Chromatin-dependent cooperativity between constitutive and inducible activation domains in CREB

Abstract

The cyclic AMP (cAMP)-responsive factor CREB induces target gene expression via constitutive (Q2) and inducible (KID, for kinase-inducible domain) activation domains that function synergistically in response to cellular signals. KID stimulates transcription via a phospho (Ser133)-dependent interaction with the coactivator paralogs CREB binding protein and p300, whereas Q2 recruits the TFIID complex via a direct association with hTAF(II)130. Here we investigate the mechanism underlying cooperativity between the Q2 domain and KID in CREB by in vitro transcription assay with naked DNA and chromatin templates containing the cAMP-responsive somatostatin promoter. The Q2 domain was highly active on a naked DNA template, and Ser133 phosphorylation had no additional effect on transcriptional initiation in crude extracts. Q2 activity was repressed on a chromatin template, however, and this repression was relieved by the phospho (Ser133) KID-dependent recruitment of p300 histone acetyltransferase activity to the promoter. In chromatin immunoprecipitation assays of NIH 3T3 cells, cAMP-dependent recruitment of p300 to the somatostatin promoter stimulated acetylation of histone H4. Correspondingly, overexpression of hTAFII130 potentiated CREB activity in cells exposed to cAMP, but had no effect on reporter gene expression in unstimulated cells. We propose that cooperativity between the KID and Q2 domains proceeds via a chromatin-dependent mechanism in which recruitment of p300 facilitates subsequent interaction of CREB with TFIID.

FIG. 1

Phospho (Ser133) KID and Q2 domains in CREB associate with p300 and hTAF_II130, respectively. (A) Schematic showing the location of the KID and Q2 domain on CREB and CREMα. CREMα lacks residues in Q2 corresponding to aa 183 to 251 of CREB. (B) Coomassie-stained gel of purified recombinant factors employed in in vitro transcription assays. Phospho (Ser133) CREB (lane 2) and phospho (Ser89) CREMα (lane 3) were phosphorylated in vitro with PKA. Recombinant Ser133Ala CREB (M1-CREB) protein is also shown (lane 4). (C) Far-Western blotting assay of recombinant P-CREB, M1-CREB, and P-CREM proteins with alkaline phosphatase-tagged KIX domain polypeptide, which contains aa 591 to 679 of CBP. (D) GST pull-down assay of ³⁵S-labeled hTAF_II130 with GST only (lane 2), GST M1 CREB (lane 4), GST phospho (Ser133) CREB (lane 3), and GST phospho (Ser89) CREMα (lane 5).

FIG. 2

Cooperativity between the Q2 domain and KID in CREB is chromatin dependent. In vitro transcription assays on naked DNA (lanes 1 to 8) and chromatin templates (lanes 9 to 16) by using the cAMP-responsive somatostatin promoter are shown. (A) The time course of chromatin assembly, nuclear factor addition, and transcription reactions is shown. (B) Micrococcal nuclease digestion of chromatin assembly reaction mixtures demonstrate regularity of nucleosome spacing on the template. M, size marker (lanes 1 and 2). Two concentrations of micrococcal nuclease are shown (lanes 3 and 4). (C) Primer extension assays of in vitro transcription reactions supplemented with purified recombinant p300, phospho (Ser133) CREB (P-CREB), Ser133Ala CREB (M1), or phospho (Ser89) CREM (P-CREM) as indicated. The relative intensity of ³²P-labeled primer extension products from individual reactions was determined by phosphorimaging. Fold induction over a control reaction containing no activators (lanes 1 and 9) is indicated. In Fig. 3 and 4, reactions were performed at least twice to confirm reproducibility.

FIG. 3

p300 HAT and C/H3 domain activities are required for target gene activation via phospho (Ser133) CREB on a chromatin template. (A) Time course of addition of Lys-CoA, activators, and template. (B) Effect of wild-type, HAT-defective (−HAT), and C/H3 region-deleted (−CH3) p300 polypeptides on transcription from the somatostatin promoter in reaction mixtures containing P-CREB. For panels B and C, duplicate reactions are shown for each condition. Naked DNA and chromatin templates are indicated. Fold induction over reaction mixture lacking p300 (lane 2) is shown in parentheses. (C) Effect of p300 HAT inhibitor Lys-CoA (10 μM) on transcription via P-CREB in vitro. Reactions with wild-type (Wt) and mutant (−CRE) somatostatin promoters containing an inactivating mutation in the consensus CRE site that disrupts binding of CREB are also shown. Fold induction over a reaction mixture lacking p300 (lane 4) is shown.

FIG. 4

p300 HAT and C/H3 domain activities are required for cAMP-dependent transcription via CREB in vivo. (A) Transient transfection assay of NIH 3T3 cells harboring integrated copies of a GAL4 luciferase reporter gene that contains four GAL4 recognition sites (KD-1 cells). Cells were transfected with a GAL4 CREB expression vector containing the GAL4 DNA binding domain fused to the N-terminal 283 residues of CREB, which contains both KID and Q2. Cotransfection of wild-type (wt), HAT defective (−HAT), and C/H3 region deletion (−CH3) mutant p300 expression vectors is indicated. Cells were treated with Forskolin (10 μm) or control vehicle as shown. Relative luciferase activity was normalized to β-galactosidase activity from cotransfected RSV β-galactosidase plasmid. Comparable expression of wild-type and mutant p300 polypeptides was verified by Western blot assay (not shown). (B, left) Chromatin immunoprecipitation assay of NIH 3T3 cells containing integrated copies of the rat somatostatin gene (D5 cells). D5 cells were treated with Forskolin (10 μM) or control vehicle for 1 h, and formaldehyde cross-linked protein DNA complexes were immunoprecipitated with anti-acetyl H4 (A-H4) or control antisera (Con). PCR analysis of each immunoprecipitate is shown, including input DNA (OP), as well as a control reaction with no antiserum (none). (Right) Intensity of PCR products estimated by densitometry, as shown in the bar graph. The average of four independent experiments ± standard error is shown. (C) The CREB Q2 domain is required for target gene activation via p300 in KD-1 cells containing integrated copies of a GAL4 luciferase reporter gene. Transfections were performed with p300, GAL4 CREB, GAL4 DNA binding domain (GAL), and GAL4 KID expression vectors. GAL4 KID contains aa 100 to 160 of CREB spanning the KID, which is sufficient for association with p300. Treatment with Forskolin (10 μM, 4 h) is indicated (Forsk).

FIG. 5

Conserved region II (CRII, aa 847 to 1083) in TAF_II130 is required for complex formation with CREB. (A) Pull-down assay of recombinant GST-TAF_II130 polypeptides with amino acid end points indicated. Interaction with CREB is summarized on the right (+ or −), and corresponding results of the pull-down assay with ³⁵S-labeled full-length CREB protein are shown on the bottom panel. (B) Pull-down assay with ³⁵S-labeled wild-type TAF_II130 and mutant TAF_II130ΔC lacking the C-terminal CREB binding domain (aa 836 to 1083) with GST-CREB (aa 1 to 283) or GST alone. OP, 10% of input protein.

FIG. 6

CRII and Q2 domains in TAF_II130 and CREB, respectively, are required for TAF_II130 to stimulate target gene expression in response to cAMP. (A) Transient transfection assay of KD-1 cells containing integrated copies of a GAL4 luciferase reporter gene. Cells were transfected with GAL4-CREB, GAL4-c-fos, or GAL4 DNA binding domain expression vectors as indicated. Cotransfection with p300 as well as wild-type and CREB interaction-defective mutant TAF_II130 (TAF_II130ΔC) constructs is shown. Treatment with cAMP agonist (Forskolin, 10 μM, 4 h) is also indicated. (B) The glutamine-rich Q2 domain in CREB is required for target gene activation via TAF_II130. Transient transfection of KD-1 cells with wild-type and mutant GAL4 CREB (GAL4-CREBΔQ2) expression vector lacking the Q2 domain, which associates with TAF_II130, is shown. Cotransfection with TAF_II130 expression vector and treatment with forskolin is indicated. The average ± standard error for triplicate samples is shown.