The Drosophila MI-2 chromatin-remodeling factor regulates higher-order chromatin structure and cohesin dynamics in vivo

Abstract

dMi-2 is a highly conserved ATP-dependent chromatin-remodeling factor that regulates transcription and cell fates by altering the structure or positioning of nucleosomes. Here we report an unanticipated role for dMi-2 in the regulation of higher-order chromatin structure in Drosophila. Loss of dMi-2 function causes salivary gland polytene chromosomes to lose their characteristic banding pattern and appear more condensed than normal. Conversely, increased expression of dMi-2 triggers decondensation of polytene chromosomes accompanied by a significant increase in nuclear volume; this effect is relatively rapid and is dependent on the ATPase activity of dMi-2. Live analysis revealed that dMi-2 disrupts interactions between the aligned chromatids of salivary gland polytene chromosomes. dMi-2 and the cohesin complex are enriched at sites of active transcription; fluorescence-recovery after photobleaching (FRAP) assays showed that dMi-2 decreases stable association of cohesin with polytene chromosomes. These findings demonstrate that dMi-2 is an important regulator of both chromosome condensation and cohesin binding in interphase cells.

Figure 1. dMi-2 increases the size of polytene chromosomes in vivo.

(A–F) Salivary gland polytene chromosomes of late third-instar UAS-LacZ/+; da-GAL4/+ control larvae (A and D) or larvae bearing one (UAS-dMi-2⁺ 3-3/+; da-GAL4/+ [1X UAS-dMi-2⁺]) (B and E) or two (UAS-dMi-2⁺ 3-3/+; UAS-dMi-2⁺ 15-1/da-GAL4 (2X UAS-dMi-2⁺)] (C and F) copies of a UAS-dMi-2⁺ transgene. (A–C) Squashes of fixed polytene chromosomes stained with DAPI. (D–F) Live analysis of chromosomes of larvae expressing His2Av-GFP. The over-expression of dMi-2 increases the size of polytene chromosomes (compare B and C to A and E and F to D) and compromises their structural integrity, as evidenced by the disruption of the banding pattern (compare B and C to A) (G) Quantification of the volume of polytene chromosomes of control, 1X UAS-dMi-2⁺, and 2X UAS-dMi-2⁺ larvae imaged by live analysis. The over-expression of dMi-2 protein causes a two to fourfold increase in chromosome volume (1X UAS-dMi-2⁺: 1.4E+04±3.3E+03 µm³ and 2X UAS-dMi-2⁺: 1.97E+04±5.5E+03 µm³) compared to UAS-Lac-Z (5.6E+03±1.4E+03 µm³). (H–I) Over-expression of KIS-L has no obvious effect on the structure of polytene chromosomes. DAPI staining of polytene chromosomes squashes from UAS-LacZ/ey-GAL4 (H) and UAS-KIS-L⁺ 20-7/ey-GAL4 (I) individuals. A, B, C, D, E F, H and I scale bars are 10 µm. Larvae were reared at 29°C.

Figure 2. The loss of dMi-2 function alters chromosome structure.

Salivary gland polytene chromosomes of late third-instar UAS-LacZ/+; da-GAL4/+ control larvae (A and C) or larvae expressing the dominant-negative dMi-2^Δ932-1158 protein (UAS-dMi-2 ^Δ932-1158 6-5/+; da-GAL4/+) (B and D). (A–D) Squashes of fixed polytene chromosomes stained with DAPI. (E–H) Live analysis of chromosomes of larvae expressing His2Av-GFP. The expression of dMi-2^Δ932-1158 reduces the size of polytene chromosomes and disrupts their banding pattern. (Compare C and D to A and B, and G and H to E and F). A, B, E and F scale bars are 10 µm.

Figure 3. dMi-2 triggers rapid changes in chromosome structure without altering DNA replication.

Third-instar UAS-dMi-2⁺ 3-3/+; UAS-dMi-2⁺ 15-1/da-GAL4, Gal80^ts larvae were shifted from 18°C to 29°C to activate UAS-dMi-2⁺ expression. (A) Changes in dMi-2 RNA levels were monitored by RT-PCR following the shift from 18° to 29°C using primers that specifically amplify the RNA encoded by UAS-dMi-2⁺ (induced) or both the induced and endogenous dMi-2 RNAs (induced+endogenous). The level of ISWI RNA was assayed as an internal control. (B and C) Live analyses of salivary gland nuclei of UAS-dMi-2⁺ 3-3/+; UAS-dMi-2⁺ 15-1/da-GAL4 GAL80^ts maintained at 18°C (B; n = 13) or shifted to 29°C for 27 hours (C; n = 14). (D) Quantification of the volume of salivary gland polytene chromosomes of larvae shown in B and C. (E) Quantification of His2Av-mRFP1 fluorescence in the same larvae shown in B and C. (F) Quantification of chromosome compaction, as assessed by the ratio of volume in µm³ to His2Av-mRFP1 fluorescence per nucleus for the larvae shown in B and C. (G) Scatter plot of the data used to generate D–F. Chromatin compaction vs. DNA content are compared for single nuclei of larvae maintained at 18°C (green triangles) or 29°C (orange circles) for 27 hours. B and C scale bars are 10 µm.

Figure 4. The loss of dMi-2 function promotes chromosome compaction.

Third-instar UAS-dMi-2 ^Δ932-1158 6-5/His2Av-mRFP1; +/da-GAL4 tubP-Gal80^ts larvae were shifted from 18°C to 29°C to activate dMi-2 ^Δ932-1158 expression. (A and B) Live analyses of salivary gland nuclei of UAS-dMi-2 ^Δ932-1158 6-5/His2Av-mRFP1; +/da-GAL4 tubP-Gal80^ts larvae maintained at 18°C (A; n = 11) or shifted to 29°C for 48 hours (B; n = 14). (C–E) Quantification of the volume (C), His2Av-mRFP1 fluorescence (D), and compaction (E) of salivary gland chromosomes of larvae maintained at 18°C or 29°C for 48 hours after the temperature shift. (F) Scatter plot of the data used to generate C–E. Chromatin compaction vs. DNA content are compared for single nuclei of larvae maintained at 18°C (green triangles) or 29°C (purple circles) for 48 hours. A and B scale bars are 10 µm.

Figure 5. dMi-2 is required for full decondensation of heat-shock loci.

w¹¹¹⁸ control and transgenic larvae expressing dominant-negative dMi-2^K761R were subjected to a 20 min heat-shock at 37°C. Polytene chromosomes were visualized by staining with DAPI (right panels) or indirect immunofluorescence using an antibody against Pol II Ser2 (left panels). Arrows indicate the hsp70 gene containing loci 87A and 87C.

Figure 6. dMi-2 does not promote chromatin decondensation by antagonizing histone H1 assembly.

(A–B) Live analysis of salivary gland nuclei of late third-instar UAS-LacZ/+; ey-GAL4/+ (UAS-LacZ) control larvae (A) and UAS-dMi-2⁺ 3-3/+; UAS-dMi-2⁺ 15-1/ey-GAL4 (UAS-dMi-2⁺) larvae (B) expressing H1-GFP. Scale bars are 10 µm. (C) Quantification of H1-GFP fluorescence in larvae shown in A and B. The exposure times used to capture the images are identical; the number of glands analyzed is noted. (D) Protein blot showing the relative levels of histones H1 and H3 in chromatin extracted from late third-instar UAS-dMi-2⁺ 3-3/+; da-GAL4/+ (UAS-dMi-2⁺) and UAS-LacZ/+; da-GAL4/+ (UAS-LacZ) larvae raised at 29°C.

Figure 7. dMi-2 regulates chromosome cohesion in the larval salivary gland.

(A) Overview of LacI-GFP/LacO tethering assay. The GFP-tagged LacI fusion protein (LacI-GFP) binds 256 tandem LacO sequences inserted at position 60F on the second chromosome allowing the detection of the locus in living cells. The HS83 heat-shock promoter (hs) that drives LacI-GFP is activated at 37°C. (B-E) Live analysis of the LacI-GFP signal at 60F in the polytene chromosomes of third-instar larvae. UAS-dMi-2⁺ 3-3/HS83-LacI-GFP LacO; UAS-dMi-2⁺ 15-1/da-GAL4 GAL80^ts individuals were cultured at 18°C until the middle of the third larval instar, maintained at 18°C (B and D) or shifted to 29°C (C and E) for 27 hours, and heat-shocked at 37°C for 1 hour to induce LacI-GFP expression. Both longitudinal (B and C) and transverse (D and E) views of the lacO array are shown.

Figure 8. dMi-2 colocalizes with cohesin.

(A–C) Magnified images of salivary gland polytene chromosomes stained with antibodies against dMi-2 (red) and Pol II Ser2 (A, green), stromalin (B, green) and Nipped B (C, green). Note the extensive overlap between the chromosomal distributions of the four proteins. (D) Reducing dMi-2 gene dosage suppresses the small wing blade phenotype of individuals heterozygous for the Nipped-B⁴⁰⁷ null allele. A minimum of twenty adult male wing blade areas was measured for each of the indicated genotypes, and the distributions of blade areas are presented as box-plots. For each genotype, the chromosome to the left of the separator (/) came from the male parent, and the chromosome to the right came from the female parent. The P57B chromosome is the wild-type chromosome in which the Nipped-B⁴⁰⁷ mutation was induced by γ rays . For the +/Nipped-B⁴⁰⁷, +/dMi-2⁴ and +/P57B genotypes, the wild-type chromosomes came from an Oregon R male parent.

Figure 9. The over-expression of dMi-2 does not decrease cohesin levels.

(A) Protein blot of salivary gland chromatin extracted from UAS-dMi-2⁺ 3-3/+; UAS-dMi-2⁺ 15-1/da-GAL4 GAL80^ts individuals raised at 18°C until the late third-instar stage and then shifted to 29°C for 24 hours to induce UAS-Mi-2⁺ expression. The blot was probed with antibodies against Smc1 and histone H3 as a control. (B, C and D) RT-PCR analysis of Smc1, SA, and Rad21 RNA levels in the salivary glands of UAS-LacZ/+; da-GAL4/+ (UAS-LacZ) control larvae and UAS-dMi-2⁺ 3-3/+; UAS-dMi-2⁺ 15-1/da-GAL4 (UAS-Mi2⁺) larvae raised at 29°C. Histone H1 RNA levels are shown as a control.

Figure 10. dMi-2 decreases the stability and extent of cohesin chromosome binding.

(A) FRAP recovery curves for EGFP-Smc1 in wild-type salivary gland nuclei, and salivary gland nuclei in which da-GAL4 drives the expression of two UAS-dMi-2⁺ transgenes (EGFP-Smc1 da-GAL4/UAS-dMi-2⁺ 3-3; UAS-dMi-2⁺ 15-1/+) at 25°C. This induced a 1.6-fold average increase in nuclear volume (see text). EGFP-Smc1 was bleached in half the nucleus, and the curves show the decreasing difference in fluorescence intensity (Δ fluorescence) between the bleached and unbleached halves over time as the unbleached EGFP-Smc1 re-equilibrates throughout the nucleus. The curves shown are the average of at least 29 nuclei, and the error bars show the standard error of the means. (B) Curve-fitting distinguishes three cohesin fractions, unbound (determined by the loss of fluorescence in the unbleached half during bleaching, not shown), a weak binding fraction, and a stable binding fraction, presumed to be bound topologically. (C) Both the half-life of the stable binding cohesin (upper right panel) and total fraction of cohesin that binds in the stable mode were decreased by dMi-2 overexpression.

Figure 11. dMi-2 alters the structure of mitotic chromosomes.

DAPI staining of mitotic chromosomes from the wing imaginal discs of UAS-LacZ/+; da-GAL4/+ (UAS-LacZ) (A) and UAS-dMi-2⁺ 3-3/+; UAS-dMi-2⁺ 3-3/da-GAL4 (UAS-dMi-2⁺) (B and C) third-instar larvae raised at 29°C. A,B and C scale bars are 5 µm.