Rebecca M. Ward

NAB2 represses transcription by interacting with the CHD4 subunit of the nucleosome remodeling and deacetylase (NuRD) complex

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 DNA-binding 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.

Direct Regulation of Myelin Protein Zero Expression by the Egr2 Transactivator

During myelination of the peripheral nervous system, the myelin protein zero (Mpz) gene is induced to produce the most abundant protein component (P0) of mature myelin. Although the basal embryonic expression of Mpz in Schwann cells has been attributed to regulation by Sox10, the molecular mechanism for the profound up-regulation of this gene during myelination has not been established. In this study, we have identified a highly conserved element within the first intron of the Mpz gene, which contains binding sites for the early growth response 2 (Egr2/Krox20) transcription factor, a critical regulator of peripheral nerve myelination. Egr2 can transactivate the intron element, and the induction is blocked by two known repressors of Egr2 activity. Using chromatin immunoprecipitation assays, we find that Egr2 binds in vivo to the intron element, but not to the Mpz promoter. Known inducers of Mpz expression such as forskolin and insulin-like growth factor-1 also activate the element in an Egr2-dependent manner. In addition, we found that Egr2 can act synergistically with Sox10 to activate this intron element, suggesting a model in which cooperative interactions between Egr2 and Sox10 mediate a large increase in Mpz expression to the high levels found in myelinating Schwann cells.

Neuropathy-associated Egr2 mutants disrupt cooperative activation of myelin protein zero by Egr2 and Sox10.

ABSTRACT Dominant mutations in the early growth response 2 (Egr2/Krox20) transactivator, a critical regulator of peripheral myelin development, have been associated with peripheral myelinopathies. These dominant mutants interfere with the expression of genes required for myelination by Schwann cells, including that for the most abundant peripheral myelin protein, Myelin protein zero (Mpz). In this study, we show that Egr2 mutants specifically affect an Egr2-responsive element within the Mpz first intron that also contains binding sites for the transcription factor Sox10. Furthermore, Egr2 activation through this element is impaired by mutation of the Sox10 binding sites. Using chromatin immunoprecipitation assays, we found that Egr2 and Sox10 bind to this element in myelinating sciatic nerve and that a dominant Egr2 mutant does not perturb Egr2 binding but rather attenuates binding of Sox10 to the Mpz intron element. Sox10 binding at other sites of Egr2/Sox10 synergy, including a novel site in the Myelin-associated glycoprotein (Mag) gene, is also reduced by the dominant Egr2 mutant. These results provide the first demonstration of binding of Egr2/Sox10 to adjacent sites in vivo and also demonstrate that neuropathy-associated Egr2 mutants antagonize binding of Sox10 at specific sites, thereby disrupting genetic control of the myelination program.

Active Gene Repression by the Egr2·NAB Complex during Peripheral Nerve Myelination

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 Egr2·NAB 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.

Interactions of Sox10 and Egr2 in Myelin Gene Regulation

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 and myelin basic protein 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.

Differential regulation of NAB corepressor genes in Schwann cells

BackgroundMyelination of peripheral nerves by Schwann cells requires not only the Egr2/Krox-20 transactivator, but also the NGFI-A/Egr-binding (NAB) corepressors, which modulate activity of Egr2. Previous work has shown that axon-dependent expression of Egr2 is mediated by neuregulin stimulation, and NAB corepressors are co-regulated with Egr2 expression in peripheral nerve development. NAB corepressors have also been implicated in macrophage development, cardiac hypertrophy, prostate carcinogenesis, and feedback regulation involved in hindbrain development.ResultsTo test the mechanism of NAB regulation in Schwann cells, transfection assays revealed that both Nab1 and Nab2 promoters are activated by Egr2 expression. Furthermore, direct binding of Egr2 at these promoters was demonstrated in vivo by chromatin immunoprecipitation analysis of myelinating sciatic nerve, and binding of Egr2 to the Nab2 promoter was stimulated by neuregulin in primary Schwann cells. Although Egr2 expression activates the Nab2 promoter more highly than Nab1, we surprisingly found that only Nab1 – but not Nab2 – expression levels were reduced in sciatic nerve from Egr2 null mice. Analysis of the Nab2 promoter showed that it is also activated by ETS proteins (Ets2 and Etv1/ER81) and is bound by Ets2 in vivo.ConclusionOverall, these results indicate that induction of Nab2 expression in Schwann cells involves not only Egr2, but also ETS proteins that are activated by neuregulin stimulation. Although Nab1 and Nab2 play partially redundant roles, regulation of Nab2 expression by ETS factors explains several observations regarding regulation of NAB genes. Finally, these data suggest that NAB proteins are not only feedback inhibitors of Egr2, but rather that co-induction of Egr2 and NAB genes is involved in forming an Egr2/NAB complex that is crucial for regulation of gene expression.

Vertebrate-like βγ-crystallins in the ocular lenses of a copepod

The diverse crystallins are water-soluble proteins that are responsible for the optical properties of cellular lenses of animal eyes. While all vertebrate lenses contain physiological stress-related α- and βγ-crystallins, some also contain taxon-specific, often enzyme-related crystallins. To date, the α- and βγ-crystallins have been found only in vertebrate lenses. Here we report lenses from an invertebrate, the pontellid copepod Anomalocera ornata, accumulate βγ-crystallin family members as judged by immunocytochemistry, western immunoblotting and microsequencing. Our data provide the first example of βγ-crystallin members in an invertebrate lens, establishing that the use of this protein family as lens crystallins is not confined to vertebrates.

ERRATUM: Vertebrate-like βγ-crystallins in the ocular lenses of a copepod

We previously reported that the ocular lenses of the pontellid copepod Anomalocera ornata possess vertebrate-like β- and γ-crystallins. We cannot repeat our earlier data suggesting that the copepod lens crystallins belong to the β- and γ-crystallin family of proteins. Our new data are consistent with the copepod crystallins being novel proteins.