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We have previously shown that parathyroid hormone (PTH) induces the phosphorylation of the DNA-binding protein Nascent polypeptide associated complex And Coregulator alpha (NACA), leading to nuclear translocation of NACA and activation of target genes. Using ChIP-Seq against NACA in parallel with RNA-sequencing, we report the identification of Ubiquitin Specific Peptidase 53 (Usp53) as a target gene of PTH-activated NACA in osteoblasts. A binding site for NACA within the ChIP fragment from the Usp53 promoter was confirmed by electrophoretic mobility shift assay. Activity of the Usp53 promoter (− 2325/+ 238 bp) was regulated by the JUN-CREB complex and this activation relied on activated PKA and the presence of NACA. Usp53 knockdown in ST2 stromal cells stimulated expression of the osteoblastic markers Bglap2 (Osteocalcin) and Alpl (Alkaline phosphatase) and inhibited expression of the adipogenic markers Pparg and Cebpa. A similar effect was measured when knocking down Naca. During osteoblastogenesis, the impact of Usp53 knockdown on PTH responses varied depending on the maturation stage of the cells. In vivo implantation of Usp53-knockdown bone marrow stromal cells in immunocompromised mice showed an increase in osteoblast number and a decrease in adipocyte counts. Our data suggest that Usp53 modulates the fate of mesenchymal cells by impacting lineage selection.

In the nucleus of differentiated osteoblasts, the alpha chain of nascent polypeptide associated complex (αNAC) interacts with cJUN transcription factors to regulate the expression of target genes, including Osteocalcin (Bglap2 ). PTH induces the phosphorylation of αNAC on serine 99 through a Gαs-PKA dependent pathway. This leads to activation of αNAC and expression of Bglap2. To identify additional target genes regulated by PTH-activated αNAC, we performed ChIP-Seq against αNAC in PTH-treated MC3T3-E1 cells. This identified Low density lipoprotein receptor-Related Protein 6 (Lrp6 ) as a potential αNAC target. LRP6 acts as a co-receptor for the PTH receptor to allow optimal activation of PTH signaling. PTH increased Lrp6 mRNA levels in primary osteoblasts. Conventional quantitative ChIP confirmed the ChIP-Seq results. To assess whether αNAC plays a critical role in PTH-stimulated Lrp6 expression, we knocked-down Naca expression in MC3T3-E1 cells. Reduction of αNAC levels decreased basal expression of Lrp6 by 30% and blocked the stimulation of Lrp6 expression by PTH. We cloned the proximal mouse Lrp6 promoter (−2523/+120 bp) upstream of the luciferase reporter. Deletion and point mutations analysis in electrophoretic mobility shift assays and transient transfections identified a functional αNAC binding site centered around −343 bp. ChIP and ChIP-reChIP against JUND and αNAC showed that they cohabit on the proximal Lrp6 promoter. Luciferase assays confirmed that PTH-activated αNAC potentiated JUND-mediated Lrp6 transcription and Jund knockdown abolished this response. This study identified a novel αNAC target gene induced downstream of PTH signaling and represents the first characterization of the regulation of Lrp6 transcription in osteoblasts.

Intermittent administration of PTH(1–34) has a profound osteoanabolic effect on the skeleton. At the cellular level, osteoblasts and osteocytes are two crucial cell types that respond to PTH stimulation in bone. The transcriptional cofactor Nascent polypeptide Associated Complex and coregulator alpha (NACA) is a downstream target of the PTH-Gαs-PKA axis in osteoblasts. NACA functions as a transcriptional cofactor affecting bZIP factor-mediated transcription of target promoters in osteoblasts, such as Osteocalcin (Bglap2). Here, we used RNA-Seq and ChIP-Seq against NACA in PTH-treated MC3T3-E1 osteoblastic cells to identify novel targets of the PTH-activated NACA. Our approach identified Nuclear factor interleukin-3-regulated (Nfil3) as a target promoter of this pathway. Knockdown of Naca reduced the response of Nfil3 to PTH(1–34) stimulation. In silico analysis of the Nfil3 promoter revealed potential binding sites for NACA (located within the ChIP fragment) and CREB. We show that following PTH stimulation, phosphorylated-CREB binds the proximal promoter of Nfil3 in osteoblasts. The activity of the Nfil3 promoter (−818/+182 bp) is regulated by CREB and this activation relies on the presence of NACA. In addition, we show that knockdown of Nfil3 enhances the expression of osteoblastic differentiation markers in MC3T3-E1 cells while it represses osteocytic marker gene expression in IDG-SW3 cells. These results show that the PTH-induced NACA axis regulates Nfil3 expression and suggest that NFIL3 acts as a transcriptional repressor in osteoblasts while it exhibits differential activity as an activator in osteocytes.

Background Cutaneous malformations are at times associated with some forms of congenital heart defects. Many a times subtle cutaneous phenotypes maybe overlooked as their significance on the lives of individuals is minimal. Lebanon represents an area of high consanguinity, where the rates can go beyond 70% in some districts. For the past 6 years, we have been studying several genodermatoses in Lebanon including those with cardiac malformations. Objectives The main aim of this study is to document the genetic basis of a familial case of Axenfeld-Rieger Syndrome (ARS) with a mild cutaneous phenotype represented histologically with degeneration/ absence of hair follicles and incomplete formation of sebaceous and eccrine glands, in addition to the cardiac and ocular phenotypes. Methods Whole exome sequencing was performed on two identical-twins with ARS along with their affected father and non-affected mother. Sanger sequencing was used to confirm the mutation, and the effects of the mutations on protein function was assessed in vitro using transient transfections. Results A novel mutation inFOXC1 designated p.L240Rfs*75 was found in both twins and their father. The affected individuals share also a rare documented variant in NFATC1 designated p.V197 M. Both were absent from 200 Lebanese exomes. Our in vitro results suggested a gain of function activity of the FOXC1/NFATC1 complex, confirming its documented role in controlling murine hair follicle stem cells quiescence and regeneration. Conclusion This is the first documented human case with a mutation inFOXC1 regulating multi-organ developmental pathways that reflect a conserved mechanism in cell differentiation and proliferation.

Background GATA transcription factors are evolutionary conserved zinc finger proteins with multiple roles in cell differentiation/proliferation and organogenesis. GATA5 is only transiently expressed in the embryonic heart, and the inactivation of both Gata5 alleles results in a partially penetrant bicuspid aortic valve (BAV) phenotype in mice. We hypothesized that only biallelic mutations in GATA5 could be disease causing. Methods A total of 185 patients with different forms of congenital heart disease (CHD) were screened along 150 healthy individuals for GATA4, 5, and 6. All patients' phenotypes were diagnosed with echocardiography. Results Sequencing results revealed eight missense variants (three of which are novel) in cases with various conotruncal and septal defects. Out of these, two were inherited in recessive forms: the p.T67P variant, which was found both in patients and in healthy individuals, and the previously described p.Y142H variant which was only found in a patient with a double outlet right ventricle (DORV). We characterized the p.Y142H variant and showed that it significantly reduced the transcriptional activity of the protein over cardiac promoters by 30–40%. Conclusion Our results do prove that p.Y142H is associated with DORV and suggests including GATA5 as a potential gene to be screened in patients with this phenotype.

Purpose: The role of GATA5 in the morphogenesis of aortic valve has been recently elucidated in the mice knock out model. Growing evidence demonstrates that genetic defects in GATA5 might be responsible for various forms of congenital heart diseases (CHDs) in humans. The published results however were neither supported by functional data, nor by proper genetic controls. The aim of this proposal is to unravel the role of GATA5 in such diseases, and establish a well-defined genotype/phenotype association. Methods: Blood was collected from children with CHD. Sanger sequencing was carried out on all coding exons of GATA5. Site directed mutagenesis was carried out on the corresponding human GATA5 cDNA and the generated expression vector was tested in transfected cells. Results: One novel mutation (T67P) and another previously reported mutation (Y142H) have been identified in four out of 150 patients screened. Unlike all previously described mutations in GATA5, our mutations were homozygous. The T67P mutation in one patient is homozygous as a consequence of one inherited allele and another de novo mutated allele. The Y142H mutation is in the trans-activating domain of the GATA5 protein suggesting a direct effect on the transcriptional properties of the protein. In vitro assays, did show a consistent reproducible 30-40% drop in transcativtional activities over the corresponding GATA binding sites on different promoters. Conclusion: We have characterized novel mutations in GATA5 and their associations with CHDs by proving that they lead to abnormal protein function. This is the first report on a homozygous mutation in a gene encoding a transcription factor linked to CHD. The results go in parallel with the knock-out mice proving that genetic results in mice are mirror-images for those in humans. ![Figure][1] [1]: pending:yes