Search

Histone deacetylases (HDACs) play a critical role in regulating gene expression and key biological processes. However, how HDACs are involved in innate immunity is little understood. Here, in this first systematic investigation of the role of HDACs in immunity, we show that HDAC inhibition by a small-molecule HDAC inhibitor (HDACi), LAQ824, alters Toll-like receptor 4 (TLR4)-dependent activation and function of macrophages and dendritic cells (DCs). Surprisingly, pan-HDAC inhibition modulates only a limited set of genes involved in distinct arms of immune responses. Specifically, it inhibited DC-controlled T helper 1 (Th1) effector but not Th2 effector cell activation and migration. It also inhibited macrophage- and DC-mediated monocyte but not neutrophil chemotaxis. These unexpected findings demonstrate the high specificity of HDAC inhibition in modulating innate and adaptive immune responses, and highlight the potential for HDACi to alter the Th1 and Th2 balance in therapeutic settings.

Many procedures have been described for treating lateral epicondylitis with good success. The purpose of this report is to compare 3 operative methods for treatment of recalcitrant lateral epicondylitis-open, arthroscopic, and percutaneous. All patients with lateral epicondylitis who were operated on over a 7-year period were retrospectively reviewed. A minimum of 3 months of conservative care before surgery had failed in these patients, and they had a minimum of 2 years of follow-up. Concomitant pathology, complications, and necessary further care were noted. The outcomes were evaluated preoperatively and postoperatively with the Andrews-Carson score and visual analog scale scores for pain at rest, worst pain, and pain with activity. We included 109 patients in the study: 24 percutaneous, 44 arthroscopic, and 41 open procedures. The mean duration of conservative care was 13.2 months, including 2.5 conservative measures and 1.35 cortisone injections. The mean follow-up was 47.8 months. The preoperative Andrews-Carson score was 160.3. The postoperative Andrews-Carson score was 195. There was a statistically significant difference between preoperative and postoperative Andrews-Carson scores for each of the groups. There were no significant differences among the populations regarding age, gender, dominance, conservative measures used, cortisone injections, recurrences, complications, failures, visual analog scale scores, and preoperative and postoperative Andrews-Carson scores. In addition, no difference in outcome scores was noted when intraarticular and concomitant pathology was addressed in comparison to the population in which tendinosis alone was addressed. Open, arthroscopic, and percutaneous treatments of lateral epicondylitis offer 3 highly effective ways for the clinician to address this common clinical problem.

Cell lineage specification depends on both gene activation and gene silencing, and in the differentiation of T helper progenitors to Th1 or Th2 effector cells, this requires the action of two opposing transcription factors, T-bet and GATA-3. T-bet is essential for the development of Th1 cells, and GATA-3 performs an equivalent role in Th2 development. We report that T-bet represses Th2 lineage commitment through tyrosine kinase-mediated interaction between the two transcription factors that interferes with the binding of GATA-3 to its target DNA. These results provide a novel function for tyrosine phosphorylation of a transcription factor in specifying alternate fates of a common progenitor cell.

The transcription factors signal transducer and activator of transcription (STAT)1 and T-bet control the differentiation of interferon (IFN)-gamma-producing T helper type (Th)1 cells. Here we compare the role of T-bet and STAT1 in the initiation and regulation of experimental autoimmune encephalomyelitis (EAE), a disease initiated by Th1 cells. T-bet-deficient mice immunized with myelin oligodendrocyte glycoprotein (MOG) were resistant to the development of EAE. This protection was also observed when T-bet(-/-) mice were crossed to the MOG-specific 2D2 T cell receptor transgenic strain. In contrast, although T-bet is downstream of STAT1, STAT1(-/-) mice were highly susceptible to EAE and developed more severe and accelerated disease with atypical neuropathologic features. The function of T-bet was dominant as mice deficient in both T-bet and STAT1 were also protected from EAE. CD4(+) CD25(+) regulatory T cells from these two mice strains were fully competent and do not explain the difference in disease susceptibility. However, enhanced EAE in STAT1(-/-) mice was associated with continued generation of IFN-gamma-producing Th1 cells and up-regulation of selective chemokines responsible for the increased recruitment of macrophages and neutrophils in the central nervous system. Although the two transcription factors, STAT1 and T-bet, both induce IFN-gamma gene transcription, our results demonstrate marked differences in their function in regulating pathogenic Th1 cell responses.

Background: The transcription factor T-bet mediates IFN-gamma production by T(H)1 cells and suppresses T(H)2 cytokine production when ectopically expressed in polarized murine T(H)2 cells. Thus T-bet-mediated inhibition of T(H)2 cytokine production might be beneficial for the treatment of allergic diseases like asthma or atopic dermatitis., Objective: We sought to investigate the effects of ectopic T-bet expression in highly polarized human T(H)2 cells obtained from skin biopsy specimens of patients with atopic dermatitis., Methods: The cytokine production of T(H)2 cells retrovirally transfected with a vector expressing human T-bet was determined by means of intracellular FACS staining and ELISA. The effects of T-bet transfection were analyzed at the mRNA level by means of real-time PCR and DNA microarrays and confirmed by using functional chemokine response assays., Results: Transfection of T-bet into T(H)2 cells induced high levels of IFN-gamma and suppressed IL-5, but IL-2 and IL-4 production remained unchanged. T-bet transfection also induced IL-12Rbeta2 and CXCR3 expression on human T(H)2 cells, whereas the IL-18 receptor was only induced as a consequence of T-bet-mediated increased responsiveness to IL-12. Furthermore, sustained T-bet expression in human T(H)2 cells induced IL-2 production and decreased the secretion of IL-4. In addition, the chemokine receptor repertoire of these cells was changed toward a T(H)1-like profile., Conclusion: The combined switch in cytokine pattern and migratory potential of highly polarized human T(H)2 cells mediated by T-bet might provide an additional advantage for the treatment of allergic diseases.

Type 1 immunity relies on the differentiation of two major subsets of T lymphocytes, the CD4+ T helper (Th) cell and the CD8+ cytotoxic T cell, that direct inflammatory and cytotoxic responses essential for the destruction of intracellular and extracellular pathogens. In contrast to CD4 cells, little is known about transcription factors that control the transition from the CD8 naïve to effector cell stage. Here, we report that the transcription factor T-bet, known to regulate Th cell differentiation, also controls the generation of the CD8+ cytotoxic effector cell. Antigen-driven generation of effector CD8+ cells was impaired in OT-I T cell receptor transgenic mice lacking T-bet, resulting in diminished cytotoxicity and a marked shift in cytokine secretion profiles. Furthermore, mice lacking T-bet responded poorly to infection with lymphocytic choriomeningitis virus. T-bet is a key player in the generation of type 1 immunity, in both Th and T cytotoxic cells.

The T helper lymphocyte is responsible for orchestrating the appropriate immune response to a wide variety of pathogens. The recognition of the polarized T helper cell subsets Th1 and Th2 has led to an understanding of the role of these cells in coordinating a variety of immune responses, both in responses to pathogens and in autoimmune and allergic disease. Here, we discuss the mechanisms that control lineage commitment to the Th1 phenotype. What has recently emerged is a rich understanding of the cytokines, receptors, signal transduction pathways, and transcription factors involved in Th1 differentiation. Although the picture is still incomplete, the basic pathways leading to Th1 differentiation can now be understood in in vitro and a number of infection and disease models.

Naïve T cells differentiate into effector cells upon stimulation with antigen, a process that is accompanied by changes in the chromatin structure of effector cytokine genes. Using histone acetylation to evaluate these changes, we showed that T cell receptor (TCR) stimulation results in early activation of the genes encoding both interleukin 4 and interferon-gamma. We found that continued culture in the presence of polarizing cytokines established a selective pattern of histone acetylation on both cytokine genes; this correlated with restricted access of the transcription factor NFAT1 to these gene regulatory regions as well as mutually exclusive gene expression by the differentiated T cells. Our data point to a biphasic process in which cytokine-driven signaling pathways maintain and reinforce chromatin structural changes initiated by the TCR. This process ensures that cytokine genes remain accessible to the relevant transcription factors and promotes functional cooperation of the inducible transcription factor NFAT with lineage-specific transcription factors such as GATA-3 and T-bet.

The balance between pro and antiinflammatory cytokines secreted by T cells regulates both the initiation and perpetuation of inflammatory bowel diseases (IBD). In particular, the balance between interferon (IFN)-gamma/interleukin (IL)-4 and transforming growth factor (TGF)-beta activity controls chronic intestinal inflammation. However, the molecular pathways that evoke these responses are not well understood. Here, we describe a critical role for the transcription factor T-bet in controlling the mucosal cytokine balance and clinical disease. We studied the expression and function of T-bet in patients with IBD and in mucosal T cells in various T helper (Th)1- and Th2-mediated animal models of chronic intestinal inflammation by taking advantage of mice that lack T-bet and retroviral transduction techniques, respectively. Whereas retroviral transduction of T-bet in CD62L(+) CD4(+) T cells exacerbated colitis in reconstituted SCID mice, T-bet-deficient T cells failed to induce colitis in adoptive transfer experiments suggesting that overexpression of T-bet is essential and sufficient to promote Th1-mediated colitis in vivo. Furthermore, T-bet-deficient CD62L(-) CD4(+) T cells showed enhanced protective functions in Th1-mediated colitis and exhibited increased TGF-beta signaling suggesting that a T-bet driven pathway of T cell activation controls the intestinal balance between IFN-gamma/IL-4 and TGF-beta responses and the development of chronic intestinal inflammation in T cell-mediated colitis. Furthermore, TGF-beta was found to suppress T-bet expression suggesting a reciprocal relationship between TGF-beta and T-bet in mucosal T cells. In summary, our data suggest a key regulatory role of T-bet in the pathogenesis of T cell-mediated colitis. Specific targeting of this pathway may be a promising novel approach for the treatment of patients with Crohn's disease and other autoimmune diseases mediated by Th1 T lymphocytes.

A molecular understanding of the regulation of IgG class switching to IL-4-independent isotypes, particularly to IgG2a, remains largely unknown. The T-box transcription factor T-bet directly regulates Th1 lineage commitment by CD4 T cells, but its role in B lymphocytes has been largely unexplored. We show here a role for T-bet in the regulation of IgG class switching, especially to IgG2a. T-bet-deficient B lymphocytes demonstrate impaired production of IgG2a, IgG2b, and IgG3 and, most strikingly, are unable to generate germ-line or postswitch IgG2a transcripts in response to IFN-gamma. Conversely, enforced expression of T-bet initiates IgG2a switching in cell lines and primary cells. This function contributes critically to the pathogenesis of murine lupus, where the absence of T-bet strikingly reduces B cell-dependent manifestations, including autoantibody production, hypergammaglobulinemia, and immune-complex renal disease and, in particular, abrogates IFN-gamma-mediated IgG2a production. Classical T cell manifestations persisted, including lymphadenopathy and cellular infiltrates of skin and liver. These results identify T-bet as a selective transducer of IFN-gamma-mediated IgG2a class switching in B cells and emphasize the importance of this regulation in the pathogenesis of humorally mediated autoimmunity.

gammadelta T cells predominantly produce IFN-gamma upon activation. To determine the basis for default production of IFN-gamma by gammadelta T cells, we analyzed the transcription factors T-box expressed in T cells (T-bet) and GATA-3. T-bet, absent in naive cells, was induced upon TCR signaling, with IFN-gamma production. T-bet also regulated IL-4 synthesis, as gammadelta cells isolated from T-bet-deficient mice displayed enhanced IL-4 levels with reduced IFN-gamma production. Notably, T-bet expression after TCR signaling in gammadelta cells was not down-regulated by IL-4, in conjunction with a higher ratio of T-bet:GATA-3 expression than that found in CD4(+) T cells. Indeed, overexpression of GATA-3 failed to inhibit IFN-gamma secretion in gammadelta cells to the degree seen in CD4(+) T cells. These results indicate that T-bet enhances IFN-gamma secretion and suppresses IL-4 secretion in gammadelta cells, and that GATA-3 fails to counterbalance T-bet-mediated IFN-gamma production, accounting for the default synthesis of IFN-gamma by these T lymphocytes.

T-bet is a member of the T-box family of transcription factors that appears to regulate lineage commitment in CD4 T helper (TH) lymphocytes in part by activating the hallmark TH1 cytokine, interferon-gamma (IFN-gamma). IFN-gamma is also produced by natural killer (NK) cells and most prominently by CD8 cytotoxic T cells, and is vital for the control of microbial pathogens. Although T-bet is expressed in all these cell types, it is required for control of IFN-gamma production in CD4 and NK cells, but not in CD8 cells. This difference is also apparent in the function of these cell subsets. Thus, the regulation of a single cytokine, IFN-gamma, is controlled by distinct transcriptional mechanisms within the T cell lineage.

Human asthma is associated with airway infiltration by T helper 2 (TH2) lymphocytes. We observed reduced expression of the TH1 transcription factor, T-bet, in T cells from airways of patients with asthma compared with that in T cells from airways of nonasthmatic patients, suggesting that loss of T-bet might be associated with asthma. Mice with a targeted deletion of the T-bet gene and severe combined immunodeficient mice receiving CD4+ cells from T-bet knockout mice spontaneously demonstrated multiple physiological and inflammatory features characteristic of asthma. Thus, T-bet deficiency, in the absence of allergen exposure, induces a murine phenotype reminiscent of both acute and chronic human asthma.

Naive T helper cells differentiate into two subsets, Th1 and Th2, each with distinct functions and cytokine profiles. Here, we report the isolation of T-bet, a Th1-specific T box transcription factor that controls the expression of the hallmark Th1 cytokine, IFNgamma. T-bet expression correlates with IFNgamma expression in Th1 and NK cells. Ectopic expression of T-bet both transactivates the IFNgamma gene and induces endogenous IFNgamma production. Remarkably, retroviral gene transduction of T-bet into polarized Th2 and Tc2 primary T cells redirects them into Th1 and Tc1 cells, respectively, as evidenced by the simultaneous induction of IFNgamma and repression of IL-4 and IL-5. Thus, T-bet initiates Th1 lineage development from naive Thp cells both by activating Th1 genetic programs and by repressing the opposing Th2 programs.

Although evidence is strong that Th cells play a major role in mediating the airway inflammation observed in asthma, the relative contributions of the Th cell subsets, Th1 and Th2, are unclear. It has been suggested that asthma is driven by Th2 predominant responses in the lung, but other data suggest a role for Th1 cells as well. Here we show by intracellular cytokine staining and flow cytometric analysis that in the murine model of OVA-induced airway inflammation, both Th1 and Th2 cells are recruited to the airways. Th1 cells predominate early in the response and Th2 cells predominate late. We further show that increasing the number of Th1 cells by passive transfer of OVA-specific Th1 cells results in increased inflammation. This effect is observed regardless of whether the T cells are transferred before sensitization or after airway inflammation is already in progress. Transfer of Th1 cells also results in increased recruitment of host T cells of both Th1 and Th2 phenotypes. Passive transfer of Th2 cells results in little change in the inflammatory response. These results demonstrate that Ag-specific Th1 cells are not protective in this model of asthma, but rather may potentiate the inflammatory response.

Much of our focus in understanding Th1/Th2 development has been on the signals delivered by IL-12 and IL-4 as final determinants of terminal T cell differentiation. Because extinction of IL-12 signaling in early Th2 development could potentially be important in imprinting a more permanent Th2 phenotype on a population of T cells, we have also examined various parameters regulating the IL-12 signaling pathway. Whereas IL-4 appears to repress functional IL-12 signaling through inhibition of IL-12R beta 2 expression, IFN-gamma in the mouse, and IFN-alpha in the human appear to induce IL-12R beta 2 expression and promote IL-12 responsiveness. We propose that Th1 development can be considered in two stages, capacitance and development. Capacitance would simply involve expression of IL-12R beta 1 and beta 2 subunits, regulated by TCR, IL-4 and IFNs. The second stage, development, we propose is the true IL-12 induced developmental stage, involving expression of Stat4 inducible proteins. In the human, this may also occur via IFN-alpha, which is able to activate Stat4. It is perhaps possible that all of Stat4 actions on Th1 development may be exert directly by Stat4 at the IFN-gamma gene, however we suggest that, more likely, Stat4 may act to induce Th1 development through the induction of other non-cytokine genes, whose stable expression maintains the transcriptional state of a Th1 cell.

Interleukin-4 is an immunomodulatory cytokine which plays a central role in the regulation of allergic and atopic immune responses. Significant progress has been made in gaining a detailed understanding of the transcriptional regulation of the interleukin-4 gene. The recent identification and characterization of several key transcription factors has helped to elucidate the molecular mechanisms of T helper cell cytokine gene expression.

The developmental commitment to a T helper 1 (Th1)- or Th2-type response can significantly influence host immunity to pathogens. Extinction of the IL-12 signaling pathway during early Th2 development provides a mechanism that allows stable phenotype commitment. In this report we demonstrate that extinction of IL-12 signaling in early Th2 cells results from a selective loss of IL-12 receptor (IL-12R) beta 2 subunit expression. To determine the basis for this selective loss, we examined IL-12R beta 2 subunit expression during Th cell development in response to T cell treatment with different cytokines. IL-12R beta 2 is not expressed by naive resting CD4+ T cells, but is induced upon antigen activation through the T cell receptor. Importantly, IL-4 and IFN-gamma were found to significantly modify IL-12 receptor beta 2 expression after T cell activation. IL-4 inhibited IL-12R beta 2 expression leading to the loss of IL-12 signaling, providing an important point of regulation to promote commitment to the Th2 pathway. IFN-gamma treatment of early developing Th2 cells maintained IL-12R beta 2 expression and restored the ability of these cells to functionally respond to IL-12, but did not directly inhibit IL-4 or induce IFN-gamma production. Thus, IFN-gamma may prevent early Th cells from premature commitment to the Th2 pathway. Controlling the expression of the IL-12R beta 2 subunit could be an important therapeutic target for the redirection of ongoing Th cell responses.

Selective cytokine gene expression by T cell subsets underlies polarization of cellular and humoral immune responses. Our interest has been to define the molecular basis for restricted cytokine expression by Th1 and Th2 cells. IL-4 is selectively expressed by Th2 cells, providing a model for Th2-specific gene expression. To allow for promoter analysis during the process of Th1/Th2 differentiation within a normal cellular context, we have taken a transgenic approach. We generated a series of murine transgenic lines harboring both the DO11.10 alphabeta-TCR transgene and the luciferase gene driven by regions of the IL-4 promoter. The results identify proximal promoter regions that provide significantly Th2-restricted IL-4 gene expression. The IL-4 -741- to +60-bp region allows, on the average, 40-fold higher inducible reporter activity in Th2 cells than in Th1 cells. When trimerized, the region spanning -88 to -61 bp, containing a composite NF-AT/AP-1 site, also confers significant Th2-specific reporter activity. These results suggest that trans-acting factors binding this NF-AT/AP-1 composite site cooperate to allow substantial Th2-selective reporter expression. Finally, because reporter expression is low relative to endogenous IL-4 mRNA in activated Th2 cells, we suggest that additional control elements outside of the IL-4 promoter may be required to enhance overall IL-4 gene activity.

The experiments described above have allowed us to define the molecular events in IL-12 signalling. Within minutes after IL-12 treatment of responsive cells, Stat1, Stat3, and Stat4 are tyrosine phosphorylated. These molecules form nuclear DNA-binding complexes consisting of homodimeric Stat1 and heterodimeric Stat3-Stat4 complexes. In a murine in vitro phenotype development model, T cells rapidly and selectively lose their capacity to respond to IL-12 upon acquisition of the Th2 phenotype. This hyporesponsiveness is manifested by the inability of IL-12 to induce IFN gamma production in differentiated Th2 cells, as well as the inability of IL-12 to induce the activation of Stat4. Despite the functional defect of IL-12 signalling in Th2 cells, all known components of the IL-12 signal transduction pathway are present. We speculate that in Th2 cells, the second receptor chain may be absent or one of the other components may be modified. Genetic experiments in Balb/c and B10.D2 strains of mice have demonstrated several differences in T helper differentiation in vitro. Stimulation of T cells under neutral conditions results in a bias of Balb/c T cells toward the Th2 extreme and B10 T cells toward the Th1 extreme of cytokine production. Following stimulation under neutral conditions, B10 T cells retain the ability to respond to IL-12 while Balb/c T cells lose IL-12 responsiveness. Mating experiments have demonstrated that the B10 genetic effect is dominant in F1 mice. Analysis of backcrossed animals has suggested that the ability to respond to IL-12 in the secondary stimulation may be controlled by a single dominant B10 gene. The results we describe may have profound implications for allergy. Since allergic responses are largely due to the activation of the Th2 subset of T lymphocytes, a better understanding of T cell phenotype development may reveal multiple targets for therapeutic intervention. First, a better understanding of Th1 phenotype induction in response to IL-12 may allow prevention of in vivo allergic responses using pharmacological tools which bias allergen-specific responses to the Th1 subset. Second, a molecular explantation of why Th2 cells fail to reverse phenotype in response to IL-12 may allow treatment of atopic individuals to remove the disease-promoting T lymphocyte compartment. Finally, a better understanding of the basis for genetic differences in murine T helper cell differentiation may allow us to identify a causative genetic element in humans, yielding better diagnostic and therapeutic methods.

Interferon gamma (IFN-gamma) responsiveness in certain cells depends on the state of cellular differentiation or activation. Here an in vitro developmental system was used to show that IFN-gamma produced during generation of the CD4+ T helper cell type 1 (TH1) subset extinguishes expression of the IFN-gamma receptor beta subunit, resulting in TH1 cells that are unresponsive to IFN-gamma. This beta chain loss also occurred in IFN-gamma-treated TH2 cells and thus represents a specific response of CD4+ T cells to IFN-gamma rather than a TH1-specific differentiation event. These results define a mechanism of cellular desensitization where a cytokine down-regulates expression of a receptor subunit required primarily for signaling and not ligand binding.

Developmental-commitment to Th1 or Th2 responses critically influences host susceptibility to particular pathogens. We describe a novel mechanism governing stable commitment to Th2 differentiation. Naive T cells develop strongly polarized Th1 and Th2 profiles by 7 days after activation. However, commitment of these developing cells differs substantially. Although IL-4 reverses early Th1 differentiation, IL-12 cannot reverse early Th2 differentiation. Th1 reversibility results from maintenance of IL-4 signal transduction, whereas Th2 commitment results from rapid loss of IL-12 signaling. The IL-12 signaling defect in Th2 cells results in failure to phosphorylate Jak2, Stat3, and Stat4. Since Th2 cells express the mRNA for the cloned murine IL-12 receptor beta subunit, the signaling defect may involve expression or function of unidentified receptor components. The rapid extinction of IL-12 signaling in Th2 cells provides a demonstration of a mechanism for the stable commitment to a T helper phenotype.

Interleukin 12 (IL-12) initiates the differentiation of naive CD4+ T cells to T helper type 1 (Th1) cells critical for resistance to intracellular pathogens such as Leishmania major. To explore the basis of IL-12 action, we analyzed induction of nuclear factors in Th1 cells. IL-12 selectively induced nuclear DNA-binding complexes that contained Stat3 and Stat4, recently cloned members of the family of signal transducers and activators of transcription (STATs). While Stat3 participates in signaling for several other cytokines, Stat4 was not previously known to participate in the signaling pathway for any natural ligand. The selective activation of Stat4 provides a basis for unique actions of IL-12 on Th1 development. Thus, this study presents the first identification of the early events in IL-12 signaling in T cells and of ligand activation of Stat4.

Activity of the murine interleukin-4 (IL-4) promoter was localized to several cis-acting elements present within the first 300 bp from the transcriptional initiation site. Five repeated elements, P0 to P4, that share the common consensus ATTTTCCNNT were located between -40 and -250, and each was shown to interact with the T-cell-specific factor NF(P). These distinct P sites appear functionally interchangeable and cooperatively confer cyclosporin A-sensitive and ionomycin-inducible promoter activity. NF(P) may be closely related to the cytoplasmic component of NF-AT (nuclear factor of activated T cells), a T-cell-specific factor essential for IL-2 gene transcription, as judged from indistinguishable molecular weights and protease fragmentation patterns of UV-photolabeled factors. Also, we identified an element in the IL-4 promoter with homology to the Y box common to all major histocompatibility complex class II gene promoters. Our data show that the IL-4 promoter Y box -114CTGATTGG-107 significantly enhances overall promoter activity, since point mutations within this element diminish promoter activity by 85%. The factor binding this region is indistinguishable from the cloned nuclear factor NF-Y, as judged from interactions with specific anti-NF-Y monoclonal and polyclonal antibodies. Last, we point out the presence of two sites that share sequence identity to the OAP region of the ARRE-1 site within the IL-2 promoter (K. S. Ullman, W. M. Flanagan, C. A. Edwards, and G. R. Crabtree, Science 254:558-562, 1991). These regions, -85GTGTAATA-78 and -245GTGTAATT-238, reside adjacent to the NF(P) binding sites P1 and P4 and bind a distinct nuclear factor.