Roles of long non coding RNAs in human embryonic stem cell pluripotency and neural differentiation 4

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

Chapter VIII – Brain lncRNA RMST Regulates

Neurogenesis by Association with SOX2

8.1 Introduction

RMST or rhabdomyosarcoma 2 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

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

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

tissue-specific 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)

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

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

(ReN-VM NSCs) differentiate into neurons

8.2.2 RMST is developmentally regulated by transcription factor REST

RMST is a spliced lncRNA, with 3 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

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

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 <0.05 and <0.01 respectively

8.2.3 RMST is indispensable for neurogenesis, but not required for maintenance

of neuronal identity

The brain-specific expression pattern, and developmental regulation of RMST indicate functionality Indeed, this was demonstrated in the loss of neurogenesis upon RMST

knockdown in ReN-VM neural stem cells (Figures 7.4, 7.5 and 8.7) Knockdown of

RMST appeared to shut down the neurogenic program of neural stem cells, with the

cells adopting a glia lineage instead (Figure 7.6) On the other hand, overexpression

of RMST enhanced neuronal differentiation efficiency (Figure 8.8) Taken together, these results indicate that RMST is indispensable for neurogenesis

Figure 8.7: Knockdown of RMST prevented neurogenesis ReN-VM neural stem

cells were transfected with the indicated siRNAs, and TUJ1, a marker for neurons, was assayed by immunofluorescence 7 days later In the non-target siRNA (si-NT) control, TUJ1+ and MAP2+ neurons were observed In RMST knockdown (si-RMST),

there were significantly fewer TUJ1+ cells, indicating a loss of neurogenesis The scale bar indicates 100 µm

Figure 8.8: Overexpression of RMST enhanced neuronal differentiation of

ReN-VM cells (A) RMST was overexpressed by transfecting the in vitro transcribed RNA

(RMST OE) As a control, total RNA extracted from ReN-VM cells were transfected (Control OE) Immunostaining of neuronal marker TUJ1 revealed that neuronal efficiency was increased when RMST was overexpressed (B) Gene expression analysis by qPCR indicating that RMST was overexpressed and neuronal markers were upregulated upon RMST overexpression

One question that arose was the role of RMST in neurons Since RMST is highly expressed in neurons, it is plausible that RMST could play a role in the

maintenance of neuronal identity To this end, ReN-VM cells were differentiated into

neurons in the N2B27 medium for 7 days, and siRNAs targeting RMST were

subsequently introduced into the neuronal cells by transfection No morphological

differences were observed in the neurons depleted of RMST, which retained TUJ1 and

synaptophysin (SYP) expression typical of neurons (Figure 8.9) In addition, there was no significant change in the expression of a panel of neurogenic and neuronal

markers assayed by qPCR (Figure 8.10) Together, this indicated that RMST was

required for neuronal differentiation, but once neuronal identity was attained, the loss

of RMST did not have apparent effects on the neurons

Figure 8.9: Loss of RMST in neurons had no apparent effect on cellular morphology A non-target siRNA and siRNA targeting RMST were transfected into

ReN-VM derived neurons Loss of RMST in the neurons did not have an apparent

effect, as the cells retained the neuronal marker TUJ1, as well as the synaptic protein SYP

Figure 8.10: No significant changes in expression of neuronal markers upon

knockdown of RMST in neurons Expression of neuronal markers was measured by

8.2.4 Nuclear-retained RMST physically associates with RNA-binding protein

hnRNPA2B1 and transcription factor SOX2

Previously, RNA fractionation studies indicated that RMST is a nuclear-localized transcript (Figures 7.7 and 8.11A) To confirm this, RNA fluorescence in-situ hybridization (RNA-FISH) using a full-length RMST fluorescent probe was used The colocalization of the RMST signal and the DAPI stain confirmed that RMST is a

nuclear-localized lncRNA (Figure 8.11B)

Figure 8.11: RMST is a nuclear-localized lncRNA (A) Cellular RNA was separated

into the nuclear and cytoplasmic fractions by RNA fractionation, and abundance of the RNA transcripts in either fraction was assayed by qPCR The nuclear/cytoplasmic

ratio was computed presented in the graph (B) To confirm that RMST is a nuclear transcript, RNA fluorescence in situ hybridization (RNA-FISH) was performed, using

a full-length fluorescent probe against RMST

As a nuclear-retained lncRNA, RMST could be physically interacting with

nuclear proteins, which may account for its importance in neurogenesis In order to

identify protein interaction partners of RMST, biotinylated RNA pulldown was performed Briefly, biotin-labeled RNA was generated by in vitro transcription The

biotin-labeled RNA was then immobilized on streptavidin beads, and subsequently incubated with pre-cleared nuclear extract from ReN-VM cells After incubation at 4

°C for 4 hours to allow the formation of protein-RNA complexes, a series of wash steps were performed and proteins were eventually eluted, ran on an SDS-PAGE gel The SDS-PAGE gel was Coomassie-stained, and two protein bands observed in the

RMST pulldown and not in the control RNA pulldowns were then sent for mass

spectrometry From the mass spectrometry, hnRNPA2B1 and SOX2 were identified (Figure 8.12) This was then confirmed by Western blot (Figure 8.13), and RNA

immunoprecipitation (RIP) established that RMST could also physically interact with hnRNPA2 and SOX2 in vivo (Figures 8.14A and 8.14B, respectively) Since an

effective hnRNPA2B1 antibody could not be found for RIP, hnRNPA2-FLAG was ectopically expressed in ReN-VM cells and RIP was performed using a FLAG antibody

Figure 8.12: Biotinylated RMST pulldown, coupled to LC-MS/MS mass

spectrometry identified hnRNPA2B1 and SOX2 as protein partners of the lncRNA Two additional protein bands corresponding to 37 kDa and 35 kDa were

observed in the RMST pulldown, and not in the controls (beads only and antisense-RMST, AS-RMST) These bands were excised for mass spectrometry and identified

to be SOX2 and hnRNPA2B1, respectively