IDH1 deficiency impaired terminal stage erythropoiesis in an enzymatic activity independent manner.

(A) Representative cytospin images on proerythroblasts, basophilic erythroblasts, polychromatic erythroblasts, and orthochromatic erythroblasts. The red arrows point to the cells that are abnormal nucleus. Scale bar, 10 μm. Quantitative analysis of the percentage of abnormal nuclear cells from three independent experiments. (B) Flow cytometry analysis showed the efficiency of enucleation on day 13 and day 15. Quantitative analysis of enucleation efficiency from three independent experiments. (C)Representative cytospin images of erythroblasts after adding GSH (50 μM) and NAC (10 μM) on day 15. Scale bar, 10 μm. Quantitative analysis of the percentage of the cells with abnormal nucleus. (D) Flow cytometry analysis showed the enucleation efficiency after adding GSH (50 μM) and NAC (10 μM) on day 15. Quantitative analysis of the enucleation efficiency after adding GSH (50 μM) and NAC (10 μM) on day 15 from three independent experiments. (E)Representative cytospin images of erythroblasts after supplement α-KG (50 μM) on day 15. Scale bar, 10 μm. Quantitative analysis of the percentage of the abnormal nucleus from three independent experiments. (F)Flow cytometry analysis showed the efficiency of enucleation after supplement α-KG (50 μM) on day 15. Quantitative analysis of the enucleation efficiency after supplement α-KG (50 μM) on day 15 from three independent experiments. Statistical analysis is from three independent experiments, and the bar plot represents mean ± SD of triplicate samples. Not significant (ns), * p < 0.05, ** p < 0.01, *** p < 0.001.

IDH1 localizes to nucleus in human erythroid cells.

(A-B) Nuclear location of IDH1 (green) on the terminal stages of erythroid cells. Normal human terminal erythroid cells induced from umbilical cord blood-derived CD34+ cells were stained with antibodies targeting IDH1 together with GPA (red) and Hoechst 33342 (blue). Scale bars, 5μm. MFI (mean fluorescent intensity) of IDH1 in nucleus and cytoplasm during the terminal stages of erythropoiesis was shown at the lower panel. (C) Representative immunofluorescence images showed the location of IDH1 at different time points in the human umbilical cord blood-derived erythroid progenitor 2 (HUDEP-2) cell line. IDH1 (green), GPA (red) and Hoechst 33342 (blue). Scale bars, 5μm. MFI of IDH1 in nucleus and cytoplasm of erythroid cells was shown at right panel. Data are presented as the mean ± SD from three independent experiments containing at least 30 cells each. (D) Representative immunofluorescence images of IDH1 (green), GPA (red) and Hoechst 33342 (blue) staining of the paraffin-embedded human bone marrow cells of AML and MDS patients with IDH1 mutation. a. AML1, b. MDS-EB1. Scale bars, 5μm. (E) Representative immunofluorescence images of IDH1 (green), GPA (red) and Hoechst 33342 (blue) staining of the 293T cells. Scale bars, 5μm. (F) Representative western blotting images showed the protein expression level of IDH1 on nucleus and cytoplasm of terminal erythroid cells, 293T cells, human HUDEP2 cell lines, K562 and HEL cell lines. RCC1 was used as nuclear loading control, while Tubulin was used as cytoplasm loading control.

Nuclear IDH1 deletion increased abnormal nuclear cells.

(A) Schematic diagram of selectively knockdown nuclear IDH1. (B) A working model for the construction of Sg-IDH1 HUDEP2 cell line and Sg-NES-IDH1 HUDEP2 cell line. (C) Representative immunofluorescence images of location of IDH1 at D8 in the human umbilical cord blood-derived erythroid progenitor 2 (HUDEP-2) cell line. IDH1 (purple), GPA (red) and Hoechst 33342 (blue). Scale bars, 5μm. (D) Representative cytospin images of control, Sg-IDH1 HUDEP2 cell line and Sg-NES-IDH1 HUDEP2 cell line. Scale bars, 5μm. (E) Quantitative analysis of the percentage of the abnormal nucleus. Statistical analysis is from 3 independent experiments, and the bar plot represents mean ± SD of triplicate samples. Not significant (ns), * p < 0.05, ** p < 0.01, *** p < 0.001.

Deficiency of IDH1 reshape chromatin landscape.

(A) Representative western blotting images showed the expression level of IDH1 in nucleus, chromatin and cytoplasm of erythroid cells cultured on day 15. (B) Representative transmission electron microscopy images showed the distribution of euchromatin and heterochromatin in nuclear on day 15 erythroid cells. (C) Quantitative analysis showed the percentage of euchromatin and heterochromatin from three independent experiments. (D) Representative immunofluorescence images of (a) H3K79me3 (green), (b) H3K9me3 (green), (c) H3K27me2 (green). GPA (red) and Hoechst 33342 (blue) staining of Luciferase-shRNA and IDH1-shRNA erythroid cells cultured on day 15. Scale bar, 10 μm. (E) Representative western blotting images showed the abundance of H3K79me3, H3K9me3, H3K27me2 in nucleus and cytoplasm of Luciferase-shRNA and IDH1-shRNA erythroid cells cultured on day 15. (F) Quantitative analysis of the abundance of H3K79me3, H3K9me3, H3K27me2 in nucleus (above) and cytoplasm (below) of Luciferase-shRNA and IDH1-shRNA erythroid cells cultured on day 15 from three independent experiments. The bar plot represents mean ± SD of triplicate samples. Not significant (ns), * p < 0.05, ** p < 0.01, *** p < 0.001.

Identification of H3K79me3 as the critical factor in response to IDH1 deficiency.

(A) Heatmaps displayed normalized ChIP signal of H3K27me2 (left), H3K79me3 (middle), and H3K9me3 (right) at gene body regions. The window represents ±1.5 kb regions from the gene body. TES, transcriptional end site; TSS, transcriptional start site. (B) Representative peaks chart image showed normalized ChIP signal of H3K27me2 (cyan), H3K79me3 (blue), and H3K9me3 (yellow) at gene body regions. (C) Statistics analysis of total peak number of H3K27me2, H3K79me3, and H3K9me3. (D) The bar plot showed the distribution of ChIPseeker-derived annotations of the genomic loci covered by peaks of H3K79me3, H3K27me2, and H3K9me3. (E) Statistics analysis of promoter peak number of H3K27me2, H3K79me3, and H3K9me3. (F) The bar plot showed GO enrichment analysis of the H3K79me3 peaks linked gene promoter.

IDH1 deletion increase the chromatin accessibility in late-stage erythroid cells.

(A) Heatmaps displayed ATAC signal of Luciferase-shRNA (left) and IDH1-shRNA (right) at TSS. The window represents ±1.5 kb regions from the TSS. (B) Representative peaks chart image showed ATAC signal of IDH1-shRNA (green) and Luciferase-shRNA (blue) at TSS. (C) The volcano map showed differentially accessible peaks of gain (red color) and loss (blue color). (D) The bar plot displayed the distribution of peaks relative to gene features for differentially accessible peaks. (E) The bar plot showed GO enrichment analysis of the gained peaks linked gene promoter. (F) The top regulatory protein-binding sites identified by the HOMER algorithm from differentially accessible peaks. The top 10 motifs were ranked by p value.

Integrated analysis of ChIP-seq, ATAC-seq and RNA-seq.

(A) GSEA analysis showed chromatin associated pathways from DEGs with promoter region marked by H3K79me3. (B) Gene overlap analysis of ATAC-seq, ChIP-seq and RNA-seq. (C) Chromatin associated genes overlap analysis of ATAC-seq, ChIP-seq and RNA-seq. (D) KLF1 binding sites of SIRT1, KMT5A and NUCKS1 locus. (E) DNA pull-down assay showed KLF1 and H3K79me3 could binding to SIRT1 gene promoter. (F) SIRT1 gene locus. Patterns of H3K79me3 modification denoted by ChIP peaks (red) are apparent in IDH1-shRNA increased chromatin accessibility (identified by ATAC-seq) (orange) and gene expression (identified by RNA-seq) (blue).

SIRT1 plays a critical role in mediating the regulatory effect of IDH1 during terminal stage erythropoiesis.

(A) Representative cytospin images of Normal-SRT1720 (0 nM, 100 nM, 500 nM, 2.5 μM) on day 13 and day 15. The red arrows point to the cells with abnormal nucleus. Scale bar, 10 μm. Statistics analysis of abnormal nuclear cells from three independent experiments. (B) Flow cytometric showing the enucleation efficiency of Normal-SRT1720 (0 nM, 100 nM, 500 nM, 2.5 μM) on day 13 and day 15. Statistics analysis of enucleation efficiency from three independent experiments. (C) Representative cytospin images of Luciferase-shRNA, IDH1-shRNA, IDH1-shRNA-EX527 (10 nM, 200 nM) on day 13 and day 15. The red arrows point to the cells that are abnormal nucleus. Scale bar, 10 μm.Statistics analysis of abnormal nuclear cells from three independent experiments. (D) Flow cytometric showing the enucleation efficiency of Luciferase-shRNA, IDH1-shRNA, IDH1-shRNA-EX527 (10 nM, 200 nM) on day 13 and day 15. Statistics analysis of enucleation efficiency from three independent experiments. Statistical analysis is from three independent experiments, and the bar plot represents mean ± SD of triplicate samples. Not significant (ns), * p < 0.05, ** p < 0.01, *** p < 0.001.