FOXG1 Regulates PRKAR2B Transcriptionally and Posttranscriptionally via miR200 in the Adult Hippocampus
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Rett syndrome is a complex neurodevelopmental disorder that is mainly caused by mutations in MECP2. However, mutations in FOXG1 cause a less frequent form of atypical Rett syndrome, called FOXG1 syndrome. FOXG1 is a key transcription factor crucial for forebrain development, where it maintains the balance between progenitor proliferation and neuronal differentiation. Using genome-wide small RNA sequencing and quantitative proteomics, we identified that FOXG1 affects the biogenesis of miR200b/a/429 and interacts with the ATP-dependent RNA helicase, DDX5/p68. Both FOXG1 and DDX5 associate with the microprocessor complex, whereby DDX5 recruits FOXG1 to DROSHA. RNA-Seq analyses of Foxg1cre/+ hippocampi and N2a cells overexpressing miR200 family members identified cAMP-dependent protein kinase type II-beta regulatory subunit (PRKAR2B) as a target of miR200 in neural cells. PRKAR2B inhibits postsynaptic functions by attenuating protein kinase A (PKA) activity; thus, increased PRKAR2B levels may contribute to neuronal dysfunctions in FOXG1 syndrome. Our data suggest that FOXG1 regulates PRKAR2B expression both on transcriptional and posttranscriptional levels.
KeywordsDROSHA Atypical Rett syndrome MECP2 Neurogenesis PKA
Rett syndrome (RTT) is a progressive neurodevelopmental disorder that affects one in 10,000 females, and it is the second leading cause of female intellectual deficiency. RTT patients show symptoms such as microcephaly, seizures, indifference to visual/auditory stimuli, and severe cognitive dysfunction . There are two forms of RTT, namely typical RTT (tRTT) and atypical RTT-like (atRTT). About 70–90% of cases are due to tRTT, which results from mutations in the Methyl CpG binding Protein 2 (MECP2) gene. Different subvariants of atRTT have been defined, one of which is caused by mutations in the Forkhead box G1 (FOXG1) gene (FOXG1 syndrome, OMIM#164874). Surprisingly, both loss- and gain-of-function mutations result in clinical phenotypes, which encompass common (e.g. seizures) but also unique features (e.g. spasms). Rett-like syndrome and epilepsy have been associated with FOXG1-haploinsufficiency . Loss- and gain-of-function mutations are reported also for both tRTT and atRTT, whereby gain-of-function is caused by gene duplication of either MECP2 or FOXG1 [3, 4]. Therefore, both gene products seem to be associated with functions that are dosage sensitive, although these functions are so far ill defined, especially for FOXG1.
FOXG1 plays a central role in forebrain development as its complete absence results in anencephaly . FOXG1 influences proliferation as well as differentiation of neural stem cells, and it is involved in migration and integration of pyramidal neurons into the cortical plate . FOXG1-deficient stem cells differentiate prematurely to Cajal–Retzius neurons, whereas overexpression of FOXG1 increases the stem cell pool and delays neurogenesis [7, 8]. On a molecular level, FOXG1 represses expression of cyclin-dependent kinase inhibitor 1A (Cdkn1a) and thereby prevents cell cycle exit of progenitor cells and promotes stem cell pool expansion [9, 10]. Cell cycle regulation through FOXG1 is mediated by its binding to Forkhead box O− (FOXO−) and SMAD− (SMA and MAD related−) protein complexes and by antagonising TGFβ-induced neuronal differentiation . In addition, FOXG1 deficiency results in the loss of the ventral telencephalon through impaired expression of ventralising signals . Also, FOXG1 interacts with one of two MECP2-isoforms (MECP2-e2), which prevents cell death of cerebellar neurons . Several mouse models were used to study the molecular basis of tRTT and atRTT, and some of these studies included non-coding RNA (ncRNA), such as miRNAs. Whereas altered expression of ncRNA is involved in MECP2-mediated RTT [14, 15, 16], a comprehensive expression study of the misregulated coding and non-coding transcriptome is missing for FOXG1 haploinsufficient adult brains.
Here, we report on altered expression of members of the miRNA200 family, namely miR200a, miR200b and miR429 in the adult Foxg1cre/+ hippocampus. Stable isotope labelling with amino acids in cell culture (SILAC) followed by quantitative mass spectrometry revealed that FOXG1 associates with the RNA helicase DDX5 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 5, p68). DDX5 recruits FOXG1 to the DROSHA complex, and FOXG1 overexpression alongside with reduced levels of DDX5 affects biogenesis of miR200 family members. Decreased expression of FOXG1 and overexpression of miR200 result in altered expression levels of protein kinase cAMP-dependent regulatory type II beta (PRKAR2B). As PRKAR2B influences synaptic function, our results reveal a novel candidate gene, whose altered expression might be implicated in FOXG1 syndrome. Additionally, we establish that FOXG1 has functions in posttranscriptional regulation besides its known role as transcription factor [6, 15].
Material and Methods
Information on cell culture conditions, transfections and plasmids used in this study, on cell fractionation, mouse hippocampus dissection, culture of neurons and viral transduction, RNA immunoprecipitation (RIP), RNA isolation, reverse transcription and quantitative real-time PCR (qRT-PCR) and luciferase assays, as well as on miRNA analysis by Northern hybridization are found in the Supplementary Material and Methods.
The animal welfare committees of the University of Freiburg and local authorities approved all mouse experiments, registered under the licence G14-096 or X14/04H. Foxg1cre/+ mice  were maintained in a C56BL/6 background. For experiments with wild-type (WT) mice, NMRI was used either at E13.5 or adult stages.
SILAC and Mass Spectrometry
N2a cells were cultured in SILAC DMEM without arginine and lysine (#89985 ThermoScientific, Bremen, Germany) supplemented with Lys0/Arg0 or Lys8/Arg10 (0.398 mM L-arginine 13C615N4, 0.798 mM L-lysine 13C615N2, Euriso-Top, Saarbrücken, Germany) and 10% dialysed FCS (ThermoScientific). N2a cells were labelled for 12 passages with SILAC medium. Further processing is described in the Supplementary Material and Methods.
Analysis of the protein groups was done with the Perseus software . Only proteins with two or more unique identified peptides, which were enriched more than 2-fold in both experiments, were considered. All raw data and original result files were deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD007040.
Immunoprecipitation (IP) was performed according to standard procedures as outlined in the Supplementary Material and Methods. For co-immunoprecipitations (co-IPs) with tagged FOXG1 (either FOXG1-Au1 or FOXG1-HA), mock conditions were either untransfected, empty vector or FOXG1 with the other tag. The following antibodies were used for IP: anti-Au1-tag (MMs-130R, Covance, Koblenz, Germany), anti-HA-tag (#3724, Cell Signaling), anti-DDX5 (rabbit, ab126730, abcam, Cambridge, UK), anti-FOXG1 (rabbit, ab18259, abcam) and anti-DROSHA (rabbit, ab12286, abcam).
Immunoblotting was performed according to standard procedures as outlined in the Supplementary Material and Methods. The following antibodies were used for immunoblots: anti-Au1-tag (1:1000, MMs-130R, Covance), anti-HA-tag (1:1000, #3724, Cell Signaling), anti-DDX5 (1:2000, rabbit, ab126730, abcam), anti-FOXG1 (1:1000, rabbit, ab18259, abcam), anti-DROSHA (1:1000, rabbit, ab12286, abcam), anti-DGCR8 (1:1000, rabbit, ab191875, abcam), anti-H3 (1:1000, goat, ab12079, abcam), anti-NPM1 (1:1000, mouse, ab10530, abcam), anti-PRKAR2B (1:1000, DAKO) and anti-GAPDH (1:3000, ab8245, abcam). Densitometric analyses were done with ImageJ.
RNA-Seq and Small RNA-Seq
Total RNA was prepared from the hippocampus with RNeasy kits (Qiagen), including on-column DNAse digestion. Samples were depleted from rRNA using RiboZero Gold kit (Illumina) before sequencing. Quality of the RNA was assessed with QIAxcel (#9001941, Qiagen, Hilden, Germany). Samples were prepared and analysed with Illumina HiSeq2500 (paired end, multiplexing run, 75 Mio/reads per sample). Bioinformatics analysis was performed using the Freiburger Galaxy Server [19, 20] as described in the Supplementary Material and Methods. For small RNA-Seq, 6-week-old Foxg1cre/+ mice hippocampi were used (n = 9). Raw data were deposited at the GEO database under the following accession numbers: small-RNAseq: GSE104169, mir-200 OE RNA-Seq: GSE106802 and Foxg1cre/+ hippocampus RNA-Seq: GSE106801.
Proximity Ligation Assay
Proximity ligation assay (PLA) was performed with the Duolink starter kit reagents (DUO92103, SIGMA) according to the manufacturer’s instruction as outlined in the Supplementary Material and Methods. The following antibodies were used: anti-Au1 (1:2000, mouse, Covance) and anti-DDX5 (1:200, goat, ab10261, abcam) for PLA and anti-Lamin B1 (1:200, rabbit, ab133741, abcam). Images were taken with a confocal microscope and analysed with the LASX software (SP8, Leica, Jena, Germany).
GraphPad Prism software was used for statistical analyses. Statistical tests are indicated in the respective figure legends. Values in bar charts are expressed as average ± SEM. In in vivo experiments, each independent N is a different animal, and in in vitro experiments, each N is a different passage of cells. One sample Student’s t test was performed (e.g. on ΔΔCt values of qRT-PCRs) if measured variables could be paired (e.g. control and treatment of the same passage of cells). Unpaired Student’s t test (equal variances) or Welch’s t test (unequal variances) was used if variables were not paired (using in these cases for example ΔCt values for each sample group).
Final figures were prepared using FIJI (ImageJ, v. 2.0.0-rc-43/1.51d ) and Inkscape (v. 0.91).
Foxg1cre/+ Animals Express Reduced Levels of Mature and Precursor miR200b/a/429 in the Hippocampus
Prkar2b Is a Target of FOXG1 and miR200 Family
Gene ontology (GO) term analysis revealed that this set of 35 genes is classified to processes like neuronal projection and development (Fig. 2b). As the Foxg1cre/+ hippocampus expressed less mature miR200b/a/429, we focused on genes with opposing expression levels after miR200 overexpression compared to Foxg1cre/+ RNA-Seq to identify putative targets. Twelve of these genes showed reduced levels after miR200 overexpression, whereas four increased in expression (highlighted in Fig. 2c). We used different target prediction tools to analyse miR200 seed sequences on the mRNAs of these 16 genes. Three of the four prediction algorithms identified putative miR200 binding sites in the 3′-untranslated region (UTR) of Prkar2b (Fig. 2d). We subsequently assessed altered expression of 13 candidates by qRT-PCR in vitro and in vivo. Prkar2b mRNA levels decreased upon overexpression of the miR200 family members and increased in Foxg1cre/+ hippocampus (Fig. 2e) as predicted. Other candidates, i.e. Serpinf1, Olfml2b and Tmem108, were either significantly altered in only one condition or were altered in an opposing direction compared to the RNA-Seq data. These expression analyses therefore rendered Prkar2b as the best candidate for altered expression through FOXG1 and/or miR200 family.
To investigate if levels of miR200 family members or Prkar2b were altered in other regions than the hippocampus of Foxg1cre/+ animals, we assessed the respective transcript levels in samples derived from the cerebral cortex and olfactory bulb. Neither mature miR200b/a/429 nor Prkar2b levels changed in the cerebral cortex or olfactory bulb in Foxg1cre/+ compared to WT animals (Fig. S1a–c).
FOXG1 Interacts with DDX5 and DROSHA Microprocessor Complex
FOXG1–DDX5 Complex Interacts with the Microprocessor in the Nucleus
We next investigated if FOXG1 mediated the binding of DDX5 to the microprocessor in neural cells, or vice versa. We performed DROSHA co-IP after overexpressing FOXG1 in N2a cells and assessed DDX5 levels. DROSHA co-precipitated similar levels of DDX5, irrespective of the presence of FOXG1 (Fig. 6d). Next, we investigated if knockdown (KD) of DDX5 affected FOXG1 binding to DROSHA. By reducing expression of DDX5, we observed decreased FOXG1 recruitment to DROSHA (Fig. 6e, f). Together, these data suggested that DDX5 associated to the microprocessor independent of the presence of FOXG1, but that FOXG1 required DDX5 to bind to DROSHA.
FOXG1 did not seem to alter pri-miR200b/a/429 transcription and it associated with the microprocessor in the nucleus. We therefore hypothesised that a FOXG1–DDX5 complex might affect miR200 biogenesis and assessed whether FOXG1 or DDX5 would bind the pri-miR200b/a/429 transcript. We applied native RIP with antibodies against the Au1-tag after FOXG1-Au1 overexpression in N2a cells, or with anti-DDX5, respectively. RIP was followed by qRT-PCR, which revealed that both FOXG1 and DDX5 precipitated the pri-miR200b/a/429 (Fig. 6g).
Decreased Levels of DDX5 in FOXG1 Overexpressing Cells Reduce Mature miR200 Levels but Increase DROSHA Processivity
We next used a pmiRGLO-miR200b/a/429 plasmid to overexpress the miR200b/a/429 family and assessed whether levels of primary miR200 transcripts changed after FOXG1 overexpression. In these conditions, the presence of FOXG1 significantly increased pri-miR200b/a/429 levels (Fig. 7d, e), which resulted in significantly increased levels of mature miR200 family expression (Fig. 7f). Moreover, the N2a cells transfected with pmiRGlo-miR200b/a/429 along with FOXG1 allowed us to observe an increased turnover of the pri-miR200b/a/429 in a luciferase reporter assay (Fig. 7g). As we observed altered mature miR200 levels only when we used pmiRGLO-miR200b/a/429, which led to increased levels of the primary transcript, but not with the pCX-miR200b/a/429 plasmid that did not change pri-miR200 levels, we concluded that FOXG1 alone is probably not sufficient to affect miR200 biogenesis. Instead, significantly altered levels of the primary transcript are necessary to observe altered expression of mature miR200 family members.
To further address whether DDX5 would play a role in the FOXG1-mediated increase in mature miR200 levels as observed in Fig. 7f, we knocked-down (KD) Ddx5 in FOXG1 and pri-miR200b/a/429 (using pmiRGLO-miR200b/a/429) expressing cells (Fig. 7i). Under these conditions, pri-miR200b/a/429 levels did not change (Fig. 7h); however, the levels of the mature miR200 family decreased significantly (Fig. 7j), despite increased activity of the microprocessor (Fig. 7k). These findings indicated that DDX5 is involved in FOXG1-mediated processing of the miR200 family members.
In this study, we identified that FOXG1 affects PRKAR2B expression at multiple levels, on one hand through direct transcriptional repression and on the other hand through the miR200 family that directly targets Prkar2b transcripts. Thereby, we discovered important new roles of FOXG1 for the function of neural cells and of hippocampal neurons in the adult CNS, independent on proliferative effects (Fig. S2). Patients with mutations in FOXG1 show impaired neuronal function and the symptoms are comparable to Rett syndrome. However, the role of FOXG1 in mature neurons and in the adult CNS is not entirely clear. In the adult hippocampus, FOXG1 prevents depletion of the progenitor pool in the dentate gyrus . In cerebellar granule neurons, FOXG1 promotes survival . Also, FOXG1 binds to a spliced, proapoptotic version of MECP2 thereby preventing neuronal death .
Apart from influencing gene transcription, Pancrazi et al. showed that FOXG1 localised within the mitochondrial matrix and regulates the mitochondrial membrane potential, mitochondrial fission and mitosis . This finding gave indication that FOXG1 might have additional functions than regulation of transcription, for example in posttranscriptional control. Here, we describe a novel interaction between FOXG1, DDX5 and the microprocessor complex that can affect maturation of miR200b/a/429. Our data show that the PKA regulator PRKAR2B is a direct target of FOXG1 as well as of miR200 family. The biogenesis of miR200 can also be influenced by FOXG1, probably in a context-dependent manner that depends on the levels of the different players. Our data robustly show that increased levels of FOXG1 in Ddx5 KD N2a and hippocampal cells decreased mature miR200. However, at least in N2a cells, we observed decreased miR200 levels despite a significantly increased turnover of the primary transcript. Therefore, we hypothesise further regulative layers downstream of DROSHA that might be affected by FOXG1. Our MS data propose different pathways that might act downstream of DROSHA, as the data suggest that FOXG1 associates for example with the exosome and therefore confers degradation of pre-miR200 family members. It is also possible that FOXG1 interacts with nuclear envelope proteins and affects the transport of pre-miR200 into the cytoplasm. The PLA signal suggests that FOXG1/DDX5 complexes localise near the membrane, which might corroborate such interpretation. However, to shed more light on FOXG1’s implication in posttranscriptional control and FOXG1/DDX5-mediated effects downstream of DROSHA, much more research is needed.
Our finding that FOXG1 influences posttranscriptional maturation of miRNAs reflects a shared cellular function between FOXG1 and MECP2, as the latter also associates to DROSHA and DGCR8 to regulate miRNA processing in the adult mouse hippocampus . However, while the involvement of FOXG1 and MECP2 in miRNA biogenesis is conserved, targets are different. MECP2 suppresses miR134, miR383, miR382 and miR182 maturation in the hippocampus . Our study revealed fewer numbers of differently expressed miRNAs that are affected in Foxg1cre/+ hippocampus compared to the data reported for MECP2 deficiency. And, we identified no commonly misregulated miRNAs between the two forms of RTT. Thus, although both MECP2 and FOXG1 associate with the microprocessor complex, they seemingly affect maturation of different miRNAs.
miR200b/a/429 transcription is regulated by different factors in a tissue-specific manner. TP53 is a transcription factor necessary for miR200b/a/429 gene expression [42, 43], whereas ZEB1 and ZEB2 repress expression of the miR200b/a/429 gene. Ovarian tumours induce miR200 expression upon DNA damage involving another RNA helicase, namely DDX1 . Control of expression and biogenesis of the miR200 is necessary as this family is implicated in diverse cellular processes, ranging from neurodegeneration, eye development, adipocyte differentiation, taste bud and tooth development to maintenance of stem cell identity (reviewed in ). The miR200 family has important regulative functions in cancer  and neural differentiation in humans , zebrafish , Drosophila  and neural PC12 cells , as well as in mice [24, 25]. Altogether, miR200 controls similar processes as FOXG1 in the developing cerebral cortex [5, 9, 22, 23]. Interestingly, Foxg1 has been proposed as miR200 target in different model systems [26, 48, 49], and varying FOXG1 expression levels might thus be regulated through a miR200-dependent feedback loop. Such feedback loop might account for the transiently reduced FOXG1 expression in differentiating progenitors in the cerebral cortex and the reinitiation of its expression in differentiated projection neurons . It is therefore tempting to speculate further that the biogenesis of miR200, influenced by FOXG1, is not restricted to the adult hippocampus but also takes place in the developing cerebral cortex. This notion is supported by our identification of FOXG1–DDX5 interaction in E13.5 embryonic cortical tissue (data not shown).
Another important finding of our study is that FOXG1 and miR200 target PRKAR2B in the CNS. PRKAR2B is the target of miR200b in platelets . PRKAR2B is a regulative type 2 subunit of protein kinase A (PKA), which is expressed in different tissues but has the highest expression in the CNS . PKA signalling is critically implicated in memory formation, and this function is evolutionary highly conserved (reviewed by ). For example, PKA regulates synaptic plasticity by phosphorylating AMPA receptor subunits , or the GluN2B subunit of NMDAR during emotional response to stress . Interference with PKA signalling in the mouse hippocampus impairs long-term spatial memory formation and elicits long-term memory deficits in contextual fear conditioning . In the latter, freezing behaviour reduces after interference with PKA signalling. Foxg1cre/+ mice show a similar behaviour of decreased contextual fear response . Thus, increasing levels of the repressive PRKAR2B subunit after decreased expression of miR200 family members might interfere with effective PKA signalling in Foxg1cre/+ mice. Strikingly, MECP2-deficient mice show the same decreased freezing behaviour , and increased levels of PRKAR2B (data not shown and ). Published data from iPS-derived neurons show that PRKAR2B expression levels are altered in some FOXG1 syndrome patients (log2FC of PRKAR2B of 1.14 with a FDR of 8E-04 ). In all, impaired PKA signalling might be a common feature in tRTT and atRTT. Altered levels of PRKAR2B might also be responsible for other phenotypic alterations in Rett syndrome, such as altered motor behaviour , or impaired vision [61, 62, 63, 64].
In summary, our data indicate that FOXG1 associates to the microprocessor complex with DDX5 and that it affects mature miR200 levels. We further suggest that FOXG1 and miR200 family are both part of a multilevel network that balances the expression of a regulative subunit of PKA, PRKAR2B. Thereby, FOXG1 and also MECP2 may affect a common PKA-dependent pathway to adjust neuronal function in the hippocampus.
The authors thank Harold Cremer (IBDM, Aix-Marseille Université, France) for pCX-miRNA200 family and pCX-D2eGFP-miR200 sponge plasmids and Nicole Gensch (BIOSS tool box, University of Freiburg) for further plasmids used in this study; Michael Müller (University Medicine Göttingen, Germany) for providing Mecp2−/y mice hippocampal tissue; Ute Baur and Monika Pätzold for technical support; and Marco Ell for his experiments during the early phase of the project.
This work was funded by the Deutsche Forschungsgemeinschaft DFG SPP1738 through grants to T. Vogel, R. Backofen and A. Fischer, by the DFG Research Training Group GRK2344 (to T. Vogel, R. Backofen, and W.R. Hess) and by the Collaborative Research Centre 992 Medical Epigenetics (T. Vogel and R. Backofen) and the German Federal Ministry of Education and Research (BMBF) through grants to R. Backofen (031A538A de. NBI) and W.R. Hess (031L0106B de. NBI Partner).
Compliance with Ethical Standards
Conflict of Interest
The authors declare that they have no conflict of interest.
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