Roles of long noncoding RNAs during early development and how their misregulation results in disease
Long Noncoding RNAs, Human Pluripotent Stem Cells, Epigenetics, X-Chromosome Inactivation
Long noncoding RNAs (lncRNAs) are young and exciting players in the field of epigenetic gene regulation, where heritable changes in expression are independent of DNA sequence. One of the remarkable findings of the Human Genome Sequencing Project was that just 2% of the genome is protein coding, yet 70-90% of the genome is transcribed. Next-Generation Sequencing experiments have sparked investigation into this ‘dark matter’ of the genome, and recent estimates suggest that the lncRNA family contains 10,000- 200,000 members. These lncRNAs exhibit cell-type and tissue specific expression, yet functional information is only available for small handful of these lncRNAs.
X-Chromosome Inactivation (XCI) is a hallmark example of epigenetic regulation completely dependent on lncRNAs. Mammals have evolved chromosome-wide silencing mechanism relying on X-linked lncRNAs to equalize X-gene dosage between genders. The master regulator of XCI is the lncRNA XIST, which is upregulated from the inactivate X-chromosome during differentiation and forms a cytologically visible RNA ‘cloud’. XIST RNA is well conserved, yet mechanisms of XCI differ between mouse and human, and the epigenetic instability of human pluripotent stem cells frequently silences XIST. We recently discovered that XIST-negative female hiPSCs have cancer-related phenotypes, compromising their use as biological models and in clinical settings.
Our lab is interested in the function and molecular mechanisms of lncRNAs and how their mis-regulation contributes to disease. We are investigating these mechanisms using human and mouse pluripotent stem cells and mouse models, using molecular, genetic, and biochemical approaches.
1. Causes and Consequences of Altered Dosage Compensation
Regenerative medicine holds great promise for treating genetic and trauma-induced disease. Pluripotent stem cells (PSCs) have the potential to differentiate into all cell types, underscoring their importance for therapeutic applications. However, human PSCs (hPSCs) are susceptible to genetic and epigenetic instability, which questions their utility and safety in clinical settings. Female hPSCs display remarkable variability of X-Chromosome Inactivation, and frequently lack XIST RNA expression. We discovered that XIST-negative female hiPSCs have cancer-related phenotypes (increased cell growth rates, poor differentiation potential, and altered gene expression for coding and long noncoding RNAs), posing a health risk for female patients. We are using these cells to determine the mechanisms that initiate undesirable epigenetic change in order to identify pathways that can be targeted to prevent epigenetic instability.
2. Mechanisms of an X-linked lncRNA (Tsx) in cell proliferation and differentiation
The Tsx gene was originally described as a protein-coding gene predominantly expressed in the testes, yet lacks protein domain homology and proteomic detection. We discovered that Tsx is robustly transcribed from a silent X-chromosome, similar to Xist RNA, escaping chromosome-wide silencing during male meiosis. We used in vivo and in vitro experiments to demonstrate that Tsx is not protein-coding, but actually a novel lncRNA. Tsx RNA is present in a variety of adult tissues, notably the brain and reproductive systems. There are different Tsx RNA isoforms, many lacking open reading frames, which are expressed in cell-specific contexts. We found that Tsx null mESCs exhibit severe growth inhibition, poor differentiation, and mis-regulated expression of ncRNAs important for XCI. Our lab is interested in investigating how Tsx deletion results in these phenotypes and identifying protein binding partners required for Tsx function.
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