In vivo detection of Egr2 binding to target genes during peripheral nerve myelination.
Egr2/Krox20 is a zinc finger transactivator that regulates a diverse array of genes required for peripheral nerve myelination. Although several studies have elucidated the Egr2-regulated gene network, it is not clear if Egr2 regulates its target genes directly or indirectly through induction of other transactivators. Moreover, very few Egr2 binding sites have been identified in regulatory elements of myelin genes. To address this issue, we have successfully adapted chromatin immunoprecipitation assays to test if Egr2 binds directly to target genes in myelinating rat sciatic nerve. These experiments demonstrate direct binding of Egr2 to previously described binding sites within the Schwann cell enhancer of the myelin basic protein gene. Furthermore, we show Egr2 binding to a conserved site within the myelin-associated glycoprotein gene. Finally, our experiments provide the first evidence that Egr2 directly regulates expression of desert hedgehog, which is critically involved in development, maintenance and regeneration of multiple nerve elements including myelinated fibers. Surprisingly, this analysis has identified an apparent preponderance of Egr2 binding sites within conserved intron sequences of several myelin genes. Application of chromatin immunoprecipitation analysis to myelination in vivo will prove to be a valuable asset in assaying transcription factor binding and chromatin modifications during activation of myelin genes.
Jang
Sung-Wook
SW
Graduate Program in Cellular and Molecular Biology, University of Wisconsin, Madison, Wisconsin, USA.
LeBlanc
Scott E
SE
Roopra
Avtar
A
Wrabetz
Lawrence
L
Svaren
John
J
D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D047369:Chromatin Immunoprecipitation / Q000379:methods; D051767:Early Growth Response Protein 2 / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology; D053823:Hedgehog Proteins; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D004676:Myelin Basic Protein / Q000378:metabolism; D009185:Myelin Proteins / Q000235:genetics / Q000378:metabolism*; D018992:Myelin-Associated Glycoprotein / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012583:Schwann Cells / Q000378:metabolism; D012584:Sciatic Nerve / Q000166:cytology* / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism
D051767:Early Growth Response Protein 2; C497327:Egr2 protein, mouse; D053823:Hedgehog Proteins; D004676:Myelin Basic Protein; D009185:Myelin Proteins; D018992:Myelin-Associated Glycoprotein; D012333:RNA, Messenger; D015534:Trans-Activators