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. 2011 Sep 25;8(11):963-8.
doi: 10.1038/nmeth.1705.

The proteomes of transcription factories containing RNA polymerases I, II or III

Svitlana Melnik  1 , Binwei Deng, Argyris Papantonis, Sabyasachi Baboo, Ian M Carr, Peter R Cook
Affiliations

The proteomes of transcription factories containing RNA polymerases I, II or III

Svitlana Melnik et al. Nat Methods. .

Abstract

Human nuclei contain three RNA polymerases (I, II and III) that transcribe different groups of genes; the active forms of all three are difficult to isolate because they are bound to the substructure. Here we describe a purification approach for isolating active RNA polymerase complexes from mammalian cells. After isolation, we analyzed their protein content by mass spectrometry. Each complex represents part of the core of a transcription factory. For example, the RNA polymerase II complex contains subunits unique to RNA polymerase II plus various transcription factors but shares a number of ribonucleoproteins with the other polymerase complexes; it is also rich in polymerase II transcripts. We also describe a native chromosome conformation capture method to confirm that the complexes remain attached to the same pairs of DNA templates found in vivo.

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Figures

Figure 1
Figure 1
Purification procedure. (a) Strategy. Cartoon (top left): chromatin loop with nucleosomes (green circles) tethered to a polymerizing complex (oval) attached to the substructure (brown). Cells are permeabilized, in some cases a run-on performed in [32P]UTP so nascent RNA can be tracked, nuclei are washed with NP40, most chromatin detached with a nuclease (here, DNase I), chromatin-depleted nuclei resuspended in NLB, and polymerizing complexes released from the substructure with caspases. After pelleting, chromatin associated with polymerizing complexes in the supernatant is degraded with DNase I, and complexes partially resolved in 2D gels (using “blue native” and “native” gels in the first and second dimensions); rough positions of complexes (and a control region, c) are shown. Finally, different regions are excised, and their content analyzed by mass spectrometry. (b) Recovery of [32P]RNA, after including a “run-on”. Fractions correspond to those at the same level in (a). (c) “Run-on” activity assayed later during fractionation (as in a, but without run-on at beginning). Different fractions, with names as in (a), were allowed to extend transcripts by < 40 nucleotides in [32P]UTP, and the amount of [32P]RNA/cell determined by scintillation counting. Fractions “2pellet” and “4pellet” were also resuspended in NLB before run-ons were performed; results indicate NLB reduces incorporation to a half or less (right). Despite this, “5super” possesses 25% run-on activity of permeabilized cells (“2pellet”) – equivalent to half the original (after correction for effects of NLB).
Figure 2
Figure 2
Resolving different polymerases in “native” 2D gels (run-ons in [32P]UTP included). (a) Resolving complexes II + III with Coomassie blue in the first dimension. The cartoon shows regions selected for mass spectrometry analysis. First, an autoradiograph of the gel was prepared; overlapping spots of (nascent) [32P]RNA are along the diagonal. ~0.03% protein, ~0.8% DNA, and ~5% nascent [32P]RNA initially present were contained in the region indicated (dotted outline). After blotting, the membrane was stained with Ponceau S; most protein is on the diagonal. Next, the membrane was immuno-probed successively for three polymerases (using antibodies against RPA194, RPB1, and RPC62); the three are partially resolved. Note that complex I is destabilized by the Coomassie blue in the first dimension, and so migrates rapidly. (b) Resolving complex I (no Coomassie in either dimension). The cartoon shows regions selected for mass spectrometry analysis. First, an autoradiograph was prepared; overlapping spots of (nascent) [32P]RNA are again along the diagonal. After staining with Coomassie, spots are seen to overlap regions rich in [32P]RNA. After blotting, the membrane was probed for the polymerases (as above); complex I now runs the slowest. (c) Proteins in regions indicated in a and b were resolved on a 4-15% SDS-acrylamide gel, and stained with Coomassie.
Figure 3
Figure 3
The content of complexes I, II, and III determined by mass spectrometry. (a) Numbers of proteins in the different complexes and their overlap. (b) Many proteins in each complex are associated with the GO term “gene expression” (GO: 0010467), and complex II contains more with “transcription from RNA polymerase II promoter” (GO: 0006366) than I and III. (c) Most proteins in each complex possess GO terms related to transcript production. Selected GO terms were incorporated into 8 groups; for example, “transcription” includes terms “RNA polymerase”, “transcription factor” and “transcription regulation”), and “other terms” includes those not in the other 7 groups. Four additional sets of proteins are included for comparison on the right. Some proteins possess terms in more than one group, and terms in each group are expressed as a fraction of the total in all groups. 2% proteins in each complex lacked any GO term, and many proteins in the complexes associated with “other terms” nevertheless turn out to play a role in transcript production (for example, actin, proteasomal constituents, nucleoporins). Each complex exhibits a characteristic pattern, which is distinct from those given by proteins with the terms “cytoplasm” and “S100”.
Figure 4
Figure 4. Isolated complexes remain associated with DNA sequences found in vivo
(a) Strategies for 3C and native 3C. Magenta and blue genes on different chromosomes are co-transcribed by one complex (oval) attached to the substructure (brown). 3C involves covalently cross-linking (turquoise lines) DNA, cutting (with HindIII here), dilution, ligation, and detection of ligated products by PCR. Note that a is joined to c, even though there was no stable molecular bridge between the two before cross-linking; such products yield an inevitable background. Native 3C omits cross-linking, and relies on pre-existing (native) contacts. As most (inactive) cellular DNA is lost during isolation (including fragment c), unwanted background is lower, and wanted 3C products are present in higher concentrations. (b) Targets of primers (grey arrows) used to monitor interactions 1-6; only contacts due to interactions 1 and 6 (purple lines) are detected by both 3C and native 3C. White arrows: primers used for loading controls. (c) 3C and native 3C yield similar bands/contacts (although less template is needed with native 3C). HUVECs were treated with TNFα (0, 30 min), and interactions 1-6 monitored by 3C and native 3C. Arrowheads indicate relevant 3C bands (all verified by sequencing; additional, non-specific, bands are amplified during the 36 PCR cycles used). “Intra-GAPDH” 3C and “loading” controls apply to all panels. Controls (with 13-50 ng template) show PCR is conducted in the linear amplification range.
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