Modulation of chromatin boundary activities by nucleosome-remodeling activities in Drosophila melanogaster

Abstract

Chromatin boundaries facilitate independent gene regulation by insulating genes from the effects of enhancers or organized chromatin. However, the mechanisms of boundary action are not well understood. To investigate whether boundary function depends on a higher order of chromatin organization, we examined the function of several Drosophila melanogaster insulators in cells with reduced chromatin-remodeling activities. We found that knockdown of NURF301 and ISWI, key components of the nucleosome-remodeling factor (NURF), synergistically disrupted the enhancer-blocking function of Fab7 and SF1 and augmented the function of Fab8. Mutations in Nurf301/Ebx and Iswi also affected the function of these boundaries in vivo. We further show that ISWI was localized on the endogenous Fab7 and Fab8 insulators and that NURF knockdown resulted in a marked increase in the nucleosome occupancy at these insulator sites. In contrast to the effect of NURF knockdown, reduction in dMi-2, the ATPase component of the Drosophila nucleosome-remodeling and deacetylation (NuRD) complex, augmented Fab7 and suppressed Fab8. Our results provide the first evidence that higher-order chromatin organization influences the enhancer-blocking activity of chromatin boundaries. In particular, the NURF and NuRD nucleosome-remodeling complexes may regulate Hox expression by modulating the function of boundaries in these complexes. The unique responses by different classes of boundaries to changes in the chromatin environment may be indicative of their distinct mechanisms of action, which may influence their placement in the genome and selection during evolution.

FIG. 1.

NURF301 and ISWI knockdown disrupts the enhancer-blocking activity of the Fab7 insulator. (A) Ratio of mRNA levels in Drosophila S2 cells with and without RNAi treatment as assayed by qRT-PCR. Transcripts of Gapdh (bars 1 and 3) and NURF301 (bar 2) or ISWI (bar 4) were quantitated in cells treated with dsRNA-NURF301 (left two bars) or dsRNA-ISWI (right two bars) and compared to a no-RNAi mock control (see Materials and Methods). (B) Duplex RT-PCR evaluation of mRNA levels of the RNAi targets and actin 88F. Left two lanes, NURF301 mRNA levels in cells with (lane 1) or without (lane 2) dsRNA-NURF301 treatment. Right two lanes, ISWI mRNA levels in cells with (lane 3) or without (lane 4) dsRNA-ISWI treatment. A DNA size marker (100-bp ladder) is shown on the left, and asterisks indicate the product of actin 88F. (C) FACS flow charts of 2×R-Fab7-containing cells before (left) and after (right) double RNAi with dsRNA-NURF301and dsRNA-ISWI. Open ovals highlight cells exhibiting changes in the GFP/RFP ratio, and dashed rectangles gate out untransfected cells. (D) Top, diagram of the 2×R-Fab7 transgene. Rectangles represent regulatory elements as indicated. F7, Fab7. Bottom, GFP/RFP ratio in 2×R-Fab7-containing cells treated with, from left to right, medium, dsRNA-NURF301, dsRNA-ISWI, and dsRNA-NURF301 together with dsRNA-ISWI. Error bars indicate standard errors.

FIG. 2.

NURF components specifically modulate the enhancer-blocking activity of multiple Drosophila insulators. (A) NURF301 and ISWI facilitate insulator function of SF1b. Top, diagram of the 2×R-SF1b transgene. Rectangles represent regulatory elements as indicated. Fb, SF1b. Bottom, GFP/RFP ratio in 2×R-SF1b-containing cells treated with, from left to right, medium, dsRNA-NURF301, dsRNA-ISWI, and dsRNA-NURF301 together with dsRNA-ISWI. (B) NURF301 and ISWI suppress insulator function of Fab8. Top, diagram of the 2×R-Fab8 transgene. F8, Fab8. Bottom, GFP/RFP ratio in 2×R-Fab8-containing cells treated with, from left to right, medium, dsRNA-NURF301, dsRNA-ISWI, and dsRNA-NURF301 together with dsRNA-ISWI. (C) Evaluation of HDAC3 transcript level before and after knockdown. Left, ratio of transcript levels of Gapdh (bar 1) and HDAC3 (bar 2) in cells with and without dsRNA-HDAC3 treatment as assayed by qRT-PCR (see Materials and Methods). Right, duplex RT-PCR assessment of HDAC3 and actin 88F mRNAs in cells with (lane 1) or without (lane 2) dsRNA-HDAC3 treatment. A DNA size marker is shown on the left, and asterisks indicate the product of actin 88F. (D) Comparison between NURF and HDAC3 knockdown effects on the enhancer-blocking function of Drosophila insulators. Left, changes in GFP/RFP ratio after double knockdown of NURF301 and ISWI in S2 cells containing Fab7, SF1b, or Fab8 transgenes. Right, changes in GFP/RFP ratio after HDAC3 knockdown in S2 cells containing the same transgenes. Error bars indicate standard errors.

FIG. 3.

Nur301 and Iswi mutations disrupt enhancer-blocking activity in transgenic Drosophila embryos. (A) SF1b activity is disrupted in NURF mutant embryos. Top, diagram of the NbbH transgene containing the lacZ reporter (blue arrow) and the NEE (N) and H1 (H) enhancers. Two copies of the SF1b insulator are inserted between N and H (2Fb, red oval). Middle, lacZ expression in NbbH transgenic embryos produced by wild-type (top), Ebx^ry122 (left), or Iswi¹ (right) females. Embryos are oriented dorsal up and anterior to the left. Lack of lacZ expression in the ventral lateral region, seen in a wild-type embryo, is indicative of a strong block of the distal NEE enhancer. NEE-directed lacZ expression in mutant embryos indicates a weaker enhancer block. Bottom, quantitation of the enhancer-blocking activity of SF1b. Fifty to 100 NbbH embryos from wild-type and NURF mutant females were scored in a double-blind fashion for strong blocking and no blocking of the NEE enhancer (see Materials and Methods). (B) Fab8 activity is enhanced in NURF mutant embryos. Top, diagram of the NF8H transgene containing the lacZ reporter (blue arrow) and the NEE (N) and H1 (H) enhancers. A single Fab8 insulator is inserted between N and H (F8, oval). Middle, lacZ expression in NF8H transgenic embryos produced by wild-type (top), Ebx^ry122 (left), or Iswi¹ (right) females. lacZ expression is seen in the ventral lateral region in a wild-type embryo, indicative of a weak enhancer block. NEE-directed lacZ expression in mutant embryos was reduced, indicative of a strong enhancer block. Bottom, quantitation of the enhancer-blocking activity of Fab8 using 50 to 100 transgenic embryos as described for panel A (see Materials and Methods).

FIG. 4.

NURF localization at endogenous insulators and knockdown effect on nucleosome occupancy at these sites. (A) Differential localization of ISWI at endogenous Fab7 and Fab8 insulator elements. Fold enrichment of the Fab7 and Fab8 sequences over that for a control coding region (Gapdh exon) after chromatin immunoprecipitation (ChIP) in S2 cells using an anti-ISWI antibody is shown. Data represent the averages and standard deviations from three independent ChIP experiments, each with triplicate qPCR quantitation. (B) NURF knockdown increases nucleosome occupancy at the endogenous Fab7 and Fab8 sites. Fold enrichment of the Fab7 and Fab8 sequences over that for a control coding region (Gapdh exon) was determined by ChIP using an anti-H3 antibody before (green bars; n = 3) and after (brown bars; n = 3) NURF knockdown.

FIG. 5.

Knockdown of NURF and dMi-2 affects Fab7 and Fab8 insulators in opposite directions. (A) Average reduction in mRNA level caused by dsRNA-mediated knockdown of NURF301, ISWI, and dMi-2 as determined by qRT-PCR. (B) Opposite effects of NURF and dMi-2 knockdown on the enhancer-blocking activity of Fab7 and Fab8. Bars show the GFP/RFP ratio in S2 cells containing Fab7 (yellow) or Fab8 (blue) transgenes before knockdown (left) and after double knockdown of NURF301 and ISWI (middle) or knockdown of dMi-2 (right). Error bars indicate standard errors.