Synergistic Activation of the Germline ε Promoter Mediated by Stat6 and C/EBPβ

Transcription of the Ig H chain germline transcripts is a prerequisite for class switching. Expression of the e germline transcript is induced by IL-4 and requires the integrity of a composite IL-4 response element. The element is bound by the IL-4-inducible transcription factor Stat6 and one or more members of the CAAT/enhancer-binding protein (C/EBP) family, a constitutively expressed class of transcription factors. Here, we show that Stat6 and C/EBPβ cooperate to synergistically activate transcription from the e element. The effect was most pronounced in lymphoid cells, and the activation domains of both proteins were required to achieve this synergy. Although other members of the C/EBP family are able to bind the element, very little cooperativity was seen with C/EBPα and none with C/EBPγ. In fact, C/EBPγ was able to inhibit IL-4-induced reporter activity. Stat6 and C/EBPβ bind the IL-4 response element simultaneously. The fast dissociation rate apparent when Stat6 binds this DNA element alone is slowed when C/EBPβ binds at the neighboring site. These data suggest a mechanism whereby C/EBPβ stabilizes Stat6 binding at this element, thereby increasing the likelihood that both of their activation domains will interact, possibly with other factors, to activate transcription in an IL-4-dependent manner.

[1]  A. Bradley,et al.  Impaired energy homeostasis in C/EBP alpha knockout mice , 1995, Science.

[2]  J. Ihle.  Cytokine receptor signalling , 1995, Nature.

[3]  K. Calame,et al.  Ig/EBP (C/EBP gamma) is a transdominant negative inhibitor of C/EBP family transcriptional activators. , 1995, Nucleic acids research.

[4]  J. Darnell.  STATs and gene regulation. , 1997, Science.

[5]  J. O’Shea.  Jaks, STATs, cytokine signal transduction, and immunoregulation: are we there yet? , 1997, Immunity.

[6]  D. Tenen,et al.  CCAAT Enhancer-Binding Protein ( C / EBP ) and AML 1 ( CBF a 2 ) Synergistically Activate the Macrophage Colony-Stimulating Factor Receptor Promoter , 1995 .

[7]  R. Cortese,et al.  Lymphoproliferative disorder and imbalanced T‐helper response in C/EBP beta‐deficient mice. , 1995, The EMBO journal.

[8]  K. Calame,et al.  Ig/EBP-1: a ubiquitously expressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP. , 1990, Genes & development.

[9]  R. Umek,et al.  Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells. , 1991, Genes & development.

[10]  K. Calame,et al.  Limited expression of C/EBP family proteins during B lymphocyte development. Negative regulator Ig/EBP predominates early and activator NF-IL-6 is induced later. , 1994, Journal of immunology.

[11]  L. Sanders,et al.  Functional differences between Stat3alpha and Stat3beta , 1997, Molecular and cellular biology.

[12]  W. Paul,et al.  Lymphocyte responses and cytokines , 1994, Cell.

[13]  M. Ptashne,et al.  Transcriptional activation by recruitment , 1997, Nature.

[14]  A. Baldwin,et al.  Functional and physical associations between NF-kappa B and C/EBP family members: a Rel domain-bZIP interaction. , 1993, Molecular and cellular biology.

[15]  J. Stavnezer,et al.  Activation of the Ig germ-line gamma 1 promoter. Involvement of C/enhancer-binding protein transcription factors and their possible interaction with an NF-IL-4 site. , 1994, Journal of immunology.

[16]  Nobuaki Yoshida,et al.  Targeted disruption of the NF-IL6 gene discloses its essential role in bacteria killing and tumor cytotoxicity by macrophages , 1995, Cell.

[17]  M. Tewari,et al.  Fas- and Tumor Necrosis Factor-induced Apoptosis Is Inhibited by the Poxvirus crmA Gene Product (*) , 1995, The Journal of Biological Chemistry.

[18]  J. Lammers,et al.  STAT3β, a Splice Variant of Transcription Factor STAT3, Is a Dominant Negative Regulator of Transcription* , 1996, The Journal of Biological Chemistry.

[19]  S. McKnight,et al.  Differentiation of T‐helper lymphocytes: selective regulation by members of the STAT family of transcription factors , 1996, Genes to cells : devoted to molecular & cellular mechanisms.

[20]  M. Kaplan,et al.  Stat6 is required for mediating responses to IL-4 and for development of Th2 cells. , 1996, Immunity.

[21]  M. Atchison,et al.  Multiple proteins physically interact with PU.1. Transcriptional synergy with NF-IL6 beta (C/EBP delta, CRP3). , 1995, Journal of immunology.

[22]  B. Chait,et al.  DNA binding of in vitro activated Stat1 alpha, Stat1 beta and truncated Stat1: interaction between NH2‐terminal domains stabilizes binding of two dimers to tandem DNA sites. , 1996, The EMBO journal.

[23]  S. McKnight,et al.  Components of a Stat recognition code: evidence for two layers of molecular selectivity. , 1995, Immunity.

[24]  S. Akira,et al.  Essential role of Stat6 in IL-4 signalling , 1996, Nature.

[25]  T. Graf,et al.  Myb and NF-M: combinatorial activators of myeloid genes in heterologous cell types. , 1993, Genes & development.

[26]  G. Stark,et al.  Complementation of a mutant cell line: central role of the 91 kDa polypeptide of ISGF3 in the interferon‐alpha and ‐gamma signal transduction pathways. , 1993, The EMBO journal.

[27]  D. Herschlag,et al.  Synergism in transcriptional activation: a kinetic view. , 1993, Genes & development.

[28]  S. McKnight,et al.  An interleukin-4-induced transcription factor: IL-4 Stat. , 1994, Science.

[29]  Kenneth M. Murphy,et al.  Functional diversity of helper T lymphocytes , 1996, Nature.

[30]  E. Ziff,et al.  cAMP stimulates the C/EBP-related transcription factor rNFIL-6 to trans-locate to the nucleus and induce c-fos transcription. , 1991, Genes & development.

[31]  R. Yamanaka,et al.  A Novel Human CCAAT/Enhancer Binding Protein Gene, C/EBPϵ, Is Expressed in Cells of Lymphoid and Myeloid Lineages and Is Localized on Chromosome 14q11.2 Close to the T-Cell Receptor α/δ Locus , 1996 .

[32]  J. Darnell,et al.  Transcriptional responses to polypeptide ligands: the JAK-STAT pathway. , 1995, Annual review of biochemistry.

[33]  M. Rosenfeld,et al.  Calcium-Regulated Phosphorylation Within the Leucine Zipper of C/EBPβ , 1992, Science.

[34]  N. Reich,et al.  Requirement of tyrosine phosphorylation for rapid activation of a DNA binding factor by IL-4. , 1993, Science.

[35]  U. Schindler,et al.  Requirements for interleukin-4-induced gene expression and functional characterization of Stat6 , 1996, Molecular and cellular biology.

[36]  J. Stavnezer,et al.  Characterization of an interleukin 4 (IL-4) responsive region in the immunoglobulin heavy chain germline epsilon promoter: regulation by NF- IL-4, a C/EBP family member and NF-kappa B/p50 , 1995, The Journal of experimental medicine.

[37]  A. Leutz,et al.  The NF‐M transcription factor is related to C/EBP beta and plays a role in signal transduction, differentiation and leukemogenesis of avian myelomonocytic cells. , 1993, The EMBO journal.