Differential tissue specific, temporal and spatial expression patterns of the Aggrecan gene is modulated by independent enhancer elements

Ian M H Li,Ke Liu,Alice Neal,George Bou-Gharios,Sarah De Val,Peter D Clegg

doi:10.1038/s41598-018-19186-4

Ian M H Li, Ke Liu + Show 4 more

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https://doi.org/10.1038/s41598-018-19186-4

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Abstract

The transcriptional mechanism through which chondrocytes control the spatial and temporal composition of the cartilage tissue has remained largely elusive. The central aim of this study was to identify whether transcriptional enhancers played a role in the organisation of the chondrocytes in cartilaginous tissue. We focused on the Aggrecan gene (Acan) as it is essential for the normal structure and function of cartilage and it is expressed developmentally in different stages of chondrocyte maturation. Using transgenic reporter studies in mice we identified four elements, two of which showed individual chondrocyte developmental stage specificity. In particular, one enhancer (−80) distinguishes itself from the others by being predominantly active in adult cartilage. Furthermore, the −62 element uniquely drove reporter activity in early chondrocytes. The remaining chondrocyte specific enhancers, +28 and −30, showed no preference to chondrocyte type. The transcription factor SOX9 interacted with all the enhancers in vitro and mutation of SOX9 binding sites in one of the enhancers (−30) resulted in a loss of its chondrocyte specificity and ectopic enhancer reporter activity. Thus, the Acan enhancers orchestrate the precise spatiotemporal expression of this gene in cartilage types at different stages of development and adulthood.

Highlights

The mammalian genome has an abundance of regulatory elements compared to coding sequences
We used the availability of data from the Encyclopaedia of DNA Elements (ENCODE) project for mouse embryo limb at embryonic day 14.5 (E14.5) enhancer marks, such as the H3K4me1/2 and H3K27Ac as well as phylogenic conservation to identify multiple cis-regulatory elements within and around the Aggrecan gene (Acan) gene
During the data collection of this study SOX9 and histone modifications ChIP-seq data from three groups, one in rats[24] and the other two in new born mouse rib chondrocytes and limb bud (E12.5) became available[23,25], this allowed us to validate that the regions we had selected had chondrocyte specific enhancer markers (Supplementary Figure S1)

Summary

Introduction

The mammalian genome has an abundance of regulatory elements compared to coding sequences. Previous studies identified a chondrocyte-specific enhancer located at −10 kb upstream of the Acan transcription start site (TSS), termed A120. The activity of this enhancer was confined to differentiated chondrocytes and was regulated by the transcription factor SOX9 with the aid of L-SOX5 and SOX620. More recently ChIP-seq experiments using SOX9 have established clusters or super enhancers that span up to 140 kb down-stream of the Acan TSS22–25 These studies provided two key insights into the Acan regulation; the first is that these enhancers are functional in differentiated chondrocytes. This is true in articular cartilage and growth plates. We identify four enhancers that exhibit overlapping spatial and temporal activities such that, together, they likely contribute to ensure the robust expression of Acan in all cartilage types throughout development and in adult tissue

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