Abstract
Transcription factors SOX9, SOX5 and SOX6 are indispensable for generation and differentiation of chondrocytes. However, molecular mechanisms to induce the SOX genes are poorly understood. To address this issue, we previously determined the human embryonic enhancer of SOX6 by 5′RACE analysis, and identified the 46-bp core enhancer region (CES6). We initially performed yeast one-hybrid assay for screening other chondrogenic factors using CES6 as bait, and identified a zinc finger protein ZNF449. ZNF449 and Zfp449, a counterpart in mouse, transactivated enhancers or promoters of SOX6, SOX9 and COL2A1. Zfp449 was expressed in mesenchyme-derived tissues including cartilage, calvaria, muscle and tendon, as well as in other tissues including brain, lung and kidney. In limb cartilage of mouse embryo, Zfp449 protein was abundantly located in periarticular chondrocytes, and decreased in accordance with the differentiation. Zfp449 protein was also detected in articular cartilage of an adult mouse. During chondrogenic differentiation of human mesenchymal stem cells, ZNF449 was increased at an early stage, and its overexpression enhanced SOX9 and SOX6 only at the initial stage of the differentiation. We further generated Zfp449 knockout mice to examine the in vivo roles; however, no obvious abnormality was observed in skeletal development or articular cartilage homeostasis. ZNF449 may regulate chondrogenic differentiation from mesenchymal progenitor cells, although the underlying mechanisms are still unknown.
Highlights
In the initial step of skeletal development, undifferentiated mesenchymal cells are recruited into condensations and differentiate into chondrocytes that produce cartilage matrix proteins including type II collagen (COL2A1) and aggrecan [1]
We confirmed the in vivo binding of the ZNF449 protein to CES6 by Chromatin immunoprecipitation (ChIP) assay (Fig. 1B)
We examined ZNF449 expression during chondrogenic differentiation of human mesenchymal stem cells (hMSCs). mRNA level of ZNF449 was increased at day 7, simultaneously with that of SOX9, while those of SOX6 and COL2A1 were gradually increased during the differentiation (Fig. 3A)
Summary
In the initial step of skeletal development, undifferentiated mesenchymal cells are recruited into condensations and differentiate into chondrocytes that produce cartilage matrix proteins including type II collagen (COL2A1) and aggrecan [1]. This process is regulated by three members of the sex-determining region Y-type high mobility group box protein (SOX) family, a transcription factor SOX9, and its co-activators SOX5 and SOX6 [2,3,4,5,6]. We further present an in vivo role of ZNF449 during skeletal development and articular cartilage homeostasis by deletion of Zfp449 gene
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