Fig 2: LANA interacts with, and inhibits, the MLL1 complex. (A) Schematic diagram of LANA. LANA proline rich (P), aspartate and glutamate (DE), glutamine (Q), putative leucine zipper (LZ), glutamate and glutamine (EQE) regions. Residues 5–13 bind histones H2A/H2B (34,77). LANA 996 –1139 self-associates to bind terminal repeat (TR) DNA and independently associates with mitotic chromosomes. (B and C) Co-immunoprecipitation (Co-IP) of GFP-LANA regions with (B) MLL1 or (C) WDR5 after expression in 293T cells. IPs were performed using anti-Flag antibody. Asterisks denote GFP-LANA fusions. (D) LANA?33–888, or WDR5 (in lanes at right) co-precipitation with GST-MLL1-SET (E) ASH2L, RbBP5, WDR5 and LANA?33–888 co-precipitation with GST-MLL1-SET. (D, E) Triangles at top indicate increasing LANA?33–888 and asterisk indicates degradation products. (F) Histone methyltransferase activity of the MLL1 complex after incubation with increasing amounts of LANA. Results are the means of three independent experiments. Standard deviation is shown. (G) Sequence alignment of MLL1WIN with LANAWIN starting at LANA residue 21. Yellow highlight indicates the conserved arginine. (H) Co-IP of GFP-LANA-1–32 or GFP LANA-1–32 containing C23P/R24A/K25A substitutions with Flag-WDR5 after co-expression in 293T cells. (I) Co-precipitation of WDR5 with MBP-LANA?33–888 in the presence or absence MM401 or enantiomer control. Two left lanes, WDR5 or MBP-LANA?33–888 alone. Box encloses WDR5 bands. (J) ASH2L, RbBP5, WDR5, or MLL1-SET co-precipitation with MBP-LANA?33–888 after incubation in the presence or absence MM401 or enantiomer control. Proteins were detected by immunoblot except MBP-LANA?33–888, which was detected by Coomassie blue. (D, E, I) Proteins were detected with Coomassie blue.

Fig 3: A subset of MLL4-bound genomic regions overlaps with H4K16ac. SV40T-immortalized Moff/f;Cre-ER brown preadipocytes were treated with 4-hydroxytamoxifen to delete Mof. Cells were collected for reverse transcriptase PCR (RT-PCR), western blot and chromatin immunoprecipitation–sequencing (ChIP-Seq) analysis. a Schematics of targeting allele and knockout allele of Moff/f mice. In the targeting allele, a single loxP site was inserted in the intron before exon 4 and a neomycin selection cassette flanked by loxP sites was inserted in the intron after exon 6. The locations of RT-PCR primers P1–P2 (used in Fig. 4b) and P3–P4 (used in Fig. 4c) are indicated by arrows. b Quantitative RT-PCR confirmation of Mof deletion in preadipocytes. The data are presented as mean ± s.d. Two technical replicates from a single experiment were performed. Source data are provided as a Source Data file. c RT-PCR confirmation of wild-type and truncated Mof mRNA. d Western blot analysis of MLL4, UTX, and MOF. PARP1 and RbBP5 were used as loading controls. e Western blot analysis of histone modifications (also see Supplementary Fig. 15). f Heat maps around MLL4-binding sites. H4K16ac ChIP-Seq data were normalized with global H4K16ac levels measured by western blot. Published H3K4me1 data were obtained from GEO database (GSE74189)49. g Pie chart illustrating that 35% of MLL4-binding sites overlap with H4K16ac. h Heat maps of MLL4 and H4K16ac around 17,457 H4K16ac+ MLL4-binding sites

Fig 4: H3K4 methylation activities of human KMT2Core complexes. (A) A schematic diagram of KMT2Core fragments used for the reconstitution of complexes and subunit associations deduced from (B). The n-SET, pre-SET, SET and post-SET (hatched box) domains and Win (red box) motif within each KMT2 protein are depicted. Numbers indicate amino acid residues of each protein. DPY30(×2) indicates the dimeric association of DPY30, which was established in recent structural analyses of KMT2 complexes (29,31,32). (B) SDS-PAGE/Coomassie blue staining (left) and immunoblot analyses (right) of purified KMT2Core complexes reconstituted with baculoviruses expressing FLAG-tagged KMT2Core protein (A), WDR5, RbBP5, ASH2L, DPY30 and CFP1. KMT2Core proteins are marked by asterisks. Note that an insect protein contaminant around 100 kDa (marked by dots) was found sometimes in the complexes prepared by single-step affinity purification and became more prominent when a large volume of samples (due to low concentration) was loaded. This protein could be removed by gel filtration and did not affect the H3K4 methylation activities of the affinity-purified complexes as demonstrated in several KMT2Core complexes (see Supplementary Figures). (C) Differential H3K4 methylation activities of KMT2Core complexes towards free histone H3 (H3) substrate. H3 was subjected to in vitro HMT assays with the indicated KMT2core complexes (7.5 nM). (D and E) Differential H3K4 methylation activities of KMT2core complexes towards chromatin substrates. Recombinant chromatin templates assembled with unmodified histone H2B (unmod)- or mono-ubiquitylated H2B at lysine 120 (H2Bub)-containing octamers were subjected to in vitro HMT assays with the indicated KMT2Core complexes at a concentration of 5 nM (D) or with the indicated concentrations of SET1ACore and SET1BCore complexes (E). H3K4 methylation status was monitored by immunoblotting with the indicated antibodies in this and other figures. Abbreviations: unmod, unmodified histone H2B; H2Bub, K120-ubiquitylated H2B; H3K4me1, K4-monomethylated H3; H3K4me2, K4-dimethylated H3; H3K4me3, K4-trimethylated H3.

Fig 5: HOTTIP lncRNA binding surface overlaps with the RbBP5 binding surface on WDR5.(A) Crystal structure of WDR5 (PDB:3P4F, visualized with PyMol) reveal that mutations abrogating HOTTIP RNA binding align along a cleft between blades 5 and 6, opposite the H3K4/MLL binding site. This binding surface overlaps the site for RbBP5. Top: schematic of tested and HOTTIP binding mutations. Bottom: magnification of HOTTIP binding cleft. Yellow: MLL peptide. Blue: RbBP5 peptide. (B) qRT-PCR results of indicated GST-WDR5 mutants tested by in vitro assay for binding to HOTTIP RNA or control Histone 1H2BG RNA. (C) RbBP5 peptide competition assay indicates that RbBP5 amino acids 371–381 can fully inhibit HOTTIP binding to WDR5. Wild type data same as in Figure 1B. (D) Average fold pulldown of full length HOTTIP by cell-based native RNA immunoprecipitation of select WDR5 mutants. Top: qRT-PCR results of RNA immunoprecipitation of FLAG-WDR5 mutants. All values are normalized to input, then to FLAG pulldown of wild type and positive control D107A. Negative RNA controls (U1, HOTAIR) and reaction without reverse transcription (-RT) show minimal enrichment (Figure 1—figure supplement 1, data not shown). Bottom: representative western blots of immunoprecipitations.DOI: http://dx.doi.org/10.7554/eLife.02046.003