MEF2D associates with Xnr1 regulatory elements.
(A) Chromatin immunoprecipitation (ChIP): Embryos were injected with mRNA encoding MEF2D-Flag and at stage 10, crosslinked sheared chromatin was prepared. Chromatin was immunoprecipitated with anti-Flag (polyclonal, Sigma) or with pre immune serum (control) and was subjected to a qPCR reaction with several pairs of primers (left). Expression of the injected MEF2D-Flag protein was analyzed by Western blot (right). (B) Upper panel: Xnr1 promoter sequence (proximal region) with highlighted putative binding sites of MEF2. PE-proximal element; IE1, 2-Intermediate element 1, 2; DE-distal element; TBX1, 2- T box binding sites (VegT) . Arrows show the two transcription start site and “M” the translation initiation codon. Lower panel: EMSA of each of the MEF2 binding elements coupled with protein extracts of stage 9 control embryos as well as embryos injected with mef2d-flag mRNA. Anti-flag antibody (1 µl, 0.1 µg/µl) was included in some reaction mixtures while unlabeled homologous double stranded oligonucleotides in 100 fold excess over the probe was included in others, as indicated. Unbound probes are not shown. Arrow indicates the MEF2D-DNA complex. Arrowhead indicates the Anti MEF2-MEF2D-DNA complex. (C) 293T HEK cells were transfected as indicated. Thirty six hours later, proteins were extracted and luciferase activity was measured and was normalized to total protein levels. Activity of the reported gene with an empty vector was set to a value of 1 and values of other treatments were standardized accordingly. Means of two independent experiments are presented.