Research ArticleCANCER GENETICS

KDM5 lysine demethylases are involved in maintenance of 3′UTR length

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Science Advances  18 Nov 2016:
Vol. 2, no. 11, e1501662
DOI: 10.1126/sciadv.1501662
  • Fig. 1 Jhd2 interacts physically with polyadenylation machinery.

    (A) Coomassie staining of TAP-Jhd2–associated protein complex. Selected proteins identified by tandem mass spectrometry are indicated. (B) Known components of the yeast polyadenylation complex and their associated subcomplexes. CPF, cleavage and polyadenylation factor. (C) Western blot analysis of TAP immunoprecipitates from the indicated TAP-tagged strains overexpressing FLAG-Jhd2 with a Jhd2 antibody. The Jhd2 antibody recognizes both Jhd2-TAP (top band, 105 kDa) and FLAG-Jhd2 (bottom band, 86 kDa) (row 2). Nonspecific bands are indicated by asterisks. Right: Copurifications performed as in the left panel but with the addition of RNase. Control indicates a yeast strain with no TAP-tagged protein. (D) Western blot analysis of TAP immunoprecipitates from Hrp1-TAP or wild-type strains expressing endogenous levels of Jhd2 with a Jhd2 antibody.

  • Fig. 2 Jhd2 recruits CF1A to 3′UTRs.

    (A) Jhd2 and Pcf11 ChIP experiments of yeast expressing endogenous levels of Jhd2-TAP or Pcf11-TAP. DNA corresponding to noted locations on PMA1 was quantified using quantitative polymerase chain reaction (qPCR) with the indicated primers (table S6). Data are represented as fold enrichment over the control ChIP using beads only. Error bars represent SEM for biological triplicate experiments. Note the trend, although the differences are not statistically different. (B) Venn diagram showing genes bound by both Jhd2 and Pcf11. (C) Rna15 ChIP experiments using wild-type and Jhd2 deletion strains. Data are represented as fold enrichment over the control. Error bars represent SEM for biological duplicate experiments. Note the trend, although the differences are not statistically different. (D) Western blot analysis of wild-type and Jhd2 deletion strains with the indicated antibodies. (E) RNA binding experiments were performed using a biotinylated RNA 20-mer conjugated to magnetic beads. Recombinant Jhd2 bound to RNA was detected using Western blotting. Beads not conjugated to RNA served as a negative control. (F) RIP experiments of yeast expressing endogenous levels of TAP-tagged Jhd2 or TAP-tagged Pcf11. RNA associated with these proteins was converted to complementary DNA (cDNA) and quantified using qPCR. Data are shown as fold enrichment over the control. Error bars represent SEM for biological duplicate experiments. **P < 0.01. Note the trend, although the other differences are not statistically different.

  • Fig. 3 Jhd2 promotes 3′UTR shortening of PMA1 in a demethylase-dependent manner.

    (A) Schematic representing amplicons used to study the 3′UTR region of PMA1. (B) Ratio of PMA1 3′UTR to CDS mRNA in a Pcf11 temperature-sensitive strain. Yeast cells grown at 30°C were transferred to 37°C for 60 min, resulting in a nonfunctional Pcf11 protein. Error bars represent SEM for biological duplicate experiments. (C) Ratio of PMA1 3′UTR to CDS mRNA in wild-type and Jhd2 deletion cells expressing empty vector (Δjhd2 + vector), FLAG-Jhd2 (Δjhd2 + FLAG-Jhd2), or demethylase-inactive FLAG-Jhd2 (Δjhd2 + FLAG-Jhd2H427A). Error bars represent SEM (n = 8). **P < 0.01. (D) Representation of the KlCYC1 3′UTR with conserved polyA motifs at its proximal polyadenylation site but with no conserved motifs at its distal polyadenylation site. EE, efficiency element; PE, positioning element; UUE, upstream U-rich enhancer element; DUE, downstream U-rich enhancer element. (E) RT-qPCR analysis of the ratio of KlCYC1 3′UTR to CDS mRNA in strains expressing KlCYC1 and either empty vector, FLAG-Jhd2, or demethylase-inactive FLAG-Jhd2H427A. All mRNA levels are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Error bars represent SEM for biological triplicate experiments. **P < 0.01.

  • Fig. 4 Jhd2 affects global polyadenylation site choice.

    (A) Schematic of the 3′READS method used to enrich for 3′UTRs before RNA-seq. (B) Volcano plot showing P value and fold change of the two most abundant pA clusters in Jhd2 deletion versus wild-type strains. Red dots indicate genes with P < 0.05 and fold change >1.5. The Jhd2 deletion strain selects for distal polyA sites for 76 lengthened transcripts (right), whereas the wild-type strain selects for distal polyA sites in 125 shortened transcripts (left). (C to E) Integrative Genomics Viewer (IGV) snapshot showing polyA clusters (the two most abundant ones in black bars and the others in gray bars) of (C) YNL289W, (D) YHR103W, and (E) YHR215W from representative wild-type and Jhd2 deletion samples. (F to H) RT-qPCR analysis validating the ratio of 3′UTR to CDS mRNA for (F) YNL289W, (G) YHR103W, and (H) YHR215W in wild-type and Jhd2 deletion samples. Error bars represent SEM for biological triplicate experiments. **P < 0.01; ***P < 0.001.

  • Fig. 5 KDM5B down-regulation leads to 3′UTR lengthening of CCND1.

    (A) Schematic representation of the amplicons used to study 3′UTR length of CCND1. Both polyA sites (red dashes) and miRNA sites (black lines) are noted. (B and C) RT-qPCR analysis of KDM5B and CCND1 with the indicated amplicons in (B) MCF7 and (C) T47D breast cancer cells infected with the indicated lentiviruses. Error bars represent SEM for biological triplicate experiments. *P < 0.05; ***P < 0.001; ****P < 0.0001; n.s., not statistically significant. (D) RT-qPCR analysis of KDM5A and CCND1 with the indicated amplicons in T47D breast cancer cells transfected with the indicated small interfering RNAs (siRNAs). Error bars represent SEM for biological duplicate experiments. *P < 0.05. (E) RNA-seq coverage of the 3′UTR region of CCND1 in patient tumors expressing low levels of KDM5B compared with patient tumors expressing high levels of KDM5B. Both polyA (green and red bars) and miRNA (green bars) sites are noted. The box highlights the difference of the two long isoforms of CCND1 between these two groups. TCGA tumor sample identifiers are listed.

  • Fig. 6 KDM5s are involved in selection of polyA sites.

    (A) Western blot and (B) RT-qPCR analyses of MCF7 cells treated with dimethyl sulfoxide (DMSO) or 10 μM KDM5-C70 for 3 days. The ratio of 3′UTR to CDS for CCND1 mRNA was plotted. Error bars represent SEM for biological triplicate experiments. (C) Western blot and (D) RT-qPCR analyses of HeLa cells treated with DMSO or 10 μM KDM5-C70 for 3 days. The ratio of 3′UTR to CDS for DICER1 mRNA was plotted. Error bars represent SEM for biological triplicate experiments. **P < 0.01. (E) RT-qPCR analysis of HeLa/iCas9-c1 cells transduced with lentiviruses carrying single-guide RNAs against KDM5A, KDM5B, KDM5C, or nontargeting control. The ratio of 3′UTR to CDS for DICER1 mRNA was plotted. KO, knockout. Error bars represent SEM for biological triplicate experiments. *P < 0.05; **P < 0.01. (F) Working model for KDM5 involvement in APA. KDM5 recruits the polyA machinery to nascent RNA to modulate polyA site choices. Demethylation or hydroxylation of certain subunits of the polyA machinery by KDM5 also contributes to selection of the polyA sites. The polyA sites (dashes) are noted on the nascent transcript.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/11/e1501662/DC1

    fig. S1. Mass spectrometry results.

    fig. S2. Western blot analysis of TAP-tagged proteins.

    fig. S3. Materials for in vitro RNA binding experiments.

    fig. S4. Loss of Jhd2 does not affect processing or polyA-dependent termination in vitro.

    fig. S5. RT-qPCR and Western blot analysis for Jhd2 mRNA and protein used in PMA1 studies.

    fig. S6. RT-qPCR and Western blot analysis for Jhd2 mRNA and protein used in KlCYC1 studies.

    fig. S7. Metagene plots of H3K4me3 levels.

    table S1. Jhd2 bound peaks identified using SICER.

    table S2. Pcf11 bound peaks identified using SICER.

    table S3. List of genes with APA after Jhd2 deletion.

    table S4. Plasmids used in this study.

    table S5. Yeast strains used in this study.

    table S6. Primers used in this study.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Mass spectrometry results.
    • fig. S2. Western blot analysis of TAP-tagged proteins.
    • fig. S3. Materials for in vitro RNA binding experiments.
    • fig. S4. Loss of Jhd2 does not affect processing or polyA-dependent termination in vitro.
    • fig. S5. RT-qPCR and Western blot analysis for Jhd2 mRNA and protein used in PMA1 studies.
    • fig. S6. RT-qPCR and Western blot analysis for Jhd2 mRNA and protein used in KlCYC1 studies.
    • fig. S7. Metagene plots of H3K4me3 levels.
    • Legends for tables S1 to S3
    • table S4. Plasmids used in this study.
    • table S5. Yeast strains used in this study.
    • table S6. Primers used in this study.

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    Other Supplementary Material for this manuscript includes the following:

    • table S1 (Microsoft Excel format). Jhd2 bound peaks identified using SICER.
    • table S2 (Microsoft Excel format). Pcf11 bound peaks identified using SICER.
    • table S3 (Microsoft Excel format). List of genes with APA after Jhd2 deletion.

    Files in this Data Supplement:

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