Transcriptional and epigenetic characterization of a new in vitro platform to model the formation of human pharyngeal endoderm.

BACKGROUND: The Pharyngeal Endoderm (PE) is an extremely relevant developmental tissue, serving as the progenitor for the esophagus, parathyroids, thyroids, lungs, and thymus. While several studies have highlighted the importance of PE cells, a detailed transcriptional and epigenetic characterization of this important developmental stage is still missing, especially in humans, due to technical and ethical constraints pertaining to its early formation. RESULTS: Here we fill this knowledge gap by developing an in vitro protocol for the derivation of PE-like cells from human Embryonic Stem Cells (hESCs) and by providing an integrated multi-omics characterization. Our PE-like cells robustly express PE markers and are transcriptionally homogenous and similar to in vivo mouse PE cells. In addition, we define their epigenetic landscape and dynamic changes in response to Retinoic Acid by combining ATAC-Seq and ChIP-Seq of histone modifications. The integration of multiple high-throughput datasets leads to the identification of new putative regulatory regions and to the inference of a Retinoic Acid-centered transcription factor network orchestrating the development of PE-like cells. CONCLUSIONS: By combining hESCs differentiation with computational genomics, our work reveals the epigenetic dynamics that occur during human PE differentiation, providing a solid resource and foundation for research focused on the development of PE derivatives and the modeling of their developmental defects in genetic syndromes.

Significant improvement of cardiac outflow tract septation defects in a DiGeorge syndrome model after minoxidil treatment.

The T-BOX transcription factor TBX1 is essential for the development of the pharyngeal apparatus and it is haploinsufficient in DiGeorge syndrome (DGS), a developmental anomaly associated with congenital heart disease and other abnormalities. The murine model recapitulates the heart phenotype and showed collagen accumulation. We first used a cellular model to study gene expression during cardiogenic differentiation of WT and Tbx1-/- mouse embryonic stem cells. Then we used a mouse model of DGS to test whether interfering with collagen accumulation using an inhibitor of lysyl hydroxylase would modify the cardiac phenotype of the mutant. We found that loss of Tbx1 in a precardiac differentiation model was associated with up regulation of a subset of ECM-related genes, including several collagen genes. In the in vivo model, early prenatal treatment with Minoxidil, a lysyl hydroxylase inhibitor, ameliorated the cardiac outflow tract septation phenotype in Tbx1 mutant fetuses, but it had no effect on septation in WT fetuses. We conclude that TBX1 suppresses a defined subset of ECM-related genes. This function is critical for OFT septation because the inhibition of collagen cross-linking in the mutant reduces significantly the penetrance of septation defects.

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.

Lymphatic endothelial transcription factor Tbx1 promotes an immunosuppressive microenvironment to facilitate post-myocardial infarction repair.

The heart is an autoimmune-prone organ. It is crucial for the heart to keep injury-induced autoimmunity in check to avoid autoimmune-mediated inflammatory disease. However, little is known about how injury-induced autoimmunity is constrained in hearts. Here, we reveal an unknown intramyocardial immunosuppressive program driven by Tbx1, a DiGeorge syndrome disease gene that encodes a T-box transcription factor (TF). We found induced profound lymphangiogenic and immunomodulatory gene expression changes in lymphatic endothelial cells (LECs) after myocardial infarction (MI). The activated LECs penetrated the infarcted area and functioned as intramyocardial immune hubs to increase the numbers of tolerogenic dendritic cells (tDCs) and regulatory T (Treg) cells through the chemokine Ccl21 and integrin Icam1, thereby inhibiting the expansion of autoreactive CD8+ T cells and promoting reparative macrophage expansion to facilitate post-MI repair. Mimicking its timing and implementation may be an additional approach to treating autoimmunity-mediated cardiac diseases.

Mesenchymal cell replacement corrects thymic hypoplasia in murine models of 22q11.2 deletion syndrome.

22q11.2 deletion syndrome (22q11.2DS) is the most common human chromosomal microdeletion, causing developmentally linked congenital malformations, thymic hypoplasia, hypoparathyroidism, and/or cardiac defects. Thymic hypoplasia leads to T cell lymphopenia, which most often results in mild SCID. Despite decades of research, the molecular underpinnings leading to thymic hypoplasia in 22q11.2DS remain unknown. Comparison of embryonic thymuses from mouse models of 22q11.2DS (Tbx1neo2/neo2) revealed proportions of mesenchymal, epithelial, and hematopoietic cell types similar to those of control thymuses. Yet, the small thymuses were growth restricted in fetal organ cultures. Replacement of Tbx1neo2/neo2 thymic mesenchymal cells with normal ones restored tissue growth. Comparative single-cell RNA-Seq of embryonic thymuses uncovered 17 distinct cell subsets, with transcriptome differences predominant in the 5 mesenchymal subsets from the Tbx1neo2/neo2 cell line. The transcripts affected included those for extracellular matrix proteins, consistent with the increased collagen deposition we observed in the small thymuses. Attenuating collagen cross-links with minoxidil restored thymic tissue expansion for hypoplastic lobes. In colony-forming assays, the Tbx1neo2/neo2-derived mesenchymal cells had reduced expansion potential, in contrast to the normal growth of thymic epithelial cells. These findings suggest that mesenchymal cells were causal to the small embryonic thymuses in the 22q11.2DS mouse models, which was correctable by substitution with normal mesenchyme.

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.

Single cell multi-omic analysis identifies a Tbx1-dependent multilineage primed population in murine cardiopharyngeal mesoderm.

The poles of the heart and branchiomeric muscles of the face and neck are formed from the cardiopharyngeal mesoderm within the pharyngeal apparatus. They are disrupted in patients with 22q11.2 deletion syndrome, due to haploinsufficiency of TBX1, encoding a T-box transcription factor. Here, using single cell RNA-sequencing, we now identify a multilineage primed population within the cardiopharyngeal mesoderm, marked by Tbx1, which has bipotent properties to form cardiac and branchiomeric muscle cells. The multilineage primed cells are localized within the nascent mesoderm of the caudal lateral pharyngeal apparatus and provide a continuous source of cardiopharyngeal mesoderm progenitors. Tbx1 regulates the maturation of multilineage primed progenitor cells to cardiopharyngeal mesoderm derivatives while restricting ectopic non-mesodermal gene expression. We further show that TBX1 confers this balance of gene expression by direct and indirect regulation of enriched genes in multilineage primed progenitors and downstream pathways, partly through altering chromatin accessibility, the perturbation of which can lead to congenital defects in individuals with 22q11.2 deletion syndrome.

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.

Pharyngeal epithelial deletion of Tbx1 causes caudal pharyngeal arch defect but not cardiac conotruncal anomaly.

TBX1 is a major disease gene of 22q11.2 deletion syndrome (22q11.2DS). It is expressed in all three germ layers of pharyngeal apparatus to control the complicated morphogenesis. The haploinsufficiency of pharyngeal endodermal or ectodermal, but not mesodermal Tbx1 causes aortic arch patterning defect. However, the mesodermal deletion of Tbx1 causes much severer pharyngeal and cardiovascular defect than either pharyngeal endodermal or ectodermal Tbx1 deletion does. It is inconsistent with the conventional thought that the invagination of pharyngeal epithelia drives pharyngeal segmentation. Therefore, we asked whether pharyngeal ectodermal and ectodermal Tbx1 can compensate the loss of each other. Here we carefully characterized pharyngeal epithelia-specific Fgf15Cre and Fgf15HspCre lines and used them to perform pharyngeal epithelia-specific deletion. Our data showed that the percentage of E18.5 Fgf15Cre;Tbx1flox/+ embryos with aortic arch patterning defects was similar to that of E10.5 Fgf15Cre;Tbx1flox/+ embryos with the 4th pharyngeal arch artery (PAA) defect, indicating that there is no significant recovery from the initial PAA defect, in contrast to germ line haploinsufficiency. Fgf15Cre;Tbx1flox/flox embryos had hypoplastic caudal pharyngeal arch and defective derivatives, but cardiac OFT development was not affected. The phenotypic spectrum of simultaneous Tbx1 deletion in both pharyngeal ectoderm and endoderm is strikingly similar to what presents with single pharyngeal endoderm or ectoderm-specific deletion of Tbx1. The absence of synergistic effect indicates intimate topographic interactions among pharyngeal endoderm and ectoderm, through which deletion of a gene in one tissue may disrupt the development of adjacent tissues and thereby lead to similar morphological phenotypes in either tissue-specific deletion.

A dual role for Tbx1 in cardiac lymphangiogenesis through genetic interaction with Vegfr3.

The transcription factor TBX1 is the major gene implicated in 22q11.2 deletion syndrome (22q11.2DS). The complex clinical phenotype includes vascular anomalies and a recent report presented new cases of primary lymphedema in 22q11.2DS patients. We have previously shown that TBX1 is required for systemic lymphatic vessel development in prenatal mice and it is critical for their survival postnatally. Using loss-of-function genetics and transgenesis in the mouse, we show here a strong genetic interaction between Tbx1 and Vegfr3 in cardiac lymphangiogenesis. Intriguingly, we found that different aspects of the cardiac lymphatic phenotype in Tbx1-Vegfr3 compound heterozygotes were regulated independently by the two genes, with Tbx1 primarily regulating vessel numbers and Vegfr3 vessel morphology. Consistent with this observation, Tbx1Cre -activated expression of a Vegfr3 transgene rescued partially the cardiac lymphatic abnormalities in compound heterozygotes. Through time-controlled genetic experiments, we show that Tbx1 is activated and required in cardiac lymphatic endothelial cell (LEC) progenitors between E10.5 and E11.5. Furthermore, we found that it is also required later in development for the growth of the cardiac lymphatics. Finally, our study revealed a differential sensitivity between ventral and dorsal cardiac lymphatics to the effects of altered Tbx1 and Vegfr3 gene dosage, and we show that this likely results from an earlier requirement for Tbx1 in ventral cardiac LEC progenitors.

Cardiopharyngeal mesoderm origins of musculoskeletal and connective tissues in the mammalian pharynx.

Cardiopharyngeal mesoderm (CPM) gives rise to muscles of the head and heart. Using genetic lineage analysis in mice, we show that CPM develops into a broad range of pharyngeal structures and cell types encompassing musculoskeletal and connective tissues. We demonstrate that CPM contributes to medial pharyngeal skeletal and connective tissues associated with both branchiomeric and somite-derived neck muscles. CPM and neural crest cells (NCC) make complementary mediolateral contributions to pharyngeal structures, in a distribution established in the early embryo. We further show that biallelic expression of the CPM regulatory gene Tbx1, haploinsufficient in 22q11.2 deletion syndrome patients, is required for the correct patterning of muscles with CPM-derived connective tissue. Our results suggest that CPM plays a patterning role during muscle development, similar to that of NCC during craniofacial myogenesis. The broad lineage contributions of CPM to pharyngeal structures provide new insights into congenital disorders and evolution of the mammalian pharynx.

TBX1 and Basal Cell Carcinoma: Expression and Interactions with Gli2 and Dvl2 Signaling.

Early events of basal cell carcinoma (BCC) tumorigenesis are triggered by inappropriate activation of SHH signaling, via the loss of Patched1 (Ptch1) or by activating mutations of Smoothened (Smo). TBX1 is a key regulator of pharyngeal development, mainly through expression in multipotent progenitor cells of the cardiopharyngeal lineage. This transcription factor is connected to several major signaling systems, such as FGF, WNT, and SHH, and it has been linked to cell proliferation and to the regulation of cell shape and cell dynamics. Here, we show that TBX1 was expressed in all of the 51 BCC samples that we have tested, while in healthy human skin it was only expressed in the hair follicle. Signal intensity and distribution was heterogeneous among tumor samples. Experiments performed on a cellular model of mouse BCC showed that Tbx1 is downstream to GLI2, a factor in the SHH signaling, and that, in turn, it regulates the expression of Dvl2, which encodes an adaptor protein that is necessary for the transduction of WNT signaling. Consistently, Tbx1 depletion in the cellular model significantly reduced cell migration. These results suggest that TBX1 is part of a core transcription network that promotes BCC tumorigenesis.

Tbx1 regulates extracellular matrix-cell interactions in the second heart field.

Tbx1, the major candidate gene for DiGeorge or 22q11.2 deletion syndrome, is required for efficient incorporation of cardiac progenitors of the second heart field (SHF) into the heart. However, the mechanisms by which TBX1 regulates this process are still unclear. Here, we have used two independent models, mouse embryos and cultured cells, to define the role of TBX1 in establishing morphological and dynamic characteristics of SHF in the mouse. We found that loss of TBX1 impairs extracellular matrix (ECM)-integrin-focal adhesion (FA) signaling in both models. Mosaic analysis in embryos suggested that this function is non-cell autonomous, and, in cultured cells, loss of TBX1 impairs cell migration and FAs. Additionally, we found that ECM-mediated integrin signaling is disrupted upon loss of TBX1. Finally, we show that interfering with the ECM-integrin-FA axis between E8.5 and E9.5 in mouse embryos, corresponding to the time window within which TBX1 is required in the SHF, causes outflow tract dysmorphogenesis. Our results demonstrate that TBX1 is required to maintain the integrity of ECM-cell interactions in the SHF and that this interaction is critical for cardiac outflow tract development. More broadly, our data identifies a novel TBX1 downstream pathway as an important player in SHF tissue architecture and cardiac morphogenesis.

Left pulmonary artery in 22q11.2 deletion syndrome. Echocardiographic evaluation in patients without cardiac defects and role of Tbx1 in mice.

INTRODUCTION AND HYPOTHESIS: Patients with 22q11 deletion syndrome (22q11.2DS) present, in about 75% of cases, typical patterns of cardiac defects, with a particular involvement on the ventricular outflow tract and great arteries. However, in this genetic condition the dimensions of the pulmonary arteries (PAs) never were specifically evaluated. We measured both PAs diameter in patients with 22q11.2DS without cardiac defects, comparing these data to a normal control group. Moreover, we measured the PAs diameter in Tbx1 mutant mice. Finally, a cell fate mapping in Tbx1 mutants was used to study the expression of this gene in the morphogenesis of PAs. METHODS: We evaluated 58 patients with 22q11.2DS without cardiac defects. The control group consisted of 54 healthy subjects, matched for age and sex. All cases underwent a complete transthoracic echocardiography. Moreover, we crossed Tbx1+/- mice and harvested fetuses. We examined the cardiovascular phenotype of 8 wild type (WT), 37 heterozygous (Tbx1+/-) and 6 null fetuses (Tbx1-/-). Finally, we crossed Tbx1Cre/+mice with R26RmT-mG Cre reporter mice to study Tbx1 expression in the pulmonary arteries. RESULTS: The echocardiographic study showed that the mean of the LPA/RPA ratio in 22q11.2DS was smaller (0.80 ± 0.12) than in controls (0.97 ± 0.08; p < 0.0001). Mouse studies resulted in similar data as the size of LPA and RPA was not significantly different in WT embryos, but in Tbx1+/- and Tbx1-/- embryos the LPA was significantly smaller than the RPA in both mutants (P = 0.0016 and 0.0043, respectively). We found that Tbx1 is expressed near the origin of the PAs and in their adventitia. CONCLUSIONS: Children with 22q11.2DS without cardiac defects show smaller LPA compared with healthy subjects. Mouse studies suggest that this anomaly is due to haploinsufficiency of Tbx1. These data may be useful in the clinical management of children with 22q11.2DS and should guide further experimental studies as to the mechanisms underlying PAs development.

Gene-environment interaction impacts on heart development and embryo survival.

Congenital heart disease (CHD) is the most common type of birth defect. In recent years, research has focussed on identifying the genetic causes of CHD. However, only a minority of CHD cases can be attributed to single gene mutations. In addition, studies have identified different environmental stressors that promote CHD, but the additive effect of genetic susceptibility and environmental factors is poorly understood. In this context, we have investigated the effects of short-term gestational hypoxia on mouse embryos genetically predisposed to heart defects. Exposure of mouse embryos heterozygous for Tbx1 or Fgfr1/Fgfr2 to hypoxia in utero increased the incidence and severity of heart defects while Nkx2-5+/- embryos died within 2 days of hypoxic exposure. We identified the molecular consequences of the interaction between Nkx2-5 and short-term gestational hypoxia, which suggest that reduced Nkx2-5 expression and a prolonged hypoxia-inducible factor 1α response together precipitate embryo death. Our study provides insight into the causes of embryo loss and variable penetrance of monogenic CHD, and raises the possibility that cases of foetal death and CHD in humans could be caused by similar gene-environment interactions.

Tbx1 represses Mef2c gene expression and is correlated with histone 3 deacetylation of the anterior heart field enhancer.

The TBX1 gene is haploinsufficient in 22q11.2 deletion syndrome (22q11.2DS), and genetic evidence from human patients and mouse models points to a major role of this gene in the pathogenesis of this syndrome. Tbx1 can activate and repress transcription, and previous work has shown that one of its functions is to negatively modulate cardiomyocyte differentiation. Tbx1 occupies the anterior heart field (AHF) enhancer of the Mef2c gene, which encodes a key cardiac differentiation transcription factor. Here, we show that increased dosage of Tbx1 correlates with downregulation of Mef2c expression and reduced acetylation of its AHF enhancer in cultured mouse myoblasts. Consistently, 22q11.2DS-derived and in vitro-differentiated human induced pluripotent stem cells (hiPSCs) expressed higher levels of MEF2C and showed increased AHF acetylation, compared with hiPSCs from a healthy donor. Most importantly, we show that in mouse embryos, loss of Tbx1 enhances the expression of the Mef2c-AHF-Cre transgene in a specific region of the splanchnic mesoderm, and in a dosage-dependent manner, providing an in vivo correlate of our cell culture data. These results indicate that Tbx1 regulates the Mef2c AHF enhancer by inducing histone deacetylation.