DNA Sequences in the Promoter Region of the NF1 Gene Are Highly Conserved between Human and Mouse

doi:10.1006/geno.1994.1328

Genomics

Volume 21, Issue 3, June 1994, Pages 649-652

https://doi.org/10.1006/geno.1994.1328 Get rights and content

Abstract

The gene for type 1 neurofibromatosis (NF1) is most highly expressed in brain and spinal cord, although low levels of mRNA can be found in nearly all tissues. As a first step in investigating the regulation of NF1 gene expression, we have cloned and sequenced the promoter regions of the human and mouse NF1 genes and mapped the transcriptional start sites in both species. We report here that the 5′ ends of the human and murine NF1 genes are highly conserved. While no discernable TATA or CCAAT box sequences are seen, transcription initiates at identical sites in both species, 484 nucleotides upstream of the ATG initiation codon in the human gene. The human and mouse NF1 genes share particularly high sequence homology (95%) between nucleotides -33 and +261 and contain several perfectly conserved transcription factor binding site motifs, including a cAMP response element, several AP2 consensus binding sites, and a serum response element. The high conservation of these sequences indicates that they are likely to be significant in the regulation of NF1 gene expression.

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Cited by (43)

Synthetic promoter for efficient and muscle-specific expression of exogenous genes
2019, Plasmid
Citation Excerpt :
To acquire synthetic promoters with activity and specificity high enough for therapeutic application, we speculated that the combination of multiple kinds of transcriptional motifs, instead of unique myogenic factors in SPc5–12 (Li et al., 2001; Zou et al., 2018), should be a better strategy. To address this issue, three kinds of transcriptional regulatory elements were rationally determined and nineteen representative motifs were carefully selected: i) eight muscle-specific elements were SRE (Coletti et al., 2016), MEF-1, MEF-2 (Li et al., 1999), MEF-3 (Hidaka et al., 1993), TEF-1 (Wang et al., 2015), Trex (Himeda et al., 2012), MPEX (Himeda et al., 2012; Himeda et al., 2010) and MAPF (Walsh, 1989); ii) five highly conserved elements were AP-1 (Schenk et al., 1994), AP-2 (Hajra et al., 1994), GATA-2 (Dorfman et al., 1992), TATA Box (Mogno et al., 2010) and CAAT box (Kadonaga et al., 1986); iii) six virus-derived motifs were 72 bp repeat sequence from SV40 enhancer (Van Gaal et al., 2011), 19 bp repeat sequence from CMV enhancer, 16 bp, 18 bp, 19 bp and 21 bp repeat sequences from CMV promoter (Stinski, 1992;Payne et al., 2017). After gradual evaluations, the promoter SP-301 was picked out from ~1200 synthetic promoters via in vitro (Fig. 1) and in vivo expression of different reporter genes (Figs. 2,3).
Synthetic promoters (SPs) have many advantages over their natural counterparts, especially with regard to transcriptional activity and tissue specificity. Here, we report a new strategy to construct SPs for efficient and muscle-specific gene expression. First, 19 nucleic acid motifs classified to 3 kinds of transcriptional regulatory elements were rationally selected. A recombinant promoter library was constructed by randomly assembling these motifs. Second, the transcriptional activities of ~1200 SPs were screened by intramuscular expression of several reporter genes in different cell lines for activity higher than that of the cytomegalovirus (CMV) promoter, with SP-301 finally identified as the strongest. A single intramuscular injection of mice with an SP-301 plasmid expressing mouse growth hormone releasing hormone accelerated mouse growth significantly over 24 days. Third, the muscle specificity of SP-301 was confirmed in transgenic mice. Finally, in comparison with the CMV promoter, SP-301 accelerated translocation and increased the level of plasmid in the nuclei of myoblast cells to a greater extent than in non-muscle cells. Altogether, the study has provided a more rational strategy to construct efficient and tissue-specific promoters, with the promoter SP-301 exhibiting promising potential for establishing an intramuscular gene expression system for therapeutic applications.
Neurofibromatosis type i learning disabilities
2007, Learning and Memory: A Comprehensive Reference
Learning disabilities are caused by genetic mutations and environmental factors that alter the normal physiology of the brain. The resulting symptoms are broad and include delayed achievement of developmental milestones, underachievement in academic and social settings, and behavioral problems. This chapter focuses on neurofibromatosis type 1 (NF1), a disorder caused by mutations in the Nf1 gene and accompanied by specific learning disabilities. The Nf1 gene encodes a negative regulator of the ras signaling pathway, which participates in multiple aspects of neuronal function. Mutation of the Nf1 gene in the mouse causes a ras-dependent increase in inhibition, reduces plasticity in the hippocampus, and impairs memory. Through a combination of clinical, genetic, cellular, and behavioral investigations, we have begun to understand the causes of NF1 and develop novel interventions to treat its symptoms.
Analysis of somatic NF1 promoter methylation in plexiform neurofibromas and Schwann cells
2005, Cancer Genetics and Cytogenetics
Citation Excerpt :
Numerous examples exist for tumor suppressor genes being transcriptionally silenced due to hypermethylation in their promoter regions in malignant and even benign tumors [8]. The NF1 promoter has several putative regulatory elements surrounding transcription start including a cAMP response element (CRE), SP1, and AP2 sites [9] (Fig. 1). Zou et al. [10] used luciferase reporter constructs to define the minimum proximal promoter region from bp −270 to +230 relative to transcription start.
Neurofibromatosis 1 (NF1) is an autosomal dominant disorder with the characteristic feature being the neurofibroma. It is believed that both NF1 alleles must be inactivated as the first step in tumorigenesis. However, often the somatic mutations are not identified, suggesting that epigenetic changes such as methylation could account for the “second hit” in some tumors. The literature reports that the region of the NF1 promoter surrounding the transcription start site is completely unmethylated in several normal tissues and some NF1-related dermal and plexiform neurofibromas. We analyzed the methylation state of the NF1 promoter in normal Schwann cells (the cell type clonally expanded in neurofibromas) and in NF1-related plexiform tumor samples with unidentified somatic mutations. In a region of 451 bp surrounding the transcription start site, a low level of methylation was found at several specific cytosines in 12 of 18 tumor samples. Overall, epigenetic silencing through methylation does not appear to be a major mechanism for the second hit. However, this study, which analyzed the largest number of NF1-related plexiform tumors and is the first to include Schwann cell-enriched tumor cultures, detected greater methylation than in any previous reports. This suggests that methylation, especially at potential transcription factor binding sites, is moderately perturbed in some plexiform neurofibromas and should be investigated further.
The interferon consensus sequence-binding protein activates transcription of the gene encoding neurofibromin 1
2004, Journal of Biological Chemistry
Citation Excerpt :
All oligonucleotides were designed with BamHI half-sites on the ends to facilitate subcloning into plasmid vectors for sequence verification of the PCR products. Oligonucleotides used to generate truncation mutants of the human NF1 promoter were designed as follows: NF1-337 F (5′-ggatcccacttccggtggggtgtcatggc-3′); NF1-315 F (5′-catggcggcgtctcggactgtgatggctgt-3′); NF1-3′ R(5′-gtcctccccgcggccgggg-3′) (22). These primers were designed with BglII half-sites to facilitate subcloning into plasmid vectors.
Deficiency of the interferon consensus sequence-binding protein (ICSBP) is associated with increased myeloid cell proliferation in response to hematopoietic cytokines. However, previously identified ICSBP target genes do not indicate a mechanism for this “cytokine hypersen-sitivity.” In these studies, we identify the gene encoding neurofibromin 1 (Nf1) as an ICSBP target gene, by chromatin immunoprecipitation. Additionally, we find decreased Nf1 expression in bone marrow-derived myeloid cells from ICSBP–/– mice. Since Nf1 deficiency is also associated with cytokine hypersensitivity, our results suggested that NF1 is a functionally significant ICSBP target gene. Consistent with this, we find that the hyper-sensitivity of ICSBP–/– myeloid cells to granulocyte monocyte colony-stimulating factor (GM-CSF) is reversed by expression of the Nf1 GAP-related domain. We also find that treatment of ICSBP-deficient myeloid cells with monocyte colony-stimulating factor (M-CSF) results in sustained Ras activation, ERK phosphorylation, and proliferation associated with impaired Nf1 expression. These M-CSF effects are reversed by ICSBP expression in ICSBP–/– cells. Consistent with this, we find that ICSBP activates the NF1 promoter in myeloid cell line transfectants and identify an ICSBP-binding NF1 cis element. Therefore, the absence of ICSBP leads to Nf1 deficiency, impairing down-regulation of Ras activation by GM-CSF or M-CSF. These results suggest that one mechanism of increased myeloid proliferation, in ICSBP-deficient cells, is decreased NF1 gene transcription. This novel ICSBP function provides insight into regulation of myelopoiesis under normal conditions and in myeloproliferative disorders.
Methylation analysis of the neurofibromatosis type 1 (NF1) promoter in peripheral nerve sheath tumours
2004, European Journal of Cancer
Peripheral nerve sheath tumours are hallmarks of neurofibromatosis type 1 (NF1). Development of plexiform neurofibromas to malignant peripheral nerve sheath tumours (MPNST) is common. The NF1 gene promoter harbours a hypomethylated CpG island. Thus, methylation changes may be involved in the development of different types of neurofibromas and malignant transformation. We investigated NF1-associated dermal (n = 9) and plexiform neurofibromas (n = 7), MPNST (n = 5) and non-NF1 leucocyte samples (n = 20) for their methylation pattern by bisulphite genomic sequencing. We could not find global hypermethylation in the NF1 promoter in our series. Nevertheless, site-specific methylation, involving transcription factor binding sites for SP1, CRE (−10), and AP-2, was observed. One region of the 5′-UTR (untranslated region) overlapping with a putative AP-2 binding site was methylated at 30–100% in 4/20 control samples. In conclusion, we did not find hypermethylation in NF1-associated tumours. Instead, low level methylation could parallel a global genomic hypomethylation in malignancy.
Mouse models of neurofibromatosis type I: Bridging the GAP
2003, Trends in Molecular Medicine
Neurofibromatosis type I (NF1) is an autosomal dominant disorder caused by mutations in the NF1 gene, leading to a variety of abnormalities in cell growth and differentiation, and to learning disabilities. The protein encoded by NF1, neurofibromin, has several biochemical functions and is expressed in a variety of different cell populations. Hence, determination of the molecular and cellular mechanisms that underlie the different NF1 symptoms is difficult. However, studies using mouse models of NF1 are beginning to unravel the mechanisms that underlie the various symptoms associated with the disease. This knowledge will aid the development of treatments for the different pathological processes associated with NF1.

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Short CommunicationDNA Sequences in the Promoter Region of the NF1 Gene Are Highly Conserved between Human and Mouse

Abstract

Short Communication
DNA Sequences in the Promoter Region of the NF1 Gene Are Highly Conserved between Human and Mouse