Sequence-specific interactions of nuclear factors with conserved sequences of human class II major histocompatibility complex genes.

Abstract

All class II major histocompatibility complex genes contain two highly conserved sequences, termed X and Y, within the promoter region(s), which may have a role in regulation of expression. To study trans-acting factors that interact with these sequences, sequence-specific DNA binding activity has been examined by the gel electrophoresis retardation assay using the HLA-DQ213 gene 5' flanking DNA and nuclear extracts derived from various cell types. Several specific protein-binding activities were found using a 45-basepair (bp) HinfI/Sau96I Two highly conserved DNA sequences have been found in all class II a-and ,-chain genes and in a third, related gene, the invariant chain gene, in the region 5' of the transcription start site (9, 10) (see Fig. 1). They are conserved box X [at -100 to -113 base pairs (bp)] and conserved box Y (at -71 to -80 bp). In addition, two regions from -67 to -70 bp and from -81 to t-99 bp are conserved but are characteristic of either a-or A-chain genes. These conserved sequences have been thought to be cis-acting elements involved in regulation of class II genes, although the function of these sequences has not been precisely determined. The analysis by transfection of a series of 5' deletion constructs of the DQ,3 gene has demonstrated the existence of both positive and negative control elements as well as a IFN-y control element in the conserved sequence region (11). To elucidate the interactions of trans-acting regulatory factors with these cis-acting elements, nuclear factors that might bind specifically to them have been sought using the gel electrophoresis DNA-binding assay. MATERIALS AND METHODSCells. Raji and T5-1 are human B-lymphoblastoid cells. RJ 2.2.5 (6) and 6.1.6 (5) are class II-negative variants of Raji and T5-1, respectively. HeLa (human carcinoma) and the human T-lymphocyte cell lines HuT 78 (class II positive), Jurkat and HPB-ALL (both class II negative), were also used. All cells were propagated in RPMI 1640 medium supplemented with 10% fetal calf serum and penicillin/streptomycin/glutamine. DNA Fragment Preparation. The 5' flanking sequences of DQ2,3 gene were from a genomic clone X-42 (12). Subclones MX159, MX128, and MX82 (11) were used for preparation of fragments. End-labeling was carried out with [a-32P]dNTPs and the Klenow fragment of DNA polymerase I, and labeled fragments were isolated by polyacrylamide gel electrophoresis. Chemically synthesized oligonucleotides were used for DNA-binding competition experiments.DNA-Binding Assay. Binding of nuclear factors to DNA probes was analyzed bn polyacrylamide gels as described (13) with minor modifications. Nuclear extracts were prepared as described (14) and incubated with 32P-end-labeled DNA fragments in 10-id, reaction mixtures containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% (vol/vol)

Sequence elements required for activity of a murine major histocompatibility complex class II promoter bind common and cell-type-specific nuclear factors.

“…2A). These sequence is found about 17 nucleotides 5' of the X-box element in all class II promoters (50; R. Dedrick, unpublished observation) and has been included in more extended sequence elements termed the Z box (56,58) or W box (39). However, since mutation of the surrounding sequences had a minimal effect on promoter activity in our experiments (LS [-119,-107] and LS[-102,-89]) and because there is some confusion about the identity of the W sequence (17, 39), we have used the H-box nomenclature to indicate the more limited element described by our data.…”

Human X-box-binding protein 1 is required for the transcription of a subset of human class II major histocompatibility genes and forms a heterodimer with c-fos.

“…Three conserved motifs (the W, X, and Y boxes) are found in the proximal promoters of all class II genes and are indispensable for their proper transcription (16)(17)(18)(19)(20). In addition, sequence-specific DNA-binding activities for these elements have been identified, and some genes encoding these proteins have been cloned (21-24).…”

MHC class II regulatory factor RFX has a novel DNA-binding domain and a functionally independent dimerization domain

“…These sequences are cis-acting elements playing a central role in class II gene expression (Dom et al 1987b;Sherman et al 1987Sherman et al , 1989Basta et al 1988;Koch et al 1988;Sakurai and Strominger 1988;Tsang et al 1988Tsang et al , 1990Dedrick and Jones 1990). We and others have identified nuclear factors binding specifically to these sequences (Dorn et al 1987a, b;Miwa et al 1987;Reith et al 1988Reith et al , 1989Sherman et al 1989;Dedrick and Jones 1990;Kobr et al 1990;Tsang et al 1990). Among these, the factor we have called RFX is of special interest because there is evidence that it is in-volved in the regulation of class II gene expression.…”

“…All class II gene promoters from man, mouse, and other species contain three conserved sequence motifs referred to as the W(or Z), X, and Y boxes (Saito et al 1983;Kelly and Trowsdale 1985;Miwa et al 1987;Tsang et al 1988). These sequences are cis-acting elements playing a central role in class II gene expression (Dom et al 1987b;Sherman et al 1987Sherman et al , 1989Basta et al 1988;Koch et al 1988;Sakurai and Strominger 1988;Tsang et al 1988Tsang et al , 1990Dedrick and Jones 1990).…”