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The C. elegans dosage compensation complex mediates interphase X chromosome compaction.

Lau AC, Nabeshima K, Csankovszki G - Epigenetics Chromatin (2014)

Bottom Line: In addition, we show that SET-1, SET-4, and SIR-2.1, histone modifiers whose activity is regulated by the DCC, need to be present for the compaction of the X chromosome territory.These results support the idea that condensin I(DC), and the histone modifications regulated by the DCC, mediate interphase X chromosome compaction.Our results link condensin-mediated chromosome compaction, an activity connected to mitotic chromosome condensation, to chromosome-wide repression of gene expression in interphase.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109 Michigan.

ABSTRACT

Background: Dosage compensation is a specialized gene regulatory mechanism which equalizes X-linked gene expression between sexes. In Caenorhabditis elegans, dosage compensation is achieved by the activity of the dosage compensation complex (DCC). The DCC localizes to both X chromosomes in hermaphrodites to downregulate gene expression by half. The DCC contains a subcomplex (condensin I(DC)) similar to the evolutionarily conserved condensin complexes which play fundamental roles in chromosome dynamics during mitosis and meiosis. Therefore, mechanisms related to mitotic chromosome condensation have been long hypothesized to mediate dosage compensation. However experimental evidence was lacking.

Results: Using 3D FISH microscopy to measure the volumes of X and chromosome I territories and to measure distances between individual loci, we show that hermaphrodite worms deficient in DCC proteins have enlarged interphase X chromosomes when compared to wild type. By contrast, chromosome I is unaffected. Interestingly, hermaphrodite worms depleted of condensin I or II show no phenotype. Therefore X chromosome compaction is specific to condensin I(DC). In addition, we show that SET-1, SET-4, and SIR-2.1, histone modifiers whose activity is regulated by the DCC, need to be present for the compaction of the X chromosome territory.

Conclusion: These results support the idea that condensin I(DC), and the histone modifications regulated by the DCC, mediate interphase X chromosome compaction. Our results link condensin-mediated chromosome compaction, an activity connected to mitotic chromosome condensation, to chromosome-wide repression of gene expression in interphase.

No MeSH data available.


Related in: MedlinePlus

X chromosome compaction is not disrupted in condensin I or II depleted animals. (A) Quantification of the percentage of nuclear volume occupied by X in vector RNAi (n = 40), smc-4(RNAi) (n = 28), capg-2(RNAi) (n = 40), hcp-6(RNAi) (n = 40), and dpy-27(RNAi) (n = 34). Error bars indicate standard deviation. Asterisks indicate level of statistical significance by t-test analysis (three asterisks, P <0.001). (B) Quantification of the percentage of nuclear volume occupied by chromosome I in vector RNAi (n = 40), smc-4(RNAi) (n = 29), capg-2(RNAi) (n = 40), hcp-6(RNAi) (n = 40), and dpy-27(RNAi) (n = 32). Error bars indicate standard deviation. (C) FISH probe pairs across the X chromosome. The position of YAC probes (red and white boxes) used in FISH is indicated. 2D projections of 3D stacked images. Representative stained diploid nuclei of vector RNAi, smc-4(RNAi) and hcp-6(RNAi) worms. Nuclei stained with probes pairs across the X chromosome (red and white) and counterstained with DAPI (blue) to label DNA. Scale bars equal 1 μm. Boxplots indicating the distribution of 3D loci distances for vector RNAi (n = 20) and smc-4(RNAi) (n = 20) and hcp-6(RNAi) (n = 20) diploid nuclei. Boxes show the median and interquartile range of the data.
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Fig5: X chromosome compaction is not disrupted in condensin I or II depleted animals. (A) Quantification of the percentage of nuclear volume occupied by X in vector RNAi (n = 40), smc-4(RNAi) (n = 28), capg-2(RNAi) (n = 40), hcp-6(RNAi) (n = 40), and dpy-27(RNAi) (n = 34). Error bars indicate standard deviation. Asterisks indicate level of statistical significance by t-test analysis (three asterisks, P <0.001). (B) Quantification of the percentage of nuclear volume occupied by chromosome I in vector RNAi (n = 40), smc-4(RNAi) (n = 29), capg-2(RNAi) (n = 40), hcp-6(RNAi) (n = 40), and dpy-27(RNAi) (n = 32). Error bars indicate standard deviation. (C) FISH probe pairs across the X chromosome. The position of YAC probes (red and white boxes) used in FISH is indicated. 2D projections of 3D stacked images. Representative stained diploid nuclei of vector RNAi, smc-4(RNAi) and hcp-6(RNAi) worms. Nuclei stained with probes pairs across the X chromosome (red and white) and counterstained with DAPI (blue) to label DNA. Scale bars equal 1 μm. Boxplots indicating the distribution of 3D loci distances for vector RNAi (n = 20) and smc-4(RNAi) (n = 20) and hcp-6(RNAi) (n = 20) diploid nuclei. Boxes show the median and interquartile range of the data.

Mentions: Previous studies have found that condensin II promotes the formation of interphase chromosome territories in Drosophila[35, 39]. Therefore, we next wanted to investigate whether the X chromosome compaction was specific to condensin IDC or if condensin I or II are also contributing to this phenotype. To test this, we depleted SMC-4, CAPG-2, or HCP-6. SMC-4 is a subunit of both condensins I and II, while CAPG-2 and HCP-6 are subunits specific to condensin II [15, 21, 31, 57, 58]. We performed a shorter, one generation RNAi feeding of SMC-4, CAPG-2, HCP-6, DPY-27, and empty vector, due to the lethality of SMC-4, CAPG-2, or HCP-6 depletion over two generations. One generation RNAi feeding depleted condensin subunits to below level of detection by western blotting (Additional file 2: Figure S2). In addition the presence of chromatin bridges between many nuclei, a hallmark of chromosome segregation defects, in SMC-4, CAPG-2, and HCP-6-depleted worms, indicated successful depletion. Depleting SMC-4, CAPG-2, or HCP-6 did not change the level of compaction compared to control vector RNAi worms (Figure 5A and Additional file 2: Figure S2). The mean volume occupied by the X chromosomes was consistently around 10.1%. However, even with the shorter one generation RNAi depletion, dpy-27(RNAi) X chromosome territories were large at 17.29 ± 2.45%. Similarly, there was no change in the volume of chromosome I when either SMC-4, CAPG-2, or HCP-6 were depleted compared to control animals (Figure 5B and Additional file 2: Figure S2). Additionally, the same analysis was performed on the diploid tail tip hypodermal cells and similar results were found (Additional file 3: Figure S3). Similar conclusions were reached when using 3D FISH with pairs of X chromosome YAC probes separated by the genomic distance of 1.2 Mb, the distance that showed the most significant difference between dpy-21(e428) mutants and wild type. 83% of smc-4(RNAi) diploid nuclei and 85% of hcp-6(RNAi) diploid nuclei had two clear spots for each probe, while no nuclei had three or four spots. At the genomic distance of 1.2 MB we did not detect a significant change in distances in smc-4(RNAi) or hcp-6(RNAi) worms compared to vector control worms (Figure 5C). Therefore, we conclude that condensin IDC, and not condensin I or condensin II, is primarily responsible for dosage compensation mediated X chromosome compaction. However we cannot rule out the possibility that condensin I or II are affecting interphase chromosome territories in C. elegans at levels undetectable by 3D FISH.Figure 5


The C. elegans dosage compensation complex mediates interphase X chromosome compaction.

Lau AC, Nabeshima K, Csankovszki G - Epigenetics Chromatin (2014)

X chromosome compaction is not disrupted in condensin I or II depleted animals. (A) Quantification of the percentage of nuclear volume occupied by X in vector RNAi (n = 40), smc-4(RNAi) (n = 28), capg-2(RNAi) (n = 40), hcp-6(RNAi) (n = 40), and dpy-27(RNAi) (n = 34). Error bars indicate standard deviation. Asterisks indicate level of statistical significance by t-test analysis (three asterisks, P <0.001). (B) Quantification of the percentage of nuclear volume occupied by chromosome I in vector RNAi (n = 40), smc-4(RNAi) (n = 29), capg-2(RNAi) (n = 40), hcp-6(RNAi) (n = 40), and dpy-27(RNAi) (n = 32). Error bars indicate standard deviation. (C) FISH probe pairs across the X chromosome. The position of YAC probes (red and white boxes) used in FISH is indicated. 2D projections of 3D stacked images. Representative stained diploid nuclei of vector RNAi, smc-4(RNAi) and hcp-6(RNAi) worms. Nuclei stained with probes pairs across the X chromosome (red and white) and counterstained with DAPI (blue) to label DNA. Scale bars equal 1 μm. Boxplots indicating the distribution of 3D loci distances for vector RNAi (n = 20) and smc-4(RNAi) (n = 20) and hcp-6(RNAi) (n = 20) diploid nuclei. Boxes show the median and interquartile range of the data.
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Fig5: X chromosome compaction is not disrupted in condensin I or II depleted animals. (A) Quantification of the percentage of nuclear volume occupied by X in vector RNAi (n = 40), smc-4(RNAi) (n = 28), capg-2(RNAi) (n = 40), hcp-6(RNAi) (n = 40), and dpy-27(RNAi) (n = 34). Error bars indicate standard deviation. Asterisks indicate level of statistical significance by t-test analysis (three asterisks, P <0.001). (B) Quantification of the percentage of nuclear volume occupied by chromosome I in vector RNAi (n = 40), smc-4(RNAi) (n = 29), capg-2(RNAi) (n = 40), hcp-6(RNAi) (n = 40), and dpy-27(RNAi) (n = 32). Error bars indicate standard deviation. (C) FISH probe pairs across the X chromosome. The position of YAC probes (red and white boxes) used in FISH is indicated. 2D projections of 3D stacked images. Representative stained diploid nuclei of vector RNAi, smc-4(RNAi) and hcp-6(RNAi) worms. Nuclei stained with probes pairs across the X chromosome (red and white) and counterstained with DAPI (blue) to label DNA. Scale bars equal 1 μm. Boxplots indicating the distribution of 3D loci distances for vector RNAi (n = 20) and smc-4(RNAi) (n = 20) and hcp-6(RNAi) (n = 20) diploid nuclei. Boxes show the median and interquartile range of the data.
Mentions: Previous studies have found that condensin II promotes the formation of interphase chromosome territories in Drosophila[35, 39]. Therefore, we next wanted to investigate whether the X chromosome compaction was specific to condensin IDC or if condensin I or II are also contributing to this phenotype. To test this, we depleted SMC-4, CAPG-2, or HCP-6. SMC-4 is a subunit of both condensins I and II, while CAPG-2 and HCP-6 are subunits specific to condensin II [15, 21, 31, 57, 58]. We performed a shorter, one generation RNAi feeding of SMC-4, CAPG-2, HCP-6, DPY-27, and empty vector, due to the lethality of SMC-4, CAPG-2, or HCP-6 depletion over two generations. One generation RNAi feeding depleted condensin subunits to below level of detection by western blotting (Additional file 2: Figure S2). In addition the presence of chromatin bridges between many nuclei, a hallmark of chromosome segregation defects, in SMC-4, CAPG-2, and HCP-6-depleted worms, indicated successful depletion. Depleting SMC-4, CAPG-2, or HCP-6 did not change the level of compaction compared to control vector RNAi worms (Figure 5A and Additional file 2: Figure S2). The mean volume occupied by the X chromosomes was consistently around 10.1%. However, even with the shorter one generation RNAi depletion, dpy-27(RNAi) X chromosome territories were large at 17.29 ± 2.45%. Similarly, there was no change in the volume of chromosome I when either SMC-4, CAPG-2, or HCP-6 were depleted compared to control animals (Figure 5B and Additional file 2: Figure S2). Additionally, the same analysis was performed on the diploid tail tip hypodermal cells and similar results were found (Additional file 3: Figure S3). Similar conclusions were reached when using 3D FISH with pairs of X chromosome YAC probes separated by the genomic distance of 1.2 Mb, the distance that showed the most significant difference between dpy-21(e428) mutants and wild type. 83% of smc-4(RNAi) diploid nuclei and 85% of hcp-6(RNAi) diploid nuclei had two clear spots for each probe, while no nuclei had three or four spots. At the genomic distance of 1.2 MB we did not detect a significant change in distances in smc-4(RNAi) or hcp-6(RNAi) worms compared to vector control worms (Figure 5C). Therefore, we conclude that condensin IDC, and not condensin I or condensin II, is primarily responsible for dosage compensation mediated X chromosome compaction. However we cannot rule out the possibility that condensin I or II are affecting interphase chromosome territories in C. elegans at levels undetectable by 3D FISH.Figure 5

Bottom Line: In addition, we show that SET-1, SET-4, and SIR-2.1, histone modifiers whose activity is regulated by the DCC, need to be present for the compaction of the X chromosome territory.These results support the idea that condensin I(DC), and the histone modifications regulated by the DCC, mediate interphase X chromosome compaction.Our results link condensin-mediated chromosome compaction, an activity connected to mitotic chromosome condensation, to chromosome-wide repression of gene expression in interphase.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109 Michigan.

ABSTRACT

Background: Dosage compensation is a specialized gene regulatory mechanism which equalizes X-linked gene expression between sexes. In Caenorhabditis elegans, dosage compensation is achieved by the activity of the dosage compensation complex (DCC). The DCC localizes to both X chromosomes in hermaphrodites to downregulate gene expression by half. The DCC contains a subcomplex (condensin I(DC)) similar to the evolutionarily conserved condensin complexes which play fundamental roles in chromosome dynamics during mitosis and meiosis. Therefore, mechanisms related to mitotic chromosome condensation have been long hypothesized to mediate dosage compensation. However experimental evidence was lacking.

Results: Using 3D FISH microscopy to measure the volumes of X and chromosome I territories and to measure distances between individual loci, we show that hermaphrodite worms deficient in DCC proteins have enlarged interphase X chromosomes when compared to wild type. By contrast, chromosome I is unaffected. Interestingly, hermaphrodite worms depleted of condensin I or II show no phenotype. Therefore X chromosome compaction is specific to condensin I(DC). In addition, we show that SET-1, SET-4, and SIR-2.1, histone modifiers whose activity is regulated by the DCC, need to be present for the compaction of the X chromosome territory.

Conclusion: These results support the idea that condensin I(DC), and the histone modifications regulated by the DCC, mediate interphase X chromosome compaction. Our results link condensin-mediated chromosome compaction, an activity connected to mitotic chromosome condensation, to chromosome-wide repression of gene expression in interphase.

No MeSH data available.


Related in: MedlinePlus