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

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. Chromatin accessibility and gene expression profile in epidermal differentiation from normal hESC, iPSC , and hESCs containing inducible expression of TFAP2C or p63 KO were generated by ATAC-seq and RNA-seq, in two replicates at each stage, using illunia Hiseq 2000 and Nextseq 500. Chromatin accessibility (NextSeq 500) and gene expression profile (HiSeq 2000) in normal human keratinocye (NHK).

人类多能干细胞 (human pluripotent stem cell, hPSC) 技术的最新进展为皮肤组织替代提供了独特资源，但目前对其调控机制的深入认知不足，限制了该技术的标准化与广泛临床应用。本研究针对hPSC来源的表皮分化过程中的染色质开放性与转录组动态变化进行探究，发现两个关键转变时期：表面外胚层起始阶段与角质细胞成熟阶段。借助推断网络建模策略，我们为每个转变阶段开发了专属计算框架，并鉴定出TFAP2与TP63及其辅因子为核心调控因子。令人意外的是，功能实验证实TFAP2C可通过激活早期转录因子网络及其染色质景观变化，诱导表面外胚层分化；而TFAP2C的缺失会抑制细胞早期定向。经TFAP2C诱导的细胞可在选择性培养基中进一步分化为功能性角质细胞，伴随角质细胞成熟网络的激活与早期网络的衰减。机制层面，TFAP2C可激活核心调控因子P63的表达，增加其结合位点的开放性并建立正自调控；而P63的缺失会导致无法关闭TFAP2诱导的早期程序，引发成熟障碍与存活缺陷，由此揭示了一条确保祖细胞向成熟组织完成转变的正负反馈环路。本研究揭示了人类表皮谱系定向过程中动态表观基因组-转录因子互作的内在逻辑，将为优化组织工程与再生医学研究提供助力。本研究通过转座酶可及性测序 (Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) 与RNA测序 (RNA-sequencing, RNA-seq) 技术，获取了正常人类胚胎干细胞 (human embryonic stem cell, hESC)、诱导多能干细胞 (induced pluripotent stem cell, iPSC) 以及携带TFAP2C诱导性表达或P63敲除 (p63 KO) 的hESC在表皮分化过程中的染色质开放性与基因表达谱；所有样本在每个分化阶段均设置两次生物学重复，测序平台采用Illumina HiSeq 2000与NextSeq 500。此外，本研究还获取了正常人类角质细胞 (normal human keratinocyte, NHK) 的染色质开放性数据（测序平台：NextSeq 500）与基因表达谱数据（测序平台：HiSeq 2000）。