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BBA - Gene Regulatory Mechanisms (v.1809, #10)

Editorial Board (pp. i).

Molecular call and response: The physiology of bacterial small RNAs by Gregory R. Richards; Carin K. Vanderpool (pp. 525-531).

The vital role of bacterial small RNAs (sRNAs) in cellular regulation is now well-established. Although many diverse mechanisms by which sRNAs bring about changes in gene expression have been thoroughly described, comparatively less is known about their biological roles and effects on cell physiology. Nevertheless, for some sRNAs, insight has been gained into the intricate regulatory interplay that is required to sense external environmental and internal metabolic cues and turn them into physiological outcomes. Here, we review examples of regulation by selected sRNAs, emphasizing signals and regulators required for sRNA expression, sRNA regulatory targets, and the resulting consequences for the cell. We highlight sRNAs involved in regulation of the processes of iron homeostasis (RyhB, PrrF, and FsrA) and carbon metabolism (Spot 42, CyaR, and SgrS).► Bacterial sRNAs are important regulators with well-described molecular mechanisms. ► Relatively less is known about the effects of sRNAs on cell physiology and behavior. ► We review environmental and metabolic cues and regulators needed for sRNA activation. ► We also emphasize sRNA targets and what is known about physiological effects. ► We focus on selected sRNAs involved in iron homeostasis and carbon metabolism.

Keywords: Abbreviations; G6P; glucose-6-phosphate; αMG6P; α-methyl glucoside-6-phosphate; αMG; α-methyl glucosideSmall RNA; RyhB; Spot 42; CyaR; SgrS

The influence of Escherichia coli Hfq mutations on RNA binding and sRNA•mRNA duplex formation in rpoS riboregulation by Taylor B. Updegrove; Roger M. Wartell (pp. 532-540).

The Escherichia coli RNA binding protein Hfq plays an important role in regulating mRNA translation through its interactions with small non-coding RNAs (sRNAs) and specific mRNAs sites. The rpoS mRNA, which codes for a transcription factor, is regulated by several sRNAs. DsrA and RprA enhance translation by pairing to a site on this mRNA, while OxyS represses rpoS mRNA translation. To better understand how Hfq interacts with these sRNAs and rpoS mRNA, the binding of wt Hfq and eleven mutant Hfqs to DsrA, RprA, OxyS and rpoS mRNA was examined. Nine of the mutant Hfq had single-residue mutations located on the proximal, distal, and outer-edge surfaces of the Hfq hexamer, while two Hfq had truncated C-terminal ends. Hfq with outer-edge mutations and truncated C-terminal ends behaved similar to wt Hfq with regard to binding the sRNAs, rpoS mRNA segments, and stimulating DsrA• rpoS mRNA formation. Proximal surface mutations decreased Hfq binding to the three sRNAs and the rpoS mRNA segment containing the translation initiation region. Distal surface mutations lowered Hfq's affinity for the rpoS mRNA segment containing the (ARN)₄ sequence. Strong Hfq binding to both rpoS mRNA segments appears to be needed for maximum enhancement of DsrA• rpoS mRNA annealing. OxyS bound tightly to Hfq but exhibited weak affinity for rpoS mRNA containing the leader region and 75 nt of coding sequence in the absence or presence of Hfq. This together with other results suggest OxyS represses rpoS mRNA translation by sequestering Hfq rather than binding to rpoS mRNA.► The binding of mutant Hfqs to sRNAs and segments of rpoS mRNA was examined. ► The ability of mutant Hfqs to enhance sRNA–mRNA binding was examined. ► Hfq with outer edge mutations or truncated C-terminal ends behaved similar to wt Hfq. ► Strong binding of Hfq to both rpoS mRNA sites is needed to enhance sRNA–rpoS mRNA hybridization. ► Results imply OxyS sequesters Hfq rather than binds rpoS mRNA to repress expression.

Keywords: Hfq protein; sRNA–rpoS mRNA regulation; DsrA; RprA; OxyS

Functional characterization of the proximal promoter of the murine pyruvate carboxylase gene in hepatocytes: Role of multiple GC boxes by Pinnara Rojvirat; Tanit Chavalit; Sureeporn Muangsawat; Ansaya Thonpho; Sarawut Jitrapakdee (pp. 541-548).

Pyruvate carboxylase (PC) catalyzes the first committed step in gluconeogenesis in the liver. The murine PC gene possesses two promoters, the proximal (P1) and the distal (P2) which mediate production of distinct tissue-specific mRNA isoforms. By comparing the luciferase activities of 5′-nested deletions of the P1-promoter in the AML12 mouse hepatocyte cell line, the critical cis-acting elements required for maintaining basal transcription were located within the 166 nucleotides proximal to the transcription start site. Three GC boxes were identified within this region and shown by gel shift and ChIP assays to bind Sp1/Sp3. Over-expression of Sp1/Sp3 in AML12 and NIH3T3 cells increased P1-promoter activity, with Sp1 being a stronger activator than Sp3. Mutation of any one of the three GC boxes dramatically reduced basal promoter activity by 60–80% suggesting that all three boxes are equally strong regulatory elements. In AML12 cells, over-expression of Sp1/Sp3 restored the transcriptional activity of GC1 and GC2 but not GC3 mutants to levels similar to that of the WT construct, suggesting that GC3 is particularly critical for Sp1/Sp3-mediated induction. In NIH3T3 cells, however, the three boxes were equally important, indicating that the GC boxes differentially contribute to transcriptional regulation of the P1-promoter in the two cell lines. Mutants harboring two disrupted GC boxes showed a further decrease in promoter activity similar to the triple GC box mutant. Neither Sp1 nor Sp3 was able to fully restore the promoter activities of these mutants to that the WT level.► The minimal elements of P1-promoter of PC gene are the first 166 nucleotides. ► The critical elements in the promoter include three GC boxes. ► All three GC boxes are equally important and are the binding sites for Sp1 and Sp3. ► GC3 is important for Sp1/Sp3-mediated transcriptional induction in AML12 cells. ► Sp1 is a stronger transcription activator of P1-promoter.

Keywords: Abbreviations; PC; pyruvate carboxylase; P1; proximal promoter, P2, distal promoter; Sp1; specific protein 1; Sp3; specific protein 3; CRE; cAMP-responsive element; PPRE; peroxisome proliferactor-activated receptor elementPyruvate carboxylase; Gluconeogenesis; Transcription; Sp1; Sp3; GC box

Gestational low protein diet selectively induces the amino acid response pathway target genes in the liver of offspring rats through transcription factor binding and histone modifications by Dan Zhou; Yuan-Xiang Pan (pp. 549-556).

The amino acid response (AAR) pathway detects a deficiency of dietary amino acid or protein. To investigate the impact of gestational protein restriction on the AAR pathway in offspring, pregnant Sprague–Dawley rats were fed a control (C) or low protein (LP) diet during gestation. Livers of female offspring were collected on postnatal d 38. The mRNA amount of Atf3 in LP offspring increased significantly compared with C offspring, while Asns did not differ between the two groups. ATF4 and p-eIF2α were both induced in LP offspring, whereas p-ERK was significantly decreased. Additionally, amino acid limitation (−AA) in HepG2 cells increased p-ERK and AAR pathway-related genes, while U0126 decreased p-ERK but did not completely reverse the activation of AAR pathway-related genes. Chromatin immunoprecipitation assay demonstrated an increased association of both RNA polymerase II (Pol II) and ATF4 at the Atf3 promoter in LP offspring, while acetylated histone H4, tri-methyl histone H3 at lysine 9, ATF4, ATF3, C/EBPβ, and CHOP, but not Pol II, were all increased at the Asns promoter in LP offspring. In −AA HepG2 cells, C/EBPβ siRNA treatment did not prevent the activation of either ATF3 or ASNS in response to −AA, while ATF4 siRNA prevented the activation of ASNS but not ATF3. Our data demonstrates that a maternal LP diet programs the AAR pathway in the liver of offspring rats. The differential priming of the downstream target genes of the AAR pathway in response to maternal LP diet presents a novel regulatory mechanism related to nutrient–gene interactions.Our study demonstrated that a gestational low protein diet programmed activation of the AAR pathway in the liver of offspring rats. ► The mRNA amount of Atf3 in LP offspring increased significantly, while Asns did not differ between the two groups. ► ATF4 and p-eIF2α were induced in LP offspring, whereas p-ERK was significantly decreased. ► RNA Pol II and ATF4 increased at Atf3 promoter. Histone modifications and transcription factors increased at Asns promoter. ► Combination of histone modifications, transcription factor bindings at Asns promoter may prevent the full-activation.

Keywords: Abbreviation; AAR pathway; amino acid response pathway; Asns; asparagine synthetase; Atf3; activating transcription factor 3; Atf4; activating transcription factor 4; bZIP; basic-region leucine zipper; PoI II; RNA polymerase II; p-eIF2α; phosphorylated eukaryotic initiation factor 2α; p-ERK; phosphorylated extracellular signal-regulated kinase; H3Ac; acetylated histone H3; H4Ac; acetylated histone H4; H3K9Me3; tri-methylated histone 3 at lysine 9 residues; LP; low protein; RT-PCR; reverse transcriptase-polymerase chain reaction; ChIP; chromatin immunoprecipitation; CARE; CCAAT-enhancer binding protein-activating transcription factor (C/EBP-ATF) response elementsPregnancy; Protein restriction; Epigenetic modification; Maternal programming

Separation-of-function mutation in HPC2, a member of the HIR complex in S. cerevisiae, results in derepression of the histone genes but does not confer cryptic TATA phenotypes by Nidhi Vishnoi; Kacie Flaherty; Leandria C. Hancock; Monica E. Ferreira; Amit Dipak Amin; Philippe Prochasson (pp. 557-566).

The HIR complex, which is comprised of the four proteins Hir1, Hir2, Hir3 and Hpc2, was first characterized as a repressor of three of the four histone gene loci in Saccharomyces cerevisiae. Using a bioinformatical approach, previous studies have identified a region of Hpc2 that is conserved in Schizosaccharomyces pombe and humans. Using a similar approach, we identified two additional domains, CDI and CDII, of the Hpc2 protein that are conserved among yeast species related to S. cerevisiae. We showed that the N terminal CDI domain (spanning amino acids 63–79) is dispensable for HIR complex assembly, but plays an essential role in the repression of the histone genes by recruiting the HIR complex to the HIR-dependent histone gene loci. The second conserved domain, CDII (spanning amino acids 452–480), is required for the stability of the Hpc2 protein itself as well as for the assembly of the HIR complex. In addition, we report a novel separation-of-function mutation within CDI of Hpc2, which causes derepression of the histone genes but does not confer other reported hir/hpc- phenotypes (such as Spt phenotypes, heterochromatin silencing defects and repression of cryptic promoters). This is the first direct demonstration that a separation-of-function mutation exists within the HIR complex.► New functional conserved Hpc2 domains CDI and CDII among fungi. ► Hpc2 N-terminal CDI essential for HIR-dependent histone gene repression. ► Hpc2 CDI domain needed for HIR complex localization to histone gene loci. ► Histone gene derepression in hpc2ΔCDI is not responsible for other hir/hpc- phenotypes. ► First report of separation-of-function mutation within the HIR complex.

Keywords: Histone gene; HIR complex; HPC2; Transcription; Chromatin; Yeast

Histone modifications in transcriptional activation during plant development by Alexandre Berr; Sarfraz Shafiq; Wen-Hui Shen (pp. 567-576).

In eukaryotic cell nuclei, chromatin states dictated by different combinations of post-translational modifications of histones, such as acetylation, methylation and monoubiquitination of lysine residues, are part of the multitude of epigenomes involved in the fine-tuning of all genetic functions and in particular transcription. During the past decade, an increasing number of ‘writers’, ‘readers’ and ‘erasers’ of histone modifications have been identified. Characterization of these factors in Arabidopsis has unraveled their pivotal roles in the regulation of essential processes, such as floral transition, cell differentiation, gametogenesis, and plant response/adaptation to environmental stresses. In this review we focus on histone modification marks associated with transcriptional activation to highlight current knowledge on Arabidopsis ‘writers’, ‘readers’ and ‘erasers’ of histone modifications and to discuss recent findings on molecular mechanisms of integration of histone modifications with the RNA polymerase II transcriptional machinery during transcription of the flowering repressor gene FLC.► Histone post-translational modifications (PTMs) involved in transcriptional activation. ► ‘Writers’, ‘readers’ and ‘erasers’ of histone PTMs. ► Histone PTMs during FLC transcription activation, initiation and elongation processes.

Keywords: Histone acetylation; Histone monoubiquitination; Histone methylation; Epigenetic regulation; FLC; transcription; Arabidopsis thaliana

The human histone H3 complement anno 2011 by Thomas H.A. Ederveen; Imke K. Mandemaker; Colin Logie (pp. 577-586).

Histones are highly basic, relatively small proteins that complex with DNA to form higher order structures that underlie chromosome topology. Of the four core histones H2A, H2B, H3 and H4, it is H3 that is most heavily modified at the post-translational level. The human genome harbours 16 annotated bona fide histone H3 genes which code for four H3 protein variants. In 2010, two novel histone H3.3 protein variants were reported, carrying over twenty amino acid substitutions. Nevertheless, they appear to be incorporated into chromatin. Interestingly, these new H3 genes are located on human chromosome 5 in a repetitive region that harbours an additional five H3 pseudogenes, but no other core histone ORFs. In addition, a human-specific novel putative histone H3.3 variant located at 12p11.21 was reported in 2011. These developments raised the question as to how many more human histone H3 ORFs there may be. Using homology searches, we detected 41 histone H3 pseudogenes in the current human genome assembly. The large majority are derived from the H3.3 gene H3F3A, and three of those may code for yet more histone H3.3 protein variants. We also identified one extra intact H3.2-type variant ORF in the vicinity of the canonical HIST2 gene cluster at chromosome 1p21.2. RNA polymerase II occupancy data revealed heterogeneity in H3 gene expression in human cell lines. None of the novel H3 genes were significantly occupied by RNA polymerase II in the data sets at hand, however. We discuss the implications of these recent developments.► Three new histone H3 protein variants have been described in the last 6months. We discuss these. ► We identify four additional human putative new H3 variant coding ORFs. ► We identify and classify 41 human H3 pseudogenes. ► We give a contemporary view on the topic of histone H3 function.

Keywords: Human histone H3; Protein variant; Pseudogene

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BBA - Gene Regulatory Mechanisms (v.1809, #10)