Fig 1: Effects of KDM1A loss in PSC differentiation to specific lineages. A, UMAP plot showing the cell types present in the day 6 embryoid body generated from Kdm1a-deficient mESCs (n = 7623) and control (n = 10,214) (29). Cells are colored based on the clusters they belong to. B, stacked violin plots of the integrated data showing the expression profile of marker genes: pluripotency (Pou5f1/OCT4), naïve pluripotency (Zfp42), formative/primed pluripotency (Pou3f1/OCT6, Fgf5), NE (Sox2, Sox1), ME (T), Meso (Pdgfra, Hand1), HG (Kdr), SMCs (Tagln), and Endo (Cldn6). C, GSEA plots showing the enrichment results of the custom gene set of the S15down genes (20 genes) in Kdm1a-deficient cells within subpopulations corresponding to naïve ESCs and formative/primed EpiLCs. D, scatter plots showing expression patterns of genes in Kdm1a-deficient cells within subpopulations corresponding to naïve ESCs and formative/primed EpiLCs. Average expression of log-normalized genes, difference between the transcriptomic profiles of WT and Kdm1a KO. E and F, half-violin plots showing (E) the distribution of the mRNA expression levels of indicated genes in either WT or Kdm1a-deficient cells within the subpopulations corresponding to naïve ESCs and formative/primed EpiLCs, and (F) the distribution of Mest mRNA expression level in either WT or Kdm1a-deficient cells within the indicated subpopulations. Each cell was treated as an independent replicate, and differences in gene expression between conditions were analyzed using the Wilcoxon rank sum test. G, model for the roles of the SMAD1/5–KDM1A complex in metastable-naïve mouse ESCs cultured in serum plus LIF (SL). SMAD1/5 recruit KDM1A to the target sites and demethylate H3K4me1/2. The SMAD1/5–KDM1A complex, which does not contain SMAD4, represses a subset of SMAD1/5 target genes related to developmental processes toward several lineages, including neuroectoderm (NE). cDNA, complementary DNA; DMR, differentially methylated region; DNMT1, DNA methyltransferase 1; EB, embryoid body; Endo, endoderm; ES, enrichment score; GEO, Gene Expression Omnibus; GO, Gene Ontology; HG, Flk1+ hemangioblast; IAD, interferon-activating domain; IP, immunoprecipitation; LSD1, lysine-specific histone demethylase 1; mAG, monomeric Azami-Green; ME, mesendoderm; Meso, mesoderm; MH2, Mad Homology 2 domain; NE, neuroectoderm (Sox1+ NE progenitor); NM, nascent mesoderm; PEG, paternally expressed gene; qRT–PCR, quantitative RT–PCR; SMC, smooth muscle cell; TF, transcription factor; TGF-β, transforming growth factor β; VE, visceral endoderm.
Fig 2: Physical interaction between SMAD1 and KDM1A. A, coimmunoprecipitation of Myc-tagged SMAD1 with the indicated FLAG-tagged coregulators in HEK293T cells. FLAG immunoprecipitates were subjected to Western blotting. Blots are representative of n = 3 independent biological replicates. B, in vitro pulldown of KDM1A (0.5 μg) and/or SMAD4 (0.5 μg) with SMAD1 (0.5 μg) as bait. Blots are representative of n = 3 independent biological replicates. Right, quantification of band intensity is shown on the right. Results of n = 3 independent biological replicates are presented as scatter plots with bar graphs, which indicate mean ± SD. Differences between the conditions were analyzed by Tukey's honestly significant difference test corrected for multiple comparisons. C, coimmunoprecipitation of endogenous SMAD1/5 protein with KDM1A in WT mESCs. SMAD1/5 immunoprecipitates were subjected to Western blotting. Blots are representative of n = 3 independent biological replicates. Right, quantification of each lane of the blots. Results of n = 3 independent biological replicates are presented as scatter plots with bar graphs, which indicate mean ± SD. Differences between the conditions were analyzed by Tukey's honestly significant difference test corrected for multiple comparisons. D and E, schematic illustrations of interaction between SMAD1 and KDM1A. F, coimmunoprecipitation of HA-tagged KDM1A with the indicated FLAG-tagged SMAD1 mutants in HEK293T cells. FLAG immunoprecipitates were subjected to Western blotting. Blots are representative of n = 3 independent biological replicates. G, coimmunoprecipitation of HA-tagged KDM1A with the indicated FLAG-tagged MH2 domain of SMAD protein or IAD of IRF3 in HEK293T cells. FLAG immunoprecipitates were subjected to Western blotting. Blots are representative of n = 3 independent biological replicates. H, coimmunoprecipitation of Myc-tagged SMAD1 with the indicated FLAG-tagged KDM1A mutants in HEK293T cells. FLAG immunoprecipitates were subjected to Western blotting. Blots are representative of n = 3 independent biological replicates. I, coimmunoprecipitation of Myc-tagged SMAD1 with the indicated FLAG-tagged KDM1A mutants in HEK293T cells. FLAG immunoprecipitates were subjected to Western blotting. Blots are representative of n = 3 independent biological replicates. ∗, ∗∗, ∗∗∗∗, and ns represent p < 0.05, 0.01, 0.0001, and not significant, respectively. HEK293T, human embryonic kidney 293T cell line; mESC, mouse embryonic stem cell; MH2, Mad Homology 2 domain.
Supplier Page from Abcam for Recombinant Human Smad4 protein (His tag N-Terminus)