We have used two strategies to identify genes that are co-regulated with MHC-II genes. First, we used a computer scan to search for genes that contain MHC-II-like S-Y enhancers in their upstream regions. Second, we performed ChIP-on-chip screens to identify genes that are direct targets of CIITA, the master regulator of MHC-II genes. These two approaches have converged on RAB4B, a gene encoding a protein implicated in endosome recycling. A combination of ChIP, ChIP-on-chip, expression analysis and functional studies in wild-type and mutant cells lacking CIITA or RFX have confirmed that RAB4B expression is indeed driven by a typical S-Y enhancer that is regulated by both CIITA and the MHC-II enhanceosome complex composed of RFX, CREB and NF-Y.
Searching for potential binding sites in genomic DNA sequences is generally of limited usefulness for identifying target genes regulated by specific transcription factors. A major problem with such approaches is that transcription factor binding sites are often short and degenerate with respect to their consensus sequence, such that computer searches for potential binding sites tend to yield overwhelmingly large numbers of irrelevant hits. Our searches with the MHC-II S-Y profile constitute an exception to this rule because they have proved to be remarkably reliable—in this report and in a previous study (20
)—for the identification of novel enhancers regulated by the MHC-II enhanceosome complex and CIITA. The success of our approach implies that similar strategies could be valuable for identifying genes regulated by higher order transcription factor complexes, particularly in systems where gene expression is controlled by well-defined composite regulatory modules.
The use of genome-scale ChIP-on-chip-based binding studies to identify target genes of specific transcription factors in eukaryotic organisms has become feasible only recently thanks to the availability of microarrays covering entire genomes, large genomic regions or the promoter regions of comprehensive sets of genes. To date, ChIP-on-chip studies have investigated the target gene specificity of DNA-binding transcription factors (48
). We demonstrate here that such ChIP-on-chip experiments can also be very powerful for studying the target genes of non-DNA-binding transcriptional co-activators such as CIITA.
Past efforts to identify new genes regulated by CIITA have led to a certain amount of controversy concerning the target gene specificity of CIITA (53
). CIITA was initially believed to be dedicated for the expression of classical MHC-II genes and related genes implicated in antigen presentation, including the Ii
, HLA–DO, HLA–DM and MHC-I genes. Subsequent reports suggested that CIITA might be more pleiotropic in its function. Several genes were suggested to be repressed by CIITA in various cell types, including those encoding IL4 and FasL in T cells, cathepsin E and IL-10 in B cells and DC, and collagen type I α2, thymidine kinase and cyclin D1 in IFN-γ induced cells (54
). A microarray experiment suggested that at least 16 other genes of diverse functions are repressed by CIITA in a human B cell line (58
). Finally, microarray experiments have suggested that CIITA enhances the expression of Plexin-A1 in mouse DC and numerous other genes, including RAB4B, in a human B cell line and in IFN-γ induced cells (58
). However, none of these candidate genes have as yet been demonstrated to be controlled by S-Y enhancers or been validated as direct targets of the MHC-II regulatory machinery by ChIP experiments. In fact, for several candidates an indirect mechanism involving sequestration of the general co-activator CBP by CIITA has been proposed (54
). Moreover, conflicting reports concerning their dependence on CIITA have been published for several candidates (60
). Therefore, with the exception of the Ii
gene, the RAB4B
gene identified here is the first example of a non-MHC gene that contains a typical S-Y enhancer and is regulated by the same regulatory machinery as MHC-II genes.
RAB4 has a well established function in recycling proteins and lipids from EE and RE back to the PM. The RAB4A and RAB4B isoforms co-localize to the same compartments and are highly homologous, suggesting that they have largely redundant functions in recycling. Enhancing RAB4B expression by placing it under the control of the MHC-II regulatory machinery may thus simply be a way of increasing the recycling capacity of APC by raising the total level of RAB4 protein. However, an alternative possibility is suggested by an intriguing difference between the two RAB4 isoforms at amino acid 199. In RAB4A there is a serine (S) at this position. Phosphorylation of S199 by the mitotic cdc2 kinase causes the dissociation of RAB4A from membranes and its accumulation in the cytoplasm (61
). This was proposed to be responsible for the inactivation of RAB4A during mitosis. Interestingly, RAB4B has a glutamine (Q) at position 199, a substitution that was shown to prevent the phosphorylation and cytoplasmic accumulation of RAB4A. RAB4B is thus likely to remain active during mitosis. This difference could underlie a specific function of RAB4B. Regulating RAB4B expression by the MHC-II regulatory machinery may thus be a way of ensuring this specific function in APC.
Endocytic recycling contributes to antigen presentation by MHC-II molecules. Cell surface MHC-II molecules can be internalized by endocytosis, loaded with new peptides in early endosomes and recycled back to the PM instead of being targeted for lysosomal degradation (9
). Certain MHC-II restricted peptides do not require extensive processing in late endosomal compartments and are generated and loaded onto MHC-II molecules in early endosomes, from which the MHC-II-peptide complexes are recycled to the cell surface (10
). This recycling-dependent antigen presentation pathway is presumably under the control of RAB4. Direct evidence for a role of RAB4 in antigen presentation has been provided by studies using a dominant negative mutant of RAB4A. Transfection of mouse A20 B cells with a dominant negative RAB4A mutant was found to inhibit the processing and presentation of certain antigens internalized by B-cell-receptor (BCR) or Fc-receptor-mediated uptake (13
). Interestingly, we have found that A20 B cells actually express only RAB4B
mRNA (data not shown). The dominant negative RAB4A mutant must therefore have interfered with the activity of RAB4B, thereby providing a functional link between the latter and antigen presentation.
In addition to MHC-II restricted antigen presentation, recycling is known to play key roles in other antigen presentation processes. Several studies have demonstrated that recycling is one of the mechanisms that accounts for the ability of DC to cross-present peptides derived from exogenous antigens in the context of MHC-I molecules (7
). DC have recently also been shown to internalize antigens and the recycle them back to PM for direct presentation to B cells (16
). Enhancing the expression of RAB4B
in APC by the MHC-II regulatory machinery is thus likely to contribute to the efficiency of several different antigen presentation processes.