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384 trends in CELL BIOLOGY (Vol. 9) October 1999

A recent trends in CELL BIOLOGY article1 reported on the involvementof the blue light photoreceptorcryptochrome in the circadian clockin mammals, flies and plants. At thattime, the available data suggestedthat, in Drosophila and Arabidopsis,cryptochrome acts as a photorecep-tor responsible for entraining theclock, whereas in mice it could formpart of the clock mechanism itself.

Two new papers have now shownmore clearly that the roles played bycryptochrome in the circadian clockin Drosophila and in mice are indeedquite distinct. In flies, the clock ‘loop’is formed by the proteins TIMELESS(TIM) and PERIOD (PER), whose levelsoscillate with a 24-hour periodicity.Levels of PER and TIM in the cyto-plasm gradually rise during the after-noon and evening. PER–TIM dimersthen enter the nucleus, where theyfeed back on their own transcriptionby inhibiting a transcriptional com-plex formed by CLOCK and BMAL. Asa consequence, levels of PER and TIMeventually fall until they no longerinhibit their own transcription, andthe cycle repeats. In the first of thenew papers, Ceriani et al. demon-strate that Drosophila cryptochrome

binds to TIM in a light-dependentmanner, rendering the PER–TIMdimer unable to negate theCLOCK–BMAL transcriptional com-plex. It therefore provides theDrosophila circadian clock with a lightresetting mechanism.

In mice, although homologues of allof the known Drosophila clock com-ponents are present, the mechanismof the clock ‘loop’ is less well charac-terized. Kume et al.3 demonstrate that,in mice, mPER and mTIM are able toinhibit the CLOCK–BMAL transcrip-tional complex independently of eachother and that cryptochrome plays akey role in localizing mPER to thenucleus. Mice possess two crypto-chromes – mCRY1 and mCRY2 – and,in this study, both were localized tothe nucleus. Kume et al. also demon-strated that the cryptochromes them-selves can inhibit the CLOCK–BMALtranscriptional complex directly.Thus, unlike in flies, where crypto-chrome acts to promote transcriptionof some clock genes, in mice cryp-tochrome acts as a repressor.Moreover, they showed that tran-scription of the mCRY genes appearsto be under the control of theCLOCK–BMAL transcriptional com-

plex, providing strong evidence thatthe cryptochromes are, in fact, centralcomponents of the clock ‘loop’ inmice.

Thus, despite the strong conser-vation of the clock components in theanimal kingdom, the roles played bycryptochrome in flies and mammalsappear to be worlds apart. In flies,cryptochrome functions primarily as a clock-resetting photoreceptor,whereas, in mammals, crypto-chromes form components of theclock itself. However, mammaliancryptochrome could also play a roleas a photoreceptor in clock resetting,and further work is required beforewe will be able to establish this.

Blues news

1 Devlin, P. F. and Kay, S. A. (1999)Trends Cell Biol. 9, 295–298

2 Ceriani, M. F. et al. (1999) Light-dependent sequestration of TIMELESSby CRYPTOCHROME, Science 285,553–556

3 Kume, K. et al. (1999) mCRY1 andmCRY2 are essential components ofthe negative limb of the circadianclock feedback loop, Cell 98, 193–205

The metaphase-to-anaphase transi-tion is marked by a sudden loss ofcohesion between chromosome armsand kinetochores. Cohesion is estab-lished during DNA replication and isrequired for correct distribution ofchromosomes to the progeny; failuresmay lead to cancer1 or death. Meioticand mitotic chromosome cohesioninvolve a multisubunit ‘cohesin’ com-plex (in budding yeast: Scc1p, Scc3p,Smc1p and Smc3p) and are morpho-logically very similar, yet different,processes. Contact between bothpaired homologs and sister-chro-matid arms is lost during meiosis I, fol-lowed by cohesion loss between cen-tromeres at meiosis II. During mitosis,cohesion is first lost between cen-tromeres and this is immediately followed by cohesion loss betweensister-chromatid arms. The six veryelegant papers1–6 summarized herethat employ the powers of buddingyeast and Xenopus report on the

molecules required for cohesion, theirregulation, DNA-sequence require-ments for cohesin binding and new assays to study chromosomecohesion.

Scc1p is a cohesin subunit known todisappear from chromosomes whenthey separate at anaphase; the loss ofScc1p requires the ‘separin’ proteinEsp1p that is kept inactive until the anaphase-promoting complex(APCcdc20) degrades its inhibitor,Pds1p/‘Securin’. In vivo and in vitroexperiments show that Esp1p regu-lates the binding of Scc1p andremoval from chromosomes.Interestingly, proteolysis of Scc1p,possibly mediated directly by the200-kDa Esp1p, is required for sister-chromatid separation2. The sites pro-teolysed in Scc1p share homologywith a region in Rec8p that isexpressed only during meiosis2.Indeed, it is shown that Rec8p (andSmc3p) are required for chromosome

cohesion during meiosis, whereScc1p is not involved3,6. Moreover,these same proteins are necessary,but not sufficient, for formation ofaxial elements and for recombi-nation3. An open question is howRec8p and Smc3p can mediate cohe-sion during both meiosis I and II. It issuggested that differential degra-dation kinetics regulate differentialchromosome arm- and centromere-associated protein degradation3;alternatively, passage through mei-osis I might ‘license’ degradation ofthe (different) centromere cohesinduring meiosis II7. In unrelated butmechanistically complementary ex-periments1, a Xenopus and a human homologue (x/h-Securins) of the yeast anaphase inhibitorPds1p/Securin was identified andcharacterized. Because there is nohomology between Pds1p andknown proteins from metazoans, thehSecurin was identified by its

Breakthroughs on mitotic and meioticchromosome cohesion

This month’sheadlines werecontributed by

Søren Andersen,Paul F. Devlin,Steve A. Kay,

Wallace F.Marshall,

Fiona Townsleyand

Cezary Wojcik.

headlines

trends in CELL BIOLOGY (Vol. 9) October 1999 385

The Wnt signalling pathway regulatescell-fate decisions in developmentthrough a complex of b-catenin andthe TCF/LEF family of transcriptionfactors. In most experimental sys-tems, Wnt signalling stabilizes cyto-solic b-catenin, which then binds toTCF and acts as a transcriptional co-activator. Thus, the loss of b-cateninand TCF results in similar phenotypes.However, in Caenorhabditis elegans,the loss of WRM-1 (a b-catenin homo-logue) has the opposite effect to theloss of POP-1 (a TCF-related protein).These papers reveal that a novel mito-gen-activated protein (MAP) kinase-like pathway converges with the Wntsignalling pathway to regulate TCF-mediated gene transcription1–3. Thispathway might be the basis for theapparent discrepancy between therole of b-catenin in C. elegans and vertebrates.

In C. elegans, Wnt signalling polar-izes the embryo by downregulatingPOP-1 activity in posterior daughtercells. The proteins MOM-1 and LIT-1are also required to downregulatePOP-11,2 and are homologous to

TAK1 (transforming-growth-factor-b-activated kinase 1) and NLK (nemo-like kinase) respectively, which arecomponents of the MAP kinase path-way. Ishitani and colleagues3 foundthat TAK1 and NLK negatively regu-late b-catenin/TCF-mediated tran-scription in mammalian cells. TAK1stimulates the kinase activity of NLK,and this in turn phosphorylateshTCF4. Phosphorylated hTCF4 canstill bind to b-catenin, but NLKphosphorylation interferes with thebinding of the b-catenin–TCF com-plex to DNA, thereby negatively regulating b-catenin–TCF-mediatedtranscription. NLK immunoprecipi-tates contain hTCF4, but the interac-tion does not appear to be direct andmight require b-catenin3. Consistentwith this, in C. elegans, WRM-1 (b-catenin) was found in a stable com-plex with LIT-1 (NLK), where it acti-vated LIT-1-dependent kinase activityto hyperphosphorylate POP-1 (TCF).However, unlike the situation in vertebrates, WRM-1 did not form a stable complex with POP-12.Interestingly, POP-1 was normally

found in the nucleus of vertebratecells (as it is in C. elegans embryos),but coexpression of POP-1 with LIT-1and WRM-1 resulted in a redistribu-tion of POP-1 to the cytoplasm2.Thus, in C. elegans, the accumulationof WRM-1 due to Wnt signallingmight promote the inactivation ofPOP-1 through LIT-1 rather than pro-moting the coactivation of POP-1-mediated transcription.

expected physical interaction withhESP1 and by its degradation duringanaphase1. Using Xenopus extracts, it is shown that nondegradablexSecurin/p25 (xSecurindm) blocksmitotic chromatid separation, com-plementing the observations by others2,3. Interestingly, xSecurin/p25is homologous to the human onco-gene PTTG, and xSecurindm does notimpede cell-cycle progression; sug-gesting that xSecurin/Pds1p is notpart of a checkpoint, at least in frog1.

How cohesins target onto chromo-somes was addressed experimentallyby asking where purified Scc1p-containing chromosome fragmentsbound onto 133 membrane-attachedPCR-generated chromosome frag-ments (covering the entire length of chromosome III)4. The Scc1p-containing fragments bound to 23evenly spaced 15 kb long chromo-somal regions, with an enhancementat the centromere region; thisenhancement might contribute tothe continued cohesion betweenchromosomes after meiosis I2. A cor-relation between high Scc1p-binding

and AT-rich chromosomal areas wasobserved4. In a complementaryapproach, an in vivo system based oncircular minichromosomes was devel-oped5 consisting of a GFP-labelledminichromosome containing a cen-tromere region flanked by site-specific recombination sites; recombi-nation, and thus centromere excision,is induced in G1 prior to DNA replication(during which chromosome cohesionis established); cells are then arrestedin G2/M, which allows microscopicexamination/quantification of co-hesion of the (now) duplicated mini-chromosomes. It is shown that theyeast centromeric CDEIII region isnecessary but not sufficient for centromere cohesion activity5.

These assays4,5 should be valuablefor a further dissection of the DNA elements required for the temporaland spatial regulation of the associ-ation and function of the protein-aceous cohesin complex1–3,6. It will be interesting to further characterizeand compare the ‘expression’ of meiotic and mitotic cohesion activity3,4,6,7.

NLK TAKs onto the Wnt signalling pathway

1 Meneghini, M. D. et al. (1999) MAPkinase and Wnt pathways converge todownregulate an HMG-domainrepressor in Caenorhabditis elegans,Nature 399, 793–797

2 Rocheleau, C. E. et al. (1999) WRM-1activates the LIT-1 protein kinase totransduce anterior/posterior polaritysignals in C. elegans, Cell 97, 717–726

3 Ishitani, T. et al. (1999) TheTAK1–NLK–MAPK-related pathwayantagonises signalling between b-catenin and transcription factor TCF, Nature 399, 798–802

1 Zou, H., McGarry, T. J., Bernal, T. andKirschner, M. W. (1999) Identification ofa vertebrate sister-chromatid separationinhibitor involved in transformation andtumorigenesis, Science 285, 418–422

2 Uhlmann, F., Lottspeich, F. andNasmyth, K. (1999) Sister-chromatidseparation at anaphase onset ispromoted by cleavage of the cohesinsubunit Scc1, Nature 400, 37–42

3 Klein, F. et al. (1999) A central role forcohesins in sister chromatid cohesion,formation of axial elements andrecombination during yeast meiosis,Cell 98, 91–103

4 Blat, Y. and Kleckner, N. (1999)Cohesins bind to preferential sites alongyeast chromosome III, with differentialregulation along arms versus the centricregion, Cell 98, 249–259

5 Megee, P. C. and Koshland, D. (1999)A functional assay for centromere-associated sister chromatid cohesion,Science 285, 254–257

6 Watanabe, Y. and Nurse, P. (1999)Cohesin Rec8 is required for reductionalchromosome segregation at meiosis,Nature 400, 461–464

7 Rieder, C. L. and Cole, R. (1999)Chromatid cohesion during mitosis:lessons from meiosis, J. Cell Sci. 112,2607–2613