Tsh

Figure 2A. The counter effects of TSH and IFN-y on the expression of HLA-DRa in thyrocytes—a proposed model (adopted from ref. [168]). (A) Effects of TSH on HLA-DRa expression in thyrocytes, as suggested by Balducci-Silano [164], TSH binds to its receptor, decreasing the amount of SSBP-1, while simultaneously increasing the amount of TSEP (the homologue of YB-1), which acts as a repressor. The decreased SSBP-1 does not bind to the single-stranded DNA elements adjacent to the W box, hence other transcription factors cannot easily associate with the HLA-DRa promoter. On the other hand, the increased TSEP represses the activity of the promoter and HLA-DRa expression. This ensures that HLA-DRa is not expressed on the surface of the thyrocyte, possible autoantigens, such as TPO and thyroglobulin cannot be presented to the immune system.

Figure 2A. The counter effects of TSH and IFN-y on the expression of HLA-DRa in thyrocytes—a proposed model (adopted from ref. [168]). (A) Effects of TSH on HLA-DRa expression in thyrocytes, as suggested by Balducci-Silano [164], TSH binds to its receptor, decreasing the amount of SSBP-1, while simultaneously increasing the amount of TSEP (the homologue of YB-1), which acts as a repressor. The decreased SSBP-1 does not bind to the single-stranded DNA elements adjacent to the W box, hence other transcription factors cannot easily associate with the HLA-DRa promoter. On the other hand, the increased TSEP represses the activity of the promoter and HLA-DRa expression. This ensures that HLA-DRa is not expressed on the surface of the thyrocyte, possible autoantigens, such as TPO and thyroglobulin cannot be presented to the immune system.

4.4. Structure of the MHC Class II Promoters

The cloning of many promoters of the genes coding for MHC class II molecules from different isotypes and species, and comparisons between these sequences, led to the identification of short, very conserved DNA sequences termed "boxes". All MHC class II promoters, as well as related promoters such as the HLA-DM [119] and the invariant chain (li) [120], contain the same boxes arranged in the same order [reviewed in 121-125]. This similarity ensures the coordinated expression of these genes, which is based on common mechanisms of transcription. Four boxes in particular are involved in mediating the transcriptional control of MHC class II molecules. The Y box is a 10-bp motif, which contains the reverse CCAAT sequence. At a conserved distance of about 19-20 bp upstream to the Y box lies the X box, which contains 14 bp. Immediately downstream to the X box, with two nucleotides overlapping, is the X2 box comprised of 8 bp, which contains a sequence similar to either the cAMP response element (CRE) or the TPA response element (TRE). Upstream to the X box a pyrimidine-rich stretch separates the X and the W boxes. The S (or H) box is a 7-bp box included within a 30 bp region called the W (or Z) box, which is 20-21 bp upstream of the X box. Apart from these four boxes, an additional unique motif in the HLA-DRa promoter, is an 8 bp sequence called octamer, which has been shown to mediate B-cell specificity. Transfection experiments, in which specific promoters or promoter elements are tested for their control of the transcription of a reporter gene, e.g., chloramphenicol acyltransferase (CAT), demonstrated that the proximal promoter of MHC class II

Figure 2B. The counter effects of TSH and IFN-y on the expression of HLA-DRa in thyrocytes—a proposed model (adopted from ref. [168]). (B) Effects of IFN-y on HLA-DRa expression in thyrocytes. IFN-y binds to its receptor and tyrosine phosphorylates the Janus kinases (Jakl and Jak2) and the STATla transcription factor. STATla increases the activity of IRF-1 and binds to the CIITA inducible promoter, where together with USF-1 it induces the expression of CIITA. IFN-j/ decreases the expression of TSEP, thereby allowing the binding of NF-Y, which serves as an activator for HLA-DRa expression. IFN-y also increases the amount of SSBP-1, which binds to the single-stranded DNA elements adjacent to the W box and allows the binding of the other double-stranded transcription factors to the HLA-DRa promoter. CIITA binds to the transcription factors, in particular to RFX, serves as a "master regulator" for the transcription of HLA-DRa (the open arrows indicate increase or decrease in the amount of the indicated protein, P represents phosphorylation).

Figure 2B. The counter effects of TSH and IFN-y on the expression of HLA-DRa in thyrocytes—a proposed model (adopted from ref. [168]). (B) Effects of IFN-y on HLA-DRa expression in thyrocytes. IFN-y binds to its receptor and tyrosine phosphorylates the Janus kinases (Jakl and Jak2) and the STATla transcription factor. STATla increases the activity of IRF-1 and binds to the CIITA inducible promoter, where together with USF-1 it induces the expression of CIITA. IFN-j/ decreases the expression of TSEP, thereby allowing the binding of NF-Y, which serves as an activator for HLA-DRa expression. IFN-y also increases the amount of SSBP-1, which binds to the single-stranded DNA elements adjacent to the W box and allows the binding of the other double-stranded transcription factors to the HLA-DRa promoter. CIITA binds to the transcription factors, in particular to RFX, serves as a "master regulator" for the transcription of HLA-DRa (the open arrows indicate increase or decrease in the amount of the indicated protein, P represents phosphorylation).

genes is sufficient and necessary for both the constitutive and the induced expression of these molecules [reviewed in 121-125]. Deletion analysis of specific promoter regions, and mutational analysis of each of the boxes have shown that not only the boxes, but also the exact spaces between them, are required the promoter activity [126], The spaces, rather than the composition of the bases in them, align the proteins that bind to the boxes on the same plenary axis of the DNA helix, and allow protein-protein interactions that lead to the activation of RNA polymerase II and to the transcription of MHC class II molecules.

4.5. Transcription Factors

Transcription factors are proteins that have the ability to recognize specific DNA sequences on gene promoters, bind to them, and then directly or indirectly activate the transcriptional apparatus, which includes RNA polymerase II and other proteins. Many DNA binding proteins have been isolated on the basis of their ability to bind to specific ¿-«-element sequences in vitro, by using methods such as affinity purification or screening of Xgtl 1 cDNA expression libraries. The multimeric nuclear complex RFX—consisting of at least two subunits—RFX5 [127] and RFXAP [128]— binds to the X box on MHC class II promoters.

TRAX1 [129], NF-X1 [130] and the family of proteins RFX1,2,3 and 4 [ 131,132] can also bind to the X box. NF-Y(also called YEBP orCBF) [133, 134] and YB-1 [135] both bind to the Y box and function as either activator or repressor, respectively. X2BP [136], NF-X2 [137], IFNEX [138], hXBPl [139], and HB16 [140] bind to the X2 box. The NF-X2 probably corresponds to the AP-1 complex, which consists of c-Jun and c-Fos, while both hXBP-1 and HB16 have been shown to be able to heterodimerize with c-Jun. The W-Bl and W-B2 complexes bind to the S box [141,142], as does NF-J [143], These are examples of proteins that have been isolated and/or characterized to-date.

As mentioned, most of these proteins were isolated on the basis of their binding to ds-elements in the MHC class II promoters in vitro. However, their role in MHC class II transcription is deduced mostly by indirect evidences, such as a correlation between the expression of the transcription factor and MHC class II genes, the effect of mutations in the promoter on its activity, the affinity of the factor to its binding site, or antisense experiments, which evaluate the effect of reducing the expression of the transcription factor on the expression of MHC class II molecules [122]. However, direct evidence, which demonstrate the role of most transcription factors in vivo is lacking. Such evidence could be provided by a genetic approach, which introduces a transcription factor into a deficient cell or into an in vitro transcription system that lacks the factor, thereby restoring MHC class II expression. Most transcription factors are ubiquitously expressed, and can bind to several other gene promoters. For example, the RFX 1-4 proteins, belonging to the RFX family of proteins, can bind to other promoters, such as the hepatitis B enhancer or the mouse ribosomal gene rpL30 [131,132,144] and are probably not involved in the regulation of MHC class II expression. Another example is the NF-Y complex, which binds to the CCAAT sequence. This sequence is located in many gene promoters, such as albumin and thymidine kinase, and is implicated in the control of the expression of these genes [145, 146], as well as in the transcription of the MHC class II genes.

Nonetheless, direct evidences or compelling indirect evidences implicate three transcription factors, in the regulation of MHC class II transcription. (A) Elec-trophoretic mobility shift assays, in which the half-life of protein complexes was measured by competition with either the X box, the X2 box or the Y box, showed that both X2BP and NF-Y bind cooperatively with RFX to the MHC class II promoters, thus stabilizing the DNA-protein complex [147-149]. The RFX complex, therefore, probably acts as an "accessibility factor" that recruits NF-Y and X2BP to the MHC class II promoters via protein-protein interactions. (B) In vitro transcription experiments demonstrate an important role for NF-Y and RFX complexes in the transcription of MHC class II molecules [145, 150]. (C) The crucial role of the RFX complex in the transcription of MHC class II genes is demonstrated in mutated cells that lack this factor and therefore do not transcribe MHC class II genes (see below).

A severe immune deficiency disease resulting in MHC class II deficiency commonly termed the bare lymphocyte syndrome (BLS) [122, 151], provides a strong functional evidence for the role of the RFX complex in the regulation of MHC class II expression. Patients with BLS, that suffer from recurrent and lethal infections, are characterized by the lack of MHC class II expression on their B cells, and by the inability of IFN-y to induce MHC class II genes in their tissue cells. Cell fusion experiments between different cell lines derived from BLS patients or MHC class II-negative cell lines divide these cells into at least four complementation groups. In one of the complementation groups (complementation group C) the molecular defect was found to be to a mutation in the RFX5 protein. This mutation abrogated the ability of the RFX complex to bind to the promoter in vivo, and resulted in an unoccupied "bare" promoter [152,153]. Trans-fection of cell lines belonging to this complementation group with cDNA of the wild type RFX5 restored the expression of all MHC class II genes [127],

Complementation cloning lead to the isolation of a new transcription factor—MHC class II transacti-vator (CIITA) [154], This protein was found to be lacking or mutated in all cell lines belonging to BLS complementation group A, and indeed transfection of these cells with the wild type CIITA restored the expression of all MHC class II genes [154, 155], Furthermore, CIITA expression was found in all MHC class II positive cells, and this expression could be induced by IFN-y [154], In addition, shutting off the expression of MHC class II molecules in plasma cells was shown to be mediated by silencing of the CIITA protein expression [156], In contrast to the RFX proteins, CIITA is thought to be a non-DNA binding transcription factor, since no binding to the MHC class

II promoters could be observed [154]. Its N-terminal region, rich in acidic amino acids, is thought to be the transactivating domain, while the C-terminal domain is responsible for mediating MHC class II specificity, probably via protein-protein interactions with the RFX complex [157,158], Based on these data a model was proposed [158], in which CIITA expression is necessary for MHC class II transcription, and serves as an "on-off switch" or a "master regulator". The complex regulation of the CIITA gene expression, which in turn determines the regulation of the MHC class II genes in terms of cellular specificity and magnitude of expression, seems to be controlled by different usage of multiple promoters of the CIITA gene [159], One of these promoters have been implicated in the regulation of the constitutive expression in dendritic cells, another is specific for the constitutive expression of CIITA in B cells, while a third promoter controls the inducible expression of CIITA in many cell types. In addition, a new protein called IK has been recently cloned, which functions as an efficient inhibitor of IFN-y-induced expression of MHC class II molecules [160]. Stable transfection of human B cells with IK lead to the total disappearance of constitutive MHC class II expression and to a marked reduction of CIITA mRNA, suggesting that IK plays a role in the regulation of CIITA and MHC class II genes [161]. All these evidences imply that an intricate mechanism exists, in which the "master regulator" CIITA is itself under tight regulation. Thus, in order to control MHC class II expression and to allow intervention and treatment of autoimmune or malignant diseases, it is necessary to regulate the expression of CIITA.

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