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Controlled renewal of the epithelium with precise cell distribution and gene expression patterns is essential for colonic function. GATA6 is expressed in the colonic epithelium, but its function in the colon is currently unknown. To define GATA6 function in the colon, we conditionally deleted Gata6 throughout the epithelium of small and large intestines of adult mice. In the colon, Gata6 deletion resulted in shorter, wider crypts, a decrease in proliferation, and a delayed crypt-to-surface epithelial migration rate. Staining techniques and electron microscopy indicated deficient maturation of goblet cells, and coimmunofluorescence demonstrated alterations in specific hormones produced by the endocrine L cells and serotonin-producing cells. Specific colonocyte genes were significantly downregulated. In LS174T, the colonic adenocarcinoma cell line, Gata6 knockdown resulted in a significant downregulation of a similar subset of goblet cell and colonocyte genes, and GATA6 was found to occupy active loci in enhancers and promoters of some of these genes, suggesting that they are direct targets of GATA6. These data demonstrate that GATA6 is necessary for proliferation, migration, lineage maturation, and gene expression in the mature colonic epithelium.

GATA4, a transcription factor expressed in the proximal small intestine but not in the distal ileum, maintains proximal–distal distinctions by multiple processes involving gene repression, gene activation, and cell fate determination. Friend of GATA (FOG) is an evolutionarily conserved family of cofactors whose members physically associate with GATA factors and mediate GATA-regulated repression in multiple tissues. Using a novel, inducible, intestine-specific Gata4 knock-in model in mice, in which wild-type GATA4 is specifically inactivated in the small intestine, but a GATA4 mutant that does not bind FOG cofactors (GATA4ki) continues to be expressed, we found that ileal-specific genes were significantly induced in the proximal small intestine (P

Background and aims The transcription factor GATA4 is expressed throughout most of the small intestine except distal ileum, and restricts expression of the apical sodium-dependent bile acid transporter (ASBT), the rate-limiting intestinal bile acid transporter, to distal ileum. The hypothesis was tested that reduction of GATA4 activity in mouse small intestine results in an induction of bile acid transport in proximal small intestine sufficient to restore bile acid absorption and homeostasis after ileocaecal resection (ICR). Methods Bile acid homeostasis was characterised in non-surgical, sham or ICR mice using two recombinant Gata4 models in which Asbt expression is induced to different levels. Results Reduction of intestinal GATA4 activity resulted in an induction of ASBT expression, bile acid absorption and expression of bile acid-responsive genes in proximal small intestine, and a reduction of luminal bile acids in distal small intestine. While faecal bile acid excretion and bile acid pool size remained unchanged, the bile acid pool became more hydrophilic due to a relative increase in tauro-β-muricholate absorption. Furthermore, proximal induction of Asbt in both Gata4 mutant models corrected ICR-associated bile acid malabsorption, reversing the decrease in bile acid pool size and increase in faecal bile acid excretion and hepatic cholesterol 7α-hydroxylase expression. Conclusions Reduction of intestinal GATA4 activity induces bile acid absorption in proximal small intestine without inducing major changes in bile acid homeostasis. This induction is sufficient to correct bile acid malabsorption caused by ICR in mice.

The terminal differentiation phases of intestinal development in mice occur during cytodifferentiation and the weaning transition. Lactase-phlorizin hydrolase (LPH), liver fatty acid binding protein (Fabp1), and sucrase-isomaltase (SI) are well-characterized markers of these transitions. With the use of gene inactivation models in mature mouse jejunum, we have previously shown that a member of the zinc finger transcription factor family (Gata4) and hepatocyte nuclear factor-1alpha (Hnf1alpha) are each indispensable for LPH and Fabp1 gene expression but are both dispensable for SI gene expression. In the present study, we used these models to test the hypothesis that Gata4 and Hnf1alpha regulate LPH, Fabp1, and SI gene expression during development, specifically focusing on cytodifferentiation and the weaning transition. Inactivation of Gata4 had no effect on LPH gene expression during either cytodifferentiation or suckling, whereas inactivation of Hnf1alpha resulted in a 50% reduction in LPH gene expression during these same time intervals. Inactivation of Gata4 or Hnf1alpha had a partial effect ( approximately 50% reduction) on Fabp1 gene expression during cytodifferentiation and suckling but no effect on SI gene expression at any time during development. Throughout the suckling period, we found a surprising and dramatic reduction in Gata4 and Hnf1alpha protein in the nuclei of absorptive enterocytes of the jejunum despite high levels of their mRNAs. Finally, we show that neither Gata4 nor Hnf1alpha mediates the glucocorticoid-induced precocious maturation of the intestine but rather are downstream targets of this process. Together, these data demonstrate that specific intestinal genes have differential requirements for Gata4 and Hnf1alpha that are dependent on the developmental time frame in which they are expressed.

Pediatric acute myeloid leukemia (AML) is a heterogeneous disease and 30-40% of the patients still die. Prognosis is dependent on relevant genetic aberrations. Although many driving genetic alterations causing AML have been defined, in ~20% of the pediatric AML patients the oncogenic events remain unidentified. The ETS-Variant gene 6 (ETV6) encodes a transcription factor that functions as a tumor suppressor gene and is required for proper hematopoiesis in the bone marrow niche. Point mutations, deletions and translocations can lead to silencing of the gene, resulting in loss of transcriptional repression activity. ETV6 aberrations strongly associate with leukemia. In pediatric B-cell precursor acute lymphoblastic leukemia, translocation ETV6/RUNX1 occurs in ~25% of cases. Mutations in ETV6 are identified in ~25% of early immature T-cell ALL and also reported as event in adult AML (Van Vlierberghe et al, J Exp Med 2011; Barjesteh van Waalwijk van Doorn-Khosrovani et al, Oncogene 2005). We previously reported that pediatric AML patients can be divided in three clusters based on HOX-expression; (1) low HOXA/B expression, (2) high HOXA and low HOXB expression, and (3) high HOXA/B expression, and identified new repetitive genetic abnormalities in the third cluster, especially in NUP98. Cluster 1 is mainly represented by core-binding factor (CBF) AML, but in ~20% of these cases we did not find specific genetic abnormalities. Helton et al presented ETV6 aberrations in pediatric CBF-AML at ASH 2011, identified with whole genome sequencing, and with poor clinical outcome. We hypothesized that ETV6 aberrations might reduce the number of patients without known driving abnormality, especially in the low HOXA/B cluster. We screened a large representative de novo pediatric AML cohort for ETV6 mutations in exons 2-8 with direct sequencing, for ETV6 deletions by multiplex ligation-dependent probe amplification and for ETV6 translocations using split signal FISH, and analyzed outcome. In a well-characterized de novo pediatric AML cases with available gene-expression data, 6/275 (2.2%) patients had mutations affecting the predicted amino acid sequence of ETV6 and one had a silent mutation, 4/259 (1.5%) had an ETV6 deletion and 6/65 (9.2%) patients an MNX1/ETV6 translocation. Additionally, we identified 3 cases with a positive split signal FISH suggestive of a break in which ETV6 is involved, and a similar gene expression profile was found in these three cases. The aberrations of ETV6 were seen in patients of all three HOX-groups; n=9, n=6 and n=4 for cluster 1, 2 and 3 respectively. In patients with an ETV6 mutation (n=6) or deletion (n=4) 13 and 38 genes, respectively, were significantly up-regulated, including CLDN5,DPEP1 and BIRC7. This is consistent with the up-regulated genes in functional studies silencing ETV6 in LOUCY cells (Van Vlierberghe et al, J Exp Med 2011). High expression of BIRC7 has been associated with poor prognosis in adult acute leukemia (El-Mesallamy et al, Leuk Res 2011). The median age of patients with an ETV6-mutation or deletion (n=10) was 11.3 years (range 4.0-15.3) and 40% were female. Median WBC was significantly lower (15.1x109/L vs 47.0x109/L, p We conclude that ETV6 aberrations are rare but recurrent in pediatric AML. ETV6 aberrations predict a poor survival, although there was no evidence for an increased relapse incidence in this small cohort. Disclosures No relevant conflicts of interest to declare.

IKAROS family zinc finger 1 (IKZF1) is a transcription factor involved in lymphoid differentiation that acts as a tumor suppressor. In B-cell precursor acute lymphoblastic leukemia (BCP-ALL) loss of IKZF1 is found in ~15% of the patients, is associated with the presence of BCR/ABL1 (t(9;22)(q34;q11)) and confers a poor clinical outcome. Recent studies suggest that IKZF1 is also involved in myeloid differentiation. The best indication that loss of IKZF1 may contribute to myeloid leukemogenesis are deletions of the short arm of chromosome 7 associated with myeloproliferative-preceded secondary acute myeloid leukemia (AML) in adults, where the commonly deleted region is mapped to the IKZF1 locus (Jager et al,Leukemia 2010). To investigate whether IKZF1 deletions play a role in pediatric AML we screened a representative well-characterized panel of 258 de novo pediatric AML samples with available gene expression data, obtained from the DCOG (The Hague, the Netherlands), the AML–Berliner-Frankfurt-Münster Study Group (Germany and Czech Republic), the Saint-Louis Hospital (Paris, France) and the Royal Hospital for Sick Children (Glasgow, UK) for deletions of the IKZF1 locus on chromosome 7p12.2 using multiplex ligation-dependent probe amplification (MLPA). Median age of the patients was 9.5 years (range 0.1-18.5 years), median white blood cell count was 46.7x109/L (range 1.2-483x109/L). All major cytogenetic subgroups were included and all patients had received intensive cytarabine-anthracycline based pediatric AML therapy. Of 11 patients with an IKZF1 deletion, 8 cases presented with monosomy 7, and 3 cases showed a focal deletion of IKZF1. These focal deletions included the complete IKZF1 gene (n=2) or exons 1-4 (n=1), leading to a loss of IKZF1 function. Focal deletions were confirmed by high-resolution array comparative genome hybridization (array-CGH). Patients with a focal deletion included a 1.5 year old boy diagnosed with AML with a fusion of MNX1/ETV6 who died from relapse; an 11.3 year old girl diagnosed with AML M5 who remains in continuous complete remission (CCR) after salvage therapy for relapse; and a 2.3 year old boy diagnosed with AML M5 in CCR. IKZF1 deleted cases (n=11) did not differ significantly from the other pediatric AML cases (n=247) with regards to age at diagnosis (median age 9.1 years compared to 9.5 years respectively, p=0.41); gender (females 55% versus 42%, p=0.41); or white blood cell count at diagnosis (median 30.2 x109/L versus 47.5 x109/L, p=0.24). No specific FAB morphology subtypes were related to IKZF1 deletions. IKZF1 deleted samples showed either none or various different additional somatic mutations, most frequently activating the RAS pathway with mutations in NRAS or PTPN11 (n=4,). In BCP-ALL IKZF1 deletions are associated with BCR/ABL1 fusions and to test if this was also true for IKZF1 deletions in AML we screened samples for the BCR/ABL1 fusion and all were negative. The 3-year pOS in IKZF1 deleted patients (n=11) was 70±14% versus 63±3% (p=0.82) in IKZF1 wild-type patients (n=231). The 3-year pEFS was 36±15% versus 46±3%, and the 3-year pCIR was 64±16% versus 36±3% (p=0.87 and p=0.09) respectively. Genes differentially expressed in monosomy 7 cases correlated significantly with gene expression changes in focal IKZF1 deleted cases when comparing significant differences to non-IKZF1-deleted cases (n=247). Genes showing increased expression in IKZF1 deleted samples included those involved in myeloid cell cycling and self-renewal, such as HEMGN, FHL2, FZD6, and SETBP1. In summary, we found focal IKZF1 deletions to be rare but recurrent events in pediatric AML. Gene expression patterns suggest that the loss of IKZF1 may be an important determinant in the biology of pediatric AML with monosomy 7. Disclosures No relevant conflicts of interest to declare.

Background & Aims GATA transcription factors regulate proliferation, differentiation, and gene expression in multiple organs. GATA4 is expressed in the proximal 85% of the small intestine and regulates the jejunal-ileal gradient in absorptive enterocyte gene expression. GATA6 is co-expressed with GATA4 but also is expressed in the ileum; its function in the mature small intestine is unknown. Methods We investigated the function of GATA6 in small intestine using adult mice with conditional, inducible deletion of Gata6 , or Gata6 and Gata4 , specifically in the intestine. Results In ileum, deletion of Gata6 caused a decrease in crypt cell proliferation and numbers of enteroendocrine and Paneth cells, an increase in numbers of goblet-like cells in crypts, and altered expression of genes specific to absorptive enterocytes. In contrast to ileum, deletion of Gata6 caused an increase in numbers of Paneth cells in jejunum and ileum. Deletion of Gata6 and Gata4 resulted in a jejunal and duodenal phenotype that was nearly identical to that in the ileum after deletion of Gata6 alone, revealing common functions for GATA6 and GATA4. Conclusions GATA transcription factors are required for crypt cell proliferation, secretory cell differentiation, and absorptive enterocyte gene expression in the small intestinal epithelium.