Fig 2: ET-1 promotes the crosstalk between BRG1/BRM and ASH2. (A and B) HASMCs were transfected with indicated siRNAs followed by treatment with ET-1 (1 nM). ChIP assays were performed with anti-H3K4Me2 (A) or anti-H3K4Me3 (B). (C and D) HASMCs were treated with or without ET-1 (1 nM). Re-ChIP assays were performed with indicated antibodies. (E) HASMCs were transfected with indicated siRNAs followed by treatment with ET-1 (1 nM). ChIP assays were performed with anti-ASH2. (F) SW-13 cells were transfected with indicated reporter constructs along with indicated expression constructs followed by treatment with or without ET-1 (1 nM). Data are expressed as relative luciferase activities. All experiments were repeated at least three times. *, P < .05.

Fig 3: 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 4: Depletion of Setd1a inhibits ß-globin gene transcription as well as recruitment of the Setd1a complex and H3K4me3 levels at the ß-globin promoter. (A) Comparison of H3K4me3 enrichment at stemness promoters and erythroid promoters in ES cells and differentiated EBs. (B) Western blot analysis of Setd1a protein levels in R1E ES cells harboring shRNA against Setd1a compared to control; a-tubulin served as a loading control. (C) Stable ESC clones harboring vector control or shSetd1a-expressing constructs were induced with EPO and other cytokines and total RNA was extracted at day 19. Expression of genes important for erythroid differentiation was analyzed by qRT-PCR. (D) Stable ESC clones harboring vector control or shSetd1a-expressing constructs were induced with EPO and other cytokines for erythroid differentiation and total RNA was extracted at day 0, 4 and 19. The ßmaj-globin transcripts were analyzed by qRT-PCR and normalized with ß-actin. (E) ChIP analysis of H3K4me1, me2 and me3 patterns over the ß-globin locus at day 19 upon the Setd1a KD. (F) ChIP analysis of ASH2L (TOP) and Setd1a (Bottom) recruitment across the ß-globin locus at days 19 upon Setd1a KD. Data are collected from at least three independent experiments and shown as mean ± SD. *P < 0.05; **P < 0.01 by student t-test.

Fig 5: Molecular interaction between GATA3 and UTX/MLL4 complex.a GST pull-down experiments were performed using bacterially expressed GST-fusion proteins and proteins prepared using in vitro transcription/translation, as indicated. b GATA3 directly interacts with JmjC domain of UTX in vitro. GST pull-down assays were performed using a series of truncation constructs of UTX and in vitro transcribed/translated GATA3. c GATA3 directly interacts with PHD-WH domain of ASH2L in vitro. GST pull-down assays were conducted using a series of truncation constructs of ASH2L and in vitro transcribed/translated GATA3. d GATA3 directly interacts with N-terminal region of RBBP5 in vitro. GST pull-down assays were performed using a series of truncation constructs of RBBP5 and in vitro transcribed/translated GATA3. e Identification of GATA3 domains required for interaction with UTX, ASH2L, or RBBP5. GST pull-down assays were conducted using a series of truncation constructs of GATA3 and in vitro transcribed/translated proteins, as indicated. f Schematic depiction of molecular interactions between GATA3 and MLL4 complex