SET1 and NURF complexes are responsible for chromatin barrier activity of the 5′HS4 insulator. FACS analysis of transgenic IL2R expression. (A) Clone 9 cells contain 13 copies of the human IL2R reporter transgene driven by the chicken βA-globin promoter and the β/ϵ enhancer and integrated stably into the genome of the chicken erythroid 6C2 cell line. After withdrawal of hygromycin selection, the transgenes became transcriptionally silent in 20 days. (B) The 809 cells contain 4 copies of the IL2R transgenes flanked by the 5′HS4 insulators, and expression of IL2R is maintained in this fully insulated line culture (809-pSuper). (C) Western blot analysis of endogenous nonerythroid or erythroid specific proteins in 6C2 809 cells harboring vector control and shSET1 (left) or shBPTF (right). The SET1 KD clone shows a significant reduction of the SET1 protein but not broadly expressed transcription factors or the erythroid-specific transcription factor GATA-1. (D) Quantitative RT-PCR of BPTF mRNA levels isolated from vector control–transfected and shBPTF-transfected cells. In 809 cells, depletion of SET1 (E) or BPTF (F) resulted in a rapid loss of chromatin barrier activity, as indicated by transcription silencing of the transgenic IL2R due to chromosomal position effect silencing. The gated bar designates IL2R-negative cells. Results from 10 000 cells are shown. The y-axis is the number of cells and the x-axis is the fluorescence intensity.