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BBA - Gene Regulatory Mechanisms (v.1779, #10)

Editorial Board (pp. ii).

NPR1 preferentially binds to the DNA-inactive form of Arabidopsis TGA2 by Christopher Johnson; Amy Mhatre; Jonathan Arias (pp. 583-589).

Systemic acquired resistance (SAR) is triggered by hormone defense cues and is associated with the onset of expression of pathogenesis-related (PR) genes that encode for anti-microbial proteins in plants. In the case of PR-1, transcriptional activation involves promoter-specific recruitment of transcription factors such as TGA2 through a mechanism that may involve transient physical interaction with the NPR1 protein. This stimulus-inducible recruitment process has yet to be fully explained at the functional and mechanistic level. To investigate this question further, we initially looked to see whether NPR1 preferentially forms a complex with the DNA bound or unbound fraction of TGA2. As shown here, NPR1 appears to preferentially interact with the non-DNA bound fraction of TGA2. We subsequently mutated this transcription factor to identify key residues in its conserved carboxyl terminal (CT) domain that mediate complex formation with NPR1. These approaches revealed that two non-overlapping regions of the CT domain of TGA2 bind independently to NPR1. The specificity and biological significance of these findings were inferred with a mutant form of NPR1 that fails to activate SAR in vivo. These and other findings raise the possibility that NPR1 may transiently interact with the DNA unbound fraction of TGA2 to promote its recruitment to an active form on cognate target promoters.

Keywords: Basic/leucine-zipper domain; bZIP; Dimerization; NPR1; TGA factor; TGA2

Transcriptional repression by leukaemia-associated ETO family members can be independent of oligomerization and coexpressed hSIN3B and N-CoR by André Olsson; Inge Olsson; Rakesh Singh Dhanda (pp. 590-598).

The leukaemia-associated eight-twenty-one (ETO) family members ETO, MTG16 (Myeloid Translocation Gene on chromosome 16) and MTGR1 (Myeloid Transforming Gene-Related protein1) are putative transcriptional repressor proteins, which form complexes with coregulatory nuclear corepressors such as SIN3 (SWI-Independent) and N-CoR (Nuclear receptor Co Repressor). In acute myeloid leukaemia (AML), fusion proteins involving the transcription factor AML1 and corepressors ETO or MTG16 are recurrently found. We investigated transcriptional repression by the ETO family members ETO and MTG16 with attention to the conserved Nervy Homology Regions (NHRs) and the interacting corepressors human SIN3B (hSIN3B) and N-CoR. Transcriptional repression was examined in a cell line by a GAL4-thymidine kinase luciferase reporter to which the corepressors were tethered through a binding domain. ETO- and MTG16-mediated repression was found to be independent of deletion of the oligomerization NHR2, but deletion of NHR4 and in particular combined deletion of NHR2 and NHR4 lowered the capacity for repression. An interaction was observed between the corepressors hSIN3B and N-CoR and these two proteins cooperated for transcriptional repression independent of co-transfected ETO and MTG16. Transcriptional repression mediated by ETO and MTG16 was only slightly strengthened by coexpression of hSIN3B or N-CoR and was dependent on HDAC activity. Our data indicate that ETO family member-mediated oligomerization and repression can be distinct events and that interaction between ETO family members and hSIN3B or N-CoR may not necessarily strengthen transcriptional repression.

Keywords: AML1-ETO; ETO; MTG16; hSIN3B; Leukaemia

Methylation of the prominin 1 TATA-less main promoters and tissue specificity of their transcript content by Victor V. Pleshkan; Tatyana V. Vinogradova; Eugene D. Sverdlov (pp. 599-605).

Prominin 1 (PROM1, CD133) is a unique transmembrane glycoprotein encoded by the PROM1 gene. It is a cell surface marker of various stem cells including hematopoietic, prostatic epithelial, pancreatic, leukemic, liver cancer, and colorectal cancer stem cells. Here, we studied tissue specificity of PROM1 transcription isoforms and the methylation level of its two main promoters (P1 and P2) in different human cell lines. Only transcripts lacking the 4th exon (the CD133.s1 form) were expressed in cell lines studied. Moreover, these transcripts, if sufficiently abundant, were initiated simultaneously and independently from both promoters P1 and P2. In cell lines with low levels of the total PROM1 transcript, the transcription was likely initiated from other promoters. Promoter P1 was hypermethylated in all cell lines under study, and therefore, methylation can hardly play an important role in its regulation. In contrast, the methylation of promoter P2 was tissue specific, and hypomethylation of this promoter is probably necessary but not sufficient for efficient transcription of the PROM1 gene. Therefore, we report an unusual instance of different mechanisms of transcription activity regulation for two closely located promoters of the same gene.

Keywords: Regulation of gene expression; Alternative promoter; Methylation; Transcriptional isoform; CD133

Suppression of EDAG gene expression by phorbol 12-myristate 13-acetate is mediated through down-regulation of GATA-1 by Chang-Yan Li; Fang Fang; Wang-Xiang Xu; Cheng-wang Xu; Yi-Qun Zhan; Zhi-Dong Wang; Ya-li Ding; Yong-Hui Li; Hui-Bin Sun; Xiao-Ming Yang (pp. 606-615).

EDAG, a hematopoietic tissue-specific protein, is involved in the regulation of proliferation, differentiation and apoptosis of hematopoietic cells. In this study, a dose-dependent inhibition of EDAG expression by PMA was observed in K562 cells. The responsive element for the PMA-induced inhibition was contained in the region between −211 and +32bp of the EDAG gene promoter. By oligonucleotide-directed mutagenesis, EMSA, ChIP and transient transfection assays, we found that two tandem repeat GATA-1 sites in the promoter of EDAG gene played an important role in the PMA-mediated down-regulation of the EDAG gene expression in K562 cells. The kinetics of EDAG expression during PMA induction showed that the levels of EDAG expression were down-regulated concomitantly with GATA-1 down-expression. Decreased GATA-1 expression by siRNA reduced expression of EDAG in K562 cells, and restored expression of GATA-1 significantly rescued EDAG expression from PMA-mediated suppression. Overexpression of EDAG in K562 cells inhibited the megakaryocytic differentiation induced by PMA which raised the interesting possibility that PMA induced K562 cells differentiation toward megakaryocytic phenotype through, at least in part, the inhibition of EDAG expression. In vivo analysis confirmed that EDAG was highly expressed in primitive progenitor cells and down-regulated in megakaryocytes which was consistent with the expression pattern of GATA-1. Furthermore, PKC and MAPK specific inhibitors treatment attenuated the down-regulation of EDAG induced by PMA. Taken together, these results suggest that the inhibition of the EDAG gene by PMA is mediated through down-regulation of transcription factor GATA-1 and involved the PKC/MAPK signaling pathway.

Keywords: Abbreviations; EDAG; Erythroid differentiation-associated gene; HSCs; hematopoietic stem cells; PMA; phorbol 12-myristate 13-acetatae; PBMC; peripheral blood mononuclear cells; PKC; protein kinase C; MAPK; mitogenic activated protein kinaseEDAG; Transcriptional regulation; GATA-1; PMA; Megakaryocytic differentiation

Analysis of the transcriptional regulation of the FABP2 promoter haplotypes by PPARγ/RXRα and Oct-1 by Mike Böhme; Inke Nitz; Frank Döring; Maja Klapper (pp. 616-621).

Variants of the human intestinal fatty acid binding protein 2 gene (FABP2) are associated with traits of the metabolic syndrome. Relevant FABP2 promoter polymorphisms c.-80_-79insT, c.-136_-132delAGTAG, c.-168_-166delAAGinsT, c.-260G>A, c.-471G>A, and c.-778G>T result in two haplotypes A and B. Activation of haplotypes by rosiglitazone stimulated PPARγ/RXRα leads to 2-fold higher activity of haplotype B than A. As shown by chimeric FABP2 promoter constructs, the higher responsiveness of FABP2 haplotype B is mainly but not solely determined by polymorphism c.-471G>A. As shown by EMSA and promoter–reporter assays, Oct-1 interacts with the −471 region of FABP2 promoters, induces the activities of both FABP2 promoter haplotypes and abolishes the different activities of haplotypes induced by rosiglitazone activated PPARγ/RXRα. In conclusion, our findings suggest a functional role of PPARγ/RXRα and Oct-1 in the regulation of the FABP2 gene.

Keywords: FABP2; PPARg; Oct-1; Promoter analysis; EMSA

Effects of poly(A)-binding protein on the interactions of translation initiation factor eIF4F and eIF4F·4B with internal ribosome entry site (IRES) of tobacco etch virus RNA by Mateen A. Khan; Hasan Yumak; Daniel R. Gallie; Dixie J. Goss (pp. 622-627).

In wheat germ, the interaction between poly(A)-binding protein and eukaryotic initiation factor eIF 4G increases the affinity of eIF4E for the cap by 20–40-fold. Recent findings that wheat germ eIF4G is required for interaction with the IRES, pseudoknot 1 (PK1), of tobacco etch virus to promote cap-independent translation led us to investigate the effects of PABP on the interaction of eIF4F with PK1. The fluorescence anisotropy data showed addition of PABP to eIF4F increased the binding affinity ∼2.0-fold for PK1 RNA as compared with eIF4F alone. Addition of both PABP and eIF4B to eIF4F enhance binding affinity to PK1 about 4-fold, showing an additive effect rather than the large increase in affinity shown for cap binding. The van't Hoff analyses showed that PK1 RNA binding to eIF4F, eIF4F·PABP, eIF4F·4B and eIF4F·4B·PABP is enthalpy-driven and entropy-favorable. PABP and eIF4B decreased the entropic contribution 65% for binding of PK1 RNA to eIF4F. The lowering of entropy for the formation of eIF4F·4B·PABP–PK1 complex suggested reduced hydrophobic interactions for complex formation. Overall, these results demonstrate the first direct effect of PABP on the interaction of eIF4F and eIF4F·4B with PK1 RNA.

Keywords: Abbreviations; m; ⁷; G; 7-methyl-guanosine; eIF; eukaryotic initiation factor; PABP; poly(A)-binding protein; TEV; tobacco etch virus; IRES; internal ribosomal entry site; nt; nucleotide; PK; pseudoknot; PMSF; phenylmethylsulfonyl fluoride; DEAE; diethylaminoethyleIF's; IRES; TEV; Equilibrium; Thermodynamic; Fluorescence anisotropy

Differential expression of microRNA species in organs of hibernating ground squirrels: A role in translational suppression during torpor by Pier Jr. Morin; Adrian Dubuc; Kenneth B. Storey (pp. 628-633).

Mammalian hibernation includes long periods of profound torpor where the rates of all metabolic processes are strongly suppressed in a reversible manner. We hypothesized that microRNAs (miRNAs), small non-coding transcripts that bind to mRNA, could play a role in the global suppression of mRNA translation when animals enter torpor. Selected miRNA species (4–9 of the following: mir-1, mir-24, mir-15a, mir-16, mir-21, mir-122a, mir-143, mir-146 and mir-206) were evaluated in four organs of euthermic versus hibernating ground squirrels, Spermophilus tridecemlineatus using RT-PCR. Levels of mir-24 transcripts were significantly reduced in heart and skeletal muscle of torpid animals as were mir-122a levels in the muscle. Mir-1 and mir-21 both increased significantly in kidney during torpor by 2.0- and 1.3-fold, respectively. No changes were found for the four miRNA species analyzed in liver. Protein levels of Dicer, an enzyme involved in miRNA processing were also quantified in heart, kidney and liver. Dicer protein levels increased by 2.7-fold in heart during hibernation but decreased by 60% in kidney. These data are the first report that differential regulation of miRNA levels occurs during mammalian hibernation and they provide a mechanism for reversible gene silencing during torpor that can be rapidly reversed to allow renewed translation of mRNA when animals arouse back to euthermia.

Keywords: MicroRNA; Hibernation; Spermophilus tridecemlineatus; Dicer; Reversible control of translation; Gene silencing

Interactions between upstream and core promoter sequences determine gene expression and nucleosome positioning in tobacco PR-1a promoter by Niraj Lodhi; Amol Ranjan; Mala Singh; Rakesh Srivastava; Sudhir Pratap Singh; Chandra Prakash Chaturvedi; Suraiya Anjum Ansari; Samir V. Sawant; Rakesh Tuli (pp. 634-644).

The expression of PR-1a gene in tobacco is accompanied by changes in the chromatin architecture over its promoter region. The transcription initiates when the gene is induced in defense response, a condition that can be simulated experimentally by external application of salicylic acid. Mutagenesis of the core promoter sequence established that the TATA-box was critical to the expression of PR-1a gene. In order to study functional specificity between the core promoter and upstream activator region, the native core promoter was exchanged with that of a heterologous salicylic acid inducible promoter, Pcec. The core promoter and the activator region of PR-1a together determine its tightly regulated expression, slow kinetics of induction by SA and several fold induction of expression. In uninduced state, a single nucleosome was present over the core promoter of PR-1a. It masked both the TATA-box and the transcription initiation region. The transcriptional activation of the promoter by SA was accompanied by shift in the position of this nucleosome. The chimeric promoters failed to show inducibility or gave very low level of induction. They showed failure in shifting the nucleosome from the core promoter region. The promoter Pcec expressed constitutively at a high uninduced level in spite of a nucleosome over the TATA-box region. However, in this case, the nucleosome did not mask the transcript initiation region. The TATA-box nucleosome was shifted as the expression increased further, following induction by SA. A fully induced Pcec had the TATA-box fully exposed, though a weak nucleosome appeared on the +1 region. The results support a close relationship among promoter sequence architecture, nucleosome positioning and PR-1a expression.

Keywords: TATA-box; Initiator; Histone modification; Chimeric promoter; Gene expression

PDX-1 interaction and regulation of the Pancreatic Derived Factor (PANDER, FAM3B) promoter by Brant R. Burkhardt; Joshua R. Cook; Robert A. Young; Bryan A. Wolf (pp. 645-651).

Pancreatic Derived Factor (PANDER) is a novel cytokine-like protein dominantly expressed within the endocrine pancreas. Our previous study demonstrated that the PANDER promoter was both tissue-specific and glucose-responsive. Surrounding the PANDER transcriptional start site are several putative A- and E-Box elements that may bind to the various pancreatic transcriptional factors of MafA, BETA2/NeuroD, and Pancreatic Duodenal Homeobox-1 (PDX-1). To characterize the transcriptional regulatory factors involved in PANDER gene expression, we performed co-transfection reporter gene analysis and demonstrated upregulation by all three transcription factors, with the greatest individual increase stemming from PDX-1. Potential binding of PDX-1 to A box (TAAT) regions of the PANDER promoter was demonstrated by chromatin immunoprecipitation (ChIP) and further corroborated by electrophoretic mobility shift assay (EMSA). Binding of PDX-1 to the A box regions was inhibited by mutagenized (TAGT) oligonucleotides. Site-directed mutagenesis of the three PDX-1 A box binding motifs revealed that A box sites 2 and 3 in combination were critical for maximal gene expression and deletion resulted in a 82% reduction in promoter activity. Furthermore, deletion of A box sites 2 and 3 completely diminished the glucose-responsiveness of the PANDER promoter. Our findings demonstrate that PANDER is a potential PDX-1 target gene and the A box sites within the promoter region are critical for basal and glucose-stimulated PANDER expression.

Keywords: Abbreviations; BETA2; BETA2 (B-cell E box transactivator 2)/NeuroD; ChIP; chromatin immunoprecipitation; DMEM; Dulbecco's modified Eagle's medium; MafA; musculoaponeurotic fibrosarcoma A; PANDER; PANcreatic DERived factor; PDX-1; pancreatic/duodenal homeobox-1PANDER; Promoter; Islet, Glucose; Reporter gene; β-TC3

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BBA - Gene Regulatory Mechanisms (v.1779, #10)