Transcription factors are attractive therapeutic targets that are considered non-druggable because they do not have binding sites for small drug-like ligands. We established a cell-free high-throughput screening assay to search for small molecule inhibitors of DNA binding by transcription factors. A screen was performed using p53 as a target, resulting in the identification of NSC194598 that inhibits p53 sequence-specific DNA binding in vitro (IC₅₀ = 180 nM) and in vivo. NSC194598 selectively inhibited DNA binding by p53 and homologs p63/p73, but did not affect E2F1, TCF1, and c-Myc. Treatment of cells with NSC194598 alone paradoxically led to p53 accumulation and modest increase of transcriptional output owing to disruption of the MDM2-negative feedback loop. When p53 was stabilized and activated by irradiation or chemotherapy drug treatment, NSC194598 inhibited p53 DNA binding and induction of target genes. A single dose of NSC194598 increased the survival of mice after irradiation. The results suggest DNA binding by p53 can be targeted using small molecules to reduce acute toxicity to normal tissues by radiation and chemotherapy.

MicroRNAs serve important functions in numerous biological processes. Whether microRNAs also act on dendritic cell (DC) differentiation and function remains unclear. In this study, both conventional DCs (cDCs) and plasmacytoid DCs (pDCs) were increased in miR-34a overexpressing bone marrow chimeric and transgenic (TG) mice. Further experiments showed that miR-34a promoted preDC differentiated into cDCs and pDCs without affecting the proliferation and apoptosis of DCs. Luciferase report assay and Western blot experiments demonstrated that WNT1 is the direct target of miR-34a in DCs. Interestingly, miR-34a overexpressing cDCs also produced a large amount of IL-17a and suppressed T cell activation because of the inhibition of TCF1 expression, thus increasing RORγT expression. Taken together, miR-34a promotes preDC to differentiate into cDCs and pDCs, as well as inhibits the function of cDCs on the activation of CD4+ T cells by producing IL-17a.

The Wiskott-Aldrich syndrome protein (WASp) family of actin-nucleating factors are present in the cytoplasm and in the nucleus. The role of nuclear WASp for T cell development remains incompletely defined.

We performed WASp chromatin immunoprecipitation and deep sequencing (ChIP-seq) in thymocytes and spleen CD4+ T cells.

WASp was enriched at genic and intergenic regions and associated with the transcription start sites of protein-coding genes. Thymocytes and spleen CD4+ T cells showed 15 common WASp-interacting genes, including the gene encoding T cell factor (TCF)12. WASp KO thymocytes had reduced nuclear TCF1TCF1TCF1TCF1-binding site and we show that WASp directly interacted with TCF1 in the nucleus.

These data place nuclear WASp in proximity with <em>TCF1</em> and <em>TCF1</em>2, essential factors for T cell development.

Current management of childhood leukemia is tailored based on disease risk determined by clinical features at presentation. Whether properties of the host immune response impact disease risk and outcome is not known. Here we combine mass cytometry, single cell genomics and functional studies to characterize the bone marrow immune environment in children with B-cell acute lymphoblastic leukemia, and acute myelogenous leukemia at presentation. T cells in leukemia marrow demonstrate evidence of chronic immune activation and exhaustion/dysfunction, with attrition of naïve T cells and TCF1+ stem-like memory T cells and accumulation of terminally-differentiated effector T cells. Marrow-infiltrating natural killer cells also exhibit evidence of dysfunction, particularly in myeloid leukemia. Properties of immune cells identified distinct immune phenotype-based clusters correlating with disease risk in acute lymphoblastic leukemia. High-risk immune signatures were associated with expression of stem-like genes on tumor cells. These data provide a comprehensive assessment of the immune landscape of childhood leukemias and identify targets potentially amenable to therapeutic intervention. These studies also suggest that properties of the host response with depletion of naïve T cells and accumulation of terminal-effector T cells may contribute to the biologic basis of disease risk. Properties of immune microenvironment identified here may also impact optimal application of immune therapies, including T cell-redirection approaches in childhood leukemia.

Keywords: Immunology; Leukemias; T cells.

While intestinal Th17 cells are critical for maintaining tissue homeostasis, recent studies have implicated their roles in the development of extra-intestinal autoimmune diseases including multiple sclerosis. However, the mechanisms by which tissue Th17 cells mediate these dichotomous functions remain unknown. Here, we characterized the heterogeneity, plasticity, and migratory phenotypes of tissue Th17 cells in vivo by combined fate mapping with profiling of the transcriptomes and TCR clonotypes of over 84,000 Th17 cells at homeostasis and during CNS autoimmune inflammation. Inter- and intra-organ single-cell analyses revealed a homeostatic, stem-like TCF1⁺ IL-17⁺ SLAMF6⁺ population that traffics to the intestine where it is maintained by the microbiota, providing a ready reservoir for the IL-23-driven generation of encephalitogenic GM-CSF⁺ IFN-γ⁺ CXCR6⁺ T cells. Our study defines a direct in vivo relationship between IL-17⁺ non-pathogenic and GM-CSF⁺ and IFN-γ⁺ pathogenic Th17 populations and provides a mechanism by which homeostatic intestinal Th17 cells direct extra-intestinal autoimmune disease.

Keywords: CNS inflammation; GM-CSF; IFNγ; IL-17; Th17 cells; autoimmunity; fate-mapping; gut-brain axis; multiple sclerosis; stem-like T cells.

The chromatin organizer SATB1 is highly enriched in thymocytes and is essential for T-cell development. Although SATB1 regulates a large number of genes important for T-cell development, the mechanism(s) regulating expression of SATB1 during this process remain elusive. Using chromatin immune precipitation-seq-based occupancy profiles of H3K4me3 and H3Kme1 at Satb1 gene locus, we predicted four different alternative promoters of Satb1 in mouse thymocytes and characterized them. The expression of Satb1 transcript variants with distinct 5' UTRs occurs in a stage-specific manner during T-cell development and is dependent on TCR signaling. The observed discrepancy between the expression levels of SATB1 mRNA and protein in developing thymocytes can be explained by the differential translatability of Satb1 transcript variants as confirmed by polysome profiling and in vitro translation assay. We show that Satb1 alternative promoters exhibit lineage-specific chromatin accessibility during T-cell development from progenitors. Furthermore, TCF1 regulates the Satb1 P2 promoter switch during CD4SP development, via direct binding to the Satb1 P2 promoter. CD4SP T cells from TCF1 KO mice exhibit downregulation of P2 transcript variant expression as well as low levels of SATB1 protein. Collectively, these results provide unequivocal evidence toward alternative promoter switch-mediated developmental stage-specific regulation of SATB1 in thymocytes.

Type 1 diabetes mellitus (T1D) is a chronic autoimmune condition in which the immune system destroys insulin-producing pancreatic β cells. In addition to well-established pathogenic effector T cells, regulatory T cells (Tregs) have also been shown to be defective in T1D. Thus, an increasing number of therapeutic approaches are being developed to target Tregs. However, the role and mechanisms of TGF-β-induced Tregs (iTregs) in T1D remain poorly understood. Here, using a streptozotocin (STZ)-induced preclinical T1D mouse model, we found that iTregs could ameliorate the development of T1D and preserve β cell function. The preventive effect was associated with the inhibition of type 1 cytotoxic T (Tc1) cell function and rebalancing the Treg/Tc1 cell ratio in recipients. Furthermore, we showed that the underlying mechanisms were due to the TGF-β-mediated combinatorial actions of mTOR and TCF1. In addition to the preventive role, the therapeutic effects of iTregs on the established STZ-T1D and nonobese diabetic (NOD) mouse models were tested, which revealed improved β cell function. Our findings therefore provide key new insights into the basic mechanisms involved in the therapeutic role of iTregs in T1D.

Keywords: TGF-β; induced regulatory T cells; mTOR and TCF1; type 1 cytotoxic T cells; type 1 diabetes mellitus.

TCF1 plays a critical role in T lineage commitment and the development of αβ lineage T cells, but its role in γδ T cell development remains poorly understood. Here, we reveal a regulatory axis where T cell receptor (TCR) signaling controls TCF1 expression through an E-protein-bound regulatory element in the Tcf7 locus, and this axis regulates both γδ T lineage commitment and effector fate. Indeed, the level of TCF1 expression plays an important role in setting the threshold for γδ T lineage commitment and modulates the ability of TCR signaling to influence effector fate adoption by γδ T lineage progenitors. This finding provides mechanistic insight into how TCR-mediated repression of E proteins promotes the development of γδ T cells and their adoption of the interleukin (IL)-17-producing effector fate. IL-17-producing γδ T cells have been implicated in cancer progression and in the pathogenesis of psoriasis and multiple sclerosis.

Keywords: E proteins; T cell receptor (TCR); TCF1; TCR signaling; development; interleukin-17; γδ T cell.

Although males and females have a variety of sexually dimorphic features related to hormonal effects, the genetic basis of dimorphism relies on early embryo development. Two pluripotent states, naïve and primed, emerge during early mammalian development. Identification of signaling pathways that induce differences between these two states can help to modulate conversion of primed cells to naïve cells. Naïve cells have a shorter doubling time and longer survival than their primed counterparts when passaged as single cells. In this study, we sought to explore the role of Y chromosome genes on human pluripotent stem cells (hPSCs) by investigating differential expressions of the male-specific region of the Y chromosome (MSY) genes in primed and naïve cells. Interestingly, we found that several MSY genes, including SRY, showed higher expression levels in primed compared to naïve human embryonic stem cells (hESCs). We hypothesize that SRY prevents WNT/β-catenin signaling by its interaction and inhibition of β-catenin activation in the nucleus. Results of the loss-of-function approach conducted by depletion of SRY indicated increased expressions of pluripotency marker genes and alkaline phosphatase (ALP) activity in the primed cells. SRY reduction was associated with over-expression of WNT signaling target genes AXIN2, Brachury, TCF1, TBX2, and TBX3. We suggest that inhibition of SRY may result in activation of β-catenin and up-regulation of the WNT signaling pathway, both of which are important to naïve conversion.

Purpose: A subset of primary prostate cancer (PCa) expresses programmed death-ligand 1 (PD-L1), but whether they have unique tumor immune microenvironment (TIME) or genomic features is unclear.

Experimental design: We selected PD-L1-positive high-grade and/or high-risk primary PCa, characterized tumor-infiltrating lymphocytes (TILS) with multiplex immunofluorescence, and identified genomic alterations in immunogenic and non-immunogenic tumor foci.

Results: One-quarter of aggressive localized PCa cases (29/115) had tumor PD-L1 expression >5%. This correlated with increased density of CD8⁺ T cells, a large fraction co-expressing PD-1, versus absent PD-1 expression on sparse CD8 T cells in unselected cases. Most CD8⁺PD-1⁺ cells did not express terminal exhaustion markers (TIM-3 or LAG-3), while a subset expressed TCF1. Consistent with these CD8⁺PD-1⁺TCF1⁺ cells being progenitors, they were found in antigen-presenting-cell niches in close proximity to MHC II⁺ cells. CD8 T cell density in immunogenic PCa and renal cell carcinoma (RCC) was nearly identical. Shallow RB1 and BRCA2 losses, and deep deletions of CHD1, were prevalent; the latter being strongly associated with a dendritic cell gene set in TCGA. Tumor mutation burden was variable; neither high microsatellite instability nor CDK12 alterations were present.

Conclusions: A subset of localized PCa is immunogenic, manifested by PD-L1 expression and CD8⁺ T cell content comparable to RCC. The CD8⁺ T cells include effector cells and exhausted progenitor cells, which may be expanded by ICIs. Genomic losses of RB1, BRCA2, and CHD1 may be drivers of this phenotype. These findings indicate that immunotherapies may be effective in biomarker-selected subpopulations of localized PCa patients.

Lef1/Tcfs family, which includes Lef1, Tcf1, Tcf3, and Tcf4, is required for the transcriptional activation induced by β-catenin. However, whether all the members play the same role in colon carcinogenesis is not clear. We found that Lef1 and Tcf1, but not Tcf3 and Tcf4, were upregulated at both mRNA and protein level with the formation of colon tumor in AOM-DSS mouse model. The same profiles were seen in human specimens with the evolvement from adenoma to adenocarcinoma. Additionally, Lef1 and Tcf1 were correlated with a subgroup of Wnt target genes, including Lgr5, a key gene of intestinal stem cell. Further studies supported the role of Tcf1 on sphere formation and transcriptional regulation of Lgr5 in vitro. Interestingly, 3' UTR of each Lef1/Tcfs member were targeted by diverse miRNAs, which were negatively correlated with respective member in human colon cancer specimens. Furthermore, these miRNAs were verified to repress Tcf1 and Lef1 in vitro. Taken together, Lef1 and Tcf1 showed oncogenic effect in colonic carcinogenesis. Cellular context of miRNAs might play important roles in carcinogenesis by altering the expression pattern of Lef/Tcfs members. © 2017 Wiley Periodicals, Inc.

Recent studies on chronic viral infections have defined a novel PD-1+ TCF1+ stem-like CD8 T cell subset that gives rise to the terminally differentiated exhausted CD8 T cells. In this study, we performed T cell receptor (TCR)β sequencing of virus-specific CD8 T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection to examine the TCR diversity and lineage relationship of these two functionally distinct subsets. We found that >95% of the TCR repertoire of the exhausted CD8 T cell subset was shared with the stem-like CD8 T cells. The TCR repertoires of both CD8 T cell subsets were composed mostly of a few dominant clonotypes but there was slightly more breadth and diversity in the stem-like CD8 T cells compared to their exhausted counterpart (∼40 versus ∼15 GP33+ clonotypes; ∼20 versus ∼7 GP276+ clonotypes). Interestingly, the breadth of the TCR repertoire was broader during the early stages (day 8) of the chronic infection compared to the later stages (day 45-60) showing that there was a narrowing of the TCR repertoire during chronic infection (∼2-fold GP33+ and GP276+ stem-like subset; ∼10-fold GP33+ and ∼5-fold GP276+ exhausted subset). In contrast, during acute LCMV infection the TCR repertoire was much broader in both GP33-specific effector (∼160 clonotypes) and memory CD8 T cells (∼160 clonotypes). Overall, our data demonstrate that the virus-specific CD8 T cell TCR repertoire is broad and remains stable after acute LCMV infection, but it contracts and is narrower during chronic infection. Our study also shows that the repertoire of the exhausted CD8 T cell subset is almost completely derived from the stem-like CD8 T cell subset during established chronic LCMV infection.IMPORTANCE CD8 TCR repertoires responding to chronic viral infections (HIV, HCV, EBV, and CMV) have limited breadth and diversity. How these repertoires change and are maintained throughout the chronic infection are unknown. We thus characterized the LCMV-specific CD8 TCR repertoires of stem-like and terminally exhausted subsets generated during chronic LCMV infections. During chronic LCMV infections, the repertoires started diverse but became more clonal at the late time point. Further, the exhausted subset was composed of dominant clonotypes that were shared with the stem-like subset. Together, we demonstrate that the TCR repertoire contracts over time and is almost exclusively derived from the stem-like subset late during the persistent viral infection. Our data suggest that dominant clonotypes in the exhausted subset are derived from a diverse pool of stem-like clonotypes which may be contributing to the clonality observed during chronic viral infections.

Maturation of lymphoid cells is controlled by the action of stage and lineage-restricted transcription factors working in concert with the general transcription and chromatin remodeling machinery to regulate gene expression. To better understand this functional interplay, we used Biotin Identification in human embryonic kidney cells to identify proximity interaction partners for GATA3, TCF7 (TCF1), SPI1, HLF, IKZF1, PAX5, ID1, and ID2. The proximity interaction partners shared among the lineage-restricted transcription factors included ARID1a, a BRG1-associated factor complex component. CUT&RUN analysis revealed that ARID1a shared binding with TCF7 and GATA3 at a substantial number of putative regulatory elements in mouse T cell progenitors. In support of an important function for ARID1a in lymphocyte development, deletion of Arid1a in early lymphoid progenitors in mice resulted in a pronounced developmental arrest in early T cell development with a reduction of CD4⁺CD8⁺ cells and a 20-fold reduction in thymic cellularity. Exploring gene expression patterns in DN3 cells from Wt and Arid1a-deficient mice suggested that the developmental block resided in the DN3a to DN3b transition, indicating a deficiency in β-selection. Our work highlights the critical importance of functional interactions between stage and lineage-restricted factors and the basic transcription machinery during lymphocyte differentiation.

Oral squamous cell carcinoma (OSCC) is among the most prevalent cancers of the head and neck. This study revealed that isoorientin attenuates OSCC cell stemness and epithelial-mesenchymal transition potential through the inhibition of JAK/signal transducer and activator of transcription 3 (STAT3) and Wnt/β-catenin signaling in cell lines. Our findings indicated that isoorientin is a potential inhibitor of β-catenin/STAT3 in vitro and in vivo. We analyzed possible synergism between isoorientin and cisplatin in OSCC. A sulforhodamine B assay, colony formation assay, tumorsphere-formation assay, and Wnt reporter activity assay were used for determining cell invasion, cell migration, drug cytotoxicity, and cell viability with potential molecular mechanisms in vitro. Isoorientin reduced the expression of p-STAT3, β-catenin, and p-GSK3 as well as downstream effectors TCF1/TCF7 and LEF1 and significantly reduced β-catenin colocalization in the nucleus. Isoorientin markedly strengthened the cytotoxic effects of cisplatin against SAS and SCC-25. Therefore, combining isoorientin and cisplatin treatments can potentially improve the anticancer effect of cisplatin. Isoorientin inhibited the tumorigenicity and growth of OSCC through the abrogation of Wnt/β-catenin/STAT3 signaling in vivo. Thus, isoorientin disrupted the β-catenin signaling pathway through the inactivation of STAT3 signaling. In conclusion, targeting OSCC-SC-mediated stemness with isoorientin to eradicate OSCC-SCs may be an effective strategy for preventing relapse and metastasis of OSCC and providing long-term survival benefits.

Keywords: Isoorientin; Oral squamous cell carcinoma; STAT3/Wnt/β-catenin pathway.

Immune checkpoint blockers (ICBs)-based immunotherapy has revolutionised oncology. However, the benefits of ICBs are limited to only a subset of patients. Herein, the biomarkers-driven application of ICBs promises to increase their efficacy. Such biomarkers include lymphocytic IFNγ-signalling and/or cytolytic activity (granzymes and perforin-1) footprints, whose levels in pre-treatment tumours can predict favourable patient survival following ICB-treatment. However, it is not clear whether such biomarkers have the same value in predicting survival of patients receiving first-line anti-CTLA4 ICB-therapy, and subsequently anti-PD1 ICB-therapy (i.e., sequential ICB-immunotherapy regimen). To address this, we applied highly integrated systems/computational immunology approaches to existing melanoma bulk-tumour transcriptomic and single-cell (sc)RNAseq data originating from immuno-oncology clinical studies applying ICB-treatment. Interestingly, we observed that CD8⁺/CD4⁺T cell-associated IFNγ-signalling or cytolytic activity signatures fail to predict tumour response in patients treated with anti-CTLA4 ICB-therapy as a first-line and anti-PD1 ICB-therapy in the second-line setting. On the contrary, signatures associated with early memory CD8⁺/CD4⁺T cells (integrating TCF1-driven stem-like transcriptional programme), capable of resisting cell death/apoptosis, better predicted objective response rates to ICB-immunotherapy, and favourable survival in the setting of sequential ICB-immunotherapy. These observations suggest that sequencing of ICB-therapy might have a specific impact on the T cell-repertoire and may influence the predictive value of tumoural immune biomarkers.

T cell factor 1 (Tcf1) is known as a critical mediator for natural killer (NK) cell development and terminal maturation. However, its essential targets and precise mechanisms involved in early NK progenitors (NKP) are not well clarified. To investigate the role of Tcf1 in NK cells at distinct developmental phases, we employed three kinds of genetic mouse models, namely, Tcf7 ^fl/fl Vav ^Cre/+, Tcf7 ^fl/fl CD122 ^Cre/+ and Tcf7 ^fl/fl Ncr1 ^Cre/+ mice, respectively. Similar to Tcf1 germline knockout mice, we found notably diminished cell number and defective development in BM NK cells from all strains. In contrast, Tcf7 ^fl/fl Ncr1 ^Cre/+ mice exhibited modest defects in splenic NK cells compared with those in the other two strains. By analyzing the published ATAC-seq and ChIP-seq data, we found that Tcf1 directly targeted 110 NK cell-related genes which displayed differential accessibility in the absence of Tcf1. Along with this clue, we further confirmed that a series of essential regulators were expressed aberrantly in distinct BM NK subsets with conditional ablating Tcf1 at NKP stage. Eomes, Ets1, Gata3, Ikzf1, Ikzf2, Nfil3, Runx3, Sh2d1a, Slamf6, Tbx21, Tox, and Zeb2 were downregulated, whereas Spi1 and Gzmb were upregulated in distinct NK subsets due to Tcf1 deficiency. The dysregulation of these genes jointly caused severe defects in NK cells lacking Tcf1. Thus, our study identified essential targets of Tcf1 in NK cells, providing new insights into Tcf1-dependent regulatory programs in step-wise governing NK cell development.

Keywords: NK cell; NKP; Tcf1; development; mice; targets.

T cell identity is established during thymic development, but how it is maintained in the periphery remains unknown. Here we show that ablating Tcf1 and Lef1 transcription factors in mature CD8⁺ T cells aberrantly induces genes from non-T cell lineages. Using high-throughput chromosome-conformation-capture sequencing, we demonstrate that Tcf1/Lef1 are important for maintaining three-dimensional genome organization at multiple scales in CD8⁺ T cells. Comprehensive network analyses coupled with genome-wide profiling of chromatin accessibility and Tcf1 occupancy show the direct impact of Tcf1/Lef1 on the T cell genome is to promote formation of extensively interconnected hubs through enforcing chromatin interaction and accessibility. The integrative mechanisms utilized by Tcf1/Lef1 underlie activation of T cell identity genes and repression of non-T lineage genes, conferring fine control of various T cell functionalities. These findings suggest that Tcf1/Lef1 control global genome organization and help form intricate chromatin-interacting hubs to facilitate promoter-enhancer/silencer contact, hence providing constant supervision of CD8⁺ T cell identity and function.

CD8 T cell responses against different tumor neoantigens occur simultaneously, yet little is known about the interplay between responses and its impact on T cell function and tumor control. In mouse lung adenocarcinoma, we found that immunodominance is established in tumors, wherein CD8 T cell expansion is predominantly driven by the antigen that most stably binds MHC. T cells responding to subdominant antigens were enriched for a TCF1⁺ progenitor phenotype correlated with response to immune checkpoint blockade (ICB) therapy. However, the subdominant T cell response did not preferentially benefit from ICB due to a dysfunctional subset of TCF1⁺ cells marked by CCR6 and Tc17 differentiation. Analysis of human samples and sequencing datasets revealed that CCR6⁺ TCF1⁺ cells exist across human cancers and are not correlated with ICB response. Vaccination eliminated CCR6⁺ TCF1⁺ cells and dramatically improved the subdominant response, highlighting a strategy to optimally engage concurrent neoantigen responses against tumors.

Keywords: CCR6; CD8 T cell; TCF1; Tc17; checkpoint blockade; immunodominance; lung cancer; neoantigen; vaccine.

Hepatocyte nuclear factor-1 alpha (HNF-1 alpha; gene symbol, TCF1) forms dimers with itself as well as with HNF-1 beta and regulates the expression of several liver-specific genes. Recently, a dimerization cofactor of hepatocyte nuclear factor-1 alpha, called DCOH, has been identified. Here, we report the chromosomal localization of the genes for this cofactor to chromosomes 10 in both humans and mice by Southern blot analyses of somatic cell hybrids.

The specificity of cytotoxic T lymphocyte (CTL) responses generated in the presence of lymphokines was studied. Thymic responder cells were activated in the presence of stimulator cells that differed in their metabolic activity. After 5 days of culture, the cytotoxic response was estimated in a 4-h 51Cr-release test. Coculture of thymic responders with irradiated splenic stimulator cells in the presence of interleukin 2(IL 2) led to preferential cytolysis of target cells that expressed the same histocompatibility antigens as the cells used for sensitization. Addition of T cell cytotoxicity-inducing factor 1 (TCF1), however, to those cultures made the presence of stimulator cells unnecessary and induced cytotoxic responses against all target cells tested, including target cells syngeneic to the responder cells. This activation was neither due to contaminating mitogen nor to the effect of heterologous serum in the assay system. The conclusion of these findings was that either polyclonal activation of CTL was induced by TCF1 or that some specific CTL clones differentiated into unrestricted killer cells under the influence of TCF1.

The identification of regulatory elements in silico is an important method for inferring function from sequence data, but it is uncertain which methods are best. We used a novel combination of expression data from a TCF1 knockout mouse (TCF1 codes for the transcription factor HNF1a), and human and mouse genome sequences, to search 2kb upstream of 28 genes downregulated in TCF1 null mice compared to wild type mice. We wrote software (http://www.BindGene.org) to search for and assign p-values to potential binding sites. This identified 8 genes as candidates for being directly regulated by HNF1a: LIPC, CRP, F13B, PRODH2, HSD17B2, SCL7A9, SLC16A7, PAH. There was evidence for conservation between human and mouse for all these regions identified as containing putative binding sites. For three of the genes identified there was experimental evidence for an HNF1a binding site. For comparison we also examined 25 genes up-regulated in TCF1 null mice; only one gene was selected and there was little evidence for conservation of this putative binding site between human and mouse. This result was consistent with HNF1a being a gene transcription activator. Another 6 up-regulated genes had unexpectedly high p-values, suggesting that possibly HNF1a sites have been suppressed from these genes. In conclusion, gene expression data from transgenic animals lacking a transcription factor can be used to identify DNA binding sites for that factor.

BACKGROUND

Monogenic forms of diabetes are often diagnosed by chance, due to the variety of clinical presentation and limited experience of the diabetologists with this kind of diabetes. Aim of this study was to evaluate clinical parameters for an efficient screening.

METHODS

Clinical parameters were: negative diabetes-specific antibodies at onset of diabetes, positive family history of diabetes, and low to moderate insulin requirements after one year of diabetes treatment. Molecular testing was performed through sequencing of the programming regions of HNF-4alpha (MODY 1), glucokinase (MODY 2) and HNF-1alpha/TCF1 (MODY 3) and in one patient the HNF-1beta/TCF2 region (MODY 5). 39 of 292 patients treated with insulin were negative for GADA and IA2A, and 8 (20.5%) patients fulfilled both other criteria.

RESULTS

Positive molecular results were found in five (63%) patients (two with MODY 2, two with MODY 3, one with MODY 5). At diabetes onset, the mean age of the 5 patients with MODY was 10.6 ± 5.3 yrs (range 2.6-15 yrs), HbA(1c) was 8.4 ± 3.1 % (6.5-13.9%), mean diabetes duration until diagnosis of MODY was 3.3 ± 3.6 yrs (0.8-9.6 yrs) with insulin requirements of 0.44 ± 0.17 U/kg/d (0.2-0.6 U/kg/d). Patients with MODY 3 were changed from insulin to repaglinide, those with MODY 2 were recommended discontinuing insulin treatment.

CONCLUSIONS

In patients with negative diabetes-specific antibodies at onset of diabetes, with a positive family history, and low to moderate insulin needs a genetic screening for MODY is indicated. Watchful consideration of these clinical parameters may lead to an early genetic testing, and to an adequate treatment.

Non-insulin-dependent diabetes mellitus (NIDDM) is the most common form of diabetes, affecting 5% of the general population. Genetic factors play an important role in the development of the disease. While in other populations NIDDM is usually diagnosed after the fifth decade of life, in Mexico a large proportion of patients develop the disease at an early age (between the third and the fourth decade). In Caucasian population, mutations in the glucokinase gene, the <em>TCF1</em>, and <em>TCF1</em>4 genes, have been identified in a subgroup of early-onset NIDDM patients denominated MODY (maturity-onset diabetes of the young), which show an autosomal dominant pattern of inheritance. As a first step in the molecular characterization of Mexican families displaying early-onset NIDDM we searched for mutations in the glucokinase gene through SSCP analysis and/or direct sequencing in 26 individuals from 22 independent families, where at least four can be classified as MODY. No mutations were detected in the exons or the intron-exon boundaries of the gene in any of the screened individuals. The phenotype and clinical profile of some of the studied patients is compatible with that of patients carrying mutations in the <em>TCF1</em> or <em>TCF1</em>4 genes, while others may carry mutations in different loci. Through computer simulation analysis we identified at least four informative families which will be used for further linkage studies.