Localisation of the SMC loading complex Nipbl/Mau2 during mammalian meiotic prophase I

Evidence from lower eukaryotes suggests that the chromosomal associations of all the structural maintenance of chromosome (SMC) complexes, cohesin, condensin and Smc5/6, are influenced by the Nipbl/Mau2 heterodimer. Whether this function is conserved in mammals is currently not known. During mammalian meiosis, very different localisation patterns have been reported for the SMC complexes, and the localisation of Nipbl/Mau2 has just recently started to be investigated. Here, we show that Nipbl/Mau2 binds on chromosomal axes from zygotene to mid-pachytene in germ cells of both sexes. In spermatocytes, Nipbl/Mau2 then relocalises to chromocenters, whereas in oocytes it remains bound to chromosomal axes throughout prophase to dictyate arrest. The localisation pattern of Nipbl/Mau2, together with those seen for cohesin, condensin and Smc5/6 subunits, is consistent with a role as a loading factor for cohesin and condensin I, but not for Smc5/6. We also demonstrate that Nipbl/Mau2 localises next to Rad51 and γH2AX foci. NIPBL gene deficiencies are associated with the Cornelia de Lange syndrome in humans, and we find that haploinsufficiency of the orthologous mouse gene results in an altered distribution of double-strand breaks marked by γH2AX during prophase I. However, this is insufficient to result in major meiotic malfunctions, and the chromosomal associations of the synaptonemal complex proteins and the three SMC complexes appear cytologically indistinguishable in wild-type and Nipbl ^+/? spermatocytes.     

Cohesin loading factor Nipbl localizes to chromosome axes during mammalian meiotic prophase

Background

Sister chromatid cohesion mediated by the cohesin complex is essential for accurate chromosome segregation during mitosis and meiosis. Loading of cohesin onto chromosomes is dependent on another protein complex called kollerin, containing Nipbl/Scc2 and Mau2/Scc4. Nipbl is an evolutionarily conserved large protein whose haploinsufficiency in humans causes a developmental disorder called Cornelia de Lange syndrome. Although the function of Nipbl homologues for chromosome cohesion in meiotic cells of non-vertebrate models has been elucidated, Nipbl has not been characterized so far in mammalian spermatocytes or oocytes.

Findings

Here we describe our analyses on the expression and localization of Nipbl in nuclei of mouse spermatocytes and oocytes at different stages of meiotic prophase. In both spermatocytes and oocytes we found that Nipbl is associated with the axial/lateral element of the synaptonemal complex (AE/LE) to which cohesin also localizes. Interestingly, Nipbl in spermatocytes, but not in oocytes, dissociates from the AE/LE at mid-pachytene stage coincident with completion of DNA double-strand break repair.

Conclusions

Our data propose that cohesin loading activity is maintained during early stages of meiotic prophase in mammalian spermatocytes and oocytes.

     

Cohesin mediates chromatin interactions that regulate mammalian β-globin expression

The β-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5' and 3' boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.     

Cornelia de Lange Syndrome and the link between chromosomal function, DNA repair and developmental gene regulation

Cornelia de Lange Syndrome (CdLS) is a rare multiple malformation disorder with characteristic facial features, growth and cognitive retardation, and many other abnormalities. CdLS individuals were recently shown to have heterozygous mutations in a previously uncharacterised gene, NIPBL, which encodes delangin, a homologue of fungal Scc2-type sister chromatid cohesion proteins and the Drosophila Nipped-B developmental regulator. Nipped-B and vertebrate delangins are also now known to regulate sister chromatid cohesion, probably as part of oligomeric complexes required to load cohesin subunits onto chromatin. CdLS is likely to be one of several developmental disorders resulting from defective expression of a multi-functional protein with roles in chromosome function, gene regulation and double-strand DNA repair - a combination of properties shared by certain bacterial proteins responsible for structural maintenance of chromatin.     

Mutations in cohesin complex members SMC3 and SMC1A cause a mild variant of cornelia de Lange syndrome with predominant mental retardation

Mutations in the cohesin regulators NIPBL and ESCO2 are causative of the Cornelia de Lange syndrome (CdLS) and Roberts or SC phocomelia syndrome, respectively. Recently, mutations in the cohesin complex structural component SMC1A have been identified in two probands with features of CdLS. Here, we report the identification of a mutation in the gene encoding the complementary subunit of the cohesin heterodimer, SMC3, and 14 additional SMC1A mutations. All mutations are predicted to retain an open reading frame, and no truncating mutations were identified. Structural analysis of the mutant SMC3 and SMC1A proteins indicate that all are likely to produce functional cohesin complexes, but we posit that they may alter their chromosome binding dynamics. Our data indicate that SMC3 and SMC1A mutations (1) contribute to approximately 5% of cases of CdLS, (2) result in a consistently mild phenotype with absence of major structural anomalies typically associated with CdLS, and (3) in some instances, result in a phenotype that approaches that of apparently nonsyndromic mental retardation.     

Neural crest cell‐specific inactivation of Nipbl or Mau2 during mouse development results in a late onset of craniofacial defects

Nipbl (Scc2) and Mau2 (Scc4) encode evolutionary conserved proteins that play a vital role for loading the cohesin complex onto chromosomes, thereby ensuring accurate chromosome segregation during cell division. While mutations in human NIPBL are known to cause the developmental disorder Cornelia de Lange syndrome, the functions of Nipbl and Mau2 in mammalian development are poorly defined. Here we generated conditional alleles for both genes in mice and show that neural crest cell‐specific inactivation of Nipbl or Mau2 strongly affects craniofacial development. Surprisingly, the early phase of neural crest cell proliferation and migration is only moderately affected in these mutants. Moreover, we found that Mau2 single homozygous mutants exhibited a more severe craniofacial phenotype when compared to that of Nipbl;Mau2 double homozygous mutants. This raises the possibility that the Mau2/Nipbl protein interaction is not only required for cohesin loading, but may also be required to restrict the level of Nipbl involved in regulating gene expression. Together, the data suggest that proliferating neural crest cells tolerate a substantial reduction of cohesin loading proteins and we propose that the successive decrease of cohesin loading proteins in neural crest cells may alter developmental gene regulation in a highly dynamic manner. genesis 52:687–694, 2014. © 2014 Wiley Periodicals, Inc.     

Structural aspects of HDAC8 mechanism and dysfunction in Cornelia de Lange syndrome spectrum disorders

Cornelia de Lange Syndrome (CdLS) encompasses a broad spectrum of phenotypes characterized by distinctive craniofacial abnormalities, limb malformations, growth retardation, and intellectual disability. CdLS spectrum disorders are referred to as cohesinopathies, with ～70% of patients having a mutation in a gene encoding a core cohesin protein (SMC1A, SMC3, or RAD21) or a cohesin regulatory protein (NIPBL or HDAC8). Notably, the regulatory function of HDAC8 in cohesin biology has only recently been discovered. This Zn²⁺‐dependent hydrolase catalyzes the deacetylation of SMC3, a necessary step for cohesin recycling during the cell cycle. To date, 23 different missense mutants in the gene encoding HDAC8 have been identified in children with developmental features that overlap those of CdLS. Enzymological, biophysical, and structural studies of CdLS HDAC8 protein mutants have yielded critical insight on compromised catalysis in vitro. Most CdLS HDAC8 mutations trigger structural changes that directly or indirectly impact substrate binding and catalysis. Additionally, several mutations significantly compromise protein thermostability. Intriguingly, catalytic activity in many HDAC8 mutants can be partially or fully restored by an N‐acylthiourea activator, suggesting a plausible strategy for the chemical rescue of compromised HDAC8 catalysis in vivo.     

Spectrum of NIPBL gene mutations in Polish patients with Cornelia de Lange syndrome

Cornelia de Lange syndrome (CdLS) is a rare multi-system genetic disorder characterised by growth and developmental delay, distinctive facial dysmorphism, limb malformations and multiple organ defects. The disease is caused by mutations in genes responsible for the formation and regulation of cohesin complex. About half of the cases result from mutations in the NIPBL gene coding delangin, a protein regulating the initialisation of cohesion. To date, approximately 250 point mutations have been identified in more than 300 CdLS patients worldwide. In the present study, conducted on a group of 64 unrelated Polish CdLS patients, 25 various NIPBL sequence variants, including 22 novel point mutations, were detected. Additionally, large genomic deletions on chromosome 5p13 encompassing the NIPBL gene locus were detected in two patients with the most severe CdLS phenotype. Taken together, 42 % of patients were found to have a deleterious alteration affecting the NIPBL gene, by and large private ones (89 %). The review of the types of mutations found so far in Polish patients, their frequency and correlation with the severity of the observed phenotype shows that Polish CdLS cases do not significantly differ from other populations.     

Functional links between <Emphasis Type="Italic">Drosophila</Emphasis> Nipped-B and cohesin in somatic and meiotic cells

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. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Gause, Webber, and Misulovin provided equal contributions.