Identification of cis sequences controlling efficient position-independent tissue-specific expression of human major histocompatibility complex class I genes in transgenic mice.

Abstract

We previously reported that genomic major histocompatibility complex class I human leukocyte antigen (HLA)-B7 gene constructs with as little as 0.66 kb of 5'-and 2.0 kb of 3'-flanking DNA were expressed efficiently and appropriately in transgenic mice. To identify and characterize the relevant cis-acting regulatory elements in more detail, we have generated and analyzed a series of transgenic mice carrying native HLA-B7 genes with further 5' truncations or intronic deletions and hybrid constructs linking the 5'-flanking region of B7 to a reporter gene. We were unable to detect a specific requirement for sequence information within introns 2 to 7 for either appropriate constitutive or inducible class I expression in adult animals. The results revealed the presence of cis-acting regulatory sequences between -0.075 kb and -0.66 kb involved in driving efficient copy number-dependent constitutive and gamma interferon-enhanced tissue-specific expression. The region from -0.11 to -0.66 kb is also sufficient to prevent integration site-specific "position effects," because in its absence HLA-B7 expression is frequently detected at significant levels at inappropriate sites. Conserved sequence elements homologous to the H-2 class I regulatory element, or enhancer A, and the interferon response sequence are located between about -151 and -228 bp of the B7 gene. Our results also indicate the existence of sequences downstream of -0.11 kb which can influence the pattern of tissue-specific expression of the HLA-B7 gene and the ability of this gene to respond to gamma interferon.

A 3′-Transcribed Region of the HLA-A2 Gene Mediates Posttranscriptional Stimulation by IFN-γ

“…A similar result was obtained in the human HLA-B7 gene, in that a truncated HLA-B7 gene lacking the 5Ј promoter region was IFN-inducible when transfected into mouse L cells (20). Further, HLA-B7 expression from constructs that lacked the 5Ј ISRE were up-regulated severalfold by IFN-␥ in transgenic mice (21). In none of these cases was it demonstrated whether IFN stimulation of class I gene expression was due to a transcriptional effect mediated by sequences downstream of the promoter, or by posttranscriptional mechanisms.…”

“…These observations raised the question of whether sequences other than the 5Ј-flanking region of the HLA-A2 gene could be necessary for IFN-␥ induction. In this regard, studies in both the mouse H-2L gene and the human HLA-B7 gene have shown that sequences located 3Ј to the transcription initiation site are both necessary for full response to IFN-␥ and sufficient to mediate some IFN-␥ response (17,18,21). Our results show that in K562 cells, which had been stably transfected with an HLA-A2 genomic clone containing 525 bp of the 5Ј promoter, all eight exons, and ϳ1300 bp of 3Ј-transcribed region, IFN-␥ stimulation increases the level of RNA from the transfected gene by 3-fold.…”

“…Although previous investigations in the murine and human MHC class I systems have suggested that a component of IFN induction of class I genes is mediated by sequences located downstream from the transcription initiation site, neither the location of the relevant sequences nor the mechanism involved have been identified (17,20,21). To determine whether the polyadenylation site, or sequences directly surrounding it, mediate IFN-␥ induction of the HLA-A2 gene, the 3Ј-flanking region of the 5.1-kb HLA-A2 clone was removed by restriction enzyme cleavage with SspI.…”

Lactacystin and clasto-Lactacystin β-Lactone Modify Multiple Proteasome β-Subunits and Inhibit Intracellular Protein Degradation and Major Histocompatibility Complex Class I Antigen Presentation

“…Unlike the proteasomes studied here, brain proteasomes normally contain very low levels of LMP2 and LMP7 ␤-subunits compared with liver or spleen (25), presumably because brain also has much lower levels of MHC molecules and is less active in antigen presentation (54). Consequently, any reaction of lactacystin with the three IFN-␥-induced subunits would have been missed in the earlier studies.…”

Cytokine regulation of expression of class I MHC antigens

“…Investigation of HLA-B7 genes showed the presence of c i s-acting regulatory sequences between -75 to -660, which are responsible for constitutive as well as IFN-γ induced tissue-specific expression of this gene (Chamberlain et al, 1991). Similarly, enhancer A, and B (Figure 1) regions present upstream of class I MHC coding sequence act as a negative element in F9 EC cells.…”