Drosophila CTCF Is Required for Fab-8 Enhancer Blocking Activity in S2 Cells

CTCF is a conserved transcriptional regulator with binding sites in DNA insulators identified in vertebrates and invertebrates. The Drosophila Abdominal-B locus contains CTCF binding sites in the Fab-8 DNA insulator. Previous reports have shown that Fab-8 has enhancer blocking activity in Drosophila transgenic assays. We now confirm the enhancer blocking capability of the Fab-8 insulator in stably transfected Drosophila S2 cells and show this activity depends on the Fab-8 CTCF binding sites. Furthermore, knockdown of Drosophila CTCF by RNAi in our stable cell lines demonstrates that CTCF itself is critical for Fab-8 enhancer blocking

Multiple elements in human β-globin locus control region 5′ HS 2 are involved in enhancer activity and position independent, transgene expression

The human β-globin Locus Control Region (LCR) has two important activities. First, the LCR opens a 200 kb chromosomal domain containing the human ε-, γ- and β-giobin genes and, secondly, these sequences function as a powerful enhancer of ε-, γ- and β-globin gene expression. Erythrold-specific, DNase I hypersensitive sites (HS) mark sequences that are critical for LCR activity. Previous experiments demonstrated that a 1.9 kb fragment containing the 5′ HS 2 site confers position-independent expression in transgenic mice and enhances human β-giobin gene expression 100-fold. Further analysis of this region demonstrates that multiple sequences are required for maximal enhancer activity; deletion of SP1, NF-E2, GATA-1 or USF binding sites significantly decrease β-globin gene expression. In contrast, no single site is required for position- independent transgene expression; all mice with site- specific mutations in 5′ HS 2 express human β-globin mRNA regardless of the site of transgene integration. Apparently, multiple combinations of protein binding sites in 5′ HS 2 are sufficient to prevent chromosomal position effects that inhibit transgene expression. © 1994 Oxford University Press

Altering the Expression in Mice of Genes by Modifying Their 3′ Regions

Polymorphic differences altering expression of genes without changing their products probably underlie human quantitative traits affecting risks of serious diseases, but methods for investigating such quantitative differences in animals are limited. Accordingly, we have developed a procedure for changing the expression in mice of chosen genes over a 100-fold range while retaining their chromosomal location and transcriptional controls. To develop the procedure, we first dissected the effects in embryonic stem (ES) cells of elements within and downstream of the 3' untranslated region (UTR) of a single copy transgene at the Hprt locus. As expected, protein expression varied with the steady-state level and half-life of the mRNA. The rank order of expression with various tested 3' regions is the same in ES cells, and in cardiomyocytes and trophoblastocytes derived from them. In mice having two functionally different native genes with modified 3'UTRs, the desired expression was obtained

Genetically Determined Severity of Anti-Myeloperoxidase Glomerulonephritis

Myeloperoxidase (MPO) is a target antigen for antineutrophil cytoplasmic autoantibodies (ANCA). There is evidence that MPO-ANCA cause necrotizing and crescentic glomerulonephritis (NCGN) and vasculitis. NCGN severity varies among patients with ANCA disease, and genetic factors influence disease severity. The role of genetics in MPO-ANCA NCGN severity was investigated using 13 inbred mouse strains, F1 and F2 hybrids, bone marrow chimeras, and neutrophil function assays. Mouse strains include founders of the Collaborative Cross. Intravenous injection of anti-MPO IgG induced glomerular crescents in >60% of glomeruli in 129S6/SvEv and CAST/EiJ mice, but <1% in A/J, DBA/1J, DBA/2J, NOD/LtJ, and PWK/PhJ mice. C57BL6J, 129S1/SvImJ, LP/J, WSB/EiJ, NZO/HILtJ, and C3H mice had intermediate severity. High-density genotypes at 542,190 single nucleotide polymorphisms were used to identify candidate loci for disease severity by identifying genomic regions that are different between 129S6/SvEv and 129S1/SvImJ mice, which are genetically similar but phenotypically distinct. C57BL/6 × 129S6 F2 mice were genotyped at 76 SNPs to capture quantitative trait loci for disease severity. The absence of a dominant quantitative trait locus suggests that differences in severity are the result of multiple gene interactions. In vivo studies using bone marrow chimeric mice and in vitro studies of neutrophil activation by anti-MPO IgG indicated that severity of NCGN is mediated by genetically determined differences in the function of neutrophils

Histone modification signature at myeloperoxidase and proteinase 3 in patients with anti-neutrophil cytoplasmic autoantibody-associated vasculitis

Abstract Background Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is a systemic autoimmune disease characterized by destructive vascular inflammation. Two prominent ANCA autoantigens are myeloperoxidase (MPO) and proteinase 3 (PR3), and transcription of MPO and PRTN3, the genes encoding the autoantigens, is associated with disease activity. We investigated whether patients with AAV have alterations in histone modifications, particularly those associated with transcriptional activation, at MPO and PRTN3. Results We identified a network of genes regulating histone modifications that were differentially expressed in AAV patients compared to healthy controls. We focused on four genes (EHMT1 and EHMT2, ING4, and MSL1) and found their expression correlated with expression of MPO and PRTN3. Methylation of histone H3K9, catalyzed by EHMT1 and EHMT2 and associated with gene silencing, was most depleted at MPO and PRTN3 in patients with active disease and the highest MPO and PRTN3 expression. Acetylation of histone H4K16, modified by complexes containing ING4 and MSL1 and associated with gene activation, was most enriched at MPO and PRTN3 in patients with active disease and the highest MPO and PRTN3 expression. Methylation at H3K4, a mark of transcriptional activation, was enriched at MPO and PRTN3 in patients and healthy controls. Conclusions MPO and PRTN3 in neutrophils of AAV patients with active disease have a distinct pattern of histone modifications, which implicates epigenetic mechanisms in regulating expression of autoantigen genes and suggests that the epigenome may be involved in AAV pathogenesis

Identification of Functional and Expression Polymorphisms Associated With Risk for Antineutrophil Cytoplasmic Autoantibodyâ Associated Vasculitis

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136726/1/art40034_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136726/2/art40034-sup-0002-suppinfo02.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136726/3/art40034-sup-0001-suppinfo01.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136726/4/art40034.pd

Isolating gene-corrected stem cells without drug selection

Progress in isolating stem cells from tissues, or generating them from adult cells by nuclear transfer, encourages attempts to use stem cells from affected individuals for gene correction and autologous therapy. Current viral vectors are efficient at introducing transgenic sequences but result in random integrations. Gene targeting, in contrast, can directly correct an affected gene, or incorporate corrective sequences into a site free from undesirable side effects, but efficiency is low. Most current targeting procedures, consequently, use positive-negative selection with drugs, often requiring ≥10 days. This drug selection causes problems with stem cells that differentiate in this time or require feeder cells, because the feeders must be drug resistant and so are not eliminated by the selection. To overcome these problems, we have developed a procedure for isolating gene-corrected stem cells free from feeder cells after 3-5 days culture without drugs. The method is still positive-negative, but the positive and negative drug-resistance genes are replaced with differently colored fluorescence genes. Gene-corrected cells are isolated by FACS. We tested the method with mouse ES cells having a mutant hypoxanthine phosphoribosyltransferase (Hprt) gene and grown on feeder cells. After 5 days in culture, gene-corrected cells were obtained free from feeder cells at a “purity” of >30%, enriched >2,000-fold and with a recovery of ≈20%. Corrected cells were also isolated singly for clonal expansion. Our FACS-based procedure should be applicable at small or large scale to stem cells that can be cultured (with feeder cells, if necessary) for ≥3 days

Additional file 3: Figure S1. of Histone modification signature at myeloperoxidase and proteinase 3 in patients with anti-neutrophil cytoplasmic autoantibody-associated vasculitis

Comparison of therapy in patients during remission on expression of histone modifying genes, EHMT1, EHMT2, ING4, and MSL1. (PDF 93.6 kb

Additional file 4: Table S3. of Histone modification signature at myeloperoxidase and proteinase 3 in patients with anti-neutrophil cytoplasmic autoantibody-associated vasculitis

Characteristics of patients with ANCA disease used for chromatin immunoprecipitation. (PDF 46.2 kb