Lena Ström

The ancient and evolving roles of cohesin in gene expression and DNA repair.

The cohesin complex, named for its key role in sister chromatid cohesion, also plays critical roles in gene regulation and DNA repair. It performs all three functions in single cell eukaryotes such as yeasts, and in higher organisms such as man. Minor disruption of cohesin function has significant consequences for human development, even in the absence of measurable effects on chromatid cohesion or chromosome segregation. Here we survey the roles of cohesin in gene regulation and DNA repair, and how these functions vary from yeast to man.

Chromosome length influences replication-induced topological stress

During chromosome duplication the parental DNA molecule becomes overwound, or positively supercoiled, in the region ahead of the advancing replication fork. To allow fork progression, this superhelical tension has to be removed by topoisomerases, which operate by introducing transient DNA breaks. Positive supercoiling can also be diminished if the advancing fork rotates along the DNA helix, but then sister chromatid intertwinings form in its wake. Despite these insights it remains largely unknown how replication-induced superhelical stress is dealt with on linear, eukaryotic chromosomes. Here we show that this stress increases with the length of Saccharomyces cerevisiae chromosomes. This highlights the possibility that superhelical tension is handled on a chromosome scale and not only within topologically closed chromosomal domains as the current view predicts. We found that inhibition of type I topoisomerases leads to a late replication delay of longer, but not shorter, chromosomes. This phenotype is also displayed by cells expressing mutated versions of the cohesin- and condensin-related Smc5/6 complex. The frequency of chromosomal association sites of the Smc5/6 complex increases in response to chromosome lengthening, chromosome circularization, or inactivation of topoisomerase 2, all having the potential to increase the number of sister chromatid intertwinings. Furthermore, non-functional Smc6 reduces the accumulation of intertwined sister plasmids after one round of replication in the absence of topoisomerase 2 function. Our results demonstrate that the length of a chromosome influences the need of superhelical tension release in Saccharomyces cerevisiae, and allow us to propose a model where the Smc5/6 complex facilitates fork rotation by sequestering nascent chromatid intertwinings that form behind the replication machinery.

The age and genomic integrity of neurons after cortical stroke in humans

It has been unclear whether ischemic stroke induces neurogenesis or neuronal DNA rearrangements in the human neocortex. Using immunohistochemistry; transcriptome, genome and ploidy analyses; and determination of nuclear bomb test–derived 14C concentration in neuronal DNA, we found neither to be the case. A large proportion of cortical neurons displayed DNA fragmentation and DNA repair a short time after stroke, whereas neurons at chronic stages after stroke showed DNA integrity, demonstrating the relevance of an intact genome for survival.

DNA damage-induced cohesion

The protein complex Cohesin, forming protein-links that hold sister chromatids together, is at the heart of chromatid cohesion. Cohesion is important both for correct chromosome segregation and double-strand break (DSB) repair, making Cohesin central for the maintenance of genome stability. Until now, establishment of Cohesin links between chromatids has been shown to occur during DNA replication only. Recently it was however observed that in cells arrested in G2/M, DSB induction not only elicits chromosomal recruitment of Cohesin, but also formation of chromatid cohesion. The establishment of cohesion outside the period of replication opens a new field of investigation. Here we present results further supporting the formation of sister chromatid cohesion in response to DNA damage, and propose a model of how damage-induced cohesion could contribute to the linkage of chromatids during normal cell cycle progression.

De novo heterozygous mutations in SMC3 cause a range of Cornelia de Lange syndrome-overlapping phenotypes.

Cornelia de Lange syndrome (CdLS) is characterized by facial dysmorphism, growth failure, intellectual disability, limb malformations, and multiple organ involvement. Mutations in five genes, encoding subunits of the cohesin complex (SMC1A, SMC3, RAD21) and its regulators (NIPBL, HDAC8), account for at least 70% of patients with CdLS or CdLS‐like phenotypes. To date, only the clinical features from a single CdLS patient with SMC3 mutation has been published. Here, we report the efforts of an international research and clinical collaboration to provide clinical comparison of 16 patients with CdLS‐like features caused by mutations in SMC3. Modeling of the mutation effects on protein structure suggests a dominant‐negative effect on the multimeric cohesin complex. When compared with typical CdLS, many SMC3‐associated phenotypes are also characterized by postnatal microcephaly but with a less distinctive craniofacial appearance, a milder prenatal growth retardation that worsens in childhood, few congenital heart defects, and an absence of limb deficiencies. While most mutations are unique, two unrelated affected individuals shared the same mutation but presented with different phenotypes. This work confirms that de novo SMC3 mutations account for ∼1%–2% of CdLS‐like phenotypes.

A regulatory role for the cohesin loader NIPBL in nonhomologous end joining during immunoglobulin class switch recombination

The cohesin loading protein is required for nonhomologous end joining of double-strand DNA breaks

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.

Binding of Ikaros to germline Ig heavy chain γ1 and ε promoters

Immunoglobulin (Ig) class switching occurs in activated B cells and results in production of antigen-specific IgA, IgE or IgG. It involves a DNA recombination event and is partly regulated by germline (GL) immunoglobulin heavy chain promoters. Ikaros is an abundant nuclear protein expressed in hematopoietic cells. Many different functions have been ascribed to Ikaros, such as transcriptional activation or repression, cell cycle control and tumor suppression. A typical feature of Ikaros is its expression in large clusters in the nucleus of activated lymphocytes. We give evidence that Ikaros can bind to several sites in the germline γ1 and e immunoglobulin heavy chain promoters, in a cooperative manner. Using a promoter reporter assay, we found evidence that Ikaros can suppress germline γ1 and e promoter activity in a B cell line. When a mutated non-DNA-binding form of Ikaros was introduced into primary activated B cells by retrovirus transduction, the endogenous Ikaros clusters were disrupted. In spite of this, there was no effect on transcription or Ig class switching. The data are discussed in relation to the different hypotheses for the function of Ikaros.

Inactivation of the budding yeast cohesin loader Scc2 alters gene expression both globally and in response to a single DNA double strand break

Genome integrity is fundamental for cell survival and cell cycle progression. Important mechanisms for keeping the genome intact are proper sister chromatid segregation, correct gene regulation and efficient repair of damaged DNA. Cohesin and its DNA loader, the Scc2/4 complex have been implicated in all these cellular actions. The gene regulation role has been described in several organisms. In yeast it has been suggested that the proteins in the cohesin network would effect transcription based on its role as insulator. More recently, data are emerging indicating direct roles for gene regulation also in yeast. Here we extend these studies by investigating whether the cohesin loader Scc2 is involved in regulation of gene expression. We performed global gene expression profiling in the absence and presence of DNA damage, in wild type and Scc2 deficient G2/M arrested cells, when it is known that Scc2 is important for DNA double strand break repair and formation of damage induced cohesion. We found that not only the DNA damage specific transcriptional response is distorted after inactivation of Scc2 but also the overall transcription profile. Interestingly, these alterations did not correlate with changes in cohesin binding.

Importance of Polη for Damage-Induced Cohesion Reveals Differential Regulation of Cohesion Establishment at the Break Site and Genome-Wide

Genome integrity depends on correct chromosome segregation, which in turn relies on cohesion between sister chromatids from S phase until anaphase. S phase cohesion, together with DNA double-strand break (DSB) recruitment of cohesin and formation of damage-induced (DI) cohesion, has previously been shown to be required also for efficient postreplicative DSB repair. The budding yeast acetyltransferase Eco1 (Ctf7) is a common essential factor for S phase and DI-cohesion. The fission yeast Eco1 ortholog, Eso1, is expressed as a fusion protein with the translesion synthesis (TLS) polymerase Polη. The involvement of Eso1 in S phase cohesion was attributed to the Eco1 homologous part of the protein and bypass of UV-induced DNA lesions to the Polη part. Here we describe an additional novel function for budding yeast Polη, i.e. formation of postreplicative DI genome-wide cohesion. This is a unique Polη function not shared with other TLS polymerases. However, Polη deficient cells are DSB repair competent, as Polη is not required for cohesion locally at the DSB. This reveals differential regulation of DSB–proximal cohesion and DI genome-wide cohesion, and challenges the importance of the latter for DSB repair. Intriguingly, we found that specific inactivation of DI genome-wide cohesion increases chromosomal mis-segregation at the entrance of the next cell cycle, suggesting that S phase cohesion is not sufficient for correct chromosome segregation in the presence of DNA damage.