fig3

Figure 3. SRF153(A3) mutant facilitates cell replication but not myocyte differentiation in the rescue of lentiviral infected SRF null murine ES cells. (A) A reproduction drawing of the key MADS box DNA contacts is shown^[7]. Note that alanine substitution at Lys154 was the most critical residue for DNA binding between Amino Acids 153 and 155. (B) A computational program^[27] predicted ΔΔG at Lys154 was -1.996, stronger than for N153 and/or L155. (C) EMSA of lentiviral expressed NKX2-5 and/or Gata4 in the context of the alpha cardiac actin promoter failed to stabilize the single alanine point mutant, Lys154Ala, a critical base contact. (D) EMSA of expressed ETS factor, ELK1, in the context of a c-fos promoter, stabilized SRF mutants, SRF154(A) and SRF153(A3), DNA binding, as revealed by the appearance of ternary complex factors (TCF). (E) Rescue of null SRF murine ES cells with lentiviral infections of SRFwt and SRF153(A3) was analyzed by heat maps of expressed genes generated on an Affymetrix array. Note that rescue with SRF153(A3) increased the expression of stem cell marker genes and cyclins but did not support the expression of many cardiac-specific genes and assembly genes needed for sarcomerogenesis.