Interactions with RNA direct the Polycomb group protein SCML2 to chromatin where it represses target genes
- Howard Hughes Medical Institute, New York University School of Medicine, United States
- Yale University School of Medicine, United States
Figures
Figure 1
Identification of the RNA-binding region of SCML2.
(A) Endogenous RIP from HeLaS3 nuclear extract for the indicated chromatin proteins or with goat (goIg) and rabbit (rbIg) control antibodies. Co-precipitating RNA was labeled in 3′ and resolved by …
Figure 2 with 2 supplements
In vitro characterization of the binding preferences of the SCML2 RBR.
(A) EMSA with 2 (+) and 4 (++) pmol GST-fused RBR and 520 fmol HOTAIR RNA 1–300, 690 fmol nucleosomes, or 275 fmol dsDNA encoding HOTAIR1–300. 5.2 pmol GST were used as a control. Complexes were …
Figure 2—figure supplement 1
Additional EMSAs.
(A) Assay performed as in Figure 2A but using the MBT–DUF fragment of SCML2. Data shown is representative of ≥4 experiments. (B) Assay performed as in Figure 2B but using the MBT–DUF fragment of …
Figure 2—figure supplement 2
Ternary complex EMSAs and pull-downs.
(A) Models for the interpretation of EMSA results shown here and in Figure 2. (I) The multiple shifted bands can be explained by multiple copies of SCML2 fragments binding to a single RNA molecule …
Figure 3 with 1 supplement
Loss of the RBR alters the subnuclear distribution of SCML2A.
(A and B) Salt fractionation of 293T-REx induced to express SCML2A WT (A) and ΔRBR (B) with increasing amounts of doxycycline (dox). The distribution of RNAPIIA and RNAPIIO are shown as …
Figure 3—figure supplement 1
Nuclear distribution of SCML2AD182A and SCML2B.
(A) Domain organization of SCML2A and SCML2B. (B) Salt fractionation experiment as in Figure 3D but using a different mutation (ala instead than gln) in the MBT domain. (C and D) Salt fractionation …
Figure 4 with 1 supplement
SCML2 interacts and shares target genes with BMI1.
(A) Proteins detected by mass spectrometry after affinity purification of SCML2A, SCML2AΔRBR, SCML2B and SCML2BΔRBR from the chromatin fraction of 293T-REx cells. The number of peptides identified …
Figure 4—figure supplement 1
Artificial recruitment of SCML2 and ChIP-seq analysis.
(A and B) ChIP-qPCR quantification of SCML2 (A) and BMI1 (B) association with a reporter locus in 293T-REx cells after artificial tethering of SCML2A or SCML2B. ChIP from cells with the tethering …
Figure 5 with 1 supplement
The role of the RBR in the localization of SCML2A and PRC1 to target genes.
(A) Normalized read density profiles for SCML2 (red), HA (green), and BMI1 (blue) in cells expressing N3-SCML2A WT (light colors) or ΔRBR (dark colors). Three RBR-dependent and one RBR-independent …
Figure 5—figure supplement 1
Validation and replication of ChIP-seq for SCML2 WT and ΔRBR.
(A) Western blot of total cellular extract from 293T-REx carrying a stable integration of an inducible shRNA for the 3′ UTR of the endogenous SCML2, and inducible N3-tagged transgenes for SCML2A WT …
Figure 6 with 1 supplement
RIP-seq for SCML2 in 293T-REx and K562 cells.
(A and B) HA IP from 293T-REx expressing the empty tag alone (N3), N3–SCML2A WT (WT) or N3–SCML2AΔRBR (Δ). After washing, the bound fraction was divided into two parts; one was analyzed by western …
Figure 6—figure supplement 1
Examples of SCML2-associated RNAs.
(A) As in Figure 6D but plotting the enrichment values for 12,124 protein-coding mRNAs with at least one mapped read in each sample. (B–E) ChIP and RIP profiles in K562 cells. Four representative …
Figure 7 with 1 supplement
Functional consequences of SCML2 depletion.
(A) Western blot for SCML2 in 293T-REx treated for 3 or 5 days with control siRNAs (Ctrl) or two different siRNAs against SCML2 (#5 and #6). The band marked with an asterisk corresponds to a …
Figure 7—figure supplement 1
Additional RNA-seq analyses.
(A) Overlap of all DEGs with p<0.05 and fold-change >1.5 and genes closest to an SCML2 ER. p-values are from the hypergeometric distribution. (B) Same as (A) but DEGs were divided in downregulated …
Author response image 1
Enrichment of SCML2-bound RNAs at SCML2 ERs on chromatin.
(A) The cis enrichment score was calculated by first counting the reads per million sequenced (RP10M) for each RIP sample in 5kb windows spanning the SCML2 ERs, dividing by the RP10M for the same …
Author response image 2
Immunoprecipitations for SCML2 and EZH2.
(A) IPs were performed in native conditions (200 mM KCl, 0.05% NP-40) with the indicated antibodies (C-20 and 86A are two different commercial antibodies against MLL). Flow-through (FT) and IP …
Author response image 3
Comparison of WT and ΔRBR ChIP-seq on unfiltered SCML2 ERs.
(A) Enrichment is defined as the normalized number of reads for the indicated sample in 1 kb regions centered on the ER summit in 293T-REx expressing N3–SCML2 WT minus the reads in the same regions …
Author response image 4
SCML2 expression in parental 293T-REx and N3 cell lines.
293T-REx either untreated (-) or treated with 1 µg/ml doxycycline for 48 h were lysed and proteins resolved on SDS-PAGE. Immunoblots for SCML2 (top) and CDK2 (bottom) as a loading control are shown. …
Additional files
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Supplementary file 1
(A) GO enrichment analysis for SCML2 targets in 293T-Rex. (B) SCML2 target genes in 293T-REx. (C) Differentially expressed genes after SCML2 KD. (D) Oligonucleotide sequences.
- https://doi.org/10.7554/eLife.02637.017
