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Differential regulation of NAB corepressor genes in Schwann cells.

Srinivasan R, Jang SW, Ward RM, Sachdev S, Ezashi T, Svaren J - BMC Mol. Biol. (2007)

Bottom Line: 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 mice.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI, USA. rsrinivasan@wisc.edu

ABSTRACT

Background: Myelination 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.

Results: To 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 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.

Conclusion: Overall, 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.

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Egr2 activates the Nab1 and Nab2 promoters. A) The plot shows percent identity of the human and mouse Nab2 loci upstream of the first exon. DNase I footprinting analysis of a 1000 bp fragment of the mouse Nab2 promoter in the presence of recombinant Egr2 protein revealed two footprinted regions (solid line) with some weaker protections (dotted lines). The footprinted regions correspond to three of the four previously identified conserved sites [filled circles, 22] that conform to the Egr2 consensus binding site. There was no apparent footprint over putative site #4 identified in previous sequence analysis of the Nab2 promoter [22] M = marker lane. B) A similar analysis of the mouse Nab1 promoter confirmed binding of Egr2 to the six previously identified conserved binding sites in the mouse Nab1 promoter [filled circles, 22]. C) JEG-3 cells were transfected with a luciferase reporter plasmid containing either the Nab1 or Nab2 promoter, and the indicated amounts of an Egr2 expression construct. The mutant Nab2 promoter contains mutations in the two upstream Egr2 binding sites. Egr2 binding sites are indicated by filled circles. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Activation of both Nab1 and Nab2 promoters by 25 ng Egr2 (compared to control) was statistically significant (P =< 0.002).
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Figure 1: Egr2 activates the Nab1 and Nab2 promoters. A) The plot shows percent identity of the human and mouse Nab2 loci upstream of the first exon. DNase I footprinting analysis of a 1000 bp fragment of the mouse Nab2 promoter in the presence of recombinant Egr2 protein revealed two footprinted regions (solid line) with some weaker protections (dotted lines). The footprinted regions correspond to three of the four previously identified conserved sites [filled circles, 22] that conform to the Egr2 consensus binding site. There was no apparent footprint over putative site #4 identified in previous sequence analysis of the Nab2 promoter [22] M = marker lane. B) A similar analysis of the mouse Nab1 promoter confirmed binding of Egr2 to the six previously identified conserved binding sites in the mouse Nab1 promoter [filled circles, 22]. C) JEG-3 cells were transfected with a luciferase reporter plasmid containing either the Nab1 or Nab2 promoter, and the indicated amounts of an Egr2 expression construct. The mutant Nab2 promoter contains mutations in the two upstream Egr2 binding sites. Egr2 binding sites are indicated by filled circles. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Activation of both Nab1 and Nab2 promoters by 25 ng Egr2 (compared to control) was statistically significant (P =< 0.002).

Mentions: Sequence analysis of the Nab1 and Nab2 promoters identified several conserved motifs that resemble Egr2 binding sites [22]. To test whether these sites bind Egr2, the mouse Nab1 and Nab2 promoters were analyzed by DNase I footprinting. In the presence of recombinant Egr2, there were strongly protected regions encompassing three previously identified sites in the Nab2 promoter, as well as some weaker protections (Figure 1A). Interestingly, previous analysis of Egr2 site specificity indicated a strong preference for T in the 4th position of the consensus sequence [GCGTGGGCG, [38]], and each of the three strongly protected sites have a T in the 4th base. A similar analysis of the mouse Nab1 promoter (Figure 1B) confirmed binding of Egr2 to the conserved binding sites identified previously [22]. The strong binding site at the 3' end of the mouse Nab2 promoter corresponds to a site of phorbol ester-induced Egr1 binding in a recent analysis of the human NAB2 promoter using electrophoretic mobility shift assays [39].


Differential regulation of NAB corepressor genes in Schwann cells.

Srinivasan R, Jang SW, Ward RM, Sachdev S, Ezashi T, Svaren J - BMC Mol. Biol. (2007)

Egr2 activates the Nab1 and Nab2 promoters. A) The plot shows percent identity of the human and mouse Nab2 loci upstream of the first exon. DNase I footprinting analysis of a 1000 bp fragment of the mouse Nab2 promoter in the presence of recombinant Egr2 protein revealed two footprinted regions (solid line) with some weaker protections (dotted lines). The footprinted regions correspond to three of the four previously identified conserved sites [filled circles, 22] that conform to the Egr2 consensus binding site. There was no apparent footprint over putative site #4 identified in previous sequence analysis of the Nab2 promoter [22] M = marker lane. B) A similar analysis of the mouse Nab1 promoter confirmed binding of Egr2 to the six previously identified conserved binding sites in the mouse Nab1 promoter [filled circles, 22]. C) JEG-3 cells were transfected with a luciferase reporter plasmid containing either the Nab1 or Nab2 promoter, and the indicated amounts of an Egr2 expression construct. The mutant Nab2 promoter contains mutations in the two upstream Egr2 binding sites. Egr2 binding sites are indicated by filled circles. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Activation of both Nab1 and Nab2 promoters by 25 ng Egr2 (compared to control) was statistically significant (P =< 0.002).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2235890&req=5

Figure 1: Egr2 activates the Nab1 and Nab2 promoters. A) The plot shows percent identity of the human and mouse Nab2 loci upstream of the first exon. DNase I footprinting analysis of a 1000 bp fragment of the mouse Nab2 promoter in the presence of recombinant Egr2 protein revealed two footprinted regions (solid line) with some weaker protections (dotted lines). The footprinted regions correspond to three of the four previously identified conserved sites [filled circles, 22] that conform to the Egr2 consensus binding site. There was no apparent footprint over putative site #4 identified in previous sequence analysis of the Nab2 promoter [22] M = marker lane. B) A similar analysis of the mouse Nab1 promoter confirmed binding of Egr2 to the six previously identified conserved binding sites in the mouse Nab1 promoter [filled circles, 22]. C) JEG-3 cells were transfected with a luciferase reporter plasmid containing either the Nab1 or Nab2 promoter, and the indicated amounts of an Egr2 expression construct. The mutant Nab2 promoter contains mutations in the two upstream Egr2 binding sites. Egr2 binding sites are indicated by filled circles. The y-axis shows the fold activation normalized to the activity of the reporter alone. Means and standard deviations of three separate transfections are shown. Activation of both Nab1 and Nab2 promoters by 25 ng Egr2 (compared to control) was statistically significant (P =< 0.002).
Mentions: Sequence analysis of the Nab1 and Nab2 promoters identified several conserved motifs that resemble Egr2 binding sites [22]. To test whether these sites bind Egr2, the mouse Nab1 and Nab2 promoters were analyzed by DNase I footprinting. In the presence of recombinant Egr2, there were strongly protected regions encompassing three previously identified sites in the Nab2 promoter, as well as some weaker protections (Figure 1A). Interestingly, previous analysis of Egr2 site specificity indicated a strong preference for T in the 4th position of the consensus sequence [GCGTGGGCG, [38]], and each of the three strongly protected sites have a T in the 4th base. A similar analysis of the mouse Nab1 promoter (Figure 1B) confirmed binding of Egr2 to the conserved binding sites identified previously [22]. The strong binding site at the 3' end of the mouse Nab2 promoter corresponds to a site of phorbol ester-induced Egr1 binding in a recent analysis of the human NAB2 promoter using electrophoretic mobility shift assays [39].

Bottom Line: 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 mice.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI, USA. rsrinivasan@wisc.edu

ABSTRACT

Background: Myelination 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.

Results: To 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 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.

Conclusion: Overall, 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.

Show MeSH
Related in: MedlinePlus