Dataset Information

Regulatory variants in a novel distal enhancer regulate the expression of CYP3A4 and CYP3A5: ATAC-Seq and 4C data from human hepatocytes [ATAC-seq]

ABSTRACT: Regulatory variants in a novel distal enhancer regulate the expression of CYP3A4 and CYP3A5: ATAC-Seq and 4C data from human hepatocytes [ATAC-seq]

PROVIDER: PRJNA856284 | ENA |

REPOSITORIES: ENA

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Project description:4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or "bait") that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes. 4C-Seq experiments from Igh and Cd83 bait in activated B cells and Tcrb (Eb) bait in double negative T cells and immature B cells. RNA-Seq and ATAC-Seq experiments in DN and Immature B cells.

Project description:The capacity to generate functional hepatocytes from renewable human pluripotent stem cells (hPSCs) could address limited supplies of primary human hepatocytes. However, hepatocytes differentiated from hPSCs in vitro are functionally immature. To understand mechanisms regulating maturation of in vitro derived hepatocytes, we developed a 3D spheroid differentiation system and compared gene regulatory elements in uncultured human primary hepatocytes with those in hepatocytes that were differentiated in 2D or 3D conditions from human PSCs by RNA-seq, ATAC-seq, and H3K27Ac ChIP-seq. Three-dimensional differentiation improved enhancer activity and expression of transcription factor ONECUT1, but was insufficient to upregulate human-specific mature hepatocytes marker gene CYP3A4 or super-enhancer regulated transcription factor gene NFIC. Regulome comparisons showed reduced enrichment of thyroid receptor THRB motifs in accessible chromatin and in active enhancers without reduced transcription of THRB, suggesting the regulation at the level of THRB ligands in PSC-differentiated hepatocytes. Addition of thyroid hormone T3 to the PSC-differentiated hepatocytes increased CYP3A4 expression. T3 increased binding of THRB to the CYP3A4 proximal enhancer and restored the super-enhancer status and gene expression of NFIC and reduced expression of AFP. The resultant hPSC-hepatocytes showed gene expression, epigenetic status and super-enhancer landscape closer to primary hepatocytes and activated regulatory regions including non-coding SNPs associated with liver-related diseases. Transplanting the 3D PSC-hepatocytes into immunocompromised mice resulted in engraftment of human hepatocytes in the mouse liver parenchyma without disrupting normal liver histology at 6 months after transplantation. This work provides insights into the functions of nuclear receptor THRB and highlights the importance of the environmental factors-nuclear receptors axis in regulating maturation of human PSC-differentiated cell types.

Dataset Information

Regulatory variants in a novel distal enhancer regulate the expression of CYP3A4 and CYP3A5: ATAC-Seq and 4C data from human hepatocytes [ATAC-seq]

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