Kinetic profiles of p300 occupancy in vivo predict common features of promoter structure and coactivator recruitment

Abstract

Understanding the language encrypted in the gene regulatory regions of the human genome is a challenging goal for the genomic era. Although customary extrapolations from steady-state mRNA levels have been effective, deciphering these regulatory codes will require additional empirical data sets that more closely reflect the dynamic progression of molecular events responsible for inducible transcription. We describe an approach using chromatin immunoprecipitation to profile the kinetic occupancy of the transcriptional coactivator and histone acetyltransferase p300 at numerous mitogen-induced genes in activated T cells. Comparison of these profiles reveals a class of promoters that share common patterns of inducible expression, p300 recruitment, dependence on selective p300 domains, and sensitivity to histone deacetylase inhibitors. Remarkably, this class also shares an evolutionarily conserved promoter composition and structure that accurately predicts additional human genes with similar functional attributes. This "reverse genomic" approach will have broad application for the genome-wide classification of promoter structure and function.

Fig. 2.

Comparison of proximal promoter structures. (A) Schematic comparison of proximal promoter regions of p21/WAF1/CDKN1A, GADD153/DDIT3, and p16/CDKN2A. Primary sequences or cis-elements are shown in Table 2. Transcription factor-binding sites found in the primary promoter sequence were identified by using matinspector 6.2.1.(B) Promoter region alignments between human and mouse are shown as VISTA identity curves. Consensus transcription factor-binding sites identified within conserved regions between the human and mouse promoter sequences are shown above the VISTA plots. The percent GC content is indicated below the VISTA plot. (C) Common transcription factor regulatory frameworks were identified within the proximal promoter regions of p21/WAF1/CDKN1A, GADD153/DDIT3 and p16/CDKN2A by using the Genomatix suite 1.2 frameworker module of gems 3.3.(D) List of genes containing framework model A. Framework model A was used to screen the human genome (embl release 71) with modelinspector 4.80 (gems 3.3) of the Genomatix suite and matched with the eight genes. Framework model B yielded no matches.

Fig. 4.

Bioinformatic analysis of predicted promoter regions. Promoter alignments between human and mouse (Top) and between human and rat (Middle) are shown as vista2.0 identity curves. Consensus transcription factor binding sites within conserved regions between the human and mouse promoter sequences were identified with matinspector 6.2.2 and are shown above VISTA plots. The percent GC content is indicated (Bottom).

Fig. 1.

The recruitment profiles of p300 to the proximal promoters of IL2 and p21/WAF1/CDKN1A are kinetically distinct. (A) RNase protection analysis shows that T cell activation induced IL2 and p21/WAF1/CDKN1A transcript levels. Cells were stimulated for 4 h with PMA (P), ionomycin (I), and anti-CD28 antibody as indicated. Protected probes specific for IL2 and p21/WAF1/CDKN1A are shown with L32 and GAPDH as loading controls. (Left) Kinetic recruitment profiles of p300 at the IL2 and p21/WAF1/CDKN1A promoters. Jurkat cells were formalin-crosslinked at 15-min intervals after P + I stimulation. ChIP was performed at each time point by using anti-p300 antibody. (Right) Ten percent of the input chromatin is shown as a loading control. (B) ChIP screen of p300 recruitment profiles at specific promoters with 10% input control. (C Upper Left) Schematic of the domain structure of p300 and the Δp300 deletion mutant expression vectors. (C Lower Left) Transient cotransfection of IL2 and p21 luciferase promoter reporters with p300 and the Δp300 deletion mutant. (C Right) Histone deacetylase inhibitor (HDACI) sensitivity of IL2, p21/WAF1/CDKN1A, GADD153, and p16/CDKN2A promoter luciferase reporters. Cells were untreated, stimulated with PMA and ionomycin alone, or stimulated with PMA and ionomycin in the presence of sodium butyrate (NaBu), suberoylanilide hydroxyamic acid (SAHA), or TSA (Right).

Fig. 3.

Analysis of framework model A predicted genes. Mitogen and mitogen/TSA sensitivity of predicted gene transcript levels. Two and 4 h after stimulation with P/I in the presence or absence of TSA, real-time RT-PCR was used to determine the relative fold induction change in transcript levels in the treated cells. (A) Comparison of framework model A gene mitogen and mitogen/TSA induction profiles by hierarchical clustering. (B) Comparison of the influence of TSA on the mitogen-induced transcript levels after 4 h of stimulation (see Table 3). ACTB was used for normalization.

Fig. 5.

Functional validation of promoter class prediction. ChIP analysis of framework model A predicted genes.