The histone chaperone TAF-I/SET/INHAT is required for transcription in vitro of chromatin templates

Abstract

To uncover factors required for transcription by RNA polymerase II on chromatin, we fractionated a mammalian cell nuclear extract. We identified the histone chaperone TAF-I (also known as INHAT [inhibitor of histone acetyltransferase]), which was previously proposed to repress transcription, as a potent activator of chromatin transcription responsive to the vitamin D3 receptor or to Gal4-VP16. TAF-I associates with chromatin in vitro and can substitute for the related protein NAP-1 in assembling chromatin onto cloned DNA templates in cooperation with the remodeling enzyme ATP-dependent chromatin assembly factor (ACF). The chromatin assembly and transcriptional activation functions are distinct, however, and can be dissociated temporally. Efficient transcription of chromatin assembled with TAF-I still requires the presence of TAF-I during the polymerization reaction. Conversely, TAF-I cannot stimulate transcript elongation when added after the other factors necessary for assembly of a preinitiation complex on naked DNA. Thus, TAF-I is required to facilitate transcription at a step after chromatin assembly but before transcript elongation.

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 assembly system (rACF and rNAP-1). (B) Fractionation of HeLa nuclear extract. (C) VDR-mediated transcription reaction mixtures were prepared with S190-assembled chromatin left unpurified (crude chromatin) or purified over an S300 spin column (purified chromatin) in the presence or absence of the PC.3 fraction (12 μg) and vitamin D₃ as indicated. (D) VDR-mediated transcription reaction mixtures were prepared on chromatin assembled in the recombinant system in the presence or absence of vitamin D₃ and buffer only or 0.25, 0.5, 0.75, 1.0, 1.5, or 2 μl of the DE330 fraction, as indicated. The relative level of transcription was quantified by Phosphorimager, with the maximal level defined as 100 U, and is indicated below each lane.

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 at ∼330 mM KCl. (C) VDR-mediated transcription reaction mixtures were prepared with 2 μl of the indicated Superdex 200 fractions or 1 μl of the load. (D) The Superdex 200 fractions were resolved by SDS-10% PAGE, and proteins were visualized by silver staining. Migration of polypeptides correlating with activity is indicated by asterisks. Mass spectrometry identified two of these as Rpb3 and Rpb7, as indicated. The values on the left are molecular sizes in kilodaltons. L, load; F, flowthrough.

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% PAGE and silver staining. Pol II subunits are indicated at the right. The values on the left are molecular sizes in kilodaltons. (C) Immunoblot, probed with anti-Rpb1 monoclonal antibody 8WG16, of equivalent volumes of DE330, the anti-Rpb1 flowthrough fraction, and immunopurified Pol II. (D) VDR-mediated transcription reaction mixtures prepared with buffer only (lane 1); 2 μl of DE330 (lane 2); 2 μl of the anti-Rpb1 flowthrough fraction (lane 3); 1, 2, or 4 μl of immunopurified Pol II, corresponding to roughly 1.5, 3, or 6 times the amount of Pol II present in DE330 (lane 4 to 6); or both 2 μl of the anti-Rpb1 flowthrough fraction and 2 μl of immunopurified Pol II (lane 7). The relative level of transcription (txn) was quantified by Phosphorimager, with the maximal level defined as 100 U, and is indicated below each lane.

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 of the indicated Superdex 200 fraction. All reaction mixtures included 0.78 μg of immunopurified Pol II. (B, bottom) The Superdex 200 fractions were resolved by SDS-12% PAGE, and proteins were visualized by silver staining. Migration of polypeptides copurifying with the activity—all identified by mass spectrometry as isoforms of TAF-I—is indicated by asterisks. In, input. The values on the left are molecular sizes in kilodaltons. (C) The active fractions from the Superdex 200 column were subjected to immunoblotting with a TAF-I antibody that recognizes both the α and β isoforms, an α-specific antibody, and a β-specific antibody, as indicated. (D) VDR-mediated transcription performed in the presence of 4 μl of pooled Superdex 200 fractions 25 and 26 from panel B, buffer only, or 0.14, 0.29, 0.57, 1.14, 2.28, 4.56, 9.12, or 18.24 μM rTAF-Iβ. The relative level of transcription was quantified by Phosphorimager, with the maximal level defined as 100 U, and is indicated below each lane.

FIG. 5.

TAF-I is a chromatin-specific coactivator that stimulates transcription mediated by both VDR and Gal4-VP16. (A) VDR-mediated transcription was performed on either naked or chromatin templates in the presence or absence of 2.28 μM rTAF-Iβ, buffer alone, or 0.024, 0.049, 0.098, 0.195, 0.39, 0.78, or 1.56 μg of immunopurified Pol II, as indicated. (B) Transcription reaction mixtures were prepared with or without 20 ng of Gal4-VP16 and 3 μM rTAF-Iβ, as indicated, and Gal4-VP16-responsive promoter-containing DNA assembled into chromatin. All reaction mixtures included 0.78 μg of immunopurified Pol II. The relative level of transcription was quantified by Phosphorimager, with the maximal level defined as 100 U, and is indicated below each lane.

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 of TAF-Iα/β heterocomplexes. His-tagged TAF-Iβ and FLAG-tagged TAF-Iα migrate identically on SDS-10% PAGE, but only TAF-Iβ can be cleaved by thrombin. Thrombin cleavage of 2.5 μg of double-affinity-purified TAF-Iα/β heterocomplexes or TAF-Iβ and separation of the products by SDS-10% PAGE reveal a 1:1 ratio of TAF-Iα to TAF-Iβ in the TAF-Iα/β heterocomplex. (C) VDR-mediated transcription performed in the presence of buffer only or 500 nM His-tagged TAF-Iα, His-tagged TAF-Iβ, FLAG-tagged TAF-Iα, or TAF-Iα/β heterocomplex. (D, left) VDR-mediated transcription performed in the presence of buffer only; 0.15, 0.3, 0.6, 1.21, 2.41, 4.82, or 9.65 μM rTAF-Iβ(1-225); or 2.28 μM wild-type rTAF-Iβ (++). (D, right) VDR-mediated transcription performed in the presence of buffer only or 0.17, 0.35, 0.69, 1.39, 2.78, 5.56, or 11.11 μM rTAF-Iβ(1-255). Wild-type rTAF-Iβ (800 nM, +) was added where indicated. (E) VDR-mediated transcription performed in the presence of buffer only or 0.15, 0.29, 0.58, 1.17, 2.34, 4.67, or 9.35 μM rTAF-Iβ or rNAP-1. The relative level of transcription was quantified by Phosphorimager, with the maximal level defined as 100 U, and is indicated below each lane.

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. Coimmunoprecipitated DNA was analyzed by semiquantitative PCR and 1% agarose gel electrophoresis. (B) Preassembled chromatin was incubated with buffer alone (−) or 15.3 (+) or 53 (++) ng of HeLa TAF-I from the peak Superdex 200 fractions per μl (Fig. 4B) and then subjected to limited micrococcal nuclease (MNase) digestion. A 15.3-ng/μl concentration of this fraction is roughly equivalent to 4 μl of fractions 25 and 26 assayed in Fig. 4B. (C) Chromatin assembly reaction mixtures were prepared without rNAP-1. Where indicated, ACF and 8 μM rTAF-Iβ were present during assembly. After assembly, the chromatin was subjected to limited micrococcal nuclease digestion. (D) HA-tagged or untagged TAF-Iβ (58 pmol) was incubated with 36 μg of the PC1 fraction for 30 min and subsequently immunoprecipitated with HA antibody. The immunoprecipitates and input (10%) were analyzed by immunoblot assay with an anti-SNF2 antibody. (E) The mock-depleted or anti-SNF2-depleted PC1 fraction was analyzed by immunoblotting with the anti-SNF2 antibody. (F) Transcription reaction mixtures were prepared with 3 μg of PC1, mock-depleted PC1, or anti-SNF2-depleted PC1 on unpurified and S300 spin column-purified chromatin (chr). rACF (170 nM) and rTAF-Iβ (3 μM) were added where indicated. The relative level of transcription was quantified by Phosphorimager, with the maximal level defined as 100 U, and is indicated below each lane.

FIG. 8.

TAF-I is required at a step subsequent to chromatin assembly and prior to transcript elongation. (A) NAP-1-assembled chromatin was preincubated with or without 7.5 μM rTAF-Iβ for 30 min as indicated. The chromatin was used directly or passed over an S300 spin column. Transcription reaction mixtures were prepared with (+) or without (−) 3 μM rTAF-Iβ. (B) Chromatin (Chr) assembled with 0.9 μM NAP-1 or 8 μM rTAF-Iβ was used directly or purified over an S300 spin column, as indicated. Transcription reaction mixtures were then prepared with (+) or without (−) 3 μM rTAF-Iβ. The relative level of transcription in panels A and B was quantified by Phosphorimager, with the maximal level defined as 100 U, and is indicated below each lane. (C) Transcription elongation assays were performed with buffer alone or with 4 μM TAF-Iβ added before the pulse (TAF-Iβ) or after the chase (TAF-Iβ postchase), as indicated. Samples were taken after the pulse (P) or at 0.5, 1, 1.5, 3, 6, or 18 min of chase and analyzed on a 6% denaturing gel. The smear of radioactivity extending up the gel indicates elongation of transcripts initiated during the pulse and was only seen when ΤΑF-Iβ was included prior to the chase. The values on the left are sizes in nucleotides.