Chapter VIII – Brain lncRNA RMST Regulates Neurogenesis by Association with SOX2 8.1 Introduction RMST or rhabdomyosarcoma associated transcript is a long non-coding RNA first found to be highly expressed in rhabdomyosarcoma, a malignant cancer of the muscles More recent studies in mice models, however, uncovered a plausible role of Rmst in the brain In a study of Pitx3-GFP knock-in mice, in which the GFP reporter is targeted into the Pitx3 gene and expressed exclusively in midbrain dopamine neurons (mDA) and their postmitotic precursors, the authors found Rmst as differentially upregulated in the Pitx3-GFP+ (mDA) compared to the Pitx3-GFP- cell population (Uhde et al., 2010) In the developing mouse brain, Rmst expression in the ventral midbrain was high in the Lmx1a expressing region during E11.5 to E13.5 (Figure 8.1) Lmx1a is a transcription factor that is important for the specification of mDA neurons during embryogenesis (Andersson et al., 2006; Nakatani et al., 2010), and is one of the three minimal factors required to reprogram fibroblasts into mDA neurons (Caiazzo et al., 2011) In the adult mice, Rmst expression is restricted to the central nervous system (Uhde et al., 2010) Taken together, the study by Uhde and colleagues showed that Rmst is developmentally regulated and exhibits tissue-specific expression patterns, which suggest a plausible role in mDA neurogenesis   125  Figure 8.1: (Reproduced from Uhde et al., 2010) Rmst is highly expressed in the midbrain and prospective dopamine neurons Rmst transcript was detected by in situ hybridization in the mouse embryonic brain (A, B) Rmst as detected in both thr roof plate (RP) and the floor plate (FP), as well as in the outermost layer of cells in the alar plate (ALP) and intermediate zone of the basal plate (BP) (C-F) Rmst expression during E11.5 to E14.5 (G-J) Lmx1a expression on an adjacent midbrain section during E11.5 to 14.5 At E14.5, Rmst expression is largely restricted to the ventral tegmental area (VTA), and not the laterally located substantia nigra (SNc) In this current study, the human RMST transcript was identified as one of the neuronal lncRNAs highly expressed in human dopamine neurons, compared to the hESCs and NPCs from which the neurons were derived from In Chapter VII, the indispensable role of RMST in human neurogenesis was confirmed in vitro, by means of an RNAi approach Neural progenitors with decreased RMST transcript expression did not differentiate into TUJ1-expressing neurons, but became glia instead (Figures 7.4 to 7.6) In this chapter, the molecular mechanism of RMST governing neurogenesis is explored   126  8.2 Results 8.2.1 RMST is highly expressed in the human brain and upregulated during neurogenesis The expression of Rmst in the mouse is restricted to the central nervous system (Uhde et al., 2010) Given the high level of conservation between mouse and human RMST transcripts (Chodroff et al., 2010), it was important to determine if a similar tissuespecific expression was observed in humans To this end, the expression of RMST was profiled in a panel of human somatic tissues, as well as in the H1 hESC line (Figure 8.2) RMST expression was found to be very highly and almost exclusively expressed in the brain tissues in this study, namely the whole brain, fetal brain, cerebellum and substantia nigra, with the highest expression observed in the substantia nigra, where mDA neurons are located This agrees with the observation in the mouse that the lncRNA is restricted to the central nervous system, particularly in the region where mDA neurons arises (Uhde et al., 2010)   127  Figure 8.2: Expression of RMST in somatic tissues measured by qPCR RMST is highly expressed in the brain tissues, including the whole brain, fetal brain, cerebellum and substantia nigra, while weakly expressed in the other somatic tissues All three isoforms of RMST were detected by qPCR, and relative expression was normalized to RMST expression in the whole brain sample Microarray analysis indicated that RMST was upregulated when NPCs differentiate into mature neurons This was confirmed by qPCR in two independent cell lines: hESC (H1)-derived NPCs and an immortalized human neural stem cell line ReN-VM In both hESC-derived cells and ReN-VM cells, RMST expression was upregulated by approximately 9-fold when the neural progenitors differentiate into neurons (Figures 8.3A and 8.3B respectively) This indicated that RMST was consistently upregulated when neural progenitors differentiate into neurons   128  Figure 8.3: RMST expression is upregulated during neurogenesis Levels of RMST were measured by qPCR during neuronal differentiation of neural progenitors (A) As H1-derived NPCs (H1-NPCs) differentiate into neurons (H1-Neuron), RMST expression increased by approximately 9-fold (B) In an identical experimental setup, RMST expression increased by more than 8-fold as ReN-VM neural stem cells (ReNVM NSCs) differentiate into neurons 8.2.2 RMST is developmentally regulated by transcription factor REST RMST is a spliced lncRNA, with alternative isoforms assigned with the following GenBank IDs: AK056164, AF429305 and AF429306 (Figure 7.1 and Figure 8.4) In humans, RMST is located on chromosome 12q23, and the ENCODE ChIP-seq data from human embryonic stem cells (H1) and neuroblastomas (PFSK-1) indicated a REST binding peak upstream of RMST, suggesting that REST may regulate the transcription of RMST (Figure 8.4) REST, also known as NRSF or neuronal restrictive silencing factor, is a transcription factor expressed in neural stem cells and non-neuronal cells, to repress neuronal gene expression To confirm that REST indeed binds upstream of RMST in the neural stem cell line ReN-VM, REST chromatin-immunoprecipitation (ChIP) was performed An   129  isotype control IgG ChIP was also performed alongside the REST ChIP to rule out non-specific chromatin binding Quantitative PCR of the ChIP DNA revealed REST occupancy at the region upstream of RMST (pRMST), confirming that REST binds upstream of RMST in the human neural stem cell line (Figure 8.5) Since transcription factor binding events may not result in target gene regulation, I performed REST knockdown, and probed for RMST expression by qPCR 48 hours after transfection of siRNAs REST was very efficiently downregulated by at least 80% following introduction of siRNAs Following REST knockdown, expression of the lncRNA RMST was 4-fold upregulated (Figure 8.6) Together with the REST ChIP data, this indicates that RMST expression is regulated by the repressive transcription factor REST Figure 8.4: ENCODE ChIP-seq database indicates the presence of a REST binding site upstream of RMST REST (or NRSF) binding sites were observed upstream of RMST in three human cell lines, namely GM12878, a lymphoblastoid cell line, H1 hESCs, and neuroblastoma line PFSK-1 The three isoforms of RMST are shown as AK056164, AF429305 and AF429306   130  Figure 8.5: ChIP-PCR indicating REST occupancy upstream of the lncRNA RMST REST chromatin immunoprecipitation (ChIP), as well as the isotype IgG ChIP experiments were performed Fold enrichment was calculated relative to the IgG ChIP Three positive controls, namely RS111, RS625 and RS774, were included in the study The promoter region of ELAV1 (pELAV1), not known to be bound by REST, was included as a negative control ** indicates a p-value of less than 0.01 Figure 8.6: RMST expression is regulated by transcription factor REST REST was efficiently knocked down by more than 80% by siRNAs Upon REST knockdown, RMST expression increased by four-fold As controls, non-REST targets showed no change in expression upon REST knockdown, while REST targets SYP and MAP2 were upregulated * and ** indicates p-values of