hnRNP K in PU.1-containing complexes recruited at the CD11b promoter: a distinct role in modulating granulocytic and monocytic differentiation of AML-derived cells
PU.1 is essential for the differentiation of haematopoietic precursors and is strongly implicated in leukemogenesis, yet the protein interactions that regulate its activity in different myeloid lineages are still largely unknown. Here, by combining fluorescent electrophoretic mobility shift assay (EMSA) with mass spectrometry, we reveal the presence of hnRNP K in molecular complexes that PU.1 forms on the CD11b promoter during the agonist-induced maturation of AML-derived cells along both the granulocytic and the monocytic lineages. WhilehnRNP K and PU.1 act synergistically during granulocytic differentiation, hnRNP K seem...
Source: BJ Gene - July 9, 2014 Category: Biochemistry Authors: E Nika, F Brugnoli, M Piazzi, E Lambertini, S Grassilli, A Bavelloni, R Piva, S Capitani, V Bertagnolo Tags: BJ Gene Source Type: research
Kinetics of promoter escape by bacterial RNA polymerase: effects of promoter contacts and transcription bubble collapse
Promoter escape by RNA polymerase, the transition between the initiation and elongation, is a critical step that defines transcription output at many promoters. We used a real-time fluorescence assay for promoter melting and escape to study the determinants of the escape. Perturbation of core promoter-polymerase contacts had opposing effects of the rates of melting and escape demonstrating a direct role of core promoter elements sequence in setting not only the kinetics of promoter melting but also the kinetics of promoter escape. The start of RNA synthesis is accompanied by an enlargement of the transcription bubble and p...
Source: BJ Gene - July 4, 2014 Category: Biochemistry Authors: J Ko, T Heyduk Tags: BJ Biomolecules Source Type: research
Archaeal MBF1 binds to 30S and 70S ribosomes via its helix-turn-helix domain
Multi-Protein Bridging factor 1 (MBF1) is a protein containing a conserved helix-turn-helix domain in both eukaryotes and archaea. Eukaryotic MBF1 has been reported to function as a transcriptional co-activator that physically bridges transcription regulators with the core transcription initiation machinery of RNA polymerase II. In addition, MBF1 has been found to be associated with poly-adenylated mRNA in yeast as well as mammalian cells. Archaeal MBF1 (aMBF1) is very well conserved among most archaeal lineages, however, its function has remained elusive thus far. To address this we have conducted a molecular characteriza...
Source: BJ Gene - May 14, 2014 Category: Biochemistry Authors: F Blombach, H Launay, A PL Snijders, V Zorraquino, H Wu, B de Koning, S J.J. Brouns, T Ettema, C Camilloni, A Cavalli, M Vendruscolo, M J Dickman, L D. Cabrita, A La Teana, D Benelli, P Londei, J Christodoulou, J van der Oost Tags: BJ Biomolecules Source Type: research
Effect of the guide strand 3{'}-end structure on the gene silencing potency of asymmetric siRNA
In this study, we explored the effect of the guide strand 3’-end structure on the gene silencing potency of asiRNA. Interestingly, asiRNAs with a 21 nucleotide (nt) guide strand solely composed of RNA resulted in gene silencing more than 6-fold more efficient compared with the corresponding asiRNA guide strand harboring a deoxythymidine dinucleotide (dTdT) at its 3'-end. We demonstrated that the molecular basis of potency of the asiRNA with a 21 nt guide strand composed solely of RNA was due to the enhanced formation of the RNA- induced silencing complex (RISC) and increased affinity towards human Argonaute2 (hAgo2)...
Source: BJ Gene - May 6, 2014 Category: Biochemistry Authors: S Hong, J Park, S Yun, C Lee, C Shin, D Lee Tags: BJ Biomolecules Source Type: research
Histone chaperone Chz1 facilitates the disfavoring property of Spt16 to H2A.Z-containing genes in Saccharomyces cerevisiae
In this study, we determined the genomic binding profiling of both Htz1 and Pol II and Spt16 using chromatin immunoprecipitation (ChIP)–microarray experiments and sequenced nucleosomal DNA using the next generation sequencing technique in wild-type and chz1 deletion strains of Saccharomyces cerevisiae. The results revealed that Spt16 and RNA polymerase II are associated, bind at nucleosome-depleted regions, and are positively correlated with transcription rate. Importantly, Spt16 disfavors the Htz1-bound genes, and this discrimination is impaired upon deletion of chz1. The anti-correlation between binding profiles o...
Source: BJ Gene - April 8, 2014 Category: Biochemistry Authors: H Liu, Z Zhou, K Luo, Y Mu, Y Wan Tags: BJ Gene Source Type: research
CCAAT/enhancer-binding protein {beta} (C/EBP{beta}) mediates progesterone production through transcriptional regulation in cooperation with steroidogenic factor 1 (SF-1)
A transcription factor, steroidogenic factor 1 (SF-1), is a master regulator for steroidogenesis. Previously, we have found that SF-1 induces the differentiation of mesenchymal stem cells into steroidogenic cells. To elucidate the molecular mechanisms of SF-1-mediated functions, we attempted to identify protein components of the SF-1 nuclear protein complex in differentiated cells. SF-1 immunoaffinity chromatography followed by MS/MS analysis was performed, and 24 proteins were identified. Among these proteins, we focused on CCAAT/enhancer-binding protein β (C/EBPβ), which is an essential transcription factor...
Source: BJ Gene - March 18, 2014 Category: Biochemistry Authors: T Mizutani, Y Ju, Y Imamichi, T Osaki, T Yazawa, S Kawabe, S Ishikane, T Matsumura, M Kanno, Y Kamiki, K Kimura, N Minamino, K Miyamoto Tags: BJ Gene Source Type: research
Ser119 phosphorylation modulates the activity and conformation of PRRXL1, a homeodomain transcription factor
In this study, we show that PRRXL1 is highly phosphorylated in vivo, and that its multiple band pattern on electrophoretic analysis is the result of different phosphorylation states. PRRXL1 phosphorylation appears to be differentially regulated along the dSC and DRG development and it is mapped to two functional domains. One region comprises amino acids 107-143, while the other one encompasses amino acids 227-263 and displays repressor activity. Using an immunoprecipitation-MS approach, two phosphorylation sites were identified: S119 and S238. Phosphorylation at S119 is shown to be determinant for PRRXL1 conformation and t...
Source: BJ Gene - February 24, 2014 Category: Biochemistry Authors: R Soares-dos-Reis, A Sofia Pessoa, M Raimundo Matos, M Falcão, V M Mendes, B Manadas, F Almeida Monteiro, D Lima, C Reguenga Tags: BJ Biomolecules Source Type: research
The NTP pyrophosphatase DCTPP1 contributes to the homeostasis and cleansing of the dNTP pool in human cells
The size and composition of deoxyribonucleotide (dNTP) pools influence the accuracy of DNA synthesis and consequently the genetic stability of nuclear and mitochondrial genomes. In order to keep the dNTP pool in balance, the synthesis and degradation of DNA precursors must be precisely regulated. One of such mechanisms involves catabolic activities that convert deoxynucleoside triphosphates into their monophosphate form. Human cells possess an all-alpha NTP pyrophosphatase named DCTPP1 (also known as XTP3-TPA). Here, we provide an extensive characterization of this enzyme which is ubiquitously distributed in the nucleus, c...
Source: BJ Gene - January 27, 2014 Category: Biochemistry Authors: C E. Requena, G Pérez-Moreno, L Ruiz-Pérez, A E. Vidal, D González-Pacanowska Tags: BJ Metabolism Source Type: research
CREB phosphorylation at Ser133 regulates transcription via distinct mechanisms downstream of cAMP and MAPK signaling
CREB is an important transcription factor for the activation of a number of immediate early genes. CREB is phosphorylated on Ser133 by PKA, promoting the recruitment of the co-activator proteins CBP and p300; this has been proposed to increase the transcription of CREB-dependent genes. CREB is also phosphorylated on Ser133 by MSK1/2 in cells in response to activation of MAPK signaling, however the relevance of this to gene transcription has been controversial. To resolve this we created a mouse with a Ser133 to Ala mutation in the endogenous CREB gene. Unlike total CREB knockout, which is perinatal lethal, these mice were ...
Source: BJ Gene - January 8, 2014 Category: Biochemistry Authors: S Naqvi, K J. Martin, J C. Arthur Tags: BJ Signal Source Type: research
Hypoxia-inducible factors regulate human and rat cystathionine {Sharp S}-synthase gene expression
Increased catalytic activity of cystathionine ß-synthase (CBS) was recently shown to mediate vasodilation of the cerebral microcirculation, which is initiated within minutes after the onset of acute hypoxia. To test whether chronic hypoxia was a stimulus for increased CBS expression, U87-MG human glioblastoma and PC12 rat pheochromocytoma cells were exposed to 1% or 20% O2 for 24 to 72 h. CBS mRNA and protein expression were increased in hypoxic cells. Hypoxic induction of CBS expression was abrogated in cells transfected with vector encoding short hairpin RNA targeting hypoxia-inducible factor (HIF) 1alpha or 2alph...
Source: BJ Gene - December 12, 2013 Category: Biochemistry Authors: N Takano, Y Peng, G K. Kumar, W Luo, H Hu, L A. Shimoda, M Suematsu, N R. Prabhakar, G L. Semenza Tags: BJ Gene Source Type: research
RAD-6: Pyrimidine synthesis and radiation sensitivity in Caenorhabditis elegans
In this study we describe the molecular identification of the rad-6 gene and reveal that it encodes the bifunctional UMP synthase protein, which carries catalytic activities for orotate phosphoribosyltransferase (OPRTase) and OMP decarboxylase (ODCase), key enzymes in the de novo pathway of pyrimidine synthesis. Mutations in genes encoding de novo pathway enzymes cause varying degrees of lethality and pleiotropic phenotypes in many organisms, including human. We have examined how the absence of rad-6 activity leads to both UV-C hypersensitivity and a decline in both metabolic rate and lifespan. We discuss how rad-6 mutants...
Source: BJ Gene - November 22, 2013 Category: Biochemistry Authors: A Merry, M Qiao, M Hasler, P E Kuwabara Tags: BJ Gene Source Type: research
Structure of RDE-4 dsRBDs and mutational studies provide insights in the dsRNA recognition in C. elegans RNAi pathway
The association of RDE-4, a protein containing two double stranded RNA binding domains (dsRBDs), with long dsRNA and Dicer (Dcr-1) initiates the siRNA pathway in C. elegans. Unlike its homologs in higher eukaryotes, RDE-4 dsRBDs possess weak (micromolar) affinity for short dsRNA. With the increasing length of dsRNA, RDE-4 exhibits enhanced affinity due to cooperativity. The linker and dsRBD2 are indispensable for RDE-4’s simultaneous interaction with dsRNA and Dcr-1. Here, we present the solution structures of RDE-4 constructs that contain both dsRBDs and the linker region. In addition to the canonical dsRBD fold, b...
Source: BJ Gene - November 21, 2013 Category: Biochemistry Authors: S Chiliveri, M V. Deshmukh Tags: BJ Biomolecules Source Type: research
A novel protein, Pho92, has a conserved YTH domain and regulates phosphate metabolism by decreasing the mRNA stability of PHO4 in Saccharomyces cerevisiae
The homologue of human YTHDF2, Ydr374c (Pho92), is the only protein that has a YT521-B homology (YTH) domain in Saccharomyces cerevisiae. Based on a microarray analysis, genes involved in the phosphate signal transduction (PHO) pathway were upregulated in the Δpho92 strain, as were genes regulated by Pho4, which is an important transcription factor in the PHO pathway. To identify the exact mechanism of Pho92 action with respect to phosphate metabolism, we investigated the effect of Pho92 on PHO4 expression. The half-life of PHO4 mRNA was increased in the Δpho92 strain; this phenotype was also observed in the ...
Source: BJ Gene - November 8, 2013 Category: Biochemistry Authors: H Kang, S Jeong, K Kim, I Baek, M Chang, C Kang, Y Park, C Yun Tags: BJ Gene Source Type: research
RAM function is dependent on Kap{beta}2{-}mediated nuclear entry
Eukaryotic gene expression is dependent on the modification of the first transcribed nucleotide of pre-mRNA by the addition of the 7-methylguanosine cap. The cap protects transcripts from exonucleases and recruits complexes which mediate transcription elongation, processing and translation initiation. The cap is synthesised by a series of reactions which link 7-methylguanosine to the first transcribed nucleotide via a 5’ to 5’ triphosphate bridge. In mammals, cap synthesis is catalysed by the sequential action of RNGTT (RNA guanylyltransferase and 5'-phosphatase) and RNMT (RNA guanine-7 methyltransferase), en...
Source: BJ Gene - November 7, 2013 Category: Biochemistry Authors: T Gonatopoulos-Pournatzis, V H. Cowling Tags: BJ Gene Source Type: research
MicroRNA-19a/b family positively regulates cardiomyocyte hypertrophy by targeting atrogin-1 and MuRF-1
ABSTRACT
Progressive cardiac hypertrophy due to pathological stimuli such as pressure overload is frequently associated with the development of heart failure, a major cause of morbidity and mortality worldwide. Growing evidence has shown that microRNAs (miRNAs) are extensively involved in the pathogenesis of cardiac hypertrophy. Here, we examined the hypothesis that miR-19a/b family acts as a key regulator of cardiac hypertrophy and apoptosis. Forced overexpression of miR-19a/b was sufficient to induce hypertrophy in rat neonatal cardiomyocytes. Luciferase assays revealed that miR-19a/b directly target the anti-hypertrophi...
Source: BJ Gene - October 14, 2013 Category: Biochemistry Authors: D Song, J Ryu, J Kim, E Kwon, D Kim Tags: BJ Signal Source Type: research
