, 2007, Sun et al , 2003, Wang et al , 2006 and Wissmuller

, 2007, Sun et al., 2003, Wang et al., 2006 and Wissmuller VE-822 solubility dmso et al., 2006). We compared the cofactor-binding properties of OLIG2S147A and OLIG2WT by coIP assays in transfected Cos-7 cells and found that, compared to OLIG2WT, OLIG2S147A had a reduced ability to form OLIG2-OLIG2 and OLIG2-OLIG1 dimers, whereas

binding to NKX2.2, SOX10, or MASH1 was unaffected (Figures 2A and S2A). In contrast, OLIG2S147A complexed more readily with NGN2 (Figure 2A). Together, these experiments indicate that S147A mutation does not destabilize OLIG2 or grossly affect its structure but, nevertheless, alters its binding to transcriptional partners. We tried to mimic constitutive phosphorylation by mutating S147 to glutamic acid (E) or aspartic acid (D). However, both OLIG2S147E and OLIG2S147D exhibited reduced homodimer formation just like OLIG2S147A (data not shown). This sort of effect is not unique to OLIG2. For example, phosphorylation/dephosphorylation of serine/threonine residues of the bHLH transcription factor HAND1 have been shown to regulate homodimer versus heterodimer formation, phosphorylation favoring heterodimers of HAND1 and E proteins and dephosphorylation selleck chemical favoring HAND1 homodimers (Firulli et al., 2003). However,

mutation of the key serine/threonine phosphate acceptors to aspartic acid did not inhibit HAND1 homodimer formation, as would have been expected if these substitutions had mimicked constitutive phosphorylation, but instead strengthened homodimer formation just like serine/threonine → alanine substitution (Firulli et al., 2003). We also compared the binding properties of OLIG2WT and OLIG2S147A using a mammalian two-hybrid system (CheckMate System, Promega) (Figures 2B–2E). This is a bipartite assay that depends on the physical whatever association

of test and target proteins at the promoter of a Luciferase reporter gene in Cos-7 cells, so activating Luciferase expression, which can be quantified by chemiluminescence. This assay confirmed the results of co-IP, namely, that association of OLIG2WT with an OLIG2S147A target was strongly reduced relative to either an OLIG2WT or OLIG1 target ( Figures 2B and 2C). In contrast, and also in agreement with the co-IP data, association of NGN2 with OLIG2S147A was enhanced ( Figure 2D). Furthermore, cotransfection of the catalytic subunit of PKA enhanced formation of OLIG2-OLIG2 and OLIG2-OLIG1 dimers while inhibiting OLIG2-NGN2 dimer formation, whereas a dnPKA had the opposite effect ( Figures 2B–2E). In summary, phosphorylation of OLIG2 on S147, possibly by PKA, has a dramatic effect on cofactor choice, favoring NGN2 over other potential partners. In addition we assessed the DNA binding activities of OLIG2WT and OLIG2S147A by electrophoretic mobility shift assay (EMSA). OLIG2S147A exhibited significantly weaker binding to the HB9/M100 E box (Lee et al., 2005) compared to OLIG2WT (Figure S2B). Given that dimerization is needed for bHLH proteins to bind to DNA targets (Murre et al.

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