SUMMARY Expression of the INK4b/ARF/INK4a tumor suppressor locus in normal and cancerous cell growth is controlled by methylation of histone H3 at lysine 27 (H3K27me) as directed by the Polycomb group proteins. The antisense non-coding RNA ANRIL of the INK4b/ARF/INK4a locus is also important for expression of the protein-coding genes in cis, but its mechanism has remained elusive. Here we report that chromobox 7 (CBX7) within the Polycomb Repressive Complex 1 binds to ANRIL, and both CBX7 and ANRIL are found at elevated levels in prostate cancer tissues. In concert with H3K27me recognition, binding to RNA contributes to CBX7 function and disruption of either interaction impacts the ability of CBX7 to repress the INK4b/ARF/INK4a locus and control senescence. Structure-guided analysis reveals the molecular interplay between non-coding RNA and H3K27me as mediated by the conserved chromodomain. Our study suggests a new mechanism by which non-coding RNA participates directly in epigenetic transcriptional repression.

Histone lysine acetylation is central to epigenetic control of gene transcription. The bromodomain, found in chromatin-associated proteins and histone acetyltranferases, functions as the sole protein module known to bind acetyl-lysine motifs. Recent structural and functional analyses of bromodomains' recognition of lysine-acetylated peptides derived from major acetylation sites in histones and cellular proteins provide new insights into differences in ligand binding selectivity as well as unifying features of histone recognition by the bromodomains. These new findings highlight the functional importance of bromodomain/acetyl-lysine binding as a pivotal mechanism for regulating protein-protein interactions in histonedirected chromatin remodeling and gene transcription. These new studies also support the notion that functional diversity of a conserved bromodomain structural fold is achieved by evolutionary changes of structurally flexible amino-acid sequences in the ligand binding site such as the ZA and BC loops.

Lysine acetylation of the tumor suppressor protein p53 in response to a wide variety of cellular stress signals is required for its activation as a transcription factor that regulates cell cycle arrest, senescence, or apoptosis. Here, we report that the conserved bromo-domain of the transcriptional coactivator CBP (CREB binding protein) binds specifically to p53 at the C-terminal acetylated lysine 382. This bromodomain/acetyl-lysine binding is responsible for p53 acetylation-dependent coactivator recruitment after DNA damage, a step essential for p53-induced transcriptional activation of the cyclin-dependent kinase inhibitor p21 in G1 cell cycle arrest. We further present the three-dimensional nuclear magnetic resonance structure of the CBP bromodomain in complex with a lysine 382-acetylated p53 peptide. Using structural and biochemical analyses, we define the molecular determinants for the specificity of this molecular recognition.

The human immunodeficiency virus type 1 (HIV-1) trans-activator protein Tat stimulates transcription of the integrated HIV-1 genome and promotes viral replication in infected cells. Tat transactivation activity is dependent on lysine acetylation and its association with nuclear histone acetyltransferases p300/CBP (CREB binding protein) and p300/CBP-associated factor (PCAF). Here, we show that the bromodomain of PCAF binds specifically to HIV-1 Tat acetylated at lysine 50 and that this interaction competes effectively against HIV-1 TAR RNA binding to the lysine-acetylated Tat. The three-dimensional solution structure of the PCAF bromodomain in complex with a lysine 50-acetylated Tat peptide together with biochemical analyses provides the structural basis for the specificity of this molecular recognition and reveals insights into the differences in ligand selectivity of bromodomains.