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Epigenome regulation during epidermal lineage commitment [RNA-seq]

ABSTRACT: Recent advances in human pluripotent stem cell (hPSC) technology provide a unique resource for skin tissue replacement, but the detailed understanding of regulatory mechanisms limits standardization and broad clinical application. Here, we interrogate chromatin accessibility and transcriptome dynamics during hPSC-derived epidermal differentiation, and discover two critical transition periods: surface ectoderm initiation and keratinocyte maturation. Using inference network modeling, we develop a computational framework for each transition, and identify TFAP2 and TP63 and their cofactors as key regulators. Surprisingly, functional studies demonstrate the sufficiency of TFAP2C to initiate surface ectoderm differentiation by activating the early TF network and its chromatin landscape changes, while loss of TFAP2C inhibits early commitment. TFAP2Cinitiated cells are competent to further differentiate into functional keratinocytes in selective media, accompanied by activation of the keratinocyte maturation network and decline of the early network. Mechanistically, TFAP2C activates the expression and increases binding site accessibility and positive autoregulation of the master regulator P63, while loss of P63 results in failure to close TFAP2-initiated early program and leads to maturation and survival defects, revealing a positive-negative feedback loop that ensures complete transition from progenitor to maturation tissue. Our work reveals the logic underlying dynamic epigenome-transcription factor interactions during human epidermal lineage commitment that will facilitate improved tissue engineering and regenerative medicine.

ORGANISM(S): Homo sapiens

PROVIDER: GSE122383 | GEO | 2019/01/28

REPOSITORIES: GEO

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Epigenome regulation during epidermal lineage commitment [ChIP-seq]

Project description:Recent advances in human pluripotent stem cell (hPSC) technology provide a unique resource for skin tissue replacement, but the detailed understanding of regulatory mechanisms limits standardization and broad clinical application. Here, we interrogate chromatin accessibility and transcriptome dynamics during hPSC-derived epidermal differentiation, and discover two critical transition periods: surface ectoderm initiation and keratinocyte maturation. Using inference network modeling, we develop a computational framework for each transition, and identify TFAP2 and TP63 and their cofactors as key regulators. Surprisingly, functional studies demonstrate the sufficiency of TFAP2C to initiate surface ectoderm differentiation by activating the early TF network and its chromatin landscape changes, while loss of TFAP2C inhibits early commitment. TFAP2Cinitiated cells are competent to further differentiate into functional keratinocytes in selective media, accompanied by activation of the keratinocyte maturation network and decline of the early network. Mechanistically, TFAP2C activates the expression and increases binding site accessibility and positive autoregulation of the master regulator P63, while loss of P63 results in failure to close TFAP2-initiated early program and leads to maturation and survival defects, revealing a positive-negative feedback loop that ensures complete transition from progenitor to maturation tissue. Our work reveals the logic underlying dynamic epigenome-transcription factor interactions during human epidermal lineage commitment that will facilitate improved tissue engineering and regenerative medicine.

2019-01-29 | GSE125857 | GEO

Epigenome regulation during epidermal lineage commitment [ATAC-seq, RNA-seq]

2019-01-28 | GSE108248 | GEO

UMI-4C in wild-type and p63-null surface ectoderm cells

Project description:Purpose: Validate HiChIP-identified long-range chromatin interactions at the TFAP2C locus, in a viewpoint-specific manner. Methods: UMI-4C libraries targeting 3 individual viewpoints at the TFAP2C locus were generated in duplicate, in surface ectoderm cells with and without p63. Replicates were sequenced on Illumina NextSeq sequencer, and reads were aligned and filtered for valid long-range interactions using HiCPro. Results: Dynamic long-range interactions at the TFAP2C locus were detected with and without p63 in surface ectoderm cells. Conclusion: Long-range interactions detected via HiChIP were confirmed in the surface ectoderm by this technique, validating the HiChIP method through an orthogonal approach.

2018-11-06 | GSE114846 | GEO

Amniogenesis occurs in two independent waves in primate embryos

Project description:In primates the amnion emerges through cavitation of the epiblast during implantation, whereas in other species by folding of the ectoderm later around gastrulation. How the mechanisms of amniogenesis diversified in evolution remains unknown. Here we employed the naïve-to-primed transition of human pluripotent stem cells (hPSC) to model peri-implantation epiblast development. We discovered that during this transition hPSC transiently gain the ability to differentiate into cavitating epithelium that transcriptionally matches the early amnion of human embryos. In contrast, primed hPSC produce cells resembling late amnion, uncovering an independent differentiation route. Unexpectedly, single-cell analysis of primate embryos revealed two transcriptionally distinct amniogenesis waves. The early wave occurs through a trophectoderm-like route and encompasses cavitation, whereas the late wave follows an ectoderm-like pathway during gastrulation. The discovery of two independent waves of amniogenesis both of which can be modelled in hPSC explains how amniogenesis through cavitation emerged during the evolution of primates.

2022-04-20 | GSE179309 | GEO

Genome-wide transcription factor binding and histone mark profiling in pluripotent and lineage-committed cells

Project description:Purpose: Assess changes in p63 binding in human embryonic stem cells and surface ectoderm; evaluate differences in the following histone marks: H3K27me3, H3K27ac, H3K4me1, and H3K4me3 in the surface ectoderm with and without p63; and examine changes in H3K27me3 between human embyronic stem cells and surface ectoderm with and without p63. Methods: p63 ChIP-seq libraries were generated in p63 gain-of-function human embryonic stem cells and wild-type surface ectoderm. H3K27me3 ChIP-seq libraries were generated in wild-type and p63 gain-of-function human embryonic stem cells as well as wild-type and p63 knockout surface ectoderm. H3K27ac, H3K4me1, and H3K4me3 ChIP-seq libraries were generated in wild-type and p63 knockout surface ectoderm. All ChIP-seq experiments were performed in duplicate and sequenced on Illumina NextSeq 500 sequencer. To ensure quality reads, fastq files were analyzed using FASTQC. Bowtie was used for read mapping and the parameters were as follows: -p 24 -S -a -m 1 --best --strata. For peak calling using MACS2, default settings were specified with a p-value of 0.05. To ensure quality peaks, IDR was run on all files, specifying FDR of 1% or 5% (or 10% for some broadPeak histone marks). Results: Limited changes in p63 binding in human embryonic stem cells and surface ectoderm; global loss of H3K37me3 without p63 in the surface ectoderm; limited differences in H3K27ac, H3K4me1, and H3K4me3 +/- p63 in the surface ectoderm; and increase in H3K27me3 with ectopic expression of p63 in human embryonic stem cells. Conclusion: p63 is able to bind it's surface ectoderm target sites regardless of the epigenetic landscape; p63 promotes H3K27me3 accumulation; and p63 does not regulate histone marks H3K27ac, H3K4me1, or H3K4me3

2018-11-06 | GSE122128 | GEO

siRNA profiling of human MCF7 cells with elimination of either TFAP2C or TFAP2A reveals TFAP2C regulates multiple pathways of estrogen signaling

Project description:We were interested in determining what genes might be controlled by TFAP2C and/or TFAP2A, either directly or indirectly through regulation of ER-alpha and potentially other signaling pathways. We performed an microarray analysis in MCF7 cells with elimination of either TFAP2C or TFAP2A. The patterns of gene expression with alteration of TFAP2 activity were compared to changes in expression induced by estrogen exposure. Knock-down of TFAP2C in the presence of estrogen altered the pattern of several known ERalpha-regulated genes and a number of genes outside the estrogen-regulated pathways. Experiment Overall Design: 6 samples were analyzed. Experiment Overall Design: 1. MCF7 cells treated with TFAP2C siRNA, without the presence of estrogen. Experiment Overall Design: 2.MCF7 cells treated with TFAP2C siRNA, with the presence of estrogen. Experiment Overall Design: 3.MCF7 cells treated with TFAP2A siRNA, without the presence of estrogen. Experiment Overall Design: 4.MCF7 cells treated with TFAP2A siRNA, with the presence of estrogen. Experiment Overall Design: 5.MCF7 cells with no siRNA treatment, without the presence of estrogen. Experiment Overall Design: 6.MCF7 cells with no siRNA treatment, with the presence of estrogen.

2008-06-16 | E-GEOD-8640 | biostudies-arrayexpress

Cohesin HiChIP datasets of pluripotent and surface ectoderm cells with and without p63

Project description:Purpose: Assess changes in the three-dimensional (3D) chromatin architecture during surface ectoderm commitment and evaluate changes in 3D chromatin architecture in human embryonic stem cells and surface ectoderm cells with and without p63. Methods: Cohesin HiChIP profiles for human embryonic stem cells and surface ectoderm cells with and without p63 were generated in triplicate with deep sequencing on the Illumina HiSeq 4000 sequencer. Using HiC-Pro, paired end reads were aligned to hg19, duplicate reads were removed, and then were assigned to MboI restriction fragments, filtered for valid interactions, and used to generate binned interaction matrices of 10 kb resolution. Contacts were called using FitHiC and filtered for counts >= 10 and FDR < 0.001. Results: The greatest change in 3D chromatin architecture was observed between human embryonic stem cells and surface ectoderm cells, while moderate changes were observed in human embryonic stem cells with and without p63, and limited changes were observed in surface ectoderm cells with and without p63. Conclusion: Both p63 and the surface ectoderm morphogens direct changes in chromatin folding to establish the 3D chromatin landscape in the surface ectoderm.

2018-11-06 | GSE120294 | GEO

RNA-seq profiles of pluripotent and surface ectoderm cells with and without p63

Project description:Purpose: Evaluate p63 regulation of gene expression in human embryonic stem cells and the surface ectoderm Methods: RNA profiles for human embyronic stem cells and surface ectoderm cells with and without p63 using deep sequencing, in duplicate, on Illumina NextSeq 500 sequencer. Quality of reads were checked by fastqc. Reads were aligned to hg19 genome using tophat and FPKM values were generated using Homer. Results: p63 is largely unable to regulate gene expression in pluripotent cells, but acts primarily as a repressor in the surface ectoderm. Conclusion: p63 regulation of gene expression is dependent on the changes the cell undergoes during surface ectoderm commitment

2018-11-06 | GSE121994 | GEO

Unbiased Proteomics Benchmarking of Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes

Project description:Rationale: Human pluripotent stem cells-derived cardiomyocytes (hPSC-CMs) exhibit the properties of fetal CMs, which limit their applications. Various methods have been used to promote maturation of hPSC-CMs; however, there is a lack of an unbiased and comprehensive method for accurate benchmarking of hPSC-CM maturation. Objective: We aim to develop an unbiased proteomics method integrating high-throughput top-down targeted proteomics and bottom-up global proteomics for accurate and comprehensive assessment of hPSC-CM maturation. Methods and Results: Utilizing hPSC-CMs from early- and late-stage two-dimensional monolayer culture and three-dimensional engineered cardiac tissue, we demonstrated high reproducibility and reliability of the top-down proteomics method, which enabled simultaneous quantification of contractile protein isoform expressions and their PTMs. This method allowed for the detection of known maturation-associated contractile protein alterations, and for the first time, identified contractile protein PTMs as promising new markers of maturation. By employing a global proteomics strategy, we identified candidate maturation markers important for sarcomere organization, cardiac excitability, and Ca2+ homeostasis; and validated these markers in the developing mouse cardiac ventricles. Conclusions: We established an unbiased proteomics method that can provide accurate and specific benchmarking of hPSC-CM maturation, and identified new markers of maturation. Furthermore, this integrated proteomics strategy laid a strong foundation for uncovering molecular basis underlying cardiac development and disease using hPSC-CMs.

| MSV000082985 | MassIVE

Genome-wide chromatin accessibility in pluripotent and surface ectoderm cells with and without p63

Project description:Purpose: Evaluate changes in chromatin accessibility between human embryonic stem cells and surface ectoderm cells with and without the transcription factor p63. Method: Chromatin accessibility profiles for human embryonic stem cells and surface ectoderm with and without p63 were generated by deep sequencing, in duplicate, with the Illumina NextSeq sequencer. Quality reads were assessed by fastq. Reads were aligned using Bowtie2, PCR duplicates and mitochondrial DNA were discarded, and peak calling was completed using MACS2. Non-reproducible peaks were filtered out by IDR with an FDR of 5%. Results: p63 fails to modify chromatin accessibility when ectopically expressed in human embryonic stem cells; differentiation of human embryonic stem cells to surface ectoderm results in the closure of some regions open in pluripotent cells and the opening of new regions; and loss of p63 in the surface ectoderm results in increased accessibility of 1/3 of the regions opened during surface ectoderm differentiation. Conclusion: p63's ability to modify chromatin accessibility is dependent upon the underlying epigenetic landscape, and when it can modify the landscape, it does so by closing regions that become open during ectoderm differentiation.

2018-11-06 | GSE119996 | GEO

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