In T-cell acute lymphoblastic leukemia (T-ALL), several members of the NK-like (NKL) homeobox genes are aberrantly expressed. Here, we have analyzed the activity of NKL homeobox gene MSX1 using pediatric T-ALL in silico data, detecting overexpression in 11% of patients. Quantification of MSX1 transcripts in a panel of 24 T-ALL cell lines demonstrated overexpression in two examples. Comparative expression profiling indicated inhibition of the bone morphogenetic protein (BMP) signaling pathway, which was shown to inhibit MSX1 transcription. In the LOUCY cell line we identified conspicuous expression of CHRDL1 encoding a BMP inhibitor which mediated activation of MSX1. Promoter analyses demonstrated activation of CHRDL1 by oncogenic PITX1. Furthermore, knockdown and overexpression studies of hematopoietic transcription factors demonstrated that GATA2 and FOXC1 mediate activation and GATA3, LEF1, TAL1 and TOX repression of MSX1 transcription. Collectively, our findings suggest that MSX1 is physiologically restricted to lymphoid progenitors. The identification of deregulated BMP signaling may provide novel therapeutic options for the treatment of T-ALL.

Immune response messenger RNAs (mRNA) were compared in liver during self-limited (resolved) and chronic neonatal woodchuck hepatitis virus (WHV) infection. At week 14 postinfection (mid-acute phase), mRNAs for leukocyte markers (CD3, CD4, CD8), type 1 cytokines and related transcription factors (IFN-gamma, TNF-alpha, STAT4, T-bet), and IL-10 were increased in livers from resolving infections, but mRNAs of other type 1 (IL-2) and type 2 (IL-4, STAT6, and GATA3 markers remained at baseline levels. Increased coexpression of IFN-gamma and TNF-alpha mRNAs correlated in most cases with lower levels of intrahepatic WHV covalently closed circular DNA (cccDNA). At the same time point postinfection, livers from woodchucks that eventually progressed to chronic infection had baseline or slightly elevated levels of CD and type 1 mRNAs, which were significantly lower (or elevated less frequently) compared with resolving woodchucks. Earlier, at week 8, there were no differences between the two outcome settings. During these early time points and at a later stage in chronic infection (15 months), type 2 mRNAs in carrier liver remained at baseline levels or, when elevated, were never in excess of those in resolving woodchucks. In conclusion, the onset and maintenance of neonatal chronic WHV infection are not associated with antagonistic type 2 immunoregulation of type 1 responses in liver. Accordingly, chronicity develops in association with a primary deficiency in the intrahepatic CD responses, especially involving CD8(+) T lymphocytes, and in both extracellular (cytokine) and intracellular (transcriptional) type 1 response mediators. This has relevant implications for future treatment of chronic hepatitis B virus (HBV) infection in humans.

GATA-binding protein 3 (GATA3) is a transcription factor and a putative tumor suppressor that is highly expressed in normal breast luminal epithelium and estrogen receptor alpha (ER)-positive breast tumors. We hypothesized that common genetic variation in GATA3 could influence breast carcinogenesis. Four tag single-nucleotide polymorphisms (SNP) in GATA3 and its 3' flanking gene FLJ4598 were genotyped in two case control studies in Norway and Poland (2,726 cases and 3,420 controls). Analyses of pooled data suggested a reduced risk of breast cancer associated with two intronic variants in GATA3 in linkage disequilibrium (rs3802604 in intron 3 and rs570613 in intron 4). Odds ratio (95% confidence interval) for rs570613 heterozygous and rare homozygous versus common homozygous were 0.85 (0.75-1.95) and 0.82 (0.62-0.96), respectively (P(trend)=0.004). Stronger associations were observed for subjects with ER-negative, than ER-positive, tumors (P(heterogeneity)=0.01 for rs3802604; P(heterogeneity)=0.09 for rs570613). Although no individual SNPs were associated with ER-positive tumors, two haplotypes (GGTC in 2% of controls and AATT in 7% of controls) showed significant and consistent associations with increased risk for these tumors when compared with the common haplotype (GATT in 46% of controls): 1.71 (1.27-2.32) and 1.26 (1.03-1.54), respectively. In summary, data from two independent study populations showed two intronic variants in GATA3 associated with overall decreases in breast cancer risk and suggested heterogeneity of these associations by ER status. These differential associations are consistent with markedly different levels of GATA3 protein by ER status. Additional epidemiologic studies are needed to clarify these intriguing relationships.

GATA3 is frequently mutated in breast cancer; these mutations are widely presumed to be loss-of function despite a dearth of information regarding their effect on disease course or their mechanistic impact on the breast cancer transcriptional network. Here, we address molecular and clinical features associated with GATA3 mutations. A novel classification scheme defines distinct clinical features for patients bearing breast tumors with mutations in the second GATA3 zinc-finger (ZnFn2). An engineered ZnFn2 mutant cell line by CRISPR-Cas9 reveals that mutation of one allele of the GATA3 second zinc finger (ZnFn2) leads to loss of binding and decreased expression at a subset of genes, including Progesterone Receptor. At other loci, associated with epithelial to mesenchymal transition, gain of binding correlates with increased gene expression. These results demonstrate that not all GATA3 mutations are equivalent and that ZnFn2 mutations impact breast cancer through gain and loss-of function.

BACKGROUND

An increasing number of epidemiological studies show that exposure to farming environment during early childhood strongly influences the development of allergic reactions later in life ('hygiene hypothesis'). Also, it had been shown that certain bacteria from this environment may have allergy-protective properties. In the present study, we further characterized one of these bacteria, namely Acinetobacter lwoffii F78, with regard to the bacteria-induced signaling and possible mechanisms of allergy protection.

METHODS

The impact of A. lwoffii F78 on human monocyte-derived dendritic cells especially with respect to their T(Helper) cell polarization capacity was investigated by ELISA and real-time PCR experiments as well as confocal microscopy. The responsible molecule for these effects was further characterized and identified using blocking experiments.

RESULTS

It was shown that A. lwoffii F78 induced a T(H)1-polarizing program in human dendritic cells which led to T(H)1 differentiation. In addition, a positive influence on the TBet/GATA3 level could be detected. Blocking experiments revealed that the lipopolysaccharide (LPS) of A. lwoffii F78 was the responsible molecule promoting these effects.

CONCLUSIONS

We found evidence that the allergy-protecting effects of A. lwoffii F78 are because of the activation of a T(H)1-polarizing program in human dendritic cells, and that the LPS of A. lwoffii F78 is responsible for these beneficial effects.

Clear cell renal cell carcinoma (ccRCC) still remains a higher mortality rate in worldwide. Obtaining promising biomakers is very crucial for improving the diagnosis and prognosis of ccRCC patients. Herein, we firstly identified eight potentially prognostic miRNAs (hsa-miR-144-5p, hsa-miR-223-3p, hsa-miR-365b-3p, hsa-miR-3613-5p, hsa-miR-9-5p, hsa-miR-183-5p, hsa-miR-335-3p, hsa-miR-1269a). Secondly, we found that a signature containing these eight miRNAs showed obviously superior to a single miRNA in the prognostic effect and credibility for predicting the survival of ccRCC patients. Thirdly, we discovered that twenty-two transcription factors (TFs) interact with these eight miRNAs, and a signature combining nine TFs (TFAP2A, KLF5, IRF1, RUNX1, RARA, GATA3, IKZF1, POU2F2, and FOXM1) could promote the prognosis of ccRCC patients. Finally, we further identified eleven genes (hsa-miR-365b-3p, hsa-miR-223-3p, hsa-miR-1269a, hsa-miR-144-5p, hsa-miR-183-5p, hsa-miR-335-3p, TFAP2A, KLF5, IRF1, MYC, IKZF1) that could combine as a signature to improve the prognosis effect of ccRCC patients, which distinctly outperformed the eight-miRNA signature and the nine-TF signature. Overall, we identified several new prognosis factors for ccRCC, and revealed a potential mechanism that TFs and miRNAs interplay cooperatively or oppositely regulate a certain number of tumor suppressors, driver genes, and oncogenes to facilitate the survival of ccRCC patients.

Umbilical cord tissue is gaining attention as a novel source of multipotent stem cells because it is easily obtainable, ethically acceptable and the cells are immunologically naïve. In this study, we have isolated and characterized a new cell type expressing MUCIN1 (CD227) from human umbilical cord lining which we termed MUCIN-expressing Cord Lining Epithelial Cell (CLEC-muc). We found that CLEC-muc is highly proliferative and had significant clonogenic ability. These cells express embryonic stem cell markers OCT-4, NANOG, SSEA-4, REX1 and SOX2. Despite the abundant expression of epithelial cell marker MUCIN1 and cytokeratins, this population is also positive to the mesenchymal stem cell (MSC) marker CD166. CLEC-muc is unique in p63 expression that shuttles from the cytoplasm to the nucleus over time in culture. To understand p63 regulation and function in CLEC-muc, cells were treated with BMP4, a potent morphogen that plays a role in epidermal differentiation via p63 upregulation in ES cell and subsequent analyses were done. We found that BMP4 does not alter cytoplasmic expression of p63 that promotes cell proliferation. However, it increases nuclear p63 expression together with several other epithelial-associated genes such as GATA3, JAGGED1, NOTCH1, HES1 and IKKα. BMP4 has also been found to weakly induce deltaNp63 expression in CLEC-muc. Our results suggest that CLEC-muc is a novel stem cell-like population that can be further differentiated by BMP4 to generate specific cell-types probably destined to form non-keratinized epithelia.

Innate lymphoid cell (ILC) development proposes that ILC precursors (ILCPs) segregate along natural killer (NK) cell versus helper cell (ILC1, ILC2, ILC3) pathways, the latter depending on expression of Id2, Zbtb16, and Gata3. We have developed an Id2-reporter strain expressing red fluorescent protein (RFP) in the context of normal Id2 expression to re-examine ILCP phenotype and function. We show that bone-marrow ILCPs were heterogeneous and harbored extensive NK-cell potential in vivo and in vitro. By multiplexing Id2^RFP with Zbtb16^CreGFP and Bcl11b^tdTomato strains, we made a single-cell dissection of the ILCP compartment. In contrast with the current model, we have demonstrated that Id2⁺Zbtb16⁺ ILCPs included multi-potent ILCPs that retained NK-cell potential. Late-stage ILC2P and ILC3P compartments could be defined by differential Zbtb16 and Bcl11b expression. We suggest a revised model for ILC differentiation that redefines the cell-fate potential of helper-ILC-restricted Zbtb16⁺ ILCPs.

OBJECTIVE

Less than 10% of registered drug intervention trials for pancreatic ductal adenocarcinoma (PDAC) include a biomarker stratification strategy. The ability to identify distinct mutation subsets via endoscopic ultrasound fine needle aspiration (EUS FNA) molecular cytology could greatly aid clinical trial patient stratification and offer predictive markers. We identified chemotherapy treatment naïve ampullary adenocarcinoma and PDAC patients who underwent EUS FNA to assess multigene mutational frequency and diversity with a surgical resection concordance assessment, where available.

METHODS

Following strict cytology smear screening criteria, targeted next generation sequencing (NGS) using a 160 cancer gene panel was performed.

RESULTS

Complete sequencing was achieved in 29 patients, whereby 83 pathogenic alterations were identified in 21 genes. Cytology genotyping revealed that the majority of mutations were identified in KRAS (93%), TP53 (72%), SMAD4 (31%), and GNAS (10%). There was 100% concordance for the following pathogenic alterations: KRAS, TP53, SMAD4, KMT2D, NOTCH2, MSH2, RB1, SMARCA4, PPP2R1A, PIK3R1, SCL7A8, ATM, and FANCD2. Absolute multigene mutational concordance was 83%. Incremental cytology smear mutations in GRIN2A, GATA3 and KDM6A were identified despite re-examination of raw sequence reads in the corresponding resection specimens.

CONCLUSIONS

EUS FNA cytology genotyping using a 160 cancer gene NGS panel revealed a broad spectrum of pathogenic alterations. The fidelity of cytology genotyping to that of paired surgical resection specimens suggests that EUS FNA represents a suitable surrogate and may complement the conventional stratification criteria in decision making for therapies and may guide future biomarker driven therapeutic development.

Inter-species somatic cell nuclear transfer (iSCNT) embryos usually fail to develop to the blastocyst stage and beyond due to incomplete reprogramming of donor cell. We evaluated whether using a karyoplast that would require less extensive reprogramming such as an embryonic blastomere or the meiotic spindle from metaphase II oocytes would provide additional insight into the development of iSCNT embryos. Our results showed that karyoplasts of embryonic or oocyte origin are no different from somatic cells; all iSCNT embryos, irrespective of karyoplast origin, were arrested during early development. We hypothesized that nuclear-cytoplasmic incompatibility could be another reason for failure of embryonic development from iSCNT. We used pig-mouse cytoplasmic hybrids as a model to address nuclear-cytoplasmic incompatibility in iSCNT embryos. Fertilized murine zygotes were reconstructed by fusing with porcine cytoplasts of varying cytoplasmic volumes (1/10 (small) and 1/5 (large) total volume of mouse zygote). The presence of pig cytoplasm significantly reduced the development of mouse zygotes to the blastocyst stage compared with control embryos at 120 h post-human chorionic gondotropin (41 vs 6 vs 94%, P<0.05; 1/10, 1/5, control respectively). While mitochondrial DNA copy numbers remained relatively unchanged, expression of several important genes namely Tfam, Polg, Polg2, Mfn2, Slc2a3 (Glut3), Slc2a1 (Glut1), Bcl2, Hspb1, Pou5f1 (Oct4), Nanog, Cdx2, Gata3, Tcfap2c, mt-Cox1 and mt-Cox2 was significantly reduced in cytoplasmic hybrids compared with control embryos. These results demonstrate that the presence of even a small amount of porcine cytoplasm is detrimental to murine embryo development and suggest that a range of factors are likely to contribute to the failure of inter-species nuclear transfer embryos.

BACKGROUND

Psoriasis is a cytokine-mediated skin disease that can be treated effectively with immunosuppressive biologic agents. These medications, however, are not equally effective in all patients and are poorly suited for treating mild psoriasis. To develop more targeted therapies, interfering with transcription factor (TF) activity is a promising strategy.

METHODS

Meta-analysis was used to identify differentially expressed genes (DEGs) in the lesional skin from psoriasis patients (n = 237). We compiled a dictionary of 2935 binding sites representing empirically-determined binding affinities of TFs and unconventional DNA-binding proteins (uDBPs). This dictionary was screened to identify "psoriasis response elements" (PREs) overrepresented in sequences upstream of psoriasis DEGs.

RESULTS

PREs are recognized by IRF1, ISGF3, NF-kappaB and multiple TFs with helix-turn-helix (homeo) or other all-alpha-helical (high-mobility group) DNA-binding domains. We identified a limited set of DEGs that encode proteins interacting with PRE motifs, including TFs (GATA3, EHF, FOXM1, SOX5) and uDBPs (AVEN, RBM8A, GPAM, WISP2). PREs were prominent within enhancer regions near cytokine-encoding DEGs (IL17A, IL19 and IL1B), suggesting that PREs might be incorporated into complex decoy oligonucleotides (cdODNs). To illustrate this idea, we designed a cdODN to concomitantly target psoriasis-activated TFs (i.e., FOXM1, ISGF3, IRF1 and NF-kappaB). Finally, we screened psoriasis-associated SNPs to identify risk alleles that disrupt or engender PRE motifs. This identified possible sites of allele-specific TF/uDBP binding and showed that PREs are disproportionately disrupted by psoriasis risk alleles.

CONCLUSIONS

We identified new TF/uDBP candidates and developed an approach that (i) connects transcriptome informatics to cdODN drug development and (ii) enhances our ability to interpret GWAS findings. Disruption of PRE motifs by psoriasis risk alleles may contribute to disease susceptibility.

BACKGROUND

The transcription factor GATA3 is involved in mammary gland development and is crucial for the maintenance of the differentiated status of luminal epithelial cells. The role of GATA3 in breast cancer as a tumor suppressor has been established, although insights into the mechanism of GATA3 expression loss are still required.

METHODS

Chromatin immunoprecipitation assays were conducted to study progestin modulation of recruitment of transcription factors to GATA3 promoter. We performed western blot and reverse RT-qPCR experiments to explore progestin regulation of GATA3 protein and mRNA expression respectively. Confocal microscopy and in vitro phosphorylation studies were conducted to examine progestin capacity to induce GATA3 serine phosphorylation in its 308 residue. GATA3 participation in progestin-induced breast cancer growth was addressed in in vitro proliferation and in vivo tumor growth experiments.

RESULTS

In this study, we demonstrate that progestin-activated progesterone receptor (PR) reduces GATA3 expression through regulation at the transcriptional and post-translational levels in breast cancer cells. In the former mechanism, the histone methyltransferase enhancer of zeste homolog 2 is co-recruited with activated PR to a putative progesterone response element in the GATA3 proximal promoter, increasing H3K27me3 levels and inducing chromatin compaction, resulting in decreased GATA3 mRNA levels. This transcriptional regulation is coupled with increased GATA3 protein turnover through progestin-induced GATA3 phosphorylation at serine 308 followed by 26S proteasome-mediated degradation. Both molecular mechanisms converge to accomplish decreased GATA3 expression levels in breast cancer cells upon PR activation. In addition, we demonstrated that decreased GATA3 levels are required for progestin-induced upregulation of cyclin A2, which mediates the G1 to S phase transition of the cell cycle and was reported to be associated with poor prognosis in breast cancer. Finally, we showed that downregulation of GATA3 is required for progestin stimulation of both in vitro cell proliferation and in vivo tumor growth.

CONCLUSIONS

In the present study, we reveal that progestin-induced PR activation leads to loss of GATA3 expression in breast cancer cells through transcriptional and post-translational regulation. Importantly, we demonstrate that GATA3 downregulation is required for progestin-induced upregulation of cyclin A2 and for progestin-induced in vitro and in vivo breast cancer cell growth.

Highly regulated expression of the negative costimulatory molecule cytotoxic T-lymphocyte antigen-4 (CTLA-4) on T cells modulates T-cell activation and proliferation. CTLA-4 is preferentially expressed in Th2 T cells, whose differentiation depends on the transcriptional regulator GATA3. Sézary syndrome (SS) is a T-cell malignancy characterized by Th2 cytokine skewing, impaired T-cell responses, and overexpression of GATA3 and CTLA-4. GATA3 is regulated by phosphorylation and ubiquitination. In SS cells, we detected increased polyubiquitinated proteins and activated GATA3. We hypothesized that proteasome dysfunction in SS T cells may lead to GATA3 and CTLA-4 overexpression. To test this hypothesis, we blocked proteasome function with bortezomib in normal T cells, and observed sustained GATA3 and CTLA-4 upregulation. The increased CTLA-4 was functionally inhibitory in a mixed lymphocyte reaction (MLR). GATA3 directly transactivated the CTLA-4 promoter, and knockdown of GATA3 messenger RNA and protein inhibited CTLA-4 induction mediated by bortezomib. Finally, knockdown of GATA3 in patient's malignant T cells suppressed CTLA-4 expression. Here we demonstrate a new T-cell regulatory pathway that directly links decreased proteasome degradation of GATA3, CTLA-4 upregulation, and inhibition of T-cell responses. We also demonstrate the requirement of the GATA3/CTLA-4 regulatory pathway in fresh neoplastic CD4+ T cells. Targeting of this pathway may be beneficial in SS and other CTLA-4-overexpressing T-cell neoplasms.

Emerging therapies for sensorineural hearing loss include replacing damaged auditory neurons (ANs) using stem cells. Ultimately, it is important that these replacement cells can be patient-matched to avoid immunorejection. As human induced pluripotent stem cells (hiPSCs) can be obtained directly from the patient, they offer an opportunity to generate patient-matched neurons for transplantation. Here, we used an established neural induction protocol to differentiate two hiPSC lines (iPS1 and iPS2) and one human embryonic stem cell line (hESC; H9) toward a neurosensory lineage in vitro. Immunocytochemistry and qRT-PCR were used to analyze the expression of key markers involved in AN development at defined time points of differentiation. The hiPSC- and hESC-derived neurosensory progenitors expressed the dorsal hindbrain marker (PAX7), otic placodal marker (PAX2), proneurosensory marker (SOX2), ganglion neuronal markers (NEUROD1, BRN3A, ISLET1, ßIII-tubulin, Neurofilament kDa 160), and sensory AN markers (GATA3 and VGLUT1) over the time course examined. The hiPSC- and hESC-derived neurosensory progenitors had the highest expression levels of the sensory neural markers at 35 days in vitro. Furthermore, the neurons generated from this assay were found to be electrically active. While all cell lines analyzed produced functional neurosensory-like progenitors, variabilities in the levels of marker expression were observed between hiPSC lines and within samples of the same cell line, when compared with the hESC controls. Overall, these findings indicate that this neural assay was capable of differentiating hiPSCs toward a neurosensory lineage but emphasize the need for improving the consistency in the differentiation of hiPSCs into the required lineages.

Current understandings of cell specification in early mammalian pre-implantation development are based mainly on mouse studies. The first lineage differentiation event occurs at the morula stage, with outer cells initiating a trophectoderm (TE) placental progenitor program. The inner cell mass arises from inner cells during subsequent developmental stages and comprises precursor cells of the embryo proper and yolk sac¹. Recent gene-expression analyses suggest that the mechanisms that regulate early lineage specification in the mouse may differ in other mammals, including human^2-5 and cow⁶. Here we show the evolutionary conservation of a molecular cascade that initiates TE segregation in human, cow and mouse embryos. At the morula stage, outer cells acquire an apical-basal cell polarity, with expression of atypical protein kinase C (aPKC) at the contact-free domain, nuclear expression of Hippo signalling pathway effectors and restricted expression of TE-associated factors such as GATA3, which suggests initiation of a TE program. Furthermore, we demonstrate that inhibition of aPKC by small-molecule pharmacological modulation or Trim-Away protein depletion impairs TE initiation at the morula stage. Our comparative embryology analysis provides insights into early lineage specification and suggests that a similar mechanism initiates a TE program in human, cow and mouse embryos.

The transcription factor GATA3 is the master regulator that drives mammary luminal epithelial cell differentiation and maintains mammary gland homeostasis. Loss of GATA3 is associated with aggressive breast cancer development. We have identified ZNF503/ZEPPO2 zinc-finger elbow-related proline domain protein 2 (ZPO2) as a transcriptional repressor of GATA3 expression and transcriptional activity that induces mammary epithelial cell proliferation and breast cancer development. We show that ZPO2 is recruited to GATA3 promoter in association with ZBTB32 (Repressor of GATA, ROG) and that ZBTB32 is essential for down-regulation of GATA3 via ZPO2. Through this modulation of GATA3 activity, ZPO2 promotes aggressive breast cancer development. Our data provide insight into a mechanism of GATA3 regulation, and identify ZPO2 as a possible candidate gene for future diagnostic and therapeutic strategies.

T cells develop from hematopoietic stem cells in the specialized microenvironment of the thymus. The main transcriptional players of T-cell differentiation such as Notch, Tcf-1, Gata3 and Bcl11b have been identified, but their role and regulation are not yet completely understood. In humans, functional experiments on T-cell development have traditionally been rather difficult to perform, but novel in vitro culture systems and in vivo xenograft models have allowed detailed studies on human T-cell development. Recent work has allowed the use of human severe combined immunodeficiency stem cells to unravel developmental checkpoints for human thymocyte development.

GATA3 mutations cause HDR (hypoparathyroidism, sensorineural deafness, and renal dysplasia) syndrome and, consistent with the presence of the second DiGeorge syndrome locus (DGS2) proximal to GATA3, distal 10p deletions often leads to HDR and DiGeorge syndromes. Here, we report on six Japanese patients with GATA3 abnormalities. Cases 1-5 had a normal karyotype, and case 6 had a 46,XX,del(10)(p15) karyotype. Cases 1-6 had two or three of the HDR triad features. Case 6 had no DiGeorge syndrome phenotype except for hypoparathyroidism common to HDR and DiGeorge syndromes. Mutation analysis showed heterozygous GATA3 mutations in cases 1-5, i.e., c.404-405insC (p.P135fsX303) in case 1, c.700T>C & c.708-709insC (p.F234L & p.S237fsX303) on the same allele in case 2, c.737-738insG (p.G246fsX303) in case 3, c.824G>T (p.W275L) in case 4, and IVS5+1G>C (splice error) in case 5. Deletion analysis of chromosome 10p revealed loss of GATA3 and preservation of D10S547 in case 6. The results are consistent with the previous finding that GATA3 mutations are usually identified in patients with two or three of the HDR triad features, and provide supportive data for the mapping of DGS2 in the region proximal to D10S547.

Cranial placodes contribute to many sensory organs and ganglia of the vertebrate head. The olfactory, otic, and lateral line placodes form the sensory receptor cells and neurons of the nose, ear, and lateral line system; the lens placode develops into the lens of the eye; epibranchial, profundal, and trigeminal placodes contribute sensory neurons to cranial nerve ganglia; and the adenohypophyseal placode gives rise to the anterior pituitary, a major endocrine control organ. Despite these differences in fate, all placodes are now known to originate from a common precursor, the preplacodal ectoderm (PPE). The latter is a horseshoe-shaped domain of ectoderm surrounding the anterior neural plate and neural crest and is defined by expression of transcription factor Six1, its cofactor Eya1, and other members of the Six and Eya families. Studies in zebrafish, Xenopus, and chick reveal that the PPE is specified together with other ectodermal territories (epidermis, neural crest, and neural plate) during early embryogenesis. During gastrulation, domains of ventrally (e.g., Dlx3/Dlx5, GATA2/GATA3, AP2, Msx1, FoxI1, and Vent1/Vent2) and dorsally (e.g., Zic1, Sox3, and Geminin) restricted transcription factors are established in response to a gradient of BMP and help to define non-neural and neural competence territories, respectively. At neural plate stages, the PPE is then induced in the non-neural competence territory by signals from the adjacent neural plate and mesoderm including FGF, BMP inhibitors, and Wnt inhibitors. Subsequently, signals from more localized signaling centers induce restricted expression domains of various transcription factors within the PPE, which specify multiplacodal areas and ultimately individual placodes. For further resources related to this article, please visit the WIREs website.

BACKGROUND

The author has declared no conflicts of interest for this article.

To evaluate the balance of interleukin IL18 and its endogenous antagonist IL18 binding protein (IL18BP) in patients with idiopathic thrombocytopenic purpura (ITP), plasma IL18, IL18BP, interferon gamma (IFNG) and IL4 levels, as well as platelet counts were measured in patients with active ITP (n = 23), ITP in remission (n = 21) and in healthy subjects (n = 24) by enzyme linked immunosorbent assay (ELISA). Using real-time quantitative polymerase chain reaction, the mRNA expression of IL18, IL18BP, IFNG, IL4, T-box (TBX21) and GATA-binding protein 3(GATA3) were studied in all subjects. The results showed that IL18 and IFNG protein and mRNA levels were significantly increased in patients with active ITP than in control subjects, but that IL18BP were not significantly elevated in ITP patients, which resulted in an elevated ratio of IL18/IL18BP in patients with active disease. During remission stages, the levels of these cytokines were comparable to those of healthy controls. The elevated levels of IL18/IL18BP in plasma during active stages of disease suggest a possible role in the pathogenesis and course of ITP.

Numerous transcription factors that regulate trophoblast developmental processes have been identified; however, the regulation of trophoblast-specific gene expression has not been definitively characterized. While a new role of Gata3 in trophoblast development was being demonstrated in mice, we examined effects of GATA transcription factors on conceptus interferon tau (IFNT), a major trophectoderm factor in ruminants. In this study, expression patterns of trophoblast ASCL2, CDX2, CSH1, ELF5, HAND1, IFNT, and TKDP1 mRNAs were initially examined, from which ASCL2, CDX2, IFNT, and TKDP1 mRNAs were found to be similar to those of GATA2 and GATA3 in days 17, 20, and 22 (day 0=day of estrus) bovine conceptuses. A chromatin immunoprecipitation (ChIP) assay revealed that endogenous GATA2 and GATA3 occupied GATA binding sites on the upstream regions of CSH1, IFNT, and TKDP1 genes and on the intron 1 region of CDX2 gene in bovine trophoblast CT-1 cells. In transient transfection analyses of the upstream region of bovine CSH1, and IFNT or the intron 1 region of CDX2 gene, over-expression of GATA2 induced transactivation of these trophoblast-specific genes in bovine non-trophoblast ear fibroblast EF cells, but over-expression of GATA3 did not substantially affect their transactivation. In CT-1 cells, endogenous CDX2 and IFNT mRNAs were down-regulated by GATA2 siRNA, while endogenous ASCL2 and CDX2 mRNAs were down-regulated by GATA3 siRNA. Our results indicate that in addition to trophectoderm lineage specification, GATA2 and/or GATA3 are involved in the regulation of trophoblast-specific gene transcription in bovine trophoblast CT-1 cells.

BACKGROUND

Multiple sclerosis (MS) is hypothetically caused by autoreactive Th1 and Th17 cells, whereas Th2 and regulatory T cells may confer protection. The development of Th subpopulations is dependant on the expression of lineage-specific transcription factors.

OBJECTIVE

The aim of this study was to assess the balance of CD4(+)T cell populations in relapsing-remitting MS.

METHODS

Blood mRNA expression of TBX21, GATA3, RORC, FOXP3 and EBI3 was assessed in 33 patients with relapsing-remitting MS and 20 healthy controls. In addition, flow cytometry was performed to assess T lymphocyte numbers.

RESULTS

In relapsing-remitting MS, diminished expression of FOXP3 (Treg) was found (p < 0.05), despite normal numbers of CD4(+)CD25(hi)Treg. Immunoregulatory EBI3 and Th2-associated GATA3 ([a-z]+) was also decreased in MS (p < 0.005 and p < 0.05, respectively). Expression of TBX21 (Th1) and RORC (Th17) did not differ between patients and controls. Similar changes were observed when analysing beta-interferon treated (n = 12) or untreated (n = 21) patients. Analysis of transcription factor ratios, comparing TBX21/GATA3 and RORC/FOXP3, revealed an increase in the RORC/FOXP3 ratio in patients with relapsing-remitting MS (p < 0.005).

CONCLUSIONS

Our findings indicate systemic defects at the mRNA level, involving downregulation of beneficial CD4(+)phenotypes. This might play a role in disease development by permitting activation of harmful T cell populations.

T-bet is an important Th1 driving transcription factor regulated by IFN-gamma/STAT1 pathway. T-bet turns on IFN-gamma transcription in CD4+ T cells and T-bet-deficient cells fail to differentiate to Th1 direction. Previous reports have characterized function of T-bet mainly in murine cells and very little is known about its functions in human cells. Here, we studied T-bet expression kinetics in parallel with GATA3 during Th1/Th2 polarization. We demonstrate that in addition to CD3/CD28 activation, cytokines IL-12 and IFN-alpha in the presence of neutralizing anti-IFN-gamma enhanced T-bet mRNA and protein expression in human CD4+ cells. T-bet is known to be a potent inducer of IFN-gamma. Even though IFN-gamma and IL-12 stimulation induced similar levels of T-bet protein in human CD4+ cells, IFN-gamma-treated cells produced considerably less IFN-gamma than cells treated with IL-12. Therefore, high T-bet protein expression does not necessarily correlate with IFN-gamma production. In addition, we show that the immunosuppressive cytokine TGF-beta inhibits T-bet and GATA3 protein expression only if it is present prior to primary T cell activation and is maintained in the cultures during the early polarization of Th1/Th2 cells. In conclusion, we report new insights into the cytokine regulation of T-bet in human CD4+ T cells.

GATA3 is a member of the GATA family of transcription factors. Heterozygous GATA3 abnormalities are associated with hypoparathyroidism, sensorineural deafness, and renal abnormality (HDR syndrome). However, this triad of symptoms does not occur in all HDR patients and other clinical features may be present in some cases. We report the clinical phenotypes and the molecular analysis of GATA3 in five Japanese HDR patients, including two familial cases. All five patients had hypoparathyroidism and sensorineural deafness, however renal abnormalities were absent in four patients. In addition, two patients with different mutations of GATA3 had female genital tract abnormalities. Sequence analysis of GATA3 demonstrated three novel (R262G, c1063delC and C318) and two reported mutations (c.432insG and c.1051-1G>T). Transient transfection assay using the GATA3 activating reporter system revealed that the transactivating activity of the R262G, c.1063delC, C318S and c.432insG mutants were markedly decreased, indicating that all four mutations are loss-of-function. In conclusion, this study reiterates the clinical variability in HDR syndrome and identifies three novel mutations of GATA3.