A conserved NR5A1-responsive enhancer regulates SRY in testis-determination.

The Y-linked SRY gene initiates mammalian testis-determination. However, how the expression of SRY is regulated remains elusive. Here, we demonstrate that a conserved steroidogenic factor-1 (SF-1)/NR5A1 binding enhancer is required for appropriate SRY expression to initiate testis-determination in humans. Comparative sequence analysis of SRY 5' regions in mammals identified an evolutionary conserved SF-1/NR5A1-binding motif within a 250 bp region of open chromatin located 5 kilobases upstream of the SRY transcription start site. Genomic analysis of 46,XY individuals with disrupted testis-determination, including a large multigenerational family, identified unique single-base substitutions of highly conserved residues within the SF-1/NR5A1-binding element. In silico modelling and in vitro assays demonstrate the enhancer properties of the NR5A1 motif. Deletion of this hemizygous element by genome-editing, in a novel in vitro cellular model recapitulating human Sertoli cell formation, resulted in a significant reduction in expression of SRY. Therefore, human NR5A1 acts as a regulatory switch between testis and ovary development by upregulating SRY expression, a role that may predate the eutherian radiation. We show that disruption of an enhancer can phenocopy variants in the coding regions of SRY that cause human testis dysgenesis. Since disease causing variants in enhancers are currently rare, the regulation of gene expression in testis-determination offers a paradigm to define enhancer activity in a key developmental process.

The ring finger protein 213 gene (Rnf213) contributes to Rift Valley fever resistance in mice.

Rift Valley fever (RVF) is an emerging viral zoonosis that primarily affects ruminants and humans. We have previously shown that wild-derived MBT/Pas mice are highly susceptible to RVF virus and that part of this phenotype is controlled by a locus located on distal Chromosome 11. Using congenic strains, we narrowed down the critical interval to a 530 kb region containing five protein-coding genes among which Rnf213 emerged as a potential candidate. We generated Rnf213-deficient mice by CRISPR/CAS9 on the C57BL/6 J background and showed that they were significantly more susceptible to RVF than control mice, with an average survival time post-infection reduced from 7 to 4 days. The human RNF213 gene had been associated with the cerebrovascular Moyamoya disease (MMD or MYMY) but the inactivation of this gene in the mouse resulted only in mild anomalies of the neovascularization. This study provides the first evidence that the Rnf213 gene may also impact the resistance to infectious diseases such as RVF.

Genetic dissection of Rift Valley fever pathogenesis: Rvfs2 locus on mouse chromosome 11 enables survival to early-onset hepatitis.

Infection of mice with Rift Valley fever virus (RVFV) reproduces major pathological features of severe human disease, notably the early-onset hepatitis and delayed-onset encephalitis. We previously reported that the Rvfs2 locus from the susceptible MBT/Pas strain reduces survival time after RVFV infection. Here, we used BALB/cByJ (BALB) mice congenic for Rvfs2 (C.MBT-Rvfs2) to investigate the pathophysiological mechanisms impacted by Rvfs2. Clinical, biochemical and histopathological features indicated similar liver damage in BALB and C.MBT-Rvfs2 mice until day 5 after infection. However, while C.MBT-Rvfs2 mice succumbed from acute liver injury, most BALB mice recovered and died later of encephalitis. Hepatocytes of BALB infected liver proliferated actively on day 6, promoting organ regeneration and recovery from liver damage. By comparison with C.MBT-Rvfs2, BALB mice had up to 100-fold lower production of infectious virions in the peripheral blood and liver, strongly decreased RVFV protein in liver and reduced viral replication in primary cultured hepatocytes, suggesting that the BALB Rvfs2 haplotype limits RVFV pathogenicity through decreased virus replication. Moreover, bone marrow chimera experiments showed that both hematopoietic and non-hematopoietic cells are required for the protective effect of the BALB Rvfs2 haplotype. Altogether, these results indicate that Rvfs2 controls critical events which allow survival to RVFV-induced hepatitis.

Pathogenic variants in the DEAH-box RNA helicase DHX37 are a frequent cause of 46,XY gonadal dysgenesis and 46,XY testicular regression syndrome.

PURPOSE: XY individuals with disorders/differences of sex development (DSD) are characterized by reduced androgenization caused, in some children, by gonadal dysgenesis or testis regression during fetal development. The genetic etiology for most patients with 46,XY gonadal dysgenesis and for all patients with testicular regression syndrome (TRS) is unknown. METHODS: We performed exome and/or Sanger sequencing in 145 individuals with 46,XY DSD of unknown etiology including gonadal dysgenesis and TRS. RESULTS: Thirteen children carried heterozygous missense pathogenic variants involving the RNA helicase DHX37, which is essential for ribosome biogenesis. Enrichment of rare/novel DHX37 missense variants in 46,XY DSD is highly significant compared with controls (P value = 5.8 × 10-10). Five variants are de novo (P value = 1.5 × 10-5). Twelve variants are clustered in two highly conserved functional domains and were specifically associated with gonadal dysgenesis and TRS. Consistent with a role in early testis development, DHX37 is expressed specifically in somatic cells of the developing human and mouse testis. CONCLUSION: DHX37 pathogenic variants are a new cause of an autosomal dominant form of 46,XY DSD, including gonadal dysgenesis and TRS, showing that these conditions are part of a clinical spectrum. This raises the possibility that some forms of DSD may be a ribosomopathy.

Expression patterns suggest that despite considerable functional redundancy, galectin-4 and -6 play distinct roles in normal and damaged mouse digestive tract.

The galectin-4 protein is mostly expressed in the digestive tract and is associated with lipid raft stabilization, protein apical trafficking, wound healing, and inflammation. While most mammalian species, including humans, have a single Lgals4 gene, some mice have two paralogues: Lgals4 and Lgals6. So far, their significant similarities have hindered the analysis of their respective expression and function. We took advantage of two antibodies that discriminate between the galectin-4 and galectin-6 proteins to document their patterns of expression in the normal and the dextran sodium sulfate (DSS)-damaged digestive tract in the mouse. In the normal digestive tract, their pattern of expression from tongue to colon is quite similar, which suggests functional redundancy. However, the presence of galectin-4, but not galectin-6, in the lamina propria of the DSS-damaged colon, its association with luminal colonic bacteria, and differences in subcellular localization of these proteins suggest that they also have distinct roles in the normal and the damaged mouse digestive tract. Our results provide a rare example of ancestral and derived functions evolving after tandem gene duplication.

Galectin-7 in the control of epidermal homeostasis after injury.

Galectins, a family of beta-galactoside binding lectins, have recently emerged as novel regulators of tissue homeostasis. Galectin-7 is predominantly expressed in stratified epithelia, especially in epidermis. We report here the generation of galectin-7-deficient mice that are viable and do not display phenotypical abnormalities in skin structure or expression of epidermal markers. However, these mice show unique defects in the maintenance of epidermal homeostasis in response to environmental challenges. First, after UVB irradiation in vivo, the apoptotic response is prematurely triggered and lasts longer in the mutant epidermis. This result contrasts with the proapoptotic role that had been proposed for galectin-7. Second, wound-healing experiments in vivo revealed that galectin-7-deficient mice displayed a reduced reepithelialization potential compared with wild-type littermates. This effect could be attributed to a defect in cell migration. Because galectin-7 is located in the podosomes of keratinocytes migrating out of skin explants in culture, we propose that this glycan-binding protein may directly influence cell/extracellular matrix interactions. Finally, we also detected an unexpected intense hyperproliferative reaction consecutive to both types of stress in galectin-7-deficient mice. Together, these studies provide the first genetic evidence showing that galectin-7 can modulate keratinocyte apoptosis, proliferation, and migration during skin repair.

Lgals6, a 2-million-year-old gene in mice: a case of positive Darwinian selection and presence/absence polymorphism.

Duplications of genes are widely considered to be a driving force in the evolutionary process. The fate of such duplicated genes (paralogs) depends mainly on the early stages of their evolution. Therefore, the study of duplications that have already started to diverge is useful to better understand their evolution. We present here the example of a 2-million-year-old segmental duplication at the origin of the Lgals4 and Lgals6 genes in the mouse genome. We analyzed the distribution of these genes in samples from 110 wild individuals and wild-derived inbred strains belonging to eight mouse species from Mus (Coelomys) pahari to M. musculus and 28 laboratory strains. Using a maximum-likelihood method, we show that the sequence of the Lgals6 gene has evolved under the influence of strong positive selection that is likely to result in its neofunctionalization. Surprisingly, despite this selection pressure, the Lgals6 gene is present in some mouse species, but not all. Furthermore, even within the species and populations where it is present, the Lgals6 gene is never fixed. To explain this paradox, we propose different hypotheses such as balanced selection and neutral retention of ancient polymophism and we discuss this unexpected result with regard to known galectin properties and response to infections by pathogens.

Phylogenetic analysis of the vertebrate galectin family.

Galectins form a family of structurally related carbohydrate binding proteins (lectins) that have been identified in a large variety of metazoan phyla. They are involved in many biological processes such as morphogenesis, control of cell death, immunological response, and cancer. To elucidate the evolutionary history of galectins and galectin-like proteins in chordates, we have exploited three independent lines of evidence: (i) location of galectin encoding genes (LGALS) in the human genome; (ii) exon-intron organization of galectin encoding genes; and (iii) sequence comparison of carbohydrate recognition domains (CRDs) of chordate galectins. Our results suggest that a duplication of a mono-CRD galectin gene gave rise to an original bi-CRD galectin gene, before or early in chordate evolution. The N-terminal and C-terminal CRDs of this original galectin subsequently diverged into two different subtypes, defined by exon-intron structure (F4-CRD and F3-CRD). We show that all vertebrate mono-CRD galectins known to date belong to either the F3- or F4- subtype. A sequence of duplication and divergence events of the different galectins in chordates is proposed.

Growth and early postimplantation defects in mice deficient for the bromodomain-containing protein Brd4.

In a gene trap screen we recovered a mouse mutant line in which an insertion generated a null allele of the Brd4 gene. Brd4 belongs to the Fsh/Brd family, a group of structurally related proteins characterized by the association of two bromodomains and one extraterminal domain. Members of this family include Brd2/Ring3/Fsrg1 in mammals, fs(1)h in Drosophila, and Bdf1 in Saccharomyces cerevisiae. Brd4 heterozygotes display pre- and postnatal growth defects associated with a reduced proliferation rate. These mice also exhibit a variety of anatomical abnormalities: head malformations, absence of subcutaneous fat, cataracts, and abnormal liver cells. In primary cell cultures, heterozygous cells also display reduced proliferation rates and moderate sensitivity to methyl methanesulfonate. Embryos nullizygous for Brd4 die shortly after implantation and are compromised in their ability to maintain an inner cell mass in vitro, suggesting a role in fundamental cellular processes. Finally, sequence comparisons suggest that Brd4 is likely to correspond to the Brd-like element of the mediator of transcriptional regulation isolated by Y. W. Jiang, P. Veschambre, H. Erdjument-Bromage, P. Tempst, J. W. Conaway, R. C. Conaway, and R. D. Kornberg (Proc. Natl. Acad. Sci. USA 95:8538-8543, 1998) and the Brd4 mutant phenotype is discussed in light of this result. Together, our results provide the first genetic evidence for an in vivo role in mammals for a member of the Fsh/Brd family.