SMC PROTEINS Flashcards
what do condensins do
SMC2-SMC4 dimers constitute the core of condensin complexes, responsible for chromosome condensation and segregation in mitosis and meiosis. These SMC proteins form ring-like structures in combination with 3 non-SMC subunits (1 kleisin protein and 2 subunits containing HEAT repeats), that entrap chromatin and facilitate ATP-dependent supercoiling and looping of DNA. This promotes the compaction of chromatin into fully resolved sister chromatids, that can be assembled onto the mitotic spindle and pulled to opposite poles of the cell in anaphase. Chromosome condensation is very important in ensuring faithful segregation of DNA in cell division, as attempting to separate entangled chromatin into daughter cells would likely result in frequent DNA damage and strand breaks
discovery of condensins
The function of condensins in this capacity was first reported by Hirano and Mitchison (1994)1
• reconstituted mitotic chromosomes from sperm chromatin using cell-free Xenopus egg extracts
• identified 2 proteins = XCAP-C and XCAP-E (also known as SMC4 and SMC2) that
- copurified with mitotic chromosomes but not sperm chromatin
- associated with each other in both coimmunoprecipitation and column chromatography experiments
• showed that localisation of XCAP-C to mitotic chromosomes correlated with phases of chromosome condensation, and that knockdown of XCAP-C inhibited the final stages of condensation; when anti-XCAP-C antibodies were added to the mitotic extract, no rod-shaped chromosomes emerged, with prophase-like chromatin fibres accumulating instead
• This last experiment was limited in that the anti-XCAP-C antibodies used did not achieve complete depletion of XCAP-C and hence may have left some aspects of protein function intact, meaning researchers could not convincingly conclude that XCAP-C did not play roles in earlier stages of chromosome condensation
Later experiments = Hirano et al, 1997
• achieved a more comprehensive depletion of condensin (over 95%) from Xenopus egg extracts using a mixture of antibodies against XCAP-C, XCAP-E, and the non-SMC subunit XCAP-G
• showed that condensin complexes are indeed required at earlier stages in condensation, with sperm chromatin incubated with the depleted extract swelling to form a fluffy interphase-like chromatin sphere, orchestrated by topoisomerase II, with no evidence of fibre formation
• differs to the prophase-like phenotype described in the 1994 paper, likely because the anti-XCAP-C antibodies used alone blocked the function of condensin but not its chromosomal targeting
• also showed that addition of condensin fractions back into the depleted extract rescued chromosome assembly, again highlighting the importance of condensins in this process
different condensins
Most multicellular organisms possess two different types of condensins, containing the same SMC proteins but different non-SMC subunits (CAP-D2, CAP-G and CAP-H in condensin I; CAP-D3, CAP-G2 and CAP-H2 in condensin II). These two complexes typically accumulate in the nucleus at different points in the cell cycle; condensin II is localised to the nucleus from G2 onwards, whilst condensin I remains sequestered in the cytoplasm until the breakdown of the nuclear membrane in prometaphase. They also exhibit differential roles, with condensin I thought to facilitate lateral condensation and condensin II to promote axial shortening.
• Hirota et al (2004) – inactivated condensin I and II by CAP-D2 and CAP-D3 depletion respectively in prophase-cells using siRNAs (CAP-D2 found in condensin I only, and CAP-D3 only in condensin II).
o DAPI staining and subsequent immunofluorescence and time-lapse confocal microscopy revealed that condensin II is required for chromosome condensation in early prophase, whereas condensin I appears to be dispensable at this stage.
o By contrast, condensin I was required for the complete dissociation of cohesin from chromosome arms, chromosome shortening and for normal timing of progression through prometaphase and metaphase, whereas normal condensin II levels are dispensable for all three of these processes.
o Moreover, after depletion of both, the onset of chromosome condensation was delayed until the end of prophase, but is then initiated rapidly before nuclear envelope breakdown, so it appears that compaction can still occur in the absence of condensin.
• Compaction occurs in absence of condensin due to small expression of condensins not fully targeted by RNAi?
• Important to note that this work is based on RNAi experiments, whilst genetic knock-downs of both condensin complexes may give a more penetrant phenotype.
• But genetic KO = difficult as is an essential gene…
how condensins achieve chromosome condensation
Terakawa et al (2017)3
• examined condensins in Saccharomyces cerevisiae using single-molecule fluorescence imaging proposed that condensin acts as a molecular motor that translocates along DNA, at maximum velocities of around 60 base pairs per second
• speed and processivity of condensin complexes varied with ATP concentrations, indicating that its translocation is dependent on ATP binding and hydrolysis.
condensin looping
Studies have also indicated that condensins condense chromatin through the extrusion of helical DNA loops on multiple levels. This was examined by
Gibcus et al (2018)4
• used chromosome conformation capture (Hi-C) to reveal changes in chromosome morphology during mitosis in DT40 cells
• found that upon release from interphase, DNA formed a central helical scaffold made up of large loops of DNA
• As cells progressed through prophase, the scaffold was progressively compressed and the DNA loops became larger (200-400kB), whilst prometaphase saw the emergence of an inner axis of smaller loops (40-60kB) nested within the larger loops
• Subsequent depletion of condensin I and II from the cells through auxin-inducible degradation of CAP-H and CAP-H2 respectively highlighted that condensin II is responsible for the earlier establishment of longer loops whilst condensin I produces shorter loops established later, as cells possessing only condensin I or condensin II could only produce narrow or wide helical axes respectively
• Whilst this study provides conclusive evidence that condensins contribute to chromosome condensation through DNA loops, it is limited in that it does not provide an explanation for the molecular mechanisms that underly this process; further studies are required to deduce how looping is actually established.
Are functions of condensin I and condensin II mutually exclusive?
- Likely not
- Kranz et al (2013) = used chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) to map genomic distribution of condensins in C. elegans embryos distributions of condensin I and II partially overlapped suggests 2 condensins could work together
cohesins loading and unloading
Interphase
- loaded by NIPBL and MAU2
- embrace of chromatids achieved by opening of SMC1-SMC3 interface (Gruber et al (2006))
- unloaded by Wapl/PDS5 = opens SMC3-SCC1 interface
S phase
- stabilised by Eco1/Esco1
- acetylates SMC3 = stable connection between SMC3 and SCC1
- binds PCNA in RF –> ensures cohesins only stably associated with DNA following replication
- acetylation also facilitates recruitment of soronin = displaces Wapl from PDS5
Prophase
- Aurora kinase B and CDK1 phosphorylate soronin on chromosome arms = soronin dissociates from PDS5 and WAPL recruited
- PLK1 also phosphorylates SA subunit
shughoshin and PP2A prevent phosphorylation of soronin on centromeric cohesins = protective
Kitajima et al (2004)
- identified Sgo2 protein in yeast
- Sgo2 KO cells = increased sensitivity to spindle-destabilising drug TBZ, increased incidence of chromosome mis-segregation at mitosis (as measured by non-disjunction of cen2-GFP gene)
- ChIP revealed Sgo2 only localises to pericentromeric regions of chromosomes in mitotic cells
Metaphase = centromeric cohesins cleaved by Esp1 seperase which cleave Scc1
why is prophase cohesin removal important
Importance of prophase cohesin removal
• Preserves cohesin rings for reloading in the subsequent cell cycle
• Prophase pathway transiently opens cohesin’s exit gate cohesin rings still intact, can be reloaded onto chromosomes
• Seperase-mediated cleavage = destroys Scc1 subunit of cohesin complexes cannot be reloaded on chromosomes
Tedeschi et al (2013)
• Generated mouse embryonic fibroblasts lacking WAPL expression = no prophase pathway
• Found that cohesin reloading in telophase and early G1 was impaired in WAPL-depleted cells
- Detected significantly less chromatin-bound Scc1 in WAPL-depleted cells using immunofluorescence microscopy
integrity of cohesin rings
Experiments by Uhlmann et al (2000)6 have shown that the integrity of cohesin rings is crucial for
their function, as proteolytic cleavage of cohesin’s Scc1 subunit was sufficient to trigger the transition from metaphase to anaphase in yeast. In these experiments, they genetically modified the Scc1 gene, exchanging the seperase cleavage site for the recognition sequence of the tobacco etch virus (TEV) protease, before introducing the TEV protease gene under the control of a galactose-inducible promoter. They then arrested the yeast cells in metaphase, noting that cells accumulated with visible replicated chromosomes and short mitotic spindles characteristic of metaphase. Induction of the TEV protease through the addition of galactose was sufficient to trigger elongation of spindles and movement of sister chromatids to opposite poles of the cell, forming 2 equal masses of DNA at each pole.
evidence that cohesins are important for chromosomal architecture
Cohesins are also integral for the organisation of wider chromosomal architecture. This was illustrated by Schwarzer et al (2017)7, who used Cre-lox techniques to selectively deplete the cohesin loader Nipbl from non-dividing hepatocytes in mice using a tamoxifen-inducible, liver-specific Cre allele. Global depletion of Nipbl was not possible as inherited Nipbl mutations are embryonically lethal, and Nipbl remains crucial for cell division in proliferating cell populations. This technique resulted in a 4-6 fold reduction in chromosome-associated cohesin in the hepatocytes, and induced drastic effects on genome organisation, including widespread loss of topologically associating domains (TADs) and associated peaks seen via Hi-C. TADs are regions of DNA where the density of interactions between DNA sequences is much higher than at other areas of the genome, and are the product of local chromatin folding. As a result, their loss on Nipbl depletion indicates that cohesins are crucial to normal chromosome organisation in interphase. However, it was noted that global compartmentalisation of DNA into active and inactive compartments was actually enhanced by Nipbl loss, suggesting that cohesin is only important for local compaction and not global chromosome structure.
what are TADs?
More detail on TADs
- TADs are a form of trans looping
- Transcriptionally active = make transcription more efficient by bringing large groups of genes together easier for RNA polymerase + transcription factors (TFs) to access promoters etc
- Also very dynamic, as needs of the cell change (e.g. phases of cell cycle, environmental factors) very important in developmental differentiation
cohesin in gene regulation
Cohesins also play important roles in the regulation of gene expression, acting in combination with a range of transcriptional regulators to promote the formation of chromatin loops, hence facilitating long-distance chromatin interactions that regulate transcription = this is cis looping. For example, cohesin has been shown to enable the insulator functions of the DNA-binding protein CTCF, which block interactions between promoters and distal enhancers to repress gene expression and prevent the spread of heterochromatin. This was illustrated in a study by Wendt et al (2008)8, who used chromatin immunoprecipitation to identify cohesin and CTCF binding sites in human DNA, deducing that 89% of cohesin sites were also bound by CTCF, providing evidence that the two proteins co-localise on DNA. They also showed that cohesin is required for the control of transcription at the imprinted H19/IGF2 locus on chromosome 11, dependent on CTCF (see figure 2). They first produced murine cells that possessed either the maternal or paternal copy of human chromosome 11, and showed that cohesin specifically binds to the H19 ICR on the maternal allele like CTCF, as the human H19 ICR could only be isolated using CTCF and SMC3 antibodies from cells expressing only the maternal copy. They then showed that depletion of the cohesin subunit SCC1 increased the number of IGF2 transcripts and reduced the number of H19 transcripts in HeLa cells in both G2 and G1, providing evidence that the insulation of the maternal IGF2 locus by CTCF is dependent on cohesin.
CTCF is a novel cohesin loader promotes association of cohesin with DNA
Other studies have shown that cohesin also regulates a range of other genes in combination with CTCF, including interferon γ, apolipoprotein gene clusters and the β globin locus. It also acts in combination with other factors in specific cell types, such as oestrogen receptor α in breast cancer cells and Mediator in embryonic stem cells, facilitating interactions between enhancers and promoters of genes required to maintain pluripotency (e.g. Oct4, Nanog).
cohesinopathies
Cohesin’s roles in gene regulation explain the phenotypes of cohesinopathies, associated with inherited mutations in cohesin or its regulators. The most common of these disorders is Cornelia de Lange syndrome (CdLS), which is usually caused by mutations in regulatory protein NIPBL or more rarely in SMC1 or SMC3 themselves, and presents with a range of signs and symptoms, including short stature, characteristic facial features, cardiac and gastrointestinal defects, and intellectual disability. Studies have found that this condition is associated with dysregulation of a wide range of different genes, particularly those involved in cell growth and proliferation and neuronal development, rather than defects in sister chromatid cohesion. This was examined by Remeseiro et al (2014)9, who produced a mouse model of CdLS through deletion of a NIPBL allele. They found that reductions in NIPBL levels had little effect on bulk condensin loading or sister chromatin cohesion, but resulted in significant reductions in cohesin loading at specific genomic loci, such as the promoters of Myc and protocadherins. This resulted in a significant decrease in the mRNA expressed from these genes, which may in part explain the growth defects and intellectual disability respectively seen in CdLS.
Patients are also very prone to carcinogenesis cohesin mutations result in poor DNA damage responses and poor sister chromatid cohesion in mitosis increased likelihood of mutations
Cohesin mutations are also implicated in the maternal age effect (increased risk of trisomy in older mothers) Angell et al (1995) = age-dependent weakening of sister-chromatid cohesion
evidence for 2 distinct pathways of cohesin removal
Waizenegger et al (2010) = proposed two distinct pathways, that remove mammalian cohesin from chromosome arms in prophase and from centromeres in anaphase
• Generated HeLa cell line stably expressing myc-tagged SCC1 = immunofluorescence microscopy revealed considerable staining of prophase chromosomes, SCC1 remained associated only with centromeric regions in metaphase
• Analysed behaviour of SCC1 by synchronising HeLa cells by double-thymidine arrest and release protocol = samples taken at different time points and analysed for SCC1 by immunoblotting with SCC1 or myc antibodies = saw emergence of mitosis-specific cleavage products in cells undergoing anaphase (coincided with disappearance of cyclin A + B, CDC20), shown to be dependent on APC and separin in vitro
• Then incubated cells with nocodazole = inhibits spindle assembly checkpoint and hence anaphase onset no mitosis-specific cleavage products in arrested cells
• Paper suggests that removal of cohesins from chromosome arms in prophase occurs without detectable SCC1 cleavage = different pathway to anaphase
• Failed to explore mechanism of action of prophase pathway
discovery of Sgo
Kitajima et al (2004) = discovery of Shugoshin
• Identified Sgo2 gene in S. pombe yeast = paralogue to Sgo1 gene implicated in protecting centromeric cohesion during meiosis
• Disrupted Sgo2 gene to produce KO cells
- Viable but sensitive to spindle-destabilising drug TBZ
- Increased incidence of chromosome mis-segregation at mitosis (as measured by rate of non-disjunction of inserted cen2-GFP gene)
• Then investigated cellular distribution of Sgo2 by tagged endogenous gene with GFP
- Immunofluorescence microscopy revealed that Sgo2 localises to nucleus (DAPI staining), and localises closely with centromeric protein Mis6
- CHIP assays showed Sgo2 only associated with chromatin at pericentromeric regions in synchronous populations of mitotic cells
• Failed to confirm results in other cell types/organisms
