Variable allelic expression of imprinted genes in human pluripotent stem cells during differentiation into specialized cell types in vitro

Genomic imprinting is an epigenetic phenomenon by which a subset of genes is asymmetrically expressed in a parent-of-origin manner. However, little is known regarding the epigenetic behaviors of imprinted genes during human development. Here, we show dynamic epigenetic changes in imprinted genes in hESCs during in vitro differentiation into specialized cell types. Out of 9 imprinted genes with single nucleotide polymorphisms, mono-allelic expression for three imprinted genes (H19, KCNQ1OT1, and IPW), and bi- or partial-allelic expression for three imprinted genes (OSBPL5, PPP1R9A, and RTL1) were stably retained in H9-hESCs throughout differentiation, representing imprinting stability. Three imprinted genes (KCNK9, ATP10A, and SLC22A3) showed a loss and a gain of imprinting in a lineage-specific manner during differentiation. Changes in allelic expression of imprinted genes were observed in another hESC line during in vitro differentiation. These findings indicate that the allelic expression of imprinted genes may be vulnerable in a lineage-specific manner in human pluripotent stem cells during differentiation.     

The cell cycle- and insulin-signaling-inhibiting miRNA expression pattern of very small embryonic-like stem cells contributes to their quiescent state

Murine Oct4⁺, very small embryonic-like stem cells (VSELs), are a quiescent stem cell population that requires a supportive co-culture layer to proliferate and/or to differentiate in vitro. Gene expression studies have revealed that the quiescence of these cells is due to changes in expression of parentally imprinted genes, including genes involved in cell cycle regulation and insulin and insulin-like growth factor signaling (IIS). To investigate the role of microRNAs (miRNAs) in VSEL quiescence, we performed miRNA studies in highly purified VSELs and observed a unique miRNA expression pattern in these cells. Specifically, we observed significant differences in the expression of certain miRNA species (relative to a reference cell population), including (i) miRNA-25_1 and miRNA-19 b, whose downregulation has the effect of upregulating cell cycle checkpoint genes and (ii) miRNA-675-3 p and miRNA-675-5 p, miRNA-292-5 p, miRNA-184, and miRNA-125 b, whose upregulation attenuates IIS. These observations are important for understanding the biology of these cells and for developing efficient ex vivo expansion strategies for VSELs isolated from adult tissues.     

Disruption of imprinted gene expression and DNA methylation status in porcine parthenogenetic fetuses and placentas

Parthenogenetically activated oocytes cannot develop to term in mammals due to the lack of paternal gene expression and failed X chromosome inactivation (XCI). To further characterize porcine parthenogenesis, the expression of 18 imprinted genes was compared between parthenogenetic (PA) and normally fertilized embryos (Con) using quantitative real-time PCR (qRT-PCR). The results revealed that maternally expressed genes were over-expressed, whereas paternally expressed genes were significantly reduced in PA fetuses and placentas. The results of bisulfite sequencing PCR (BSP) demonstrated that PRE-1 and Satellite were hypermethylated in both Con and PA fetuses and placentas, while XIST DMRs were hypomethylated only in PA samples. Taken together, these results suggest that the aberrant methylation profile of XIST DMRs and abnormal imprinted gene expression may be responsible for developmental failure and impaired growth in porcine parthenogenesis.     

Similar variation in carbon storage between deciduous and evergreen treeline species across elevational gradients

Background and Aims

The most plausible explanation for treeline formation so far is provided by the growth limitation hypothesis (GLH), which proposes that carbon sinks are more restricted by low temperatures than by carbon sources. Evidence supporting the GLH has been strong in evergreen, but less and weaker in deciduous treeline species. Here a test is made of the GLH in deciduous–evergreen mixed species forests across elevational gradients, with the hypothesis that deciduous treeline species show a different carbon storage trend from that shown by evergreen species across elevations.

Methods

Tree growth and concentrations of non-structural carbohydrates (NSCs) in foliage, branch sapwood and stem sapwood tissues were measured at four elevations in six deciduous–evergreen treeline ecotones (including treeline) in the southern Andes of Chile (40°S, Nothofagus pumilio and Nothofagus betuloides; 46°S, Nothofagus pumilio and Pinus sylvestris) and in the Swiss Alps (46°N, Larix decidua and Pinus cembra).

Key Results

Tree growth (basal area increment) decreased with elevation for all species. Regardless of foliar habit, NSCs did not deplete across elevations, indicating no shortage of carbon storage in any of the investigated tissues. Rather, NSCs increased significantly with elevation in leaves (P < 0·001) and branch sapwood (P = 0·012) tissues. Deciduous species showed significantly higher NSCs than evergreens for all tissues; on average, the former had 11 % (leaves), 158 % (branch) and 103 % (sapwood) significantly (P < 0·001) higher NSCs than the latter. Finally, deciduous species had higher NSC (particularly starch) increases with elevation than evergreens for stem sapwood, but the opposite was true for leaves and branch sapwood.

Conclusions

Considering the observed decrease in tree growth and increase in NSCs with elevation, it is concluded that both deciduous and evergreen treeline species are sink limited when faced with decreasing temperatures. Despite the overall higher requirements of deciduous tree species for carbon storage, no indication was found of carbon limitation in deciduous species in the alpine treeline ecotone.     

Regulatory links between imprinted genes: evolutionary predictions and consequences

Genomic imprinting is essential for development and growth and plays diverse roles in physiology and behaviour. Imprinted genes have traditionally been studied in isolation or in clusters with respect to cis-acting modes of gene regulation, both from a mechanistic and evolutionary point of view. Recent studies in mammals, however, reveal that imprinted genes are often co-regulated and are part of a gene network involved in the control of cellular proliferation and differentiation. Moreover, a subset of imprinted genes acts in trans on the expression of other imprinted genes. Numerous studies have modulated levels of imprinted gene expression to explore phenotypic and gene regulatory consequences. Increasingly, the applied genome-wide approaches highlight how perturbation of one imprinted gene may affect other maternally or paternally expressed genes. Here, we discuss these novel findings and consider evolutionary theories that offer a rationale for such intricate interactions among imprinted genes. An evolutionary view of these trans-regulatory effects provides a novel interpretation of the logic of gene networks within species and has implications for the origin of reproductive isolation between species.     

Fruit load modulates flowering-related gene expression in buds of alternate-bearing ‘Moncada’ mandarin

Background and Aims

Gene determination of flowering is the result of complex interactions involving both promoters and inhibitors. In this study, the expression of flowering-related genes at the meristem level in alternate-bearing citrus trees is analysed, together with the interplay between buds and leaves in the determination of flowering.

Methods

First defruiting experiments were performed to manipulate blossoming intensity in ‘Moncada’ mandarin, Citrus clementina. Further defoliation was performed to elucidate the role leaves play in the flowering process. In both cases, the activity of flowering-related genes was investigated at the flower induction (November) and differentiation (February) stages.

Key Results

Study of the expression pattern of flowering-genes in buds from on (fully loaded) and off (without fruits) trees revealed that homologues of FLOWERING LOCUS T (CiFT), TWIN SISTER OF FT (TSF), APETALA1 (CsAP1) and LEAFY (CsLFY) were negatively affected by fruit load. CiFT and TSF activities showed a marked increase in buds from off trees through the study period (ten-fold in November). By contrast, expression of the homologues of the flowering inhibitors of TERMINAL FLOWER 1 (CsTFL), TERMINAL FLOWER 2 (TFL2) and FLOWERING LOCUS C (FLC) was generally lower in off trees. Regarding floral identity genes, the increase in CsAP1 expression in off trees was much greater in buds than in leaves, and significant variations in CsLFY expression (approx. 20 %) were found only in February. Defoliation experiments further revealed that the absence of leaves completely abolished blossoming and severely affected the expression of most of the flowering-related genes, particularly decreasing the activity of floral promoters and of CsAP1 at the induction stage.

Conclusions

These results suggest that the presence of fruit affects flowering by greatly altering gene-expression not only at the leaf but also at the meristem level. Although leaves are required for flowering to occur, their absence strongly affects the activity of floral promoters and identity genes.     

Vernalization response of Phleum pratense and its relationships to stem lignification and floral transition

Background

Timothy is a long-day grass species well adapted for cultivation in northern latitudes. It produces elongating tillers not only in spring growth but also later in summer. As the quantity and quality of harvested biomass is dictated by canopy architecture and the proportion of stem-forming flowering tillers, the regulation of flowering is of great interest in forage grass production.

Methods

Canopy architecture, stem morphology and freezing tolerance of vernalized timothy were investigated in greenhouse and field experiments. The molecular control of development was examined by analysing the relationship between apex development and expression of timothy homologues of the floral inducer VRN1 and repressor VRN2.

Key Results

True stem formation and lignification of the sclerenchyma ring occur in both vernalized and regrowing stems irrespective of the developmental stage of the apex. The stems had, however, divergent morphology. Vernalization enhanced flowering, and the expression of the VRN1 homologue was elevated when the apex had passed into the reproductive stage. High VRN1 homologue expression was not associated with reduction in freezing tolerance and the expression coincided with increased levels of the floral repressor VRN2 homologue. Field experiments supported the observed linkage between the upregulation of the VRN1 homologue and the transition to the reproductive stage in vernalized tillers. The upregulation of putative VRN1 or VRN2 genes was restricted to vernalized tillers in the spring yield and, thus, not detected in non-vernalized tillers of the second yield; so-called regrowth.

Conclusions

The formation of a lignified sclerenchyma ring that efficiently reduces the digestibility of the stem was not related to apex development but rather to a requirement for mechanical support. The observed good freezing tolerance of reproductive timothy tillers could be one important adaptation mechanism ensuring high yields in northern conditions. Both VRN1 and VRN2 homologues required a vernalization signal for expression so the development of yield-forming tillers in regrowth was regulated independently of the studied genes.