PURIFICATION AND CHARACTERIZATION OF NUCLEAR FACTORS BINDING TO THE NEGATIVE REGULATORY ELEMENT-D OF HUMAN APOLIPOPROTEIN A-II PROMOTER - ANEGATIVE REGULATORY EFFECT IS REVERSED BY GABP, AN ETS-RELATED PROTEIN

We have previously shown that transcription of the human apolipoprotei n A-II (apoA-II) gene is controlled by a complex set of regulatory ele ments [Cardot et al. (1943) Biochemistry 32, 9080-9093] We have also i dentified previously described, as well as new activities which bind t o these elements and influence to varying degrees the transcription of the apoA-II gene. DNA binding and competition assays indicated that e lement D binds three hew activities, designated AIID1, AIID2, and AIID 4, as well as an activity related to C/EBP. Activities AIID1, AIID2, a nd AIID4 were purified aad characterized further in order to determine their function on the transcriptional regulation of human apoA-II gen e. SDS-PAGE analysis as well as photoaffinity cross-linking of the aff inity-purified AIID2 showed that it consists of three proteins with mo lecular masses ranging between 54 and 63 kDa. The amino acid sequence of tryptic peptides obtained from AIID2 protein bands revealed that it is homologous to GABP, an Ets-related protein. Similar analysis showe d that affinity-purified AIIID4 has an apparent molecular mass of 130 kDa. AIID1 activity was purified partially; in addition to binding to the apoA-II promoter, AIID1 also binds to the regulatory element C of apoCIII and may play a role in the transcriptional regulation of both genes. Methylation interference of G residues and permanganate modific ation of T residues indicated that the binding sites of AIID2 and AIID 4 were contiguous on element D. However, the binding site of AIID1 ove rlaps with the binding sites of both AIID2 and AIID4. This suggests th at the binding of AIID1 and AIID2 or of AIID1 and AIID4 may be mutuall y exclusive, whereas AIID2 and AIID4 may bind simultaneously. Transcri ption from a minimal promoter containing elements AB, C, and D of apoA -II increased 1.5- to 1.6-fold when element D is deleted, as well as b y promoter mutations which eliminated the binding of both AIID1 and/or AIID4 to element D, but permitted the binding of AIID2/GABP. The find ings suggest that element D has a negative regulatory role on apoA-II gene transcription when it is occupied by protein AIID1 and/or AIID4. This negative effect is reversed when element D is occupied only by th e regulatory factor AIID2/GABP.