The transcription factor hXBP-1 belongs to the family of basic region/leucine zipper (bZIP) proteins and interacts with the cAMP responsive element (CRE) of the major histocompatibility complex (MHC) class II A alpha, DR alpha and DP beta genes. However, the developmental expression of hXBP-1 as revealed by in situ hybridization in mouse embryos, has suggested that it interacts with the promoter of additional genes. To identify other potential target genes of this factor, we performed binding site selection experiments with recombinant hXBP-1 protein. The results indicated that hXBP-1 binds preferably to the CRE-like element GAT-GACGTG(T/G)NNN(A/T)T, wherein the core sequence ACGT is highly conserved, and that it also binds to some TPA response elements (TRE). hXBP-1 can transactivate multimers of the target sequences to which it binds in COS cells, and the level of transactivation directly correlates with the extent of binding as observed in gel retardation experiments. One target sequence that is strongly bound by hXBP-1 is the 21 bp repeat in the HTLV-1 LTR, and we demonstrate here that hXBP-1 can transactivate the HTLV-1 LTR. Further, the transactivation domain of hXBP-1 encompasses a large C-terminal region of the protein, containing domains rich in glutamine, serine and threonine, and proline and glutamine residues, as shown in transient transfection experiments using hXBP-1-GAL4 fusion proteins and a reporter gene under the control of GAL4-binding sites.

In order to investigate the molecular basis of the regulation of interferon-inducible genes, we isolated the promoter region of two such genes coding for the (2'-S')oligo(adenylate) synthetase and a 56-kDa protein (IFI-56K). The regions surrounding the cap site were sequenced and compared with the sequences of vertebrate and viral DNA present in the Genbank data bank. Small DNA segments were found in both genes which are homologous to part of the promoter region of other genes, such as those of interferon-p, tumor necrosis factor / 3, interleukin-2 and its receptor. Since these homologies were found located in functionally important regions of these genes, we tested whether their inducers also enhance the (2'-5')oligo(adenylate) synthetase and IFI-56K gene expression. We found that poly(r1) . poly(rC) and interleukin-1, activators of the interferon-p gene and of T lymphocytes respectively, are both able to enhance IFI-56K mRNA accumulation in all cell lines tested.Cycloheximide even superinduces this gene when added together with poly(r1) . poly(rC) and interleukin-1 (but not when added with interferon). We showed that these inductions are direct and not mediated by interferon produced by cells in response to poly(r1) . poly(rC) or interleukin-1 . The promoter sequence analyses have thus led to the discovery of unexpected inducers, i.e. an interferon inducer such as poly(r1) . poly(rC) is also able to directly induce a gene that is under the control of interferon.Interferons are proteins or glycoproteins secreted by various cells in response to viral infection or other stimuli. They are defined by their antiviral activity, i.e. protection against subsequent infection by different viruses of pretreated, metabolically active cells. Beside the establishment of an antiviral state, interaction between interferon and its receptor on the cell surface also leads to a wide range of biological effects, such as the inhibition of cell growth and modulations of the immune system (for reviews see [l, 21. In that way interferons, as other cytokines, act as pleiotropic effectors, since they modify not only the biochemistry of some target cells but also the physiology of the entire organism. Conversely it has been suggested that some other cytokines (tumor necrosis factors c( and p [3,41) have intrinsic antiviral activities.Numerous cDNAs corresponding to interferon-inducible genes have been cloned but the biological function of these genes has been discovered in a few cases only. Their transcriptional regulation appears to be rather complex. Indeed, all the genes studied so far seem to behave differently with respect to their kinetics of mRNA accumulation after interferon treatment. Moreover, for a given gene, the response to interferon treatment is dependent on the type of interferon and target cell used [5 -111. Finally, amongst these genes, only some remain inducible in interferon-resistant lymphoblastoid (Daudi) cells, demonstrating the existence of at least two different pathways of induction downstream from the interacti...

The spatial and temporal distribution of transcripts for the TREERE-binding basic region-leucine zipper protein hXBP-1 was determined by in situ hybridization. Analysis of embryos from day 10.5 to 18.5 pc revealed high level expression of hXBP-1 RNA in two developing organ systems: 1) in bone and cartilage cells of the developing skeleton and toothbuds, and 2) in exocrine glands including the pancreas and the submandibular and salivary glands. High level expression was also found in whisker follicles and in selected cells in brown adipose tissue. In the developing skeleton, hXBP-1 RNA was expressed starting on day 11.5 pc in osteoblasts of newly formed intramembranous bone. Thereafter, hXBP-1 was expressed in both osteoblasts and preosteoblasts in bone formed directly by intramembranous formation as well as in bone formed during endochondral ossification. The most intense signal was observed in preosteoblasts and osteoblasts of newly forming bone. At day 11.5 pc low level hXBP-1 expression was also observed in matrix secreting chondroblasts of bones which are formed initially of cartilage, at the stage where they consist entirely of cartilage. Signal was also present in matrix producing chondroblasts of the mature zone of the growth region during endochondral ossification although at significantly lower level than in osteoblasts. hXBP-1 is thus the first transcription factor described, to our knowledge, whose level of expression is modulated during the osteoblast developmental sequence in vivo. The pattern of expression of hXBP-1 in the developing skeleton was found to be very similar to that of the genes encoding the tissue inhibitor of metalloproteinase and alkaline phosphatase throughout development. These observations suggest that hXBP-1 may play a role in regulating the expression of tissue specific genes (TIMP, osteonectin, osteopontin, osteocalcin) expressed in osteoblasts. It is intriguing that the promoter regions of several such genes contain po-

The IFI-56K and IFI-54K genes are transcriptionally stimulated when cells are treated by interferon. We have previously shown that the IFI-56K gene is in addition directly induced by poly(r1) . poly(rC), and inducer of interferon-P. Since the regulation of the IFI-56K and IFI-54K genes by interferon are very much alike, we tested whether the IFI-54K gene is also directly regulated by poly(r1) . poly(rC). Treatment of various cell lines with poly(r1) . poly(rC) leads to a clear accumulation of the IFI-54K mRNA to a level which sometimes even exceeds that obtained with high doses of interferon. Several interferon-resistant cell lines were investigated for the inducibility of both the IFI-56K and IFI-54K genes by interferons, poly(r1) . poly(rC) and viruses (which are the natural inducers of interferon-a and -p). Both genes appear to be coordinately regulated by these inducers. It was thus interesting to search for common regulatory element(s) in the control region of these two genes. The IFI-54K gene promoter region was isolated, from which a 520-base-pair segment was sequenced and compared with the promoter region of the IFI-56K gene that we had previously sequenced. The only homology was found is a well conserved 19-bp segment located just upstream of the TATA box of these genes; interestingly, this sequence is also homologous to the minimal region needed for the inducibility by poly(r1) . poly(rC) of the interferon-p gene. This conserved sequence might be responsible for the coordinate induction of the IFI-56K and IFI-54K genes by interferon, poly(r1) . poly(rC) and viruses.Interferons (IFNs) are a class of proteins or glycoproteins produced by vertebrate cells triggered by various stimuli such as viral infections or poly(r1) . poly(rC) treatment. IFNs act as pleiotropic effectors, which protect cells from viral infections, exert and antimitogenic effect and modulate the immune system (reviewed in [l, 21). At least some of these various biological responses to IFNs are probably mediated by the new proteins induced by IFNs. A number of these proteins appear in IFN-treated cells as a consequence of an increased transcription rate of the corresponding gene. The regulation of the IFN-inducible genes is rather complex; indeed, the set of proteins that are induced, as well as their kinetics of accumulation, differ from one cell line to the other, and depend upon the IFN type used [3 -141. Moreover, amongst these genes, only some remain inducible in IFN-resistant Daudi cells, demonstrating the existence of at least two different pathways of induction downstream from the interaction of IFN with its receptor [15 -171. Finally, some of the IFN-inducible genes are also regulated by other inducers: e. g. the metallothionein gene is not only controlled by IFNs, but also by heavy metals, glucocorticoids, interleukin 1 and other factors [18 -201.We have previously described the cDNA cloning of two human genes inducible by IFNs, namely the 42-kDa (2'-5')oligo(adenylate) synthetase and the IFI-56K genes [21, 221, We have also i...