Proximity-dependent Mapping of the Androgen Receptor Identifies Kruppel-like Factor 4 as a Functional Partner.

Prostate cancer (PCa) is the most frequently diagnosed cancer in men and the third cause of cancer mortality. PCa initiation and growth are driven by the androgen receptor (AR). The AR is activated by androgens such as testosterone and controls prostatic cell proliferation and survival. Here, we report an AR signaling network generated using BioID proximity labeling proteomics in androgen-dependent LAPC4 cells. We identified 31 AR-associated proteins in nonstimulated cells. Strikingly, the AR signaling network increased to 182 and 200 proteins, upon 24 h or 72 h of androgenic stimulation, respectively, for a total of 267 nonredundant AR-associated candidates. Among the latter group, we identified 213 proteins that were not previously reported in databases. Many of these new AR-associated proteins are involved in DNA metabolism, RNA processing, and RNA polymerase II transcription. Moreover, we identified 44 transcription factors, including the Kru¨ppel-like factor 4 (KLF4), which were found interacting in androgen-stimulated cells. Interestingly, KLF4 repressed the well-characterized AR-dependent transcription of the KLK3 (PSA) gene; AR and KLF4 also colocalized genome-wide. Taken together, our data report an expanded high-confidence proximity network for AR, which will be instrumental to further dissect the molecular mechanisms underlying androgen signaling in PCa cells.

Dysregulation of cotranscriptional alternative splicing underlies CHARGE syndrome.

CHARGE syndrome-which stands for coloboma of the eye, heart defects, atresia of choanae, retardation of growth/development, genital abnormalities, and ear anomalies-is a severe developmental disorder with wide phenotypic variability, caused mainly by mutations in CHD7 (chromodomain helicase DNA-binding protein 7), known to encode a chromatin remodeler. The genetic lesions responsible for CHD7 mutation-negative cases are unknown, at least in part because the pathogenic mechanisms underlying CHARGE syndrome remain poorly defined. Here, we report the characterization of a mouse model for CHD7 mutation-negative cases of CHARGE syndrome generated by insertional mutagenesis of Fam172a (family with sequence similarity 172, member A). We show that Fam172a plays a key role in the regulation of cotranscriptional alternative splicing, notably by interacting with Ago2 (Argonaute-2) and Chd7. Validation studies in a human cohort allow us to propose that dysregulation of cotranscriptional alternative splicing is a unifying pathogenic mechanism for both CHD7 mutation-positive and CHD7 mutation-negative cases. We also present evidence that such splicing defects can be corrected in vitro by acute rapamycin treatment.

Crucial requirement of ERK/MAPK signaling in respiratory tract development.

There was an error published in Development 141, 3197-3211. In the key for Fig. 3C, the grey bars were labelled with the incorrect genotype name. The correct genotype is Mek1+/flox;Mek2−/−; Dermo1+/cre. This error does not affect the conclusions of the paper. The authors apologise to readers for this mistake.

Epithelial inactivation of Yy1 abrogates lung branching morphogenesis.

Yin Yang 1 (YY1) is a multifunctional zinc-finger-containing transcription factor that plays crucial roles in numerous biological processes by selectively activating or repressing transcription, depending upon promoter contextual differences and specific protein interactions. In mice, Yy1 null mutants die early in gestation whereas Yy1 hypomorphs die at birth from lung defects. We studied how the epithelial-specific inactivation of Yy1 impacts on lung development. The Yy1 mutation in lung epithelium resulted in neonatal death due to respiratory failure. It impaired tracheal cartilage formation, altered cell differentiation, abrogated lung branching and caused airway dilation similar to that seen in human congenital cystic lung diseases. The cystic lung phenotype in Yy1 mutants can be partly explained by the reduced expression of Shh, a transcriptional target of YY1, in lung endoderm, and the subsequent derepression of mesenchymal Fgf10 expression. Accordingly, SHH supplementation partially rescued the lung phenotype in vitro. Analysis of human lung tissues revealed decreased YY1 expression in children with pleuropulmonary blastoma (PPB), a rare pediatric lung tumor arising during fetal development and associated with DICER1 mutations. No evidence for a potential genetic interplay between murine Dicer and Yy1 genes during lung morphogenesis was observed. However, the cystic lung phenotype resulting from the epithelial inactivation of Dicer function mimics the Yy1 lung malformations with similar changes in Shh and Fgf10 expression. Together, our data demonstrate the crucial requirement for YY1 in lung morphogenesis and identify Yy1 mutant mice as a potential model for studying the genetic basis of PPB.

Crucial requirement of ERK/MAPK signaling in respiratory tract development.

The mammalian genome contains two ERK/MAP kinase genes, Mek1 and Mek2, which encode dual-specificity kinases responsible for ERK/MAP kinase activation. In order to define the function of the ERK/MAPK pathway in the lung development in mice, we performed tissue-specific deletions of Mek1 function on a Mek2 null background. Inactivation of both Mek genes in mesenchyme resulted in several phenotypes, including giant omphalocele, kyphosis, pulmonary hypoplasia, defective tracheal cartilage and death at birth. The absence of tracheal cartilage rings establishes the crucial role of intracellular signaling molecules in tracheal chondrogenesis and provides a putative mouse model for tracheomalacia. In vitro, the loss of Mek function in lung mesenchyme did not interfere with lung growth and branching, suggesting that both the reduced intrathoracic space due to the dysmorphic rib cage and the omphalocele impaired lung development in vivo. Conversely, Mek mutation in the respiratory epithelium caused lung agenesis, a phenotype resulting from the direct impact of the ERK/MAPK pathway on cell proliferation and survival. No tracheal epithelial cell differentiation occurred and no SOX2-positive progenitor cells were detected in mutants, implying a role for the ERK/MAPK pathway in trachea progenitor cell maintenance and differentiation. Moreover, these anomalies were phenocopied when the Erk1 and Erk2 genes were mutated in airway epithelium. Thus, the ERK/MAPK pathway is required for the integration of mesenchymal and epithelial signals essential for the development of the entire respiratory tract.

HLX is a candidate gene for a pattern of anomalies associated with congenital diaphragmatic hernia, short bowel, and asplenia.

Isolated congenital diaphragmatic hernia is often a sporadic event with a low recurrence risk. However, underlying genetic etiologies, such as chromosome anomalies or single gene disorders, are identified in a small number of individuals. We describe two fetuses with a unique pattern of multiple congenital anomalies, including diaphragmatic hernia, short bowel and asplenia, born to first-cousin parents. Whole exome sequencing showed that both were homozygous for a missense variant, c.950A>C, predicting p.Asp317Ala, in the H.20-Like Homeobox 1 (HLX1) gene. HLX is a homeobox transcription factor gene which is relatively conserved across species. Hlx homozygous null mice have a short bowel and reduced muscle cells in the diaphragm, closely resembling the anomalies in the two fetuses and we therefore suggest that the HLX mutation in this family could explain the fetal findings.

Cardiomyocyte lipotoxicity is mediated by Il-6 and causes down-regulation of PPARs.

Here we sought to evaluate the effect of palmitate on cytokine and PPAR activity/expression. We investigated the effect of BSA conjugated palmitate and oleate on PPAR activity, PPAR-α and δ expression, as well as the expression of cytokines and key factors responsible for ß-oxidation by qRT-PCR and western blotting in primary rat neonatal cardiomyocytes (NCMs). Furthermore we evaluated the effect of anti-inflammatory actions of AICAR and PPAR agonists on cytokine expression and cell death in palmitate treated NCMs. We found that palmitate caused down regulation of PPARs and increased cytokine expression and cell death, all of which was significantly attenuated by the co-administration of either AICAR or PPAR agonists. This work supports the pro-inflammatory actions of intracellular lipid and provides further insight into the pathological mechanism of cardiac lipotoxicity as occurs in diabetic hearts.

Hoxa5/Cre transgenic mice: novel tools for regional deletion along the anterior-posterior axis.

The Hoxa5 homeobox gene encodes a transcription factor that plays a critical role in specifying the identity of the cervico-thoracic region along the anterior-posterior embryo axis and in orchestrating organ morphogenesis. The loss of Hoxa5 function results in skeletal transformations, lethality at birth due to lung defects, and organ anomalies affecting the digestive tract, the mammary gland and the ovary. Study of Hoxa5 gene regulation has revealed the interplay of several control regions that direct Hoxa5 developmental expression. Enhancers targeting expression in the CNS, the paraxial and lateral plate mesoderm at the cervico-thoracic level, and in the mesenchymal compartment of the respiratory and digestive tracts have been identified. Using these molecular tools, we have generated two Hoxa5/Cre transgenic mouse lines carrying different combinations of Hoxa5 regulatory enhancers and allowing site-specific recombination in subsets of Hoxa5 expression sites as tested with the Rosa26/lacZ reporter mice. Further validation of the recombination efficiency of the Hoxa5/Cre transgenic lines was performed with mice carrying a Hoxa5 conditional allele. Hoxa5 deletion with the Hoxa5/Cre mouse lines recapitulates Hoxa5 mutant phenotypes, such as skeletal defects, neonatal lethality, and lung malformations. Hoxa5/Cre mouse lines provide novel genetic tools for gene function analysis in defined tissues along the anterior-posterior axis.

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.

The CHARGE syndrome-associated protein FAM172A controls AGO2 nuclear import.

CHARGE syndrome is a neural crest-related disorder mainly caused by mutation of the chromatin remodeler-coding gene CHD7 Alternative causes include mutation of other chromatin and/or splicing factors. One of these additional players is the poorly characterized FAM172A, which we previously found in a complex with CHD7 and the small RNA-binding protein AGO2 at the chromatin-spliceosome interface. Focusing on the FAM172A-AGO2 interplay, we now report that FAM172A is a direct binding partner of AGO2 and, as such, one of the long sought-after regulators of AGO2 nuclear import. We show that this FAM172A function mainly relies on its classical bipartite nuclear localization signal and associated canonical importin-α/ß pathway, being enhanced by CK2-induced phosphorylation and abrogated by a CHARGE syndrome-associated missense mutation. Overall, this study thus strengthens the notion that noncanonical nuclear functions of AGO2 and associated regulatory mechanisms might be clinically relevant.

Glucocorticoid stimulation induces regionalized gene responses within topologically associating domains.

Transcription-factor binding to cis-regulatory regions regulates the gene expression program of a cell, but occupancy is often a poor predictor of the gene response. Here, we show that glucocorticoid stimulation led to the reorganization of transcriptional coregulators MED1 and BRD4 within topologically associating domains (TADs), resulting in active or repressive gene environments. Indeed, we observed a bias toward the activation or repression of a TAD when their activities were defined by the number of regions gaining and losing MED1 and BRD4 following dexamethasone (Dex) stimulation. Variations in Dex-responsive genes at the RNA levels were consistent with the redistribution of MED1 and BRD4 at the associated cis-regulatory regions. Interestingly, Dex-responsive genes without the differential recruitment of MED1 and BRD4 or binding by the glucocorticoid receptor were found within TADs, which gained or lost MED1 and BRD4, suggesting a role of the surrounding environment in gene regulation. However, the amplitude of the response of Dex-regulated genes was higher when the differential recruitment of the glucocorticoid receptor and transcriptional coregulators was observed, reaffirming the role of transcription factor-driven gene regulation and attributing a lesser role to the TAD environment. These results support a model where a signal-induced transcription factor induces a regionalized effect throughout the TAD, redefining the notion of direct and indirect effects of transcription factors on target genes.

Modulating HSF1 levels impacts expression of the estrogen receptor α and antiestrogen response.

Master transcription factors control the transcriptional program and are essential to maintain cellular functions. Among them, steroid nuclear receptors, such as the estrogen receptor α (ERα), are central to the etiology of hormone-dependent cancers which are accordingly treated with corresponding endocrine therapies. However, resistance invariably arises. Here, we show that high levels of the stress response master regulator, the heat shock factor 1 (HSF1), are associated with antiestrogen resistance in breast cancer cells. Indeed, overexpression of HSF1 leads to ERα degradation, decreased expression of ERα-activated genes, and antiestrogen resistance. Furthermore, we demonstrate that reducing HSF1 levels reinstates expression of the ERα and restores response to antiestrogens. Last, our results establish a proof of concept that inhibition of HSF1, in combination with antiestrogens, is a valid strategy to tackle resistant breast cancers. Taken together, we are proposing a mechanism where high HSF1 levels interfere with the ERα-dependent transcriptional program leading to endocrine resistance in breast cancer.

Molecular dissection of CHARGE syndrome highlights the vulnerability of neural crest cells to problems with alternative splicing and other transcription-related processes.

CHARGE syndrome is characterized by co-occurrence of multiple malformations due to abnormal development of neural crest cells. Here, we review the phenotypic and molecular overlap between CHARGE syndrome and similar pathologies, and further discuss the observation that neural crest cells appear especially sensitive to malfunction of the chromatin-transcription-splicing molecular hub.

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.

Multiple promoters and alternative splicing: Hoxa5 transcriptional complexity in the mouse embryo.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. SIGNIFICANCE: 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.

Functional resolution of duplicated hoxb5 genes in teleosts.

The duplication-degeneration-complementation (DDC) model predicts that subfunctionalization of duplicated genes is a common mechanism for their preservation. The additional Hox complexes of teleost fish constitute a good system in which to test this hypothesis. Zebrafish have two hoxb complexes, with two hoxb5 genes, hoxb5a and hoxb5b, the expression patterns of which suggest subfunctionalization of an ancestral hoxb5 gene. We characterized conserved non-coding elements (CNEs) near the zebrafish hoxb5 genes. One CNE, J3, is only retained in the hoxb5a locus, whereas the others, J1 and J2, are present in both hoxb5 loci. When tested individually, the enhancer activity of individual CNEs, including J3, extensively overlapped and did not support a role in subfunctionalization. By contrast, reporter transgene constructs encompassing multiple CNEs were able to target reporter gene expression to unique domains of hoxb5a and hoxb5b expression. The deletion of J3 from the hoxb5a locus resulted in expression that approached that of hoxb5b, whereas its insertion in the hoxb5b locus increased reporter expression and rendered it more similar to that of hoxb5a. Our results highlight the importance of interactions between CNEs in the execution of complementary subfunctions of duplicated genes.