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. 2015 Nov 23;6:8869. doi: 10.1038/ncomms9869

Figure 2. The pSER domain associates with SL1 on chromatin and activates transcription.

Figure 2

(a) Transactivation activities of the AF4 subdomains. The schematic structures of various FLAG-tagged GAL4 fusion proteins are shown. Various GAL4 fusion genes were cloned into pCMV5 vector and used for transient expression. fG, FLAG-tagged GAL4 DNA-binding domain. 293T cells were transiently transfected with the expression vectors for various GAL4 fusion proteins with the pFR–LUC and pRL–TK reporter plasmids. Promoter activity was assessed with the dual luciferase reporter assay. The transcription activation activity, normalized to the RL activity, is shown with error bars (s.d. from triplicates) relative to the value of fG–AF4-2C (arbitrarily set at 1). UAS, upstream activation sequence. (b) The protein scores of SL1 components identified by mass spectrometry as co-precipitates with fG–AF4-2C. (c) Factors associated with various AF4 subdomains. Various FLAG-tagged GAL4–AF4 fusion proteins were expressed in 293T cells and subjected to fanChIP–WB. The sample shown in the input lane is indicated by an asterisk. (d) Association of the SL1 complex with the pSER domain. All SL1 components were co-expressed with fG-AF4-2C in 293T cells and subjected to fanChIP–WB. The pcDNA4 HisMax-TAF1A/B/D and -TBP vectors and the pCMV5-TAF1C-HA vector were used for transient expression of Xpress-tagged or HA-tagged SL1 components. (e) Chromatin-specific association of SL1 with the pSER domain. IP–WB analyses were performed with the chromatin fraction (CHR) or the chromatin-unbound soluble faction (SOL) from 293T cells expressing fG-AF4-2C. (f) DNA-independent association of SL1 with the pSER domain. FanChIP analysis, as described in c, was performed, followed by DNaseI treatment. (g) Association of SL1 with AEP. FLAG-tagged AF4 was transiently expressed in 293T cells and subjected to fanChIP–WB. The pBICEP2–AF4 vector was used for transient expression. (h) Association of SL1 with MLL–AEP fusion proteins. FLAG-tagged MLL–AEP fusion proteins were co-expressed with Xpress-tagged AF4 (xAF4) and HA-tagged MENIN (MENINh) in 293T cells and subjected to fanChIP–WB. The pCMV5-MLL fusion vectors, the pcDNA 3.1 hygro (+)-MEN1-HA vector and the pcDNA4 HisMax-AF4 vector were used for transient expression.