Limits...
PDX1 binds and represses hepatic genes to ensure robust pancreatic commitment in differentiating human embryonic stem cells.

Teo AK, Tsuneyoshi N, Hoon S, Tan EK, Stanton LW, Wright CV, Dunn NR - Stem Cell Reports (2015)

Bottom Line: ChIP-seq also revealed PDX1 occupancy at hepatic genes.In HepG2 cells and differentiating hESCs, we found that PDX1 binds and suppresses expression of endogenous liver genes.These findings rebrand PDX1 as a context-dependent transcriptional repressor and activator within the same cell type.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Biology, A(∗)STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648, Singapore.

Show MeSH

Related in: MedlinePlus

PDX1 Represses Liver Marker Genes(A) Microarray gene expression heatmap showing increasing levels of liver lineage-related genes between days 0 and 17 of pancreatic differentiation.(B) ChIP-qPCR provides independent confirmation that PDX1 binds in proximity to liver genes. Error bars indicate the SD of three technical replicates from a single ChIP-qPCR experiment. These data were independently confirmed in repeat pull-down qPCR experiments (data not shown). Normal goat IgG is the negative control.(C) AFP and PDX1 immunostaining on day 17 of pancreatic differentiation. AFP and PDX1 label distinct cell populations that are often observed in close proximity. Scale bar represents 100 μm.(D) Expression of hepatic genes (AFP, ALB, TTR, APOA2, FOXA1, FOXA2, FOXA3, and HHEX) by qPCR in HepG2 cells after transient overexpression of GFP, PDX1, or the PDX1(N196S) mutant that cannot bind DNA. Both WT PDX1 and PDX1(N196S) are robustly overexpressed after transfection. All error bars indicate SD of three biological replicates. For all genes, data are shown relative to the GFP-transfected controls. p values are indicated.(E) Western blot analyses for PDX1 and AFP on days 0 and 17 of pancreatic differentiation in GFP- or human PDX1-overexpressing hESC clones.(F) Expression of liver markers (AFP, ALB, TTR, APOA2, FOXA1, HHEX) in hESCs stably overexpressing either GFP or human PDX1 and differentiated for 17 days. Error bars indicate the SD of three biological replicates. p values were calculated when compared with GFP control.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4400640&req=5

fig4: PDX1 Represses Liver Marker Genes(A) Microarray gene expression heatmap showing increasing levels of liver lineage-related genes between days 0 and 17 of pancreatic differentiation.(B) ChIP-qPCR provides independent confirmation that PDX1 binds in proximity to liver genes. Error bars indicate the SD of three technical replicates from a single ChIP-qPCR experiment. These data were independently confirmed in repeat pull-down qPCR experiments (data not shown). Normal goat IgG is the negative control.(C) AFP and PDX1 immunostaining on day 17 of pancreatic differentiation. AFP and PDX1 label distinct cell populations that are often observed in close proximity. Scale bar represents 100 μm.(D) Expression of hepatic genes (AFP, ALB, TTR, APOA2, FOXA1, FOXA2, FOXA3, and HHEX) by qPCR in HepG2 cells after transient overexpression of GFP, PDX1, or the PDX1(N196S) mutant that cannot bind DNA. Both WT PDX1 and PDX1(N196S) are robustly overexpressed after transfection. All error bars indicate SD of three biological replicates. For all genes, data are shown relative to the GFP-transfected controls. p values are indicated.(E) Western blot analyses for PDX1 and AFP on days 0 and 17 of pancreatic differentiation in GFP- or human PDX1-overexpressing hESC clones.(F) Expression of liver markers (AFP, ALB, TTR, APOA2, FOXA1, HHEX) in hESCs stably overexpressing either GFP or human PDX1 and differentiated for 17 days. Error bars indicate the SD of three biological replicates. p values were calculated when compared with GFP control.

Mentions: In the developing mouse embryo, both explant culture experiments and comprehensive lineage tracing show that the liver and ventral pancreas arise from bipotent, and possibly multipotent, precursors in the foregut endoderm (Angelo et al., 2012; Deutsch et al., 2001; Miki et al., 2012; Tremblay and Zaret, 2005). Consistent with this tight lineage relationship, closer inspection of our microarray data revealed companion upregulation of early liver lineage genes, including APOA2, TBX3 FOXA1, TTR, AFP, APOB, HHEX, HNF4A, APOC3, ALB, SERPINA1 (A1AT), and TDO2 on day 17 (Figure 4A). We reasoned that this finding reflected the heterogeneous nature of our pancreatic differentiation protocol, unmasking the identity of the roughly ∼35% PDX1− (Figure 1D), yet endodermally derived cells present in a given differentiation. We thus confirmed by qPCR that a series of canonical hepatic markers (FOXA1, FOXA3, HNF4A, AFP, ALB, TTR, APOA2) were indeed upregulated in an independent differentiation experiment (Figure S4C). We were surprised to find that PDX1 bound a number of these genes (±20 kb from the TSS), including HHEX, FOXA1, FOXA3, TBX3, TTR, AFP, ALB, FABP1, APOA2, PHKA2, GYS2, ARG1, LEAP2, and CDH17 (Figures 4B and S4A; Table S1, part B). Early Pdx1 expression in the mouse embryo exclusively labels the dorsal and ventral pancreatic buds, the caudal stomach, bile duct, and rostral duodenum (Jonsson et al., 1994; Jørgensen et al., 2007; Offield et al., 1996). Recent high-resolution immunohistochemistry and whole-mount in situ hybridization further demonstrate that Pdx1 and Afp do not co-localize in the AIP in the early-somite-stage mouse embryo (Miki et al., 2012). Consistent with this, PDX1 and AFP labeled distinct cell populations on day 17 of differentiation, with small nests of AFP+ cells often abutting or near PDX1 labeled ridges (Figure 4C). This result raises the possibility that a critical aspect of PDX1 function is to bind and repress hepatic genes in pancreatic progenitor (PP) cells, ensuring stable commitment to the pancreatic lineage.


PDX1 binds and represses hepatic genes to ensure robust pancreatic commitment in differentiating human embryonic stem cells.

Teo AK, Tsuneyoshi N, Hoon S, Tan EK, Stanton LW, Wright CV, Dunn NR - Stem Cell Reports (2015)

PDX1 Represses Liver Marker Genes(A) Microarray gene expression heatmap showing increasing levels of liver lineage-related genes between days 0 and 17 of pancreatic differentiation.(B) ChIP-qPCR provides independent confirmation that PDX1 binds in proximity to liver genes. Error bars indicate the SD of three technical replicates from a single ChIP-qPCR experiment. These data were independently confirmed in repeat pull-down qPCR experiments (data not shown). Normal goat IgG is the negative control.(C) AFP and PDX1 immunostaining on day 17 of pancreatic differentiation. AFP and PDX1 label distinct cell populations that are often observed in close proximity. Scale bar represents 100 μm.(D) Expression of hepatic genes (AFP, ALB, TTR, APOA2, FOXA1, FOXA2, FOXA3, and HHEX) by qPCR in HepG2 cells after transient overexpression of GFP, PDX1, or the PDX1(N196S) mutant that cannot bind DNA. Both WT PDX1 and PDX1(N196S) are robustly overexpressed after transfection. All error bars indicate SD of three biological replicates. For all genes, data are shown relative to the GFP-transfected controls. p values are indicated.(E) Western blot analyses for PDX1 and AFP on days 0 and 17 of pancreatic differentiation in GFP- or human PDX1-overexpressing hESC clones.(F) Expression of liver markers (AFP, ALB, TTR, APOA2, FOXA1, HHEX) in hESCs stably overexpressing either GFP or human PDX1 and differentiated for 17 days. Error bars indicate the SD of three biological replicates. p values were calculated when compared with GFP control.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400640&req=5

fig4: PDX1 Represses Liver Marker Genes(A) Microarray gene expression heatmap showing increasing levels of liver lineage-related genes between days 0 and 17 of pancreatic differentiation.(B) ChIP-qPCR provides independent confirmation that PDX1 binds in proximity to liver genes. Error bars indicate the SD of three technical replicates from a single ChIP-qPCR experiment. These data were independently confirmed in repeat pull-down qPCR experiments (data not shown). Normal goat IgG is the negative control.(C) AFP and PDX1 immunostaining on day 17 of pancreatic differentiation. AFP and PDX1 label distinct cell populations that are often observed in close proximity. Scale bar represents 100 μm.(D) Expression of hepatic genes (AFP, ALB, TTR, APOA2, FOXA1, FOXA2, FOXA3, and HHEX) by qPCR in HepG2 cells after transient overexpression of GFP, PDX1, or the PDX1(N196S) mutant that cannot bind DNA. Both WT PDX1 and PDX1(N196S) are robustly overexpressed after transfection. All error bars indicate SD of three biological replicates. For all genes, data are shown relative to the GFP-transfected controls. p values are indicated.(E) Western blot analyses for PDX1 and AFP on days 0 and 17 of pancreatic differentiation in GFP- or human PDX1-overexpressing hESC clones.(F) Expression of liver markers (AFP, ALB, TTR, APOA2, FOXA1, HHEX) in hESCs stably overexpressing either GFP or human PDX1 and differentiated for 17 days. Error bars indicate the SD of three biological replicates. p values were calculated when compared with GFP control.
Mentions: In the developing mouse embryo, both explant culture experiments and comprehensive lineage tracing show that the liver and ventral pancreas arise from bipotent, and possibly multipotent, precursors in the foregut endoderm (Angelo et al., 2012; Deutsch et al., 2001; Miki et al., 2012; Tremblay and Zaret, 2005). Consistent with this tight lineage relationship, closer inspection of our microarray data revealed companion upregulation of early liver lineage genes, including APOA2, TBX3 FOXA1, TTR, AFP, APOB, HHEX, HNF4A, APOC3, ALB, SERPINA1 (A1AT), and TDO2 on day 17 (Figure 4A). We reasoned that this finding reflected the heterogeneous nature of our pancreatic differentiation protocol, unmasking the identity of the roughly ∼35% PDX1− (Figure 1D), yet endodermally derived cells present in a given differentiation. We thus confirmed by qPCR that a series of canonical hepatic markers (FOXA1, FOXA3, HNF4A, AFP, ALB, TTR, APOA2) were indeed upregulated in an independent differentiation experiment (Figure S4C). We were surprised to find that PDX1 bound a number of these genes (±20 kb from the TSS), including HHEX, FOXA1, FOXA3, TBX3, TTR, AFP, ALB, FABP1, APOA2, PHKA2, GYS2, ARG1, LEAP2, and CDH17 (Figures 4B and S4A; Table S1, part B). Early Pdx1 expression in the mouse embryo exclusively labels the dorsal and ventral pancreatic buds, the caudal stomach, bile duct, and rostral duodenum (Jonsson et al., 1994; Jørgensen et al., 2007; Offield et al., 1996). Recent high-resolution immunohistochemistry and whole-mount in situ hybridization further demonstrate that Pdx1 and Afp do not co-localize in the AIP in the early-somite-stage mouse embryo (Miki et al., 2012). Consistent with this, PDX1 and AFP labeled distinct cell populations on day 17 of differentiation, with small nests of AFP+ cells often abutting or near PDX1 labeled ridges (Figure 4C). This result raises the possibility that a critical aspect of PDX1 function is to bind and repress hepatic genes in pancreatic progenitor (PP) cells, ensuring stable commitment to the pancreatic lineage.

Bottom Line: ChIP-seq also revealed PDX1 occupancy at hepatic genes.In HepG2 cells and differentiating hESCs, we found that PDX1 binds and suppresses expression of endogenous liver genes.These findings rebrand PDX1 as a context-dependent transcriptional repressor and activator within the same cell type.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Biology, A(∗)STAR (Agency for Science, Technology and Research), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648, Singapore.

Show MeSH
Related in: MedlinePlus