Identification of a fraction required for VDR-mediated transcription on chromatin.
We set out to define the components of a HeLa cell nuclear extract required to support transcription on a chromatin template. When we incubated a reporter plasmid containing four VDREs upstream of an adenovirus E1B minimal core promoter with purified core histones and a Drosophila embryonic S190 extract, nucleosomes were deposited in a physiologically spaced array, as revealed by limited micrococcal nuclease digestion (Fig. , left). In the presence of VDR, a fractionated transcription system derived from the phosphocellulose-bound fraction of a HeLa nuclear extract was capable of ligand-responsive transcription from crude chromatin assembled in the S190 extract (Fig. , lanes 1 and 2). However, when the chromatin was partially purified from the S190 extract by gel filtration, ligand-dependent transcription was nearly abolished (Fig. , lanes 5 and 6). Therefore, components of the S190 extract not stably incorporated into the chromatin are apparently required for efficient transcription. Transcription on the purified chromatin was restored by adding the phosphocellulose-unbound fraction of the nuclear extract (PC.3) to the reaction mixture (Fig. , lanes 7 and 8), indicating that PC.3 contained an activity or activities required for VDR-activated transcription on chromatin templates. We next fractionated the PC.3 fraction by DEAE anion-exchange chromatography; transcription-stimulatory activity eluted in a single peak at ~330 mM KCl, a fraction we designated DE330 (Fig. ).
FIG. 1. Identification of activities required for chromatin transcription. (A) Titration of micrococcal nuclease in a limited digestion of promoter-containing DNA assembled into chromatin with crude chromatin assembly extract (S190) or with a recombinant chromatin (more ...)
FIG. 2. Pol II copurifies with chromatin transcription activity. (A) Scheme for the further fractionation of PC.3. (B) VDR-mediated transcription reaction mixtures were prepared with 1.5 μl of the indicated DEAE fractions. The peak of activity eluted (more ...)
Because the S190 extract contains potentially confounding activities capable of stimulating chromatin transcription, we next tested the ability of the DE330 fraction to stimulate transcription on chromatin assembled in a system consisting of purified core histones and recombinant NAP-1 and ACF (a complex of the catalytic subunit ISWI and the accessory factor Acf1). Chromatin assembled in the recombinant system also contained well-spaced nucleosomes (Fig. , right). Just as PC.3 was required to transcribe partially purified chromatin assembled in the S190 extract, DE330 was required for transcription from chromatin assembled in the recombinant system (Fig. ), which was the source of the chromatin used in all subsequent assays. We measured ~25-fold stimulation of activated transcription in the presence of vitamin D3 by the DE330 fraction at the maximal concentration attainable in the assay. Transcription in the absence of vitamin D3 also increased with increasing amounts of the DE330 fraction, but there was an essentially constant ratio of activated-to-basal transcription at all concentrations for which a signal could be measured above the background in the absence of ligand, indicating that the DE330 fraction facilitated but did not circumvent VDR-mediated activation of transcription on chromatin templates.
Pol II cofractionates with chromatin transcription activity.
We further purified the stimulatory activity by Q Sepharose anion-exchange and Superdex 200 gel filtration chromatography (Fig. ). We observed a peak of activity in Superdex 200 fractions 20 to 22, corresponding to an apparent molecular mass of ~500 kDa (Fig. ), although ~70% of the activity was lost during the gel filtration step. Four polypeptides of 38, 22, 18, and 16 kDa cofractionated with the activity (Fig. ). Mass spectrometric analysis identified the 38- and 22-kDa species as the Rpb3 and Rpb7 subunits of Pol II, respectively. Immunoblotting confirmed the copurification of Rpb1, the largest subunit of Pol II, with transcription-stimulatory activity (data not shown).
To determine whether the Pol II enzyme purified from the DE330 fraction is necessary and/or sufficient to reconstitute transcription on chromatin, we subjected the fraction to anti-Rpb1 immunoaffinity chromatography (Fig. ). Pol II was efficiently depleted from the DE330 fraction by passage over an anti-Rpb1 monoclonal antibody (8WG16) column and could be eluted with a peptide corresponding to the epitope: the carboxyl-terminal domain (CTD) of Rpb1 (Fig. ). The CTD peptide eluate contained highly purified Pol II, as judged by silver staining after SDS-PAGE (Fig. ); we detected all known subunits of Pol II in apparently equal stoichiometry, as well as several high-molecular-weight polypeptides that were apparently substoichiometric (Fig. ).
FIG. 3. Pol II is required but not sufficient to reconstitute the chromatin transcription activity in the DE330 fraction. (A) Pol II immunoaffinity chromatography scheme to purify Pol II from the DE330 fraction. (B) Immunopurified Pol II was analyzed by SDS-12% (more ...)
Depleting the DE330 fraction of Pol II nearly abolished activity, indicating that this source of Pol II was indeed required for efficient transcription from chromatin templates (Fig. , compare lanes 2 and 3). Whether this represented a quantitative effect of increasing the Pol II concentration relative to those of the other PC1 components or a specific requirement for the form of Pol II that flows through phosphocellulose remains to be determined. The purified Pol II enzyme was insufficient, however, to reconstitute the full activity of the parent DE330 fraction. In fact, roughly six times more purified Pol II, relative to the Pol II in the starting material, was required to produce equivalent transcription signals (Fig. , compare lanes 2 and 6).
TAF-I/SET is required for transcription from chromatin.
Because neither the anti-Rpb1 flowthrough fraction nor purified Pol II alone could fully reconstitute the activity of the starting fraction, we performed mixing experiments. When we added both the anti-Rpb1 flowthrough fraction and immunopurified Pol II, we observed full reconstitution of the input activity. The stimulation of transcription by the two fractions was more than additive (Fig. , compare lanes 3 and 5 to lane 7), indicating that both Pol II and a distinct activity in the anti-Rpb1 unbound fraction are required for chromatin-templated transcription.
We further purified the activity in the anti-Rpb1 flowthrough fraction by Q Sepharose anion-exchange and Superdex 200 gel filtration chromatography (Fig. ). The activity in the anti-Rpb1 flowthrough fraction was only detected in the presence of immunopurified Pol II; we therefore assayed the column fractions in the presence of saturating amounts of Pol II. Under these conditions, we measured a >100% yield during gel filtration (Fig. , upper part). Also, in contrast to the sizing column run prior to depletion of Pol II (Fig. ), the transcription-stimulatory activity migrated with an apparent size of ~200 kDa (rather than ~500 kDa). Two major polypeptides of 44 and 42 kDa cofractionated with activity during gel filtration (Fig. , lower part). Mass spectrometric analysis determined that both of these polypeptides were derived from TAF-I/SET (hereafter referred to as TAF-I).
FIG. 4. TAF-I stimulates activated transcription on chromatin templates. (A) Scheme for the further fractionation of the anti-Rpb1 flowthrough fraction. (B, top) VDR-mediated transcription performed in the presence of buffer, 1 μl of load, or 4 μl (more ...)
TAF-I was originally identified as the product of a gene fused to the CAN
gene in an acute undifferentiated myeloid leukemia (43
) and was first characterized biochemically as a cellular factor required for the replication of purified adenovirus core particles in vitro (31
). TAF-I contains a central domain homologous to NAP-1 and other members of the NAP family of histone chaperones. TAF-I exists in two isoforms generated by variable splicing—TAF-Iα and TAF-Iβ—that are identical except for unique stretches of 37 and 24 amino acids, respectively, at the amino terminus. Immunoblot assays with antibodies specific for either TAF-Iα or TAF-Iβ showed that both were present in the peak fraction obtained by Superdex 200 chromatography (Fig. ). We purified TAF-Iβ tagged with hexahistidine (His) at the amino terminus and expressed in bacteria. The recombinant TAF-Iβ (rTAF-Iβ) obtained was sufficient to reconstitute the activity in the Superdex 200 peak fractions (Fig. ), indicating that TAF-I is the only required factor in those fractions and that TAF-Iβ suffices for full activity.
TAF-I is a chromatin-specific coactivator for multiple activators.
As demonstrated above (Fig. ), the DE330 fraction contained two components required to reconstitute chromatin-templated transcription in vitro: Pol II and TAF-I. To determine the factor(s) in which chromatin specificity resides, we tested the effect of each component, alone or in combination, on transcription performed with naked or chromatin-assembled DNA templates (Fig. ). The immunopurified Pol II enzyme was capable of stimulating transcription by at least 10-fold on both chromatin and naked templates, regardless of the presence of TAF-Iβ. On the other hand, rTAF-Iβ stimulated transcription on chromatin by ~10-fold but had a less-than-2-fold effect on transcription of naked DNA over the range of Pol II concentrations tested. Thus, rTAF-Iβ preferentially stimulated transcription of chromatin templates.
We next sought to determine whether TAF-I acts as a specific coactivator for nuclear receptors or plays a more general role. We tested the effect of TAF-I on transcription activated by Gal4-VP16—a fusion of the yeast Gal4 DNA-binding domain with the activation domain of the viral transactivator VP16—of a chromatin template containing five Gal4-binding sites upstream of the E4 viral promoter. The level of Gal4-VP16-mediated transcription was enhanced about sixfold by rTAF-Iβ (Fig. ). Therefore, stimulation of chromatin transcription by TAF-I is not restricted to promoters activated by nuclear receptors, suggesting a more general role in facilitating transcription.
Characterization of TAF-I in transcription.
The TAF-I protein we purified from HeLa cells was a nearly stoichiometric mixture of TAF-Iα and TAF-Iβ. To examine the effect of TAF-Iα on chromatin transcription, TAF-Iα was expressed in bacteria and purified identically to rTAF-Iβ. rTAF-Iα was also able to stimulate chromatin transcription, albeit with a specific activity more than twofold lower than that of TAF-Iβ (Fig. ).
FIG. 6. Characterization of the TAF-I requirement in chromatin transcription. (A) VDR-mediated transcription performed in the presence of buffer only or 0.16, 0.33, 0.67, 1.32, or 2.64 μM rTAF-Iβ or rTAF-Iα, as indicated. (B) Purification (more ...)
TAF-Iα and TAF-Iβ have been shown to form dimers in extracts and when expressed in bacteria (32
). To compare the activity of TAF-Iβ with that of a presumptive TAF-Iα/β dimer, we constructed rTAF-Iα in which the His tag was replaced with a FLAG tag. We expressed FLAG-rTAF-Iα together with His-TAF-Iβ in E. coli
and purified, by sequential cobalt and FLAG affinity chromatography, a complex containing TAF-Iα and TAF-Iβ in equal proportions, consistent with a heterodimer (Fig. ). In transcription reaction mixtures, the relative activities of the three complexes were as follows: TAF-Iα/TAF-Iβ = TAF-Iβ alone > TAF-Iα alone (Fig. ).
The acidic carboxyl terminus of TAF-I is important for several of its previously reported functions (34
). We expressed and purified a truncation mutant form that lacked the 52 amino acids corresponding to the acidic carboxyl terminus [rTAF-Iβ(1-225)]. The truncated protein was completely inactive in transcription, indicating that the carboxyl terminus is required to stimulate transcription on chromatin (Fig. , left part). The rTAF-Iβ(1-225) mutant construct was also capable of inhibiting the activity of wild-type TAF-I when added to the same reaction mixture (Fig. , right part).
Because TAF-I is closely related to NAP-1, we investigated whether NAP-1 could also stimulate transcription. NAP-1 was indeed capable of substituting for TAF-I but had about twofold lower specific activity (Fig. ). Thus, stimulation of transcription from chromatin templates may be a general property of the NAP-1 family of histone chaperones.
TAF-I interactions with chromatin and chromatin remodeling factors.
Like other histone chaperones, TAF-I is able to bind histones in solution (30
). Because TAF-I is required for transcription on chromatin, we asked whether it could interact with chromatin under transcription conditions. For this experiment, we purified a version of TAF-Iβ tagged at its amino terminus with the HA epitope, as well as with His. HA-His-TAF-Iβ was as active as His-TAF-Iβ in the chromatin transcription assay (data not shown). We performed chromatin immunoprecipitations to determine if TAF-I was interacting with chromatin during transcription reactions in vitro. Transcription was allowed to proceed for 30 min in the presence of HA-tagged or untagged TAF-I. The chromatin was then digested with micrococcal nuclease to yield an average fragment size of 0.5 kb and immunoprecipitated with anti-HA antibody. We quantified the DNA recovered by PCR with primers flanking the promoter and measured about sixfold enrichment when the reaction mixture contained HA-His-TAF-Iβ, relative to control reaction mixtures containing His-TAF-Iβ (Fig. ). Thus, TAF-Iβ can indeed associate with chromatin in the context of transcription.
FIG. 7. TAF-I interacts with chromatin and chromatin-remodeling factors. (A) Transcription reaction mixtures containing HA-tagged or untagged TAF-Iβ were incubated for 30 min and then subjected to chromatin immunoprecipitation (IP) in vitro with HA antibody. (more ...)
Because TAF-I is a histone chaperone, one possible explanation for its ability to enhance transcription is that it disrupts or disassembles chromatin, in effect rendering the plasmid DNA naked. To test this possibility, different amounts of TAF-I were incubated with preassembled chromatin prior to limited micrococcal nuclease digestion (Fig. ). At concentrations equal to or greater than those that stimulate transcription, TAF-I did not cause a gross change in chromatin structure detectable by increased accessibility to micrococcal nuclease.
TAF-I belongs to the same family of histone chaperones as NAP-1, which can participate in chromatin assembly in cooperation with the ACF complex. Indeed, TAF-I could substitute for NAP-1 in the ACF-dependent assembly of chromatin, but with a 10-fold higher optimal concentration compared to NAP-1 (Fig. ). We also tested whether TAF-I and ACF physically interact. In pulldown assays with purified proteins, TAF-Iβ interacted with the recombinant ISWI-containing ACF complex (data not shown). SNF2H is the human homolog of Drosophila ISWI, the catalytic subunit of the ACF complex. Incubation of HA-His-TAF-Iβ with the PC1 fraction, followed by anti-HA immunoprecipitation and immunoblotting with an SNF2H-specific antibody, confirmed an interaction of SNF2H with TAF-I (Fig. ). To our knowledge direct binding between a NAP family histone chaperone and an ATP-dependent chromatin-remodeling enzyme has not previously been described.
We explored a possible role of the SNF2H-TAF-I interaction in chromatin transcription by depleting SNF2H from the PC1 fraction with an anti-SNF2H antibody (Fig. ). Compared to mock depletion, depletion of SNF2H had a very small positive effect on TAF-I-mediated transcription (Fig. , SNF2 depleted). Adding back recombinant ACF to the PC1 fraction depleted of endogenous SNF2H strongly inhibited transcription (Fig. , SNF2 depleted + rACF). Therefore, SNF2H, a factor with which TAF-I cooperates to assemble chromatin, is dispensable (and possibly even inhibitory) for chromatin-templated transcription.
Defining the timing of TAF-I action.
Although chromatin incubated with TAF-I after assembly or chromatin assembled with TAF-I could not be distinguished from NAP-1-assembled chromatin by micrococcal nuclease digestion, it remained possible that TAF-I caused subtle and/or local changes in chromatin structure. To test whether TAF-I alone could stably modify the chromatin to render it transcriptionally competent, we preincubated rTAF-Iβ with preassembled chromatin for 30 min prior to S300 spin column chromatography to remove the TAF-I protein. The pretreated chromatin still required addition of TAF-I during the transcription reaction, suggesting that TAF-I was incapable of performing its function in the absence of other components of the transcription machinery (Fig. ).
We also tested the possibility of a functional difference between chromatin assembled with TAF-I and chromatin assembled with NAP-1 by performing transcription of either template with or without additional TAF-I. Without purification of the templates over S300 spin columns, addition of TAF-I during the transcription reaction was only required for the chromatin assembled with NAP-1 (Fig. ). When the chromatin was purified to remove factors not stably incorporated during assembly, however, TAF-I was required during the transcription reaction regardless of whether NAP-1 or TAF-I mediated assembly (Fig. ). Therefore, TAF-I facilitates two temporally distinct processes, assembly of DNA into a chromatin structure and activation of transcription on a chromatin template.
Because neither incubation of TAF-I with preassembled chromatin nor assembly de novo with TAF-I could render chromatin competent for transcription, TAF-I must act at a subsequent step to facilitate either preinitiation complex formation, initiation of transcription, or elongation by Pol II through chromatin. Interestingly, two known Pol II elongation factors, Spt6 and FACT, are histone chaperones (6
). To test whether TAF-I executes its required function during transcript elongation—as does FACT—or at a prior step, we performed pulse-chase transcription on chromatin templates (Fig. ). When TAF-I was added prior to a pulse with [α-32
P]CTP, the elongation of labeled transcripts initiated during the pulse could be observed as a smear extending up the gel after chasing with an excess of unlabeled CTP. We estimated an elongation rate of 130 nucleotides/min, comparable to rates previously measured in vitro on chromatin templates (16
). However, if TAF-I was added only after the reaction mixture had been supplemented with unlabeled CTP, no elongating transcripts appeared. Therefore, the requirement for TAF-I is at a step after the assembly of chromatin but prior to the elongation phase of transcription.