Mutational analysis of Ikaros binding sites in human gamma-satellite DNA. (A) Analysis of Ikaros transcripts in RL5 cells by RT-PCR using a set of primers specific for alternatively spliced variants. Sequences of primers for RT–PCR are described in Supplemental Table S10. (B) Western analysis of MEL cells extract using IK-CTS antibodies that recognize most of Ikaros isoform and IK-H antibodies that specifically recognize the largest isoform. Note that in MEL cells IK-H is approximately fivefold less abundant than IK-VI isoform. (C) Schemes of three variants of a 4-mer-based gamma-satellite array used for the analysis of antisilencing activity at the RL5 locus. Two conservative bipartite Ikaros binding motifs, “a” and “b,” are present in monomers 3 and 4. In a monomer 5, the motifs are highly diverged and cannot bind Ikaros. Two mutant forms of the array were generated, IHBSM and IBSM, resulting in failure of binding of IK-H isoform and all isoforms of Ikaros, correspondingly. (D) Sequences of original and mutated Ikaros binding sites in 4-mer-based arrays. (E) Effect of IHBSM and IBSM mutations on antisilencing activity of gamma-satellite DNA at the locus RL5. (F,G) ChIP analysis of wild type and IHBSM and IBSM gamma-satellite arrays using IK-CTS and IK-H antibodies. Sequences of primers that specifically amplify two nearest monomers in the arrays are presented in Supplemental Table S10.