Nested Irreducible Complexity | Evolution News

In previous articles, I have described the molecular marvel that is mitotic cell division, with a view towards imparting to readers a sense of the engineering prowess and, indeed, genius behind this phenomenal process. Here, I will zoom in on the process of chromosome condensation, which occurs during prophase. The compaction of chromatin to form the recognizable mitotic chromosome structures would serve little value until the evolution of the machinery for facilitating mitotic segregation. And yet, it is essential for mitotic division to proceed — that is, the process of chromosome condensation resides within an irreducibly complex system. As we shall see, however, the process of chromosome condensation itself exhibits irreducible complexity. Thus, this represents an example of irreducible complexity that is nested within a larger irreducibly complex apparatus. It is, therefore, difficult to envision such a process coming together through a blind process, without foresight of the target. This aspect of cell division is  best explained by teleology – i.e., conscious design.

What Happens During Prophase? The Big Picture

During prophase, the replicated chromosomes (each consisting of two sister chromatids) condense and are recognizable under the microscope. This process of condensation reduces the length of a typical interphase chromosome by approximately ten-fold and gene expression shuts down. The two sister chromatids of each mitotic chromosome have been disentangled from one another and are joined at the centromere. This facilitates the later separation of the sister chromatids as they are segregated between the two daughter cells. The compaction also protects the fragile DNA molecules during this process. Each pair of sister chromatids are genetically identical to one another. Outside of the cell nucleus, the mitotic spindle (which is made up of microtubules) begins to form between the two centrosomes, which have already duplicated during S and G2 phases of the cell cycle. The mitotic spindle will be responsible later (during anaphase) for facilitating the separation of the two sister chromatids.

Phosphorylation of Histone H3

Phosphorylation of histone H3, by Aurora B kinase, has been shown to play an important role in the condensation of mitotic chromosomes — in particular, at residues Ser10 and Ser28 (for short, H3S10 and H3S28).^1,2 This disrupts electrostatic interactions between the DNA and histones, loosening chromatin structure and making it more accessible for the condensin complexes to bind. Phosphorylation of H3S10 and H3S28 also promotes the binding of topoisomerase IIα and the chromosomal passenger complex (CPC). These phosphorylations of Histone 3 are essential for chromosome condensation and segregation. Without them, the result would be defective chromosome condensation and,  consequently, impaired segregation of the genetic material. In fact, deregulation of these epigenetic histone markings has been linked to cancer.³Moreover, when Aurora B kinase is depleted using RNA interference, the localization of the CPC to centromeres is impaired, disrupting mitotic progression.⁴

Chromatin Condensation

Electron micrograph images reveal that each chromatid is arranged into loops of chromatin, which emanate from a central scaffolding. Condensins work to coil the two DNA molecules, using the energy from ATP hydrolysis.⁵ The result is the two sister chromatids associated with the mitotic chromosome.

Condensin proteins are made up of five constituent parts.⁶ Among those are the SMC (Structural Maintenance of Chromosomes) proteins, SMC2 and SMC4 (which have ATPase activity). The SMC proteins have coiled-coil domains (i.e., long, flexible arms that fold back on themselves-creating a V-shaped structure), a hinge domain (facilitating the dimerization of the two SMC proteins), and head domains (possessing ATP-binding and ATPase sites, which energizes condensing activity). There are also three non-SMC subunits, which associate with specific regions of DNA and assist in regulating the activity of the condensins.

Condensin complexes load onto the chromatin in a stepwise manner, directed by the non-SMC subunits, which create loops in the DNA (energized by their ATPase activity). These loops are subsequently condensed into mitotic chromosomes. The condensin proteins are critical for cell division to occur. In their absence, the consequence would be chromosomal disorganization, as well as great difficulty in achieving proper segregation during mitosis. This is borne out by condensin knockout studies. For example, one paper reported that “CAP-D3 (condensin II) knockout results in masses of chromatin-containing anaphase bridges. CAP-H (condensin I)-knockout anaphases have a more subtle defect, with chromatids showing fine chromatin fibres that are associated with failure of cytokinesis and cell death.”⁷ The authors conclude that “condensin II alone can support mitotic chromosome rigidity, whereas condensin I is clearly not able to do so.” Though having both condensin I and II is apparently unnecessary, it is surely the case that having at least condensin II is essential for successful condensation to achieve the mitotic chromosome.

The Chromosome Passenger Complex

The Chromosome Passenger Complex (CPC) is a complex involving four proteins, which are essential to ensuring proper chromosome condensation, alignment, and segregation. The four main components are Aurora B kinase, Survivin, Borealin, and INCENP.^8,9

As mentioned previously, Aurora B kinase plays a crucial role in phosphorylating histone H3.¹⁰ Aurora B kinase also phosphorylates Heterochromatin Protein 1 (HP1), a protein that normally maintains chromatin in its relaxed interphase state. Inhibitory phosphorylation of this protein promotes chromosome condensation.¹¹Aurora B kinase is also responsible for phosphorylating and activating subunits of condensin.¹² When Aurora B is depleted, condensin activity is significantly impaired — for example, in Xenopus (clawed frogs, pictured at the top) egg extracts, removal of Aurora B has been shown to result in a 50 percent decrease in condensin I association with chromosomes.¹³

Survivin is responsible for targeting the CPC to chromatin by associating with histone modifications, and thereby assisting with the positioning of Aurora B kinase.^14,15,16  Depletion of survivin has been shown to result in “significant reduction of endogenous phosphorylated histone H3 and mislocalization of Aurora-B.”¹⁷

The CPC is itself stabilized at chromosomes by Borealin, ensuring the continued activity of Aurora B kinase and the efficient recruitment of condensins.¹⁸ When Borealin is knocked-out by RNA interference, mitotic progression is delayed and the consequence is “kinetochore-spindle misattachments and an increase in bipolar spindles associated with ectopic asters.”¹⁹

The Inner Centromere Protein (INCENP) serves a scaffolding role, and activates Aurora B kinase so that it can phosphorylate substrates (in particular, histones, condensins, and Topoisomerase IIα).²⁰

Topoisomerase IIα

Topoisomerase IIα is crucial for successful mitotic division. In its absence, the typical result is chromosomal mis-segregation, aneuploidy, and cell death. In particular, Topoisomerase IIα is essential for resolving chromosomal catenations. Catenation refers to the physical intertwining of the sister chromatids following DNA replication (resulting from the DNA becoming topologically linked during copying). Failure to disentangle the sister chromatids can result in their improper separation during anaphase, the consequence of which is mis-segregation and aneuploidy. Catenation can also result in the chromatids being subject to tensile stress during the process of mitosis — this can result in chromosomal breakage. When Topoisomerase IIα has been experimentally deleted prior to mitosis, the effect is a failure of chromatin condensation, and exit of the cell from mitosis without chromosome segregation having occurred.²¹ In addition, it was found that “removal of TOP2A from cells arrested in prometaphase or metaphase cause dramatic loss of compacted mitotic chromosome structure,” indicating that Topoisomerase IIα is “crucial for maintenance of mitotic chromosomes.”²²

How does Topoisomerase IIα resolve catenation to allow mitosis to proceed successfully?^23,24,25  First, it recognizes and binds to catenated regions where the sister chromatids are entangled. Two DNA-binding domains recognize two DNA segments — i.e., the G and T segments (Gate and Transport Segments respectively). The G-segment is on the DNA duplex that will be cleaved, while the T-segment is on the duplex that will be passed through the break. Using the energy from ATP hydrolysis, Topoisomerase IIα induces a conformational change that places the G-segment within its active site. Both strands of the G-segment are then cleaved, and the T-segment is passed through the break. The result of this process is the untangling of the interlinked chromatids. Topoisomerase IIα then relegates the broken G-segment, and the enzyme is released from DNA (energized by another round of ATP hydrolysis), and the enzyme is reset.

The function of Topoisomerase enzymes is, of course, not limited to resolving catenation in preparation for mitosis — they are also important for alleviating supercoils during DNA replication. However, following genome duplication, some catenation remains, especially in centromeric and heterochromatic regions — this must be fully resolved in advance of mitosis (otherwise, it will hinder proper chromosome segregation). To ensure that this happens, Topoisomerase IIα is phosphorylated by Cyclin-dependent kinase (Cdk) 1 and Aurora B kinase, enhancing its activity in late G2/M phase for the task of catenation-resolution that is needed for chromosome condensation.^26,27

There is even a checkpoint in late G2 that determines that catenation has been completely resolved.²⁸ If topological stress is detected, Ataxia Telangiectasia and Rad3-Related kinase (ATR) are recruited to chromatin. This phosphorylates Checkpoint kinase 1 (Chk1), which in turn phosphorylates Cdc25C, a phosphatase that activates Cdk1. This results in the inhibition of Cdc25C, preventing activation of Cdk1, thereby delaying entry of the cell into mitosis. In fact, experimental knockout of ATR in mice has been shown to result in early embryonic lethality due to massive apoptosis and mitotic defects, demonstrating the essential role of ATR in mitotic fidelity.^29,30 A similar study also revealed that Chk1 deletion in mice results in embryonic lethality.³¹

Intelligent Design

The process of chromosomal condensation is absolutely essential to successful mitotic cell division — that is to say, it is a part of a larger irreducibly complex system. Moreover, the highly condensed mitotic chromosome structures (with sister chromatids joined at the centromere) do not serve a purpose apart from in the context of mitosis. And yet, as we have seen, various components are themselves indispensable for effective chromosome condensation. Thus, we have an example of an irreducibly complex apparatus that is nested within a larger irreducibly complex system. It is highly implausible that such a wonder of engineering arose by means of an unguided evolutionary process. It is, in my judgment, far better explained on the hypothesis of the involvement of a conscious mind.

Notes

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3. Komar D, Juszczynski P. Rebelled epigenome: histone H3S10 phosphorylation and H3S10 kinases in cancer biology and therapy. Clin Epigenetics. 2020 Oct 14;12(1):147. doi: 10.1186/s13148-020-00941-2. PMID: 33054831; PMCID: PMC7556946.
4. Honda R, Körner R, Nigg EA. Exploring the functional interactions between Aurora B, INCENP, and survivin in mitosis. Mol Biol Cell. 2003 Aug;14(8):3325-41. doi: 10.1091/mbc.e02-11-0769. Epub 2003 May 29. PMID: 12925766; PMCID: PMC181570.
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6. Hirano T. Condensin-Based Chromosome Organization from Bacteria to Vertebrates. Cell. 2016 Feb 25;164(5):847-57. doi: 10.1016/j.cell.2016.01.033. PMID: 26919425.
7. Green LC, Kalitsis P, Chang TM, Cipetic M, Kim JH, Marshall O, Turnbull L, Whitchurch CB, Vagnarelli P, Samejima K, Earnshaw WC, Choo KH, Hudson DF. Contrasting roles of condensin I and condensin II in mitotic chromosome formation. J Cell Sci. 2012 Mar 15;125(Pt 6):1591-604. doi: 10.1242/jcs.097790. Epub 2012 Feb 17. PMID: 22344259; PMCID: PMC3336382.
8. Carmena M, Wheelock M, Funabiki H, Earnshaw WC. The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis. Nat Rev Mol Cell Biol. 2012 Dec;13(12):789-803. doi: 10.1038/nrm3474. PMID: 23175282; PMCID: PMC3729939.
9. Tan L, Kapoor TM. Examining the dynamics of chromosomal passenger complex (CPC)-dependent phosphorylation during cell division. Proc Natl Acad Sci U S A. 2011 Oct 4;108(40):16675-80. doi: 10.1073/pnas.1106748108. Epub 2011 Sep 26. PMID: 21949386; PMCID: PMC3189036.
10. Hirota T, Lipp JJ, Toh BH, Peters JM. Histone H3 serine 10 phosphorylation by Aurora B causes HP1 dissociation from heterochromatin. Nature. 2005 Dec 22;438(7071):1176-80. doi: 10.1038/nature04254. Epub 2005 Oct 12. PMID: 16222244.
11. Williams MM, Mathison AJ, Christensen T, Greipp PT, Knutson DL, Klee EW, Zimmermann MT, Iovanna J, Lomberk GA, Urrutia RA. Aurora kinase B-phosphorylated HP1α functions in chromosomal instability. Cell Cycle. 2019 Jun;18(12):1407-1421. doi: 10.1080/15384101.2019.1618126. Epub 2019 May 26. PMID: 31130069; PMCID: PMC6592258.
12. Lipp JJ, Hirota T, Poser I, Peters JM. Aurora B controls the association of condensin I but not condensin II with mitotic chromosomes. J Cell Sci. 2007 Apr 1;120(Pt 7):1245-55. doi: 10.1242/jcs.03425. Epub 2007 Mar 13. PMID: 17356064.
13. Takemoto A, Murayama A, Katano M, Urano T, Furukawa K, Yokoyama S, Yanagisawa J, Hanaoka F, Kimura K. Analysis of the role of Aurora B on the chromosomal targeting of condensin I. Nucleic Acids Res. 2007;35(7):2403-12. doi: 10.1093/nar/gkm157. Epub 2007 Mar 28. PMID: 17392339; PMCID: PMC1874644.
14. Wheatley SP, Altieri DC. Survivin at a glance. J Cell Sci. 2019 Apr 4;132(7):jcs223826. doi: 10.1242/jcs.223826. PMID: 30948431; PMCID: PMC6467487.
15. Li F, Ling X. Survivin study: an update of “what is the next wave”? J Cell Physiol. 2006 Sep;208(3):476-86. doi: 10.1002/jcp.20634. PMID: 16557517; PMCID: PMC2821201.
16. Garg H, Suri P, Gupta JC, Talwar GP, Dubey S. Survivin: a unique target for tumor therapy. Cancer Cell Int. 2016 Jun 23;16:49. doi: 10.1186/s12935-016-0326-1. PMID: 27340370; PMCID: PMC4917988.
17. Chen J, Jin S, Tahir SK, Zhang H, Liu X, Sarthy AV, McGonigal TP, Liu Z, Rosenberg SH, Ng SC. Survivin enhances Aurora-B kinase activity and localizes Aurora-B in human cells. J Biol Chem. 2003 Jan 3;278(1):486-90. doi: 10.1074/jbc.M211119200. Epub 2002 Nov 4. PMID: 12419797.
18. ^[1] Gassmann R, Carvalho A, Henzing AJ, Ruchaud S, Hudson DF, Honda R, Nigg EA, Gerloff DL, Earnshaw WC. Borealin: a novel chromosomal passenger required for stability of the bipolar mitotic spindle. J Cell Biol. 2004 Jul 19;166(2):179-91. doi: 10.1083/jcb.200404001. Epub 2004 Jul 12. PMID: 15249581; PMCID: PMC2172304.
19. Ibid.
20. Samejima K, Platani M, Wolny M, Ogawa H, Vargiu G, Knight PJ, Peckham M, Earnshaw WC. The Inner Centromere Protein (INCENP) Coil Is a Single α-Helix (SAH) Domain That Binds Directly to Microtubules and Is Important for Chromosome Passenger Complex (CPC) Localization and Function in Mitosis. J Biol Chem. 2015 Aug 28;290(35):21460-72. doi: 10.1074/jbc.M115.645317. Epub 2015 Jul 14. PMID: 26175154; PMCID: PMC4571873.
21. Nielsen CF, Zhang T, Barisic M, Kalitsis P, Hudson DF. Topoisomerase IIα is essential for maintenance of mitotic chromosome structure. Proc Natl Acad Sci U S A. 2020 Jun 2;117(22):12131-12142. doi: 10.1073/pnas.2001760117. Epub 2020 May 15. PMID: 32414923; PMCID: PMC7275761.
22. Ibid.
23. Roca J, Wang JC. DNA transport by a type II DNA topoisomerase: evidence in favor of a two-gate mechanism. Cell. 1994 May 20;77(4):609-16. doi: 10.1016/0092-8674(94)90222-4. PMID: 8187179.
24. Berger JM, Gamblin SJ, Harrison SC, Wang JC. Structure and mechanism of DNA topoisomerase II. Nature. 1996 Jan 18;379(6562):225-32. doi: 10.1038/379225a0. Erratum in: Nature 1996 Mar 14;380(6570):179. PMID: 8538787.
25. Champoux JJ. DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem. 2001;70:369-413. doi: 10.1146/annurev.biochem.70.1.369. PMID: 11395412.
26. Morrison C, Henzing AJ, Jensen ON, Osheroff N, Dodson H, Kandels-Lewis SE, Adams RR, Earnshaw WC. Proteomic analysis of human metaphase chromosomes reveals topoisomerase II alpha as an Aurora B substrate. Nucleic Acids Res. 2002 Dec 1;30(23):5318-27. doi: 10.1093/nar/gkf665. PMID: 12466558; PMCID: PMC137976.
27. Escargueil AE, Larsen AK. Mitosis-specific MPM-2 phosphorylation of DNA topoisomerase IIalpha is regulated directly by protein phosphatase 2A. Biochem J. 2007 Apr 15;403(2):235-42. doi: 10.1042/BJ20061460. PMID: 17212588; PMCID: PMC1874246.
28. Deming PB, Cistulli CA, Zhao H, Graves PR, Piwnica-Worms H, Paules RS, Downes CS, Kaufmann WK. The human decatenation checkpoint. Proc Natl Acad Sci U S A. 2001 Oct 9;98(21):12044-9. doi: 10.1073/pnas.221430898. Epub 2001 Oct 2. PMID: 11593014; PMCID: PMC59764.
29. Brown EJ, Baltimore D. ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev. 2000 Feb 15;14(4):397-402. PMID: 10691732; PMCID: PMC316378.
30. Brown EJ, Baltimore D. Essential and dispensable roles of ATR in cell cycle arrest and genome maintenance. Genes Dev. 2003 Mar 1;17(5):615-28. doi: 10.1101/gad.1067403. PMID: 12629044; PMCID: PMC196009.
31. Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge SJ. Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. Genes Dev. 2000 Jun 15;14(12):1448-59. PMID: 10859164; PMCID: PMC316686.