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Tinman/Nkx2-5 acts via miR-1 and upstream of Cdc42 to regulate heart function across species.

7 November 2018

Unraveling the gene regulatory networks that govern development and function of the mammalian heart is critical for the rational design of therapeutic interventions in human heart disease. Using the Drosophila heart as a platform for identifying novel gene interactions leading to heart disease, we found that the Rho-GTPase Cdc42 cooperates with the cardiac transcription factor Tinman/Nkx2-5. Compound Cdc42, tinman heterozygous mutant flies exhibited impaired cardiac output and altered myofibrillar architecture, and adult heart-specific interference with Cdc42 function is sufficient to cause these same defects. We also identified K(+) channels, encoded by dSUR and slowpoke, as potential effectors of the Cdc42-Tinman interaction. To determine whether a Cdc42-Nkx2-5 interaction is conserved in the mammalian heart, we examined compound heterozygous mutant mice and found conduction system and cardiac output defects. In exploring the mechanism of Nkx2-5 interaction with Cdc42, we demonstrated that mouse Cdc42 was a target of, and negatively regulated by miR-1, which itself was negatively regulated by Nkx2-5 in the mouse heart and by Tinman in the fly heart. We conclude that Cdc42 plays a conserved role in regulating heart function and is an indirect target of Tinman/Nkx2-5 via miR-1.

MULTIPLE REGIONS OF THE HUMAN CARDIAC ACTIN GENE ARE NECESSARY FOR MATURATION-BASED EXPRESSION IN STRIATED-MUSCLE

7 November 2018

HIF-1α deletion partially rescues defects of hematopoietic stem cell quiescence caused by Cited2 deficiency.

7 November 2018

Cited2 is a transcriptional modulator involved in various biologic processes including fetal liver hematopoiesis. In the present study, the function of Cited2 in adult hematopoiesis was investigated in conditional knockout mice. Deletion of Cited2 using Mx1-Cre resulted in increased hematopoietic stem cell (HSC) apoptosis, loss of quiescence, and increased cycling, leading to a severely impaired reconstitution capacity as assessed by 5-fluorouracil treatment and long-term transplantation. Transcriptional profiling revealed that multiple HSC quiescence- and hypoxia-related genes such as Egr1, p57, and Hes1 were affected in Cited2-deficient HSCs. Because Cited2 is a negative regulator of HIF-1, which is essential for maintaining HSC quiescence, and because we demonstrated previously that decreased HIF-1α gene dosage partially rescues both cardiac and lens defects caused by Cited2 deficiency, we generated Cited2 and HIF-1α double-knockout mice. Additional deletion of HIF-1α in Cited2-knockout BM partially rescued impaired HSC quiescence and reconstitution capacity. At the transcriptional level, deletion of HIF-1α restored expression of p57 and Hes1 but not Egr1 to normal levels. Our results suggest that Cited2 regulates HSC quiescence through both HIF-1-dependent and HIF-1-independent pathways.

TBX6 null variants and a common hypomorphic allele in congenital scoliosis.

7 November 2018

BACKGROUND: Congenital scoliosis is a common type of vertebral malformation. Genetic susceptibility has been implicated in congenital scoliosis. METHODS: We evaluated 161 Han Chinese persons with sporadic congenital scoliosis, 166 Han Chinese controls, and 2 pedigrees, family members of which had a 16p11.2 deletion, using comparative genomic hybridization, quantitative polymerase-chain-reaction analysis, and DNA sequencing. We carried out tests of replication using an additional series of 76 Han Chinese persons with congenital scoliosis and a multicenter series of 42 persons with 16p11.2 deletions. RESULTS: We identified a total of 17 heterozygous TBX6 null mutations in the 161 persons with sporadic congenital scoliosis (11%); we did not observe any null mutations in TBX6 in 166 controls (P<3.8×10(-6)). These null alleles include copy-number variants (12 instances of a 16p11.2 deletion affecting TBX6) and single-nucleotide variants (1 nonsense and 4 frame-shift mutations). However, the discordant intrafamilial phenotypes of 16p11.2 deletion carriers suggest that heterozygous TBX6 null mutation is insufficient to cause congenital scoliosis. We went on to identify a common TBX6 haplotype as the second risk allele in all 17 carriers of TBX6 null mutations (P<1.1×10(-6)). Replication studies involving additional persons with congenital scoliosis who carried a deletion affecting TBX6 confirmed this compound inheritance model. In vitro functional assays suggested that the risk haplotype is a hypomorphic allele. Hemivertebrae are characteristic of TBX6-associated congenital scoliosis. CONCLUSIONS: Compound inheritance of a rare null mutation and a hypomorphic allele of TBX6 accounted for up to 11% of congenital scoliosis cases in the series that we analyzed. (Funded by the National Basic Research Program of China and others.).

Advances in the Genetics of Congenital Heart Disease: A Clinician's Guide.

7 November 2018

Our understanding of the genetics of congenital heart disease (CHD) is rapidly expanding; however, many questions, particularly those relating to sporadic forms of disease, remain unanswered. Massively parallel sequencing technology has made significant contributions to the field, both from a diagnostic perspective for patients and, importantly, also from the perspective of disease mechanism. The importance of de novo variation in sporadic disease is a recent highlight, and the genetic link between heart and brain development has been established. Furthermore, evidence of an underlying burden of genetic variation contributing to sporadic and familial forms of CHD has been identified. Although we are still unable to identify the cause of CHD for most patients, recent findings have provided us with a much clearer understanding of the types of variants and their individual contributions and collectively mark an important milestone in our understanding of both familial and sporadic forms of disease.

Transcriptional activating activity of Smad4: roles of SMAD hetero-oligomerization and enhancement by an associating transactivator.

7 November 2018

Smad4 plays a pivotal role in signal transduction of the transforming growth factor beta superfamily cytokines by mediating transcriptional activation of target genes. Hetero-oligomerization of Smad4 with the pathway-restricted SMAD proteins is essential for Smad4-mediated transcription. We provide evidence that SMAD hetero-oligomerization is directly required for the Smad4 C-terminal domain [Smad4(C)] to show its transcriptional transactivating activity; this requirement obtains even when Smad4(C) is recruited to promoters by heterologous DNA-binding domains and in the absence of the inhibitory Smad4 N-terminal domain. Defined mutations of GAL4 DNA-binding domain fusion of Smad4(C) that disrupt SMAD hetero-oligomerization suppressed transcriptional activation. Importantly, we found that an orphan transcriptional activator MSG1, a nuclear protein that has strong transactivating activity but apparently lacks DNA-binding activity, functionally interacted with Smad4 and enhanced transcription mediated by GAL4 DNA-binding domain-Smad4(C) and full-length Smad4. Transcriptional enhancement by MSG1 depended on transforming growth factor beta signaling and was suppressed by Smad4(C) mutations disrupting SMAD hetero-oligomerization or by the presence of Smad4 N-terminal domain. Furthermore, Smad4(C) did not show any detectable transactivating activity in yeast when fused to heterologous DNA-binding domains. These results demonstrate additional roles of SMAD hetero-oligomerization in Smad4-mediated transcriptional activation. They also suggest that the transcriptional-activating activity observed in the presence of Smad4 in mammalian cells may be derived, at least in part, from endogenously expressed separate transcriptional activators, such as MSG1.

Cited2 gene controls pluripotency and cardiomyocyte differentiation of murine embryonic stem cells through Oct4 gene.

7 November 2018

Cited2 (CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich tail 2) is a transcriptional modulator critical for the development of multiple organs. Although many Cited2-mediated phenotypes and molecular events have been well characterized using in vivo genetic murine models, Cited2-directed cell fate decision in embryonic stem cells (ESCs) remains elusive. In this study, we examined the role of Cited2 in the maintenance of stemness and pluripotency of murine ESCs by a gene-targeting approach. Cited2 knock-out (Cited2(Δ/-), KO) ESCs display defective differentiation. Loss of Cited2 in differentiating ESCs results in delayed silencing of the genes involved in the maintenance of pluripotency and self-renewal of stem cells (Oct4, Klf4, Sox2, and c-Myc) and the disturbance in cardiomyocyte, hematopoietic, and neuronal differentiation. In addition, Cited2 KO ESCs experience a delayed induction of cardiomyocyte differentiation-associated proteins, NFAT3 (along with the reduced expression of NFAT3 target genes, Nkx2.5 and β-MHC), N-cadherin, and smooth muscle actin. CITED2 is recruited to the Oct4 promoter to regulate its expression during early ESC differentiation. This is the first demonstration that Cited2 controls ESC pluripotency and differentiation via direct regulation of Oct4 gene expression.

Targeted next-generation sequencing identifies pathogenic variants in familial congenital heart disease.

7 November 2018

BACKGROUND: Many genes have been implicated in the development of congenital heart disease (CHD). Next-generation sequencing offers opportunities for genetic testing but is often complicated by logistic and interpretative hurdles. OBJECTIVES: This study sought to apply next-generation sequencing technology to CHD families with multiple affected members using a purpose-designed gene panel to assess diagnostic potential for future clinical applications. METHODS: We designed a targeted next-generation sequencing gene panel for 57 genes previously implicated in CHD. Probands were screened in 16 families with strong CHD histories and in 15 control subjects. Variants affecting protein-coding regions were classified in silico using prediction programs and filtered according to predicted mode of inheritance, minor allele frequencies, and presence in databases such as dbSNP (Single Nucleotide Polymorphism Database) and ESP (Exome Sequencing Project). Disease segregation studies were conducted in variants identified in CHD cases predicted to be deleterious and with minor allele frequencies <0.1%. RESULTS: Thirteen potential disease-causing variants were identified in 9 families. Of these, 5 variants segregated with disease phenotype, revealing a likely molecular diagnosis in 31% of this cohort. Significant increases in the number of "indels, nonsense, and splice" variants, as well as variants classified as "probably damaging" were identified in CHD cases but not in control subjects. Also, there was a significant increase in the total number of "rare" and "low" frequency variants (minor allele frequencies <0.05) in the CHD cases. CONCLUSIONS: When multiple relatives are affected by CHD, a gene panel-based approach may identify its cause in up to 31% of families. Identifying causal variants has implications for clinical care and future family planning.

De novo, deleterious sequence variants that alter the transcriptional activity of the homeoprotein PBX1 are associated with intellectual disability and pleiotropic developmental defects

7 November 2018

Sp5, a new member of the Sp1 family, is dynamically expressed during development and genetically interacts with Brachyury.

7 November 2018

We describe the identification, biochemical characterisation, and mutation of a novel mouse gene: Sp5. Sp5 encodes a protein having a C-terminal C(2)H(2) zinc finger domain closely related to that of the transcription factor Sp1. In vitro, DNA binding studies show that it binds to the GC box, a DNA motif present in the promoter of a very large number of genes, including Brachyury, and recognised by members of the Sp1 family. However, outside of its DNA binding domain, Sp5 has little homology with any other member of the Sp1 family. In contrast to the ubiquitous expression of Sp1, Sp5 exhibits a remarkably dynamic pattern of expression throughout early development. This is suggestive of a role in numerous tissue patterning events, including gastrulation and axial elongation; differentiation and patterning of the neural tube, pharyngeal region, and somites; and formation of skeletal muscle in the body and limbs. Mice homozygous for a targeted mutation in Sp5 show no overt phenotype. However, the enhancement of the T/+ phenotype in compound mutant mice (Sp5(lacZ)/Sp5(lacZ), T/+) indicates a genetic interaction between Sp5 and Brachyury. These observations are consistent with a role for Sp5 in the coordination of changes in transcription required to generate pattern in the developing embryo.