NF1 Transcription Research Articles

Analysis of 200 unrelated individuals with a constitutional NF1 deep intronic pathogenic variant reveals that variants flanking the alternatively spliced NF1 exon 31 [23a] cause a classical neurofibromatosis type 1 phenotype while altering predominantly NF1 isoform type II

Neurofibromatosis type 1 results from loss-of-function NF1 pathogenic variants (PVs). Up to 30% of all NF1 PVs disrupt mRNA splicing, including deep intronic variants. Here, we retrospectively investigated the spectrum of NF1 deep intronic PVs in a cohort of 8,090 unrelated individuals from the University of Alabama at Birmingham (UAB) dataset with a molecularly confirmed neurofibromatosis type 1. All variants were identified through their effect on the NF1 transcript, followed by variant characterization at the DNA-level. A total of 68 distinct variants, which were ≥ 20 nucleotides away from the closest exon–intron junction, were identified in 2.5% unrelated individuals with NF1 (200/8,090). Nine different pathogenic splice variants, identified in 20 probands, led to exonization of different parts of intron 30 [23.2] or 31 [23a]. The two major NF1 transcript isoforms, distinguished by the absence (type I) or presence (type II) of the alternatively spliced cassette exon 31 [23a], are equally expressed in blood in control individuals without NF1 or NF1-affected individuals carrying their PV not in the introns flanking exon 31 [23a]. By fragment and cloning analysis we demonstrated that the exonization of intron 31 [23a] sequences due to deep intronic PV predominantly affects the NF1 isoform II. Seven additional (likely) pathogenic NF1 deep intronic variants not observed in the UAB dataset were found by classification of 36 variants identified by a literature search. Hence, the unique list of these 75 deep intronic (likely) PVs should be included in any comprehensive NF1 testing strategy.

A human Myogenin promoter modified to be highly active in alveolar rhabdomyosarcoma drives an effective suicide gene therapy

Rhabdomyosarcoma is a rare childhood soft tissue cancer whose cells resemble poorly differentiated skeletal muscle, expressing myogenic proteins including MYOGENIN. Alveolar rhabdomyosarcoma (ARMS) accounts for ~40% of cases and is associated with a poorer prognosis than other rhabdomyosarcoma variants, especially if containing the chromosomal translocation generating the PAX3-FOXO1 hybrid transcription factor. Metastasis is commonly present at diagnosis, with a five-year survival rate of <30%, highlighting the need for novel therapeutic approaches. We designed a suicide gene therapy by generating an ARMS-targeted promoter to drive the herpes simplex virus thymidine kinase (HSV-TK) suicide gene. We modified the minimal human MYOGENIN promoter by deleting both the NF1 and MEF3 transcription factor binding motifs to produce a promoter that is highly active in ARMS cells. Our bespoke ARMS promoter driving HSV-TK efficiently killed ARMS cells in vitro, but not skeletal myoblasts. Using a xenograft mouse model, we also demonstrated that ARMS promoter-HSV-TK causes apoptosis of ARMS cells in vivo. Importantly, combining our suicide gene therapy with standard chemotherapy agents used in the treatment of rhabdomyosarcoma, reduced the effective drug dose, diminishing deleterious side effects/patient burden. This modified, highly ARMS-specific promoter could provide a new therapy option for this difficult-to-treat cancer.

Isoform-specific NF1 mRNA levels correlate with disease severity in Neurofibromatosis type 1

BackgroundNeurofibromatosis type 1 (NF1) is characterized by an extreme clinical variability both within and between families that cannot be explained solely by the nature of the pathogenic NF1 gene mutations. A proposed model hypothesizes that variation in the levels of protein isoforms generated via alternative transcript processing acts as modifier and contributes to phenotypic variability.ResultsHere we used real-time quantitative PCR to investigate the levels of two major NF1 mRNA isoforms encoding proteins differing in their ability to control RAS signaling (isoforms I and II) in the peripheral blood leukocytes of 138 clinically well-characterized NF1 patients and 138 aged-matched healthy controls. As expected, expression analysis showed that NF1 isoforms I and II levels were significantly lower in patients than controls. Notably, these differences were more evident when patients were stratified according to the severity of phenotype. Moreover, a correlation was identified when comparing the levels of isoform I mRNA and the severity of NF1 features, with statistically significant lower levels associated with a severe phenotype (i.e., occurrence of learning disability/intellectual disability, optic gliomas and/or other neoplasias, and/or cerebrovascular disease) as well as in patients with cognitive impairment.ConclusionsThe present findings provide preliminary evidence for a role of circuits controlling NF1 transcript processing in modulating NF1 expressivity, and document an association between the levels of neurofibromin isoform I mRNA and the severity of phenotype and cognitive impairment in NF1.

Methylome profiling reveals distinct alterations in phenotypic and mutational subgroups of myeloproliferative neoplasms.

Even though mutations in epigenetic regulators frequently occur in myeloproliferative neoplasms, their effects on the epigenome have not been well studied. Furthermore, even though primary myelofibrosis (PMF) has a markedly worse prognosis than essential thrombocytosis or polycythemia vera, the molecular distinctions between these subgroups are not well elucidated. We conducted the HELP (HpaII tiny fragment enriched by LM-PCR) assay to study genome-wide methylation in polycythemia vera, essential thrombocytosis, and PMF samples compared with healthy controls. We determined that polycythemia vera and essential thrombocytosis are characterized by aberrant promoter hypermethylation, whereas PMF is an epigenetically distinct subgroup characterized by both aberrant hyper- and hypomethylation. Aberrant hypomethylation in PMF was seen to occur in non-CpG island loci, showing further qualitative differences between the disease subgroups. The differentially methylated genes in polycythemia vera and essential thrombocytosis were involved predominantly in cell signaling pathways and were enriched for binding sites of GATA1 and other transcription factors. In contrast, aberrantly methylated genes in PMF were involved in inflammatory pathways and were enriched for NF1, LEF1, and other transcription factors. Within the PMF subgroup, cases with ASXL1 disruptions formed an epigenetically distinct subgroup with relatively increased methylation. Cases of myeloproliferative neoplasms (MPN) with TET2 mutations showed decreased levels of hydroxymethylation and distinct set of hypermethylated genes. In contrast, the JAK2V617F mutation did not drive epigenetic clustering within MPNs. Finally, the significance of aberrant methylation was shown by sensitivity of MPN-derived cell lines to decitabine. These results show epigenetic differences between PMF and polycythemia vera/essential thrombocytosis and reveal methylomic signatures of ASXL1 and TET2 mutations.

Epigenetic Regulation by Z-DNA Silencer Function Controls Cancer-Associated ADAM-12 Expression in Breast Cancer: Cross-talk between MeCP2 and NF1 Transcription Factor Family

A disintegrin and metalloprotease domain-containing protein 12 (ADAM-12) is upregulated in many human cancers and promotes cancer metastasis. Increased urinary level of ADAM-12 in breast and bladder cancers correlates with disease progression. However, the mechanism of its induction in cancer remains less understood. Previously, we reported a Z-DNA-forming negative regulatory element (NRE) in ADAM-12 that functions as a transcriptional suppressor to maintain a low-level expression of ADAM-12 in most normal cells. We now report here that overexpression of ADAM-12 in triple-negative MDA-MB-231 breast cancer cells and breast cancer tumors is likely due to a marked loss of this Z-DNA-mediated transcriptional suppression function. We show that Z-DNA suppressor operates by interaction with methyl-CpG-binding protein, MeCP2, a prominent epigenetic regulator, and two members of the nuclear factor 1 family of transcription factors, NF1C and NF1X. While this tripartite interaction is highly prevalent in normal breast epithelial cells, both in vitro and in vivo, it is significantly lower in breast cancer cells. Western blot analysis has revealed significant differences in the levels of these 3 proteins between normal mammary epithelial and breast cancer cells. Furthermore, we show, by NRE mutation analysis, that interaction of these proteins with the NRE is necessary for effective suppressor function. Our findings unveil a new epigenetic regulatory process in which Z-DNA/MeCP2/NF1 interaction leads to transcriptional suppression, loss of which results in ADAM-12 overexpression in breast cancer cells.

Aberrant Epigenetic and Genetic Marks Are Seen in Myelodysplastic Leukocytes and Reveal Dock4 as a Candidate Pathogenic Gene on Chromosome 7q

Myelodysplastic syndromes (MDS) are characterized by abnormal and dysplastic maturation of all blood lineages. Even though epigenetic alterations have been seen in MDS marrow progenitors, very little is known about the molecular alterations in dysplastic peripheral blood cells. We analyzed the methylome of MDS leukocytes by the HELP assay and determined that it was globally distinct from age-matched controls and was characterized by numerous novel, aberrant hypermethylated marks that were located mainly outside of CpG islands and preferentially affected GTPase regulators and other cancer-related pathways. Additionally, array comparative genomic hybridization revealed that novel as well as previously characterized deletions and amplifications could also be visualized in peripheral blood leukocytes, thus potentially reducing the need for bone marrow samples for future studies. Using integrative analysis, potentially pathogenic genes silenced by genetic deletions and aberrant hypermethylation in different patients were identified. DOCK4, a GTPase regulator located in the commonly deleted 7q31 region, was identified by this unbiased approach. Significant hypermethylation and reduced expression of DOCK4 in MDS bone marrow stem cells was observed in two large independent datasets, providing further validation of our findings. Finally, DOCK4 knockdown in primary marrow CD34(+) stem cells led to decreased erythroid colony formation and increased apoptosis, thus recapitulating the bone marrow failure seen in MDS. These findings reveal widespread novel epigenetic alterations in myelodysplastic leukocytes and implicate DOCK4 as a pathogenic gene located on the 7q chromosomal region.

Protective effect of the dopamine D3 receptor agonist (7-OH-PIPAT) against apoptosis in malignant peripheral nerve sheath tumor (MPNST) cells

Emerging evidence indicates that the dopamine D(3) receptor (D(3)R) mediates protective roles both in neuronal and non-neuronal cell lines. In a previous study we proposed that neurofibromin, a large tumor suppressor protein encoded by the neurofibromatosis type 1 gene (NF1), may increase susceptibility to apoptosis after serum deprivation in malignant peripheral nerve sheath tumor (MPNST) cells, thus acting as a proapoptotic gene. In addition, it has been observed that D(3)Rs are functionally correlated to neurofibromin. In this study, we examined whether 7-OH-PIPAT, a potent dopamine D(3)R agonist, exerts an antiapoptotic role under the same culture conditions and then correlated this effect to changes in NF1 expression. Results showed that serum deprivation caused a significant reduction of cell viability (MTT assay) both after 24 and 48 h (p<0.001). Treatment with increasing concentrations of 7-OH-PIPAT (10(-9)-10(-5) M) induced a progressive increase in cell viability both after 24 and 48 h as compared to vehicle-treated cells, with significant changes at the highest concentrations tested (10(-6) and 10(-5) M). Consistently, at the latter two concentrations, a significant reduction in oligonucleosomes formation was observed, thus suggesting an antiapoptotic role of 7-OH-PIPAT. These results were confirmed by Hoechst 33254 nuclear staining. To investigate whether these effects were correlated to changes in NF1 transcript and protein expression, quantitative real-time PCR, Western blot and immunofluorescence analyses were performed. Results demonstrated that the upregulation of NF1 transcripts and protein levels induced by serum withdrawal were remarkably attenuated by 10(-6) and 10(-5) M agonist treatment within 24 h (p<0.01 and p<0.001, respectively), whereas similar effects were observed already at a lower concentration (10(-7) M) after 48 h treatment (p<0.001). In conclusion, these results suggest that D(3)R might mediate the protective response to serum deprivation in MPNST cells through the inhibition of NF1 gene expression, further underlying a subtle role of these receptors in MPNST development.

Highly Compacted Chromatin Formed In Vitro Reflects the Dynamics of Transcription Activation In Vivo

High-order chromatin was reconstituted in vitro. This species reflects the criteria associated with transcriptional regulation in vivo. Histone H1 was determinant to formation of condensed structures, with deacetylated histones giving rise to highly compacted chromatin that approximated 30 nm fibers as evidenced by electron microscopy. Using the PEPCK promoter, we validated the integrity of these templates that were refractory to transcription by attaining transcription through the progressive action of the pertinent factors. The retinoic acid receptor binds to highly compacted chromatin, but the NF1 transcription factor binds only after histone acetylation by p300 and SWI/SNF-mediated nucleosome mobilization, reflecting the in vivo case. Mapping studies revealed the same pattern of nucleosomal repositioning on the PEPCK promoter in vitro and in vivo, correlating with NF1 binding and transcription. The reconstitution of such highly compacted "30 nm" chromatin that mimics in vivo characteristics should advance studies of its conversion to a transcriptionally active form.

Transcriptional regulation of the 5′-flanking region of the human transcription factor Sp3 gene by NF-1, c-Myb, B-Myb, AP-1 and E2F

We analyzed in detail the proximal promoter of transcription factor Sp3, which expands 281 bp from the translational start. This sequence contains putative binding sites for Sp1, NF-Y, NF-1, Myb, AP-1 and E2F transcription factors. In this work, we further explored the role of these boxes on the regulation of the Sp3 gene. Gel-shift and competition assays showed specific binding of NF-1, Myb, AP-1 and E2F. Furthermore, chromatin immunoprecipitation assays demonstrated that Sp1, Sp3, NF-Y, NF-1, c-Myb, B-Myb, c-Jun and E2F1 actually occupied the Sp3 promoter in HeLa cells. Transient transfections and luciferase assays revealed activation of the Sp3 proximal promoter upon overexpression of NF-1, c-Myb, B-Myb, c-Jun and c-Fos, and repression after overexpression of E2F/DP1. Point mutation of the binding sites for NF1, Myb, AP1 and E2F and cell incubation with specific siRNAs further confirmed the role of these transcription factors in the regulation of the Sp3 promoter. The regulation of the endogenous Sp3 gene was also observed at the mRNA level when the studied transcription factors were overexpressed or knocked down by siRNA incubation. These results help to explain the complex regulation of the Sp3 gene, which depends, at least in part, on the relative amount of Sp1, Sp3, NF-Y, NF-1, c-Myb, B-Myb, AP-1, and E2F proteins in the cell.

Transcriptional regulation of human CYP11A1 in gonads and adrenals

The CYP11A1 gene encodes the cholesterol side-chain cleavage enzyme, also termed cytochrome P450scc, which catalyzes the conversion of cholesterol to pregnenolone in the first step of steroid biosynthesis in mitochondria. The adrenal- and gonad-selective, hormonally and developmentally regulated expression of CYP11A1 is principally driven by its 2.3 kb promoter. Multiple trans-acting factors like SF-1, Sp1, AP-2, TReP-132, LBP-1b, LBP-9, AP-1, NF-1, and Ets control CYP11A1 transcription either through DNA-protein interaction with their specific cis-acting elements or through protein-protein interaction between each other, wherein SF-1 plays a central role in adrenals and testes. In addition to binding with its proximal and upstream motifs, SF-1 also physically interacts with TFIIB, CBP/p300, TReP-132, and c-Jun/AP-1 to specifically transmit the regulatory signals of cAMP. Other factors like Sp1 family members, AP-2, and LBP-1b/LBP-9 may be other factors that play a role in CYP11A1 transcription, particularly in placental cells. The TATA sequence could also contribute to tissue-specificity and hormonal regulation of CYP11A1 transcription. This article reviews recent studies focusing on adrenals and gonads.

PU.1, Interferon Regulatory Factor (IRF) 2, and the Interferon Consensus Sequence-binding Protein (ICSBP/IRF8) Cooperate to Activate NF1 Transcription in Differentiating Myeloid Cells

Nf1 (neurofibromin 1) is a Ras-GAP protein that regulates cytokine-induced proliferation of myeloid cells. In previous studies, we found that the interferon consensus sequence-binding protein (ICSBP; also referred to as interferon regulatory factor 8) activates transcription of the gene encoding Nf1 (the NF1 gene) in differentiating myeloid cells. We also found that NF1 activation requires cytokine-stimulated phosphorylation of a conserved tyrosine residue in the interferon regulatory factor (IRF) domain of ICSBP/IRF8. In this study, we found that ICSBP/IRF8 cooperates with PU.1 and interferon regulatory factor 2 to activate a composite ets/IRF-cis element in the NF1 promoter. We found that PU.1 binds directly to the NF1-cis element, and DNA-bound PU.1 interacts with IRF2, recruiting IRF2 to the cis element. This interaction requires cytokine-induced phosphorylation of specific serine residues in the PU.1 PEST domain and of a conserved tyrosine residue in the IRF domain of IRF2. We found that ICSBP/IRF8 interaction with the NF1-cis element requires pre-binding of PU.1 and IRF2. The conserved IRF domain tyrosine in ICSBP/IRF8 is required for interaction with the DNA-bound PU.1-IRF2 heterodimer. NF1 deficiency in myeloid progenitor cells results in cytokine hypersensitivity and myeloproliferation. Therefore, these studies identify a target gene for the previously observed tumor-suppressor effect of PU.1. Additionally, these studies identify a tumor-suppressor function for the "oncogenic" transcription factor, IRF2.

Gonadotroph-specific expression of the human follicle stimulating hormone β gene in transgenic mice

A paucity of in vitro models has hampered studies of molecular mechanisms of FSH subunit gene expression. Consequently, we used an in vivo transgenic strategy to map the location of regulatory elements in the cloned 10 kb human FSHβ gene. Analyses of transgenic mouse lines revealed that successive 5′ truncations of the hFSHβ promoter region to −350 bp relative to the transcriptional initiation site retained gonadotroph-specific expression and the sexually dimorphic pattern of male greater than female FSHβ mRNA levels found normally in rodent pituitary. Truncation of the 3′ flanking sequences from positions +3142 to +2138 bp relative to the translational stop codon in exon 3 resulted in a complete loss of transgene expression, suggesting the presence of critical regulatory elements mapping to the 1 kb genomic segment downstream of position +2138, in addition to the proximal 5′ promoter elements. In silico phylogenetic comparisons of mammalian FSHβ genes revealed five islands of highly conserved sequence homology corresponding precisely to the proximal 5′ promoter region, exon 2, the 5′ translated region of exon 3, and two regions at the 3′ untranslated end of exon 3 that include putative polyadenylation and transcriptional termination signals. Sequence analyses of the 5′ proximal promoter revealed the presence of several putative homeodomain binding sites as well as GATA, SMAD, AP-1, NF-1, NF-Y and steroid hormone transcription factor binding sites within the highly conserved −350 bp promoter region. Notably absent from these 5′ sequences, however, are consensus binding sites for either Egr-1 or Lim-2 transcription factors known to be critical for the gonadotroph-specific expression of the LHβ gene. These findings support the hypothesis that one of the mechanisms underlying the differential regulation of the LHβ, FSHβ, and common α-gonadotropin subunits within pituitary gonadotrophs may be differences in sequence-specific binding requirements for distinct combinations of transcription factors.

ICSBP Activates Transcription of the Gene Encoding Neurofibromin 1 in Differentiating Myeloid Cells.

Previous studies in murine models and human myeloid malignancies indicate that the interferon consensus sequence binding protein (ICSBP) functions as a leukemia tumor suppressor. Previously identified ICSBP target genes include genes encoding gp91phox and p67phox (components of the phagocyte respiratory burst oxidase), the Toll like receptor 4, and other genes involved in mediating the phagocyte inflammatory response. Transcription of these target genes involves interaction of ICSBP with PU.1 and/or interferon regulatory factor 1 (IRF1), and is restricted to mature phagocytic cells. These results predict that ICSBP-deficiency would be associated with terminal differentiation block and immune dysfunction. However, the phenotype of ICSBP −/− mice is dominated by development of a myeloproliferative disorder which evolves to acute myeloid leukemia. Therefore, we used the chromatin immuno-precipitation technique to identify novel ICSBP target genes which might be involved in the ICSBP tumor suppressor effect. We identified the gene encoding neurofibromin 1 (the NF1 gene) as an ICSBP-target-gene. We found that ICSBP activates NF1 transcription during cytokine induced differentiation of myeloid progenitor cells. Because Nf1 protein has ras-GAP activity, increased Nf1-expression down regulates cytokine-induced ras activity, resulting in proliferation arrest during myeloid cell differentiation. In the current studies, we investigate the mechanism by which ICSBP activates NF1-transcription. We find that ICSBP is part of a multi protein complex that binds to and activates a cis element in the NF1 gene. This complex also includes the ets protein PU.1 and interferon regulatory factor 2 (IRF2). We find that tyrosine phosphorylation of ICSBP in response to hematopoietic cytokines is required for assembly of this transcriptional activation complex and therefore NF1-transcription. Identification of NF1 as an ICSBP target gene represents a mechanism for ICSBP-leukemia-suppression. These studies also provide the first indication of the importance of NF1-transcriptional regulation to the control of cytokine-induced proliferation in differentiating myeloid cells.

Identification of molecular variants at the promoter region of the human α7 neuronal nicotinic acetylcholine receptor subunit gene but lack of association with schizophrenia

The human α7 neuronal nicotinic receptor subunit gene has been considered as a candidate gene for P50 sensory gating deficit in schizophrenic patients. Because P50 sensory gating deficit is a common neurophysiological dysfunction in patients with schizophrenia and schizophrenia spectrum disorders, it is conceivable to hypothesize that the human α7 neuronal nicotinic receptor subunit gene might be a susceptible gene for schizophrenia. Researchers have reported that mutations in the protein-coding sequences of the human α7 neuronal nicotinic receptor subunit gene are very rare. Therefore, we searched for mutations at the promoter region of the human α7 neuronal nicotinic receptor subunit gene and performed a genetic association study in 249 unrelated Han Chinese schizophrenic patients and 273 non-psychotic subjects from Taiwan. Two molecular variants were identified and designated g.−213G>A and g.−324A>G, respectively. The g.−213G>A variant was found to obliterate a putative NF-1 transcription factor binding site using computer analysis. One out of 249 patients was detected to be a heterozygote for this variant, but none of 273 control subjects was. The g.−324A>G variant was also very rare in both patients and control subjects, only one heterozygote of this variant was identified in 249 patients and 273 control subjects, respectively. Hence, in this study, we did not find mutations in the human α7 neuronal nicotinic receptor subunit gene that are associated with schizophrenia in our population.

Characterization of functional elements in the neurofibromatosis (NF1) proximal promoter region

An essential requirement to understand how genes contribute to genetic disease is the thorough knowledge of the transcriptional regulation of gene expression. Here, we have characterized transcription factor binding sites within the type 1 neurofibromatosis (NF1) proximal regulatory region, and addressed the molecular mechanisms that regulate NF1 transcription. Overlapping regions of the NF1 proximal promoter have been cloned and characterized for use in luciferase reporter assays. These assays have identified a 500 bp region displaying activities up to 80-fold higher than control reporter levels. Mutations at putative CRE and SP1-binding sites immediately 5' to the transcription start site have dramatic effects that lead to a 70-90% decrease in reporter activity in all cell lines tested. Gelshift assays confirm binding of CREB and SP1/KLF family members to their putative recognition sequences, and provide the first evidence identifying functional sites likely involved in regulating NF1 transcription. These assays have also revealed a putative repressor region within the NF1 promoter region corresponding to CCCTC-rich sequences between the transcription and translation start sites. This work provides new information concerning the transcriptional regulation of the NF1 gene, and is the most thorough attempt to date to map functionally relevant regions within the NF1 proximal promoter region.

Genomic organization and expression of parkin in Drosophila melanogaster.

We report here the isolation, characterization on genomic structure and expression of the D. melanogaster homolog of human parkin. The 2,122 bp parkin gene sequence contains six exons that form a 1,449 bp transcript encoding a protein of 482 amino acids. 151 bp of 5' and 112 bp of 3' untranslated regions were identified by a combination of 5'-RACE/primer extension and 3'-RACE, respectively. The 5' UTR contains three transcription initiation sites. Neither a classical TATA nor a CAAT box was found in the putative promoter sequence. However, binding sites for AhR-Arnt, AP4, NF1 and GATA transcription factors were identified. Transient transfection analysis of the 5' UTR confirmed its promoter activity in HEK 293 cells and SH-SY5Y neuronal cells using a dual luciferase reporting system. The amino acid sequence of D. melanogaster Parkin exhibits 42%, 43% and 43% identity to that of human, mouse and rat, respectively, representing a 54 kDa protein band via western blot analysis. It shows a high degree of conservation in the Ubiquitin-like domain at the N-terminus (34%), the In-Between RING finger domains (IBR, 65-69%), and the RING finger domains at the C-terminus (56-57%). The expression pattern of D. melanogaster parkin varies during the developmental stages, with the highest expression in the adult stage as measured by competitive RT-PCR. From immunostainings of the embryo, D. melanogaster parkin was expressed slightly higher in the central nervous system (brain and nerve cord) during the late embryonic stage.

N-myc oncogene expression in neuroblastoma is driven by Sp1 and Sp3

Regulation of N-myc oncogene expression is an important determinant of the biological behavior of neuroblastoma. The N-myc promoter contains several potential binding sites for transcription factors of the Sp1 family. Mutation of a CT-box motif contained within a 26 bp region required for N-myc downregulation by retinoic acid decreased basal transcriptional activity and altered DNA–protein interactions of the promoter, while mutations flanking this motif did neither. On super-shift, this region was shown to recruit Sp1 and Sp3 transcription factor proteins, while a functionally significant CT-box mutation resulted in their replacement by NF-1 transcription factor. Lysates from Drosophila S2 cells expressing exogenous Sp1, Sp3, and NF-1 proteins were able to partially mimic gel shift complexes seen with neuroblastoma nuclear extract and either wild type or mutant probes. Transient transfections of S2 cells showed that both individually and together, Sp1 and Sp3 were able to trans-activate a wild type CT-box-driven luciferase reporter construct in a dose-dependent manner. Transfection of the wild type but not mutant CT-box oligonucleotide was able to decrease endogenous N-myc expression in neuroblastoma cells. Together these results suggest that the CT-box element serves a critically functional role, and in the basal state, allows for N-myc trans-activation by Sp1 and Sp3. Moreover when mutated, the CT-box may still function as a binding motif for alternate transcription factors such as NF-1 that can allow persistent N-myc expression.

Mutational and expression analysis of the NF1 gene argues against a role as tumor suppressor in sporadic pilocytic astrocytomas.

Children with neurofibromatosis type I (NF1) have a highly increased risk for developing optic nerve gliomas. Several lines of evidence support the notion that the NF1 gene functions as tumor suppressor in these pilocytic astrocytomas and therefore it is tempting to hypothesize that the NF1 gene plays a similar role in sporadic pilocytic astrocytomas. We searched for possible mechanisms of inactivation of the NF1 gene in pilocytic astrocytomas of different locations. Protein truncation testing (PTT) did not render indication for inactivating mutations in 10 analyzed tumors. Further, loss of heterozygosity analysis revealed maintenance of heterozygosity for 3 intragenic markers in 11 informative cases. Using a real-time PCR-based assay we showed that total NF1 transcript levels are high in pilocytic astrocytomas and that the NF1 type I and type II expression ratios in pilocytic astrocytomas are comparable to ratios in normal brain tissue and high-grade gliomas. Consequently, the data presented here argue against altered NF1 gene expression and the involvement of the NF1 gene in the tumorigenesis of sporadic pilocytic astrocytomas.

Members of the Nuclear Factor 1 Family Reduce the Transcriptional Potential of the Nuclear Receptor LXRα Promoter

Expression of the LXRα nuclear receptor in liver is predicted to affect cholesterol and lipid metabolism. Here we show that a short fragment from the LXRα gene promoter spanning the region from −144 to +43 relative to the mRNA initiation site can drive transcription of a reporter gene. Under basal conditions, in vitro DNase I footprinting demonstrated interaction between nuclear proteins and an NF1 recognition site in close vicinity to the transcriptional initiation. Both supershift, mutational analyses in EMSA and transfections provided evidence that the NF1 (nuclear factor I) transcription factor interacts with the LXRα promoter. All four members of the NF1 family were found to suppress the transcriptional activity indicating a general inhibitory effect on LXRα expression. A similar regulation by NF1 was also observed when using a fragment from the LXRα promoter extending up to position −3033 therefore giving the inhibitory effect of NF1 a significant impact on LXRα gene expression.

Cloning and functional characterization of the human heterogeneous nuclear ribonucleoprotein type I promoter

We have cloned and functionally characterized a portion of the human hnRNP I (heterogeneous nuclear ribonucleoprotein type I) gene containing the promoter elements. HnRNP I is an alternative splicing modulator of tissue-specific transcripts that is expressed in three different isoforms. The DNA sequence at the transcription start site, identified by 5′-rapid amplification of cDNA ends, shows a high ‘GC’ content, lacks canonical TATA sequences and contains multiple putative Sp1 and NF1 transcription factor-binding sites, a GATA box and a CAAT box. By means of a chloramphenicol acetyltransferase reporter construct and deletion analyses, we have identified two regions between −770 bp and −206 bp that had a positive effect on expression activity in HeLa cells.