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Biochemical screen identifies LEDGF and HDGF2 as FACT-like factors

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Presentation on theme: "Biochemical screen identifies LEDGF and HDGF2 as FACT-like factors"— Presentation transcript:

1 Fig. 1 Biochemical screen identifies LEDGF and HDGF2 as FACT-like factors.
Biochemical screen identifies LEDGF and HDGF2 as FACT-like factors. (A) Venn diagram showing occupancy of RNAPII and SPT16 (FACT) on active genes (at least five transcripts) in mouse embryonic stem cells (mESCs). Hypergeometric test, P = 0. (B) Top: Fractions from the Superdex 200 step were separated by SDS–polyacrylamide gel electrophoresis (PAGE), stained with Coomassie Blue, and analyzed by mass spectrometry (MS) for protein identification. Bottom: Fractions from the Superdex 200 step analyzed using the in vitro chromatin transcription assay. (C) Schematic of the predicted domain structure of the LEDGF/HDGF2 family of proteins. PWWP, methyl-lysine binding domain; NLS, nuclear localization sequence; AT, AT-hook domain; HMGB, high mobility group box domain; IBD, integrase binding domain; a.a., amino acid. (D) Highly purified recombinant versions of the LEDGF/HDGF2 family proteins were separated by SDS-PAGE and stained with Coomassie Blue (left) and analyzed using the in vitro chromatin transcription assay (right). (E) Titration of FACT, LEDGF, and HDGF2 in defined RNAPII transcription assays with nucleosomal templates. Molar ratio of protein (X):nucleosome in assays is indicated on top. (F) Graph of transcription quantified from (H). y axis is the relative activities quantified with ImageJ software. x axis is the molar ratio of protein (X):nucleosome in assays. (G) Schematic depicting the nucleosome transfer assay. (H) Nucleosome transfer assays containing purified oligonucleosome chains and 32P-labeled 601 nucleosome trapping DNA and ±purified HDGF2 as indicated. The first lane is a control nucleosome, and the second lane is control tetrasome. These controls were assembled by decreasing salt dialysis. Gary LeRoy et al. Sci Adv 2019;5:eaay3068 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

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