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Egr genes as targets of ETS transcription factors

ETS transcription factors (See Drosophila Pointed) play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example, in Ewing's sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, two approaches were used. In the first approach, genes were isolated by the RNA differential display technique. Three known genes isolated by differential display are identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, taken to isolate ETS target promoters directly, ETS1 binding was performed with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned. Of the large number of clones isolated, 43 represent unique sequences not previously identified. Three clones turn out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II and Egr1 genes. The ETS binding sites derived from these three regulatory sequences show specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene (Robinson, 1997).

Egr genes and Schwann cells

The transcription factors Krox-20 and SCIP each play important roles in the differentiation of Schwann cells. However, the genes encoding these two proteins exhibit distinct time courses of expression and yield distinct cellular phenotypes upon mutation. SCIP is expressed prior to the initial appearance of Krox-20, and is transient in both the myelinating and non-myelinating Schwann cell lineages; in contrast, Krox-20 appears ~24 hours after SCIP and then only within the myelinating lineage, where its expression is stably maintained into adulthood. Similarly, differentiation of SCIP-/- Schwann cells appears to transiently stall at the promyelinating stage that precedes myelination, whereas Krox-20(-/-) cells are, by morphological criteria, arrested at this stage. These observations led to an examination of SCIP regulation and Schwann cell phenotype in Krox-20 mouse mutants. In Krox-20(-/-) Schwann cells, SCIP expression is converted from transient to sustained. Both Schwann cell proliferation and apoptosis, which are normal features of SCIP+ cells, are also markedly increased late in postnatal development in Krox-20 mutants relative to wild type, and the levels of cell division and apoptosis are balanced to yield a stable number of Schwann cells within peripheral nerves. These data demonstrate that the loss of Krox-20 in myelinating Schwann cells arrests differentiation at the promyelinating stage, as assessed by SCIP expression, mitotic activity and susceptibility to apoptosis (Zorick, 1999).

The identification of EGR2 mutations in patients with neuropathies and the phenotype Egr2/Krox20-/- have demonstrated that the Egr2 transcription factor is critical for peripheral nerve myelination. However, the mechanism by which these mutations cause disease remains unclear, because most patients present with disease in the heterozygous state, whereas Egr2+/- mice are phenotypically normal. To understand the effect of aberrant Egr2 activity on Schwann cell gene expression, microarray expression profiling was performed to identify genes regulated by Egr2 in Schwann cells. These include genes encoding myelin proteins and enzymes required for synthesis of normal myelin lipids. Using these newly identified targets, it has been shown that neuropathy-associated EGR2 mutants dominant-negatively inhibit wild-type Egr2-mediated expression of essential myelin genes to levels sufficiently low to result in the abnormal myelination observed in these patients (Nararajan, 2001).

In Schwann cells (SC), myelination is controlled by the transcription factor gene Krox20/Egr2. Analysis of cis-acting elements governing Krox20 expression in SC reveals the existence of two separate elements. The first, designated immature Schwann cell element (ISE), is active in immature but not myelinating SC, whereas the second, designated myelinating Schwann cell element (MSE), is active from the onset of myelination to adulthood in myelinating SC. In vivo sciatic nerve regeneration experiments have demonstrated that both elements are activated during this process, in an axon-dependent manner. Together the activity of these elements reproduced the profile of Krox20 expression during development and regeneration. Genetic studies have shownthat both elements are active in a Krox20 mutant background, while the activity of the MSE, but likely not of the ISE, requires the POU domain transcription factor Oct6 at the time of myelination. The MSE has been localized to a 1.3 kb fragment, 35 kb downstream of Krox20. The identification of multiple Oct6 binding sites within this fragment suggests that Oct6 directly controls Krox20 transcription. Taken together, these data indicate that, although Krox20 is expressed continuously from 15.5 dpc in SC, the regulation of its expression is a biphasic, axon-dependent phenomenon involving two cis-acting elements that act in succession during development. In addition, they provide insight into the complexity of the transcription factor regulatory network controlling myelination (Ghislain, 2002).

Egr genes and the peripheral nervous system

Adrenomedullary chromaffin cells express at least two subtypes of acetylcholine nicotinic receptors, which differ in their sensitivity to the snake toxin alpha-bungarotoxin. One subtype is involved in the activation step of the catecholamine secretion process and is not blocked by the toxin. The other is alpha-bungarotoxin-sensitive, and its functional role has not yet been defined. The alpha7 subunit is a component of this subtype. Autoradiography of bovine adrenal gland slices with alpha-bungarotoxin indicates that these receptors are restricted to medullary areas adjacent to the adrenal cortex and colocalize with the enzyme phenylethanolamine N-methyl transferase (PNMT), which confers the adrenergic phenotype to chromaffin cells. Transcripts corresponding to the alpha7 subunit are localized exclusively to adrenergic cells. The alpha7 subunit gene 5' flanking region has putative binding sites for the immediate early gene transcription factor Egr-1, which is known to activate PNMT expression. Egr-1 increases alpha7 promoter activity by up to sevenfold. Activation is abolished when the most promoter-proximal of the Egr-1 sites is mutated, whereas modification of a close upstream site produces a partial decrease of the Egr-1 response. Because Egr-1 is found to be expressed exclusively in adrenergic cells, it has been suggested that this transcription factor may be part of a common mechanism involved in the induction of the adrenergic phenotype and the differential expression of alpha-bungarotoxin-sensitive nicotinic receptors in the adrenal gland (Criado 1997).

Muscle spindles are skeletal muscle mechanoreceptors that mediate the stretch reflex and provide axial and limb position information (proprioception) to the central nervous system. Spindles consist of encapsulated muscle fibers (intrafusal fibers) that are innervated by specialized sensory (groups Ia and II) and motor (gamma) neurons. The Egr family of zinc-finger transcription factors, consisting of Egr1, Egr2, Egr3, and Egr4, are involved in cellular growth and differentiation. Adult Egr3-deficient mice are ataxic and lack muscle spindle proprioceptors that normally develop at the sites of Ia afferent-myotube contacts during embryogenesis. To resolve whether spindles form and then degenerate, or whether they never form in the absence of Egr3, the spatiotemporal expression of Egr3 was examined relative to spindle development. In wild type mice, Egr3 is expressed in developing myotubes shortly after they are innervated by Ia afferents and its expression is controlled by innervation because it dissipates following nerve transection. In Egr3-deficient mice, myotubes receive Ia afferent innervation and assemble normally into spindles during embryogenesis. However, newborn Egr3-deficient spindles have few internal myonuclei in intrafusal fibers and thin capsules. Moreover, slow-developmental myosin heavy chain is not induced in embryonic Egr3-deficient spindles suggesting that impairments in differentiation are present before they can be detected morphologically. After birth, sensory and motor innervation withdraws from the Egr3-deficient spindles, and the spindles disassemble. In spite of the spindle disassembly and retraction of afferents from muscles, the cell bodies of proprioceptive neurons within dorsal root ganglia are retained. It is concluded that Egr3 has an essential role in regulating genes required for the transformation of undifferentiated myotubes into intrafusal fibers, and hence for the phenotypic differentiation of spindles (Tourtellotte, 2001).

Egr3 appears to play a role as an intermediate signaling molecule that regulates the expression of genes required for spindle morphogenesis. Additional work is needed to identify 'upstream' and 'downstream' signaling molecules in spindle morphogenetic processes. Presumably, Ia afferents produce a nerve-derived factor that activates a signal transduction cascade to induce the expression of Egr3 in target myotubes. Since Egr gene expression is coupled to Ras/MAP kinase signaling pathways, it is possible that this 'upstream' regulatory pathway is used to transduce the morphogenetic signals imparted by Ia afferents. In all likelihood, Egr3 regulates additional 'down-stream' genes that are specifically involved in directing intrafusal muscle fiber phenotypes and the overall spindle structure. Mechanoreceptor morphogenesis, controlled by sensory innervation, is not unique to spindles since Merkel cells (light touch skin receptors), Pacinian corpuscles (vibratory receptors), and GTOs (muscle tension receptors) all depend upon sensory innervation for their development. Egr3 function appears to be restricted to spindles and Ia afferents since GTOs and Pacinian corpuscles are intact in Egr3-deficient mice. Egr3 is the first transcription factor known to play a critical role in signal transduction events involved in mechanoreceptor morphogenesis (Tourtellotte, 2001).

Egr genes, neural circuits, learning and behavior

Mammalian circadian rhythms are regulated by a pacemaker in the suprachiasmatic nucleus of the hypothalamus. Light induces the immediate early genes (IEGs) c-fos and jun-B, in the rodent suprachiasmatic nucleus. In hamsters, there is a strong correlation between circadian entrainment and the induction of c-fos and jun-B in the suprachiasmatic nucleus by light. The IEGs nur77 and zif268, both of which encode transcription factors, are also light-inducible in the rat suprachiasmatic nucleus. To characterize the photic-regulation of these genes in the suprachiasmatic nucleus of golden hamsters, in situ hybridization was used to measure nur77 and zif268 mRNA levels. 5-min monochromatic light pulses induce a dramatic increase in both nur77 and zif268 mRNA levels. Peak mRNA levels occur 45-60 min after light onset for both nur77 and zif268. The induction of both nur77 and zif268 mRNA levels is gated by the circadian pacemaker. Light pulses during subjective day (CT3 and CT9), which do not cause behavioral phase-shifts, do not significantly alter mRNA levels of either nur77 or zif268; light pulses during the subjective night (CT14 and CT19), which do induce phase-shifts, dramatically increase both nur77 and zif268 mRNA levels. In contrast to c-fos induction, which has a photic threshold indistinguishable from that of the behavioral phase-shifting response, nur77 and zif268 mRNA induction are found to have visual sensitivities greater than the phase-shifting response by 1-2 log units (10-100-fold). Although light and circadian phase regulates nur77 and zif268 expression in the SCN, these results demonstrate that their induction is not rate-limiting for photic entrainment of the hamster circadian system (Lin, 1997).

Photic induction of NGFI-A gene expression was investigated in the rat suprachiasmatic nucleus (SCN) using in situ hybridization histochemistry. Following light exposure for 30 min, NGFI-A mRNA appears in the ventral portion of the rostral SCN, in the ventrolateral and in part of the dorsomedial portion at the middle level, and in the lateral portion of the caudal SCN. The distribution of NGFI-A mRNA is wider than that of c-fos mRNA which is confined to the ventrolateral portion at the middle level of the SCN. Approximately half of NGFI-A mRNA-positive cells in the SCN coexpress vasoactive intestinal peptide (VIP) mRNA, while 16% of cells positive for c-fos mRNA coexpressed VIP mRNA. These findings indicate that the broadness of NGFI-A mRNA and c-fos mRNA expression after photic stimulation are different. The NGFI-A gene, induced in these cells of the SCN (including VIP neurons) may be involved in circadian entrainment by light (Tanaka, 1997).

Immediate early gene (IEG) expression in the cat visual cortex is highly responsive to visual input and may initiate genetic mechanisms responsible for neuronal plasticity. Immunoreactivity of the two IEG proteins was compared between 5-week-old and adult cats, under three conditions of visual input: ambient light to assess basal levels of expression, 1 week of darkness to assess the effect of reduced activity, and exposure to light after 1 week of darkness to determine rapid changes in expression as a result of visual input. At both ages, there are marked differences in the expression of the two IEG proteins. EGR-1 responds to visual input with sustained changes in its level of expression. It shows high basal levels, reduced expression in darkness, and a rapid return to high constitutive levels with the introduction of light. Fos shows a markedly different profile. It has very low basal expression which is not demonstrably affected by darkness and its principal response is a marked transient induction upon exposure to light after darkness. These unique changes in expression highlight the complex response across IEGs to environmental input and suggest a genetic "on/off" signaling mechanism. There are marked differences in the laminar distribution of EGR-1 and Fos proteins between young and adult cats. In young animals, cells in all visual cortical layers show high levels of EGR-1 and Fos proteins. In adults, immunostaining is largely specific to cells located above and below layer IV; only very faint labeling occurred within layer IV. These differences in laminar distribution between ages are inconsistent with a simple explanation of IEG expression in terms of neural activity level; rather, they suggest a relation between IEG expression and the state of plasticity in visual cortex (Kaplan, 1996).

Long-term potentiation-inducing stimulation of the perforant path was followed in dentate gyrus granule cells by a dramatic increase of mRNA and protein for Krox20, a zinc-finger-containing transcription factor. Induction of Krox20 requires stimulation sufficient to induce LTP and is prevented by NMDA antagonists CPP and MK-801, which block LTP induction. Krox20 protein increases within 20 min of tetanization, is maximal between 1 and 8 h, and is still significantly elevated at 24 h after LTP induction. This prolonged appearance is in striking contrast to the more transient induction of the related molecule, Krox24. The elevation in the mRNA for Krox20 and Krox24 is of similar duration, suggesting that the Krox20 protein has a greater stability and may play a key role in the stabilization of long-term potentiation (Williams, 1995).

Changes in the distribution pattern of mRNA encoding the zif268 transcription factor (also referred to as NGFI-A, Krox-24 or EGR-1) were investigated by in situ hybridization histochemistry during postnatal rat brain development. Marked changes in zif268 expression patterns are seen in particular in the cerebral cortex and the hippocampal formation during the first three weeks. In the first postnatal week, zif268 mRNA levels are highest in the corpus striatum and the piriform cortex. In the neocortex, expression rises sharply in the sensorimotor area between postnatal days (PNDs) 10 and 12. In the frontal and occipital cortex, in contrast, an increase in zif268 mRNA levels is first seen on PND 14. After PND 17, levels decrease in the sensorimotor and the frontal cortex but remain high in the occipital and the piriform cortex. In the hippocampus, an initially uniform increase in expression during the second week is followed by a marked dissociation in expression levels among CA1 (with continuously high expression levels) CA3, CA4 and the dentate gyrus, in contrast to a strong decline of expression during the third week. These results indicate that zif268 expression displays a highly dynamic expression pattern during plastic adaptations of different cerebral subregions during postnatal development, suggesting a possible involvement in gene regulatory processes during these phases (Herms, 1994).

Forebrain neuronal connections associated with the cardiovascular response to unilateral, low-intensity, electrical stimulation of the mesencephalic cuneiform nucleus were examined in rats for c-fos and nerve growth factor inducible-A gene (NGFI-A) messenger RNAs. Stimulation of the cuneiform nucleus leads to increases in mean arterial pressure and heart rate, whereas no cardiovascular response is observed in animals stimulated in the inferior colliculus or in sham-operated animals. Cuneiform nucleus stimulation is associated with increased c-fos and NGFI-A messenger RNA levels bilaterally in the ventromedial, dorsomedial and lateroanterior hypothalamic nuclei, lateral and anterior hypothalamic areas, and ipsilaterally in the medial amygdaloid nucleus, at levels significantly greater than those in inferior colliculus-stimulated, sham-operated and naive, unoperated animals. C-fos, but not NGFI-A, messenger RNA expression is increased bilaterally in the piriform cortex and subparafascicular thalamic nucleus. These results are consistent with the existence of direct and indirect projections between the cuneiform nucleus and the aforementioned activated areas, the functions of which may include the control of reproduction and metabolism, as well as cardiovascular regulation. The ipsilateral nature of responses in certain brain areas may be explained by the absence of decussating pathways and/or the presence of multisynaptic connections that attenuate bilateral signal transmission. The existence of structures that are known to receive afferent projections from the cuneiform nucleus, but that are not activated, may be explained by synaptic depolarization not reaching the threshold for immediate early gene expression or by a net inhibitory effect on innervated neurons. Characterization of these activated forebrain regions using other compatible labelling techniques should further elucidate the mechanisms by which these central nervous system structures are integrated in the response to stimulation of the cuneiform nucleus (Lam, 1997).

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huckebein: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 5 August 97  

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