Promiscuous RNA binding by Polycomb Repressive Complex 2

Transcription

1 Promiscuous RNA binding by Polycomb Repressive Complex 2 Chen Davidovich 1,2, Leon Zheng 3, Karen J. Goodrich 1,2, and Thomas R. Cech 1,2,4 1 Department of Chemistry & Biochemistry, Howard Hughes Medical Institute, University of Colorado, Boulder, Colorado, 80309, USA 2 BioFrontiers Institute, University of Colorado, Boulder, Colorado, 80309, USA 3 Medical Scientist Training Program, University of Colorado School of Medicine, Aurora, Colorado, 80045, USA 4 Correspondence to: thomas.cech@colorado.edu Supplementary Materials Supplementary Figure 1. In vitro histone methyltransferase (HMTase) assay Supplementary Figure 2. Sequence source for HOTAIR RNA and HOTAIR 400 RNA Supplementary Figure 3. PRC2-RNA direct binding assay after prolonged incubation Supplementary Figure 4. Direct binding assay of PRC2 with various RNAs Supplementary Figure 5. Secondary structure prediction of MBP Supplementary Figure 6. Gene Ontology (GO) analysis for genes that were up-regulated following SUZ12 knockdown Supplementary Figure 7. Original western blots used to generate Figure 7a Supplementary Table 1. Genes observed with Ezh2-FE larger than 3 Supplementary Table 2. GO analysis for genes with high Ezh2-FE and coverage Supplementary Table 3. Data for Fig 5b Supplementary Table 4. PRC2-Regulated and EZH2-Associated genes Supplementary Table 5. GO analysis for genes up-regulated by SUZ12 KD Supplementary Table 6. ChIP-seq datasets used for Figure 6 Supplementary Table 7. NGS datasets generated in the scope of this study

2 Supplementary Figure 1. In vitro histone methyltransferase (HMTase) assay. Assays carried out under identical conditions, except for the exclusion of PRC2, H3.1 or both as highlighted. The higher radioactive signal following TCA precipitation, appearing only in the presence of both PRC2 and H3.1 histone, confirmed HMTase activity of PRC2. Some low signal was observed in the absence of histone substrate, possibly because of loaded SAM that was co-precipitated with PRC2 or a weak auto HMTase activity of PRC2, as previously observed 4. Error bars represents standard deviations that were generated based on 3 independent samples.

3 Supplementary Figure 2. Sequence source for HOTAIR RNA and HOTAIR 400 RNA. The sequence of HOTAIR (in red) originated from a previously generated cdna clone 58 and is in accord with the HOTAIR RNA examined in previous studies 24,34. We found this RNA construct prone to aggregation under various conditions, evidenced by the presence of radiolabeled RNA in wells and across lanes following non-denaturing gel electrophoresis, in the absence or presence of protein (Fig 1b). RNA aggregation was eliminated by adding approximately 50 nucleotides upstream and downstream of HOTAIR 1-300, forming HOTAIR 400 RNA (see Supplementary Methods for complete details of DNA templates used for in vitro RNA transcription). Importantly, all bases that were added (in light blue) were not arbitrarily tailored but appear within cellular HOTAIR transcripts (in blue).

4 Supplementary Figure 3. PRC2-RNA direct binding assay after prolonged incubation. To verify that dissociation constants were derived under equilibrium conditions, incubation time was increased eightfold, from 30 min to 240 min. The fraction of bound RNA was not increased following this prolonged incubation (panels a). An increase of 43% in equilibrium dissociation constant (panel b) is most likely due to loss of similar fraction of RNA binding activity by PRC2 due to the prolonged incubation period (4 hours at 30 ⁰C) prior to the EMSA experiment.

5 Supplementary Figure 4. Direct binding assay of PRC2 with various RNAs. (a) EMSA of different RNAs after incubation in the presence or absence of PRC2 complex confirms similar affinity of PRC2 to the following: a 400 base RNA from the 5 domain of HOTAIR RNA (HOTAIR 400 for sense RNA and ashotair 400 for antisense RNA), an approximately 500 base RNA representing the entire A-region from the RepA gene including all tandem repeats (A-region for sense and asa-region for antisense strand), the 397 base mouse telomerase RNA (mouse TR), a 157 base RNA representing the wild type (wt) P4-P6 region within the Tetrahymena group I intron, and a mutant P4-P6 RNA that cannot form tertiary structure 49. (b) Direct binding EMSA of the antisense strand of HOTAIR (ashotair 1-300, binding curve presented in Fig 1c).

6 Supplementary Figure 5. Secondary structure prediction of MBP Secondary structure prediction of MBP performed by mfold using default settings. Presented are the top three hits, those with the lowest Gs. These predictions failed to identify the two-hairpin motif (in dashed frame) that was previously suggested to be enriched in short ncrnas associated with PRC2 28.

7 Supplementary Figure 6. Gene Ontology (GO) analysis for genes that were up-regulated following SUZ12 knockdown. GO analysis yielded a notable network of significant GO terms for developmental processes, with specification at neuron development. GO term IDs indicated in brackets, p-values indicated under each GO term name.

8 Supplementary Figure 7. Original western blots used to generate Figure 7a. Full lanes shown as captured, without further cropping.

9 Supplementary Table 6 ChIP-seq datasets used for Figure 6 Dataset (cell line, method, additional information) GEO Accession Number Data source and/or reference Human Dnd41, ChIP seq, Input GSM ENCODE 59,60 Human Dnd41, ChIP seq, EZH2 GSM ENCODE 59,60 Human Dnd41, ChIP seq, H3K4me3 GSM ENCODE 59,60 Human Dnd41, ChIP seq, H3K27me3 GSM ENCODE 59,60 Human Dnd41, ChIP seq, H3K36me3 GSM ENCODE 59,60 Human HSMMtube, ChIP seq, Control GSM ENCODE 59,60 Human HSMMtube, ChIP seq, EZH2 GSM ENCODE 59,60 Human HSMMtube, ChIP seq, H3K4me3 GSM ENCODE 59,60 Human HSMMtube, ChIP seq, H3K27me3 GSM ENCODE 59,60 Human HSMMtube, ChIP seq, H3K36me3 GSM ENCODE 59,60 Human NHEK, ChIP seq, Input GSM ENCODE 59,60 Human NHEK, ChIP seq, EZH2 GSM ENCODE 59,60 Human NHEK, ChIP seq, H3K4me3 GSM ENCODE 59,60 Human NHEK, ChIP seq, H3K27me3 GSM ENCODE 59,60 Human NHEK, ChIP seq, H3K36me3 GSM ENCODE 59,60 Human NHLF, ChIP seq, Input GSM ENCODE 59,60 Human NHLF, ChIP seq, EZH2 GSM ENCODE 59,60 Human NHLF, ChIP seq, H3K4me3 GSM ENCODE 59,60 Human NHLF, ChIP seq, H3K27me3 GSM ENCODE 59,60 Human NHLF, ChIP seq, H3K36me3 GSM ENCODE 59,60 Mouse E14, RNA Seq GSM Mouse E14, ChIP seq, H3K36me3 GSM Mouse E14, ChIP seq, H3K27me3 GSM Mouse E14, ChIP seq, H3K4me3 GSM Mouse E14, ChIP seq, EZH2 GSM

10 Supplementary Table 7 NGS datasets generated in the scope of this study Cell line Treatment Method Specifications Number of reads aligned to the human genome HEK293T/17 untreated ChIP seq H3K4me2/3 30 M HEK293T/17 untreated ChIP seq EZH2 13 M HEK293T/17 sisuz12 RNA seq biological replicate 1, Poly A isolated 33 M HEK293T/17 sisuz12 RNA seq biological replicate 2, Poly A isolated 37 M HEK293T/17 sictrl RNA seq biological replicate 1, Poly A isolated 29 M HEK293T/17 sictrl RNA seq biological replicate 2, Poly A isolated 33 M

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