AF10 (MLLT10) prevents somatic cell reprogramming through regulation of DOT1L-mediated H3K79 methylation

Abstract

Background: The histone H3 lysine 79 (H3K79) methyltransferase DOT1L is a key chromatin-based barrier to somatic cell reprogramming. However, the mechanisms by which DOT1L safeguards cell identity and somatic-specific transcriptional programs remain unknown.

Results: We employed a proteomic approach using proximity-based labeling to identify DOT1L-interacting proteins and investigated their effects on reprogramming. Among DOT1L interactors, suppression of AF10 (MLLT10) via RNA interference or CRISPR/Cas9, significantly increases reprogramming efficiency. In somatic cells and induced pluripotent stem cells (iPSCs) higher order H3K79 methylation is dependent on AF10 expression. In AF10 knock-out cells, re-expression wild-type AF10, but not a DOT1L binding-impaired mutant, rescues overall H3K79 methylation and reduces reprogramming efficiency. Transcriptomic analyses during reprogramming show that AF10 suppression results in downregulation of fibroblast-specific genes and accelerates the activation of pluripotency-associated genes.

Conclusions: Our findings establish AF10 as a novel barrier to reprogramming by regulating H3K79 methylation and thereby sheds light on the mechanism by which cell identity is maintained in somatic cells.

Keywords: AF10; BioID; DOT1L; H3K79 methylation; Reprogramming; iPSC.

Fig. 1

Identification of proximal interactors of DOT1L via BioID. a Schematic of BirA*-DOT1L fusion protein-expressing vector constructs. b H3K79 di-methylation (H3K79me2) levels in control (gControl) and DOT1L knock-out (gDOT1L) cells expressing either WT or MUT BirA*-DOT1L fusion constructs. Total histone H3 was used as a loading control. c Proximal-protein interactions of DOT1L as revealed via proteomic analyses of biotinylated proteins. Proteins were ordered according to their average coverage and PSM (peptide spectrum matches) values in BirA*-DOT1L wt expressing cells. Asterisk indicates proteins detected only in wt-DOT1L samples. d Schematic of shRNA-mediated somatic cell reprogramming timeline. e Bar graph represents fold change in reprogramming efficiency upon shRNA-mediated gene silencing. Tra-1-60 positive colony numbers of each experiment were normalized to shControl sample. Average of fold changes from independent experiments are indicated (circles). Representative Tra-1-60 stained well images for each shRNA-infected sample are displayed under the bar graph. Error bars represent SEM. *P < 0.05

Fig. 2

AF10 regulates H3K79 methylation and is a barrier to reprogramming. a Schematic for AF10 (MLLT10) gene indicating target sites for the AF10 sgRNAs. b H3K79me2 upon sgRNA-mediated AF10 knock-out. iDOT1L (EPZ004777) was used as a positive control of H3K79me2 depletion. Fibroblasts were treated with DMSO or 3 μM iDOT1L for 10 days. sgAF10 infected dH1fs were selected with puromycin and cultured for 1 week. Total H3 levels are used as loading control. Quantifications were normalized to sgControl sample. c Fold change in the number of Tra-1-60 positive colonies upon sgAF10 expression. P values were determined by one sample t-test; *P < 0.05. Bar graphs show the mean and error bars represent SEM in independent biological replicates (each circle). Representative Tra-1-60 stained wells are shown below the graph. P values were 0.009 for sgAF10-1 and 0.016 for sgAF10-2. d Immunoblot for H3K79me2 in single-cell clones of sgControl and sgAF10 iPSC lines. Total H3 levels were used as loading control. e OCT4, SSEA4 and NANOG immunofluorescence of iPSCs derived from sgControl and sgAF10 expressing fibroblasts. DAPI was used to stain the nuclei. Scale bars represent 50 μm. f Hematoxylin and eosin stained sections of teratomas generated by iPSCs derived from sgControl and sgAF10-1 cells. Black arrowheads show glandular epithelium (endoderm, top), cartilage tissue (mesoderm, middle), and pigmented neural tissue (ectoderm, bottom). Representative images are from one of two independent teratomas. Scale bars represent 20 μm

Fig. 3

AF10 prevents reprograming through its interaction with DOT1L. a Domain organization of wild type and mutant AF10s used in d. L107A mutation abolishes Histone H3K27 binding and OM-LZ deletion impairs DOT1L binding. b Immunoblots for AF10 following streptavidin pulldown from cells expressing BioID-DOT1L and GFP, WT-AF10 or OM-LZ∆ AF10. Top panels show 2% of input samples and bottom panels show Streptavidin pull-down samples. (+) Biotin cells were treated with 50 μM D-Biotin 24 h. c Immunoblots for AF10 following immunoprecipitation with IgG or HA antibodies form cells expressing DOT1L-HA and GFP, WT-AF10 or OM-LZ∆ AF10. Left panels show 2% of input samples and right panel shows HA pull-down samples. d Fold change in the number of Tra-1-60-positive colonies derived from control or AF10 knock-out cells expressing WT, L107A or OM-LZ∆ AF10 cDNAs. P values were determined by one sample t test; *P < 0.05. Bar graph shows the mean and error bars represent SEM in 3 independent biological replicates. Bottom panel shows the H3K79me2 levels with H3 total as a loading control. e Relative cell viability of dH1f cells expressing WT, L107A or OM-LZ∆ AF10. Cell Titer Glo Assay measurements were normalized to uninfected dH1f cells across the indicated time-points. Error bars indicate standard deviation of triplicate samples

Fig. 4

AF10 expression maintains somatic cell identity. a Sample distance matrix of RNA-sequencing replicate samples. b Gene set enrichment analysis (GSEA) of transcriptome data of sgAF10 cells with respect to pluripotency-related and fibroblast-related gene sets. NES: normalized enrichment score, q: false discovery rate (FDR) q-value. c Gene set enrichment analysis (GSEA) of transcriptome data of sgAF10 cells with respect to iDOT1L_DOWN and iDOT1L_UP gene sets. NES: normalized enrichment score, q: false discovery rate (FDR) q-value. d mRNA levels for a set of DOT1L-regulated genes in AF10 knock-out fibroblasts as determined by qRT-PCR. β-actin was used as an internal control. Gene expression levels were normalized to sgControl expressing fibroblasts for sgAF10 samples and DMSO treated cells for iDOT1L samples. Two biological replicates are indicated for sgAF10 samples and bar graph indicates the average of replicates. e Fold change in the number of Tra-1-60-positive colonies derived from AF10 sgRNA expressing cells in the presence of DMSO or a DOT1L inhibitor (iDOT1L; EPZ004777). P values were 0.001 for sgAF10-1 and 0.004 for sgAF10-2. n.s. not significant. f Fold change in the number of Tra-1-60-positive colonies derived from control or AF10 knock-down cells (shAF10) treated with either vehicle (DMSO) or iDOT1L (EPZ004777). P values were 0.034 for shAF10-1 and 0.007 for shAF10-2. n.s., not significant. g Immunoblot for H3K79me2 in double KO cells. Total H3 levels were used as loading control. Replicate of this experiment in Additional file 1: Figure S3c. Quantifications were normalized to gNT sample. h Fold change in the number of Tra-1-60-positive colonies derived from double knock-out cells expressing DOT1L and AF10 targeting sgRNAs. P values were 0.031 for sgAF10-1 and 0.014 for sgAF10-2. n.s. not significant

Fig. 5

Model for AF10’s role in maintaining somatic cell identity and reprogramming. In the presence of AF10, DOT1L-mediated H3K79 methylation and expression of somatic specific genes are high. Upon silencing of AF10, H3K79me2 is reduced, and somatic specific gene expression is downregulated, resulting in higher reprogramming efficiency. This effect is dependent on AF10–DOT1L interaction but not on AF10 histone binding