β-Arrestin1/miR-326 Transcription Unit Is Epigenetically Regulated in Neural Stem Cells Where It Controls Stemness and Growth Arrest

Cell development is regulated by a complex network of mRNA-encoded proteins and microRNAs, all funnelling onto the modulation of self-renewal or differentiation genes. How intragenic microRNAs and their host genes are transcriptionally coregulated and their functional relationships for the control of neural stem cells (NSCs) are poorly understood. We propose here the intragenic miR-326 and its host gene β-arrestin1 as novel players whose epigenetic silencing maintains stemness in normal cerebellar stem cells. Such a regulation is mediated by CpG islands methylation of the common promoter. Epigenetic derepression of β-arrestin1/miR-326 by differentiation signals or demethylating agents leads to suppression of stemness features and cell growth and promotes cell differentiation. β-Arrestin1 inhibits cell proliferation by enhancing the nuclear expression of the cyclin-dependent kinase inhibitor p27. Therefore, we propose a new mechanism for the control of cerebellar NSCs where a coordinated epigenetic mechanism finely regulates β-arrestin1/miR-326 expression and consequently NSCs stemness and cell growth.


Introduction
Neural stem cells (NSCs) are believed to foster a hierarchical developmental program in which self-renewal and pluri/multipotency are responsible for the expansion and/or the maintenance of an uncommitted cell population pool.NSCs are ready to undergo the cascade of lineage restriction and subsequent terminal differentiation under the action of regulatory morphogenic cues [1].The early postnatal murine cerebellum contains multipotent NSCs, which can be isolated and expanded in vitro.NSCs maintain their undifferentiated phenotype under mitogenic signals and are able to differentiate into the different kind of neurons [2].
Recent evidences have highlighted the crucial role of microRNAs (miRNAs) in conferring neural cell identities during neural induction, neuronal differentiation, and subtype specification [3].MiRNAs are widespread throughout the genome, where they can be found in either intergenic or intragenic (especially intronic) regions [4].Transcription of both intergenic and intragenic miRNAs may be regulated by their own promoters, whether some intragenic miRNAs share promoters with their host genes that generate pre-miRNA and mRNA, both arising from the same transcript [5,6].The consequent spatial and temporal coexpression implies a functional relationship between the intragenic miRNA and its host gene.

Stem Cells International
We previously identified miR-326 as a miRNA that is required to differentiate cerebellar granule cell progenitors (GCPs) to mature granule cells [7].This miRNA is embedded within the first intron of -arrestin1 (arr-1) gene and shares with its host gene the same regulatory sequences [8,9].Since stem cell commitment to GCPs is a crucial event in cerebellar development [2,10] we raised the question of miR-326 expression and regulation in cerebellar NSC.We report here that miR-326 expression is further downregulated in NSC prior to progenitor commitment, along with the downregulation of its host gene.Therefore, we investigated the expression, functions, and regulation of miR-326/arr-1 transcription unit in NSCs.

Materials and Methods
Unless otherwise indicated, media and supplements were purchased from Gibco-Invitrogen (Carlsbad, CA), chemicals were purchased from Sigma-Aldrich (St. Louis, MO), and commercial products were used according to the manufacturers' instructions/protocols.

Knockdown Experiments.
Silencing of -arrestin1 was performed using ON-TARGETplus SMARTpool (L40976-00-005) from Thermo Scientific, after testing each single siRNA of the pool, alone or in combination, for its specificity to avoid OFF-target effects.

Cell Proliferation. Cell proliferation was evaluated by
BrdU incorporation as previously described [2].More in detail, after 24-hour pulse with BrdU, NSCs were plated on poly-lysine-coated Lab-Tek chamber slides (coverslips) and allowed to adhere for 3 hours.Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and BrdU detection (Roche) was performed.Nuclei were counterstained with the Hoechst reagent.Cells were counted in triplicate and the number of BrdU-positive nuclei was annotated.
NSC growth was measured by MTT assay (Promega) according to manufacturer's instructions.Each sample was measured in triplicate and repeated at least three times.

RNA Isolation and Gene Expression Analysis.
Total RNA was purified and reverse transcribed as previously described [12].Quantitative RT-PCR (qRT-PCR) analysis was performed using the ABI Prism 7900HT Sequence Detection System (Thermo Scientific), using best coverage TaqMan gene expression assays, specific for each analyzed mRNA.Each amplification was performed in triplicate and the average of the three threshold cycles was used to calculate the amount of transcripts (Thermo Scientific).mRNA quantification was expressed, in arbitrary units, as previously described [13].mRNA quantification was expressed, in arbitrary units, as the ratio of the sample quantity to the calibrator or to the mean values of control samples.All values were normalized to three endogenous controls, actin, 2-microglobulin, and Hprt.miRNA expression levels were evaluated on RNA samples using specific stem-loop primers to achieve retrotranscription (Thermo Scientific) and was followed by a quantitative RT-PCR using miRNAspecific TaqMan probes (Thermo Scientific) in a ABI Prism 7900HT Sequence Detection System (Thermo Scientific).miRNA expression levels were normalized to RNAU6B [14].

In Situ Hybridization and Immunofluorescence Assay.
All reagents used prior to probe hybridization were prepared with DEPC-treated water (diethyl pyrocarbonate, Sigma D5758) to prevent RNAse contamination.
Cells were fixed with 4% PFA for 10 minutes at room temperature.After 3 washes with PBS, cells were incubated for 2 minutes with 10 g/mL proteinase K (Sigma P2308) at 37 ∘ C.
Samples were incubated with 0.1x SSC buffer for 3 hours at 67 ∘ C and washed 3 times with 0.1 M Tris-HCl pH 75 and 0.15 M NaCl.Nonspecific antibody binding was performed with 0.5% blocking reagent (Roche 11096176001), 5% Sheep Serum (Sigma S3772), in 50 mM Tris-HCl pH 7.5, and 5 mM EDTA for 2 hours at room temperature in a humidified chamber.Fluorescein conjugated anti-DIG was incubated at the dilution of 1 : 200 in blocking buffer for 16 hours at 4 ∘ C.After antibody incubation, samples were washed 3 times with 0.1 M Tris-HCl pH 75 and 0.15 M NaCl and coverslips were mounted using fluorescent mounting (DAKO S3023).
Immunofluorescence was performed as previously described [13].More in detail, NSCs were cultured in Lab-Tek chamber slides fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.1% Triton X-100 cells, and incubated in blocking buffer (PBS with 1% BSA) for 30 min and then with anti-HA (sc-7392 Santa Cruz) overnight in blocking solution at 4 ∘ C. 488-conjugated anti-rabbit secondary antibody was purchased from Molecular Probes (Invitrogen).Nuclei were counterstained with Hoechst (H6024 Sigma).At least 300 nuclei were counted in triplicate.Fluorescence was visualized and images were acquired with Carl Zeiss microscope (Axio Observer Z1) using ApoTome technology and AxioVision Digital Image Processing Software.
CpG islands were identified in the regulatory regions of miR-326/arr-1 by using database of CpG islands and Analytical Tool (DBCAT) software.
The primers for methylation specific PCR were designed by using Methyl Primer Express Software v1.0, Thermo Fisher Scientific.Primers were first tested and validated using mouse Universal Methylated Mouse DNA Standard (Zymo Research).

miR-326 and 𝛽arr-1 Expression Inversely Correlates with
Stemness.First we evaluated expression levels of miR-326 and of its host gene arr-1 in NSCs (Figures 1(a)-1(d)).NSCs displayed, as compared to the bulk preneurosphere cell population (T0), very low levels of both mature and precursor miR-326 forms and of arr-1 (Figures 1(a)-1(d)), together with an enhanced expression of the Nanog stemness marker (Figure 1(d)).
These results suggest that loss of miR-326/arr-1 locus expression is associated with a "stem-like phenotype."This notion was further supported by the observation that the expression of miR-326 and arr-1 increased in NSCs exposed to differentiation stimuli, for example, retinoic acid (RA) or platelet-derived growth factor (PDGF) (Figures 2(a)-2(d)).Under these conditions NSCs were able to differentiate into distinct lineages as indicated by the increased expression of both neuronal differentiation markers, III-tubulin (IIItub) and NeuN, and astrocytic differentiation markers Gfap and S100 (Figures 2(c

Epigenetic Inactivation of miR-326/𝛽arr-1 Locus.
Next we investigated the mechanism responsible for miR-326/arr-1 locus inactivation in NSCs.There is increasing evidence that epigenetic regulation of stem cells, including "CpG island" methylation, is crucial in the preservation of their stemness by controlling the transcription switch on/off of specific developmental genes [15].Indeed, the presence of several CpG islands in the region spanning from the first exon to part of the first intron (Figure 3(a) and Supplementary Figure 1: see Supplementary Figure 1 in the Supplementary Material available online at https://doi.org/10.1155/2017/5274171)suggested a DNA methylation-dependent control.Treatment of NSCs with the 5  -azacytidine (5-AZA) demethylating agent induced a significant increase of miR-326, its precursor transcripts, and arr-1 levels (Figure 3(b)) and impaired stemness features while it increased cell differentiation (Figures 3(c) and 3(d)).Using methylation specific PCR we evaluated the methylation status of selected CpG islands.We found that these CpG islands were methylated in NSCs but not in the starting bulk population (T0) (Figure 3(e)).Moreover, when NCSs were treated with 5-AZA, the analyzed CpG islands lost their methylation status (Figure 3(f)), strongly suggesting that the upregulation of arr-1 and miR-326 was due to the demethylation of their promoter region (Figure 3(b)).
These findings highlight that the methylation status of arr-1/miR-326 CpG islands is a mechanism to silence their expression in cerebellar NSCs.On the other hand, previous studies have shown that arr-1 functions as a cytoplasm-nucleus shuttling protein that interacts with p300/CBP to activate the transcription of the cyclin-dependent kinase (CDK) inhibitor Cdkn1b/p27kip (p27), a major determinant of cell cycle exit [16].To gain insight into arr-1 functions in neuronal cells stemness and differentiation, we overexpressed arr-1 (Figures 4(a found that, when shifted to differentiation medium, NSCs increased arr-1 level was paralleled by an activation of p27 transcription and the increase of p27 protein in the nucleus as observed in nucleous/cytoplasmic fractionation experiment (Figure 5).Accordingly, p27 mRNA and the nuclear protein cell fraction levels were increased by exogenously  Altogether these results show that arr-1 expression inhibits the cell cycle via activation of p27 thus blocking cell stemness features in NSCs.

Discussion
In this study we propose a model in which epigenetic silencing of the intragenic miR-326 and its host gene, arr-1, maintains physiological neuronal stemness.While miR-326 and arr-1 are highly expressed in differentiated cells where they induce growth arrest, in NSCs they are kept at low levels through CpG hypermethylation.Coherently, modulation of the expression of arr-1 through overexpression regulated the differentiation and growth rate of cerebellar NSCs.The reactivation of the locus miR-326/arr-1 enhances the cell cycle inhibitor p27 while inhibiting proliferative signalling (e.g., Hh or Notch by miR-326), thus resulting in stem cell differentiation and growth arrest (Figure 7).
These findings suggest a new circuitry composed of miR-326/Arr-1, morphogenic molecules, and cell cycle modifiers which control neural stemness status.
In particular, here we identify arr-1 as a new player in the coordinated control of cerebellar NSCs.arr-1 was first identified as a gene encoding a scaffold protein that regulates G-protein-coupled receptor (GPCR), through interaction with cytoplasmic proteins linking GPCRs to intracellular signaling pathways [17,18].Our observations of the role of arr-1 in NSCs are consistent with the growth arrest observed in GCPs where arr-1 directly affects gene expression, translocating into the nucleus where it interacts with transcription cofactors at the promoters of the target gene p27 [19].
Consistently, previous studies investigated the role of this CDK-inhibitor in suppressing self-renewal and proliferation while driving differentiation of NSCs [17,18,20,21] and cerebellar progenitors [22,23].A further link between p27 and stem/progenitor cell differentiation is provided by the p27-destabilizing effect induced by REST, a repressor of neuronal differentiation, resulting in maintenance of cell proliferation and blockade of neuronal differentiation [24].In this way, loss of p27 maintains a high turnover of selfrenewing cells, by coupling the ability to control both cell cycle and undifferentiated status.Of note, arr-1 overexpression in NSCs affected also cell viability and increased cell apoptosis suggesting that such scaffold adaptor protein could have other possible functions in controlling different mechanisms besides the cell growth arrest.
Our findings underline a coordinated epigenetic mechanism that finely regulates miR-326/arr-1 expression and cell growth in neural stem cells.Indeed, the same promoter drives the expression of arr-1 and miR-326 through an epigenetic control in cerebellar NSC.The ability of miR-326/arr-1 to be epigenetically regulated also links this transcript to the processes preserving the stemness status.Indeed, increasing evidence supports the crucial role of epigenetic regulation of stem cells, including NSCs [25].Such a regulation includes the mechanisms responsible for the maintenance of repressive chromatin states at specific loci, consisting of CpG islands methylation [26].
Accordingly, in our study miR-326 and arr-1 are among the silenced genes in NSCs, whose regulation contributes to balance the opposing forces of cell growth and differentiation.Indeed the miR-326/arr-1 promoter is characterized by methylated or demethylated CpG islands in NSC or differentiated cells, respectively.Interestingly, in line with this, miR-326 and arr-1 have been described to be key molecules in multiple sclerosis pathogenesis, being crucial for CD4+ T cell survival and differentiation.Indeed arr-1 enhanced the expression of the protooncogene Bcl2 via the acetylation of histone H4 at the Bcl2 promoter [27] while miR-326 promoted interleukin 17-(IL-17-) producing T helper cells (TH-17) differentiation by targeting Ets-1, a negative regulator of TH-17 differentiation [28].Of note CD4+ T cells from patients with multiple sclerosis had much higher Arrb1 expression [27] and miR-326 expression was highly correlated with disease severity in patients with multiple sclerosis [28].
Even if no specific studies of arr-1/miR-326 coregulation have been conducted in multiple sclerosis our results together with the abovementioned observations allow speculating that the arr-1/miR-326 transcription unit is regulated in other stem or progenitor cells during differentiation, participating in the control of cell fate, development, and disease.

Conclusions
We describe in the present work the role and regulation of the locus miR-326/arr-1.miR-326 and its host gene arr-1 represent a novel miRNA/protein network that controls cerebellar NSCs through the modulation of morphogenic signals and cell cycle modifiers, both regulating stemness and suggested to be involved in the maintenance of self-renewal feature of NSCs.
In conclusion, our findings describe a bivalent signal (miRNA and hosting protein encoding gene) converging upon the coordinated inhibition of normal stem cell functions.

Disclosure
Federica Begalli's current address is Center for Cell Signalling and Inflammation (CCSI), Department of Medicine, Imperial College London.Elisabetta Ferretti and Evelina Miele are colast authors.

2. 6 .
Statistical Analysis.Statistical analysis of cellular experimental triplicates was performed using StatView 4.1 software (Abacus Concepts, Berkeley, CA).Statistical differences were analyzed by Mann-Whitney  test for nonparametric values and a  value of ≤0.05 was considered significant.The results are expressed as mean ± SD from at least three experiments.

Figure 2 :
Figure 2: miR-326 and arr-1 are upregulated in differentiated NSCs.(a) miR-326 levels evaluated in NSC exposed to differentiation stimuli (DFM) with retinoic acid (RA) or platelet-derived growth factor (PDGF) for 48 hours.Data are means ± SD from 3 independent experiments.*  < 0.05.(b) Fluorescent in situ hybridization (ISH) staining of miR-326 of NSCs grown in stemness conditions (SM) and after RA-induced differentiation (DFM) for 48 hours.Nuclei are counterstained with Hoechst.Scale bar: 10 m.Representative ISH images from 4 independent experiments.(c) mRNA expression levels of arr-1 along with stemness and differentiation markers of NSC in SM and after differentiation (DFM).(d) Western blot (WB) analysis of endogenous arr-1 and markers of stemness (Nanog, Sox2, and Oct4) and differentiation (IIItub and Gfap) of NSCs in SM and after differentiation (DFM).Gapdh as loading control (LC).((c) and (d)) Data are means ± SD from 3 independent experiments.*  < 0.05.