Mathias Jenal

Scavenger Chemokine (CXC Motif) Receptor 7 (CXCR7) Is a Direct Target Gene of HIC1 (Hypermethylated in Cancer 1)

The tumor suppressor gene HIC1 (Hypermethylated in Cancer 1) that is epigenetically silenced in many human tumors and is essential for mammalian development encodes a sequence-specific transcriptional repressor. The few genes that have been reported to be directly regulated by HIC1 include ATOH1, FGFBP1, SIRT1, and E2F1. HIC1 is thus involved in the complex regulatory loops modulating p53-dependent and E2F1-dependent cell survival and stress responses. We performed genome-wide expression profiling analyses to identify new HIC1 target genes, using HIC1-deficient U2OS human osteosarcoma cells infected with adenoviruses expressing either HIC1 or GFP as a negative control. These studies identified several putative direct target genes, including CXCR7, a G-protein-coupled receptor recently identified as a scavenger receptor for the chemokine SDF-1/CXCL12. CXCR7 is highly expressed in human breast, lung, and prostate cancers. Using quantitative reverse transcription-PCR analyses, we demonstrated that CXCR7 was repressed in U2OS cells overexpressing HIC1. Inversely, inactivation of endogenous HIC1 by RNA interference in normal human WI38 fibroblasts results in up-regulation of CXCR7 and SIRT1. In silico analyses followed by deletion studies and luciferase reporter assays identified a functional and phylogenetically conserved HIC1-responsive element in the human CXCR7 promoter. Moreover, chromatin immunoprecipitation (ChIP) and ChIP upon ChIP experiments demonstrated that endogenous HIC1 proteins are bound together with the C-terminal binding protein corepressor to the CXCR7 and SIRT1 promoters in WI38 cells. Taken together, our results implicate the tumor suppressor HIC1 in the transcriptional regulation of the chemokine receptor CXCR7, a key player in the promotion of tumorigenesis in a wide variety of cell types.

The scavenger chemokine (C-X-C motif)receptor7 CXCR7 is a direct target gene of hypermethylated in cancer 1 HIC1

The tumor suppressor gene HIC1 (Hypermethylated in Cancer 1) that is epigenetically silenced in many human tumors and is essential for mammalian development encodes a sequence-specific transcriptional repressor. The few genes that have been reported to be directly regulated by HIC1 include ATOH1, FGFBP1, SIRT1, and E2F1. HIC1 is thus involved in the complex regulatory loops modulating p53-dependent and E2F1-dependent cell survival and stress responses. We performed genome-wide expression profiling analyses to identify new HIC1 target genes, using HIC1-deficient U2OS human osteosarcoma cells infected with adenoviruses expressing either HIC1 or GFP as a negative control. These studies identified several putative direct target genes, including CXCR7, a G-protein-coupled receptor recently identified as a scavenger receptor for the chemokine SDF-1/CXCL12. CXCR7 is highly expressed in human breast, lung, and prostate cancers. Using quantitative reverse transcription-PCR analyses, we demonstrated that CXCR7 was repressed in U2OS cells overexpressing HIC1. Inversely, inactivation of endogenous HIC1 by RNA interference in normal human WI38 fibroblasts results in up-regulation of CXCR7 and SIRT1. In silico analyses followed by deletion studies and luciferase reporter assays identified a functional and phylogenetically conserved HIC1-responsive element in the human CXCR7 promoter. Moreover, chromatin immunoprecipitation (ChIP) and ChIP upon ChIP experiments demonstrated that endogenous HIC1 proteins are bound together with the C-terminal binding protein corepressor to the CXCR7 and SIRT1 promoters in WI38 cells. Taken together, our results implicate the tumor suppressor HIC1 in the transcriptional regulation of the chemokine receptor CXCR7, a key player in the promotion of tumorigenesis in a wide variety of cell types.

HIC1 tumour suppressor gene is suppressed in acute myeloid leukaemia and induced during granulocytic differentiation

A hallmark of acute myeloid leukaemia (AML) is a block in differentiation caused by deregulated gene expression. The tumour suppressor Hypermethylated In Cancer 1 (HIC1) is a transcriptional repressor, which is epigenetically silenced in solid cancers. HIC1 mRNA expression was found to be low in 128 patient samples of AML and CD34+ progenitor cells when compared with terminally differentiated granulocytes. HIC1 mRNA was induced in a patient with t(15;17)‐positive acute promyelocytic leukaemia receiving all‐trans retinoic acid (ATRA) therapy. We therefore investigated whether HIC1 plays a role in granulocytic differentiation and whether loss of function of this gene might contribute to the differentiation block in AML. We evaluated HIC1 mRNA levels in HL‐60 and U‐937 cells upon ATRA‐induced differentiation and in CD34+ progenitor cells after granulocyte colony‐stimulating factor‐induced differentiation. In both models of granulocytic differentiation, we observed significant HIC1 induction. When HIC1 mRNA was suppressed in HL‐60 cells using stably expressed short hairpin RNA targeting HIC1, granulocytic differentiation was altered as assessed by CD11b expression. Bisulphite sequencing of GC‐rich regions (CpG islands) in the HIC1 promoter provided evidence that the observed suppression in HL‐60 cells was not because of promoter hypermethylation. Our findings indicate a role for the tumour suppressor gene HIC1 in granulocytic differentiation. Low expression of HIC1 may very well contribute to pathogenic events in leukaemogenesis.

DAPK2 is a novel E2F1/KLF6 target gene involved in their proapoptotic function.

Death-associated protein kinase 2 (DAPK2) belongs to a family of proapoptotic Ca2+/calmodulin-regulated serine/threonine kinases. We recently identified DAPK2 as an enhancing factor during granulocytic differentiation. To identify transcriptional DAPK2 regulators, we cloned 2.7 kb of the 5′-flanking region of the DAPK2 gene. We found that E2F1 and Krüppel-like factor 6 (KLF6) strongly activate the DAPK2 promoter. We mapped the E2F1 and KLF6 responsive elements to a GC-rich region 5′ of exon 1 containing several binding sites for KLF6 and Sp1 but not for E2F. Moreover, we showed that transcriptional activation of DAPK2 by E2F1 and KLF6 is dependent on Sp1 using Sp1/KLF6-deficient insect cells, mithramycin A treatment to block Sp1-binding or Sp1 knockdown cells. Chromatin immunoprecipitation revealed recruitment of Sp1 and to lesser extent that of E2F1 and KLF6 to the DAPK2 promoter. Activation of E2F1 in osteosarcoma cells led to an increase of endogenous DAPK2 paralleled by cell death. Inhibition of DAPK2 expression resulted in significantly reduced cell death upon E2F1 activation. Similarly, KLF6 expression in H1299 cells increased DAPK2 levels accompanied by cell death that is markedly decreased upon DAPK2 knockdown. Moreover, E2F1 and KLF6 show cooperation in activating the DAPK2 promoter. In summary, our findings establish DAPK2 as a novel Sp1-dependent target gene for E2F1 and KLF6 in cell death response.

The tumor suppressor gene hypermethylated in cancer 1 is transcriptionally regulated by E2F1

The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened a set of transcription factors for regulation of a human HIC1 promoter reporter. We found that E2F1 strongly activates the full-length HIC1 promoter reporter. Promoter deletions and mutations identified two E2F responsive elements in the HIC1 core promoter region. Moreover, in vivo binding of E2F1 to the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53−/− hepatocellular carcinoma cell line Hep3B led to an increase of endogenous HIC1 mRNA, although bisulfite genomic sequencing of the HIC1 promoter revealed that the region bearing the two E2F1 binding sites is hypermethylated. In addition, endogenous E2F1 induced by etoposide treatment bound to the HIC1 promoter. Moreover, inhibition of E2F1 strongly reduced the expression of etoposide-induced HIC1. In conclusion, we identified HIC1 as novel E2F1 transcriptional target in DNA damage responses. (Mol Cancer Res 2009;7(6):916–22)

CLEC5A (MDL-1) is a novel PU.1 transcriptional target during myeloid differentiation

C-type lectin domain family 5, member A (CLEC5A), also known as myeloid DNAX activation protein 12 (DAP12)-associating lectin-1 (MDL-1), is a cell surface receptor strongly associated with the activation and differentiation of myeloid cells. CLEC5A associates with its adaptor protein DAP12 to activate a signaling cascade resulting in activation of downstream kinases in inflammatory responses. Currently, little is known about the transcriptional regulation of CLEC5A. We identified CLEC5A as one of the most highly induced genes in a microarray gene profiling experiment of PU.1 restored myeloid PU.1-null cells. We further report that CLEC5A expression is significantly reduced in several myeloid differentiation models upon PU.1 inhibition during monocyte/macrophage or granulocyte differentiation. In addition, CLEC5A mRNA expression was significantly lower in primary acute myeloid leukemia (AML) patient samples than in macrophages and granulocytes from healthy donors. Moreover, we found activation of a CLEC5A promoter reporter by PU.1 as well as in vivo binding of PU.1 to the CLEC5A promoter. Our findings indicate that CLEC5A expression in monocyte/macrophage and granulocytes is regulated by PU.1.

The anti-apoptotic gene BCL2A1 is a novel transcriptional target of PU.1.

Carillo for JAK2 exon 12 mutation analysis; to Dr Isabelle Corre for expert advice; and to Mrs Danielle Pineau for excellent technical help. The study was supported by grants from the Ligue Nationale contre le Cancer (Comité de Loire-Atlantique, Comité du Morbihan, Comité d’Ille-et-Vilaine) and from the Association pour la Recherche contre le Cancer (ARC). CC and MB benefited from scholarships from the French Ministry of Research.

Transcriptional regulation of MIR29B by PU.1 (SPI1) and MYC during neutrophil differentiation of acute promyelocytic leukaemia cells

Lineage commitment of haematopoietic cells is tightly regulated through an intricate network of molecular pathways and cascades of genes including microRNAs (miRNAs). Moreover, recent studies show that abnormal expression of miRNAs directly contributes to haematological malignancies, such as acute myeloid leukaemia (AML), chronic myeloid leukaemia (CML) and myelodysplastic syndromes (MDS) (Fabbri et al, 2008; Starczynowski et al, 2011). miRNAs are 19–25 nucleotide long, non-protein coding RNAs that regulate gene expression by binding to partially complementary sequences in the 3¢-untranslated regions (3¢-UTR) of their target mRNAs. Acute promyelocytic leukaemia (APL) is characterized by the t(15;17) translocation resulting in the oncogenic PML-RARA fusion gene. Pharmacologial doses of all-trans retinoic acid (ATRA) are used to directly target PML-RARA for degradation and, in combination with chemotherapy, have been successfully used in the treatment of APL patients (de The & Chen, 2010). A discrete number of miRNAs modulated upon ATRA treatment of APL cells in vitro have been published (Garzon et al, 2007; Saumet et al, 2009). Among them MIR29B, a key miRNA induced during neutrophil differentiation of APL cells. The tumour suppressor MIR29B plays a role in targeting leukaemic oncogenes such as DNA methyltransferase 3A/B (DNMT3A/DNMT3B) in AML cells (Garzon et al, 2009). In addition, high MIR29B levels were associated with a response to the demethylating agent decitabine in AML (Blum et al, 2010). The present study aimed to confirm a role for MIR29B in the neutrophil differentiation of APL cells and to analyse the transcriptional regulation of MIR29B during this process. We first measured MIR29B expression during ATRAinduced neutrophil differentiation of NB4 and HT93 APL cell lines. MIR29B was clearly induced in both APL cell lines (Fig 1A). Furthermore, MIR29B expression was markedly induced in an APL patient undergoing ATRA therapy (Fig 1B). We then measured MIR29B in blast cells of primary AML patients of different French-American-British (FAB) subtypes (M0-M4) as well as in mature neutrophils from healthy donors. We observed significantly lower MIR29B expression levels in AML patient samples as compared to normal granulocytes from healthy individuals (Fig 1C). Lastly, to further define the role of MIR29B in neutrophil differentiation, we inhibited its expression by antisense MIR29B (antiMIR29B) in HT93 APL cells. Neutrophil differentiation in anti-MIR29B expressing HT93 cells was significantly reduced compared to control cells as evidenced by lower CD11b levels (Fig 1D,E). As a control for the functionality of our antisense MIR29B, we analysed the protein expression of the reported MIR29B target gene DNMT3B. We found markedly elevated DNMT3B protein levels in anti-MIR29B expressing AML cells indicating successful inhibition of MIR29B (Fig 1F). In general, our results indicated that MIR29B expression was not only suppressed in APL but generally in AML patients, indicating that the low expression is associated with an immature myeloid phenotype. In summary, these data suggest that MIR29B is associated with granulocytic differentiation and further support a tumour suppressor function of MIR29B in AML. To assess transcriptonal regulation of MIR29B, we analysed its promoter region for myeloid transcription factor binding sites. We identified two putative responsive elements of the myeloid master regulator PU.1 (SPI1) upstream of the MIR29B2/MIR29C (MIR29B2/C) gene (Fig 2A). First, to test if PU.1 activates MIR29B2/C transcription, we cloned a 1Æ4-kb putative MIR29B2/C promoter carrying the two PU.1 response elements and a shorter 0Æ7-kb construct encompassing only one PU.1 binding element into luciferase reporter vectors. PU.1 specifically increased promoter activity of both constructs to the same levels in a dose-dependent manner (Fig 2B). Our experiments indicate that the PU.1 binding site between the MIR29B2 and MIR29C coding regions is the major PU.1 responsive element. Next, we showed direct binding of PU.1 to the MIR29B2/C promoter in vivo using chromatin immunoprecipitation (ChIP) assays (Fig 2C). To analyse PU.1-dependent MIR29B induction during neutrophil differentiation, we assessed its regulation in HT93 PU.1 knockdown cells upon ATRA treatment. Indeed, inhibiting PU.1 in HT93 cells resulted in 50% reduction of ATRAinduced MIR29B expression (Fig 2D, left panel). Successful PU.1 knockdown was confirmed by real-time quantitative polymerase chain reaction and Western blotting in HT93 shPU.1_1 and _2 knockdown cells (Figs 2D, right panel and 2E). PU.1 induced MIR29B by directly interacting with its promoter and we confirmed an essential role for PU.1 in MIR29B2/C regulation upon myeloid differentiation by ChIP, luciferase reporter and PU.1 knockdown experiments. Chang et al (2008) placed the transcriptional start site of MIR29B2/C within a conserved regulatory region located 20 kb upstream of the MIR29B2/C cluster on chromosome 1. However, we identified a functional PU.1 binding site in the proximal MIR29B2/C promoter, indicating additional regulatory eleCorrespondence

Inactivation of the hypermethylated in cancer 1 tumour suppressor--not just a question of promoter hypermethylation?

The chromosomal region 17p13.3 is frequently deleted or epigenetically silenced in a variety of human cancers. It includes the hypermethylated in cancer 1 (HIC1) gene placed telomerically to the p53 tumour suppressor gene. HIC1 encodes a transcriptional repressor, and its targets identified to date are genes involved in proliferation, tumour growth and angiogenesis. In addition, HIC1 functionally cooperates with p53 to suppress cancer development. Frequent allelic loss at position 17p13.1 in human cancers often points to mutations of the tumour suppressor p53. However, in a variety of cancer types, allelic loss of the short arm of chromosome 17 may hit regions distal to p53 and, interestingly, without leading to p53 mutations. Furthermore, the neighbouring region 17p13.3 often shows loss of heterozygosity or DNA hypermethylation in various types of solid tumours and leukaemias. In line with this concept, Wales et al. described a new potential tumour suppressor in this region and named it hypermethylated in cancer 1 (HIC1). Further, it was shown that in the majority of cases hypermethylation of this chromosomal region leads to epigenetic inactivation of HIC1. A role for HIC1 in tumour development is further supported by a mouse model, since various spontaneous, age- and gender-specific malignant tumours occur in heterozygous Hic1+/- knockout mice. Furthermore, exogenously delivered HIC1 leads to a significant decrease in clonogenic survival in cancer cell lines. This review highlights the role of HIC1 inactivation in solid tumours and particularly in leukaemia development.