Transcription factor Ap2delta associates with Ash2l and ALR, a trithorax family histone methyltransferase, to activate Hoxc8 transcription.

The family of Ap2 transcription factors comprises five members with highly conserved DNA-binding domains. Among the family members, Ap2delta is the most divergent, because it lacks highly conserved residues within the transactivation domain (TAD) and has weak affinity for known Ap2 binding sites. To identify specific Ap2delta coactivators/regulators during development, we performed a yeast two-hybrid screen, using Ap2delta's TAD. We identified the trithorax superfamily member, Ash2l, as a binding partner that interacts exclusively with Ap2delta. We showed that Ash2l positively mediates Ap2delta transactivation in a dose-dependent manner. Given the known role of Ash2l in histone modification, we determined whether Ap2delta was able to form a complex with that activity. Our results showed that Ap2delta associates with endogenous ASH2L and a member of the MLL family of histone lysine methyltransferases (HKMTs), MLL2 (ALR), forming a complex that methylates lysine 4 of histone H3 (H3K4). Additionally, we showed that Ap2delta is necessary for recruitment of Ash2l and Alr to the Hoxc8 locus and that recruitment of this complex leads to H3K4 trimethylation (H3K4me3) and subsequent gene activation. Altogether, we provide evidence of an association between a highly restricted gene-specific transcription factor and a Su(var), Enhancer of Zeste, Trithorax (SET)1/trithorax-like complex with H3K4 methyltransferase activity. Our studies also document a functional role for Ap2delta in recruiting histone methyltransferases (HMTs) to specific gene targets, such as Hoxc8. This role provides a mechanism through which these transcription factors can have diverse effects despite nearly identical DNA-binding motifs.

The ZNF217 oncogene is a candidate organizer of repressive histone modifiers.

The zinc finger protein 217 (ZNF217) is an important oncogene based on the high frequency of amplification and overexpression in many cancer types, but its molecular mode of gene regulation is poorly understood. We purified a complex of nuclear ZNF217-binding proteins by affinity chromatography and identified its components by mass spectrometry as Jarid1b/Plu-1, G9a, LSD1, CoREST and CtBP1. Individual binding of these with ZNF217 was confirmed by co-immunoprecipiation (IP). Known activities of these proteins suggested a role of the ZNF217 complex in histone modification. Using in vitro assays the following activities were demonstrated: Histone H3 lysine 4 trimethyl (H3K4me3) demethylase activity, which co-fractionated with Jarid1b/Plu-1 in anion-exchange chromatography; H3K9 methylation, the known principal activity of G9a; and H3K27 methylation. The latter suggested EZH2 as another ZNF217 binding candidate, which could be confirmed by co-IP. Taken together, these findings suggest that ZNF217 assembles a distinct set of histone modifying proteins at target DNA sites that act synergistically in transcriptional repression.

The MMSET protein is a histone methyltransferase with characteristics of a transcriptional corepressor.

MMSET, identified by its fusion to the IgH locus in t(4;14)-associated multiple myeloma, possesses domains found within chromatin regulators, including the SET domain. MMSET protein is overexpressed and highly associated with chromatin in myeloma cell lines carrying t(4;14). MMSET possesses methyltransferase activity for core histone H3 lysine 4 and histone 4 lysine 20, whereas MMSET made in cells only modified H4. Segments of MMSET fused to the Gal4 DNA binding domain repressed transcription of a chromatin-embedded Gal4 reporter gene. MMSET-mediated repression was associated with increased H4K20 methylation gene and loss of histone acetylation. Consistent with this repressive activity, MMSET could form a complex with HDAC1 and HDAC2, mSin3a, and the histone demethylase LSD1, suggesting that it is a component of corepressor complexes. Furthermore, MMSET coexpression enhances HDAC1- and HDAC2-mediated repression in transcriptional reporter assays. Finally, shRNA-mediated knockdown of MMSET compromised viability of a myeloma cell line, suggesting a biologic role for the protein in malignant cell growth. Collectively, these data suggest that, by acting directly as a modifier of chromatin as well as through binding of other chromatin-modifying enzymes, MMSET influences gene expression and potentially acts as a pathogenic agent in multiple myeloma.

CCAAT displacement protein/cut homolog recruits G9a histone lysine methyltransferase to repress transcription.

CCAAT displacement protein/cut homolog (CDP/cut) is a highly conserved homeodomain protein that contains three cut repeat sequences. CDP/cut is a transcriptional factor for many diverse cellular and viral genes that are involved in most cellular processes, including differentiation, development, and proliferation. Here, we report that CDP/cut interacts with a histone lysine methyltransferase (HKMT), G9a, in vivo and in vitro. The deletion of the cut repeats within CDP/cut abrogates the interaction with G9a. The transcriptional repressor function of CDP/cut is mediated through HKMT activity of G9a associated with CDP/cut. We show that the recruitment of G9a to the human p21(waf1/cdi1) promoter is contingent on the interaction with CDP/cut, and CDP/cut is directly associated with an increase in the methylation in vivo of Lys-9 in histone H3 within the CDP/cut-regulatory region of the p21(waf1/cdi1) promoter. The endogenous level of p21(waf1/cdi1) expression is repressed through CDP/cut and mediated by HKMT activity of G9a. Furthermore, we report the identification of G9a as a component of CDP/cut complex. G9a colocalizes with CDP/cut in the nucleus. These results indicate that G9a functions as a transcriptional corepressor in association with a CDP/cut complex. These studies now reveal the interaction of G9a with a sequence-specific transcription factor that regulates gene repression through CDP/cut.