Promoter melting and TFIID complexes on Drosophila genes in vivo.

Giardina, Charles; Pérez‐Riba, Mercè; Lis, John T.

doi:10.1101/gad.6.11.2190

Cited by 146 publications

(178 citation statements)

References 53 publications

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“…Another basal factor, GAGA is also involved in enhancing promoter accessibility in unstressed conditions. Further, it has been well established that in unstressed cells, RNA polymerase II is poised on the Hsp70 promoter and paused preceding activation by stress-induced Hsf-1, priming for transcription and allowing a quick induction (Giardina et al, 1992;Lis and Wu, 1993). It is possible that differences in any of these binding sequences or basal activities on the endogenous Hsp70B 0 promoter may contribute to differences in basal expression of this protein in different cell types.…”

Section: Discussionmentioning

confidence: 99%

Cell number‐dependent regulation of Hsp70B′ expression: Evidence of an extracellular regulator

Noonan

Place

Rasoulpour

et al. 2006

Journal Cellular Physiology

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Hsp70B 0 is a unique member of the human Hsp70 family of chaperones about which information is scarce. Unlike the major inducible Hsp72 protein, Hsp70B 0 is strictly inducible having little or no basal expression levels in most cells. We observed that Hsp70B 0 appears transiently in response to heat stress whereas Hsp72 levels persist for many days. Also, Hsp70B 0 is optimally induced when cell numbers are low, whereas Hsp72 levels are greatest at higher cell number. Hsp70B 0 promoter activation was measured by flow cytometry using an Hsp70B 0 promoter-driven GFP construct. In heat stressed cells, promoter activation is cell number independent over a broad range. However, when cell number increases beyond a certain population size, cells are less stress inducible for Hsp70B 0 and induction becomes highly cell number-dependent. Cell number differences in Hsp70 activation cannot be explained by changes in Hsf-1 DNA-binding activity or hyperphosphorylation. Cells with few or no cell matrix attachments (laminin-coated and low attachment plates, respectively) appear to be more sensitive to cell number-dependent inhibition. Medium conditioned by the low cell number (LCN) populations supports increased Hsp70B 0 promoter activation in high cell number (HCN) cultures. Likewise, medium conditioned in HCN culture conditions causes decreased activation of Hsp70B 0 promoter in LCN cultures. As HCN-conditioned medium has all the components necessary for cell growth, two possibilities for the activation of Hsp70B 0 gene expression exist: an inhibitory component that accumulates in culture medium at HCN, or an activator that accumulates at LCN.

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Section: Discussionmentioning

confidence: 99%

Cell number‐dependent regulation of Hsp70B′ expression: Evidence of an extracellular regulator

Noonan

Place

Rasoulpour

et al. 2006

Journal Cellular Physiology

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“…Additional support for the existence and characterization of the nature of this Pol II complex came from the mapping of promoter melting with KMnO 4 (ref. 23), which revealed melted DNA in the regions of heat shock genes residing 20-50 base pairs downstream of the transcription start sites. Also, the isolation and sizing of the chain-terminated run-on transcripts 24 provided nearnucleotide-resolution mapping of pause sites to this same region, as two peaks separated by 10 base pairs.…”

Section: Molecular Imaging Of Proteins On Genes In Vivomentioning

confidence: 99%

“…24). Promoter-melting assays using KMnO 4 can be performed on intact cells for short periods (30 s) to provide a snapshot of the reactivity of T residues, the hyper-reactivity of which is indicative of Pol II-melted DNA 23 . Whereas these melted residues tend to cluster in the region of 20-50 base pairs and highly correlate with paused Pol II, the pattern of reactivity can be influenced by other proteins either protecting or altering the reactivity of T residues.…”

Section: Defining a Pol II As 'Promoter Paused'mentioning

confidence: 99%

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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“…Previous experiments had discovered a Pol II molecule arrested at the 5' end of the uninduced hsp70 transcription unit (Gilmour and Lis 1986), apparently having initiated an RNA chain but having stopped after polymerizing -25 nucleotides (Rougvie and Lis 1988;Giardina et al 1992). Like the endogeneous loci, the hsp70-lacZ transgene construct in transformant Bg9,61 had also been shown to carry a 5'-arrested Pol II (Lee et al 1992); therefore, we attempted to observe Pol II on the hybrid transgene before heat shock.…”

Section: Pol Iia At the Hsp70-1acz Transgene Insertion Site Before Inmentioning

confidence: 99%

Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing.

Weeks¹,

Hardin²,

Shen³

et al. 1993

Genes & Development

172

146

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To investigate functional differences between RNA polymerases IIA and II0 (Pol IIA and Pol II0), with hypoand hyperphosphorylated carboxy-terminal repeat domains (CTDs), respectively, we have visualized the in vivo distributions of the differentially phosphorylated forms of Pol II on Drosophila polytene chromosomes.Using phosphorylation state-sensitive antibodies and immunofluorescence microscopy with digital imaging, we find Pol IIA and Pol II0 arrayed in markedly different, locus-and condition-specific patterns. Major ecdysone-induced puffs, for example, stain exclusively for Pol II0, indicating that hyperphosphorylated Pol II is the transcriptionally active form of the enzyme on these genes. In striking contrast, induced heat shock puffs stain strongly for both Pol IIA and Pol II0, suggesting that heat shock genes are transcribed by a mixture of hypo-and hyperphosphorylated forms of Pol II. At the insertion sites of a transposon carrying a hybrid hsp70-lacZ transgene, we observe only Pol IIA before heat shock induction, consistent with the idea that Pol II arrested on the hsp70 gene is form IIA. After a 90-sec heat shock, we detect heat shock factor (HSF) at the transposon insertion sites; and after a 5-min shock its spatial distribution on the induced transgene puffs is clearly resolved from that of Pol II. Finally, using antibodies to hnRNP proteins and splicing components, we have discerned an apparent overall correlation between the presence and processing of nascent transcripts and the presence of Pol II0.[Key Words: RNA polymerase IIA/II0; CTD phosphorylation; transcription factors; in situ localization; immunofluorescence microscopy; digital imaging] Received July 28, 1993; revised version accepted September 29, 1993.The carboxy-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is an unusual entity composed of multiple repeats of the 7-amino-acid consensus sequence YSPTSPS (for review, see Corden 1990;Young 1991). The number of repeats ranges from 26 in yeast, to 42 in Drosophila, to 52 in mammals. The CTD has been shown to carry out essential in vivo roles in yeast (Nonet et al. 1987), Drosophila (Zehring et al. 1988), and mammalian cells (Bartolomei et al. 1988), but precisely what those roles are is not well understood. Suggested roles for the CTD include interacting with transcription initiation factors, serving as a molecular "cowcatcher" to facilitate movement of polymerase on chromatin templates, providing a link between transcription and RNA processing, and localizing polymerase to specific nuclear compartments (Corden 1990 (Allison et al. 1988;Scafe et al. 1990;Peterson et al. 1991), suggesting that the CTD may be involved in receiving regulatory signals at certain promoters. In vivo and in vitro experiments suggest that these responses might be mediated through the TATA-binding protein (TBP) component of TFIID (Koleske et al. 1992;Usheva et al. 1992;Thompson et al. 1993). On the other hand, the precise nature of the CTD involvement in initiation is not known, ...

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Promoter melting and TFIID complexes on Drosophila genes in vivo.

Cited by 146 publications

References 53 publications

Cell number‐dependent regulation of Hsp70B′ expression: Evidence of an extracellular regulator

Cell number‐dependent regulation of Hsp70B′ expression: Evidence of an extracellular regulator

Imaging Drosophila gene activation and polymerase pausing in vivo

Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing.

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