Nascent RNA antagonises the interaction of a set of regulatory proteins with chromatin
A number of regulatory factors are recruited to chromatin by specialised RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonised the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors including BAF, NuRD, EHMT1 and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonising the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.
Steps to reproduce
Cell fractions and extracts were quantified by BCA (Pierce). Samples were boiled in Laemmli buffer or NuPAGE buffer and equal amounts (10 µg) of cell fractions (equal volumes for immunoprecipitates) were loaded for each treatment. Proteins were detected with primary antibodies to DNMT3A (Abcam ab2850), TRRAP (Abcam ab73546), SET (Abcam ab181990), LMNA (Abcam ab26300), HMGN1 (Bethyl Laboratories A302-263), UTF1 (Abcam ab24273), CDK9 (Abcam ab6544), beta-Tubulin (Abcam ab6064), RUVBL2 (Abcam ab36569), SF3A3 (Bethyl Laboratories A302-506A), ZFP57 (Abcam ab45341), FUS (Novus Biologicals 100-565), LARP7 (Novus biologicals A303-723A), ILF3 (Abcam ab92355), CyclinT1 (Abcam ab184703), UBTF (Santa Cruz sc-13125), KAP1/TRIM28 (Abcam ab3831), SUZ12 (Santa Cruz sc-46264), LEO1 (Bethyl Laboratories A300-174A), HNRNPU (Abcam ab10297), EHMT1 (Abcam ab41969), STAG1 (Abcam ab4457), STAG2 (Abcam 4463), SMARCC1 (Abcam ab172638), BRD4 (Santa Cruz sc-48772), INO80 (ProteinTech 18810-1-AP), CHD1 (Cell Signalling D8C2), CHD4 (Abcam ab70369), CHD8 (Bethyl A301-224A), P300 (Santa Cruz sc-585), KDM2A/JHDM1A (Bethyl A301-475A), MPP8 (Santa Cruz sc-398598), Pol II S2P (Abcam ab5095), Pol II S5P (Millipore 05-623), total Pol II (Santa Cruz sc-899), Beta-actin (Cell Signalling 4967S), histone H3 (Abcam ab1791) and H2A.X (Ser139) (clone JBW301) (Sigma 05-636-I). Proteins were visualised using Amersham ECL western blotting detection reagent (GE) and detected using an ImageQuantLAS 4000 imager and ImageQuantTL (GE). CLIP was performed as described (Huppertz et al., 2014) with the following differences: cells were irradiated with 0.2 J/cm2 of 254 nm UV light in a Stratalinker 2400 (Stratagene). 5x106 cells were used per IP and were lysed in 1 ml of lysis buffer. Lysates were passed through a 27 G needle, 4 U/ml of DNase Turbo (Ambion AM2238) and RNase I (Ambion AM2294, range between 1-20 U/ml) added, and incubated in a thermomixer at 37 °C and 1100 rpm for 3 minutes. 5 μl of α-RUVBLl2 (Abcam ab36569), 5 μg α-UBTF (Santa Cruz sc-13125), 5 μg α-INO80 (ProteinTech 18810-1-AP), α-CHD4 (Abcam ab70369), α-SMARCC1 (Abcam ab172638), α-EHMT1 (Abcam ab41969), α-CDK9 (Santa Cruz sc-484), α-LARP7 (Bethyl A303-723A), α-SMARCA5/SNF2H (Abcam ab3749), α-INTS11 (Bethyl A301-274A), α-CCNT1 (Abcam ab238940), α-CDK9 (Santa Cruz sc-484), α-LARP7 Bethyl A303-723A) or non-specific IgG (Abcam ab46540) antibody was used per experiment and bound to 50 µl of pre-washed Dynabeads protein G beads (Invitrogen 10003D) for 1 hr at RT. Antibody-bound beads were then incubated with lysate for 5 hrs at 4 °C. Beads were washed 3 times with 900 µl of high-salt buffer (supplemented with 1 M urea) and twice with 900 µl of wash buffer. After transfer, the membrane was washed twice with 1x PBS, exposed overnight to a phosphoimager screen (Fuji), and visualised with an Amersham Typhoon Trio image scanner.