A competitive mechanism of CArG element regulation by YY1 and SRF: implications for assessment of Phox1/MHox transcription factor interactions at CArG elements.

In the promoters of many immediate early genes, including c-fos, CArG DNA regulatory elements mediate basal constituitive expression and rapid and transient serum induction. CArG boxes also occur in the promoters of muscle-specific genes, including skeletal alpha-actin, where it confers muscle-specific expression. These elements are regulated, at least in part, by the ubiquitous transcription factors serum response factor (SRF) and YY1. The homeobox transcription factor Phox1/MHox has also been implicated in regulation of the c-fos CArG element and is thought to function by facilitating SRF binding to DNA. Here, we provide in vitro and in vivo evidence that the mechanism of YY1 repression of CArG elements results from competition with SRF for overlapping binding sites. We describe in detail the binding sites of YY1 and SRF through serial point mutations of the skeletal alpha-actin proximal CArG element and identify a mutation that dramatically reduces YY1 binding but retains normal SRF binding. YY1 competes with SRF for binding to wild-type CArG elements, but not to this point mutant in vitro. This mutant is sufficient for muscle-specific expression in vivo but is much less sensitive to repression by YY1 overexpression. We utilized the YY1/SRF competition to address the role of Phox1 at these elements. Phox1 overexpression did not diminish YY1-mediated repression, suggesting that transcriptional activation by Phox1 does not result from enhanced SRF binding to these elements. These methods may prove to be useful for assessing interactions between other CArG element regulatory factors.

[1]  R. Treisman.  Identification and purification of a polypeptide that binds to the c‐fos serum response element. , 1987, The EMBO journal.

[2]  M. Gilman,et al.  YY1 facilitates the association of serum response factor with the c-fos serum response element , 1995, Molecular and cellular biology.

[3]  T. Cooper,et al.  Analysis of the upstream regions governing expression of the chicken cardiac troponin T gene in embryonic cardiac and skeletal muscle cells , 1988, The Journal of cell biology.

[4]  D. Grueneberg,et al.  Human and Drosophila Homeodomain Proteins That Enhance the DNA-Binding Activity of Serum Response Factor , 1992, Science.

[5]  V. Rangnekar,et al.  The serum and TPA responsive promoter and intron-exon structure of EGR2, a human early growth response gene encoding a zinc finger protein. , 1990, Nucleic acids research.

[6]  Richard Treisman,et al.  Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5′ element and c-fos 3′ sequences , 1985, Cell.

[7]  J. Ye,et al.  Characterization of the human granulocyte-macrophage colony-stimulating factor gene promoter: an AP1 complex and an Sp1-related complex transactivate the promoter activity that is suppressed by a YY1 complex , 1996, Molecular and cellular biology.

[8]  E. Olson,et al.  MHox: a mesodermally restricted homeodomain protein that binds an essential site in the muscle creatine kinase enhancer. , 1992, Development.

[9]  K. Walsh,et al.  Cardiac Muscle , 2019, Keynes & Aidley's Nerve and Muscle.

[10]  D. Nathans,et al.  Functional serum response elements upstream of the growth factor-inducible gene zif268 , 1989, Molecular and cellular biology.

[11]  T. Lee,et al.  Bifunctional transcriptional properties of YY1 in regulating muscle actin and c-myc gene expression during myogenesis. , 1994, Oncogene.

[12]  R. Weinberg,et al.  Multiple protein-binding sites in the 5'-flanking region regulate c-fos expression , 1986, Molecular and cellular biology.

[13]  Dana L. Smith,et al.  A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an α2 dimer , 1992, Cell.

[14]  T. Lee,et al.  Displacement of BrdUrd-induced YY1 by serum response factor activates skeletal alpha-actin transcription in embryonic myoblasts. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[15]  D. Grueneberg,et al.  Sequence-specific targeting of nuclear signal transduction pathways by homeodomain proteins , 1995, Molecular and cellular biology.

[16]  S. Gangopadhyay,et al.  Molecular and functional analysis of the muscle-specific promoter region of the Duchenne muscular dystrophy gene , 1990, Molecular and cellular biology.

[17]  E. Olson,et al.  Homeodomain protein MHox and MADS protein myocyte enhancer-binding factor-2 converge on a common element in the muscle creatine kinase enhancer. , 1994, The Journal of biological chemistry.

[18]  E. Ziff,et al.  The helix-loop-helix protein rE12 and the C/EBP-related factor rNFIL-6 bind to neighboring sites within the c-fos serum response element. , 1991, Oncogene.

[19]  T. Curran,et al.  Transcriptional activation and repression by Fos are independent functions: the C terminus represses immediate-early gene expression via CArG elements , 1990, Molecular and cellular biology.

[20]  M. Atchison,et al.  Isolation of a candidate repressor/activator, NF-E1 (YY-1, delta), that binds to the immunoglobulin kappa 3' enhancer and the immunoglobulin heavy-chain mu E1 site. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[21]  R J Schwartz,et al.  Identification of Novel DNA Binding Targets and Regulatory Domains of a Murine Tinman Homeodomain Factor, nkx-2.5(*) , 1995, The Journal of Biological Chemistry.

[22]  W. Liao,et al.  YY1 represses rat serum amyloid A1 gene transcription and is antagonized by NF-kappa B during acute-phase response , 1994, Molecular and cellular biology.

[23]  K. Chien,et al.  A conserved 28-base-pair element (HF-1) in the rat cardiac myosin light-chain-2 gene confers cardiac-specific and alpha-adrenergic-inducible expression in cultured neonatal rat myocardial cells , 1991, Molecular and cellular biology.

[24]  N. Rosenthal,et al.  The myosin light chain enhancer and the skeletal actin promoter share a binding site for factors involved in muscle-specific gene expression , 1991, Molecular and cellular biology.

[25]  B. Raught,et al.  YY1 represses beta-casein gene expression by preventing the formation of a lactation-associated complex , 1994, Molecular and cellular biology.

[26]  R. Treisman.  The serum response element. , 1992, Trends in biochemical sciences.

[27]  R. Schwartz,et al.  A combination of closely associated positive and negative cis-acting promoter elements regulates transcription of the skeletal alpha-actin gene , 1990, Molecular and cellular biology.

[28]  Richard Treisman,et al.  Isolation and properties of cDNA clones encoding SRF, a transcription factor that binds to the c-fos serum response element , 1988, Cell.

[29]  M. Seiki,et al.  Interaction of HTLV-1 Tax1 with p67SRF causes the aberrant induction of cellular immediate early genes through CArG boxes. , 1992, Genes & development.

[30]  K. Calame,et al.  An analysis of genes regulated by the multi-functional transcriptional regulator Yin Yang-1. , 1994, Nucleic acids research.

[31]  K. Walsh,et al.  Natural and synthetic DNA elements with the CArG motif differ in expression and protein-binding properties , 1991, Molecular and cellular biology.

[32]  K. Walsh.  Cross-binding of factors to functionally different promoter elements in c-fos and skeletal actin genes , 1989, Molecular and cellular biology.

[33]  V. N. Roa,et al.  Ets-related protein Elk-1 is homologous to the c-fos regulatory factor p62TCF , 1991, Nature.

[34]  B. Groner,et al.  The nuclear factor YY1 participates in repression of the beta-casein gene promoter in mammary epithelial cells and is counteracted by mammary gland factor during lactogenic hormone induction , 1994, Molecular and cellular biology.

[35]  T. Kawamoto,et al.  Identification of the human beta-actin enhancer and its binding factor , 1988, Molecular and cellular biology.

[36]  R. Treisman,et al.  Casein kinase II phosphorylation increases the rate of serum response factor‐binding site exchange. , 1992, The EMBO journal.

[37]  G. Spizz,et al.  Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene , 1988, Molecular and cellular biology.

[38]  R. Treisman,et al.  Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element , 1992, Cell.

[39]  T. Lee,et al.  Transforming growth factor-beta response elements of the skeletal alpha-actin gene. Combinatorial action of serum response factor, YY1, and the SV40 enhancer-binding protein, TEF-1. , 1994, The Journal of biological chemistry.