RESUMEN
Several gene families are known in which member genes are expressed in variegated patterns in differentiated cell types. Mechanisms responsible for imposition of a variegated pattern of gene expression are unknown. Members of the closely linked Ly49 inhibitory receptor gene family are expressed in a variegated fashion by NK cells. Variegated expression of these genes results in subsets of NK cells that differ in specificity for MHC class I molecules. To address the mechanisms underlying variegation, a 30-kb genomic fragment containing a single Ly49 gene was used to generate a panel of murine transgenic lines. The results demonstrated that, in almost all of the lines, the isolated Ly49A gene was expressed in a variegated pattern, remarkably similar in nearly all respects to the expression pattern of the endogenous Ly49A gene. Furthermore, the developmental timing of gene expression and regulation by host MHC molecules closely mirrored that of the endogenous Ly49A gene. Therefore, Ly49 variegation does not require competition in cis between different Ly49 genes, and the sequences imposing variegation are located proximally to Ly49 genes. Efforts to define regulatory elements of the Ly49A gene led to the identification of a DNase I hypersensitive site 4.5 kb upstream of the Ly49A gene transcription initiation site, which was shown to be essential for transgene expression. Highly related sequence elements were found upstream of other Ly49 genes, suggesting that a similar regulatory element controls each Ly49 gene.
Asunto(s)
Antígenos Ly/biosíntesis , Antígenos Ly/genética , Regulación de la Expresión Génica/inmunología , Secuencias Reguladoras de Ácidos Nucleicos/inmunología , Transgenes/inmunología , Regiones no Traducidas 5'/genética , Regiones no Traducidas 5'/metabolismo , Animales , Animales Recién Nacidos , Secuencia Conservada/inmunología , Desoxirribonucleasa I/genética , Desoxirribonucleasa I/metabolismo , Variación Genética/inmunología , Inmunidad Celular/genética , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Lectinas Tipo C , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Familia de Multigenes/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Regiones Promotoras Genéticas/inmunología , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Receptores Similares a Lectina de Células NKRESUMEN
The establishment of clonally variable expression of MHC class I-specific receptors by NK cells is not well understood. The Ly-49A receptor is used by approximately 20% of NK cells, whereby most cells express either the maternal or paternal allele and few express simultaneously both alleles. We have previously shown that NK cells expressing Ly-49A were reduced or almost absent in mice harboring a single or no functional allele of the transcription factor T cell factor-1 (TCF-1), respectively. In this study, we show that enforced expression of TCF-1 in transgenic mice yields an expanded Ly-49A subset. Even though the frequencies of Ly-49A(+) NK cells varied as a function of the TCF-1 dosage, the relative abundance of mono- and biallelic Ly-49A cells was maintained. Mono- and biallelic Ly-49A NK cells were also observed in mice expressing exclusively a transgenic TCF-1, i.e., expressing a fixed amount of TCF-1 in all NK cells. These findings suggest that Ly-49A acquisition is a stochastic event due to limiting TCF-1 availability, rather than the consequence of clonally variable expression of the endogenous TCF-1 locus. Efficient Ly-49A acquisition depended on the expression of a TCF-1 isoform, which included a domain known to associate with the TCF-1 coactivator beta-catenin. Indeed, the proximal Ly-49A promoter was beta-catenin responsive in reporter gene assays. We thus propose that Ly-49A receptor expression is induced from a single allele in occasional NK cells due to a limitation in the amount of a transcription factor complex requiring TCF-1.