Analysis of chromatin boundary activity in Drosophila cells

Abstract

Background: Chromatin boundaries, also known as insulators, regulate gene activity by organizing active and repressive chromatin domains and modulate enhancer-promoter interactions. However, the mechanisms of boundary action are poorly understood, in part due to our limited knowledge about insulator proteins, and a shortage of standard assays by which diverse boundaries could be compared.

Results: We report here the development of an enhancer-blocking assay for studying insulator activity in Drosophila cultured cells. We show that the activities of diverse Drosophila insulators including suHw, SF1, SF1b, Fab7 and Fab8 are supported in these cells. We further show that double stranded RNA (dsRNA)-mediated knockdown of SuHw and dCTCF factors disrupts the enhancer-blocking function of suHw and Fab8, respectively, thereby establishing the effectiveness of using RNA interference in our cell-based assay for probing insulator function.

Conclusion: The novel boundary assay provides a quantitative and efficient method for analyzing insulator mechanism and can be further exploited in genome-wide RNAi screens for insulator components. It provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements.

Figure 1

Enhancer-mediated GFP activation is blocked by the suHw and the SF1 insulators in Drosophila S2 cells. A. Induction of S2 cells containing the GFP transgene. Top: a diagram of MT-GFP: key regulatory components shown include: the MT enhancer (yellow), the evenskipped basal promoter (light blue) and the GFP reporter gene (green). Middle: fluorescence microscopy images of S2 cell containing MT-GFP before (left) and after (right) induction. Bottom: FACS histogram of uninduced (left) and induced (right) S2 cells. X-axes: log scale of GFP level; Y-axes: cells number at indicated GFP level. Red bar: percentage of total cells with GFP level above 10². B. Induction of S2 containing MT-sp-GFP (grey box). Bottom: FACS histogram of uninduced (left) and induced (right) S2 cells. C-D. GFP induction in S2 cells containing enhancer-blocking transgenes. Top: a diagram of MT-suHw-GFP (C) or MT- SF1-GFP (D) transgenes. Insulator elements are represented by the red ovals. Bottom: FACS histogram of uninduced (left) and induced (right) S2 cells. E. Quantitation of GFP induction (I) in S2 cells transfected with insulator-containing transgenes. Number of replicates for each experiment is shown in parentheses. See methods for calculation of I and standard error of mean (SEM). F. Comparison of enhancer-blocking activity of suHw in S2 and Kc cells. Bar graph shows percentage of GFP induction in S2 (left) or Kc (right) cells transfected with MT-suHw-GFP (red bars) and MT-GFP (green bars). Transfection and induction were done in parallel.

Figure 2

Enhancer-blocking assay using two reporter transgenes. Reporter expression in S2 cells cotransfected with separate RFP and GFP transgenes. A. Left, diagrams of MT-RFP (top) and MT-GFP (bottom). Right, FACS chart of GFP and RFP induction in S2 cells cotransfected with both transgenes. Level of GFP or RFP is shown in logarithm scale (Y or X axis, respectively). The lower-left quadrant contains cell with both GFP and RFP levels <80 (double-negative cells); and the top-right quadrant contains cells with both GFP and RFP levels >80. Percentage of cells indicated at the corner in each quadrant. B. Induction of S2 cells cotransfected with MT-RFP and MT-sp-GFP, which contains a spacer (grey box) between MT and the eve promoter. Right: FACS chart of GFP and RFP induction in S2 cells cotransfected with both plasmids. C. Induction of S2 cells cotransfected with MT-RFP and the MT-suHw-GFP, which contains the 340-bp suHw element (red oval) between MT and the eve promoter. Right: FACS chart of GFP and RFP induction in S2 cells cotransfected with both plasmids. D. Relative induction level (I') of the RFP (red bars) and GFP (green bars) in cotransfection experiments in Panels A-C, see Methods).

Figure 3

Enhancer-blocking assay using a dual-reporter transgene. Reporter expression in S2 cells transfected with a single 2xR dual reporter transgene. A. A diagram of the 2xR dualreporter transgene vector. B. Top: RFP and GFP expression ofS2 cell containing 2xR before (top) and after (middle) induction. Bottom: FACS chart of 2xR-transfected S2 cells before (left) andafter (right panel) induction. C. Top: a diagram of 2xR transgene with a test insulator (In, red oval) inserted between MTand the eve-GFP reporter. Bottom: FACS chart of induced S2 cells containing 2xR-spacer (left) and 2xR-SF1 (right). Insets containing double positive cells are shown at identical position. D. Ratio (R) between GFP and RFP induction in S2 cells containing 2xR-suHw, SF1, SF1b, Fab7, and Fab8, respectively. Number of replicates for each experiment is shown in parentheses. Standard error of mean (SEM) is indicated.

Figure 4

RNAi -mediated disruption of insulator function in the S2 assay. A. Diagram of 2xR transgenes used in RNAi knockdown tests. B. RT-PCR assessment of SuHw, GAF and dCTCF transcript level in S2 cells containing 2xR-insulator transgenes. Transgenes used in transfection are indicated on top of the panel. Double-strand RNA (dsRNA) used in knockdown treatment and mRNA-specific primers used in RT-PCR reactions are indicated on the left. S2 cells not treated (-, left lanes) or treated (+, right lanes) with dsRNA were used in RT-PCR using gene-specific primers and Actin 88f primers. The asterisk indicates the expected actin product at 370 bp. C. Western blot analysis of SuHw protein level in S2 cells. Left lane, molecular weight standard in kilodalton (KD); middle lane, untreated cells; and right, dsRNA-SuHw-treated cells. Arrowhead points to the position of SuHw at ~145 KD. Asterisk indicates a non-specific band reactive to the antibody. D. Summary of mRNA and protein reduction in the RNAi-mediated knockdown. N indicates the number of replicate of RT-PCR used in the assessment. E. Changes in GFP/RFP ratio as a result of knockdown (untreated cell = 100%). The dsRNA used in the knockdown is indicated below the bar graph. Number of replicates is indicated in parentheses.