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. 2008 Feb;117(1):51-66.
doi: 10.1007/s00412-007-0125-5. Epub 2007 Oct 2.

Functional links between Drosophila Nipped-B and cohesin in somatic and meiotic cells

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Functional links between Drosophila Nipped-B and cohesin in somatic and meiotic cells

Maria Gause et al. Chromosoma. 2008 Feb.

Abstract

Drosophila Nipped-B is an essential protein that has multiple functions. It facilitates expression of homeobox genes and is also required for sister chromatid cohesion. Nipped-B is conserved from yeast to man, and its orthologs also play roles in deoxyribonucleic acid repair and meiosis. Mutation of the human ortholog, Nipped-B-Like (NIPBL), causes Cornelia de Lange syndrome (CdLS), associated with multiple developmental defects. The Nipped-B protein family is required for the cohesin complex that mediates sister chromatid cohesion to bind to chromosomes. A key question, therefore, is whether the Nipped-B family regulates gene expression, meiosis, and development by controlling cohesin. To gain insights into Nipped-B's functions, we compared the effects of several Nipped-B mutations on gene expression, sister chromatid cohesion, and meiosis. We also examined association of Nipped-B and cohesin with somatic and meiotic chromosomes by immunostaining. Missense Nipped-B alleles affecting the same HEAT repeat motifs as CdLS-causing NIPBL mutations have intermediate effects on both gene expression and mitotic chromatid cohesion, linking these two functions and the role of NIPBL in human development. Nipped-B colocalizes extensively with cohesin on chromosomes in both somatic and meiotic cells and is present in soluble complexes with cohesin subunits in nuclear extracts. In meiosis, Nipped-B also colocalizes with the synaptonemal complex and contributes to maintenance of meiotic chromosome cores. These results support the idea that direct regulation of cohesin function underlies the diverse functions of Nipped-B and its orthologs.

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Figures

Fig. 1
Fig. 1
Nipped-B mutations and predicted mutant proteins. The diagrams at the top indicate the predicted protein products of the sequenced Nipped-B mutant alleles, and the table at the bottom gives the changes in nucleotide sequence from the parental wild-type cn bw sequence and the associated change in protein coding. Orange boxes show the positions of the HEAT repeats
Fig. 2
Fig. 2
Alignment of Nipped-B missense mutations to CdLS-causing NIPBL mutations. Nipped-BNC71 and Nipped-BNC78 alter conserved residues in HEAT repeats 2 and 3, respectively, and are close to conserved residues altered in NIPBL missense mutants. Nipped-BNC39 affects a conserved residue between HEAT repeats 4 and 5 that is immediately adjacent to a conserved residue altered by a NIPBL missense mutation
Fig. 3
Fig. 3
Effects of Nipped-B mutations on su(Hw)e2 bx34e and ctK mutant phenotypes. The dominant effects of the indicated Nipped-B alleles on the bithorax phenotype displayed by su(Hw)e2 bx34e males and the cut wing phenotype displayed by ctK males were quantified as described in the text. The error bars on the bithorax phenotype data are standard errors. The cut wing phenotype data are presented as box plots with the vertical lines indicating the 10th, 25th, 50th (median), 75th, and 90th percentiles for each genotype. The circles are data points that fall outside the 10th and 90th percentiles. Asterisks indicate phenotypes that differ significantly from the wild-type control (cn bw-a) by the Bonferroni–Dunn post-hoc test
Fig. 4
Fig. 4
Examples of precocious sister chromatid separation (PSCS) in Nipped-B mutants. The panels show an example wild-type second instar neuroblast metaphase spread and examples of PSCS in Nipped-BNC6/Nipped-B02047 and Nipped-BNC41/Nipped-B02047 second instar metaphase spreads. Arrowheads indicate instances of PSCS. For Table 1, a metaphase was scored as having PSCS if one or more chromosomes show PSCS
Fig. 5
Fig. 5
Immunostaining of salivary gland polytene chromosomes for Nipped-B, Smc1, Pds5, and HP1 proteins. The upper panels show double immunostaining of Oregon R wild-type salivary gland chromosomes for Nipped-B and the Smc1 cohesin subunits. The upper left panel shows a whole nucleus, and the right shows a higher magnification view of the tip of the X chromosome from the same nucleus. The Nipped-B and Smc1 proteins colocalize, and there is a strong correlation in Nipped-B and Smc1 staining intensity. The bottom right panel shows double immunostaining of Oregon R wild-type chromosomes for Pds5 and Smc1, which also colocalize almost completely. The lower left panel shows double immunostaining for Nipped-B and HP1, which do not colocalize. The bright HP1 staining is the heterochromatic chromocenter. White scale bars in all panels indicate 5 μm
Fig. 6
Fig. 6
Immunostaining of female meiotic chromosome spreads for Nipped-B, C(3)G, Smc1/3, Ord, and histone H3 variant, CID. The panels show that Nipped-B colocalizes extensively along the arms with the Smc1 and Smc3 cohesin subunits, the SC component C(3)G and the cohesion protein Ord. Little or no Nipped-B signal is detected at centromeric regions (arrows), as confirmed by staining for the centromere-specific histone H3 variant, CID (bottom panels)
Fig. 7
Fig. 7
Dominant effect of the Nipped-B10E mutation on chromosome cores and synaptonemal complex during female meiosis. Meiotic chromosomes at the indicated stages of meiosis and oogenesis were visualized by staining for Smc1/3 and C(3)G protein in wild-type and Nipped-B10E heterozygous mutant ovarioles (whole-mount preparation). Nipped-B10E is a null-like allele generated by P element excision. The developmental stages (germarium regions 2A, 2B, 3, and stage 2 of oogenesis) are organized in temporal order from the top down. During wild-type prophase I, the synaptonemal complex first forms in region 2A of the germarium, becomes restricted to the oocyte by region 3, and remains stable until stage 6 of oogenesis. In heterozygous Nipped-B mutants, formation of the chromosome cores in region 2A appears to occur normally, but the cores fragment (lose their linear structure) in germarium region 3 to stage 2 of oogenesis, which is substantially earlier than in the wild type. Despite the early fragmentation, Smc1/3 staining is retained at the centromeres. The bright Smc1/3 spots in the stage 2 oocyte also stain for CID, a centromere-specific histone H3 variant (Fig. 8). Temporal progression of fragmentation defects was quantified in 42 Nipped-B10E/+ ovarioles and revealed that more than 50% of region 3 and more than 90% of stage 2 oocytes exhibit extensive fragmentation of chromosome cores. Similar results were obtained with other Nipped-B alleles (see text). The wild type never shows chromosome disintegration at these early stages
Fig. 8
Fig. 8
Colocalization of Smc1/3 and CID in Nipped-B mutant meiotic chromosomes. The upper panels show the staining of the same stage 2 oocyte in Fig. 7 for Smc1/3 and C(3)G in color, and the lower left panel shows the staining for the CID centromere-specific histone. The lower right panel is a merge of the Smc1/3 and CID staining, showing that the bright Smc1/3 spots occur at centromeres
Fig. 9
Fig. 9
Immunoprecipitation of cohesin subunits and Nipped-B from Kc cell nuclear extracts. The serum used for precipitation is indicated above each Western blot. For each blot, pre indicates a preimmune serum control precipitation, imm indicates immune serum precipitation, and extract is a control lane containing input nuclear extract. The left panels show that precipitation with anti-SA or anti-Smc1 precipitates significant amounts of other cohesin subunits. We were unable to detect Nipped-B or Pds5 in these precipitates in multiple experiments. The panels on the right show that precipitation of Nipped-B coprecipitates small amounts of Rad21 and SA. In all nine repeated Nipped-B immunoprecipitation experiments with different extract preparations, we detected Rad21 and SA but were unable to detect Smc1. The rabbit anti-Nipped-B in these experiments precipitates all Nipped-B from the extract (Supplementary Fig. 1). In some experiments, such as those on the right, we detected multiple forms of Rad21 or SA but do not know if these represent protein modifications or partial proteolysis

References

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