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Our understanding of mechanisms by which the expression of IFN-[gamma] is regulated is limited. Herein, we identify two evolutionarily conserved noncoding sequence elements (IFNgCNS1 and IFNg CNS2) located [approximately equal to]5 kb upstream and [approximately equal to]18 kb downstream of the initiation codon of the murine Ifng gene. When linked to the murine Ifng gene (-3.4 to +5.6 kb) and transiently transfected into EL-4 cells, these elements clearly enhanced IFN-[gamma], expression in response to ionomycin and phorbol 12-myristate 13-acetate and weakly enhanced expression in response to T-bet. A DNase I hypersensitive site and extragenic transcripts at IFNgCNS2 correlated positively with the capacity of primary T cell subsets to produce IFN-[gamma]. Transcriptionally favorable histone modifications in the Ifng promoter, intronic regions, IFNgCNS2, and, although less pronounced, IFNgCNS1 increased as naive T cells differentiated into IFN-[gamma],-producing effector [CD8.sup.+] and T helper (TH) 1 T cells, but not into TH2 T cells. Like IFN-[gamma], expression, these histone modifications were T-bet-dependent in [CD4.sup.+] cells, but not [CD8.sup.+] T cells. These findings define two distal regulatory elements associated with T cell subset-specific IFN-[gamma], expression.

Influenza virus infection activates cytolytic T lymphocytes (CTL) that contribute to viral clearance by releasing perforin and granzymes from cytoplasmic granules. Virus-specific, perforin-dependent CD[8.sup.+] CTL were detected in freshly isolated cells from the mouse lung parenchyma but not from the mediastinal lymph nodes (MLN), where they are primed, or from the spleen during primary influenza virus infection. To determine whether this difference was due to the low frequency or incomplete maturation of effector CTL in MLN, we measured expression of perforin, granzymes A, B, and C, and IFN-[gamma] mRNAs in CD[8.sup.+] populations and single cells immediately after isolation from virus-infected mice. Quantitative PCR revealed significant expression of perforin, granzyme A, granzyme B, and IFN-[gamma] in activated CD[8.sup.+] cells from MLN, spleen, and lung parenchyma. Granzyme C expression was not detected. Individual activated or nucleoprotein peptide/class I tetramer-binding CD[8.sup.+] cells from the three tissues expressed diverse combinations of perforin, granzyme, and IFN-[gamma] mRNAs. Although cells from lung expressed granzymes A and B at higher frequency, each of the tissues contained cells that coexpressed perforin with granzymes A and/or B. The main difference between MLN and lung was the elevated frequency of activated CD[8.sup.+] T cells in the lung, rather than their perforin/granzyme expression profile. The data suggest that some CTL mature into perforin/granzyme-expressing effector cells in MLN but reach detectable frequencies only when they accumulate in the infected lung.

Adenomas are the precursors of most colorectal cancers. Hyperplastic polyps have been linked to the subset of colorectal cancers showing DNA microsatellite instability, but little is known of their underlying genetic etiology. Using a strategy that isolates differentially methylated sequences from hyperplastic polyps and normal mucosa, we identified a 370-bp sequence containing the 5' untranslated region and the first exon of a gene that we have called HPP1. Rapid amplification of cDNA ends was used to isolate HPP1 from normal mucosa. Using reverse transcription-PCR, HPP1 was expressed in 28 of 30 (93%) normal colonic samples but in only seven of 30 (23%) colorectal cancers (P [is less than] 0.001). The 5' region of HPP1 included a CpG island containing 49 CpG sites, of which 96% were found to be methylated by bisulfite sequencing of DNA from colonic tumor samples. By COBRA analysis, methylation was detected in six of nine (66%) adenomas, 17 of 27 (63%) hyperplastic polyps, and 46 of 55 (84%) colorectal cancers. There was an inverse relationship between methylation level and mRNA expression in cancers (r = -0.67; P [is less than] 0.001), and 5-aza-2-deoxycytidine treatment restored HPP1 expression in two colorectal cancer cell lines. In situ hybridization of HPP1 indicated that expression occurs in epithelial and stromal elements in normal mucosa but is silenced in both cell types in early colonic neoplasia. HPP1 is predicted to encode a transmembrane protein containing follistatin and epidermal growth factor-like domains. Silencing of HPP1 by methylation may increase the probability of neoplastic transformation.

Our understanding of mechanisms by which the expression of IFN-γ is regulated is limited. Herein, we identify two evolutionarily conserved noncoding sequence elements (IFNgCNS1 and IFNg CNS2) located ≈5 kb upstream and ≈18 kb downstream of the initiation codon of the murine Ifng gene. When linked to the murine Ifng gene (-3.4 to +5.6 kb) and transiently transfected into EL-4 cells, these elements clearly enhanced IFN-γ expression in response to ionomycin and phorbol 12-myristate 13-acetate and weakly enhanced expression in response to T-bet. A DNase I hypersensitive site and extragenic transcripts at IFNgCNS2 correlated positively with the capacity of primary T cell subsets to produce IFN-γ. Transcriptionally favorable histone modifications in the Ifng promoter, intronic regions, IFNgCNS2, and, although less pronounced, IFNgCNSI increased as naïve T cells differentiated into IFN-γ-producing effector CD8+ and T helper (TH) 1 T cells, but not into TH2 T cells. Like IFN-γ expression, these histone modifications were T-bet-dependent in CD4+ cells, but not CD8+ T cells. These findings define two distal regulatory elements associated with T cell subset-specific IFN-γ expression. [ABSTRACT FROM AUTHOR]

Influenza virus infection activates cytolytic T lymphocytes (CTL) that contribute to viral clearance by releasing perforin and granzymes from cytoplasmic granules. Virus-specific, perforindependent CD8[sup +] CTL were detected in freshly isolated cells from the mouse lung parenchyma but not from the mediastinal lymph nodes (MLN), where they are primed, or from the spleen during primary influenza virus infection. To determine whether this difference was due to the low frequency or incomplete maturation of effector CTL in MLN, we measured expression of perforin, granzymes A, B, and C, and IFN-γ, mRNAs in CD8[sup +] populations and single cells immediately after isolation from virus-infected mice. Quantitative PCR revealed significant expression of perforin, granzyme A, granzyme B, and IFN-γ in activated CD8[sup +] cells from MLN, spleen, and lung parenchyma. Granzyme C expression was not detected. Individual activated or nucleoprotein peptide/class I tetramerbinding CD8[sup +] cells from the three tissues expressed diverse combinations of perforin, granzyme, and IFN-γ mRNAs. Although cells from lung expressed granzymes A and B at higher frequency, each of the tissues contained cells that coexpressed perforin with granzymes A and/or B. The main difference between MLN and lung was the elevated frequency of activated CD8[sup +] T cells in the lung, rather than their perforin/granzyme expression profile. The data suggest that some CTL mature into perforin/granzyme-expressing effector cells in MLN but reach detectable frequencies only when they accumulate in the infected lung. [ABSTRACT FROM AUTHOR]