Journal: Blood

Article Title: The HRI-regulated transcription factor ATF4 activates BCL11A transcription to silence fetal hemoglobin expression

doi: 10.1182/blood.2020005301

Figure Lengend Snippet: CRISPR screen identifies ATF4 as a novel γ-globin regulator. (A) Scatter plot of the transcription factor DNA binding domain–focused CRISPR screen results. Each dot represents an sgRNA. BCL11A, LRF, and NuRD components MBD2 and MTA are expected targets that validated the screen. (B) γ-globin mRNA measured by RT-qPCR relative to AHSP (top) and as percentage of total β-like globin transcripts (bottom). Control: nontargeting sgRNA. Results are shown as mean ± standard deviation (n = 3). (C) Representative flow cytometric analysis of cells stained with anti-HbF antibody. **P < .01 by Student t test. FSC, forward scatter; RPM, reads per million; RT-qPCR, quantitative reverse transcription polymerase chain reaction; WT, wild type.

Article Snippet: Primary antibodies used for western blot were as follows: HRI (MBS2526743; Biosource), phosphorylated eIF2α (ab32157; Abcam), total eIF2α ab5369; Abcam), BCL11A (19487; Abcam), ATF4 (11815S; Cell Signaling Technology), γ-globin (NB110-41084; Novus Biologicals), and B-actin (47778; Santa Cruz Biotechnology).

Techniques: CRISPR, Binding Assay, Quantitative RT-PCR, Control, Standard Deviation, Staining, Reverse Transcription, Polymerase Chain Reaction

Journal: Blood

Article Title: The HRI-regulated transcription factor ATF4 activates BCL11A transcription to silence fetal hemoglobin expression

doi: 10.1182/blood.2020005301

Figure Lengend Snippet: ATF4 links HRI to BCL11A and γ-globin production. (A) Immunoblot analysis of differentiated HUDEP2 after depletion of HRI. (B) BCL11A mRNA levels measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) in indicated cells. Results are shown as mean ± standard deviation (SD; n = 3). (C) Immunoblot analysis of HUDEP2 upon depletion of ATF4. (D) Immunoblot analysis of undifferentiated HUDEP2 cells treated with 2.5 μg/mL of tunicamycin for 3 days. (E) BCL11A and γ-globin mRNA levels measured by RT-qPCR in parental or ATF4 overexpressing (ATF4 OE) undifferentiated HUDEP2 cells. Results are shown as mean ± SD (n = 4). (F) γ-globin mRNA levels measured by RT-qPCR with overexpression of BCL11A-ER in differentiated HUDEP2 cells. Results are shown as mean ± SD (n = 3). **P < .01 by Student t test. WT, wild type.

Article Snippet: Primary antibodies used for western blot were as follows: HRI (MBS2526743; Biosource), phosphorylated eIF2α (ab32157; Abcam), total eIF2α ab5369; Abcam), BCL11A (19487; Abcam), ATF4 (11815S; Cell Signaling Technology), γ-globin (NB110-41084; Novus Biologicals), and B-actin (47778; Santa Cruz Biotechnology).

Techniques: Western Blot, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Standard Deviation, Over Expression

Journal: Blood

Article Title: The HRI-regulated transcription factor ATF4 activates BCL11A transcription to silence fetal hemoglobin expression

doi: 10.1182/blood.2020005301

Figure Lengend Snippet: ATF4 regulates BCL11A through the +55 enhancer. (A) BCL11A mRNA (top) and γ-globin mRNA (bottom) levels measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) relative to AHSP mRNA in BCL11A +55 sgRNA-treated and control HUDEP2 cells. Results are shown as mean ± standard deviation (SD; n = 3). (B) BCL11A mRNA (top) and γ-globin mRNA (bottom) levels measured by RT-qPCR relative to AHSP mRNA in BCL11A +55 sgRNA-treated and control primary erythroid cells. An sgRNA against the +58 enhancer served as positive control. Results are shown as mean ± SD (n = 3). (C) Representative high-performance liquid chromatography (HPLC) analysis of Hb in primary erythroid cells. HbF peaks are indicated by red arrows, and HbF peak area is displayed as percentage of total HbF + HbA. (D) Capture-C using the BCL11A promoter as anchor. The erythroid specific enhancers +55, +58, and +62 are highlighted in purple, and the BCL11A promoter anchor in cyan. The segment used for quantification of reads is indicated by the blue bar (supplemental Figure 4G). H3K27ac and DNaseI were obtained from published data.41 *P < .05, **P < .01 by Student t test. WT, wild type.

Article Snippet: Primary antibodies used for western blot were as follows: HRI (MBS2526743; Biosource), phosphorylated eIF2α (ab32157; Abcam), total eIF2α ab5369; Abcam), BCL11A (19487; Abcam), ATF4 (11815S; Cell Signaling Technology), γ-globin (NB110-41084; Novus Biologicals), and B-actin (47778; Santa Cruz Biotechnology).

Techniques: Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Control, Standard Deviation, Positive Control, High Performance Liquid Chromatography, Capture-C

Journal: Blood

Article Title: The HRI-regulated transcription factor ATF4 activates BCL11A transcription to silence fetal hemoglobin expression

doi: 10.1182/blood.2020005301

Figure Lengend Snippet: Bcl11a and human γ-globin transgene levels remain unchanged in HRI knockout Townes mice. (A) Complete blood cell count of HRI+/+, HRI+/−, and HRI−/− Townes βAβS mice. Results are shown as mean ± standard deviation (SD; n = 3). (B) HRI (left), Bcl11a (middle), and γ-globin (right) mRNA levels as measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) of sorted CD71+Ter119+ bone marrow cells in HRI+/+, HRI+/−, and HRI−/− Townes βAβS mice. HRI and Bcl11a are displayed as absolute values normalized to RPS18, whereas γ-globin is displayed as percentage of total β-like globin transcripts. Results are shown as mean ± SD (n = 3). *P < .05, **P < .01 by Student t test. MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; n.s., not significant; PLT, platelet count; WBC, white blood cell count.

Article Snippet: Primary antibodies used for western blot were as follows: HRI (MBS2526743; Biosource), phosphorylated eIF2α (ab32157; Abcam), total eIF2α ab5369; Abcam), BCL11A (19487; Abcam), ATF4 (11815S; Cell Signaling Technology), γ-globin (NB110-41084; Novus Biologicals), and B-actin (47778; Santa Cruz Biotechnology).

Techniques: Knock-Out, Cell Counting, Standard Deviation, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Blood

Article Title: The HRI-regulated transcription factor ATF4 activates BCL11A transcription to silence fetal hemoglobin expression

doi: 10.1182/blood.2020005301

Figure Lengend Snippet: ATF4 regulates BCL11A in a species-selective manner. (A) Venn diagrams of the ATF4 ChIP-seq peaks in G1E-ER4 cells and the most enriched ATF4 binding motif. (B) ATF4 binding profile at mouse Bcl11a +55 enhancer. The sgRNA targeting the mouse ATF4 consensus motif is underlined, and the PAM sequence is shown in bold. (C) Representative ATF4 ChIP-seq track of the murine Bcl11a locus. The region orthologous to the human BCL11A +55 enhancer is highlighted in purple. ATAC-seq was obtained from ENCODE (ENCSR428BSK), and H3K27ac was obtained from published data.42 (D) Bcl11a (left) and Hbb-bh1 (right) mRNA levels in indicated cells. Note: the loss of function of Bcl11a by the sgRNA targeting exon 2 did not diminish transcript levels but depleted Bcl11a protein levels. Results are shown as mean ± standard deviation (n = 3). (E) Model depicting HRI and ATF4 regulation of BCL11A and γ-globin levels. **P < .01 by Student t test. n.s., not significant.

Article Snippet: Primary antibodies used for western blot were as follows: HRI (MBS2526743; Biosource), phosphorylated eIF2α (ab32157; Abcam), total eIF2α ab5369; Abcam), BCL11A (19487; Abcam), ATF4 (11815S; Cell Signaling Technology), γ-globin (NB110-41084; Novus Biologicals), and B-actin (47778; Santa Cruz Biotechnology).

Techniques: ChIP-sequencing, Binding Assay, Sequencing, Standard Deviation

Journal:

Article Title: Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System ▿

doi: 10.1128/AEM.01291-08

Figure Lengend Snippet: Hemoglobin production in BL21(DE3) transformed with various plasmids. (A) Absorbance scans of live cultures of BL21(DE3) with and without hemoglobin or P. shigelloides heme transport genes. Solid line, E. coli containing hemoglobin and heme transport genes [(BL21(DE3)/pHB0.0, pHUG21]; dotted line, E. coli containing hemoglobin genes alone [BL21(DE3)/pHB0.0, pWSK29]; dashed line, E. coli containing heme transport genes alone [BL21(DE3)/pDSV1, pHUG21]. Values on the x axis are wavelengths in nanometers. (B) First derivative spectrum obtained from absorbance scans. Derivative values are shown beside each arrow. Solid line, BL21(DE3) containing heme transport and hemoglobin genes; dotted line, BL21(DE3) containing hemoglobin genes alone. (C) Immunoblot of soluble (S) and insoluble (I) fractions from 0.004 OD unit of cells at a wavelength of 700 nm. Lanes 1 and 4, BL21(DE3) containing heme transport genes alone; lanes 2 and 5, BL21(DE3) containing hemoglobin genes alone; lanes 3 and 6, BL21(DE3) containing hemoglobin and heme transport genes; lane 7, 0.45 μg of hemoglobin (Sigma Chemicals).

Article Snippet: Immunoblots using goat anti-human hemoglobin antibody (BiosPacific Fortron Bio Science, Inc.) and horseradish peroxidase-labeled rabbit anti-goat antibody (Bio-Rad) were performed on protein samples that had been electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometry of autoradiograms was performed using the UVP AutoChemi system.

Techniques: Transformation Assay, Western Blot

Journal:

Article Title: Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System ▿

doi: 10.1128/AEM.01291-08

Figure Lengend Snippet: Hemoglobin production in BL21(DE3) containing the one-plasmid or two-plasmid system. (A) Genetic and partial restriction enzyme map of genes on pHB0.0hug. Genes are indicated with horizontal arrows, and promoters are indicated with vertical bent arrows. Restriction enzyme sites are as follows: B, BamHI; H, HindIII; N, NheI; S, SalI; and W, BsiWI. (B) Immunoblot of soluble (S) and insoluble (I) fractions. Lanes 1 and 3, BL21(DE3) containing the two-plasmid system (pHB0.0, pHUG21); lanes 2 and 4, BL21(DE3) containing the one-plasmid system (pHB0.0hug); lane 5, 0.45 μg of hemoglobin. (C) Densitometry analysis of immunoblots of soluble fractions. Data are from three independent sets of samples analyzed on the same immunoblot; soluble fractions from 0.004 OD unit of cells at a wavelength of 700 nm were loaded. Open circles, hemoglobin control; filled squares, BL21(DE3) containing the two-plasmid system; filled triangles, BL21(DE3) containing the one-plasmid system.

Article Snippet: Immunoblots using goat anti-human hemoglobin antibody (BiosPacific Fortron Bio Science, Inc.) and horseradish peroxidase-labeled rabbit anti-goat antibody (Bio-Rad) were performed on protein samples that had been electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometry of autoradiograms was performed using the UVP AutoChemi system.

Techniques: Plasmid Preparation, Western Blot, Control

Journal:

Article Title: Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System ▿

doi: 10.1128/AEM.01291-08

Figure Lengend Snippet: Analysis of hemoglobin production in BL21(DE3) containing various plasmids

Article Snippet: Immunoblots using goat anti-human hemoglobin antibody (BiosPacific Fortron Bio Science, Inc.) and horseradish peroxidase-labeled rabbit anti-goat antibody (Bio-Rad) were performed on protein samples that had been electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometry of autoradiograms was performed using the UVP AutoChemi system.

Techniques: Plasmid Preparation