Co-transcriptional folding orchestrates sequential multi-effector sensing by a glycine tandem riboswitch.

BIOLOGICAL SIGNIFICANCE OF mRNA TURNOVER It has become clear that mRNA turnover can play a major role in the control of gene expression. In eukaryotic cells, the decay rates of individual mRNAs vary by more than two orders of magnitude (Singer and Penman 1973; Spradling et al. 1975; Cabrera et al. 1984). There have also been a growing number of examples wherein the regulation of gene expression in response to an environmental cue is attained by altering the rates of degradation for specific mRNAs (for review, see Ross 1995). In addition, a specialized system of mRNA decay, referred to as mRNA surveillance, functions to ensure that mRNA biogenesis has been completed correctly by degrading aberrant transcripts (for reviews, see Chapters 29 and 30). Finally, specific mechanisms of mRNA degradation function in the genespecific silencing induced by double-stranded RNA (for review, see Fire 1999). The numerous and diverse roles for mRNA degradation suggest that the modulation of mRNA stability is a critical step in the regulation of eukaryotic gene expression. PATHWAYS OF mRNA DEGRADATION Four distinct pathways of mRNA turnover have been identified in eukaryotic organisms (Fig. 1). In yeast, degradation begins by shortening of the 3′ poly(A) tail followed by removal of the 5′ cap structure (decapping), thus allowing 5′ to 3′ exonucleolytic digestion of the body of the transcript (Muhlrad and Parker 1992; Decker and Parker 1993; Hsu and Stevens 1993; Muhlrad et al. 1994, 1995). Alternatively, mRNAs can be degraded 3′ to 5′ following deadenylation (Muhlrad et al....

Regulated Translation Initiation Controls Stress-Induced Gene Expression in Mammalian Cells

Antioxidant-response element (ARE) and nuclear factor Nrf2-mediated expression and coordinated induction of genes encoding chemopreventive proteins, including NQO1, are critical mechanisms in chemoprotection. Recently, Nrf3, a new member of the Nrf family with substantial homology to Nrf2, was identified and cloned. In this report, we have investigated the role of Nrf3 in ARE-mediated gene expression and induction of NQO1 in response to antioxidants. Overexpression of Nrf3 in Hep-G2 cells led to a concentration-dependent decrease in transfected and endogenous NQO1 gene expression and induction in response to antioxidant tert-butylhydroquinone (t-BHQ). Deletion mutation analysis revealed that Nrf3 repression of NQO1 gene expression required heterodimerization and DNA binding domains but not transcriptional activation domain of Nrf3. Bandshift and supershift assays with in vitro transcribed and translated proteins and nuclear extracts from Hep-G2 cells treated with Me2SO and t-BHQ and immunoprecipitation assays demonstrated that Nrf3 associates with small Maf proteins to bind to the ARE. RNA interference specific to Nrf3 reduced intracellular Nrf3 leading to increased expression and induction of transfected and endogenous NQO1 gene expression in response to t-BHQ. These results combined suggest that Nrf3 is a negative regulator of ARE-mediated gene expression.

The Drosophila Dachshund (Dac) gene, cloned as a dominant inhibitor of the hyperactive growth factor mutant, ellipse, encodes a key component of the Retinal Determination Gene Network (RDGN) that governs cell fate. DACH1 (Dachshund homolog 1) regulates gene expression either indirectly as a co-integrator or through direct DNA binding. CAST identified a DACH1 DNA-binding sequence which resembles the FOX (Forkhead Box containing protein) binding site. Genome-wide in-silico promoter analysis of DACH1 binding sites identified gene clusters populating cellular pathways associated with the cell cycle and growth factor signaling. ChIP-Seq mapped DACH1 binding sites to corresponding gene clusters predicted in silico and identified as weight matrix resembling the CAST-defined sequence. DACH1 antagonized FOXM1 target gene expression, promoter occupancy in the context of local chromatin, and contact-independent growth. Further studies were conducted to understand, at a higher level of resolution, the mechanisms governing DACH1-mediated transcriptional repression via DNA-sequence specific binding. DACH1 repressed, while FOX proteins enhanced, both DRE and FOXA responsive element-driven gene expression. Reduced DACH1 expression using a shRNA approach enhanced FOX protein activity. As DACH1 antagonized FOX target gene expression and attenuated FOX signaling, we sought to identify limiting co-integrator proteins governing DACH1 signaling. Proteomic analysis identified transcription elongation regulator 1 (TCERG1) as the transcriptional co-regulator of DACH1 activity. The FF2 domain of TCERG1 was required for DACH1 binding, and the deletion of FF2 abolished DACH1 trans-repression function. The carboxyl terminus of DACH1 was necessary and sufficient for TCERG1 binding. Thus, DACH1 represses gene transcription through direct DNA binding to the promoter region of target genes by recruiting the transcriptional co-regulator, TCERG1. Attenuation of FOX function by the cell fate determination pathway has broad implications given the diverse role of FOX proteins in cellular biology and tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 269. doi:10.1158/1538-7445.AM2011-269

TPP riboswitch aptamer: Role of Mg2+ ions, ligand unbinding, and allostery

Coastal fish populations are threatened by multiple anthropogenic impacts, including the accumulation of industrial contaminants and the increasing frequency of hypoxia. Some populations of the Atlantic killifish (Fundulus heteroclitus), like those in New Bedford Harbor (NBH), Massachusetts, USA, have evolved a resistance to dioxin-like polychlorinated biphenyls (PCBs) that may influence their ability to cope with secondary stressors. To address this question, we compared hepatic gene expression and DNA methylation patterns in response to mild or severe hypoxia in killifish from NBH and Scorton Creek (SC), a reference population from a relatively pristine environment. We hypothesized that NBH fish would show altered responses to hypoxia due to trade-offs linked to toxicant resistance. Our results revealed substantial differences between populations. SC fish demonstrated dose-dependent changes in gene expression in response to hypoxia, while NBH fish exhibited a muted transcriptional response to severe hypoxia. Interestingly, NBH fish showed significant DNA methylation changes in response to hypoxia, while SC fish did not exhibit notable epigenetic alterations. These findings suggest that toxicant-adapted killifish may face trade-offs in their molecular response to environmental stress, potentially impacting their ability to survive severe hypoxia in coastal habitats. Further research is needed to elucidate the functional implications of these epigenetic modifications and their role in adaptive stress responses.

Despite extensive investigations, an effective treatment for sepsis remains elusive and a better understanding of the inflammatory response to infection is required to identify potential new targets for therapy. In this study we have used RNAi technology to show, for the first time, that the inducible lysophosphatidylcholine acyltransferase 2 (LPCAT2) plays a key role in macrophage inflammatory gene expression in response to stimulation with bacterial ligands. Using siRNA- or shRNA-mediated knockdown, we demonstrate that, in contrast to the constitutive LPCAT1, LPCAT2 is required for macrophage cytokine gene expression and release in response to TLR4 and TLR2 ligand stimulation but not for TLR-independent stimuli. In addition, cells transfected to overexpress LPCAT2 exhibited increased expression of inflammatory genes in response to LPS and other bacterial ligands. Furthermore, we have used immunoprecipitation and Western blotting to show that in response to LPS, LPCAT2, but not LPCAT1, rapidly associates with TLR4 and translocates to membrane lipid raft domains. Our data thus suggest a novel mechanism for the regulation of inflammatory gene expression in response to bacterial stimuli and highlight LPCAT2 as a potential therapeutic target for development of anti-inflammatory and anti-sepsis therapies.

In Pseudomonas putida, the expression of the pWW0 plasmid genes for the toluene/xylene assimilation pathway (the TOL pathway) is subject to complex regulation in response to environmental and physiological signals. This includes strong inhibition via catabolite repression, elicited by the carbon sources that the cells prefer to hydrocarbons. The Crc protein, a global regulator that controls carbon flow in pseudomonads, has an important role in this inhibition. Crc is a translational repressor that regulates the TOL genes, but how it does this has remained unknown. This study reports that Crc binds to sites located at the translation initiation regions of the mRNAs coding for XylR and XylS, two specific transcription activators of the TOL genes. Unexpectedly, eight additional Crc binding sites were found overlapping the translation initiation sites of genes coding for several enzymes of the pathway, all encoded within two polycistronic mRNAs. Evidence is provided supporting the idea that these sites are functional. This implies that Crc can differentially modulate the expression of particular genes within polycistronic mRNAs. It is proposed that Crc controls TOL genes in two ways. First, Crc inhibits the translation of the XylR and XylS regulators, thereby reducing the transcription of all TOL pathway genes. Second, Crc inhibits the translation of specific structural genes of the pathway, acting mainly on proteins involved in the first steps of toluene assimilation. This ensures a rapid inhibitory response that reduces the expression of the toluene/xylene degradation proteins when preferred carbon sources become available.

De-Etiolated 1 and Damaged DNA Binding Protein 1 Interact to Regulate Arabidopsis Photomorphogenesis

Plant productivity is greatly affected by environmental stresses such as drought, salt loading, and freezing. We reported previously that a cis-acting promoter element, the dehydration response element (DRE), plays an important role in regulating gene expression in response to these stresses. The transcription factor DREB1A specifically interacts with the DRE and induces expression of stress tolerance genes. We show here that overexpression of the cDNA encoding DREB1A in transgenic plants activated the expression of many of these stress tolerance genes under normal growing conditions and resulted in improved tolerance to drought, salt loading, and freezing. However, use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of DREB1A also resulted in severe growth retardation under normal growing conditions. In contrast, expression of DREB1A from the stress inducible rd29A promoter gave rise to minimal effects on plant growth while providing an even greater tolerance to stress conditions than did expression of the gene from the CaMV promoter.

Gene expression profiles have been mainly determined by analysis of transcript abundance. However, these analyses cannot capture posttranscriptional gene expression control at the level of translation, which is a key step in the regulation of gene expression, as evidenced by the fact that transcript levels often poorly correlate with protein levels. Furthermore, genome-wide transcript profiling of distinct cell types is challenging due to the fact that lysates from tissues always represent a mixture of cells. This study aimed to develop a new experimental method that overcomes both limitations and to apply this method to perform a genome-wide analysis of gene expression on the translational level in response to pressure overload. By combining ribosome profiling (Ribo-seq) with a ribosome-tagging approach (Ribo-tag), it was possible to determine the translated transcriptome in specific cell types from the heart. After pressure overload, we monitored the cardiac myocyte translatome by purifying tagged cardiac myocyte ribosomes from cardiac lysates and subjecting the ribosome-protected mRNA fragments to deep sequencing. We identified subsets of mRNAs that are regulated at the translational level and found that translational control determines early changes in gene expression in response to cardiac stress in cardiac myocytes. Translationally controlled transcripts are associated with specific biological processes related to translation, protein quality control, and metabolism. Mechanistically, Ribo-seq allowed for the identification of upstream open reading frames in transcripts, which we predict to be important regulators of translation. This method has the potential to (1) provide a new tool for studying cell-specific gene expression at the level of translation in tissues, (2) reveal new therapeutic targets to prevent cellular remodeling, and (3) trigger follow-up studies that address both, the molecular mechanisms involved in the posttranscriptional control of gene expression in cardiac cells, and the protective functions of proteins expressed in response to cellular stress.

Invasive lobular carcinoma (ILC) represents ~10% of newly diagnosed breast tumors, or ~30,000 cases annually in the US. However, ILC-specific signaling and endocrine responsiveness are not well characterized. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than invasive ductal carcinoma (IDC) patients with similar biomarkers, and that ILC patients may not benefit from adjuvant tamoxifen. We hypothesize that estrogen receptor-alpha (ER) regulated gene expression is unique in ILC cells and drives endocrine resistance. The ER-positive ILC cell lines MDA MB 134VI and SUM44PE were used as in vitro models of cell growth and ER-regulated gene expression in response to estradiol (E2). To examine the ER-regulated transcriptome, we performed gene expression microarray analyses and ER ChIP-Seq following E2 treatment. In parallel, E2 response was assessed in vivo in the primary ILC xenograft HCI-013. Response to endocrine therapies, tamoxifen (Tam), 4-hydroxytamoxifen (4OHT), endoxifen (Bx), and fulvestrant (ICI), were also examined in ILC cell lines. We observed that E2 induced growth and ER target gene expression in MDA MB 134VI and SUM44PE. We compared our ILC microarray data to published data from ER-positive IDC cell lines (MCF-7, T47D, BT474), and identified 254 genes that were E2-regulated in all 5 cell lines (e.g. GREB1, MYC). 915 genes were E2-regulated only in both ILC cell lines. Consistent with this, roughly half of ER binding sites identified in MDA MB 134VI ChIP-Seq were unique versus published MCF-7 data. We chose a subset of ILC-specific and common E2-regulated genes (n=107) to assess in vivo using Nanostring gene expression analyses of HCI-013. E2-regulation was observed for 32/107 target genes (30%), suggesting that these genes may be E2-regulated in vivo in ILC patient tumors. Consistent with clinical data, both ILC cell lines presented de novo tamoxifen resistance. SUM44PE were growth-inhibited by ICI, but unaffected by Tam, 4OHT, or Bx. Similarly, ICI blocked E2-induced growth in MDA MB 134VI, but Tam, 4OHT, and Bx acted as partial agonists, inducing ~25% growth. Partial agonism was not limited to tamoxifen, as other SERMs (e.g. raloxifene) also induced growth. We then measured ER-regulated gene expression in MDA MB 134VI following tamoxifen treatment. 4OHT acted as an agonist for E2-induced genes including SNAI1 and MYC. Further, for the majority of E2-repressed genes assessed including FOXO1, 4OHT also repressed gene expression, suggesting that ER-mediated gene repression may be critical to tamoxifen-resistance in ILC. Finally, we observed that FGFR1, frequently amplified in ILC, may be critical for ILC cell survival in the presence of tamoxifen. These data support the hypothesis that unique ER-mediated gene expression in ILC cells drives endocrine resistance. The de novo tamoxifen resistance observed in ILC cells may correlate with the worse outcomes in ILC patients recently reported. We hypothesize that genes regulated by tamoxifen as an agonist may play a role in tamoxifen-induced growth or serve as biomarkers of resistance. Targeting growth factor signaling using FGFR1 inhibitors may block survival pathways required by ILC cells and reverse tamoxifen resistance. Citation Format: Matthew J. Sikora, Kristine L. Cooper, Amir Bahreini, Soumya Luthra, Uma R. Chandran, Guoying Wang, David J. Dabbs, Alana L. Welm, Steffi Oesterreich. Invasive lobular carcinoma cells express unique estrogen-mediated genes and are de novo tamoxifen resistant. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B060.

Structure/Function Relationships of CREB/ATF Proteins

DDB1 binds histone reader BRWD3 to activate the transcriptional cascade in adipogenesis and promote onset of obesity.

The 'X4' accession of zoysiagrass demonstrated superior drought tolerance compared to other accessions. Integration analysis of transcriptomics and epigenomics revealed a positive correlation between ATAC-seq peak intensity and gene expression levels. Six motifs involved in regulating drought responses were identified, which are similar to the domains of the ERF and C2H2 transcription factor families. Heterologous expression of Zja11G000860 in yeast enhanced tolerance to drought stress, allowing robust growth even at high PEG6000 concentrations. Zoysiagrass is renowned for its drought tolerance and serves as an exceptional domestic turfgrass in China. However, the changes in chromatin accessibility during drought in zoysiagrass are not well understood. We conducted a preliminary evaluation of the phenotypic changes in drought tolerance for six zoysiagrass cultivars, taking into account their growth characteristics and physiological traits under drought conditions. Additionally, we utilized an integrated multi-omics strategy, encompassing RNA sequencing (RNA-seq), Assay for Transposase Accessible Chromatin using high-throughput sequencing (ATAC-seq), and reverse transcription quantitative PCR (RT-qPCR) verification experiments, to gain deeper understanding of the chromatin accessibility patterns linked to gene expression in response to drought stress in zoysiagrass. Preliminary analysis of the trends in relative water content and proline content suggested that the variety 'X4' exhibited superior drought tolerance compared to the other five accessions. The KEGG pathway enrichment analysis revealed that zoysiagrass responded to environmental stress by regulating stress response and antioxidant defense pathways. Notably, the expression levels of genes Zja03G031540 and Zja11G000860 were significantly increased in the 'X4' zoysiagrass genotype, which exhibited improved drought tolerance, compared to the 'X1' zoysiagrass genotype with reduced drought tolerance. This study suggested that 63 high-confidence genes are related to drought stress, including Zja03G031540 and Zja11G000860. Additionally, six motifs regulating drought responses were unearthed. Furthermore, the heterologous expression of Zja11G000860 in yeast enhanced tolerance to drought stress. The study discovered a positive correlation between ATAC-seq peak intensity and gene expression levels. The expression of high-confidence genes was linked to zoysiagrass resistance evaluation and phenotypic traits, implying that these genes are involved in responding to external drought stress. This study combined ATAC-seq and RNA-seq technologies for the first time to identify drought-related gene expression in zoysiagrass, elucidating the grass adaptation to environmental stress and the regulatory mechanisms underlying stress responses, and laying the groundwork for zoysiagrass improvement and breeding.