Zhi-zi-chi decoction mitigates depression by enhancing lncRNA Six3os1 expression and promoting histone H3K4 methylation at the BDNF promoter.

Traditional Chinese medicine, particularly Zhi-zi-chi decoction (ZZCD), is gaining recognition as a potential treatment for depression. This study aimed to uncover the molecular mechanisms behind ZZCD's antidepressant effects, focusing on lncRNA Six3os1 and histone H3K4 methylation at the BDNF promoter. Network pharmacology and in vivo experiments were conducted to identify ZZCD targets and evaluate its impact on depression-related behaviours and neuron injury. The role of Six3os1 in recruiting KMT2A to the BDNF promoter and its effects on oxidative stress and neuron injury were investigated. ZZCD reduced depression-like behaviours and neuron injury in mice subjected to chronic stress. It upregulated Six3os1, which facilitated KMT2A recruitment to the BDNF promoter, leading to increased histone H3K4 methylation and enhanced BDNF expression. ZZCD also inhibited CORT-induced neuron injury, inflammatory response and oxidative stress in vitro. ZZCD's antidepressant properties involve Six3os1 upregulation, which exerts neuroprotective effects by inhibiting oxidative stress and neuron injury, thereby alleviating depressive symptoms. Targeting Six3os1 upregulation may offer a potential therapeutic intervention for depression.

Mechanical Tensions Regulate Gene Expression in the Xenopus laevis Axial Tissues.

During gastrulation and neurulation, the chordamesoderm and overlying neuroectoderm of vertebrate embryos converge under the control of a specific genetic programme to the dorsal midline, simultaneously extending along it. However, whether mechanical tensions resulting from these morphogenetic movements play a role in long-range feedback signaling that in turn regulates gene expression in the chordamesoderm and neuroectoderm is unclear. In the present work, by using a model of artificially stretched explants of Xenopus midgastrula embryos and full-transcriptome sequencing, we identified genes with altered expression in response to external mechanical stretching. Importantly, mechanically activated genes appeared to be expressed during normal development in the trunk, i.e., in the stretched region only. By contrast, genes inhibited by mechanical stretching were normally expressed in the anterior neuroectoderm, where mechanical stress is low. These results indicate that mechanical tensions may play the role of a long-range signaling factor that regulates patterning of the embryo, serving as a link coupling morphogenesis and cell differentiation.

Geniposide improves depression by promoting the expression of synapse-related proteins through the Creb1/Six3os1 axis.

The present study aims to investigate the mechanism of Geniposide in the treatment of depression. By screening the effective components and targets of Zhi-zi-chi decoction, 140 candidate targets related to depression were identified. Further transcriptome sequencing was conducted to screen differentially expressed mRNAs and lncRNAs; 7 candidate Geniposide treatment targets for depression were obtained. KEGG/GO enrichment analysis and molecular docking were performed to select the optimal drug target, revealing that Creb1 is an important target. Additionally, Six3os1 is the lncRNA with the smallest P-value among the differentially expressed lncRNAs, and the JASPAR database revealed a binding site between Creb1 and the Six3os1 promoter. The intersection of Synapse-related genes obtained from the GeneCards database and differentially expressed mRNAs produced 6 synaptic-related genes. RNA-protein interaction prediction revealed that Six3os1 interacts with the protein encoded by these genes. Geniposide upregulates the expression of Creb1 and Six3os1. Creb1 can transcriptionally activate Six3os1, thereby upregulating the expression of the synaptic-related proteins Htr3a and Htr2a, improving depression.

Cell fate decisions, transcription factors and signaling during early retinal development.

The development of the vertebrate eyes is a complex process starting from anterior-posterior and dorso-ventral patterning of the anterior neural tube, resulting in the formation of the eye field. Symmetrical separation of the eye field at the anterior neural plate is followed by two symmetrical evaginations to generate a pair of optic vesicles. Next, reciprocal invagination of the optic vesicles with surface ectoderm-derived lens placodes generates double-layered optic cups. The inner and outer layers of the optic cups develop into the neural retina and retinal pigment epithelium (RPE), respectively. In vitro produced retinal tissues, called retinal organoids, are formed from human pluripotent stem cells, mimicking major steps of retinal differentiation in vivo. This review article summarizes recent progress in our understanding of early eye development, focusing on the formation the eye field, optic vesicles, and early optic cups. Recent single-cell transcriptomic studies are integrated with classical in vivo genetic and functional studies to uncover a range of cellular mechanisms underlying early eye development. The functions of signal transduction pathways and lineage-specific DNA-binding transcription factors are dissected to explain cell-specific regulatory mechanisms underlying cell fate determination during early eye development. The functions of homeodomain (HD) transcription factors Otx2, Pax6, Lhx2, Six3 and Six6, which are required for early eye development, are discussed in detail. Comprehensive understanding of the mechanisms of early eye development provides insight into the molecular and cellular basis of developmental ocular anomalies, such as optic cup coloboma. Lastly, modeling human development and inherited retinal diseases using stem cell-derived retinal organoids generates opportunities to discover novel therapies for retinal diseases.

Comprehensive Analysis of lncRNAs, miRNAs and mRNAs in Mouse Hippocampus With Hepatic Encephalopathy.

Hepatic encephalopathy (HE) often presents with varying degrees of cognitive impairment. However, the molecular mechanism of its cognitive impairment has not been fully elucidated. Whole transcriptome analysis of hippocampus between normal and HE mice was performed by using RNA sequencing. 229 lncRNAs, 49 miRNAs and 363 mRNAs were differentially expressed in HE mice. The lncRNA-miRNA-mRNA interaction networks were established, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Dysregulated RNAs in interaction networks were mainly involved in synaptic plasticity and the regulation of learning and memory. In NH4Cl-treated hippocampal neurons, the dendritic spine density and maturity decreased significantly, the amplitude and frequency of mIPSC increased, while the amplitude and frequency of mEPSC decreased. These manifestations can be reversed by silencing SIX3OS1. Further research on these no-coding RNAs may lead to new therapies for the treatment and management of brain dysfunction caused by HE.

Deletion of the homeodomain gene Six3 from kisspeptin neurons causes subfertility in female mice.

The homeodomain transcription factor SIX3 is a known regulator of eye, nose, and forebrain development, and has recently been implicated in female reproduction. Germline heterozygosity of SIX3 is sufficient to cause subfertility, but the cell populations that mediate this role are unknown. The neuropeptide kisspeptin is a critical component of the reproductive axis and plays roles in sexual maturation, ovulation, and the maintenance of gonadotropin secretion. We used Cre-Lox technology to remove Six3 specifically from kisspeptin neurons in mice to test the hypothesis that SIX3 in kisspeptin neurons is required for reproduction. We found that loss of Six3 in kisspeptin neurons causes subfertility and estrous cycle irregularities in females, but no effect in males. Overall, we find that SIX3 expression in kisspeptin neurons is an important contributor to female fertility.

Dlx1/2-dependent expression of Meis2 promotes neuronal fate determination in the mammalian striatum.

The striatum is a central regulator of behavior and motor function through the actions of D1 and D2 medium-sized spiny neurons (MSNs), which arise from a common lateral ganglionic eminence (LGE) progenitor. The molecular mechanisms of cell fate specification of these two neuronal subtypes are incompletely understood. Here, we found that deletion of murine Meis2, which is highly expressed in the LGE and derivatives, led to a large reduction in striatal MSNs due to a block in their differentiation. Meis2 directly binds to the Zfp503 and Six3 promoters and is required for their expression and specification of D1 and D2 MSNs, respectively. Finally, Meis2 expression is regulated by Dlx1/2 at least partially through the enhancer hs599 in the LGE subventricular zone. Overall, our findings define a pathway in the LGE whereby Dlx1/2 drives expression of Meis2, which subsequently promotes the fate determination of striatal D1 and D2 MSNs via Zfp503 and Six3.

New SHH and Known SIX3 Variants in a Series of Latin American Patients with Holoprosencephaly.

Holoprosencephaly (HPE) is the failure of the embryonic forebrain to develop into 2 hemispheres promoting midline cerebral and facial defects. The wide phenotypic variability and causal heterogeneity make genetic counseling difficult. Heterozygous variants with incomplete penetrance and variable expressivity in the SHH, SIX3, ZIC2, and TGIF1 genes explain ∼25% of the known causes of nonchromosomal HPE. We studied these 4 genes and clinically described 27 Latin American families presenting with nonchromosomal HPE. Three new SHH variants and a third known SIX3 likely pathogenic variant found by Sanger sequencing explained 15% of our cases. Genotype-phenotype correlation in these 4 families and published families with identical or similar driver gene, mutated domain, conservation of residue in other species, and the type of variant explain the pathogenicity but not the phenotypic variability. Nine patients, including 2 with SHH pathogenic variants, presented benign variants of the SHH, SIX3, ZIC2, and TGIF1 genes with potential alteration of splicing, a causal proposition in need of further studies. Finding more families with the same SIX3 variant may allow further identification of genetic or environmental modifiers explaining its variable phenotypic expression.

Homeobox Gene Six3 is Required for the Differentiation of D2-Type Medium Spiny Neurons.

Medium spiny neurons (MSNs) in the striatum, which can be divided into D1 and D2 MSNs, originate from the lateral ganglionic eminence (LGE). Previously, we reported that Six3 is a downstream target of Sp8/Sp9 in the transcriptional regulatory cascade of D2 MSN development and that conditionally knocking out Six3 leads to a severe loss of D2 MSNs. Here, we showed that Six3 mainly functions in D2 MSN precursor cells and gradually loses its function as D2 MSNs mature. Conditional deletion of Six3 had little effect on cell proliferation but blocked the differentiation of D2 MSN precursor cells. In addition, conditional overexpression of Six3 promoted the differentiation of precursor cells in the LGE. We measured an increase of apoptosis in the postnatal striatum of conditional Six3-knockout mice. This suggests that, in the absence of Six3, abnormally differentiated D2 MSNs are eliminated by programmed cell death. These results further identify Six3 as an important regulatory element during D2 MSN differentiation.

miR-4306 Suppresses Proliferation of Esophageal Squamous Cell Carcinoma Cell by Targeting SIX3.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers and the primary cause of cancer-related mortality in China. micoRNA plays a vital role during tumor initiation and malignant progression. miR-4306 has been reported to negatively regulate aggressive cell phenotypes in triple-negative breast cancer (TNBC). Nevertheless, the function of miR-4306 in ESCC was still not clear. In this study, we detected miR-4306 expression by quantitative real-time reverse transcription-PCR (qRT-PCR) and found that miR-4306 expression was downregulated in human ESCC tissue samples and cell lines. Moreover, miR-4306 overexpression could restrain ESCC cell proliferation, migratory and invasive ability and epithelial-mesenchymal transition (EMT), promote cell apoptosis after treatment with or without cisplatin. In contrast, inhibiting the expression of miR-4306 showed the opposing results. Furthermore, we explored the molecular mechanism of effects of miR-4306 and found that miR-4306 inhibited the expression of SIX3 by interaction with SIX3 3'UTR in ESCC cells, and SIX3 overexpression significantly reversed the effect of miR-4306-mediated ESCC cells proliferation. The current study provided evidence of miR-4306 as a tumor suppression gene in ESCC.

Homeobox Gene Six3 is Required for the Differentiation of D2-Type Medium Spiny Neurons / 神经科学通报·英文版

Medium spiny neurons (MSNs) in the striatum, which can be divided into D1 and D2 MSNs, originate from the lateral ganglionic eminence (LGE). Previously, we reported that Six3 is a downstream target of Sp8/Sp9 in the transcriptional regulatory cascade of D2 MSN development and that conditionally knocking out Six3 leads to a severe loss of D2 MSNs. Here, we showed that Six3 mainly functions in D2 MSN precursor cells and gradually loses its function as D2 MSNs mature. Conditional deletion of Six3 had little effect on cell proliferation but blocked the differentiation of D2 MSN precursor cells. In addition, conditional overexpression of Six3 promoted the differentiation of precursor cells in the LGE. We measured an increase of apoptosis in the postnatal striatum of conditional Six3-knockout mice. This suggests that, in the absence of Six3, abnormally differentiated D2 MSNs are eliminated by programmed cell death. These results further identify Six3 as an important regulatory element during D2 MSN differentiation.

TRIM27 acts as an oncogene and regulates cell proliferation and metastasis in non-small cell lung cancer through SIX3-ß-catenin signaling.

The Wnt/ß-catenin pathway plays vital roles in diverse biological processes, including cell differentiation, proliferation, migration, and insulin sensitivity. A recent study reported that the DNA-binding transcriptional factor SIX3 is essential during embryonic development in vertebrates and capable of downregulating target genes of the Wnt/ß-catenin pathway in lung cancer, indicating negative regulation of Wnt/ß-catenin activation. However, regulation of the SIX3-Wnt/ß-catenin pathway axis remains unknown. We measured the expression of TRIM27 and SIX3 as well as investigated whether there was a correlation between them in lung cancer tissue samples. Herein, we found that the E3 ubiquitin ligase, TRIM27, ubiquitinates, and degrades SIX3. TRIM27 induces non-small cell lung cancer (NSCLC) cell proliferation and metastasis, and the expression of ß-catenin, S100P, TGFB3, and MMP-9 were significantly inhibited by SIX3. Furthermore, XAV939 is a selective ß-catenin-mediated transcription inhibitor that inhibited TRIM27- and SIX3-mediated NSCLC cell proliferation, migration, and invasion. Clinically, lung tissue samples of cancer patients showed increased TRIM27 expression and decreased SIX3 expression. Taken together, these data demonstrate that TRIM27 acts as an oncogene regulating cell proliferation and metastasis in NSCLC through SIX3-ß-catenin signaling.

Geniposide Alleviates Oxidative Stress of Mice With Depression-Like Behaviors by Upregulating Six3os1.

Depression is a major cause of disease burden and severely impairs well-being of patients around the globe. Geniposide (GP) has been revealed to play a significant role in depression treatment. Of note, RNA sequencing of this study identified highly expressed long non-coding RNA Six3os1 in response to GP treatment. Thus, we aim to explore how GP affected chronic unpredictable mild stress (CUMS)-induced depression-like behaviors in mice in vivo and in vitro and the downstream molecular mechanism related to Six3os1. The relationship of Six3os1, miR-511-3p and Fezf1 was evaluated by dual-luciferase reporter gene assay, RIP assay, and RNA pulling down assay. Ectopic expression and knockdown experiments were developed in CUMS-induced mice and neurons with or without GP treatment. In vitro experiments and behavioral tests were conducted to examine alteration of CUMS-triggered oxidative stress following different interferences. The experimental data validated that GP treatment resulted in high expression of Six3os1 and Fezf1 and poor expression of miR-511-3p in CUMS-induced neurons. Six3os1 activated the AKT signaling pathway by upregulating miR-511-3p-targeted Fezf1. Either GP treatment or overexpression of Six3os1 or Fezf1 alleviated depression-like behaviors of CUMS-induced mice. GP treatment, miR-511-3p inhibition or overexpression of Six3os1 or Fezf1 not only reduced oxidative stress in CUMS-induced mice and neurons, but also reduced CUMS-induced neuronal apoptosis. Collectively, GP treatment-mediated Six3os1 upregulation ameliorated oxidative stress of mice with depression-like behaviors via the miR-511-3p/Fezf1/AKT axis.

SIX3 and SIX6 interact with GEMININ via C-terminal regions.

The histoarchitecture and function of eye and forebrain depend on a well-controlled balance between cell proliferation and differentiation. For example, the binding of the cell cycle regulator GEMININ to CDT1, which is a part of the pre-replication complex, promotes cell differentiation. Homeodomain transcription factors SIX3 and SIX6 also interact with GEMININ of which SIX3-GEMININ interaction promotes cell proliferation, whereas the nature of SIX6-GEMININ interaction has not been studied to date. We investigated SIX3/SIX6 and GEMININ interactions using bimolecular fluorescence complementation, surface plasmon resonance and isothermal titration calorimetry. Interactions between SIX3/SIX6 and GEMININ were detected in mammalian cells in culture. The presence of the C-terminal regions of SIX3 and SIX6 proteins, but not their SIX domains or homeodomains as previously thought, were required for interaction with GEMININ. Interestingly, the disordered C- and N- terminal regions of GEMININ were involved in binding to SIX3/SIX6. The coiled-coil region of GEMININ, which is the known protein-binding domain and also interacts with CDT1, was not involved in GEMININ-SIX3/SIX6 interaction. Using SPR and ITC, SIX3 bound GEMININ with a micromolar affinity and the binding stoichiometry was 1:2 (SIX3 - GEMININ). The present study gives new insights into the binding properties of SIX proteins, especially the role of their variable and disordered C-terminal regions.

Six3 and Six6 Are Jointly Required for the Maintenance of Multipotent Retinal Progenitors through Both Positive and Negative Regulation.

Gene regulation of multipotent neuroretinal progenitors is partially understood. Through characterizing Six3 and Six6 double knockout retinas (DKOs), we demonstrate Six3 and Six6 are jointly required for the maintenance of multipotent neuroretinal progenitors. Phenotypes in DKOs were not found in either Six3 nulls or Six6 nulls. At the far periphery, ciliary margin (CM) markers Otx1 and Cdon together with Wnt3a and Fzd1 were ectopically upregulated, whereas neuroretinal progenitor markers Sox2, Notch1, and Otx2 were absent or reduced. At the mid periphery, multi-lineage differentiation was defective. The gene set jointly regulated by Six3 and Six6 significantly overlapped with the gene networks regulated by WNT3A, CTNNB1, POU4F2, or SOX2. Stimulation of Wnt/ß-catenin signaling by either Wnt-3a or a GS3Kß inhibitor promoted CM progenitors at the cost of neuroretinal identity at the periphery of eyecups. Therefore, Six3 and Six6 together directly or indirectly suppress Wnt/ß-catenin signaling but promote retinogenic factors for the maintenance of multipotent neuroretinal progenitors.

SP8 and SP9 coordinately promote D2-type medium spiny neuron production by activating Six3 expression.

Dopamine receptor DRD1-expressing medium spiny neurons (D1 MSNs) and dopamine receptor DRD2-expressing medium spiny neurons (D2 MSNs) are the principal projection neurons in the striatum, which is divided into dorsal striatum (caudate nucleus and putamen) and ventral striatum (nucleus accumbens and olfactory tubercle). Progenitors of these neurons arise in the lateral ganglionic eminence (LGE). Using conditional deletion, we show that mice lacking the transcription factor genes Sp8 and Sp9 lose virtually all D2 MSNs as a result of reduced neurogenesis in the LGE, whereas D1 MSNs are largely unaffected. SP8 and SP9 together drive expression of the transcription factor Six3 in a spatially restricted domain of the LGE subventricular zone. Conditional deletion of Six3 also prevents the formation of most D2 MSNs, phenocopying the Sp8/9 mutants. Finally, ChIP-Seq reveals that SP9 directly binds to the promoter and a putative enhancer of Six3 Thus, this study defines components of a transcription pathway in a regionally restricted LGE progenitor domain that selectively drives the generation of D2 MSNs.

A novel long noncoding RNA lncWDR26 suppresses the growth and metastasis of hepatocellular carcinoma cells through interaction with SIX3.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Long noncoding RNAs (lncRNAs) are involved in the tumorigenesis and progression of some cancers. However, only a handful of lncRNAs have been functionally identified in HCC. In the present study, we identified a novel functional lncRNA in HCC, termed lncWDR26 (GenBank Accession no. RP11-365O16). Here, we reported that lncWDR26 was significantly downregulated in HCC tissues and cells. Moreover, decreased lncWDR26 expression correlates with larger tumor size, higher clinical stage, and tumor metastasis, and also predicts poor prognosis in patients with HCC. In HCC cells, overexpression of lncWDR26 inhibited growth and metastasis, both in vitro and in vivo. Mechanistically, lncWDR26 suppressed HCC growth and metastasis by inhibiting WDR26 transcription. Notably, lncWDR26 was associated with SIX homeobox 3 (SIX3), and this association was required for the repression of WDR26 transcription. Together, these results indicate that lncWDR26 is a tumor suppressor lncRNA that promotes tumor progression, leading us to propose that lncRNAs may serve as key regulatory hubs in HCC progression.

The Homeotic Protein SIX3 Suppresses Carcinogenesis and Metastasis through Recruiting the LSD1/NuRD(MTA3) Complex.

The homeodomain transcription factor SIX3 was recently reported to be a negative regulator of the Wnt pathway and has an emerging role in cancer. However, how SIX3 contributes to tumorigenesis and metastasis is poorly understood. METHODS: We employed affinity purification and mass spectrometry (MS) to identify the proteins physically associated with SIX3. Genome-wide analysis of the SIX3/LSD1/NuRD(MTA3) complex using a chromatin immunoprecipitation-on-chip approach identified a cohort of target genes including WNT1 and FOXC2, which are critically involved in cell proliferation and epithelial-to-mesenchymal transition. Also, we used flow cytometry, growth curve analysis, EdU incorporation assay, colony formation assays, trans-well invasion assays, immunohistochemical staining and in vivo bioluminescence assay to investigate the function of SIX3 in tumorigenesis. RESULTS: We demonstrate that the SIX3/LSD1/NuRD(MTA3) complex inhibits carcinogenesis in breast cancer cells and suppresses metastasis in breast cancer. SIX3 expression is downregulated in various human cancers and high SIX3 is correlated with improved prognosis. CONCLUSION: Our study revealed an important mechanistic link between the loss of function of SIX3 and tumor progression, identified a molecular basis for the opposing actions of MTA1 and MTA3, and may provide new potential prognostic indicators and targets for cancer therapy.

SIX3 deletions and incomplete penetrance in families affected by holoprosencephaly.

Holoprosencephaly (HPE) is failure of the forebrain to divide completely during embryogenesis. Incomplete penetrance has not been reported previously in SIX3 whole gene deletions, which are known to cause HPE. Both chromosomal microarray and whole exome sequencing (WES) were used to evaluate families with inherited HPE. Two families showed inherited deletions that contain SIX3 and were incompletely penetrant for HPE. Using WES, we ruled out parental mosaicism, a SIX3 hypomorph, and clinically significant variants in genes that are known to interact with SIX3 as causes of incomplete penetrance. We demonstrate the importance of molecular cascade testing in families with HPE and we answer important questions about incomplete penetrance.

An Eye Organoid Approach Identifies Six3 Suppression of R-spondin 2 as a Critical Step in Mouse Neuroretina Differentiation.

Recent advances in self-organizing, 3-dimensional tissue cultures of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) provided an in vitro model that recapitulates many aspects of the in vivo developmental steps. Using Rax-GFP-expressing ESCs, newly generated Six3-/- iPSCs, and conditional null Six3delta/f;Rax-Cre ESCs, we identified Six3 repression of R-spondin 2 (Rspo2) as a required step during optic vesicle morphogenesis and neuroretina differentiation. We validated these results in vivo by showing that transient ectopic expression of Rspo2 in the anterior neural plate of transgenic mouse embryos was sufficient to inhibit neuroretina differentiation. Additionally, using a chimeric eye organoid assay, we determined that Six3 null cells exert a non-cell-autonomous repressive effect during optic vesicle formation and neuroretina differentiation. Our results further validate the organoid culture system as a reliable and fast alternative to identify and evaluate genes involved in eye morphogenesis and neuroretina differentiation in vivo.