Specific Interaction of Egr1 and c/EBPβ Leads to the Transcriptional Activation of the Human Low Density Lipoprotein Receptor Gene

Abstract

The sterol-independent regulatory element (SIRE) of the LDL receptor (LDLR) promoter mediates oncostatin M (OM)-induced transcription of the LDLR gene through a cholesterol-independent pathway. Our prior studies have detected specific associations of the zinc finger transcription factor Egr1 with the SIRE sequence in OM-stimulated HepG2 cells. Because the SIRE motif is composed of a c/EBP binding site and a cAMP response element, both of which are quite divergent from the classical GC-rich Egr1 recognition sequences, we hypothesized that Egr1 may regulate LDLR transcription through interacting with members of the c/EBP and CREB families. Here, we show that treating HepG2 cells with OM specifically leads to prominent increases of the levels of c/EBP␤ and Egr1 bound to the LDLR promoter in vivo. In vitro, the binding of Egr1 to the SIRE sequence is weak, but is strikingly enhanced in the presence of HepG2 nuclear extract. Mammalian two-hybrid assays demonstrate that the N-terminal transactivation domain of Egr1 specifically interacts with c/EBP␤ but not with c/EBP␣ or CREB. The OM treatment further enhances this interaction, resulting in a large increase in the Egr1 transactivating activity. The direct protein to protein contact between Egr1 and c/EBP␤ is also demonstrated by co-immunoprecipitation experiments. Furthermore, we show that a mutation of the phosphorylation motif of c/EBP␤ diminished the OM-stimulated interaction of Egr1 and c/EBP␤. Taken together, we provide strong evidence that Egr1 regulates LDLR transcription via a novel mechanism of protein-protein interaction with c/EBP␤. Regulation of the human LDL receptor (LDLR)1 gene transcription in liver cells involves cholesterol-dependent and -independent mechanisms. The sterol-regulatory element (SRE-1) plays a key role in the cholesterol-mediated negative feedback regulation of the LDLR transcription by interacting with the SRE-1-binding proteins (SREBPs), whose nuclear translocation is controlled by intracellular cholesterol levels (1). The sterol-independent regulatory element (SIRE) has been recently identified and shown to mediate the stimulatory effects of cytokine oncostatin M (OM), cAMP, and c/EBP␤ on LDLR transcription in HepG2 cells in a cholesterol-independent fashion (2). The SIRE motif (TGCTGTAAATGACGTGG) is located at the promoter region of Ϫ17 to Ϫ1, downstream of the SRE-1 and Sp1 binding sites. It is composed of a c/EBP binding site (Ϫ17 to Ϫ9) and a CRE site (Ϫ8 to Ϫ1). Mutations within the SIRE sequence do not affect the cholesterol-mediated suppression and only slightly lower basal promoter activity to levels 60 -80% of the wild-type sequence. However, alterations of nucleotides (even a single base) within the SIRE motif can totally prevent the OM-induced increase in the LDLR promoter activity.In vitro DNA binding assays (EMSA) have detected the binding of several basic leucine zipper DNA-binding proteins including c/EBP␤, CREB, ATF1, ATF2, ATF3, and c-Jun to the SIRE sequence (2). These DNA-protein complexes can be detected in unsti...

Early Growth Response 1 (Egr1) Regulates Cholesterol Biosynthetic Gene Expression

“…Our data provide a potential mechanistic explanation for the reduced lipid synthesis in Egr1 null mice in response to ethanol. In addition, Egr1 was previously implicated in the induction of the LDLR gene by oncostatin M, specifically through an interaction with a sterol-independent regulatory element in the LDLR promoter (42)(43)(44). Finally, our data are consistent with a bioinformatics analysis implicating Egr1 in induction of cholesterol biosynthetic genes by growth factors (45).…”

Egr-1 TransactivatesBimGene Expression to Promote Neuronal Apoptosis

“…Bim, Egr-1, and specificity protein 1 (Sp1) constructs were cloned from rat cDNA and incorporated into the pshuttle-Flag-IRES-hrGFP-1 expression vector. The dominantnegative (dn) Egr plasmid was generated by PCR amplification of the zinc finger region (amino acids 322-427) of mouse Egr-1 from pCMV-Flag-NLS-dnEgr (amino acids 322-533) as described previously (Zhang et al, 2003). Briefly, the PCR product (forward primer, 5Ј-GTTTAGTGAAC-CGTCAGAAT-3Ј and reverse primer, 5Ј-AATTGATATCTTAGTCT-GCTTTCTTGTCCTTCT-3Ј) was cloned into the HindIII and EcoRV sites of the pCMV-Flag-NLS expression vector.…”

Transcriptional and epigenetic profiling of nutrient-deprived cells to identify novel regulators of autophagy

“…EGR1 is an immediate-early response gene, of which its expression can be induced within minutes after stimulation [30]. Mitogens [31,32], growth factors [33], and stress stimuli, such as cigarette smoke [34–36], hypoxia [37,38], and nutrient deprivation [39] regulate EGR1. For example, in agreement with our data, glucose restriction rapidly increases EGR1 protein levels in multiple cell lines [39].…”