Ayraethazide

Biochemist. MSci at Gonville and Caius, University of Cambridge. Infographic designer. STEM education advocate.

[ayraethazide]

Subcellular mRNA localisation is desirable because not all transcripts are translated homogenously throughout the cell in which they are transcribed. Subcellular localisation allows the complex patterning of expression in specialised regions of the cell, as is evident in the hierarchical development of the Drosophila oocyte. Subcellular localisation of mRNA also means that the proteins that they encode are also localised - this in turn allows for complex patterning by combinatorial control of gene expression. Localisation is also regulatory: removal of ash1 mRNA from the mother cell into the daughter bud is required to relieve the repression on mating type switching machinery in S. cerevisiae. In multinucleate cells, subcellular localisation allows the localisation of some proteins around a particular cellular region where they are needed.

Mechanisms

Diffusion-entrapment works by allowing messenger ribonucleoprotein particles (mRNPs) diffuse freely around the cell. Already localised at a subcellular region is an anchor protein, which captures the mRNPs to be localised there. This is how Drosophila nanos mRNA localises at the posterior pole of the oocyte. Clearly though, this requires a mechanism for the anchor itself to be localised at that region.

Active transport works by mobilising mRNPs using the cytoskeleton. Transcripts that have zipcodes in the 3' UTRs can bind cytoskeletal motor proteins via one or more adaptor proteins and zipcode binding proteins (ZBPs). She2/3 is the adaptor for ash1 mRNA mobilisation on myosin. It requires the cell to be polarised.

Local degradation works by degrading the transcript everywhere in the cell, except for at its desired location. This is how Drosophila hsp83 mRNA is localised. The transcript needs two cis sequences to be recognised: one for protection within the protection zone, and another for degradation elsewhere. The trans-actors need to be pre-localised in the same manner for this to work.

Local synthesis works in multinucleate cells. The transcript is only expressed at the nuclei closest to the desired location. This means that the mRNP is already localised to the correct subcellular region when exported from the nucleus, and is not found elsewhere in the cell because it is not transcribed anywhere else. This is how synaptogenesis works: mRNA encoding the acetylcholine receptor is only transcribed on nuclei near the neuromuscular junction, and translation is therefore also localised there.

Further reading:

Parton, R.M.; Davidson, A.; Davis, I.; Weil, T.T. 2014. "Subcellular mRNA localisation at a glance." Journal of Cell Science 127:2127-2133.

Ayraethazide. 2015. "Patterning in Drosophila melanogaster." [Accessed 2016-03-26.]

Ayraethazide. 2015. "Saccharomyces cerevisiae mating type switching." [Accessed 2016-03-26.]

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what happens to nitrogen when the sun rises

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it becomes daytrogen

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I’m going to bed.

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good nitrogen

[faggot-about-it]

sleep tightrogen

[accentuateawesome-deactivated20]

don’t let the bed bugs bitrogen

[asapscience]

guys

[scienceshenanigans]

Is everything alrightrogen?

[sagansense]

I tried to resist sharing this but I couldn’t fightrogen…

[ayraethazide]

Examples of experimental models of human cancer and their use. Modelling human cancer in other organisms allows us to observe and monitor the effect of therapies against the cancer before actually administering it to patients. This is best done in animal models as their biology is most similar to humans'. Non-animal, eukaryotic models are still useful for studying oncogenic processes in pathways that are conserved between the model and humans.

Genetic strategies

Classical transgenic mice use a tissue-specific promoter to drive the expression of an gene. The aim is to express the gene only in a particular tissue, and see if it causes tumours to form. It should also identify driver and passenger mutations. The downfall of this strategy is that oncogenes usually acts to de-differentiate the cell, so that would switch off the promoter that is driving its expression, resulting in a self-inhibitory feedback loop. Classical knockout mice have one or both copies of a gene knocked out in the germline, so all cells have the knockout. This is good for studying sporadic versus familial loss of heterozygosity events, but there is no control over where and when that gene is deactivated.

Inducible systems include the tet on and tet off systems, where tetracycline/doxycyline results in the induction (tet on) or inhibition (tet off) of the transcription of a gene. In tet on, doxycyline binds a transactivator that drives transcription; in its absence, there is no transcription. In tet off, the transactivator binds the locus without doxycycline; in its presence, the transactivator dissociates from the gene.

Cre-lox systems work by recombining DNA across lox sites. Any sequences occurring in between the lox sites are said to be "floxed", and is removed upon Cre presence.

The ER-Tam system is a switchable system that works on the protein level. Proteins fused with the estrogen recetor (ER) are not functional unless tamoxifen is present. Tamoxifen can be removed to make the protein nonfunctional again.

in vivo models

Mouse models are widely used due to their evolutionary and genetic similarity to humans. Differences do exist, such as telomere length and mutation rate. They can be the recipient of xenografts and their cancers largely resemble human cancers.

Flies and yeast do not get cancer, but instead serve as models for oncogenic pathways. They can be subject to genetic and chemical screens to identify putative driver mutations causing pathway deregulation, and to identify drugs that can combat such mutations.

Culture

Cell cultures are generally not useful for monitoring tumours, because tumours are complex organs made of many cell types, and culture conditions usually maximise prolferation. Organoid culture allows growth of organs in 3D from a patient biopsy. This can be subjected to functional assays, drug screens, and genome editing for truly personalised therapy that is specific to the patient. However, this shares a common downfall with cell culture in that there is no contribution from the tumour microenvironment in a culture.

Further reading:

Sharpless, N.E.; DePinho, R.A. 2006. "The mighty mouse: genetically engineered mouse models in cancer drug development." Nature Reviews Drug Discovery 5 (9):741-754.

Vidal, M.; Cagan, R.L. 2006. "Drosophila models for cancer research." Current Opinion in Genetics & Development 16:10-16.

Xu, H.; Tomaszewski, J.M.; McKay, M.J. 2011. "Can corruption of chromosome cohesion create a conduit to cancer?" Nature Reviews Cancer 11 (3):199-210.

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Pseudomonas aeruginosa is an opportunistic human pathogen. It is found in the cystic fibrosis (CF) lung, where it exerts chronic infection, but can also cause acute infection leading to bacteremia, necrosis, and gangrene. P. aeruginosa employs many different strategies to regulate the chronic/acute switch, signal to its neighbours, kill off competing bacteria, and evade antibiotic action.

Intrinsic drug resistance

P. aeruginosa assumes multidrug resistance through its many resistance-nodulation-division (RND) pumps. RND pumps are broad-specificity efflux pumps which extrude antibacterial agents out of the cell. Their expression is regulated by an unknown signal, which dissociates NalC from the armR locus. ArmR inhibits MexR and dissociates it from the mexAB-oprM locus. The RND pump is then expressed.

Latent pumps are RND pumps which are insensitive to any signalling. An example is the mexCD-oprJ locus, which is held repressed by NfxB. Natural selection potentiates the MexCD-OprJ pump by loss-of-function mutation in NfxB.

Resistance genes can be switched on in response to the drug itself. Normal cell wall turnover naturally produces 1,6-anhydro-MurNAc cell wall fragments, which are processed and recycled by AmpD in the normal case. In the normal case, the β-lactamase gene ampC is repressed by AmpR. In the presence of β-lactam antibiotics, cell wall degradation increases and AmpD is overwhelmed by 1,6-anhydro-MurNac fragments. It cannot process them all, so they are allowed to bind AmpR, switching it from a transcriptional repressor to a transcriptional activator of ampC. The β-lactamase AmpC is expressed and degrades the β-lactam.

Iron homeostasis

P. aeruginosa chelate iron using siderophores. Siderophore biosynthesis clusters are regulated by the cellular concentration of iron. The ferric uptake regulator (Fur) acts as a sensor for this regulator. When cellular iron is high, Fur represses the transcription of the pyoverdine biosynthesis cluster σ factor PvdS. When cellular iron is low, Fur does not do this and the pvd cluster is expressed. Pyoverdine shuttles back and forth between the cell exterior and the periplasmic space, scavenging iron from its surroundings. Pyochelin moves between the cell exterior and the cytoplasm.

Secretion

Type III secretion is used to inject toxins into host cells during acute infection. Type VI secretion is used to wipe out the competition from other bacteria during a chronic infection.

Quorum sensing

P. aeruginosa possess three quorum sensing systems, which are interlinked with each other and to regulation of biofilm formation. las and rhl are most similar to the canonical lux pathway in that the autoinducer synthase (LasI/RhlI) produces the quorum sensing molecule which is bound by LasR/RhlR. The third quorum sensing system in P. aeruginosa is the Pseudomonas quinalone signal (PQS), which is synthesised along a biosynthetic cluster. The final precursor to PQS is called HHQ; the final step for HHQ conversion into PQS is catalysed by PqsH. HHQ is more diffusible and so works well as a quorum sensing molecule; PQS induces a larger downstream signal as it is the more tightly bound by PqsR. However, there is evidence that PQS itself is transmitted between cells by means of extracellular vesicles.

Activated LasR feeds into both rhlR and pqsH transcription to coordinate switching into biofilm production for a chronic infection. Activated RhlR switches on rhamnolipid biosynthesis genes (see below), among other targets; while activated PqsR turns on pqsE for increased virulence.

Biofilms and cystic fibrosis

P. aeruginosa attack the CF lung by attaching onto the lung epithelium and producing a thick biofilm. Biofilms confer intrinsic resistance to antimicrobials as their thickness retards their movement into lower layers, and nutrient-poor, excretion product-rich regions can act to antagonise their action. Additionally, biofilms can promote the differentiation of cells to take on a resistant phenotype.

Biofilms are formed by reversible and then irreversible attachment of P. aeruginosa onto the substratum, followed by formation of mushroom structures. Mushrooms rupture, causing massive dispersal of cells to nucleate a new biofilm at another site. Flagella- and type IV pili mutants are unable to form a biofilm: flagella are needed to swim to explore a new site and establish the mushroom stalk; pili are needed to twitch to establish the mushroom cap.

The formation of biofilms is regulated with the acute/chronic switch via a two-component signalling system. Input signals cause phosphorylation of membrane-bound GacS, which transfers it phosphoryl group onto diffusible GacR. Phospho-GacR turns on the genes for the small RNAs rsmX, Y, and Z. These small RNAs bind and inhibit pro-plankonic (acute infection) RsmA, repressing type III secretion system expression and promoting biofilm formation, chronic infection, and type VI secretion system expression.

GacS is regulated positively and negatively by the homologous proteins LadS and RetS respectively. LadS transfers a phosphoryl group onto GacS, thus activating the pathway, while RetS binds GacS as a dominant-negative. (GacS functions normally as a homodimer.)

A second input comes from cyclic di-GMP (c-di-GMP), which is created by WspSR and destroyed by SadSR two-component systems, whose input signals are unknown.

The GacS system feeds positively into the LasR system. This upregulates rhamnolipid biosynthesis via RhlR. Rhamnolipids are used as a biological surfactant, aiding sliding of cells to find new nucleation sites for biofilm formation. Rhamnolipids are also used as a biological detergent, keeping channels clear of dirt.

Further reading:

Mao, W.; Warren, M.S.; Black, D.S.; Satou, T.; Murata, T.; Nishino, T.; Gotoh, N.; Lomovskaya, O. 2002. "On the mechanism of substrate specificity by resistance nodulation division (RND)-type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition." Molecular Microbiology 46 (3):889-901.

Hassett, D.J.; Sokol, P.A.; Howell, M.L.; Ma, J.-F.; Schweizer, H.T.; Ochsner, U.; Vasil, M.L. 1996. "Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities." Journal of Bacteriology 178 (14):3996-4003.

van Delden, C.; Iglewski, B.H. 1998. "Cell-to-cell signaling and Pseudomonas aeruginosa infections" Emerging Infectious Diseases 4 (4):551-560.

de Kierit, T.R. 2009. "Quorum sensing in Pseudomonas aeruginosa biofilms." Environmental Microbiology 11 (2):279-288.

Klausen, M. et al. 2003. "Biofilm formation by Pseudomonas aeruginosa wild type, flagella and type IV pili mutants."Molecular Microbiology 48 (6):1511-1524.

Moscoso, J.A. et al. 2011. "The Pseudomonas aeruginosa sensor RetS switches Type III and Type VI secretion via c-di-GMP signallinge" Environmental Microbiology 13 (12):3128-3138.

Ayraethazide. 2015. “Export and secretion in gram-negative bacteria.”

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Oncogenes and tumour suppressors are mutation targets promoting the onset and maintenance of cancer. Oncogenic mutations result in gain-of-function and deregulation of the function of the oncoprotein that they encode. Tumour suppressors act to run quality checking of DNA, keep cell cycle checkpoints, and shut down mitogenic signals; mutations in genes encoding tumour suppressors can lead to absence of these checks and give activated oncoproteins the chance to run riot in a cell. Co-incidence of mutations in oncogenes and tumour suppressor genes potentially leads to cancer.

Oncogenes

Ras is a small G protein involved in a whole host of cellular functions. Mutation of Ras at a functional site can lead to a pleiotropic phenotype. Oncogenic Ras causes inappropriate signalling through its three pathways: MAPK, PI3K, and RalGEF. Signalling through the PI3K activates antiapoptotic Akt (PKB), which acts to promote cell survival. Signalling through RalGEF causes cell motility by formation of filopodia (Cdc42) and lamellipodia (Rac), which may be associated with metastasis. Signalling through MAPK actually causes the expression of some Ras signalling inhibitors (Sproutys, SPREDs, GAPs) which shuts down the signal in normal cells.

Myc is a transcription factor with more than 8000 transcription targets. Deregulated Myc leads to cell proliferation, but does not block apoptosis. Thus, it leads to a modest amount of growth before it is eradicated by apoptosis. Inhibition of apoptosis by antiapoptotic Bcl-xL is tumourigenic in cells expressing Myc highly. Additionally, Myc is thought to contribute to the tumour microenvironment, immune evasion, and inhibition of differentiation.

Ras and Myc work together by combining their abilities. Myc promotes cell proliferation and disfavours differentiation, and Ras inhibits apoptosis. The combination of the two means that cells are allowed to proliferate without triggering apoptosis. Ras actually activates Myc in normal cells - but in normal cells, activation is transient. Ras stabilises Myc by phosphorylation on S62 through MEK signalling, but also promotes its degradation by phosphorylation on T58 through PI3K signalling. The result is transient activation of Myc by Ras. Mutations which ultimately block phosphorylation at T58 will switch activation by Ras from a transient to a constitutive response.

Tumour suppressors

p53 is the so-called guardian of the genome. High Myc and oncogenic Ras cause stabilisation and activation of p53. p53 gets two bites at the cherry to combat the inheritance of damaged genomes: at the point of DNA damage, p53 arrests the cell until the DNA is repaired. p53 decides whether the cell enters senescence or apoptosis - its own state of post-translational modifications and the genomic context of its target genes (p53 is also a TF) on the genome in that particular cell both play a role in which way the scale tips. In this way, p53's second bite of the cherry is the selection of apoptosis in cells whose DNA is damaged beyond repair.

Rb is the keeper of the G1/S checkpoint. Loss of both copies of the Rb gene leads to retinoblastoma. Familial retinoblastoma predisposes heterozygotes with a heightened risk of retinoblastoma by loss of heterozygosity - loss of their only functional copy. This can occur by mutation, but also by mitotic recombination, gene conversion, and nondisjunction. Cells null for Rb can still enter G0 phase, as p107 and p130 share some redundant functions with Rb.

NF1 displays the phenomenon of haploinsufficiency. Nf1-/- Schwann cells can be complemented for the wild-type by Nf1+/+ mast cells, but not Nf1+/- mast cells. The former gives the wild-type; the latter causes neurofibromas.

VHL suppresses the hypoxic response in normoxia by mediating the ubiquitin-associated degradation of HIF-1α in normoxia. Loss of VHL leads to a hypoxic response no matter the oxygen level.

Further reading:

Hanahan, D.; Weinberg, R.A. 2011. "Hallmarks of cancer: The next generation." Cell 144:646-674.

Lowe, S.W.; Cepero, E.; Evan, G. 2004. "Intrinsic tumour suppression." Nature 432:307-315.

Pylayeva-Gupta, Y.; Grabocka, E.; Bar-Sagi, D. 2011. "RAS oncogenes: Weaving a tumorigenic web." Nature Reviews Cancer 11:761-774.

Soucek, L.; Evan, G.I. 2010. "The ups and downs of Myc biology." Current Opinion in Genetics and Development 20:91-95.

Vousden, K.H.; Prives, C.; 2009. "Blinded by the light: The growing complexity of p53." Cell 137:413-431.

Burkhart, D.L.; Sage, J. 2008. "Cellular mechanisms of tumour suppression by the retinoblastoma gene." Nature Reviews Cancer 8:671-682.

[ayraethazeigen]

Meine Freundin hat mir für Valentinstag Schokolade gegeben. ♥

[ayraethazide]

The regulation and stimulation of apoptosis. Apoptosis is driven by internal (intrinsic pathway) and external (extrinsic pathway) signals, and can also be induced by immune cells. Cells undergoing apoptosis tend to shrink, condense, and detach from surfaces including other cells. They develop blebs, fragment their nuclei and DNA (karyorrhexis), and recruit macrophages by displaying phosphatidylserine on their surface.

The extrinsic pathway funnels signals to the cell from its environmental context, while the intrinsic pathway tells the cell to die when it is under stress. Internal death stimuli include DNA damage, unfolded proteins, reactive oxygen species, oncogene activation, infection, toxic insult, and heat shock. Both pathways funnel through to the caspase proteins, which lie at the core of apoptosis by proteolytically cleaving their many targets to either deactivate them or give them specific apoptotic functions.

Caspases

The cysteine-dependent, aspartate-directed proteases (caspases) exist as zymogens when inactive. When conditions are favourable, (that is, during a death response,) procaspases dimerise and reciprocally activate by mutual proteolytic cleavage. The resulting caspases are then fully competent to cleave its downstream targets.

There are two types of caspase: initiators and executioners. Initiator caspases are the first ones to respond to the death signal, and their activity is to activate other caspases - both initiators and executioners. They contain CARDs (caspase recruitment domains) and DEDs (death execution domains). Executioner caspases have many downstream targets which are responsible for the morphological changes that apoptotic cells undergo. For example, executioner caspases can target cell-cell and cell-extracellular matrix contacts, leading to detachment; they can target ROCK, activating its function to increase actinomyosin force and leading to blebbing; they can cleave nuclear lamin B, leading to nuclear envelope breakdown; and they can destroy the inhibitor of caspase-activated DNase (iCAD), allowing the caspase-activated DNase (CAD) to perform karyorrhexis.

Extrinsic activation

Two pathways in extrinsic activation of apoptosis are through the Fas receptor and the TNFα receptor. Signalling through a Fas ligand-bound Fas receptor leads to recruitment of FADD on the cytosolic face. FADD docks onto the Fas receptor through homotypic interactions between death domains (DDs). FADD also contains a DED, which recruits and concentrates procaspase 8, thereby leading to a caspase cascade. The last step is inhibited by FLIP, which competes with procaspase 8 for procaspase 8 binding. It blocks the formation of procaspase dimers, and so blocks the caspase cascade.

Signalling through a TNFα-bound TNFα receptor leads to recruitment of TRADD and assembly of complex I on the cytosolic face - also through a series of DD homotypic binding interactions. This can go one of two ways: it can be internalised, leading to release of complex I and processing into complex II. Complex II then uses its DDs to recruit FADD, where signals intersect with the Fas pathway. Alternatively, the receptor does not internalise, and complex I leads to signalling through NF-κB, which signals for survival, not apoptosis. cIAP disfavours complex II formation and promotes complex I retention.

Intrinsic activation

The intrinsic pathway involves leakage of cytochrome c via mitochrondial outer membrane permeabilisation (MOMP), formation of the apoptosome, and concentration of procaspase 9. In response to intrinsic death stimuli, homodimers of Bax and of Bak form pores in the mitochrondrial outer membrane. Cytochrome c is then allowed to leak out of the mitochondria and bind Apaf1. In the absence of cytochrome c, Apaf1 autoinhibits by interaction between its WD40 domains. In the presence of cytochrome c, Apaf1 is allowed to oligomerise, forming the apoptosome. This interaction reveals the CARD on Apaf1, and recruits and concentrates procaspase 9, leading to a caspase cascade.

There is regulation at each of these steps. Bcl-2 is an antiapoptotic protein, which disfavours homodimerisation of Bax and of Bak, thereby preventing the formation of pores. It does this by favouring the conformation of Bax and of Bak where their BH3 domain is hidden - it is through the BH3 domain that Bax anad Bak homodimerise. XIAP is another antiapoptotic protein which binds and sequesters caspases through its BIR (bacculovirus inhibitor of apoptosis (IAP) repeat) domain. Through its RING domain, it can recruit an E3 ubiqitin ligase, and mark its binding partners for proteasomal destruction. Finally, proapoptotic Smac (second mitochondria-derived activator of caspases) and DIABLO (direct IAP-binding protein with low pI) bind XIAP on its BIR domains, thereby inactivating it.

Immune system

Both cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells can induce cell death, by apoptosis or otherwise. T cells recognise cells decorated with major histocompatibility complexes (MHCs) which display foreign antigens. T cells provide the Fas ligand to these cells, ensuing death. If the cell does not have a Fas receptor, the T cell can invoke death by perforin and granzymes - perforin punches holes in the cell membrane while granzymes induce a caspase cascade, or can alternatively kill the cell independently of apoptosis.

Natural killer cells recognise MHC class I proteins on the surfaces of cells. They also recognise activating ligands on the cell surface which act to promote perforin/granzyme-induced death. The activatory signal is suppressed if the cell bears an MHC class I protein which is recognised as 'self' - absence of this elicits death by perforin and granzymes.

This infographic looks like candy. Please do not try to eat it. Happy Valentine's Day (for tomorrow)!

Further reading

Riedl, S.J.; Shi, Y. 2004. "Molecular mechanisms of caspase regulation during apoptosis." Nature Reviews Molecular Cell Biology 5:897-907.

Trapani, J.A.; Smyth, M.J. 2002. "Functional significance of the perforin/granzyme cell death pathway." Nature Reviews Immunology 2:735-747.

Ljunggren, H.-G.; Malmberg, K.-J. 2007. "Prospects for the use of NK cells in immunotherapy of human cancer." Nature Reviews Immunology 7:329-339.

Holcik, M.; Korneluk, R.G. 2001. "XIAP, the guardian angel." Nature Reviews Molecular Cell Biology 2:550-556.

Ayraethazide. 2014. “Apoptosis”. [Accessed 2016-02-13.]

[ayraethazide]

DNA damage and repair are necessary mechanisms put in place by the cell to ensure genomic stability and inheritance of only undamaged DNA. Our DNA is constantly being challenged by spontaneous and environmental sources of damage, and multiple pathways are invoked by the cell to reverse the damage, if possible. If that is not possible, the cell can choose to become senescent or to undergo apoptosis.

Telomeres are at the ends of chromosomes. When not bound by a protein complex called shelterin, they cause cell senescence in a pathway that converges with the DNA damage response (DDR). Telomeres also get shorter with each DNA replication cycle; this gives a mechanism of cellular ageing and cancer control. Telomeres prevent cells from replicating from too many times, and when they reach their limit (the Hayflick limit), the cell exits from the cell cycle forever.

On a grander scale, nature has made DNA not invincible for a reason. Replication errors and environmental mutagenesis introduce the mutations required for evolution. If DNA was not susceptible to damage, it could never evolve.

Types of DNA damage and repair

Spontaneous sources of DNA damage are those that originate from within the cell: replication error, reactive oxygen species, or mutagenic metabolites. Base tautomerisation allows A-C base-pairs, and base deamination turns Cs into Us - or more dangerously, 5Me-Cs into Ts (5Me-Us). These are fixed by mismatch repair (MMR). Oxidative damage includes formation of abasic sites and single-strand breaks - these are fixed by base excision repair (BER).

Environmental damage comes from ultraviolet light, ionising radition, and chemicals. Ultraviolet light causes formation of adducts between bases, which can be reversed directly, or repaired by nucleotide excision repair (NER). Left unchecked, these adducts can cause helix distortions and can block a progressing DNA polymerase or RNA polymerase machine. In fact, NER is sometimes coupled to transcription; TFIIH has some activity in repairing DNA. Ionising radiation crosslink DNA or cause double- or single-strand breaks. Double-strand breaks are the most cytotoxic type of DNA damage to the cell as DNA ends are highly recombinagenic. They are repaired by homologous recombination (HR) or nonhomologous end joining (NHEJ). Alkylating agents add alkyl groups to DNA - these are usually fixed by direct reversal or BER. Crosslinking agents introduce intra- or interstrand crosslinks - repaired by NER or via the Fanconi anaemia pathway.

Double-strand break repair

Double-strand breaks are detected by the MRN complex or the Ku complex - the two are in competition. The MRN complex contains Mre11, Rad50, and Nbs1. The Ku complex contains Ku70 and Ku86. MRN activates ATM, which repairs the damage by HR through BRCA1/2 in S- or G2 phase, when a sister chromatid is present. If not, ATM repairs by NHEJ, which is also the favoured pathway of the Ku pathway. NHEJ is available at any time in the cell cycle.

Telomeres and the DNA damage response

Naked telomeric DNA is recognised as a double-strand break because there is no DNA past the very terminal base-pairs on the chromosome. If not for the shelterin complex, this would elicit the same DDR as for any double-strand break. In the absence of shelterin, DDR against telomere ends is constitutive, and eventually leads to cell cycle exit and senescence.

Shelterin, at its core, is composed of TRF1, TRF2, and POT1. The TRFs bind telomeric dsDNA while POT1 binds the ssDNA - that is, the 3' overhang. Loss of shelterin elicits the DDR. This happens naturally as telomeres get shorter, and the amount of bound shelterin decreases accordingly. Experimentally, it has been demonstrated that a dominant-negative allele of TRF2, which can assemble shelterin but cannot bind DNA, also causes DDR.

Telomeres get shorter with each replicative cycle. This gives an indication of cellular age. The molecular basis of telomere shortening is encapsulated in the end-replication problem. At a replication fork approaching the end of a chromosome, the leading strand can run cleanly off the end of the chromosome, but the lagging strand cannot fully replicate its template. This is because the final Okazaki fragment cannot be primed - even if the final RNA primer binds flush with the 3' end of the lagging strand template, there is no downstream 3'OH from which to elongate DNA into it. Thus, the lagging strand is shorter than the parental lagging strand template, which now features a 3' overhang.

Critically short telomeres inevitably trigger the DDR. Double-strand breaks are recognised by the MRN complex, which recruits ATM to the telomeres. ATM phosphorylates histone variant H2A.X on serine 139 - the histone is now called γH2A.X. γH2A.X recruits MDC1, which recruits more MRN; in this manner, the signal propagates along the telomere into subtelomeric regions. ATM activates checkpoint kinases which arrest the cell at cell cycle checkpoints until the DNA damage is repaired... But it can never be repaired, so the cell enters senescence.

Further reading:

Houtgraaf, J.H.; Versmissen, J.; van der Giessen, W.J. 2006. "A concise review of DNA damage checkpoints and repair in mammalian cells." Cardiovascular Revascularization Medicine 7:165-172.

Maser, R.S.; Monsen, K.J.; Nelms, B.E.; Petrini, J.H.J. 1997. "hMre11 and hRad50 nuclear foci are induced during the normal cellular response to DNA double-strand breaks." Molecular and Cellular Biology 17 (10):6087-6096.

d'Adda di Fagagna, F.; Reaper, P.M.; Clay-Farrace, L.; Fiegler, H.; Carr, P.; von Zglinichi, T.; Saretzki, G.; Carter, N.P.; Jackson, S.P. 2003. "A DNA damage checkpoint response in telomere-initiated senescence." Nature 423:194-198.

van Steensel, B.; Smogorzewska, A.; de Lange, T. 1998. "TRF2 protects human telomeres from end-to-end fusions." Cell 92:401-413.

de Lange, T. 2009. "How telomeres solve the end-protection problem." Science 326 (5955):948-952.

Fumagalli, M.; Rossiello, F.; Clerici, M.; Barozzi, S.; Cittaro, D.; Kaplunov, J.M.; Bucci, G.; Dobreva, M.; Matti, V.; Beausejour, C.M.; Herbig, U.; Longhese, M.P.; d'Adda di Fagagna, F. 2014. "Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation." Nature Cell Biology 14 (4):355-365.

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"DNA pre-replication" is supposed to refer to the reactions leading up to the formation of the pre-initiation complex (PIC) of DNA replication and the regulation therein. I debated for a while whether the title should instead read "Pre-replication of DNA" or "Pre-DNA replication", but I settled on this in the end.

Licensing reaction

Origin recognition complexes (ORCs) are present on origins of replication throughout the cell cycle, but pre-replicative complexes (pre-RCs) are only assembled during G1-phase. Assembly of the pre-RC gives the origin the credentials to initiate DNA replication (fire) from that origin. Pre-RCs are assembled by Cdt1 and Cdc6, which use ATP to load a double hexamer of the helicase MCM2-7. The pre-RC is visualised as a DNA footprint at the A, B1, and B2 consensus sites on origin sequences. When ORC is alone, the footprint is only at A.

Regulation of licensing

Licensing is only possible in G1-phase, when S-Cdk activity is low. Elevated S-Cdk activity in late G1 onwards is inhibitory on pre-RC formation: it places inhibitory phosphate groups on MCM2-7; targets Cdc6 for ubiquitination by SCF, and then degradation by the proteasome; and exports Cdt1 out of the nucleus. In addition to this, Geminin protein blocks the interaction between Cdc6/Cdt1 and MCM2-7, preventing licensing until mitotic exit, when Geminin is ubiquitinated by the APC/C. The multiplicity in these factors is essential for preventing re-replication and maintaining genomic stability in all cells, as errors in regulating even a single origin can be catastrophic for the cell.

Pre-initiation complex formation

When S-Cdk levels rise, licensing ends. Origins which already bear a replication licence (are loaded with a pre-RC) are processed by two kinases for binding more interacting partners. Dbf4-dependent kinase (DDK), comprised of Dbf4 and Cdc7, activates the MCM2-7 hexamers by phosphorylation. S-Cdk phosphorylates proteins Sld2 and Sld3, which recruit Dpb11, which in turn recruits Pol ε.

MCM2-7 is a 3'→5' ssDNA helicase, a behaviour which was elucidated in experiments in which artificial roadblocks were placed on single strands of DNA. MCM2-7 has a "cracked ring" structure, which is completed by its interacting partners Cdc45 and GINS. This complex is called the CMG (Cdc45/MCM2-7/GINS) complex, and offers a model of strand passage from dsDNA sliding to ssDNA processing. Sld3 binds MCM2-7 and Cdc45, while Sld2 binds GINS. In this way, Dpb11 and thus Pol ε are brought to the pre-initation complex (PIC).

Once or zero times per cell cycle

Origin firing occurs once or zero times per cell cycle. Late firing of an origin can be worse than not firing at all, and is in fact inhibited. Processing replication forks displace and push along pre-RCs on unfired origins, meaning that their chance of initiating is irrevocably lost. This is a vital step, as it prevents origins on replicated DNA from firing.

Pre-RC formation and origin firing are events separated by time. S-Cdk activity regulates the ordering of events and ensures that one follows the other - both cannot be allowed to occur at once. Low S-Cdk activity allows pre-RC assembly in G1-phase (recall that S-Cdk inhibits the licensing reaction); high S-Cdk activity allows PIC formation and origin firing in S-phase (recall that S-Cdk is needed for the recruitment of Pol ε).

Ultrasensitivity contributes to biphasic-like behaviour in PIC formation at the G1→S transition. Sic1 (an S-Cdki) is inhibited by phosphorylation; Sld2 is activated by phosphorylation. Both display ultrasensitivity to phosphorylation as they have many phosphorylation sites, but only a few confer function to the protein. Thus, as kinase activity rises, more and more "masquerade" "buffer" phosphorylation sites are being titrated, until eventually, a functional phosphorylation site is occupied. In the case of Sic1, superthreshold G1-Cdk levels inhibit its S-Cdki activity, and S-Cdk activity in the cell rises. In the case of Sld2, superthreshold S-Cdk levels cause proper folding of the protein for its interaction with GINS.

Further reading:

Arias, E.E.; Walter, J.C. 2007. "Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells." Genes & Development 21:497-518.

Labib, K. 2010. "How do Cdc7 and cyclin-dependent kinases trigger the initiation of chromosome replication in eukaryotic cells?" Genes & Development 24:1208-1219.

Bell, S.P.; Stillman, B. 1992. "ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex." Nature 357:128-134.

Diffley, J.F.X.; Cocker, J.H. 1992. "Protein-DNA interactions at a yeast replication origin." Nature 357:169-172.

Remus, D.; Beuron, F.; Tolun, G.; Griffith, J.D.; Morris, E.P.; Diffley, J.F.X. 2009. "Concerted loading of Mcm2–7 double hexamers around DNA during DNA replication origin licensing." Cell 139:719-730.

Fu, Y.V.; Yardimci, H.; Long, D.T.; Ho, T.V.; Guainazzi, A.; Bermudez, V.P.; Hurwitz, J.; van Oijen, A.; Schärer, O.D.; Walter, J.C. 2011. "Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase." Cell 143:931-941.

Costa, A.; Ilves, I.; Tamberg, N.; Petojevic, T.; Nogales, E.; Botchan, M.R.; Berger, J.M. 2011. "The structural basis for MCM2–7 helicase activation by GINS and Cdc45." Nature Structural and Molecular Biology 18 (4):471-477.

Ayraethazide. 2014. “DNA Replication.” [Accessed 2016-01-30].

[ayraethazide]

The spindle attachment checkpoint is a vital regulatory check in mitosis to ensure that daughter cells receive exactly one genome. It keeps the cell from moving from metaphase to anaphase until all kinetochores are attached onto the mitotic spindle. Clearly, disruption of the spindle attachment checkpoint would cause improper segregation of sister chromatids, and lead to genomic instability.

Coupling spindle attachment to sister chromatid resolution

Sister chromatid resolution is the first event that occurs after the checkpoint has been passed. Passing the checkpoint requires completion of spindle attachment. Unattached kinetochores are bound by Mad1 along with Mad2 in the closed conformation (cMad2). The cMad2:Mad1 complex catalyses the formation of the so-called mitotic checkpoint complex (MCC), which comprises of open-conformation Mad2 (oMad2), Mad3 (a.k.a. BubR1), and Cdc20. While Cdc20 is in the MCC, it cannot bind as a coactivator of the anaphase-promoting complex/cyclosome (APC/C). The APC/C is thus inactive, and cannot release separase from inhibition from securin. Separase cannot cause digestion of cohesin when inhibited, so sister chromatid resolution does not occur.

When the final kinetochore is bound, there are no remaining cMad2:Mad1 complexes on kinetochores. Cdc20 inhibition is relieved, and it activates APC/C. Activated APC/C attaches a K48 polyubiquitin tail onto securin, marking it for destruction by the proteasome. Loss of securin lifts the inhibition on separase, which then digests cohesin, freeing the sisters. The story is told in reverse in the infographic.

Structural biology of APC/C and coactivator regulation

The keeper of the checkpoint needs regulating so as to ensure abrupt, timely, and committed entry into anaphase. APC/C is activated by one of two possible coactivators: Cdc20 for entry into anaphase, and then Cdh1 for mitotic exit. The two coactivators also confer substrate specificity - clearly, the specificities are different for each coactivator.

Binding of a coactivator to the APC/C leads to completion of a bipartite destruction box receptor. This ensures that the APC/C cannot recognise, bind, and ligate ubiquitin onto destruction box-containing substrates (such as cyclin B) on its own. On top of this, binding of a coactivator to the APC/C also activates it through a series of conformational changes in APC2 and APC11 subunits. This brings the E2 ubiquitin-conjugating enzyme (charged with ubiquitin) closer to the substrate, making ubiquitin ligation possible.

The fact that coactivator binding is so instrumental for the APC/C's function means it is not surprising to find that Mad2 and Mad3 kill APC/C function by binding Cdc20. Mad3 binds the lysine/glutamate/asparagine (KEN) box receptor on Cdc20, meaning that KEN-containing substrates cannot bind. Mad2 binds the KILR motif on Cdc20, which is where it binds the APC/C. Recall that the MCC contains Mad2, Mad3, and Cdc20. Cdc20 is being inhibited by both of its binding partners; it is this double-hit action which means that alleviation of inhibition (i.e. completion of spindle attachment) leads to an abrupt increase in APC/C action and sister chromatid resolution.

Panels describing background to ubiquitination and RING E3 ubiquitin ligases are also offered in the infographic.

Further reading:

Lara-Gondalez, P.; Westhorpe, F.G.; Taylor, S.S. 2012. "The spindle assembly checkpoint." Current Biology 22:R966-R980.

Duda, D.M.; Borg, L.A.; Scott, D.C.; Hunt, H.W.; Hammel, M.; Schulman, B.A. 2008. "Structural insights into NEDD8 activation of cullin-RING ligases: Conformational control of conjugation." Cell 134:995-1006.

Chang, L.; Zhang, Z.; Yang, J.; McLaughlin, S.H.; Barford, D. 2014. "Molecular architecture and mechanism of the anaphase-promoting complex." Nature 513:388-393.

Chao, W.; Kulkarni, K.; Zhang, Z.; Kong, E.H.; Barford, D. 2012. "Structure of the mitotic checkpoint complex." Nature 484:208-213.

Ayraethazide. 2015. “G2→M”.

[theverge]

Scientists revive microscopic water bears after 30 years of deep freeze    

These eight-legged, microscopic creatures are probably the hardiest animals in existence, able to live through extreme heat, cold, pressure, radiation, and even the vacuum of space. Now, these water-dwelling critters have set a new personal best, with cryobiologists from Japan successfully reviving a tardigrade after it had been frozen for more than 30 years. What’s more, the defrosted creatures even managed to reproduce; with one laying 19 eggs of which 14 successfully hatched.

[currentsinbiology]

Scientists discover why X chromosome lacks ‘housekeeping genes’

Men have one copy, women have two, but scientists have long puzzled over why the human X chromosome mostly contains genes that are active in a small number of tissues. Now, a team of researchers led by the University of Bath studying the evolution of this X chromosome has discovered why it contains such an unusual mixture of genes.

In humans, males have XY chromosomes, females have XX but only one of these is active, meaning that both sexes only have one active copy of the X chromosome.

Scientists discovered in 2002 that the X chromosome is unusual because it contains very few of the most important genes needed for basic cell function - dubbed “housekeeping” genes.

Now the team, a collaboration between researchers at the University of Bath and Uppsala University, along with members of the FANTOM consortium, have found out why.

They analysed the world’s largest compendium of data on gene activity (expression) and looked at how activity on the X chromosome compares with that on other chromosomes.

In a paper published in the journal PLoS Biology, they found that the peak level of gene expression on the X chromosome was under half that of other chromosomes where we have two functioning copies.

He explained: “Since we showed that X-linked genes tend to be relatively tissue specific over a decade ago, the reason as to why the X chromosome is so odd has bugged me.

"In the end, we have found the answer to be quite simple. Whereas most chromosomes operate in pairs, meaning there are two copies of each gene in every cell, in contrast, we only have one active copy of the X chromosome.

"This means it is not sustainable for highly active genes to be on the X chromosome. Housekeeping genes tend also to be highly active - they just couldn’t survive on the X.”

Read more about this research: https://theconversation.com/why-is-the-x-chromosome-so-odd-traffic-analogy-helped-us-crack-the-mystery-51942

[ayraethazide]

Translation initiation and reinitiation are achieved by cap-dependent and cap-independent manners. Cap-dependent initiation requires all the eukaryotic initiation factors (eIFs), an initiator Met-tRNAi, the poly(A) binding protein (PABP), and both the 40S and 60S ribosomal subunits. Ribosomes can initiate at internal ribosome entry sites (IRESs) which do not depend on the 5' cap. Reinitiation is possible after translation of a short upstream ORF (sORF or uORF) providing that eIF4F participated in the initial initiation of the uORF.

Cap-dependent initiation relies on recognition of the initiation codon AUG. More than that, local flanking sequence, the Kozak sequence, plays an important role in recognition of the initiation codon. A strong enough Kozak sequence can initiate from near-cognate initiation codons. Members of the 43S pre-initiation complex (PIC) recognise the initiation codon, deliver the initiator tRNA to the initiation codon, and bind the 40S ribosomal subunit - eIF1/1A, eIF2 ternary complex (TC), and eIF3 respectively. Scanning is dependent on the eIF4F complex, whose member eIF4A contains an ATP-dependent bidirectional helicase. The helicase unwinds secondary structures, helping eIF1/1A to scan for the AUG. It stands to reason that eIF1/1A can scan without eIF4A if the 5' leader is completely unstructured. Other members of the eIF4F complex cap-binding protein eIF4E and scaffold protein eIF4G. Once the recognition event has occured, eIF5 hydrolyses GTP, eIF2, -1, -1A, and 3 dissociate, and eIF5B recruits the 60S ribosomal subunit. The elongation phase is entered.

Internal ribosome entry occurs at IRESs. Picornaviruses do not require eIF4E and may possess proteases against eIF4G to prevent it from associating with host eIF4E, thereby blocking host translation. Classical swine fever virus (CSFV) do not require eIF4F, eIF1, or eIF1A. Cricket paralysis virus (CrPV) does not require any initiation factors, but bizzarely begins translation from elongation. CrPV IRES pseudoknot 1 contains a mimic codon/anticodon pair, which inserts in the ribosomal P-site. The next codon in the A-site is translated, and then a pseudotranslocation event occurs. From there, translation is productive as normal!

Reinitiation refers to re-scanning for an AUG following termination from a uORF. Reinitiation is better when uORF translation time is short - this means on shorter uORFs and more unstructured regions. Termination from a uORF leaves behind the 48S scanning complex; reinitiation only works if the complex can regain a eIF2 TC and a 60S ribosomal subunit. Reinitiation does not work if eIF4F did not participate in the initial initiation at the uORF - so that means there is no reinitiation following CrPV or CSFV IRESs.

Further reading:

Hinnesbusch, A.G. 2014. "The scanning mechanism of eukaryotic translation initiation." Annual Review of Biochemistry 83:779-812.

Jackson, R.J. 2005. "Alternative mechanisms of initiating translation of mammalian mRNAs." Biochemical Society Transactions 33 (6):1231-1241.

Pöyry, T.A.A.; Kaminski, A.; Jackson, R.J. 2004. "What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame?" Genes & Development 18:62-75.

Ayraethazide. 2015. "Translation elongation." [Accessed 2016-01-13.]

[ayraethazide]

Examples of translational expression regulation. Gene expression on the level of translation refers to modulation of the rate of translation of transcripts into protein. Illustrated are cap-dependent and cap-independent modes of such regulation in iron homeostasis; and a well-studied reversible mode of regulation in Xenopus laevis development. Also described is the current closed loop model to account for synergistic and differential interactions between the 5' cap and regions in the 3' untranslated region (UTR).

Iron homeostasis

Two proteins that control cellular iron concentration are ferritin and the transferrin receptor (TfR). Ferritin sequesters iron and lowers its concentration; TfR promotes uptake of iron and raises its concentration. Both ferritin and TfR mRNA transcripts are regulated by the iron response proteins (IRPs) 1 and 2. IRP1 has aconitase activity in iron-replete cells because its cubic Fe-S cluster is complete. In iron-deplete cells, IRP1 lacks its Fe-S cluster and behaves as an RNA-binding protein instead. IRP2 has no aconitase activity.

In the iron-depleted state, IRP1/2 bind iron response elements (IREs). On the ferritin transcript, IREs are 5' cap-proximal, and binding causes repression of translation, leading to less ferritin in the cell, less iron sequestration, and increased iron concentration. It has been shown that the binding interaction is sufficient for the effect: U1A protein bound to MS2 stem-loops in a tether function assay replicates this effect. What is happening is that the interaction prevents the 43S ribosomal complex from being recruited.

On the TfR transcript, IREs are on the 3' UTR. Binding of IRP1/2 causes decreased 3' to 5' degradation of the mRNA, meaning that more TfR is translated, leading to more iron uptake. In the iron-replete state, neither transcript is bound by IRP1/2 because IRP1, with its completed cubic Fe-S cluster, now acts as an aconitase instead.

Xenopus early translation

A phenomenon observed in Xenopus laevis is that polyadenylation can occur in the cytoplasm. GLD2 catalyses this reaction, and requires the cleavage and polyadenylation specificity factor (CPSF) and cytoplasmic polyadenylation element binding proteins (CPEBs) bound to mRNA at cytoplasmic polyadenylation elements (CPEs). The canonical polyadenylation hexanucleotide (Hex) AAUAAA is also needed. There also exists a poly(A) ribonuclease (PARN), which acts to shorten poly(A) tails. Prior to germinal vesicle breakdown (GVBD), PARN is trapped in the germinal vesicle (nucleus) and cannot act on cytosolic poly(A) tails.

Prior to GVBD, GLD2 stimulates translation by lengthening the poly(A) tails. The translation rate of actin, tubulin, and ribosomal proteins is high. Following GVBD, PARN becomes cytosolic, poly(A) tails shorten, and translation rates fall. Experiments with luciferase fused to the cyclin B1 3' UTR have shown that it is the sequences in the 3' UTR which are necessary and sufficient for this type of regulation.

Repression by PARN or the eIF4E transporter (4E-T) (repressive closed loops, see below) can be reversed by phosphorylation. Phosphorylation of PARN by Aurora A causes its dissociation; GLD2 can then re-elongate the poly(A) tail and enhance translation once more. Phosphorylation of 4E-T by Cdk1 causes its dissociation from eIF4E, allowing a productive closed loop (see below) to form and translation to increase.

Closed loop model

The closed loop model is the current model for 5' cap/poly(A) tail synergy and has been visualised by atomic force microscopy. Proteins bridge between the 5' cap and poly(A) tail - traditionally, these are eIF4E, eIF4G, and PABP, in that order. eIF4E binds the 5' cap, PABP binds poly(A), and eIF4G acts as a scaffold between the two (as well as others such as eIF3). Depletion of eIF4E or PABP causes massive decrease in translation rate.

Rotavirus protein NSP3 competes with PABP for eIF4E binding, but binds UGACC rather than poly(A). The result is that viral mRNA is translated efficiently, and host mRNA is knocked down considerably: viral transcripts contain UGACC in their 3' UTRs and thus form a closed loop, whereas host transcripts do not.

Exclusion of PABP from the closed loop can also cause a repressive closed loop. The XYZ model of cap/tail bridging is observed in Xenopus (above) and Drosophila and appears to repress translation of the transcript.

Further reading:

Abaza, A.; Gebauer, F. 2008. "Trading translation with RNA-binding proteins." RNA 14:404-409.

Rouault, T.A. 2006. "The role of iron regulatory proteins in mammalian iron homeostasis and disease." Nature Chemical Biology 2 (8):406-414.

Radford, H.E.; Meijer, H.A.; de Moor, C.H. 2008. "Translational control by cytoplasmic polyadenylation in Xenopus oocytes." Biochimica et Biophysica Acta 1779:217-229.

Barkoff, A.F.; Dickson, K.S.; Gray, N.K.; Wickens, M. 2000. "Translational control of cyclin B1 mRNA during meiotic maturation: Coordinated repression and cytoplasmic polyadenylation." Developmental Biology 220:97-109.

Kahvejian, A.; Svitkin, Y.V.; Sukarieh, R.; M'Boutchou, M.-N.; Sonenberg, N. 2005. "Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms." Genes & Development 19:104-113.

Wells, S.E.; Hillner, P.E.; Vale, R.D.; Sachs, A.B. 1998. "Circularization of mRNA by eukaryotic translation initiation factors." Molecular Cell 2:135-140.

Piron, M.; Vende, P.; Cohen, J.; Poncet, D. 1998. "Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F." EMBO Journal 17 (19):5811-5821.

Jackson, R.J. Hellen, C.U.T.; Pestova, T.V. 2010. "The mechanism of eukaryotic translation initiation and principles of its regulation." Nature Reviews Molecular Cell Biology 10:113-127.

[sixpenceee]

These cartoons depict the relative scale of things. 

[ayraethazide]

An epigenetic histone modification associated with heterochromatic state of nuclear chromatin is methyl-lysine 9 on histone H3 (H3K9Me). Certain proteins can recognise dimethyl-lysine via their chromodomains and recruit effectors to the chromatin via their shadow chromodomains. Heterochromatin protein 1 (HP1), known as mouse chromatin modifier protein 1 (MoMOD1) in mice and Swi6 in yeast, is one such protein, and is thought to cause the heterochromatic state through compaction of polynucleosomes and recruitment of interacting partners. Genes within heterochromatin are repressed - this is a gene expression regulation feature that is unique to eukaryotes.

Structure and sequence

Chromodomains and shadow chromodomains resemble each other in structure and sequence. They form an N-terminal three-strand antiparallel β-sheet overlaid by C-terminal α-helix. In shadow chromodomains, the residues I161, Y164, and W170 show elevated levels of conservation compared to the equivalent positions in the chromodomain - this is probably due to retain the dimerisation function of the C-terminal region, where those residues lie.

One monomer of HP1 contains an N-terminal chromodomain and a C-terminal shadow chromodomain. HP1 exists in vivo as a homodimer. This means that one homodimer of HP1 contains two chromodomains and two shadow chromodomains. The dimerisation interface is between the two shadow chromodomains at the unstructured C-terminus and α-helix. N-termini are also unstructured, which gives the overall structure a high degree of structural flexibility.

Interaction of the shadow chromodomain

Through a variety of GST-pulldown assays and LexA/VP16 two-hybrid assays of MoMOD1 with CAF-1, the minimal interacting region (MIR) was identified. This contains the motif PxVxL. NMR structural studies show that proteins bearing the PxVxL bind HP1 at the dimerisation interface between the two shadow chromodomains - explaining why monomers of MoMOD1 did not display binding with CAF-1. Binding creates an unorthadox three-standed β-sheet with the unstructured C-terminal regions on each of the shadow chromodomains; formally, it is two separate β-sheets: one parallel, and one antiparallel.

Interaction of the chromodomain

Structural insight elucidated that the all-important H3K9Me2 epigenetic mark is bound by a hydrophobic groove in the chromodomain through a combination of hydrophobic and π aromatic interactions. Mutation at Y21, W42, or F45 reduce these interactions. Binding is specific to substrates with the tetrapeptide Q5, A7, R8, K9 - histone H3 has QTARK. Mutation in this region extinguishes binding of chromodomain substrates.

Compaction of chromatin

HP1 dimerises via shadow chromodomains. HP1 dimers therefore have two chromodomains, which can each bind a different H3K9Me2 mark on two different nucleosomes. Each nucleosome has two H3 histones, and if one HP1 dimer binds only once to a single nucleosome, then this gives a mechanism for heterochromatin spreading by 'sticky ends'. Swi6 has been shown to bind longer polynucleosomes with greater specificity than shorter polynucleosomes. In 1N and 2N stretches, Swi6 bound nonspecifically to linker DNA and not histone H3. HP1 binding the same nucleosome twice is futile and is not compatible with the sticky end mechanism.

Further reading:

Ball, L.J.; Murzina, N.V.; Broadhurst, R.W.; Raine, A.R.C.; Archer, S.J.; Stott, F.J.; Murzin, A.G.; Singh, P.B.; Domaille, P.J.; Laue, E.D. 1997. "Structure of the chromatin binding (chromo) domain from mouse modifier protein 1." EMBO Journal 16:2473-2481.

Smothers, J.F.; Henikoff, S. 2000. "The HP1 chromo shadow domain binds a consensus peptide pentamer." Current Biology 10:27-30.

Thiru, A.; Nietlispach, D.; Mott, H.R.; Okuwaki, M.; Lyon, D.; Nielsen, P.R.; Hirshberg, M.; Verreault, A.; Murzina, N.V.; Laue, E.D. 2004. "Structural basis of HP1/PXVXL motif peptide interactions and HP1 localisation to heterochromatin." EMBO Journal 23:489-499.

Nielsen, P.R.; Nietlispach, D.; Mott, H.R.; Callaghan, J.; Bannister, A.; Kouzarides, T.; Murzin, A.G.; Murzina, N.; Laue, E.D. 2002. "Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9." Nature 416:103-107.

Canzio, D.; Chang, E.Y.; Shankar, S.; Kuchenbecker, K.M.; Simon, M.D.; Madhani, H.D.; Narlikar, G.J.; Al-Sady, B. 2011. "Chromodomain-mediated oligomerization of HP1 suggests a nucleosome-bridging mechanism for heterochromatin assembly." Molecular Cell 47:67-81.

[ayraethazide]

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