Control of mouse limb initiation and antero-posterior patterning by Meis transcription factors.

Meis1 and Meis2 are homeodomain transcription factors that regulate organogenesis through cooperation with Hox proteins. Elimination of Meis genes after limb induction has shown their role in limb proximo-distal patterning; however, limb development in the complete absence of Meis function has not been studied. Here, we report that Meis1/2 inactivation in the lateral plate mesoderm of mouse embryos leads to limb agenesis. Meis and Tbx factors converge in this function, extensively co-binding with Tbx to genomic sites and co-regulating enhancers of Fgf10, a critical factor in limb initiation. Limbs with three deleted Meis alleles show proximal-specific skeletal hypoplasia and agenesis of posterior skeletal elements. This failure in posterior specification results from an early role of Meis factors in establishing the limb antero-posterior prepattern required for Shh activation. Our results demonstrate roles for Meis transcription factors in early limb development and identify their involvement in previously undescribed interaction networks that regulate organogenesis.

Pioneer and nonpioneer factor cooperation drives lineage specific chromatin opening.

Pioneer transcription factors are characterized by having the unique property of enabling the opening of closed chromatin sites, for implementation of cell fates. We previously found that the pioneer Pax7 specifies melanotrope cells through deployment of an enhancer repertoire, which allows binding of Tpit, a nonpioneer factor that determines the related lineages of melanotropes and corticotropes. Here, we investigate the relation between these two factors in the pioneer mechanism. Cell-specific gene expression and chromatin landscapes are defined by scRNAseq and chromatin accessibility profiling. We find that in vivo deployment of the melanotrope enhancer repertoire and chromatin opening requires both Pax7 and Tpit. In cells, binding of heterochromatin targets by Pax7 is independent of Tpit but Pax7-dependent chromatin opening requires Tpit. The present work shows that pioneer core properties are limited to the ability to recognize heterochromatin targets and facilitate nonpioneer binding. Chromatin opening per se may be provided through cooperation with nonpioneer factors.

Regulatory integration of Hox factor activity with T-box factors in limb development.

In tetrapods, Tbx4, Tbx5 and Hox cluster genes are crucial for forelimb and hindlimb development and mutations in these genes are responsible for congenital limb defects. The molecular basis of their integrated mechanisms of action in the context of limb development remains poorly understood. We studied Tbx4 and Hoxc10 owing to their overlapping loss-of-function phenotypes and colocalized expression in mouse hindlimb buds. We report an extensive overlap between Tbx4 and Hoxc10 genome occupancy and their putative target genes. Tbx4 and Hoxc10 interact directly with each other, have the ability to bind to a previously unrecognized T-box-Hox composite DNA motif and show synergistic activity when acting on reporter genes. Pitx1, the master regulator for hindlimb specification, also shows extensive genomic colocalization with Tbx4 and Hoxc10. Genome occupancy by Tbx4 in hindlimb buds is similar to Tbx5 occupancy in forelimbs. By contrast, another Hox factor, Hoxd13, also interacts with Tbx4/Tbx5 but antagonizes Tbx4/Tbx5-dependent transcriptional activity. Collectively, the modulation of Tbx-dependent activity by Hox factors acting on common DNA targets may integrate different developmental processes for the balanced formation of proportionate limbs.

Pioneer factor Pax7 deploys a stable enhancer repertoire for specification of cell fate.

Pioneer transcription factors establish new cell-fate competence by triggering chromatin remodeling. However, many features of pioneer action, such as their kinetics and stability, remain poorly defined. Here, we show that Pax7, by opening a unique repertoire of enhancers, is necessary and sufficient for specification of one pituitary lineage. Pax7 binds its targeted enhancers rapidly, but chromatin remodeling and gene activation are slower. Enhancers opened by Pax7 show a loss of DNA methylation and acquire stable epigenetic memory, as evidenced by binding of nonpioneer factors after Pax7 withdrawal. This work shows that transient Pax7 expression is sufficient for stable specification of cell identity.

Nanoparticle-Based Dressing: The Future of Wound Treatment?

Reconstructing functional skin after a wound remains a challenge due to the complexity of healing. In this regard, biocompatible nanoparticles (NPs) carrying and releasing bioactive drugs in a controlled and sustained manner may significantly improve the efficacy of wound therapies compared with current treatments. Topical administration of drug-loaded NPs allows optimal delivery to the dermis and improves product efficacy. Furthermore, associating NPs with scaffolds represents a new concept of 'dressing'. Experimental in vivo, ex vivo, and in vitro models have been developed in preclinical assays to evaluate the beneficial effects of nanoparticulate dressings. Drug-loaded NPs are promising tools for innovative wound healing treatment, especially with regard to their multifunctional properties.

Loss-of-function mutations in the CABLES1 gene are a novel cause of Cushing's disease.

The CABLES1 cell cycle regulator participates in the adrenal-pituitary negative feedback, and its expression is reduced in corticotropinomas, pituitary tumors with a largely unexplained genetic basis. We investigated the presence of CABLES1 mutations/copy number variations (CNVs) and their associated clinical, histopathological and molecular features in patients with Cushing's disease (CD). Samples from 146 pediatric (118 germline DNA only/28 germline and tumor DNA) and 35 adult (tumor DNA) CD patients were screened for CABLES1 mutations. CNVs were assessed in 116 pediatric CD patients (87 germline DNA only/29 germline and tumor DNA). Four potentially pathogenic missense variants in CABLES1 were identified, two in young adults (c.532G > A, p.E178K and c.718C > T, p.L240F) and two in children (c.935G > A, p.G312D and c.1388A > G, and p.D463G) with CD; no CNVs were found. The four variants affected residues within or close to the predicted cyclin-dependent kinase-3 (CDK3)-binding region of the CABLES1 protein and impaired its ability to block cell growth in a mouse corticotropinoma cell line (AtT20/D16v-F2). The four patients had macroadenomas. We provide evidence for a role of CABLES1 as a novel pituitary tumor-predisposing gene. Its function might link two of the main molecular mechanisms altered in corticotropinomas: the cyclin-dependent kinase/cyclin group of cell cycle regulators and the epidermal growth factor receptor signaling pathway. Further studies are needed to assess the prevalence of CABLES1 mutations among patients with other types of pituitary adenomas and to elucidate the pituitary-specific functions of this gene.

The selector gene Pax7 dictates alternate pituitary cell fates through its pioneer action on chromatin remodeling.

The anterior and intermediate lobes of the pituitary gland derive from the surface ectoderm. They provide a simple system to assess mechanisms of developmental identity established by tissue determinants. Each lobe contains a lineage expressing the hormone precursor pro-opiomelanocortin (POMC): the corticotropes and melanotropes. The T-box transcription factor Tpit controls terminal differentiation of both lineages. We now report on the unique role of Pax7 as a selector of intermediate lobe and melanotrope identity. Inactivation of the Pax7 gene results in loss of melanotrope gene expression and derepression of corticotrope genes. Pax7 acts by remodeling chromatin and allowing Tpit binding to a new subset of enhancers for activation of melanotrope-specific genes. Thus, the selector function of Pax7 is exerted through pioneer transcription factor activity. Genome-wide, the Pax7 pioneer activity is preferentially associated with composite binding sites that include paired and homeodomain motifs. Pax7 expression is conserved in human and dog melanotropes and defines two subtypes of pituitary adenomas causing Cushing's disease. In summary, expression of Pax7 provides a unique tissue identity to the pituitary intermediate lobe that alters Tpit-driven differentiation through pioneer and classical transcription factor activities.

Differential role of the interleukin-17 axis and neutrophils in resolution of inhalational anthrax.

The roles of interleukin-17 (IL-17) and neutrophils in the lung have been described as those of two intricate but independent players. Here we identify neutrophils as the primary IL-17-secreting subset of cells in a model of inhalation anthrax using A/J and C57BL/6 mice. With IL-17 receptor A knockout (IL-17RA-/-) mice, we confirmed that IL-17A/F signaling is instrumental in the self-recruitment of this population. We also show that the IL-17A/F axis is critical for surviving pulmonary infection, as IL-17RA-/- mice become susceptible to intranasal infection by Bacillus anthracis Sterne spores. Strikingly, infection with a fully virulent strain did not affect IL-17RA-/- mouse survival. Eventually, by depleting neutrophils in wild-type and IL-17RA-/- mice, we demonstrated the crucial role of IL-17-secreting neutrophils in mouse survival of infection by fully virulent B. anthracis. This work demonstrates the important roles of both IL-17 signaling and neutrophils in clearing this pathogen and surviving pulmonary B. anthracis infection.

Rapid identification of Burkholderia pseudomallei and Burkholderia mallei by fluorescence in situ hybridization (FISH) from culture and paraffin-embedded tissue samples.

We evaluated newly developed probes for rapid identification of Burkholderia (B.) pseudomallei and B. mallei and differentiation from B. thailandensis by fluorescence in situ hybridization (FISH). FISH correctly identified 100% of the tested B. pseudomallei (11), B. mallei (11), and B. thailandensis (1) strains, excluded 100% of all tested negative controls (61), and allowed demonstration of B. pseudomallei infection in a paraffin-embedded spleen tissue sample of an experimentally infected mouse.

Efficacy of a vaccine based on protective antigen and killed spores against experimental inhalational anthrax.

Protective antigen (PA)-based anthrax vaccines acting on toxins are less effective than live attenuated vaccines, suggesting that additional antigens may contribute to protective immunity. Several reports indicate that capsule or spore-associated antigens may enhance the protection afforded by PA. Addition of formaldehyde-inactivated spores (FIS) to PA (PA-FIS) elicits total protection against cutaneous anthrax. Nevertheless, vaccines that are effective against cutaneous anthrax may not be so against inhalational anthrax. The aim of this work was to optimize immunization with PA-FIS and to assess vaccine efficacy against inhalational anthrax. We assessed the immune response to recombinant anthrax PA from Bacillus anthracis (rPA)-FIS administered by various immunization protocols and the protection provided to mice and guinea pigs infected through the respiratory route with spores of a virulent strain of B. anthracis. Combined subcutaneous plus intranasal immunization of mice yielded a mucosal immunoglobulin G response to rPA that was more than 20 times higher than that in lung mucosal secretions after subcutaneous vaccination. The titers of toxin-neutralizing antibody and antispore antibody were also significantly higher: nine and eight times higher, respectively. The optimized immunization elicited total protection of mice intranasally infected with the virulent B. anthracis strain 17JB. Guinea pigs were fully protected, both against an intranasal challenge with 100 50% lethal doses (LD(50)) and against an aerosol with 75 LD(50) of spores of the highly virulent strain 9602. Conversely, immunization with PA alone did not elicit protection. These results demonstrate that the association of PA and spores is very much more effective than PA alone against experimental inhalational anthrax.

Burkholderia pseudomallei aerosol infection results in differential inflammatory responses in BALB/c and C57Bl/6 mice.

Melioidosis is caused by the Gram-negative bacterium Burkholderia pseudomallei, whose portals of entry into the body include subcutaneous, ingestion and inhalation routes. Animal models play an important role in furthering our understanding of this disease, which is associated with high morbidity and mortality in susceptible subjects. Previous studies using intranasal inoculation showed a differential susceptibility to inhalational melioidosis in BALB/c and C57Bl/6 mice and attributed the difference to genetic factors and host response. However, a recent study found no difference in susceptibility when the two species of mice were exposed to nebulized bacteria. We sought to address this discrepancy by using a nasal route only, instead of whole-body aerosol exposure system. Employing three different clinical strains of B. pseudomallei and following the progression of disease development in both BALB/c and C57Bl/6 mice, we found that BALB/c mice were at least 10- to 100-fold more susceptible to infection than C57Bl/6 mice. Comparison of bacterial burdens in aerosol-challenged mice, at both the pulmonary and distant sites of infection, suggests that C57Bl/6 mice were more efficient in clearing the bacteria than BALB/c mice. In addition, a comprehensive study of a wide panel of chemokines and cytokines at the protein level demonstrated that hyperproduction of proinflammatory cytokines in aerosol-challenged BALB/c mice did not translate into better protection and survival of these mice, whereas a moderate increase in these proteins in aerosol-challenged C57Bl/6 mice was more beneficial in clearing the infection. This suggests that high levels of proinflammatory cytokines are detrimental and contribute to the immunopathogenesis of the infection.

Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model.

The human pathogen Pseudomonas aeruginosa has been shown previously to use similar virulence factors when infecting mammalian hosts or Dictyostelium amoebae. Here we randomly mutagenized a clinical isolate of P. aeruginosa, and identified mutants with attenuated virulence towards Dictyostelium. These mutant strains also exhibited a strong decrease in virulence when infecting Drosophila and mice, confirming that P. aeruginosa makes use of similar virulence traits to confront these very different hosts. Further characterization of these bacterial mutants showed that TrpD is important for the induction of the quorum-sensing circuit, while PchH and PchI are involved in the induction of the type III secretion system. These results demonstrate the usefulness and the relevance of the Dictyostelium host model to identify and analyse new virulence genes in P. aeruginosa.

The TPIT gene mutation M86R associated with isolated adrenocorticotropin deficiency interferes with protein: protein interactions.

CONTEXT: Tpit is a T-box transcription factor important for terminal differentiation of pituitary proopiomelanocortin-expressing cells. We previously showed that human and murine mutations in the gene encoding this highly cortico/melanotrope-specific transcription factor cause a neonatal onset form of congenital isolated ACTH deficiency (IAD). We characterized the largest series of neonatal IAD patients caused by TPIT mutations, and this revealed a highly homogeneous clinical presentation. So far, 12 different loss-of-function TPIT mutations have been identified. The methionine 86 arginine (M86R) TPIT mutation was recently identified in compound heterozygosity with the 782delA frame-shift mutation in two siblings with early-onset IAD. OBJECTIVE: We conducted a functional analysis of the missense M86R mutation to assess transcriptional activity, DNA binding activity, and nuclear location, as well as protein-protein interactions. RESULTS: Although the M86 residue is located within the T-box DNA-binding domain, it did not affect monomer DNA-binding activity per se, but it impaired DNA binding with other DNA-bound proteins, including itself (homodimers) and pituitary homeobox 1 (Pitx1). The M86 residue is at the interface between T domains in the T dimers crystal structure, and it appears that the same residue is involved in heterodimer formation with pituitary Pitx1. Furthermore, TPIT M86R is deficient in the recruitment of the coactivator SRC2 that partly mediates the CRH stimulation of proopiomelanocortin transcription. CONCLUSION: Thus, the M86R TPIT mutation is defining an important surface of the T domain for multiple protein interactions and for transcription.

Role of Brg1 and HDAC2 in GR trans-repression of the pituitary POMC gene and misexpression in Cushing disease.

Negative feedback regulation of the proopiomelanocortin (POMC) gene by the glucocorticoid (Gc) receptor (GR) is a critical feature of the hypothalamo-pituitary-adrenal axis, and it is in part exerted by trans-repression between GR and the orphan nuclear receptors related to NGFI-B. We now show that Brg1, the ATPase subunit of the Swi/Snf complex, is essential for this trans-repression and that Brg1 is required in vivo to stabilize interactions between GR and NGFI-B as well as between GR and HDAC2. Whereas Brg1 is constitutively present at the POMC promoter, recruitment of GR and HDAC2 is ligand-dependent and results in histone H4 deacetylation of the POMC locus. In addition, GR-dependent repression inhibits promoter clearance by RNA polymerase II. Thus, corecruitment of repressor and activator at the promoter and chromatin modification jointly contribute to trans-repression initiated by direct interactions between GR and NGFI-B. Loss of Brg1 or HDAC2 should therefore produce Gc resistance, and we show that approximately 50% of Gc-resistant human and dog corticotroph adenomas, which are the hallmark of Cushing disease, are deficient in nuclear expression of either protein. In addition to providing a molecular basis for Gc resistance, these deficiencies may also contribute to the tumorigenic process.

Rb enhances p160/SRC coactivator-dependent activity of nuclear receptors and hormone responsiveness.

The retinoblastoma tumor suppressor protein (Rb) is best known as a repressor of genes involved in cell cycle progression. Rb has also been implicated in activation of transcription, in particular by nuclear receptors (NRs) and by differentiation-related transcription factors, but the relevance of this activity is unclear. We show that Rb and the related proteins p107 and p130 enhance the activity of NRs related to NGFI-B (Nur factors) through direct interactions with NGFI-B and SRC-2. Although recruitment of SRC/p160 coactivators to the NGFI-B AF1 domain is independent of Rb, its presence enhances SRC-dependent transcription. Rb potentiation of SRC coactivators is exerted on a subset (Nur factors, hepatocyte nuclear factor-4 (HNF-4), SF-1, and ER) but not all NRs. The levels of Rb-related proteins modulate hormone responsiveness of the NGFI-B-dependent pituitary proopiomelanocortin gene and HNF-4-dependent transcription during enterocyte differentiation. Increased Rb expression upon cell differentiation may promote differentiated functions, at least in part, by potentiation of NR activity.

Protein-protein interactions and transcriptional antagonism between the subfamily of NGFI-B/Nur77 orphan nuclear receptors and glucocorticoid receptor.

Glucocorticoids (Gc) act through the glucocorticoid receptor (GR) to enhance or repress transcription of glucocorticoid-responsive genes depending on the promoter and cellular context. Repression of proopiomelanocortin (POMC) gene expression by Gc was proposed to use different mechanisms. We described the POMC promoter Nur response element (NurRE) as a target for Gc repression. NGFI-B (Nur77), an orphan nuclear receptor, and two related factors, Nurr1 and NOR1, bind the NurRE as homo- or heterodimers to enhance POMC gene expression in response to CRH. Gc antagonize CRH-stimulated as well as NGFI-B-dependent transcription. We now show that GR antagonizes NurRE-dependent transcription induced by all members of the Nur77 subfamily and that these nuclear receptors can all interact directly with GR. Transcriptional antagonism as well as direct protein-protein interaction between NGFI-B and GR take place primarily via their respective DNA binding domains, although DNA binding itself and the GR homodimerization interface are not involved. In vivo, GR and Nur factors can be coimmunoprecipitated whereas GR is recruited to the POMC promoter upon glucocorticoid action. Thus, our data suggest a mechanism for transrepression between two nuclear receptors, GR and NGFI-B, that is unique, although quite similar to that proposed for transrepression between GR and activator protein 1 (AP-1) or nuclear factor-kappaB (NFkappaB).

Attempted passive prophylaxis with a monoclonal anti-Burkholderia pseudomallei exopolysaccharide antibody in a murine model of melioidosis.

Melioidosis is a severe gram-negative infection caused by the facultative intracellular bacterium Burkholderia pseudomallei, which is responsible for a broad spectrum of symptoms in both humans and animals. No licensed vaccine currently exists. This study evaluated the protective effect of a monoclonal antibody (Mab Ps6F6) specific to B. pseudomallei exopolysaccharide in an outbred murine model of sub-acute melioidosis. When administered before the infectious challenge, Ps6F6 significantly increased resistance to infection and restrained bacterial burden in the spleen over a 30-days period. Patterns of IFN-gamma production were similar in the treated and non treated groups of mice. However, Ps6F6 lowered IFN-gamma levels over the duration of the assay period, except on day 1, suggesting a transient and rapid production of IFN-gamma under Ps6F6 control. Minor but persisting increases occurred in IL-12 levels while TNF-alpha was detected only in the controls at the later stages of infection. No IL-10 secretion was detected in both groups of mice. These data suggest that passive prophylaxis with Mab Ps6F6 provide a moderate and transient induction of inflammatory responses in infected mice but failed to trigger a sterilizing protective immunity.

Pitx1 in vivo promoter activity and mechanisms of positive autoregulation.

During early mouse embryogenesis, Pitx1 (pituitary homeobox 1), a member of the bicoid subgroup of PAIRED homeobox-containing transcription factors, marks the stomodeum, oral ectoderm, pituitary and first branchial arch in the anterior part of the embryo and lateral plate mesoderm only in the posterior half of the embryo. We have now defined PITX1 promoter fragments that mimic the anterior but not posterior expression of PITX1 in transgenic mice. In addition, we show positive regulation of this promoter in transfection studies by three members of the Pitx1 family (Pitx1, Pitx1b, Pitx2), as well as by a related factor, Otx1. PITX1 autoregulation depends on DNA-binding and trans-activation domains of Pitx1 and it may be responsible for establishment and/or maintenance of the Pitx1 expression domain.

Human and mouse TPIT gene mutations cause early onset pituitary ACTH deficiency.

Tpit is a highly cell-restricted transcription factor that is required for expression of the pro-opiomelanocortin (POMC) gene and for terminal differentiation of the pituitary corticotroph lineage. Its exclusive expression in pituitary POMC-expressing cells has suggested that its mutation may cause isolated deficiency of pituitary adrenocorticotropin (ACTH). We now show that Tpit-deficient mice constitute a model of isolated ACTH deficiency (IAD) that is very similar to human IAD patients carrying TPIT gene mutations. Through genetic analysis of a panel of IAD patients, we show that TPIT gene mutations are associated at high frequency with early onset IAD, but not with juvenile forms of this deficiency. We identified seven different TPIT mutations, including nonsense, missense, point deletion, and a genomic deletion. This work defines congenital early onset IAD as a relatively homogeneous clinical entity caused by recessive transmission of loss-of-function mutations in the TPIT gene.