Research ArticleMOLECULAR BIOLOGY

LEDGF and HDGF2 relieve the nucleosome-induced barrier to transcription in differentiated cells

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Science Advances  02 Oct 2019:
Vol. 5, no. 10, eaay3068
DOI: 10.1126/sciadv.aay3068
  • Fig. 1 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.

  • Fig. 2 LEDGF and HDGF2 bind to clusters of active chromatin.

    (A) Venn diagram showing occupancy of RNAPII and SPT16 (FACT) on active genes (at least five transcripts) in human 293T cells. Hypergeometric test, P = 0. (B) Western blots performed with whole-cell protein extracts from mESCs, 293T cells, myoblasts (MBs), and myotubes (MTs) using the antibodies indicated. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) ChIP-seq genomic tracks of the indicated factors and histone modifications at a (~14 Mb) region of chromosome 6 with corresponding RNA-seq (location = chromosome 6 q-arm; position, ~70 to 84 Mb). (D) Left: Cartoon depicting LEDGF- or HDGF2-purified chromatin used to quantify histone modifications by MS. Right: Histone H3K27 and H3K36 methylations quantified by MS from the input (293T whole-genome chromatin), FLAG-LEDGF, and FLAG-HDGF2 ChIPs.

  • Fig. 3 LEDGF and HDGF2 are required for the induction of some genes in stem cells.

    (A and B) ChIP-seq tracks for LEDGF, HDGF2, SPT16, and RNAPII with corresponding RNA-seq tracks in ESCs and in EBs at the Tpm1 gene (A) and Essrb gene (B). (C) Pie chart showing the percentage of genes exhibiting increased HDGF2 and/or FACT binding on genes with an increase in RNAPII during ESC differentiation into EB. (D) Mean average (MA) plots showing the number of differentially expressed genes (twofold, BH-corrected P < 0.05) in LEDGF KO and LEDGF/HDGF2 double KO (dKO) in ESC to EB differentiation. (E) Venn diagram depicting the number of up-regulated genes (twofold) from ESC to EB in wild-type (WT) cells overlaid with the down-regulated genes in the LEDGF/HDGF2 dKO EBs. Of the 2308 genes normally up-regulated in the WT cells (ESC to EB), 618 were found to be down-regulated in the dKO EB cells. The overlap with dKO down-regulated genes is significant (P = 1.5 × 10−13, hypergeometric test). (F) Boxplots with confidence intervals of expression of the 618 genes selected from (E) at WT ESC, WT EB, and dKO EB. Wilcoxon rank sum test: WT ESCs versus WT EBs, P = 4.5 × 10−13; WT ESCs versus dKO EBs, P = 3.8 × 10−3. (G) Top: Average density ChIP-seq profiles of HDGF2, SPT16, and LEDGF on the 618 genes that are up-regulated in WT EBs and down-regulated in dKO EBs. Bottom: Average density ChIP-seq profiles of HDGF2, SPT16, and LEDGF on the 4999 genes identified as not having any change in expression between ESCs and EBs.

  • Fig. 4 LEDGF and HDGF2 substitute for FACT in differentiated cells.

    (A) Western blots of whole-cell protein extracts from MBs differentiated to MTs obtained from days 0 (D0), 3 (D3), and 6 (D6) using the antibodies indicated. (B) Average density ChIP-seq profiles for HDGF2, LEDGF, and SPT16 based on genes exhibiting RNAPII binding in MB (top) and MT (bottom). Levels based on genes with RNAPII binding: highest (purple; top 5%), high (blue; top 5 to 20%), medium (dark green; 20 to 80%), low (bright green; bottom 5 to 20%), and lowest (red; bottom 5%). (C) Venn diagram depicting the number of genes up-regulated twofold in MB to MT cells overlaid with genes down-regulated twofold in HDGF2 KO MT cells. (D) Average density ChIP-seq profiles for HDGF2 and LEDGF in MB and MT cells. Top: The 488 genes that are up-regulated in MB to MT and fail to induce in the HDGF2 KO cells. Bottom: The 334 genes that still induce in the HDGF2 KO. (E to G) ChIP-seq tracks for H3K27me3, SPT16 LEDGF, HDGF2, H3K36me2, H3K36me3, and RNAPII with the corresponding RNA-seq tracks for MB and MT at the myosin heavy chain (MHC) cluster (E), the NES locus (F), and the C20orf166 locus (G). (H and I) Metagene profiles of precision nuclear run-on sequencing (PRO-seq) RNA data plotted for the top 20% HDGF2-bound genes in WT and HDGF2 KO and HDGF2 KO rescue MB cell lines as indicated on the panels. (H) Plotted by 1 kb-pause site-1 kb-(scaled gene bodies)-1 kb-TES-1 kb. (I) Plotted with data centered on the TSS (±1 kb). First panel has assay for transposase-accessible chromatin sequencing (ATAC-seq) data overlayed with PRO-seq from WT cells showing the +1 nucleosome position (+1 nuc). (J) Schematic depiction showing the replacement of FACT by HDGF2 and/or LEDGF on chromatin during cellular differentiation, when FACT expression is reduced.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaay3068/DC1

    Fig. S1. Identification of a novel FACT-like chromatin transcription chaperone.

    Fig. S2. In vitro transcription assays.

    Fig. S3. Bioinformatics analysis of genes within categories defined by levels of FACT, LEDGF, and HDGF2 binding.

    Fig. S4. HDGF2/LEDGF colocalize with H3K36me2 and moderate levels of RNAPII excluding H3K27me3 domains.

    Fig. S5. Validation of LEDGF and HDGF2 antibodies.

    Fig. S6. Genes in MB and MT are categorized on the basis of RNAPII occupancy.

    Fig. S7. Highly transcribed genes with SPT16 (FACT) occupancy in MBs.

    Fig. S8. HDGF2 is required to activate MT-specific genes.

    Fig. S9. Genomic characterization of FACT, LEDGF, and HDGF2 in ESCs, 293T cells, MBs, and MTs.

    Table S1. MS sequencing results for S-200 (fraction 5) as shown in Fig. 1D.

    Table S2. Table of histone modification quantified by MS in LEDGF and HDGF2 ChIPs.

    Table S3. Table of gRNA dropout results from CRISPR-CAS9 screens in mESCs.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Identification of a novel FACT-like chromatin transcription chaperone.
    • Fig. S2. In vitro transcription assays.
    • Fig. S3. Bioinformatics analysis of genes within categories defined by levels of FACT, LEDGF, and HDGF2 binding.
    • Fig. S4. HDGF2/LEDGF colocalize with H3K36me2 and moderate levels of RNAPII excluding H3K27me3 domains.
    • Fig. S5. Validation of LEDGF and HDGF2 antibodies.
    • Fig. S6. Genes in MB and MT are categorized on the basis of RNAPII occupancy.
    • Fig. S7. Highly transcribed genes with SPT16 (FACT) occupancy in MBs.
    • Fig. S8. HDGF2 is required to activate MT-specific genes.
    • Fig. S9. Genomic characterization of FACT, LEDGF, and HDGF2 in ESCs, 293T cells, MBs, and MTs.
    • Table S1. MS sequencing results for S-200 (fraction 5) as shown in Fig. 1D.
    • Table S2. Table of histone modification quantified by MS in LEDGF and HDGF2 ChIPs.
    • Table S3. Table of gRNA dropout results from CRISPR-CAS9 screens in mESCs.

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