The PHD type zinc finger is an integral part of the CBP acetyltransferase domain

Abstract

Histone acetyltransferases (HATs) such as CBP and p300 are regarded as key regulators of RNA polymerase II-mediated transcription, but the critical structural features of their HAT modules remain ill defined. The HAT domains of CBP and p300 are characterized by the presence of a highly conserved putative plant homeodomain (PHD) (C4HC3) type zinc finger, which is part of the functionally uncharacterized cysteine-histidine-rich region 2 (CH2). Here we show that this region conforms to the PHD type zinc finger consensus and that it is essential for in vitro acetylation of core histones and the basal transcription factor TFIIE34 as well as for CBP autoacetylation. PHD finger mutations also reduced the transcriptional activity of the full-length CBP protein when tested on transfected reporter genes. Importantly, similar results were obtained on integrated reporters, which reflect a more natural chromatinized state. Taken together, our results indicate that the PHD finger forms an integral part of the enzymatic core of the HAT domain of CBP.

FIG. 1.

A conserved PHD finger in the acetyltransferase domain of CBP and p300. (A) Schematic representation of mouse CBP (mCBP), showing the position of the bromodomain (BD), the acetyltransferase domain (HAT), the PHD finger, and the CH1 through -3. Numbers refer to amino acid positions in mCBP. The putative CoA binding site is indicated by a lightly shaded box, and the positions of two HAT mutants, the F1541A (46) and L1690K C1691L (34) mutants, are shown. An alignment of the PHD fingers in the HAT domains of mCBP (GenBank accession number P45481), human CBP (hCBP) (Q92793), human p300 (hp300) (A54277), Xenopus CBP (xCBP) (20), Drosophila CBP (dCBP) (T13828), and Caenorhabditis elegans CBP (ceCBP) (P34545) is given. Putative zinc-coordinating cysteines and histidine are shaded; other amino acids that have been changed in this study are boxed. The PHD consensus sequence is shown below the alignment, with strongly conserved hydrophobic amino acids indicated (#). The lengths of the loops between the second and third and between the sixth and seventh zinc-coordinating residues vary between PHD fingers, as indicated. (B) Putative structure of the PHD finger in mCBP, based on the structures of the PHD fingers in KAP-1 (13) and WSTF (52). The zinc-coordinating cysteines and histidine and the zinc atoms (Zn) are indicated, as well as other amino acids that have been changed in this study.

FIG. 2.

The PHD finger is essential for the in vitro HAT activity of the CBP HAT domain. (A) Gal4 DBD fusions of wild-type CBP HAT or various mutants, overexpressed in U-2 OS cells and immunoprecipitated with an antibody against the Gal4 DBD, were incubated with purified Drosophila core histones in the presence of [¹⁴C]acetyl-CoA. Reaction products were separated by SDS-PAGE and stained with Coomassie brilliant blue to verify the presence of equal amounts of histones in each lane. Gels were subsequently dried, and labeled proteins were detected by fluorography. An aliquot from the same immunoprecipitation was used for Western blot analysis with an antibody against the Gal4 DBD. (B) Quantification of the data presented in panel A, as determined by PhosphorImager analysis. Data are presented relative to the activity of wild-type CBP (taken as 100%). Solid bars, wild-type CBP and HAT mutants F1541A and L1690K/C1691L; shaded bars, zinc-coordinating PHD finger mutants; open bars, other PHD finger mutants.

FIG. 3.

Zinc ligation and coordination are essential for the in vitro HAT activity of CBP. (A) GST fusions of the HAT domains of CBP and PCAF were preincubated with 1,10-phenanthroline (5 or 10 mM) and subsequently incubated with purified Drosophila core histones in the presence of [¹⁴C]acetyl-CoA. Reaction products were processed as described in the legend to Fig. 2. (B) GST fusions of the CBP HAT domain isolated on glutathione-Sepharose beads were separated by SDS-PAGE and stained with Coomassie brilliant blue. (C) GST-CBP HAT fusion proteins were incubated with purified Drosophila core histones in the presence of [¹⁴C]acetyl-CoA. Reaction products were processed as described above.

FIG. 4.

The PHD finger is essential for acetylation of nonhistone proteins. Gal4 DBD fusions of wild-type CBP HAT or various mutants, isolated as described in the legend to Fig. 2, were incubated with bacterially expressed TFIIE34 in the presence of [¹⁴C]acetyl-CoA. Reaction products were processed as described in the legend to Fig. 2. An aliquot from the same immunoprecipitation was used for Western blot analysis with antibodies against the Gal4 DBD or acetylated lysine residues (AcLys), as indicated. The asterisk on the right indicates aspecific binding of the anti-acetyl antibody.

FIG. 5.

Mutations in the PHD finger disrupt the transcriptional activity of the CBP HAT domain. U-2 OS cells were transfected with expression vectors for Gal4 DBD fusions of the HAT domain of CBP and a 5xGal4-AdMLTATA-Luc reporter, schematically represented at the top. Activation of the luciferase reporter, normalized for β-galactosidase activity, is shown as fold induction over that with the Gal4 DBD alone. Solid bars, wild-type CBP and the HAT mutants F1541A and L1690K/C1691L; shaded bars, zinc-coordinating PHD finger mutants; open bars, other PHD finger mutants. Results are averages of a minimum of three independent experiments assayed in duplicate ± standard errors of the means.

FIG. 6.

Transcriptional activity of the CBP HAT domain on various promoters and cell lines. U-2 OS (top and center panels) and 911 (bottom panel) cells were transfected with expression vectors for Gal4 DBD fusions of the HAT domain of CBP and a 5xGal4-E1BTATA-Luc (top), a 5xGal4-TK-Luc (center), or a 5xGal4-AdMLTATA-Luc (bottom) reporter. Data are presented as described in the legend to Fig. 5.

FIG. 7.

Effects of PHD mutations on the HAT activity and transcriptional activity of full-length CBP. (A) Gal4 DBD fusions of full-length wild-type CBP HAT or various mutants, overexpressed in 911 cells, were incubated with purified Drosophila core histones in the presence of [¹⁴C]acetyl-CoA. Reaction products were processed as described in the legend to Fig. 2. (B) 911 cells (top panel) were transiently transfected with expression vectors for Gal4 DBD fusions of full-length CBP and a 5xGal4-AdMLTATA-Luc reporter. Similar experiments were performed on 911 cells stably transfected with the 5xGal4-AdMLTATA-Luc reporter (clone 10) (center panel) or the 5xGal4-E1BTATA-Luc reporter (clone 8) (bottom panel). Data are presented as described in the legend to Fig. 5.

FIG. 8.

PHD mutations reduce the ability of CBP to coactivate CREB. 911 cells stably transfected with a 5xGal4-E1BTATA-Luc reporter (clone 8) were transfected with either Gal4 DBD alone, Gal4 DBD-CREB, or the S133A mutant, with or without the catalytic subunit of PKA, and with or without an expression vector containing full-length CBP or the C1241A or C1287A PHD mutant, as indicated. Data are presented as described in the legend to Fig. 5.