Although a critical role for developmentally regulated transcription factors in patterning and differentiation has been established for some time, the role of long non-coding RNAs (lncRNAs) is only beginning to be understood. My lab discovered novel non-coding RNAs, Evf2 and Evf1, that regulate transcription factors essential for proper GABAergic interneuron development. Our work on Evf2, an lncRNA transcribed as a spliced anti-sense transcript shows that lncRNAs play specific gene regulatory functions. Evf2 is conserved across vertebrates (ultraconserved), and activated by sonic hedgehog in the developing forebrain. We show that Evf2 forms a complex with the DLX2 homeodomain transcription factor and regulates Dlx5/6 enhancer activity. Through analysis of an Evf2 loss-of-function mouse model developed in my lab, we find that Evf2 is not only required for proper transcriptional regulation of Dlx5 and Dlx6, but also for proper GABAergic interneuron development in embryonic brain and adult hippocampal circuitry . While much of my past work has focused on the biological role of Evf2 lncRNA in interneuron development, the precise mechanisms involved in Evf2 long- non-coding RNA-dependent transcriptional regulation remain unknown. The proposed studies will use mouse genetics, RNA and protein biochemistry, and RNA cloud visualization in nuclei of embryonic brain to determine the mechanism of Evf2 lncRNA transcriptional regulation. Some of the techniques are novel, such as the use of RNA cloud visualization in 3 dimensions, mono and bi-allelic analysis, and in vitro DNA-dependent RNA-protein complex assembly;the approach as a whole is novel, as this combination of methods to study lncRNA gene regulation has not been reported. These studies will provide an understanding of a critical aspect of gene regulation with particular relevance to neural development and function, as well as growth control and cancer, where multiple ultraconserved lncRNAs have been identified.
This grant proposes to investigate how the ultraconserved long non-coding RNA Evf2 regulates gene expression in GABAergic interneuron precursors. Since altered GABAergic interneuron function has been linked to epilepsy, autism, schizophrenia, and mental retardation, studies on the normal development of GABAergic interneurons are critical to understanding the molecular basis for multiple neuropsychiatric disorders. In addition, the recent identification of ultraconserved long non-coding RNAs in cancer has made studies of how this class of molecules controls gene regulation relevant to growth control. Ultimately, it is hoped that a better understanding of gene regulation in neuronal subpopulations and cellular growth control will lead to the development of novel therapies for treatment of human neurological diseases and cancer.
|Himmelstein, Diana S; Cajigas, Ivelisse; Bi, Chunming et al. (2017) SHH E176/E177-Zn2+ conformation is required for signaling at endogenous sites. Dev Biol 424:221-235|
|Cajigas, Ivelisse; Leib, David E; Cochrane, Jesse et al. (2015) Evf2 lncRNA/BRG1/DLX1 interactions reveal RNA-dependent inhibition of chromatin remodeling. Development 142:2641-52|
|Kohtz, Jhumku D (2014) Long non-coding RNAs learn the importance of being in vivo. Front Genet 5:45|
|Berghoff, Emily G; Clark, Mary F; Chen, Sean et al. (2013) Evf2 (Dlx6as) lncRNA regulates ultraconserved enhancer methylation and the differential transcriptional control of adjacent genes. Development 140:4407-16|