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. 2005 Nov;132(21):4743-53.
doi: 10.1242/dev.02064. Epub 2005 Oct 5.

Effects of sister chromatid cohesion proteins on cut gene expression during wing development in Drosophila

Dale Dorsett  1 , Joel C Eissenberg, Ziva Misulovin, Andrew Martens, Bethany Redding, Kim McKim
Affiliations

Effects of sister chromatid cohesion proteins on cut gene expression during wing development in Drosophila

Dale Dorsett et al. Development. 2005 Nov.

Abstract

The cohesin protein complex is a conserved structural component of chromosomes. Cohesin binds numerous sites along interphase chromosomes and is essential for sister chromatid cohesion and DNA repair. Here, we test the idea that cohesin also regulates gene expression. This idea arose from the finding that the Drosophila Nipped-B protein, a functional homolog of the yeast Scc2 factor that loads cohesin onto chromosomes, facilitates the transcriptional activation of certain genes by enhancers located many kilobases away from their promoters. We find that cohesin binds between a remote wing margin enhancer and the promoter at the cut locus in cultured cells, and that reducing the dosage of the Smc1 cohesin subunit increases cut expression in the developing wing margin. We also find that cut expression is increased by a unique pds5 gene mutation that reduces the binding of cohesin to chromosomes. On the basis of these results, we posit that cohesin inhibits long-range activation of the Drosophila cut gene, and that Nipped-B facilitates activation by regulating cohesin-chromosome binding. Such effects of cohesin on gene expression could be responsible for many of the developmental deficits that occur in Cornelia de Lange syndrome, which is caused by mutations in the human homolog of Nipped-B.

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Figures

Fig. 1
Fig. 1
An smc1 null mutation increases expression of the ctK allele of cut. (Lower left) A ctK wing with margin nicks. (Lower right) Schematic of the cohesin complex. (Upper left) The distribution of wing nicks in ctK males heterozygous for the viable P insertion used to generate smc1exc46 (blue), and in ctK males heterozygous for smc1exc46 (red). The curved lines show normal distributions calculated from the histograms. (Upper right) Box plots of the wing nick distribution for both genotypes; horizontal lines for each box represent the tenth, twenty-fifth, fiftieth, seventy-fifth and ninetieth percentiles. The difference between the two distributions is significant using the Bonferroni/Dunn test (P<0.0001). The smc1exc46 mutation gives fewer nicks, indicating greater cut expression.
Fig. 2
Fig. 2
Mutations in the san, deco and Sse genes do not affect ctK expression. Shown are box plots of the wing nick distributions obtained for ctK males heterozygous for the indicated mutations. Parental chromosomes for the san2, deco1 and Sse13m mutations are not available, so a cn bw stock was used as a control. None of the distributions differ from each other in Bonferroni/Dunn tests. Similar results were obtained with san1 and deco2 mutations (not shown).
Fig. 3
Fig. 3
Cohesin associates with the cut locus in salivary gland chromosomes. (A) Wild-type (Oregon R) polytene chromosomes double immunostained for the Smc1 (green) and Stromalin (red) cohesin subunits. The merged image shows that both subunits bind the same sites. Identities of the chromosome arms are indicated in the phase-contrast micrograph. The staining pattern is reproducible in several spreads. Neither pre-immune serum showed staining, and the secondary antibodies did not show cross-species reactivity. (B) Higher magnification of the anti-Stromalin staining (red), showing cohesin association with the 7B3-4 region (arrows) containing cut. The phase-contrast micrograph is a photographic negative to make it easier to see cohesin staining in the merge.
Fig. 4
Fig. 4
Cohesin binds multiple sites in the cut regulatory region in Kc cells. Chromatin immunoprecipitation was performed with pre-immune and immune serum for Smc1 and Stromalin, and PCR amplicons spaced 1 kbp apart starting 0.6 kbp upstream of the wing margin enhancer (salmon-colored bar) extending into the transcribed region (blue-green bar) 2.8 kbp downstream of the transcription start site. Enrichment of each amplicon is plotted as the ratio of the amount of PCR product obtained with the immune serum relative to the amount obtained with the pre-immune serum. Most points represent the average of two or three measurements. Enrichment by Smc1 immune serum is plotted in blue, enrichment by Stromalin serum in red, and the black line is the average of the Smc1 and Stromalin values. This reveals cohesin-binding sites at 0.5, 4, 30.5, and 68 kbp upstream of the promoter. These peaks are recognized by an increase in the immune to pre-immune ratio relative to the baseline, which as expected, is close to 1.
Fig. 5
Fig. 5
Drosophila pds5 mutations cause chromosome segregation and cohesion abnormalities. (A) The panels show sample third instar neuroblast metaphases for the indicated genotypes. For heterozygous pds5e3/+ mutants, 117 metaphases were scored: 15.4% showed aneuploidy and 12.8% showed precocious sister chromatid separation (PSCS), similar to wild type (Rollins et al., 2004). Fifty-one metaphases were scored for homozygous pds5e3 mutants: all showed aneuploidy and 65% displayed PSCS (arrows). Thirty-two homozygous pds5e6 metaphases were scored: 87.5% showed aneuploidy and 93.8% showed PSCS. (B) Predicted structure of the pds5 gene (CG17509). The site of the viable P transposon insertion (P{EPgy2}CG17509EY06473) used to generate pds5e3 and pds5e6 is shown by the red circle. Exons are numbered boxes, with blue indicating the open reading frame.
Fig. 6
Fig. 6
The pds5e3 and pds5e6 mutations have different effects on ctK expression. Shown are box plots of the distributions of wing nicks for ctK males heterozygous for each pds5 mutation or the parental P transposon insertion used to generate the pds5 mutations. By the Bonferroni/Dunn test, the difference between the pds5e3 and pds5e6 distributions is significant (P<0.0001), as is the difference between the parental chromosome (P/+) and the pds5e6 distribution (P=0.0008). The difference between pds5e3 and the parental chromosome is not significant (P=0.0562).
Fig. 7
Fig. 7
The pds5e6 mutant produces a transcript lacking exons 1 through 5. (A) Northern blot of wild-type (Oregon R) total RNA from different developmental stages: EE, early embryo (0 to 30 minutes post egg-laying); L1, first instar larvae; L2, second instar larvae; LL3, late third instar larvae; P1, 0- to 1-day-old pupae. Each lane contained 5 μg of total RNA and the blot was probed sequentially for pds5 (4.6 kb) and rp49 transcripts. The bar graph shows phosphorimager quantification of the northern, with pds5 transcript levels normalized to rp49 levels, and the amount of pds5 transcript present in second instar larvae was set to 1 unit. (B) Northern blot of total RNA from the indicated genotypes: WT, y w; P/P, homozygotes for the viable insertion (P{EPgy2}CG17509EY06473) used to generate the pds5 mutations; e3/+, pds5e3/+; e6/+, pds5e6/+; e3/e3, pds5e3 homozygotes; e6/e6, pds5e6 homozygotes. Each lane contained 5 μg of total RNA and the blot was probed for pds5 exon 9. The bar graph to the right shows phosphorimager quantification with pds5 transcript levels normalized to rp49 levels and the amount of 4.6 kb transcript present in wild type (WT) set to 1 unit. Black bars indicate the levels of the 4.6 kb transcript, and gray bars indicate the level of 3.65 kb transcript in heterozygous and homozygous pds5e6 mutants. (C) Diagram of the pds5 gene and the pds5e6 mutant 3.65 kb transcript determined by 5′ RACE. The 3.65 kb transcript begins 67 nucleotides upstream of the pds5 start site predicted by EST sequences. The 3.65 kb transcript includes the first few nucleotides of exon 1 and 28 nucleotides of P element sequence fused to sequence near the start of pds5 exon 6. The 5′ RACE sequence is shown in Fig. S1 in the supplementary material.
Fig. 8
Fig. 8
The pds5e6 mutation reduces the binding of cohesin to salivary gland chromosomes. The panels show double immunostaining of wild-type and homozygous pds5 mutant polytene chromosomes for Stromalin and Smc1. Chromosome morphology is altered in both mutants. The pds5e3 chromosomes are slightly thicker and less extended, and the pds5e6 chromosomes (arrows) are thinner. Chromosomes from both show banding and developmental puffs, indicating that they are transcribed. Immunostaining of chromosomes from size-matched mutants and wild type was performed at the same time with the same antibody dilution; digital micrographs were taken using identical exposures and were adjusted identically for reproduction. Examination of multiple nuclei from multiple glands showed that the staining intensity of the pds5e3 mutant is indistinguishable from that of wild type, whereas the pds5e6 mutants show strongly reduced staining, similar to background. Staining in pds5e3 mutants appears somewhat less discrete than in wild type.
Fig. S1
Fig. S1
See this image and copyright information in PMC

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