Multiple forms of C/EBP beta bind the EFII enhancer sequence in the Rous sarcoma virus long terminal repeat.

177

240

Expression of human asparagine synthetase (ASNS), which catalyzes asparagine and glutamate biosynthesis, is transcriptionally induced following amino acid deprivation. Previous overexpression and electrophoresis mobility shift analysis showed the involvement of the transcription factors ATF4, C/EBP␤, and ATF3-FL through the nutrient-sensing response element-1 (NSRE-1) within the ASNS promoter. Amino acid deprivation caused an elevated mRNA level for ATF4, C/EBP␤, and ATF3-FL, and the present study established that the nuclear protein content for ATF4 and ATF3-FL were increased during amino acid limitation, whereas C/EBP␤-LIP declined slightly. The total amount of C/EBP␤-LAP protein was unchanged, but changes in the distribution among multiple C/EBP␤-LAP forms were observed. Overexpression studies established that ATF4, ATF3-FL, and C/EBP␤-LAP could coordinately modulate the transcription from the human ASNS promoter. Chromatin immunoprecipitation demonstrated that amino acid deprivation increased ATF3-FL, ATF4, and C/EBP␤ binding to the ASNS promoter and enhanced promoter association of RNA polymerase II, TATA-binding protein, and TFIIB of the general transcription machinery. A time course revealed a markedly different temporal order of interaction between these transcription factors and the ASNS promoter. During the initial 2 h, there was a 20-fold increase in ATF4 binding and a rapid increase in histone H3 and H4 acetylation, which closely paralleled the increased transcription rate of the ASNS gene, whereas the increase in ATF3-FL and C/EBP␤ binding was considerably slower and more closely correlated with the decline in transcription rate between 2 and 6 h. The data suggest that ATF3-FL and C/EBP␤ act as transcriptional suppressors for the ASNS gene to counterbalance the transcription rate activated by ATF4 following amino acid deprivation.

Section: Atf3-fl Atf4 and C/ebp␤ Protein Content Responds To Aminosupporting

confidence: 57%

Amino Acid Deprivation Induces the Transcription Rate of the Human Asparagine Synthetase Gene through a Timed Program of Expression and Promoter Binding of Nutrient-responsive Basic Region/Leucine Zipper Transcription Factors as Well as Localized Histone Acetylation

Chen¹,

Pan²,

Dudenhausen

et al. 2004

177

240

“…It is known that the regulation of gene transcription by C/EBP␤ involves a precise balance of both activator and inhibitor forms (43,48,106). When p20/C/EBP␤ (LIP) was transfected along with RhoA, the activation of the SRE was efficiently inhibited.…”

Section: Discussionmentioning

confidence: 95%

Activation of Serum Response Factor by RhoA Is Mediated by the Nuclear Factor-κB and C/EBP Transcription Factors

Montaner¹,

Perona²,

Saniger³

et al. 1999

The activity of the transcription factor NF-B can be modulated by members of the Rho family of small GTPases (Perona, R., Montaner, S., Saniger, L., Sá nchez-Pé rez, I., Bravo, R., and Lacal, J. C. (1997) Genes Dev. 11, 463-475). Ectopic expression of RhoA, Rac1, and Cdc42Hs proteins induces the translocation of NF-B dimers to the nucleus, triggering the transactivation of the NF-B-dependent promoter from the human immunodeficiency virus. Here, we demonstrate that activation of NF-B by RhoA does not exclusively promote its nuclear translocation and binding to the specific B sequences. NF-B is also involved in the regulation of the transcriptional activity of the c-fos serum response factor (SRF), since the activation of a SRE-dependent promoter by RhoA can be efficiently interfered by the double mutant IB␣S32A/S36A, an inhibitor of the NF-B activity. We also present evidence that RelA and p50 NF-B subunits cooperate with the transcription factor C/EBP␤ in the transactivation of the 4 ؋ SRE-CAT reporter. Furthermore, RhoA increases the levels of C/EBP␤ protein, facilitating the functional cooperation between NF-B, C/EBP␤, and SRF proteins. These results strengthen the pivotal importance of the Rho family of small GTPases in signal transduction pathways which modulate gene expression and reveal that NF-B and C/EBP␤ transcription factors are accessory proteins for the RhoA-linked regulation of the activity of the SRF.Gene expression is regulated by the interplay of different transcription factors which bind to specific DNA recognition motifs and cooperate with the basal machinery to initiate transcription. During the last few years, an emerging body of evidence is revealing the importance of crossed interactions between members of distinct families of transcription factors to form higher complexes, enabling the accurate regulation of this process. The serum response element (SRE) 1 is a specific DNA sequence which is found in the promoter of several immediateearly genes (2). The prototypic c-fos SRE binds a ternary complex composed of a homodimer of p67 SRF (serum response factor) and a third subunit, p62 TCF (ternary complex factor), which belongs to the Ets family of accessory proteins. These TCF factors have the ability to bind a purine-rich motif 5Ј to the SRF-binding site, known as the Ets recognition domain, only when SRF is bound to DNA, and include Elk-1, SAP-1, and SAP-2/ERP/NET proteins. The formation of this SRE bindingternary complex requires the conserved B-box motif of the Ets subunits and sequences located in the core domain of the SRF protein (core SRF ), which is a region that is also responsible of its DNA binding and dimerization capabilities (2-8).The SRE has shown to be necessary and sufficient for the rapid induction of the c-fos proto-oncogen in response to different external stimuli such as serum, growth factors, and phorbol esters (2, 9). Furthermore, this DNA motif is a point of convergence of different signal transduction cascades activated by an extensive range of agonists. The regulat...

“…The amplified fragments with EcoRI ends were inserted into the EcoRI site of pGAL4. pVP16-LIP was constructed by digestion of pRSETA-LIP (14) with BamHI, treatment with DNA polymerase I at 4°C to generate blunt ends, and subsequent digestion with HindIII to release a 582-bp fragment. This fragment was inserted into a pVP16 plasmid, digested with MluI, treated with DNA polymerase I at 4°C, and digested with HindIII.…”

Section: Methodsmentioning

confidence: 99%

“…The N-terminal half of p38 and p35 contains a strong transactivation domain, whereas p20-C/EBP␤ lacks this transactivation domain and acts as an inhibitor of transcription. Many fibroblast cell types, such as NIH 3T3 cells, express both the long (35 kDa) and short (20 kDa) forms of C/EBP␤ at approximately equal levels (14). Both p35 and p20 homodimers as well as p35-p20 heterodimers bind to the SRE with equivalent affinity.…”

mentioning

confidence: 99%

Ras Regulates the Association of Serum Response Factor and CCAAT/Enhancer-binding Protein β

Hanlon¹,

Sealy

1999

The serum response element (SRE) is a promoter element essential for transcriptional activation of immediate early genes, such as c-fos and early growth response-1, by mitogenic signals. Several transcription factors bind the SRE, including the serum response factor (SRF), the ternary complex factor, and the CCAAT/ enhancer-binding protein ␤ (C/EBP␤). The C/EBP␤ mRNA encodes three translation products of 38, 35, and 20 kDa. p35-C/EBP␤ activates transcription of the SRE in an SRF-dependent fashion, whereas p20-C/EBP␤, which initiates at an internal in-frame methionine, lacks a transactivation domain and inhibits transcription. We show that SRF and C/EBP␤ interact in vivo through the DNA binding domain of SRF and the C terminus of C/EBP␤ common to p35/38 and p20. Therefore, like the ternary complex factor, C/EBP␤ may be recruited to the SRE not only by binding to the DNA, which is not a high affinity site, but also by proteinprotein interactions with SRF. Strikingly, in both the mammalian two-hybrid assay and in vivo coimmunoprecipitations, the association of SRF and p35-C/EBP␤ but not p20-C/EBP␤ is dramatically stimulated by activated Ras. Furthermore, mutation of the threonine within a mitogen-activated protein kinase consensus motif in the C terminus of C/EBP␤ eliminates the response to Ras. These results suggest a new mechanism by which mitogenic signals may influence transcription activity of the SRE by selectively promoting protein-protein interactions between SRF and the transactivator p35-C/EBP␤.The c-fos proto-oncogene is a prototypic member of the set of "immediate early" genes whose transcription is rapidly activated in the absence of protein synthesis by mitogenic signals (1). Within the promoter of the c-fos gene is a 26-bp 1 regulatory sequence, the serum response element (SRE), that mediates the rapid and transient induction of the c-fos gene by serum and growth factors (2). The SRE binds a ubiquitous transcription factor, the serum response factor (SRF), which is essential but not sufficient for serum and growth factor activation of SRE (3-5). SRF is an obligatory member of a multiprotein complex that occupies the SRE and serves as a convergence point for multiple signal transduction pathways. Thus, growth factors that activate the Ras-Raf-extracellular signal-regulated kinase cascade and phorbol esters target a family of transcription factors containing an N-terminal Ets domain that form a ternary complex with SRF and SRE DNA (6). c-fos promoter mutants that cannot bind the ternary complex factor (TCF) remain responsive to serum induction through a second TCFindependent pathway that still requires SRF and can involve certain Rho family GTPases (5,7,8). The role of accessory factors in the TCF-independent pathway has yet to be completely defined.We have observed that maximal TCF-independent serum induction of the SRE requires SRF and another transcription factor, C/EBP␤ (9, 10). C/EBP␤, which binds to the SRE overlapping and immediately 3Ј to SRF, is a member of the basic leucine zipper family of t...