Patent Application
20210155905
The invention relates to a method for obtaining haploid cells, in particular for obtaining haploid cells from somatic cells.
Cells are classified according to the ploidy level of their DNA content: haploid, etc.
Some animals such as yeasts or males of social insects are haploid, i.e. they carry a single set of chromosomes, while haploidy in mammals is exclusively restricted to mature germ cells.
A single copy of the genome provides the basis for genetic analyses where any recessive mutation of essential genes will show a clear phenotype due to the absence of a second gene copy. Most prominently, haploidy in yeast has been utilized for recessive genetic screens that have markedly contributed to the inventors' understanding of development, basic physiology, and disease.
Somatic mammalian cells carry two copies of chromosomes (diploidy) that obscure genetic analysis.
Near haploid human leukemic cells however have been developed as a high throughput screening tool. Although deemed impossible, some researchers have generated mammalian haploid embryonic stem cells from parthenogenetic mouse embryos. Haploid stem cells open the possibility of combining the power of a haploid genome with pluripotency of embryonic stem cells to uncover fundamental biological processes in defined cell types at a genomic scale. Haploid genetics has thus become a powerful alternative to RNAi or CRISPR based screens.
However, both near haploid human cells and mammalian haploid embryonic stem cells are extremely difficult to obtain. Such cells would be an invaluable tool in life sciences, both for genetic screening, but also as a new tool to modify the genome.
It would be also useful to obtain haploid cells in order to study some inherited pathologies, the genotype of which being autosomal dominant, in cells carrying the single-allele mutation. Another aspect could be the necessity to obtain tissue specific haploid cells in order to study the physiology of these cells by simplest genetics techniques.
The invention therefore intends to obviate these drawbacks and deficiencies.
One aim of the invention is to provide a method for producing haploid cells from somatic cells.
Another aim of the invention is to provide haploid somatic cells.
One other aim of the invention is to provide compounds or molecules that can stimulate the formation of haploid cells from differentiated non-germinal cells. The present invention relates to the use of an inhibitor of the activity or stability of the pre-replication complex, also called pre-RC, for the preparation, preferably in vitro, of haploid eukaryotic somatic cells from eukaryotic somatic diploid cells, said inhibitor completely inhibiting the activity or stability of said pre-RC.
In the invention, Eukaryotic cells encompass all the eukaryotic cells such as metazoan cells, including plant cells, in particular Chlorobionta, yeast cells, insect cells, worm cells, frog cells, mammal cells . . . .
Advantageously, the invention relates to the use of an inhibitor of the activity or stability of the pre-replication complex, also called pre-RC, for the preparation, preferably in vitro, of haploid mammal somatic cells from mammal somatic diploid cells, said inhibitor completely inhibiting the activity or stability of said pre-RC.
The invention is based on the unexpected observation made by the inventors that a complete blockage of the DNA replication licensing, by destabilizing or inhibiting the activity of the pre-replication complex, does not kill cells but only inhibit their ability to duplicate DNA. These cells remain able to enter into mitosis and further complete cell division, providing two haploid sister cells from a mother diploid cell.
This observation is a breakthrough, since the prior art recited that blockage of DNA replication would only induce cell death or senescence, but it was absolutely unexpected that the cells can survive to this DNA replication blockage.
Within the context of the invention, “DNA replication blockage” means that cells are not able to carry out DNA replication (i.e. producing two identical replicas of DNA from one original DNA molecule), in particular by inhibiting the initiation of the DNA replication (i.e. by inhibiting the formation of the replication origin complex or replication origin activation). Thus, in the invention, a DNA replication process which has begun but is blocked before the complete duplication of the parental DNA molecule is not considered as a “DNA replication blockage”. The “DNA replication blockage” according to the invention in particular completely blocks initiation of DNA replication.
By “pre-replication complex”, it is meant in the invention the protein complex that forms at the origin of replication during the initiation step of DNA replication. Formation of the pre-RC is required for DNA replication to occur. Accordingly, formation of the pre-RC is a very important part of the cell cycle.
Pre-RC complex comprises so far the following proteins: the Origin replication complex (ORC) 1-6 proteins, CDCl6 protein, the chromatin licensing and DNA replication factor 1 (Cdt1) protein and minichromosome maintenance 2-7 proteins. Eukaryotic cells have the most complex pre-RC. After ORC1-6 bind the origin of replication, Cdc6 is recruited. Cdc6 recruits therefore the licensing factor Cdt1 and MCM2-7 complex. Cdt1 binding and ATP hydrolysis by the ORC and Cdc6 load MCM2-7 onto DNA.
It is known in the art that cyclin dependent kinases CDKs prevent formation of the replication complex during late G1, S, and G2 phases by excluding MCM2-7 and Cdt1 from the nucleus, targeting Cdc6 for degradation by the proteasome, and dissociating ORC1-6 from chromatin via phosphorylation.
Proteolytic regulation of Cdt1 is also a mean to block pre-RC activity. This proteolysis is shared by higher eukaryotes including Caenorhabditis elegans, Drosophila melanogaster, Xenopus laevis, and mammals. In metazoans, another mechanism to prevent re-replication; during S and G2, involves the geminin protein which binds to Cdt1 and inhibits Cdt1 from loading MCM2-7 onto the origin of replication. Thus advantageously, the invention relates to the use of an inhibitor as defined above, this inhibitor being, without limitation
Said inhibitor, used in the invention completely affects DNA replication. It is particularly relevant to block DNA replication initiation in synchronized cells, in order to simultaneously block the replication in all the cells before the initiation of the replication. When cells are not synchronized, i.e. all the cells are in same step (G1, S, G2 or M phase) of the cell cycle, the cells which are in the G1 phase when the inhibitor is applied would be the one that could efficiently give rise to haploid cells further to the M phase.
If replication has started and then blocked, cells will activate the DNA damage checkpoint and be blocked at the G2/M transition. If the licensing of replication is partially inhibited, some DNA replication will fire but not enough: in this case, cells will enter into M phase with partially replicated chromatids that will induce mitotic catastrophes.
The senescence is induced by cells that did not replicate but do not manage to divide (only a fraction of unlicensed cells managed to divide).
Advantageously, the invention relates to the use as defined above, for the preparation, in particular in vitro, of haploid eukaryotic somatic cells from eukaryotic somatic diploid cells which are in the G1 phase of the cell cycle, said inhibitor completely inhibiting the activity or stability of said pre-RC.
Advantageously, the invention relates to the use as defined above, for the preparation, in particular in vitro, of haploid mammal somatic cells from mammal somatic diploid cells which are in the G1 phase of the cell cycle, said inhibitor completely inhibiting the activity or stability of said pre-RC.
The above use of an inhibitor of the activity or stability of the pre-RC complex allows to obtain, from somatic diploid cells which are particularly in the G1 phase of the cell cycle, haploid cells. These cells are called “haploid somatic cells” because they arbor all the features of a somatic differentiated cell, but the DNA content is reduced by half compared to the somatic diploid cells.
According to one aspect of the invention, the “haploid somatic cells” according to the invention are completely different from the natural germinal haploid cells, and cannot directly, when fused to the nucleus of a spermatozoid or an oocyte, carry out the early steps of development, i.e. reproduce fecundation and embryo development.
Advantageously, the invention relates to the use as defined above, wherein said inhibitor is an inhibitor of the activity or expression of the chromatin licensing or of the CDT1 protein, or an inhibitor of one of the following proteins: ORC1-6. CDCl6, MCM2-7 proteins or the usage of any inhibitor of these latter factors, like geminin, inhibitor of CDT1.
In one another advantageous embodiment, the invention relates to the use as defined above, wherein said inhibitor is a formulation of geminin protein, said formulation comprising an amount of geminin protein at least 1-fold higher compared to the cellular endogenous amount of geminin protein.
Advantageously, the invention relates to the use as defined above wherein said inhibitor is a formulation of geminin protein, said geminin protein being expressed during G1 phase of the cell cycle whereas the natural endogenous geminin is not expressed during G1 phase of the cell cycle.
Indeed, as mentioned above, expressing a geminin protein during G1 phase will inhibit PreRC formation and thus inhibit cell division.
Advantageously, the invention relates to the use as defined above, wherein said inhibitor is a non proteasome-degradable form of the Geminin protein.
The inventors demonstrated, as shown in the Example section, that a non-degradable mutant of the Geminin protein inhibits Cdt1 and induces a DNA replication blockage by inhibiting the Pre-RC complex.
Advantageously, the invention relates to the use as defined above, wherein said non-proteasome-degradable form of the Geminin protein lacks the following sequence: RxTLKxzQx. (SEQ ID NO: 2), wherein x represents any amino acid and z represent a leucine (L) or an isoleucine (I).
The inventors show that advantageously, a mutated form of the Geminin protein, in which the destruction box domain is deleted, acts as an efficient inhibitor of DNA replication, and thus is useful to produce haploid somatic cells from somatic diploid cells.
In the invention the Geminin protein, which is to be mutated in order to block DNA replication according to the invention, can be any Geminin of eukaryotes, such as
Another way to inhibit the degradation of geminin protein is to delete the amino acid sequences SEQ ID NO: 2 in the sequence of wild type proteins, for instance as represented in SEQ ID NO: 3-8. In this context, and further a deletion, it is also possible to insert one or more amino acids.
An example of a geminin mutant particularly advantageous in the invention is the mutant consisting essentially of the amino acid sequence as set forth in SEQ ID NO: 9, which is the result of the deletion of SEQ ID NO: 2, followed by an insertion at the same site within the sequence SEQ ID NO: 4. Another example of a geminin mutant particularly advantageous in the invention is the mutant consisting essentially of the amino acid sequence as set forth in SEQ ID NO: 10, which is the result of the deletion of SEQ ID NO: 2, followed by an insertion at the same site, within the sequence SEQ ID NO: 3.
Thus, advantageously, the invention relates to use as defined above, wherein said non proteasome-degradable geminin protein consist essentially of the protein as set forth in SEQ ID NO: 9 or 10, or any protein having at least 70% identity and which is not degraded by a cellular degradation process.
The above mentioned inhibitors can be added to a diploid somatic cell by injection of purified protein, or lipofection of protein, or by transfection or infection with a DNA molecule, or a RNA molecule coding for these protein.
Use according to claim 2, wherein said inhibitor is a small interfering molecule inhibiting the expression of said Cdt 1 protein.
As Ctd1 is a target of a non-degradable Geminin, for instance as defined above, it is possible to provide inhibitors of Ctd1 to block DNA replication. For instance an inhibition by RNA interference using siRNA, shRNA or miRNA can be carry out. The skilled person could easily determine the best small interfering molecule that can completely block DNA replication.
Advantageously, the invention relates to the use according to the above definition, wherein said haploid eukaryotic somatic cells are able to carry out mitosis.
Advantageously, the invention relates to the use according to the above definition, wherein said haploid mammal somatic cells are able to carry out mitosis.
One important point in the process according to the invention, is that the resulting haploid cells, when treated with the inhibitor as defined above, can carry out one or more new complete cell cycle, including DNA replication, when the inhibitor as defined above is removed.
Thus for this reason, it could be advantageous that the inhibitor defined above be “activable” (for instance inducible) when necessary. Activation of the inhibitor may for instance the result of:
The invention also relates to a protein consisting essentially of the amino acid sequence as set forth in SEQ ID NO: 9 or 10.
These mutant of Geminin are novel.
The invention also relates to a method for producing, in particular in vitro, eukaryotic somatic haploid cells from eukaryotic somatic diploid cells, said method comprising a step of contacting eukaryotic somatic diploid cells with an inhibitor of the activity or stability of pre-RC, said inhibitor completely inhibiting the activity or stability of said pre-RC. The method according to the invention may further comprise a step of isolation of the resulting somatic haploid cells, for instance by flow cytometry, by isolating the cells according to their DNA content.
Advantageously, the invention relates to a method for producing, in particular in vitro, mammal somatic haploid cells from mammal somatic diploid cells, said method comprising a step of contacting mammal somatic diploid cells with an inhibitor of the activity or stability of pre-RC, said inhibitor completely inhibiting the activity or stability of said pre-RC. The method according to the invention may further comprise a step of isolation of the resulting somatic haploid cells, for instance by flow cytometry, by isolating the cells according to their DNA content.
Advantageously, the invention relates to the method as defined above, wherein said inhibitor is either an inhibitor of the activity or expression of the Cdt1 protein, or said inhibitor is a small interfering molecule inhibiting the expression of said Cdt 1 protein. More advantageously, the invention relates to the method as defined above, wherein said inhibitor is a non degradable form of the Geminin protein, in particular a Geminin protein lacks the following sequence: RxTLKxzQx. (SEQ ID NO: 2).
Advantageously, the invention relates to the method as defined above, wherein said cells are mammal somatic cells and wherein said inhibitor is a non-degradable form of the Geminin protein, in particular a Geminin protein lacks the following sequence: RxTLKxzQx. (SEQ ID NO: 2).
More advantageously, the invention relates to the method as defined above, wherein the somatic diploid cells are transfected or infected with a DNA or a RNA molecule allowing a conditional expression of a mutated Cdt1 protein, said mutated Cdt1 protein being possibly represented by the amino acid sequence SEQ ID NO: 9 or SEQ ID NO: 10, or any amino acid sequence having at least 70% identity with said sequences SEQ ID NO: 9 or SEQ ID NO: 10, provided that they have lost their ability to be degraded by the proteasome pathway.
The conditional expression may be the inducible systems Tet-On or Tet-Off well known in the art. The tetracycline-controlled Tet-Off and Tet-On gene expression systems are used to regulate the activity of genes in eukaryotic cells in diverse settings, varying from basic biological research to biotechnology and gene therapy applications. These systems are based on regulatory elements that control the activity of the tetracycline-resistance operon in bacteria. The Tet-Off system allows silencing of gene expression by administration of tetracycline (Tc) or tetracycline-derivatives like doxycycline (dox), whereas the Tet-On system allows activation of gene expression by dox. Since the initial design and construction of the original Tet-system, these bacterium-derived systems have been significantly improved for their function in eukaryotic cells. Tetracycline-controlled gene expression is based upon the mechanism of resistance to tetracycline antibiotic treatment found in gram-negative bacteria. In nature, the Ptet promoter expresses TetR, the repressor, and TetA, the protein that pumps tetracycline antibiotic out of the cell. The difference between Tet-On and Tet-Off is not whether the transactivator turns a gene on or off, as the name might suggest; rather, both proteins activate expression. The difference relates to their respective response to tetracycline or doxycycline (Dox, a more stable tetracycline analogue); Tet-Off activates expression in the absence of Dox, whereas Tet-On activates in the presence of Dox.
The invention also relates to an isolated eukaryotic somatic haploid cell liable to be obtained by the method as defined above.
The invention also relates to an isolated mammal somatic haploid cell liable to be obtained by the method as defined above.
The somatic haploid cells according to the invention are novel, and particularly because they contain a mutated Geminin protein which is not degradable via the proteasome degradation pathway.
The invention relates to an isolated eukaryotic somatic haploid cell provided that said eukaryotic somatic haploid cell is not an embryonic haploid stem cells, said somatic cell being characterized in that it harbors differentiation epigenetic marks, and possibly contain in their genome a sequence coding for a non-degradable Geminin protein.
The invention relates to an isolated plant somatic haploid cell, said somatic cell being characterized in that it harbors differentiation marks.
The invention relates to an isolated mammal somatic haploid cell provided that said mammal somatic haploid cell is not an embryonic haploid stem cells, said somatic cell being characterized in that it harbors differentiation marks, and possibly contain in their genome a sequence coding for a non-degradable Geminin protein.
The main difference compared to the prior art haploid cells is that the haploid cells of the invention would not be derived from a haploid cell. In the already existing haploid ES cells or in vitro differentiated cells coming from these haploid ES cells, the ES cells come from an oocyte that is not fertilized and activated: so, cells are primarily haploid. These cells are novel, and particularly, contrary to the prior art, are not originated from a tumor.
In one another advantageous embodiment, the invention relates to isolated mammal somatic haploid cell, said haploid cell deriving from HCT116, DLD1, 3T3 cell lines, or primary fibroblasts.
The invention also relates to a nucleus of an isolated eukaryotic somatic haploid cell as defined above.
The invention also relates to a nucleus of an isolated plant somatic haploid cell as defined above.
The invention also relates to a nucleus of an isolated mammal somatic haploid cell as defined above.
Moreover, the invention relates to a method for carrying out fecundation (fertilization), in particular in vitro, comprising a step of introducing into a female germinal egg a nucleus of an isolated mammal somatic haploid cell as defined above. This method is advantageously carried out provided that it does not intend to provide processes for cloning human beings, or it does not intend to produce human embryos for industrial or commercial purposes.
The method defined above is based on the ability to obtain haploid cells, the nucleus of which could be fused to the nucleus of a recipient germinal female cell.
By this method, it is artificially reproduced the mechanism of fecundation (fertilization) and could help couples having fertility issues due to a lack of production of gametes.
In one other advantageous aspect, the invention relates to a method for carrying out, advantageously in vitro, fertilization comprising a step of introducing into a female germinal cell a nucleus of an isolated plant somatic haploid cell as defined above.
The invention also relates to a method for the production, possibly in vitro, of homozygote cells for at least a determined locus, said method comprising a step of genetically modifying at least one locus of a n eukaryotic somatic haploid cell as defined above, a step of inducing DNA replication of said eukaryotic somatic haploid cell and a step of inhibiting cell division.
The invention also relates to a method for the production, possibly in vitro, of homozygote cells for at least a determined locus, said method comprising a step of genetically modifying at least one locus of a mammal somatic haploid cell as defined above, a step of inducing DNA replication of said mammal somatic haploid cell and a step of inhibiting cell division.
In the invention, when producing an haploid cell, it could be easy to modify a determined locus within the genome compared to diploid cells, in which it is necessary for some studies to obtain a bialletic modification.
In this context, the genetic manipulation becomes easier, such as the genetics carried out in Yeasts.
According to this method, it is possible to modify the genome of a somatic haploid cells by any molecular biology technic, including CRISP/cas9 technic. When the modification is carried out, it is sufficient to block cell division, by using drugs inhibition the chromosomal segregation, or inhibition of mitosis (such as inhibitor of chromosome segregation). Therefore, when entering to the S phase, the DNA content of the cell will be duplicated, and if the mitosis is not effective, the haploid will become diploid. This cell would have therefore pairs of chromosomes, each member of the pair being strictly identical.
The invention also relates to a nucleic acid molecule coding for a mutated geminin protein, said protein containing a deletion of the sequence RxTLKxzQx. (SEQ ID NO: 2), said nucleic acid sequence being possibly contained in a vector allowing the expression of said nucleic acid sequence further to a conditional activation.
More advantageously, the invention relates to a nucleic acid molecule coding for the mutated geminin as set forth in SEQ ID NO: 9 or 10.
More advantageously, the invention relates to a nucleic acid molecule consisting essentially of the nucleic acid molecules as forth in SEQ ID NO: 11 or 12.
The invention will be better understood from the following examples and figures.
Lower panel represents a bi-parametric analysis images of DNA content (propidium iodide; PI) and BrdU incorporation in non-induced 3T3 cells that were transfected with empty vector or the CDT1-encoding plasmid at T1 and incubated with BrdU for 15 minutes prior to harvesting at T2 (to visualize replicating cells, 24 hours after transfection) or for 24 hours (to quantify the proportion of cells that have replicated DNA during the last 24 hours). Cells were fixed and stained for DNA (PI) and BrdU incorporation assessed.
Results show that compared to vector alone, CDT1 overexpression did not dramatically change the cell cycle features of 3T3 cells in which ΔDboxGeminin was not induced.
Lower panel: fates of normal cells in which licensing is inhibited by overexpression of ΔDboxGeminin. The absence of the Dbox (destruction box) stabilizes Geminin, normally degraded in G1 by the action of the APC/C. The ΔDboxGeminin prevents binding of CDT1 onto origins and inhibits formation of the pre-RCs. Unlicensed cells enter into mitosis with unreplicated chromatids (2C chromosomes). The majority of these mitoses fail to complete, generating one single cell with one (mitotic slippage) or two (cytokinesis failure) nuclei. These cells display nucleolar γH2AX staining and enter into senescence. A discrete population of unlicensed cells manages to divide and gives rise to two daughter cells, some of them containing 10 chromosomes.
Plasmids and Constructs
Deletion of the destruction box (Dbox) and addition of N-terminal Flag-HA epitopes were performed by PCR. ΔDboxGeminin was finally cloned into pTRIPZ using the Age1-Mlu1 cloning sites (Supplementary Information 1). The plasmid encoding the shRNA against CDT1 was from Sigma-Aldrich (TRCN0000174484). The CDT1-pCDNA3 plasmid is described in Coulombe et al. [Coulombe et al. 2013, Nature Communications 4, pp 1-10].
Preparation of Viral Particles and Infection
HIV-derived vectors pseudo-typed with the VSV-G envelope protein were produced by transient co-transfection of the Gag-Pol packaging construct psPAX2, the envelop plasmid pMD.2G and the self-inactivating (SIN) HIV-1-derived vector coding for the ΔDboxGeminin or shRNA of interest. 36 hours after transfection, particles were harvested and filtered before infection of different cells.
Cells and Media
Mouse embryonic fibroblasts (MEFs) were derived as previously described [Ganier et al. Proc Natl Acad Sci USA. 2011 Oct. 18; 108(42):17331-6]. Briefly, 13.5-day-post-coitum wild type C57BL6 mouse embryos were dissected and gonads, internal organs and head were removed before MEF isolation. Tissues were then dissociated with 0.05% trypsin/EDTA (Gibco, Invitrogen) at 37° C. for 15 minutes to isolates single cells. To obtain tail tip fibroblasts, tail tips from adult mice were sliced into small pieces, trypsinized and plated to derive fibroblast cultures. All fibroblasts were expanded for three passages in Dulbecco's Modified Eagle Medium (DMEM) (Invitrogen) with 10% fetal bovine serum (FBS) (catalog no. S1810; Biowest) before being used for experiments.
3T3, TTF, MEFs and T98G cells were grown in high-glucose DMEM (Invitrogen) supplemented with 10% FBS, 2 mM L-glutamine (Invitrogen) and 1 mM sodium pyruvate (Sigma). HCT116 p53+/+ cells were grown in McCoy's 5a medium modified with 10% FBS. The mouse ES cell line CGR8 was from C. Crozet (Institut de Génétique Humaine, Montpellier, France) and was cultured as described in Ganier et al. [Ganier et al. Proc Natl Acad Sci USA. 2011 Oct. 18; 108(42):17331-6]. Specifically, ES cells were grown on 0.1% gelatin without feeders in ES cell medium [Glasgow minimum essential medium (Invitrogen) with 10% FBS, 0.1 mM β-mercaptoethanol, 1 mM sodium pyruvate, 1% nonessential amino acids (Gibco), 2 mM L-glutamine and 1,000 U/mL Leukemia inhibitory factor (LIF) (ES-GRO)] at 37° C. in 5% CO2.
To obtain stable cell lines that express inducible ΔDboxGeminin, exponentially growing cells were infected with HIV-derived viral particles that were previously filtered and concentrated by ultracentrifugation. 48 hours after infection, cells were selected with 2 μg·mL−1 puromycin. For ES cells, clonal selection was performed to ensure ΔDboxGeminin expression upon doxycycline induction.
Primary TTFs and MEFs were infected at sub-confluence with viral particles containing pTRIPZ-ΔDboxGeminin and processed immediately for experiments.
Expression of ΔDboxGeminin was induced by adding 1-2 μg·mL−1 doxycycline (DOX, Clontech) in the medium.
Immunofluorescence
Cells were plated on coverslips, washed twice in PBS and then fixed in 3% paraformaldehyde (PFA) at room temperature (RT) for 10 min, washed with PBS, and permeabilized with PBS/0.2% Triton X-100 for 5 min except for CREST and γ-tubulin staining for which cells were fixed in cold Methanol for 5 min. Then, cells were washed three times in PBS/2% BSA for 10 min, incubated with primary antibodies diluted in blocking buffer (PBS/0.5% Tween/2% BSA) at 4° C. overnight, except for the CREST antibody (diluted in PBS/0.5% Tween/5% non-fat milk and added at RT for 1 hour). After, three washes in PBS/0.5% Tween, cells were incubated with secondary antibodies diluted in blocking buffer for 1 hour and DNA stained with Hoechst. Cells were mounted on glass slides with Prolong (Sigma-Aldrich). For co-staining with EdU, EdU was detected prior to immune-detection according to the manufacturer's instructions.
Stainings were observed using widefield fluorescence microscopes (Leica, Germany and Carl Zeiss, Germany) and 63×1.4NA PL APO lenses. Filter sets allowing quadruple staining were used (Carl Zeiss FS49, FS38HE, FS43 and FS50). Images were acquired using a Hamamatsu ORCA Flash4 sCMOS camera. For chromosomes counting using CREST staining, ES cells were fixed and stained as described above for EdU and CREST 24 hours after ΔDboxGeminin induction. Images were acquired with the Zeiss Axioimager Z3 Apotome and stacks were acquired according to the Nyquist criterion. For better contrast, Grid Projection Illumination Microscopy (aka Apotome) was used. 3D reconstruction was performed using Imarls software (Bitplane, Oxford Instruments). Briefly the nuclear envelope was modelized using isosurface detection and CREST foci were detected using spot detection. Number of spots/envelope were automatically derived using the split into surface object Xtension.
3D-SIM imaging was performed using an OMX-V3 microscope (General Electrics) equipped with 405 nm, 488 nm and 561 nm lasers and the corresponding dichroic and filter sets. Far Red channel, used to detect EdU incorporation, was acquired in widefield mode only. Reconstruction and alignment of the 3D-SIM images was carried out with softWoRx v 5.0 (General Electrics). 100 nm green fluorescent beads (Life Technologies) were used to measure the optical transfer function (o.t.f.) used for the 405 and 488 channels, and 100 nm red fluorescent beads (Life Technologies) were used to measure the o.t.f. used for the 561 channel. 170 nm TetraSpeck beads (Life Technologies) were employed to measure the offsets and rotation parameters used in the image registration. Reconstructed 3D-SIM images were analyzed using Image J 1.4.7v software.
Metaphase Spreads
pTRIPZ-ΔDboxGeminin-infected MEFs were synchronized in G0 by confluence and then released by splitting them in fresh medium containing 15% FBS+10 μM EdU with or without DOX. 15 hours later, 100 nM Karyomax was added in the medium for 7 hours. Then, metaphase spreads were prepared as described in Eot-Houllier et al. [Eot-Houllier et al Genes Dev. 2008 Oct. 1; 22(19):2639-44], fixed and processed for EdU detection and DNA staining.
Videomicroscopy
For video microscopy experiments, ΔDboxGeminin 3T3 cells were infected with viruses encoding H2B-GFP and processed for time lapse experiments without selection. 3T3 cells were seeded on μ-Dish 35 mm, high Grid-50 Glass Bottom gridded coverslips and were synchronized by serum starvation, as described above, and released in the presence of 10 μM EdU and DOX or not. Tilescan Timelapse images were acquired every 5 minutes for 20 h. To ensure a large sample size, the four, whole quadrants of the Ibidi dish were recorded, then fixed and processed for EdU detection as described above.
Flow Cytometry
For PI, BrdU/PI and ser10PH3/PI experiments, cells were fixed in cold 70% Ethanol/PBS and then processed for indirect immunofluorescence, as described above. To detect cyclin B1/PI, MPM2/PI and FLAG/PI, cells were fixed in 3% PFA in PBS at RT for 15 min, permeabilized in 0.5% Triton X-100 for 5 min and processed for indirect immunofluorescence. For DNA staining, cells were treated with 50 μg·mL−1 RNase A (Sigma, R6513) and stained with 25 μg·mL−1 propidium iodide.
For BrdU detection, cells were incubated for the indicated times before harvesting and fixation. Then, cells were treated with 2N HCl for 30 min and washed in PBS 0.2% Tween (PBS-T) with 5% BSA before BrdU detection.
For EdU/ser10PH3/DRAQ5 detection, fixed cells were first processed for EdU detection following the manufacturer's instructions, and then incubated with the anti-ser10PH3 antibody in PBS-T 5% BSA at 4° C. overnight. Cells were then washed twice in PBS-T and incubated at RT with the anti-rabbit PE antibody (1/200 in PBS-T/5% BSA) and after two washes in PBS-T, stained with 2.5 μm DRAQ5 (DNA) and 50 μg·mL−1 RNase A at RT for 1 hour.
Cells were analyzed with a FACSCalibur flow cytometer using the CellQuestPro software and a Miltenyi MACS quant cytometer.
Cell Synchronization and Treatments
To synchronize 3T3 cells in G0/G1, cells were incubated in serum-depleted medium (0.5% FBS) for 36 hours. Cells were then released in medium supplemented with 15% FBS+/−DOX and +/−10 μM EdU. Primary TTFs and MEFs were synchronized by confluence 16 hours after infection and released by splitting them (1:4 ratio).
To express CDT1, 3T3 cells were transfected with 5 μg of plasmid pCDNA3-CDT1 or empty vector using Nucleofector TN II; Amaxa Biosystem; AAD-1001N. To induce DNA damage, synchronized cells were exposed to UV (0.002 joules) using the Bioblock Scientific BLX-254 upon release or incubated with 2 mM hydroxyurea for 20 hours. To block mitotic progression, 9 μM RO-3306 was added in the medium upon G0/G1 release.
Cell Proliferation Curves:
At day 0, 103 cells (3T3, T98G, HCT116) were seeded at sub-confluence and then harvested every day for cell counting. Values represent the average of three experiments±SD.
Senescence-Associated β-Galactosidase Staining
Sub-confluent asynchronous mouse TTFs were infected with ΔDboxGeminin-encoding viral particles and cultured in the presence, or not, of DOX for 14 days. Senescence-associated β-galactosidase (SA-β-gal) activity was assessed as described [Debacq-Chainiaux et al., 2009, Nature Protocols 4, 1798-1806]. Briefly, cells were washed twice in PBS, fixed in 2% formaldehyde-0.2% glutaraldehyde at RT for 5 min, washed twice with PBS and incubated at 37° C. in freshly prepared SA-β-gal stain solution (1 mg·mL−1 of X-gal; 40 mM citric acid; sodium phosphate, pH 6.0; 5 mM K4[Fe(CN)6]3H2O, 5 mM K3[Fe(CN)6]; 150 mM NaCl and 2 mM MgCl2) overnight. Cells were washed with PBS, fixed with methanol and air-dried before analysis.
Western Blotting
To prepare total cell extracts, cells were harvested, washed twice with PBS, lysed in 2× Laemmli buffer, sonicated (3×30 seconds) and boiled for 5 min. To obtain chromatin-enriched fractions, a protocol adapted from Lutzmann et al. [Lutzmann et al. 2012 Molecular Cell, 47, 523-534] was used. Briefly, cells were harvested, washed with PBS and lysed on ice in CSK buffer (150 mM NaCl, 10 mM HEPES pH 7.5, 300 mM Sucrose, 1 mM MgCl2, 1 mM EDTA, 1 mM ATP.MgCl2, 1 mM DTT, 0.5% Triton X-100, phosphatase-inhibitors [Calbiochem], the protease inhibitors leupeptin, aprotinin, and pepstatin at a final concentration of 10 μg/mL) for 30 min. Lysed cells were then centrifuged at 3800 g, 4° C. for 5 min to obtain the soluble fraction. Pellets were washed twice with CSK buffer on ice for 5 min, centrifuged at 3800 g, 4° C. for 5 min, solubilized in 2× Laemmli Sample Buffer, then sonicated (3×30 s) and boiled for 5 min.
Testis Extracts
Sv129/B6 mice were euthanized at the indicated ages and testis protein extracts were prepared in RIPA buffer (25 mM Tris, pH 7-8; 150 mM NaCl; 0.1% SDS; 0.5% sodium deoxycholate; 1% Triton X-100; the protease inhibitors leupeptin, aprotinin and pepstatin at a final concentration of 10 μg/mL).
Introduction
The tight coordination between DNA replication and cell division allows the replication of the whole genome before each cell division, thus ensuring its accurate transmission.
This is achieved through the ordered succession of the different cell cycle phases that is regulated by several checkpoints to prevent mitotic entry during genome replication or in the case of DNA damage. DNA damage is sensed by the DNA damage checkpoint that relies on the kinases ATM and ATR to activate CHK1 or CHK2. This results in the arrest of cell cycle progression through inhibition of cyclin-dependent kinases (CDKs) and p53 stabilization that promotes transcription of cell cycle inhibitors, such as p21 and p27 (reviewed in) (Zhou and Elledge, 2000). Proper coupling of M phase to S phase is also ensured by a G2-M checkpoint that prevents premature cell entry into mitosis until DNA replication is completed (Eykelenboom et al., 2013; Sorensen et al., 2003; Taylor et al., 1999; Zachos et al., 2005; Zuazua-Villar et al., 2014). More recently, the concept of a licensing checkpoint during G1 has emerged (reviewed in Ge and Blow, 2009; Hills and Diffley, 2014; McIntosh and Blow, 2012). The assembly of a prereplication complex (pre-RC) on origins of replication (DNA replication origin licensing) occurs in late mitosis-early G1 and is the first step towards DNA replication. During pre-RC assembly, first Origin Recognition Complex (ORC) binds to replication origins. This is followed by CDCl6 and CDT1 recruitment, thus enabling the loading of the replicative MCM2-7 helicase (reviewed in Fragkos et al., 2015). Upon S phase entry, CDKs activate replicative complexes, while inhibiting further licensing, thus ensuring one single round of DNA replication per cell cycle. CDT1 is one of the most regulated pre-RC components. Upon replication fork activation, chromatin-bound CDT1 is degraded and CDT1 binding to chromatin is prevented by geminin, an APC/C target that is actively degraded in G1 and only expressed from S phase to mitosis. This contributes to prevent DNA replication re-initiation during S phase by restricting licensing to G1 (reviewed in Fragkos et al., 2015; Machida et al., 2005a). During the S phase, cells delay the activation of mitotic cyclins/CDKs until DNA replication has been completed (Eykelenboom et al., 2013).
The licensing checkpoint concept arose from experiments showing that in primarymammalian cells, inhibition of the licensing reaction induces the p53-p21 axis and blocks cell cycle progression in a G1-like state in somatic cells, whereas it triggers the death of cancer cells (Lau and Jiang, 2006; Nevis et al., 2009; Shreeram et al., 2002, reviewed in McIntosh and Blow, 2012). This led to the hypothesis that this postulated checkpoint ensures the licensing of the correct number of origins in G1, ready to be activated before S phase onset (Lau and Jiang, 2006; Lau et al., 2006; Liu et al., 2009; Lunn et al., 2010; Machida et al., 2005b; Nevis et al., 2009; Shreeram et al., 2002; Teer et al., 2006; Yoshida et al., 2004). The licensing checkpoint, like several other checkpoints, seems to be absent also during early development, (reviewed in Kermi et al., 2017). As observed in cancer cells, inhibition of DNA licensing during early development in Xenopus laevis or Drosophila does not lead to cell cycle arrest, but rather to death of cells that enter mitosis with an incompletely replicated genome (McCleland et al., 2009; McGarry and Kirschner, 1998; Whittaker et al., 2000). Furthermore, in haploid yeast cells, a partial DNA licensing block causes abortive replication and DNA damage checkpoint induction, whereas complete inhibition of DNA licensing in haploid yeast cells that carry a null allele of CDT1 or CDCl6 leads to the occurrence of reductive mitoses and the generation of daughter cells with a reduced number of chromosomes (less than 1C) (Hofmann and Beach, 1994; Kelly et al., 1993; Piatti et al., 1995). The absence of licensing checkpoint in yeast has been however proposed to be due to the lack p53 and geminin in yeast cells (Hills and Diffley, 2014; Machida et al., 2005a; Nevis et al., 2009).
Here, the inventors show that, in mammalian somatic cells, complete inhibition of DNA licensing obtained by inducible expression of a non-degradable geminin variant is not sensed by any checkpoint. Consequently, unlicensed cells skip the S phase and enter mitosis despite the absence of DNA replication. Most of these cells cannot complete cell division and subsequently stop proliferating and remain in a senescent G1 state. However, some can successfully terminate cell division, thus producing daughter cells that contain only half of the normal diploid complement of chromosomes (1C). Importantly, these 1C cells can re-enter into the cell cycle upon restoration of the licensing reaction by CDT1 overexpression. These observations might pave the way to new approaches to generate haploid cells from a wide range of somatic cells. To date, it is possible to obtain haploid cells by derivating haploid embryonic stem cells from an activated blastocyst (Leeb 2011, Nature). If such haploid embryonic stem cells survive and can be propagated in vitro, they appeared to be incapable to sustain an efficient and proper differentiation (Leeb 2011). Therefore, obtaining viable and stable haploid somatic cells from different cell types—or already existing stable cell lines—has not been achieved so far. The present invention aims to counteract this roadblock by producing haploid from somatic cells that are already differentiated—or engaged into differentiation.
Results
ΔDboxGeminin Expression Stops Cell Proliferation of Normal Cells, but does not Induce Checkpoints
To study the molecular mechanisms that trigger the licensing checkpoint in normal mammalian cells, the inventors generated different stable cell lines where expression of a geminin mutant lacking the Destruction box (ΔDboxGeminin) can be induced by doxycycline (DOX) (
As previously reported (Shreeram et al., 2002; Yoshida et al., 2004), ΔDboxGeminin expression led to massive cell death in different cancer cell lines, such as HCT116, T98G, HeLa and HEK 293 cells, as indicated by the cell number decrease, the sub-G1 population observed by cytometry and the disappearance of cells 72 hours after induction (+DOX), as observed by microscopy (
To understand the earliest mechanisms leading to the ΔDboxGeminin-triggered cell cycle arrest in normal cells, the inventors induced or not (+/− DOX) ΔDboxGeminin expression in synchronized 3T3 cells released from quiescence (
It has been reported that inhibition of DNA replication licensing leads to cell cycle arrest that relies on p53 and p21 induction (Shreeram et al., 2002; Yoshida et al., 2004).
However, despite the efficient inhibition of licensing and replication upon ΔDboxGeminin expression, the inventors could not detect induction of the licensing checkpoint, p53 phosphorylation and p21 upregulation, at least during the first cell cycle after DOX addition (
Mitotic Progression in the Absence of DNA Replication Licensing in Somatic Cells
In line with the absence of licensing checkpoint induction, cell cycle was not blocked in ΔDboxGeminin-expressing cells. Markers of cell cycle entry showed similar kinetics in ΔDboxGeminin-expressing and control cells (no DOX) (
The inventors then asked whether unlicensed cells could enter mitosis despite the absence of DNA replication. They synchronized 3T3 fibroblasts in G0/G1 and released them in the presence of the nucleotide analogue EdU, to monitor DNA replication during the whole cell cycle (
To further understand how mitosis could happen in cells in which licensing was inhibited, the inventors analyzed the DNA content of EdU-negative mitotic cells (as visualized by ser10PH3 expression) by flow cytometry at different time points after release from G0. In control cells (−DOX), the inventors detected ser10PH3 only in 4C cells, but never in the 2C population (
As geminin blocks initiation of DNA replication by inhibiting CDT1 (Wohlschlegel et al., 2000), the inventors then asked whether CDT1 inhibition by RNA interference (shCDT1) would result in the same phenotype as upon ΔDboxGeminin expression induction (
Taken together, these data show that in somatic mammalian cells, geminin stabilization or CDT1 inhibition during the G1 phase can trigger mitotic entry of unlicensed cells, despite the inhibition of DNA replication.
A Discrete Population of Unlicensed Cells can Successfully Divide
The inventors then investigated whether unlicensed cells that entered mitosis could divide. To this aim, the inventors prepared metaphase spreads of MEFs 10 hours after synchronization in G0/G1 and release in the presence of EdU (with or without DOX). Control cells (−ΔDboxGem) had 40 pairs of chromosomes with the two replicated (EdU-positive) sister chromatids attached by the kinetochore (
In summary, although most unlicensed cells cannot complete cell division, about one quarter can produce two daughter cells. The inventors then assessed the fate of these two cell populations.
Mitotic Division Failure in Unlicensed Mitotic Cells Leads to a Long-Term Irreversible G1 Arrest with Features of Senescence
To investigate the fate of unlicensed 3T3 cells that entered mitosis but did not complete cell division, the inventors analyzed them by immunofluorescence at different time points after release from G0 in the presence or not of DOX. Quantification of the percentage of ser10PH3-positive cells showed that the first wave of mitoses started 14 h post-release with a peak after 17 h (
The inventors' data indicate that the population of unlicensed mitotic cells that failed cell division is subsequently blocked in G1. This is associated with atypical nucleolar DNA damage, induction of the DNA damage checkpoint and features of senescence that only appear after the first mitotic wave. Therefore, induction of p53 and p21 in unlicensed cells is not primarily triggered by lack of licensed origins, but rather by mitotic failure-induced DNA damage, as observed after abortive completion of normal (40 DNA content, postreplicative) mitoses (Hayashi and Karlseder, 2013; Panopoulos et al., 2014).
Division of Unlicensed Cells Leads to a Cell Population with 1C DNA Content.
The fraction of unlicensed cells (24%) that managed to divide produced two daughter cells, despite the complete absence of DNA replication. If the unlicensed genome were equally distributed in each of the two daughter cells originating from a mitotic cell with 2C DNA content, these cells should have 10 DNA content. Indeed, the inventors flow cytometry analyses always highlighted the presence of a distinct cell population among ΔDboxGeminin-expressing cells with 10 DNA content that appeared after the first cell cycle (arrows in
To determine the number of chromosomes in this cell population, the inventors counted the number of kinetochores stained by the CREST polyclonal antibody in mouse embryonic stem cells that exhibit a more stable diploid karyotype than 3T3 fibroblasts and MEFs. In the inventors conditions, the inventors found an average of 31.5 dots per nucleus in control diploid cells (theoretically, 40 dots, one per chromosome, are expected) (
The 1C Cell Population can Re-Enter S Phase
Finally, the inventors investigated whether 10 cells could re-enter the cell cycle after stopping ΔDboxGeminin action. First, the inventors removed DOX from the medium 24 hours after induction, to stop ΔDboxGeminin expression (T1,
To rule out the possibility that these BrdU-positive 10 cells corresponded to diploid cells that replicated and died during the 24 hours preceding cell harvesting, the inventors performed a short BrdU pulse (15 minutes) just before cell collection at T2. In these conditions, 8.3% of 10 cells transfected with CDT1 were still actively replicating compared with 2.7% of cells transfected with empty vector (
Taken together, these data show that in mammalian cells, reductive division of unlicensed mitotic cells can lead to the appearance of 10 daughter cells that can survive and re-enter into the cell cycle if the licensing reaction is rescued.
Discussion
Revisiting the Licensing Checkpoint
The existence of a mammalian somatic cell-specific licensing checkpoint has been suggested by the G1-like block observed in normal cells in which licensing was inhibited by siRNA-based approaches (Lau et al., 2009; Liu et al., 2009; Lunn et al., 2010; Machida et al., 2005b; Nevis et al., 2009; Teer et al., 2006). As most of the pre-RC components are highly stable in G1 as soon as they are bound to origins, it is challenging to completely stop the licensing reaction in a single cell cycle by interfering with their expression during pre-RC formation. This aspect is crucial because previous observations made in yeast show that a licensed origin will fire regardless of the number of licensed origins (Piatti et al., 1995; Shimada et al., 2002). Therefore, only a complete pre-RC inhibition can prevent the activation of the intra S-phase checkpoint. Here, the inventors took advantage of different mammalian cell lines that express an inducible and not degradable geminin to completely block replication licensing in a single cell cycle. The inventors data show that in mammalian somatic cells, complete inhibition of CDT1 action and therefore of licensing in G1 is not sensed by any cell cycle checkpoint during the first cell cycle, leading to the entry of unlicensed cells into mitosis. This suggests that in mammalian somatic cells, a licensing checkpoint is not activated when CDT1 action is fully prevented. As the DNA content of unlicensed cells remains unchanged despite their cell cycle progression, these cells cannot be distinguished from a typical G0/G1 population by quantifying the DNA content, and could easily be missed when only measuring the DNA content using a single flow cytometry approach.
G1-Like Cell Cycle Arrest Upon Licensing Inhibition
As illustrated in
Licensing Inhibition to Generate Haploid Mammalian Cells
The inventors' data show that some unlicensed cells can establish a mitotic spindle and divide despite the absence of DNA replication. This is in line with the observation that the spindle assembly checkpoint can be satisfied even in the absence of DNA replication in cancer cells after DNA replication and checkpoint inhibition (Brinkley et al., 1988; O'Connell et al., 2009) and in immortalized Drosophila embryonic S2 cells (Drpic et al., 2015). Importantly, some unlicensed mammalian somatic cells can eventually complete mitosis, giving raise to two daughter cells. It is also worth noting that geminin expression is strongly upregulated during testis gametogenesis, particularly when meiosis is induced (
The 1C daughter cells derived from unlicensed somatic mammalian cells are viable and show a relative homogenous DNA content. Some of them contain up to 20 chromosomes, suggesting that they could be fully haploid. Moreover, after reestablishment of the licensing reaction, these cells can enter the cell cycle and even replicate DNA. The inventors' demonstrate a new experimental approach to generate haploid cells from a wide range of somatic mammalian cells that could become a powerful tool for the development of genetic and drug screens (Carette et al., 2009; Sagi et al., 2016).
The invention is not limited to the embodiments described above and other embodiments will clearly appear from the specification to the skilled person.
| Number | Date | Country | Kind |
|---|---|---|---|
| 17305841.3 | Jun 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/067811 | 7/2/2018 | WO | 00 |
