Promiscuous RNA binding by WDR5 remodels the KMT2A (MLL1) histone methyltransferase complex to an inactive state

Description

Chromatin modifying complexes are critical in gene regulation, yet their proposed interactions with RNA remain poorly understood. Counter to prior models of specific recruitment, we find that WDR5, an essential subunit of the MLL1/KMT2A histone methyltransferase complex, binds RNA promiscuously. Using a stringent approach, we demonstrate that WDR5 directly engages a diverse pool of cellular RNAs, dictated primarily by RNA abundance rather than specific motifs. Equilibrium binding assays show that RNA length, rather than sequence, governs high affinity. WDR5 binds to RNA through multiple surfaces, some of which overlap with MLL1 complex subunit interfaces. Strikingly, we find that RNA binding disrupts the MLL1 complex by competitively displacing WDR5 from these critical protein-protein interactions, leading to a marked inhibition of MLL1 catalytic activity. Further experiments in human cell lines suggest that this disassembly mechanism may operate to homeostatically down-regulate MLL1 complex activity, thereby integrating transcriptional activity with chromatin state.

Steps to reproduce

For Western Blots: Samples were loaded for SDS-PAGE and run at 90V for 1.5 hours. Gels were transferred to PVDF (Millipore, Immobilon-P SQ) in modified Towbin’s buffer (25 mM Tris pH 8.0, 192 mM glycine, 10% Methanol, 0.04% SDS [w/v]). Membranes were then blocked for at least one hour in 2% (w/v) Amersham ECL Prime blocking agent (Cytiva) in TBST. Primary antibodies in the same blocking solution were used as follows: 1:1000 α-ASH2L (Bethyl A300-107A), 1:1000 α-FLAG (Sigma M2), 1:1000 α-GAPDH (Cell Signaling 5174), 1:20,000 α-H3 C-term (Epicypher 13-0001), 1:1000 α-MLL1-C (Cell Signaling 14197), 1:1000 α-MLL2-C (Cell Signaling 63735), 1:2000 α-Pol II S5P (Cosmo Bio, MCA-MABI0603-100-EX), and 1:1000 α-RBBP5 (Bethyl, A300-109A). Secondary goat anti-mouse (1:10,000; Thermo 31432) and goat anti-rabbit (1:10,000; Sigma A6154) HRP-conjugated antibodies were detected using ECL Select (Cytiva; RPN2235) on a Fuji LAS-4000 imager. Microscopy of RNA perfusion in isolated nuclei: One 10 cm plate of FLAG-WDR5 T-REx™ 293 cells at 70-80% confluency was used per replicate for the nuclear fractionation. Cells were washed twice with PBS, scraped in 1 ml PBS on ice and centrifuged at 500g for 10 minutes at 4°C. Cells were resuspended in 3 packed cell volumes (PCV) of hypotonic buffer HB (10 mM HEPES pH 7.8, 10 mM KCl, 1.5 mM MgCl2, 340 mM sucrose, 10% glycerol (v/v), 0.5 mM PMSF, 0.5 mM DTT, and 1X protease inhibitor cocktail). Cells were lysed by adding an equal volume of HB supplemented with 0.2% Triton-X100 (v/v) with incubation at 4°C with gentle rotation for 12 minutes. Nuclei were collected by centrifugation at 500g for 10 minutes at 4°C. The supernatant was saved as the cytoplasmic fraction. The crude nuclei were resuspended in 3 PCV of hypotonic buffer HB and cleaned through a sucrose cushion (5 ml; 30% w/v sucrose, HEPES, pH 7.8, 1.5 mM MgSO4) with centrifugation at 1300g for 12 minutes at 4˚C. Recovered nuclei were resuspended in 6 PCV of hypotonic buffer HB supplemented to 150 mM KCl and divided equally in three tubes. In vitro transcribed fluorescein-labeled RNA was added to a final concentration of 1 µM. Nuclei suspensions were then subjected to gentle rotation at 4°C for 1.5 hours to isolate soluble nuclear extract. An aliquot of nuclear suspension was stained with DAPI and images were acquired using a Stellaris 5 microscope.

Institutions

• University of Chicago

  Illinois, Chicago

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