XB-ART-52486Development January 1, 2016; 143 (19): 3573-3581.
The histone methyltransferase Setd7 promotes pancreatic progenitor identity.
Cell fate specification depends on transcriptional activation driven by lineage-specific transcription factors as well as changes in chromatin organization. To date, the interplay between transcription factors and chromatin modifiers during development is not well understood. We focus here on the initiation of the pancreatic program from multipotent endodermal progenitors. Transcription factors that play key roles in regulating pancreatic progenitor state have been identified, but the chromatin regulators that help to establish and maintain pancreatic fate are less well known. Using a comparative approach, we identify a crucial role for the histone methyltransferase Setd7 in establishing pancreatic cell identity. We show that Setd7 is expressed in the prospective pancreatic endoderm of Xenopus and mouse embryos prior to Pdx1 induction. Importantly, we demonstrate that setd7 is sufficient and required for pancreatic cell fate specification in Xenopus Functional and biochemical approaches in Xenopus and mouse endoderm support that Setd7 modulates methylation marks at pancreatic regulatory regions, possibly through interaction with the transcription factor Foxa2. Together, these results demonstrate that Setd7 acts as a central component of the transcription complex initiating the pancreatic program.
PubMed ID: 27578787
Article link: Development
Genes referenced: eef1a1o epha8 fabp2 foxa2 golga2 hhex ins kcnt1 myc odc1 pdx1 ptf1a sdha setd7 sox2 tcf3 tff3.8
Antibodies: Acetylated H3f3a Ab20 Tri-Methyl H3f3a Ab19 methyl-H3f3a Ab13 polr2a Ab8
Morpholinos: setd7 MO1
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|Fig. 1. Expression of Setd7 in the developing pancreas is conserved across vertebrate species. (A) RT-qPCR analysis of indicated transcription factors in anterior (AE) and posterior (PE) endodermal explants isolated from gastrula stage (st.) Xenopus embryos and cultured ex vivo until indicated stages. AE but not PE gives rise to pancreatic endoderm that expresses foxa2, pdx1 and ptf1a (Fig. S2) (Kelly and Melton, 2000; Horb and Slack, 2001; Spagnoli and Brivanlou, 2008). Data were normalized to that of odc and represented as fold change compared with PE sample (set to 1). n=4; error bars represent ±s.e.m. *P<0.05, **P<0.01. (B-E) In situ hybridization analysis of setd7 (B,E), pdx1 (C) and insulin (D) expression in cross-sectioned Xenopus tadpole (st. 30) embryos and dissected gut at st. 41 (E). Yellow star (*) indicates setd7 expression in the neural tube. dp, dorsal pancreatic bud; lv, liver bud; pa, pancreas. (F-J) Immunofluorescence analysis of Setd7 in the mouse endoderm and pancreas. Setd7 colocalizes with Foxa2 in the foregut endodermat E8.5 (8- to 9-somite stages) (F; dotted line demarcates the ventral foregut epithelium) and with Pdx1 in pancreatic progenitors at E10.5 (G). At E12.5 and 14.5 (H,H′,I), Setd7 is retained in the cytoplasm of pancreatic epithelium (arrowheads) and nuclear localization is detected only in delaminating cells (arrows). At E17.5 (J), Setd7 protein colocalizes with Pdx1 and insulin (Ins) in β-cell progenitors (arrows) clustering into islets. Arrowheads indicate Setd7 nuclear staining in neighbouring islet cells. Scale bars: 50 μm.|
|Fig. 2. Setd7 promotes pancreatic fate in the endoderm. (A) RT-qPCR analysis of indicated genes in Xenopus PE explants injected with Xenopus setd7 mRNA (1 ng). Embryos were injected into PE region at 4- to 8-cell stage, dissected at early gastrula stage, cultured until early tadpole stage and assayed for indicated genes; n=10. (B,C) Whole-mount ISH analysis of ptf1a (B) and pdx1 (C) in uninjected (ctrl) and setd7 mRNAinjected (1 ng) Xenopus embryos. Arrowheads indicate dorsal (dp) and ventral pancreatic (vp) buds. Brackets indicate ptf1a expression in the eye and hindbrain. Arrows indicate ectopic ptf1a and pdx1 expression in the endoderm of setd7-injected embryos. Insets show higher magnification of regions indicated by dashed frame. Scale bars: 1 mm. (D) RT-qPCR analysis of indicated genes in Xenopus PE explants injected with mouse Setd7- Myc mRNA. All explants were assayed at tadpole stage; n=6. In A and D, data were normalized to that of odc and represented as fold changes compared with uninjected PE sample (set to 1). Error bars represent ±s.e.m. *P<0.05, **P<0.01, ***P<0.001.|
|Fig. 3. Setd7 is required for specification of pancreatic progenitor cells. (A-F) Whole-mount ISH analysis of pdx1 (A,B), ptf1a (C,D) and hhex (E,F) in uninjected (ctrl) and setd7-MO-injected (5 ng) Xenopus embryos. Arrowheads indicate dorsal (dp) and ventral pancreatic (vp) buds. Brackets indicate ptf1a expression in the eye and hindbrain. Scale bars: 1 mm. (G) RT-qPCR analysis of indicated genes in Xenopus AE explants injected with increasing doses of setd7- MO. n=10. (H) RT-qPCR analysis shows that co-injection of mouse Setd7 mRNA (1 ng) together with setd7-MO in AE restores the expression of indicated endodermal and pancreatic genes; n=4. Data were normalized to that of odc and represented as fold changes compared with AE samples (set to 1). Error bars represent ±s.e.m. *P<0.05, **P<0.01, ***P<0.001.|
|Fig. 4. Setd7-H297A histone methyltransferase mutant lacks pancreatic fate-inducing activity. (A) Whole-mount ISH of pdx1, ptf1a and hhex in uninjected (ctrl) and setd7-H297A-injected (1 ng) Xenopus embryos. Arrowheads indicate dorsal (dp) and ventral pancreatic (vp) buds. Brackets indicate ptf1a expression in the eye and hindbrain. Scale bar: 1 mm. lv, liver. (B) RT-qPCR analysis of setd7-H297A-injected AE explants for indicated markers. Data were normalized to that of odc and represented as fold changes compared with uninjectedAEsample (set to 1). n=8; error bars represent ±s.e.m. *P<0.05, **P<0.01, ***P<0.001.|
|Fig. 5. Epigenetic changes upon modulation of Setd7 levels. (A) Global protein levels of H3K4me1, H3K4me3 and H3K27ac in setd7-myc (1 ng)- or setd7-MO (5 ng)-injected Xenopus lysates were determined by western blot. (B) Schematic illustration of sample preparation for ChIP-qPCR or Co-IP using setd7 mRNA- or setd7-MO-injected endodermal explants. (C) ChIP experiments were performed from setd7-MO-injected and uninjected (control) Xenopus explants with the indicated histone modification antibodies. ChIP material was analysed by qPCR for selected pancreatic and hepatic regulatory regions. Data were normalized to % of input (y-axis). n=6, error bars represent ±s.d. *P<0.05, **P<0.01, ***P<0.001. (D) Ex vivo differentiation of mESCs into PaE. RT-qPCR showing expression levels of Setd7, pluripotency markers and pancreatic genes in PaE cells at day (d) 5 relative to undifferentiated mESCs at d0. Data were normalized to Sdha and represented as fold changes compared with mESC samples (set to 1). Error bars represent ±s.e.m. **P<0.01, ***P<0.001. PaE, pancreatic endoderm. (E) ChIP experiments were performed on mESCs chromatin with the indicated antibodies. ChIP material was analysed by qPCR for selected endodermal and pancreatic regulatory regions. Data were normalized to % of input (y axis) and represented as fold changes compared with mESC undifferentiated samples (set to 1). n=4; error bars represent ±s.d. *P<0.05, **P<0.01, ***P<0.001. (F) Immunoprecipitation (IP) of Setd7 and Foxa2. Left panel, 4-cell st. embryos were injected with Xenopus setd7-myc, lysates were prepared at tadpole st., immunoprecipitated with anti-Myc antibody and analysed by immunoblot (IB) with anti-Foxa2 antibodies. Ectopic Setd7-Myc was checked by IB on the crude extracts used for the IP reaction. Right panel, IP of endogenous Setd7 and Foxa2 in differentiated mESCs. Lysates were prepared from undifferentiated mESCs and definitive endoderm (DE) and PaE cells, immunoprecipitated with anti-Setd7 antibody and analysed by IB with the indicated antibodies. Expression of Setd7 and Foxa2 was checked by IB on the crude extracts used for the IP reaction. As a loading control, all membranes were stripped and reprobed with anti-α-tubulin. (G) Model for Setd7-mediated activation of pancreatic genes. The model suggests that Setd7 is co-recruited by a cell-specific TF, such as Foxa2, to promoters of pancreatic genes, such as Pdx1, where it is required for the establishment of H3K4me3.|
|Figure S1. Expression of Setd7 in the endoderm and developing pancreas is conserved across vertebrate species. (A) Expression level of setd7 in Xenopus embryos at indicated stages. A progressive increase in setd7 transcript levels is measured at neurula and tadpole stages. Data were normalized to that of odc and represented as fold changes compared with gastrula stage sample (set to 1). Error bars represent ± SEM. *P < 0.05. (B) Expression level of Setd7 in the mouse foregut endoderm (E8.5) and pancreatic rudiments at indicated stages. Data were normalized to that of Sdha and represented as fold changes compared with E8.5 (set to 1). (C, D) Whole mount in situ hybridization (ISH) for setd7 in gastrula (st. 10+) and neurula stage (st. 14) Xenopus embryos. In C, yellow arrowheads indicate setd7 transcript in the dorsal cells adjacent to the lip. In D, hemisected st. 14 embryo shows expression of setd7 in the anterior ventral endoderm and middle dorsal portion of the archenteron roof (see yellow open arrowheads). Both regions of the endoderm are fated to give rise to pancreas. (E) Whole mount ISH analysis for foxa2 in hemisected neurula stage Xenopus embryos. foxa2 expression partially overlaps with setd7 in the endoderm. (F-H) Immunofluorescence analysis of Setd7, Pdx1, Lamp1 and GM130 in E12.5 mouse pancreas. Arrows indicate cytoplasmic Setd7 and GM130. Size bar, 50 μm. (G) Immunofluorescence analysis of Setd7, Pdx1, and Insulin in E17.5 mouse pancreas. Dotted circles demarcate acinar clusters negative for Setd7. Size bar, 50 μm. Abbreviations, Ecadherin, E-cad; Hoechst, Hoe; Insulin, Ins.|
|Figure S2. Setd7 is sufficient for the specification of pancreatic progenitor cells. (A) Schematic illustration of posterior endoderm (PE) injection into 4-cell stage Xenopus embryos. Injection of setd7-myc mRNA was confirmed by Western Blot analysis. The antibody against Setd7 does not recognize endogenous Setd7 protein in Xenopus embryo extracts. (B) Semiquantitative RT-PCR of PE and anterior endoderm (AE) explants at tadpole stage (st. 28). Expression of pdx1 is detected exclusively in AE explants, while setd7 is expressed in both regions of the endoderm, though at higher levels in AE. (C-D) Phenotypic analysis of setd7- injected Xenopus embryos. Setd7-injected embryos showed slight dorsalization in a dosedependent manner. (E) RT-qPCR analysis of indicated genes in Xenopus PE explants injected with setd7 mRNA (1 ng), uninjected PE and AE. Embryos were injected into PE region at 2-4 cell st., dissected at early gastrula st., cultured until early tadpole st. and assayed for indicated genes. Data were normalized to that of odc and represented as fold changes compared to uninjected PE sample (set to 1). Error bars represent ± SEM. *P < 0.05.|
|Figure S3. Setd7-MO knockdown approach in Xenopus embryos. (A) Schematic illustration of anterior endoderm (AE) injection into 4-cell stage Xenopus embryos. (B) Western blot analysis shows reduction of Setd7-myc protein in the presence of setd7-MO in Xenopus embryonic lysates, confirming the functionality of the designed morpholino oligonucleotide. (C) Setd7-MO-injected embryos showed slight ventralization in a dose-dependent manner. (D) Immunofluorescence analysis of skeletal muscle marker (SMM) showed disrupted muscle fibers in tadpole stage Xenopus embryos, which is consistent with the phenotype previously reported upon setd7- knockdown in zebrafish embryos (Tao et al., 2011). (E) Pdx1 expression shown in transverse section of control (ctrl) and setd7-MO-injected Xenopus embryos processed by whole mount ISH for pdx1. Arrowheads indicate ventral (vp) and dorsal (dp) pancreatic buds in setd7-MO-injected embryos. (F) Quantification of the pdx1+ expression area in the ventral and dorsal pancreatic rudiments on transverse sections of control and setd7-MO injected Xenopus embryos (n=4). Error bars represent ± SD. *P < 0.05. (G) RT-qPCR analysis of sox2 and ifabp expression in Xenopus AE-explants injected with setd7-MO (10 ng). Data were normalized to that of odc and represented as fold changes compared with AE samples (set to 1). Error bars represent ± SEM. ** P < 0.01.|
|Figure S4. Control experiments for setd7-MO knockdown approach in Xenopus embryos. (A) Whole mount ISH analysis of ptf1a and pdx1 in uninjected (ctrl), 5bp-mismatched setd7 morpholino oligonucleotide (setd7-5MM MO)-injected (5 ng) and setd7-MO-injected (5 ng) Xenopus embryos. A 5bp-mismatched setd7 MO was designed introducing 5 G to C or C to G substitutions distributed evenly through the sequence and used as additional specificity control in the knockdown experiments (Eisen, Smith, 2008) (see Material and Methods for the sequences). While injection of 5 ng of setd7-MO led to strong reduction of pancreatic genes, the equivalent dose of setd7-5MM MO did not perturb pancreas formation, providing further evidence of morpholino specificity. Arrowheads indicate dorsal and ventral pancreatic buds. Brackets indicate ptf1a expression in the eye and hindbrain. Size bar, 1mm. n=3. (B) Whole mount ISH analysis using ptf1a and insulin antisense probes. Tadpole embryos (st. 36) injected with setd7-MO showed reduction of ptf1a and insulin expression domains (see arrowheads; 70%). ptf1a and insulin expression was rescued in embryos injected with setd7-MO and mouse Setd7 (500 pg) mRNA (~70-80%). Embryos left untreated (ctrl) show normal ptf1a expression in both pancreatic buds, as indicated by the arrowheads, and hindbrain (white bracket), as well as insulin expression in the dp (yellow arrowheads). n=3. Development • Supplementary information|
|Figure S5. Activity of Setd7-H297A mutant in Xenopus embryos. (A) Schematic of Setd7 protein structure and aminoacid allignment of the highly conserved region in the SET domain (288-306), harboring the histonemethyltransferase activity of SET proteins (Nishioka et al., 2002). The SET7 domain is highly conserved across vertebrate species, with 81% identity between the human and Xenopus laevis homologues. In Red, the key conserved histidine residue within the SET domain; its single amino acid substitution (H297A) results in the loss of methyltransferase activity (Nishioka et al., 2002). Xenopus setd7-H297A was generated by site-directed mutagenesis PCR. (B-C) Xenopus embryos injected with setd7-H297A mRNA recapitulate the same pancreatic phenotype observed in setd7-MO-injected embryos. Transverse sections of control (ctrl) and setd7-H297A-injected Xenopus embryos processed by whole mount ISH for hhex (B) and ptf1a (C). Hhex+ and ptf1a+ expression areas were quantified on transverse sections. Error bars represent ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations, dp, dorsal pancreas; vp, ventral pancreas; lv, liver.|
|Figure S6. ChIP-qPCR control experiments. (A) ChIP-qPCR analysis of H3K4me1, H3K4me3, H3K27ac and GFP at the indicated regulatory regions in Xenopus embryos at tadpole stage. Eef1a1o (eukaryotic translation elongation factor 1 alpha 1, promoter) and tff2 (trefoil factor 2) loci were chosen as positive and negative controls primers for H3K4me3 binding, respectively (Akkers et al., 2012). Data were normalized to % of input (y axis). Error bars represent ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (B) Occupancy of H3K4me1, H3K4me3 and H3K27ac at the tff2 locus, used as negative control locus, in control and setd7-MO-injected embryos. No changes in non-specific enrichment at the negative control locus were detected in setd7-MO-injected embryos when compared to controls. (C) ChIP-qPCR analysis of GFP at the indicated regulatory regions in control and setd7-MO-injected embryos at tadpole stage. As expected, GFP binding was not detected at any of the regions tested by ChIP assays. ChIP-qPCR signals were calculated as percentage of input as previously described (Blythe et al. 2009; Akkers et al., 2012; Kartikasari et al., 2013).|
|setd7(SET domain containing (lysine methyltransferase) 7) gene expression in dissected gut of Xenopus laevis embryo, assayed via in situ hybridization, NF stage 41, lateral view, anterior left, dorsal up.|