ATF-2 and C/EBPα Can Form a Heterodimeric DNA Binding Complex in Vitro

244

214

The CHOP gene is transcriptionally induced by amino acid starvation. We have previously identified a genomic cis-acting element (amino acid response element (AARE)) involved in the transcriptional activation of the human CHOP gene by leucine starvation and shown that it binds the activating transcription factor 2 (ATF2). The present study was designed to identify other transcription factors capable of binding to the CHOP AARE and to establish their role with regard to induction of the gene by amino acid deprivation. Electrophoretic mobility shift assay and transient transfection experiments show that several transcription factors that belong to the C/EBP or ATF families bind the AARE sequence and activate transcription. Among all these transcription factors, only ATF4 and ATF2 are involved in the amino acid control of CHOP expression. We show that inhibition of ATF2 or ATF4 expression impairs the transcriptional activation of CHOP by amino acid starvation. The transacting capacity of ATF4 depends on its expression level and that of ATF2 on its phosphorylation state. In response to leucine starvation, ATF4 expression and ATF2 phosphorylation are increased. However, induction of ATF4 expression by the endoplasmic reticulum stress pathway does not fully activate the AARE-dependent transcription. Taken together our results demonstrate that at least two pathways, one leading to ATF4 induction and one leading to ATF2 phosphorylation, are necessary to induce CHOP expression by amino acid starvation. This work was extended to the regulation of other amino acid regulated genes and suggests that ATF4 and ATF2 are key components of the amino acid control of gene expression.

Section: Methodsmentioning

confidence: 99%

Section: Transcription Factors That Belong To the C/ebp And Atf Familmentioning

confidence: 99%

Induction of CHOP Expression by Amino Acid Limitation Requires Both ATF4 Expression and ATF2 Phosphorylation

Avérous

Bruhat

Jousse

et al. 2004

244

214

“…For example, in the CD11c and rat cytochrome CYP2D5 genes, C/EBP acts synergistically with the transcription factor Sp1 to enhance gene expression through a mechanism whereby Sp1 facilitates the binding of C/EBP to a low affinity site in the DNA (48,49). C/EBP also interacts with retinoblastoma protein and various other transcriptional activators or co-activators such as c-Myb, PU.1, and ATF-2 (41,50). In the osteocalcin gene, C/EBP␤ binds to DNA and, in turn, binds to the transcription factor Runx2/Cbfa1 to greatly enhance gene expression (32).…”

Section: Figmentioning

confidence: 99%

CCAAT/Enhancer-binding Proteins β and δ Mediate the Repression of Gene Transcription of Cartilage-derived Retinoic Acid-sensitive Protein Induced by Interleukin-1β

Okazaki

et al. 2002

Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a secreted protein expressed by chondrocytes; the expression is repressed by interleukin 1␤ (IL-1␤). To investigate the transcriptional mechanism, by which CD-RAP expression is suppressed by IL-1␤, deletion constructs of the mouse CD-RAP promoter were transfected into rat chondrocytes treated with or without IL-1␤. The results revealed an IL-1␤-responsive element located between ؊2138 and ؊2068 bp. As this element contains a CAAT/enhancer-binding protein (C/EBP) motif, the function of C/EBP␤ and C/EBP␦ was examined. IL-1␤ stimulated the expression of C/EBP␤ and -␦, and the direct binding of C/EBP␤ to the C/EBP motif was confirmed. The ؊2251-bp CD-RAP promoter activity was down-regulated by co-transfection with C/EBP expression vectors. Mutation of the C/EBP motif abolished the inhibitory response to IL-1␤. Additionally, C/EBP expression vectors were found to down-regulate the construct containing the promoter and enhancer of the type II collagen gene. Finally, the enhancer factor, Sox9, was shown to bind adjacent to the C/EBP site competing with C/EBP binding. Taken together, these results suggest that C/EBP␤ and -␦ may play an important role in the IL-1␤-induced repression of cartilage-specific proteins and that expression of matrix proteins will be influenced by the availability of positive and negative trans-acting factors.

“…Osteoblast markers (OC, Runx), which are not present in soft tissues, are shown for comparison. The abundance of C/EBP␤ and -␦ in bone prompted examination of expression of the C/EBP family members from growth to differentiation stages of osteoblasts in vitro, representing proliferation (days 3-5), matrix maturation (days [7][8][9][10][11][12], and the mineralization stage (days 19 -22), reflected by peak levels of histone H4, alkaline phosphatase (ALP), and OC, respectively (Fig. 1B).…”

Section: Site-directed Mutagenesis and Expression Constructs-constructsmentioning

confidence: 99%

CCAAT/Enhancer-binding Proteins (C/EBP) β and δ Activate Osteocalcin Gene Transcription and Synergize with Runx2 at the C/EBP Element to Regulate Bone-specific Expression

Gutiérrez

Javed

Tennant

et al. 2002

238

231

CCAAT/enhancer-binding proteins (C/EBP) are critical determinants for cellular differentiation and cell typespecific gene expression. Their functional roles in osteoblast development have not been determined. We addressed a key component of the mechanisms by which C/EBP factors regulate transcription of a tissue-specific gene during osteoblast differentiation. Expression of both C/EBP␤ and C/EBP␦ increases from the growth to maturation developmental stages and, like the bone-specific osteocalcin (OC) gene, is also stimulated 3-6-fold by vitamin D 3 , a regulator of osteoblast differentiation. We characterized a C/EBP enhancer element in the proximal promoter of the rat osteocalcin gene, which resides in close proximity to a Runx2 (Cbfa1) element, essential for tissuespecific activation. We find that C/EBP and Runx2 factors interact together in a synergistic manner to enhance OC transcription (35-40-fold) in cell culture systems. We show by mutational analysis that this synergism is mediated through the C/EBP-responsive element in the OC promoter and by a direct interaction between Runx2 and C/EBP␤. Furthermore, we have mapped a domain in Runx2 necessary for this interaction by immunoprecipitation. A Runx2 mutant lacking this interaction domain does not exhibit functional synergism. We conclude that, in addition to Runx2 DNA binding functions, Runx2 can also form a protein complex at C/EBP sites to regulate transcription. Taken together, our findings indicate that C/EBP is a principal transactivator of the OC gene and the synergism with Runx2 suggests that a combinatorial interaction of these factors is a principal mechanism for regulating tissue-specific expression during osteoblast differentiation.