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

Abstract

The appropriate development of the small intestine (SI) is critical for efficient nutrient absorption and barrier function after birth. Most of the molecular features and regional patterning of the SI are programmed very early in prenatal development. However, the chromatin regulatory dynamics that underpin early SI development in humans is largely unknown. To fill this knowledge void, we apply a cutting-edge genomic technology to a state-of-the-art model of human SI development. Specifically, we leverage chromatin run-on sequencing (ChRO-seq) to define the landscape of active transcriptional regulatory elements across early stages of directed differentiation of human pluripotent stem cells (hPSCs) into SI organoids. Through comprehensive bioinformatic analysis of the data we provide the first-ever view of the changing chromatin regulatory landscape and define stage-specific key enhancer hotspots during human SI development. We also identify candidate transcription factors and their cistromes that are associated with the acquisition of SI identity and the initiation of regional patterning. This work offers a rich resource for studying transcriptional regulation of early human SI development.

Super-enhancer signature reveals key mechanisms associated with resistance to non-alcoholic steatohepatitis in humans with obesity

“…Accumulating evidence shows that enhancer hotspots serve to control the transcription of genes especially critical for cell fate identity during development and cell behavior in disease progression(22). To identify enhancer regions, stitched enhancer regions, and enhancer hotspots in NP, we used an algorithm that we have recently developed(31, 32) (Methods; Figure 3A ), which itself is adapted from a previous pipeline for super enhancer detection(23).…”

“…To identify enhancer regions, stitched enhancer regions, and enhancer hotspots in NP, we used an algorithm that we have recently developed (31,32) (Methods; Figure 3A), which itself is adapted from a previous pipeline for super enhancer detection (23). Among all 364 stitched enhancers in NP, we identified 42 that contain multiple NP-specific enhancers ("NP-specific multi-enhancer regions", Figure 3B).…”

“…NASH-resistant and NASH-prone enhancer hotspots were defined using an analysis pipeline described previously (32). The enhancer hotspots in this study were identified by the criteria similar to the studies describing 'super-enhancers'.…”

“…Also, a major advantage of ChRO-seq relative to its previous iterations, known as GRO-seq (29) and PRO-seq (30), is that it can be applied to archived, frozen tissues, making it available for profiling human liver biopsy specimens. As an example of the utility of this method, recent studies have leveraged ChRO-seq to define transcriptional programs that underpin human cancer pathogenesis (28,31) as well as gut development in a human organoid model (32).…”

Multiomic analysis defines the first microRNA atlas across all small intestinal epithelial lineages and reveals novel markers of almost all major cell types

“…ChRO-seq was performed as previously described (51,52) To construct ChRO-seq libraries, the RNAs were processed through the following procedures: (i) 3′ adapter ligation with T4 RNA Ligase 1 (NEB, M0204), (ii) Streptavidin bead binding followed by Trizol extraction, (iii) 5′ de-capping with RNA 5′ pyrophosphohydrolase (RppH, NEB, M0356), (iv) 5′ end phosphorylation using T4 polynucleotide kinase (NEB, M0201), (v) 5′ adapter ligation with T4 RNA Ligase 1 (NEB, M0204). The 5′ adaptor contained a 6-nucleotide unique molecular identifier (UMI) to allow for bioinformatic detection and elimination of PCR duplicates in data processing steps, (vi) Streptavidin bead binding followed by Trizol extraction, (vii) cDNA synthesis by reverse transcription using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific, 18090010) and (viii) library amplification by PCR using the Q5 High-Fidelity DNA Polymerase (NEB, M0491).…”