The -6.1-kilobase chicken lysozyme enhancer is a multifactorial complex containing several cell-type-specific elements.
In the chromatin domain of the chicken lysozyme gene of myeloid and oviduct cells, which both have the potential to activate the gene, a developmentally stable DNase I-hypersensitive site is formed around 6.1 kb upstream of the gene. This implies that this DNA region, which has previously been demonstrated to function as a transcriptional enhancer element in myeloid cells, is intimately involved in the cell-type-specific activation of the lysozyme gene locus. Deletion analysis identifies a 157-bp minimal fragment that confers the same promacrophage-specific enhancer activity as the originally described 562-bp -6.1-kb enhancer fragment. By introducing specific point mutations, we demonstrate in transient gene transfer experiments that the minimal fragment consists of at least six adjacent elements, each substantially contributing to enhancer function. The compact multifactorial enhancer complex includes a nuclear factor I (NF-I)/TGGCA binding site, homologies to AP1, and octanucleotide or enhancer core consensus motifs. Point mutation of the NF-I binding site results in the loss of NF-I binding in vitro and enhancer activity in vivo after gene transfer. Surprisingly, four overlapping oligonucleotides, each consisting of at least two elements of the -6.1-kb enhancer, confer myeloid-cell-specific enhancer activity. We found several myeloid-cell-specific DNA-binding proteins interacting with the -6.1-kb enhancer, a result consistent with that described above. Therefore, we suggest that more than a single trans-acting factor mediates the cell type specificity of the -6.1-kb enhancer.
Grewal
T
T
Institut für Biologie III, Albert-Ludwigs Universität, Freiburg, Germany.
Theisen
M
M
Borgmeyer
U
U
Grussenmeyer
T
T
Rupp
R A
RA
Stief
A
A
Qian
F
F
Hecht
A
A
Sippel
A E
AE
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000502:physiology; D002872:Chromosome Deletion; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000201:enzymology; D008969:Molecular Sequence Data; D009113:Muramidase / Q000235:genetics* / Q000378:metabolism; D009132:Muscles / Q000201:enzymology; D016297:Mutagenesis, Site-Directed; D010057:Oviducts / Q000201:enzymology; D010957:Plasmids; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic; D014162:Transfection
D002843:Chromatin; D008156:Luciferases; D015500:Chloramphenicol O-Acetyltransferase; D003850:Deoxyribonuclease I; D009113:Muramidase