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

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.

Development and functional characterization of a lncRNA‐HIT conditional loss of function allele

“…Characterization the GT epithelial marker, Sox4 (Dy et al, ), revealed strong expression in the UPE of homozygous shRNA active embryos, suggesting that the malformation of the PUPE is not the result of epithelial cell loss (Figure c–e). The malformation of the PUPE in shRNA active /shRNA active ; Shh‐Cre/+ mice was surprising as our previous analysis of lncRNA‐HIT indicates that the lncRNA can regulate Hoxa13 expression (Carlson et al, ). Based on this analysis, we predicted that lncRNA‐HIT deficient GTs would exhibit a subset of the Hoxa13 null phenotype, which we previously reported affects distal GT structures including the meatus and distal urethral plate epithelium (DUPE) (Morgan, Nguyen, Scott, & Stadler, ).…”

“…LncRNA‐HIT is highly expressed in multiple tissues during murine development including the distal limb, gut, genital tubercle, central nervous system, and bladder (Figure ) (Carlson et al, ). To determine the efficacy of the shRNA allele to knock‐down endogenous lncRNA‐HIT throughout the embryo, a CMV‐Cre allele was used to broadly activate shRNA expression from the Rosa26 locus (Figure ).…”

“…Next, to assess the efficacy of the Rosa26 LncRNA‐HIT shRNA allele to reduce lncRNA levels in a specific cell type, a Col2‐Cre allele was used to activate shRNA expression in chondrocytes which we previously demonstrated highly express lncRNA‐HIT (Carlson et al, ). Characterization of the shRNA active /shRNA active ; Col2‐Cre/+ embryos revealed strong activation of the mCherry reporter in the E12.5 and E14.5 skeletal anlagen (Figure ).…”

Emerging roles for long noncoding RNAs in skeletal biology and disease

“…An elegant study published recently by Stadler’s group has identified another IncRNA located within the HOXA locus, named IncRNA-HIT , which appears to function as a critical epigenetic regulator of chondrogenesis (104). LncRNA-HIT (HOXA Transcript Induced by TGF-β) was initially characterized as a TGF-P-responsive IncRNA during epithelial-to-mesenchymal transition in mammary epithelia (105).…”

The long non‐coding RNA Paupar promotes KAP 1‐dependent chromatin changes and regulates olfactory bulb neurogenesis

“…Studies of cis ‐acting lncRNAs such as Haunt and Hottip have shown that lncRNA transcript accumulation at their sites of expression can effectively recruit regulatory complexes (Yin et al , ; Pradeepa et al , ). LncRNAs, however, have also been reported to directly bind and regulate genes across multiple chromosomes away from their sites of synthesis (Chu et al , ; Chalei et al , ; Vance et al , ; West et al , ; Carlson et al , ). By way of contrast, the mechanisms by which such trans ‐acting lncRNAs mediate transcription and chromatin regulation at distal bound target genes are less clear.…”