LncRNA-HIT Functions as an Epigenetic Regulator of Chondrogenesis through Its Recruitment of p100/CBP Complexes.

Hanqian L Carlson,Jeffrey J Quinn,Yul W Yang,H Scott Stadler,Howard Y Chang,Chelsea K Thornburg,John F Bateman

doi:10.1371/journal.pgen.1005680

Abstract

Gene expression profiling in E 11 mouse embryos identified high expression of the long noncoding RNA (lncRNA), LNCRNA-HIT in the undifferentiated limb mesenchyme, gut, and developing genital tubercle. In the limb mesenchyme, LncRNA-HIT was found to be retained in the nucleus, forming a complex with p100 and CBP. Analysis of the genome-wide distribution of LncRNA-HIT-p100/CBP complexes by ChIRP-seq revealed LncRNA-HIT associated peaks at multiple loci in the murine genome. Ontological analysis of the genes contacted by LncRNA-HIT-p100/CBP complexes indicate a primary role for these loci in chondrogenic differentiation. Functional analysis using siRNA-mediated reductions in LncRNA-HIT or p100 transcripts revealed a significant decrease in expression of many of the LncRNA-HIT-associated loci. LncRNA-HIT siRNA treatments also impacted the ability of the limb mesenchyme to form cartilage, reducing mesenchymal cell condensation and the formation of cartilage nodules. Mechanistically the LncRNA-HIT siRNA treatments impacted pro-chondrogenic gene expression by reducing H3K27ac or p100 activity, confirming that LncRNA-HIT is essential for chondrogenic differentiation in the limb mesenchyme. Taken together, these findings reveal a fundamental epigenetic mechanism functioning during early limb development, using LncRNA-HIT and its associated proteins to promote the expression of multiple genes whose products are necessary for the formation of cartilage.

Highlights

In the animal kingdom, embryogenesis proceeds through the coordinated expression of genes whose products mediate the formation of complex tissues and structures
A fundamental problem studied by skeletal biologists is the development of regenerative therapies to replace cartilage tissues impacted by injury or disease, which for individuals affected by osteoarthritis represents nearly half of all of all adults over the age of sixty five
The LncRNA-HIT transcript was first identified as the full length cDNA, 9530018H14RIK, by the RIKEN Mouse Gene Encyclopedia Project and was mapped as a single exon to mouse chromosome 6 between Hoxa11 and Hoxa13 by genome sequencing (S1 Fig) [36]

Summary

Introduction

Embryogenesis proceeds through the coordinated expression of genes whose products mediate the formation of complex tissues and structures. While proteins encoded by mRNAs contribute extensively to the regulation of these developmental processes, recent studies of the human and mouse genomes suggest that long noncoding RNAs (lncRNAs) play an essential role in coordinating the expression of genes required for tissue formation and organ development [1,2,3,4,5]. LncRNAs modulate target gene expression using a variety of mechanisms, in the nucleus where they can function as decoys, scaffolds, guides, or even enhancers [6]. Nuclear lncRNAs may function as guides to localize protein complexes to specific chromosomal regions. A growing body of evidence indicates that lncRNAs may function as enhancer RNAs, using chromosomal looping to place proteins bound by the lncRNA proximal to genes to facilitate their regulation [5, 20,21,22]

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