Selective repression of the locus control region is then seen whenever multipotent stem cells differentiate into cells other than the erythroid lineage (Jimenez et al., 1992). These results suggest that inhibition is directed at the level of transactivation activity, which is dependent on Ca2+ influx (Ullman et al., 1992). In the first section of the introduction, I discussed the first acquisition of IL-2 gene inducibility early during T cell development.
For example, methylation of the muscle-specific α-actin promoter results in transcriptional repression in both nonmuscle and muscle cells (Paroush et al., 1990). Unfortunately, little is known about the methylation status of the IL-2 gene, particularly with regard to its inducibility. The negative regulation of the IL-2 gene by a suppressor or a negative regulator can be implied at two levels.
During myocyte differentiation, downregulation of the Id protein allows dimerization of MyoD family member proteins, which can now bind DNA (Benezra et al., 1990). Most of the transcription factors described above are not T-cell specific or IL-2 gene specific. Recent studies have shown that the factor(s) bound to one of the NF-AT-like sites in the IL-4 gene promoter did not contain an AP-1 factor (Rooney et al., 1994), in other words AP-1 is possibly not involved in IL-4 gene activation.
Promoter region of the IL-2 gene undergoes chromatin structure changes and confers inducibility to chloramphenicol.
TCR CD28
-I by itself did not induce any detectable IL-2 production. the effect of TL-1 in these cells was to potentiate the effects of TPA and A23187 stimuli on IL-2 synthesis. Arrows indicate the mobilities of the main AP-1p complex (left) and the AP-10 complex (right). Two of the factors detected by these assays appeared to be constitutively expressed in EL4.E1 thymoma cells.
IL-l-mediated induction coincides with the activation of a protein that can bind to the NF-κB consensus site in the 5'-flanking region of the K-chain genes (40). This inhibition is especially sharp at the beginning. time~ when IL-1 is strongest in its stimulatory activity. .. the cooperative effect of I L-1 with phosphoino:.itide pathway stimulation and that of cAMP appears quite clearly. The effects of IL-1 would then be additive with those of the pho phoino itide pathway agonists.
IL-1 alone is remarkably weak in affecting any increase or decrease in the binding activity of any of the factors studied here. Ubiquitous and lymphocyte-specific factors are involved in the induction of the murine interleukin 2 gene in T lymphocytes. It is then abrogated during the phase when the cells undergo positive and negative selection, i.e., the fate-determining processes that select the cells on maturity or death.
Levels of binding activity in different cell types were compared after being normalized to Oct-1 protein levels determined in parallel in aliquots of the same extracts. The pattern of inducibility of the DNA-binding proteins studied here is qualitatively summarized in Fig. Extracts from the thymocyte samples shown in panel a were analyzed for binding to the AP-1P oligonucleotide in the presence of the following antibodies: anti-CREB (C ).
While the characteristics of the three phases are established by our results. the proposed connections between them are hypothetical. as mentioned in the text. Cyclosporin A suppresses the expression of the interleukin 2 gene by inhibiting the binding of lymphocyte-specific factors to the IL-2 enhancer. Because much of the regulation of IL-2 is transcriptional (3, 22), the IL-2 gene has been extensively studied to learn how multiple signal transduction pathways are integrated to elicit a specific transcriptional response (45).
Finally, the diversity of factors suggests that only a subset of the factors detected in vitro are assembled into the active complex in vivo. Its intensity did not correlate in any way with the identity of the DNA sample (data not shown).
TPA/A23
Thus, CsA blocks the establishment and maintenance of the protein-DNA complex at the IL-2 promoter/enhancer. Thus, the cell-specific competence of the inducible IL-2 locus could not be detected at the level of major groove protein-DNA contacts. Activation of cells in the presence of forskolin prevents the maintenance of stable protein-DNA interactions in vivo, not only at NF-κB and TGGGC sites of the IL-2 enhancer, but also at NF-AT, AP-1, and other sites.
Furthermore, while TNF-α could “rescue” NF-κB binding activity in cells stimulated in the presence of forskolin, there was no rescue of the TGGGC factor (data not shown). When cells were activated in the presence of forskolin, mobilization of the factors that bind to TGGGC. Only after at least 4 h of stimulation in the presence of forskolin did we see the characteristic decrease in NF-κB site binding activity (Fig. 3 A, compare lanes 5 and 7).
Consistent with its effects on IL-2 mRNA expression, expression of a dominant negative mutant of PKA also facilitated the inhibitory effects of forskolin on NF-κB and TGGGC binding activity (Figure 4 B). Therefore, it is clear that PKA mediates the inhibitory effects of forskolin on DNA binding activities. By more closely examining protein-DNA interactions by in vivo imprinting, we discovered that the loss of NF-κB and TGGGC binding activities is associated with the disappearance of stable pro-.
Forskolin affects the formation of specific DNA binding complexes at multiple sites in the IL-2 promoter/enhancer region. A close-up of the effects of forskolin treatment on footprints at the distal and proximal NF-AT sites. In vivo footprints in three regions of the IL2 regulatory sequence (sense strand) are shown.
Activation of DNA binding activity in an apparently cytoplasmic precursor of the transcription factor NF-κB. Another developmental stage-dependent control of the IL-2 gene is in immature CD4-CD8-TCR cells. Second, upon examination of the IL-2 promoter region by in vivo footprinting analysis, two forms of the promoter are seen.
It has been demonstrated that blocking protein synthesis with cycloheximide leads to loss of protein-DNA contact in vivo (Weih et al., 1990). The site (AACAAAG) at -130 is immediately downstream of the NF-AT site, while the site -95 (CTITGAA) is immediately upstream of the Oct/OAP40 site.
