Immunogenetics 2000) 51:882±886 Digital Object Identifier DOI) 10.1007/s002510000213 BRIEF COMMUNICATION Yunfei Chen ´ Susan L. Carpenter ´ Susan J. Lamont A functional role for the Y box in regulating an MHC class II B gene promoter in chicken lymphocytes Received: 23 December 1999 / Revised: 10 May 2000 / Published online: 30 June 2000  Springer-Verlag 2000 Key words MHC ´ Promoter ´ Y box ´ Gene regulation ´ Chicken The conserved S, X, X2, and Y boxes located in the proximal regions of MHC class II gene promoters are necessary and sufficient to control both constitutive and induced MHC class II expression in mammals Benoist and Mathis 1990; Glimcher and Kara 1992; Mach et al. 1996). The chicken immune system shares many of the features possessed by its mammalian counterparts, but has also evolved major points of divergence, such as the mechanism of antibody diver- sification Reynaud et al. 1985, 1987; Thompson and Neiman 1987), and a different MHC genomic struc- ture with a separate Rfp-Y region Briles et al. 1993; Miller et al. 1994) and interspersed class I and class II genes Guillemot et al. 1989; Kaufman and Lamont 1996). Sequence analysis of the promoters from chicken class II B genes Xu et al. 1989; Zoorob et al. 1990) detected S, X, X2, and Y boxes in the proximal promoter region similar to those in mammals. Surpris- ingly, deletion of these conserved sequences did not significantly affect promoter activity in a chicken mac- rophage cell line Chen et al. 1997). By characterizing the role of these DNA elements in class II gene regu- lation in chicken lymphocyte cell lines, the current study investigated whether the lack of function of these conserved boxes represents another major evolu- tionary divergence of the chicken immune system or is a macrophage-specific phenomenon. A Marek©s disease virus-transformed MSB1 chicken T-cell line Akiyama and Kato 1974), an avian leuko- sis virus-induced DT40 chicken pre-B-cell line Baba et al. 1985), and a lymphoid leukosis virus-induced RP9 cell line were used to study class II regulation in lymphocytes. Cells were labeled with an anti-chicken class II monoclonal antibody mAb) Ewert et al. 1984) to examine the expression of surface class II molecules by flow cytometry. The class II-positive RP9 chicken B-cell line Okazaki et al. 1980) was used as positive control. Figure 1 shows that MSB1 cells were positive for surface class II antigens. The DT40 cells, which were at an early differentiation stage, had little class II expression. The CCII-7-1 chicken class II b-chain gene Xu et al. 1989) proximal promoter region Chen et al. 1997) was analyzed for transcription factor binding by DNase footprinting Im and Muzyczka 1989) and elec- trophoretic mobility shift assay EMSA). DNase foot- printing of the ±130/+19 fragment from the CCII-7-1 upstream region revealed a strong footprint covering the Y box and the immediate downstream GC box on both the sense and the antisense strands if nuclear extracts from MSB1 or RP9 cells were used Fig. 2A). The footprint was competed by a YGC oligonucleo- tide, which contained the Y box and the GC box sequences. Protein binding to the Y-box sequence was further investigated by EMSA. Oligonucleotides see legend to Fig.2) were synthesized and used as probes and competitors. Nuclear extracts from the lympho- cyte cell lines were incubated with 32 P-labeled oligo- nucleotide probe as in DNase footprinting, except that competitor oligonucleotides were added at 100-fold excess to the labeled probe. The binding mixture was then separated in a 4% nondenaturing polyacrylamide gel and autoradiographed. The Y-box probe detected a strong shifted band with nuclear extracts from the DT40 and MSB1 cell lines Fig. 2B). This binding activity was competed by excess unlabeled Y oligonu- cleotide, but not by an unrelated GRE oligonucleo- Y. Chen ´ S.J. Lamont  ) ) Department of Animal Science, 2255 Kildee Hall, Iowa State University, Ames, IA 50011, USA E-mail: sjlamont@iastate.edu Phone: +1-515-2944100 Fax: +1-515-2942401 S.L. Carpenter Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA 50011, USA