RUNX1 and GATA2 Regulate the Expression of the SET Oncogene in Acute Myeloid Leukemia

INTRODUCTION. The protein SET (I2PP2A), a potent protein phosphatase 2A (PP2A) inhibitor, has been implicated in many cell processes such as DNA replication, chromatin remodeling and gene transcription, differentiation, migration, and cell-cycle regulation. In fact, SET has been described as an oncogene that regulates important signaling pathways. Our group reported that PP2A inhibition is a common event in AML, and that SET is overexpressed in 28% of acute myeloid leukemia (AML) cases, where it is associated with short overall survival. Moreover, the anti-leukemic effects of the FTY720 and OP449 PP2A-activating drugs in AML cells depend on interaction/sequestration of SET. However, despite the importance of SET overexpression and its prognostic impact in both hematological and solid tumors, there are few data about the mechanisms involved in its regulation. AIM. To characterize the functional promoter region of the SET gene, and to identify transcription factors (TFs) involved in its regulation. RESULTS. Luciferase reporter assays with five truncatedconstructs allowed us to determine a 163bp-region as the minimal promoter region of SET that contains consensus sites for several TFs. Chromatin immunoprecipitation (ChIP) assays confirmed the binding of RUNX1, GATA2, MYC, and SP1. RUNX1 and GATA2 are two essential TFs in hematopoiesis, and localized on the SET promoter when the acetylation state of both histone H3 and H4 and the tri-methylation on H3K4 is high, confirming that they both could act as positive regulators of SET transcription. In silico analysis in large series of adult patient samples with de novo AML recently published by The Cancer Genome Atlas Research Network showed a significant positive correlation between SET and RUNX1 and GATA2 at mRNA level. Furthermore, knockdown of RUNX1 and/or GATA2 triggered SET downregulation, whereas only a simultaneous overexpression of these two TFs caused a significant up-regulation of SET. Interestingly, RUNX1 interacts with GATA2 in both HL-60 and HEL cell lines. Moreover, we found that SP1 is also part of this transcription complex. Altogether, these results show that RUNX1 and GATA2, together with SP1, regulate the transcription of the SET gene. CONCLUSIONS We have defined the minimal promoter region of the SET gene, and have demonstrated that RUNX1 and GATA2 regulate its expression in AML. Moreover, our functional studies demonstrate that RUNX1 and GATA2 form a complex with SP1 that activates the transcription of SET in AML cells. This study opens new directions to further understand the mechanisms of SET overexpressing leukemias. Disclosures No relevant conflicts of interest to declare.

Repression of the RHOH gene by JunD

RhoH is a member of the Rho family of small GTP-binding proteins that lacks GTPase activity. Since RhoH is constantly bound by GTP, it is thought to be constitutively active and controlled predominantly by changes in quantitative expression. RhoH is produced specifically in haematopoietic cells and aberrant expression has been linked to various forms of leukaemia. Transcription of the RHOH gene is the first level at which the quantitative levels of the RhoH protein are regulated. Previous studies have demonstrated that RHOH gene transcription is initiated by three distinct promoter regions designated P1, P2 and P3 that define the 5′ end of exons 1, 2 and 4 respectively. In the present study we report that the P3 promoter is largely responsible for RHOH gene transcription in the B-lymphocytic cell line Raji. The P3 promoter contains a minimal promoter region and a repressor region extending from −236 to +67 and +68 to +245 respectively, relative to the 5′ end of exon 4. Chromatin immunoprecipitation demonstrated that two AP1 (activator protein 1) sites in the minimal promoter region bind JunD. When JUND is overexpressed, the endogenous RHOH gene is repressed; however, when JUND is inhibited, expression of endogenous RHOH is induced both in the Raji cell line and AML (acute myeloid leukaemia) cells. In the HCL (hairy cell leukaemia) cell line JOK-1, induction of RHOH increases expression of the α isoform of protein kinase C. This downstream target of RHOH is also induced in AML cells by JUND inhibition. Collectively, these data indicate that JunD is an inhibitor of RHOH gene expression.

Sp1 and Sp3 mediate NHE2 gene transcription in the intestinal epithelial cells

Our previous studies have identified a minimal Sp1-driven promoter region (nt −36/+116) directing NHE2 expression in mouse renal epithelial cells. However, this minimal promoter region was not sufficient to support active transcription of NHE2 gene in the intestinal epithelial cells, suggesting the need for additional upstream regulatory elements. In the present study, we used nontransformed rat intestinal epithelial (RIE) cells as a model to identify the minimal promoter region and transcription factors necessary for the basal transcription of rat NHE2 gene in the intestinal epithelial cells. We identified a region within the rat NHE2 gene promoter located within nt −67/−43 upstream of transcription initiation site as indispensable for the promoter function in intestinal epithelial cells. Mutations at nt −56/−51 not only abolished the DNA-protein interaction in this region, but also completely abolished NHE2 gene promoter activity in RIE cells. Supershift assays revealed that Sp1 and Sp3 interact with this promoter region, but, contrary to the minimal promoter indispensable for renal expression of NHE2, both transcription factors expressed individually in Drosophila SL2 cells activated rat NHE2 gene promoter. Moreover, Sp1 was a weaker transactivator and when coexpressed in SL2 cells it reduced Sp3-mediated NHE2 basal promoter activity. Furthermore, DNase I footprinting confirmed that nt −58/−51 is protected by nuclear protein from RIE cells. We conclude that the mechanism of basal control of rat NHE2 gene promoter activity is different in the renal and intestinal epithelium, with Sp3 being the major transcriptional activator of NHE2 gene transcription in the intestinal epithelial cells.

Characterization of the 5′-regulatory region of the human thiamin transporter SLC19A3: in vitro and in vivo studies

Transcriptional regulation of expression of the human thiamin transporter-2 (the product of the SLC19A3 gene) is unknown. In this study, we cloned the 5′-regulatory region of the human SLC19A3 gene (2,016 bp), identified the minimal promoter region required for basal activity, demonstrated a critical role for specific cis-regulatory elements in determining the promoter activity, and confirmed activity and physiological relevance of the cloned SLC19A3 promoter in vivo. With the use of transiently transfected human intestinal epithelial Caco-2 cells and 5′-deletion analysis, the minimal promoter region required for basal activity of the SLC19A3 promoter was found to be encoded in a sequence between −77 and +59 by using the start of transcription initiation as position 1. This minimal region was found to contain a number of putative cis-regulatory elements, with a critical role for a stimulating protein-1 (SP1)/GC-box binding site (at position −48/−45 bp) established by means of mutational analysis. With the use of EMSA and supershift assays, the binding of SP1 and SP3 to the minimal promoter region was also demonstrated. In transiently transfected Drosophila SL2 cells, both SP1 and SP3 transactivated the SLC19A3 minimal promoter in a dose-dependent manner and in combination demonstrated an additive stimulatory effect. Functionality of the full-length SLC19A3 promoter was confirmed in vivo in transgenic mice expressing the promoter-luciferase reporter gene. These studies report the first characterization of the SLC19A3 promoter in vitro and in vivo and demonstrate the importance of an SP1 cis-regulatory element in regulating promoter activity of this important human gene.

Identification and Genetic Characterization of a Novel Proteinase, PrtR, from the Human Isolate Lactobacillus rhamnosus BGT10

ABSTRACT A novel proteinase, PrtR, produced by the human vaginal isolate Lactobacillus rhamnosus strain BGT10 was identified and genetically characterized. The prtR gene and flanking regions were cloned and sequenced. The deduced amino acid sequence of PrtR shares characteristics that are common for other cell envelope proteinases (CEPs) characterized to date, but in contrast to the other cell surface subtilisin-like serine proteinases, it has a smaller and somewhat different B domain and lacks the helix domain, and the anchor domain has a rare sorting signal sequence. Furthermore, PrtR lacks the insert domain, which otherwise is situated inside the catalytic serine protease domain of all CEPs, and has a different cell wall spacer (W) domain similar to that of the cell surface antigen I and II polypeptides expressed by oral and vaginal streptococci. Moreover, the PrtR W domain exhibits significant sequence homology to the consensus sequence that has been shown to be the hallmark of human intestinal mucin protein. According to its αS1- and β-casein cleavage efficacy, PrtR is an efficient proteinase at pH 6.5 and is distributed throughout all L. rhamnosus strains tested. Proteinase extracts of the BGT10 strain obtained with Ca2+-free buffer at pH 6.5 were proteolytically active. The prtR promoter-like sequence was determined, and the minimal promoter region was defined by use of prtR-gusA operon fusions. The prtR expression is Casitone dependent, emphasizing that nitrogen depletion elevates its transcription. This is in correlation with the catalytic activity of the PrtR proteinase.

Cooperative Action of the Catabolite Activator Protein and AraC In Vitro at the araFGH Promoter

ABSTRACT Full activation of transcription of the araFGHpromoter, pFGH , requires both the catabolite activator protein (CAP) and AraC protein. AtpFGH , the binding site for CAP is centered at position −41.5, an essential binding site for AraC is centered at position −79.5, and a second, nonessential binding site is centered at position −154.5. In this work, we used the minimal promoter region required for in vivo activation of pFGH to examine the roles of CAP and AraC in stimulating formation of open complexes at pFGH . Migration retardation assays of open complexes showed that RNA polymerase binds exceptionally tightly to the AraC-CAP-pFGH complex and that the order of addition of proteins to the initiating complex is important. Similar assays with RNA polymerase containing truncated alpha subunits suggest that AraC interacts with the C-terminal domain of the alpha subunit. Finally, AraC protein also acts to prevent the improper binding of RNA polymerase at a pseudo promoter near the truepFGH promoter.