In the Sense of Transcription Regulation by G-Quadruplexes: Asymmetric Effects in Sense and Antisense Strands

G-Quadruplexes occupy important regulatory regions in the genome. DNA G-quadruplexes in the promoter regions and RNA quadruplexes in the UTRs (untranslated regions) have been individually studied and variously implicated at different regulatory levels of gene expression. However, the formation of G-quadruplexes in the sense and antisense strands and their corresponding roles in gene regulation have not been studied in much detail. In the present study, we have elucidated the effect of strand asymmetry in this context. Using biophysical methods, we have demonstrated the formation of stable G-quadruplex structure in vitro using CD and UV melting. Additionally, ITC was employed to demonstrate that a previously reported selective G-quadruplex ligand was able to bind and stabilize the G-quadruplex in the present sequence. Further, we have shown using reporter constructs that although the DNA G-quadruplex in either strand can reduce translation efficiency, transcriptional regulation differs when G-quadruplex is present in the sense or antisense strand. We demonstrate that the G-quadruplex motif in the antisense strand substantially inhibits transcription, while when in the sense strand, it does not affect transcription, although it does ultimately reduce translation. Further, it is also shown that the G-quadruplex stabilizing ligand can enhance this asymmetric transcription regulation as a result of the increased stabilization of the G-quadruplex.

MTERF1 Binds mtDNA to Prevent Transcriptional Interference at the Light-Strand Promoter but Is Dispensable for rRNA Gene Transcription Regulation

Mitochondrial transcription termination factor 1, MTERF1, has been reported to couple rRNA gene transcription initiation with termination and is therefore thought to be a key regulator of mammalian mitochondrial ribosome biogenesis. The prevailing model is based on a series of observations published over the last two decades, but no in vivo evidence exists to show that MTERF1 regulates transcription of the heavy-strand region of mtDNA containing the rRNA genes. Here, we demonstrate that knockout of Mterf1 in mice has no effect on mitochondrial rRNA levels or mitochondrial translation. Instead, loss of Mterf1 influences transcription initiation at the light-strand promoter, resulting in a decrease of de novo transcription manifested as reduced 7S RNA levels. Based on these observations, we suggest that MTERF1 does not regulate heavy-strand transcription, but rather acts to block transcription on the opposite strand of mtDNA to prevent transcription interference at the light-strand promoter.

Transcription regulation by inflexibility of promoter DNA in a looped complex.

The gal operon of Escherichia coli is negatively regulated by repressor binding to bipartite operators separated by 11 helical turns of DNA. Synergistic binding of repressor to separate sites on DNA results in looping, with the intervening DNA as a topologically closed domain containing the two promoters. A closed DNA loop of 11 helical turns, which is in-flexible to torsional changes, disables the promoters either by resisting DNA unwinding needed for open complex formation or by impeding the processive DNA contacts by an RNA polymerase in flux during transcription initiation. Interaction between two proteins bound to different sites on DNA modulating the activity of the intervening segment toward other proteins by allostery may be a common mechanism of regulation in DNA-multiprotein complexes.

Transcriptional regulation of IRS5/DOK4 expression in non-small-cell lung cancer cells

The insulin-receptor substrate family plays important roles in cellular growth, signaling, and survival. Two new members of this family have recently been isolated: IRS5/Dok4 and IRS6/Dok5. This study examines the expression of IRS5/DOK4 in a panel of lung cancer cell lines and tumor specimens. The results demonstrate that expression of IRS5/DOK4 is frequently altered with both elevated and decreased expression in non-small-cell lung cancer (NSCLC) tumor specimens. The altered expression of IRS5/DOK4 observed in tumor samples is not due to aberrant methylation. In vitro cell culture studies demonstrate that treatment of NSCLC cell lines with the histone deacetylase inhibitor trichostatin A (TSA) upregulates IRS5/DOK4. This finding indicates that expression is regulated epigenetically at the level of chromatin remodeling. Chromatin immunoprecipitation experiments confirm that the IRS5/DOK4 promoter has enhanced histone hyperacetylation following treatments with TSA. Finally, hypoxia was demonstrated to downregulate IRS5/DOK4 expression. This expression was restored by TSA. The clinical relevance of altered IRS5/DOK4 expression in NSCLC requires fur ther evaluation.

Transcription factories : gene expression in unions?

Transcription is a fundamental step in gene expression, yet it remains poorly understood at a cellular level. Visualization of transcription sites and active genes has led to the suggestion that transcription occurs at discrete sites in the nucleus, termed transcription factories, where multiple active RNA polymerases are concentrated and anchored to a nuclear substructure. However, this concept is not universally accepted. This Review discusses the experimental evidence in support of the transcription factory model and the evidence that argues against such a spatially structured view of transcription. The transcription factory model has implications for the regulation of transcription initiation and elongation, for the organization of genes in the genome, for the co-regulation of genes and for genome instability.

Integrated genome-wide chromatin occupancy and expression analyses identify key myeloid pro-differentiation transcription factors repressed by Myb

To gain insight into the mechanisms by which the Myb transcription factor controls normal hematopoiesis and particularly, how it contributes to leukemogenesis, we mapped the genome-wide occupancy of Myb by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) in ERMYB myeloid progenitor cells. By integrating the genome occupancy data with whole genome expression profiling data, we identified a Myb-regulated transcriptional program. Gene signatures for leukemia stem cells, normal hematopoietic stem/progenitor cells and myeloid development were overrepresented in 2368 Myb regulated genes. Of these, Myb bound directly near or within 793 genes. Myb directly activates some genes known critical in maintaining hematopoietic stem cells, such as Gfi1 and Cited2. Importantly, we also show that, despite being usually considered as a transactivator, Myb also functions to repress approximately half of its direct targets, including several key regulators of myeloid differentiation, such as Sfpi1 (also known as Pu.1), Runx1, Junb and Cebpb. Furthermore, our results demonstrate that interaction with p300, an established coactivator for Myb, is unexpectedly required for Myb-mediated transcriptional repression. We propose that the repression of the above mentioned key pro-differentiation factors may contribute essentially to Myb's ability to suppress differentiation and promote self-renewal, thus maintaining progenitor cells in an undifferentiated state and promoting leukemic transformation. © 2011 The Author(s).

The multiple sclerosis whole blood mRNA transcriptome and genetic associations indicate dysregulation of specific T cell pathways in pathogenesis

Multiple sclerosis (MS) is an autoimmune disease with a genetic component, caused at least in part by aberrant lymphocyte activity. The whole blood mRNA transcriptome was measured for 99 untreated MS patients: 43 primary progressive MS, 20 secondary progressive MS, 36 relapsing remitting MS and 45 age-matched healthy controls. The ANZgene Multiple Sclerosis Genetics Consortium genotyped more than 300 000 SNPs for 115 of these samples. Transcription from genes on translational regulation, oxidative phosphorylation, immune synapse and antigen presentation pathways was markedly increased in all forms of MS. Expression of genes tagging T cells was also upregulated (P < 10-12) in MS. A T cell gene signature predicts disease state with a concordance index of 0.79 with age and gender as co-variables, but the signature is not associated with clinical course or disability. The ANZgene genome wide association screen identified two novel regions with genome wide significance: one encoding the T cell co-stimulatory molecule, CD40; the other a region on chromosome 12q13-14. The CD40 haplotype associated with increased MS susceptibility has decreased gene expression in MS (P < 0.0007). The second MS susceptibility region includes 17 genes on 12q13-14 in tight linkage disequilibrium. Of these, only 13 are expressed in leukocytes, and of these the expression of one, FAM119B, is much lower in the susceptibility haplotype (P tdthomlt; 10-14). Overall, these data indicate dysregulation of T cells can be detected in the whole blood of untreated MS patients, and supports targeting of activated T cells in therapy for all forms of MS.

Genetic, functional, and histopathological evaluation of two C-terminal BRCA1 missense variants

Background: The vast majority of BRCA1 missense sequence variants remain uncharacterised for their possible effect on protein expression and function, and therefore are unclassified in terms of their pathogenicity. BRCA1 plays diverse cellular roles and it is unlikely that any single functional assay will accurately reflect the total cellular implications of missense mutations in this gene. Objective: To elucidate the effect of two BRCA1 variants, 5236G>C (G1706A) and 5242C>A (A1708E) on BRCA1 function, and to survey the relative usefulness of several assays to direct the characterisation of other unclassified variants in BRCA genes. Methods and Results: Data from a range of bioinformatic, genetic, and histopathological analyses, and in vitro functional assays indicated that the 1708E variant was associated with the disruption of different cellular functions of BRCA1. In transient transfection experiments in T47D and 293T cells, the 1708E product was mislocalised to the cytoplasm and induced centrosome amplification in 293T cells. The 1708E variant also failed to transactivate transcription of reporter constructs in mammalian transcriptional transactivation assays. In contrast, the 1706A variant displayed a phenotype comparable to wildtype BRCA1 in these assays. Consistent with functional data, tumours from 1708E carriers showed typical BRCA1 pathology, while tumour material from 1706A carriers displayed few histopathological features associated with BRCA1 related tumours. Conclusions: A comprehensive range of genetic, bioinformatic, and functional analyses have been combined for the characterisation of BRCA1 unclassified sequence variants. Consistent with the functional analyses, the combined odds of causality calculated for the 1706A variant after multifactorial likelihood analysis (1:142) indicates a definitive classification of this variant as "benign". In contrast, functional assays of the 1708E variant indicate that it is pathogenic, possibly through subcellular mislocalisation. However, the combined odds of 262:1 in favour of causality of this variant does not meet the minimal ratio of 1000:1 for classification as pathogenic, and A1708E remains formally designated as unclassified. Our findings highlight the importance of comprehensive genetic information, together with detailed functional analysis for the definitive categorisation of unclassified sequence variants. This combination of analyses may have direct application to the characterisation of other unclassified variants in BRCA1 and BRCA2.

Promoting developmental transcription.

Animal growth and development depend on the precise control of gene expression at the level of transcription. A central role in the regulation of developmental transcription is attributed to transcription factors that bind DNA enhancer elements, which are often located far from gene transcription start sites. Here, we review recent studies that have uncovered significant regulatory functions in developmental transcription for the TFIID basal transcription factors and for the DNA core promoter elements that are located close to transcription start sites.

Regulation of the alpha-fetoprotein promoter: Ku binding and DNA spatial conformation

This work shows that the proximal promoter of the mouse Afp gene contains a Ku binding site and that Ku binding is associated with down-regulation of the transcriptional activity of the Afp promoter. The Ku binding site is located in a segment able to adopt a peculiar structured form, probably a hairpin structure. Interestingly, the structured form eliminates the binding sites of the positive transcription factor HNF1. Furthermore, a DNAse hypersensitive site is detected in footprinting experiments done with extracts of AFP non-expressing hepatoma cells. These observations suggest that the structured form is stabilised by Ku and is associated with extinction of the gene in AFP non-expressing hepatic cells.

A tobacco cDNA reveals two different transcription patterns in vegetative and reproductive organs

In order to identify genes expressed in the pistil that may have a role in the reproduction process, we have established an expressed sequence tags project to randomly sequence clones from a Nicotiana tabacum stigma/style cDNA library. A cDNA clone (MTL-8) showing high sequence similarity to genes encoding glycine-rich RNA-binding proteins was chosen for further characterization. Based on the extensive identity of MTL-8 to the RGP-1a sequence of N. sylvestris, a primer was defined to extend the 5′ sequence of MTL-8 by RT-PCR from stigma/style RNAs. The amplification product was sequenced and it was confirmed that MTL-8 corresponds to an mRNA encoding a glycine-rich RNA-binding protein. Two transcripts of different sizes and expression patterns were identified when the MTL-8 cDNA insert was used as a probe in RNA blots. The largest is 1,100 nucleotides (nt) long and markedly predominant in ovaries. The smaller transcript, with 600 nt, is ubiquitous to the vegetative and reproductive organs analyzed (roots, stems, leaves, sepals, petals, stamens, stigmas/styles and ovaries). Plants submitted to stress (wounding, virus infection and ethylene treatment) presented an increased level of the 600-nt transcript in leaves, especially after tobacco necrosis virus infection. In contrast, the level of the 1,100-nt transcript seems to be unaffected by the stress conditions tested. Results of Southern blot experiments have suggested that MTL-8 is present in one or two copies in the tobacco genome. Our results suggest that the shorter transcript is related to stress while the larger one is a flower predominant and nonstress-inducible messenger.

Ligand-signaled upregulation of Enterococcus faecalis ace transcription, a mechanism for modulating host-E. faecalis interaction.

Enterococcus faecalis, the third most frequent cause of bacterial endocarditis, appears to be equipped with diverse surface-associated proteins showing structural-fold similarity to the immunoglobulin-fold family of staphylococcal adhesins. Among the putative E. faecalis surface proteins, the previously characterized adhesin Ace, which shows specific binding to collagen and laminin, was detectable in surface protein preparations only after growth at 46 degrees C, mirroring the finding that adherence was observed in 46 degrees C, but not 37 degrees C, grown E. faecalis cultures. To elucidate the influence of different growth and host parameters on ace expression, we investigated ace expression using E. faecalis OG1RF grown in routine laboratory media (brain heart infusion) and found that ace mRNA levels were low in all growth phases. However, quantitative reverse transcription-PCR showed 18-fold-higher ace mRNA amounts in cells grown in the presence of collagen type IV compared to the controls. Similarly, a marked increase was observed when cells were either grown in the presence of collagen type I or serum but not in the presence of fibrinogen or bovine serum albumin. The production of Ace after growth in the presence of collagen type IV was demonstrated by immunofluorescence microscopy, mirroring the increased ace mRNA levels. Furthermore, increased Ace expression correlated with increased collagen and laminin adhesion. Collagen-induced Ace expression was also seen in three of three other E. faecalis strains of diverse origins tested, and thus it appears to be a common phenomenon. The observation of host matrix signal-induced adherence of E. faecalis may have important implications on our understanding of this opportunistic pathogen.

Genetic Engineering of Induced Pluripotent Stem Cells and Reprogramming to Motor Neurons to Elucidate Selective Motor Neuron Death in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease, fatal within 1 to 5 years after onset of symptoms. About 3 out of 100’000 persons are diagnosed with ALS and there is still no cure available [1, 2]. 95% of all cases occur sporadically and the aetiology remains largely unknown [XXXX]. However, up to now 16 genes were identified to play a role in the development of familial ALS. One of these genes is FUS that encodes for the protein fused in sarcoma/translocated in liposarcoma (FUS/TLS). Mutations in this gene are responsible for some cases of sporadic as well as of inherited ALS [3]. FUS belongs to the family of heterogeneous nuclear ribonucleoproteins and is predicted to be involved in several cellular functions like transcription regulation [4], RNA splicing [5, 6], mRNA transport in neurons [7] and microRNA processing [8]. Aberrant accumulation of mutated FUS has been found in the cytoplasm of motor neurons from ALS patients [9]. The mislocalization of FUS is based on a mutation in the nuclear localization signal of FUS [10]. However, it is still unclear if the cytoplasmic localization of FUS leads to a toxic gain of cytoplasmic function and/or a loss of nuclear function that might be crucial in the course of ALS. The goal of this project is to characterize the impact of ALS-associated FUS mutations on in vitro differentiated motor neurons. To this end, we edit the genome of induced pluripotent stem cells (iPSC) using transcription activator-like effector nucleases (TALENs) [11,12] to create three isogenic cell lines, each carrying an ALS-associated FUS mutation (G156E, R244C and P525L). These iPSC’s will then be differentiated to motor neurons according to a recently establishe protocol (Ref Wichterle) and serve to study alterations in the transcriptome, proteome and metabolome upon the expression of ALS-associated FUS. With this approach, we hope to unravel the molecular mechanism leading to FUS-associated ALS and to provide new insight into the emerging connection between misregulation of RNA metabolism and neurodegeneration, a connection that is currently implied in a variety of additional neurological diseases, including spinocerebellar ataxia 2 (SCA-2), spinal muscular atrophy (SMA), fragile X syndrome, and myotonic dystrophy.