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BBA - Gene Regulatory Mechanisms (v.1809, #4-6)

Editorial Board (pp. i).

Induced malignant genome reprogramming in somatic cells by testis-specific factors by Jin Wang; Anouk Emadali; Aurore Le Bescont; Mary Callanan; Sophie Rousseaux; Saadi Khochbin (pp. 221-225).

Germline cell differentiation is controlled by a specific set of genes whose expression is tightly locked into the repressed state in somatic cells. Large-scale epigenome alterations, now evidenced in nearly all cancers, lead to aberrant activation of these normally silenced genes, as attested by the many reports describing the expression of testis-specific factors, known as cancer-testis genes, in various cancer cells. Here, based on the literature, we argue that off-context activity of some of the testis-specific epigenome regulators can reprogram the somatic cell epigenome toward a malignant state by favoring self-renewal and sustaining cell proliferation under stressful conditions, thereby constituting a major oncogenic mechanism.► This review discusses the very exciting possibility that the off-context activity of germline-specific genes initiates a process of epigenetic reprogramming and could strongly contribute to malignant transformation.

Keywords: BRD4-NUT; NUT; BRDT; ATAD2; iPS

Juvenile hormone binding protein core promoter is TATA-driven with a suppressory element by Anna Niewiadomska-Cimicka; Marcin Schmidt; Ozyhar Andrzej Ożyhar; Davy Jones; Grace Jones; Marian Kochman (pp. 226-235).

The hemolymph juvenile hormone binding protein (JHBP) plays a key role in transporting juvenile hormone (JH) to target tissues and in protecting JH from the degradation by nonspecific esterases. Juvenile hormone esterase (JHE) removes JH signal at precisely defined insect developmental stages. The functional analysis of regulatory elements in the core promoter has been described only for the jhe gene. In this report we define the core promoter functional elements in the Galleria mellonella jhbp gene. It appears that jhbp core promoter is under strong control of TATA box and the transcription site (tss). In contrast to regulation of the jhe gene, the jhbp core promoter contains a sequence which directly suppresses jhbp expression. Evidences are provided for the contribution of the will die slowly (WDS) suppressory protein in jhbp basal transcription. We have also shown that the activity of the jhbp core promoter can be regulated by JHIII, and lesser so by 20E.► Jhbp core promoter is TATA box driven and suppressed by will die slowly protein. ► Core DPE-like element participates in suppressing jhbp gene expression. ► Tss and Inr-like elements play an opposite effect in jhbp gene control. ► In contrast to jhbp the jhe core promoter is controlled by activatory elements only

Keywords: Abbreviations; 20E; 20-hydroxyecdysone; BR-C; Broad-Complex; BRE; TFIIB recognition element; DCE; downstream core element; DEE; downstream enhancing element; DPE; downstream promoter element; EcR; ecdysone receptor; EMSA; electrophoretic mobility shift assay; Hi5; High Five cells; Inr; Initiator element; JH; juvenile hormone; JHBP; juvenile hormone binding protein; PRE; proximal response element; SCP 1; super core promoter 1; tsp; transcription start point; tss; transcription start site; MTE; motif ten element; USP; utraspiracle; WDS; will die slowly; XCPE1; X Core Promoter Element 1 jhbp; WDS; JH; Juvenile hormone; Core promoter

Functional and sequence analysis of human neuroglobin gene promoter region by Wei Zhang; Zhipeng Tian; Sha Sha; Lydia Yuk Luen Cheng; Sjaak Philipsen; Kian-Cheng Tan-Un (pp. 236-244).

Neuroglobin (Ngb), a recently found oxygen-binding protein belonging to the vertebrate globin family, is mainly expressed in neurons of brains and eyes. Current studies have revealed diverse potential functions of Ngb and it was found to be able to reduce the severity of stroke and Alzheimer's disease, implying its importance in brains. However, the mechanism of Ngb regulation of transcription has not been elucidated yet. In this study, we analyzed the 5′-flanking region of human neuroglobin gene ( NGB) and identified a transcription start site (TSS) located at −306bp relative to the translation start site ATG. We characterized the proximal promoter of NGB and found two GC-boxes located at −16 and +30bp relative to the TSS which are bound by transcription factor Sp1 and Sp3. Mutation of either GC-box led to a significant reduction in NGB promoter activity, while overexpression of Sp1 and Sp3 resulted in activation of the promoter. However, two putative NRSE sites (−359 and −127bp relative to the TSS) apparently showed no influence on NGB tissue-specific expression. Treatment of two non-neuronal cell lines HeLa and BEAS-2B with 5-aza-2′-deoxycytidine remarkably induced NGB expression, suggesting a potential role of DNA methylation in regulating NGB tissue-specific expression.► One transcription start site of human neuroglobin ( NGB) locates at −306bp relative to the translation start codon ATG. ► The proximal promoter of NGB lies between −164 to +306bp relative to the transcription start site of NGB. ► Both Sp1 and Sp3 are transactivators of NGB. ► Treatment of HeLa and BEAS-2B with 5-aza-2′-deoxycytidine induces NGB transcription.

Keywords: Neuroglobin; (; NGB; ); Transcription start site; Promoter; Sp1; Sp3; Methylation

Retinal expression of the X-linked juvenile retinoschisis (RS1) gene is controlled by an upstream CpG island and two opposing CRX-bound regions by Dominik Kraus; Marcus Karlstetter; Yana Walczak; Daniela Hilfinger; Thomas Langmann; Bernhard H.F. Weber (pp. 245-254).

X-linked juvenile retinoschisis (XLRS) is an orphan retinal disease in males caused by mutations in the RS1 gene. Previously we have characterized cone-rod homeobox (CRX)-responsive elements in the promoter region of RS1 driving selective gene expression in the retina. Here, we expanded our identification and functional analysis of cis-regulatory elements controlling quantitative expression of RS1 in vitro and in vivo. Sequence analysis identified a CpG island 3kb upstream of the transcription start site (TSS). In addition, chromatin immunoprecipitation coupled to microarrays (ChIP-Chip) targeting the retinal transcription factor CRX was performed. Thereby, we identified a second CRX-bound region (CBR2) in the first intron of RS1 which contains six evolutionarily conserved CRX binding motifs. In vitro luciferase reporter gene assays and dsRed reporter electroporation of mouse retinal organ cultures demonstrated a strong constitutive and orientation-independent enhancing effect of the upstream CpG island. The intronic CBR2 potently suppressed CBR1-driven RS1 promoter activity in vitro but failed to regulate a CBR1-reporter in short-term cultured mouse retinae. We conclude that a CpG island enhancer and two CBRs may act in a combinatorial fashion to fine-tune RS1 transcript levels in the retina.► We identified an upstream CpG island in the human retinoschisin (RS1) gene known to be associated with X-linked juvenile retinoschisis. ► We further identified two highly conserved CRX-bound regions (CBRs) at the RS1 gene locus. ► Electroporations of reporters into ex vivo mouse retinal organ cultures were performed and confirmed our in vitro data. ► CBR1 in the proximal promoter drives basic RS1 expression. ► RS1 expression was enhanced by the CpG island and modulated by the intronic CBR2.

Keywords: X-linked juvenile retinoschisis; RS1 gene; cis; -regulatory elements; CRX-responsive element; CpG island

The p150 subunit of the histone chaperone Caf-1 interacts with the transcriptional repressor Gfi1 by Florian Heyd; Riyan Chen; Khashayar Afshar; Ingrid Saba; Claude Lazure; Katharina Fiolka; Moroy Tarik Möröy (pp. 255-261).

Modification of histones is critically involved in regulating chromatin structure and gene expression. The zinc finger protein Gfi1 silences transcription by recruiting a complex of histone modifying enzymes such as LSD-1/CoRest and HDAC-1 to target gene promoters. Here we present evidence that Gfi1 forms a complex with the p150 subunit of the histone chaperone chromatin assembly factor-1 (Caf-1). Gfi1 and p150 interact at endogenous expression levels and co-localize in distinct sub-nuclear structures. We show that p150 enhances Gfi1-mediated transcriptional repression and that it occupies Gfi1 target gene promoters in transfected cells and primary murine T cells only in the presence of Gfi1. Finally, size exclusion chromatography shows a fraction of p150 to coelute with Gfi1, LSD-1 and HDAC-1 and thus provides evidence that p150 is part of the Gfi1 repression complex. Since p150 binds directly to histones H3 and H4, our findings suggest that p150 may link the DNA-bound Gfi1 repressor complex to histones enabling modifications required for transcriptional silencing.► The DNA binding transcriptional repressor Gfi1 binds to the p150 subunit of the histone chaperone Caf-1. ► Both Gfi1 and Caf-1 co-localize in human cell nuclei. ► Caf-1 is required for the transcriptional repressor activity of Gfi1. ► Caf-1 is recruited to Gfi1 target gene promoters. ► Caf-1 is contained in a larger repressor complex that contains Gfi1 and histone modifying enzymes such as HDAC1 and LSD1.

Keywords: Transcriptional repression; Gfi1; p150 subunit of Caf-1

Induction of hypoxia inducible factor (HIF-1α) in rat kidneys by iron chelation with the hydroxypyridinone, CP94 by Jin Hyen Baek; Chad E.N. Reiter; Dominador J. Manalo; Paul W. Buehler; Robert C. Hider; Abdu I. Alayash (pp. 262-268).

Hypoxia inducible factor (HIF-1α) is a master regulator of tissue adaptive responses to hypoxia whose stability is controlled by an iron containing prolyl hydroxylase domain (PHD) protein. A catalytic redox cycle in the PHD's iron center that results in the formation of a ferryl (Fe+⁴) intermediate has been reported to be responsible for the hydroxylation and subsequent degradation of HIF-1α under normoxia. We show that induction of HIF-1α in rat kidneys can be achieved by iron reduction by the hydroxypyridin-4 one (CP94), an iron chelator administered intraperitoneally in rats. The extent of HIF protein stabilization as well as the expression of HIF target genes, including erythropoietin (EPO), in kidney tissues was comparable to those induced by known inhibitors of the PHD enzyme, such as desferrioxamine (DFO) and cobalt chloride (CoCl₂). In human kidney cells and in vitro PHD activity assay, we were able to show that the HIF-1α protein can be stabilized by addition of CP94. This appears to inactivate PHD; and thus prevents the hydroxylation of HIF-1α. In conclusion, we have identified the inhibition of iron-binding pocket of PHD as an underlying mechanism of HIF induction in vivo and in vitro by a bidentate hydroxypyridinone.► 1,2-Diethyl-hydroxypyridin-4-one (CP94) dosing results in HIF-1α stabilization and activity in rat kidney. ► CP94 is among a class of bidentate hydroxypyridinone compounds with iron-chelating properties. ► CP94 inhibits the catalytic iron pocket domain of prolyl hydroxylase (PHD), which blocks HIF-1α hydroxylation. ► Unlike HIF-1 mimicker, desferrioxamine (DFO) that target PHD, CP94 can be administered orally or intraperitoneally.

Keywords: Iron chelator; Hypoxia inducible factor; Erythropoietin

EVI1 up-regulates the stress responsive gene SIRT1 which triggers deacetylation and degradation of EVI1 by Anjan Kumar Pradhan; Nivedita Kuila; Sneha Singh; Soumen Chakraborty (pp. 269-275).

EVI1 (Ecotropic Viral Integration site I), which was originally identified as a site of viral integration in murine myeloid tumors, encodes a complex protein required for embryogenesis. The gene is known to express inappropriately in many types of human myeloid leukemias and solid tumors. Forced expression of EVI1 in murine hematopoietic precursor cells lead to abnormal differentiation and increased proliferation. EVI1 encodes two sets of zinc finger domains due to which it behaves as a transcriptional factor. However, except a few, the targets of EVI1 are not well understood and hence also the mechanism by which it initiates oncogenesis is not very clear. In this report, we show that SIRT1, a histone deacetylase is a direct target of EVI1. In vivo chromatin immunoprecipitation assay revealed that EVI1 binds to the promoter region of SIRT1 approximately 1kb upstream of the transcription start site. The functionality of the site was deduced by luciferase assay which showed that EVI1 significantly increases the SIRT1 promoter activity. SIRT1 was also found to be up regulated in cell lines and in chronic myeloid leukemia patient samples where EVI1 was detected. Over expression of SIRT1 in cells shows that it interacts with EVI1 and this interaction lead to the deacetylation of the protein. Upon deacetylation the stability of EVI1 was found to be affected which was negatively regulated by nicotinamide (NAM). Our results thus identify an EVI1-SIRT1 axis in the regulation of EVI1 activity suggesting a possible role of SIRT1 in EVI1 positive neoplasms.► EVI1 binds to the SIRT1 promoter element and up regulates SIRT1 transactivity. ► CML cases with EVI1 positivity showed higher SIRT1 expression. ► SIRT1 and EVI1 formed a complex in cells. ► SIRT1 deacetylated EVI1 and also rendered the protein susceptible to degradation. ► EVI1-SIRT1 axis can also regulate p53 activity.

Keywords: EVI1; SIRT1; PCAF; Deacetylation; CML

DNA-binding and regulatory properties of the transcription factor and putative tumor suppressor p150^Sal2 by Hongcang Gu; Dawei Li; Chang K. Sung; Hyungshin Yim; Philip Troke; Thomas Benjamin (pp. 276-283).

The product of the SALL2 protein p150^Sal2 is a multi-zinc finger transcription factor with growth arrest and proapoptotic functions that overlap those of p53. Its DNA-binding properties are unknown. We have used a modified SELEX procedure with purified p150^Sal2 and a pool of oligonucleotides of random sequence to identify those that are bound preferentially by p150^Sal2. The consensus sequence for optimal binding in vitro is GGG(T/C)GGG, placing p150^Sal2 among a large group of GC box-binding proteins including the Sp1 family of transcription factors. A triple zinc finger motif in p150^Sal2 similar to that in Sp1 is required for DNA binding. p150^Sal2 and Sp1 show evidence of co-operative binding in vitro and of interaction in vivo. p150^Sal2, a known activator of the CDK inhibitor p21^Cip1/Waf1 (p21), binds to regions of the human p21 promoter that contain variations of the consensus sequence in multiple copies. p150^Sal2 is also shown to bind to the BAX promoter with similar elements and to activate its expression following an apoptotic stimulus. These results demonstrate binding of p150^Sal2 to two natural promoters with GC elements related to the optimal binding sequence defined in vitro and whose regulation is important for suppression of tumor growth.► DNA-binding properties of p150^Sal2 have been defined in vitro and in vivo. ► p150^Sal2 binds to GC-rich elements in the p21^Cip1/Waf1 and BAX promoters. ► p150^Sal2 activates the BAX promoter following an apoptotic stimulus. ► These DNA-binding properties of p150^Sal2 underlie its tumor suppressor-like functions.

Keywords: Abbreviations; ChIP; chromatin immunoprecipitation; CS; optimal consensus sequence for DNA binding; in vitro; EMSA; electrophoretic mobility shift assay; GST; glutathione S-transferase; HOSE cells; established human ovarian surface epithelial cells; p21; the cyclin-dependent kinase inhibitor p21; ^Cip1/Waf1; SALL2; the Spalt-like gene 2; SELEX; systematic evolution of ligands by exponential enrichmentSALL2 transcription factor; DNA binding; Growth arrest; Apoptosis; Polyoma virus

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BBA - Gene Regulatory Mechanisms (v.1809, #4-6)

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BBA - Gene Regulatory Mechanisms (v.1809, #4-6)