Endothelial gene regulatory elements associated with cardiopharyngeal lineage differentiation.

Endothelial cells (EC) differentiate from multiple sources, including the cardiopharyngeal mesoderm, which gives rise also to cardiac and branchiomeric muscles. The enhancers activated during endothelial differentiation within the cardiopharyngeal mesoderm are not completely known. Here, we use a cardiogenic mesoderm differentiation model that activates an endothelial transcription program to identify endothelial regulatory elements activated in early cardiogenic mesoderm. Integrating chromatin remodeling and gene expression data with available single-cell RNA-seq data from mouse embryos, we identify 101 putative regulatory elements of EC genes. We then apply a machine-learning strategy, trained on validated enhancers, to predict enhancers. Using this computational assay, we determine that 50% of these sequences are likely enhancers, some of which are already reported. We also identify a smaller set of regulatory elements of well-known EC genes and validate them using genetic and epigenetic perturbation. Finally, we integrate multiple data sources and computational tools to search for transcriptional factor binding motifs. In conclusion, we show EC regulatory sequences with a high likelihood to be enhancers, and we validate a subset of them using computational and cell culture models. Motif analyses show that the core EC transcription factors GATA/ETS/FOS is a likely driver of EC regulation in cardiopharyngeal mesoderm.

A phenotypic rescue approach identifies lineage regionalization defects in a mouse model of DiGeorge syndrome.

TBX1 is a key regulator of pharyngeal apparatus (PhAp) development. Vitamin B12 (vB12) treatment partially rescues aortic arch patterning defects of Tbx1+/- embryos. Here, we show that it also improves cardiac outflow tract septation and branchiomeric muscle anomalies of Tbx1 hypomorphic mutants. At the molecular level, in vivo vB12 treatment enabled us to identify genes that were dysregulated by Tbx1 haploinsufficiency and rescued by treatment. We found that SNAI2, also known as SLUG, encoded by the rescued gene Snai2, identified a population of mesodermal cells that was partially overlapping with, but distinct from, ISL1+ and TBX1+ populations. In addition, SNAI2+ cells were mislocalized and had a greater tendency to aggregate in Tbx1+/- and Tbx1-/- embryos, and vB12 treatment restored cellular distribution. Adjacent neural crest-derived mesenchymal cells, which do not express TBX1, were also affected, showing enhanced segregation from cardiopharyngeal mesodermal cells. We propose that TBX1 regulates cell distribution in the core mesoderm and the arrangement of multiple lineages within the PhAp.

Pharmacological Rescue of the Brain Cortex Phenotype of Tbx1 Mouse Mutants: Significance for 22q11.2 Deletion Syndrome.

OBJECTIVES: Tbx1 mutant mice are a widely used model of 22q11.2 deletion syndrome (22q11.2DS) because they manifest a broad spectrum of physical and behavioral abnormalities that is similar to that found in 22q11.2DS patients. In Tbx1 mutants, brain abnormalities include changes in cortical cytoarchitecture, hypothesized to be caused by the precocious differentiation of cortical progenitors. The objectives of this research are to identify drugs that have efficacy against the brain phenotype, and through a phenotypic rescue approach, gain insights into the pathogenetic mechanisms underlying Tbx1 haploinsufficiency. EXPERIMENTAL APPROACH: Disease model: Tbx1 heterozygous and homozygous embryos. We tested the ability of two FDA-approved drugs, the LSD1 inhibitor Tranylcypromine and Vitamin B12, to rescue the Tbx1 mutant cortical phenotype. Both drugs have proven efficacy against the cardiovascular phenotype, albeit at a much reduced level compared to the rescue achieved in the brain. METHODS: In situ hybridization and immunostaining of histological brain sections using a subset of molecular markers that label specific cortical regions or cell types. Appropriate quantification and statistical analysis of gene and protein expression were applied to identify cortical abnormalities and to determine the level of phenotypic rescue achieved. RESULTS: Cortical abnormalities observed in Tbx1 mutant embryos were fully rescued by both drugs. Intriguingly, rescue was obtained with both drugs in Tbx1 homozygous mutants, indicating that they function through mechanisms that do not depend upon Tbx1 function. This was particularly surprising for Vitamin B12, which was identified through its ability to increase Tbx1 gene expression. CONCLUSION: To our knowledge, this is only the second example of drugs to be identified that ameliorate phenotypes caused by the mutation of a single gene from the 22q11.2 homologous region of the mouse genome. This one drug-one gene approach might be important because there is evidence that the brain phenotype in 22q11.2DS patients is multigenic in origin, unlike the physical phenotypes, which are overwhelmingly attributable to Tbx1 haploinsufficiency. Therefore, effective treatments will likely involve the use of multiple drugs that are targeted to the function of specific genes within the deleted region.

EZH2 is required for parathyroid and thymic development through differentiation of the third pharyngeal pouch endoderm.

The Ezh2 gene encodes a histone methyltransferase of the polycomb repressive complex 2 that methylates histone H3 lysine 27. In this study, we investigated whether EZH2 has a role in the development of the pharyngeal apparatus and whether it regulates the expression of the Tbx1 gene, which encodes a key transcription factor required in pharyngeal development. To these ends, we performed genetic in vivo experiments with mouse embryos and used mouse embryonic stem cell (ESC)-based protocols to probe endoderm and cardiogenic mesoderm differentiation. Results showed that EZH2 occupies the Tbx1 gene locus in mouse embryos, and that suppression of EZH2 was associated with reduced expression of Tbx1 in differentiated mouse ESCs. Conditional deletion of Ezh2 in the Tbx1 expression domain, which includes the pharyngeal endoderm, did not cause cardiac defects but revealed that the gene has an important role in the morphogenesis of the third pharyngeal pouch (PP). We found that in conditionally deleted embryos the third PP was hypoplastic, had reduced expression of Tbx1, lacked the expression of Gcm2, a gene that marks the parathyroid domain, but expressed FoxN1, a gene marking the thymic domain. Consistently, the parathyroids did not develop, and the thymus was hypoplastic. Thus, Ezh2 is required for parathyroid and thymic development, probably through a function in the pouch endoderm. This discovery also provides a novel interpretational key for the finding of Ezh2 activating mutations in hyperparathyroidism and parathyroid cancer.

Chromatin and Transcriptional Response to Loss of TBX1 in Early Differentiation of Mouse Cells.

The T-box transcription factor TBX1 has critical roles in the cardiopharyngeal lineage and the gene is haploinsufficient in DiGeorge syndrome, a typical developmental anomaly of the pharyngeal apparatus. Despite almost two decades of research, if and how TBX1 function triggers chromatin remodeling is not known. Here, we explored genome-wide gene expression and chromatin remodeling in two independent cellular models of Tbx1 loss of function, mouse embryonic carcinoma cells P19Cl6, and mouse embryonic stem cells (mESCs). The results of our study revealed that the loss or knockdown of TBX1 caused extensive transcriptional changes, some of which were cell type-specific, some were in common between the two models. However, unexpectedly we observed only limited chromatin changes in both systems. In P19Cl6 cells, differentially accessible regions (DARs) were not enriched in T-BOX binding motifs; in contrast, in mESCs, 34% (n = 47) of all DARs included a T-BOX binding motif and almost all of them gained accessibility in Tbx1 -/- cells. In conclusion, despite a clear transcriptional response of our cell models to loss of TBX1 in early cell differentiation, chromatin changes were relatively modest.

c-Myc Modulation and Acetylation Is a Key HDAC Inhibitor Target in Cancer.

Purpose: Histone deacetylase inhibitors (HDACi) are promising anticancer drugs. Although some HDACi have entered the clinic, the mechanism(s) underlying their tumor selectivity are poorly understood.Experimental Design and Results: Using gene expression analysis, we define a core set of six genes commonly regulated in acute myeloid leukemia (AML) blasts and cell lines. MYC, the most prominently modulated, is preferentially altered in leukemia. Upon HDACi treatment, c-Myc is acetylated at lysine 323 and its expression decreases, leading to TRAIL activation and apoptosis. c-Myc binds to the TRAIL promoter on the proximal GC box through SP1 or MIZ1, impairing TRAIL activation. HDACi exposure triggers TRAIL expression, altering c-Myc-TRAIL binding. These events do not occur in normal cells. Excitingly, this inverse correlation between TRAIL and c-Myc is supported by HDACi treatment ex vivo of AML blasts and primary human breast cancer cells. The predictive value of c-Myc to HDACi responsiveness is confirmed in vivo in AML patients undergoing HDACi-based clinical trials.Conclusions: Collectively, our findings identify a key role for c-Myc in TRAIL deregulation and as a biomarker of the anticancer action of HDACi in AML. The potential improved patient stratification could pave the way toward personalized therapies. Clin Cancer Res; 23(10); 2542-55. ©2016 AACR.

Vitamin B12 ameliorates the phenotype of a mouse model of DiGeorge syndrome.

Abstract: Pathological conditions caused by reduced dosage of a gene, such as gene haploinsufficiency, can potentially be reverted by enhancing the expression of the functional allele. In practice, low specificity of therapeutic agents, or their toxicity reduces their clinical applicability. Here, we have used a high throughput screening (HTS) approach to identify molecules capable of increasing the expression of the gene Tbx1, which is involved in one of the most common gene haploinsufficiency syndromes, the 22q11.2 deletion syndrome. Surprisingly, we found that one of the two compounds identified by the HTS is the vitamin B12. Validation in a mouse model demonstrated that vitamin B12 treatment enhances Tbx1 gene expression and partially rescues the haploinsufficiency phenotype. These results lay the basis for preclinical and clinical studies to establish the effectiveness of this drug in the human syndrome.

TBX1 Represses Vegfr2 Gene Expression and Enhances the Cardiac Fate of VEGFR2+ Cells.

The T-box transcription factor TBX1 has critical roles in maintaining proliferation and inhibiting differentiation of cardiac progenitor cells of the second heart field (SHF). Haploinsufficiency of the gene that encodes it is a cause of congenital heart disease. Here, we developed an embryonic stem (ES) cell-based model in which Tbx1 expression can be modulated by tetracycline. Using this model, we found that TBX1 down regulates the expression of VEGFR2, and we confirmed this finding in vivo during embryonic development. In addition, we found a Vegfr2 domain of expression, not previously described, in the posterior SHF and this expression is extended by loss of Tbx1. VEGFR2 has been previously described as a marker of a subpopulation of cardiac progenitors. Clonal analysis of ES-derived VEGFR2+ cells indicated that 12.5% of clones expressed three markers of cardiac lineage (cardiomyocyte, smooth muscle and endothelium). However, a pulse of Tbx1 expression was sufficient to increase the percentage to 20.8%. In addition, the percentage of clones expressing markers of multiple cardiac lineages increased from 41.6% to 79.1% after Tbx1 pulse. These results suggest that TBX1 plays a role in maintaining a progenitor state in VEGFR2+ cells.

Partial rescue of the Tbx1 mutant heart phenotype by Fgf8: genetic evidence of impaired tissue response to Fgf8.

Tbx1 is the candidate gene of DiGeorge syndrome and is required in humans and mice for the development of the cardiac outflow tract (OFT) and aortic arch arteries. Loss of function mutants present with reduced cell proliferation and premature differentiation of cardiac progenitor cells of the second heart field (SHF). Tbx1 regulates Fgf8 expression hence the hypothesis that the proliferation impairment may contribute to the heart phenotype of mutants. Here we show that forced Fgf8 expression modifies and partially rescues the OFT septation defects of Tbx1 mutants but only if there is some residual expression of Tbx1. This genetic experiment suggests that Tbx1, directly or indirectly, affects tissue response to Fgf8. Indeed, Tbx1(-/-) mouse embryonic fibroblasts were unable to respond to Fgf8 added to the culture media and showed defective response of Erk1/2 and Rsk1. Our data suggest a coordinated pathway modulating Fgf8 ligand expression and tissue response to it in the SHF.

Early thyroid development requires a Tbx1-Fgf8 pathway.

The thyroid develops within the pharyngeal apparatus from endodermally-derived cells. The many derivatives of the pharyngeal apparatus develop at similar times and sometimes from common cell types, explaining why many syndromic disorders express multiple birth defects affecting different structures that share a common pharyngeal origin. Thus, different derivatives may share common genetic networks during their development. Tbx1, the major gene associated with DiGeorge syndrome, is a key player in the global development of the pharyngeal apparatus, being required for virtually all its derivatives, including the thyroid. Here we show that Tbx1 regulates the size of the early thyroid primordium through its expression in the adjacent mesoderm. Because Tbx1 regulates the expression of Fgf8 in the mesoderm, we postulated that Fgf8 mediates critical Tbx1-dependent interactions between mesodermal cells and endodermal thyrocyte progenitors. Indeed, conditional ablation of Fgf8 in Tbx1-expressing cells caused an early thyroid phenotype similar to that of Tbx1 mutant mice. In addition, expression of an Fgf8 cDNA in the Tbx1 domain rescued the early size defect of the thyroid primordium in Tbx1 mutants. Thus, we have established that a Tbx1->Fgf8 pathway in the pharyngeal mesoderm is a key size regulator of mammalian thyroid.

Interplay of negative and positive signals controls endoderm-specific expression of the ascidian Cititf1 gene promoter.

Cititf1 is an early and specific marker of endoderm development in Ciona intestinalis [ Development 126, 5149]. Here, we examine Cititf1 transcriptional regulation focusing in particular on its endodermal restricted expression. Through the analysis of Ciona embryos, electroporated with different portions of Cititf1 5'-flanking region fused to lacZ, we characterized a minimal 300-bp cis-regulatory sequence able to closely reproduce the spatial and temporal expression pattern of the endogenous gene. This enhancer contains at least three distinct regulatory regions, two of which are responsible for activation of transcription in the endoderm and in the mesenchyme, respectively, while the third is a negative control element that represses mesenchyme transcription. We have further defined the sequences responsible for transcriptional activation in the endoderm by clustered point mutations and DNA-binding assays.