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

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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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Acknowledgements

The authors thank Steve Myster and Mark Peifer for the EMS-generated Nipped-B mutants, Scott Hawley for C(3)G antibodies, and Gary Karpen for the J21A fly stocks. We also thank Patrick Morcillo for advice on statistical analysis and Ian Krantz and Matt Deardorff for information on human NIPBL mutations. This work was supported by NIH Grants R01 GM055683 (D.D.), R01 GM059354 (S.E.B.); P01 HD058260 (D.D., Project III Director), and March of Dimes FY05-103 (D.D.).

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  1. Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO, 63104, USA

    Maria Gause, Ziva Misulovin, Gabe Haller, Joel C. Eissenberg & Dale Dorsett

  2. Department of Biological Sciences, Dartmouth College, Hanover, NH, USA

    Hayley A. Webber & Sharon E. Bickel

  3. Wyeth Research, Pearl River, NY, USA

    Robert A. Rollins

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  1. Maria Gause
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Correspondence to Dale Dorsett.

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Communicated by F. Uhlmann

Gause, Webber, and Misulovin provided equal contributions.

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Supplementary Fig. 1

Nipped-B Western blots. All blots were probed with the guinea pig anti-Nipped-B serum used in the immunostaining experiments shown in Figs. 5 and 6. The upper left panel shows an overexposed Western blot of whole cell extracts of cultured Sg4 cells, whole adult ovaries, and whole third instar imaginal disks, showing that a band close to the expected size of 237 kDa is the major reacting protein. The right panels are a Western blot of whole-Sg4-cells extracts treated with no RNAi or RNAi directed against Nipped-B (Rollins et al. 2004), showing that the major band is strongly reduced by RNAi. The blot was reprobed for actin as a loading control. The middle left panel is a Western blot of whole early embryos (0 to 12 h after egg laying) from wild-type mothers and mothers heterozygous for the indicated mutant Nipped-B alleles. All lanes contained 100 embryos, and the truncation alleles all give lower levels than the wild type, while the two missense alleles shown have similar levels. The asterisks show very faint double bands consistent with the predicted sizes for the NC7- and NC59-truncated proteins. The bottom panel is a Western blot showing immunoprecipitation of the Kc nuclear extract with the rabbit anti-Nipped-B serum used for the experiment in Fig. 9. The preimmune serum (pre) did not precipitate Nipped-B and left it in the postprecipitation supernatant. The immune serum (imm) precipitates Nipped-B and removes it entirely from the supernatant. All lanes all are from the same gel but have been reordered from the original for clarity (PDF 131 kb)

Supplementary Fig. 2

Locations of Nipped-B wild-type amino acid polymorphisms and EMS-induced mutations. The top is a map of the exons (blue boxes, coding; orange boxes, noncoding) of the previously reported Nipped-B cDNA clone (Rollins et al. 1999; GenBank accession no. AF114160) mapped onto the Drosophila genome scaffold with the putative initiation (ATG) and termination codons (TGA). The positions of polymorphisms that cause changes in amino acid sequence (a–h) and EMS-induced mutations (NC) are indicated. Distances are indicated in kilobases (1K–41K). The table at the bottom lists the differences in amino acid sequence predicted by the wild-type Nipped-B polymorphisms (a–h). cn bw-a is a stock with a wild-type Nipped-B allele kept in our laboratory, and cn bw-b is the wild-type allele used for the mutagenesis (Myster et al. 2004) (PDF 147 kb)

Supplementary Fig. 3

N-terminal alternative splicing of Nipped-B transcripts The maps (a–d) show the first six exons of the standard Nipped-B cDNA (GenBank accession no. AF114160) with alternative splicing variants detected by direct sequencing of PCR-amplified reverse transcription products of total RNA from second instar larvae. a Two closely spaced alternative splice donors occur in exon 1, before the start of the open reading frame (dashed lines). b Splicing from the exon 2 donor site to alternative acceptor site in exon 4, bypassing exon 3, creates a short open reading frame (ATG–TAA). Initiation of translation at the next ATG would produce a Nipped-B protein lacking the conserved N-terminal residues. c Splicing from the exon 2 donor site to the exon 5 acceptor skips exons 3 and 4 but maintains the reading frame. d Splicing of a small intron within exon 6 preserves the reading frame. The predicted protein sequences produced by splicing patterns a–d are given in Supplementary Fig. 4. We do not know if these alternative splicing events occur independently or in combination, although patterns b and c are incompatible with each other (PDF 62.9 kb)

Supplementary Fig. 4

Predicted N-terminal protein sequences for the Nipped-B alternative splicing patterns. The protein sequences for the splicing patterns (a–d) shown in Supplementary Fig. 3 are aligned, with dashes indicating absence of the residues (PDF 488 kb)

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Gause, M., Webber, H.A., Misulovin, Z. et al. Functional links between Drosophila Nipped-B and cohesin in somatic and meiotic cells. Chromosoma 117, 51–66 (2008). https://doi.org/10.1007/s00412-007-0125-5

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