scholarly journals Nascent transcript folding plays a major role in determining RNA polymerase elongation rates

2020 ◽  
Author(s):  
Tomasz W Turowski ◽  
Elisabeth Petfalski ◽  
Benjamin D Goddard ◽  
Sarah L French ◽  
Aleksandra Helwak ◽  
...  
Keyword(s):  
Folding Energy ◽  
Rna Structures ◽  
Nascent Transcript ◽  
Transcription Bubble ◽  
Uv Crosslinking ◽  
Specific Regulation ◽  
Splice Site Usage ◽  
Regulatory Significance

Transcription elongation rates are important for RNA processing, but sequence-specific regulation is poorly understood. We addressed this in vivo, analyzing RNAPI in S.cerevisiae. Analysis of Miller chromatin spreads and mapping RNAPI using UV crosslinking, revealed a marked 5' bias and strikingly uneven local polymerase occupancy, indicating substantial variation in transcription speed. Two features of the nascent transcript correlated with RNAPI distribution; folding energy and G+C content. In vitro experiments confirmed that strong RNA structures close to the polymerase promote forward translocation and limit backtracking, whereas high G+C within the transcription bubble slows elongation. We developed a mathematical model for RNAPI elongation, which confirmed the importance of nascent RNA folding in transcription. RNAPI from S.pombe was similarly sensitive to transcript folding, as were S.cerevisiae RNAPII and RNAPIII. For RNAPII, unstructured RNA, which favors slowed elongation, was associated with faster cotranscriptional splicing and proximal splice site usage indicating regulatory significance for transcript folding.

scholarly journals Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

2016 ◽  
Vol 113 (21) ◽  
pp. E2899-E2905 ◽  
Author(s):  
Irina O. Vvedenskaya ◽  
Hanif Vahedian-Movahed ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
Richard H. Ebright ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Transcription Initiation ◽  
Specific Protein ◽  
Start Site ◽  
Transcription Start ◽  
Transcription Bubble ◽  
Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

scholarly journals Regulation of SH3PX1 by dNedd4-long at the Drosophila neuromuscular junction

2018 ◽  
Vol 294 (5) ◽  
pp. 1739-1752 ◽  
Author(s):  
Samantha S. Wasserman ◽  
Alina Shteiman-Kotler ◽  
Kathryn Harris ◽  
Konstantin G. Iliadi ◽  
Avinash Persaud ◽  
...  
Keyword(s):  
Neurotransmitter Release ◽  
Neuromuscular Junctions ◽  
Cell Periphery ◽  
Middle Region ◽  
Binding Partners ◽  
Direct Binding ◽  
Specific Regulation ◽  
Neuromuscular Synaptogenesis

Drosophila Nedd4 (dNedd4) is a HECT E3 ubiquitin ligase present in two major isoforms: short (dNedd4S) and long (dNedd4Lo), with the latter containing two unique regions (N terminus and Middle). Although dNedd4S promotes neuromuscular synaptogenesis (NMS), dNedd4Lo inhibits it and impairs larval locomotion. To explain how dNedd4Lo inhibits NMS, MS analysis was performed to find its binding partners and identified SH3PX1, which binds dNedd4Lo unique Middle region. SH3PX1 contains SH3, PX, and BAR domains and is present at neuromuscular junctions, where it regulates active zone ultrastructure and presynaptic neurotransmitter release. Here, we demonstrate direct binding of SH3PX1 to the dNedd4Lo Middle region (which contains a Pro-rich sequence) in vitro and in cells, via the SH3PX1-SH3 domain. In Drosophila S2 cells, dNedd4Lo overexpression reduces SH3PX1 levels at the cell periphery. In vivo overexpression of dNedd4Lo post-synaptically, but not pre-synaptically, reduces SH3PX1 levels at the subsynaptic reticulum and impairs neurotransmitter release. Unexpectedly, larvae that overexpress dNedd4Lo post-synaptically and are heterozygous for a null mutation in SH3PX1 display increased neurotransmission compared with dNedd4Lo or SH3PX1 mutant larvae alone, suggesting a compensatory effect from the remaining SH3PX1 allele. These results suggest a post-synaptic–specific regulation of SH3PX1 by dNedd4Lo.

scholarly journals Reconstitution of prospermatogonial specification in vitro from human induced pluripotent stem cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Young Sun Hwang ◽  
Shinnosuke Suzuki ◽  
Yasunari Seita ◽  
Jumpei Ito ◽  
Yuka Sakata ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cell ◽  
Postnatal Period ◽  
Germline Development ◽  
Specific Regulation ◽  
Induced Pluripotent

Abstract Establishment of spermatogonia throughout the fetal and postnatal period is essential for production of spermatozoa and male fertility. Here, we establish a protocol for in vitro reconstitution of human prospermatogonial specification whereby human primordial germ cell (PGC)-like cells differentiated from human induced pluripotent stem cells are further induced into M-prospermatogonia-like cells and T1 prospermatogonia-like cells (T1LCs) using long-term cultured xenogeneic reconstituted testes. Single cell RNA-sequencing is used to delineate the lineage trajectory leading to T1LCs, which closely resemble human T1-prospermatogonia in vivo and exhibit gene expression related to spermatogenesis and diminished proliferation, a hallmark of quiescent T1 prospermatogonia. Notably, this system enables us to visualize the dynamic and stage-specific regulation of transposable elements during human prospermatogonial specification. Together, our findings pave the way for understanding and reconstructing human male germline development in vitro.

scholarly journals PIN*POINT analysis on the endogenous MnSOD promoter: specific demonstration of Sp1 binding in vivo

2003 ◽  
Vol 284 (2) ◽  
pp. C528-C534 ◽  
Author(s):  
Shiuhyang Kuo ◽  
Ann L. Chokas ◽  
Richard J. Rogers ◽  
Harry S. Nick
Keyword(s):  
Binding Sites ◽  
Manganese Superoxide Dismutase ◽  
Consensus Sequence ◽  
Dimethyl Sulfate ◽  
Site Directed Mutagenesis ◽  
Cellular Respiration ◽  
Point Analysis ◽  
Specific Regulation

Manganese superoxide dismutase (MnSOD) is a critical antioxidant enzyme that protects against superoxide anion generated as a consequence of normal cellular respiration, as well as during the inflammatory response. By employing dimethyl sulfate in vivo footprinting, we have previously identified ten basal protein binding sites within the MnSODpromoter. On the basis of consensus sequence comparison and in vitro footprinting data, one would predict that Sp1 might occupy five of these binding sites. To address these findings in the context of the nucleoprotein environment, we first utilized chromatin immunoprecipitation (ChIP) to demonstrate the nuclear association of Sp1 with the MnSOD promoter region. To identify the precise location of Sp1 binding, we have modified the original protein position identification with nuclease tail (PIN*POINT) methodology, providing an approach to establish both the identity and binding occupancy of Sp1 in the context of the endogenous MnSOD promoter. These data, coupled with site-directed mutagenesis, demonstrate the functional importance of two of the Sp1 binding sites in the stimulus-specific regulation of MnSOD gene expression. We feel that the combination of ChIP and PIN*POINT analysis allows unequivocal identification and localization of protein/DNA interactions in vivo, specifically the demonstration of Sp1 with the MnSODpromoter.

scholarly journals Stimulation of collagen galactosyltransferase and glucosyltransferase activities by lysophosphatidylcholine

1976 ◽  
Vol 160 (1) ◽  
pp. 29-35 ◽  
Author(s):  
H Anttinen
Keyword(s):  
Chick Embryo ◽  
High Speed ◽  
Membrane Structures ◽  
Embryo Extract ◽  
Chick Embryo Extract ◽  
Triton X ◽  
Regulatory Significance ◽  
Stimulation Of

Lysophosphatidylcholine stimulated the activities of collagen galactosyl- and glucosyl-transferases in chick-embryo extract and its particulate fractions in vitro, whereas essentially no stimulation was noted in the high-speed supernatant, where the enzymes are soluble and membrane-free. The stimulatory effect of lysophosphatidylcholine was masked by 0.1% Triton X-100. In kinetic experiments lysophosphatidylcholine raised the maximum velocities with respect to the substrates and co-substrates, whereas no changes were observed in the apparant Km values. Phospholipase A preincubation of the chick-embryo extract resulted in stimulation of both transferase activities, probably gy generating lysophosphatides from endogenous phospholipids. No stimulation by lysophosphatidylcholine was found when tested with 500-fold-purified glycosyltransferase. The results suggest that collagen glycosyltransferases must be associated with the membrane structures of the cell in order to be stimulated by lysophosphatidylcholine. Lysophosphatidylcholine could have some regulatory significance in vivo, since its concentration in the cell is comparable with that which produced marked stimulation in vitro.

scholarly journals The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes.

1990 ◽  
Vol 10 (3) ◽  
pp. 982-990 ◽  
Author(s):  
D G Johnson ◽  
L Carayannopoulos ◽  
J D Capra ◽  
P W Tucker ◽  
J H Hanke
Keyword(s):  
B Cell ◽  
Protein S ◽  
Cell Types ◽  
In Vitro Transcription ◽  
Immunoglobulin Gene ◽  
Immunoglobulin Genes ◽  
Specific Expression ◽  
Specific Regulation

All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression.

scholarly journals Modulation of the tumour promoting functions of cancer associated fibroblasts by phosphodiesterase type 5 inhibition increases the efficacy of chemotherapy in human preclinical models of esophageal adenocarcinoma

2020 ◽  
Author(s):  
Annette Hayden ◽  
Antigoni Manousopoulou ◽  
Andrew Cowie ◽  
Robert Walker ◽  
Benjamin P. Sharpe ◽  
...  
Keyword(s):  
Esophageal Adenocarcinoma ◽  
Standard Of Care ◽  
Shotgun Proteomics ◽  
Model Systems ◽  
Cancer Associated Fibroblasts ◽  
Phosphodiesterase Type 5 Inhibitors ◽  
Specific Regulation ◽  
Phosphodiesterase Type

AbstractBackground and aimsEsophageal adenocarcinoma (EAC) is chemoresistant in the majority of cases. The tumor-promoting biology of cancer associated fibroblasts (CAF) make them a target for novel therapies. Phosphodiesterase type 5 inhibitors (PDE5i) have been shown to regulate the activated fibroblast phenotype in benign disease. We investigated the potential for CAF modulation in EAC using PDE5i to enhance the efficacy of chemotherapy.MethodsEAC fibroblasts were treated with PDE5i and phenotypic effects examined using immunoblotting, immunohistochemistry, gel contraction, transwell invasion, organotypics, single cell RNAseq and shotgun proteomics. The combination of PDE5i with standard-of-care chemotherapy (Epirubicin, 5-Fluorouracil and Cisplatin) was tested for safety and efficacy in validated near-patient model systems (3D tumor growth assays (3D-TGAs) and patient derived xenograft (PDX) mouse models).ResultsPDE5i treatment reduced α–SMA expression in CAFs by 50% (p<0.05), associated with a significant reduction in the ability of CAFs to contract collagen-1 gels and induce cancer cell invasion, (p<0.05). RNAseq and proteomic analysis of CAF and EAC cell lines revealed PDE5i specific regulation of pathways related to fibroblast activation and tumor promotion. 3D-TGA assays confirmed the importance of stromal cells to chemoresistance in EAC, which could be attenuated by PDE5i. Chemotherapy+PDE5i in PDX-bearing mice was safe and significantly reduced PDX tumor volume (p<0.05).ConclusionPDE5 is a candidate for clinical trials to alter the fibroblast phenotype in esophageal cancer. We demonstrate the specificity of PDE5i for fibroblasts to prevent transdifferentiation and revert the CAF phenotype. Finally, we confirm the efficacy of PDE5i in combination with chemotherapy in close-to-patient in vitro and in vivo PDX-based model systems.

scholarly journals Reconstitution of prospermatogonial specification in vitro from human induced pluripotent stem cells

2020 ◽  
Author(s):  
Young Sun Hwang ◽  
Shinnosuke Suzuki ◽  
Yasunari Seita ◽  
Jumpei Ito ◽  
Yuka Sakata ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cell ◽  
Postnatal Period ◽  
Germline Development ◽  
Specific Regulation ◽  
Induced Pluripotent

ABSTRACTEstablishment of spermatogonia throughout the fetal and postnatal period is essential for production of spermatozoa and male fertility. Here, we established a protocol for in vitro reconstitution of human prospermatogonial specification whereby human primordial germ cell (PGC)-like cells (hPGCLCs) differentiated from human induced pluripotent stem cells were further induced into M-prospermatogonia-like cells (MLCs) and T1 prospermatogonia-like cells (T1LCs) using long-term cultured xenogeneic reconstituted testes. Single cell RNA-sequencing was used to delineate the lineage trajectory leading to T1LCs, which closely resemble human T1-prospermatogonia in vivo and exhibited gene expression related to spermatogenesis and diminished proliferation, a hallmark of quiescent T1 prospermatogonia. Notably, this system enabled us to visualize the dynamic and stage-specific regulation of transposable elements during human prospermatogonial specification. Together, our findings pave the way for understanding and reconstructing human male germline development in vitro.

Cyclin A1 directly interacts with B-myb and cyclin A1/cdk2 phosphorylate B-myb at functionally important serine and threonine residues: tissue-specific regulation of B-myb function

Blood ◽  
2001 ◽  
Vol 97 (7) ◽  
pp. 2091-2097 ◽  
Author(s):  
Carsten Müller-Tidow ◽  
Wenbing Wang ◽  
Gregory E. Idos ◽  
Sven Diederichs ◽  
Rong Yang ◽  
...  
Keyword(s):  
Leukemic Cells ◽  
Glutathione S Transferase ◽  
Cyclin A1 ◽  
Tissue Specific ◽  
Cdk2 Complex ◽  
Cyclin A2 ◽  
Specific Regulation ◽  
Phosphopeptide Mapping

Abstract Cyclin A1 is tissue-specifically expressed during spermatogenesis, but it is also highly expressed in acute myeloid leukemia (AML). Its pathogenetic role in AML and in the cell cycle of leukemic blasts is unknown. B-myb is essential for G1/S transition and has been shown to be phosphorylated by the cyclin A2/cdk2 complex. Here it is demonstrated that cyclin A1 interacts with the C-terminal portion of B-myb as shown by glutathione S-transferase (GST) precipitation. This interaction is confined to cyclin A1 because binding could not be detected between cyclin A2 and B-myb. Also, cdk2 was not pulled down by GST–B-myb from U937 lysates. In addition, co-immunoprecipitation of cyclin A1 and B-myb in leukemic cells evidenced protein interaction in vivo. Baculovirus-expressed cyclin A1/cdk2 complexes were able to phosphorylate human as well as murine B-myb in vitro. Tryptic phosphopeptide mapping revealed that cyclin A1/cdk2 complexes phosphorylated the C-terminal part of B-myb at several sites including threonine 447, 490, and 497 and serine 581. These phosphorylation sites have been demonstrated to be important for the enhancement of B-myb transcriptional activity. Further studies showed that cyclin A1 cooperated with B-myb to transactivate myb binding site containing promoters including the promoter of the human cyclin A1 gene. Taken together, the data suggest that cyclin A1 is a tissue-specific regulator of B-myb function and activates B-myb in leukemic blasts.

scholarly journals Cell-intrinsic regulation of murine epidermal Langerhans cells by protein S

2018 ◽  
Vol 115 (25) ◽  
pp. E5736-E5745 ◽  
Author(s):  
Yaara Tabib ◽  
Nora S. Jaber ◽  
Maria Nassar ◽  
Tal Capucha ◽  
Gabriel Mizraji ◽  
...  
Keyword(s):  
Langerhans Cells ◽  
Developmental Stages ◽  
Protein S ◽  
Altered Expression ◽  
Genetic Ablation ◽  
Adult Mice ◽  
Specific Regulation ◽  
Epidermal Langerhans Cells

AXL, a member of the TYRO3, AXL, and MERTK (TAM) receptor tyrosine kinase family, has been shown to play a role in the differentiation and activation of epidermal Langerhans cells (LCs). Here, we demonstrate that growth arrest-specific 6 (GAS6) protein, the predominant ligand of AXL, has no impact on LC differentiation and homeostasis. We thus examined the role of protein S (PROS1), the other TAM ligand acting primarily via TYRO3 and MERTK, in LC function. Genetic ablation of PROS1 in keratinocytes resulted in a typical postnatal differentiation of LCs; however, a significant reduction in LC frequencies was observed in adult mice due to increased apoptosis. This was attributed to altered expression of cytokines involved in LC development and tissue homeostasis within keratinocytes. PROS1 was then excised in LysM+ cells to target LCs at early embryonic developmental stages, as well as in adult monocytes that also give rise to LCs. Differentiation and homeostasis of LCs derived from embryonic precursors was not affected following Pros1 ablation. However, differentiation of LCs from bone marrow (BM) precursors in vitro was accelerated, as was their capability to reconstitute epidermal LCs in vivo. These reveal an inhibitory role for PROS1 on BM-derived LCs. Collectively, this study highlights a cell-specific regulation of LC differentiation and homeostasis by TAM signaling.

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