Reshaped DNA methylation cooperating with homoeolog-divergent expression promotes improved root traits in synthesized tetraploid wheat.

Polyploidization is a major event driving plant evolution and domestication. However, how reshaped epigenetic modifications coordinate gene transcription to generate phenotypic variations during wheat polyploidization is currently elusive. Here, we profiled transcriptomes and DNA methylomes of two diploid wheat accessions (SlSl and AA) and their synthetic allotetraploid wheat line (SlSlAA), which displayed elongated root hair and improved root capability for nitrate uptake and assimilation after tetraploidization. Globally decreased DNA methylation levels with a reduced difference between subgenomes were observed in the roots of SlSlAA. DNA methylation changes in first exon showed strong connections with altered transcription during tetraploidization. Homoeolog-specific transcription was associated with biased DNA methylation as shaped by homoeologous sequence variation. The hypomethylated promoters showed significantly enriched binding sites for MYB, which may affect gene transcription in response to root hair growth. Two master regulators in root hair elongation pathway, AlCPC and TuRSL4, exhibited upregulated transcription levels accompanied by hypomethylation in promoter, which may contribute to the elongated root hair. The upregulated nitrate transporter genes, including NPFs and NRTs, also are significantly associated with hypomethylation, indicating an epigenetic-incorporated regulation manner in improving nitrogen use efficiency. Collectively, these results provided new insights into epigenetic changes in response to crop polyploidization and underscored the importance of epigenetic regulation in improving crop traits.

Whole-Genome Duplication and Genome Instability in Cancer Cells: Double the Trouble.

Whole-genome duplication (WGD) is one of the most common genomic abnormalities in cancers. WGD can provide a source of redundant genes to buffer the deleterious effect of somatic alterations and facilitate clonal evolution in cancer cells. The extra DNA and centrosome burden after WGD is associated with an elevation of genome instability. Causes of genome instability are multifaceted and occur throughout the cell cycle. Among these are DNA damage caused by the abortive mitosis that initially triggers tetraploidization, replication stress and DNA damage associated with an enlarged genome, and chromosomal instability during the subsequent mitosis in the presence of extra centrosomes and altered spindle morphology. Here, we chronicle the events after WGD, from tetraploidization instigated by abortive mitosis including mitotic slippage and cytokinesis failure to the replication of the tetraploid genome, and finally, to the mitosis in the presence of supernumerary centrosomes. A recurring theme is the ability of some cancer cells to overcome the obstacles in place for preventing WGD. The underlying mechanisms range from the attenuation of the p53-dependent G1 checkpoint to enabling pseudobipolar spindle formation via the clustering of supernumerary centrosomes. These survival tactics and the resulting genome instability confer a subset of polyploid cancer cells proliferative advantage over their diploid counterparts and the development of therapeutic resistance.

Protein kinase C family evolution in jawed vertebrates.

Protein kinase C (PKC) was one of the first kinases identified in human cells. It is now known to constitute a family of kinases that respond to diacylglycerol, phosphatidylserine and for some family members, Ca2+. They have a plethora of different functions, such as cell cycle regulation, immune response and memory formation. In mammals, 12 PKC family members have been described, usually divided into 4 different subfamilies. We present here a comprehensive evolutionary analysis of the PKC genes in jawed vertebrates with special focus on the impact of the two tetraploidizations (1R and 2R) before the radiation of jawed vertebrates and the teleost tetraploidization (3R), as illuminated by synteny and paralogon analysis including many neighboring gene families. We conclude that the vertebrate predecessor had five PKC genes, as tunicates and lancelets still do, and that the PKC family should therefore ideally be organized into five subfamilies. The 1R and 2R events led to a total of 12 genes distributed among these five subfamilies. All 12 genes are still present in some of the major lineages of jawed vertebrates, including mammals, whereas birds and cartilaginous fishes have lost one member. The 3R event added another nine genes in teleosts, bringing the total to 21 genes. The zebrafish, a common experimental model animal, has retained 19. We have found no independent gene duplications. Thus, the genome doublings completely account for the complexity of this gene family in jawed vertebrates and have thereby had a huge impact on their evolution.

A tetraploidization event shaped the Aquilaria sinensis genome and contributed to the ability of sesquiterpenes synthesis.

BACKGROUND: Agarwood, generated from the Aquilaria sinensis, has high economic and medicinal value. Although its genome has been sequenced, the ploidy of A. sinensis paleopolyploid remains unclear. Moreover, the expression changes of genes associated with agarwood formation were not analyzed either. RESULTS: In the present work, we reanalyzed the genome of A. sinensis and found that it experienced a recent tetraploidization event ~ 63-71 million years ago (Mya). The results also demonstrated that the A. sinensis genome had suffered extensive gene deletion or relocation after the tetraploidization event, and exhibited accelerated evolutionary rates. At the same time, an alignment of homologous genes related to different events of polyploidization and speciation were generated as well, which provides an important comparative genomics resource for Thymelaeaceae and related families. Interestingly, the expression changes of genes related to sesquiterpene synthesis in wounded stems of A. sinensis were also observed. Further analysis demonstrated that polyploidization promotes the functional differentiation of the key genes in the sesquiterpene synthesis pathway. CONCLUSIONS: By reanalyzing its genome, we found that the tetraploidization event shaped the A. sinensis genome and contributed to the ability of sesquiterpenes synthesis. We hope that these results will facilitate our understanding of the evolution of A. sinensis and the function of genes involved in agarwood formation.

Hyperactivation of cyclin A-CDK induces centrosome overduplication and chromosome tetraploidization in mouse cells.

Mammalian cyclin A-CDK (cyclin-dependent kinase) activity during mitotic exit is regulated by two redundant pathways, cyclin degradation and CDK inhibitors (CKIs). Ectopic expression of a destruction box-truncated (thereby stabilized) mutant of cyclin A in the mouse embryonic fibroblasts nullizygous for three CKIs (p21, p27, and p107) results in constitutive activation ("hyperactivation") of cyclin A-CDK and induces rapid tetraploidization, suggesting loss of the two redundant pathways causes genomic instability. To elucidate the mechanism underlying teraploidization by hyperactive cyclin A-CDK, we first examined if the induction of tetraploidization depends on specific cell cycle stage(s). Arresting the cell cycle at either S phase or M phase blocked the induction of tetraploidization, which was restored by subsequent release from the arrest. These results suggest that both S- and M-phase progressions are necessary for the tetraploidization by hyperactive cyclin A-CDK and that the tetraploidization is not caused by chromosome endoreduplication but by mitotic failure. We also observed that the induction of tetraploidization is associated with excessive duplication of centrosomes, which was suppressed by S-phase but not M-phase block, suggesting that hyperactive cyclin A-CDK promotes centrosome overduplication during S phase. Time-lapse microscopy revealed that hyperactive cyclin A-CDK can lead cells to bypass cell division and enter pseudo-G1 state. These observations implicate that hyperactive cyclin A-CDK causes centrosome overduplication, which leads to mitotic slippage and subsequent tetraploidization.

Femtosecond laser-induced blastomere fusion results in embryo tetraploidy by common metaphase plate formation.

The cell fusion is a widespread process, which takes place in many systems in vivo and in vitro. Fusion of cells is frequently related to tetraploidy, which can be found within natural physiological conditions, e.g., placentation, and in pathophysiological conditions, such as cancer and early pregnancy failure in humans. Here we investigate the mechanism of tetraploidization with help of femtosecond laser-induced mouse blastomere fusion by the means of Hoechst staining, GFP, BODIPY dyes and fluorescent species generated intracellularly by a femtosecond laser. We establish diffusive mixing of cytosol, whereas the large components of a cytoplasm (organelles, cytoskeleton) are poorly diffusible and are not completely mixed after cell fusion and a subsequent division. We show that mechanisms which are responsible for the formation of a common metaphase plate triggered tetraploidization in fused mouse embryos and could be a significant factor in polyploidy formation in vivo. Thus, our results suggest that microtubules play a critical role in tetraploidization.

Evolution of vertebrate nicotinic acetylcholine receptors.

BACKGROUND: Many physiological processes are influenced by nicotinic acetylcholine receptors (nAChR), ranging from neuromuscular and parasympathetic signaling to modulation of the reward system and long-term memory. Due to the complexity of the nAChR family and variable evolutionary rates among its members, their evolution in vertebrates has been difficult to resolve. In order to understand how and when the nAChR genes arose, we have used a broad approach of analyses combining sequence-based phylogeny, chromosomal synteny and intron positions. RESULTS: Our analyses suggest that there were ten subunit genes present in the vertebrate predecessor. The two basal vertebrate tetraploidizations (1R and 2R) then expanded this set to 19 genes. Three of these have been lost in mammals, resulting in 16 members today. None of the ten ancestral genes have kept all four copies after 2R. Following 2R, two of the ancestral genes became triplicates, five of them became pairs, and three seem to have remained single genes. One triplet consists of CHRNA7, CHRNA8 and the previously undescribed CHRNA11, of which the two latter have been lost in mammals but are still present in lizards and ray-finned fishes. The other triplet consists of CHRNB2, CHRNB4 and CHRNB5, the latter of which has also been lost in mammals. In ray-finned fish the neuromuscular subunit gene CHRNB1 underwent a local gene duplication generating CHRNB1.2. The third tetraploidization in the predecessor of teleosts (3R) expanded the repertoire to a total of 31 genes, of which 27 remain in zebrafish. These evolutionary relationships are supported by the exon-intron organization of the genes. CONCLUSION: The tetraploidizations explain all gene duplication events in vertebrates except two. This indicates that the genome doublings have had a substantial impact on the complexity of this gene family leading to a very large number of members that have existed for hundreds of millions of years.

Asymmetrically reduced expression of hand1 homeologs involving a single nucleotide substitution in a cis-regulatory element.

During vertebrate evolution, whole genome duplications resulted in a number of duplicated genes, some of which eventually changed their expression patterns and/or levels via alteration of cis-regulatory sequences. However, the initial process involved in such cis-regulatory changes remains unclear. Therefore, we investigated this process by analyzing the duplicated hand1 genes of Xenopus laevis (hand1.L and hand1.S), which were generated by allotetraploidization 17-18 million years ago, and compared these with their single ortholog in the ancestral-type diploid species X. tropicalis. A dN/dS analysis indicated that hand1.L and hand1.S are still under purifying selection, and thus, their products appear to retain ancestral functional properties. RNA-seq and in situ hybridization analyses revealed that hand1.L and hand1.S have similar expression patterns to each other and to X. tropicalis hand1, but the hand1.S expression level was much lower than the hand1.L expression level in the primordial heart. A comparative sequence analysis, luciferase reporter analysis, ChIP-PCR analysis, and transgenic reporter analysis showed that a single nucleotide substitution in the hand1.S promoter was responsible for the reduced expression in the heart. These findings demonstrated that a small change in the promoter sequence can trigger diversification of duplicated gene expression prior to diversification of their encoded protein functions in a young duplicated genome.

Phenotypic effects of allotetraploidization of wild Arachis and their implications for peanut domestication.

PREMISE OF THE STUDY: Several species of Arachis have been cultivated for their edible seeds, historically and to the present day. The diploid species that have a history of cultivation show relatively small signatures of domestication. In contrast, the tetraploid species A. hypogaea evolved into highly domesticated forms and became a major world crop, the cultivated peanut. It seems likely that allotetraploidization (hybridity and/or tetraploidization) in some way enhanced attractiveness for cultivation. Here we investigate this using six different hybridization and tetraploidization events, from distinct Arachis diploid species, including one event derived from the same wild species that originated peanut. METHODS: Twenty-six anatomical, morphological, and physiological traits were examined in the induced allotetraploid plants and compared with their wild diploid parents. KEY RESULTS: Nineteen traits were transgressive (showed strong response to hybridization and chromosome duplication): allotetraploids had larger leaves, stomata and epidermal cells than did their diploid parents. In addition, allotetraploids produced more photosynthetic pigments. These traits have the same trend across the different hybrid combinations, suggesting that the changes are more likely due to ploidy rather than hybridity. In contrast, seed dimensions and seed mass did not significantly change in response to hybridization or tetraploidization. CONCLUSIONS: We suggest that the original allotetraploid that gave rise to cultivated peanut may have been attractive because of an increase in plant size, different transpiration characteristics, higher photosynthetic capacity, or other characteristics, but contrary to accepted knowledge, increased seed size was unlikely to have been important in the initial domestication.

Proliferation following tetraploidization regulates the size and number of erythrocytes in the blood flow during medaka development, as revealed by the abnormal karyotype of erythrocytes in the medaka TFDP1 mutant.

BACKGROUND: For the delivery of oxygen, the correct size/number of erythrocytes is required for proper blood flow. RESULTS: By combined analyses of wild-type (WT) medaka and the kyoho (kyo) mutant, we found proliferation-mediated adaptation for size/number of erythrocytes in the blood flow during medaka development. Before the start of heart beating in the WT medaka, the karyotype of erythrocytes was 2N-4N. After the start of blood flow, the karyotype changed to 4N-8N with tetraploidization, and the cell size became larger. After the start of intersegmental and pharyngeal blood flow, the erythrocytes became smaller. The medaka mutant kyo showed erythrocytes of large size, and positional cloning of kyo demonstrated the candidate gene TFDP1, indicating higher polyploidization due to arrest in S-phase in erythrocytes of the kyo mutant. CONCLUSIONS: From our findings, we uncovered a previously unrecognized system for the regulation of the size/number in the blood flow:proliferation of erythrocytes following tetraploidization during embryonic development.

Evolution of the gastrin-cholecystokinin gene family revealed by synteny analysis.

Gastrin (GAST) and cholecystokinin (CCK) are two structurally and functionally related peptide hormones that exert many functions, including regulation of gastric and pancreatic secretion, feeding behaviour and energy homeostasis. GAST and CCK genes are assumed to have diverged from a common ancestral gene, over 500 million years ago in the vertebrate lineage. However, although a large number of GAST and CCK-related sequences have been identified both in vertebrate and non-vertebrate species, the evolutionary history of the GAST/CCK family remains little understood. To address this issue, we used extensive genome synteny comparisons of vertebrate chromosomes, in particular to evaluate the impact of whole-genome duplications. In the present study, we confirm that the GAST/CCK family in vertebrates is composed of two paralogous genes, namely GAST and CCK, and even three in teleosts, namely GAST, CCK1 and CCK2. We also show that the GAST and CCK genes arose by duplications of a single ancestral gene through the 2R and that the two copies of the CCK gene found in teleosts have probably been generated through the 3R. Finally, our results suggest that the vertebrate ancestor possessed four members of the GAST/CCK family, of which two have likely been lost during evolution.

Phylogenetic analysis of AGAMOUS sequences reveals the origin of the diploid and tetraploid forms of self-pollinating wild buckwheat, Fagopyrum homotropicum Ohnishi.

Fagopyrum homotropicum Ohnishi is a self-pollinating wild buckwheat species indigenous to eastern Tibet and the Yunnan and Sichuan Provinces of China. It is useful breeding material for shifting cultivated buckwheat (F. esculentum ssp. esculentum Moench) from out-crossing to self-pollinating. Despite its importance as a genetic resource in buckwheat breeding, the genetic variation of F. homotropicum is poorly understood. In this study, we investigated the genetic variation and phylogenetic relationships of the diploid and tetraploid forms of F. homotropicum based on the nucleotide sequences of a nuclear gene, AGAMOUS (AG). Neighbor-joining analysis revealed that representative individuals clustered into three large groups (Group I, II and III). Each group contained diploid and tetraploid forms of F. homotropicum. We identified tetraploid plants that had two diverged AG sequences; one belonging to Group I and the other belonging to Group II, or one belonging to Group II and the other belonging to Group III. These results suggest that the tetraploid form originated from at least two hybridization events between deeply differentiated diploids. The results also imply that the genetic diversity contributed by tetraploidization of differentiated diploids may have allowed the distribution range of F. homotropicum to expand to the northern areas of China.

Dynamic evolution of transient receptor potential vanilloid (TRPV) ion channel family with numerous gene duplications and losses.

The transient receptor potential vanilloid (TRPV) ion channel family is involved in multiple sensory and physiological functions including thermosensing and temperature-dependent neuroendocrine regulation. The objective of the present study was to investigate the number, origin and evolution of TRPV genes in metazoans, with special focus on the impact of the vertebrate whole-genome duplications (WGD). Gene searches followed by phylogenetic and synteny analyses revealed multiple previously undescribed TRPV genes. The common ancestor of Cnidaria and Bilateria had three TRPV genes that became four in the deuterostome ancestor. Two of these were lost in the vertebrate ancestor. The remaining two genes gave rise to two TRPV subfamilies in vertebrates, consisting of subtypes 1, 2, 3, 4, 9 and 5, 6, 7, 8, respectively. This gene expansion resulted from the two basal vertebrate WGD events (1R and 2R) and three local duplications before the radiation of gnathostomes. TRPV1, 4 and 5 have been retained in all gnathostomes investigated, presumably reflecting important functions. TRPV7 and 8 have been lost independently in various lineages but are still retained in cyclostomes, actinistians (coelacanth), amphibians, prototherians and basal actinopterygians (Polypteridae). TRPV3 and 9 are present in extant elasmobranchs, while TRPV9 was lost in the osteichthyan ancestor and TRPV3 in the actinopterygian ancestor. The coelacanth has retained the ancestral osteichthyan repertoire of TRPV1, 3, 4, 5, 7 and 8. TRPV2 arose in the tetrapod ancestor. Duplications of TRPV5 occurred independently in various lineages, such as cyclostomes, chondrichthyans, anuran amphibians, sauropsids, mammals (where the duplicate is called TRPV6), and actinopterygians (Polypteridae and Esocidae). After the teleost-specific WGD (3R) only TRPV1 retained its duplicate, whereas TRPV4 and 5 remained as single genes. Both 3R-paralogs of TRPV1 were kept in some teleost species, while one paralog was lost in others. The salmonid-specific WGD (4R) duplicated TRPV1, 4, and 5 leading to six TRPV genes. The largest number was found in Xenopus tropicalis with no less than 15 TRPV genes. This study provides a comprehensive evolutionary scenario for the vertebrate TRPV family, revealing additional TRPV types and proposing a phylogeny-based classification of TRPV across metazoans.

Somatic Hypomethylation of Pericentromeric SST1 Repeats and Tetraploidization in Human Colorectal Cancer Cells.

Somatic DNA hypomethylation and aneuploidy are hallmarks of cancer, and there is evidence for a causal relationship between them in knockout mice but not in human cancer. The non-mobile pericentromeric repetitive elements SST1 are hypomethylated in about 17% of human colorectal cancers (CRC) with some 5-7% exhibiting strong age-independent demethylation. We studied the frequency of genome doubling, a common event in solid tumors linked to aneuploidy, in randomly selected single cell clones of near-diploid LS174T human CRC cells differing in their level of SST1 demethylation. Near-diploid LS174T cells underwent frequent genome-doubling events generating near-tetraploid clones with lower levels of SST1 methylation. In primary CRC, strong SST1 hypomethylation was significantly associated with global genomic hypomethylation and mutations in TP53. This work uncovers the association of the naturally occurring demethylation of the SST1 pericentromeric repeat with the onset of spontaneous tetraploidization in human CRC cells in culture and with TP53 mutations in primary CRCs. Altogether, our findings provide further support for an oncogenic pathway linking somatic hypomethylation and genetic copy number alterations in a subset of human CRC.

Corticotropin-Releasing Hormone (CRH) Gene Family Duplications in Lampreys Correlate With Two Early Vertebrate Genome Doublings.

The ancestor of gnathostomes (jawed vertebrates) is generally considered to have undergone two rounds of whole genome duplication (WGD). The timing of these WGD events relative to the divergence of the closest relatives of the gnathostomes, the cyclostomes, has remained contentious. Lampreys and hagfishes are extant cyclostomes whose gene families can shed light on the relationship between the WGDs and the cyclostome-gnathostome divergence. Previously, we have characterized in detail the evolution of the gnathostome corticotropin-releasing hormone (CRH) family and found that its five members arose from two ancestral genes that existed before the WGDs. The two WGDs resulted, after secondary losses, in one triplet consisting of CRH1, CRH2, and UCN1, and one pair consisting of UCN2 and UCN3. All five genes exist in representatives for cartilaginous fishes, ray-finned fishes, and lobe-finned fishes. Differential losses have occurred in some lineages. We present here analyses of CRH-family members in lamprey and hagfish by comparing sequences and gene synteny with gnathostomes. We found five CRH-family genes in each of two lamprey species (Petromyzon marinus and Lethenteron camtschaticum) and two genes in a hagfish (Eptatretus burgeri). Synteny analyses show that all five lamprey CRH-family genes have similar chromosomal neighbors as the gnathostome genes. The most parsimonious explanation is that the lamprey CRH-family genes are orthologs of the five gnathostome genes and thus arose in the same chromosome duplications. This suggests that lampreys and gnathostomes share the same two WGD events and that these took place before the lamprey-gnathostome divergence.

Karyotype Analysis of Diploid and Spontaneously Occurring Tetraploid Blood Orange [Citrus sinensis (L.) Osbeck] Using Multicolor FISH With Repetitive DNA Sequences as Probes.

Blood orange [Citrus sinensis (L.) Osbeck] has been increasingly appreciated by consumers worldwide owing to its brilliant red color, abundant anthocyanin and other health-promoting compounds. However, there is still relatively little known about its cytogenetic characteristics, probably because of the small size and similar morphology of metaphase chromosomes and the paucity of chromosomal landmarks. In our previous study, a naturally occurring tetraploid blood orange plant was obtained via seedling screening. Before this tetraploid germplasm can be manipulated into a citrus triploid seedless breeding program, it is of great importance to determine its chromosome characterization and composition. In the present study, an integrated karyotype of blood orange was constructed using sequential multicolor fluorescence in situ hybridization (FISH) with four satellite repeats, two ribosomal DNAs (rDNAs), a centromere-like repeat and an oligonucleotide of telomere repeat (TTTAGGG)3 as probes. Satellite repeats were preferentially located at the terminal regions of the chromosomes of blood orange. Individual somatic chromosome pairs of blood orange were unambiguously identified by repetitive DNA-based multicolor FISH. These probes proved to be effective chromosomal landmarks. The karyotype was formulated as 2n = 2x = 18 = 16m+2sm (1sat) with the karyotype asymmetry degree belonging to 2B. The chromosomal distribution pattern of these repetitive DNAs in this spontaneously occurring tetraploid was identical to that of the diploid, but the tetraploid carried twice the number of hybridization sites as the diploid, indicating a possible pathway involving the spontaneous duplication of chromosome sets in nucellar cells. Our work may facilitate the molecular cytogenetic study of blood orange and provide chromosomal characterization for the future utilization of this tetraploid germplasm in the service of seedless breeding programs.

Nucleolar Dominance in a Tetraploidy Hybrid Lineage Derived From Carassius auratus red var. () × Megalobrama amblycephala ().

Nucleolar dominance is related to the expression of 45S rRNA genes inherited from one progenitor due to the silencing of the other progenitor's rRNA genes. To investigate nucleolar dominance associated with tetraploidization, we analyzed the changes regarding the genetic traits and expression of 45S rRNA genes in tetraploidy hybrid lineage including F1 allotetraploids (4n = 148) and F2 autotetraploids (4n = 200) derived from the distant hybridization of Carassius auratus red var. (2n = 100) () ×Megalobrama amblycephala (2n = 48) (). Results showed that nucleolar dominance from the females was established in F1 hybrids and it was inherited in F2 hybrids, suggesting that tetraploidization can lead to rapid establishment of nucleolar dominance in the hybrid origin's tetraploid lineage. These results extend the knowledge of nucleolar dominance in polyploidy hybrid animals, which are of significance for the evolution of hybrids in vertebrates.

Evolution of the Muscarinic Acetylcholine Receptors in Vertebrates.

The family of muscarinic acetylcholine receptors (mAChRs) consists of five members in mammals, encoded by the CHRM1-5 genes. The mAChRs are G-protein-coupled receptors, which can be divided into the following two subfamilies: M2 and M4 receptors coupling to Gi/o; and M1, M3, and M5 receptors coupling to Gq/11. However, despite the fundamental roles played by these receptors, their evolution in vertebrates has not yet been fully described. We have combined sequence-based phylogenetic analyses with comparisons of exon-intron organization and conserved synteny in order to deduce the evolution of the mAChR receptors. Our analyses verify the existence of two ancestral genes prior to the two vertebrate tetraploidizations (1R and 2R). After these events, one gene had duplicated, resulting in CHRM2 and CHRM4; and the other had triplicated, forming the CHRM1, CHRM3, and CHRM5 subfamily. All five genes are still present in all vertebrate groups investigated except the CHRM1 gene, which has not been identified in some of the teleosts or in chicken or any other birds. Interestingly, the third tetraploidization (3R) that took place in the teleost predecessor resulted in duplicates of all five mAChR genes of which all 10 are present in zebrafish. One of the copies of the CHRM2 and CHRM3 genes and both CHRM4 copies have gained introns in teleosts. Not a single separate (nontetraploidization) duplicate has been identified in any vertebrate species. These results clarify the evolution of the vertebrate mAChR family and reveal a doubled repertoire in zebrafish, inviting studies of gene neofunctionalization and subfunctionalization.

Losmapimod Overcomes Gefitinib Resistance in Non-small Cell Lung Cancer by Preventing Tetraploidization.

The epidermal growth factor receptor (EGFR) is known to play a critical role in non-small cell lung cancer (NSCLC). Constitutively active EGFR mutations, including in-frame deletion in exon 19 and L858R point mutation in exon 21, contribute about 90% of all EGFR-activating mutations in NSCLC. Although oral EGFR-tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, show dramatic clinical efficacy with significantly prolonged progression-free survival in patients harboring these EGFR-activating mutations, most of these patients will eventually develop acquired resistance. Researchers have recently named genomic instability as one of the hallmarks of cancer. Genomic instability usually involves a transient phase of polyploidization, in particular tetraploidization. Tetraploid cells can undergo asymmetric cell division or chromosome loss, leading to tumor heterogeneity and multidrug resistance. Therefore, identification of signaling pathways involved in tetraploidization is crucial in overcoming drug resistance. In our present study, we found that gefitinib could activate YAP-MKK3/6-p38 MAPK-STAT3 signaling and induce tetraploidization in gefitinib-resistance cells. Using p38 MAPK inhibitors, SB203580 and losmapimod, we could eliminate gefitinib-induced tetraploidization and overcome gefitinib-resistance. In addition, shRNA approach to knockdown p38α MAPK could prevent tetraploidy formation and showed significant inhibition of cancer cell growth. Finally, in an in vivo study, losmapimod could successfully overcome gefitinib resistance using an in-house established patient-derived xenograft (PDX) mouse model. Overall, these findings suggest that losmapimod could be a potential clinical agent to overcome gefitinib resistance in NSCLC.

Tetraploidization or autophagy: The ultimate fate of senescent human endometrial stem cells under ATM or p53 inhibition.

Previously we demonstrated that endometrium-derived human mesenchymal stem cells (hMESCs) via activation of the ATM/p53/p21/Rb pathway enter the premature senescence in response to oxidative stress. Down regulation effects of the key components of this signaling pathway, particularly ATM and p53, on a fate of stressed hMESCs have not yet been investigated. In the present study by using the specific inhibitors Ku55933 and Pifithrin-α, we confirmed implication of both ATM and p53 in H(2)O(2)-induced senescence of hMESCs. ATM or p53 down regulation was shown to modulate differently the cellular fate of H(2)O(2)-treated hMESCs. ATM inhibition allowed H(2)O(2)-stimulated hMESCs to escape the permanent cell cycle arrest due to loss of the functional ATM/p53/p21/Rb pathway, and induced bypass of mitosis and re-entry into S phase, resulting in tetraploid cells. On the contrary, suppression of the p53 transcriptional activity caused a pronounced cell death of H(2)O(2)-treated hMESCs via autophagy induction. The obtained data clearly demonstrate that down regulation of ATM or p53 shifts senescence of human endometrial stem cells toward tetraploidization or autophagy.