Intestinal stem cell (ISC) plasticity is thought to be regulated by broadly-permissive chromatin shared between ISCs and their progeny. Here, we utilize a Sox9EGFP reporter to examine chromatin across ISC differentiation. We find that open chromatin regions (OCRs) can be defined as broadly-permissive or dynamic in a locus-specific manner, with dynamic OCRs found primarily in loci consistent with distal enhancers. By integrating gene expression with chromatin accessibility at transcription factor (TF) motifs in context of Sox9EGFP populations, we classify broadly-permissive and dynamic chromatin relative to TF usage. These analyses identify known and potential regulators of ISC differentiation via association with dynamic changes in chromatin. Consistent with computational predictions, Id3-null mice exhibit increased numbers of cells expressing ISC-specific biomarker OLFM4. Finally, we examine the relationship between gene expression and 5-hydroxymethylcytosine (5hmC) in Sox9EGFP populations, which reveals 5hmC enrichment in absorptive lineage specific genes. Our data demonstrate that intestinal chromatin dynamics can be quantitatively defined in a locus-specific manner, identify novel potential regulators of ISC differentiation, and provide a chromatin roadmap for further dissecting cis regulation of cell fate in the intestine.

Background & Aims Defining the genetic heterogeneity of intrahepatic biliary epithelial cells (BECs) is challenging, and tools for identifying BEC subpopulations are limited. Here, we characterize the expression of a Sox9 EGFP transgene in the liver and demonstrate that green fluorescent protein (GFP) expression levels are associated with distinct cell types. Methods Sox9 EGFP BAC transgenic mice were assayed by immunofluorescence, flow cytometry, and gene expression profiling to characterize in vivo characteristics of GFP populations. Single BECs from distinct GFP populations were isolated by fluorescence-activated cell sorting, and functional analysis was conducted in organoid forming assays. Intrahepatic ductal epithelium was grown as organoids and treated with a Yes-associated protein (Yap) inhibitor or bile acids to determine upstream regulation of Sox9 in BECs. Sox9 EGFP mice were subjected to bile duct ligation, and GFP expression was assessed by immunofluorescence. Results BECs express low or high levels of GFP, whereas periportal hepatocytes express sublow GFP. Sox9 EGFP+ BECs are differentially distributed by duct size and demonstrate distinct gene expression signatures, with enrichment of Cyr61 and Hes1 in GFP high BECs. Single Sox9 EGFP+ cells form organoids that exhibit heterogeneous survival, growth, and HNF4A activation dependent on culture conditions, suggesting that exogenous signaling impacts BEC heterogeneity. Yap is required to maintain Sox9 expression in biliary organoids, but bile acids are insufficient to induce BEC Yap activity or Sox9 in vivo and in vitro. Sox9 EGFP remains restricted to BECs and periportal hepatocytes after bile duct ligation. Conclusions Our data demonstrate that Sox9 EGFP levels provide readout of Yap activity and delineate BEC heterogeneity, providing a tool for assaying subpopulation-specific cellular function in the liver.

Intestinal stem cell (ISC) plasticity is thought to be regulated by broadly-permissive chromatin shared between ISCs and their progeny. Here, we utilize a Sox9 EGFP reporter to examine chromatin across ISC differentiation. We find that open chromatin regions (OCRs) can be defined as broadly-permissive or dynamic in a locus-specific manner, with dynamic OCRs found primarily in loci consistent with distal enhancers. By integrating gene expression with chromatin accessibility at transcription factor (TF) motifs in context of Sox9 EGFP populations, we classify broadly-permissive and dynamic chromatin relative to TF usage. These analyses identify known and potential regulators of ISC differentiation via their association with dynamic changes in chromatin. We observe ISC expansion in Id3-null mice, consistent with computational predictions. Finally, we examine the relationship between gene expression and 5hydroxymethylcytosine (5hmC) in Sox9 EGFP populations, which reveals 5hmC enrichment in absorptive lineage specific genes. Our data demonstrate that intestinal chromatin dynamics can be quantitatively defined in a locus-specific manner, identify novel potential regulators of ISC differentiation, and provide a chromatin roadmap for further dissecting the role of cis regulation of cell fate in the intestine. Tetteh et al., 2016). The genetic mechanisms that allow IECs to adopt metastable differentiated fates while exhibiting facultative ISC function are not fully understood.Chromatin landscapes consist of histone post-translational modifications, DNA modifications, and higher-order structural organization, and are known to exert regulatory control on cell fate.

Intrahepatic bile ducts are lined by biliary epithelial cells (BECs). However, defining the genetic heterogeneity of BECs remains challenging, and tools for identifying BEC subpopulations are limited. Here, we characterize Sox9EGFP transgene expression in the liver and demonstrate that GFP expression levels are associated with distinct cell types. BECs express “low” or “high” levels of GFP, while periportal hepatocytes express “sublow” GFP. Sox9EGFP distribution varies by duct size, with GFPhigh BECs found at greater numbers in smaller ducts. RNA-seq reveals distinct gene expression signatures for Sox9EGFP populations and enrichment of Notch and Yap signaling in GFPlow and GFPhigh BECs. All GFP+ populations are capable of forming organoids, but demonstrate interpopulation differences in organoid survival and size, dependent on media conditions. Organoids derived from Sox9EGFP populations also demonstrate differential activation of HNF4A protein in hepatocyte media conditions, suggesting variable potency in BEC subpopulations. We find that Yap signaling is required to maintain Sox9 expression in biliary organoids, and that bile acids are insufficient to induce Yap activity or Sox9 in vivo and in vitro. Our data demonstrate that Sox9EGFP levels provide a readout of Yap activity and delineate BEC heterogeneity, providing a tool for assaying subpopulation-specific cellular function in the liver.

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