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Cell Cycle 10:14, 2248-2254; July 15, 2011; © 2011 Landes Bioscience

CeLL CyCLe news & views

2248 Cell Cycle volume 10 issue 14

in a recent issue of Cell Cycle, wei et al.1 dis-played a novel and exciting tool to study the effect of mutant p53 variants in stem cells with genetically a uniform background. Although presented in the context of p53 in mice, this new approach could be applied to any mutated gene at large.

p53 is a tumor suppressor gene that orchestrates cellular responses to various stress insults.2 The loss of p53 is often coupled with the appearance of a mutated form, which, as opposed to other tumor suppressor genes, takes on new oncogenic roles known as “gain-of-function.”3 Furthermore, epidemiological studies have identified dozens of mutant p53 forms, implying that each of these mutants has a unique biological significance and may project on diagnosis and prognosis.

studying the function of p53 and its mutants mostly relied upon in vitro studies as well as on mice models. The first p53-knockout mice have indicated that p53 functions as a tumor suppressor gene.4 This was followed by studies utilizing transgenic mice expressing different mutant p53 forms, which led to several fasci-nating discoveries regarding the physiological functions of p53 (reviewed in Donehower and Lozano).5 For example, Malkin et. al. noticed that p53-null mice develop the same tumor spectrum exhibited by Li-Fraumnei patients, who develop sporadic cancers throughout their lives. indeed, a significant proportion of these patients carried a germ-line mutation in their p53 allele.6 Although initially overlooked, the emerging role for p53 in development and differentiation was first discovered in a p53-null mouse model, as 20–30% of the female embryos exhibited a brain developmental defect.7 Another important tool was the gen-erations of mice expressing a “humanized” p53 knock-in (Hupki) protein, in which the p53-DnA binding domain was replaced by the cor-responding human sequence, thus increasing the authenticity of the mouse model.8 A more recent development in the mutant p53 field

Cell Cycle News & Views

Same stem cells but different: Isogenic embryonic stem cells harboring different p53 mutantsComment on: Wei QX, et al. Cell Cycle 2011; 10:1261–70Shalom Madar†, Hilla Solomon† and Varda Rotter*; Weizmann Institute of Science; Rehovot, Israel; †These authors contributed equally; *Email: [email protected]; DOI: 10.4161/cc.10.14.15933

is the establishment of mutant p53 knock-in human cell lines.9

studying the effect of any mutant form is quite an elaborate task that encompasses sev-eral challenges, among them are the need for a uniform genetic background and the capac-ity to produce mutant variants in a timely and effective manner. Thus, although the above mentioned approaches significantly extended our knowledge as to the expression and behavior of numerous p53 mutants, additional refinements are required.

in their recent work, wei et al. managed to generate an experimental setup that suc-cessfully deals with the aforementioned chal-lenges.1 This setup consists of three principal steps, as follows (also depicted in Fig. 1). First, by homologous recombination, a mouse strain is generated that expresses a drug resistance sequence instead of exons 2–9 of the p53 locus, giving rise to a p53-null mouse. The “null” region is flanked by specific sites that

serve as a platform to receive other expression cassettes.

An important issue that must be prop-erly addressed is the assessment of insertion efficiency. To achieve this, the authors used a cassette with a promoter-less marker on one direction and a resistance sequence on the other. Cells that receive the cassette in the proper platform site will express the marker (under p53 promoter) as well as the resistance, while a non-random integration will result in drug resistance alone. The cells dramatically favored an insertion into the docking position compared to random events (~80% and ~1%, respectively). The third and last step is to intro-duce Hupki-based, p53-mutated plasmids8

harboring matching integrating sites and a plasmid encoding the integrase enzyme into the platform-bearing mouse cells. The authors went on to show a high yield of positive clones, which were further expanded in culture and validated for p53 expression and function.

Figure 1. A schematic representation of the experimental setup. (1) A p53-‘null’ cassette flanked with ‘docking’ sites (purple squares) was introduced into a wild-type mouse by homologous recombination (green squares) and, following proper breeding, gave rise to a platform-carrying mouse. (2) To assess the efficiency of the insertion, a Red Fluorescent Protein (RFP) and resistant drug in the opposite orientation were introduced to the cells. (3) A plasmid-containing mutated p53 sequence was integrated into the platform-carrying mouse.


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Mutations within the TP53 locus, which encodes the important tumor suppressor p53, are found in the majority of human cancers. The wide variety of TP53 mutations that are seen in human cancers can cause a range of effects, not simply through loss of normal p53 function, but also via gain of function.1 Mutant p53 may aberrantly interact with a wide range of different proteins, and its high levels of expression within tumor tissue can result in a cancer-promoting action.

Both constitutive and conditional p53 knockouts have been exceptionally useful in elucidating the tumor suppressive function of normal p53, but they are not truly reflec-tive of human p53 mutations. Humanized p53 knock-in (Hupki) mice, on the other hand, are invaluable in the study of mutations arising from exposure to a variety of carcinogens,2 and Hupki mutant strains have been impor-tant in increasing our understanding of the gain-of-function properties of some mutant p53 variants.3

The wealth of knowledge regarding the function of p53 and many of its mutant forms has only been made possible by the range of p53 models available. The production of new mutants, however, is extremely labor intensive, imposing a limiting factor on even the most useful models.4

One approach that potentially permits the rapid generation of a range of novel alleles at a given locus is the use of recombinase-mediated

Expanding the p53 toolkit: A new platform for rapid engineering of the p53 locus in vivoComment on: Wei QX, et al. Cell Cycle 2011; 10:1261–70Madeleine Young and Alan R. Clarke*; Cardiff University; Wales, UK; *Email: [email protected]; DOI: 10.4161/cc.10.14.16205

cassette exchange (RMCe), a method by which DnA cassettes can be introduced into genomic DnA at specific loci engineered to contain “docking sites.” For example, FLP recombi-nase derived from Sacharomyces cerevisia can be used to introduce DnA cassettes into FRT docking sites,5 whereas more efficient recombination can be performed using the P1-bacteriophage-derived Cre recombinase to act on LoxP docking sites.6 The real disadvan-tage of RMCe is that it leaves the docking sites unaltered after recombinase action, thereby permitting further recombination. This has led to the predominant use of this technique for DnA excision.

The problem of reversibility has been over-come by the use of iMCe, integrase-mediated cassette exchange. Using PhiC31 integrase from Streptomyces lividans, iMCe functions in a similar way to RMCe but results in altered docking sites, preventing further activity.7 iMCe enables sections of genomic DnA flanked by att recognition sites to be replaced by any newly introduced sequences that are also flanked by the specific att sites. This method has been used to enable site-specific insertions into the mouse genome and to produce transgenic animals using the embryonic stem (es) cell modification and blastocyst injection system.8

Hollstein et al. recently described in Cell Cycle,9 the generation of a new p53 mouse strain called p53 PLF (Platform) mice, which contain the att sites and so can be used to

rapidly engineer the p53 locus through the use of iMCe. The PLF mouse was created by recom-bination between sequences of murine es genomic DnA at the Trp53 locus and homolo-gous sequences in an introduced targeting construct. The targeting construct replaced exons 2–9 of Trp53 with a drug cassette con-ferring neomycin resistance to the successfully transformed es cells flanked by specific att sites. The removal of exons 2–9 renders the allele null, and the att sites now provide a plat-form for replacement by any new mutated p53 sequences (which are also flanked by att sites) mediated by PhiC31 integrase.

PLF mice were generated from the PLF es cell line by blastocyst injection followed by generation and breeding of chimeric mice. Using p53 PLF es cell lines generated from these PLF mice, the efficiency of iMCe was shown to be high using an indicator vector, with > 60% of integrations shown to be specific.

The high efficiency of iMCe enabled the easy generation of a range of cell lines with genetically uniform backgrounds expressing different mutations of p53 under the control of the endogenous Trp53 promoter. The authors are currently attempting to generate mice from some of these cell lines to assess the ease by which this method can be used to produce a range of p53 mutants.

The new model described by Holstein et al., enables the easy, quick and cheap genera-tion of cell lines and, potentially, mice that

so what could be the ramifications of this newly reported method? First, the human p53 mutant-expressing pluripotent cells share a common genetic background, therefore can be used to generate corresponding mice that can be further exploited in an in vivo model. it should be noted that hitherto the “gain-of-function” of various forms of mutant p53 was mainly studied in somatic cells. The availability of stem cells expressing differ-ent mutant p53 provides a new model that recapitulates the formation of cancer stem cells and may improve our understanding of cancer initiation. indeed, it was recently shown that p53 and its mutated forms affect

the reprogramming of embryonic cells.10-11 Another emerging research field that might benefit from this newly presented work is the involvement of mutant p53 in the tumor stroma crosstalk.12 Finally, the most copious task is to unravel the clinical implications of mutant p53, which calls for improved strate-gies for generating endogenously expressing mutant p53 mice and cell lines.

The work by wei et. al. could cer-tainly benefit these research challenges by providing a highly efficient, inexpen-sive and rapid technique, one that could be employed in the research of any given mutant.

References1. Quan-Xiang wei AFO, et al. Cell Cycle 2011;

10:1261-70.2. vousden KH, et al. nat Rev Mol Cell Biol 2007;

8:275-83.3. Oren M, et al. Cold spring Harb Perspect Biol 2010;

2:a001107.4. Donehower LA, et al. nature 1992; 356:215-21.5. Donehower LA, et al. nat Rev Cancer 2009; 9:831-41.6. Malkin D, et al. science 1990; 250:1233-8.7. sah vP, et al. nature genetics 1995; 10:175-80.8. Luo JL, et al. Oncogene 2001; 20:320-8.9. Rago C, et al. nat Protoc 2007; 2:2734-46.10. Deng w, et al. Trends Genet 2009; 25:425-7.11. sarig R, et al. J exp Med 2010; 207:2127-40.12. Bar J, et al. semin Cell Dev Biol 2010; 21:47-54.13. wei QX, et al. Cell Cycle 2011; 10:1261-70.



and angiogenesis. A few studies have shown that transplantation of bone marrow-derived mononuclear cells (BMC) may be useful in the context of experimental T. cruzi infection,4 and limited clinical observations show that humans may also benefit from BMC trans-plantation.6 Despite the clear limitation of rodent models in mimicking the full extent of the chronic human disease, it appears that BMC transplantation was beneficial even many months after infection in mice, a time at which fibrosis had already been established.4 These results suggest that BMC therapy may be use-ful in the context of Chagas disease, and that clear understanding of how stem cells func-tion to ameliorate heart fibrosis and function is necessary.

in a previous issue of Cell Cycle, soares et al.7 found that BMC transplantation at the chronic stage of T. cruzi infection had a major effect on the expression profile of genes in the heart of infected mice. indeed, over 90% of genes that were altered by infection and chronic disease returned to baseline expres-sion. This change in expression profile was translated into improvement of inflammation and tissue fibrosis but no detectable changes in angiogenesis. The extent of gene repro-gramming incurred after bone marrow trans-plant was indeed very marked, highlighting the potential of this strategy in resetting gene expression to previous levels. several ques-tions remain and should guide future work:

what are the trafficking signals for BMC to enter fibrotic tissues, and how many cells are necessary for effects to be observed? Does reprogramming rely on migrated BMC, or is their systemic presence sufficient? what is the molecular mechanism of gene reprogram-ming? is the ability of BMC transplantation to cause genetic reprogramming a feature of Chagas disease, or will it occur in other kinds of heart diseases, such as after infarc-tion? How long-lasting is reprogramming? in other words, does this strategy reset the gene expression profile to that found in healthy organs for long periods, or are the effects tran-sient and in need of repetitive administration? in the context of Chagas disease, is antimi-crobial treatment necessary for any sustained effect? There is an ongoing trial to investi-gate the potential usefulness of BMC therapy in the context of Chagas disease.5 However, answering the questions above should lead to optimal use of BMC therapy in Chagas and potentially other fibrotic diseases of the heart and other tissues.

References1. Dutra wO, et al. Trends Parasitol 2005; 21:581-7.2. Machado Fs, et al. Br J Pharmacol 2010; 160:258-9.3. Ou L, et al. Open Cardiovasc Med J 2010; 4:231-9.4. soares MB, et al. Am J Pathol 2004; 164:441-7.5. Tura BR, et al. Trials 2007; 8:2.6. vilas-Boas F, et al. Arq Bras Cardiol 2011; 96:1-52.7. soares MB, et al. Cell Cycle 2011; 10:1448-55.

Chagas disease is caused by infection with the protozoan parasite Trypanosoma cruzi. Chagas disease does not usually cure spon-taneously, but most patients undergo a silent phase, known as indeterminate form, in which chronic infection is not associated with clinical disease. However, in a significant proportion of cases (up to 30%), infection causes heart disease that can be very severe. The major pathological finding in the heart of infected individuals is that of a chronic progressive and fibrosing myocarditis, which causes two major clinical syndromes: heart failure and complex arrhythmias (Dutra et al., 2005). vaccines are not available, and specific treatment with anti-parasite drugs is recommended during acute infection and is likely to be useful in patients with the indeterminate form of the disease. in patients with severe heart disease, medical treatment of heart failure and arrhythmia is necessary, and heart transplant may be carried out as a last resort.2

several studies have now shown that stem cells may differentiate into specialized cell types and/or exert paracrine actions that exert regenerative properties and repair tissue dam-age. These studies have fostered the investiga-tion of cell transplantation as a possible therapy to improve cardiac function in many different disease settings.3 in the context of Chagas disease, any envisaged therapy would have to remove existing fibrosis, deal with chronic inflammation and stimulate cardiomyogenesis

Resetting gene expression in chronic Chagas heart disease Comment on: Soares MB, et al. Cell Cycle 2011; 10:1448–55Lucíola S. Barcelos and Mauro M. Teixeira*; Universidade Federal de Minas Gerais (UFMG); Belo Horizonte, Brazil; *Email: [email protected]; DOI: 10.4161/cc.10.14.15852

endogenously express any p53 mutant of inter-est. Potentially, direct injection of plasmids into mouse zygotes harboring the PLF allele could even further increase the speed and ease of mutant derivation. This will not only enable the study of specific mutant p53 interactions

and network involvement, but also provide new models in which to assess the efficacy of potential therapeutics as cancer treatments for a subtle range of p53 mutations in vitro and in vivo that are essential for the understanding and treatment of human cancers.

Reference1. Brosh R, et al. nat Rev Cancer 2009; 9:701-13.2. Luo JL, et al. Oncogene 2001; 20:320.3. song H, et al. nat Cell Biol 2007; 9:573-80.4. Besaratinia A, et al. FAseB J 2010; 24:2612.5. seibler J, et al. Biochemistry 1998; 37:6229-34.6. Bouhassira ee, et al. Blood 1997; 90:3332.7. Thyagarajan B, et al. Mol Cell Biol 2001; 21:3926.8. Belteki G, et al. nature Biotechnol 2003; 21:321-4.9. wei QX, et al. Cell Cycle 2011; 10:1261-70.



inflammatory dilated cardiomyopathy (iDCM) is characterized by chronic inflammation of the myocardium (myocarditis), degeneration of cardiomyocytes, fibrosis, ventricular dila-tion and progressively impaired myocardial contraction. iDCM is frequently triggered by microbes, most prominently, Trypanosoma cruzi and coxsackievirus B3, and is usually asso-ciated with autoimmune responses against heart tissue antigens.1,2 At present, there is no effective treatment for iCDM.

T. cruzi infects the heart and other organs, causing a chronic debilitating illness (Chagas disease) that affects about 8 million people, predominantly in Latin America, 20–30% of whom have or will develop chronic chagasic cardiomyopathy (CCC).1 The main histological features of CCC are widespread and excessive cardiac inflammatory infiltrates and extra-cellular matrix (eCM) (fibrotic scar). excess inflammation is believed to tip the balance from protective responses to tissue degen-eration and maladaptive fibrosis. The heart in CCC bears an abnormal expression of genes associated with inflammation, fibrosis and angiogenesis.3 CCC manifests clinically as arrhythmias, right bundle block, apical aneurysm, stroke, heart failure and sudden death years after the initial infection. Current therapies for CCC only delay disease progres-sion; the best of them, cardiac transplant, is extremely limited due to the scarcity of donor organs, high risk of disease reactivation and prohibitive cost.

Hence, much excitement has been gen-erated by recent studies in Brazil and the UsA showing that a single dose of bone marrow cells (BMCs), either from uninfected or infected syngeneic donors, dramatically reduces cardiac inflammatory infiltrates and fibrosis in mice with CCC, significantly improv-ing heart ventricular function.4,5 Transplanted BMCs cause massive apoptosis (~10-fold

increase relative to untreated chagasic mice) in the cardiac inflammatory infiltrates, most of which are of bone marrow origin. Presumably, transplanted BMCs initiate, directly or indi-rectly, the destruction of excess inflammatory cells once they reach the heart parenchyma and interstitium. But it does not have to be only this way: recent studies show that virus-expressed matricellular protein CCn1 potently attenuates experimental autoimmune myo-carditis, not from the heart but from the spleen and without altering cardiac chemo-kine and cytokine profiles.6

it was recently reported in Cell Cycle that BMC therapy brings the number of genes that were upregulated in experimental CCC, most of them related to inflammation and fibrosis, down from 1222 to 103.7 Conversely, BMC therapy restores to background levels the majority of the genes downregulated in CCC, many associated with mitochondrial func-tion, supporting the idea that bone marrow stem and/or progenitor cells enhance cardiac regeneration and improve cardiac function. These findings are important, for they raise hope for a low-cost and effective treatment of CCC.

However, despite the therapeutic promise of BMC transplant for CCC (and for myocar-dial infarction and other types of inflamma-tory degenerative cardiomyopathy),8 many challenges and uncertainties remain.9 First, the extent, if any, of BMC therapy in T. cruzi heart burden must be formally scrutinized,4 as the sharp, BMC-induced reduction in inflam-mation may, in principle, maintain or even enhance tissue parasitism, restarting CCC. This underscores the importance of eliminating or attenuating T. cruzi parasitism of the heart, which requires understanding how T. cruzi invades and uses cardiomyocytes as long-term habitat. second, the cellular and molecu-lar basis underlying cardiac repair after BMC

transplantation is poorly understood.9 Among other issues, future studies should define the nature of the cells and/or diffusible factors that selectively trigger apoptosis of cardiac inflammatory cells.8,9

And third, it is critical to understand the signals that result in BMC differentiation into cells that cause cardiac repair rather than cardiac malfunction. About 25–60% of eCM-producing cardiac myofibroblasts (positive for vimentin and a-smooth muscle cell actin and negative for CD45) in mouse models of myocardial infarction, fibrotic ischemia/reperfusion and autoimmune myocarditis are of bone-marrow origin.2 Myofibroblasts can promote cardiac repair or, if chronically and excessively activated, pathological scars. Furthermore, it has been established that heart-infiltrating BMCs differentiate into either myofibroblasts or inflammatory cells depending upon the local cytokine/chemo-kine pattern.2 Therefore, avoidance of BMC differentiation into myofibroblasts or inflam-matory cells is critical for repair, and not exac-erbation, of CCC. This concern is particularly vexing in Chagas disease, for the cellular and molecular basis underlying fibrosis respon-sible for, among other pathological features, the abnormal electrical conduction system of the heart, is largely unknown10 in contrast to other types of iDCM.2

References1. Rassi A, Jr., et al. Lancet 2010; 375:1388-402.2. Kania G, et al. Trends Cardiovasc Med 2009;

19:247-52.3. soares MB, et al. J infect Dis 2010; 202:416-26.4. soares MB, et al. Am J Pathol 2004; 164:441-7.5. Goldenberg RC, et al. J infect Dis 2008; 197:544-7.6. Rother M, et al. Circulation 2010; 122:2688-98.7. soares MBP, et al. Cell Cycle 2011; 10;1448-55. 8. segers vF, et al. nature 2008; 451:937-42.9. Tongers J, et al. eur Heart J 2011; 32:1197-206.10. Araujo-Jorge TC, et al. Cytokine Growth Factor Rev

2008; 19:405-13.

That’s amore: Bone marrow cells compete for the heart in Chagas diseaseComment on: Soares MB, et al. Cell Cycle 2011; 10:1448–55Mercio A. Perrin; Tufts University School of Medicine; Boston, MA USA; Email: [email protected]; DOI: 10.4161/cc.10.14.15854



Figure 1. illustrated is a potential integration of nanog regulation by ezh2 protein, as described by villasante et al. (green boxes), in the context of a two-step differentiation process, as suggested by Glauche et al. (blue). in this concept, pluripotent mes cells reversibly change between an Oct4-sox2-high/nanog-high and an Oct4-sox2-high/nanog-low state (vertical dimension). The transi-tion between these two states is controlled either by a sufficiently high level of transcriptional noise (e.g., fluctuating nanog levels) or by a negative feedback loop acting on the nanog expres-sion. As nanog-low cells are prone to differentiation, some of the nanog-low cells lose the ability to revert back into the nanog-high state by further downregulation of nanog and other pluripotency genes, such as Oct4 and sox2 (horizontal dimension).

stem cells are characterized by their poten-tial to self-renew their own population while retaining the capacity to differentiate into more specialized cells. it is still a central ques-tion how the balance between self-renewal and differentiation is regulated. Although several lines of evidence suggest that differ-ent types of intercellular heterogeneity might play a decisive role,1,2 many details about the molecular basis of cell fate decisions and the dynamics of the relevant interaction processes are still unknown.

For mouse embryonic stem (mes) cells, it has been demonstrated that their propen-sity for differentiation correlates with specific expression patterns and chromatin modifica-tions.3,4 One prominent example is the tran-scription factor nanog. experimental findings reveal that continuously high expression levels of nanog are not ultimately required to main-tain pluripotency. Under culture conditions promoting both self-renewal and differentia-tion (e.g., LiF/serum conditions), a bimodal distribution of cells with high and low nanog expression levels is established. in accordance with the known roles of nanog for induction of pluripotency and for preventing differentiation, nanog-low cells are more prone to differentiate than nanog-high cells.3 interestingly, mes cells are not captured in one of these states. in fact, they are capable of reversible change between the nanog-low and the nanog-high state.3 These findings suggest that nanog heteroge-neity is an intrinsic property of mes cells to facilitate self-renewal and differentiation.

in a previous issue of Cell Cycle, villasante and colleagues described an epigenetic mechanism regulating the nanog expres-sion in pluripotent stem cells.5 in particular, they demonstrated that the methyltransfer-ase ezh2 directly alters the epigenetic status of the nanog promotor and, therefore, the proportion of nanog-high (self-renewing) and nanog-low (differentiating) cells. ezh2 is the catalytic subunit of the Polycomb repres-sive complex 2 (PCR2), which is required for long-term epigenetic silencing of chromatin (repressive mechanism) and is involved in the establishment of gene expression patterns during development. Using ezh2-null iPs and

Epigenetic Nanog regulation and the role of functional heterogeneityComment on: Villasante A, et al. Cell Cycle 2011; 10:1488–98Maria Herberg and Ingo Roeder*; Technische Universität Dresden; Dresden, Germany; *Email: [email protected]; DOI: 10.4161/cc.10.14.16203

TnG-A es cells, villasante et al. demonstrated that the absence of ezh2 increases the fraction of nanog-high cells and reduces the nanog-low compartment. They further examined by ChiP analysis that ezh2 directly binds to the nanog promotor and produces the repressive histone mark H3K27me3. The expression of ezh2 itself is considerably higher in nanog-low cells. Consistent with the findings on revers-ible, biased nanog states, villasante et al. revealed that the nanog promotor exists in two univalent epigenetic configurations, one characterized by the active epigenetic mark H3K4me3 (nanog-high state) and the other

with the repressive mark H3K27me3 (nanog-low state). To conclude, ezh2 has been identi-fied by villasante et al. as a potential candidate to shift the balance between self-renewal and differentiation toward differentiation.

This result should also be seen in the context of theoretical concepts that explain general regulatory principles underlying the determi-nation of cellular states. in 2006, Chickarmane et al. described a bistable On/ OFF switch for self-renewal vs. differentiation in the pluripo-tency network, which arises due to several positive feedback loops.6 However, in the sug-gested model, cells are fixed in either of the



Pluripotent cells of the early blastocyst inner cell mass (iCM) and embryonic stem cells (esCs), their in vitro counterpart, have the remarkable capacity to differentiate into all three germ layers. while the exact mecha-nisms governing pluripotency maintenance and cell commitment are still being elucidated, epigenetics have been shown to play a vital role in the regulation of appropriate gene expression. A group of proteins known as polycomb group (PcG) proteins are essential to regulate cell specification during devel-opment and differentiation. These proteins form multiprotein complexes, termed poly-comb repressive complexes (PRCs), which con-tribute to transcription repression through chromatin modification. Polycomb repressive complex 2 (PRC2) members eed and suz12 proteins cooperate the histone methyltrans-ferase ezh2 protein, which catalyzes trimeth-ylation of histone 3 lysine 27 (H3K27me3), a mark associated with repressed chromatin.1 strikingly, these protein complexes bind and repress the promoters of hundreds of genes in esCs that are important for cell specifica-tion.2 The mechanism for proper and efficient PRC recruitment to specific promoters remains largely unknown, though recently, PRC bind-ing elements have been identified in the HOXD cluster in humans3 and the MafB locus in mice.4

PRCs can also be recruited through DnA bind-ing factors, including yy1 and JARiD2 and non-coding RnA, such as HOTAiR1. As such, there is much investigation still required to determine

which factors are responsible for directing recruitment and dissociation of PRCs during cell differentiation.

while the iCM of the blastocyst or esCs was originally thought to be a homogeneous population, there is a growing body of evi-dence that these cells are actually hetero-geneous in expression of various proteins, including the pluripotency-associated fac-tors nanog, Pecam1 and Rex1 and lineage-restricted markers, including Gata6, Hex and sox17.5 interestingly, nanog expression fluc-tuates in pluripotent iCM6 and esCs,7,8 but it is not known how gene regulation occurs in such a rapid manner. in a previous issue of Cell Cycle, villasante et al.9 uncovered a novel connection between PRC2 member ezh2 and nanog heterogeneity. Using mouse induced pluripotent stem cells (iPsCs), they observed an inverse relationship in expression of Nanog and Ezh2 and further demonstrated binding of ezh2 to the nanog promoter region. induction of ezh2-null iPsCs using 4-hydroxytamoxifen demonstrated that these cells display global reduction in H3K27me3, impaired differen-tiation and an unexpectedly higher level of nanog expression. Further, using sequential ChiP, the authors revealed that the Nanog locus does not display bivalent H3K4me3 and H3K27me3 marks, but that high or low nanog expressing cells show enrichment of H3K4me3 or H3K27me3, respectively. These results sug-gest a model where nanog expression is epi-genetically regulated by ezh2.

This study clearly shows, for the first time, that PRC2 is involved in the regulation of genes necessary for maintenance of pluripotency, in addition to their known role in regulating lin-eage specification genes. These findings raise a number of interesting questions regarding the mechanism of PRC recruitment to and dis-sociation from the pluripotency-related genes, which are dynamically expressed. it is currently unclear whether ezh2 directly interacts with a PRC binding element in the nanog promoter, or whether it associates indirectly through other factors which control Nanog transcrip-tion. it should also be interesting to study how the FGFR pathway, a known signaling cascade triggering nanog heterogeneity in iCM and esC,6,8 induces PRC2 association to the nanog gene. in a more developmental context, are these epigenetic marks influenced by cell posi-tion during early embryonic cleavage divisions to regulate pluripotency and lineage specifica-tion genes, and what is the developmental significance of heterogeneous and fluctuating expression of key factors? interestingly, PRC2 components are required for reprogramming B cells using esC fusion,10 so it will be informa-tive to examine whether these components are also critical for iPsC generation. Thus, a greater understanding of epigenetic control of pluripotency-related genes could provide insight beyond esC biology and impact our understanding of lineage specification dur-ing development and induced reprogramming during iPsC generation.

An Ezh way to turn off NanogComment on: Villasante A, et al. Cell Cycle 2011; 10:1488–98Katherine E. Hankowski and Naohiro Terada*; University of Florida; Gainesville, FL USA; *Email: [email protected] ; DOI: 10.4161/cc.10.14.16388

two stable states. Reversible state changes, as observed for nanog concentrations, require additional/external forces. in 2010, Glauche et al. developed an alternative network model suggesting two potential mechanisms that could possibly be linked to the epigenetic modulator ezh2 studied by villasante et al. The theoretical results described by Glauche et al. demonstrate that the variability in nanog expression can be explained either by transcriptional noise inducing fluctua-tions between two coexisting stable cellular

states or by an additional (currently unknown) nanog-repressor, leading to oscillating nanog levels via a negative feedback loop.7 ezh2 (shown to interfere the nanog transcription) might be a potential mediator for both of these mechanisms.

summarizing, it turns out that nanog het-erogeneity is most likely a functional element to control the differentiation propensity of mes cells supporting the idea that nanog acts as a gatekeeper of pluripotency. ezh2 represent an interesting potential mechanism

for the initial destabilization (transient down-regulation) of the nanog levels to render mes cells responsive for external signals (Fig 1).

References:

1. Graf T, et al. Cell 2008; 3:480-3.2. silva J, et al. Cell.2008; 132:532-6.3. Chambers i, et al. nature 2007; 450:1230-4.4. niwa H, Genes Dev. 2007; 21: 2671-6.5. villasante A et al. Cell Cycle 2011; 1488-98.6. Chickarmane v, et al. PLos Comput Biol 2006;

2:e123.7. Glauche i, et al. PLos One 2010; 5:e1123.



Timeless makes some time for itselfComment on: Smith-Roe SL, et al. Cell Cycle 2011; 10:1618–24Laura A. Diaz-Martinez1 and Duncan J. Clarke2; 1University of Texas Southwestern Medical Center; Dallas, TX USA; 2University of Minnesota Medical School; Minneapolis, MN USA; Email: [email protected] and [email protected] ; DOI: 10.4161/cc.10.14.15853

References: 1. Margueron R, et al. nature 2011; 469:343-9.2. Boyer LA, et al. nature 2006; 441:349-53.3. sing A, et al. Cell 2009; 138:885-97.4. woo CJ, et al. Cell 2010; 140:99-110.5. Lanner F, et al. Development 2010; 137:3351-60.6. Chazaud C, et al. Dev Cell 2006; 10; 615-24.7. Chambers i, et al. nature 2007; 450:1230-48. singh AM, et al. stem Cells 2007; 25:2534-42.9. villasante A, et al. Cell Cycle 2011;1488-98.10. Pereira CF, et al. Cell stem Cell 2010; 6:547-56.

First identified as a key player in circadian rhythms in Drosophila,1,2 members of the Timeless family of proteins have been iden-tified in all eukaryotes studied, with most organisms containing a single Timeless gene, except Drosophila and other insects, which contain a Timeless paralog named Timeout/Timeless2. interestingly, Timeless genes from non-insect eukaryotes, including yeasts and humans, are more closely related to the Drosophila paralog Timeout than to Drosophila Timeless.3 Furthermore, while a role for mammalian Timeless in the control of circadian rhythmicity is still unclear,3 Timeless proteins and the Timeout paralog in flies have been implicated in roles as diverse as DnA rep-lication and repair,4,5 chromosome structure (reviewed in ref. 6) and apoptosis,7 consistent with evolutionarily conserved roles for the Timeless family that are independent of a role in circadian rhythms.

A role for Timeless in chromosome cohe-sion was first described in Caenorhabditis elegans using a biochemical approach that identified the worm Timeless protein (Tim-1) as a cohesin-binding partner. similar to depletion of cohesins by siRnA, TiM-1 siRnA results in embryonic lethality, while TiM-1 hypomorphs show chromosome disorganiza-tion and loss of cohesion during the meiotic divisions.8 This role for Timeless in sister chro-matid cohesion is conserved in Xenopus9,10

and humans.11 However, the mechanism by which Timeless contributes to sister chroma-tid cohesion remains to be determined. it has been proposed that Timeless contributes to sister chromatid cohesion establishment dur-ing s phase by interaction with both cohesins and components of the replication fork.9-11 Timeless is thought to form an obligate het-erodimer with Tipin, as depletion of either protein by siRnA results in a severe reduc-tion of its binding partner.12 Furthermore, Tipin, but not Timeless, interacts directly with Replication Protein A (RPA).13 These results have led to a model in which the Timeless-Tipin heterodimer is the functional unit that somehow stabilizes cohesin at the replication forks.

interestingly, in a previous issue of Cell Cycle, smith-Roe and collaborators suggest the intriguing possibility that Timeless pro-motes sister chromatid cohesion in a Tipin-independent manner.14 Through a detailed analysis of chromosome cohesion after Timeless and Tipin depletion in human cells, the authors found that siRnAs directed against either Timeless or Tipin resulted in chromo-some cohesion defects, but the frequency of chromosome cohesion defects was ten times higher when using Timeless siRnAs com-pared to Tipin siRnAs. Consistent with previ-ous studies,12 depletion of either Timeless or Tipin resulted in a marked decrease in the

other binding partner. However, smith-Roe et al. noted that, although severely decreased, Timeless protein was still present after Tipin siRnA at slightly higher levels than when depleted by Timeless siRnA. Thus, a small pool of Timeless, which cannot be depleted by Tipin siRnA, might be responsible for its role in sister chromatid cohesion. intriguingly, these findings suggest that Timeless’s role in cohe-sion might be completely independent of its role at the replication fork, even though they both occur during s phase. These results open a new door towards our understanding of the multiple functions of the Timeless family of proteins, and indicate that some of these roles might be studied in further detail through the use of separation of function mutations.

References1. vosshall LB, et al. science 1994; 263:1606-9.2. sehgal A, et al. science 1994; 263:1603-6.3. Gotter AL. neuroreport 2006; 17:1229-33.4. Unsal-Kacmaz K, et al. Mol Cell Biol 2005;

25:3109-16.5. Benna C, et al. Curr Biol 2010; 20:346-52.6. McFarlane RJ, et al. Cell Cycle 2010; 9:700-5.7. O’Reilly LP, et al. PLos One 2011; 6:e17157.8. Chan RC, et al. nature 2003; 423:1002-9.9. errico A, et al. eMBO J 2009; 28:3681-92.10. Tanaka H, et al. Genes Cells 2009; 14:949-63.11. Leman AR, et al. J Cell sci 2010; 123:660-70.12. Chou DM, et al. Proc natl Acad sci UsA 2006;

103:18143-7.13. nakaya R, et al. J Biochem 2010; 148:539-47.14. smith-Roe et al. Cell Cycle 2011; 10:1618-24.

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