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BBA - Gene Regulatory Mechanisms (v.1799, #5-6)

Editorial Board (pp. i).

Modulation of transcription factor function by O-GlcNAc modification by Ozcan Sabire Özcan; Sreenath S. Andrali; Jamie E.L. Cantrell (pp. 353-364).

O-linked beta-N-acetylglucosamine (O-GlcNAc) modification of nuclear and cytoplasmic proteins is important for many cellular processes, and the number of proteins that contain this modification is steadily increasing. This modification is dynamic and reversible, and in some cases competes for phosphorylation of the same residues. O-GlcNAc modification of proteins is regulated by cell cycle, nutrient metabolism, and other extracellular signals. Compared to protein phosphorylation, which is mediated by a large number of kinases, O-GlcNAc modification is catalyzed only by one enzyme called O-linked N-acetylglucosaminyl transferase or OGT. Removal of O-GlcNAc from proteins is catalyzed by the enzyme beta-N-acetylglucosaminidase (O-GlcNAcase or OGA). Altered O-linked GlcNAc modification levels contribute to the establishment of many diseases, such as cancer, diabetes, cardiovascular disease, and neurodegeneration. Many transcription factors have been shown to be modified by O-linked GlcNAc modification, which can influence their transcriptional activity, DNA binding, localization, stability, and interaction with other co-factors. This review focuses on modulation of transcription factor function by O-linked GlcNAc modification.

Keywords: O-GlcNAc; Transcription factor; OGT; OGA; Hexosamine; Transcription

Conserved and divergent features of the structure and function of La and La-related proteins (LARPs) by Mark A. Bayfield; Ruiqing Yang; Richard J. Maraia (pp. 365-378).

Genuine La proteins contain two RNA binding motifs, a La motif (LAM) followed by a RNA recognition motif (RRM), arranged in a unique way to bind RNA. These proteins interact with an extensive variety of cellular RNAs and exhibit activities in two broad categories: i) to promote the metabolism of nascent pol III transcripts, including precursor-tRNAs, by binding to their common, UUU-3′OH containing ends, and ii) to modulate the translation of certain mRNAs involving an unknown binding mechanism. Characterization of several La–RNA crystal structures as well as biochemical studies reveal insight into their unique two-motif domain architecture and how the LAM recognizes UUU-3′OH while the RRM binds other parts of a pre-tRNA. Recent studies of members of distinct families of conserved La-related proteins (LARPs) indicate that some of these harbor activity related to genuine La proteins, suggesting that their UUU-3′OH binding mode has been appropriated for the assembly and regulation of a specific snRNP (e.g., 7SK snRNP assembly by hLARP7/PIP7S). Analyses of other LARP family members suggest more diverged RNA binding modes and specialization for cytoplasmic mRNA-related functions. Thus it appears that while genuine La proteins exhibit broad general involvement in both snRNA-related and mRNA-related functions, different LARP families may have evolved specialized activities in either snRNA or mRNA-related functions. In this review, we summarize recent progress that has led to greater understanding of the structure and function of La proteins and their roles in tRNA processing and RNP assembly dynamics, as well as progress on the different LARPs.

Keywords: La protein; La-related protein (LARP); Ribonucleoprotein (RNP); La motif; RNA recognition motif (RRM); 3′ end binding; RNA processing

The Paf1 complex: Platform or player in RNA polymerase II transcription? by Judith A. Jaehning (pp. 379-388).

The Paf1 complex (Paf1C), composed of the proteins Paf1, Ctr9, Cdc73, Rtf1, and Leo1, accompanies RNA polymerase II (pol II) from the promoter to the 3′ end formation site of mRNA and snoRNA encoding genes; it is also found associated with RNA polymerase I (pol I) on rDNA. The Paf1C is found in simple and complex eukaryotes; in human cells hSki8 is also part of the complex. The Paf1C has been linked to a large and growing list of transcription related processes including: communication with transcriptional activators; recruitment and activation of histone modification factors; facilitation of elongation on chromatin templates; and the recruitment of 3′ end-processing factors necessary for accurate termination of transcription. Absence of, or mutations in, Paf1C factors result in alterations in gene expression that can result in misregulation of developmental programs and loss of control of cell division leading to cancer in humans. This review considers recent information that may help to resolve whether the Paf1C is primarily a “platform” on pol II that coordinates the association of many critical transcription factors, or if the complex itself plays a more direct role in one or more steps in transcription.

Keywords: RNA polymerase II; Paf1 complex; Transcription elongation; Transcription termination; Histone modification; RNA 3′ end formation

Translocation by multi-subunit RNA polymerases by Maria Kireeva; Mikhail Kashlev; Zachary F. Burton (pp. 389-401).

DNA template and RNA/DNA hybrid movement through RNA polymerase (RNAP) is referred to as “translocation”. Because nucleic acid movement is coupled to NTP loading, pyrophosphate release, and conformational changes, the precise ordering of events during bond addition is consequential. Moreover, based on several lines of experimental evidence, translocation, pyrophosphate release or an associated conformational change may determine the transcription elongation rate. In this review we discuss various models of translocation, the data supporting the hypothesis that translocation rate determines transcription elongation rate and also data that may be inconsistent with this point of view. A model of the nucleotide addition cycle accommodating available experimental data is proposed. On the basis of this model, the molecular mechanisms regulating translocation and potential routes for NTP entry are discussed.

Keywords: Abbreviations; RNAP; RNA polymerase; TEC; ternary elongation complex; TFII; transcription factor RNA polymerase II; BH; bridge helix; TH; trigger helices; TL; trigger loop; FL2; fork loop 2; P; pre-insertion site; A; insertion siteRNA polymerase; Elongation; Translocation; Pyrophosphate release; Allostery; NTP-driven translocation

Induction of peroxiredoxin I gene expression by LPS involves the Src/PI3K/JNK signalling pathway by Antje Bast; Katja Fischer; Saskia F. Erttmann; Reinhard Walther (pp. 402-410).

Peroxiredoxin I (Prx I) belongs to a family of proteins with thiol-dependent peroxidase activity and is involved in the cellular protection against oxidative stress, the modulation of intracellular signalling cascades as well as the regulation of cell proliferation and apoptosis. In RAW 264.7 mouse macrophage cells Prx I was up-regulated on the mRNA and protein level by lipopolysaccharide (LPS). Treatment of cells with LPS increased the phosphorylation of c-Jun-NH₂ terminal kinase (JNK) and protein kinase B (PKB). Both SP600125, an inhibitor of JNK, and LY294002, an inhibitor of phosphoinositide 3-kinase (PI3K), dose-dependently decreased LPS-induced Prx I mRNA expression. Furthermore, up-regulation of Prx I mRNA by LPS was diminished by the Src tyrosine kinase inhibitor PP2 and the iNOS inhibitor L-NMMA. LPS-dependent induction of Prx I is likely mediated by an activator protein-1 site within the Prx I promoter region binding JunB and c-Fos. In contrast, NFκB was not involved in the activation of Prx I transcription. Our results suggest that the up-regulation of Prx I gene expression by LPS is part of the cellular response to stress and may protect against oxidative stress-related injury in RAW 264.7 cells.

Keywords: Abbreviations; AD; actinomycin D; AP-1; activator protein-1; CHX; cycloheximide; CREB; cAMP response element-binding protein; EMSA; electrophoretic mobility shift assay; ERK; extracellular signal-regulated kinase; GAPDH; glycerinaldehyde-3-phosphate dehydrogenase; IFNγ; interferon γ; iNOS; inducible nitric oxide synthase; JNK; c-Jun NH; ₂; -terminal kinase; Keap-1; Kelch-like ECH-associated protein 1; L-NMMA; N; ^G; -monomethyl-; l; -arginine; LPS; lipopolysaccharide; MAPK; mitogen-activated protein kinase; NFκB; nuclear factor κB; Nrf2; nuclear-erythroid derived 2-related factor-2; PI3K; phosphoinositide 3-kinase; PKB; protein kinase B; Prx; peroxiredoxin; RPLP0; ribosomal protein large P0; rRNA; ribosomal RNA; SAPK; stress-activated protein kinase; TNFα; tumour necrosis factor αAP-1; JNK; Macrophage; Peroxiredoxin; PI3K; Src

Tissue-specific Forkhead protein FOXA2 up-regulates SOX14 gene expression by Jelena Popovic; Andrijana Klajn; Isidora Petrovic; Milena Stevanovic (pp. 411-418).

The expression of Sox14 gene in spinal cord explants was found to be regulated by Sonic hedgehog (SHH) in a dose-dependent manner, indicating that this signaling molecule might act as a regulator of Sox14-expressing interneuron differentiation. In the present study we identified the positive control element and provided the first evidence that FOXA2 is involved in up-regulation of SOX14 expression in HepG2 and U87MG cell lines. By functional analysis we demonstrated that mutation in FOXA2 binding site reduced the SOX14 reporter construct activity, and that FOXA2 over-expression increased endogenous SOX14 protein expression. Further, we have shown that human SOX14 expression is GLI1 dependent in U87MG cells and SHH-N dependent in U87MG and HepG2 cell lines. By applying siRNA silencing of FOXA2, we have demonstrated that upregulation of endogenous SOX14 gene expression by SHH is, at least in part, mediated by FOXA2. However, our data revealed that a positive regulatory region, containing functional FOXA2 site analyzed in this study, is not involved in mediation of SHH dependent SOX14 activation. Data presented here provide the initial insight into molecular mechanism underlying tissue and developmentally specific regulation of the SOX14 gene expression.

Keywords: SOX14; Foxa2; SHH; GLI

hnRNP H1 and intronic G runs in the splicing control of the human rpL3 gene by Annapina Russo; Gabriella Siciliano; Morena Catillo; Chiara Giangrande; Angela Amoresano; Pietro Pucci; Concetta Pietropaolo; Giulia Russo (pp. 419-428).

By generating mRNA containing a premature termination codon (PTC), alternative splicing (AS) can quantitatively regulate the expression of genes that are degraded by nonsense-mediated mRNA decay (NMD). We previously demonstrated that AS-induced retention of part of intron 3 of rpL3 pre-mRNA produces an mRNA isoform that contains a PTC and is targeted for decay by NMD. We also demonstrated that overexpression of rpL3 downregulates canonical splicing and upregulates the alternative splicing of its pre-mRNA. We are currently investigating the molecular mechanism underlying rpL3 autoregulation. Here we report that the heterogeneous nuclear ribonucleoprotein (hnRNP) H1 is a transacting factor able to interact in vitro and in vivo with rpL3 and with intron 3 of the rpL3 gene. We investigated the role played by hnRNP H1 in the regulation of splicing of rpL3 pre-mRNA by manipulating its expression level. Depletion of hnRNP H1 reduced the level of the PTC-containing mRNA isoform, whereas its overexpression favored the selection of the cryptic 3′ splice site of intron 3. We also identified and characterized the cis-acting regulatory elements involved in hnRNP H1-mediated regulation of splicing. RNA electromobility shift assay demonstrated that hnRNP H1 specifically recognizes and binds directly to the intron 3 region that contains seven copies of G-rich elements. Site-directed mutagenesis analysis and in vivo studies showed that the G3 and G6 elements are required for hnRNP H1-mediated regulation of rpL3 pre-mRNA splicing. We propose a working model in which rpL3 recruits hnRNP H1 and, through cooperation with other splicing factors, promotes selection of the alternative splice site.

Keywords: Abbreviations; AS; alternative splicing; hnRNP; heterogeneous nuclear ribonucleoprotein; NMD; nonsense-mediated mRNA decay; PTC; premature termination codon; r-protein; ribosomal protein; rp; ribosomal protein; rp-mRNA; mRNA for ribosomal protein; RT-PCR; reverse transcriptase-PCR; SH-PTP1; SH-protein-tyrosine phosphatase 1; siRNA; small interfering RNA; SR; serine-richAlternative splicing; hnRNP H1; NMD; Ribosomal protein; Splicing regulation

Transcriptional upregulation of calcineurin Aβ by endothelin-1 is partially mediated by calcium/calmodulin-dependent protein kinase IIδ3 in rat cardiomyocytes by Ying-Mei Lu; Norifumi Shioda; Yui Yamamoto; Feng Han; Kohji Fukunaga (pp. 429-441).

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) are positive regulators of cardiac hypertrophy, but the nature of cross-talk between CaMKII and CaN signaling pathways in hypertrophic cardiomyocytes remains unclear. Here we documented that CaMKIIδ3 activation enhances transcription of the CaN gene through activation of the CaN-Aβ subunit (CnAβ) promoter in rat cultured cardiomyocytes. Co-immunoprecipitation assays showed that MEF2 forms a complex with GATA4 following transfection of an active CaMKIIδ3 (T278D) mutant in neonatal cardiomyocytes. Inversely, transfection of a dominant negative CaMKIIδ3 mutant failed to promote a MEF2–GATA4 complex. Consistent with these observations, immunocytochemistry indicated nuclear co-localization of MEF2 with GATA4 after hypertrophic agonist stimulation or CaMKIIδ3 (T278D) transfection. These data demonstrate that CaMKII can enhance CnAβ promoter activity by enhancing MEF2–GATA4 synergy, suggesting a novel mechanism for CaMKII-mediated hypertrophic signaling, which contributes to induction and development of the hypertrophic response through CaN activation.

Keywords: Hypertrophy; CaMKII; Calcineurin; MEF2c; GATA4

Sp1 and Sp3 regulate transcription of the chicken GAS41 gene by Hubner Katrin Hübner; Loc Phi-van (pp. 442-447).

The 5′-flanking region of the chicken glioma-amplified sequence (GAS) 41 gene is close to the 3′ end of the lysozyme gene and contains no typical TATA box, but several GC boxes. In this study, we have localized the GAS 41 promoter to this narrow region. Electrophoretic mobility shift assays and chromatin immunoprecipitation analyses revealed that Sp1 and Sp3 bind to this promoter. Mapping by a technique of indirect end labeling demonstrated that the Sp1-binding sites contained in this region exactly co-map with two previously identified DNase I hypersensitive (HS) sites, which suggests the important role of Sp1 binding in maintaining an open chromatin structure of the GAS41 promoter. We further found that Sp1 and Sp3 strongly activate CAT expression controlled by the putative GAS41 promoter in Drosophila Schneider S2 cells and that deletion of the Sp1 sites resulted in a loss of promoter activity in chicken HD11 cells. The results indicate that transcription factors of the Sp family play an important role in the transcriptional regulation of the chicken GAS41 gene.

Keywords: Glioma-amplified sequence (GAS)41; Sp1; Sp3; Lysozyme GAS41 locus; GAS41 promoter

DARPP-32 binds to tra2-beta1 and influences alternative splicing by Natalya Benderska; Kristina Becker; Jean-Antoine Girault; Cord-Michael Becker; Athena Andreadis; Stefan Stamm (pp. 448-453).

The majority of human genes undergo alternative splicing, which is frequently altered in response to physiological stimuli. DARPP-32 (dopamine and cAMP regulated phosphoprotein, 32kDa) is a component of PKA-dependent signaling pathways. Here we show that DARPP-32 binds directly to the splicing factor tra2-beta1 (transformer 2). DARPP-32 changes the usage of tra2-beta1 dependent alternative exons in a concentration-dependent manner, suggesting that the DARPP-32:tra2-beta1 interaction is a molecular link between signaling pathways and pre-mRNA processing.

Keywords: RNA processing; Alternative splicing; Protein phosphatase 1; DARPP-32; Signaling

DNA binding by the Arabidopsis CBF1 transcription factor requires the PKKP/RAGRxKFxETRHP signature sequence by Donatella Canella; Sarah J. Gilmour; Leslie A. Kuhn; Michael F. Thomashow (pp. 454-462).

The CBF/DREB1 transcriptional activators are key regulators of plant freezing tolerance. They are members of the AP2/ERF multi-gene family, which in Arabidopsis comprises about 145 members. Common to these proteins is the AP2/ERF DNA-binding domain, a 60-amino-acid fold composed of a three-stranded β-sheet followed by a C-terminal α-helix. A feature that distinguishes the CBF proteins from the other AP2/ERF proteins is the presence of “signature sequences,” PKKP/RAGRxKFxETRHP (abbreviated PKKPAGR) and DSAWR, which are located immediately upstream and downstream, respectively, of the AP2/ERF DNA-binding domain. The signature sequences are highly conserved in CBF proteins from diverse plant species suggesting that they have an important functional role. Here we show that the PKKPAGR sequence of AtCBF1 is essential for its transcriptional activity. Deletion of the sequence or mutations within it greatly impaired the ability of CBF1 to induce expression of its target genes. This impairment was not due to the mutations eliminating CBF1 localization to the nucleus or preventing protein accumulation. Rather, we show that this loss of function was due to the mutations greatly impairing the ability of the CBF1 protein to bind to its DNA recognition sequence, the CRT/DRE element. These results establish that the ability of the CBF proteins to bind to the CRT/DRE element requires amino acids that extend beyond the AP2/ERF DNA-binding domain and raise the possibility that the PKKPAGR sequence contributes to determining the DNA-binding specificity of the CBF proteins.

Keywords: AP2/ERF domain; Arabidopsis; CBF/DREB1 transcription factor; DNA binding; Signature sequence

The NIZP1 KRAB and C2HR domains cross-talk for transcriptional regulation by Regine Losson; Anders L. Nielsen (pp. 463-468).

The NSD1 histone methyltransferase is involved in the outgrowth disorders Sotos and Weaver syndromes and childhood acute myeloid leukemia. NSD1 is a bona fida transcriptional co-repressor for Nizp1 which is a protein including SCAN, KRAB, C2HR and zinc-finger domains. In this study the Nizp1 KRAB-domain was identified to possess an intrinsic transcriptional activation capacity suppressed in cis by the presence of the C2HR domain. Oppositely, the KRAB-domain supported C2HR domain mediated transcriptional repression. The presence of the KRAB-domain resulted in increased NSD1 co-repressor association with the C2HR domain. This study shows a new function of the KRAB-domain, C2HR-domain, and the associated factors to confer Nizp1 mediated transcriptional regulation.

Keywords: Epigenetics; Gene regulation; Silencing; Zinc finger; TIF1β/KAP1

Estrogen receptor-dependent regulation of CYP2B6 in human breast cancer cells by Raymond Lo; Lyle Burgoon; Laura MacPherson; Shaimaa Ahmed; Jason Matthews (pp. 469-479).

Estrogen receptor α (ERα) mediates the biological actions of estrogens and also contributes to the development and progression of breast cancer. To gain a more comprehensive understanding of ERα-mediated transcription, we used chromatin immunoprecipitation and promoter focused microarrays (ChIP-chip) to identify ERα binding sites in T-47D human breast cancer cells. Transcription factor binding site analysis revealed that the estrogen response element (ERE) was significantly over-represented and was found in 50% of the 243 ERα-bound regions identified. Interestingly, multiple ERα-bound regions were detected in the upstream regulatory sequences of the CYP2B gene cluster. Because ERα has been reported to regulate the expression of other cytochrome P450 enzymes and CYP2B6 is highly expressed in ERα-positive breast tumors, we focused on characterizing the ERα-dependent regulation of CYP2B6. Reporter gene assays revealed that ERα and ERβ increased C YP2B6-regulated gene expression through a functional ERE located at −1669 to −1657 in the upstream regulatory region of CYP2B6. E2 increased ERα and nuclear receptor coactivator 3 (NCoA3) recruitment to the 5′-flanking region of CYP2B6, and increased CYP2B6 mRNA levels in T-47D but not in MCF-7 human breast cancer cells. RNAi-mediated knockdown of ERα in the T-47D cells resulted in a significant decrease in CYP2B6 mRNA levels. Taken together, our study provides evidence for cell-type specific transcriptional regulation of the CYP2B6 gene by ERs.

Keywords: Abbreviations; ER; estrogen receptor; ChIP; chromatin immunoprecipitation; ChIP-chip; chromatin immunoprecipitation on microarray chip; NCoA3; nuclear coactivator 3; ERE; estrogen response element; CYP; cytochrome P450; CAR; constitutive androstane receptor; RXR; retinoid X receptor; PBREM; phenobarbital response enhancer module; PXR; pregnane X receptor; GR; glucocorticoid receptor; GRE; glucocorticoid response element; DEX; dexamethasone TFF1, trefoil factor 1; E2; 17β-estradiol; ICI 182,780; fulvestrant; PWM; position weight matrix; FDR; false detection rateClassification codes; 50.070: nuclear receptor; 50.100: response element; 50.150: transcription factor; 50.160: transcription factor binding sitesChromatin immunoprecipitation and microarray chip (ChIP-chip); Estrogen receptor; Estrogen; Transcription factor binding analysis; Gene regulation; Cytochrome P450 2B6

Structural characterization of H3K56Q nucleosomes and nucleosomal arrays by Shinya Watanabe; Michael Resch; Wayne Lilyestrom; Nicholas Clark; Jeffrey C. Hansen; Craig Peterson; Karolin Luger (pp. 480-486).

The post-translational modification of histones is a key mechanism for the modulation of DNA accessibility. Acetylated lysine 56 in histone H3 is associated with nucleosome assembly during replication and DNA repair, and is thus likely to predominate in regions of chromatin containing nucleosome-free regions. Here we show by X-ray crystallography that mutation of H3 lysine 56 to glutamine (to mimic acetylation) or glutamate (to cause a charge reversal) has no detectable effects on the structure of the nucleosome. At the level of higher order chromatin structure, the K to Q substitution has no effect on the folding of model nucleosomal arrays in cis, regardless of the degree of nucleosome density. In contrast, defects in array–array interactions in trans (‘oligomerization’) are selectively observed for mutant H3 lysine 56 arrays that contain nucleosome-free regions. Our data suggests that H3K56 acetylation is one of the molecular mechanisms employed to keep chromatin with nucleosome-free regions accessible to the DNA replication and repair machinery.

Keywords: Nucleosome; Histone acetylation; X-ray crystallography; Chromatin folding; Oligomerization; H3K56Ac

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BBA - Gene Regulatory Mechanisms (v.1799, #5-6)