scholarly journals c-Jun Controls Histone Modifications, NF-κB Recruitment, and RNA Polymerase II Function To Activate the ccl2 Gene

2008 ◽  
Vol 28 (13) ◽  
pp. 4407-4423 ◽  
Author(s):  
Sabine Wolter ◽  
Anneke Doerrie ◽  
Axel Weber ◽  
Heike Schneider ◽  
Elke Hoffmann ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Interleukin 1 ◽  
Histone Deacetylase 3 ◽  
H3 Acetylation ◽  
Ccl2 Gene ◽  
Dependent Interaction ◽  
In Cells

ABSTRACT Interleukin-1 (IL-1)-induced mRNA expression of ccl2 (also called MCP-1), a prototypic highly regulated inflammatory gene, is severely suppressed in cells lacking c-Jun or Jun N-terminal protein kinase 1 (JNK1)/JNK2 genes and is only partially restored in cells expressing a c-Jun(SS63/73AA) mutant protein. We used chromatin immunoprecipitation to identify three c-Jun-binding sites located in the far 5′ region close to the transcriptional start site and in the far 3′ region of murine and human ccl2 genes. Mutational analysis revealed that the latter two sites contribute to ccl2 transcription in response to the presence of IL-1 or of ectopically expressed c-Jun-ATF-2 dimers. Further experiments comparing wild-type and c-Jun-deficient cells revealed that c-Jun regulates Ser10 phosphorylation of histone H3, acetylation of histones H3 and H4, and recruitment of histone deacetylase 3 (HDAC3), NF-κB subunits, and RNA polymerase II across the ccl2 locus. c-Jun also coimmunoprecipitated with p65 NF-κB and HDAC3. Based on DNA microarray analysis, c-Jun was required for full expression of 133 out of 162 IL-1-induced genes. For inflammatory genes, these data support the idea of an activator function of c-Jun that is executed by multiple mechanisms, including phosphorylation-dependent interaction with p65 NF-κB and HDAC3 at the level of chromatin.

scholarly journals Functional dissection of the catalytic mechanism of mammalian RNA polymerase II

2005 ◽  
Vol 83 (4) ◽  
pp. 497-504 ◽  
Author(s):  
Benoit Coulombe ◽  
Marie-France Langelier
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Structural Elements ◽  
Catalytic Mechanisms ◽  
Rnap Ii ◽  
Affinity Peptide

High resolution X-ray crystal structures of multisubunit RNA polymerases (RNAP) have contributed to our understanding of transcriptional mechanisms. They also provided a powerful guide for the design of experiments aimed at further characterizing the molecular stages of the transcription reaction. Our laboratory used tandem-affinity peptide purification in native conditions to isolate human RNAP II variants that had site-specific mutations in structural elements located strategically within the enzyme's catalytic center. Both in vitro and in vivo analyses of these mutants revealed novel features of the catalytic mechanisms involving this enzyme.Key words: RNA polymerase II, transcriptional mechanisms, mutational analysis, mRNA synthesis.

scholarly journals Signaling Pathways Differentially Affect RNA Polymerase II Initiation, Pausing, and Elongation Rate in Cells

2013 ◽  
Vol 50 (2) ◽  
pp. 212-222 ◽  
Author(s):  
Charles G. Danko ◽  
Nasun Hah ◽  
Xin Luo ◽  
André L. Martins ◽  
Leighton Core ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Signaling Pathways ◽  
Rna Polymerase Ii ◽  
Elongation Rate ◽  
In Cells

scholarly journals Mutational Analysis of an RNA Polymerase II Elongation Factor in Drosophila melanogaster

2005 ◽  
Vol 25 (17) ◽  
pp. 7803-7811 ◽  
Author(s):  
Mark A. Gerber ◽  
Ali Shilatifard ◽  
Joel C. Eissenberg
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Cultured Cells ◽  
Mutational Analysis ◽  
Elongation Factor ◽  
Regulatory Function ◽  
Polymerase Binding ◽  
Necessary And Sufficient ◽  
Elongation Activity

ABSTRACT The ELL family of proteins function in vitro as elongation factors for RNA polymerase II. Deletion studies have defined domains in mammalian ELL required for transcription elongation activity and RNA polymerase binding in vitro, for transformation of cultured cells when overexpressed, and for leukemogenesis and cell proliferation as part of a leukemic fusion protein. The goal of this study was to identify domains required for chromosome targeting and viability in the unique Drosophila ELL (dELL) protein. Here, we show that an N-terminal domain of dELL is necessary and sufficient for targeting to transcriptionally active puff sites in chromatin, supporting a role for this domain in recruiting dELL to elongating RNA polymerase II. We demonstrate that a central domain of dELL is required for rapid mobilization of ELL during the heat shock response, suggesting a regulatory function for this domain. Unexpectedly, transgenic dELL in which the N-terminal chromosome binding domain is deleted can complement the recessive lethality of mutations in ELL, suggesting that Drosophila ELL has an essential activity in development distinct from its role as an RNA polymerase II elongation factor.

scholarly journals Three Key Subregions Contribute to the Function of the Downstream RNA Polymerase II Core Promoter

2010 ◽  
Vol 30 (14) ◽  
pp. 3471-3479 ◽  
Author(s):  
Joshua W. M. Theisen ◽  
Chin Yan Lim ◽  
James T. Kadonaga
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mutational Analysis ◽  
Core Promoter ◽  
Regulatory Element ◽  
Consensus Sequence ◽  
Transcription Start ◽  
The Core ◽  
Close Proximity ◽  
Core Promoter Activity

ABSTRACT The RNA polymerase II core promoter is a diverse and complex regulatory element. To gain a better understanding of the core promoter, we examined the motif 10 element (MTE), which is located downstream of the transcription start site and acts in conjunction with the initiator (Inr). We found that the MTE promotes the binding of purified TFIID to the core promoter and that the TAF6 and TAF9 subunits of TFIID appear to be in close proximity to the MTE. To identify the specific nucleotides that contribute to MTE activity, we performed a detailed mutational analysis and determined a functional MTE consensus sequence. These studies identified favored as well as disfavored nucleotides and demonstrated the previously unrecognized importance of nucleotides in the subregion of nucleotides 27 to 29 (+27 to + 29 relative to A+1 in the Inr consensus) for MTE function. Further analysis led to the identification of three downstream subregions (nucleotides 18 to 22, 27 to 29, and 30 to 33) that contribute to core promoter activity. The three binary combinations of these subregions lead to the MTE (nucleotides 18 to 22 and 27 to 29), a downstream core promoter element (nucleotides 27 to 29 and 30 to 33), and a novel “bridge” core promoter motif (nucleotides 18 to 22 and 30 to 33). These studies have thus revealed a tripartite organization of key subregions in the downstream core promoter.

scholarly journals Anatomy of an unusual RNA polymerase II promoter containing a downstream TATA element.

1992 ◽  
Vol 12 (6) ◽  
pp. 2884-2897 ◽  
Author(s):  
Y Kasai ◽  
H Chen ◽  
S J Flint
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Mutational Analysis ◽  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Cell Extracts ◽  
Tata Element ◽  
Tata Sequence

The adenovirus type 2 IVa2 promoter lacks a conventional TATA element yet directs transcription from two closely spaced initiation sites. To define elements required for in vitro transcription of this promoter, IVa2 templates carrying 5' deletions or linker-scanning mutations were transcribed in HeLa whole-cell extracts and the transcripts were analyzed by primer extension. Mutation of the sequence centered on position -47, which is specifically recognized by a cellular factor, reduced the efficiency of IVa2 transcription two- to threefold, whereas mutation of the sequence centered on position -30 selectively impaired utilization of the minor in vivo initiation site. Utilization of the major in vivo site was decreased no more than fivefold by deletion of all sequences upstream of position -15. By contrast, mutation of the region from +13 to +19 or of the initiation region severely impaired IVa2 transcription. The sequence spanning the initiation sites was sufficient to direct accurate initiation by RNA polymerase II from the major in vivo site. Thus, the two initiation sites of the IVa2 promoter are specified by independent elements, and a downstream element is the primary determinant of efficient transcription from both of these sites. The downstream element identified by mutational analysis altered the TATA element-like sequence TATAGAAA lying at positions +21 to +14 in the coding strand. Transcription from the wild-type IVa2 promoter was severely inhibited when endogenous TFIID was inactivated by mild heat treatment. Exogenous human TATA-binding protein (TBP) synthesized in Escherichia coli restored specific IVa2 transcription from both initiation sites when added to such heat-treated extracts. Although efficient IVa2 transcription requires both the downstream TATA sequence and active TFIID, bacterially synthesized TBP also stimulated the low level of IVa2 transcription observed when the TATA sequence was mutated to a sequence that failed to bind TBP.

scholarly journals Use of the nuclear walk-on methodology to determine sites of RNA polymerase II initiation and pausing and quantify nascent RNAs in cells

Methods ◽  
2019 ◽  
Vol 159-160 ◽  
pp. 165-176 ◽  
Author(s):  
Christopher B. Ball ◽  
Kyle A. Nilson ◽  
David H. Price
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
In Cells
Sign in / Sign up

Export Citation Format

Share Document