Drosophila CTCF is required for Fab-8 enhancer blocking activity in S2 cells

Abstract

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.

Fig. 1

Fab-8 insulator blocks the OpIE2 enhancer in Drosophila S2 cells. Diagrams at left depict constructs diagrammed between parentheses. The OpIE2 enhancer and Fab-8 insulator were amplified from plasmid pIZ/V5-EGFP (Invitrogen) and a fly lysis preparation, respectively, using primers listed in supplemental data. The OpIE2 enhancer and Fab-8 insulator were cloned into a pBluescript plasmid upstream of a minimal promoter EGFP SV40 pA fragment, which was amplified from plasmid pIZ/V5-EGFP (Invitrogen) using the primers listed in supplemental data. The black wavy line represents genomic DNA surrounding the site of integration. The “n” signifies that at the site of integration there may be multiple copies of the reporter construct in our stable polyclonal cell lines. Stable lines were made by co-transfecting Drosophila S2 cells, that were cultured at room temperature in Schneider’s medium (Invitrogen) with 10% FBS, 100 units penicillin, and 100 μg streptomycin, in a 6 well dish with 1 μg of reporter plasmid DNA and 250 ng of pCoHygromycin plasmid DNA. Four days after transfection the cells were passaged and hygromycin was added to the cultures at a final concentration of 500 μg/ml. Cells were maintained in selection for 6 weeks. Panels a, b, and c are fluorescent images, taken with the same exposure settings, that show GFP expression in the stably transfected cell lines. Inserts are bright field images of the same field showing similar cell densities.

Fig. 2

Knockdown of Drosophila CTCF reduces enhancer blocking activity of Fab-8. DNA templates for dsRNA were amplified using the primers listed in supplemental data. Two dsRNA templates were amplified from dCTCF cDNA: dCTCF1 spanned nucleotides 755 to 1051 and dCTCF2 spanned nucleotides 1452 to 1954 of the dCTCF cDNA. DsRNA was produced using Promega T7 RiboMax Express RNAi System. RNAi was performed as previously described . Briefly, 1 million cells were incubated in 1 ml of serum free medium with 20 μg of dsRNA. After 30 minutes 1 ml of serum containing medium was added. Three or four days later the cells were analyzed by fluorescence microscopy and FACS. Fluorescent images of live cells at similar cell densities were captured with a Leica DML fluorescence microscope and SPOT RT software using auto exposure setting to capture the images of cells with the F8enhGFP construct. This exposure time was used to capture subsequent images of cells with other constructs or dsRNA treatments. The 8-bit grayscale images were pseudo-colored and the dynamic range adjusted to the same levels with SPOT RT software. Single cell suspensions of live stably transfected S2 cell lines were analyzed with a DakoCytomation, Inc. CyAn ADP flow cytometer. Percent of GFP positive cells and GFP fluorescence intensity were determined by analyzing histograms with DakoCytomation Summit software version 4.3. Statistics for the percentage of GFP positive cells and mean level of GFP fluorescence were performed using a paired Student’s t-test. RNA was extracted from stably transfected S2 cell lines using Trizol reagent. 5 μg of total RNA was separated by formaldehyde-agarose gel electrophoresis, transferred to Hybound-N nylon membrane from Amersham, and probed with ³²P labeled dCTCF fragment. The dCTCF fragment used as a probe was amplified using the primers listed in supplemental data; it spanned nucleotides 363 to 861 of the dCTCF cDNA. The probed membrane was exposed to a Molecular Dynamics phosphor imager screen and scanned with an Amersham Typhoon variable mode imager. A. Fluorescent images of F8enhGFP and F8enhF8GFP cell lines, taken with the same exposure settings, show GFP expression after treatment with dsRNAs indicated to the left of images. Panels a, c, e, and g are of cell line F8enhGFP following mock dsRNA treatment or treatment with dsRNAs against Drosophila CTCF (dCTCF), EGFP, or mouse Ctcf (mCtcf) mRNAs, respectively. Panels b, d, f, and h are of cell line F8enhF8 GFP following treatment identical to cell line F8enhGFP. B. Flow cytometry analysis used to determine percentage of GFP positive cells and the mean GFP fluorescence is summarized from multiple independent RNAi experiments. Solid bars represent cell line F8enhGFP; open bars, F8enhF8GFP. Values for the mock treated cells were set at one. The number of independent experiments for different dsRNAs are as follows: mock=5, dCTCF1=5, dCTCF2=3, EGFP=4, mCtcf=2. The percentage of GFP positive cells and mean level of GFP fluorescence are significantly different in F8enhF8GFP cell line treated with dsRNA against dCTCF compared to the non-treated cell line with the following p-values: *0.0065, #0.0029, §0.0037, and ‡0.024. C. Northern blot shows dCTCF mRNA (2.87kb) from F8enhGFP and F8enhF8GFP cell lines 4 days after mock dsRNA treatment, treatment with dsRNA dCTCF1, and dsRNA against mouse Ctcf mRNA (mCtcf). The ethidium bromide stained gel, showing the 18S and processed 28S rRNA bands, indicates that total RNA loaded was similar for each sample.

Fig. 3

Recovery of dCTCF mRNA restores Fab-8 enhancer blocking. A. Fluorescent images of F8enhGFP and F8enhF8GFP cell lines show GFP expression at two time points after treatment with dsRNA against dCTCF mRNA. Panels a, c, and e are of cell line F8enhGFP 4 days after mock dsRNA treatment, 4 days after treatment with dsRNA dCTCF1, and 10 days after treatment with dsRNA dCTCF1, respectively. Panels b, d, and f are of cell line F8enhF8 GFP following treatment identical to cell line F8enhGFP. All images were taken with the same exposure settings. B. Northern blot shows levels dCTCF mRNA from F8enhGFP and F8enhF8GFP cell lines 4 days after mock dsRNA treatment, 4 days after treatment with dsRNA dCTCF1, and 10 days after treatment with dsRNA dCTCF1. The ethidium bromide stained gel, showing the 18S and processed 28S rRNA bands, indicates that total RNA loaded was similar for each sample.