Analysis of EAP histone methyltransferase and CDK kinase activities in vitro. (A) EAP contains a H3K79 dimethylating activity. Left panel: an EAP preparation or a mock control that was done with unliganded agarose as precipitation agent were incubated with core histones and methyl-³H-S-adenosylmethionine. The reaction products and a histone input sample (100% input) were separated on a 18% SDS gel, stained with Coomassie blue, and treated with a fluorographic enhancer solution before exposure to x-ray film (fluorography). Right panel: in an analogous experiment, core histones were incubated with EAP or a mock control as in panel A, and nonradioactive S-adenosylmethionine. After separation of the reaction products by 18% SDS-PAGE, the proteins were blotted and analyzed by immunodetection first with a H3K79 dimethyl–specific antibody and subsequently on the same membrane with a pan-H3–reactive antibody. As a further control, aliquots of the EAP and mock reactions were probed by Western blot for changes of dimethylation at H3K9. The densitometrically obtained concentration values are given underneath the respective lanes. (B) EAP has RNAPolII CTD kinase and pTEFb activity in vitro. Left panel: EAP works as RNAPolII CTD kinase in vitro. Recombinant GST–RNAPolII CTD fusion protein was incubated in the presence of γ³²P ATP and either an EAP preparation or a mock control as described. Reactions were separated by SDS-PAGE followed by Coomassie staining and autoradiography. Right panel: EAP supplies pTEFb activity for in vitro transcription. Standard run-off transcription reactions in HeLa nuclear extracts were primed with a template allowing the transcription of the first 244 bp of the Hoxa9 coding sequence under control of the CMV immediate early promoter. The reaction was carried out with α³²P-UTP to allow labeling of the transcript. The reaction products were phenolized and separated on a denaturing 6 M urea 6% PAA gel followed by autoradiography. The transcription assays were supplemented either with a mock precipitate as described, or with an EAP preparation. Whereas transcript levels were reduced by addition of the pTEFb inhibitor DRB in unsupplemented samples (left), the presence of EAP rescues a significant amount of transcriptional activity under the same conditions (right).