Snail Transcriptionally Represses Brachyury to Promote the Mesenchymal-Epithelial Transition in Ascidian Notochord Cells.

Mesenchymal-epithelial transition (MET) is a widely spread and evolutionarily conserved process across species during development. In Ciona embryogenesis, the notochord cells undergo the transition from the non-polarized mesenchymal state into the polarized endothelial-like state to initiate the lumen formation between adjacent cells. Based on previously screened MET-related transcription factors by ATAC-seq and Smart-Seq of notochord cells, Ciona robusta Snail (Ci-Snail) was selected for its high-level expression during this period. Our current knockout results demonstrated that Ci-Snail was required for notochord cell MET. Importantly, overexpression of the transcription factor Brachyury in notochord cells resulted in a similar phenotype with failure of lumen formation and MET. More interestingly, expression of Ci-Snail in the notochord cells at the late tailbud stage could partially rescue the MET defect caused by Brachyury-overexpression. These results indicated an inverse relationship between Ci-Snail and Brachyury during notochord cell MET, which was verified by RT-qPCR analysis. Moreover, the overexpression of Ci-Snail could significantly inhibit the transcription of Brachyury, and the CUT&Tag-qPCR analysis demonstrated that Ci-Snail is directly bound to the upstream region of Brachyury. In summary, we revealed that Ci-Snail promoted the notochord cell MET and was essential for lumen formation via transcriptionally repressing Brachyury.

Identification of potential biomarkers for aging diagnosis of mesenchymal stem cells derived from the aged donors.

BACKGROUND: The clinical application of human bone-marrow derived mesenchymal stem cells (MSCs) for the treatment of refractory diseases has achieved remarkable results. However, there is a need for a systematic evaluation of the quality and safety of MSCs sourced from donors. In this study, we sought to assess one potential factor that might impact quality, namely the age of the donor. METHODS: We downloaded two data sets from each of two Gene Expression Omnibus (GEO), GSE39035 and GSE97311 databases, namely samples form young (< 65 years of age) and old (> 65) donor groups. Through, bioinformatics analysis and experimental validation to these retrieved data, we found that MSCs derived from aged donors can lead to differential expression of gene profiles compared with those from young donors, and potentially affect the function of MSCs, and may even induce malignant tumors. RESULTS: We identified a total of 337 differentially expressed genes (DEGs), including two upregulated and eight downregulated genes from the databases of both GSE39035 and GSE97311. We further identified 13 hub genes. Six of them, TBX15, IGF1, GATA2, PITX2, SNAI1 and VCAN, were highly expressed in many human malignancies in Human Protein Atlas database. In the MSCs in vitro senescent cell model, qPCR analysis validated that all six hub genes were highly expressed in senescent MSCs. Our findings confirm that aged donors of MSCs have a significant effect on gene expression profiles. The MSCs from old donors have the potential to cause a variety of malignancies. These TBX15, IGF1, GATA2, PITX2, SNAI1, VCAN genes could be used as potential biomarkers to diagnosis aging state of donor MSCs, and evaluate whether MSCs derived from an aged donor could be used for therapy in the clinic. Our findings provide a diagnostic basis for the clinical use of MSCs to treat a variety of diseases. CONCLUSIONS: Therefore, our findings not only provide guidance for the safe and standardized use of MSCs in the clinic for the treatment of various diseases, but also provide insights into the use of cell regeneration approaches to reverse aging and support rejuvenation.

Exhaustive identification of genome-wide binding events of transcriptional regulators.

Genome-wide binding assays aspire to map the complete binding pattern of gene regulators. Common practice relies on replication-duplicates or triplicates-and high stringency statistics to favor false negatives over false positives. Here we show that duplicates and triplicates of CUT&RUN are not sufficient to discover the entire activity of transcriptional regulators. We introduce ICEBERG (Increased Capture of Enrichment By Exhaustive Replicate aGgregation), a pipeline that harnesses large numbers of CUT&RUN replicates to discover the full set of binding events and chart the line between false positives and false negatives. We employed ICEBERG to map the full set of H3K4me3-marked regions, the targets of the co-factor ß-catenin, and those of the transcription factor TBX3, in human colorectal cancer cells. The ICEBERG datasets allow benchmarking of individual replicates, comparing the performance of peak calling and replication approaches, and expose the arbitrary nature of strategies to identify reproducible peaks. Instead of a static view of genomic targets, ICEBERG establishes a spectrum of detection probabilities across the genome for a given factor, underlying the intrinsic dynamicity of its mechanism of action, and permitting to distinguish frequent from rare regulation events. Finally, ICEBERG discovered instances, undetectable with other approaches, that underlie novel mechanisms of colorectal cancer progression.

Development of a Small Molecule Downmodulator for the Transcription Factor Brachyury.

Brachyury is an oncogenic transcription factor whose overexpression drives chordoma growth. The downmodulation of brachyury in chordoma cells has demonstrated therapeutic potential, however, as a transcription factor it is classically deemed "undruggable". Given that direct pharmacological intervention against brachyury has proven difficult, attempts at intervention have instead targeted upstream kinases. Recently, afatinib, an FDA-approved kinase inhibitor, has been shown to modulate brachyury levels in multiple chordoma cell lines. Herein, we use afatinib as a lead to undertake a structure-based drug design approach, aided by mass-spectrometry and X-ray crystallography, to develop DHC-156, a small molecule that more selectively binds brachyury and downmodulates it as potently as afatinib. We eliminated kinase-inhibition from this novel scaffold while demonstrating that DHC-156 induces the post-translational downmodulation of brachyury that results in an irreversible impairment of chordoma tumor cell growth. In doing so, we demonstrate the feasibility of direct brachyury modulation, which may further be developed into more potent tool compounds and therapies.

Heat shock protein gp96 drives natural killer cell maturation and anti-tumor immunity by counteracting Trim28 to stabilize Eomes.

The maturation process of natural killer (NK) cells, which is regulated by multiple transcription factors, determines their functionality, but few checkpoints specifically targeting this process have been thoroughly studied. Here we show that NK-specific deficiency of glucose-regulated protein 94 (gp96) leads to decreased maturation of NK cells in mice. These gp96-deficient NK cells exhibit undermined activation, cytotoxicity and IFN-γ production upon stimulation, as well as weakened responses to IL-15 for NK cell maturation, in vitro. In vivo, NK-specific gp96-deficient mice show increased tumor growth. Mechanistically, we identify Eomes as the downstream transcription factor, with gp96 binding to Trim28 to prevent Trim28-mediated ubiquitination and degradation of Eomes. Our study thus suggests the gp96-Trim28-Eomes axis to be an important regulator for NK cell maturation and cancer surveillance in mice.

TBX3 reciprocally controls key trophoblast lineage decisions in villi during human placenta development in the first trimester.

Human trophoblastic lineage development is intertwined with placental development and pregnancy outcomes, but the regulatory mechanisms underpinning this process remain inadequately understood. In this study, based on single-nuclei RNA sequencing (snRNA-seq) analysis of the human early maternal-fetal interface, we compared the gene expression pattern of trophoblast at different developmental stages. Our findings reveal a predominant upregulation of TBX3 during the transition from villous cytotrophoblast (VCT) to syncytiotrophoblast (SCT), but downregulation of TBX3 as VCT progresses into extravillous trophoblast cells (EVT). Immunofluorescence analysis verified the primary expression of TBX3 in SCT, partial expression in MKi67-positive VCT, and absence in HLA-G-positive EVT, consistent with our snRNA-seq results. Using immortalized trophoblastic cell lines (BeWo and HTR8/SVneo) and human primary trophoblast stem cells (hTSCs), we observed that TBX3 knockdown impedes SCT formation through RAS-MAPK signaling, while TBX3 overexpression disrupts the cytoskeleton structure of EVT and hinders EVT differentiation by suppressing FAK signaling. In conclusion, our study suggests that the spatiotemporal expression of TBX3 plays a critical role in regulating trophoblastic lineage development via distinct signaling pathways. This underscores TBX3 as a key determinant during hemochorial placental development.

On the genetic basis of tail-loss evolution in humans and apes.

The loss of the tail is among the most notable anatomical changes to have occurred along the evolutionary lineage leading to humans and to the 'anthropomorphous apes'1-3, with a proposed role in contributing to human bipedalism4-6. Yet, the genetic mechanism that facilitated tail-loss evolution in hominoids remains unknown. Here we present evidence that an individual insertion of an Alu element in the genome of the hominoid ancestor may have contributed to tail-loss evolution. We demonstrate that this Alu element-inserted into an intron of the TBXT gene7-9-pairs with a neighbouring ancestral Alu element encoded in the reverse genomic orientation and leads to a hominoid-specific alternative splicing event. To study the effect of this splicing event, we generated multiple mouse models that express both full-length and exon-skipped isoforms of Tbxt, mimicking the expression pattern of its hominoid orthologue TBXT. Mice expressing both Tbxt isoforms exhibit a complete absence of the tail or a shortened tail depending on the relative abundance of Tbxt isoforms expressed at the embryonic tail bud. These results support the notion that the exon-skipped transcript is sufficient to induce a tail-loss phenotype. Moreover, mice expressing the exon-skipped Tbxt isoform develop neural tube defects, a condition that affects approximately 1 in 1,000 neonates in humans10. Thus, tail-loss evolution may have been associated with an adaptive cost of the potential for neural tube defects, which continue to affect human health today.

BAF45D-binding to HOX genes was differentially targeted in H9-derived spinal cord neural stem cells.

Chromatin accessibility has been used to define how cells adopt region-specific neural fates. BAF45D is one of the subunits of a specialised chromatin remodelling BAF complex. It has been reported that BAF45D is expressed in spinal cord neural stem cells (NSCs) and regulates their fate specification. Within the developing vertebrate spinal cord, HOX genes exhibit spatially restricted expression patterns. However, the chromatin accessibility of BAF45D binding HOX genes in spinal cord NSCs is unclear. In the present study, we found that in H9-derived spinal cord NSCs, BAF45D targets TBX6, a gene that regulates spinal cord neural mesodermal progenitors. Furthermore, BAF45D binding to the NES gene is much more enriched in H9-derived spinal cord NSCs chromatin compared to ESCs chromatin. In addition, BAF45D binding to anterior and trunk/central HOX genes, but not to lumbosacral HOX genes, was much more enriched in NSCs chromatin compared to ESCs chromatin. These results may shed new light on the role of BAF45D in regulating region-specific spinal cord NSCs by targeting HOX genes.

Genetic and functional variants of the TBX20 gene promoter in dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a major cause of heart failure and sudden cardiac death. As DCM is a genetically heterogeneous disease, genetic variants of cardiac transcription factor genes may play an important role. Transcription factor TBX20, an indispensable factor in normal heart development, is involved in the regulation of cardiac structure and function. Although the TBX20 gene is associated with the occurrence and development of DCM, the influence of genetic variants of the TBX20 gene promoter region on DCM has not been reported. METHODS: We conducted a case-control study consisting of 107 DCM patients and 210 healthy controls. Genetic variants within TBX20 gene promoter region were identified using sequencing techniques and were functionally analyzed by dual-luciferase reporting assay. Electrophoretic mobility shift assay (EMSA) was used to investigate DNA-protein interactions. RESULTS: In this study cohort (n = 317), we identified eight variants within TBX20 gene promoter. One novel DNA sequence variants (DSV) (g.4275G>T) and four single-nucleotide polymorphisms (SNPs) [g.4169G>A (rs1263874255), g.4949C>T (rs1191745927), g.5114G>A (rs112076877), g.5252C>T (rs1356932911)] were identified in DCM patients, but in none of controls. Among them, the DSV (g.4275G>T) and three SNPs [g.4949C>T (rs1191745927), g.5114G>A (rs112076877) and g.5252C>T (rs1356932911)] significantly altered the transcription activity of TBX20 gene promoter by dual-luciferase reporting assay (p < 0.05). Further, EMSA assay indicated that the DSV (g.4275G>T) and three SNPs [g.4949C>T (rs1191745927), g.5114G>A (rs112076877) and g.5252C>T (rs1356932911)] affected the binding of transcription factors. CONCLUSIONS: These data indicate that the DSV (g.4275G>T) and three SNPs [g.4949C>T (rs1191745927), g.5114G>A (rs112076877) and g.5252C>T (rs1356932911)] increase transcription activity of TBX20 gene promoter in both HEK-293 and neonatal rat cardiomyocytes (NRCMs) cell lines by affecting the binding of transcription factors. But the mechanism remains to be verified in vivo.

Exosomal miR-23a-3p derived from human umbilical cord mesenchymal stem cells promotes remyelination in central nervous system demyelinating diseases by targeting Tbr1/Wnt pathway.

Oligodendrocyte precursor cells are present in the adult central nervous system, and their impaired ability to differentiate into myelinating oligodendrocytes can lead to demyelination in patients with multiple sclerosis, accompanied by neurological deficits and cognitive impairment. Exosomes, small vesicles released by cells, are known to facilitate intercellular communication by carrying bioactive molecules. In this study, we utilized exosomes derived from human umbilical cord mesenchymal stem cells (HUMSCs-Exos). We performed sequencing and bioinformatics analysis of exosome-treated cells to demonstrate that HUMSCs-Exos can stimulate myelin gene expression in oigodendrocyte precursor cells. Functional investigations revealed that HUMSCs-Exos activate the Pi3k/Akt pathway and regulate the Tbr1/Wnt signaling molecules through the transfer of miR-23a-3p, promoting oligodendrocytes differentiation and enhancing the expression of myelin-related proteins. In an experimental autoimmune encephalomyelitis model, treatment with HUMSCs-Exos significantly improved neurological function and facilitated remyelination. This study provides cellular and molecular insights into the use of cell-free exosome therapy for central nervous system demyelination associated with multiple sclerosis, demonstrating its great potential for treating demyelinating and neurodegenerative diseases.

Long-Term Effect of TBX4 Germline Mutation on Pulmonary Clinico-Histopathologic Phenotype.

Tbx4 protein, expressed in mesenchyme of the developing lung, contributes to airway branching and distal lung growth. An association between pediatric onset of pulmonary arterial hypertension (PAH) and genetic variations coding for the T-box transcription factor 4 gene (TBX4) has been increasingly recognized. Tbx4-related PAH onset has a bimodal age distribution, including severe to lethal PAH in newborns and later onset PAH. We present an autopsy study of a 24-year-old male with a heterozygous TBX4 variant, who developed pulmonary arterial hypertension at age 12 years. This unique case highlights the complex pulmonary histopathology leading to lethal cardiopulmonary failure in the setting of TBX4 mutation.

NADPH Oxidase 2-Derived Reactive Oxygen Species Promote CD8+ T Cell Effector Function.

Oxidants participate in lymphocyte activation and function. We previously demonstrated that eliminating the activity of NADPH oxidase 2 (NOX2) significantly impaired the effectiveness of autoreactive CD8+ CTLs. However, the molecular mechanisms impacting CD8+ T cell function remain unknown. In the present study, we examined the role of NOX2 in both NOD mouse and human CD8+ T cell function. Genetic ablation or chemical inhibition of NOX2 in CD8+ T cells significantly suppressed activation-induced expression of the transcription factor T-bet, the master transcription factor of the Tc1 cell lineage, and T-bet target effector genes such as IFN-γ and granzyme B. Inhibition of NOX2 in both human and mouse CD8+ T cells prevented target cell lysis. We identified that superoxide generated by NOX2 must be converted into hydrogen peroxide to transduce the redox signal in CD8+ T cells. Furthermore, we show that NOX2-generated oxidants deactivate the tumor suppressor complex leading to activation of RheB and subsequently mTOR complex 1. These results indicate that NOX2 plays a nonredundant role in TCR-mediated CD8+ T cell effector function.

Evaluation of the presence of Th1 response through T-bet and IFN-gamma immunohistochemical expression in lower lip and oral tongue squamous cell carcinomas.

INTRODUCTION: Evaluate the immunohistochemical expression of T-bet and IFN-γ in lower lip (LLSCC) and oral tongue squamous cell carcinoma (OTSCC), verifying the presence of Th1 responses in lesions with different clinical conditions. METHODS AND MATERIALS: Thirty OTSCC and 30 LLSCC were analyzed by immunohistochemistry. T-bet was quantitatively assessed by parenchyma cell and stroma quantification, and IFN-γ was semi-quantitatively analyzed: 1:0-25%; 2:26-50%; 3:51-75%; 4:> 75% immunopositive cells. Histological differentiation degrees were categorized as well differentiated (WD), moderately differentiated (MD), or poorly differentiated (PD). RESULTS: OTSCC presented the highest number of T-bet+, parenchyma (p: 0.006), stroma (p: 0.156), parenchyma/stroma (p: 0.015), with no relationship to histological malignancy grade. IFN-γ higher concentrations in LLSCC were detected in parenchyma, stroma and in parenchyma/stroma (p: 0.000), as well as greater immunoreactivity in WD and MD (p: 0.001). In OTSCC, a positive and statistically significant correlation was observed between T-bet+ in parenchyma and IFN-γ in stroma(r: 0.388; p: 0.034), in addition to a statistically significant positive correlation between T-bet in parenchyma compared to stroma(r: 0.411; p: 0.024) and for IFN-γ in both parenchyma and stroma(r: 0.775; p: 0.000) in LLSCC. Higher T-bet+ was observed in OTSCCs, although higher IFN-γ was detected in LLSCCs. CONCLUSION: Thus, we suggest that, even though LLSCC presented lower T-bet+, the favorable microenvironment in these lesions led to an expressive activation of IFN-γ by T-bet+, considerably acting on Th1 differentiation and in antitumor activity, which, admittedly, present less aggressive behavior, reinforcing once again the important role of this cytokine and its use in strategy to fight cancer.

Dissecting the autism-associated 16p11.2 locus identifies multiple drivers in neuroanatomical phenotypes and unveils a male-specific role for the major vault protein.

BACKGROUND: Using mouse genetic studies and systematic assessments of brain neuroanatomical phenotypes, we set out to identify which of the 30 genes causes brain defects at the autism-associated 16p11.2 locus. RESULTS: We show that multiple genes mapping to this region interact to regulate brain anatomy, with female mice exhibiting far fewer brain neuroanatomical phenotypes. In male mice, among the 13 genes associated with neuroanatomical defects (Mvp, Ppp4c, Zg16, Taok2, Slx1b, Maz, Fam57b, Bola2, Tbx6, Qprt, Spn, Hirip3, and Doc2a), Mvp is the top driver implicated in phenotypes pertaining to brain, cortex, hippocampus, ventricles, and corpus callosum sizes. The major vault protein (MVP), the main component of the vault organelle, is a conserved protein found in eukaryotic cells, yet its function is not understood. Here, we find MVP expression highly specific to the limbic system and show that Mvp regulates neuronal morphology, postnatally and specifically in males. We also recapitulate a previously reported genetic interaction and show that Mvp+/-;Mapk3+/- mice exhibit behavioral deficits, notably decreased anxiety-like traits detected in the elevated plus maze and open field paradigms. CONCLUSIONS: Our study highlights multiple gene drivers in neuroanatomical phenotypes, interacting with each other through complex relationships. It also provides the first evidence for the involvement of the major vault protein in the regulation of brain size and neuroanatomy, specifically in male mice.

OCT4 regulates WNT/ß-catenin signaling and prevents mesoendoderm differentiation by repressing EOMES in porcine pluripotent stem cells.

The regulatory network between signaling pathways and transcription factors (TFs) is crucial for the maintenance of pluripotent stem cells. However, little is known about how the key TF OCT4 coordinates signaling pathways to regulate self-renewal and lineage differentiation of porcine pluripotent stem cells (pPSCs). Here, we explored the function of OCT4 in pPSCs by transcriptome and chromatin accessibility analysis. The TFs motif enrichment analysis revealed that, following OCT4 knockdown, the regions of increased chromatin accessibility were enriched with EOMES, GATA6, and FOXA1, indicating that pPSCs differentiated toward the mesoendoderm (ME) lineage. Besides, pPSCs rapidly differentiated into ME when the WNT/ß-catenin inhibitor XAV939 was removed. However, the ME differentiation of pPSCs caused by OCT4 knockdown did not rely on the activation of WNT/ß-catenin signaling because the target gene of WNT/ß-catenin signaling, AXIN2 was not upregulated after OCT4 knockdown, despite significant upregulation of WLS and some WNT ligands. Importantly, OCT4 is directly bound to the promoter and enhancers of EOMES and repressed its transcription. Overexpression of EOMES was sufficient to induce ME differentiation in the presence of XAV939. These results demonstrate that OCT4 can regulate WNT/ß-catenin signaling and prevent ME differentiation of pPSCs by repressing EOMES transcription.

Conserved enhancers control notochord expression of vertebrate Brachyury.

The cell type-specific expression of key transcription factors is central to development and disease. Brachyury/T/TBXT is a major transcription factor for gastrulation, tailbud patterning, and notochord formation; however, how its expression is controlled in the mammalian notochord has remained elusive. Here, we identify the complement of notochord-specific enhancers in the mammalian Brachyury/T/TBXT gene. Using transgenic assays in zebrafish, axolotl, and mouse, we discover three conserved Brachyury-controlling notochord enhancers, T3, C, and I, in human, mouse, and marsupial genomes. Acting as Brachyury-responsive, auto-regulatory shadow enhancers, in cis deletion of all three enhancers in mouse abolishes Brachyury/T/Tbxt expression selectively in the notochord, causing specific trunk and neural tube defects without gastrulation or tailbud defects. The three Brachyury-driving notochord enhancers are conserved beyond mammals in the brachyury/tbxtb loci of fishes, dating their origin to the last common ancestor of jawed vertebrates. Our data define the vertebrate enhancers for Brachyury/T/TBXTB notochord expression through an auto-regulatory mechanism that conveys robustness and adaptability as ancient basis for axis development.

A degron-based approach to manipulate Eomes functions in the context of the developing mouse embryo.

The T-box transcription factor Eomesodermin (Eomes), also known as Tbr2, plays essential roles in the early mouse embryo. Loss-of-function mutant embryos arrest at implantation due to Eomes requirements in the trophectoderm cell lineage. Slightly later, expression in the visceral endoderm promotes anterior visceral endoderm formation and anterior-posterior axis specification. Early induction in the epiblast beginning at day 6 is necessary for nascent mesoderm to undergo epithelial to mesenchymal transition (EMT). Eomes acts in a temporally and spatially restricted manner to sequentially specify the yolk sac haemogenic endothelium, cardiac mesoderm, definitive endoderm, and axial mesoderm progenitors during gastrulation. Little is known about the underlying molecular mechanisms governing Eomes actions during the formation of these distinct progenitor cell populations. Here, we introduced a degron-tag and mCherry reporter sequence into the Eomes locus. Our experiments analyzing homozygously tagged embryonic stem cells and embryos demonstrate that the degron-tagged Eomes protein is fully functional. dTAG (degradation fusion tag) treatment in vitro results in rapid protein degradation and recapitulates the Eomes-null phenotype. However in utero administration of dTAG resulted in variable and lineage-specific degradation, likely reflecting diverse cell type-specific Eomes expression dynamics. Finally, we demonstrate that Eomes protein rapidly recovers following dTAG wash-out in vitro. The ability to temporally manipulate Eomes protein expression in combination with cell marking by the mCherry-reporter offers a powerful tool for dissecting Eomes-dependent functional roles in these diverse cell types in the early embryo.

Genome-wide identification of notochord enhancers comprising the regulatory landscape of the brachyury locus in mouse.

The node and notochord are important signaling centers organizing the dorso-ventral patterning of cells arising from neuro-mesodermal progenitors forming the embryonic body anlage. Owing to the scarcity of notochord progenitors and notochord cells, a comprehensive identification of regulatory elements driving notochord-specific gene expression has been lacking. Here, we have used ATAC-seq analysis of FACS-purified notochord cells from Theiler stage 12-13 mouse embryos to identify 8921 putative notochord enhancers. In addition, we established a new model for generating notochord-like cells in culture, and found 3728 of these enhancers occupied by the essential notochord control factors brachyury (T) and/or Foxa2. We describe the regulatory landscape of the T locus, comprising ten putative enhancers occupied by these factors, and confirmed the regulatory activity of three of these elements. Moreover, we characterized seven new elements by knockout analysis in embryos and identified one new notochord enhancer, termed TNE2. TNE2 cooperates with TNE in the trunk notochord, and is essential for notochord differentiation in the tail. Our data reveal an essential role of Foxa2 in directing T-expressing cells towards the notochord lineage.

The Tbx20-TLE interaction is essential for the maintenance of the second heart field.

T-box transcription factor 20 (Tbx20) plays a multifaceted role in cardiac morphogenesis and controls a broad gene regulatory network. However, the mechanism by which Tbx20 activates and represses target genes in a tissue-specific and temporal manner remains unclear. Studies show that Tbx20 directly interacts with the Transducin-like Enhancer of Split (TLE) family of proteins to mediate transcriptional repression. However, a function for the Tbx20-TLE transcriptional repression complex during heart development has yet to be established. We created a mouse model with a two amino acid substitution in the Tbx20 EH1 domain, thereby disrupting the Tbx20-TLE interaction. Disruption of this interaction impaired crucial morphogenic events, including cardiac looping and chamber formation. Transcriptional profiling of Tbx20EH1Mut hearts and analysis of putative direct targets revealed misexpression of the retinoic acid pathway and cardiac progenitor genes. Further, we show that altered cardiac progenitor development and function contribute to the severe cardiac defects in our model. Our studies indicate that TLE-mediated repression is a primary mechanism by which Tbx20 controls gene expression.

Eomes restricts Brachyury functions at the onset of mouse gastrulation.

Mammalian specification of mesoderm and definitive endoderm (DE) is instructed by the two related Tbx transcription factors (TFs) Eomesodermin (Eomes) and Brachyury sharing partially redundant functions. Gross differences in mutant embryonic phenotypes suggest specific functions of each TF. To date, the molecular details of separated lineage-specific gene regulation by Eomes and Brachyury remain poorly understood. Here, we combine mouse embryonic and stem-cell-based analyses to delineate the non-overlapping, lineage-specific transcriptional activities. On a genome-wide scale, binding of both TFs overlaps at promoters of target genes but shows specificity for distal enhancer regions that is conferred by differences in Tbx DNA-binding motifs. The unique binding to enhancer sites instructs the specification of anterior mesoderm (AM) and DE by Eomes and caudal mesoderm by Brachyury. Remarkably, EOMES antagonizes BRACHYURY gene regulatory functions in coexpressing cells during early gastrulation to ensure the proper sequence of early AM and DE lineage specification followed by posterior mesoderm derivatives.