Role of the CBP catalytic core in intramolecular SUMOylation and control of histone H3 acetylation

Abstract

The histone acetyl transferases CREB-binding protein (CBP) and its paralog p300 play a critical role in numerous cellular processes. Dysregulation of their catalytic activity is associated with several human diseases. Previous work has elucidated the regulatory mechanisms of p300 acetyltransferase activity, but it is not known whether CBP activity is controlled similarly. Here, we present the crystal structure of the CBP catalytic core encompassing the bromodomain (BRD), CH2 (comprising PHD and RING), HAT, and ZZ domains at 2.4-Å resolution. The BRD, PHD, and HAT domains form an integral structural unit to which the RING and ZZ domains are flexibly attached. The structure of the apo-CBP HAT domain is similar to that of acyl-CoA-bound p300 HAT complexes and shows that the acetyl-CoA binding site is stably formed in the absence of cofactor. The BRD, PHD, and ZZ domains interact with small ubiquitin-like modifier 1 (SUMO-1) and Ubc9, and function as an intramolecular E3 ligase for SUMOylation of the cell cycle regulatory domain 1 (CRD1) of CBP, which is located adjacent to the BRD. In vitro HAT assays suggest that the RING domain, the autoregulatory loop (AL) within the HAT domain, and the ZZ domain do not directly influence catalytic activity, whereas the BRD is essential for histone H3 acetylation in nucleosomal substrates. Several lysine residues in the intrinsically disordered AL are autoacetylated by the HAT domain. Upon autoacetylation, acetyl-K1596 (Ac-K1596) binds intramolecularly to the BRD, competing with histones for binding to the BRD and acting as a negative regulator that inhibits histone H3 acetylation.

Enhancer Activity Requires CBP/P300 Bromodomain-Dependent Histone H3K27 Acetylation

“…Consistent with published data using a structurally distinct compound, we detected minimal changes in acetylation of nonhistone proteins after GNE-049 treatment, as assessed by pan-acetyl immunoprecipitation (IP) mass spectrometry (Weinert et al, 2018) ( Figure S1D; Table S1). Structural data imply a direct role for the CBP bromodomain in catalytic activity (Park et al, 2017). Consistent with this, addition of GNE-049 to an in vitro acetylation reaction containing a fragment of CBP or P300 protein consisting of the bromo and HAT (BD-HAT) domains (p300 BD-HAT, aa 965-1,810; CBP BD-HAT, aa 1,075-1,873) inhibited acetylation of a recombinant polynucleosome substrate ( Figure 1F).…”

SUMO1 SUMOylates and SENP3 deSUMOylates NLRP3 to orchestrate the inflammasome activation

“…Yeast two-hybrid (Y2H) analysis revealed that UBC9 interacted with full-length NLRP3, PYD, and LRR, but not with NACHT ( Figure 1B, bottom), suggesting that NLRP3 is highly associated with UBC9. To confirm the role of SUMO1 in the SUMOylation of NLRP3, we carried out biochemical analyses using an in vitro constructed SUMOylation system as described previously, 35,36 which contains purified SUMO1 (modifier), SUMO-activating enzyme subunit 1/2 (SAE1/ SAE2) (E1 activating enzyme), UBC9 (E2 conjugating enzyme), and NLRP3 (substrate). Three SUMO paralogs (SUMO1, 2, and 3) were identified in mammal tissues tested 20 and NLRP3 was SUMOylated by SUMO2/3.…”

“…19 Here, we revealed that SUMOylation of NLRP3 was detected in the presence of SUMO1 ( Figure 1C, lane 4) and further promoted in the presence of SUMO1 and UBC9 ( Figure 1C, lane 6), indicating that NLRP3 could be SUMOylated by SUMO1. To confirm the role of SUMO1 in the SUMOylation of NLRP3, we carried out biochemical analyses using an in vitro constructed SUMOylation system as described previously, 35,36 which contains purified SUMO1 (modifier), SUMO-activating enzyme subunit 1/2 (SAE1/ SAE2) (E1 activating enzyme), UBC9 (E2 conjugating enzyme), and NLRP3 (substrate). NLRP3 SUMOylation was detected in the presence of all components, SUMO1, SAE1/ SAE2, UBC9, and adenosine triphosphate (ATP) ( Figure 1D Similarly, p53, a typical SUMO1/2/3 modification protein as reported previously, 37,38 was SUMOylated in the presence of SUMO1, SAE1/SAE2, UBC9, and ATP ( Figure 1E, bottom lane 2).…”

Hat1-Dependent Lysine Acetylation Targets Diverse Cellular Functions

“…Interestingly, neither of these residues is present at lysines 5 and 12 of H4. However, one or both of the -7 glycine or the +5 alanine are found in canonical mammalian histone H2A and the H2A variants could directly interact with CBP and alter CBP structure or activity to facilitate the autoacetylation of the AL by CBP (27). Hat1 could also interact with other factors that regulate the autoacetylation of the AL, such as RNAs that bind CBP and activate its acetyltransferase activity (41).…”

“…Importantly, the Hat1-dependent sites of acetylation on CBP are all located in the auto-regulatory loop (AL) in the CBP HAT domain ( Figure 3B). The acetylation state of the AL of CBP is critical for its acetyltransferase activity (27).…”