PAF1 binding is necessary for neutralization of polycomb mediated repression. (A) Schematic overview of ENL and crystal structure of a YEATS domain. Known binding partners of ENL are listed. Numbers denote amino acids. The mutations introduced for this study are indicated. The crystal structure (PDB: 4TMP) represents the YEATS domain of the close ENL homolog AF9 with a cocrystalized H3 tail peptide (blue). Highlighted amino acids are from ENL. (In AF9 the sequence reads KKPTVE). (B) Interactions of YEATS-domain mutants with histone H3. Extracts of 293T cells expressing flag-tagged versions of wild-type (wt)-ENL, ENL∆21-26, or ENL∆N were supplemented with recombinant histone H3, followed by flag-specific immunoprecipitation and immunoblotting. Flag- and H3-specific western blots are displayed. Because equal amounts of H3 were added manually, no separate input sample is shown. (C) Binding of PAF1 to YEATS mutants. Extracts from cells expressing HA-tagged PAF1 and flag-labeled wt-ENL, ENL∆N (Ci), or ENL∆21-26 (Cii) were precipitated with antiflag and analyzed on western blots, as indicated. (D) Elongation reporter assay. The impact of regulators on transcriptional elongation can be detected by a reporter system based on a HIV LTR that regulates transcription by elongation control. Proteins can be recruited selectively to a short RNA trailing behind a stalled RNA polymerase II by fusing them to the RNA binding protein Rev. In this way their impact on elongation can be measured by luciferase output. Either Rev-only as control (light tan bars) or Rev-CBX8 (dark blue bars) was recruited, and the impact of addition of wt-ENL or a YEATS mutant was recorded, as indicated. Values were normalized to the Rev-only control. The bar chart shows averages and standard deviations of triplicate experiments. (E) Chromatin immunoprecipitation on reporter plasmids. Elongation reporter plasmid, Rev-CBX8, and wt-ENL, ENL∆21-26, or a vector control were coexpressed and cells subjected to crosslinking and H2Bub-specific immunoprecipitation. Quantitation was done by qPCR, with primers spanning the luciferase coding sequence. Results shown are averages and standard deviations of PCR triplicates and represent a typical example of a duplicate. (F) Effect of increasing PAF1 concentration on ENL and ENL∆21-26-mediated derepression. Rev-CBX8 was coexpressed with limiting amounts of wt-ENL or ENL∆21-26 and increasing concentrations of PAF1, as indicated. The impact on elongation was measured and depicted as described for panel D. (G) Disruption of the PAF1 ENL interaction. An internal part of PAF1 encompassing amino acids 266 to 400 (PAF266) is sufficient for interaction with ENL in cells (Gi). A flag-specific immunoprecipitation of flag-ENL and HA-tagged PAF266 is shown. The ENL interaction domain is located between the previously identified¹⁸  LEO and histone H3-binding regions, as schematically depicted. Results of a reporter assay performed as described in panel F, exchanging wt-PAF1 for PAF266 (Gii). *P < .05. CDS, luciferase coding sequence; f, flag; H-PAF, HA-tagged PAF1; IP, immunoprecipitation; luci, luciferase; ppt, precipitation; recomb., recombinant; rel., relative; smpls., samples; TAR, transactivation response element; vec, vector; WB, western blot.