BMP/SMAD signaling is a crucial regulator of intestinal differentiation 1 – 4 . However, the molecular underpinnings of the BMP pathway in this context are unknown. Here, we characterize the mechanism by which BMP/SMAD signaling drives enterocyte differentiation. We establish that the transcription factor HNF4A acts redundantly with an intestine-restricted HNF4 paralog, HNF4G, to activate enhancer chromatin and upregulate the majority of transcripts enriched in the differentiated epithelium; cells fail to differentiate upon double knockout of both HNF4 paralogs. Furthermore, we show that SMAD4 and HNF4 function via a reinforcing feed-forward loop, activating each other’s expression and co-binding to regulatory elements of differentiation genes. This feed-forward regulatory module promotes and stabilizes enterocyte cell identity; disruption of the HNF4-SMAD4 module results in loss of enterocyte fate in favor of progenitor and secretory cell lineages. This intersection of signaling and transcriptional control provides a framework to understand regenerative tissue homeostasis, particularly in tissues with inherent cellular plasticity 5 .

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The brush border is comprised of microvilli surface protrusions on the apical surface of epithelia. This specialized structure greatly increases absorptive surface area and plays crucial roles in human health. However, transcriptional regulatory networks controlling brush border genes are not fully understood. Here, we identify that hepatocyte nuclear factor 4 (HNF4) transcription factor is a conserved and important regulator of brush border gene program in multiple organs, such as intestine, kidney and yolk sac. Compromised brush border gene signatures and impaired transport were observed in these tissues upon HNF4 loss. By ChIP-seq, we find HNF4 binds and activates brush border genes in the intestine and kidney. H3K4me3 HiChIP-seq identifies that HNF4 loss results in impaired chromatin looping between enhancers and promoters at gene loci of brush border genes, and instead enhanced chromatin looping at gene loci of stress fiber genes in the intestine. This study provides comprehensive transcriptional regulatory mechanisms and a functional demonstration of a critical role for HNF4 in brush border gene regulation across multiple murine epithelial tissues.

18As an embryo matures into a fetus, cells undergo remarkable transitions, accompanied by shifts 19 in transcription factor regulatory networks and chromatin landscapes. The mechanisms of these 20 developmental transitions are not completely understood. The embryonic intestine transitions 21 from a rapidly proliferating tube with pseudostratified epithelium prior to embryonic day (E) 22 14.5, to an exquisitely folded columnar epithelium in the fetus. We sought to define factors that 23 drive fetal maturation of the intestine. ATAC-seq profiling revealed a dramatic restructuring of 24intestinal chromatin during the embryonic-to-fetal transition, with CDX2 transcription factor 25 motifs abundant at chromatin-accessible regions of the embryo, and hepatocyte nuclear factor 26 4 (HNF4) transcription factor motifs the most abundant in the fetal stages. Genetic inactivation 27of Hnf4and its paralog, Hnf4, revealed that HNF4 factors are redundantly and vitally required 28 for fetal maturation. In the embryo, CDX2 binds to and activates Hnf4 gene loci to drive HNF4 29 expression at fetal stages. HNF4 and CDX2 transcription factors then occupy shared genomic 30 regulatory sites and are required for chromatin accessibility at genes expressed in the maturing 31 fetal intestine. Thus, intestinal transcription factor regulatory networks shift to accompany 32 changing chromatin landscapes and gene expression profiles that occur during the transition of 33 an embryonic tissue to its mature state. 34 35 KEY WORDS 36 HNF4 transcription factors, Developing intestine, Maturation, Chromatin 37 38 39

As embryos mature, cells undergo remarkable transitions that are accompanied by shifts in transcription factor regulatory networks. Mechanisms driving developmental transitions are incompletely understood. The embryonic intestine transitions from a rapidly proliferating tube with pseudostratified epithelium prior to murine embryonic day (E) 14.5 to an exquisitely folded columnar epithelium in fetal stages. We sought to identify factors driving mouse fetal intestinal maturation by mining chromatin accessibility data for transcription factor motifs. ATAC-seq accessible regions shift during tissue maturation, with CDX2 transcription factor motifs abundant at chromatin-accessible regions of the embryo. Hepatocyte nuclear factor 4 (HNF4) transcription factor motifs are the most abundant in the fetal stages (>E16.5). Genetic inactivation of Hnf4a and its paralog Hnf4g revealed that HNF4 factors are redundantly required for fetal maturation. CDX2 binds to and activates Hnf4 gene loci to elevate HNF4 expression at fetal stages. HNF4 and CDX2 transcription factors then occupy shared genomic regulatory sites to promote chromatin accessibility and gene expression in the maturing intestine. Thus, HNF4 paralogs are key components of an intestinal transcription factor network shift during the embryonic to fetal transition.