George Blanck

Co-occupancy of the interferon regulatory element of the class II transactivator (CIITA) Type IV promoter by interferon regulatory factors 1 and 2

Class II transactivator (CIITA) activates the expression of major histocompatibility class II genes, which encode antigen-presenting molecules recognized by the T-cell receptor of CD4+ T cells. IFN-γ induced CIITA transcription in many cell types is directed by the CIITA Type IV promoter. Here we report that the human CIITA Type IV promoter IRF-E binds IRF-1 and can be activated by exogenous expression of IRF-1. Surprisingly, the CIITA Type IV promoter IRF-E is also activated by IRF-2, another member of the IRF family that generally acts as a transcriptional repressor. In addition, we found that IRF-1 and IRF-2 synergistically activate the CIITA Type IV promoter. Electrophoretic mobility shift assays revealed that IRF-1 and IRF-2 can simultaneously occupy the IRF-E of the CIITA Type IV promoter, suggesting a novel mechanism for the role of these two proteins in promoter activation. Our results also indicate that IRF-1 and IRF-2 can cooperatively activate and co-occupy the IRF-E of the guanylate binding protein (GBP) promoter. Finally, CIITA induction by IFN-γ does not occur in a pancreatic tumor cell line that expresses a mutated IRF-2, representing the first IRF-2 mutation identified in a human tumor cell line.

Histone Deacetylase Activity Represses Gamma Interferon-Inducible HLA-DR Gene Expression following the Establishment of a DNase I-Hypersensitive Chromatin Conformation

ABSTRACT Expression of the retinoblastoma tumor suppressor protein (Rb) is required for gamma interferon (IFN-γ)-inducible major histocompatibility complex class II gene expression and transcriptionally productive HLA-DRA promoter occupancy in several human tumor cell lines. Treatment of these Rb-defective tumor cell lines with histone deacetylase (HDAC) inhibitors rescued IFN-γ-inducible HLA-DRA and -DRB mRNA and cell surface protein expression, demonstrating repression of these genes by endogenous cellular HDAC activity. Additionally, Rb-defective, transcriptionally incompetent tumor cells retained the HLA-DRA promoter DNase I-hypersensitive site. Thus, HDAC-mediated repression of the HLA-DRA promoter occurs following the establishment of an apparent nucleosome-free promoter region and before transcriptionally productive occupancy of the promoter by the required transactivators. Repression of HLA-DRA promoter activation by HDAC activity likely involves a YY1 binding element located in the first exon of the HLA-DRA gene. Chromatin immunoprecipitation experiments localized YY1 to the HLA-DRA gene in Rb-defective tumor cells. Additionally, mutation of the YY1 binding site prevented repression of the promoter by HDAC1 and partially prevented activation of the promoter by trichostatin A. Mutation of the octamer element also significantly reduced the ability of HDAC1 to confer repression of inducible HLA-DRA promoter activation. Treatment of Rb-defective tumor cells with HDAC inhibitors greatly reduced the DNA binding activity of Oct-1, a repressor of inducible HLA-DRA promoter activation. These findings represent the first evidence that HDAC activity can repress IFN-γ-inducible HLA class II gene expression and also demonstrate that HDAC activity can contribute to promoter repression following the establishment of a DNase I-hypersensitive chromatin conformation.

Substantially reduced expression of PIAS1 is associated with colon cancer development.

PurposeProtein inhibitors of activated STATs (PIAS) regulate the interferon-gamma (IFN-γ) signaling pathway, which has numerous effects on tumor development and tumor cell biology. PIAS’s also regulate STAT family members not directly involved in IFN-γ signaling. This project was designed to assess PIAS1 expression in colon cancer.MethodsTo determine whether PIAS1, one of the PIAS family members, or IFN-γ signaling pathway components could be used to stratify colon tumors, we stained tissue microarrays for PIAS1, interferon regulatory factor-1 (IRF-1) and STAT1α.ResultsPIAS1 staining of the colon cancer tissue microarrays indicated a strong correlation of normal colon cells, and adenomas, with high expression of both PIAS1 and IRF-1.ConclusionThe PIAS1 results in particular may represent a basis for new approaches for efficiently distinguishing adenomas from colon cancer.

Activation of a Methylated Promoter Mediated by a Sequence-specific DNA-binding Protein, RFX

The roles of eukaryotic DNA methylation in the repression of mRNA transcription and in the formation of heterochromatin have been extensively elucidated over the past several years. However, the role of DNA methylation in transcriptional activation remains a mystery. In particular, it is not known whether the transcriptional activation of methylated DNA is promoter-specific, depends directly on sequence-specific DNA-binding proteins, or is facilitated by the methylation. Here we report that the sequence-specific DNA-binding protein, RFX, previously shown to mediate the transition from an inactive to an active chromatin structure, activates a methylated promoter. RFX is capable of mediating enhanceosome formation on a methylated promoter, thereby mediating a transition from a methylation-dependent repression of the promoter to a methylation-dependent activation of the promoter. These results indicate novel roles for DNA methylation and sequence-specific DNA-binding proteins in transcriptional activation.

The IRF-2 DNA binding domain facilitates the activation of the class II transactivator (CIITA) type IV promoter by IRF-1

IFN-gamma induced transcription of class II transactivator (CIITA), a major regulator of MHC class II gene expression, is directed by the CIITA type IV promoter. The IFN-gamma activation of the CIITA type IV promoter is mediated by STAT1 and IRF-1, which bind to the GAS and IRF-E of the promoter, respectively. We and others have determined that IRF-2, another member of the IRF family, also activates the CIITA type IV promoter, by binding to the IRF-E. Also, IRF-2 cooperates with IRF-1 to activate the promoter. DNA binding analyses determined that IRF-1 and IRF-2 can co-occupy the IRF-E of the CIITA type IV promoter. To further understand the mechanism of IRF-1 and IRF-2 cooperativity in the activation of CIITA type IV promoter, we characterized the binding of IRF-1 and IRF-2 to the CIITA IRF-E and mapped the domains of IRF-2 required for the cooperative transactivation. Off-rate experiments revealed that the IRF-2/IRF-E complex was more stable than the IRF-1/IRF-E complex and that the affinity of IRF-1 for the IRF-E was increased when IRF-1 co-occupied the IRF-E with IRF-2. Deletion analysis of functional domains of IRF-2 revealed that a previously described latent activation domain of IRF-2 was essential for IRF-2 transactivation and participated in cooperative activation of the CIITA promoter by IRF-1 and IRF-2. However, the DNA binding domain of IRF-2 was sufficient for cooperativity with IRF-1 in the activation of the CIITA type IV promoter. DNA binding assay demonstrated that, like the full-length IRF-2, the IRF-2 DNA binding domain could co-occupy the CIITA IRF-E with IRF-1.

IFN-gamma inducibility of class II transactivator is specifically lacking in human tumour lines: relevance to retinoblastoma protein rescue of IFN-gamma inducibility of the HLA class II genes.

We have previously reported that HLA class II induction by IFN‐γ is rescuable by reconstitution of functional retinoblastoma protein (RB) in two RB‐defective tumour lines: the breast carcinoma line. MDA‐ 468‐S4 (S4) and the non‐small cell lung carcinoma line. H2009. To determine the range of tumours and tumour types in which RB rescues HLA class II inducibility, we examined another RB‐defective tumour line. the retinoblastoma line. WERI‐Rb1. As in the case of S4 and H2009, HLA‐DRA and ‐DRB were non‐inducible by IFN‐γ in WERI‐Rb1. However, neither inducibility of DRA nor DRB mRNA was restored in an RB‐positive stable transformant of WERI‐Rb1. WLRB‐S, While guanylate‐binding protein (GBP) inducibility indicated that the basic IFN‐7 signal transduction pathway remained intact in WERI‐Rb1, mRNA for class II transactivator (CIITA), a mediator of the IFN‐γ activation of the HLA class II genes and several other genes related to immune function, was not detectable in IFN‐γ‐treated WERI‐Rbl. indicating that the lack of CIITA expression was responsible, at least in pan. for the inability of RB to rescue HLA class Il‐inducibility. The HLA class Il‐associated invariant chain (Ii), the expression of which is also up‐regulated by CIITA. was non‐inducible in WERI‐Rb1. consistent with non‐inducible CIITA. Also. IFN‐γ failed to activate the DRA. DRB and Ii promoters in WERI‐Rb1. However, exogenous CIITA expression in WERI‐Rb1 activated the DRA. DRB and Ii promoter‐chloramphinocol acetyltransferase constructs, confirming that CIITA was not induced in WERI‐Rb1 and indicating that other proteins required for activation of the class II and Ii promoters were functional in this cell lines. Examination of additional cell lines for GBP and CIITA induction revealed that a specific lack of the CIITA IFN‐γ response is common in human tumour lines. The possible role of CIITA defects in tumorigenesis is discussed.

Impaired class II transactivator expression in mice lacking interferon regulatory factor-2.

Class II transactivator (CIITA) is required for both constitutive and inducible expression of MHC class II genes. IFN-γ induced expression of CIITA in various cell types is directed by CIITA type IV promoter. The two transactivators, STAT1 and IRF-1, mediate the IFN-γ activation of the type IV promoter by binding to the GAS and IRF-E of the promoter, respectively. In addition to IRF-1, IRF-2, another member of the IRF family, also activates the human CIITA type IV promoter, and IRF-2 cooperates with IRF-1 to activate the promoter in transient transfection assays. IRF-1 and IRF-2 can co-occupy the IRF-E of the human CIITA type IV promoter. To understand the effect of loss of IRF-2 on the endogenous CIITA expression, we assayed for CIITA expression in IRF-2 knock-out mice. Both basal and IFN-γ induced CIITA expression were reduced in IRF-2 knock-out mice. At least half of the amount of inducible CIITA mRNA depends on IRF-2. The reduction of IFN-γ induced CIITA mRNA in IRF-2 knock-out mice was due to the reduction of the type IV CIITA mRNA induction. The reduction of basal CIITA mRNA was apparently due to the reduction of CIITA mRNA originating from other promoters. These data indicate that IRF-2, like IRF-1, plays a critical role in the regulation of the endogenous CIITA gene. The implications in understanding the previously described phenotypes of IRF-2 defective mice are discussed.

High Level Class II trans-Activator Induction Does Not Occur with Transient Activation of the IFN-γ Signaling Pathway

Gene activation in early development is highly dependent on precise concentrations of trans-acting factors for the activation of different genes at differing points in the embryo. Thus, not only is the presence or absence of a particular trans-activator or repressor relevant in determining gene activation, but also the concentration of the regulatory protein must be above or below a certain threshold for proper gene regulation. Signaling pathways in somatic cells are thought to represent cascades of on/off switches, mediated most commonly by phosphorylation. Here we demonstrate a quantitative mechanism for regulating the level of a component of the IFN-γ signaling pathway that in effect represents the differential sensitivities of STAT1, IFN-regulatory factor-1, and class II trans-activator (CIITA) to IFN-γ. Unlike developmental gene regulation, in which specificity of gene activation is a function of regulatory protein concentrations, specificity of gene activation in the IFN-γ signaling pathway is regulated by the duration of the activation of the primary IFN-γ-regulatory protein, STAT1. This result most likely explains previously reported data indicating that a minimum amount of IFN-γ is required for MHC class II gene activation despite the fact that the level of the IFN-γ-inducible factor directly required for MHC class II induction, CIITA, directly correlates with the level of MHC class II expression. The induction of a high level of CIITA is dependent on sustained IFN-γ signaling. The possible implications of this result for tumorigenesis are discussed.

Genes that contribute to cancer fusion genes are large and evolutionarily conserved

Numerous cancer fusion genes have been identified and studied, and in some cases, therapy or diagnostic techniques have been designed that are specific to the fusion protein encoded by the fusion gene. There has been little progress, however, in understanding the general features of cancer fusion genes in a way that could provide the foundation for an algorithm for predicting the occurrence of a fusion gene once the chromosomal translocation points have been identified by karyotype analyses. In this study, we used publicly available data sets to characterize 59 cancer fusion genes. The results indicate that all but 17% of the genes involved in fusion events are either relatively large, compared to neighboring genes, or are highly conserved in evolution. These results support a basis for designing algorithms that could have a high degree of predictive value in identifying fusion genes once conventional microscopic analyses have identified the chromosomal breakpoints.

Interferon regulatory factor‐2 point mutations in human pancreatic tumors

Interferon regulatory factor (IRF)‐2, a member of the IRF family, is a transcription factor involved in the regulation of various interferon and virus‐stimulated genes and other genes. For example, IRF‐2 is an activator of the interferon (IFN)‐γ‐inducible MHC class II transactivator (CIITA) type IV promoter. It cooperates with IRF‐1 in the activation of the CIITA type IV promoter and can co‐occupy the IRF‐E of the promoter with IRF‐1. In a previous study, we identified an inactivating point mutation in the DNA binding domain of IRF‐2 expressed in a human pancreatic tumor cell line that does not express CIITA or MHC class II in response to IFN‐γ. To further assess the potential impact of IRF‐2 mutations in tumorigenesis, we screened fresh pancreatic tumor explants and identified 2 IRF‐2 point mutations in the 2 alleles of IRF‐2 from a single tumor specimen. Both mutations occurred in the DNA binding domain of IRF‐2. DNA binding assays demonstrated that the IRF‐2 point mutations impaired IRF‐2 DNA binding. The transactivation function of the mutant IRF‐2s was similarly impaired. This is the first report of IRF‐2 mutations in human tumor explants. Int. J. Cancer 87:803–808, 2000.