Journal: Nature Cell Biology

Article Title: Expansion of ventral foregut is linked to changes in the enhancer landscape for organ-specific differentiation

doi: 10.1038/s41556-022-01075-8

Figure Lengend Snippet: a , RT-qPCR of HHEX (left) and FOXA1 (right) in the HHEX or FOXA1 KD cells. Expression was normalized relative to the scrambled shRNA controls. Triangles and circles mark cells derived from HUES4 and H9 ESCs respectively. Data are represented as mean ± SEM ( N = 4 independent experiments). ** P < 0.01 (unpaired two-tailed t-test, only significant comparisons are shown). b , Differentiation of scrambled control, HHEX KD, FOXA1 KD and HHEX/FOXA1 double KDs VFG cells to PE. Relative fold change of pancreatic genes ( PDX1 , GLIS3 , SOX9 and NKX6-2 ) was assayed by RT-qPCR and normalized relative to the scrambled shRNA controls. Data are represented as mean ± SEM ( N = 3 independent experiments). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way ANOVA Dunnett’s multiple comparison test compared with scramble control and HHEX/FOXA1 double KDs). c-d , FOXA1 ( c ) and HHEX ( d ) binding enrichment by ChIP-qPCR at enhancer regions of PDX1 (area IV), SFRP5 (peak32665), GLIS3 (peak246749), and TBX3 (peak60307) in HHEX and FOXA1 shRNA KD VFG and scrambled control cell lines. An intragenic region of NCAPD2 served as non-bound control. Data are represented as mean ± SEM ( N = 3 independent experiments). Statistical analysis was performed between KD and control VFG cells (* P < 0.05, ** P < 0.01, unpaired one-tailed t-test, only significant comparisons are shown). e-f , H3K4me1 ( e ) and H3K27ac ( f ) enrichment by ChIP-qPCR at enhancer regions of PDX1 (area IV), SFRP5 (peak32665), GLIS3 (peak246749), and TBX3 (peak60307) in HHEX shRNA KD VFG and scrambled control cell lines. An intragenic region of NCAPD2 served as a non-bound control. Data are represented as mean ± SEM ( N = 3 independent experiments). Statistical analysis was performed between the KD and control VFG cells (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, unpaired one-tailed t-test, only significant comparisons are shown). g , HHEX signal plotted on VFG-specific enhancer classes (PE-PP1 common, PE-not-PP, VFG OFF -in-DE-PP1, and VFG TR -in-PP1) at the FG and PP1 stages of directed differentiation (ChIP-seq dataset ) (from two independent experiments).

Article Snippet: For immunoprecipitation, the following antibodies and amounts of antibody were used for the 50,000-cell ChIP: 2 µg of FOXA1 (1:50) (Abcam, ab170933), 2 µg of H3K4me1 (1:50) (Abcam, ab8895), 2 µg of H3K27ac (1:50) (Abcam, ab4279) and 2 µg of HHEX (1:100) (R&D, MAB83771).

Techniques: Quantitative RT-PCR, Expressing, shRNA, Derivative Assay, Two Tailed Test, Binding Assay, One-tailed Test, ChIP-sequencing