Journal: Nucleic Acids Research
Article Title: Expression of a large LINE-1-driven antisense RNA is linked to epigenetic silencing of the metastasis suppressor gene TFPI-2 in cancer
Figure Lengend Snippet: A human TFPI-2 transgene is sensitive to antisense RNA repression in mouse ES cells. (A) Schematic diagram of constructs introduced into mouse ES cells: pTFPI-2as is designed to transcribe antisense to TFPI-2 from a CMV promoter, while pTFPI-2pa has a poly-A signal insertion downstream of the CMV promoter to block antisense transcription. Arrows indicate direction of transcription. Regions analysed by ChIP are annotated as ‘prom’ and ‘ex-in2’. ( B ) Strand-specific RT–PCR analysis of TFPI-2 antisenese (TFPI-2as) expression in transgenic mouse ES cell lines demonstrates increased levels in pTFPI-2as lines (L2 and L12) relative to pTFPI-2pa cells (L7 and L9), mouse Aprt acts as a positive control for RNA quality and quantity. This correlates with a reduction in TFPI-2 expression as shown by real-time PCR normalized to mouse Gapdh . ( C ) ChIP analysis followed by real-time PCR. Left panel: Antibodies to H3K9me3 reveal localized enrichment of H3K9me3 in the promoter region in the antisense expressing cell line, pTFPI-2as (L2), compared to cells transfected with pTFPI-2pa (L9), which express low levels of TFPI-2as. Right panel: Antibodies to H4K20me3 also show enrichment at the TFPI-2 promoter in pTFPI-2as compared to pTFPI-2pa.
Article Snippet: Generation of constructs and ES cell clones For pTFPI-2as and pTFPI-2pa constructs, a 4.93-kb human genomic DNA fragment including the full-length TFPI-2 gene obtained by long-range PCR (Expand Long PCR kit, Roche) on human genomic DNA with primers HC63f and HC63g and was cloned into the BamHI and KpnI sites of pcDNA3 (Invitrogen) and pcDNA3p(A)for, respectively. pcDNA3p(A)for was derived from pcDNA3 by cloning a 262 bp BGHp(A) fragment, obtained by PCR on pcDNA3 with primers Hind-p(A)-for and Hind-p(A)-rev, into the HindIII site of pcDNA3.
Techniques: Construct, Blocking Assay, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction, Expressing, Transgenic Assay, Positive Control, Real-time Polymerase Chain Reaction, Transfection