Structure, chromosome mapping and regulation of the mouse zinc-finger gene Krox-24; evidence for a common regulatory pathway for immediate-early serum-response genes

doi:10.1016/0378-1119(89)90296-5

Gene

Volume 80, Issue 2, 15 August 1989, Pages 325-336

https://doi.org/10.1016/0378-1119(89)90296-5 Get rights and content

Abstract

The structure of Krox-24, a mouse zinc-finger-encoding gene that is transiently activated during G0/G1 transition, has been established. Krox-24 is located on mouse chromosome 18, bands C-D. The gene product, as anticipated for a putative DNA-binding protein, is localized within the cell nucleus. The Krox-24 5'-flanking region contains a series of serum response elements (SREs) similar to the SRE observed upstream of the c-fos proto-oncogene. These elements can substitute for the c-fos SRE, their effect is cumulative and they bind the same cellular factor, the serum response factor (SRF), as the c-fos SRE. This suggests that the SRE and its cognate protein are likely to be involved in the regulation of Krox-24 and presumably of other immediate-early serum response genes. SRE and SRF therefore constitute key components in the regulatory pathway leading from mitogenic stimulation to cellular proliferation.

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We are interested in examining the sequence in which the different modifications of histones are acquired in the different nucleosomes, as well as to study the structural outcome of remodeling of individual nucleosomes during transcription. To do this we have selected the murine Egr1 gene, an immediate-early gene (26), which codes for a zinc finger transcriptional factor. We have previously found that this gene is rapidly activated in response to the treatment of MLP29 mouse progenitor hepatocyte cells with phorbol esters, and we have determined the time course of factor assembly in its promoter.
Histone post-translational modifications and nucleosome remodeling are coordinate events involved in eukaryotic transcriptional regulation. There are relatively few data on the time course with which these events occur in individual nucleosomes. As a contribution to fill this gap, we first describe the nature and time course of structural changes in the nucleosomes −2, −1, and +1 of the murine Egr1 gene upon induction. To initiate the transient activation of the gene, we used the stimulation of MLP29 cells with phorbol esters and the in vivo activation after partial hepatectomy. In both models, nucleosomes −1 and +1 are partially evicted, whereas nucleosomes +1 and −2 slide downstream during transcription. The sliding of the latter nucleosome allows the EGR1 protein to bind its site, resulting in the repression of the gene. To decide whether EGR1 is involved in the sliding of nucleosome −2, Egr1 was knocked down. In the absence of detectable EGR1, the nucleosome still slides and remains downstream longer than in control cells, suggesting that the product of the gene may be rather involved in the returning of the nucleosome to the basal position. Moreover, the presence of eight epigenetic histone marks has been determined at a mononucleosomal level in that chromatin region. H3S10phK14ac, H3K4me3, H3K9me3, and H3K27me3 are characteristic of nucleosome +1, and H3K9ac and H4K16ac are mainly found in nucleosome −1, and H3K27ac predominates in nucleosomes −2 and −1. The temporal changes in these marks suggest distinct functions for some of them, although changes in H3K4me3 may result from histone turnover.Chromatin structure and histone modifications regulate transcription in eukaryotes.
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Remodeling mechanisms and histone modifications are specific for each nucleosome.
Mononucleosomal level studies give unique information on chromatin functions.
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HEK293 cells were grown in Dulbecco's modified Eagle's medium containing 5% fetal calf serum and supplemented with glutamine. The plasmids used were 1) bicistronic vectors (pIRES) expressing Ha-Ras (Val-12), Ki-Ras (Val-12), and green fluorescent protein (GFP) genes under the control of the cytomegalovirus (CMV) promoter (1); 2) Ki-Ras (Val-12) mutants in the cysteine of the CAAX box and the polybasic region previously described (1, 2); 3) Ki-Ras4B (wild type), including 100 nucleotides from the 3′-untranslated region (K-100); 4) pNGF1-A-CAT, containing sequences from −1150 to +200 bp relative to the NGF1-A transcription start site (14) fused to the CAT gene (kindly provided by A. Levi, Institute of Neurobiology, CNR, Rome, Italy); 5) wild type rat MnSOD (accession number NM_017051), cloned in pcDNA 3.0 and MnSOD S82A mutant, generated by using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) (the primers used for rat MnSOD Ser82Ala mutagenesis were forward (5′-ACAAACCTGGCCCCTAAGGGT-3′) and reverse (5′-ACCCTTAGGGGCCAGGTTTGT-3′)); 6) wild type Ki-Ras4B and Ki-Ras mutants fused to the COOH terminus of enhanced cyan fluorescent protein (ECFP).3 To obtain the constructs expressing the protein chimera ECFP:Ki-Ras4B and the Cys− and Lys− mutants, the ECFP coding sequence was amplified by PCR from the pECFP-N1 (Invitrogen) and subcloned in pcDNA 3.1 vector.
Nerve growth factor (NGF) induces terminal differentiation in PC12, a pheochromocytoma-derived cell line. NGF binds a specific receptor on the membrane and triggers the ERK1/2 cascade, which stimulates the transcription of neural genes. We report that NGF significantly affects mitochondrial metabolism by reducing mitochondrial-produced reactive oxygen species and stabilizing the electrochemical gradient. This is accomplished by stimulation of mitochondrial manganese superoxide dismutase (MnSOD) both transcriptionally and post-transcriptionally via Ki-Ras and ERK1/2. Activation of MnSOD is essential for completion of neuronal differentiation because 1) expression of MnSOD induces the transcription of a neuronal specific promoter and neurite outgrowth, 2) silencing of endogenous MnSOD by small interfering RNA significantly reduces transcription induced by NGF, and 3) a Ki-Ras mutant in the polylysine stretch at the COOH terminus, unable to stimulate MnSOD, fails to induce complete differentiation. Overexpression of MnSOD restores differentiation in cells expressing this mutant. ERK1/2 is also downstream of MnSOD, as a SOD mimetic drug stimulates ERK1/2 with the same kinetics of NGF and silencing of MnSOD reduces NGF-induced late ERK1/2. Long term activation of ERK1/2 by NGF requires SOD activation, low levels of hydrogen peroxide, and the integrity of the microtubular cytoskeleton. Confocal immunofluorescence shows that NGF stimulates the formation of a complex containing membrane-bound Ki-Ras, microtubules, and mitochondria. We propose that active NGF receptor induces association of mitochondria with plasma membrane. Local activation of ERK1/2 by Ki-Ras stimulates mitochondrial SOD, which reduces reactive oxygen species and produces H₂O₂. Low and spatially restricted levels of H₂O₂ induce and maintain long term ERK1/2 activity and ultimately differentiation of PC12 cells.
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Formins, proteins defined by the presence of an FH2 domain and their ability to nucleate linear F-actin de novo, play a key role in the regulation of the cytoskeleton. Initially thought to primarily regulate actin, recent studies have highlighted a role for formins in the regulation of microtubule dynamics, and most recently have uncovered the ability of some formins to coordinate the organization of both the microtubule and actin cytoskeletons. While biochemical analyses of this family of proteins have yielded many insights into how formins regulate diverse cytoskeletal reorganizations, we are only beginning to appreciate how and when these functional properties are relevant to biological processes in a developmental or organismal context. Developmental genetic studies in fungi, Dictyostelium, vertebrates, plants and other model organisms have revealed conserved roles for formins in cell polarity, actin cable assembly and cytokinesis. However, roles have also been discovered for formins that are specific to particular organisms. Thus, formins perform both global and specific functions, with some of these roles concurring with previous biochemical data and others exposing new properties of formins. While not all family members have been examined across all organisms, the analyses to date highlight the significance of the flexibility within the formin family to regulate a broad spectrum of diverse cytoskeletal processes during development.
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Citation Excerpt :
Dopamine agonists can stimulate an array of IEGs from at least two gene families (Cole et al., 1992; Graybiel et al., 1990; Moratalla et al., 1992, 1993; Young et al., 1991). Further, the known genes of these families have different collections of upstream elements in their promotors and their products are capable of acting in different combinatorial patterns (Janssen-Timmen et al., 1989; Treisman, 1995; Tsai-Morris et al., 1988). Several studies using knockouts of c-fos, zif268, and an upstream kinase (MSK-1), have demonstrated that locomotor sensitization to cocaine requires the coordinated expression of both genes whereas cocaine CPP appears to require only zif268 expression (Brami-Cherrier et al., 2005; Valjent et al., 2006; Zhang et al., 2006).
Adolescence may be a critical period for drug addiction. Young adolescent male rats have greater locomotor responses than adults after acute low dose cocaine administration. Further, repeated cocaine administration produces as much or more conditioned place preference but reduced locomotor sensitization in adolescents compared to adults. Acute activation of neurons by cocaine induces long-term changes in behavior by activating transcriptional complexes. The purpose of the present study was to correlate cocaine-induced locomotor activity with neuronal activation in subregions of the striatum and cortex by acute cocaine in young adolescent (postnatal (PN) 28) and adult (PN 65) male rats by measuring the induction of the plasticity-associated immediate early genes (IEGs) c-fos and zif268 using in situ hybridization. Animals were treated with saline, low (10 mg/kg), or high (40 mg/kg) dose cocaine in locomotor activity chambers and killed 30 min later. Low dose cocaine induced more locomotor activity and striatal c-fos expression in adolescents than adults whereas high dose cocaine induced more locomotor activity, striatal c-fos, and striatal zif268 expression in adults. Locomotor activity correlated with the expression of both genes in adults but correlated with striatal c-fos only in adolescents. Finally, there was a significant correlation between the expression of c-fos and zif268 in the adult striatum but not in adolescents. Our results suggest that the coordinated expression of transcription factors by cocaine continues to develop during adolescence. The immature regulation of transcription factors by cocaine could explain why adolescents show unique sensitivity to specific long-term behavioral alterations following cocaine treatment.
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Zif268 is a transcription regulatory protein, the product of an immediate early gene. Zif268 was originally described as inducible in cell cultures; however, it was later shown to be activated by a variety of stimuli, including ongoing synaptic activity in the adult brain. Recently, mice with experimentally mutated zif268 gene have been obtained and employed in neurobiological research. In this review we present a critical overview of Zif268 expression patterns in the naive brain and following neuronal stimulation as well as functional data with Zif268 mutants. In conclusion, we suggest that Zif268 expression and function should be considered in a context of neuronal activity that is tightly linked to neuronal plasticity.
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^∗: Present address: CNRS D 1302, Département de Biologie, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris Cedex 05 (France).

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Gene

Abstract

References (32)

Zinc fingers: gilt by association

Cell

Xenopus 5S gene transcription factor, TFIIIA: characterization of a cDNA clone and measurement of RNA levels throughout development

Cell

Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts

Cell

Isolation of cDNA encoding transcription factor Sp1 and functional analysis of the DNA binding domain

Cell

A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization

Cell

Transient accumulation of c-fos mRNA following serum stimulation requires a conserved 5' element and c-fos 3' sequences

Cell

Identification of a protein-binding site that mediates transcriptional response of the c-fos gene to serum factors

Cell

Identification of two distinct regulatory regions adjacent to the human betainterferon gene

Cell

Complexity of the early genetic response to growth factors in mouse fibroblasts

Mol. Cell. Biol.

The Biology of Cell Reproduction

A gene encoding a protein with zinc fingers is activated during G0/G1 transition in cultured cells

EMBO J.

Characterization of a mouse multigene family encoding zinc finger structures

Mol. Cell. Biol.

Structure, chromosome location and expression of the mouse zinc finger gene Krox-20: multiple gene products and coregulation with the proto-oncogene c-fos

Mol. Cell. Biol.

Supercoil sequencing: a fast and simple method for sequencing plasmid DNA

DNA

A gene activated in mouse 3T3 cells by serum growth factors encodes a protein with ‘zinc finger’ sequences

Cited by (67)

Nucleosome-specific, time-dependent changes in histone modifications during activation of the early growth response 1 (Egr1) gene

Reactive oxygen species, Ki-Ras, and mitochondrial superoxide dismutase cooperate in nerve growth factor-induced differentiation of PC12 cells

Formins in development: Orchestrating body plan origami

Maturation of coordinated immediate early gene expression by cocaine during adolescence

A gene for neuronal plasticity in the mammalian brain: Zif268/Egr-1/NGFI-A/ Krox-24/TIS8/ZENK?

Molecular cloning, chromosomal mapping, and characteristic expression in tooth organ of rat and mouse Krox-25