Journal: Science Advances

Article Title: Dual mechanism of inflammation sensing by the hematopoietic progenitor genome

doi: 10.1126/sciadv.adv3169

Figure Lengend Snippet: ( A ) Strategy for attenuating TLR signaling. Embryonic day 14.5 (E14.5) fetal livers from two timed mating schemes (−77 +/− and −77 +/− ; Myd88 −/− ) were harvested, and lineage-depleted progenitors were isolated and treated with IFN-γ (1 ng/ml), Pam 3 CSK 4 (100 ng/ml), or both for 4 hours. In parallel, fetal livers were harvested for flow cytometry to quantify the cellular composition and GP versus MP ratios. ( B ) Representative E14.5 embryos obtained from the mating schemes in (A). ( C ) Total cellularity of E14.5 fetal livers. ( D ) The expression of select genes was quantitated using RT-qPCR. ( E ) The responsiveness of Tnf and Cxcl10 to IFN-γ and Pam 3 CSK 4 was quantitated using RT-qPCR. Statistics in (C) and (D) were one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test and in (E) were multiple unpaired t tests. ( F ) Quantitation of MP and GP frequency within the Ly6C + GMP population in E13.5 to E14.5 fetal liver obtained from eight litters. ( G ) Quantitation of progenitor populations in E14.5 fetal livers obtained from seven litters. Error bars for all plots represent means ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001; Welch’s unequal variance t tests. ( H ) Model depicting Irf8 , but not Myd88 , ablation reverses the GP:MP imbalance resulting from Gata2 −77 enhancer deletion. Although the levels of GPs and MPs were reduced by Myd88 ablation, the ratio was not altered.

Article Snippet: Affinity-purified rabbit polyclonal anti-GATA2 antibody (1.2 μg) ( , ), anti-PU.1 mouse antibody (1.2 μg; Santa Cruz Biotechnology, sc-390405), H3K4me3 rabbit antibody (1 μg; Abcam, ab213224), or rabbit IgG isotype-matched control (1 μg; Invitrogen, 10500C) was added to each tube.

Techniques: Isolation, Flow Cytometry, Expressing, Quantitative RT-PCR, Quantitation Assay

Journal: Science Advances

Article Title: Dual mechanism of inflammation sensing by the hematopoietic progenitor genome

doi: 10.1126/sciadv.adv3169

Figure Lengend Snippet: ( A ) Strategy for gene expression analysis in ER-HOXB8–immortalized (hi) fetal liver myeloid progenitors. Wild-type (hi-77 +/+ ), GATA2 low (hi-77 −/− ), and GATA2/PU.1 low (hi-77 −/− ; Spi1 URE −/− ) progenitors were treated with vehicle, IFN-γ (1 ng/ml), Pam 3 CSK 4 (100 ng/ml), or both for 4 hours ( n = 4 biological replicates). Total RNA was isolated for RNA-seq. ( B ) RT-qPCR analysis of Gata2 and Spi1 expression ( n = 4 to 6 biological replicates). ( C ) Left: Representative Western blot of GATA2 and PU.1 expression. β-Actin was used as a loading control. Right: Densitometric analysis of band intensities normalized to β-Actin ( n = 4). M r is the apparent molecular mass in kDa. Statistics in (B) and (C): One-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001. ( D ) Left: Overlap of activated differentially expressed genes (DEGs) in hi-77 −/− and hi-77 −/− ; Spi1 URE −/− cells in response to Pam 3 CSK 4 . A DEG had |log 2 (fold change)| ≥ 1, adjusted P -value <0.05, and transcripts per million (TPM) ≥ 1 in all replicates in at least one of the two conditions compared. Right: Heatmap depicting expression of activated DEGs in response to Pam 3 CSK 4 and presented as z -scores of TPMs. Representative genes are indicated. ( E ) Left: Overlap of activated DEGs in hi-77 −/− and hi-77 −/− ; Spi1 URE −/− cells in response to IFN-γ. Right: Heatmap depicting expression of activated DEG in response to IFN-γ and presented as z -scores of TPMs. ( F ) Left: Overlap of activated DEGs in hi-77 −/− and hi-77 −/− ; Spi1 URE −/− cells in response to IFN-γ and Pam 3 CSK 4 . Right: Heatmap depicting expression of activated DEGs upon combinatorial signaling and presented as z -scores of TPMs. Each section of the heatmaps in (D) and (E) corresponds to each section of Venn diagrams in the same order. ( G ) TLR gene ( Tlr1 and Tlr2 ) and IFN-γ receptor subunit ( Ifngr1 and Ifngr2 ) expression in GATA2 low and GATA2/PU.1 low progenitors.

Article Snippet: Affinity-purified rabbit polyclonal anti-GATA2 antibody (1.2 μg) ( , ), anti-PU.1 mouse antibody (1.2 μg; Santa Cruz Biotechnology, sc-390405), H3K4me3 rabbit antibody (1 μg; Abcam, ab213224), or rabbit IgG isotype-matched control (1 μg; Invitrogen, 10500C) was added to each tube.

Techniques: Gene Expression, Isolation, RNA Sequencing, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Science Advances

Article Title: Dual mechanism of inflammation sensing by the hematopoietic progenitor genome

doi: 10.1126/sciadv.adv3169

Figure Lengend Snippet: ( A ) GATA2-repressed genes that were activated synergistically in hi-77 −/− cells by IFN-γ and Pam 3 CSK 4 were parsed into PU.1-activated, PU.1-repressed, and PU.1-insensitive cohorts. The synergistic genes were defined as inflammation-activated DEGs with a larger |log 2 (fold change)| in combination versus vehicle than that of IFN-γ versus vehicle and Pam 3 CSK 4 versus vehicle. ( B ) Top: Models depicting three different modes of regulation of signal-dependent genes by GATA2 and PU.1. Bottom: RT-qPCR analysis of representative synergistic genes with different GATA2/PU.1 regulatory modes was quantitated with RT-qPCR ( n = 4). ( C ) The responsiveness of prioritized synergistic genes in primary lineage-depleted progenitors isolated from E14.5 fetal livers of −77 +/+ ( n = 6) and −77 −/− ( n = 4) embryos (pooled from three litters). Statistics in (B) and (C): Multiple unpaired t tests. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Affinity-purified rabbit polyclonal anti-GATA2 antibody (1.2 μg) ( , ), anti-PU.1 mouse antibody (1.2 μg; Santa Cruz Biotechnology, sc-390405), H3K4me3 rabbit antibody (1 μg; Abcam, ab213224), or rabbit IgG isotype-matched control (1 μg; Invitrogen, 10500C) was added to each tube.

Techniques: Quantitative RT-PCR, Isolation

Journal: Science Advances

Article Title: Dual mechanism of inflammation sensing by the hematopoietic progenitor genome

doi: 10.1126/sciadv.adv3169

Figure Lengend Snippet: ( A ) Schematic depicting the chromatin occupancy analysis. hi-77 +/+ , hi-77 −/− , and hi-77 −/− ; Spi1 URE −/− cells were stimulated with vehicle or IFN-γ (1 ng/ml) and Pam 3 CSK 4 (100 ng/ml) for 4 hours. Cells were cross-linked with 0.1% formaldehyde for 2 min before CUT&Tag analysis with antibodies against GATA2 ( n = 4), PU.1 ( n = 4), or H3K4me3 ( n = 2). IgG with hi-77 +/+ served as a negative control. ( B ) Comparison of median PU.1 (left) and GATA2 (right) CUT&Tag signals at 217 DEGs in untreated versus inflammation-activated hi-77 −/− and hi-77 +/+ cells, respectively. DEGs were colored by their cluster identities. The median signal is the median of signals from 1 kb upstream to 1 kb downstream of the transcription start site (TSS) of each DEG ( n = 4). ( C ) Heatmaps depicting PU.1 CUT&Tag signals in untreated versus inflammation-treated hi-77 +/+ (WT), hi-77 −/− (G low ), and hi-77 −/− ; Spi1 URE −/− (GP low ) progenitors. A total of 217 DEGs were clustered as inflammation-activated and inflammation-independent and ranked by their log 2 (fold change) on the basis of RNA-seq data of hi-77 −/− (inflammation) versus hi-77 −/− (vehicle). FC, fold change. ( D ) Range of signals for three categories of DEGs. The range is defined by the average signals of the four replicates from the same condition and presented as lines in the figure. Average signals are calculated over (TSS − 1 kb, TSS + 1 kb) with a bin width of 100 bp. ( E ) Representative CUT&Tag profiles (replicate 2) for genes with inflammation-induced GATA2 and PU.1 occupancy ( Cd40 and Gbp5 ) and genes prebound by GATA2 and PU.1 ( Ccl3 and Cd69 ). ( F ) Model of signal-dependent GATA2 and PU.1 occupancy at loci in GATA2 low progenitors. ( G ) Model depicting inflammation-independent GATA2 and PU.1 occupancy at inflammation-activated genes.

Article Snippet: Affinity-purified rabbit polyclonal anti-GATA2 antibody (1.2 μg) ( , ), anti-PU.1 mouse antibody (1.2 μg; Santa Cruz Biotechnology, sc-390405), H3K4me3 rabbit antibody (1 μg; Abcam, ab213224), or rabbit IgG isotype-matched control (1 μg; Invitrogen, 10500C) was added to each tube.

Techniques: Negative Control, Comparison, RNA Sequencing

Journal: Science Advances

Article Title: Dual mechanism of inflammation sensing by the hematopoietic progenitor genome

doi: 10.1126/sciadv.adv3169

Figure Lengend Snippet: ( A ) Schematic of promoters and introns used for motif enrichment analysis of inflammation-activated genes. The promoter was defined as 1 kb upstream and 100 bp downstream of the TSS. ( B ) Heatmap showing motif enrichment, by MEME analysis, at promoters (top) and introns (bottom) of IFN-γ– and TLR-activated genes in hi-77 +/+ , hi-77 −/− , and hi-77 −/− ; Spi1 URE −/− cells. Motifs binding canonical inflammation-activated transcription factors (STAT, IRF, and NF-κB) are presented as positive controls. Motifs enriched in hi-77 −/− but not hi-77 +/+ or hi-77 −/− ; Spi1 URE −/− progenitors were ranked on the basis of −log 10 ( P value) and a P value cutoff of 0.005. Motifs with adjusted P value > 0.05 were set to 1 Complete heatmaps of all enriched motifs were presented in fig. S8 (A and B). ( C ) Schematic depicting positions of guide RNA for CRISPR-Cas9 gene editing tool to delete Runx1 gene in hi-77 −/− progenitors. ( D ) PCR-based genotyping assay for Runx1 −/− allele in hi-77 −/− progenitors. Two hi-77 −/− ; Runx1 −/− clonal lines are denoted as C1 and C2. ( E ) Runx1 , Gata2 , and Spi1 expression in hi-77 +/+ , hi-77 −/− , and hi-77 −/− ; Runx1 −/− progenitors ( n = 4). ( F ) Representative Western blot analysis of RUNX1 in hi-77 +/+ , hi-77 −/− , and hi-77 −/− ; Runx1 −/− progenitors with β-Actin as a control. M r is the apparent molecular mass in kDa. ( G ) The responsiveness of genes to IFN-γ and Pam 3 CSK 4 was quantified using RT-qPCR. hi-77 +/+ , hi-77 −/− , and hi-77 −/− ; Runx1 −/− progenitors were treated with or without IFN-γ (1 ng/ml), Pam 3 CSK 4 (100 ng/ml), or both for 4 hours ( n = 4 to 6). Statistics in (E) and (F): One-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Affinity-purified rabbit polyclonal anti-GATA2 antibody (1.2 μg) ( , ), anti-PU.1 mouse antibody (1.2 μg; Santa Cruz Biotechnology, sc-390405), H3K4me3 rabbit antibody (1 μg; Abcam, ab213224), or rabbit IgG isotype-matched control (1 μg; Invitrogen, 10500C) was added to each tube.

Techniques: Binding Assay, CRISPR, Genotyping Assay, Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: Science Advances

Article Title: Dual mechanism of inflammation sensing by the hematopoietic progenitor genome

doi: 10.1126/sciadv.adv3169

Figure Lengend Snippet: ( A ) PCR-based genotyping assay for Runx1 −/− allele in hi-77 +/+ progenitors. ( B ) Runx1 , Gata2 , and Spi1 expression was quantified using RT-qPCR. ( C ) The responsiveness of select genes to TLR1/2 agonist (Pam 3 CSK 4 ) and TLR4 agonist (LPS) were quantitated using RT-qPCR. hi-77 +/+ , hi-77 −/− , and hi- Runx1 −/− progenitors were treated with vehicle, Pam 3 CSK 4 (100 ng/ml), or LPS (100 ng/ml) for 4 hours ( n = 6 biological replicates). ( D ) Tlr1 , Tlr2 , and Tlr4 expression in hi-77 +/+ , hi-77 −/− , and hi- Runx1 −/− progenitors was quantified using RT-qPCR. Statistics: One-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Affinity-purified rabbit polyclonal anti-GATA2 antibody (1.2 μg) ( , ), anti-PU.1 mouse antibody (1.2 μg; Santa Cruz Biotechnology, sc-390405), H3K4me3 rabbit antibody (1 μg; Abcam, ab213224), or rabbit IgG isotype-matched control (1 μg; Invitrogen, 10500C) was added to each tube.

Techniques: Genotyping Assay, Expressing, Quantitative RT-PCR

Journal: Science Advances

Article Title: Dual mechanism of inflammation sensing by the hematopoietic progenitor genome

doi: 10.1126/sciadv.adv3169

Figure Lengend Snippet: The hematopoietic progenitor genome uses a dual PU.1-dependent mechanism to sense and respond to inflammation. A gene cohort has inaccessible chromatin in the steady state, and inflammation increases chromatin accessibility and occupancy of the hematopoietic transcription factors GATA2 and PU.1 to activate transcription via an IKKβ-dependent mechanism. At another inflammation-activated gene cohort, GATA2 and PU.1 occupancy precede inflammation, and this mechanism is not compromised by IKKβ inhibition. RUNX1 co-occupies chromatin with GATA2 and PU.1, yet GATA2 and RUNX1 differentially control inflammation-activated transcription, and transcriptional responses involving distinct TLR signaling pathways activated by unique pathogen-associated molecular patterns .

Article Snippet: Affinity-purified rabbit polyclonal anti-GATA2 antibody (1.2 μg) ( , ), anti-PU.1 mouse antibody (1.2 μg; Santa Cruz Biotechnology, sc-390405), H3K4me3 rabbit antibody (1 μg; Abcam, ab213224), or rabbit IgG isotype-matched control (1 μg; Invitrogen, 10500C) was added to each tube.

Techniques: Inhibition, Control, Protein-Protein interactions

Journal: bioRxiv

Article Title: PU.1 and TGF-β signaling transactivate CD103 expression in mast cells and dendritic cells: Opposite roles of GATA2 in the expression of mucosal mast cell-specific genes

doi: 10.1101/2025.02.10.637371

Figure Lengend Snippet: a. mRNA levels of Gata2 , Itgae , Itgb7 , and Mcpt2 in Gata2 siRNA-transfected BMMCs. b. Western blotting profiles of Gata2 KD and control BMMCs. Whole cell lysates containing 20 mg of protein were loaded onto each lane. After the detection of GATA2 proteins, the stripped membrane was re-probed with anti-β-actin Ab. c. Cell surface CD103 levels on Gata2 siRNA-transfected BMMCs. d. mRNA levels of Itgae and Mcpt2 in Gata2 siRNA-transfected and TGF-β-treated BMMCs. e. Cell surface CD103 levels on Gata2 siRNA-transfected and TGF-β-treated BMMCs. BMMCs transfected with Gata2 siRNA or its control siRNA were harvested 48 h after transfection ( a - c ), Gata2 siRNA transfectants or its controls were collected 48 h after electroporation and replaced in medium with or without TGF-β for an additional 48-h incubation ( d , e ). Data represent the mean ± SEM of independent experiments. Dunnett’s multiple comparison test ( d , e ) and the two-tailed paired t -test ( a, c ) were used for statistical analyses. *, p < 0.05; **, p <0.01; n.s., not significant.

Article Snippet: Western blot analyses were performed as previously described [ , ] with anti-GATA2 Ab (CG2-96, sc-267, and H-116, sc-9008, Santa Cruz Biotechnology), anti-PU.1 Ab (C-3, sc-390405, and D19, sc-5949, Santa Cruz Biotechnology), anti-β-actin Ab (AC-15, A5441, Sigma-Aldrich), and anti-GAPDH (14C-10, 2118, Cell Signaling Technology).

Techniques: Transfection, Western Blot, Control, Membrane, Electroporation, Incubation, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: PU.1 and TGF-β signaling transactivate CD103 expression in mast cells and dendritic cells: Opposite roles of GATA2 in the expression of mucosal mast cell-specific genes

doi: 10.1101/2025.02.10.637371

Figure Lengend Snippet: a. mRNA levels of Spi1 and Irf4 in Gata2 KD BMMCs. b. Itgae mRNA levels in Spi1 or Irf4 KD BMMCs. c. mRNA levels of MMC-related molecules in Spi1 KD BMMCs. d. Western blotting profile of PU.1 protein levels in Spi1 KD BMMCs. e. and f. Effects of Spi1 KD on the Gata2 KD-induced up-regulation of Itgae mRNA ( e ) and of cell surface CD103 ( f ). A typical dot-plot profile of staining with anti-CD103 Ab and anti-c-kit (left in f ) and MFIs of surface CD103 obtained from three independent experiments (right in f ). Data represent the mean ± SEM of independent experiments. Dunnett’s multiple comparison test ( e , f ) and the two-tailed paired t -test ( a , b , c , d ) were used for statistical analyses. *, p < 0.05; n.s., not significant.

Article Snippet: Western blot analyses were performed as previously described [ , ] with anti-GATA2 Ab (CG2-96, sc-267, and H-116, sc-9008, Santa Cruz Biotechnology), anti-PU.1 Ab (C-3, sc-390405, and D19, sc-5949, Santa Cruz Biotechnology), anti-β-actin Ab (AC-15, A5441, Sigma-Aldrich), and anti-GAPDH (14C-10, 2118, Cell Signaling Technology).

Techniques: Western Blot, Staining, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: PU.1 and TGF-β signaling transactivate CD103 expression in mast cells and dendritic cells: Opposite roles of GATA2 in the expression of mucosal mast cell-specific genes

doi: 10.1101/2025.02.10.637371

Figure Lengend Snippet: a. The profiles of H3K27 acetylation, ATAC-seq, and PU.1-binding around the Itgae gene in MCs. H3K27 acetylation ( https://chip-atlas.dbcls.jp/data/mm9/eachData/bw/SRX1456419 ), ATAC-seq ( https://chip-atlas.dbcls.jp/data/mm9/eachData/bw/SRX7750160 ), PU.1-binding ( https://chip-atlas.dbcls.jp/data/mm9/eachData/bw/SRX5026215 ), and GATA2-binding ( https://chip-atlas.dbcls.jp/data/mm9/eachData/bw/SRX9030838 ) were obtained via the ChIP-Atlas analysis ( http://chip-atlas.org ). b. Amount of PU.1 binding to 6 selected regions around the Itgae gene in BMMCs. c. Effects of Gata2 KD on the amount of PU.1 binding to region3 in the Itgae gene. d. Surface CD103 level on Spi1 -overexpressed BMMCs. A typical dot plot profile detected with GFP and anti-CD103 Ab (left) and MFIs of surface CD103 obtained from three independent experiments (right). Empty; BMMCs transfected with mock vector (pIRES2-AcGFP), PU.1; BMMCs transfected with pIRES2-AcGFP-3FL-rPU.1. e. mRNA level of Itgae in total BMMCs subjected to electroporation with pIRES2-AcGFP (empty) or pIRES2-AcGFP-3FL-rPU.1 (PU.1). Data represent the mean ± SEM of independent experiments. Dunnett’s multiple comparison test ( c ) and the two-tailed paired t -test ( b , d , e ) were used for statistical analyses. *, p < 0.05; n.s., not significant.

Article Snippet: Western blot analyses were performed as previously described [ , ] with anti-GATA2 Ab (CG2-96, sc-267, and H-116, sc-9008, Santa Cruz Biotechnology), anti-PU.1 Ab (C-3, sc-390405, and D19, sc-5949, Santa Cruz Biotechnology), anti-β-actin Ab (AC-15, A5441, Sigma-Aldrich), and anti-GAPDH (14C-10, 2118, Cell Signaling Technology).

Techniques: Binding Assay, Transfection, Plasmid Preparation, Electroporation, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: PU.1 and TGF-β signaling transactivate CD103 expression in mast cells and dendritic cells: Opposite roles of GATA2 in the expression of mucosal mast cell-specific genes

doi: 10.1101/2025.02.10.637371

Figure Lengend Snippet: a. ChIP assay data showing acetyl histone H4 on the Itgae and Mcpt2 genes in TGF-β-treated BMMCs. b. ChIP assay data showing PU.1 binding to the region3 in the Itgae gene in TGF-β-treated BMMCs. c. and d. mRNA levels of Spi1 and Gata2 ( c ) and protein levels of PU.1 and GATA2 ( d ) in TGF-β-treated BMMCs. BMMCs were incubated in the presence or absence of TGF-β for 48 h ( a - d ). e . Surface CD103 levels on Spi1 siRNA-transfected and TGF-β-treated BMMCs. f. mRNA levels of MMC-related genes in Spi1 siRNA-transfected and TGF-β-treated BMMCs. BMMCs transfected with Spi1 siRNA or its control were stimulated with TGF-β with the same time schedule as that in . Data represent the mean ± SEM of independent experiments. Dunnett’s multiple comparison test ( b, e ) and the two-tailed paired t -test ( c , d ) were used for statistical analyses. *, p < 0.05; n.s., not significant.

Article Snippet: Western blot analyses were performed as previously described [ , ] with anti-GATA2 Ab (CG2-96, sc-267, and H-116, sc-9008, Santa Cruz Biotechnology), anti-PU.1 Ab (C-3, sc-390405, and D19, sc-5949, Santa Cruz Biotechnology), anti-β-actin Ab (AC-15, A5441, Sigma-Aldrich), and anti-GAPDH (14C-10, 2118, Cell Signaling Technology).

Techniques: Binding Assay, Incubation, Transfection, Control, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: PU.1 and TGF-β signaling transactivate CD103 expression in mast cells and dendritic cells: Opposite roles of GATA2 in the expression of mucosal mast cell-specific genes

doi: 10.1101/2025.02.10.637371

Figure Lengend Snippet: a. and b. mRNA levels of MMC-related genes in ( a ) or surface CD103 on ( b ) peritoneal MCs incubated with or without TGF-β for 48 h. c. and d. Peritoneal MCs transfected with Gata2 siRNA and its control ( c ) or with Spi1 siRNA and its control ( d ) were stimulated with TGF-β for 48 h with same time schedule as those in and . Data represent the mean ± SEM of independent experiments. Dunnett’s multiple comparison test ( c , d ) and two-tailed paired t -test ( a , b ) were used for statistical analyses. *, p < 0.05; **, p < 0.01; n.s., not significant.

Article Snippet: Western blot analyses were performed as previously described [ , ] with anti-GATA2 Ab (CG2-96, sc-267, and H-116, sc-9008, Santa Cruz Biotechnology), anti-PU.1 Ab (C-3, sc-390405, and D19, sc-5949, Santa Cruz Biotechnology), anti-β-actin Ab (AC-15, A5441, Sigma-Aldrich), and anti-GAPDH (14C-10, 2118, Cell Signaling Technology).

Techniques: Incubation, Transfection, Control, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: RNA methylation controls stress granule formation and function during erythropoiesis

doi: 10.1101/2024.11.07.622459

Figure Lengend Snippet: a-h , m 6 A analysis of human CD34+ cells during erythropoiesis. hCD34+ cells were subjected to invitro erythropoiesis, and poly-A tail RNAs were subjected to m 6 A pull down using anti-m 6 A antibody and sequencing. Blue plots represent the total RNA input sequence. Green and red plots represent m 6 A methylation marks along the gene for (Proerythroblasts) day 7 and (late erythroid-polychromatic erythroblast) day 10, respectively. Erythropoiesis-specific genes ( a ) HBB , ( b ) TFRC , and ( c ) GAPDH shows m 6 A modifications along gene body and 3’UTR. d , Quantification of m 6 A levels at 3’UTR of genes HBB , TFRC , and GAPDH. e , Transcriptome-wide analysis identified consensus motif U/AGGAC as an m 6 A modification site. f , Venn diagram showing the overlap of m 6 A peaks identified in mRNA of CD34+ cells at Day 7 and Day 10. g , Differential m 6 A peaks during erythropoiesis. Blue dots represent decreased m 6 A peaks (hypo-m 6 A), and red dots represent increased m 6 A peaks (hyper-m 6 A). Grey dots correspond to m 6 A peaks with no significant change. h , GO terms of genes with hypo m 6 A peaks. i , ALKBH5 transcript levels during hCD34+ cell erythropoiesis. j , Enhancer analysis showing GATA1 and GATA2 binding at ALKBH5 locus during hCD34+ cell erythropoiesis. p-values ** <0.001, ** <0.01 from unpaired t-test. H-hour, D-Day.

Article Snippet: For Gata 1 and Gata 2 ChIP-seq experiments, we used Gata1 (Santa Cruz sc265X), Gata2 (Santa Cruz sc9008X) antibodies.

Techniques: Sequencing, Methylation, Modification, Binding Assay