The scaffolding protein JADE1 physically links the acetyltransferase subunit HBO1 with its histone H3-H4 substrate

Abstract

The human enzyme histone acetyltransferase binding to ORC1 (HBO1) regulates DNA replication, cell proliferation, and development. HBO1 is part of a multiprotein histone acetyltransferase (HAT) complex that also contains inhibitor of growth family member (ING) 4/5, MYST/Esa1-associated factor (MEAF) 6, and the scaffolding proteins Jade family PHD finger (JADE) 1/2/3 or bromodomain and PHD finger-containing protein (BRPF) 2/3 to acetylate histone H4 H4K5/8/12 or H3K14, respectively. Within this four-protein complex, JADE1 determines histone H4 substrate specificity of the HBO1-HAT complex. However, the mechanism by which JADE1 controls the H4-specific acetyltransferase activity of HBO1 is unknown. Here we used recombinant proteins in vitro to dissect the specific regions and activities of HBO1 and JADE1 that mediate histone H3-H4 acetylation via the HBO1-HAT domain. We found that JADE1 increases the catalytic efficiency of HBO1 acetylation of an H3-H4 substrate by about 5-fold through an N-terminal, 21-residue HBO1- and histone-binding domain and a nearby second histone core-binding domain. We also demonstrate that HBO1 contains an N-terminal histone-binding domain (HBD) that makes additional contacts with H3-H4 independent of JADE1 interactions with histones and that the HBO1 HBD does not significantly contribute to HBO1's overall HAT activity. Experiments with JADE1 deletions in vivo recapitulated these in vitro interactions and their roles in HBO1 histone acetylation activity. Together, these results indicate that the N-terminal region of JADE1 functions as a platform that brings together the catalytic HBO1 subunit with its cognate H3-H4 substrate for histone acetylation.

Keywords: acetyltransferase; chromatin regulation; epigenetics; histone acetylation; protein complex.

Figure 1.

An N-terminal region of JADE1 binds HBO1. A, sequence alignment of the N-terminal region of JADE1/2/3 paralogs, with the highest degree of conservation shaded in red and conservative substitutions colored in red. The HBO1 and HHBD are highlighted. B, MBP-JADE1 pulldowns with various N-terminal and C-terminal deletion constructs of the HBO1-HAT domain and H3/H4 complex with results resolved on SDS-PAGE. MW Std., molecular weight standard. C, MBP-JADE1 pulldowns of the HBO-HAT with JADE1 triple alanine mutations with results resolved on SDS-PAGE. D, ITC studies of JADE1 constructs titrated into the HBO1-HAT, showing the heat profile (top) and the calculated binding isotherm (bottom). Thermodynamic data calculated from this data are shown at the right.

Figure 2.

JADE1 requires an additional N-terminal region for H3/H4 binding and HBO1 HAT potentiation. Shown is the titration of JADE1 constructs (55–85 and 1–188) from 0- to 16-fold molar excess of HBO1 HAT domain tested for acetyltransferase activity on the H3/H4 complex. Radioactive counts are converted to enzyme rate as described under “Experimental procedures.”

Figure 3.

The additional JADE1 N-terminal HCBD specifically binds the histone core region. A, MBP-JADE1 pulldowns of four different H3/H4 complexes (H3/H4, H3(TL)/H4, H3/H4(TL), H3(TL)/H4(TL)) with results resolved on SDS-PAGE. MW Std., molecular weight standard. B, GST-H4(1–19) pulldowns of HBO1-HAT in the presence and absence of JADE1(1–188) with results resolved on SDS-PAGE. C, ITC studies of JADE1 constructs titrated into the H3/H4 complex showing the heat profile (top) and the calculated binding isotherm (bottom). Thermodynamic data calculated from these data are shown at the right. D, MBP-JADE1 pulldowns of HBO1-HAT and H3/H4 complex in the presence and absence of each other, with results resolved on SDS-PAGE.

Figure 4.

JADE1 selectively binds to H3/H4 over H2A/H2B and the nucleosome core particle. A, MBP-JADE1 pulldowns of HBO1 HAT and (H3/H4 or H2A/H2B) in the presence or absence of each other, with results resolved on SDS-PAGE. MW Std., molecular weight standard. B, MBP-JADE1 pulldowns of HBO1 HAT and H3/H4, H2A/H2B and mixtures of the two with results resolved on SDS-PAGE. C, MBP-JADE1 pulldowns of H3/H4 or nucleosome core particles with results resolved on SDS-PAGE. D, activity comparison of HBO1 HAT and HBO1 full-length in the presence and absence of a saturating concentration of JADE1(1–188) as either H3H4 complex or nucleosome core particles as the substrate. The activity of these proteins on various substrates was compared using radioactive counts.

Figure 5.

JADE1-HHBD and -HCBD work cooperatively to activate HBO1 HAT activity. A, titration of JADE1 constructs (1–188, 60–188, 80–188, 1–80, and 1–80/80–188) from 0 to 25 molar excess over the HBO1 HAT domain tested for acetyltransferase activity on the H3/H4 complex. Radioactive counts are converted to enzyme rate as described under “Experimental procedures.” B, plot of the linear range of the Michaelis–Menten plot of HBO1-HAT and HBO1-FL ± JADE1 with H3/H4 as the substrate to calculate the catalytic efficiency (slope, K_cat/K_m). Radioactive counts are converted to enzyme rate as described under “Experimental procedures.”

Figure 6.

HBO1-FL contains an N-terminal H3/H4 binding domain that binds histones independently of JADE1. A, MBP-HBO1-HAT pulldown of H3/H4 of H3(TL)/H4(TL) in the presence and absence of JADE1-HCBD, with results resolved on SDS-PAGE. MW Std., molecular weight standard. B, GST-HBO-FL and GST control pulldown of the H3/H4 complex, with results resolved on SDS-PAGE. Note that JADE1(80–188) and H3.1 co-migrate on SDS-PAGE. C, MBP-JADE1(1–188) and (108–188) pulldown of HBO1-FL ± H3/H4 complex, with results resolved on SDS-PAGE.

Figure 7.

JADE1 HHBD and HCBD are both required for HBO1 acetyltransferase activity on histones in cells. A, Western blot (WB) analysis of JADE1 complexes with HBO1, H3, ING4, and EAF6 with different N-terminal JADE1 truncations. An anti-HA immunoblot was done first to visualize the HA-tagged subunits of the FLAG-JADE1 complex overexpressed in HEK 293T cells. The membrane was then reprobed with the HBO1 antibody (showing endogenous HBO1 association beside HA-HBO1) and finally with the anti-FLAG antibody. IP, immunoprecipitation; MW Std., molecular weight standard. B, HBO1 association with the C-terminal truncated forms of JADE1. Anti-HA was followed by anti-FLAG immunoblot on FLAG-JADE1 complex overexpressed in 293T cells (the asterisk denotes the remaining HBO1 HA signal in the anti-FLAG blot). C, HAT assay of immunopurified complexes from A. HAT assays were performed on free histones (0.5 μg) with JADE1 complex using the same ratio as for the immunoblotting. Shown is a graphical representation of counts per minute, measured by scintillation, and a fluorogram of the radioactive HAT assay on free histones.

Figure 8.

Schematic of the HBO1, JADE1, H3/H4 complex based on the data obtained in this study. A, schematic of the JADE1 and HBO1 domains. B, schematic of JADE1-HBO1 domain interactions.