Volume 22, Issue 6, Pages 1374-1383 (February 2018) Crosstalk between the RNA Methylation and Histone-Binding Activities of MePCE Regulates P-TEFb Activation on Chromatin  Samantha B. Shelton, Nakul M. Shah, Nathan S. Abell, Sravan K. Devanathan, Marvin Mercado, Blerta Xhemalçe  Cell Reports  Volume 22, Issue 6, Pages 1374-1383 (February 2018) DOI: 10.1016/j.celrep.2018.01.028 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 22, 1374-1383DOI: (10.1016/j.celrep.2018.01.028) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 MePCE Knockdown Alleviates Tumorigenic Features in Breast Cancer Cells (A) Validation by western blot of MePCE knockdown in MCF10A, MCF7, and MDA-MB-231 cells stably expressing scrambled negative control (shNC) and shMePCE. (B) MePCE knockdown significantly decreases 7SK RNA levels. Upper panels correspond to northern blots with an anti-7SK probe, and lower panels to SYBR-Gold-stained gels to verify equal loading. (C) Representative images of cells grown in soft agar for ∼4 weeks. MCF10A cells are untransformed and constitute a control for cells unable to form colonies under anchorage-independent conditions. (D) Analysis of the migration and invasion ability of MDA-MB-231 shNC and shMePCE cells. Shown are the ratios of cells able to go through an 8-μM pore size membrane (migration assay) or an 8-μM pore size membrane coated with matrigel solution (invasion assay). The data are represented as mean ± SEM, n = 2 replicates, after normalization to cellular proliferation and shNC. (E) Volcano plot representing the fold change (FC) of normalized RNA read counts (shMePCE over shNC) on the x axis (log2) and the false discovery rate (FDR) on the y axis (log10). Green dots indicate significantly down- or upregulated genes in shMePCE over shNC with a FC ≥ 1.5 and FDR ≤ 0.05. (F) Validation of RNA-seq data obtained in MDA-MB-231-shNC and -shMePCE cells by RT-qPCR analysis. MePCE and four downregulated and four upregulated genes in shMePCE versus shNC cells are shown. The data are represented as mean ± SEM, n = 2 replicates, after normalization to the ALAS1 and TUBA1A genes and shNC. (G) Immunofluorescence analysis with an anti-Myc primary antibody and an anti-rabbit secondary antibody coupled to Alexa Fluor 594 (AF594) in shMePCE stably expressing eGFP (Control) or Myc-Flag-tagged ID1, ID2, and ID3. (H) Migration assay of the cells shown in (G), represented as mean ± SEM, n = 2 replicates, after normalization to shMePCE. See also Figure S1 and Table S1. Cell Reports 2018 22, 1374-1383DOI: (10.1016/j.celrep.2018.01.028) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Genes Downregulated upon MePCE Knockdown Display RNAPII-S2 Phosphorylation Defect (A) Graphical representation of the significantly deregulated genes ranked by fold change (FC; log2) of shMePCE over shNC. (B) Distribution of CDK9, RNAPII-S2P, and MePCE on cellular fractionation in MDA-MB-231-shNC and -shMePCE cells. (C) Heatmaps of RNAPII-S2P occupancy around the TSS (±2 kb) in MDA-MB-231-shMePCE and -shNC cells. Rows are sorted by decreasing RNAPII-S2P occupancy in shNC cells. Color scale intensities are in units of reads per million (rpm). The darkest shade of blue is for rpm ≥ 1. RNAPII-S5P and positive (H3K4me3) and negative (GFP) controls for these experiments are shown in Figure S2A. (D) Meta-analysis of RNAPII-S2P and -S5P occupancy around the TSS (±1 kb) of the significantly downregulated genes in shMePCE versus shNC cells as defined in Figure 1E. GFP signal serves as a negative control. The full set of meta-analysis plots for downregulated, upregulated, or not significantly changed genes is shown in Figure S2B. (E) Genome browser track of RNAPII-S2P ChIP-seq at the ID1 gene in MDA-MB-231-shNC and -shMePCE cells. The purple segment indicates the position of the ID1 qPCR amplicon in (G). (F) Dot blot analysis of unmodified, S2P, and S5P RNAPII-CTD peptides with the anti RNAPII-S2P and RNAPII-S5P antibodies used in the ChIP-seqs. The dot blot with anti-RNAPII (8WG16) is a control showing that this antibody’s recognition of the CTD is sensitive to CTD phosphorylation. (G) ChIP-qPCR with a CDK9 antibody (Cell Signaling Technology) in MDA-MB-231-shNC and -shMePCE cells. The position of the qPCR amplicon for the ID1 gene is indicated by the purple segment in (E). The data are represented as mean ± SEM, n = 2 replicates, after normalization to input (%). See also Figure S2. Cell Reports 2018 22, 1374-1383DOI: (10.1016/j.celrep.2018.01.028) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 MePCE Binds to the Tail of Histone H4 (A) Diagram of human MePCE, in which SAM-BD, S-adenosyl-methionine-binding domain; GLY-rich, glycine-rich domain of unknown function; C-ter, 220-amino-acid C-terminal fragment of MePCE; and G451/G455A, catalytic-inactive mutant determined based on homology with BCDIN3D (Xhemalce et al., 2012). (B) In vitro methyltransferase assay using recombinant GST-MePCE, 3H-radioactive SAM as the methyl group donor, and in-vitro-transcribed 7SK RNA as the substrate. FL, full length; CM, catalytic mutant. (C) In vitro GST pull-down assays using the indicated recombinant proteins and each of the human canonical histones or the 7SK RNA. (D) Meta-analysis of MePCE occupancy around the TSS (± 1kb) in MDA-MB-231-shNC and -shMePCE cells. GFP signal serves as a negative control. (E) Meta-analysis of MePCE occupancy around the TSS (± 1kb) in HeLa-S3-FlpIn cells. Beads and anti-Flag signal serve as negative controls. (F) Genome browser track of MePCE ChIP-seq at the ID1 gene. Beads alone and Flag signal serve as negative controls. See also Figure S3. Cell Reports 2018 22, 1374-1383DOI: (10.1016/j.celrep.2018.01.028) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 MePCE Binds to Chromatin Independently of the 7SK snRNP (A) Schematic of the biochemical purification of chromatin-bound MePCE using HeLa-S3-FlpIn-MePCE-Flag cells. (B) Isogenic HeLa-S3-FlpIn cells containing Flag-tagged MePCE or GFP inserts were used in anti-Flag co-immunoprecipitations followed by elutions with a Flag peptide. Lysates were treated with RNase or MNase to reveal RNA-dependent interactions or to solubilize chromatin-bound MePCE, respectively. Inputs and Flag eluates were analyzed with the indicated antibodies. Levels of MePCE-Flag in the Flag eluates are shown by silver staining. (C) In vitro GST pull-down assays using the indicated GST-tagged recombinant proteins and full-length His-tagged CDK9/cyclin T1 holoenzyme (Thermo Fisher Scientific catalog no. PV4131). (D) In vitro GST pull-down assays using the indicated GST-tagged recombinant proteins and histone H4 and/or the 7SK RNA. The Coomassie stain of histone H4 and GST-MePCE are from the same image of the same gel to show that MePCE is not in excess in these experiments. (E) MePCE’s 7SK methyltransferase activity is inhibited by the tail of histone H4. In vitro RNA methyltransferase assay using recombinant GST-MePCE, 3H-SAM, 7SK RNA, and the indicated proteins. (F) RT-qPCR analysis in MDA-MB-231-shNC and –shMePCE cells and mock- or 200-nM JQ1-treated for 24 hr. The data are represented as mean ± SEM, n = 3 replicates, after normalization to the ALAS1 and TUBA1A genes and shNC. (G) Model for the effect of histone H4 binding on 7SK snRNP stability and P-TEFb activity. See also Figure S4 and Table S2. Cell Reports 2018 22, 1374-1383DOI: (10.1016/j.celrep.2018.01.028) Copyright © 2018 The Author(s) Terms and Conditions