Multitasking C2H2 zinc fingers link Zac DNA binding to coordinated regulation of p300-histone acetyltransferase activity

Abstract

Zac is a C(2)H(2) zinc finger protein that regulates apoptosis and cell cycle arrest through DNA binding and transactivation. The coactivator proteins p300/CBP enhance transactivation through their histone acetyltransferase (HAT) activity by modulating chromatin structure. Here, we show that p300 increases Zac transactivation in a strictly HAT-dependent manner. Whereas the classic recruitment model proposes that coactivation simply depends on the capacity of the activator to recruit the coactivator, we demonstrate that coordinated binding of Zac zinc fingers and C terminus to p300 regulates HAT function by increasing histone and acetyl coenzyme A affinities and catalytic activity. This concerted regulation of HAT function is mediated via the KIX and CH3 domains of p300 in an interdependent manner. Interestingly, Zac zinc fingers 6 and 7 simultaneously play key roles in DNA binding and p300 regulation. Our findings demonstrate, for the first time, that C(2)H(2) zinc fingers can link DNA binding to HAT signaling and suggest a dynamic role for DNA-binding proteins in the enzymatic control of transcription.

FIG. 1.

p300/CBP enhance Zac transactivation. (A) Scheme of Zac proteins. Numbers denote amino acids, and domains are boxed. Mouse Zac and human ZAC protein contain virtually identical zinc finger domains (ZF) and a strongly conserved linker (L) and N-terminal region within the C terminus (C), whereas the central proline-rich transactivation domain (PR) is specific to mice. Identity (%) is indicated. (B) Pulldown assay. Equal amounts of in vitro-translated p300 or CBP were incubated with adjusted amounts of GST-Zac, GST-ZAC, or GST alone. The fraction of the input (100%) bound by each GST protein [BD (%)] is indicated. (C) The Coomassie blue stain shows adjusted amounts of GST-Zac, GST-ZAC, or GST used in pulldown assays. Asterisks mark bands of predicted molecular mass (kDa). (D) Coimmunoprecipitation experiment. Zac (0.5 μg of pRK7Flag-Zac) and p300 (3 μg of pCMV-p300-HA) were cotransfected in 293T cells as indicated in the text. Immunoprecipitation (IP) and immunoblotting (IB) were done with anti-Flag and anti-p300 antibodies. (E to G) Reporter assays. Zac (0.1 μg of pRK7Flag-Zac) was cotransfected with the DR element, PAL element, and cytokeratin 14 promoter (CK14) (2 μg each) in the absence (dark gray) or presence (light gray) of Zac. Activity of the reporter alone was set to 100% and compared to cotransfection of p300 or the p300 HAT-deficient mutant (p300-HATmt). Activity of the reporter in the presence of Zac was set to 100% and compared to the activity in the presence of wild-type p300, CBP, PCAF, SRC-1, or HAT-deficient mutants (1 μg each). (H) Immunoblot. Expression of Zac (0.1 μg of pRK7Flag-Zac) in the presence or absence of coactivators (1 μg each) as indicated. Coexpressions did not alter Zac or coactivator protein levels. Antibodies were anti-p300 (sc-584), anti-CBP (sc-369), anti-SRC (sc-8995), anti-Flag (F3165), and anti-Zac.

FIG. 2.

p300 preferentially mediates Zac coactivation. (A) RNA interference. The DR reporter (2 μg) was cotransfected in the absence (dark gray) or in the presence (light gray) of Flag-Zac (0.1 μg of pRK7Flag-Zac) with p300 or control siRNA into HeLa cells. Activity of the reporter alone or of the reporter plus Zac in the absence of siRNA was each set to 100% and compared to conditions containing siRNA. Columns represent means and standard deviations from three experiments. Representative immunoblots of p300, Zac and actin proteins are shown. (B) CBP or control siRNA was tested as described in the text. (C and D) Cotransfection of 293T and PA-TU cells. Given amounts of Zac (0.1 μg of pRK7Flag-Zac) were cotransfected with the DR or PAL reporters (2 μg each) in the presence of increasing doses of wild-type or HAT-deficient p300 as indicated.

FIG. 3.

p300-Zac interactions. (A) Mapping p300 domains. A scheme of p300 is shown. Numbers denote amino acids, and domains are boxed. Abbreviations: CH, cystidine-histidine-rich region; BD, bromodomain; HAT, histone acetyltransferase domain; SID, steroid receptor interacting domain. Zac binding sites are underlined. Equal amounts of in vitro-translated p300 segments were incubated with adjusted amounts of GST-Zac or GST alone; representative autoradiograms are shown. The fraction of the input (100%) bound by each GST protein is given as [BD (%)]. (B) p300 derivatives singly deleted (p300ΔKIX and p300ΔCH3) bound indistinguishably from p300 to GST-Zac, while combined deletion (p300ΔKIXΔCH3) reduced binding by 60%. The absence of either Zac binding site (p300ΔKIXΔHATΔCH3) abolished the interaction. p300 binding was set to 100%. (C) Mapping Zac domains. A scheme of Zac is shown. Abbreviations: ZF, zinc finger domain; L, linker domain; PR, proline repeat domain; C, C terminus, further subdivided in C1 to C3. Equal amounts of in vitro-translated Zac segments were incubated with adjusted amounts of GST-KIX, -HAT, -CH3, and GST alone. Fraction of the input (100%) bound by each GST protein is given [BD (%)], with the binding of Zac set to 100%. (D) The Coomassie blue stain shows adjusted amounts of GST-KIX, GST-HAT, GST-CH3, and GST used in pulldown assays. Asterisks mark bands of predicted molecular mass (kDa). (E) Equal amounts of in vitro-translated Zac, zinc fingers, linker-proline domain, or C terminus were incubated with GST-KIX, -HAT, -CH3, or GST alone. The binding of Zac was set to 100%. (F) The conserved C1 region binds to GST-KIX. Equal amounts of in vitro-translated Zac, ZacΔC1, ZacΔC2, and ZacΔC3 were incubated with GST-KIX or GST alone. Zac binding was set to 100%.

FIG. 4.

Zinc fingers interact cooperatively and selectively with p300. (A) In vitro competition experiment. Binding of the in vitro-translated zinc fingers to GST-KIX (aa 566 to 650) protein was competed by the indicated amounts of in vitro-translated C terminus (C) or by itself. Similarly, binding of the in vitro-translated C terminus was competed by the indicated amounts of in vitro-translated zinc fingers or by itself. In control experiments, a 15-fold excess of in vitro-translated luciferase (Luc) protein failed to compete binding of the zinc fingers (open diamonds) or of the C terminus (filled diamonds) to the KIX domain. (B) Model of Zac-p300 interaction. Zinc fingers and C1 region cooperatively bind to the KIX domain. The zinc fingers additionally bind to the CH3 domain. SID, steroid receptor interacting domain; BD, bromodomain. (C) Pulldown assays. Zac and successive deletion mutants of the zinc fingers were in vitro translated and tested for binding to GST fusions of the KIX, HAT, or CH3 domains. Zac binding was set to 100%.

FIG. 5.

Zac zinc fingers and p300 form a DNA-bound complex. (A) Scheme of p300. Fragments used for immunization are underlined; numbers denote amino acids. The corresponding antibodies are labeled α-p300-N and α-p300-C. SID, steroid receptor interacting domain; BD, bromodomain. (B) EMSA. PA-TU cells were transfected with Zac (0.2 μg of pRK7Flag-Zac) in the absence or presence of p300-HA (1.0 μg of pCMV-p300-HA). Zac-DNA complexes on the DR element () are supershifted by anti-Flag antibodies (). Anti-p300 antibodies (α-p300-N and α-p300-C) were ineffective in the absence of ectopic p300 (lanes 3 and 4) but strongly supershifted Zac-DNA complexes () upon p300 cotransfection (lanes 7 and 8). Preimmune sera were ineffective (lanes 9 and 10). (C) EMSA. Adjusted amounts of Zac (0.2 μg of pRK7Flag-Zac) and ZacΔC1 (0.04 μg of pRK7Flag-ZacΔC1) were transfected in PA-TU cells and tested as described above.

FIG. 6.

The C1 region mediates coactivation via p300. (A) Zac or ZacΔC1 were transfected into LLC-PK1 cells, and different amounts of cell extracts (left lanes, 50 μg; middle lanes, 20 μg; and right lanes, 5 μg) were immunoblotted with α-Zac. Indicated amounts of DNA refer to Zac with ZacΔC1 adjusted appropriately. (B) The indicated doses of Zac or adjusted amounts of ZacΔC1 were cotransfected with the DR or PAL reporter plasmids (2 μg each) into LLC-PK1 cells. (C) Zac or ZacΔC1 was cotransfected with the indicated reporters in the absence or presence of a given amount of p300 (0.5 μg of pCMV-p300-HA) into PA-TU cells. (D) Colony formation assay. Zac (0.2 μg of pRK7Flag-Zac) or ZacΔC1 (0.04 μg of pRK7Flag-ZacΔC1) were cotransfected with a puromycin resistance vector (pRK7Pur) at a ratio of 3:1 into LLC-PK1 and PA-TU cells. Selection (puromycin, 2 μg/ml) began on day 2, and medium was renewed every third day. Colonies were stained with MTT (methylthiazolyldiphenyl-tetrazolium bromide; 1 mg/ml) on day 10 and counted. Growth inhibition by Zac was set to 100%.

FIG. 7.

KIX and CH3 domains are necessary for Zac coactivation. (A) Immunoblot of LLC-PK1 cells expressing adjusted amounts of single p300 domains (0.5 μg of pRK7Flag-CH1 [aa 302 to 528], 1.0 μg of pRK7Flag-KIX [aa 566 to 650], and 3.0 μg of pRK7Flag-CH3 [aa 1197 to 1673]) as detected by α-Flag. (B) Competition scheme. Broken lines show binding of Zac ZF6 and ZF7 and of the C1 region to the KIX and CH3 domains of endogenous p300. Intact lines symbolize competition by overexpression of the isolated KIX and CH3 domains (circles). (C and D) In vivo competition experiment. Zac and DR or PAL reporters were cotransfected with increasing amounts of the CH1, KIX, and CH3 domains singly or together into LLC-PK1 cells.

FIG. 8.

KIX and CH3 domains interdependently regulate Zac coactivation. (A) Immunoblot of PA-TU cells expressing adjusted amounts of p300 or derivatives as detected by α-Flag (10 μg of pCI.Flag-p300, 5 μg of pCI.Flag-p300ΔKIX, 3 μg of pCI.Flag-p300ΔCH3, or 1 μg of pCI.Flag-p300ΔKIX ΔCH3). (B) EMSA with DR element. PA-TU cells were transfected with Zac in the absence or presence of adjusted amounts of p300, p300ΔKIX, p300ΔCH3, or p300ΔKIXΔCH3. Zac-DNA complexes () were unaffected by preimmune-N or α-p300-N sera in the absence of p300 (lanes 1 and 2). They were indistinguishably supershifted () by α-p300-N but not by preimmune-N serum in the presence of p300, p300ΔKIX, or p300ΔCH3 (lanes 3 to 8), while p300ΔKIXΔCH3 was ineffective (lanes 9 and 10). (C and D) Zac (0.1 μg of pRK7Flag-Zac) and DR or PAL reporters (2 μg each) were cotransfected with increasing amounts of p300, p300ΔKIX, and p300ΔCH3 into PA-TU cells. Indicated amounts of DNA refer to p300 and appropriately adjusted amounts of the derivatives.

FIG. 9.

p300 does not acetylate Zac. Acetylation assay. GST-p300-HAT (25 nM) was incubated with GST fusions of Zac, Zac zinc fingers, or the general transcription factors TFIIF or TFIIE (1 μM each). Reaction mixtures were fractionated on a 10% SDS-polyacrylamide gel. A representative autoradiogram following 2,5-diphenyloxazol treatment and exposure for 2 days is shown. Open arrows indicate predicted positions of Zac and zinc fingers; filled arrows mark positions of acetylated TFIIF, TFIIE, and autoacetylated p300-HAT.

FIG. 10.

Coordinated Zac binding regulates in vitro p300-HAT substrate affinities. Data represent means and standard deviations from at least three experiments done in duplicate. (A) Kinetic analysis of acetylation reaction. Saturating amounts of core histones, H4 peptide, and [³H]acetyl-CoA were incubated with in vitro-translated p300. ³H acetylation was measured for the indicated times. (B) Various concentrations of core histones and H4 peptide were assayed with in vitro-translated p300 and saturating concentrations of [³H]acetyl-CoA. (C) Zac or derivatives dose-dependently regulate p300-HAT activity. p300 (1 nM) was incubated for 2 min with saturating amounts of substrates and with differing concentrations of recombinant Zac, ZacΔC1, or ZacΔZF6 at a ratio of 3:1, 1:1, or 1:3, respectively. (D) Saturation analysis. p300 (1 nM) was incubated for 2 min with differing concentrations of H4 peptide (left) and acetyl-CoA (right) in the absence or presence of equimolar amounts of recombinant Zac, ZacΔC1, or ZacΔZF6. All reactions contained the DR element. Acetylation products and substrate concentrations were plotted in a double-reciprocal graph. (E and F) p300ΔKIX and p300ΔCH3 (1 nM each) were tested as detailed for panel D in the absence or presence of equimolar amounts of Zac.

FIG. 11.

Coordinated Zac binding regulates in vitro the catalytic activity of p300-HAT. (A) p300 (1 nM) was incubated in the absence or presence of equimolar amounts of Zac, ZacΔC1, or ZacΔZF6 with saturating amounts of H4 peptide and [³H]acetyl-CoA for the indicated times. All reactions contained the DR element. Linear least-squares fit was used to calculate maximal v_z. (B and C) v_z of p300ΔKIX or of p300ΔCH3 (1 nM each) in the absence or presence of equimolar amounts of Zac was determined as described above. (A through C) Data represent means and standard deviations from at least three experiments done in duplicate. (D) Survey of K_m(h), K_m(a), and v_z values for p300 and derivatives in the absence or presence of Zac, ZacΔC1, and ZacΔZF6.

FIG. 12.

Coordinated Zac binding regulates in vivo p300-HAT activity. (A) Scheme of Zac reporter plasmid. Arrows indicate positions of PCR primers next to the direct repeat DNA elements (DR). (B) Zac-dependent p300-mediated histone H4 acetylation. p300-negative PA-TU cells transfected with Zac (20 ng of pRK7.HA-Zac) or p300 (0.5 μg of pCI.Flag-p300) alone or together were subjected to ChIP analysis with antibodies against p300 (α-Flag) or acetylated histone H4 (α-acH4) or without antibodies (−AB). PCR analysis with input chromatin confirmed that equal amounts were used for all reactions. (C) Coordinated Zac binding to p300 regulates H4 acetylation. p300 was transfected singly or together with adjusted amounts of Zac, ZacΔC1, or ZacΔZF6 (20 ng of pRK7.HA-Zac, 4 ng of pRK7.HA-ZacΔC1, or 20 ng of pRK7.HA-ZacΔZF6). ChIP analysis was done as described above. (D) KIX and CH3 domains interdependently regulate Zac-induced acetylation. Adjusted amounts of p300, p300ΔKIX, or p300ΔCH3 (0.5 μg of pCI.Flag-p300, 0.25 μg of pCI.Flag-p300 ΔKIX, or 0.15 μg of pCI.Flag-p300 ΔCH3) were transfected singly or together with Zac (20 ng of pRK7.HA-Zac). ChIP analysis was done as described above. (B through D) Results are representative of four independent experiments.