Patsie Polly

VDR-Alien: a novel, DNA-selective vitamin D3 receptor-corepressor partnership

The vitamin D receptor (VDR) is a transcription factor that transmits incoming 1,25‐dihy‐droxyvitamin D3 (1α,25(OH)2D3) signaling via combined contact with coactivator proteins and specific DNA binding sites (VDREs), which ultimately results in activation of transcription. In contrast, the mechanisms of transcriptional repression via the VDR are less well understood. This study documents VDR‐dependent transcriptional repression largely via histone deacety‐lase (HDAC) activity. Direct, ligand‐sensitive protein‐protein interaction of the VDR with the nuclear receptor corepressor (NCoR) and a novel corepressor, called Alien, was demonstrated to be comparable but independent of the VDR AF‐2 trans‐activation domain. Functional assays indicated that Alien, but not NCoR, displays selectivity for different VDRE structures for transferring these repressive effects into gene regulatory activities. Moreover, superrepression via Alien was found to be affected only in part by HDAC inhibitors such as trichostatin A. Finally, for a dissociation of VDR‐Alien complexes in vitro and in vivo, higher ligand concentrations were needed than for a dissociation of VDR‐NCoR complexes. This suggests that Alien and NCoR are using different interfaces for interaction with the VDR and different pathways for mediating superrepression, which in turn characterizes Alien as a representative of a new class of corepressors. Taken together, association of the VDR with corepressor proteins provides a further level of transcriptional regulation, which is emerging as a complex network of protein‐protein interaction‐mediated control.—Polly, P., Herdick, M., Moehren, U., Baniahmad, A., Heinzel, T., Carlberg, C. VDR‐Alien: a novel, VDR‐Alien: a novel, DNA‐selective vitamin D3 receptor‐corepressor partnership. FASEBJ. 14, 1455–1463 (2000)

Dominantly Inherited Constitutional Epigenetic Silencing of MLH1 in a Cancer-Affected Family Is Linked to a Single Nucleotide Variant within the 5′UTR

Constitutional epimutations of tumor suppressor genes manifest as promoter methylation and transcriptional silencing of a single allele in normal somatic tissues, thereby predisposing to cancer. Constitutional MLH1 epimutations occur in individuals with young-onset cancer and demonstrate non-Mendelian inheritance through their reversal in the germline. We report a cancer-affected family showing dominant transmission of soma-wide highly mosaic MLH1 methylation and transcriptional repression linked to a particular genetic haplotype. The epimutation was erased in spermatozoa but reinstated in the somatic cells of the next generation. The affected haplotype harbored two single nucleotide substitutions in tandem; c.-27C > A located near the transcription initiation site and c.85G > T. The c.-27C > A variant significantly reduced transcriptional activity in reporter assays and is the probable cause of this epimutation.

All natural DR3-type vitamin D response elements show a similar functionality in vitro

The vitamin D(3) receptor (VDR), which is the nuclear receptor for 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)], acts primarily as a heterodimer with the retinoid X receptor (RXR) and binds preferentially to directly repeated arrangements of two hexameric binding sites with three spacing nucleotides [DR3-type vitamin D response elements (VDREs)]. In this study, all presently known natural DR3-type VDREs have been compared and classified on the basis of their complex-formation with VDR-RXR heterodimers and their ability to stabilize VDR-RXR heterodimer conformations. Based on the affinity of each VDRE for VDR-RXR heterodimers, the DR3-type VDREs were divided into three classes. The ligand sensitivity of this complex-formation and conformational stabilization was determined to be in the range of 0.1 nM. No significant differences in the 1alpha,25(OH)(2)D(3)-modulated interactions of the DR3-type VDRE-complexed VDR-RXR heterodimer with the co-activator SRC-1 (steroid receptor co-activator-1) or the co-repressor NCoR (nuclear receptor co-repressor) were found. Taken together, the affinity for VDR-RXR heterodimers appears to be the major discriminating parameter between natural DR3-type VDREs. This will not only facilitate further investigation of the principles of DR3-type-VDRE-mediated gene regulation, but also strongly suggests that DR3-type VDREs alone cannot explain the pleiotropic genomic action of 1alpha,25(OH)(2)D(3).

DIFFERENTIAL APOPTOTIC RESPONSE OF HUMAN MELANOMA CELLS TO 1 ALPHA , 25-DIHYDROXYVITAMIN D3 AND ITS ANALOGUES

Pleiotropic actions of the biologically active form of vitamin D3, 1α,25-dihydroxyvitamin D3 (VD), include antiproliferative effects in both normal human melanocytes and malignant melanoma cell lines. In this study the actions of VD and its low calcemic analogues EB1089 and CB1093, have been examined in two human melanoma cell lines MeWo and WM1341. Both cell lines express similar amounts of vitamin D receptor mRNA and show functional gene regulatory effects in response to VD and its analogues. VD, EB1089 and CB1093 induced apoptosis only in WM1341 cells and not in MeWo cells, even though both cell lines responded well to etoposide, a strong inducer of apoptosis. Additionally, these results were confirmed by analysis of cell morphology. Interestingly in WM1341 cells, CB1093 was found to be more potent in inducing apoptosis than EB1089 and the natural hormone. Moreover, CB1093 appeared to induce apoptosis at a relatively low concentration of 0.1 nM, whereas greater than tenfold higher concentrations of VD and EB1089 were needed to obtain comparable effects. These observations highlight CB1093 as a promising drug for a future treatment against specific types of melanoma.

Central role of VDR conformations for understanding selective actions of vitamin D3 analogues

The vitamin D(3) receptor (VDR) acts primarily as a heterodimer with the retinoid X receptor (RXR) on different types of 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) response elements (VDREs). Therefore, DNA-bound VDR-RXR heterodimers can be considered as the molecular switches of 1alpha,25(OH)(2)D(3) signalling. Functional conformations of the VDR within these molecular switches appear to be of central importance for describing the biologic actions of 1alpha,25(OH)(2)D(3) and its analogues. Moreover, VDR conformations provide a molecular basis for understanding the potential selective profile of VDR agonists, which is critical for a therapeutic application. This review discusses VDR conformations and their selective stabilization by 1alpha,25(OH)(2)D(3) and its analogues, such as EB1089 and Gemini, as a monomer in solution or as a heterodimer with RXR bound to different VDREs and complexed with coactivator or corepressor proteins.

Identification of a vitamin D3 response element in the fibronectin gene that is bound by a vitamin D3 receptor homodimer

Fibronectin (FN) is an important adhesive noncollagenous glycoprotein involved in maintenance of the extracellular matrix and cell adhesiveness, loss of which has been implicated in the metastatic potential of cells. Regulation of FN occurs at the transcriptional level by the active metabolite of vitamin D3, 1,25‐dihydroxyvitamin D3 (1,25‐(OH)2D3). Transient transfection of homologous and heterologous promoter reporter constructs into ROS 17/2,8 (rat osteosarcoma), NIH 3T3 (mouse fibroblast), and MCF‐7 (human mammary carcinoma) cell lines showed a consistent two‐ to threefold induction of transcription when stimulated with 1,25‐(OH)2D3. These heterologous promoter transfection studies with gel shift analysis locate a third, natural DR6‐type vitamin D responsive element (VDRE) at nucleotide positions ‐171 to ‐154 in the murine FN promoter. Interestingly, this VDRE is also present in rat and human FN promoters. This study shows that 1,25‐(OH)2D3 induces FN transcription from an existing elevated basal transcriptional activity by acting through two putative hexameric core binding motifs which bind VDR homodimers. Furthermore, the FN VDRE is the first homodimer‐type VDRE that is not overlaid by a DR3‐type structure.

Effects of a Novel Long Noncoding RNA, lncUSMycN, on N-Myc Expression and Neuroblastoma Progression

BACKGROUND Patients with neuroblastoma due to the amplification of a 130-kb genomic DNA region containing the MYCN oncogene have poor prognoses. METHODS Bioinformatics data were used to discover a novel long noncoding RNA, lncUSMycN, at the 130-kb amplicon. RNA-protein pull-down assays were used to identify proteins bound to lncUSMycN RNA. Kaplan-Meier survival analysis, multivariable Cox regression, and two-sided log-rank test were used to examine the prognostic value of lncUSMycN and NonO expression in three cohorts of neuroblastoma patients (n = 47, 88, and 476, respectively). Neuroblastoma-bearing mice were treated with antisense oligonucleotides targeting lncUSMycN (n = 12) or mismatch sequence (n = 13), and results were analyzed by multiple comparison two-way analysis of variance. All statistical tests were two-sided. RESULTS Bioinformatics data predicted lncUSMycN gene and RNA, and reverse-transcription polymerase chain reaction confirmed its three exons and two introns. The lncUSMycN gene was coamplified with MYCN in 88 of 341 human neuroblastoma tissues. lncUSMycN RNA bound to the RNA-binding protein NonO, leading to N-Myc RNA upregulation and neuroblastoma cell proliferation. High levels of lncUSMycN and NonO expression in human neuroblastoma tissues independently predicted poor patient prognoses (lncUSMycN: hazard ratio [HR] = 1.87, 95% confidence interval [CI] = 1.06 to 3.28, P = .03; NonO: HR = 2.48, 95% CI = 1.34 to 4.57, P = .004). Treatment with antisense oligonucleotides targeting lncUSMycN in neuroblastoma-bearing mice statistically significantly hindered tumor progression (P < .001). CONCLUSIONS Our data demonstrate the important roles of lncUSMycN and NonO in regulating N-Myc expression and neuroblastoma oncogenesis and provide the first evidence that amplification of long noncoding RNA genes can contribute to tumorigenesis.

Glutamine repeat variants in human RUNX2 associated with decreased femoral neck BMD, broadband ultrasound attenuation and target gene transactivation.

RUNX2 is an essential transcription factor required for skeletal development and cartilage formation. Haploinsufficiency of RUNX2 leads to cleidocranial displaysia (CCD) a skeletal disorder characterised by gross dysgenesis of bones particularly those derived from intramembranous bone formation. A notable feature of the RUNX2 protein is the polyglutamine and polyalanine (23Q/17A) domain coded by a repeat sequence. Since none of the known mutations causing CCD characterised to date map in the glutamine repeat region, we hypothesised that Q-repeat mutations may be related to a more subtle bone phenotype. We screened subjects derived from four normal populations for Q-repeat variants. A total of 22 subjects were identified who were heterozygous for a wild type allele and a Q-repeat variant allele: (15Q, 16Q, 18Q and 30Q). Although not every subject had data for all measures, Q-repeat variants had a significant deficit in BMD with an average decrease of 0.7SD measured over 12 BMD-related parameters (p = 0.005). Femoral neck BMD was measured in all subjects (−0.6SD, p = 0.0007). The transactivation function of RUNX2 was determined for 16Q and 30Q alleles using a reporter gene assay. 16Q and 30Q alleles displayed significantly lower transactivation function compared to wild type (23Q). Our analysis has identified novel Q-repeat mutations that occur at a collective frequency of about 0.4%. These mutations significantly alter BMD and display impaired transactivation function, introducing a new class of functionally relevant RUNX2 mutants.

The role of vitamin D in skeletal and cardiac muscle function

Myopathy is a feature of many inflammatory syndromes. Chronic inflammation has been linked to pathophysiological mechanisms which implicate 1,25 dihydroxyvitamin D3 (1,25-(OH)2D3)-mediated signaling pathways with emerging evidence supporting a role for the vitamin D receptor (VDR) in contractile and metabolic function of both skeletal and cardiac muscle. Altered VDR expression in skeletal and cardiac muscle has been reported to result in significant effects on metabolism, calcium signaling and fibrosis in these tissues. Elevated levels of serum inflammatory cytokines, such as IL-6, TNF-α and IFNγ, have been shown to impact myogenic and nuclear receptor signaling pathways in cancer-induced cachexia. The dysregulation of nuclear receptors, such as VDR and RXRα in muscle cells, has also been postulated to result in myopathy via their effects on muscle structural integrity and metabolism. Future research directions include generating transcriptome-wide information incorporating VDR and its gene targets and using systems biology approaches to identify altered molecular networks in human tissues such as muscle. These approaches will aid in the development of novel therapeutic targeting strategies for inflammation-induced myopathies.

1α,25-Dihydroxyvitamin D3 receptor as a mediator of transrepression of retinoid signaling

The receptors for retinoic acid (RA) and for 1α,25‐dihydroxyvitamin D3 (VD), RAR, RXR, and VDR are ligand‐inducible members of the nuclear receptor superfamily. These receptors mediate their regulatory effects by binding as dimeric complexes to response elements located in regulatory regions of hormone target genes. Sequence scanning of the tumor necrosis factor‐α type I receptor (TNFαRI) gene identified a 3′ enhancer region composed of two directly repeated hexameric core motifs spaced by 2 nucleotides (DR2). On this novel DR2‐type sequence, but not on a DR5‐type RA response element, VD was shown to act through its receptor, the vitamin D receptor (VDR), as a repressor of retinoid signalling. The repression appears to be mediated by competitive protein–protein interactions between VDR, RAR, RXR, and possibly their cofactors. This VDR‐mediated transrepression of retinoid signaling suggests a novel mechanism for the complex regulatory interaction between retinoids and VD. J. Cell. Biochem. 67:287–296, 1997.