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

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 and the scaffolding proteins Jade family 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 independently 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.

Histone acetylation has been shown to promote transcription in at least two ways. First, acetylation neutralizes the lysine positive charge, thus altering its physiochemical properties and making the local chromatin environment more accessible for gene transcription activation (1).
Second, lysine acetylation generates a docking recognition sites for various gene-activating proteins containing acetyl-lysine recognition domains such as bromodomains (5)(6)(7)(8)(9). Therefore, the tight regulation of acetylation on lysine residues within histones, as well as the enzymes that mediate these modifications, are critical to maintain normal gene expression and regulation.
The enzymes that mediate histone acetylation are histone acetyltransferases (HATs), while the enzymes that convert acetyl-lysine residues within histone back to lysine are histone deactylases (HDACs) (10) where their substrate specificities and activities are modulated by the other subunits of the respective HAT complexes (11,12).
complexes are localized at the transcription start sites and coding regions of active genes in the genome (16), and is responsible for the majority of H4(14, 17,18) and H3K14 (19,20) acetylation.
HBO1 has also been shown to play more specific roles in activating the expression of key genes in development and embryonic patterning (19). The aberrant activity of HBO1 has been correlated with several cancers. HBO1 has been shown to be over-expressed in MCF7 and Saos-2 established cancer cell lines and in a subset of human primary cancers such as carcinomas of testis, breast, and ovary (21,22). Finally, a HBO1 catalytic defective mutant inhibits MCM complex loading onto the origin, indicating that HBO1 functions as a DNA replication co-activator in addition to its transcriptional roles (5,13,18).
HBO1 functions in the context of two multiprotein histone acetyltransferase complexes containing ING4/5, MEAF6 and paralogs JADE1/2/3 for targeting the H4 tail or BRPF1/2/3 for H3K14 acetylation (11,20). The activity and substrate selectivity of HBO1 is dependent on the proteins within the HBO1 HAT complex, which also have other functions in various cellular proliferation and tumor suppression pathways.  (11,15). We also We next asked if residues of 60-80 of JADE1 are also able to interact with histones to potentially bridge the HBO1-HAT acetylation of histones.
To do this, we carried out pull-down studies of MBP-JADE1 N-and C-terminal deletion constructs with HBO1-HAT plus H3/H4 ( Figure   1B). These studies revealed that only JADE1 constructs harboring residues 60-80 of JADE1 were able to interact with both the HBO1-HAT and H3/H4, confirming that JADE1 residues 60-80 bridge HBO1-HAT/histone interactions.
Notably, JADE1 constructs starting at residue 60 and extending to residue 188, showed more robust pull down with HBO1-HAT and histones than JADE1 constructs harboring only residues 60-80, suggesting that additional regions of JADE1 between residues 108 and 188 also contribute to HBO1/histone interactions ( Figure   1B). Based on the HBO1-HAT and histone binding properties of residues 60-80 of JADE1, we refer to it as the HBO1 and Histone Binding Domain (JADE1-HHBD).
We then asked if the minimal region of JADE1 that facilitates HBO1 and histone binding is sufficient to potentiate HBO1-HAT activity toward histone tail acetylation. To do this, we titrated JADE1 (55-85) and JADE1(1-188) into the HBO1-HAT and H3-H4 complex and assayed for histone acetyltransferase activity ( Figure 2A).  Previous studies indicate that JADE1 facilitates HBO1 H4-specific tail acetylation (11).  HAT activity and that these two regions of JADE1 must be connected to mediate this activity.  histones.

Discussion
Histone tail acetylation events have been linked to many important cellular processes through contacts within the histone core region ( Figure   8A). The N-terminal region of JADE1 therefore functions as a platform to bring the catalytic HBO1 subunit and the free histone H3/H4 substrate tail together for catalysis. 500mM NaCl, 5mM BME, 0.1mg/ml PMSF).        C. HAT assay of immunopurified complexes from 7A. HAT assay were performed on free histones (0.5µg) with JADE1 complex using the same ratio as for the immunoblotting. The graphical representation of counts per minutes (CPM) measured by scintillation and a fluorogram of radioactive HAT assay on free histones.