Chromatin structural analyses of the mouse Igkappa gene locus reveal new hypersensitive sites specifying a transcriptional silencer and enhancer.
To identify new regulatory elements within the mouse Igkappa locus, we have mapped DNase I hypersensitive sites (HSs) in the chromatin of B cell lines arrested at different stages of differentiation. We have focused on two regions encompassing 50 kilobases suspected to contain new regulatory elements based on our previous high level expression results with yeast artificial chromosome-based mouse Igkappa transgenes. This approach has revealed a cluster of HSs within the 18-kilobase intervening sequence, which we cloned and sequenced in its entirety, between the Vkappa gene closest to the Jkappa region. These HSs exhibit pro/pre-B cell-specific transcriptional silencing of a Vkappa gene promoter in transient transfection assays. We also identified a plasmacytoma cell-specific HS in the far downstream region of the locus, which in analogous transient transfection assays proved to be a powerful transcriptional enhancer. Deletional analyses reveal that for each element multiple DNA segments cooperate to achieve either silencing or enhancement. The enhancer sequence is conserved in the human Igkappa gene locus, including NF-kappaB and E-box sites that are important for the activity. In summary, our results pinpoint the locations of presumptive regulatory elements for future knockout studies to define their functional roles in the native locus.
Liu
Zhi-Mei
ZM
Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148, USA.
George-Raizen
Julia B
JB
Li
Shuyu
S
Meyers
Katherine C
KC
Chang
Mee Young
MY
Garrard
William T
WT
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry* / Q000378:metabolism; D018244:Chromosomes, Artificial, Yeast; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D020868:Gene Silencing; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D013997:Time Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D019076:Transgenes
D002843:Chromatin; D007145:Immunoglobulin kappa-Chains; D016328:NF-kappa B; D004247:DNA; D008156:Luciferases; D003850:Deoxyribonuclease I