scholarly journals Immunoglobulin switch μ sequence causes RNA polymerase II accumulation and reduces dA hypermutation

2009 ◽  
Vol 206 (6) ◽  
pp. 1237-1244 ◽  
Author(s):  
Deepa Rajagopal ◽  
Robert W. Maul ◽  
Amalendu Ghosh ◽  
Tirtha Chakraborty ◽  
Ahmed Amine Khamlichi ◽  
...  
Keyword(s):  
B Cells ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Polymerase ◽  
Dna Sequences ◽  
Repetitive Sequence ◽  
Ex Vivo ◽  
Dna Structure ◽  
Switch Region ◽  
Activation Induced Deaminase

Repetitive DNA sequences in the immunoglobulin switch μ region form RNA-containing secondary structures and undergo hypermutation by activation-induced deaminase (AID). To examine how DNA structure affects transcription and hypermutation, we mapped the position of RNA polymerase II molecules and mutations across a 5-kb region spanning the intronic enhancer to the constant μ gene. For RNA polymerase II, the distribution was determined by nuclear run-on and chromatin immunoprecipitation assays in B cells from uracil-DNA glycosylase (UNG)–deficient mice stimulated ex vivo. RNA polymerases were found at a high density in DNA flanking both sides of a 1-kb repetitive sequence that forms the core of the switch region. The pileup of polymerases was similar in unstimulated and stimulated cells from Ung−/− and Aid−/−Ung−/− mice but was absent in cells from mice with a deletion of the switch region. For mutations, DNA was sequenced from Ung−/− B cells stimulated in vivo. Surprisingly, mutations of A nucleotides, which are incorporated by DNA polymerase η, decreased 10-fold before the repetitive sequence, suggesting that the polymerase was less active in this region. We propose that altered DNA structure in the switch region pauses RNA polymerase II and limits access of DNA polymerase η during hypermutation.

scholarly journals Spt5 accumulation at variable genes distinguishes somatic hypermutation in germinal center B cells from ex vivo–activated cells

2014 ◽  
Vol 211 (11) ◽  
pp. 2297-2306 ◽  
Author(s):  
Robert W. Maul ◽  
Zheng Cao ◽  
Lakshmi Venkataraman ◽  
Carol A. Giorgetti ◽  
Joan L. Press ◽  
...  
Keyword(s):  
B Cells ◽  
Rna Polymerase Ii ◽  
Germinal Center ◽  
Cultured Cells ◽  
Somatic Hypermutation ◽  
Ex Vivo ◽  
Transcription Cofactor ◽  
V Genes ◽  
Activation Induced Deaminase ◽  
Germinal Center B Cells

Variable (V) genes of immunoglobulins undergo somatic hypermutation by activation-induced deaminase (AID) to generate amino acid substitutions that encode antibodies with increased affinity for antigen. Hypermutation is restricted to germinal center B cells and cannot be recapitulated in ex vivo–activated splenic cells, even though the latter express high levels of AID. This suggests that there is a specific feature of antigen activation in germinal centers that recruits AID to V genes which is absent in mitogen-activated cultured cells. Using two Igh knock-in mouse models, we found that RNA polymerase II accumulates in V regions in B cells after both types of stimulation for an extended distance of 1.2 kb from the TATA box. The paused polymerases generate abundant single-strand DNA targets for AID. However, there is a distinct accumulation of the initiating form of polymerase, along with the transcription cofactor Spt5 and AID, in the V region from germinal center cells, which is totally absent in cultured cells. These data support a model where mutations are prevalent in germinal center cells, but not in ex vivo cells, because the initiating form of polymerase is retained, which affects Spt5 and AID recruitment.

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals A55 Molecular systematics of sturgeon nucleocytoplasmic large DNA viruses

2019 ◽  
Vol 5 (Supplement_1) ◽  
Author(s):  
S Clouthier ◽  
E Anderson ◽  
G Kurath ◽  
R Breyta
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Small Subunit ◽  
Sequence Length ◽  
Dna Viruses ◽  
Mrna Capping ◽  
Capping Enzyme ◽  
Nucleocytoplasmic Large Dna Virus ◽  
Nucleocytoplasmic Large Dna Viruses

Abstract Namao virus (NV) is a sturgeon nucleocytoplasmic large DNA virus (sNCLDV) that can cause a lethal disease of the integumentary system in lake sturgeon Acipenser fulvescens. As a group, the sNCLDV have not been assigned to any currently recognized taxonomic family of viruses. In this study, a dataset of NV DNA sequences was generated and assembled as two non-overlapping contigs of 306 and 448 base pairs (bp) and then used to conduct a comprehensive systematics analysis using Bayesian phylogenetic inference for NV, other sNCLDV, and representative members of six families of the NCLDV superfamily. The phylogeny of NV was reconstructed using protein homologues encoded by nine nucleocytoplasmic virus orthologous genes (NCVOGs): NCVOG0022—mcp, NCVOG0038—DNA polymerase B elongation subunit, NCVOG0076—VV A18-type helicase, NCVOG0249—VV A32-type ATPase, NCVOG0262—AL2 VLTF3-like transcription factor, NCVOG0271—RNA polymerase II subunit II, NCVOG0274—RNA polymerase II subunit I, NCVOG0276—ribonucleotide reductase small subunit, and NCVOG1117—mRNA capping enzyme. The accuracy of our phylogenetic method was evaluated using a combination of Bayesian statistical analysis and congruence analysis. Stable tree topologies were obtained with datasets differing in target molecule(s), sequence length, and taxa. Congruent topologies were obtained in phylogenies constructed using individual protein datasets and when four proteins were used in a concatenated approach. The major capsid protein phylogeny indicated that ten representative sNCLDV form a monophyletic group comprised of four lineages within a polyphyletic Mimi-Phycodnaviridae group of taxa. Overall, the analyses revealed that Namao virus is a member of the Mimiviridae family with strong and consistent support for a clade containing NV and CroV as sister taxa.

DNA structure in human RNA polymerase II promoters 1 1Edited by J. Karn

1998 ◽  
Vol 281 (4) ◽  
pp. 663-673 ◽  
Author(s):  
Anders Gorm Pedersen ◽  
Pierre Baldi ◽  
Yves Chauvin ◽  
Søren Brunak
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Structure

scholarly journals DNA polymerase ε associates with the elongating form of RNA polymerase II and nascent transcripts

FEBS Journal ◽  
2006 ◽  
Vol 273 (24) ◽  
pp. 5535-5549 ◽  
Author(s):  
Anna K. Rytkönen ◽  
Tomi Hillukkala ◽  
Markku Vaara ◽  
Miiko Sokka ◽  
Maarit Jokela ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Polymerase ◽  
Nascent Transcripts

scholarly journals Ex vivo rescue of recombinant very virulent IBDV using a RNA polymerase II driven system and primary chicken bursal cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Liliana L. Cubas-Gaona ◽  
Romane Trombetta ◽  
Céline Courtillon ◽  
Kai Li ◽  
Xiaole Qi ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ex Vivo ◽  
Driven System

scholarly journals Dexamethasone increases the number of RNA polymerase II molecules transcribing integrated mouse mammary tumor virus DNA and flanking mouse sequences.

1984 ◽  
Vol 4 (6) ◽  
pp. 1057-1062 ◽  
Author(s):  
J M Firzlaff ◽  
H Diggelmann
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mammary Tumor ◽  
Dna Sequences ◽  
Mouse Mammary Tumor Virus ◽  
Hormone Treatment ◽  
Mrna Levels ◽  
Mammary Tumor Virus ◽  
Mouse Mammary Tumor ◽  
Tumor Virus

In mouse Ltk- cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the transcribed MMTV DNA as well as on the flanking mouse DNA sequences. We found that the specific increase in the polymerase loading after hormone treatment is proportional to the increase in the amount of stable MMTV mRNA. When the DNA sequences which are responsible for hormone-receptor binding and for the increased MMTV mRNA levels were deleted, no increase in RNA polymerase II loading on MMTV DNA was observed. Nuclear RNA chains which were transcribed in response to hormone treatment were detected not only from the transfected MMTV DNA but also from the mouse DNA sequences adjacent to the 3' end of the provirus.

scholarly journals E3-ubiquitin ligase Nedd4 determines the fate of AID-associated RNA polymerase II in B cells

2013 ◽  
Vol 27 (16) ◽  
pp. 1821-1833 ◽  
Author(s):  
J. Sun ◽  
C. D. Keim ◽  
J. Wang ◽  
D. Kazadi ◽  
P. M. Oliver ◽  
...  
Keyword(s):  
B Cells ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase

scholarly journals Control of Transcription Initiation by Biased Thermal Fluctuations on Repetitive Genomic Sequences

2020 ◽  
Author(s):  
Masahiko Imashimizu ◽  
Yuji Tokunaga ◽  
Ariel Afek ◽  
Hiroki Takahashi ◽  
Nobuo Shimamoto ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Repetitive Sequence ◽  
Transcription Initiation ◽  
High Throughput Sequencing ◽  
Thermal Fluctuations ◽  
A Genome ◽  
Sequence Elements ◽  
Promoter Dna

In the process of transcription initiation by RNA polymerase, promoter DNA sequences affect multiple reaction pathways determining the productivity of transcription. However, the question of how the molecular mechanism of transcription initiation depends on sequence properties of promoter DNA remains poorly understood. Here, combining the statistical mechanical approach with high-throughput sequencing results, we characterize abortive transcription and pausing during transcription initiation by Escherichia coli RNA polymerase at a genome-wide level. Our results suggest that initially transcribed sequences enriched with thymine bases represent the signal inducing abortive transcription. On the other hand, certain repetitive sequence elements broadly embedded in promoter regions constitute the signal inducing pausing. Both signals decrease the productivity of transcription initiation. Based on solution NMR and in vitro transcription measurements, we also suggest that repetitive sequence elements of promoter DNA modulate the rigidity of its double-stranded form, which profoundly influences the reaction coordinates of the productive initiation via pausing.

scholarly journals Role of Dot1L and H3K79 methylation in regulating somatic hypermutation of immunoglobulin genes

2021 ◽  
Vol 118 (29) ◽  
pp. e2104013118
Author(s):  
Zhi Duan ◽  
Linda B. Baughn ◽  
Xiaohua Wang ◽  
Yongwei Zhang ◽  
Varun Gupta ◽  
...  
Keyword(s):  
B Cells ◽  
Somatic Hypermutation ◽  
Ex Vivo ◽  
Pol Ii ◽  
Class Switch ◽  
Igh Locus ◽  
Gene Treatment ◽  
Activation Induced Deaminase ◽  
V Region ◽  
Nascent Transcripts

Somatic hypermutation (SHM) and class-switch recombination (CSR) of the immunoglobulin (Ig) genes allow B cells to make antibodies that protect us against a wide variety of pathogens. SHM is mediated by activation-induced deaminase (AID), occurs at a million times higher frequency than other mutations in the mammalian genome, and is largely restricted to the variable (V) and switch (S) regions of Ig genes. Using the Ramos human Burkitt’s lymphoma cell line, we find that H3K79me2/3 and its methyltransferase Dot1L are more abundant on the V region than on the constant (C) region, which does not undergo mutation. In primary naïve mouse B cells examined ex vivo, the H3K79me2/3 modification appears constitutively in the donor Sμ and is inducible in the recipient Sγ1 upon CSR stimulation. Knockout and inhibition of Dot1L in Ramos cells significantly reduces V region mutation and the abundance of H3K79me2/3 on the V region and is associated with a decrease of polymerase II (Pol II) and its S2 phosphorylated form at the IgH locus. Knockout of Dot1L also decreases the abundance of BRD4 and CDK9 (a subunit of the P-TEFb complex) on the V region, and this is accompanied by decreased nascent transcripts throughout the IgH gene. Treatment with JQ1 (inhibitor of BRD4) or DRB (inhibitor of CDK9) decreases SHM and the abundance of Pol II S2P at the IgH locus. Since all these factors play a role in transcription elongation, our studies reinforce the idea that the chromatin context and dynamics of transcription are critical for SHM.

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