Dynamic transcriptional and epigenetic changes define postnatal tendon growth

Tendons are dynamic structures that efficiently transmit forces and enable movement. From birth, tendons undergo dramatic changes from a principally cellular tissue to a hypocellular one characterized by a dense and highly ordered extracellular matrix. During this time, tendon cells change morphology from rounded to stellate in appearance and their proliferative rates decline. There is also significant expansion and maturation of the extracellular matrix (ECM) as tendons grow in length and diameter and alter their biomechanical properties to sustain increased physical activities. Surprisingly, for such an important stage of tendon maturation, we understand very little about the transcriptional and epigenetic regulators that direct these processes. Here, we present a roadmap of genes that are differentially regulated during the early neonatal and postnatal time period. We find differentially expressed genes fall into specific transcriptional modules, representing expression increases, decreases, or gene sets undergoing dynamic changes over postnatal time. By pairing our transcriptomic data with epigenetic data, we were able to perform an integrative analysis of the datasets and further define modules with highly correlated changes in gene expression and chromatin accessibility. From this analysis, several new pathways emerge. Among them, we focus on Yap1, a transcriptional co-activator of the Hippo signaling pathway. We observe accessible regions near to differentially expressed genes, containing motifs for TEAD, the transcription factor that binds Yap to regulate transcription. Conditional loss of Yap1 at postnatal stages results in altered expression of Col1a1 and disrupted matrix organization and density, suggesting that Yap is important for refining tendon ECM maturation. Together, our analyses identify a regulator of matrix maturation and provides a rich dataset with which to interrogate transcriptional networks and pathways during this poorly understood time in tendon growth. Tendon samples from weekly timepoints during postnatal development were subjected to both RNAseq and ATACseq to profile transcriptional and chromatin level changes during postnatal tendon growth and development.肌腱是一类高效传递力学信号、驱动机体运动的动态结构。从出生伊始，肌腱便经历显著的发育转变：从最初以细胞成分为主的组织，逐渐演变为以致密且高度有序的细胞外基质（extracellular matrix, ECM）为核心的细胞稀少型组织。在此过程中，肌腱细胞的形态从圆形逐步转变为星状，增殖速率亦随之下降。随着肌腱的长度与直径不断增加，其细胞外基质还会发生显著的扩增与成熟，并通过调整自身生物力学特性以适配日益增强的生理活动负荷。令人意外的是，作为肌腱成熟的关键阶段，我们对调控上述发育过程的转录与表观遗传调控因子却知之甚少。本研究构建了新生早期及出生后阶段差异调控基因的表达全景图谱。研究发现，差异表达基因可划分为特定的转录模块，分别对应出生后不同时期的表达上调、下调，或呈现动态时序变化的基因集。通过将转录组数据与表观遗传数据进行整合分析，本研究进一步明确了基因表达与染色质开放度变化高度相关的转录模块。经此分析，多条全新的调控通路得以显现，其中我们重点关注了Hippo信号通路的转录共激活因子Yap1。我们在差异表达基因的邻近开放染色质区域中，发现了TEAD的结合基序——TEAD是一类可与Yap结合并调控转录的转录因子。出生后阶段特异性敲除Yap1会导致Col1a1的表达异常，并破坏细胞外基质的组织结构与密度，提示Yap1对于优化肌腱细胞外基质的成熟过程至关重要。综上，本研究不仅鉴定了肌腱细胞外基质成熟的关键调控因子，还提供了一套高质量的多组学数据集，可用于深入探究肌腱生长发育这一长期研究匮乏阶段的转录调控网络与信号通路。本研究采集了出生后发育过程中每周时间节点的肌腱样本，通过RNA测序（RNAseq）与ATAC测序（ATACseq）技术，对肌腱出生后生长发育过程中的转录组与染色质水平变化进行了系统表征分析。