Neurofilament Light Gene Research Articles

Characterization of the mouse neurofilament light (NF-L) gene promoter by in vitro transcription

We have used in vitro transcription to access the basic sequences and factors required for the transcription of the mouse neurofilament light promoter (pNF-L) in the absence of chromatin structure. Deletion from −1.7 to −154 results in little change in NF-L promoter activity using nuclear extracts from either brain (expressing) or liver (non-expressing) tissues. Further deletion to −29 results in a gradual five-fold drop in promoter activity in both extracts. Only replacement of the entire −148 to −29 region results in a drop in NF-L promoter activity to basal levels. Thus, the NF-L promoter differs from the mouse NF heavy (NF-H) and mid-sized (NF-M) promoters in that no specific sequence within the immediate upstream NF-L promoter region (−154 to −29) appears to be responsible for enhancement or brain-specific transcription. We show that the order of strength of the three NF promoters is NF-H>NF-M>NF-L and identify sequences that can increase or reduce transcription when placed in front of heterologous NF promoters. We conclude that the NF-L promoter is a modular, weak and promiscuous promoter whose regulation differs from NF-H or NF-M. Our data suggest that chromatin structure may play an important role in the regulation of the NF-L promoter.

Run-on gene transcription in human neocortical nuclei. Inhibition by nanomolar aluminum and implications for neurodegenerative disease.

The incorporation of [alpha-32P]-uridine triphosphate into DNA transcription products was examined in short post-mortem interval (PMI) human brain neocortical nuclei (n, 22; PMI, 0.5-24 h) using run-on-gene transcription. Reverse Northern dot-blot hybridization of newly synthesized RNA against either total cDNA or Alu repetitive DNA indicated that human brain neocortical nuclei of up to 4-h PMI were efficient in incorporating radiolabel into new transcription products, after which there was a graded decline in de novo RNA biosynthetic capacity. To test the effects of 0-3000 nM concentrations of ambient aluminum on RNA polymerase I (RNAP I) and RNA polymerase II (RNAP II) transcription, dot blots containing 0.5, 1.0, 2.0, and 5.0 micrograms of DNA for (1) the human-specific Alu repetitive element (2) the neurofilament light (NFL) chain, and (3) glial fibrillary acidic protein (GFAP) were Northern hybridized against newly synthesized radiolabeled total RNA. These DNAs represent heterogeneous nuclear RNA (hnRNA), neuronal-, and glial-specific markers, respectively. We report here a dose-dependent repression in the biosynthetic capabilities of brain RNAP II in the range of 50-100 nM aluminum, deficits similar to those previously described using a rabbit neocortical nuclei transcription system and at concentrations that have been reported in Alzheimer's disease (AD) euchromatin. Transcription from RNAP II and the neuron-specific NFL gene in the presence of aluminum was found to be particularly affected. These findings support the hypothesis that brain gene transcription in the presence of trace amounts of ambient aluminum impairs mammalian brain DNA to adequately read out genetic information.

Multiple Neuron-specific Enhancers in the Gene Coding for the Human Neurofilament Light Chain

To define DNA regions involved in the neuron-specific expression of the neurofilament light (NF-L) gene, we generated transgenic mice bearing different NF-L constructs. A 4.9-kilobase human NF-L fragment including -292 base pairs of 5'-flanking sequences contained sufficient elements for nervous system expression in transgenic mice. Deletion of introns 1 and 2 from this 4.9-kilobase DNA fragment resulted in reduced levels of transgene expression in the cortex, while deletion of intron 3 had little effect. Both introns 1 and 2 could act independently as enhancers to confer neuronal expression of the basal heat shock promoter (hsp68) fused to lacZ in transgenic mice. The hNF-L basal promoter (-292 base pairs) was found to contain elements for directing neuronal expression of either the lacZ reporter gene or an intronless hNF-L construct. Sequence comparison revealed that intron 1, intron 2, and the basal human NF-L promoter all contain an ETS-like motif, CAGGA, present in a variety of genes expressed in the nervous system.

Neuronal expression of human immunodeficiency virus type 1 env proteins in transgenic mice: distribution in the central nervous system and pathological alterations.

It is now well documented that human immunodeficiency virus type 1 (HIV-1) induces encephalopathy in patients with AIDS. In vitro studies have implicated the envelope protein (gp120) as a factor which causes neuronal death. To better evaluate the role and elucidate the mechanisms of gp120 neurotoxicity, we have developed transgenic mice carrying a segment of the HIV-1 genome that expresses the viral gp160 protein under the control of the human neurofilament light gene promoter. In two separate lines of transgenic mice, the Env protein was found to be expressed in several nuclei of the brain stem and in the anterior horns of the spinal cord. The two lines showed identical patterns of Env expression. Neuropathological evaluation revealed numerous abnormal dendritic swellings in the immunostained motor neuron structures. Large and numerous neuritic swellings were also prominent in the nucleus gracilis and in the gracilis and cuneate fascicles. In addition, reactive astrocytosis was observed in several immunoreactive areas of the central nervous system. These transgenic mice offer a unique model to further investigate the role of HIV-1 Env protein in neuronal toxicity and to help elucidate the mechanisms that are involved.

Neuron Specificity of the Neurofilament Light Promoter in Transgenic Mice Requires the Presence of DNA Unwinding Elements

Three reporter genes, the chloramphenicol acetyltransferase (CAT), the lacZ, and the intronless NF-L DNA, were used to test the activity of the proximal promoter region (-292 bp) of the human neurofilament light (hNF-L) gene in transgenic mice. Surprisingly, the hNF-L/CAT construct was highly sensitive to position effect, and its expression was found at low levels in several tissues of adult transgenic mice (Beaudet, L., Charron, G., Houle, D., Tretjakoff, I. Peterson, A., and Julien, J.-P. (1992) Gene (Amst.) 116, 205-214). In contrast, the hNF-L/lacZ or the hNF-L/intronless constructs were expressed exclusively in the nervous system during embryonic development and in adult animals. The DNA sequences analysis of the different reporter genes revealed the presence of matrix attachment regions (MARs) within the 3'-untranslated regions of all three transgenes. DNA unwinding elements were found within the MARs of lacZ and hNF-L gene constructs but not in the CAT gene construct. When this element was removed from the lacZ construct, expression of the hNF-L/lacZ transgene became susceptible to position effect and was no longer tissue-specific. These results indicate that DNA unwinding elements are essential for position effect independence conferred by MARs to the hNF-L basal promoter.

The first intron of the mouse neurofilament light gene (NF-L) increases gene expression.

Neurofilament expression is developmentally and post-transcriptionally controlled. Using transient transfection assays in mouse L cells, we demonstrate that the expression of the mouse neurofilament light subunit (NF-L) is influenced by intron sequences. NF-L expression was decreased twenty fold upon deletion of the three intron sequences. Elements contained principally within a 350 bp region of intron 1 were responsible for enhanced NF-L expression. Enhancement of expression did not occur when intron I was placed 3' to a heterologous chloramphenicol acetyl transferase (CAT) gene whose expression was driven by NF-L 5' sequences. The intron enhancement of NF-L expression was not promoter-specific and also occurred with the mouse sarcoma virus (MSV) LTR promoter. These data suggest intron sequences may be important in regulating NF gene expression.

Both upstream and intragenic sequences of the human neurofilament light gene direct expression of lacZ in neurons of transgenic mouse embryos.

Initial expression of the neurofilament light gene coincides with the appearance of postmitotic neurons. To investigate the molecular mechanisms involved in neuron-specific gene expression during embryogenesis, we generated transgenic mice carrying various regions of the human neurofilament light gene (hNF-L) fused to the lacZ reporter gene. We found that 2.3 or 0.3 kb of the hNF-L promoter region directs expression of lacZ in neurons of transgenic embryos. Addition of 1.8 kb hNF-L intragenic sequences (IS) enlarges the neuronal pattern of transgene expression. The 2.3-kb hNF-L promote lacZ-IS construct contains all regulatory elements essential for both spatial and temporal expression of the hNF-L gene during embryogenesis and in the adult. The use of a heterologous promoter demonstrated that the 1.8-kb hNF-L intragenic sequences are sufficient to direct the expression of lacZ in a NF-L-specific manner both temporally and spatially during development and in the adult. We conclude that these hNF-L intragenic sequences contain cis-acting DNA regulatory elements that specify neuronal expression. Taken together, these results show that the neurofilament light gene contains separate upstream and intragenic elements, each of which directs lacZ expression in embryonic neurons.

Different posttranscriptional controls for the human neurofilament light and heavy genes in transgenic mice

To investigate the mechanisms regulating neurofilament gene expression, we generated transgenic mice with high copy number of the intact human neurofilament light (NF-L) and heavy (NF-H) genes. Overexpression in transgenic mice of NF-L mRNA from 3- to 5-fold in different regions of the central nervous system (CNS) resulted only in a mild increase of 10–50% in the levels of NF-L proteins. The failure to enhance NF-L protein content was not due to interspecies differences in posttranscriptional NF-L regulation. For instance, based on specific immunodetection, it is estimated that human NF-L proteins composed 80% of total NF-L content in the spinal cord of transgenics. In contrast to the situation with NF-L, the CNS of transgenic mice bearing multiple copies of the human NF-H gene showed comparable increases in the levels of NF-H mRNA and proteins. These results suggest that the NF-L and NF-H genes are subject to different posttranscriptional regulation in the CNS. In vivo labeling of newly synthesized proteins by injection of [ 35S]methionine in the spinal cords of normal and transgenic mice provided evidence that the posttranscriptional regulation of NF-L expression in the CNS must occur, at least in part, at the level of translation.

Intragenic regulatory elements contribute to transcriptional control of the neurofilament light gene

To date, no DNA regions involved in the neuron-specific expression of the neurofilament light gene ( NF-L) have been defined using transfection assays in cultured cells. To identify those regulatory regions in the human NF-L gene, we generated transgenic mice with a construct containing the basal NF-L promoter (−292 to +15) fused to the cat gene and with three DNA fragments of 21.5, 7.6 and 4.9 kb each, including NF-L with different lengths of either 5′- or 3′-flanking sequences. We show that the proximal NF-L 5′ region (0.3 kb) constitutes a weak promoter and that it lacks information to confer neural specificity. However, appropriate expression in the nervous system occurred when this minimal promoter was combined with either 7.3 or 4.6 kb of NF-L sequences downstream from the transcription start point. We conclude that the intragenic NF-L region contains cis-acting elements conferring cell-type-specific regulation on the basal activity of the NF-L promoter. Interestingly, AP-2 motifs were found within homologously placed introns of all three NF genes, as well as in the promoter regulatory regions of many neuronal genes. We propose that the acquisition of introns by an ancestral intronless IF gene may have contributed to the emergence of a lineage of IF genes expressed in the nervous system.

Origin of the two mRNA species for the human neurofilament light gene.

The human neurofilament light (hNF-L) gene yields two major species of mRNAs of 2.4 and 3.8 kilobases (kb) in size. To investigate the origin of these two mRNAs, we have analyzed in transgenic mice the expression of hNF-L DNA fragments including different lengths of 5'-flanking regions. The finding that the 3.8-kb mRNA species is produced by a hNF-L transgene that includes only the proximal promoter region (-0.3 kb) demonstrates that both the 2.4- and 3.8-kb mRNAs are derived from the same site of transcription initiation. Sequencing of the 3' untranslated region of the hNF-L gene revealed the presence of multiple AATAAA polyadenylation signals. We conclude from Northern blotting experiments using probes spanning various regions of the hNF-L gene that the 2.4- and 3.8-kb mRNAs originate from the selective use of polyadenylation signals located 1.4 kb apart.