The molecular mechanisms underlying multi-brain region origins and sexual dimorphism of anxiety remain unclear. Here, we leverage large-scale transcriptomics from seven brain regions in mouse models of anxiety and extensive experiments to dissect brain-region- and sex-specific gene networks. We identify 4,840 genes with sex-specific expression alterations across seven brain regions, organized into ten network modules with sex-biased expression patterns. Modular analysis prioritizes 86 sex-specific mediators of anxiety susceptibility, including myocyte-specific enhancer factor 2c (Mef2c) in the CA3 region of male mice. Mef2c expression is decreased in the pyramidal neurons (PyNs) of susceptible male mice. Up-regulating Mef2c in CA3 PyNs significantly alleviates anxiety-like behavior, whereas down-regulating Mef2c induces anxiety-like behavior in male mice. The anxiolytic effect of Mef2c up-regulation is associated with enhanced neuronal excitability and synaptic transmission. In summary, this study uncovers brain-region- and sex-specific networks and identifies Mef2c in CA3 PyNs as a critical mediator of anxiety in male mice.

Fas ligand (FasL, CD178) belongs to classical apoptotic molecules, however, recent evidence expands the spectrum of FasL functions into non-apoptotic processes which also applies for the bone. Tgfb subfamily members (Tgfb1, Tgfb2, Tgfb3) represent major components in osteogenic pathways and extracellular matrix. Their possible association with FasL has not yet been investigated but can be postulated. To test such a hypothesis, FasL deficient (gld) calvaria-derived cells were examined with a focus on the expression of Tgfb receptor ligands. The qPCR analysis revealed significantly increased expression of Tgfb1, Tgfb2 and Tgfb3 in gld cells. To check the vice versa effect, the gld cells were stimulated by soluble FasL. As a consequence, a dramatic decrease in expression levels of all three ligands was observed. This phenomenon was also confirmed in IDG-SW3 (osteoblastic cells of endochondral origin). TFLink gateway identified Fosl2 as an exclusive candidate of FasL capable to impact expression of all three Tgfb ligands. However, Fosl2 siRNA did not cause any significant changes in expression of Tgfb ligands. Therefore, the upregulation of the three ligands is likely to occur separately. In this respect, we tested the only exclusive candidate transcription factor for Tgfb3, Prrx1. Additionally, an overlapping candidate for Tgfb1 and Tgfb2, Mef2c capable to modulate expression of sclerostin, was examined. Prrx1 as well as Mef2c were found upregulated in gld samples and their expression decreased after addition of FasL. The same effect of FasL treatment was observed in the IDG-SW3 model. Taken together, FasL deficiency causes an increase in the expression of Tgfb ligands and stimulation by FasL reduces Tgfb expression in osteoblastic cells. The candidates mediating the effect comprise Prrx1 for Tgfb3 and Mef2c for Tgfb1/2. These results indicate FasL as a novel cytokine interfering with Tgfb signaling and thus the complex osteogenic network. The emerging non-apoptotic functions of FasL in bone development and maintenance should also be considered in treatment strategies such as the anti-osteoporotic factor.

Transcriptional regulation constitutes a key step in gene expression regulation. Myocyte enhancer factor 2C (MEF2C) is a transcription factor of the MADS box family involved in the early development of several cell types, including muscle cells. Over the last decade, a novel layer of complexity modulating gene regulation has emerged as non-coding RNAs have been identified, impacting both transcriptional and post-transcriptional regulation. microRNAs represent the most studied and abundantly expressed subtype of small non-coding RNAs, and their functional roles have been widely documented. On the other hand, our knowledge of the transcriptional and post-transcriptional regulatory mechanisms that drive microRNA expression is still incipient. We recently demonstrated that MEF2C is able to transactivate the long, but not short, regulatory element upstream of the miR-23a-miR-27a-miR-24-2 transcriptional start site. However, MEF2C over-expression and silencing, respectively, displayed distinct effects on each of the miR-23a-miR-27a-miR-24-2 mature cluster members without affecting pri-miRNA expression levels, thus supporting additional MEF2C-driven regulatory mechanisms. Within this study, we demonstrated a complex post-transcriptional regulatory mechanism directed by MEF2C in the regulation of miR-23a-miR-27a-miR-24-2 cluster members, distinctly involving different domains of the MEF2C transcription factor and the physical interaction with pre-miRNAs and Ksrp, HnRNPa3 and Ddx17 transcripts.

Blood-brain barrier (BBB) changes are acknowledged as early indicators of Alzheimer\'s disease (AD). The permeability and integrity of the BBB rely significantly on the essential role played by the tight junction proteins (TJPs) connecting endothelial cells. This study found the reduced RNA binding motif protein 3 (RBM3) expression in brain microvascular endothelial cells (BMECs) incubated with Aβ1-42. This downregulation of RBM3 caused a decrease in the levels of ZO-1 and occludin and increased the permeability of BBB cell model in AD microenvironment. Myocyte enhancer factor 2C (MEF2C) expression was also inhibited in BMECs incubated with Aβ1-42. A decrease in MEF2C expression led to increased permeability of BBB cell model in AD microenvironment and reductions in the levels of ZO-1 and occludin. Further analysis of the underlying mechanism revealed that RBM3 binds to and stabilizes MEF2C mRNA. MEF2C binds to the promoters of ZO-1 and occludin, enhancing their transcriptional activities and modulating BBB permeability. RBM3 increases the stability of MEF2C mRNA and subsequently modulates BBB permeability through the paracellular pathway of TJPs. This may provide new insights for AD research.

Neurons are post-mitotic cells, with microtubules playing crucial roles in axonal transport and growth. Kinesin family member 2c (KIF2C), a member of the Kinesin-13 family, possesses the ability to depolymerize microtubules and is involved in remodelling the microtubule lattice. Myocyte enhancer factor 2c (MEF2C) was initially identified as a regulator of muscle differentiation but has recently been associated with neurological abnormalities such as severe cognitive impairment, stereotyping, epilepsy and brain malformations when mutated or deleted. However, further investigation is required to determine which target genes MEF2C acts upon to influence neuronal function as a transcription regulator. Our data demonstrate that knockdown of both Mef2c and Kif2c significantly impacts spinal motor neuron development and behaviour in zebrafish. Luciferase reporter assays and chromosome immunoprecipitation assays, along with down/upregulated expression analysis, revealed that MFE2C functions as a novel transcription regulator for the Kif2c gene. Additionally, the knockdown of either Mef2c or Kif2c expression in E18 cortical neurons substantially reduces the number of primary neurites and axonal branches during neuronal development in vitro without affecting neurite length. Finally, depletion of Kif2c eliminated the effects of overexpression of Mef2c on the neurite branching. Based on these findings, we provided novel evidence demonstrating that MEF2C regulates the transcription of the Kif2c gene thereby influencing the axonal branching.

UNASSIGNED: Heterozygous mutations or deletions of MEF2C cause a neurodevelopmental disorder termed MEF2C haploinsufficiency syndrome (MCHS), characterized by autism spectrum disorder and neurological symptoms. In mice, global Mef2c heterozygosity has produced multiple MCHS-like phenotypes. MEF2C is highly expressed in multiple cell types of the developing brain, including GABAergic (gamma-aminobutyric acidergic) inhibitory neurons, but the influence of MEF2C hypofunction in GABAergic neurons on MCHS-like phenotypes remains unclear.
UNASSIGNED: We employed GABAergic cell type-specific manipulations to study mouse Mef2c heterozygosity in a battery of MCHS-like behaviors. We also performed electroencephalography, single-cell transcriptomics, and patch-clamp electrophysiology and optogenetics to assess the impact of Mef2c haploinsufficiency on gene expression and prefrontal cortex microcircuits.
UNASSIGNED: Mef2c heterozygosity in developing GABAergic cells produced female-specific deficits in social preference and altered approach-avoidance behavior. In female, but not male, mice, we observed that Mef2c heterozygosity in developing GABAergic cells produced 1) differentially expressed genes in multiple cell types, including parvalbumin-expressing GABAergic neurons, 2) baseline and social-related frontocortical network activity alterations, and 3) reductions in parvalbumin cell intrinsic excitability and inhibitory synaptic transmission onto deep-layer pyramidal neurons.
UNASSIGNED: MEF2C hypofunction in female, but not male, developing GABAergic cells is important for typical sociability and approach-avoidance behaviors and normal parvalbumin inhibitory neuron function in the prefrontal cortex of mice. While there is no apparent sex bias in autism spectrum disorder symptoms of MCHS, our findings suggest that GABAergic cell-specific dysfunction in females with MCHS may contribute disproportionately to sociability symptoms.

RNA binding proteins have been shown to regulate heart development and cardiac diseases. However, the detailed molecular mechanisms is not known. In this study, we identified Wilms\' tumor 1-associating protein (WTAP, a key regulatory protein of the m6A RNA methyltransferase complex) as a key regulator of heart function and cardiac diseases. WTAP is associated with heart development, and its expression is downregulated in both human and mice with heart failure. Cardiomyocyte-specific knockout of Wtap (Wtap-CKO) induces dilated cardiomyopathy, heart failure and neonatal death. Although WTAP deficiency in the heart decreases METTL3 (methyltransferase-like 3) protein levels, cardiomyocyte-specific overexpression of Mettl3 in Wtap-CKO mice does not rescue the phenotypes of Wtap-CKO mice. Instead, WTAP deficiency in the heart decreases chromatin accessibility in the promoter regions of Mef2a (myocyte enhancer factor-2α) and Mef2c, leading to reduced mRNA and protein levels of these genes and lower expression of their target genes. Conversely, WTAP directly binds to the promoter of the Mef2c gene and increases its promoter luciferase activity and expression. These data demonstrate that WTAP plays a key role in heart development and cardiac function by maintaining the chromatin accessibility of cardiomyocyte specific genes.

Long noncoding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) alleviates the progression of diabetic nephropathy by inhibiting inflammation and fibrosis. This study investigated how CASC2 impacts renal interstitial fibrosis (RIF) through regulating M1 macrophage (M1) polarization.
Nine-week-old mice underwent unilateral ureteral obstruction (UUO) establishment. Macrophages were induced toward M1 polarization using lipopolysaccharide (LPS) in vitro and cocultured with fibroblasts to examine how M1 polarization influences RIF. LnCeCell predicted that CASC2 interacted with myocyte enhancer factor 2 C (MEF2C), which was validated by dual-luciferase reporter assay. CASC2/MEF2C overexpression was achieved by lentivirus-expressing lncRNA CASC2 injection in vivo or CASC2 and MEF2C transfection in vitro. Renal injury was evaluated through biochemical analysis and hematoxylin-eosin/Masson staining. Macrophage infiltration and M1 polarization in the kidney and/or macrophages were detected by immunofluorescence, flow cytometry, and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR). Expressions of CASC2, MEF2C, and markers related to inflammation/M1/fibrosis in the kidney/macrophages/fibroblasts were analyzed by qRT-PCR, fluorescence in situ hybridization, enzyme-linked immunosorbent assay, and/or Western blot.
In the kidneys of mice, CASC2 was downregulated and macrophage infiltration was promoted time-dependently from days 3 to 14 post-UUO induction; CASC2 overexpression alleviated renal histological abnormalities, hindered macrophage infiltration and M1 polarization, downregulated renal function markers serum creatinine and blood urea nitrogen and inflammation/M1/fibrosis-related makers, and offset UUO-induced MEF2C upregulation. LncRNA CASC2 overexpression inhibited fibroblast fibrosis and M1 polarization in cocultured fibroblasts with LPS-activated macrophages. Also, CASC2 bound to MEF2C and inhibited its expression in LPS-activated macrophages. Furthermore, MEF2C reversed the inhibitory effects of lncRNA CASC2 overexpression.
CASC2 alleviates RIF by inhibiting M1 polarization through directly downregulating MEF2C expression. CASC2 might represent a promising value of future investigations on treatment for RIF.

Mutations in the activity-dependent transcription factor MEF2C have been associated with several neuropsychiatric disorders. Among these, autism spectrum disorder (ASD)-related behavioral deficits are manifested. Multiple animal models that harbor mutations in Mef2c have provided compelling evidence that Mef2c is indeed an ASD gene. However, studies in mice with germline or global brain knock-out of Mef2c are limited in their ability to identify the precise neural substrates and cell types that are required for the expression of Mef2c-mediated ASD behaviors. Given the role of hippocampal neurogenesis in cognitive and social behaviors, in this study we aimed to investigate the role of Mef2c in the structure and function of newly generated dentate granule cells (DGCs) in the postnatal hippocampus and to determine whether disrupted Mef2c function is responsible for manifesting ASD behaviors. Overexpression of Mef2c (Mef2cOE ) arrested the transition of neurogenesis at progenitor stages, as indicated by sustained expression of Sox2+ in Mef2cOE DGCs. Conditional knock-out of Mef2c (Mef2ccko ) allowed neuronal commitment of Mef2ccko cells; however, Mef2ccko impaired not only dendritic arborization and spine formation but also synaptic transmission onto Mef2ccko DGCs. Moreover, the abnormal structure and function of Mef2ccko DGCs led to deficits in social interaction and social novelty recognition, which are key characteristics of ASD behaviors. Thus, our study revealed a dose-dependent requirement of Mef2c in the control of distinct steps of neurogenesis, as well as a critical cell-autonomous function of Mef2c in newborn DGCs in the expression of proper social behavior in both sexes.

Mef2c is a transcription factor that mediates key cellular behaviors that promote endochondral ossification and bone formation. Previously, Mef2c has been shown to regulate Sost transcription via its osteocyte-specific enhancer, ECR5, and conditional deletions of Mef2cfl/fl with either Col1-Cre or Dmp1-Cre produced generalized high bone mass (HBM) consistent with Van Buchem Disease phenotypes. However, Sost-/-; Mef2cfl/fl; Dmp1-Cre mice produced a significantly higher bone mass phenotype that Sost-/- alone suggesting that Mef2c modulates bone mass through additional mechanisms, independent of Sost. To identify new Mef2c transcriptional targets important in bone metabolism, we profiled gene expression by single-cell RNA sequencing in subpopulations of cells isolated from Mef2cfl/fl; Dmp1-Cre and Mef2cfl/fl; Bglap-Cre femurs, both strains exhibiting similar high bone mass phenotypes. However, we found Mef2cfl/fl; Bglap-Cre to also display a growth plate defect characterized by an expansion of several osteoprogenitor subpopulations. Differential gene expression analysis identified a total of 96 up- and 2434 down- regulated genes in Mef2cfl/fl; Bglap-Cre and 176 up- and 1041 down- regulated genes in Mef2cfl/fl; Dmp1-Cre bone cell subpopulations compared to wildtype mice. Mef2c deletion affected the transcriptomes across several cell types including mesenchymal progenitors (MP), osteoprogenitors (OSP), osteoblast (OB), and osteocyte (OCY) subpopulations. Several energy metabolism genes such as Uqcrb, Ndufv2, Ndufs3, Ndufa13, Ndufb9, Ndufb5, Cox6a1, Cox5a, Atp5o, Atp5g2, Atp5b, Atp5 were significantly down regulated in Mef2c-deficient OBs and OCYs, in both strains. Binding motif analysis of promoter regions of differentially expressed genes identified Mef2c binding in Bone Sialoprotein (BSP/Ibsp), a gene known to cause increased trabecular BV/TV in the femurs of Ibsp-/- mice. Immunohistochemical analysis confirmed the absence of Ibsp protein in OBs and OCYs. These findings suggests that the HBM in Sost-/-; Mef2cfl/fl; Dmp1-Cre is caused by a multitude of transcriptional changes in genes that regulate bone formation, two of which are Sost and Ibsp.