Human AP-endonuclease 1 and hnRNP-L interact with a nCaRE-like repressor element in the AP-endonuclease 1 promoter

Abstract

The major human AP-endonuclease 1 (APE1) is a multifunctional protein that plays a central role in the repair of damaged DNA by acting as a dual-function nuclease in the base excision repair pathway. This enzyme was also independently identified as a redox activator of AP-1 DNA-binding activity and has subsequently been shown to activate a variety of transcription factors via a redox mechanism. In a third distinct role, APE1 was identified as a component of a trans-acting complex that acts as a repressor by binding to the negative calcium responsive elements (nCaRE)-A and nCaRE-B, which were first discovered in the promoter of the human parathyroid gene and later in the APE1 promoter itself. Here we show that the nuclear protein complex which binds to the nCaRE-B2 of the hAPE1 gene contains APE1 itself and the heterogeneous nuclear ribonucleoprotein L (hnRNP-L). The interaction between the APE1 and hnRNP-L proteins does not require the presence of nCaRE-B2. Our results support the possibility that the APE1 gene is down-regulated by its own product, which would be the first such example of the regulation of a DNA repair enzyme, and identify a novel function of hnRNP-L in transcriptional regulation.

Figure 1

Purification of nCaRE-B2-binding proteins. (A) EMSA of fractions (1–14) eluted from a nCaRE-B2–Sepharose column with 0.5 NaCl. Lanes with probe alone (free) and control (con) unfractionated HeLa nuclear extracts are indicated. (B) (Left) Coomassie brilliant blue stained PVDF membrane. Lane 1, marker proteins; lane 2, eluted fraction 5. Bands at 66 and 36 kDa are indicated by arrows. (Right) Western analysis of eluted fraction 5 (lane 3) and control HeLa nuclear extract (lane 4) using anti-APE1 antibody. (C) Sequences of three internal peptides from the 66 kDa species and the numbers indicate the positions of the terminal residues in the hnRNP-L polypeptide.

Figure 2

Competitive EMSA using mutant nCaRE-B2 sequence. Sequence-specific binding of HeLa nuclear extract to nCaRE-B2. Lanes 1 and 6, free probe; lanes 2–5 and 7–9, 20 µg HeLa extract with 20-fold molar excess each of unlabeled nCaRE-B2 (lane 3), mutant nCaRE-B2 (lane 8), nCaRE-A probe (lane 9) or after preincubation of extract with 4 µl of anti-APE1 (lane 4) or anti-hnRNP-L antibody (lane 5). Lanes 2 and 7, HeLa nuclear extract alone.

Figure 3

EMSA with extracts of transiently transfected COS-1 cells. (A) Formation of the nCaRE-B2-binding complex with increasing amounts of nuclear extract of untransfected COS-1 cells. (B) Western analysis of transfected cell extracts. Lane 1, control (C); lane 2, cells transfected with empty vector (EV); lane 3, APE1 vector (A); lane 4, hnRNP-L vector (L); lane 5, both APE1 and hnRNP-L expression vectors (A + L). The protein bands were detected with anti-Xpress antibody. (C) nCaRE-B2 binding with 3.5 µg nuclear extract. Lane 1, free probe; lane 2, control cells; lane 3, cells transfected with empty vector; lane 4, APE1 cDNA; lane 5, hnRNP-L cDNA; lane 6, both APE1 and hnRNP-L cDNAs. The spots observed in control lanes 1 and 2 in the same position as the gel shifted band are artifacts. (D) Densitometric analysis (arbitrary scale) of nCaRE-B2-binding complexes shown in (C); nd, not detected. Error bars represent standard deviation (six samples).

Figure 4

Reconstitution of the nCaRE-B2-binding complex and peptide interference assay. (A) Domain organization of the GST–hnRNP-L fusion protein. (B) Purification of recombinant hnRNP-L protein from Sf9 cells. (Left) Coomassie brilliant blue staining: lanes 1 and 4, protein standards; lane 2, uninfected Sf9 extract; lane 3, Sf9 extract expressing GST–hnRNP-L; lanes 5 and 6, purified GST–hnRNP-L before (lane 5) and after (lane 6) cleavage with thrombin. (Right) Western analysis of the proteins in lanes 5 and 6 with anti-hnRNP-L antibody. (C) In vitro reconstitution of nCaRE-B2-binding activity by EMSA. Lane 1, no protein; lane 2, HeLa nuclear extract; lane 3, APE1 and hnRNP-L; lane 4, APE1 alone; lane 5, hnRNP-L alone.

Figure 5

Interaction between hnRNP-L and APE1. HeLa nuclear extract (NE) (100 µg) was incubated with recombinant GST–hnRNP-L in the presence or absence of nCaRE-B2 oligo, mixed with glutathione–agarose resin and the bound proteins eluted with glutathione for western analysis. Lane 1, HeLa extract alone; lane 2, with GST–hnRNP-L fusion protein; lane 3, both GST–hnRNP-L and nCaRE-B2 oligo.

Figure 6

Partial sequence alignment of basic regions in hnRNP-L, HLF, c-Fos and c-Jun. The sequence containing the Cys residue involved in redox regulation is boxed. The numbers indicate residue numbers of these proteins.