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The recognition of β cell dedifferentiation in type 2 diabetes raises the translational relevance of mechanisms that direct and maintain β cell identity. LIM domain-binding protein 1 (LDB1) nucleates multimeric transcriptional complexes and establishes promoter-enhancer looping, thereby directing fate assignment and maturation of progenitor populations. Many terminally differentiated endocrine cell types, however, remain enriched for LDB1, but its role is unknown. Here, we have demonstrated a requirement for LDB1 in maintaining the terminally differentiated status of pancreatic β cells. Inducible ablation of LDB1 in mature β cells impaired insulin secretion and glucose homeostasis. Transcriptomic analysis of LDB1-depleted β cells revealed the collapse of the terminally differentiated gene program, indicated by a loss of β cell identity genes and induction of the endocrine progenitor factor neurogenin 3 (NEUROG3). Lineage tracing confirmed that LDB1-depleted, insulin-negative β cells express NEUROG3 but do not adopt alternate endocrine cell fates. In primary mouse islets, LDB1 and its LIM homeodomain-binding partner islet 1 (ISL1) were coenriched at chromatin sites occupied by pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), forkhead box A2 (FOXA2), and NK2 homeobox 2 (NKX2.2)--factors that co-occupy active enhancers in 3D chromatin domains in human islets. Indeed, LDB1 was enriched at active enhancers in human islets. Thus, LDB1 maintains the terminally differentiated state of β cells and is a component of active enhancers in both murine and human islets., Introduction All forms of diabetes are characterized by reduced numbers and/ or dysfunction of insulin-producing pancreatic islet β cells (1, 2). The β cell is unique in its ability to [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2010.10.033 Byline: Liat Harel-Sharvit (1), Naama Eldad (1), Gal Haimovich (1), Oren Barkai (1), Lea Duek (1), Mordechai Choder (1) Keywords: RNA; CELLBIO Abstract: Little is known about crosstalk between the eukaryotic transcription and translation machineries that operate in different cell compartments. The yeast proteins Rpb4p and Rpb7p represent one such link as they form a heterodimer that shuttles between the nucleus, where it functions in transcription, and the cytoplasm, where it functions in the major mRNA decay pathways. Here we show that the Rpb4/7 heterodimer interacts physically and functionally with components of the translation initiation factor 3 (eIF3), and is required for efficient translation initiation. Efficient translation in the cytoplasm depends on association of Rpb4/7 with RNA polymerase II (Pol II) in the nucleus, leading to a model in which Pol II remotely controls translation. Hence, like in prokaryotes, the eukaryotic translation is coupled to transcription. We propose that Rpb4/7, through its interactions at each step in the mRNA lifecycle, represents a class of factors, 'mRNA coordinators,' which integrate the various stages of gene expression into a system. Author Affiliation: (1) Department of Molecular Microbiology, Rappaport Faculty of Medicine, Technion - Israel Institute of Technology, Haifa, 31096, Israel Article History: Received 17 December 2009; Revised 28 July 2010; Accepted 25 October 2010 Article Note: (miscellaneous) Published: November 11, 2010

Genome sequencing of Helicobacter pylori has revealed the potential proteins and genetic diversity of this prevalent human pathogen, yet little is known about its transcriptional organization and noncoding RNA output. Massively parallel cDNA sequencing (RNA-seq) has been revolutionizing global transcriptomic analysis. Here, using a novel differential approach (dRNA-seq) selective for the 5' end of primary transcripts, we present a genome-wide map of H. pylori transcriptional start sites and operons. We discovered hundreds of transcriptional start sites within operons, and opposite to annotated genes, indicating that complexity of gene expression from the small H. pylori genome is increased by uncoupling of polycistrons and by genome-wide antisense transcription. We also discovered an unexpected number of ~60 small RNAs including the e-subdivision counterpart of the regulatory 6S RNA and associated RNA products, and potential regulators of cis- and trans-encoded target messenger RNAs. Our approach establishes a paradigm for mapping and annotating the primary transcriptomes of many living species., About 50% of all humans are infected with Helicobacter pylori, a microaerophilic, Gram-negative e-proteobacterium that thrives in the acidic environment of the stomach, and is associated with severe inflammation' peptic [...]

Atonal is a Drosophila proneural protein required for the proper formation of the R8 photoreceptor cell, the founding photoreceptor cell in the developing retina. Proper expression and refinement of the Atonal protein is essential for the proper formation of the Drosophila adult eye. In vertebrates, expression of transcription factors orthologous to Drosophila Atonal (MATH5/Atoh7, XATH5, and ATH5) and their progressive restriction are also involved in specifying the retinal ganglion cell, the founding neural cell type in the mammalian retina. Thus, identifying factors that are involved in regulating the expression of Atonal during development are important to fully understand how retinal neurogenesis is accomplished. We have performed a chemical mutagenesis screen for autosomal dominant enhancers of a loss-of-function atonal eye phenotype. We report here the identification of five genes required for proper Atonal expression, three of which are novel regulators of Atonal expression in the Drosophila retina. We characterize the role of the daughterless, kismet, and roughened eye genes on atonal transcriptional regulation in the developing retina and show that each gene regulates atonal transcription differently within the context of retinal development. Our results provide additional insights into the regulation of Atonal expression in the developing Drosophila retina.

In genomewide mapping of expression quantitative trait loci (eQTL), it is widely believed that thousands of genes are trans-regulated by a small number of genomic regions called 'regulatory hotspots,' resulting in 'trans-regulatory bands' in an eQTL map. As several recent studies have demonstrated, technical confounding factors such as batch effects can complicate eQTL analysis by causing many spurious associations including spurious regulatory hotspots. Yet little is understood about how these technical confounding factors affect eQTL analyses and how to correct for these factors. Our analysis of data sets with biological replicates suggests that it is this intersample correlation structure inherent in expression data that leads to spurious associations between genetic loci and a large number of transcripts inducing spurious regulatory hotspots. We propose a statistical method that corrects for the spurious associations caused by complex intersample correlation of expression measurements in eQTL mapping. Applying our intersample correlation emended (ICE) eQTL mapping method to mouse, yeast, and human identifies many more cis associations while eliminating most of the spurious trans associations. The concordances of cis and trans associations have consistently increased between different replicates, tissues, and populations, demonstrating the higher accuracy of our method to identify real genetic effects.

bZIP-type transcription factors AREBs/ABFs bind an abscisic acid (ABA)-responsive cis-acting element named ABRE and transactivate downstream gene expression in Arabidopsis. Because AREB1 overexpression could not induce downstream gene expression, activation of AREB1 requires ABA-dependent posttranscriptional modification. We confirmed that ABA activated 42-kDa kinase activity, which, in turn, phosphorylated Ser/Thr residues of R-X-X-S/T sites in the conserved regions of AREB1. Amino acid substitutions of R-X-X-S/T sites to Ala suppressed transactivation activity, and multiple substitution of these sites resulted in almost complete suppression of transactivation activity in transient assays. In contrast, substitution of the Ser/Thr residues to Asp resulted in high transactivation activity without exogenous ABA application. A phosphorylated, transcriptionally active form was achieved by substitution of Ser/Thr in all conserved R-X-X-S/T sites to Asp. Transgenic plants overexpressing the phosphorylated active form of AREB1 expressed many ABA-inducible genes, such as RD29B, without ABA treatment. These results indicate that the ABA-dependent multisite phosphorylation of AREB1 regulates its own activation in plants. transactivation activity | transcription factorAREB1 | transgenic Arabidopsis

An insertion between iscA and hscB of the Xenorhabdus nematophila iscRSUA-hscBA-fdx locus, predicted to encode Fe-S assembly machinery, prevented colonization of Steinernema carpocapsae nematodes. The insertion disrupted cotranscription of iscA and hscB, but did not reduce hscBA expression, suggesting that X.nematophila requires coordinated expression of the isc.hsc.fdx locus for colonization.

Isolation and characterization of a poly-beta-hydroxybutyrate mutant of Azotobacter vinelandii are presented. Four promoters within the phbR-phbB intergenic region comprise two promoters each transcribing phbB and phbR. Results indicate that PhbR is involved in the transcription activaion, which occurs from one of the phbB promoters.

cDNA array technology has proven to be a powerful way to monitor global changes in gene expression patterns. Here, we present an approach that extends the current utility of cDNA arrays to allow the evaluation of the relative roles of transcription and mRNA turnover in governing gene expression on a global basis, compared with current individual gene-by-gene analyses. This method, which involves comparison of large-scale hybridization patterns generated with steady-state mRNA versus newly transcribed (nuclear run-on) RNA, was used to demonstrate the importance of mRNA turnover in regulating gene expression following several conditions of stress. transcription | stress response | gene expression

The aldehyde/alcohol dehydrogenase (AADH) coding gene adhE2 of Clostridium acetobutylicum ATC 824 is the second gene identified in this bacterium besides adhE. The adhE2 is localized to the pSOL1 megaplasmid, is preferentially expressed in alcohologenic cultures, and is a NADH-dependent bifunctional AADH.

Research discusses scoC gene mutational effects in Bacillus subtilis by comparing its expression in both wild-tpe and mutant cells using DNA microarray technique. Phenotypic-genotypic correlation analysis suggests that scoC gene regulates the expression of at least 560 genes in the B. subtilis genome.

Research has been conducted on the ftsZ expression in Streptomyces griseus. Results indicate that this expression is developmentally regulated at the transcriptional level in S. griseus.

Results show that the matB fimbrial gene is expressed only in a geneticlly conserved, virulent Escherichica coli strain. Data suggest that regulation of Mat fimbria expression involves transcriptional and posttranslational events.

Transcription analyses of Deinococcus radiodurans promoters reveal that the -10 and -35 sequences of the promoters resemble Escherichia coli sigma(sup)70 sequences.

Research demonstrates that transcription modulation of the sequence unmodified pilE gene alters gonococcal piliation phenotype, affecting the number of pili per cell, pilus bundling, DNA transformation competence, and epithelial cell adherence.

Of the eight chitinase genes of Streptomyces coelicolor A3(2), five are induced by colloidal chitin and chitobiose with the latter inducing chitinase gene transcription sooner than the former. Data further suggest that glucose represses the expression of the five chitinase genes.

Results show that yabG gene is expressed only during sporulation of Bacillus subtilis. Data further indicate that the gene encodes a sporulation specific protein which has role in the coat protein composition.

Ribosomes possess an innate capacity for open reading frames coupling by translational bypassing suggesting to its common prevalence in cells. Data show that T(sub)4 gene 60 uses this route for bypassing 50 nucleotides between two open reading frames.

Results show that involvement of double-stranded RNA interference (RNAi) in the gene-silencing events is sequence specific and mediated only by double stranded RNAi targeting specific mRNA for degradation.

The [Beta]3 tubulin gene ([Beta]Tub60D) is a structural gene expressed during mesoderm development from the extended germ band stage onward. Expression within the individual mesodermal derivatives is guided by different control elements. The upstream regions allow expression in the dorsal vessel and the somatic mesoderm while enhancers localized in the first intron guide expression in the visceral mesoderm. Deletion analysis carried out in transgenic flies revealed independent regulatory elements for the dorsal vessel and the somatic mesoderm. For expression in the somatic mesoderm, a 279-bp region is absolutely essential. This region contains a binding site for the Drosophila myocyte-specific enhancer binding factor 2 (D-MEF2), a MADS-box transcription factor known to be essential for mesoderm development. Deletion or mutation of this D-MEF2 binding site strongly reduces transcription. This pattern is consistent with the strongly reduced expression of [Beta]3 tubulin in D-mef2 mutant embryos. This analysis furthermore reveals that the D-MEF2 binding site acts in concert with nearby cis regulatory elements. These data show that the upstream control region of the [Beta]3 tubulin gene is an early target of the D-MEF2 transcriptional activator. Key Words: tubulin gene; mesoderm; regulation; MEF2.

Tag1 is an autonomous transposable element of Arabidopsis thaliana. Tag1 expression was examined in two ecotypes of Arabidopsis (Columbia and No-O) that were transformed with CaMV 35S-Tag1-GUS DNA. These ecotypes contain no endogenous Tag1 elements. A major 2.3-kb and several minor transcripts were detected in all major organs of the plants. The major transcript encoded a putative transposase of 84.2 kD with two nuclear localization signal sequences and a region conserved among transposases of the Ac or hAT family of elements. The abundance of Tag1 transcripts varied among transgenic lines and did not correlate with somatic excision frequency or germinal reversion rates, suggesting that factors other than transcript levels control Tag1 excision activity. In untransformed plants of the Landsberg ecotype, which contain two endogenous Tag1 elements, no Tag1 transcripts were detected. Agrobacterium-mediated transformation of these Landsberg plants with a defective 1.4-kb Tag1 element resulted in the appearance of full-length Tag1 transcripts from the endogenous elements. Transformation with control DNA containing no Tag1 sequences did not activate endogenous Tag1 expression. These results indicate that Agrobacterium-mediated transformation with dTag1 can activate the expression of Tag1.

The mechanism behind the regulation of dimethyl sulfoxide (DMSO) reductase genes of the phototroph Rhodobacter sphaeroides by both DMSO and oxygen was studied by investigating the transcriptional regulation of the dorC, dorR and dorS promoters. The dorC::lacZ expression was minimized by up to one-third when the cells were grown photosynthetically with DMSO and with medium to high lighting. The induction also depends on the DorR and DorS proteins.

The egr-type zinc-finger transcription factor encoded by the Drosophila gene stripe (sr) is expressed in a subset of epidermal cells to which muscles attach during late stages of embryogenesis. We report loss-of-function and gain-of-function experiments indicating that sr activity provides ectodermal cells with properties required for the establishment of a normal muscle pattern during embryogenesis and for the differentiation of tendon-like epidermal muscle attachment sites (EMA). Our results show that sr encodes a transcriptional activator which acts as an autoregulated developmental switch gene. sr activity controls the expression of EMA-specific target genes in cells of ectodermal but not of mesodermal origin. sr-expressing ectodermal cells generate long-range signals that interfere with the spatial orientation of the elongating myotubes.

The effects of mutations on the -45 region sequence of the Escherichia coli lac promoter were analyzed to characterize the interaction between the gene and catabolite gene activator-adenosine cyclic monophosphate (CAP). Randomized mutations of the DNA located the in -45 region of the Escherichia coli lac promoter significantly altered promoter activity and its activation by CAP. However, only a small percentage of changes in the -45 region inhibited CAP stimulation of lac.

Analysis of the isolated characteristics of marked and unmarked mutations of mutated Paracoccus denitricans shows the MxaX response regulator is crucial to enable the mox genes to express themselves. The activation of MxaX requires more than one pathway. P. denitrificans oxidizes methanol, and forms formaldehyde when methanol substrates form. MxaX can be activated even in the absence of the signal sensor MxaY on a parallel signal transduction pathway.

A correlation between a transgenic reporter gene's tissue-specific expression and an octamer and CCAAT motif binding nuclear proteins, with the help of in vitro and in vivo studies, indicate that the presence of sequence elements in the -1,824-bp region of the promoter is essential for in vivo tissue-specific transcription of lipoprotein lipase. Gel-retention analysis and transient transfection methods reveal maximum expression of the luciferase reporter gene in transgenic mice having the -1,824-bp promoter constructs.

A comparative study of the spatiotemporal arrangement of epsilon- subunit transcripts of the acetylcholine receptor during synapsis in adult and fetal muscle using in situ hybridization and reverse transcription-PCR techniques shows that motor neurons, and not the developing muscle nuclei, control the in vivo epsilon-subunit gene transcription during synapsis. The motor neuron also induces the mRNA expression of the epsilon-subunit in subsynaptic nuclei during 'ectopic' and 'ontogenic' synaptogenesis in adult and fetal muscles, respectively. The muscle and neurons retain their plasticity during their active involvement in the gene expression and induction.

Northern and Southern RNA blot analyses, primer extension and primed DNA labeling with 32P of the DNA fragment clone from Anabaena sp. strain PCC 7120 reveal that the argD gene, which encodes N-acetylornithine aminotransferase, and the argC gene are found constitutively as monocistronic mRNA transcripts. The transcription initiation sites of the genes follow the sequences resembling that of the RNA polymerase sigma 70 consensus promoter of Escherichia coli. Dinitrogen-grown cultures exhibit a transcription start site for argD gene that does not follow the sigma 70 consensus. Exogenous insertions of arginine have no effect on the genes.

Transcriptional fusions between a lacZ reporter gene and the 399 bp intergenic region of the two operons that transcribe the tagA and tagD Bacillus subtilis genes to study phosphate limitation, chromosomal location and germination of the bacterial cells reveal that tagD and tagA expressions occur coordinately, with tagD exhibiting a higher level of expression than tagA. Chromosomal location has no effect on the expression level while a decrease in tag gene expression induces phosphate limitation. The gene expression is mild during germination but completely disappears during sporulation. Impairments in cell DNA sequences or incorporation of SOS activity generates no influence on the tag gene expression.

Agrobacterium tumefaciens incorporated with VirGN54D plasmids contain constitutive virulence (Vir) gene expressions and enhance the transfer properties of transferred DNA (T-DNA). Beta-glucuronidase (GUS) reporter gene used to determine post-transfer T-DNA expression reveals an increase in GUS-expressing foci. Wild-type VirG gene transcripts enhance T-DNA expression in tobacco but not in maize.

Gene expressions and promoter functions are analyzed by using transcriptional fusion in genes. Reporter genes increase promoter activity. The luxA gene inhibits or activates transcription of promoters due to the curved DNA fragment in 5' sequence of the gene. The DNA sequence is eliminated by inducing multiple mutations. LuxAB is a single reporter gene used in promoter activity.

An analysis of the spatial distribution and translation of the alpha subunit of mRNA reveals that, unlike the alpha subunit, it accumulates in all cells of the early blastocysts, confirming the post-transcriptional regulation of spatial distribution of the subunit. The presence of alpha subunit mRNA in polyribosome is evident from the 4-cell stage.

DNA methylation regulates the transition between phase ON and OFF pyelonephritis-associated pili (Pap) expression states in Escherichia coli. Methylation states of the GATC(super 1028) and GATC(super 1130) sites limit the pap operon transcriptional state. Proper methylation configuration of these sites is essential for transcription. These regulatory interactions form an epigenetic switch for the expression states of E. coli.