Uhrf1 is indispensable for normal limb growth by regulating chondrocyte differentiation through specific gene expression
Uhrf1 is indispensable for normal limb growth by regulating chondrocyte differentiation through specific gene expression
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DOI：
10.1242/dev.157412
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发表时间：
2018-01
期刊：
影响因子：
4.6
通讯作者：
M. Yamashita;Kazuki Inoue;Noritaka Saeki;Maky Ideta-Otsuka;Yuta Yanagihara;Y. Sawada;Iori Sakakibara-Iori-Sakakib
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文献类型：
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作者：
M. Yamashita;Kazuki Inoue;Noritaka Saeki;Maky Ideta-Otsuka;Yuta Yanagihara;Y. Sawada;Iori Sakakibara-Iori-Sakakib
摘要：表观遗传调控因子可严密调控转录。其中，含PHD和无名指结构域的泛素样蛋白1（Uhrf1）据报道具有多种表观遗传功能，包括对DNA甲基化的调控。然而，Uhrf1在骨骼组织中的生理功能仍不清楚。在此，我们发现肢间充质细胞特异性Uhrf1条件性敲除小鼠（Uhrf1ΔLimb/ΔLimb）表现出长骨显著缩短，且由于软骨细胞分化和增殖失调而出现形态畸形。对从Uhrf1ΔLimb/ΔLimb小鼠原代培养的软骨细胞进行的RNA测序显示软骨细胞分化异常。此外，利用RNA测序和甲基化DNA结合蛋白测序（MBD - seq）进行的综合分析表明，Uhrf1缺失降低了全基因组DNA甲基化水平，并通过降低28个基因（包括Hspb1，据报道它是一种白细胞介素1相关基因并影响软骨细胞分化）启动子区域的DNA甲基化而增加基因表达。在条件性敲除（cKO）软骨细胞中敲低Hspb1可使参与软骨细胞分化的基因（如Mmp13）的异常表达恢复正常。这些结果表明，Uhrf1通过控制全基因组DNA甲基化状态以及调控后续的细胞分化和骨骼成熟来调控细胞类型特异性转录。总结：利用Uhrf1ΔLimb/ΔLimb小鼠和全基因组测序进行的综合分析揭示，Uhrf1改变全基因组DNA甲基化状态，进而改变特定基因的表达，并且对正常肢体生长是必不可少的。
ABSTRACT Transcriptional regulation can be tightly orchestrated by epigenetic regulators. Among these, ubiquitin-like with PHD and RING finger domains 1 (Uhrf1) is reported to have diverse epigenetic functions, including regulation of DNA methylation. However, the physiological functions of Uhrf1 in skeletal tissues remain unclear. Here, we show that limb mesenchymal cell-specific Uhrf1 conditional knockout mice (Uhrf1ΔLimb/ΔLimb) exhibit remarkably shortened long bones that have morphological deformities due to dysregulated chondrocyte differentiation and proliferation. RNA-seq performed on primary cultured chondrocytes obtained from Uhrf1ΔLimb/ΔLimb mice showed abnormal chondrocyte differentiation. In addition, integrative analyses using RNA-seq and MBD-seq revealed that Uhrf1 deficiency decreased genome-wide DNA methylation and increased gene expression through reduced DNA methylation in the promoter regions of 28 genes, including Hspb1, which is reported to be an IL1-related gene and to affect chondrocyte differentiation. Hspb1 knockdown in cKO chondrocytes can normalize abnormal expression of genes involved in chondrocyte differentiation, such as Mmp13. These results indicate that Uhrf1 governs cell type-specific transcriptional regulation by controlling the genome-wide DNA methylation status and regulating consequent cell differentiation and skeletal maturation. Summary: Integrative analyses using Uhrf1ΔLimb/ΔLimb mice and genome-wide sequencing reveal that Uhrf1 alters genome-wide DNA methylation status and, in turn, expression of specific genes, and is indispensable for normal limb growth.