Oxidative stress drives disulfide bond formation between basic helix-loop-helix transcription factors.
Basic helix-loop-helix (bHLH) transcription factors including Twist1 and E2a proteins regulate essential processes. These factors bind DNA as homo- or heterodimers and the choice of binding partners determines their functional output. To investigate potential regulators of bHLH dimerization, cells were exposed to the oxidative agent hydrogen peroxide (H(2)O(2)). Western blot analysis in the presence or absence of reducing agents, revealed that H(2)O(2) induces the rapid formation of an intermolecular disulfide bond between Twist1 homodimers and Twist/E2a proteins heterodimers. The disulfide bond is first observed between Twist1 homodimers at 25 mM H(2)O(2) and between Twist1 heterodimers at 75 mM H(2)O(2). This response is dependent upon cell density as H(2)O(2) did not induce disulfide bridge formation between bHLH proteins in cells seeded at high density. In the presence of E proteins, the formation of Twist1/E2a proteins heterodimers is favored over Twist1 homodimers, identifying an oxidative stimulus as an important factor in modulating binding partner specificity. We further demonstrated that a cysteine residue located at the C-terminus of Twist1 and E2a proteins is involved in this response. Disulfide bond formation between Twist1 homodimers significantly reduced its ability to interact with two of its binding partners, Runx2 and HDAC4, indicating that disulfide dimerization in response to H(2)O(2) has functional significance. These data support the conclusion that disulfide bond formation in response to an oxidative stimulus contributes to Twist1 homo- and heterodimerization and raises the possibility that the redox status of a cell may represent an important step in bHLH transcriptional regulation.
Danciu
Theodora E
TE
Department of Developmental Biology, Harvard School of Dental Medicine, Boston, Massachusetts 02115, USA.
Whitman
Malcolm
M
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002452:Cell Count; D002460:Cell Line; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D003545:Cysteine; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D018257:Helix-Loop-Helix Motifs / Q000502:physiology; D006655:Histone Deacetylases / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide; D051379:Mice; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D012097:Repressor Proteins / Q000378:metabolism; D051879:Twist-Related Protein 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D051792:Basic Helix-Loop-Helix Proteins; D050659:Core Binding Factor Alpha 1 Subunit; D003545:Cysteine; D004220:Disulfides; D006655:Histone Deacetylases; D006861:Hydrogen Peroxide; D009687:Nuclear Proteins; D012097:Repressor Proteins; D051879:Twist-Related Protein 1; C407909:TCF3 protein, human; C067250:Twist1 protein, mouse; C400300:HDAC4 protein, human