Nuclear distribution of transcription factors in relation to sites of transcription and RNA polymerase II

We have investigated the spatial relationship between sites containing newly synthesized RNA and domains containing proteins involved in transcription, such as RNA polymerase II and the transcription factors TFIIH, Oct1, BRG1, E2F-1 and glucocorticoid receptors, using dual immunofluorescence labelling followed by confocal microscopy on cultured cells. As expected, a high degree of colocalisation between the RNA polymerase II and sites containing newly synthesised RNA was observed. Like the newly synthesised RNA and the RNA polymerase II, we found that all the transcription factors that we studied are distributed more or less homogeneously throughout the nucleoplasm, occupying numerous small domains. In addition to these small domains, TFIIH was found concentrated in coiled bodies and Oct1 in a single large domain of about 1.5 microm in 30% of the cells in an asynchronous HeLa cell culture. Remarkably, we found little or no relationship between the spatial distribution of the glucocorticoid receptor, Oct1 and E2F-1 on the one hand and RNA polymerase II and transcription sites on the other hand. In contrast, a significant but incomplete overlap was observed between the spatial distributions of transcription sites and BRG1 and TFIIH. These results indicate that many of the transcription factor-rich nuclear domains are not actively involved in transcription. They may represent incomplete transcription initiation complexes, inhibitory complexes, or storage sites.

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RNA polymerase II transcription is concentrated outside replication domains throughout S-phase

Journal of Cell Science ◽

10.1242/jcs.107.6.1449 ◽

1994 ◽

Vol 107 (6) ◽

pp. 1449-1456 ◽

Cited By ~ 3

Author(s):

D.G. Wansink ◽

E.E. Manders ◽

I. van der Kraan ◽

J.A. Aten ◽

R. van Driel ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Spatial Relationship ◽

S Phase ◽

Double Labelling ◽

Confocal Immunofluorescence Microscopy ◽

Nuclear Domain ◽

Confocal Immunofluorescence ◽

Nuclear Domains ◽

Transcription And Replication

Transcription and replication are, like many other nuclear functions and components, concentrated in nuclear domains. Transcription domains and replication domains may play an important role in the coordination of gene expression and gene duplication in S-phase. We have investigated the spatial relationship between transcription and replication in S-phase nuclei after fluorescent labelling of nascent RNA and nascent DNA, using confocal immunofluorescence microscopy. Permeabilized human bladder carcinoma cells were labelled with 5-bromouridine 5′-triphosphate and digoxigenin-11-deoxyuridine 5′-triphosphate to visualize sites of RNA synthesis and DNA synthesis, respectively. Transcription by RNA polymerase II was localized in several hundreds of domains scattered throughout the nucleoplasm in all stages of S-phase. This distribution resembled that of nascent DNA in early S-phase. In contrast, replication patterns in late S-phase consisted of fewer, larger replication domains. In double-labelling experiments we found that transcription domains did not colocalize with replication domains in late S-phase nuclei. This is in agreement with the notion that late replicating DNA is generally not actively transcribed. Also in early S-phase nuclei, transcription domains and replication domains did not colocalize. We conclude that nuclear domains exist, large enough to be resolved by light microscopy, that are characterized by a high activity of either transcription or replication, but never both at the same time. This probably means that as soon as the DNA in a nuclear domain is being replicated, transcription of that DNA essentially stops until replication in the entire domain is completed.

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The General Transcription Factors IIA, IIB, IIF, and IIE Are Required for RNA Polymerase II Transcription from the Human U1 Small Nuclear RNA Promoter

Molecular and Cellular Biology ◽

10.1128/mcb.19.3.2130 ◽

1999 ◽

Vol 19 (3) ◽

pp. 2130-2141 ◽

Cited By ~ 39

Author(s):

T. C. Kuhlman ◽

H. Cho ◽

D. Reinberg ◽

N. Hernandez

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Small Nuclear Rna ◽

Initiation Complex ◽

General Transcription Factors ◽

Nuclear Rna ◽

Transcription Initiation Complex ◽

Rna Polymerase Ii Transcription

ABSTRACT RNA polymerase II transcribes the mRNA-encoding genes and the majority of the small nuclear RNA (snRNA) genes. The formation of a minimal functional transcription initiation complex on a TATA-box-containing mRNA promoter has been well characterized and involves the ordered assembly of a number of general transcription factors (GTFs), all of which have been either cloned or purified to near homogeneity. In the human RNA polymerase II snRNA promoters, a single element, the proximal sequence element (PSE), is sufficient to direct basal levels of transcription in vitro. The PSE is recognized by the basal transcription complex SNAPc. SNAPc, which is not required for transcription from mRNA-type RNA polymerase II promoters such as the adenovirus type 2 major late (Ad2ML) promoter, is thought to recruit TATA binding protein (TBP) and nucleate the assembly of the snRNA transcription initiation complex, but little is known about which GTFs other than TBP are required. Here we show that the GTFs IIA, IIB, IIF, and IIE are required for efficient RNA polymerase II transcription from snRNA promoters. Thus, although the factors that recognize the core elements of RNA polymerase II mRNA and snRNA-type promoters differ, they mediate the recruitment of many common GTFs.

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Dynamic sumoylation of promoter-bound general transcription factors facilitates transcription by RNA polymerase II

PLoS Genetics ◽

10.1371/journal.pgen.1009828 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1009828

Author(s):

Mohammad S. Baig ◽

Yimo Dou ◽

Benjamin G. Bergey ◽

Russell Bahar ◽

Justin M. Burgener ◽

...

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Ribosomal Proteins ◽

Protein Coding ◽

General Transcription Factors ◽

Genes Encoding ◽

Associated Proteins ◽

Related Proteins

Transcription-related proteins are frequently identified as targets of sumoylation, including multiple subunits of the RNA polymerase II (RNAPII) general transcription factors (GTFs). However, it is not known how sumoylation affects GTFs or whether they are sumoylated when they assemble at promoters to facilitate RNAPII recruitment and transcription initiation. To explore how sumoylation can regulate transcription genome-wide, we performed SUMO ChIP-seq in yeast and found, in agreement with others, that most chromatin-associated sumoylated proteins are detected at genes encoding tRNAs and ribosomal proteins (RPGs). However, we also detected 147 robust SUMO peaks at promoters of non-ribosomal protein-coding genes (non-RPGs), indicating that sumoylation also regulates this gene class. Importantly, SUMO peaks at non-RPGs align specifically with binding sites of GTFs, but not other promoter-associated proteins, indicating that it is GTFs specifically that are sumoylated there. Predominantly, non-RPGs with SUMO peaks are among the most highly transcribed, have high levels of TFIIF, and show reduced RNAPII levels when cellular sumoylation is impaired, linking sumoylation with elevated transcription. However, detection of promoter-associated SUMO by ChIP might be limited to sites with high levels of substrate GTFs, and promoter-associated sumoylation at non-RPGs may actually be far more widespread than we detected. Among GTFs, we found that TFIIF is a major target of sumoylation, specifically at lysines 60/61 of its Tfg1 subunit, and elevating Tfg1 sumoylation resulted in decreased interaction of TFIIF with RNAPII. Interestingly, both reducing promoter-associated sumoylation, in a sumoylation-deficient Tfg1-K60/61R mutant strain, and elevating promoter-associated SUMO levels, by constitutively tethering SUMO to Tfg1, resulted in reduced RNAPII occupancy at non-RPGs. This implies that dynamic GTF sumoylation at non-RPG promoters, not simply the presence or absence of SUMO, is important for maintaining elevated transcription. Together, our findings reveal a novel mechanism of regulating the basal transcription machinery through sumoylation of promoter-bound GTFs.

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DNA wrapping in transcription initiation by RNA polymerase II

Biochemistry and Cell Biology ◽

10.1139/o99-028 ◽

1999 ◽

Vol 77 (4) ◽

pp. 257-264 ◽

Cited By ~ 5

Author(s):

Benoit Coulombe

Keyword(s):

Transcription Factors ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Initiation ◽

Dna Bending ◽

Transcriptional Initiation ◽

General Transcription Factors ◽

Transcript Elongation ◽

Dna Wrapping

The DNA wrapping model of transcription stipulates that DNA bending and wrapping around RNA polymerase causes an unwinding of the DNA helix at the enzyme catalytic center that stimulates strand separation prior to initiation and during transcript elongation. Recent experiments with mammalian RNA polymerase II indicate the significance of DNA bending and wrapping in transcriptional mechanisms. These findings have important implications in our understanding of the role of the general transcription factors in transcriptional initiation and the mechanisms underlying transcriptional regulation.Key words: mRNA synthesis, transcription initiation, RNA polymerase II, DNA wrapping, general transcription factors.

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