Genomic structure and transcription start site (TSS) of the EEN gene. (A) Schematic diagram of the structure of the contig-containing EEN gene and head-to-head–located CAF1 gene. (B) Identification of the TSS of the EEN gene by RNase protection assay. The sequencing result of M13mp18 is shown as the length reference (left 4 lanes). The corresponding M13mp18 sequence is shown at the bottom. The protected RNA band is indicated by the arrow. (C) Further verification of the EEN TSS by primer extension assay. A specific reverse transcription primer EEN-exten was end-labeled with γ-³²P by T4 polynucleotide kinase, annealed to 20 μg total RNA from HL60 cells, and extended by reverse transcriptase. The extension product (lane 3) was analyzed on 7M urea, 8% polyacrylamide gel along with a ³²P-labeled 140 bp PCR product (lane 2) and MspI-digested pBR322 (lane 1). Lane 4 was loaded with blank control without RNA in the reverse transcription reaction. (D) The genomic sequence containing the TSS of the EEN gene. The first exon is underlined and the start codon is bolded and boxed. TSS is used as the reference point (+1, with an enlarged G) for numbering the EEN promoter sequence. The positions of the SP1 and AML1 binding sites are also boxed.