CD45 isoform expression on human neonatal T cells: expression and turnover of CD45 isoforms on neonatal versus adult T cells after activation.

Neonatal T cells are phenotypically similar to "naive" T cells from adult donors in the CD45 isoform expression. Despite the phenotypic similarity, large differences were found between neonatal and adult T cells when T cells were activated. After activation with PHA, adult CD45RA+ T cells began to express CD45RO and no loss of CD45RA expression had yet occurred at Day 3 post-stimulation. Three days after activation, CD45RA+ neonatal T cells also coexpressed CD45RO; however, in contrast to adult T cells, a marked loss of CD45RA was observed. We analyzed the rapid loss of CD45RA found in neonatal T cells. The de novo synthesis of CD45 isoforms in neonatal T cells was essentially the same as that in the adult T cells. Turnover of the CD45RA was very rapid in both resting adult and neonatal T cells. After activation with PHA, the turnover of CD45RA on adult T cells was decreased significantly, while the turnover of CD45RA on neonatal T cells was not changed after activation. Therefore, the regulation of CD45 isoform expression not only involves switches in alternative splicing, but also involves different regulation of turnover of these isoforms from the cell membrane.     

Substitution rate variation at human CpG sites correlates with non-CpG divergence,methylation level and GC content

Background  

A major goal in the study of molecular evolution is to unravel the mechanisms that induce variation in the germ line mutation rate and in the genome-wide mutation profile. The rate of germ line mutation is considerably higher for cytosines at CpG sites than for any other nucleotide in the human genome, an increase commonly attributed to cytosine methylation at CpG sites. The CpG mutation rate, however, is not uniform across the genome and, as methylation levels have recently been shown to vary throughout the genome, it has been hypothesized that methylation status may govern variation in the rate of CpG mutation.     

CpG and non-CpG oligodeoxynucleotides directly costimulate mouse and human CD4+ T cells through a TLR9- and MyD88-independent mechanism

TLR ligands are known to activate APCs, but direct T cell responsiveness to TLR ligands is controversial. Because of their clinical relevance, we performed in-depth studies of the effects of the TLR9-associated ligands, oligodeoxynucleotides (ODNs), on highly purified T lymphocytes. Both CpG and non-CpG ODNs directly costimulate mouse and human CD4(+) T cells, resulting in activation marker upregulation, cytokine secretion, elevated TCR phosphorylation, and proliferation. Surprisingly, ODN costimulation occurred independently of TLR9 and MyD88, as well as ICOS, CD28, and TRIF. TLR9-antagonist ODNs likewise promoted T cell activation, which has important implications for the study of these "inhibitory" ODNs in inflammatory diseases. Cytokine profiling revealed that ODNs promote polarization of distinct Th subsets, and that ODNs differentially affect human naive and memory T cells. Our studies reveal a striking and unexpected ability of ODNs to directly activate and polarize T cells, presenting an opportunity to enhance the paradigm for selection of therapeutic ODNs in humans.     

Dynamic CpG and non-CpG methylation of the Peg1/Mest gene in the mouse oocyte and preimplantation embryo

In somatic tissues, the CpG island of the imprinted Peg1/Mest gene is methylated on the maternal allele. We have examined the methylation of CpG and non-CpG sites of this differentially methylated CpG island in freshly ovulated oocytes, in vitro aged oocytes, and preimplantation embryos. The CpG methylation pattern was heterogeneous in freshly ovulated oocytes, despite the fact that they all were arrested in metaphase II. After short in vitro culture, Peg1/Mest became hypermethylated, whereas prolonged in vitro culture resulted in demethylation in a fraction of oocytes. Non-CpG methylation also occurred in a stage-specific manner. On alleles that were fully methylated at CpG sites, this modification was found, and it became reduced in two-cell stage embryos and blastocysts. These observations suggest that the process of establishment of the methylation imprint at this locus is more dynamic than previously thought.     

Tissue-specific methylation patterns and expression of the human apolipoprotein AI gene.

To better understand the tissue-specific expression of the human apolipoprotein (apo)AI gene, we performed a detailed analysis of the pattern of methylation of the gene in various human adult and embryonic tissues and in tissues of transgenic mice harboring the human apo-AI gene. In addition, the gene was analyzed also in liver and intestine-derived human cell lines (HepG2 and Caco2, respectively). Using methyl-sensitive restriction enzymes (HpaII, HhaI, and SmaI) and the appropriate radioactive probes, we were able to determine separately the status of methylation of the 5'-end, the body of the gene, and 3'-end flanking sequences. The apo-AI gene in tissues that express the gene was undermethylated at the 5'-end. However, the 5'-end of the gene in sperm and in all adult tissues that do not express the gene was heavily methylated. The body of the gene which contains a CpG island and the 3'-end flanking sequences were, in general, hypomethylated except for specific sites that showed partial methylation. In contrast, while the gene showed tissue-specific expression already in a 12-week-old embryo, the 5'-end was invariably hypomethylated in all tissues of the embryo. A human apo-AI transgene has recently been shown to be active exclusively in the liver, while the endogenous gene is expressed in both liver and intestine (6). We show here that the 5'-end of the apo-AI transgene was methylated in all tissues of the mouse (including intestine) except liver. The results presented here demonstrate a clear correlation between hypomethylation of the 5'-end and activity of the apo-AI gene. However, the observed methylation pattern of the gene in embryonic tissues suggests that tissue-specific expression precedes formation of the tissue-specific methylation pattern.     

Mycobacteria inhibition of IFN-gamma induced HLA-DR gene expression by up-regulating histone deacetylation at the promoter region in human THP-1 monocytic cells

Infection of macrophages with mycobacteria has been shown to inhibit the macrophage response to IFN-gamma. In the current study, we examined the effect of Mycobacteria avium, Mycobacteria tuberculosis, and TLR2 stimulation on IFN-gamma-induced gene expression in human PMA-differentiated THP-1 monocytic cells. Mycobacterial infection inhibited IFN-gamma-induced expression of HLA-DRalpha and HLA-DRbeta mRNA and partially inhibited CIITA expression but did not affect expression of IFN regulatory factor-1 mRNA. To determine whether inhibition of histone deacetylase (HDAC) activity could rescue HLA-DR gene expression, butyric acid and MS-275, inhibitors of HDAC activity, were added at the time of M. avium or M. tuberculosis infection or TLR2 stimulation. HDAC inhibition restored the ability of these cells to express HLA-DRalpha and HLA-DRbeta mRNA in response to IFN-gamma. Histone acetylation induced by IFN-gamma at the HLA-DRalpha promoter was repressed upon mycobacteria infection or TLR2 stimulation. HDAC gene expression was not affected by mycobacterial infection. However, mycobacterial infection or TLR2 stimulation up-regulated expression of mammalian Sin3A, a corepressor that is required for MHC class II repression by HDAC. Furthermore, we show that the mammalian Sin3A corepressor is associated with the HLA-DRalpha promoter in M. avium-infected THP-1 cells stimulated with IFN-gamma. Thus, mycobacterial infection of human THP-1 cells specifically inhibits HLA-DR gene expression by a novel pathway that involves HDAC complex formation at the HLA-DR promoter, resulting in histone deacetylation and gene silencing.     

Effect of in vitro methylation at CpG sites on gene expression in a genome functioning autonomously in a vertebrate host

The effect of in vitro methylation at the HpaII sites in polyoma DNA on viral gene expression and the maintenance of the methyl groups upon replication in vivo were examined. Most of the methylatable sites are located in the early region coding for the viral large T antigen which is essential for the replication and infectivity of the viral DNA. Methylated or mock-methylated polyoma DNA produced the same number of virus plaques appearing at the same time post-transfection in either case. The lack of effect on the infectivity of the viral DNA indicates that the expression of the T antigen gene was not inhibited by methylation. Replication in vivo of the DNA also resulted in a total loss of the methyl groups introduced in vitro. These results underscore basic differences between the behavior of an autonomously functioning papovavirus DNA and the animal cell DNA vis-a-vis methylation at CpG sites. These differences might be due to subtle variations in the mechanism of regulation of gene expression and replication in the two systems.     

Correlation between patterns of DNase I-hypersensitive sites and upstream promoter activity of the human epsilon-globin gene at different stages of erythroid development.

DNA 5' to the human epsilon-globin gene exhibits unique patterns of DNase I-hypersensitive sites (DHS) in three human erythroleukemic cell lines which represent the embryonic (K562), fetal (HEL), and adult (KMOE) stages of erythroid development. We have mapped 10 epsilon-globin DHS in K562 cells, in which the epsilon-globin gene is maximally active. Major sites are located -11.7, -10.5, -6.5, -2.2 kilobase pairs (kbp) and -200 base pairs (bp) upstream of the gene and directly over the major cap site. Minor sites are located -5.5, -4.5, and -1.48 kbp and -900 bp upstream of the cap site. In HEL cells, in which the epsilon-globin gene is expressed at extremely low levels, the -11.7-, -10.5-, -5.5-, -4.5-, and -2.2-kbp DHS are no longer detectable; the -200-bp site is approximately 300-fold less sensitive to DNase I; and the -1.48-kbp, -900-bp, and major cap site DHS are 3- to 4-fold less sensitive. Only the DHS located -6.5 kbp relative to the major cap site is detectable at all three stages of erythroid development, including KMOE cells in which epsilon-globin synthesis is undetectable. We suggest that this site may be implicated in maintaining the entire beta-globin cluster in an active chromatin conformation. The five DHS downstream of the -6.5-kbp element possess associated promoters. Thus two distinct types of DHS exist--promoter positive and promoter negative. In HEL cells, all the upstream promoters are inactivated, although the -1.48-kbp and -900- and -200-bp DHS are still present. This suggests that the maintenance of DHS and regulation of their associated promoters occur by independent mechanisms. The inactivation of the upstream promoters in HEL cells while the major cap site remains active represents a unique pattern of expression and suggests that HEL cells possess regulatory factors which specifically down regulate the epsilon-globin upstream promoters.