Aberrant epigenetic changes and gene expression in cloned cattle dying around birth

Background  

Aberrant reprogramming of donor somatic cell nuclei may result in many severe problems in animal cloning. To assess the extent of abnormal epigenetic modifications and gene expression in clones, we simultaneously examined DNA methylation, histone H4 acetylation and expression of six genes (β-actin, VEGF, oct4, TERT, H19 and Igf2) and a repetitive sequence (art2) in five organs (heart, liver, spleen, lung and kidney) from two cloned cattle groups that had died at different stages. In the ED group (early death, n = 3), the cloned cattle died in the perinatal period. The cattle in the LD group (late death, n = 3) died after the perinatal period. Normally reproduced cattle served as a control group (n = 3).     

Genomic imprinting of H19 in naturally reproduced and cloned cattle

Animals produced from assisted reproductive technologies suffer from developmental abnormalities and early fetal death at a higher frequency than that observed in those produced by natural breeding. These symptoms are reminiscent of imprinting disruptions in the human and mouse, suggesting the possibility of perturbations in the expression of imprinted genes such as biallelic expression or silencing. H19 is one of the imprinted genes first identified in mice and humans, but its sequence and imprinting status have not been determined in cattle. In the present study, we obtained the majority of the bovine H19 gene sequence (approximately 2311 base pairs), identified a single nucleotide polymorphism (SNP) in exon 5 and determined the frequencies of different alleles containing the SNP. Our analysis demonstrated that, in cattle produced by natural breeding, H19 was indeed imprinted as shown by either predominant or exclusive expression of the maternal allele. We also analyzed the imprinting pattern of H19 in organs of four animals produced by somatic cell nuclear transfer that died shortly after birth or had developed abnormalities that necessitated immediate killing at birth. Three out of four cloned animals showed biallelic expression of H19, supporting our hypothesis that imprinting disruption is present in cloned animals that suffered from developmental abnormalities at birth. Examination of the expression of H19 in the offspring of a cloned animal produced by artificial insemination showed that the imprinting pattern in this animal was indistinguishable from those of control animals, suggesting that either imprinting disruptions in cloned animals are corrected through natural reproduction or that they are not present in healthy cloned animals capable of undergoing natural reproduction.     

Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene

Maintenance of genomic methylation patterns in mammalian somatic cells depends on DNA methyltransferase-1 (Dnmt1). Mouse oocytes and preimplantation embryos lack Dnmt1 but express a variant of this protein called Dnmt1o. We eliminated Dnmt1o by deletion of the oocyte-specific promoter and first exon from the Dnmt1 locus. Homozygous animals were normal, but most heterozygous fetuses of homozygous females died during the last third of gestation. Although genomic methylation patterns were established normally in Dnmt1o-deficient oocytes, embryos derived from such oocytes showed a loss of allele-specific expression and methylation at certain imprinted loci. Transient nuclear localization of Dnmt1o in 8-cell embryos suggests that this variant of Dnmt1 provides maintenance methyltransferase activity specifically at imprinted loci during the fourth embryonic S phase.     

Variation in gene expression and aberrantly regulated chromosome regions in cloned mice

DNA microarray analysis was used to determine the precise genome-wide gene expression profiles of somatic cloned mice derived from Sertoli and cumulus cells. It demonstrated unexpectedly large epigenetic diversity in neonatal cloned mice, despite their normal appearance and genetic identity. In three neonatal tissues of the cloned mice, the expression of 9-40% of the genes examined was more than two times higher or lower in donor cell-dependent or -independent manners compared with normal controls. Relatively few (0.4-4%) of the genes exhibited up- or downregulation in the same manner in both types of clone. A cluster analysis of the variation in gene expression led to the identification of several chromosome regions in which gene expression was aberrantly controlled in the somatic clones. These results provide a more complete understanding of how somatic clones differ from each other and from normal individuals produced by sexual reproduction and indicate the significant difficulties that face the application of somatic cloning in regenerative medicine.     

The effect of genetic conflict on genomic imprinting and modification of expression at a sex-linked locus

We examine how genomic imprinting may have evolved at an X-linked locus, using six diallelic models of selection in which one allele is imprintable and the other is not. Selection pressures are generated by genetic conflict between mothers and their offspring. The various models describe cases of maternal and paternal inactivation, in which females may be monogamous or bigamous. When inactivation is maternal, we examine the situations in which only female offspring exhibit imprinting as well as when both sexes do. We compare our results to those previously obtained for an autosomal locus and to four models in which a dominant modifier of biallelic expression is subjected to the same selection pressures. We find that, in accord with verbal predictions, maternal inactivation of growth enhancers and paternal inactivation of growth inhibitors are more likely than imprinting in the respective opposite directions, although these latter outcomes are possible for certain parameter combinations. The expected outcomes are easier to evolve than the same outcomes for autosomal loci, contradicting the available evidence concerning the direction of imprinting on mammalian sex chromosomes. In most of our models stable polymorphism of imprinting status is possible, a behavior not predicted by verbal accounts.     

Enhancing mammary differentiation by overcoming lineage-specific epigenetic modification and signature gene expression of fibroblast-derived iPSCs

Recent studies have shown that induced pluripotent stem cells (iPSCs) retain a memory of their origin and exhibit biased differentiation potential. This finding reveals a severe limitation in the application of iPSCs to cell-based therapy because it means that certain cell types are not available for reprogramming for patients. Here we show that the iPSC differentiation process is accompanied by profound gene expression and epigenetic modifications that reflect cells'' origins. Under typical conditions for mammary differentiation, iPSCs reprogrammed from tail-tip fibroblasts (TF-iPSCs) activated a fibroblast-specific signature that was not compatible with mammary differentiation. Strikingly, under optimized conditions, including coculture with iPSCs derived from the mammary epithelium or in the presence of pregnancy hormones, the fibroblast-specific signature of TF-iPSCs obtained during differentiation was erased and cells displayed a mammary-specific signature with a markedly enhanced ability for mammary differentiation. These findings provide new insights into the precise control of differentiation conditions that may have applications in personalized cell-based therapy.The mammary gland is a primary target for carcinogenesis. Breast cancer occurs at a high rate and affects one in eight women in Western countries during their lifetime.^{1, 2} In the United States alone, 232 340 new invasive breast cancer cases were reported for women in 2013 and 39 620 patients died.³ Regenerative therapy of the damaged mammary gland tissues is the best way to restore breast functions; therefore, the creation of stem cells that are capable of developing into fully functional mammary glands is desirable. There are two distinct types of pluripotent stem cells that may be used for this purpose. The first is embryonic stem cells (ESCs) derived from the inner cell mass of embryonic blastocysts,⁴ and the second is induced pluripotent stem cells (iPSCs) obtained by reprogramming somatic cells.⁵ Although, in theory, both ESCs and iPSCs can be differentiated into any type of mature cell, use of the latter is more desirable because it does not require the killing of embryos, and the cells can be derived from virtually any type of tissue. In addition, because iPSCs can be generated from the same patient, the use of iPSCs avoids the immunosuppressive reactions that have long hampered organ and tissue transplantation.^{6, 7, 8} However, recent studies have shown that some iPSCs seem to retain a memory of their origin and exhibit skewed potential during differentiation for tissue/organ formation.^{9, 10, 11, 12, 13, 14} This feature may represent a limitation if certain cell types from diseased tissues or organs are not available for reprogramming.Numerous studies about the use of ESCs have indicated that, although these cells have the potential to generate all cell types, their differentiation depends critically on many factors.^{14, 15, 16} Precise conditions are required for driving cells into specific pathways leading to new lineage formation (reviewed in Murry and Keller¹⁷ and Cahan and Daley¹⁸). Based on these observations, we hypothesized that the skewed differentiation of iPSCs could be overcome by providing favorable conditions for differentiation. To test this hypothesis, we have generated iPSCs from mouse mammary epithelial cells (ME-iPSCs) and mouse-tail fibroblasts (TF-iPSCs), and have studied the gene expression profiles and epigenetic modifications during differentiation. We found that, although these iPSCs activate distinct signature memories that are reflective of their origins during the differentiation process, the fate of iPSCs could be redirected under optimized conditions in favor of the formation of a desired tissue/organ.     

Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH₄)₂SO₄ as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.Abbreviation GS glutamine synthetase     

DNA甲基化对牛Igf-2r表达的影响及其在克隆牛发育中的作用

目前认为克隆效率低的主要原因是供体核的不完全重编程导致发育过程中一些重要的基因异常表达。运用DNA甲基化转移酶抑制剂5′-脱氧胞苷(5′-azacytidine,5′-aza)处理MDBK细胞(牛肾上皮细胞),并通过实时荧光定量PCR方法对Igf-2r基因的表达进行了定量分析;在此基础上,应用亚硫酸盐甲基化测序法检测正常牛及克隆牛脑、肺、心、肝组织Igf-2r印迹调控区DMR2(DNA differentially methylated region,DMR)及非印迹调控区3′-UTR(3′-untranslated region,UTR)的DNA甲基化水平。研究发现,5′-aza处理MDBK细胞后,Igf-2r基因的表达上调。正常牛各组织中Igf-2r DMR2区的DNA甲基化程度差异较大,3′-UTR区较稳定;与正常牛相比,克隆牛DMR2区的甲基化程度变化较大,3′-UTR区无显著性变化。结果表明,DNA甲基化修饰影响Igf-2r基因的表达。正常牛不同组织中Igf-2r基因DMR2区的DNA甲基化程度不同,提示Igf-2r基因的印迹调控方式在不同组织中可能不同。克隆牛发育过程中,调控Igf-2r基因印迹的DMR2表观结构被明显改变,而非印迹调控区3′-UTR则无明显变化,提示Igf-2r基因印迹调控区被破坏,很可能是导致克隆牛发育异常的一个重要原因。     

Allele-specific modification of the expression of the adh2 gene in maize by ethylene

The effect of ethylene on the induction of alcohol dehydrogenase-2 in seedling roots of maize is reported. Allele-specific differences are observed in the response to the hormone. Hormonal treatment acts to eliminate the difference in gene expression that is characteristic of the alleles in untreated roots.     

Upregulation of DLEU1 expression by epigenetic modification promotes tumorigenesis in human cancer

The function of DLEU1 in human cancer is largely unknown. The Cancer Genome Atlas data were applied to identify the landscape of differential genes between tumor tissues and normal tissues, which was further validated by our cohort data and pan-cancer data including 33 cancer types with 11,060 patients. Next, DLEU1 was selected to validate the novel finding and result showed that it promoted tumorigenesis in vitro and in vivo. Mechanistically, DLEU1 promotes SRP4 expression via increasing H3K27ac enrichment to SRP4 locus epigenetically. Moreover, epigenetic modification leads to upregulation of DLEU1 expression via decreased DNA methylation and increased H3K27ac and H3K4me3 histone modification in its locus. Finally, high expression of DLEU1 correlates with worse prognosis not only in specific cancer type patients but also in patients in the pan-cancer cohort. In summary, the work broadens the function landscape of known long noncoding RNAs in human cancer and provides novel insights into their roles in tumorigenesis.     

Correction of aberrant imprinting of IGF2 in human tumors by nuclear transfer-induced epigenetic reprogramming

Loss of genomic imprinting of insulin-like growth factor II (IGF2) is a hallmark of many human neoplasms. We attempted to correct this aberrant epigenotype by transferring nuclei from human tumor cells that showed loss of IGF2 imprinting into enucleated mouse and human fibroblasts that had maintained normal IGF2 imprinting. After nuclear transfer, the abnormal biallelic expression of IGF2 in tumor nuclei transiently converted to normal monoallelic imprinted expression in the reconstructed diploid cells. In tetraploid hybrid cells, however, normal IGF2 imprinting was permanently restored in the tumor genome. Inhibition of the synthesis of putative trans imprinting factors with cycloheximide led to loss of IGF2 imprinting in normal cultured fibroblasts, suggesting that normal cells produce proteins that act in trans to induce or maintain genomic imprinting. These data demonstrate that an abnormal tumor epigenotype can be corrected by in vitro reprogramming, and suggest that loss of imprinting is associated with the loss of activity of non-CTCF trans imprinting factor(s) that are either inactivated or mutated in tumors.