Upstream stimulatory factor regulates major histocompatibility complex class I gene expression: the U2DeltaE4 splice variant abrogates E-box activity

Abstract

The tissue-specific expression of major histocompatibility complex class I genes is determined by a series of upstream regulatory elements, many of which remain ill defined. We now report that a distal E-box element, located between bp -309 and -314 upstream of transcription initiation, acts as a cell type-specific enhancer of class I promoter activity. The class I E box is very active in a neuroblastoma cell line, CHP-126, but is relatively inactive in the HeLa epithelial cell line. The basic helix-loop-helix leucine zipper proteins upstream stimulatory factor 1 (USF1) and USF2 were shown to specifically recognize the class I E box, resulting in the activation of the downstream promoter. Fine mapping of USF1 and USF2 amino-terminal functional domains revealed differences in their abilities to activate the class I E box. Whereas USF1 contained only an extended activation domain, USF2 contained both an activation domain and a negative regulatory region. Surprisingly, the naturally occurring splice variant of USF2 lacking the exon 4 domain, U2DeltaE4, acted as a dominant-negative regulator of USF-mediated activation of the class I promoter. This latter activity is in sharp contrast to the known ability of U2DeltaE4 to activate the adenovirus major late promoter. Class I E-box function is correlated with the relative amount of U2DeltaE4 in a cell, leading to the proposal that U2DeltaE4 modulates class I E-box activity and may represent one mechanism to fine-tune class I expression in various tissues.

FIG. 1

The MHC class I reporter construct -400CAT is shown with selected regulatory motifs indicated. The E box is located between bp −309 and −314 upstream of the point of transcription initiation. The proximal promoter extends from bp −203 to −68 and contains an enhancer, enhancer A, and an overlapping interferon response element (IRE). The basal promoter extends from bp −68 to +1 and includes the CCAAT box, promoter elements, and the site of transcription initiation. The derivation of the 107-bp and 3E oligonucleotide probes used in gel shift analysis is also shown; there is a detailed description in Materials and Methods. The core hexamer sequence of the 3E probe is shown.

FIG. 2

The MHC class I E box forms complexes with USF proteins. The 3E oligonucleotide was end labeled, and DNA binding assay mixtures were incubated for 30 min at 4°C prior to the separation of bound and free probe in native 4% PAGE. Each lane contains 4.5 μg of HeLa WCE and competitor oligonucleotides or antisera (1 μg), as indicated, in a total volume of 20 μl. (A) A specific complex (indicated by the arrowhead) was shown to be bound to the class I E box. Lane 1, probe alone; lane 2, HeLa WCE; lanes 3 and 4, HeLa WCE (WT) and a 1,000-fold excess of self and nonspecific (NS) oligonucleotide competitors, respectively. (B) Antibody supershift examination of the 3E specific complex (represented by the arrowhead). Lane 1, probe alone; lane 2, HeLa WCE; lane 3, HeLa WCE and control (C) antisera (directed against the p50 subunit of NF-κB); lanes 4 to 6, antisera against USF1, USF2, and USF1 plus USF2, respectively, were added to DNA binding assay mixtures. Anti-USF1 antisera generated two supershifted complexes (indicated by the circles). Antisera to USF2 generated only one supershifted complex (closed circle). Combining anti-USF1 and anti-USF2 antisera together resulted in a further supershift of the USF2 containing complex (arrow).

FIG. 3

USF1 and USF2 activate the class I promoter. HeLa cells were cotransfected with 10 μg of -400CAT class I reporter construct (Fig. 1) and 3 μg of the vector control, pSG5, or the indicated USF expression plasmids. Representative maps of the expressed USF sequences and deleted regions are given on the left-hand side of the figure: upstream regulatory region (USR), binding region (BR), and HLH-Z interaction domain (HLH-LZ). Numbers in parentheses refer to deleted amino acids. Basal -400CAT activity (cotransfected with pSG5) is shown at the top of each panel. (A) The ability of USF1 and derivative truncations to trans-activate MHC class I promoter expression. (B) The ability of USF2 and truncated derivatives to activate class I expression. Data are expressed as relative percentages of acetylation normalized to the transfection control, pSV2LUC. Error bars indicate standard errors.

FIG. 4

U2ΔE4 activates an AdMLP E box-containing reporter construct. The AdMLP E-box construct U2E1bCAT contains two copies of the AdMLP E box upstream of the E1b TATA promoter. HeLa cells were cotransfected with 3 μg of control or USF expression vectors and 10 μg of reporter construct. Data are expressed as relative percentages of acetylation normalized to the transfection control, pSV2LUC. Error bars indicate standard errors.

FIG. 5

U2ΔE4 can bind the class I E box. Equivalent amounts of in vitro-translated USF proteins (determined by SDS-PAGE of ³⁵S-labeled proteins; data not shown) were added to end-labeled class I E-box probe, and binding reactions were analyzed as described in the legend to Fig. 2 and in Materials and Methods. (A) Homodimer of U2ΔE4 can bind the class I E box. Lane 1, probe alone; lanes 2 to 4, USF1; lanes 5 to 7, USF2; lanes 8 to 10, U2ΔE4. Antisera against USF1 or USF2 were included to verify the identities of the translated proteins and demonstrate the specificity of the individual antisera. Lanes 3, 6, and 9 contain the anti-USF1 antiserum. Lanes 4, 7, and 10 contain the anti-USF2 antiserum. Specific supershifted complexes are present in lanes 3 (USF1), 7 (USF2), and 10 (U2ΔE4). Note the slower mobility of USF2 (due to its higher molecular weight) than that of USF1 and the faster mobility of U2ΔE4. (B) U2ΔE4 can generate class I E box-binding dimers with USF1 and USF2. U2ΔE4 was cotranslated with either USF1 or USF2, and the specific binding complexes generated were examined by antibody supershift analyses. Lane 1, probe alone; lanes 2 to 5, U2ΔE4 and USF1; lanes 6 to 9, U2ΔE4 and USF2. The antiserum against either USF2 or USF1 was added to distinguish homodimers from heterodimers. Control (C) antisera against the p50 subunit of NF-κB was added to the binding reaction mixtures shown in lanes 3 and 7. The antiserum against either USF1 or USF2 was added to the binding reaction mixtures shown in lanes 4 and 8 and 5 and 9, respectively.

FIG. 6

U2ΔE4 abrogates USF activation of MHC class I E-box activity. HeLa cells were cotransfected with 10 μg of -400CAT class I reporter construct (Fig. 1) and 3 μg of either USF1 (right panel) or USF2 (left panel) expression construct. Fold stimulation of the class I reporter, relative to the control and in the absence of added U2ΔE4, is indicated on the ordinate. Increasing amounts of the U2ΔE4 expression construct were included (shown on the abscissa as 1, 3, and 9 μg). All assays were normalized to the cotransfected internal control plasmid pSV2LUC.

FIG. 7

CHP-126 neuroblastoma cell line contains less U2ΔE4 relative to HeLa epithelial cells. Approximately 150 μg of either CHP-126 or HeLa WCE was separated by SDS–12.5% PAGE and transferred to nitrocellulose membranes, and USF proteins were distinguished by Western blotting with antisera against USF1 and USF2. Densitometric values to quantitate the relative ratios of USF1 and USF2 to U2ΔE4 between the two cell lines are provided to the right.

FIG. 8

A region of the class I promoter containing the E box is active in the CHP-126 neuroblastoma cell line but is inactive in HeLa epithelial cells. HeLa epithelial cells and CHP-126 neuroblastoma cell lines were transfected with 10 μg of the indicated MHC class I reporter constructs, and promoter function was assessed. Note that the actual class I promoter activities in HeLa and CHP-126 cells were normalized to facilitate this comparison; class I expression in the CHP-126 cell line (although significant) is approximately 40-fold less than in HeLa cells.

FIG. 9

The ability of the class I E box to stimulate transcription is cell type specific. Wild-type (WT) or mutant (Mut) class I E boxes, in sense and antisense orientations, were cloned upstream of -294CAT (which lacks an E box). HeLa and CHP-126 cell lines were transfected with 10 μg of the indicated reporter construct, and promoter activity was determined.