Class I MHC complexes (MHCI) are essential in mediating immune response. The transport of antigenic peptides (TAP) to MHCI and the stable expression of MHCI on the cell surface require the presence of a dedicated TAP. In this study we report that IFN-γ and thrombopoietin (TPO) strongly increase TAP1 protein expression in megakaryocytes, followed by an enhanced expression of MHCI on the cell surface. This expression parallels the enhanced TAP1 promoter activity and TAP1 mRNA expression, which are independent of protein synthesis. We also show that this cytokine-dependent expression of TAP1 transcripts depends on STAT1 and IFN regulatory factor-2 (IRF-2), but not on IRF-1, and provide evidence that IRF-2 constitutively binds to the TAP1 gene promoter and enhances TAP1 promoter activity. We show that IRF-2 forms a complex with STAT1 and the cytokine-responsive region of the TAP1 promoter in any TPO or IFN-γ target cells tested. Interaction of IRF-2 and STAT1 on the promoter depends on the DNA-binding domain of IRF-2. Overall, our data indicate that TPO and IFN-γ activate the expression of TAP1 via a new mechanism that involves functional cooperation between STAT1 and IRF-2 on the TAP1 promoter.

Retinitis pigmentosa (RP) is the most common form of inherited retinopathy, with an approximate incidence of 1 in 3700 individuals worldwide. Mutations in the retinitis pigmentosa 1 (RP1) gene are responsible for about 5 -10% cases of autosomal dominant RP. The RP1 gene is specifically expressed in the photoreceptor layers of the postnatal retina and encodes a predicted protein characterised by the presence of two doublecortin (DC) domains, known to be implicated in microtubule binding. We identified and characterised, both in human and in mouse, a novel mammalian gene, termed Retinitis Pigmentosa1-like1 (RP1L1), because of its significant sequence similarity to the RP1 gene product. The sequence homology between RP1 and RP1L1 was found to be mostly restricted to the DC domains and to the N-terminal region, including the first 350 amino acids. The RP1L1 gene was also found to be conserved in distant vertebrates, since we identified a homologue in Fugu rubripes (pufferfish). Similar to RP1, RP1L1 expression is restricted to the postnatal retina, as determined by semiquantitative reverse transcriptase-PCR and Northern analysis. The retina-specific expression and the sequence similarity to RP1 render RP1L1 a potential candidate for inherited retinal disorders.

The minimal muscle-specific dystrophin promoter contains the consensus sequence CC(A/T) 6 GG, or the CArG element, which can be found in serum-inducible or muscle-specific promoters. The serum response factor (SRF), which mediates the transcriptional activation of the c-fos gene in response to serum stimulation, can bind to different CArG box elements, suggesting that it could be involved in muscle-constitutive transcription. Here we show that SRF binds to the dystrophin promoter and regulates its muscle-specific transcription. In transient transfections, an altered-binding-specificity SRF mutant restores the muscle-constitutive transcription of a dystrophin promoter with a mutation in its CArG box element. The muscle-constitutive transcription of the dystrophin promoter also requires the sequence GAAACC immediately downstream of the CArG box. This sequence is recognized by a novel DNA bending factor which was named dystrophin promoterbending factor (DPBF). Mutations of the CArG flanking sequence abolish both DPBF binding and the promoter activity in muscle cells. Its replacement with a p62/ternary complex factor binding site changes the promoter specificity from muscle constitutive to serum responsive. These results show that, on the dystrophin promoter, the transcriptional activation induced by SRF requires the DNA bending induced by DPBF. The bending, next to the CArG box, could promote interactions between SRF and other proteins in the transcriptional complex.

The antiviral distamycin A and its phenyl mustard derivative FCE24517 possessing antitumor activity were tested for their ability to inhibit macromolecular synthesis in three human and one murine cell line. While distamycin A was poorly active in these systems, FCE24517 inhibited DNA synthesis efficiently, RNA synthesis to a lower extent and had little effect on protein synthesis. These findings suggest that the in vivo activity of FCE24517 derives from the specific inhibition of DNA synthesis. When the two drugs were tested on several enzymes involved in human DNA metabolism a strikingly similar pattern of inhibition appeared, with distamycin A being the more potent. Both drugs showed: A), no inhibitory activity against thymidine kinase and DNA primase; B), low activity against DNA topoisomerases I and II and the 3'-5' exonuclease associated with the DNA polymerase epsilon; C), high activity against DNA polymerases alpha and epsilon, uracil-DNA glycosylase and the joining activity of the replicative DNA ligase; D), the highest inhibitory activity against the AMP-dependent DNA relaxing activity of DNA ligase. The strong in vitro inhibition of several DNA enzymatic activities, including DNA ligase, do not match with the in vivo activities of the two drugs. However a unique difference was observed: only FCE24517 inhibited the DNA-independent reaction of adenylation of human DNA ligase while the adenylation reaction of T4 and E. coli DNA ligase was unaffected by either drug. It is still unclear whether these properties are relevant for modulating the killing activity of FCE24517 against proliferating cells both in culture and in vivo. Nevertheless FCE24517 is the first known molecule capable of interacting directly and specifically with human DNA ligase.

Bacteriophage-T4 and human type I DNA ligases were found capable of self-adenylating upon exposure to both ribo- and deoxyribo-[alpha-35S]thio-ATP. However, the joining reaction does not take place in the presence of the deoxyribotriphosphates. Enzyme adenylation is reversed in all cases by an excess of PPi, but the rate of reversion is lower with thio derivatives. Therefore thio derivatives can be used to study the adenylation of DNA ligases and to search for specific inhibitors of the first step of the ligation reaction. In addition we show that thio derivatives can be used to detect DNA ligase adenylation activity covalently bound to a solid matrix.