One cell-specific and three ubiquitous nuclear proteins bind in vitro to overlapping motifs in the domain B1 of the SV40 enhancer.

We have used the gel retardation assay to investigate the binding of nuclear proteins to the domain B1 of the SV40 enhancer, which contains the GT-II motif. Four proteins (GT-IIA, GT-IIB alpha, GT-IIB beta and GT-IIC) were detected, three of which were present in nuclear extracts from several cell lines. The fourth protein (GT-IIC) showed a clear cell-specificity, being absent from the lymphoid cell extracts tested. The results of methylation interference assays and of the binding of the proteins to mutated templates indicate that the domain B1 contains three distinct, but overlapping, protein-binding motifs (GT-IIA, B and C). The cell-specific binding of protein GT-IIC in vitro correlates with the in vivo enhancer activity of its cognate motif, strongly suggesting that this protein acts as a positive trans-acting enhancer factor. Two of the proteins also recognize other enhancer motifs; protein GT-IIB alpha binds to the microE3 motif present in the immunoglobulin heavy chain enhancer; protein GT-IIC binds to an enhancer motif of the polyomavirus mutant PyEC9.1 adapted to growth in F9 embryonal carcinoma cells, but not to the corresponding wild-type sequence.     

Activity and interleukin 1 responsiveness of SV40 enhancer motifs in a rodent immature T cell line.

We have analysed the enhancer activity and the interleukin 1 (IL1) responsiveness of individual motifs of the SV40 enhancer in an immature rodent T cell line, PC60. Transient transfection assays showed that tetramers of GT-I plus GT-IIC motifs, the TC-II or the P motif have significant enhancer activity in PC60, while neither Octamer nor SphI+II motifs have a detectable effect on promoter strength. Two motifs, TC-II and P, strongly respond to stimulation by IL1. DNase I and methylation protection experiments with nuclear extracts show specific footprints in the TC-II region of the SV40 enhancer. Exposure of PC60 cells to IL1 increases their intensity. The TC-II sequence forms several complexes detected in band shift assays. The molecules involved all have similar sequence specificity as NF-kappa B. Surprisingly, band shifts with extracts from control or IL1 treated cells differ only slightly. However, if GTP is added to the binding reactions the intensity of bands formed by extracts from control cells is strongly reduced, whereas extracts from IL1 treated cells form a single retarded complex that co-migrates with NF-kappa B from a pre-B cell line. The results suggest that in PC60 IL1 induces NF-kappa B activity by activating molecules that are already in the nucleus.     

The myosin light chain enhancer and the skeletal actin promoter share a binding site for factors involved in muscle-specific gene expression.

The myosin light chain (MLC) 1/3 enhancer (MLC enhancer), identified at the 3' end of the skeletal MLC1/3 locus, contains a sequence motif that is homologous to a protein-binding site of the skeletal muscle alpha-actin promoter. Gel shift, competition, and footprint assays demonstrated that a CArG motif in the MLC enhancer binds the proteins MAPF1 and MAPF2, previously identified as factors interacting with the muscle regulatory element of the skeletal alpha-actin promoter. Transient transfection assays with constructs containing the chloramphenicol acetyltransferase reporter gene demonstrated that a 115-bp subfragment of the MLC enhancer is able to exert promoter activity when provided with a silent nonmuscle TATA box. A point mutation at the MAPF1/2-binding site interferes with factor binding and abolishes the promoter activity of the 115-bp fragment. The observation that an oligonucleotide encompassing the MAPF1/2 site of the MLC enhancer alone cannot serve as a promoter element suggests that additional factor-binding sites are necessary for this function. The finding that MAPF1 and MAPF2 recognize similar sequence motifs in two muscle genes, simultaneously activated during muscle differentiation, implies that these factors may have a role in coordinating the activation of contractile protein gene expression during myogenesis.