During cellular reprogramming, the pioneer transcription factor GATA3 binds chromatin, and in a context-dependent manner directs local chromatin remodeling and enhancer formation. Here, we use high-resolution nucleosome mapping in human cells to explore the impact of the position of GATA motifs on the surface of nucleosomes on productive enhancer formation, finding productivity correlates with binding sites located near the nucleosomal dyad axis. Biochemical experiments with model nucleosomes demonstrate sufficiently stable transcription factor-nucleosome interaction to empower cryo-electron microscopy structure determination of the complex at 3.15 Å resolution. The GATA3 zinc fingers efficiently bind their target 5'-GAT-3' sequences in the nucleosome when they are located in solvent accessible, consecutive major grooves without significant changes in nucleosome structure. Analysis of genomic loci bound by GATA3 during reprogramming suggests a correlation of recognition motif sequence and spacing that may distinguish productivity of new enhancer formation.

Abnormalities in pyloric development or in contractile function of the pylorus cause reflux of duodenal contents into the stomach and increase the risk of gastric metaplasia and cancer. Abnormalities of the pyloric region are also linked to congenital defects such as the relatively common neonatal hypertrophic pyloric stenosis, and primary duodenogastric reflux. Therefore, understanding pyloric development is of great clinical relevance. Here, we investigated the role of the LIM homeodomain transcription factor Isl1 in pyloric development.

The transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-α (ERα)-positive breast tumors in which it participates with ERα and FOXA1 in a complex transcriptional regulatory program driving tumor growth. GATA3 mutations are frequent in breast cancer and have been classified as driver mutations. To elucidate the contribution(s) of GATA3 alterations to cancer, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus.

Lymphoid tissue inducer (LTi) cells are regarded as a subset of innate lymphoid cells (ILCs). However, these cells are not derived from the ILC common progenitor, which generates other ILC subsets and is defined by the expression of the transcription factor PLZF. Here, we examined transcription factor(s) determining the fate of LTi progenitors versus non-LTi ILC progenitors. Conditional deletion of Gata3 resulted in the loss of PLZF+ non-LTi progenitors but not the LTi progenitors that expressed the transcription factor RORγt. Consistently, PLZF+ non-LTi progenitors expressed high amounts of GATA3, whereas GATA3 expression was low in RORγt+ LTi progenitors. The generation of both progenitors required the transcriptional regulator Id2, which defines the common helper-like innate lymphoid progenitor (ChILP), but not cytokine signaling. Nevertheless, low GATA3 expression was necessary for the generation of functionally mature LTi cells. Thus, differential expression of GATA3 determines the fates and functions of distinct ILC progenitors.

During the development of the inner ear, auditory and vestibular ganglion neurons are generated in a highly regulated sequential process. First, neuroblasts are specified, delaminate from the epithelium of the otocyst, and migrate to form the auditory-vestibular ganglion (AVG). These neuroblasts then undergo proliferation and differentiate into afferent neurons of the auditory and vestibular ganglia. The zinc finger transcription factor Gata3 has been shown to play a role in cell proliferation and differentiation in various regions of the inner ear. Here we profile the spatiotemporal expression pattern of Gata3 in the developing auditory and vestibular ganglia of the chick embryo. Gata3 is expressed in a distinct population of sensorineural precursor cells within the otic epithelium, but is not expressed in migrating or proliferating neuroblasts. Following terminal mitosis, Gata3 expression is restricted to very few cells in the auditory ganglion and is not expressed in any cells of the vestibular ganglion. Gata3 expression levels then increase in auditory neurons as they mature. The increase of Gata3 in auditory ganglion neurons is accompanied by decreased expression of NeuroD. Our results suggest that Gata3 may be specifically involved in the differentiation of auditory ganglion neurons.

Innate lymphoid cells (ILCs) are critical in innate immune responses to pathogens and lymphoid organ development. Similar to CD4(+) T helper (Th) cell subsets, ILC subsets positive for interleukin-7 receptor α (IL-7Rα) produce distinct sets of effector cytokines. However, the molecular control of IL-7Rα(+) ILC development and maintenance is unclear. Here, we report that GATA3 was indispensable for the development of all IL-7Rα(+) ILC subsets and T cells but was not required for the development of classical natural killer cells. Conditionally Gata3-deficient mice had no lymph nodes and were susceptible to Citrobactor rodentium infection. After the ILCs had fully developed, GATA3 remained important for the maintenance and functions of ILC2s. Genome-wide gene expression analyses indicated that GATA3 regulated a similar set of cytokines and receptors in Th2 cells and ILC2s, but not in ILC3s. Thus, GATA3 plays parallel roles in regulating the development and functions of CD4(+) T cells and IL-7Rα(+) ILCs.

We have studied the function of the zinc finger transcription factor gata3 in auditory system development by analysing temporal profiles of gene expression during differentiation of conditionally immortal cell lines derived to model specific auditory cell types and developmental stages. We tested and applied a novel probabilistic method called the gamma Model for Oligonucleotide Signals to analyse hybridization signals from Affymetrix oligonucleotide arrays. Expression levels estimated by this method correlated closely (p<0.0001) across a 10-fold range with those measured by quantitative RT-PCR for a sample of 61 different genes. In an unbiased list of 26 genes whose temporal profiles clustered most closely with that of gata3 in all cell lines, 10 were linked to Insulin-like Growth Factor signalling, including the serine/threonine kinase Akt/PKB. Knock-down of gata3 in vitro was associated with a decrease in expression of genes linked to IGF-signalling, including IGF1, IGF2 and several IGF-binding proteins. It also led to a small decrease in protein levels of the serine-threonine kinase Akt2/PKBbeta, a dramatic increase in Akt1/PKBalpha protein and relocation of Akt1/PKBalpha from the nucleus to the cytoplasm. The cyclin-dependent kinase inhibitor p27(kip1), a known target of PKB/Akt, simultaneously decreased. In heterozygous gata3 null mice the expression of gata3 correlated with high levels of activated Akt/PKB. This functional relationship could explain the diverse function of gata3 during development, the hearing loss associated with gata3 heterozygous null mice and the broader symptoms of human patients with Hearing-Deafness-Renal anomaly syndrome.

Lineage-determining transcription factors (LD-TFs) drive the differentiation of progenitor cells into a specific lineage. In CD4+ T cells, T-bet dictates differentiation of the TH1 lineage, whereas GATA3 drives differentiation of the alternative TH2 lineage. However, LD-TFs, including T-bet and GATA3, are frequently co-expressed but how this affects LD-TF function is not known. By expressing T-bet and GATA3 separately or together in mouse T cells, we show that T-bet sequesters GATA3 at its target sites, thereby removing GATA3 from TH2 genes. This redistribution of GATA3 is independent of GATA3 DNA binding activity and is instead mediated by the T-bet DNA binding domain, which interacts with the GATA3 DNA binding domain and changes GATA3's sequence binding preference. This mechanism allows T-bet to drive the TH1 gene expression program in the presence of GATA3. We propose that redistribution of one LD-TF by another may be a common mechanism that could explain how specific cell fate choices can be made even in the presence of other transcription factors driving alternative differentiation pathways.

The regulation of memory CD4(+) helper T (Th) cell function, such as polarized cytokine production, remains unclear. Here we show that memory T helper 2 (Th2) cells are divided into four subpopulations by CD62L and CXCR3 expression. All four subpopulations produced interleukin-4 (IL-4) and IL-13, whereas only the CD62L(lo)CXCR3(lo) population produced IL-5 accompanied by increased H3-K4 methylation at the Il5 gene locus. The transcription factor Eomesodermin (encoded by Eomes) was highly expressed in memory Th2 cells, whereas its expression was selectively downregulated in the IL-5-producing cells. Il5 expression was enhanced in Eomes-deficient cells, and Eomesodermin was shown to interact with the transcription factor GATA3, preventing GATA3 binding to the Il5 promoter. Memory Th2 cell-dependent airway inflammation was attenuated in the absence of the CD62L(lo)CXCR3(lo) population but was enhanced by Eomes-deficient memory Th2 cells. Thus, IL-5 production in memory Th2 cells is regulated by Eomesodermin via the inhibition of GATA3 activity.

Differentiation of naive CD4+ T cells into helper T (Th) cells is controlled by a combination of several transcriptional factors. In this study, we examined the functional role of the Runx1 transcription factor in Th cell differentiation. Naive T cells from transgenic mice expressing a dominant interfering form of Runx1 exhibited enhanced interleukin 4 production and efficient Th2 differentiation. In contrast, transduction of Runx1 into wild-type T cells caused a complete attenuation of Th2 differentiation and was accompanied by the cessation of GATA3 expression. Furthermore, endogenous expression of Runx1 in naive T cells declined after T cell receptor stimulation, at the same time that expression of GATA3 increased. We conclude that Runx1 plays a novel role as a negative regulator of GATA3 expression, thereby inhibiting the Th2 cell differentiation.

AP-2β is a new mammary epithelial differentiation marker and its expression is preferentially retained and enhanced in lobular carcinoma in situ and invasive lobular breast cancer. In normal breast epithelium AP-2β is expressed in a scattered subpopulation of luminal cells. So far, these cells have not been further characterized. Co-expression of AP-2β protein and luminal epithelium markers (GATA3, CK8/18), hormone receptors [estrogen receptor (ER), androgen receptor (AR)] and candidate stem cells markers (CK5/14, CD44) were assessed by double-immunofluorescence staining in normal mammary gland epithelium. The subpopulation of AP-2β-positive mammary epithelial cells showed an almost complete, superimposable co-expression with GATA3 and a peculiar intense, ring-like appearing immunoreactivity for CK8/18. Confocal immunofluorescence microscopy revealed an apicobasal staining for CK8/18 in AP-2β-positive cells, which was not seen in in AP-2β-negative cells. Furthermore, AP-2β-positive displayed a partial co-expression with ER and AR, but lacked expression of candidate stem cell markers CK5/14 and CD44. In summary, AP-2β is a new luminal mammary epithelial differentiation marker, which is expressed in the GATA3-positive subpopulation of luminal epithelial cells. These AP-2β-positive/GATA3-positive cells also show a peculiar CK8/18-expression which may indicate a previously unknown functionally specialized mammary epithelial cell population.

Type 2 diabetes mellitus is a multifactorial metabolic disorder caused by environmental factors and has a strong association with hereditary issues. These hereditary issues result in an imbalance in CD4+T cells and a decreased level of naïve CD4+T cells, which may be critical in the pathogenesis of type 2 diabetes. Transcription factors GATA3 and STAT4 mediate the cytokine-induced development of naïve T cells into Th1 or Th2 types. In the present study, genetic analyses of GATA3 SNP rs3824662 and STAT4 SNP rs10181656 were performed to investigate the association of allelic and genotypic variations with the risk of T2D in the Bangladeshi population. A total of 297 unrelated Bangladeshi patients with type 2 diabetes and 247 healthy individuals were included in the study. The allelic and genotypic frequencies of rs10181656 located in the STAT4 gene were not found to be associated with risk of type 2 diabetes. The GATA3 rs3824662 T allele and mutant TT genotype had a significant association with the risk of T2D [OR: 1.52 (1.15-2.02), X2 = 8.66, p = 0.003 and OR: 2.98 (1.36-6.55), X2 = 7.98, p = 0.04, respectively]. Thus, the present study postulates that the genetic variation of the transcription factor GATA3, not STAT4, is associated with the risk of type 2 diabetes in the Bangladeshi population.

Metastasis associates with late stages of lung cancer progression and remains the main cause of patient death due to the lack of clinically effective therapeutics. Here we report that the transcription factor GATA3 and its co-factor FOG2 commonly promote the expression of the lysyl hydroxylase (LH) family members, including LH2 and LH3, which in turn drive lung adenocarcinoma cell migration, invasion, and metastasis. We show evidence that both LH2 and LH3 are direct transcription targets for GATA3. Knockdown of either LH2 or LH3 suppresses migration and invasion; on the contrary, forced expression of LH2 or LH3 promotes growth and migration, suggesting that the two LHs exert redundant oncogenic functions. Importantly, re-expression of LH2 is sufficient to restore the metastatic capacity of GATA3-depleted cells, suggesting a role for LHs as the downstream mediators of GATA3. Collectively, our data reveal a pro-metastatic GATA3-LHs axis for lung cancer, supporting the notion that targeting LHs may be useful for treating lung cancer.

USP21 is a kind of deubiquitinating enzymes involved in the malignant progression of various cancers, while its role in gastric cancer (GC) and the specific molecular mechanism are still unclear. This study probed into the function of USP21 in vitro and in vivo, and discussed the regulatory mechanism of USP21 in GC in vitro. We reported that USP21 promoted GC cell proliferation, migration, invasion, and stemness in vitro, and regulated GC tumor growth and cell stemness in mice in vivo. USP21 stabilized the expression of GATA3 by binding to GATA3. Besides, GATA3 also regulated the expression of MAPK1 at the transcriptional level. A series of in vitro experiments testified that USP21 regulated the expression of MAPK1 by binding to transcription factor GATA3, thereby regulating the tumor growth and cell stemness of GC. Overall, this study identified a new USP21/GATA3/MAPK1 axis, which plays a pivotal role in promoting the malignant progression of GC and might provide a potential target for treatment.

The transcription factor PU.1 is predominantly expressed in dendritic cells (DCs) and is essential for DC differentiation. Although there are several reports that PU.1 positively regulates the expression of DC-specific genes, whether PU.1 also has a suppressive effect on DCs is largely unknown. Here we demonstrate that PU.1 suppresses the expression of Th2 cytokines including IL-13 and IL-5 in bone marrow-derived DCs (BMDCs), through repression of the expression of GATA3, which is a master regulator of Th2 differentiations. When PU.1 siRNA was introduced into BMDCs, LPS-induced expression of IL-13 and IL-5 was increased along with upregulation of the constitutive expression of GATA2 and GATA3. The additional introduction of GATA3 siRNA but not of GATA2 siRNA abrogated PU.1 siRNA-mediated upregulation of IL-13 and IL-5. A chromatin immunoprecipitation assay showed that PU.1 bound to Gata3 proximal promoter region, which is more dominant than the distal promoter in driving GATA3 transcription in DCs. The degree of histone acetylation at the Gata3 promoter was decreased in PU.1 siRNA-introduced DCs, suggesting the involvement of PU.1 in chromatin modification of the Gata3 promoter. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, increased the degree of histone H3 acetylation at the Gata3 promoter and induced the subsequent expression of GATA3. Experiments using HDAC inhibitors and siRNAs showed that HDAC3 suppressed GATA3 expression. The recruitment of HDAC3 to the Gata3 promoter was decreased by PU.1 knockdown. LPS-induced IL-13 expression was dramatically reduced in BMDCs generated from mice lacking the conserved GATA3 response element, termed CGRE, which is an essential site for the binding of GATA3 on the Il-13 promoter. The degree of H3K4me3 at CGRE was significantly increased in PU.1 siRNA-transfected stimulated DCs. Our results indicate that PU.1 plays pivotal roles in DC development and function, serving not only as a transcriptional activator but also as a repressor.

Picroside II isolated from Pseudolysimachion rotundum var. subintegrum has been used as traditional medicine to treat inflammatory diseases. In this study, we assessed whether picroside II has inhibitory effects on airway inflammation in a mouse model of house dust mite (HDM)-induced asthma. In the HDM-induced asthmatic model, picroside II significantly reduced inflammatory cell counts in the bronchoalveolar lavage fluid (BALF), the levels of total immunoglobulin (Ig) E and HDM-specific IgE and IgG1 in serum, airway inflammation, and mucus hypersecretion in the lung tissues. ELISA analysis showed that picroside II down-regulated the levels of Th2-related cytokines (including IL-4, IL-5, and IL-13) and asthma-related mediators, but it up-regulated Th1-related cytokine, IFNγ in BALF. Picroside II also inhibited the expression of Th2 type cytokine genes and the transcription factor GATA3 in the lung tissues of HDM-induced mice. Finally, we demonstrated that picroside II significantly decreased the expression of GATA3 and Th2 cytokines in developing Th2 cells, consistent with in vivo results. Taken together, these results indicate that picroside II has protective effects on allergic asthma by reducing GATA3 expression and Th2 cytokine bias.

Differentiation of preadipocytes into adipocytes and the formation of the subsequent adipose tissue are critical for mammalian growth and development. The molecular mechanism relating to preadipocyte differentiation and adipogenesis from the perspective of miRNAs is not yet completely understood. Here we investigated whether miR-183 functioned in the differentiation process. Both gain-of-function and loss-of-function assays demonstrated that miR-183 positively regulated 3T3-L1 differentiation by enhancing the expression of adipogenic marker genes such as CCAAT/enhancer binding protein α (C/EBPα), peroxisome proliferator-activated receptor γ (PPARγ), adiponectin and fatty acid synthase (FAS), as well as the triglyceride content and accumulation of lipid droplets. Meanwhile, low-density lipoprotein receptor-related protein 6 (LRP6) was known to impair the canonical Wnt/β-catenin signaling pathway and thereafter reduce c-myc and nuclear β-catenin protein. We showed that the inhibition of LRP6 by siRNA promoted 3T3-L1 adipogenic differentiation and adipogenesis. Further analysis showed that mouse miR-183 gene had its own transcription unit containing CpG islands, transcription start site (TSS), coding sequence (CDS) and polyA signal within the flanking sequences 2500 nt upstream and downstream of mouse miR-183 in genome. The core promoter of miR-183 gene was identified and transcription factor GATA3 (GATA binding protein 3) significantly inhibited the expression of mature miR-183 by binding to its core promoter in vivo, as indicated by the chromatin immunoprecipitation (ChIP) assay. These results suggest that miR-183, though negatively regulated by transcription factor GATA3, enhances 3T3-L1 preadipocyte differentiation and adipogenesis through the inhibition of the canonical Wnt/β-catenin signaling pathway by targeting LRP6.

Estrogen receptor α (ERα) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcriptional events that promote tumor growth. ERα binding activity largely depends on access to binding sites on chromatin, which is facilitated in part by Pioneer Factors (PFs). Transcription factors operate in complexes through thousands of genomic binding sites in a combinatorial fashion to control the expression of genes. However, the extent of crosstalk and cooperation between ERα pioneer factors and more collaborative transcription factors in breast cancer still remains to be elucidated systematically. Methods: Here, we determined the genomic binding information of 40 transcription-related factors and histone modifications with ChIP-seq in ENCODE and integrated it with other genomic information (RNA-seq, ATAC-seq, Gene microarray, 450k methylation chip, GRO-seq), forming a multi-dimension network to illuminate ERα associated transcription. Results: We show that transcription factor, NR2F2 binds to most sites independently of estrogen. Perturbation of NR2F2 expression decreases ERα DNA binding, chromatin openning, and estrogen-dependent cell growth. In the genome-wide analysis, we show that most binding events of NR2F2 and known pioneer factors FOXA1, GATA3 occur together, covering 85% of the ERα binding sites. Regions bound by all the three TFs appeared to be the most active, to have the strongest ERα binding and to be enriched for the super enhancers. Conclusions: The ERα binds to pre-accessible sites containing ERE elements bound by the three transcription factors (NR2F2, FOXA1 and GATA3).The three genes were also identified to correlate with decreased metastatic potential in patient cohorts and co-regulate each other. Together, our results suggest that NR2F2 is a cofactor with FOXA1 and GATA3 in ERα-mediated transcription.

T helper-2 (Th2) cells and type 2 innate lymphoid cells (ILC2s) play crucial roles during type 2 immune responses; the transcription factor GATA3 is essential for the differentiation and functions of these cell types. It has been demonstrated that GATA3 is critical for maintaining Th2 and ILC2 phenotype in vitro; GATA3 not only positively regulates type 2 lymphocyte-associated genes, it also negatively regulates many genes associated with other lineages. However, such functions cannot be easily verified in vivo because the expression of the markers for identifying Th2 and ILC2s depends on GATA3. Thus, whether Th2 cells and ILC2s disappear after Gata3 deletion or these Gata3-deleted "Th2 cells" or "ILC2s" acquire an alternative lineage fate is unknown. In this study, we generated novel GATA3 reporter mouse strains carrying the Gata3 ZsG or Gata3 ZsG-fl allele. This was achieved by inserting a ZsGreen-T2A cassette at the translation initiation site of either the wild type Gata3 allele or the modified Gata3 allele which carries two loxP sites flanking the exon 4. ZsGreen faithfully reflected the endogenous GATA3 protein expression in Th2 cells and ILC2s both in vitro and in vivo. These reporter mice also allowed us to visualize Th2 cells and ILC2s in vivo. An inducible Gata3 deletion system was created by crossing Gata3 ZsG-fl/fl mice with a tamoxifen-inducible Cre. Continuous expression of ZsGreen even after the Gata3 exon 4 deletion was noted, which allows us to isolate and monitor GATA3-deficient "Th2" cells and "ILC2s" during in vivo immune responses. Our results not only indicated that functional GATA3 is dispensable for regulating its own expression in mature type 2 lymphocytes, but also revealed that GATA3-deficient "ILC2s" might be much more stable in vivo than in vitro. Overall, the generation of these novel GATA3 reporters will provide valuable research tools to the scientific community in investigating type 2 immune responses in vivo.

Autism is a complex heterogeneous neurodevelopmental disorder; previous studies have identified altered immune responses among individuals diagnosed with autism. An imbalance in the production of pro- and anti-inflammatory cytokines and transcription factors plays a role in neurodevelopmental behavioral and autism disorders. BTBR T+ Itpr3tf/J (BTBR) mice are used as a model for autism, as they exhibit social deficits, communication deficits, and repetitive behaviors compared with C57BL/6J (B6) mice. The adenosine A2A receptor (A2AR) appears to be a potential target for the improvement of behavioral, inflammatory, immune, and neurological disorders. We investigated the effects of the A2AR antagonist SCH 5826 (SCH) and agonist CGS 21680 (CGS) on IL-21, IL-22, T-bet, T-box transcription factor (T-bet), GATA3 (GATA Binding Protein 3), and CD152 (CTLA-4) expression in BTBR mice. Our results showed that BTBR mice treated with SCH had increased CD4+IL-21+, CD4+IL-22+, CD4+GATA3+, and CD4+T-bet+ and decreased CD4+CTLA-4+ expression in spleen cells compared with BTBR control mice. Moreover, CGS efficiently decreased CD4+IL-21+, CD4+IL-22+, CD4+GATA3+, and CD4+T-bet+ and increased CD4+CTLA-4 production in spleen cells compared with SCH-treated and BTBR control mice. Additionally, SCH treatment significantly increased the mRNA and protein expression levels of IL-21, IL-22, GATA3, and T-bet in brain tissue compared with CGS-treated and BTBR control mice. The augmented levels of IL-21/IL-22 and GATA3/T-bet could be due to altered A2AR signaling. Our results indicate that A2AR agonists may represent a new class of compounds that can be developed for use in the treatment of autistic and neuroimmune dysfunctions.