D1 Promoter Research Articles

RAVL1 Activates IDD3 to Negatively Regulate Rice Resistance to Sheath Blight Disease

Sheath blight disease (ShB) has a severe impact on the production of rice. ABI3/VP1-like 1 (RAVL1) negatively regulated the rice defense mechanism against ShB, however, this regulatory mechanism is not clearly understood. In this study, we identified that indeterminate domain 3 (IDD3) was positively regulated by RAVL1. Further, chromatin immunoprecipitation (ChIP) assay, yeast one-hybrid assay and transient expression assay indicated a direct binding between RAVL1 and the IDD3 promoter region. IDD3 was ubiquitously expressed in different tissues and at different stages, and its expression was significantly enhanced by Rhizoctonia solani infection. IDD3 exhibited transcription activation activity in yeast and IDD3-GFP was found to be localized in the nucleus. IDD3 mutants exhibited no significant differences in response to ShB, while IDD3 overexpressors were more susceptible to ShB compared with wild type (WT) plants. Furthermore, IDD3 repressors were less susceptible to R. solani than WT plants. Interestingly, the expression of brassinosteroid-related genes (D2, D11 and BRI1) was lower in IDD3 repressors and higher in IDD3 overexpressors compared with WT. However, the ChIP assay revealed that IDD3 did not directly bind to the D2 and D11 promoters. Overexpression of IDD3 in BRI1 mutant d61-1 inhibited the activity of IDD3, reducing its susceptibility to ShB compared with IDD3 overexpressor and WT plants, indicating that IDD3 negatively regulated the rice defense mechanism against ShB by activating the BR signaling pathway. Thus, our analyses provided information to enhance the understanding of the rice defense mechanism against ShB.

Measurement of Cyclin D2 (CCND2) Gene Promoter Methylation in Plasma and Peripheral Blood Mononuclear Cells and Alpha-Fetoprotein Levels in Patients with Hepatitis B Virus-Associated Hepatocellular Carcinoma.

BackgroundAlpha-fetoprotein (AFP) is widely used to screen for hepatocellular carcinoma (HCC). However, the use of this biomarker has been challenged due to its low sensitivity and high rate of false negatives. In this study, we evaluated the diagnostic capability of cyclin D2 (CCND2) promoter methylation in patients with HCC related to hepatitis B virus (HBV).Material/MethodsUsing methylation-specific PCR and quantitative real-time PCR, we measured methylation status and mRNA levels of CCND2 in plasma and peripheral blood mononuclear cells (PBMCs) from 275 subjects: 75 patients with chronic hepatitis B (CHB), 47 with liver cirrhosis (LC), 118 with HCC, and 35 healthy controls (HCs).ResultsThe methylation rate of the CCND2 promoter was significantly higher in HCC patients than in patients without HCC (P<0.001). Furthermore, advanced HCC (TNM III/IV) was associated with a significantly higher frequency of CCND2 methylation and lower CCND2 mRNA levels than early-stage disease (TNM I/II; P<0.05). Combined measurement of CCND2 methylation and AFP yielded significantly higher sensitivity and area under the curve (AUC) than AFP alone in distinguishing patients with HCC from subjects with LC and CHB (P<0.001).ConclusionsCCND2 methylation may be useful for predicting HCC progression. In addition, combined measurement of CCND2 methylation and AFP could serve as a non-invasive diagnostic marker for patients with HBV-related HCC.

The NANOG Transcription Factor Induces Type 2 Deiodinase Expression and Regulates the Intracellular Activation of Thyroid Hormone in Keratinocyte Carcinomas.

Type 2 deiodinase (D2), the principal activator of thyroid hormone (TH) signaling in target tissues, is expressed in cutaneous squamous cell carcinomas (SCCs) during late tumorigenesis, and its repression attenuates the invasiveness and metastatic spread of SCC. Although D2 plays multiple roles in cancer progression, nothing is known about the mechanisms regulating D2 in cancer. To address this issue, we investigated putative upstream regulators of D2 in keratinocyte carcinomas. We found that the expression of D2 in SCC cells is positively regulated by the NANOG transcription factor, whose expression, besides being causally linked to embryonic stemness, is associated with many human cancers. We also found that NANOG binds to the D2 promoter and enhances D2 transcription. Notably, blockage of D2 activity reduced NANOG-induced cell migration as well as the expression of key genes involved in epithelial–mesenchymal transition in SCC cells. In conclusion, our study reveals a link among endogenous endocrine regulators of cancer, thyroid hormone and its activating enzyme, and the NANOG regulator of cancer biology. These findings could provide the basis for the development of TH inhibitors as context-dependent anti-tumor agents.

Social defeat stress-specific increase in c-Fos expression in the extended amygdala in mice: Involvement of dopamine D1 receptor in the medial prefrontal cortex

We recently reported that dopamine D1 receptor in the medial prefrontal cortex (mPFC) is activated by subthreshold social defeat stress and suppresses the induction of depressive-like behavior in mice. However, which mPFC projection(s) mediates this antidepressant-like effect remains poorly understood. Here we show that social defeat stress specifically increased c-Fos expression, a marker for neuronal activity, in distinct brain regions involved in emotional regulation, relative to novelty-induced exploration. Among these brain areas, D1 knockdown in the mPFC decreased social defeat stress-induced c-Fos expression in the interstitial nucleus of the posterior limb of the anterior commissure (IPAC), a subregion of the extended amygdala. Using retrograde adeno-associated virus vectors and transgenic mice expressing Cre recombinase under the D1 promoter, we also found that D1-expressing deep-layer pyramidal neurons in the mPFC send direct projections to the IPAC. These findings indicate that social defeat stress specifically activates neurons in distinct brain areas, among which the IPAC is regulated by dopamine D1 receptor in the mPFC perhaps through direct projections. Thus, this study provides hints toward identifying neural circuits that underlie antidepressant-like effects of stress-induced dopamine D1 receptor signaling in the mPFC.

The JAK2/STAT3/CCND2 Axis promotes colorectal Cancer stem cell persistence and radioresistance

BackgroundRadiotherapy (RT) is a highly effective multimodal nonsurgical treatment that is essential for patients with advanced colorectal cancer (CRC). Nevertheless, cell subpopulations displaying intrinsic radioresistance survive after RT. The reactivation of their proliferation and successful colonization at local or distant sites may increase the risk of poor clinical outcomes. Recently, radioresistant cancer cells surviving RT were reported to exhibit a more aggressive phenotype than parental cells, although the underlying mechanisms remain unclear.MethodsBy investigating public databases containing CRC patient data, we explored potential radioresistance-associated signaling pathways. Then, their mechanistic roles in radioresistance were investigated through multiple validation steps using patient-derived primary CRC cells, human CRC cell lines, and CRC xenografts.ResultsJanus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling was activated in radioresistant CRC tissues in correlation with local and distant metastases. JAK2 was preferentially overexpressed in the CRC stem cell subpopulation, which was accompanied by the phosphorylation of STAT proteins, especially STAT3. JAK2/STAT3 signaling played an essential role in promoting tumor initiation and radioresistance by limiting apoptosis and enhancing clonogenic potential. Mechanistically, the direct binding of STAT3 to the cyclin D2 (CCND2) promoter increased CCND2 transcription. CCND2 expression was required for persistent cancer stem cell (CSC) growth via the maintenance of an intact cell cycle and proliferation with low levels of DNA damage accumulation.ConclusionHerein, we first identified JAK2/STAT3/CCND2 signaling as a resistance mechanism for the persistent growth of CSCs after RT, suggesting potential biomarkers and regimens for improving outcomes among CRC patients.

Opposing roles of TCF7/LEF1 and TCF7L2 in cyclin D2 and Bmp4 expression and cardiomyocyte cell cycle control during late heart development

Bone morphogenetic protein (BMP) and Wnt pathways regulate cell proliferation and differentiation, but how these two pathways interact and mediate their nuclear actions in the heart, especially during late cardiac development, remains poorly defined. T-cell factor (TCF) and lymphoid enhancer factor (LEF) family transcriptional factors, including Lef1, Tcf7, Tcf7l1, and Tcf7l2, are important nuclear mediators of canonical Wnt/β-catenin signaling throughout cardiac development. We reveal that these TCF/LEF family members direct heart maturation through distinct temporal and spatial control. TCF7 and LEF1 decrease while TCF7L1 and TCF7L2 remain relatively stable during heart development. LEF1 is mainly expressed in mesenchymal cells in valvular regions. TCF7 and TCF7L1 are detected in the nucleus of mesothelial and endothelial cells, but not in cardiomyocytes or mesenchymal cells. Tcf7l2 is the primary TCF/LEF family member in cardiomyocytes and undergoes alternative splicing during heart development. A TCF7L2 intensity gradient opposite to that of β-catenin and cardiomyocyte proliferative activity is present in fetal hearts. Wnt activation by cardiac deletion of APC, a negative Wnt regulator, dramatically increases Cyclin D2 and Bmp4 expression. BMP signal transducing transcription factors, the mothers against decapentaplegic homologs (SMADs) are increasingly phosphorylated upon Wnt activation. LEF1/TCF7 displaces TCF7L2 and cooperates with pSMAD 1/5/8 in the regulatory elements of Cyclin D2 and Bmp4 promoters to promote β-catenin recruitment and transcriptional activation. Finally, we demonstrate that TCF7L2 is a transcriptional suppressor of Cyclin D2 and Bmp 4 in a cardiac cell line by overexpression and knockdown experiments.

The effect of early life stress on human dopamine receptor D2 (DRD2) and parvalbumin (PVALB) promoter methylation and the association with cognition

Prenatal stress (PS) contributes to depression-like behavior in the offspring. PDLIM5 is involved in the onset of mental disorders. This study is to investigate the role and mechanism of PDLIM5 in depression-like behavior of PS offspring rats.PS model was used to analyze the effects of different treatments to PS offspring rats with different sex, including PDLIM5, PDLIM5 shRNA and 5-aza-2’ -deoxycytidine (5-azaD). The depression-like behavior was assessed by the sucrose preference test (SPT) and forced swimming test (FST). The mRNA and protein expression levels of PDLIM5 in the hippocampus of PS offspring rats were detected by qRT-PCR and western blot, respectively. The methylation of PDLIM5 promoter were analyzed by bisulfite sequencing.Our data revealed that PS offspring rats showed a significant decrease in sucrose preference and a prolonged immobility time. Injection of PDLIM5 significantly improved the depression-like behavior in PS offspring rats, whereas administration of PDLIM5 shRNA aggravated it. In addition, PDLIM5 expression was decreased at the mRNA and protein levels, and the methylation level of PDLIM5 promoter was increased in hippocampus of PS male but not female offspring rats. Furthermore, microinjection of 5-azaD improved the PS induced depression-like behavior in offspring rats. Moreover, in male PS offspring rats, microinjection of 5-azaD reversed the effect of PS on PDLIM5 expression and promoter methylation.PDLIM5 can significantly improve the depression-like behavior of both male and female PS offspring rats, while the PDLIM5 promoter methylation is only observed in male PS offspring rats. Our study may provide new mechanism for the pathogenesis of depression and experimental evidence for sex-based precise treatment.

Histone deacetylases differentially regulate the proliferative phenotype of mouse bone marrow stromal and hematopoietic stem/progenitor cells

Mouse bone marrow stromal stem/progenitor cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) and Hematopoietic Stem and Progenitor Cells (HSPCs) with differential proliferative potentials were investigated for identifying epigenetic signals that can modulate their growth. In the present study, immunodepletion of granulo-monocytic (CD11b) and erythroid (Ter119) population yielded CD11b−/Ter119− cells, capable of differentiating into chondrogenic, osteogenic and adipogenic cells. Enrichment of the CD11b+ population by positive selection of multipotent stem/progenitor marker (CD133) yielded CD11b+/CD133+ cells, efficiently differentiated into hematopoietic lineages. Molecular characterization revealed the expression of BMSC and HSPC markers in CD11b−/Ter119− and CD11b+/CD133+ sorted populations, respectively. Cell expansion studies depicted a higher growth rate and percentage of proliferating cells in G2/M phase of cell cycle in BMSCs (13.9±2.9%) as compared with HSPCs (5.8±0.8%). Analysis of the HDACs gene expression revealed a differential expression pattern in BMSCs and HSPCs that modulates the cell cycle genes. Trichostatin A (TSA)-mediated HDAC inhibition led to an increased level of AcH3 and AcH4 along with cyclins B1 and D2. Chromatin immunoprecipitation revealed alleviation of HDAC2 and HDAC3 binding by TSA on cyclins B1 and D2 promoter, thereby enhancing cell proliferation. This study identifies epigenetic modulation on the proliferative potential of BMSCs and HSPCs for stem cell transplantation therapies.

Genetic background and epigenetic modifications in the core of the nucleus accumbens predict addiction-like behavior in a rat model

This study provides a demonstration in the rat of a clear genetic difference in the propensity for addiction-related behaviors following prolonged cocaine self-administration. It relies on the use of selectively bred high-responder (bHR) and low-responder (bLR) rat lines that differ in several characteristics associated with "temperament," including novelty-induced locomotion and impulsivity. We show that bHR rats exhibit behaviors reminiscent of human addiction, including persistent cocaine-seeking and increased reinstatement of cocaine seeking. To uncover potential underlying mechanisms of this differential vulnerability, we focused on the core of the nucleus accumbens and examined expression and epigenetic regulation of two transcripts previously implicated in bHR/bLR differences: fibroblast growth factor (FGF2) and the dopamine D2 receptor (D2). Relative to bHRs, bLRs had lower FGF2 mRNA levels and increased association of a repressive mark on histones (H3K9me3) at the FGF2 promoter. These differences were apparent under basal conditions and persisted even following prolonged cocaine self-administration. In contrast, bHRs had lower D2 mRNA under basal conditions, with greater association of H3K9me3 at the D2 promoter and these differences were no longer apparent following prolonged cocaine self-administration. Correlational analyses indicate that the association of H3K9me3 at D2 may be a critical substrate underlying the propensity to relapse. These findings suggest that low D2 mRNA levels in the nucleus accumbens core, likely mediated via epigenetic modifications, may render individuals more susceptible to cocaine addiction. In contrast, low FGF2 levels, which appear immutable even following prolonged cocaine exposure, may serve as a protective factor.

Essential Role of GATA2 in the Negative Regulation of Type 2 Deiodinase Gene by Liganded Thyroid Hormone Receptor β2 in Thyrotroph.

The inhibition of thyrotropin (thyroid stimulating hormone; TSH) by thyroid hormone (T3) and its receptor (TR) is the central mechanism of the hypothalamus-pituitary-thyroid axis. Two transcription factors, GATA2 and Pit-1, determine thyrotroph differentiation and maintain the expression of the β subunit of TSH (TSHβ). We previously reported that T3-dependent repression of the TSHβ gene is mediated by GATA2 but not by the reported negative T3-responsive element (nTRE). In thyrotrophs, T3 also represses mRNA of the type-2 deiodinase (D2) gene, where no nTRE has been identified. Here, the human D2 promoter fused to the CAT or modified Renilla luciferase gene was co-transfected with Pit-1 and/or GATA2 expression plasmids into cell lines including CV1 and thyrotroph-derived TαT1. GATA2 but not Pit-1 activated the D2 promoter. Two GATA responsive elements (GATA-REs) were identified close to cAMP responsive element. The protein kinase A activator, forskolin, synergistically enhanced GATA2-dependent activity. Gel-shift and chromatin immunoprecipitation assays with TαT1 cells indicated that GATA2 binds to these GATA-REs. T3 repressed the GATA2-induced activity of the D2 promoter in the presence of the pituitary-specific TR, TRβ2. The inhibition by T3-bound TRβ2 was dominant over the synergism between GATA2 and forskolin. The D2 promoter is also stimulated by GATA4, the major GATA in cardiomyocytes, and this activity was repressed by T3 in the presence of TRα1. These data indicate that the GATA-induced activity of the D2 promoter is suppressed by T3-bound TRs via a tethering mechanism, as in the case of the TSHβ gene.

Redox regulation of cardiomyocyte cell cycling via an ERK1/2 and c-Myc-dependent activation of cyclin D2 transcription

Adult mammalian cardiomyocytes have a very limited capacity to proliferate, and consequently the loss of cells after cardiac stress promotes heart failure. Recent evidence suggests that administration of hydrogen peroxide (H2O2), can regulate redox-dependent signalling pathway(s) to promote cardiomyocyte proliferation in vitro, but the potential relevance of such a pathway in vivo has not been tested. We have generated a transgenic (Tg) mouse model in which the H2O2-generating enzyme, NADPH oxidase 4 (Nox4), is overexpressed within the postnatal cardiomyocytes, and observed that the hearts of 1–3week old Tg mice pups are larger in comparison to wild type (Wt) littermate controls. We demonstrate that the cardiomyocytes of Tg mouse pups have increased cell cycling capacity in vivo as determined by incorporation of 5-bromo-2′-deoxyuridine. Further, microarray analyses of the transcriptome of these Tg mouse hearts suggested that the expression of cyclin D2 is significantly increased. We investigated the molecular mechanisms which underlie this more proliferative phenotype in isolated neonatal rat cardiomyocytes (NRCs) in vitro, and demonstrate that Nox4 overexpression mediates an H2O2-dependent activation of the ERK1/2 signalling pathway, which in turn phosphorylates and activates the transcription factor c-myc. This results in a significant increase in cyclin D2 expression, which we show to be mediated, at least in part, by cis-acting c-myc binding sites within the proximal cyclin D2 promoter. Overexpression of Nox4 in NRCs results in an increase in their proliferative capacity that is ablated by the silencing of cyclin D2. We further demonstrate activation of the ERK1/2 signalling pathway, increased phosphorylation of c-myc and significantly increased expression of cyclin D2 protein in the Nox4 Tg hearts. We suggest that this pathway acts to maintain the proliferative capacity of cardiomyocytes in Nox4 Tg pups in vivo and so delays their exit from the cell cycle after birth.

Breast cancer nodal metastasis correlates with tumour and lymph node methylation profiles of Caveolin-1 and CXCR4

DNA methylation is the best characterised epigenetic change so far. However, its role in breast cancer metastasis has not as yet been elucidated. The aim of this study was to investigate the differences between the methylation profiles characterising primary tumours and their corresponding positive or negative for metastasis lymph nodes (LN) and correlate these with tumour metastatic potential. Methylation signatures of Caveolin-1, CXCR4, RAR-β, Cyclin D2 and Twist gene promoters were studied in 30 breast cancer primary lesions and their corresponding metastasis-free and tumour-infiltrated LN with Methylation-Specific PCR. CXCR4 and Caveolin-1 expression was further studied by immunohistochemistry. Tumours were typified by methylation of RAR-β and hypermethylation of Cyclin-D2 and Twist gene promoters. Tumour patterns were highly conserved in tumour-infiltrated LN. CXCR4 and Caveolin-1 promoter methylation patterns differentiated between node-negative and metastatic tumours. Nodal metastasis was associated with tumour and lymph node profiles of extended methylation of Caveolin-1 and lack of CXCR4 hypermethylation. Immunodetection studies verified CXCR4 and Caveolin-1 hypermethylation as gene silencing mechanism. Absence of Caveolin-1 expression in stromal cells associated with tumour aggressiveness while strong Caveolin-1 expression in tumour cells correlated with decreased 7-year disease-free survival. Methylation-mediated activation of CXCR4 and inactivation of Caveolin-1 was linked with nodal metastasis while intratumoral Caveolin-1 expression heterogeneity correlated with disease progression. This evidence contributes to the better understanding and, thereby, therapeutic management of breast cancer metastasis process.

Differential Synaptic and Extrasynaptic Glutamate-Receptor Alterations in Striatal Medium-Sized Spiny Neurons of Aged YAC128 Huntington's Disease Mice.

Huntington’s disease (HD) is a late-onset, slowly progressing neurodegenerative disorder caused by an expansion of glutamine repeats. The YAC128 mouse model has been widely used to study the progression of HD symptoms, but little is known about synaptic alterations in very old animals. The present experiments examined synaptic properties of striatal medium-sized spiny neurons (MSNs) in 16 month-old YAC128 mice. These mice were crossed with mice expressing enhanced green fluorescent protein (EGFP) under the control of either D1 or D2 dopamine receptor promoters to identify MSNs originating the direct and indirect pathways, respectively. The input-output curves of evoked excitatory postsynaptic currents mediated by activation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or N-methyl-D-aspartate (NMDA) receptors were reduced in MSNs in both pathways. In the presence of DL-threo-β-Benzyloxyaspartic acid (DL-TBOA), a glutamate transporter blocker used to increase activation of extrasynaptic receptors, NMDA receptor-mediated currents displayed altered amplitudes, longer decay times, and greater charge (response areas) in both direct and indirect pathway MSNs in YAC128 mice compared to wildtype controls. Amplitudes were significantly increased, primarily in direct pathway MSNs while normalized areas were significantly increased only in indirect pathway MSNs, suggesting that the two types of MSNs are affected in different ways. It may be that indirect pathway neurons are more susceptible to changes in glutamate transport. Taken together, the present findings demonstrate differential alterations in synaptic versus extrasynaptic NMDA receptors in both direct and indirect pathway MSNs in late HD, which may contribute to the dysfunction and degeneration in both pathways.

V. Molecular classification and risk stratification of myeloma

Multiple myeloma (MM) cells are the malignant counterpart of post-germinal centre (GC) long-lived plasma cells (PCs), characterized by strong bone marrow (BM) dependence, somatic hypermutation of immunoglobulin (Ig) genes and isotype class-switch resulting in absence of IgM expression in all but 1% of tumours. However, MM cells differ from healthy PCs because they retain the potential for a low rate of proliferation (1–3% of cycling cells). Virtually every case of MM is preceded by a premalignant PC tumour called monoclonal gammopathy of undetermined significance (MGUS) that, like MM, produces a typical M-spike (almost always non-IgM) by serum protein electrophoresis (SPEP) or free light chain in the urine [1].

Sa1681 Inhibition of the Prostaglandin EP1 Receptor Suppresses Colon Cancer Cell Invasion and Metastasis.

Background: Kruppel-like factor 5 (KLF5) is a zinc-finger transcription factor that is highly expressed in the crypt cells of the intestinal epithelia and plays a critical role in regulating proliferation of both normal intestinal epithelial cells and colorectal cancer cells. We previously showed that KLF5 is a critical mediator for intestinal tumorigenesis resulted from either ApcMin mutation or oncogenic KRAS activation (McConnell et al. [2009] Cancer Res. 69:4125, Nandan et al. [2010] Molecular Cancer 9:63, respectively). KLF5 is regulated at the transcriptional, translational, and post-translational level. Previous studies indicate that the protein kinase GSK3β and E3 ubiquitin ligase FBW7 facilitate proteosomal degradation of KLF5 protein via phosphorylation (GSK3β) and recognition (FBW7) of a phosphodegron sequence surrounding Ser303 in human KLF5. A recent genomic analysis of colorectal cancer tissues identified a somatic mutation (P301S) in KLF5 within the phosphodegron sequence (Sjoblom et al. [2006] Science 314:268). Aim: To investigate the impact of P301S mutation on KLF5 protein stability. Methods: Plasmids containing human wildtype (WT) and mutant (S303A, P301S, P301A, and P301E) KLF5 under the control of the CMV promoter were transiently transfected into HEK293 cells. The effect of FBW7 on the stability of KLF5 produced from the various constructs was investigated by cycloheximide chase and Western blot analysis. Transcriptional activity of various KLF5 constructs was examined by luciferase reporter assay with Cyclin D1 and CDC2 reporter plasmids. Results: All four KLF5 mutants examined are more resistant to FBW7-mediated proteosomal degradation as compared to WT KLF5. Proteasome inhibition using MG132 resulted in the accumulation of WT KLF5 but had little impact on P301S KLF5 protein levels. Co-immunoprecipitation experiments show that, in contrast to WT KLF5, P301S and S303A KLF5 do not interact with FBW7. Concomitantly, the P301S KLF5 mutant displayed reduced levels of phosphorylation at Ser303 in comparison with WT KLF5. Furthermore, luciferase assays using Cyclin D1 and CDC2 promoter constructs demonstrate significantly elevated transcriptional activity of P301S KLF5 as compared to WT KLF5. Conclusion: Our data indicate that residue 301 of KLF5 is critical for proper recognition of the phosphodegron sequence. Furthermore, we show that the colon cancer-derived P301S mutation in KLF5 inhibits FBW7 recognition of the phosphodegron, yielding a degradation-resistant KLF5 protein, elevated protein levels, and enhanced KLF5 transcriptional activity. These results suggest that the P301S mutation in KLF5 is potentially oncogenic in colon cancer.

Regulation of thyroid hormone sensitivity by differential expression of the thyroid hormone receptor during Xenopus metamorphosis

During amphibian metamorphosis, a series of dynamic changes occur in a predetermined order. Hind limb morphogenesis begins in response to low levels of thyroid hormone (TH) in early prometamorphosis, but tail muscle cell death is delayed until climax, when TH levels are high. It takes about 20 days for tadpoles to grow from early prometamorphosis to climax. To study the molecular basis of the timing of tissue-specific transformations, we introduced thyroid hormone receptor (TR) expression constructs into tail muscle cells of Xenopus tadpoles. The TR-transfected tail muscle cells died upon exposure to a low level of thyroxine (T4). This cell death was suggested to be mediated by type 2 iodothyronine deiodinase (D2) that converts T4 to T3-the more active form of TH. D2 mRNA was induced in the TR-overexpressing cells by low levels of TH. D2 promoter contains a TH-response element (TRE) with a lower affinity for TR. These results show that the TR transfection confers the ability to respond to physiological concentrations of TH at early prometamorphosis to tail muscle cells through D2 activity and promotes TH signaling. We propose the positive feedback loop model to amplify the cell's ability to respond to low levels of T4.

Promoter de-methylation of cyclin D2 by sulforaphane in prostate cancer cells

Sulforaphane (SFN), an isothiocyanate derived from cruciferous vegetables, induces potent anti-proliferative effects in prostate cancer cells. One mechanism that may contribute to the anti-proliferative effects of SFN is the modulation of epigenetic marks, such as inhibition of histone deacetylase (HDAC) enzymes. However, the effects of SFN on other common epigenetic marks such as DNA methylation are understudied. Promoter hyper-methylation of cyclin D2, a major regulator of cell cycle, is correlated with prostate cancer progression, and restoration of cyclin D2 expression exerts anti-proliferative effects on LnCap prostate cancer cells. Our study aimed to investigate the effects of SFN on DNA methylation status of cyclin D2 promoter, and how alteration in promoter methylation impacts cyclin D2 gene expression in LnCap cells. We found that SFN significantly decreased the expression of DNA methyltransferases (DNMTs), especially DNMT1 and DNMT3b. Furthermore, SFN significantly decreased methylation in cyclin D2 promoter regions containing c-Myc and multiple Sp1 binding sites. Reduced methlyation of cyclin D2 promoter corresponded to an increase in cyclin D2 transcript levels, suggesting that SFN may de-repress methylation-silenced cyclin D2 by impacting epigenetic pathways. Our results demonstrated the ability of SFN to epigenetically modulate cyclin D2 expression, and provide novel insights into the mechanisms by which SFN may regulate gene expression as a prostate cancer chemopreventive agent.

Regulation of sonic hedgehog-GLI1 downstream target genes PTCH1, Cyclin D2, Plakoglobin, PAX6 and NKX2.2 and their epigenetic status in medulloblastoma and astrocytoma

BackgroundThe Sonic hedgehog (Shh) signaling pathway is critical for cell growth and differentiation. Impairment of this pathway can result in both birth defects and cancer. Despite its importance in cancer development, the Shh pathway has not been thoroughly investigated in tumorigenesis of brain tumors. In this study, we sought to understand the regulatory roles of GLI1, the immediate downstream activator of the Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6 in medulloblastoma and astrocytic tumors.MethodsWe silenced GLI1 expression in medulloblastoma and astrocytic cell lines by transfection of siRNA against GLI1. Subsequently, we performed RT-PCR and quantitative real time RT-PCR (qRT-PCR) to assay the expression of downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We also attempted to correlate the pattern of expression of GLI1 and its regulated genes in 14 cell lines and 41 primary medulloblastoma and astrocytoma tumor samples. We also assessed the methylation status of the Cyclin D2 and PTCH1 promoters in these 14 cell lines and 58 primary tumor samples.ResultsSilencing expression of GLI1 resulted up-regulation of all target genes in the medulloblastoma cell line, while only PTCH1 was up-regulated in astrocytoma. We also observed methylation of the cyclin D2 promoter in a significant number of astrocytoma cell lines (63%) and primary astrocytoma tumor samples (32%), but not at all in any medulloblastoma samples. PTCH1 promoter methylation was less frequently observed than Cyclin D2 promoter methylation in astrocytomas, and not at all in medulloblastomas.ConclusionsOur results demonstrate different regulatory mechanisms of Shh-GLI1 signaling. These differences vary according to the downstream target gene affected, the origin of the tissue, as well as epigenetic regulation of some of these genes.

Evidence of a role for the novel zinc-finger transcription factor ZKSCAN3 in modulating Cyclin D2 expression in multiple myeloma

Dysregulation of cyclin D2 contributes to the pathogenesis of multiple myeloma, and can occur through translocations that activate MAF/MAFB or MMSET/FGFR3. However, cyclin D2 induction can also be seen in the absence of such translocations, such as in patients with hyperdiploid disease, through unknown mechanisms. In UniGene cluster data-mining and ECgene analysis, we found that zinc-finger with KRAB and SCAN domains 3 (ZKSCAN3), a novel transcription factor, is overrepresented in this malignancy, and three consensus ZKSCAN3 binding sites were found in the cyclin D2 promoter. Analysis of a panel of myeloma cell lines, primary patient samples and datasets from Oncomine and the Multiple Myeloma Genomics Portal (MMGP) revealed expression of ZKSCAN3 messenger RNA (mRNA) in a majority of samples. Studies of cell lines by western blotting, and of primary tissue microarrays by immunohistochemistry, showed ZKSCAN3 protein expression in a majority, and in a manner that paralleled messenger levels in cell lines. ZKSCAN3 overexpression was associated with increased gene copy number or genomic DNA gain/amplification in a subset based on analysis of data from the MMGP, and from fluorescence in situ hybridization studies of cell lines and primary samples. Overexpression of ZKSCAN3 induced cyclin D2 promoter activity in a MAF/MAFB-independent manner, and to an extent that was influenced by the number of consensus ZKSCAN3 binding sites. Moreover, ZKSCAN3 protein expression correlated with cyclin D2 levels in cell lines and primary samples, and its overexpression induced cyclin D2. Conversely, ZKSCAN3 suppression using small hairpin RNAs (shRNAs) reduced cyclin D2 levels, and, importantly, inhibited myeloma cell line proliferation. Finally, ZKSCAN3 was noted to specifically bind to oligonucleotides representing sequences from the cyclin D2 promoter, and to the endogenous promoter itself in myeloma cells. Taken together, the data support the conclusion that ZKSCAN3 induction represents a mechanism by which myeloma cells can induce cyclin D2 dysregulation, and contribute to disease pathogenesis.

Epigenetic changes play critical role in age‐associated dysfunctions of the liver

CCAAT/Enhancer Binding Proteins family proteins are important regulators of liver functions. Here, we show the critical role of C/EBPα-mediated chromatin remodeling in the age-associated dysfunctions of the liver and in the maintenance of physiological homeostasis. Because ph-S193 isoform of C/EBPα is increased in livers of old mice, we have generated C/EBPα-S193D knockin mice, which mimic the ph-S193 isoform of C/EBPα. Analyses of these mice showed that the S193D mutation causes chromatin remodeling leading to histological appearance of 'foci-like' nodules, which are also observed in livers of old mice. These 'foci-like' structures contain K9 trimethylated histone H3, a marker of heterochromatin. The increase of heterochromatin regions in S193D mice correlates with the elevation of S193D-C/EBPα-HDAC1 complexes and with dys-regulation of gene expression including epigenetic silencing of cyclin D1 and D2 promoters and the inhibition of liver proliferation. The elimination of C/EBPα-HDAC1 complexes in S193D mice by inhibition of HDAC1 corrects chromatin structure and normalizes expression of cyclin D1 and D2. We found that epigenetic dys-regulation is also associated with the elevation of C/EBPβ and with the increase of C/EBPα/β heterodimers in S193D mice. The C/EBPα/β heterodimers activate transcription of Glut4 and increase the levels of Glut4. As the result, S193D livers have accelerated uptake of glucose and accumulation of glycogen in the liver. Thus, this study demonstrates that the phosphorylation of C/EBPα at S193 leads to the appearance of heterochromatin regions, which correlates with the development of age-related dysfunctions of the liver.