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BBA - Gene Regulatory Mechanisms (v.1799, #7)
Mediator subunits: Gene expression pattern, a novel transcript identification and nuclear localization in human endothelial progenitor cells by Monica Rienzo; Jens Nagel; Amelia Casamassimi; Alfonso Giovane; Steffen Dietzel; Claudio Napoli (pp. 487-495).
Mediator of RNA polymerase II transcription subunits (MEDs) serve to promote the assembly, activation, and regeneration of transcription complexes on core promoters during the initiation and re-initiation phases of transcription. There are no studies on the Mediator complex during development of endothelial progenitors (EPCs). Here, we have analysed all known MEDs during the differentiation of EPCs, by expression profile studies at RNA level and, for a limited subset of MED subunits, also at protein level. Since beneficial effects ofl-arginine on EPCs have been described, we have also examined its effect on the expression of Mediator subunit coding genes. Through RT-PCR we have found increased expression for MED12 and decreased levels for MED30 afterl-arginine treatment; Western blot analysis do not agree entirely with the RNA data in the identification of a putative protein product. Furthermore, we have analysed the three-dimensional nuclear positions of MED12 and MED30 genes in the presence ofl-arginine treatment. Our major finding is the identification of a novel transcript of MED30, termed MED30 short (MED30s) generating by alternative splicing. Our results showed that the mRNA of this novel isoform is present only in circulating cells, but it is not expressed in cultured adherent cells.These findings are broadly relevant and will contribute to our understanding of the role of Mediator in eukaryotic gene expression. Despite the need to confirm the in vivo presence of the protein of this novel isoform, the presence of this novel RNA raises the possibility of regulating pathophysiological mechanism in progenitors.
Keywords: Key words; Mediator subunits; Alternative splicing; Endothelial progenitor cells
Reexamination of the electrophile response element sequences and context reveals a lack of consensus in gene function by Hongqiao Zhang; Henry Jay Forman (pp. 496-501).
The electrophile response element (EpRE) is essential for regulation of many genes involved in protection against toxic agents. Putative EpRE core sequences (TGAnnnnGC) are localized in 5′-flanking regions (5′-UTR) of these genes but specificity of the internal bases and whether location affects function has not been refined. The catalytic subunit of human glutamate cysteine ligase (GCLC) gene is well documented to be under EpRE regulation and four sequences having an EpRE “consensus” sequence were reported with only one (EpRE 4) responsive to electrophiles. Using GCLC as a model, we asked whether the internal variable or flanking nucleotides and the location of the sequence were required for functional activity in response to 4-hydroxenonenal (HNE). We found that thirteen putative EpRE core sequences (TGAnnnnGC) were localized in 5′-UTR of GCLC and confirmed that EpRE 4 showed both constitutive and HNE-inducible activity. Four other sequences exhibited only constitutive activity while other putative EpREs demonstrated no activity. Nucleotide mutagenesis demonstrated specific requirements for internal and flanking nucleotides that were specific for the electrophilic response and that a TRE-like sequence within EpRE was essential for basal (non-electrophile-dependent) activity. Furthermore, EpRE 4 relocated to positions of other putative EpREs maintained activity but moving other EpREs to the EpRE 4 location did not. Thus in GCLC, specific flanking and internal nucleotides within EpRE were far more important for function than previously described while location did not influence activity. These two findings bring into question the meaning of the phrase, “consensus sequence” for this important cis element.
Keywords: EpRE; Nrf2; Consensus sequence; Oxidative stress; Antioxidant response element; Glutamate cysteine ligase
Mechanisms of p53-mediated repression of the human polycystic kidney disease-1 promoter by Diederik van Bodegom; Wijnand Roessingh; Andrew Pridjian; Samir S. El Dahr (pp. 502-509).
We previously reported that the tumor suppressor protein p53 participates in a negative feedback loop to fine-tune PKD1 gene expression. This physiological pathway is believed to prevent polycystin-1 overexpression and thus renal cysts. The present study examined the mechanisms of p53-mediated repression of PKD1. The 5′-upstream region of the human PKD1 gene is TATA-less, GC-rich, and contains four consensus p53 binding sites at positions −2.7kb (BS4), −1.2kb (BS3), −0.8kb (BS2), and −0.2kb (BS1), respectively. PKD1 BS1 – 4 are bound to endogenous p53 in vivo and in vitro. Transient transfection assays in inner medullary collecting duct cells revealed that disruption of PKD1 BS1 enhances baseline PKD1 promoter activity; in contrast, disruption of PKD1 BS4 suppressed PKD1 transcription. PKD1 BS1 confers p53-mediated repression when substituted for the p53 enhancer element in the bradykinin B2 receptor gene, indicating that PKD1 BS1 is a bona fide p53 repressor element. Moreover, PKD1 BS1 requires intact BS2-4 and cellular histone deacetylase activity for full functional activity. Indeed, the PKD1 BS1/4 regions are occupied by a complex containing HDAC1/2 and mSin3. These findings suggest a model whereby p53 exerts a biphasic control on PKD1 gene transcription, depending on cellular context and the cognate cis-acting element.
Keywords: Abbreviations; ADPKD; adult polycystic kidney disease; ChIP; chromatin immunoprecipitation; HDAC; histone deacetylasePolycystic kidney disease gene; p53; Gene transcription; Promoter
Regulation of the Drosophila p38b gene by transcription factor DREF in the adult midgut by Joung-Sun Park; Young-Shin Kim; Joong-Gook Kim; Shin-Hae Lee; So-Young Park; Masamitsu Yamaguchi; Mi-Ae Yoo (pp. 510-519).
The Drosophila midgut is an excellent model for evaluation of gene networks that regulate adult stem cell proliferation and differentiation. The Drosophila p38b ( D-p38b) gene has been shown to be involved in intestinal stem cell (ISC) proliferation and differentiation in the adult midgut. Here, we report that D-p38b gene expression is regulated by DREF (DNA replication-related element binding factor) in the adult midgut. We have identified a DRE in the 5′-flanking region of the D-p38b gene and showed that DREF could bind to this DRE via a gel mobility shift assay and a ChIP assay. Base-substitution mutations of the D-p38b promoter DRE and analyses of transformants carrying D-p38b-lacZ or D-p38b-DREmut -lacZ indicated that this DRE is required for the activity of the D-p38b gene promoter. Furthermore, by using the GAL4-UAS system, we showed that DREF regulates the activity of the D-p38b gene promoter in adult ISCs and progenitors. In addition, the D-p38b knockdown phenotypes in the midgut were rescued by DREF overexpression, suggesting a functional link between these two factors. Our results suggest that the D-p38b gene is regulated by the DREF pathway and that DREF is involved in the regulation of proliferation and differentiation of Drosophila ISCs and progenitors.
Keywords: Drosophila; p38b MAPK; DREF; Transcriptional regulation; Intestinal stem cell; Midgut
Transcription of mouse Sp2 yields alternatively spliced and sub-genomic mRNAs in a tissue- and cell-type-specific fashion by Haifeng Yin; Teresa D. Nichols; Jonathan M. Horowitz (pp. 520-531).
The Sp-family of transcription factors is comprised by nine members, Sp1-9, that share a highly conserved DNA-binding domain. Sp2 is a poorly characterized member of this transcription factor family that is widely expressed in murine and human cell lines yet exhibits little DNA-binding or trans-activation activity in these settings. As a prelude to the generation of a “knock-out” mouse strain, we isolated a mouse Sp2 cDNA and performed a detailed analysis of Sp2 transcription in embryonic and adult mouse tissues. We report that (1) the 5′ untranslated region of Sp2 is subject to alternative splicing, (2) Sp2 transcription is regulated by at least two promoters that differ in their cell-type specificity, (3) one Sp2 promoter is highly active in nine mammalian cell lines and strains and is regulated by at least five discrete stimulatory and inhibitory elements, (4) a variety of sub-genomic messages are synthesized from the Sp2 locus in a tissue- and cell-type-specific fashion and these transcripts have the capacity to encode a novel partial-Sp2 protein, and (5) RNA in situ hybridization assays indicate that Sp2 is widely expressed during mouse embryogenesis, particularly in the embryonic brain, and robust Sp2 expression occurs in neurogenic regions of the post-natal and adult brain.
Keywords: Abbreviations; Sp; specificity protein; Cys; cysteine; His; histidine; GC; guanine and cytosine; kb; kilobases; Mb; megabases; HOX; homeobox gene; DMEM; Dulbecco's modified Eagle's medium; PBS; phosphate-buffered saline; EDTA; ethylenediaminetetraacetic acid; PCR; polymerase chain reaction; dATP; deoxyadenosine triphosphate; dI/dC; deoxyinosine/deoxycytosine; Leu; leucine; Trp; tryptophan; SD; synthetic dropout; mM; millimolar; SSC-DEPC; standard saline citrate prepared with diethylpyrocarbonate-treated water; Fab-AP; fragment antigen binding-alkaline phosphate; NBT/BCIP; nitro-blue tetrazolium chloride/5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt; RT-PCR; reverse transcription-polymerase chain reaction; 5′RACE; rapid amplification of 5′ complementary ends; EGL; external granule layer; IGL; internal granule layer; PCL; Purkinje cell layer; OLB; olfactory bulb; RMS; rostral-migratory stream; DG; dentate gyrus; SVZ; subventricular zone; ML; molecular layer; GCL; granule cell layer; MCL; mitral cell layer; GL; glomerular layerSp2; Promoter analysis; Exonic promoter; Alternative splicing; In situ; hybridization