Journal: Communications Biology

Article Title: Erythroid lineage chromatin accessibility maps facilitate identification and validation of NFIX as a fetal hemoglobin repressor

doi: 10.1038/s42003-023-05025-4

Figure Lengend Snippet: a ATAC-seq profiles spanning Chr19:13,122,000–13,138,000 (hg19) of sorted BM (HbF low) and CB (HbF high) cell populations showing increased chromatin accessibility at the NFIX promoter in BM cells (red boxes). NFIX splice variants NM_002501 (line 1), NM_001271044 (line 2), and NM_001365985 (line 3) are shown at the top. b , c NFIX mRNA quantification in BM versus CB sorted cell populations and in HUDEP-2 (HbF low) versus HUDEP-1 (HbF high). Bars represent mean of two biological replicates. Asterisk denotes statistically significant data using Student’s t -test [ P -value < 0.05 for BM versus CB cells (paired t -test, b )]. d Western blot for NFIX protein. Histone H3 served as a normalizing control. Representative immunoblots from two biological replicates are shown.

Article Snippet: TaqMan® Probe IDs used in this study: Hs_00362215_g1 ( HBE ), Hs00361131_g1 ( HBG ), Hs_00426283_m1 ( HBD ), Hs_00747223_g1 ( HBB ), Hs_00958843_m1 ( NFIX ), Hs01093198_m1 ( BCL11A ), Hs_00252415_s1 ( ZBTB7A ), and Hs99999903_m1 ( ACTB ).

Techniques: Western Blot, Control

Journal: Communications Biology

Article Title: Erythroid lineage chromatin accessibility maps facilitate identification and validation of NFIX as a fetal hemoglobin repressor

doi: 10.1038/s42003-023-05025-4

Figure Lengend Snippet: a RT-qPCR validation of NFIX knockdown in BM cells shows a 90% reduction in NFIX transcripts. b ATAC-seq profiles spanning Chr11:5,245,000–5,277,000 (hg19) of BM control and NFIX knockdown cells showing reduced chromatin accessibility at the HBB promoter and increased chromatin accessibility at the HBG promoter relative to the control cells (BM day 7 of differentiation, red boxes). c Percent HBG promoter DNA methylation at CpG -162 and an average of all 6 CpGs tested shows decreased DNA methylation in NFIX knockdown BM cells relative to the control. d Knockdown of NFIX in BM cells results in induction of HBG mRNA. e NFIX knockdown increases the number of F-cells (BM day 10 of differentiation). f HPLC chromatograms show increased absolute HbF levels in NFIX knockdown cells (BM day 14 of differentiation). g Knockdown of NFIX in HUDEP-2 cells increases HBG mRNA, F-cells, and HbF relative to the control. Data shown are representative of N = 3 independent experiments ( a , e , f ) and N = 2 independent experiments ( b , c , d , g ) using distinct BM and CB donors and independent HUDEP-2 transductions.

Article Snippet: TaqMan® Probe IDs used in this study: Hs_00362215_g1 ( HBE ), Hs00361131_g1 ( HBG ), Hs_00426283_m1 ( HBD ), Hs_00747223_g1 ( HBB ), Hs_00958843_m1 ( NFIX ), Hs01093198_m1 ( BCL11A ), Hs_00252415_s1 ( ZBTB7A ), and Hs99999903_m1 ( ACTB ).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Knockdown, Control, DNA Methylation Assay

Journal: Communications Biology

Article Title: Erythroid lineage chromatin accessibility maps facilitate identification and validation of NFIX as a fetal hemoglobin repressor

doi: 10.1038/s42003-023-05025-4

Figure Lengend Snippet: a RT-qPCR validation of NFIX overexpression in CB cells shows approximately 10-fold increase in NFIX transcripts relative to empty vector control cells. Bars represent mean of two biological replicates. b Western blot validation of NFIX overexpression in CB cells as determined by detection of the FLAG tag on day 10 of erythroid differentiation. c CB cells overexpressing NFIX have a reduction in HBG mRNA levels relative to other beta-like globins on day 7 of erythroid differentiation. Bars represent mean of two biological replicates. d CB cells overexpressing NFIX show reduced percentage of F-cells relative to empty vector control on day 14 of erythroid differentiation. e HPLC profiles demonstrate reduction in HbF protein in NFIX overexpressing CB cells relative to the empty vector control on day 14 of erythroid differentiation. Data are representative of N = 2 biological replicates using distinct CB donors and independent NFIX transductions.

Article Snippet: TaqMan® Probe IDs used in this study: Hs_00362215_g1 ( HBE ), Hs00361131_g1 ( HBG ), Hs_00426283_m1 ( HBD ), Hs_00747223_g1 ( HBB ), Hs_00958843_m1 ( NFIX ), Hs01093198_m1 ( BCL11A ), Hs_00252415_s1 ( ZBTB7A ), and Hs99999903_m1 ( ACTB ).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Over Expression, Plasmid Preparation, Control, Western Blot, FLAG-tag