Summary Genomic studies demonstrate that while the majority of the mammalian genome is transcribed, only about 2% of these transcripts are protein coding. We have been investigating how the long, polyadenylated Evf2 non-coding RNA regulates transcription of homeodomain transcription factors DLX5 and DLX6 in the developing mouse forebrain. Here we show that in developing ventral forebrain, Evf2 recruits DLX and MECP2 transcription factors to key DNA regulatory elements in the Dlx 5/6 intergenic region and controls Dlx5, Dlx6, and GAD67 expression through trans and cis-acting mechanisms. Evf2 mouse mutants have reduced numbers of GABAergic interneurons in early post-natal hippocampus and dentate gyrus. Although the numbers of GABAergic interneurons and GAD67 RNA levels return to normal in Evf2 mutant adult hippocampus, reduced synaptic inhibition occurs. These results suggest that non-coding RNA-dependent balanced gene regulation in embryonic brain is critical for proper formation of GABA-dependent neuronal circuitry in adult brain.

SUMMARYSeveral lines of evidence suggest that long non-coding RNA (lncRNA)-dependent mechanisms regulate transcription and CpG DNA methylation. Whereas CpG island methylation has been studied in detail, the significance of enhancer DNA methylation and its relationship with lncRNAs is relatively unexplored. Previous experiments proposed that the ultraconserved lncRNA Evf2 represses transcription through Dlx6 antisense (Dlx6as) transcription and methyl-CpG binding protein (MECP2) recruitment to the Dlx5/6 ultraconserved DNA regulatory enhancer (Dlx5/6ei) in embryonic day 13.5 medial ganglionic eminence (E13.5 MGE). Here, genetic epistasis experiments show that MECP2 transcriptional repression of Evf2 and Dlx5, but not Dlx6, occurs through antagonism of DLX1/2 in E13.5 MGE. Analysis of E13.5 MGE from mice lacking Evf2 and of partially rescued Evf2 transgenic mice shows that Evf2 prevents site-specific CpG DNA methylation of Dlx5/6ei in trans, without altering Dlx5/6 expression. Dlx1/2 loss increases CpG DNA methylation, whereas Mecp2 loss does not affect Dlx5/6ei methylation. Based on these studies, we propose a model in which Evf2 inhibits enhancer DNA methylation, effectively modulating competition between the DLX1/2 activator and MECP2 repressor. Evf2 antisense transcription and Evf2-dependent balanced recruitment of activator and repressor proteins enables differential transcriptional control of adjacent genes with shared DNA regulatory elements.

Prolongation of cell survival through prevention of apoptosis is considered to be a significant factor leading to anabolic responses in bone. The current studies were carried out to determine the role of the small GTPase, RhoA, in osteoblast apoptosis, since RhoA has been found to be critical for cell survival in other tissues. We investigated the effects of inhibitors and activators of RhoA signaling on osteoblast apoptosis. In addition, we assessed the relationship of this pathway to parathyroid hormone (PTH) effects on apoptotic signaling and cell survival. Rho A is activated by geranylgeranylation, which promotes its membrane anchoring. In serum-starved MC3T3-E1 osteoblastic cells, inhibition of geranylgeranylation with geranylgeranyl transferase I inhibitors increased activity of caspase-3, a component step in the apoptosis cascade, and increased cell death. Dominant negative RhoA and Y27632, an inhibitor of the RhoA effector Rho kinase, also increased caspase-3 activity. A geranylgeranyl group donor, geranylgeraniol, antagonized the effect of the geranylgeranyl tranferase I inhibitor GGTI-2166, but could not overcome the effect of the Rho kinase inhibitor. PTH 1-34, a potent antiapoptotic agent, completely antagonized the stimulatory effects of GGTI-2166, dominant negative RhoA, and Y27632, on caspase-3 activity. The results suggest that RhoA signaling is essential for osteoblastic cell survival but that the survival effects of PTH 1-34 are independent of this pathway. Keywordsosteoblast; RhoA; apoptosis; parathyroid hormone Anti-apototic actions are a mechanism through which a number of stimuli have been proposed to increase osteoblastic activity, leading to anabolic responses in bone. These anti-apoptotic factors include parathyroid hormone (PTH) [Jilka, 2007;Jilka et al., 1999;Sowa et al., 2003;Wang et al., 2007], insulin-like growth factor-I [Grey et al., 2003], endothelin [Van Sant et al., 2007], mechanical loading, [Bran et al., 2008;Pavalko et al., 2003;Weyts et al., 2003] and matrix environment [Buxton et al., 2008]. Activation of protein kinase A appears to be a critical component of the anti-apoptotic response to parathyroid hormone [Chen et al., 2007;Swarthout et al., 2002]. However, other signaling pathways could contribute to anti-apoptotic actions. Our previous studies had shown that parathyroid hormone activates the small G protein RhoA in osteoblastic cells [Radeff et al., 2004]. RhoA signaling promotes survival in other tissues, [Gallagher, 2004;Kobayashi et al., 2004;Reuveny et al., 2004;Stepan et al., 2004;Zhu et al., 2008]. RhoA inactivation by statins leads to apoptosis of human osteosarcoma cells [Fromigue et al., 2006]. However, in some tissues RhoA can induce apoptosis [Del Re et al., 2007;Wu , 2006]. In view of these diverse findings, we were interested in determining whether the pathway is important for survival of osteoblastic cells, and whether it plays a role in the effects of PTH to promote osteoblast survival.RhoA signaling can be manipulated with genetic and pharma...