Chromatin regulatory dynamics of early human small intestinal development using a directed differentiation model

Abstract

The establishment of the small intestinal (SI) lineage during human embryogenesis ensures functional integrity of the intestine after birth. The chromatin dynamics that drive SI lineage formation and regional patterning in humans are essentially unknown. To fill this knowledge void, we apply a cutting-edge genomic technology to a state-of-the-art human model of early SI development. Specifically, we leverage chromatin run-on sequencing (ChRO-seq) to define the landscape of active promoters, enhancers and gene bodies across distinct stages of directed differentiation of human pluripotent stem cells into SI spheroids with regional specification. Through comprehensive ChRO-seq analysis we identify candidate stage-specific chromatin activity states, novel markers and enhancer hotspots during the directed differentiation. Moreover, we propose a detailed transcriptional network associated with SI lineage formation or regional patterning. Our ChRO-seq analyses uncover a previously undescribed pattern of enhancer activity and transcription at HOX gene loci underlying SI regional patterning. We also validated this unique HOX dynamics by the analysis of single cell RNA-seq data from human fetal SI. Overall, the results lead to a new proposed working model for the regulatory underpinnings of human SI development, thereby adding a novel dimension to the literature that has relied almost exclusively on non-human models.

Cells of the human intestinal tract mapped across space and time

“…Fetal-specific subsets included proximal progenitors (FGG, BEX5) as described in refs. [1][2][3]52 , distal progenitors enriched for colon genes (CKB, AKAP7, GPC3) and CLDN10 cells that are possibly pancreatic progenitors based on expression of DLK1, PDX1, RBPJ, CPA1 and SOX9 53 . This population also highly expressed CLU, a marker for intestinal revival stem cells that has previously been described 54 .…”

Integrative genome-scale analyses reveal post-transcriptional signatures of early human small intestinal development in a directed differentiation organoid model

“…Indeed, we observed high transcriptional activity around the miR-375 locus only in the DE stage, but not in other stages including Duo spheroids ( Figure 6B ). We mined previously defined transcriptional regulatory elements (TREs) in the hPSC-HIO model (26) and found that four out of the six TREs adjacent to the miR-375 locus (TRE#2, #3, #4 and #6) are uniquely active in DE ( Figure 6B ). We also observed that the summed transcriptional activity of putative enhancer TREs (TRE#1-4) is highly correlated with the transcriptional activity of TREs overlapping with the putative promoter region of miR-375 (TRE#5-6) across all four stages of the directed differentiation model ( Figure 6C ).…”

“…Chromatin run-on sequencing: Chromatin isolation for chromatin run-on sequencing (ChRO-seq) was performed as previously described (25,26). To perform run-on reaction with the isolated chromatin, samples were mixed with an equal volume of 2X run-on reaction mix [10 mM Tris-HCl pH 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 400 μM ATP, 0.8 μM CTP, 400 μM GTP, 400 μM UTP, 40 μM Biotin-11-CTP (Perkin Elmer, Waltham, MA; NEL542001EA), 100 ng yeast tRNA (VWR, 80054-306), 0.8 units/μL SUPERase In RNase Inhibitor, 1% (w/v) Sarkosyl].…”

“…Reads from transcriptional regulatory elements (TREs) that are present within gene bodies were also excluded to avoid bias of intragenic TRE activity. The TREs that are present in specific stage of directed differentiation experiments were defined previously in Hung et al (26) using dREG (82). BEDTools was used to remove reads mapping to these TREs within gene bodies.…”

“…These SI spheroids can be regionally patterned (duodenum vs. ileum) by controlling the length of time that they are exposed to FGF and WNT signaling (23). We have previously applied chromatin run-on sequencing (ChROseq) (25) in this model system to successfully profile enhancer dynamics and define transcriptional programs relevant to SI developmental events in human (26). Here we aimed to define post-transcriptional programs that underlie human SI development and identify candidate master miRNA regulators in this context.…”