Early growth response (EGR) transactivators act as critical regulators of several physiological processes, including peripheral nerve myelination and progression of prostate cancer. The NAB1 and NAB2 (NGFI-A/EGR1-binding protein) transcriptional corepressors directly interact with three EGR family members (Egr1/NGFI-A/zif268, Egr2/Krox20, and Egr3) and repress activation of their target promoters. To understand the molecular mechanisms underlying NAB repression, we found that EGR activity is modulated by at least two repression domains within NAB2, one of which uniquely requires interaction with the CHD4 (chromodomain helicase DNAbinding protein 4) subunit of the NuRD (nucleosome remodeling and deacetylase) chromatin remodeling complex. Both NAB proteins can bind either CHD3 or CHD4, indicating that the interaction is conserved among these two protein families. Furthermore, we show that repression of the endogenous Rad gene by NAB2 involves interaction with CHD4 and demonstrate colocalization of NAB2 and CHD4 on the Rad promoter in myelinating Schwann cells. Finally, we show that interaction with CHD4 is regulated by alternative splicing of the NAB2 mRNA.By virtue of their ability to regulate the early growth response (EGR) 3 family of transactivators, NAB (NGFI-A-binding protein) corepressors play an important role in regulating inflammation, nervous system function, and prostate cancer development. The NAB1 and NAB2 corepressors interact with a conserved domain found within Egr1 (also called NGFI-A/zif268), Egr2/Krox20, and Egr3 (1-3). The remaining family member, Egr4/NGFI-C, shares substantial homology with other EGR family members but lacks the NAB interaction domain and is therefore resistant to NAB repression.Members of the EGR family play diverse physiological roles, including having both positive and negative effects on growth. For example, Egr1 null fibroblasts bypass senescence because of reduced expression of the p53 gene (4). On the other hand, EGR1 overexpression is also involved in the development of prostate cancer (5, 6), as it regulates several growth factor genes (7-10). The other EGR family member that has been studied intensively is Egr2/Krox20. Targeted disruption of the mouse Egr2 gene resulted in defects in hindbrain segmentation, bone development, and peripheral nerve myelination by Schwann cells (11)(12)(13)(14). A number of Egr2 target genes in the hindbrain and Schwann cells have been identified, including several Hox family members, EphA4, and myelin-associated genes such as myelin protein zero and myelin basic protein (13,(15)(16)(17)(18)(19)(20)(21).Several experiments in various systems have established that NAB corepressors are important regulators of EGR activity. NAB1 and NAB2 both repress EGR activation of several promoters (3, 22-26). Interestingly, although EGR1 is overexpressed in prostate cancer (5, 6), NAB2 expression is reduced in a majority of prostate cancer samples (27). This observation is consistent with the idea that derepression of EGR1 activity is a progression f...

Myelination of peripheral nerves by Schwann cells requires a large amount of lipid and cholesterol biosynthesis. To understand the transcriptional coordination of the myelination process, we have investigated the developmental relationship between early growth response 2 (Egr2)/Krox20 -a pivotal regulator of peripheral nerve myelination -and the sterol regulatory element binding protein (SREBP) pathway, which controls expression of cholesterol/lipid biosynthetic genes. During myelination of sciatic nerve, there is a very significant induction of SREBP1 and SREBP2, as well as their target genes, suggesting that the SREBP transactivators are important regulators in the myelination process. Egr2/Krox20 does not appear to directly regulate the levels of SREBP pathway components, but rather, we found that Egr2/Krox20 and SREBP transactivators can synergistically activate promoters of several SREBP target genes, indicating that direct induction of cholesterol/lipid biosynthetic genes by Egr2/ Krox20 is a part of the myelination program regulated by this transactivator.

Myelination in the PNS is accompanied by a large induction of the Myelin Protein Zero (Mpz) gene to produce the most abundant component in peripheral myelin. Analyses of knockout mice have shown that the EGR2/Krox20 and SOX10 transcription factors are required for Mpz expression. Our recent work has shown that the dominant EGR2 mutations associated with human peripheral neuropathies cause disruption of EGR2/SOX10 synergy at specific sites, including a conserved enhancer element in the first intron of the Mpz gene. Further investigation of Egr2/Sox10 interactions reveals that activation of the Mpz intron element by Egr2 requires both Sox10-binding sites. In addition, both Egr1 and Egr3 cooperate with Sox10 to activate this element, which indicates that this capacity is conserved among Egr family members. Finally, a conserved composite structure of Egr2/ Sox10-binding sites in the genes encoding Mpz, Myelin-associated glycoprotein (Mag), and Myelin Basic Protein (Mbp) genes was used to screen for similar modules in other myelin genes, revealing a potential regulatory element in the periaxin gene. Overall, these results elucidate a working model for developmental regulation of Mpz expression, several facets of which extend to regulation of other peripheral myelin genes.

The Egr2/Krox20 transactivator is required for activation of many myelin-associated genes during peripheral nerve myelination by Schwann cells. However, recent work has indicated that Egr2 not only activates genes required for peripheral nerve myelination but may also be involved in gene repression. The NAB (NGFI-A/Egr-binding) corepressors interact with Egr2 and are required for proper coordination of myelin formation. Therefore, NAB proteins could mediate repression of some Egr2 target genes, although direct repression by Egr2 or NAB proteins during myelination has not been demonstrated. To define the physiological role of NAB corepression in gene repression by Egr2, we tested whether the Egr2⅐NAB complex directly repressed specific target genes. A screen for NAB-regulated genes identified several (including Id2, Id4, and Rad) that declined during the course of peripheral nerve myelination. In vivo chromatin immunoprecipitation analysis of the myelinating sciatic nerve was used to show developmental association of both Egr2 and NAB2 on the Id2, Id4, and Rad promoters as they were repressed during the myelination process. In addition, NAB2 represses transcription by interaction with the chromodomain helicase DNA-binding protein 4 (CHD4) subunit of the nucleosome remodeling and deacetylase chromatin remodeling complex, and we demonstrate that CHD4 occupies NAB-repressed promoters in a developmentally regulated manner in vivo. These results illustrate a novel aspect of genetic regulation of peripheral nerve myelination by showing that Egr2 directly represses genes during myelination in conjunction with NAB corepressors. Furthermore, repression of Id2 was found to augment activation of Mpz (myelin protein zero) expression.The early growth response-2 (Egr2/Krox20) 3 transcriptional activator is critically required for peripheral nerve myelination by Schwann cells. Peripheral nerve from Egr2/Krox20-deficient mice is characterized by arrested myelination and decreased myelin protein levels (1, 2), and recent analysis has indicated that Egr2/Krox20 is required for maintenance of peripheral myelin (3). In addition, mutations in EGR2 have been identified in several patients with myelin disorders such as CharcotMarie-Tooth (CMT) disease, Dejerine-Sottas syndrome, and congenital hypomyelinating neuropathy (4 -6) (reviewed in Ref. 7). Egr2 is induced in Schwann cells at the onset of myelination (1, 8) and is required for activation of a variety of genes involved in peripheral nerve myelination in vivo including Mpz (myelin protein zero) and Pmp22 (peripheral myelin protein-22), as well as genes involved in lipid biosynthesis (1, 2, 9). Recent work has demonstrated sites of Egr2 occupancy within activated myelin genes in vivo (10, 11).One of the EGR2 mutations associated with a very severe congenital hypomyelinating neuropathy (I268N (4)) prevents binding of EGR2 to NGFI-A/Egr-binding (NAB1 and NAB2) corepressors (12), which repress Egr-mediated transcription (13-15). The importance of NAB corepressors to the regulation of ...

We studied mesenteric arterial arcades from 3- and 35-day-old swine to determine the relationship between perfusate flow rate and release of nitric oxide (NO) into mesenteric effluent. Mesenteric arterial arcades were perfused under controlled-flow conditions with a peristaltic pump using warm oxygenated Krebs buffer. Basal rates of NO production were 43.6 +/- 4.2 vs. 12.1 +/- 2.5 nmol/min in 3- vs. 35-day-old mesentery during perfusion at in vivo flow rates (9 vs. 20 ml/min, respectively). Rate of NO production was directly related to flow rate over a wide range of flows (5-40 ml/min) in 3- but not 35-day-old mesentery. Both age groups demonstrated a brisk, albeit brief, increase in NO production in response to infusion of NO-dependent vasodilator substance P (10(-8) M/min). Tyrosine kinase inhibitor herbimycin A and L-arginine analog L-NMMA significantly attenuated flow-induced increase in NO production, and phosphatase inhibitor phenylarsine oxide increased magnitude of flow-induced increase in NO production in 3-day-olds. Removal of extracellular Ca(2+) and depletion of intracellular Ca(2+) stores (Ca(2+)-free Krebs with EGTA plus thapsigargin) had no effect on NO production in either group. Thus, basal rate of NO production is greater in mesenteric arterial arcades from 3- than from 35-day old swine, a direct relationship between flow rate and NO production rate is present in mesentery from 3- but not 35-day-olds, and phosphorylation events are necessary for this interaction to occur.