Nucleosomal Structures of c-<i>myc</i> Promoters with Transcriptionally Engaged RNA Polymerase II

Albert, Thomas K; Mautner, Josef; Funk, Jens Oliver; Hörtnagel, Konstanze; Pullner, Andrea; Eick, Dirk

doi:10.1128/mcb.17.8.4363

Cited by 22 publications

(18 citation statements)

References 71 publications

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“…Interestingly, chromatin analyses of templates activated by TSA treatment also revealed only minor variations in chromatin structure. These findings are in agreement with other studies reporting little difference in the chromatin structure of nonexpressing, uninduced c-myc genes and that of highly expressing alleles, induced by sodium butyrate (1,42). In combination, our results implicate histone acetylation as a primary regulator of c-myc transcription in the Raji cell line.…”

Section: Discussionsupporting

confidence: 82%

The Immunoglobulin Heavy Chain Locus Control Region Increases Histone Acetylation along Linked c-myc Genes

Madisen

Krumm

Hebbes

et al. 1998

Molecular and Cellular Biology

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In chromosome translocations characteristic of Burkitt lymphomas (BL) and murine plasmacytomas, c-myc genes become juxtaposed to immunoglobulin heavy-chain (IgH) sequences, resulting in aberrant c-myc transcription. Translocated c-myc alleles that retain the first exon exhibit increased transcription from the normally minor c-myc promoter, P1, and increased transcriptional elongation through inherent pause sites proximal to the major c-myc promoter, P2. We recently demonstrated that a cassette derived from four DNase I-hypersensitive sites (HS1234) in the 3C␣ region of the IgH locus functions as an enhancer-locus control region (LCR) and directs a similar pattern of deregulated expression of linked c-myc genes in BL and plasmacytoma cell lines. Here, we report that the HS1234 enhancer-LCR mediates a widespread increase in histone acetylation along linked c-myc genes in Raji BL cells. Significantly, the increase in acetylation was not restricted to nucleosomes within the promoter region but also was apparent upstream and downstream of the transcription start sites as well as along vector sequences. Histone hyperacetylation of control c-myc genes, which was induced by the deacetylase inhibitor trichostatin A, mimics the effect of the HS1234 enhancer on expression from the c-myc P2 promoter, but not that from the P1 promoter. These results suggest that the HS1234 enhancer stimulates transcription of c-myc by a combination of mechanisms. Whereas HS1234 activates expression from the P2 promoter through a mechanism that includes increased histone acetylation, a general increase in histone acetylation is not sufficient to explain the HS1234-mediated activation of transcription from P1.

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Section: Discussionsupporting

confidence: 82%

The Immunoglobulin Heavy Chain Locus Control Region Increases Histone Acetylation along Linked c-myc Genes

Madisen

Krumm

Hebbes

et al. 1998

Molecular and Cellular Biology

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“…Additionally, evidence supporting some form of elongational control in specific vertebrate genes has existed since the early 1980s, for example, greater nuclear run-on signals from 59 portions than 39 regions of chicken b-globin 32 . Similar data for mammalian c-Myc and c-Fos were augmented with in vitro studies that suggested initially a termination control further downstream, but more thorough characterizations in vivo showed these genes to have properties like those of Drosophila Hsp70 paused Pol II 33,34 .…”

Section: How General Is Paused Pol Ii?supporting

confidence: 56%

Imaging Drosophila gene activation and polymerase pausing in vivo

Lis

2007

Nature

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Since the early 1960s, imaging studies of Drosophila sp. polytene chromosomes have provided unique views of gene transcription in vivo. The dramatic changes in chromatin structure that accompany gene activation can be visualized as chromosome puffs. Now, live-cell imaging techniques coupled with protein-DNA crosslinking assays on a genome-wide scale allow more detailed mechanistic questions to be addressed and are prompting the re-evaluation of models of transcription regulation in both Drosophila and mammals.D etermining the complete genome sequence of humans, Drosophila and other model higher eukaryotes was an important step in cataloguing the complete code that programmes the development and maintenance of multicellular organisms. A critical challenge that remains is to determine in molecular terms how this code is read and regulated in higher eukaryotes 1 . The regulation of transcription of the genome is a major mode by which an organism controls both its homeostasis and development. This regulation is executed mainly through the interactions of a plethora of transcription factor proteins and RNAs with each other and with DNA and associated histones 2 . These interactions then dictate when, where, and to what level specific genes are transcribed. Although biochemical and genetic approaches have identified many of the macromolecular players and their activities, a mechanistic understanding of how they operate in gene regulation can be guided and tested by direct imaging of these molecular interactions and the resulting biochemical processes in living cells.Two developments in the imaging of transcription factors at specific endogenous gene loci in vivo are providing new views of transcription mechanisms and regulation. One, currently specific to Drosophila, makes use of state-of-the-art optics combined with the natural amplification of signals in tissue containing polytene chromosomes, allowing investigation of molecular interactions and dynamics at specific loci in real time in living nuclei 3 . The other, although having a history in Drosophila 4-6 , is species-general and uses crosslinking in vivo-that is, chromatin immunoprecipitation (ChIP) assays and variants thereof-to produce a 'molecular image' of specific protein interactions and chromatin modifications with particular DNA sequences in the genome 7 . Here I describe how these optical and molecular imaging approaches are providing new insights into transcription and the rate-limiting steps in its regulation in multicellular organisms. In particular, recent genome-wide ChIP assays in Drosophila 8,9 and mammals 10 support a paradigm shift in gene regulation by indicating that control of transcription elongation by RNA polymerase II (Pol II) in a promoter-proximal pausing model (Box 1), rather than control of the recruitment or initiation of Pol II, is the rate-limiting step for a large fraction of highly regulated genes. Early insights from spread polytene nucleiOver the past several decades, Drosophila has provided extraordinary views of chromosome s...

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“…They found that they could drastically decrease both CTD phosphorylation and Pol II transcript accumulation in vivo using a highly specific inhibitor of P-TEFb, suggesting that P-TEFb is globally required for transcription. In vivo studies by Lis et al have demonstrated that P-TEFb is present at transcriptionally active loci in Drosophila (24), where it is located over the extent of the entire gene (2). This distribution contrasts with that reported for TFIIH, which, at least in vitro, is known to dissociate from the elongation complex soon after initiation (46).…”

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confidence: 44%

“…The guinea pig antiserum from the second or third bleed was used for immunoprecipitation and polytene immunofluorescence assays. Rabbit anti-heat shock factor (HSF) antibodies were produced as previously described (2). Rabbit anti-cyclin T antibodies were a gift from David Price.…”

Section: Fly Stocks Cdk7mentioning

confidence: 99%

Cdk7 Is Required for Full Activation of Drosophila Heat Shock Genes and RNA Polymerase II Phosphorylation In Vivo

Schwartz

Larochelle

Suter

et al. 2003

Molecular and Cellular Biology

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TFIIH has been implicated in several fundamental cellular processes, including DNA repair, cell cycle progression, and transcription. In transcription, the helicase activity of TFIIH functions to melt promoter DNA; however, the in vivo function of the Cdk7 kinase subunit of TFIIH, which has been hypothesized to be involved in RNA polymerase II (Pol II) phosphorylation, is not clearly understood. Using temperature-sensitive and null alleles of cdk7, we have examined the role of Cdk7 in the activation of Drosophila heat shock genes. Several in vivo approaches, including polytene chromosome immunofluorescence, nuclear run-on assays, and, in particular, a protein-DNA cross-linking assay customized for adults, revealed that Cdk7 kinase activity is required for full activation of heat shock genes, promoter-proximal Pol II pausing, and Pol II-dependent chromatin decondensation. The requirement for Cdk7 occurs very early in the transcription cycle. Furthermore, we provide evidence that TFIIH associates with the elongation complex much longer than previously suspected.Accurate transcription of most eukaryotic genes requires the coordinated recruitment of RNA polymerase II (Pol II), along with a host of general transcription factors (GTFs), sequencespecific transcription factors, and chromatin-remodeling factors. The assembly of these factors culminates in the formation of a Pol II promoter complex that initiates transcription, begins elongation, and matures into an elongationally competent state. This maturation is characterized by the phosphorylation of the carboxy-terminal domain (CTD) in the largest subunit of Pol II and the association of a set of elongation and RNAprocessing factors.One of the GTFs, TFIIH, has been implicated in both ATPdependent promoter melting and phosphorylation of the Pol II CTD at serine 5 (Ser5) within the heptapeptide repeat. TFIIH is comprised of two subcomplexes, core IIH and CAK (Cdkactivating kinase) (41). Core IIH contains six subunits, two of which are ATP-dependent DNA helicases. One of these helicases, ERCC3, has been shown to be required for promoter melting in vitro (41) and in vivo (13). CAK is a tripartite complex composed of MAT-1, cyclin H, and the CTD kinase, Cdk7. In higher eukaryotes, CAK also occurs as a free complex, which is primarily involved in cell cycle regulation (19).Cdk7 has been proposed to be one of possibly several kinases responsible for phosphorylating the CTD and thus contributing to the formation of the mature, elongating form of polymerase. Indeed, in vitro assays have demonstrated that Cdk7 kinase activity is required for transcription from the dihydrofolate reductase (DHFR) promoter (1, 41). However, this requirement may depend on the promoter that is used and the purity of the transcription system. For example, Makela et al. (30) found that a kinase-deficient TFIIH supported transcription from the adenovirus major late promoter. More recently, Tirode et al. (41) confirmed these results but found that the mere physical presence of the CAK complex could ...

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Nucleosomal Structures of c-myc Promoters with Transcriptionally Engaged RNA Polymerase II

Cited by 22 publications

References 71 publications

The Immunoglobulin Heavy Chain Locus Control Region Increases Histone Acetylation along Linked c-myc Genes

The Immunoglobulin Heavy Chain Locus Control Region Increases Histone Acetylation along Linked c-myc Genes

Imaging Drosophila gene activation and polymerase pausing in vivo

Cdk7 Is Required for Full Activation of Drosophila Heat Shock Genes and RNA Polymerase II Phosphorylation In Vivo

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