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Fig. 4 | BMC Biology

Fig. 4

From: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation

Fig. 4

A defined 42 nucleotide stretch within the ICln promoter represents the minimal region essential for TIS7-regulated ICln transcription. a Proliferating TIS7 WT MSCs were transiently co-transfected with indicated ICln promoter-luciferase reporter constructs and with β-galactosidase expression plasmid for transfection control. The activity of the WT full length ICln promoter-driven luciferase reporter was set as 100 %. Values represent the mean ± SEM of at least three biological replicates. b Activities of luciferase reporter constructs driven by two or four (–154/–113) repeats from ICln promoter. Bars marked + TIS7 represent samples co-transfected with GFP-TIS7 plasmid vector. Control samples were co-transfected with GFP plasmid. c TIS7 binds to the minimal essential region necessary for the TIS7-regulated transcription of ICln. GST (lane 3) or GST-TIS7 (lanes 2, 4, and 5) recombinant proteins were incubated with the fluorescently labeled oligonucleotide probe containing the –154/–113 sequence of the ICln promoter. d Specific TIS7 binding to ICln minimal essential region (MER) determined by surface plasmon resonance measurements (SPR). Oligonucleotides representing either the ICln MER or its scrambled version were incubated with increasing concentrations of GST-TIS7 or GST alone as indicated. SPR steady-state response values (single values) are presented as a function of protein concentration (lines). Kd = dissociation constant. e ChIP analysis of TIS7 binding to the ICln promoter region. Chromatin from TIS7 WT and KO MSCs was immunoprecipitated as above using either specific antibody against TIS7 or control rabbit IgG

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