Journal: Acta Biochimica et Biophysica Sinica

Article Title: MYB represses ζ-globin expression through upregulating ETO2

doi: 10.3724/abbs.2024239

Figure Lengend Snippet: The absence of ETO2 leads to ineffective silencing of the embryonic globin gene (A,B) qPCR analysis was conducted to evaluate the expression levels of ETO2 and ζ-globin and the percentage of embryonic ζ-globin mRNA relative to total α-like globin (α-globin + ζ-globin) expression in ETO2-KO HUDEP-2 cell lines on days 0 and 7 of differentiation. (C,D) Western blot analysis of ETO2 and ζ-globin in ETO2-KO HUDEP-2 cells on days 0 and 7 of differentiation; GAPDH served as a negative control. (E) qPCR analysis was performed to detect ζ-globin expression in ETO2-KO CD34+ cells mobilized from both healthy donors and individuals diagnosed with HbH disease (--SEA/αcsα) during 12 days of differentiation. (F) ChIP-qPCR for LMO2 and CHD4 occupancy at the LCR HSs and ζ-globin gene promoter in WT and ETO2-KO HUDEP-2 cells. (G) ChIP-qPCR for H3K9ac and H3K27ac occupancy at the LCR HSs and ζ-globin gene promoter in WT and ETO2-KO HUDEP-2 cells. The abscissa represents the LCR HSs, ζ-globin promoter and Necdin (negative control), whereas the ordinate represents the relative enrichment level of the occupied protein. (H) ATAC-seq screenshots of MYB-KO (red, bottom-most) and ETO2-KO (blue, middle) HUDEP-2 cells indicating the chromatin accessibility of MYB and ETO2 loss. WT (grey, upper) was used as a negative control. The ζ-globin (HBZ) gene is indicated by the light blue bar. Each experiment was conducted in triplicate. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

Article Snippet: Specific antibody or rabbit IgG was used to precipitate the crosslinked DNA overnight at 4°C, then magnetic beads (56383; CST) were added and incubated for 1 h. After wash with washing buffer, elution was done at room temperature for 15 min. DNA was extracted following reversal of crosslinks using NaCl at 65°C for 5 h.The following antibodies were used at the indicated staining dilutions: ETO2 (A303-616A-T, 1:50; Proteintech, Wuhan, China), LMO2 (ab91652, 1:100; Abcam), MED1 (A300-793A, 1:100; Abcam), CHD4 (ab240640, 1:50; Abcam), MTA2 (ab8106, 1:100; Abcam), H3K9ac (39137, 1:100; Active Motif, Carlsbad, USA), and H3K27ac (39133, 1:100; Active Motif).

Techniques: Expressing, Western Blot, Negative Control, ChIP-qPCR

Journal: Acta Biochimica et Biophysica Sinica

Article Title: MYB represses ζ-globin expression through upregulating ETO2

doi: 10.3724/abbs.2024239

Figure Lengend Snippet: Identification of ETO2 as a regulator of ζ-globin repression mediated by MYB (A) Venn diagram illustrating Myb target selection. (B) MYB enrichment at the gene promoter of Eto2 from MEL cells obtained from ENCODE. The de novo motif logo of MYB in the mouse genome is provided below. (C) RT-qPCR analysis was performed to assess the expression levels of selected genes in population S3 of Myb–/– fetal liver cells at E12.5. (D) ChIP-qPCR analysis of MYB occupancy at the promoter region of the ETO2 gene in WT and MYB-KO HUDEP-2 cells. The abscissa represents the ζ-globin promoter and Necdin (negative control), whereas the ordinate represents the relative enrichment level of MYB. (E) Left panel: Intersection analysis of genes whose expression level changed in Myb–/– and Eto2–/– fetal liver cells at E12.5. Fold change > 2 and P < 0.05 were used as cutoffs for identifying DEGs. Right panel: Intersection analysis of genes co-regulated by Eto2 and Myb, as well as erythroid-specific genes. The Eto2 knockout (KO) data originated from the GEO database ( GSE156306 ). (F) GO analysis of genes co-regulated by Eto2 and Myb. P < 0.05 were used as cutoffs for identifying DEGs. ***P < 0.001, ****P < 0.0001.

Article Snippet: Specific antibody or rabbit IgG was used to precipitate the crosslinked DNA overnight at 4°C, then magnetic beads (56383; CST) were added and incubated for 1 h. After wash with washing buffer, elution was done at room temperature for 15 min. DNA was extracted following reversal of crosslinks using NaCl at 65°C for 5 h.The following antibodies were used at the indicated staining dilutions: ETO2 (A303-616A-T, 1:50; Proteintech, Wuhan, China), LMO2 (ab91652, 1:100; Abcam), MED1 (A300-793A, 1:100; Abcam), CHD4 (ab240640, 1:50; Abcam), MTA2 (ab8106, 1:100; Abcam), H3K9ac (39137, 1:100; Active Motif, Carlsbad, USA), and H3K27ac (39133, 1:100; Active Motif).

Techniques: Selection, Quantitative RT-PCR, Expressing, ChIP-qPCR, Negative Control, Knock-Out

Journal: Acta Biochimica et Biophysica Sinica

Article Title: MYB represses ζ-globin expression through upregulating ETO2

doi: 10.3724/abbs.2024239

Figure Lengend Snippet: The downregulation of MYB expression regulates a reduction in ζ-globin expression by modulating ETO2 (A) qPCR detection of MYB, ETO2, and ζ-globin expression, along with the percentage of ζ-globin mRNA relative to the total α-like globin expression of MYB-KO HUDEP-2 cell lines. (B) Western blot analysis of ζ-globin, MYB and ETO2 in MYB-KO HUDEP-2 cells. GAPDH served as a negative control. (C) ChIP-qPCR for ETO2 occupancy at locus control region hypersensitive sites (LCR HSs) and the ζ-globin promoter in WT and MYB-KO HUDEP-2 cells. The abscissa represents the LCR HSs, ζ-globin promoter and Necdin (negative control), whereas the ordinate represents the relative enrichment level of ETO2. The de novo motif logo of MYB in the human genome is provided on the right. (D) RT-qPCR results showing the expressions of MYB, ETO2 and ζ-globin in CD34+ cells mobilized from HbH disease patients (--SEA/αcsα) on D12 versus D6 of differentiation. Individuals with the GG genotype of rs7776054 were classified as MYB(–/–) individuals, whereas those with the T/T genotype were labeled as MYB(+/+) individuals, with both groups carrying the αcsα/--SEA deletion. The “G” allele of rs7776054 is correlated with elevated ζ-globin expression and reduced MYB and ETO2 expression levels. (E) qPCR detection of MYB, ETO2, and ζ-globin expression in MYB-KO ETO2-overexpressing HUDEP-2 cell lines. Each experiment was conducted in triplicate. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.

Article Snippet: Specific antibody or rabbit IgG was used to precipitate the crosslinked DNA overnight at 4°C, then magnetic beads (56383; CST) were added and incubated for 1 h. After wash with washing buffer, elution was done at room temperature for 15 min. DNA was extracted following reversal of crosslinks using NaCl at 65°C for 5 h.The following antibodies were used at the indicated staining dilutions: ETO2 (A303-616A-T, 1:50; Proteintech, Wuhan, China), LMO2 (ab91652, 1:100; Abcam), MED1 (A300-793A, 1:100; Abcam), CHD4 (ab240640, 1:50; Abcam), MTA2 (ab8106, 1:100; Abcam), H3K9ac (39137, 1:100; Active Motif, Carlsbad, USA), and H3K27ac (39133, 1:100; Active Motif).

Techniques: Expressing, Western Blot, Negative Control, ChIP-qPCR, Control, Quantitative RT-PCR, Labeling

Journal: Acta Biochimica et Biophysica Sinica

Article Title: MYB represses ζ-globin expression through upregulating ETO2

doi: 10.3724/abbs.2024239

Figure Lengend Snippet: The absence of ETO2 leads to ineffective silencing of the embryonic globin gene (A,B) qPCR analysis was conducted to evaluate the expression levels of ETO2 and ζ-globin and the percentage of embryonic ζ-globin mRNA relative to total α-like globin (α-globin + ζ-globin) expression in ETO2-KO HUDEP-2 cell lines on days 0 and 7 of differentiation. (C,D) Western blot analysis of ETO2 and ζ-globin in ETO2-KO HUDEP-2 cells on days 0 and 7 of differentiation; GAPDH served as a negative control. (E) qPCR analysis was performed to detect ζ-globin expression in ETO2-KO CD34+ cells mobilized from both healthy donors and individuals diagnosed with HbH disease (--SEA/αcsα) during 12 days of differentiation. (F) ChIP-qPCR for LMO2 and CHD4 occupancy at the LCR HSs and ζ-globin gene promoter in WT and ETO2-KO HUDEP-2 cells. (G) ChIP-qPCR for H3K9ac and H3K27ac occupancy at the LCR HSs and ζ-globin gene promoter in WT and ETO2-KO HUDEP-2 cells. The abscissa represents the LCR HSs, ζ-globin promoter and Necdin (negative control), whereas the ordinate represents the relative enrichment level of the occupied protein. (H) ATAC-seq screenshots of MYB-KO (red, bottom-most) and ETO2-KO (blue, middle) HUDEP-2 cells indicating the chromatin accessibility of MYB and ETO2 loss. WT (grey, upper) was used as a negative control. The ζ-globin (HBZ) gene is indicated by the light blue bar. Each experiment was conducted in triplicate. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant.

Article Snippet: Specific antibody or rabbit IgG was used to precipitate the crosslinked DNA overnight at 4°C, then magnetic beads (56383; CST) were added and incubated for 1 h. After wash with washing buffer, elution was done at room temperature for 15 min. DNA was extracted following reversal of crosslinks using NaCl at 65°C for 5 h.The following antibodies were used at the indicated staining dilutions: ETO2 (A303-616A-T, 1:50; Proteintech, Wuhan, China), LMO2 (ab91652, 1:100; Abcam), MED1 (A300-793A, 1:100; Abcam), CHD4 (ab240640, 1:50; Abcam), MTA2 (ab8106, 1:100; Abcam), H3K9ac (39137, 1:100; Active Motif, Carlsbad, USA), and H3K27ac (39133, 1:100; Active Motif).

Techniques: Expressing, Western Blot, Negative Control, ChIP-qPCR

Journal: Acta Biochimica et Biophysica Sinica

Article Title: MYB represses ζ-globin expression through upregulating ETO2

doi: 10.3724/abbs.2024239

Figure Lengend Snippet: MYB and ETO2 downregulate ζ-globin expression through similar effects on erythroid differentiation (A) Progressive erythropoiesis was assessed by flow cytometry analysis of CD71 and CD235a antibodies. The cells can be separated into four subsets (Q4-Q1-Q2-Q3), corresponding to increased erythroid differentiation. Erythroid differentiation in WT, MYB-KO and ETO2-KO HUDEP-2 cells on day 0 and day 3 and staining with CD71 and CD235a antibodies. Flow cytometry analysis was conducted on MYB-KO HUDEP-2 cells (middle panel), ETO2-KO HUDEP-2 cells (right panel) and WT HUDEP-2 cells (left panel). (B) Quantification of each population (Q4-Q1-Q2-Q3) of WT, MYB-KO and ETO2-KO HUDEP-2 cells. The calculation of Q3 is not performed because of the low number of cells. (C,D) Left panel: Intersection analysis of genes co-regulated by Klf1 and Myb, as well as erythroid-specific genes. Right panel: Intersection analysis of genes co-regulated by Eto2 and Myb, as well as erythroid-specific genes. Lower panel: Venn diagram showing the intersection analysis of genes co-regulated by MYB, ETO2 and KLF1 as well as erythroid-specific genes. The Klf1 KO and Eto2 KO data for E14.5 fetal liver cells were obtained from the GEO database ( GSE71396 and GSE142226 ). Fold change > 2 and adjusted P < 0.05 were used as cutoffs for identifying differentially expressed genes in Myb–/– and Eto2–/– fetal liver cells at E12.5. P < 0.05 were used as cutoffs for identifying differentially expressed genes in Klf1–/– and fetal liver cells at E14.5. (E) qPCR detection of ETO2, BCL11A and ζ-globin in ETO2-KO, BCL11A-KO, and double-KO HUDEP-2 cells. Each experiment was conducted in triplicate. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001, ns, not significant.

Article Snippet: Specific antibody or rabbit IgG was used to precipitate the crosslinked DNA overnight at 4°C, then magnetic beads (56383; CST) were added and incubated for 1 h. After wash with washing buffer, elution was done at room temperature for 15 min. DNA was extracted following reversal of crosslinks using NaCl at 65°C for 5 h.The following antibodies were used at the indicated staining dilutions: ETO2 (A303-616A-T, 1:50; Proteintech, Wuhan, China), LMO2 (ab91652, 1:100; Abcam), MED1 (A300-793A, 1:100; Abcam), CHD4 (ab240640, 1:50; Abcam), MTA2 (ab8106, 1:100; Abcam), H3K9ac (39137, 1:100; Active Motif, Carlsbad, USA), and H3K27ac (39133, 1:100; Active Motif).

Techniques: Expressing, Flow Cytometry, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Pharmacological Disruption of Phosphorylated Eukaryotic Initiation Factor-2α/Activating Transcription Factor 4/Indian Hedgehog Protects Intervertebral Disc Degeneration via Reducing the Reactive Oxygen Species and Apoptosis of Nucleus Pulposus Cells

doi: 10.3389/fcell.2021.675486

Figure Lengend Snippet: A proposed model depicting the p-eIF2α/ATF4/Ihh in regulating hypertrophy and apoptosis of NP cell. TNF-α and BTd promote the phosphorylation of eIF2α and upregulate the ATF4 expression, continuously resulting in the Ihh activation. Suppression of eIF2α phosphorylation by ISRIB decreases the ATF4 and Ihh levels and alleviates the hypertrophy and apoptosis of NP cell. Additionally, CPE treatment suppresses the hypertrophy and apoptosis of NP cell. Thus, disrupting the pathways ultimately protect against the development of IDD. p-eIF2α, phosphorylated eukaryotic initiation factor-2α; ATF4, activating transcription factor 4; Ihh, Indian hedgehog; NP, nucleus pulposus; TNF, tumor necrosis factor; BTd, BTdCPU; eIF2α, eukaryotic initiation factor-2α; CPE, cyclopamine.

Article Snippet: Collagenase XI, dispase II, tumor necrosis factor (TNF)-α, bovine serum albumin (BSA), and biotinylated IgG were from Sigma (Ohio, United States); DMEM/F12 medium, mixture of penicillin/streptomycin, and fetal bovine serum (FBS) were from Thermo (Massachusetts, United States); Masson’s Trichrome Stain Kit and Safranin O solution were from Solarbio (Shanghai, China); anti-ATF4 (ab216839 and ab184909), anti-Ihh (ab52919), anti-Col-X (ab49945, and ab260040), anti-superoxide dismutase 1 (SOD1, ab51254), anti-MMP13 (ab219620 and ab51072), anti-IgG (ab171870), anti-eIF2α (ab169528), anti-p-eIF2α (ab32157), anti-β-actin (ab8227), goat anti-mouse IgG H&L (Alexa Fluor ® 488) (ab150113), and goat anti-rabbit IgG H&L (Alexa Fluor 647) (ab150083) were from Abcam (Cambridge, United Kingdom); Vectastain Elite ABC reagent, Annexin V-EGFP/PI kit (KGA102), and ROS detection kit(KGAF018) were obtained from KeyGen (Nanjing, China); cyclopamine (CPE), BTdCPU (BTd), and ISRIB were from Selleck (Shanghai, China); Collagen II Polyclonal Antibody (Col-II, PA5-99159), cleaved Caspase3 antibody (PA5-114687), Lipofectamine 2000 transfection reagent, and protein isolation (89900, 78510) were from Thermo Fisher Scientific (Waltham, United States); SimpleChIP Plus Sonication Chromatin IP Kit (Magnetic Beads) was from Cell Signaling Technology (56383, Massachusetts, United States); other reagents not precisely specified were from Beyotime (Shanghai, China).

Techniques: Phospho-proteomics, Expressing, Activation Assay