, Hoxa5 and Hoxc5, control diverse aspects of PMC development including their clustering, intramuscular branching, and survival. In mice lacking Hox5 genes in motor neurons, axons extend to the diaphragm, but fail to arborize, leading to respiratory failure. Genetic rescue of cell death fails to restore columnar organization and branching patterns, indicating these defects are independent of neuronal loss. Unexpectedly, late Hox5 removal preserves columnar organization but depletes PMC number and branches, demonstrating a continuous requirement for Hox function in motor neurons. These findings indicate that Hox5 genes orchestrate PMC development through deployment of temporally distinct wiring programs.

Loss of Hoxa5 gene function in mice perturbs intestinal maturation

The Hox gene family of transcription factors constitutes candidate regulators in the molecular cascade of events that governs establishment of normal terminal differentiation along the duodenum to colon axis. One member of this family, Hoxa5, displays a dynamic pattern of expression during gut development. Hoxa5 transcripts are present in midgut mesenchyme at the time of remodeling, supporting a role for this gene in digestive tract specification. To study the role of Hoxa5 in proper intestinal development and maturation, we examined whether Hoxa5 mutant mice exhibit any defect in this process. We report here that even though Hoxa5 is not required for midgut morphogenesis, its loss of function perturbs the acquisition of adult mode of digestion, which normally is temporally coordinated with the process of spontaneous weaning. Impaired maturation of the digestive tract might be related to altered specification of intestinal epithelial cells. Our findings provide evidence that Hoxa5 expression in the gut mesoderm is important for the region-specific differentiation of the adjacent endoderm.

Perturbed thyroid morphology and transient hypothyroidism symptoms in Hoxa5 mutant mice

The Hox family of transcriptional regulators has been extensively studied for their role in axial and appendicular patterning. Genetic analyses have also unveiled Hox gene function in organogenesis and postnatal development. A phenotypical survey of the Hoxa5−/− mutant mice shows that the surviving mutants display symptoms of hypothyroidism, including transient growth retardation, and delayed eye opening and ear elevation. Thyroid gland morphogenesis initiates normally, but follicle formation and thyroglobulin processing are abnormal at late gestation. The expression of several molecular markers essential for thyroid gland formation and function, namely Nkx2.1, Pax8, and Titf2, is affected in the developing thyroid gland of Hoxa5−/− mutants. As a consequence, the expression of thyroid effector genes, including the thyroglobulin and thyroperoxidase genes, is perturbed. Our characterization reveals that the loss of Hoxa5 function transiently affects thyroid development in a non–cell autonomous manner. Developmental Dynamics 227:367–378, 2003.

Multiple cis-acting regulatory regions are required for restricted spatio-temporal Hoxa5 gene expression.

Genetic analyses have revealed the essential role of the murine Hoxa5 gene for the correct specification of the cervical and upper thoracic region of the skeleton, and for the normal organogenesis and function of the respiratory tract, both structures expressing Hoxa5 during embryogenesis. To understand how the expression domains of the Hoxa5 gene are established during development, we have analyzed the cis‐acting control regions mediating Hoxa5 gene expression using a transgenic approach. Four transcripts are derived from the Hoxa5 locus. The shortest and most abundant one displays a specific spatio‐temporal profile of expression at earlier stages and in more anterior structures along the embryonic axis than the larger forms. We established that an 11.1 kilobase pair (kb) genomic fragment, extending from position −3.8 kb to +7.3 kb relative to Hoxa5 transcription initiation site, was sufficient to reproduce the temporal expression and substantially reconstitute the spatial pattern of the major Hoxa5 transcript. By deletion analyses, we identified a 2.1 kb fragment located downstream of the Hoxa5 gene that possesses mesodermal enhancer activity. Overall, the findings demonstrate that cis‐acting regulatory elements essential for the correct expression of the major Hoxa5 transcript are located both upstream and downstream of the Hoxa5 coding sequences. Dev Dyn 1999;214:127–140. 

Cdx Protein Interaction with Hoxa5 Regulatory Sequences Contributes to Hoxa5 Regional Expression along the Axial Skeleton

ABSTRACT Hox gene functions are intimately linked to correct developmental expression of the genes. The identification of cis-acting regulatory sequences and their associated trans-acting factors constitutes a key step in deciphering the mechanisms underlying the correct positioning of the functional domain of Hox genes along the anterior-posterior axis. We have identified DNA elements driving Hoxa5 regionalized expression in mice, using the 2.1-kb mesodermal enhancer (MES) localized in Hoxa5 3′ flanking sequences as a starting point. The MES sequence comprises regulatory elements targeting Hoxa5 expression in the limbs, the urogenital and gastrointestinal tracts, and the cervical-upper thoracic region of the prevertebral column. A 164-bp DNA fragment within the MES caudally restricts Hoxa5 expression at the level of prevertebra 10, corresponding to the posterior limit of its functional domain. Cdx proteins directly bind to this element in vitro via two conserved sites. Preventing Cdx binding by mutating the sites causes caudal expansion of the transgene expression domain. Of all three murine Cdx proteins that bind this element in vitro, Cdx4 has emerged as a potential regional posterior repressor of Hoxa5 expression. The restrictive control provided by Cdx interactions with Hoxa5 regulatory sequences may be one of the critical events in cervicothoracic axial specification.

The loss of Hoxa5 function promotes Notch-dependent goblet cell metaplasia in lung airways

Summary Hox genes encode transcription factors controlling complex developmental processes in various organs. Little is known, however, about how HOX proteins control cell fate. Herein, we demonstrate that the goblet cell metaplasia observed in lung airways from Hoxa5−/− mice originates from the transdifferentiation of Clara cells. Reduced CC10 expression in Hoxa5−/− embryos indicates that altered cell specification occurs prior to birth. The loss of Hoxa5 function does not preclude airway repair after naphthalene exposure, but the regenerated epithelium presents goblet cell metaplasia and less CC10-positive cells, demonstrating the essential role of Hoxa5 for correct differentiation. Goblet cell metaplasia in Hoxa5−/− mice is a FOXA2-independent process. However, it is associated with increased Notch signaling activity. Consistent with these findings, expression levels of activated NOTCH1 and the effector gene HEY2 are enhanced in patients with chronic obstructive pulmonary disease. In vivo administration of a &ggr;-secretase inhibitor attenuates goblet cell metaplasia in Hoxa5−/− mice, highlighting the contribution of Notch signaling to the phenotype and suggesting a potential therapeutic strategy to inhibit goblet cell differentiation and mucus overproduction in airway diseases. In summary, the loss of Hoxa5 function in lung mesenchyme impacts on epithelial cell fate by modulating Notch signaling.

Reduced fertility in male mice deficient in the zinc metallopeptidase NL1.

ABSTRACT Members of the M13 family of zinc metalloendopeptidases have been shown to play critical roles in the metabolism of various neuropeptides and peptide hormones, and they have been identified as important therapeutic targets. Recently, a mouse NL1 protein, a novel member of the family, was identified and shown to be expressed mainly in the testis as a secreted protein. To define its physiological role(s), we used a gene targeting strategy to disrupt the endogenous murine Nl1 gene by homologous recombination and generate Nl1 mutant mice. The Nl1−/− mice were viable and developed normally, suggesting that zygotic expression of Nl1 is not required for development. However, Nl1−/− males produced smaller litters than their wild-type siblings, indicating specific male fertility problems. Reduced fertility may be explained by two impaired processes, decreased egg fertilization and perturbed early development of fertilized eggs. These two phenotypes did not result from gross anatomical modifications of the testis or from impaired spermatogenesis. Basic sperm parameters were also normal. Thus, our findings suggest that one of the roles of NL1 in mice is related to sperm function and that NL1 modulates the processes of fertilization and early embryonic development in vivo.

Partial functional redundancy between Hoxa5 and Hoxb5 paralog genes during lung morphogenesis

Hox genes encode transcription factors governing complex developmental processes in several organs. A subset of Hox genes are expressed in the developing lung. Except for Hoxa5, the lack of overt lung phenotype in single mutants suggests that Hox genes may not play a predominant role in lung ontogeny or that functional redundancy may mask anomalies. In the Hox5 paralog group, both Hoxa5 and Hoxb5 genes are expressed in the lung mesenchyme whereas Hoxa5 is also expressed in the tracheal mesenchyme. Herein, we generated Hoxa5;Hoxb5 compound mutant mice to evaluate the relative contribution of each gene to lung development. Hoxa5;Hoxb5 mutants carrying the four mutated alleles displayed an aggravated lung phenotype, resulting in the death of the mutant pups at birth. Characterization of the phenotype highlighted the role of Hoxb5 in lung formation, the latter being involved in branching morphogenesis, goblet cell specification, and postnatal air space structure, revealing partial functional redundancy with Hoxa5. However, the Hoxb5 lung phenotypes were less severe than those seen in Hoxa5 mutants, likely because of Hoxa5 compensation. New specific roles for Hoxa5 were also unveiled, demonstrating the extensive contribution of Hoxa5 to the developing respiratory system. The exclusive expression of Hoxa5 in the trachea and the phrenic motor column likely underlies the Hoxa5-specific trachea and diaphragm phenotypes. Altogether, our observations establish that the Hoxa5 and Hoxb5 paralog genes shared some functions during lung morphogenesis, Hoxa5 playing a predominant role.