DMH-1 Consistent with our in vitro data two recent genetic

Consistent with our in vitro data, two recent genetic studies in the mouse provide strong in vivo evidence that Pdx1 is a bona fide transcriptional repressor. By E11.5, the pancreatic buds in Pdx1 null mutant embryos arrest and begin to regress (Ahlgren et al., 1996; Offield et al., 1996). Recently, Seymour et al. (2012) performed high-resolution quantitative immunohistochemistry on Pdx1−/− embryos and observed significant numbers of ectopic Afp+ DMH-1 within the dorsal pancreatic bud. This suggestion of partial conversion to the hepatic cell fate is consistent with prior work demonstrating that hepatic competence is not restricted to the region of the ventral foregut where the liver normally forms (Bossard and Zaret, 1998, 2000; Gualdi et al., 1996) and with our data that PDX1 represses AFP in vitro (Figures 4D–4F). Seymour et al. (2012) also showed that Pdx1 deficiency caused varying degrees of Sox9 downregulation and that focal loss of Sox9 in the developing pancreas led to elevated expression of hepatic markers. Our results show that in hESC-derived ePP cells PDX1 binds SOX9 between exons 2 and 3 (Table S1, part A), which suggests that SOX9 is positively regulated by PDX1, as expected for these principal regulators of the pancreatic program. Phylogenetic sequence conservation in this region of mouse and human SOX9 genes (data not shown) fits the idea that this Pdx1-Sox9 regulatory relationship is central to the pro-pancreatic gene regulatory network. Taken together, these findings indicate that early-stage Pdx1+ progenitor cells are not stably determined (“metastable”), with Pdx1 positively regulating Sox9 and actively repressing liver potential during a substantial period of early pancreas organogenesis. In a second and very recent study, Gao et al. (2014) inactivated Pdx1 in the adult β cell using Cre-Lox methods with concurrent indelible YFP labeling of the derived Pdx1−/− cells (Gao et al., 2014). Expectedly, these mice became rapidly hyperglycemic—a result consistent with prior work (Ahlgren et al., 1998; Gannon et al., 2008)—but unexpectedly lineage-labeled Pdx1−/− cells contained glucagon and expressed MafB, a transcription factor that in the adult mouse is restricted to the islet α-cells. These authors used ChIP from mouse insulinoma cell lines to detect PDX1 binding within 1.5 kb of the MafB TSS. Taken together, these findings strongly suggest that Pdx1 directly represses MafB transcription in adult β cells. Interestingly, MafB is required for the production of both α and β cells during pancreas development, but its expression is extinguished in β cells soon after birth (Hang and Stein, 2011). Consistent with this in vivo expression kinetic, we observed MAFB levels increasing from days 0 to 17 of hESC differentiation (Figure 3B). However, MAFB transcriptional regulation is apparently independent of PDX1 at these stages, as PDX1 binding was observed a great distance from the MAFB TSS (≥150 kb) (Table S1, part A). In addition, it is important to highlight recent data showing that, in contrast to mice, MAFB persists in a subset of human adult β cells (∼9%) (Dai et al., 2012). This finding suggests that in mice Pdx1 adopts its role as a transcription repressor late in β cell ontogeny and that in humans PDX1+MAFB+ and PDX1+MAFB− represent distinct β cell subtypes. Our findings raise an important outstanding question: what is the mechanism underlying PDX1 transcriptional repression? We speculate that one answer lies in the top-ranking motifs enriched in our ChIP-seq data—PBX1 and FOXA1/A2. Nearly 20 years ago, Pdx1 was shown to bind the HOX-cofactor Pbx1 (pre-B cell leukemia factor 1), a member of the TALE (three-amino-acid loop extension) family of atypical homeodomain-containing proteins (Peers et al., 1995). Pbx1 alternative splicing yields two isoforms differing at their C termini, the longer Pbx1a and shorter Pbx1b. Pdx1:Pbx1b complexes transcriptionally activate target genes, while Pdx1:Pbx1a forms a repressor complex through the recruitment of co-repressor proteins such as NCoR-SMRT (or HDAC) (Asahara et al., 1999; Saleh et al., 2000). PBX1A and PBX1B are both expressed during human ePP differentiation (Figure S1A; A.K.K.T. and N.R.D., unpublished data), raising the possibility of differential recruitment within the same cell to activate or repress appropriate gene targets to direct lineage choice and stabilization. Similarly, FOXA transcription factors can also recruit HDAC via the co-repressor Groucho-related protein 3 (Grg3; formally Tle3), which is highly expressed in the pancreas during embryonic development, to silence genes central to hepatic differentiation (Lam et al., 2013; Santisteban et al., 2010). Our data also show that FOXA1/A2 binding sites were significantly enriched in sequence reads from day 17 PDX1 ChIP-seq (Figure 2A), and FOXA2:PDX1 co-binding was observed frequently in nearly 2,000 loci in mouse islets (Hoffman et al., 2010). Canonical FOXA1/A2 motifs exist close to PDX1 binding in both AFP and TTR, also suggesting context-dependent functions of FOXA proteins. Finally, it is important to note that approximately 100 genes are bound by PDX1 on day 17, but their expression is significantly downregulated by microarray (comparing day 17 to day 0) (Figure 3C; Table S2, part E). Obvious candidates for direct repression among these ∼100 include FGF8, TWIST2, ETV4, and ZIC3, whose mouse orthologs are typically expressed in mesoderm or mesodermally derived tissues during embryonic development (http://www.informatics.jax.org/genes.shtml) (Table S2, part E). Methods to address the issue of direct repression or activation of PDX1 target loci include the development of PDX1-deficient hESC lines or lines carrying inducible knockdown tools for context and time-dependent inactivation.