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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.

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