NF1-L Is the DNA-binding Component of the Protein Complex at the Peripherin Negative Regulatory Element

Abstract

The peripherin gene, which encodes a neuronal-specific intermediate filament protein, is transcriptionally induced with a late time course when nerve growth factor stimulates PC12 cells to differentiate into neurons. We have defined a negative regulatory element (NRE) that has a functional role in repressing peripherin expression in undifferentiate and nonneuronal cells. Nerve growth factor-induced derepression of peripherin gene expression is associated with alterations in proteins binding to a GC-rich DNA sequence in the NRE as detected by the DNA electrophoretic mobility shift assay (EMSA). We have utilized DNA affinity chromatography to purify from rat liver a 33-kDa DNA-binding protein that specifically recognizes the NRE. Microsequencing reveals identity with NF1-L, a member of the CTF/NF-1 transcription factor family. This protein forms a single complex when incubated with the NRE probe using EMSA analysis. The more slowly migrating complexes characteristic of crude undifferentiated PC12 cell extract are reconstituted by mixing the purified protein with the flow-through from the DNA affinity column, thereby demonstrating that protein-protein interactions are involved in complex formation. Supershift experiments incubating anti-CTF-1 antibody with undifferentiated PC12 cell extract prior to EMSA analysis confirm that NF1-L, or a closely related family member, is the DNA-binding protein component of the multiprotein complex at the NRE.

Transcriptional Regulation of α1b Adrenergic Receptors (α1bAR) by Nuclear Factor 1 (NF1): a Decline in the Concentration of NF1 Correlates with the Downregulation of α1bAR Gene Expression in Regenerating Liver

“…First, SDS-PAGE of the purified protein yielded two bands with molecular masses of 32 and 34 kDa. The pattern is very similar to that observed with NF1 identified in hamster (21), chicken (52), and rat (1,47) Relative rates of transcription of the ␣ 1b AR and GAPDH genes in livers from partially hepatectomized (PH) and sham-operated (SH) rats, as measured by nuclear run-on assays. Equal amounts of radioactive mRNA transcribed in vitro by nuclei isolated from the livers of PH or SH rats were hybridized to the indicated cDNA probes (␣ 1b AR, 10 g; GAPDH, 1 g) immobilized on nitrocellulose membranes.…”

“…In earlier studies, only a single major 32-kDa polypeptide was purified from the rat liver by DNA affinity chromatography using the human albumin promoter sequence, which contains a TGGCA site (47), or the peripherin negative regulator element, which contains GGCAGGGCGCC (1), and the polypeptide was identified as NF1/L (1,47). However, when the 3-hydroxy-3-methylglutaryl-coenzyme A reductase promoter sequence (which contains TGGN 7 CCA) was coupled to the affinity column, two NF1 factors, including NF1/L and NF1/ Red1, were purified from the hamster liver (21).…”

Cell type-specific Transcriptional Activation and Suppression of the α1B Adrenergic Receptor Gene Middle Promoter by Nuclear Factor 1

“…3) Subtle variations in the nucleotide sequence of the NF1 binding site might represent another mechanism by which the cell can switch a particular NF1 isoform from a positive to a negative transcriptional regulator (37), as has been shown for other transcription factors, such as the glucocorticoid receptor, where the presence of an adenine at position 12 in the consensus glucocorticoid response element is the likely cause for a switch from positive to negative transcriptional regulation (37). 4) It has been also proposed that the C terminus of NF1 may be involved in protein-protein interactions (13), by analogy to a similar mechanism in the case of the negative regulator element of the peripherin gene (18). However, there is no published information on any specific domain of NF1 that might be involved in transcriptional repression.…”

“…NF1 has been reported to act as a transcriptional silencer for some genes, such as the genes encoding retinol-binding protein (12), 3-hydroxy-3-methylglutaryl-coenzyme A reductase (13), AP1 (14), growth hormone (15), mouse ␣2(I) collagen (16), von Willebrand factor (17), and peripherin (18), but it acts as a transcriptional activator for other genes, including the ␣-globin gene (19), human hepatitis B virus S gene (20), P53 gene (21), and the gene encoding myelin basic protein (22). The molecular basis for this duality remains unknown.…”

A Role for Nuclear Factor I in the Intrinsic Control of Cerebellar Granule Neuron Gene Expression

“…Thus, NFI-A is a critical regulator of the in vivo expression of the GABRA6 gene in the mouse cerebellum. DISCUSSION NFI interacts with the promoters for numerous cell-specific and developmentally expressed genes in a diverse range of cell types, including neurons (31)(32)(33). Furthermore, gene knockout studies have demonstrated the necessity for Nfia in CNS midline glia formation (12).…”