| Abstract |
Proteins encoded by Major Histocompatibility Complex class I genes bind, transport and present oligopeptides to T lymphocytes, thus playing a crucial role in immune response against pathogens and neoplastic cells but, also, in graft rejection and in some immune disorders. Their expression level is regulated by external stimuli such as virus, mitogens and, most important, cytokines (TNFα, IFNα, β and γ). CIITA is the major regulatory factor of MHC class II genes. In the present study, we have shown that CIITA also plays an important role in MHC class I gene expression, since its overexpression leads to an increase of MHC class I and II molecules on the cell membrane. Our data shows that this increase is due to an transcriptional activation of MHC promoters which is followed by all processes necessary for the translation and presentation of these molecules on the cell surface. Class I superinduction by IFNγ and TNFα on HeLa and Κ562 cell lines suggest a compiled and cooperative action of these cytokines with CIITA. It seems that both cytokines act through a different mechanism than CIITA. In NT2 cells, CIITA is capable to induce class I gene transcription in higher levels than Retinoic acid (RA). The mechanisms of action of these two factors do not show any synergy, are completely independent, they seem not to use common factors and they act through distinct sequences, enhancer A and ICS-α-B elements respectively. According to our results, CIITA has a partial involvement in basal and IFNγ-induced class I activation. IFNγ action is mediated by ICS and, on the other hand, by a mechanism in which CIITA is directly involved. The latter is responsible for 50% of the IFNγ inducing capacity and shows an absolute requirement for α and B boxes. Analytically, our data suggest that IFNγ action on class I promoter is mediated by two distinct regions with equal contribution in the total inducer capacity: ICS element and region 121-116. Both require functional 111-105, α and B boxes. 111-105 sequence is a new element in class I regulation. We have shown that enhancer B is an important regulatory element in constitutive and inducible by IFNγ and CIITA expression and that NFY-A is a common binding factor to Y and B elements of MHC class I and II genes respectively. Thus, our result converge to the existence of a common activation pathway of MHC class I and II promoters, acting through CIITA, α-B and X-Y elements. In conclusion, MHC class I transcriptional regulation is mediated by two groups of proximal promoter elements : enhancer A, that mediates RA, TNFα and IFNγ action though a CIITA-independent mechanism, and enhancer B-ICS element that mediates IFNγ and CIITA action. We have demonstrated the important role of α box in H2-Kb promoter response to IFNγ and CIITA. We identified a Tre-like and a X2BF-like binding activity. We found that the former is a CREB/ATF1 heterodimer and the latter is a new member of X2-binding proteins that shows synergistic binding with CREB/ATF. In our system, as in MHC class II, RFX5 seems to require synergy for binding and its in vivo recruitment depend on CIITA presence. These findings are consistent with the general belief that MHC genes are regulated in transcriptional level and that CIITA is a master control gene for MHC and, also, for the factors required for their presentation on the cell membrane. Moreover, CIITA seems to stabilize other important factors such as RFX5, thus playing an important role in the complex formation that leads to the transcriptional regulation of MHC genes.
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