TCF7L2 acts as a molecular switch in midbrain to control mammal vocalization through its DNA binding domain but not transcription activation domain.

Vocalization is an essential medium for social signaling in birds and mammals. Periaqueductal gray (PAG) a conserved midbrain structure is believed to be responsible for innate vocalizations, but its molecular regulation remains largely unknown. Here, through a mouse forward genetic screening we identified one of the key Wnt/ß-catenin effectors TCF7L2/TCF4 controls ultrasonic vocalization (USV) production and syllable complexity during maternal deprivation and sexual encounter. Early developmental expression of TCF7L2 in PAG excitatory neurons is necessary for the complex trait, while TCF7L2 loss reduces neuronal gene expressions and synaptic transmission in PAG. TCF7L2-mediated vocal control is independent of its ß-catenin-binding domain but dependent of its DNA binding ability. Patient mutations associated with developmental disorders, including autism spectrum disorders, disrupt the transcriptional repression effect of TCF7L2, while mice carrying those mutations display severe USV impairments. Therefore, we conclude that TCF7L2 orchestrates gene expression in midbrain to control vocal production through its DNA binding but not transcription activation domain.

Long Noncoding RNAs in CNS Myelination and Disease.

Myelination by oligodendrocytes is crucial for neuronal survival and function, and defects in myelination or failure in myelin repair can lead to axonal degeneration and various neurological diseases. At present, the factors that promote myelination and overcome the remyelination block in demyelinating diseases are poorly defined. Although the roles of protein-coding genes in oligodendrocyte differentiation have been extensively studied, the majority of the mammalian genome is transcribed into noncoding RNAs, and the functions of these molecules in myelination are poorly characterized. Long noncoding RNAs (lncRNAs) regulate transcription at multiple levels, providing spatiotemporal control and robustness for cell type-specific gene expression and physiological functions. lncRNAs have been shown to regulate neural cell-type specification, differentiation, and maintenance of cell identity, and dysregulation of lncRNA function has been shown to contribute to neurological diseases. In this review, we discuss recent advances in our understanding of the functions of lncRNAs in oligodendrocyte development and myelination as well their roles in neurological diseases and brain tumorigenesis. A more systematic characterization of lncRNA functional networks will be instrumental for a better understanding of CNS myelination, myelin disorders, and myelin repair.

Developmental origins and oncogenic pathways in malignant brain tumors.

Brain tumors such as adult glioblastomas and pediatric high-grade gliomas or medulloblastomas are among the leading causes of cancer-related deaths, exhibiting poor prognoses with little improvement in outcomes in the past several decades. These tumors are heterogeneous and can be initiated from various neural cell types, contributing to therapy resistance. How such heterogeneity arises is linked to the tumor cell of origin and their genetic alterations. Brain tumorigenesis and progression recapitulate key features associated with normal neurogenesis; however, the underlying mechanisms are quite dysregulated as tumor cells grow and divide in an uncontrolled manner. Recent comprehensive genomic, transcriptomic, and epigenomic studies at single-cell resolution have shed new light onto diverse tumor-driving events, cellular heterogeneity, and cells of origin in different brain tumors. Primary and secondary glioblastomas develop through different genetic alterations and pathways, such as EGFR amplification and IDH1/2 or TP53 mutation, respectively. Mutations such as histone H3K27M impacting epigenetic modifications define a distinct group of pediatric high-grade gliomas such as diffuse intrinsic pontine glioma. The identification of distinct genetic, epigenomic profiles and cellular heterogeneity has led to new classifications of adult and pediatric brain tumor subtypes, affording insights into molecular and lineage-specific vulnerabilities for treatment stratification. This review discusses our current understanding of tumor cells of origin, heterogeneity, recurring genetic and epigenetic alterations, oncogenic drivers and signaling pathways for adult glioblastomas, pediatric high-grade gliomas, and medulloblastomas, the genetically heterogeneous groups of malignant brain tumors. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Gene Networks and Genomics Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cell Differentiation and Reversion Signaling Pathways > Cell Fate Signaling.

Rapamycin promotes the anticancer action of dihydroartemisinin in breast cancer MDA-MB-231 cells by regulating expression of Atg7 and DAPK.

There is limited knowledge regarding the influence of autophagy on the anticancer effect of dihydroartemisinin (DHA). The present study aimed to investigate this influence within human breast cancer cells. Changes in cell viability, cell cycle distribution, apoptosis and associated genes were analyzed in MDA-MB-231 cells subjected to DHA following alteration in autophagy levels; the autophagy level was decreased following autophagy-related 7 (Atg7) knockdown or increased using rapamycin. The data indicated that rapamycin had the ability to notably enhance the anticancer effect of DHA on MDA-MB-231 cells. Autophagy induction may be key in mediating the anticancer effects of DHA, and rapamycin may regulate the death-associated protein kinase via the alteration of Atg7 expression, which would influence cell apoptosis. The present study presented a novel insight into enhancing the effectiveness of future treatment regimens for breast cancer using DHA.

Transcriptional Regulator ZEB2 Is Essential for Bergmann Glia Development.

Bergmann glia facilitate granule neuron migration during development and maintain the cerebellar organization and functional integrity. At present, molecular control of Bergmann glia specification from cerebellar radial glia is not fully understood. In this report, we show that ZEB2 (aka, SIP1 or ZFHX1B), a Mowat-Wilson syndrome-associated transcriptional regulator, is highly expressed in Bergmann glia, but hardly detectable in astrocytes in the cerebellum. The mice lacking Zeb2 in cerebellar radial glia exhibit severe deficits in Bergmann glia specification, and develop cerebellar cortical lamination dysgenesis and locomotion defects. In developing Zeb2-mutant cerebella, inward migration of granule neuron progenitors is compromised, the proliferation of glial precursors is reduced, and radial glia fail to differentiate into Bergmann glia in the Purkinje cell layer. In contrast, Zeb2 ablation in granule neuron precursors or oligodendrocyte progenitors does not affect Bergmann glia formation, despite myelination deficits caused by Zeb2 mutation in the oligodendrocyte lineage. Transcriptome profiling identified that ZEB2 regulates a set of Bergmann glia-related genes and FGF, NOTCH, and TGFß/BMP signaling pathway components. Our data reveal that ZEB2 acts as an integral regulator of Bergmann glia formation ensuring maintenance of cerebellar integrity, suggesting that ZEB2 dysfunction in Bergmann gliogenesis might contribute to motor deficits in Mowat-Wilson syndrome.SIGNIFICANCE STATEMENT Bergmann glia are essential for proper cerebellar organization and functional circuitry, however, the molecular mechanisms that control the specification of Bergmann glia remain elusive. Here, we show that transcriptional factor ZEB2 is highly expressed in mature Bergmann glia, but not in cerebellar astrocytes. The mice lacking Zeb2 in cerebellar radial glia, but not oligodendrocyte progenitors or granular neuron progenitors, exhibit severe defects in Bergmann glia formation. The orderly radial scaffolding formed by Bergmann glial fibers critical for cerebellar lamination was not established in Zeb2 mutants, displaying motor behavior deficits. This finding demonstrates a previously unrecognized critical role for ZEB2 in Bergmann glia specification, and points to an important contribution of ZEB2 dysfunction to cerebellar motor disorders in Mowat-Wilson syndrome.

The cHS4 Chromatin Insulator Reduces the Rate of Retroviral Vector-Mediated Gene Dysregulation Associated with Aberrant Vector Transcription.

Integrating gammaretroviral vectors can dysregulate the expression of cellular genes through a variety of mechanisms, leading to genotoxicity and malignant transformation. Although most attention has focused on the activation of cellular genes by vector enhancers, aberrant fusion transcripts involving cellular gene sequences and vector promoters, vector splice elements, and vector transcription termination sequences have also been mechanistically associated with dysregulated expression of cellular genes. Chromatin insulators have emerged as an effective tool for reducing the frequency of vector-mediated genotoxicity and malignant transformation and have been shown to block the activation of cellular genes by vector enhancers. We report here evidence that flanking a gammaretroviral reporter vector with the cHS4 chromatin insulator also reduces the frequency of vector-mediated cellular gene dysregulation associated with aberrant vector transcripts, including vector transcription run-through and aberrant splicing. We demonstrate that the cHS4 element does not function to terminate transcription directly, implicating other mechanisms for this activity.

Hdac3 Interaction with p300 Histone Acetyltransferase Regulates the Oligodendrocyte and Astrocyte Lineage Fate Switch.

Establishment and maintenance of CNS glial cell identity ensures proper brain development and function, yet the epigenetic mechanisms underlying glial fate control remain poorly understood. Here, we show that the histone deacetylase Hdac3 controls oligodendrocyte-specification gene Olig2 expression and functions as a molecular switch for oligodendrocyte and astrocyte lineage determination. Hdac3 ablation leads to a significant increase of astrocytes with a concomitant loss of oligodendrocytes. Lineage tracing indicates that the ectopic astrocytes originate from oligodendrocyte progenitors. Genome-wide occupancy analysis reveals that Hdac3 interacts with p300 to activate oligodendroglial lineage-specific genes, while suppressing astroglial differentiation genes including NFIA. Furthermore, we find that Hdac3 modulates the acetylation state of Stat3 and competes with Stat3 for p300 binding to antagonize astrogliogenesis. Thus, our data suggest that Hdac3 cooperates with p300 to prime and maintain oligodendrocyte identity while inhibiting NFIA and Stat3-mediated astrogliogenesis, and thereby regulates phenotypic commitment at the point of oligodendrocyte-astrocytic fate decision.

Hepatoma upregulated protein expression is involved in the pathogenesis of human breast carcinogenesis.

In the last decade, the overexpression of hepatoma upregulated protein (HURP) has been reported in hepatocellular carcinoma, adrenocortical tumors and urogenital carcinoma. However, the role of HURP in breast cancer remains unknown. In the present study, a comprehensive analysis was performed to examine the HURP expression level in 43 breast cancer tumor samples and paired adjacent normal tissues. The correlation between the HURP expression level and the clinicopathological characteristics was evaluated. The role of HURP in breast cancer was investigated by quantitative polymerase chain reaction, western blot analysis and cell proliferation assays. HURP expression was found to be significantly increased in the breast cancer samples. The HURP expression level was higher in the tumors with advanced-grade metastasis and was strongly associated with tumor-node-metastasis staging (P=0.003). Transfection and cell proliferation assays suggested that the suppression of HURP expression or the interference in HURP activity in the breast cancer cells inhibited cell proliferation significantly. These data suggest that HURP is associated with the degree of malignancy and the proliferation of breast cancer. HURP could be a tumor biomarker for prognosis and a potential therapeutic drug target for human breast cancer.

Promoter hypermethylation may be an important mechanism of the transcriptional inactivation of ARRDC3, GATA5, and ELP3 in invasive ductal breast carcinoma.

Hypermethylation of promoter CpG islands represents an alternative mechanism to inactivate tumor suppressor genes. This study was to detect promoter methylation status and mRNA expression levels of ARRDC3, ELP3, GATA5, and PAX6, and to explore the association between methylation and expression in invasive ductal carcinomas (IDCs) and matched normal tissues (MNTs) from breast cancer patients. Aberrant gene methylation was observed as follows: ARRDC3 in 38.5 %, ELP3 in 73.1 %, GATA5 in 48.1 %, and PAX6 in 50.0 % of IDCs. mRNA expression of ARRDC3, ELP3, and GATA5 in IDCs showed a lower level than that in MNTs (P < 0.001, P = 0.001 and P < 0.001, respectively). For ARRDC3, both methylated and unmethylated IDCs showed significantly lower expression values compared to MNTs (P = 0.001 and P = 0.007, respectively). For ELP3 and GATA5, methylated tumors only showed significantly lower expression values compared to MNTs (P = 0.001 and P < 0.001, respectively). For ARRDC3 and GATA5, methylation was associated with their less fold change in IDCs (P = 0.049 and P = 0.020, respectively). Methylation of ARRDC3 was significantly associated with grades and lymph node status of IDCs (P = 0.036 and P = 0.002, respectively). Methylation frequency of ELP3 was higher in lymph node positive versus lymph node negative tumors (P = 0.020); whereas methylation frequency of PAX6 was lower in tumors with the ER negative samples (P = 0.025). Our data suggested that promoter hypermethylation may be an important mechanism of the transcriptional inactivation of ARRDC3, GATA5, and ELP3 in IDCs.

Effect of autophagy inhibition on cell viability and cell cycle progression in MDA­MB­231 human breast cancer cells.

Atg7 is an autophagy­related gene, and is involved in two ubiquitin­like conjugation systems in the process of autophagy. It is well established that 3­methyladenine (3Ma) is an autophagy inhibitor. The present study aimed to investigate the effect of autophagy inhibition on the cell viability and cell cycle progression of human breast cancer cells. MDA­MB­231 human breast cancer cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with high glucose, then divided into six groups. The six groups included the three fundamental groups as follows: The control group (untreated); the starvation group (high­glucose DMEM replaced with glucose­free minimal essential medium); and the starvation 3Ma group (maintained in glucose­free culture medium and treated with the autophagy inhibitor 3Ma). The three fundamental groups were further divided into Atg7 siRNA­transfected and non­transfected groups. The cell viability and apoptosis of each group was determined by MTT assay and flow cytometry. The results of the current study demonstrated that Atg7 deficiency alone had no statically significant effect on the cell viability of MDA­MB­231 human breast cancer cells, while 3Ma reduced the cell viability and its effect was potentiated by Atg7 deficiency. Atg7 deficiency was more intense than 3Ma in the promotion of apoptosis and cell arrest in G0/G1­phase in the absence of glucose and its effect was reduced by 3Ma. In conclusion, 3Ma and Atg7 may be involved in different pathways in the process of autophagy. Inhibition of autophagy may influence the cell viability and cell cycle through different pathways in MDA­MB­231 human breast cancer cells.

Transgenic wheat expressing Thinopyrum intermedium MYB transcription factor TiMYB2R-1 shows enhanced resistance to the take-all disease.

The disease take-all, caused by the fungus Gaeumannomyces graminis, is one of the most destructive root diseases of wheat worldwide. Breeding resistant cultivars is an effective way to protect wheat from take-all. However, little progress has been made in improving the disease resistance level in commercial wheat cultivars. MYB transcription factors play important roles in plant responses to environmental stresses. In this study, an R2R3-MYB gene in Thinopyrum intermedium, TiMYB2R-1, was cloned and characterized. The gene sequence includes two exons and an intron. The expression of TiMYB2R-1 was significantly induced following G. graminis infection. An in vitro DNA binding assay proved that TiMYB2R-1 protein could bind to the MYB-binding site cis-element ACI. Subcellular localization assays revealed that TiMYB2R-1 was localized in the nucleus. TiMYB2R-1 transgenic wheat plants were generated, characterized molecularly, and evaluated for take-all resistance. PCR and Southern blot analyses confirmed that TiMYB2R-1 was integrated into the genomes of three independent transgenic wheat lines by distinct patterns and the transgene was heritable. Reverse transcription-PCR and western blot analyses revealed that TiMYB2R-1 was highly expressed in the transgenic wheat lines. Based on disease response assessments for three successive generations, the significantly enhanced resistance to take-all was observed in the three TiMYB2R-1-overexpressing transgenic wheat lines. Furthermore, the transcript levels of at least six wheat defence-related genes were significantly elevated in the TiMYB2R-1 transgenic wheat lines. These results suggest that engineering and overexpression of TiMYB2R-1 may be used for improving take-all resistance of wheat and other cereal crops.

An R2R3 MYB transcription factor in wheat, TaPIMP1, mediates host resistance to Bipolaris sorokiniana and drought stresses through regulation of defense- and stress-related genes.

In this study, we report new insights into the function of a wheat (Triticum aestivum) MYB gene TaPIMP1 through overexpression and underexpression, and its underlying mechanism in wheat. Electrophoretic mobility shift and yeast-one-hybrid assays indicated that TaPIMP1 can bind to five MYB-binding sites including ACI, and activate the expression of the genes with the cis-element, confirming that TaPIMP1 is an MYB transcription activator. TaPIMP1-overexpressing transgenic wheat exhibited significantly enhanced resistance to the fungal pathogen Bipolaris sorokiniana and drought stresses, whereas TaPIMP1-underexpressing transgenic wheat showed more susceptibility to the stresses compared with untransformed wheat, revealing that TaPIMP1 positively modulates host-defense responses to B. sorokiniana and drought stresses. Microarray analysis showed that a subset of defense- and stress-related genes were up-regulated by TaPIMP1. These genes, including TaPIMP1, RD22, TLP4 and PR1a, were regulated by ABA and salicylic acid (SA). TaPIMP1-underexpressing transgenic wheat showed compromised induction of these stress-responsive genes following ABA and SA treatments. In summary, TaPIMP1, as a positive molecular linker, mediates resistance to B. sorokiniana and drought stresses by regulation of stress-related genes in ABA- and SA-signaling pathways in wheat. Furthermore, TaPIMP1 may provide a transgenic tool for engineering multiple-resistance wheat in breeding programs.

Expression of a wheat MYB gene in transgenic tobacco enhances resistance to Ralstonia solanacearum, and to drought and salt stresses.

MYB transcription factors play diverse roles in plant growth, developmental processes and stress responses. A full-length cDNA sequence of a MYB gene, namely TaPIMP1, was isolated from wheat (Triticum aestivum L.). The TaPIMP1 transcript level was significantly up-regulated by inoculation with a fungal pathogen Bipolaris sorokiniana and by drought treatment. TaPIMP1 encodes the MYB protein TaPIMP1 consisting of 323 amino acids. TaPIMP1 contains two MYB DNA binding domains (R2, R3), two putative nuclear localization sites and two putative transcription activation domains. TaPIMP1 is a new member of the R2R3-MYB transcription factor subfamily. Transient expression in onion epidermal cells of GFP fused with TaPIMP1 proved that subcellular localization of TaPIMP1 occurred in the nucleus. The TaPIMP1 gene was transferred into tobacco (Nicotiana tabacum L.) cultivar W38 by Agrobacterium-mediated transformation. After screening through PCR and RT-PCR analyses, transgenic tobacco lines expressing TaPIMP1 were identified and evaluated for pathogen resistance, and drought and salt tolerance. Compared to untransformed tobacco host plants, TaPIMP1 expressing plants displayed significantly enhanced resistance to Ralstonia solanacearum and exhibited improved tolerances to drought and salt stresses. In these transgenic lines, the activities of phenylalanine ammonia-lyase (PAL) and superoxide dismutase (SOD) were significantly increased relative to wild-type tobacco plants. The results suggested that the wheat R2R3-MYB transcription factor plays an important role in modulating responses to biotic and abiotic stresses.