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BBA - Gene Regulatory Mechanisms (v.1819, #5)

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The mouse gene encoding the carnitine biosynthetic enzyme 4-N-trimethylaminobutyraldehyde dehydrogenase is regulated by peroxisome proliferator-activated receptor α by Gaiping Wen; Robert Ringseis; Christine Rauer; Klaus Eder (pp. 357-365).

Genes involved in carnitine uptake and synthesis, such as organic cation transporter-2 (OCTN2) and γ-butyrobetaine dioxygenase (BBD), have been shown to be regulated by peroxisome proliferator-activated receptor (PPAR)α directly. Whether other genes encoding enzymes involved in the carnitine synthesis pathway, such as 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABA-DH) and trimethyllysine dioxygenase (TMLD), are also direct PPARα target genes is less clear. In silico-analysis of the mouse TMLD promoter and first intron and the TMABA-DH promoter revealed several putative peroxisome proliferator response elements (PPRE) with high similarity to the consensus PPRE. Luciferase reporter gene assays using either a 2kb TMLD promoter or a 4kb TMLD first intron reporter constructs revealed no functional PPRE. In contrast, reporter gene assays using wild-type and mutated 5´-truncation TMABA-DH promoter reporter constructs showed that one PPRE located at position −132 in the proximal promoter is probably functional. Using gel shift assays we observed in vitro-binding of PPARα to this PPRE. Moreover, using chromatin immunoprecipitation assays we found that PPARα also binds in vivo to a nucleotide sequence spanning the PPRE at −132, which confirms that this PPRE is functional. In conclusion, the present study shows that the mouse TMABA-DH gene is a direct PPARα target gene. Together with the recent identification of the mouse BBD and the mouse OCTN2 genes as PPARα target genes this finding confirm that PPARα plays a key role in the regulation of carnitine homeostasis by controlling genes involved in carnitine synthesis and carnitine uptake.► The mouse TMABA-DH promoter is transcriptionally activated by PPARα. ► EMSA demonstrates in vitro-binding of PPARα to one PPRE in the TMABA-DH promoter. ► ChIP assay shows in vivo-binding of PPARα to the PPRE of TMABA-DH promoter. ► the TMLD promoter and first intron contains no functional PPRE. ► The mouse TMLD gene is not regulated by PPARα.

Keywords: Carnitine synthesis; PPARα; Functional PPRE; Target gene

Hho1p, the linker histone of Saccharomyces cerevisiae, is important for the proper chromatin organization in vivo by Milena Georgieva; Assen Roguev; Konstantin Balashev; Jordanka Zlatanova; George Miloshev (pp. 366-374).

Despite the existence of certain differences between yeast and higher eukaryotic cells a considerable part of our knowledge on chromatin structure and function has been obtained by experimenting on Saccharomyces cerevisiae. One of the peculiarities of S. cerevisiae cells is the unusual and less abundant linker histone, Hho1p. Sparse is the information about Hho1p involvement in yeast higher-order chromatin organization. In an attempt to search for possible effects of Hho1p on the global organization of chromatin, we have applied Chromatin Comet Assay (ChCA) on HHO1 knock-out yeast cells. The results showed that the mutant cells exhibited highly distorted higher-order chromatin organization. Characteristically, linker histone depleted chromatin generally exhibited longer chromatin loops than the wild-type. According to the Atomic force microscopy data the wild-type chromatin appeared well organized in structures resembling quite a lot the “30-nm” fiber in contrast to HHO1 knock-out yeast.Display Omitted► We trace the role of Saccharomyces cerevisiae linker histone, Hho1p. ► We study the global chromatin organization at the higher-levels of its compaction. ► The lack of Hho1p leads to longer chromatin loops. ► Hho1p is essential for the maintenance of the “30nm” fiber.

Keywords: Saccharomyces cerevisiae; Linker histone; Chromatin structure; “30; nm” chromatin fiber

Mapping acetylation sites in E2A identifies a conserved lysine residue in activation domain 1 that promotes CBP/p300 recruitment and transcriptional activation by Brandy D. Hyndman; Patrick Thompson; Christopher M. Denis; Seth Chitayat; Richard Bayly; Steven P. Smith; David P. LeBrun (pp. 375-381).

E-proteins are basic helix-loop-helix transcription factors that function in cell type specification. The gene E2A encodes two E-proteins, E12 and E47, which are required in B-lymphopoiesis. E2A proteins can interact directly with the transcriptional co-activators and lysine acetyltranferases (KATs) CBP, p300 and PCAF to induce target gene transcription. Prior investigations have shown that the E2A-encoded isoform E2-5 is acetylated by CBP, p300 or PCAF in vitro or in vivo. However, E2-5 lacks the important N-terminal activation domain AD1. Furthermore, the acetylated residues in E-proteins have not been mapped, and the functional consequences of acetylation are largely unknown. Here, we use mutagenesis to show that a lysine residue at position 34 within AD1 of E12/E47 is acetylated by CBP/p300 and PCAF. Lys34 lies adjacent to a conserved helical LXXLL motif that interacts directly with the KIX domain of CBP/p300. We show that acetylation at Lys34 increases the affinity of AD1 for the KIX domain and enhances AD1-driven transcriptional induction. Our results illustrate for the first time that AD1 can both recruit, and be acetylated by, KATs and that KAT recruitment may promote transcriptional induction in part through acetylation of AD1 itself.► We map acetylated, phylogenetically conserved amino acids in E2A for the first time. ► Lys34, a residue within transcriptional activation domain 1, is acetylated. ► Mimicking acetylation of Lys34 increases E2A‐mediated transcriptional activation. ► Acetylation of Lys34 promotes binding of an E2A peptide to the KIX domain of CBP. ► Acetylation of Lys34 promotes CBP recruitment and transcriptional activation by E2A.

Keywords: E-proteins; Acetylation; Gene transcription; E2A; p300; CREB binding protein

Insights into the mechanism of activation of the phosphorylation-independent response regulator NblR. Role of residues Cys69 and Cys96 by Javier Espinosa; Maria-Luisa López-Redondo; Laura Miguel-Romero; José L. Neira; Alberto Marina; Asunción Contreras (pp. 382-390).

Cyanobacteria respond to environmental stress conditions by adjusting their photosynthesis machinery. In Synechococcus sp. PCC 7942, phycobilisome degradation and other acclimation responses after nutrient or high light stress require activation by the phosphorylation-independent response regulator NblR. Structural modelling of its receiver domain suggested a role for Cys69 and Cys96 on activation of NblR. Here, we investigate this hypothesis by engineering Cys to Ala substitutions. In vivo and in vitro analyses indicated that mutations Cys69Ala and/or Cys96Ala have a minor impact on NblR function, structure, size, or oligomerization state of the protein, and that Cys69 and Cys96 do not seem to form disulphide bridges. Our results argue against the predicted involvement of Cys69 and Cys96 on NblR activation by redox sensing.► The C96A and C69A NblR mutant proteins are native-like. ► Residues Cys69 and Cys96 are not involved in redox sensing in the protein. ► Residues Cys69 and Cys96 do not form a disulphide bridge. ► The Y104A NblR mutant strain presents a null mutant phenotype

Keywords: Abbreviations; CD; circular dichroism; nblA; non-bleaching protein A; nblR; non-bleaching protein R; NblR; ^C69A; the alanine mutant of NblR at position 69; NblR; ^C96A; the alanine mutant of NblR at position 96; NblR; ^C69A/C96A; the double alanine mutant of NblR at positions 69 and 96; NblR; ^Y104A; the alanine mutant of NblR at position 104; RR; response regulator; PIARR; phosphorylation-independent activation of response regulatorStress response; NblR; Cyanobacterium; Cysteine; Response regulator; Structure

The transcription cycle in eukaryotes: From productive initiation to RNA polymerase II recycling by Jayasha Shandilya; Stefan G.E. Roberts (pp. 391-400).

The cycle of eukaryotic transcription, from initiation to elongation and termination is regulated at multiple steps. Coordinated action of regulatory factors keeps in check the transcriptional competence of RNA polymerase II (RNAPII) at different stages. Productive transcription requires the escape of the paused RNAPII from the promoter and transition to rapid elongation of the transcript. Numerous studies have identified diverse mechanisms of initiating transcription by overriding inhibitory signals at the gene promoter. The general theme that has emerged is that the balance between positive and negative regulatory factors determines the overall rate of transcription. Recently transcription termination has emerged as an important area of transcriptional regulation that is coupled with the efficient recycling of RNAPII. The factors associated with transcription termination can also mediate gene looping and thereby determine the efficiency of re-initiation. This review highlights these regulatory steps, the key modulators involved in transcription dynamics, and the emerging tools to analyze them.► Eukaryotic transcription is a highly coordinated and stringently regulated event. ► Multiple rounds of initiation–elongation–termination occur in a cyclic pattern. ► Alleviation of pausing by diverse mechanisms stimulates the transcription process. ► Gene looping facilitates RNA polymerase II recycling via promoter–terminator contact. ► A new dimension in transcriptional regulation is revealed by single molecule studies.

Keywords: Transcription initiation; Transcriptional pausing; Transcription termination; Gene looping; Promoter; RNA polymerase II

Discovering genome regulation with 3C and 3C-related technologies by Sylvain D. Ethier; Hisashi Miura; Josée Dostie (pp. 401-410).

It has been known for some time that eukaryotic genomic DNA is packaged in the form of highly organized chromatin in vivo. This organization is important not only to reduce the length of chromosomes during interphase but also because it represents a type of higher-order genome regulation mechanism. Indeed, spatial chromatin architecture is known to be important for transcription, DNA replication and repair. Chromosome structure can be observed at different scales and studied with a variety of complementary techniques. For example, microscopy can provide single cell information while technologies such as the chromosome conformation capture (3C) method and its derivatives can yield higher-resolution data from cell populations. In this review, we report on the biological questions addressed with 3C and 3C-related techniques and what has been uncovered to date. We also explore what these methods may further reveal about the regulation of genomic DNA activities.► This review describes how 3C technologies can probe functional genome organization. ► 3C and 3C-related techniques map physical chromatin contacts in vivo. ► Physical chromatin contacts regulate transcription, DNA replication and repair. ► Functional genome organization relies on physical chromatin contacts.

Keywords: Chromatin; Gene regulation; Chromosome conformation capture; Epigenetics; Transcription

Effect of the methyltransferase domain of Japanese encephalitis virus NS5 on the polymerase activity by Qiang Wang; Leiyun Weng; Xiao Tian; Dorian Counor; Jin Sun; Yingying Mao; Vincent Deubel; Hidechika Okada; Tetsuya Toyoda (pp. 411-418).

Japanese encephalitis virus (JEV) NS5 consists of an N-terminal guanylyltransferase/methyltransferase (MTase) domain and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. We purified JEV NS5 from bacteria and examined its RdRp activity in vitro. It showed exclusive specificity for Mn²⁺ and alkaline conditions (pH 8–10) for RdRp activity. It showed strong RdRp activity with dinucleotide primers, and the order of template strength was poly(U)>(I)>(A)>(C). It showed weak transcription activity without primers, but could not transcribe poly(I) without primers. It bound homopolymeric RNA templates, but weakly bound poly(C). The Km (μM) values were 22.13±1.11 (ATP), 21.94±3.88 (CTP), 21.27±1.23 (GTP), and 9.91±0.30 (UTP), indicating low substrate affinity. Vmax (/min) values were 0.216±0.017 (ATP), 0.781±0.020 (CTP), 0.597±0.049 (GTP), and 0.347±0.022 (UTP), indicating high polymerization activity. The RdRp domain alone did not show RdRp activity; a structural and functional interaction between the MTase and RdRp domains via 299-EHPYRTWTYH-308 (MTase domain) and 739-LIGRARISPG-748 (RdRp domain) was predicted, because mutations in the MTase domain affected RdRp activity.► JEV NS5 consists of an N-terminal MTase domain and a C-terminal RdRp domain. ► JEV NS5 showed both dinucleotide primer-dependent and de novo transcription activity. ► MTase domain is important for RdRp activity.

Keywords: Abbreviations; DV; dengue virus; E. coli; Escherichia coli; JEV; Japanese encephalitis virus; KV; Kunjin virus; MTase; methyltransferase; nt; nucleotides; preM; pre-membrane; RdRp; RNA-dependent RNA polymerase; SAM; S-adenosyl-; l; -methionine; UTR; untranslated regions; WNV; West Nile virus; YFV; yellow fever virusJapanese encephalitis virus; NS5; RNA polymerase; K; m; V; max; Methyltransferase

Snf1/AMPK regulates Gcn5 occupancy, H3 acetylation and chromatin remodelling at S. cerevisiae ADY2 promoter by Georgia Abate; Emanuela Bastonini; Katherine A. Braun; Loredana Verdone; Elton T. Young; Micaela Caserta (pp. 419-427).

The ability of cells to respond to changes in their environment is mediated by transcription factors that remodel chromatin and reprogram expression of specific subsets of genes. In Saccharomyces cerevisiae, changes in carbon source lead to gene induction by Adr1 and Cat8 that are known to require the upstream function of the Snf1 protein kinase, the central regulator of carbon metabolism, to exert their activating effect. How Snf1 facilitates transcription activation by Adr1 and Cat8 is not known. Here we show that under derepressing conditions, deletion of SNF1 abolishes the increase of histone H3 acetylation at the promoter of the glucose-repressed ADY2 gene, and as a consequence profoundly affects the chromatin structural alterations accompanying transcriptional activation. Adr1 and Cat8 are not required to regulate the acetylation switch and show only a partial influence on chromatin remodelling at this promoter, though their double deletion completely abolishes mRNA accumulation. Finally, we show that under derepressing conditions the recruitment of the histone acetyltransferase Gcn5 is abolished by SNF1 deletion, possibly explaining the lack of increased histone H3 acetylation and nucleosome remodelling.The results highlight a mechanism by which signalling to chromatin provides an essential permissive signal that is required for activation by glucose-responsive transcription factors.► The main role of Snf1 is to control chromatin structure and function. ► The specific promoter architecture of each gene is a key element in determining the mechanism of transcription activation. ► Cell metabolism is controlled by epigenetic mechanisms.

Keywords: Adr1; ADY2; Cat8; Nucleosome remodeling; Histone acetylation; Snf1

Regulation of the human prostacyclin receptor gene in megakaryocytes: Major roles for C/EBPδ and PU.1 by Garret L. Keating; Elizebeth C. Turner; B. Therese Kinsella (pp. 428-445).

The prostanoid prostacyclin plays a central role in haemostasis and vascular repair. Recent studies investigating the regulation of the human prostacyclin receptor (hIP) gene identified an upstream repressor region (URR) within its regulatory promoter, herein termed the PrmIP. This study aimed to identify the main trans-acting factors that bind within the URR to transcriptionally repress PrmIP-directed gene expression in the megakaryoblastic human erythroleukemia (HEL) 92.1.7 cell line. Of the putative cis-acting elements examined, disruption of C/EBP and PU.1 elements within the URR substantially increased PrmIP-directed gene expression. Chromatin immunoprecipitation (ChIP) confirmed that C/EBPδ and PU.1, but not C/EBPβ, bind to the URR in vivo, while ectopic expression of C/EBPδ substantially reduced hIP mRNA levels and PrmIP-directed gene expression. Phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation increased hIP mRNA and PrmIP-directed reporter gene expression and hIP-mediated cAMP generation in HEL cells. Two PMA-responsive regions, termed PRR1 and PRR2, were identified within PrmIP. Disruption of C/EBPδ and PU.1 cis-elements within the overlapping PRR1/URR and of Sp1, PU.1 and Oct-1 cis-elements within the overlapping PRR2/core PrmIP, revealed that both PRR1 and PRR2 contribute to the PMA- induction of hIP mRNA and gene expression in HEL cells. Furthermore, ChIP analysis established that induction of PrmIP-directed gene expression during megakaryocytic differentiation is largely regulated by PMA-induced dissociation of C/EBPδ and enhanced binding of PU.1 to PRR1 in addition to increased binding of Sp1, PU.1 and Oct-1 to elements within the core promoter/PRR2 in vivo. Taken together, these data provide critical insights into the transcriptional regulation of the hIP gene within the vasculature, including during megakaryocytic differentiation.► An upstream repressor region (URR) identified in the prostacyclin receptor (IP) promoter. ► C/EBPδ and PU.1 can bind to cis-elements in the URR to repress IP gene expression. ► Human (h)IP expression is induced in response to PMA-differentiation of HEL cells. ► Differentiation coincides with a switch from C/EBPδ to mainly PU.1 binding to the URR. ► Differentiation also increases Sp1, PU.1 and Oct-1 binding to the Core IP promoter.

Keywords: Abbreviations; CAD; coronary artery disease; C/EBP; CCAAT/enhancer binding protein; ChIP; chromatin immunoprecipitation; COX; cyclooxygenase; C-LH; cut-like homeodomain; CVD; cardiovascular disease; DMSO; dimethyl sulfoxide; ERE; estrogen response element; FBS; fetal bovine serum; HEL; human erythroleukemia; hIP; human prostacyclin receptor; IgG; immunoglobulin G; IP; prostacyclin receptor; Nrf; Nuclear respiratory factor; PMA; phorbol 12-myristate 13-acetate; pGL3B; pGL3Basic; pRL-TK; pRL-thymidine kinase; PRR; PMA-responsive region; QT; quantitative reverse-transcriptase; RLU; relative luciferase unit; SEM; standard error of the mean; SNP; single-nucleotide polymorphism; STAT; Signal Transduction And Transcription; TI; transcription initiation; SDM; site-directed mutagenesis; TX; thromboxane; URR; upstream repressor regionProstacyclin receptor; C/EBP; Sp1; PU.1; Oct-1; Promoter

E2A proteins enhance the histone acetyltransferase activity of the transcriptional co-activators CBP and p300 by Brandy D. Hyndman; Patrick Thompson; Richard Bayly; Cote Graham P. Côté; David P. LeBrun (pp. 446-453).

The E2A gene encodes the E-protein transcription factors E12 and E47 that play critical roles in B-lymphopoiesis. A somatic chromosomal translocation detectable in 5% of cases of acute lymphoblastic leukemia (ALL) involves E2A and results in expression of the oncogenic transcription factor E2A-PBX1. CREB binding protein (CBP) and its close paralog p300 are transcriptional co-activators with intrinsic histone acetyltransferase (HAT) activity. We and others have shown that direct binding of an N-terminal transcriptional activation domain present in E12/E47 and E2A-PBX1 to the KIX domain of CBP/p300 contributes to E2A protein function. In the current work we show for the first time that the catalytic HAT activity of CBP/p300 is increased in the presence of residues 1–483 of E2A (i.e., the portion present in E2A-PBX1). The addition of purified, recombinant E2A protein to in vitro assays results in a two-fold augmentation of CBP/p300 HAT activity, whereas in vivo assays show a ten-fold augmentation of HAT-dependent transcriptional induction and a five-fold augmentation of acetylation of reporter plasmid-associated histone by CBP in response to co-transfected E2A. Our results indicate that the HAT-enhancing effect is independent of the well-documented E2A–CBP interaction involving the KIX domain and suggest a role for direct, perhaps low affinity binding of E2A to a portion of CBP that includes the HAT domain and flanking elements. Our findings add to a growing body of literature indicating that interactions between CBP/p300 and transcription factors can function in a specific manner to modulate HAT catalytic activity.► We show that E2A protein increases the catalytic activity of the histone acetyltransferase CBP/p300. ► This effect enhances HAT-dependent transcriptional activation by CBP in vivo. ► The effect is selective for E2A since other proteins assayed had no effect on CBP/p300 catalytic activity. ► The effect does not require the known, direct interaction between E2A and the KIX domain of CBP/p300. ► CBP/p300 stably-bound at enhancers could be regulated through interactions with transcription factors that bind nearby.

Keywords: Histone acetyltransferase; E2A; Gene transcription; CBP; p300; Chromatin immunoprecipitation

From plant gene regulatory grids to network dynamics by Maria Katherine Mejia-Guerra; Marcelo Pomeranz; Kengo Morohashi; Erich Grotewold (pp. 454-465).

The regulation of gene expression is the most basic level at which genotypes encoded in DNA can manifest themselves into observable phenotypes. In eukaryotes, gene regulatory networks (GRNs) describe the regulatory web through which transcription factors and microRNAs tightly regulate the spatial and temporal expression of genes. In yeast, Escherichia coli, and animals the study of GRNs has uncovered many of the network properties responsible for creating complex regulatory behavior such as organism growth, development, and response to environmental stimuli. In plants, the study of GRNs is just starting to gain momentum thanks to new high quality genomes and the development of new tools for GRN mapping. Here, we review the latest advancements in the study of plant GRNs and describe the tools and techniques used to produce them. We also discuss the emerging field of network dynamics and the methods currently being developed to measure network dynamics and function in plants.►The study of plant GRNs is lagging behind that of animal and unicellular model systems but is quickly gaining momentum. ►The functional definition of a promoter is evolving from positional to dynamic. ►We discuss techniques for GRN mapping, comparing TF-centered and gene-centered approaches.

Keywords: Arabidopsis; Network motif; Network hierarchy; Network dynamics; Systems biology

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BBA - Gene Regulatory Mechanisms (v.1819, #5)