Upstream Cis Research Articles

Comprehensive in silico analysis of Phospholipase D gene family in economically important orchids

• A total of 25 putative PLD proteins from the genomes of Phalaenopsis equestris (7) , Dendrobium catenatum (9) and Apostasia shenzhenica (9) were identified. • These proteins are comprised of two HKD domains and two additional domains; C2 and PXPH domains at N-terminal region. • The multiple sequence alignment, domain and motifs analysis revealed their conserved nature and homology structure simulation and Ramachadran plot analysis revealed the abundance of beta sheets in tertiary structures. • The presence of Root-specific element, DOF proteins binding domain, scaffold or matrix attachment region, pollen-specific, dehydration-responsive, light-responsive, gibberellin-responsive, ABA-responsive elements and Meja-responsive upstream cis- regulatory promoter elements indicated their role in various physiological processes. Phospholipase D (PLD) plays a fundamental role in various aspects of plant growth, development and stress responses. In present study, 25 putative PLD proteins from the genomes of Phalaenopsis equestris (7) , Dendrobium catenatum (9) and Apostasia shenzhenica (9) were identified on the basis of presence of two HKD domains and two additional domains; C2 and PXPH domains at N-terminal region. These proteins were clustered into five sub-groups (α, β, δ, ζ and φ) along with their orthologs in Arabidopsis thaliana and Oryza sativa upon phylogenetic analysis. Gene characterization divulged that most of the PePLD, DcPLD and AsPLD genes within the same sub-group exhibited similar exon-intron organization and highly conserved motifs. RNA-seq based spatio-temporal studies indicated differential expression in various vegetative and reproductive tissues. PePLDα 1 , DcPLDα 1 and AsPLDα 1 were showing significant expression in all the tissues, while for other genes the expression was limited either in vegetative or reproductive tissues. Homology structure simulation revealed the abundance of random coils in secondary structures and beta sheets in tertiary structures. The presence of specific upstream cis- regulatory promoter elements emphasized the role of PLD s in various physiological processes. This report is the first genome-wide characterization of the PLD gene family in orchids and shall provide new insight into the functional characterization of these genes.

Distinct Evolutionary Origins of Intron Retention Splicing Events in NHX1 Antiporter Transcripts Relate to Sequence Specific Distinctions in Oryza Species.

The genome of Asian cultivated rice (Oryza sativa L.) shows the presence of six organelle-specific and one plasma membrane (OsNHX1-7) NHX-type cation proton antiporters. Of these, vacuolar-localized OsNHX1 is extensively characterized. The genus Oryza consists of 27 species and 11 genome-types, with cultivated rice, diploid O. sativa, having an AA-type genome. Oryza NHX1 orthologous regions (gene organization, 5′ upstream cis elements, amino acid residues/motifs) from closely related Oryza AA genomes cluster distinctly from NHX1 regions from more ancestral Oryza BB, FF and KKLL genomes. These sequence-specific distinctions also extend to two separate intron retention (IR) events involving Oryza NHX1 transcripts that occur at the 5′ and 3′ ends of the NHX1 transcripts. We demonstrate that the IR event involving the 5′ UTR is present only in more recently evolved Oryza AA genomes while the IR event governing retention of the 13th intron of Oryza NHX1 (terminal intron) is more ancient in origin, also occurring in halophytic wild rice, Oryza coarctata (KKLL). We also report presence of a retro-copy of the OcNHX1 cDNA in the genome of O. coarctata (rOcNHX1). Preferential species and tissue specific up- or down-regulation of the correctly spliced NHX1 transcript/5′ UTR/13th intron-retaining splice variants under salinity was observed. The implications of IR on NHX1 mRNA stability and ORF diversity in Oryza spp. is discussed.

Inhibitor of DNA Binding 2 (ID2) Plays a Key Tumor Suppressor Role in Promoting Oncogenic Transformation in Multiple Myeloma

Dysregulation of transcriptional control is a common phenomenon associated with oncogenesis. Inhibitors of DNA binding (ID) proteins are critical actors in lymphopoiesis, acting as regulators of transcription through a helix-loop-helix (HLH) domain which enables heterodimerization with basic HLH (bHLH) proteins inhibiting their binding to DNA. ID proteins have been implicated in malignant transformation, but their role in multiple myeloma (MM) is unknown. Here, we evaluated the role of ID proteins in biology and transcriptional dysregulation in MM.We first evaluated the expression of the four ID proteins in normal and malignant plasma cells using RNA sequencing data from a cohort of 360 newly diagnosed MM patients and 16 normal plasma cells. We observed significant downregulation of ID2 in primary patient MM cells in comparison to normal plasma cells (p 0.0013).To study ID2 function in MM cells, we next overexpressed ID2 in 2 MM cell lines (MM1S and NCIH929) and observed a significant decrease in proliferation rate, together with G0/G1 phase cell cycle arrest. We performed RNA-sequencing to evaluate the transcriptomic changes following ID2 overexpression. Gene set enrichment analysis (GSEA) revealed significant downregulation of genes involved in E2F pathway and significant changes in pathways related to immune response, regulation of cell death and cell proliferation. In addition, analysis of upstream cis-regulatory motifs of genes significantly dysregulated in both cell lines (>1.5 fold change) showed a highly significant enrichment for bHLH class I transcription factors (E proteins) binding motifs. Conversely, stable ID2 knockdown in 4 MM cell lines (MM1S, NCIH929, RPMI8226 and KMS11) expressing intermediate levels of ID2, showed an increased proliferation rate, assessed by cell counting, H3-thymidine incorporation and ATP production. RNA-sequencing after ID2 knockdown in MM1S and NCIH929 cells showed 600 common genes upregulated in both cell lines (>1.5 fold change). GSEA revealed upregulation of pathways involved in inflammatory response and epithelial-to-mesenchymal transition, while upstream cis regulatory motifs analysis showed a highly significant enrichment for binding motifs of bHLH class I transcription factors E proteins, in particular Tcf3 (p <0.0001).Next, we sought to investigate the mechanisms involved in ID2 downregulation in MM. Since the role of the microenvironment is critical in myelomagenesis, we evaluated the impact of BM microenvironment on ID2 expression in a co-culture system. Using bone marrow stromal cells (BMSC) derived from MM patients and stromal cell line (HS5) in co-culture with various MM cell lines, we observed that both cell-cell interactions and soluble factors secreted by BMSC or HS5 were able to significantly downregulate ID2 expression at the RNA and protein level. Furthermore, ID2 overexpression in MM cell lines (MM1S and NCIH929) abrogated the impact of BMSC on MM cell proliferation. Next, we evaluated ID2 promoter methylation profile and binding motifs using Sequenom mass array and the assay for transposase-accessible chromatin sequencing (ATAC-seq), respectively. While we didn't observe any increase in methylation of CpG islands located in ID2 promoter in co-culture, explaining ID2 downregulation, we identified several binding motifs corresponding to known driver transcription factors in MM. Especially, we identified SP1 binding motif and we confirmed SP1 binding to ID2 promoter by ChIP-sequencing in MM1S, NCIH929 and U266.These data demonstrate that in MM, ID2 acts as a tumor suppressor by promoting major transcriptomic changes and cell cycle arrest. Bone marrow stromal cells further induce significant downregulation of ID2 in myeloma cells suggesting that ID2/bHLH axis and other ID2 related pathways represent a potential new therapeutic target in myeloma. DisclosuresAnderson:Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; C4 Therapeutics: Equity Ownership, Other: Scientific founder; OncoPep: Equity Ownership, Other: Scientific founder; Millennium Takeda: Consultancy. Munshi:OncoPep: Other: Board of director.

Photosynthetic redox regulation of the plastocyanin promoter in tobacco

Persistent light quality gradients in dense plant populations induce imbalances in the distribution of excitation energy between the photosystems. Plants counteract such conditions by re-adjusting the stoichiometry of photosystems, which involves control of photosynthesis gene expression both in chloroplasts and in the nucleus. Decisive control parameters are redox signals from the photosynthetic electron transport chain, one prominent is the plastoquinone (PQ) pool. In a recent study, a plastocyanin (PC)-promoter::beta-glucuronidase reporter gene construct in tobacco demonstrated reversible redox regulation in response to varying light qualities. Here, northern and Western analyses demonstrate that this promoter regulation also accounts for the accumulation of the endogenous tobacco PetE gene transcripts and the protein amounts of the encoded PC. Hence, the reporter gene construct reflects the natural regulation of this nuclear gene in tobacco. In kinetic experiments, the response of the construct to either oxidation or reduction of the PQ pool was tested by defined light quality shifts. The construct displayed upregulation in response to a reduction signal and downregulation in response to an oxidation signal, both with a half-time of about 24 h. The response was finished after 48 h. DCMU application abolished the upregulation in response to the reduction signal, indicating the dependence on thylakoid membrane electron transport. To study the redox-responsive promoter region in more detail, several promoter deletion constructs were tested for their responsiveness. All constructs displayed a reversible response to light-induced oxidation and reduction signals; however, a minimal promoter region localised between -168 to -79 bp upstream of the transcription start site was sufficient to confer this redox regulation. This indicates that photosynthetic redox signals act on distinct regions in the PC promoter in a manner independent from photoreceptors and upstream cis elements conferring high basic expression in the light.

Identification of cis-regulatory elements from the C. elegans Hox gene lin-39 required for embryonic expression and for regulation by the transcription factors LIN-1, LIN-31 and LIN-39

Expression of the Caenorhabditis elegans Hox gene lin-39 begins in the embryo and continues in multiple larval cells, including the P cell lineages that generate ventral cord neurons (VCNs) and vulval precursor cells (VPCs). lin-39 is regulated by several factors and by Wnt and Ras signaling pathways; however, no cis-acting sites mediating lin-39 regulation have been identified. Here, we describe three elements controlling lin-39 expression: a 338-bp upstream fragment that directs embryonic expression in P5-P8 and their descendants in the larva, a 247-bp intronic region sufficient for VCN expression, and a 1.3-kb upstream cis-regulatory module that drives expression in the VPC P6.p in a Ras-dependent manner. Three trans-acting factors regulate expression via the 1.3-kb element. A single binding site for the ETS factor LIN-1 mediates repression in VPCs other than P6.p; however, loss of LIN-1 decreases expression in P6.p. Therefore, LIN-1 acts both negatively and positively on lin-39 in different VPCs. The Forkhead domain protein LIN-31 also acts positively on lin-39 in P6.p via this module. Finally, LIN-39 itself binds to this element, suggesting that LIN-39 autoregulates its expression in P6.p. Therefore, we have begun to unravel the cis-acting sites regulating lin-39 Hox gene expression and have shown that lin-39 is a direct target of the Ras pathway acting via LIN-1 and LIN-31.

The lac repressor

Few proteins have had such a strong impact on a field as the lac repressor has had in Molecular Biology. Over 40 years ago, Jacob and Monod [Genetic regulatory mechanisms in the synthesis of proteins, J. Mol. Biol. 3 (1961) 318] proposed a model for gene regulation, which survives essentially unchanged in contemporary textbooks. It is a cogent depiction of how a set of ‘structural’ genes may be coordinately transcribed in response to environmental conditions and regulates metabolic events in the cell. In bacteria, the genes required for lactose utilization are negatively regulated when a repressor molecule binds to an upstream cis activated operator. The repressor and its operator together form a genetic switch, the lac operon. The switch functions when inducer molecules alter the conformation of the repressor in a specific manner. In the presence of a particular metabolite, the repressor undergoes a conformational change that reduces its affinity for the operator. The structures of the lac repressor and its complexes with operator DNA and effector molecules have provided a physical platform for visualizing at the molecular level the different conformations the repressor and the molecular basis for the switch. The structures of lac repressor, bound to its operator and inducer, have also been invaluable for interpreting a plethora of biochemical and genetic data. To cite this article: M. Lewis, C. R. Biologies 328 (2005).

Bidirectionalization of polar promoters in plants.

A typical eukaryotic promoter consists of a minimal promoter and other upstream cis elements. The minimal promoter is essentially a TATA box region where RNA polymerase II, TATA-binding protein (TBP), and TBP-associated factors (TAFs) bind to initiate transcription, but minimal promoters alone have no transcriptional activity. The cis elements, to which tissue-specific or development-specific transcription factors bind, individually or in combination, determine the spatio-temporal expression pattern of a promoter at the transcriptional level. The arrangement of upstream cis elements followed by a minimal promoter sets the polarity of the promoter. Promoters in plants that have been cloned and widely used for both basic research and biotechnological application are generally unidirectional, directing only one gene that has been fused at its 3' end (downstream). It is often necessary to introduce multiple genes into plants for metabolic engineering and trait stacking. It is also desirable to minimize or avoid repeated use of a single promoter that may cause transcriptional gene silencing. Here we describe a strategy to make polar promoters bidirectional so that one promoter can direct the expression of two genes, one on each end of the promoter.

Long-distance chromatin mechanisms controlling tissue-specific gene locus activation.

Several different types of regulatory mechanisms contribute to the tissue- and development-specific regulation of a gene. It is now well established that, in addition to promoters, upstream cis-regulatory elements, which bind a variety of trans-acting factors, are essential for correct gene activation. In the last few years, however, it has become evident that the chromatin structure of eukaryotic genes is an important additional regulatory layer that is essential for correct gene expression during development. Chromatin is essentially a repressive environment for transcription factors; hence, much effort in recent years has been devoted to the elucidation of how these repressive forces are overcome during the process of gene locus activation. A particular interesting question in this context is: what are the molecular mechanisms by which extensive regions of chromatin, in many cases far outside the coding region, are reorganized during development? In this review, I summarize data from recent investigations that have uncovered a surprising variety of factors involved in this process.

Regulation of endothelin 1 gene by fluid shear stress is transcriptionally mediated and independent of protein kinase C and cAMP.

Fluid shear stress induces a number of morphological and functional changes in vascular endothelium, including a rapid and significant down-regulation of endothelin 1 (ET-1) mRNA and peptide release in bovine aortic endothelial cells. We show here that both the cell alignment and ET-1 down-regulation depend on on-going protein synthesis, and that the latter is the result of a decrease in transcription, as shown by nuclear run-off assay, and not the result of changes in ET-1 mRNA half-life. The treatment of endothelial cells with either phorbol 12-myristate 13-acetate (100 nM) to activate protein kinase C (PKC) or forskolin (10 microM) to stimulate adenylate cyclase sharply decreased ET-1 mRNA. However, the phorbol-induced ET-1 decrease was, unlike the shear-induced down-regulation, independent of active protein synthesis. Physiological shear stress (20 dynes/cm2) did not significantly activate PKC, as assessed by PKC translocation and enzymatic activity assay and failed to increase intracellular cAMP content. Furthermore treatment with calphostin C (1 microM) did not prevent the shear-induced down-regulation of ET-1. DNA transfection experiments suggest that the shear stress-responsive element of the ET-1 gene is contained in the sequence between -2.5 kb and -2.9 kb of the 5'-upstream region. Neither the transcription factor AP-1 binding site nor the GATA-2-factor binding site, necessary for the basal level of transcription of ET-1 gene, is sufficient to confer shear-responsiveness to the reporter gene. These results suggest that shear stress regulates the transcription of the ET-1 gene via an upstream cis element by a distinct mechanism not dependent on the PKC or cAMP pathways.

Nef Protein of HIV-1 Is a Transcriptional Repressor of HIV-1 LTR

In studies of the genetics of human immunodeficiency virus type 1 (HIV-1), the product of the nef gene, formerly known as F, 3'-orf, or B-ORF, was a negative regulator of HIV-1 replication. Proviruses with mutations in the nef gene replicated better than their standard counterparts during transient expression, and the mutant virus maintained its enhanced replication even after serial passages in T lymphocytes. The nef protein trans-suppressed, in a dose-dependent manner, the replication of wild-type and nef mutant proviruses and the expression of reporter genes linked to the HIV-1 long terminal repeat (LTR). The repression induced by the nef protein was mediated by inhibition of transcription from the HIV-1 LTR, which contains a far upstream cis element (previously recognized to be a negative regulatory element) between 340 and 156 nucleotides upstream of the RNA initiation site.