Epigenetic states of donor cells significantly affect the development of somatic cell nuclear transfer (SCNT) embryos in pigs

The type and pattern of epigenetic modification in donor cells can significantly affect the developmental competency of somatic cell nuclear transfer (SCNT) embryos. Here, we investigated the developmental capacity, gene expression, and epigenetic modifications of SCNT embryos derived from porcine bone marrow‐derived mesenchymal stem cells (BMSCs) and fetal fibroblasts (FFs) donor cells compared to embryos obtained from in vitro fertilization (IVF). Compared to FFs, the donor BMSCs had more active epigenetic markers (Histone H3 modifications: H3K9Ac, H3K4me3, and H3K4me2) and fewer repressive epigenetic markers (H3K9me3, H3K9me2, and DNA methyltransferase 1). Embryos derived from BMSC nuclear‐transfer (BMSC‐NT embryos) and IVF embryos had significantly higher cleavage and blastocyst rates (BMSC‐NT: 71.3 ± 3.4%, 29.1 ± 2.3%; IVF: 69.2 ± 2.2%, 30.2 ± 3.3%; respectively) than FF‐NT embryos (58.1 ± 3.4%, 15.1 ± 1.5%, respectively). Bisulfite sequencing revealed that DNA methylation at the promoter regions of NANOG and POU5F1 was lower in BMSC‐NT embryos (30.0%, 9.8%, respectively) than those in FF‐NT embryos (34.2%, 28.0%, respectively). We also found that BMSC‐NT embryos had more H3K9Ac and less H3K9me3 and 5‐methylcytosine than FF‐NT embryos. In conclusion, our finding comparing BMSCs versus FFs as donors for nuclear transfer revealed that differences in the initial epigenetic state of donor cells have a remarkable effect on overall nuclear reprogramming of SCNT embryos, wherein donor cells possessing a more open chromatin state are more conducive to nuclear reprogramming.     

Posttranslational Modifications of the Histone 3 Tail and Their Impact on the Activity of Histone Lysine Demethylases In Vitro

Posttranslational modifications (PTMs) of the histone H3 tail such as methylation, acetylation and phosphorylation play important roles in epigenetic signaling. Here we study the effect of some of these PTMs on the demethylation rates of methylated lysine 9 in vitro using peptide substrates mimicking histone H3. Various combinations with other PTMs were employed to study possible cross-talk effects by comparing enzyme kinetic characteristics. We compared the kinetics of histone tail substrates for truncated histone lysine demethylases KDM4A and KDM4C containing only the catalytic core (cc) and some combinations were characterized on full length (FL) KDM4A and KDM4C. We found that the substrates combining trimethylated K4 and K9 resulted in a significant increase in the catalytic activity for FL-KDM4A. For the truncated versions of KDM4A and KDM4C a two-fold increase in the catalytic activity toward bis-trimethylated substrates could be observed. Furthermore, a significant difference in the catalytic activity between dimethylated and trimethylated substrates was found for full length demethylases in line with what has been reported previously for truncated demethylases. Histone peptide substrates phosphorylated at T11 could not be demethylated by neither truncated nor full length KDM4A and KDM4C, suggesting that phosphorylation of threonine 11 prevents demethylation of the H3K9me3 mark on the same peptide. Acetylation of K14 was also found to influence demethylation rates significantly. Thus, for truncated KDM4A, acetylation on K14 of the substrate leads to an increase in enzymatic catalytic efficiency (k _cat/K _m), while for truncated KDM4C it induces a decrease, primarily caused by changes in K _m. This study demonstrates that demethylation activities towards trimethylated H3K9 are significantly influenced by other PTMs on the same peptide, and emphasizes the importance of studying these interactions at the peptide level to get a more detailed understanding of the dynamics of epigenetic marks.     

Activation of p53 stimulates proteasome-dependent truncation of eIF4E-binding protein 1 (4E-BP1)

Background information. The translational inhibitor protein 4E‐BP1 [eIF4E (eukaryotic initiation factor 4E)‐binding protein 1] regulates the availability of polypeptide chain initiation factor eIF4E for protein synthesis. Initiation factor eIF4E binds the 5′ cap structure present on all cellular mRNAs. Its ability to associate with initiation factors eIF4G and eIF4A, forming the eIF4F complex, brings the mRNA to the 43S complex during the initiation of translation. Binding of eIF4E to eIF4G is inhibited in a competitive manner by 4E‐BP1. Phosphorylation of 4E‐BP1 decreases the affinity of this protein for eIF4E, thus favouring the binding of eIF4G and enhancing translation. We have previously shown that induction or activation of the tumour suppressor protein p53 rapidly leads to 4E‐BP1 dephosphorylation, resulting in sequestration of eIF4E, decreased formation of the eIF4F complex and inhibition of protein synthesis. Results. We now report that activation of p53 also results in modification of 4E‐BP1 to a truncated form. Unlike full‐length 4E‐BP1, which is reversibly phosphorylated at multiple sites, the truncated protein is almost completely unphosphorylated. Moreover, the latter interacts with eIF4E in preference to full‐length 4E‐BP1. Inhibitor studies indicate that the p53‐induced cleavage of 4E‐BP1 is mediated by the proteasome and is blocked by conditions that inhibit the dephosphorylation of full‐length 4E‐BP1. Measurements of the turnover of 4E‐BP1 indicate that the truncated form is much more stable than the full‐length protein. Conclusions. The results suggest a model in which proteasome activity gives rise to a stable, hypophosphorylated and truncated form of 4E‐BP1, which may exert a long‐term inhibitory effect on the availability of eIF4E, thus contributing to the inhibition of protein synthesis and the growth‐inhibitory and pro‐apoptotic effects of p53.     

The histone methyltransferase ESET is required for the survival of spermatogonial stem/progenitor cells in mice

Self-renewal and differentiation of spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout a male''s life. SSC transplantation will be a valuable solution for young male patients to preserve their fertility. As SSCs in the collected testis tissue from the patients are very limited, it is necessary to expansion the SSCs in vitro. Previous studies suggested that histone methyltransferase ERG-associated protein with SET domain (ESET) represses gene expression and is essential for the maintenance of the pool of embryonic stem cells and neurons. The objective of this study was to determine the role of ESET in SSCs using in vitrocell culture and germ cell transplantation. Cell transplantation assay showed that knockdown of ESET reduced the number of seminiferous tubules with spermatogenesis when compared with that of the control. Knockdown of ESET also upregulated the expression of apoptosis-associated genes (such as P53, Caspase9, Apaf1), whereas inhibited the expression of apoptosis-suppressing genes (such as Bcl2l1, X-linked inhibitor of apoptosis protein). In addition, suppression of ESET led to increase in expression of Caspase9 and activation of Caspase3 (P17) as well as cleavage of poly (ADP-ribose) polymerase. Among the five ESET-targeting genes (Cox4i2, spermatogenesis and oogenesis Specific Basic Helix-Loop-Helix 2, Nobox, Foxn1 and Dazl) examined by ChIP assay, Cox4i2 was found to regulate SSC apoptosis by the rescue experiment. BSP analyses further showed that DNA methylation in the promoter loci of Cox4i2was influenced by ESET, indicating that ESET also regulated gene expression through DNA methylation in addition to histone methylation. In conclusion, we found that ESET regulated SSC apoptosis by suppressing of Cox4i2 expression through histone H3 lysine 9 tri-methylation and DNA methylation. The results obtained will provide unique insights that would broaden the research on SSC biology and contribute to the treatment of male infertility.     

Analysis of lissencephaly-causing LIS1 mutations.

Mutations in the LIS1 gene may result in severe abnormalities of brain cortical layering known as lissencephaly. Most lissencephaly-causing LIS1 mutations are deletions that encompass the entire gene, therefore the mechanism of the disease is regarded as haploinsufficiency. So far, 13 different intragenic mutations have been reported: one point mutation, H149R; deletion of exon 9, which results in deleted acids Delta301-334; deletion of exon 4, which results in deleted amino acids Delta40-64; 10 mutations resulting in truncated proteins and one predicted to result in extra amino acids. We studied the consequences of the point mutation, deletion mutation and one of the reported truncations. In order to study LIS1 structure function, we introduced an additional point mutation and other truncations in different regions of the protein. The consequences of these mutations to protein folding were studied by gel filtration, sucrose density gradient centrifugation and measuring resistance to trypsin cleavage. On the basis of our results, we suggest that all truncation mutations and lissencephaly-causing point mutations or internal deletion result in a reduction in the amount of correctly folded LIS1 protein.     

Identification of three alternatively spliced variants of human CD28 mRNA.

CD28, expressed by T cells, plays a central role in providing costimulatory signals to T cells. The cd28 gene is organized into 4 exons. An alternatively spliced CD28 mRNA lacking most of the exon 2 has been previously evidenced. We report here that non stimulated human T cells express three additional alternatively spliced variants of CD28 mRNA (CD28a-c) in. The CD28a variant, expressed at similar levels to that of the full length CD28 mRNA encoding for the membrane form, lacks exon 3. This deletion introduces (i) a frame shift resulting in the addition of two extra amino acids and a premature stop codon and, (ii) induces the loss of the transmembrane region, suggesting that it could encodes for a soluble monomeric molecule which conserves the binding sites of CD28. The CD28b and CD28c variants, expressed at a low level compared with CD28a, are generated by deletion of most of the 3' end of exon 2 plus exon 3 and exon 2 plus exon 3, respectively. Activated T cells express only the membrane CD28 mRNA. These results suggest that resting human T cells may constitutively express both membrane and soluble CD28 which can differentially regulate the outcome of the T cell response.     

Generation of a conditional allele for the mouse endothelial nitric oxide synthase gene

Mice with endothelial nitric oxide synthase (eNOS) deletions have defined the crucial role of eNOS in vascular development, homeostasis, and pathology. However, cell specific eNOS function has not been determined, although an important role of eNOS has been suggested in multiple cell types. Here, we have generated a floxed eNOS allele in which exons 9–12, encoding the sites essential to eNOS activity, are flanked with loxP sites. Mice homozygous for the floxed allele showed normal eNOS protein levels and no overt phenotype. Conversely, homozygous mice with Cre‐deleted alleles displayed truncated eNOS protein, lack of vascular NO production, and exhibited similar phenotype to eNOS knockout mice, including hypertension, low heart rate, and focal renal scarring. These findings demonstrate that the floxed allele is normal and it can be converted to a non‐functional eNOS allele through Cre recombination. This mouse will allow time‐ and cell‐specific eNOS deletion. genesis 50:685–692, 2012. © 2012 Wiley Periodicals, Inc.     

RHD mRNA替代剪接区域研究

目的2007年国内报道一例弱D型个体存在第4—9外显子选择性剪接的转录子,我们探讨正常Rh（D）阳性个体的RHD基因mRNA的选择性剪接区域。方法随机选择3名Rh（D）阳性个体,从新鲜全血中提取总RNA,通过特异性引物,采用“一步法”逆转录-PCR（1iT—PCR）,扩增RHDmRNA第1～7外显子区域,以及第6-10外显子区域,然后cDNA琼脂糖凝胶电泳和成像分析。结果未发现第1～7外显子区域存在mRNA的选择性剪接条带,仅存在由特异性引物所扩增的第1—7外显子全长的序列条带;而第6～10外显子区域观察到5种替代剪切条带,序列分析显示分别为无缺失片段,以及完整缺失第7、第9、第7和9、第7—9外显子5种RHD转录子。结论正常Rh（D）抗原阳性个体的RHD基因mRNA的选择性剪接仅存在于第7～9外显子区域。     

Mesenchyme-specific Knockout of ESET Histone Methyltransferase Causes Ectopic Hypertrophy and Terminal Differentiation of Articular Chondrocytes

The exact molecular mechanisms governing articular chondrocytes remain unknown in skeletal biology. In this study, we have found that ESET (an ERG-associated protein with a SET domain, also called SETDB1) histone methyltransferase is expressed in articular cartilage. To test whether ESET regulates articular chondrocytes, we carried out mesenchyme-specific deletion of the ESET gene in mice. ESET knock-out did not affect generation of articular chondrocytes during embryonic development. Two weeks after birth, there was minimal qualitative difference at the knee joints between wild-type and ESET knock-out animals. At 1 month, ectopic hypertrophy, proliferation, and apoptosis of articular chondrocytes were seen in the articular cartilage of ESET-null animals. At 3 months, additional signs of terminal differentiation such as increased alkaline phosphatase activity and an elevated level of matrix metalloproteinase (MMP)-13 were found in ESET-null cartilage. Staining for type II collagen and proteoglycan revealed that cartilage degeneration became progressively worse from 2 weeks to 12 months at the knee joints of ESET knock-out mutants. Analysis of over 14 pairs of age- and sex-matched wild-type and knock-out mice indicated that the articular chondrocyte phenotype in ESET-null mutants is 100% penetrant. Our results demonstrate that expression of ESET plays an essential role in the maintenance of articular cartilage by preventing articular chondrocytes from terminal differentiation and may have implications in joint diseases such as osteoarthritis.     

A novel variant of t-PA resistant to plasminogen activator inhibitor-1; expression in CHO cells based on in silico experiments

Resistance to PAI-1 is a factor which confers clinical benefits in thrombolytic therapy. The only US FDA approved PAI-1 resistant drug is Tenecteplase?. Deletion variants of t-PA have the advantage of fewer disulfide bonds in addition to higher plasma half lives. A new variant was developed by deletion of the first three domains in t-PA in addition to substitution of KHRR 128-131 amino acids with AAAA in truncated t-PA. The specific activity of this new variant, 570 IU/μg, was found to be similar to those found in full length t-PA (Alteplase?), 580 IU/μg. A 65% and 85% residual activity after inhibition by rPAI-1 was observed for full length and truncated-mutant form, respectively. This new variant as the first PAI-1 resistant truncated t-PA may offer more advantages in clinical conditions in which high PAI-1 levels makes the thrombolytic system prone to re-occlusion.     

The Role of the Carbohydrate Recognition Domain of Placental Protein 13 (PP13) in Pregnancy Evaluated with Recombinant PP13 and the DelT221 PP13 Variant

Introduction

Placental protein 13 (PP13), a placenta specific protein, is reduced in the first trimester of pregnancy in women who subsequently develop preeclampsia. A naturally occurring PP13 deletion of thymidine at position 221 (DelT₂₂₁ or truncated variant) is associated with increased frequency of severe preeclampsia. In this study we compared the full length (wildtype) PP13 and the truncated variant.

Methods

Full length PP13 or its DelT₂₂₁ variant were cloned, expressed and purified from E-Coli. Both variants were administrated into pregnant rats at day 8 of pregnancy for slow release (>5 days) through osmotic pumps and rat blood pressure was measured. Animals were sacrificed at day 15 or day 21 and their utero-placental vasculature was examined.

Results

The DelT₂₂₁ variant (11 kDA) lacked exon 4 and a part of exon 3, and is short of 2 amino acids involved in the carbohydrate (CRD) binding of the wildtype (18 kDA). Unlike the wildtype PP13, purification of DelT₂₂₁ variant required special refolding. PP13 specific poly- clonal antibodies recognized both PP13 and DelT₂₂₁ but PP13 specific monoclonal antibodies recognized only the wildtype, indicating the loss of major epitopes. Wildtype PP13 mRNA and its respective proteins were both lower in PE patients compared to normal pregnancies. The DelT₂₂₁ mutant was not found in a large Caucasian cohort. Pregnant rats exposed to wildtype or DelT₂₂₁ PP13 variants had significantly lower blood pressure compared to control. The wildtype but not the DelT₂₂₁ mutant caused extensive vein expansion.

Conclusion

This study revealed the importance of PP13 in regulating blood pressure and expanding the utero-placental vasculature in pregnant rats. PP13 mutant lacking amino acids of the PP13 CRD domain fails to cause vein expansion but did reduce blood pressure. The study provides a basis for replenishing patients at risk for preeclampsia by the full length but not the truncated PP13.     

Generation of a germ cell nuclear factor conditional allele in mice

Two recombination steps in embryonic stem (ES) cells were adopted to generate a floxed Germ Cell Nuclear Factor (GCNF) allele. First, a targeting vector containing a loxP site upstream of exon 4, encoding the DNA binding domain (DBD), and a floxed NeoTK double selection cassette downstream of exon 4 was integrated into the GCNF locus by homologous recombination. Second, a Cre-expressing vector was transiently introduced to remove the floxed NeoTK cassette via site-specific recombination. Heterogeneous ES cell populations were found in a single colony after Cre transfection and were separated using an ES cell re-pick step. Floxed GCNF mice were generated and had normal GCNF expression in the adult gonads. Using the Msx2Cre transgenic mice, the floxed GCNF can be completely deleted in the female germline. Taken together, the floxed GCNF mice were successfully generated and female germline deletion of the floxed GCNF allele was achieved using Msx2Cre mice.     

ESET histone methyltransferase regulates osteoblastic differentiation of mesenchymal stem cells during postnatal bone development

To investigate the effects of histone methyltransferase ESET (also known as SETDB1) on bone metabolism, we analyzed osteoblasts and osteoclasts in ESET knockout animals, and performed osteogenesis assays using ESET-null mesenchymal stem cells. We found that ESET deletion severely impairs osteoblast differentiation but has no effect on osteoclastogenesis, that co-transfection of ESET represses Runx2-mediated luciferase reporter while siRNA knockdown of ESET activates the luciferase reporter in mesenchymal cells, and that ESET is required for postnatal expression of Indian hedgehog protein in the growth plate. As the bone phenotype in ESET-null mice is 100% penetrant, these results support ESET as a critical regulator of osteoblast differentiation during bone development.     

Mitotic destruction of the cell cycle regulated NIMA protein kinase of Aspergillus nidulans is required for mitotic exit.

NIMA is a cell cycle regulated protein kinase required, in addition to p34cdc2/cyclin B, for initiation of mitosis in Aspergillus nidulans. Like cyclin B, NIMA accumulates when cells are arrested in G2 and is degraded as cells traverse mitosis. However, it is stable in cells arrested in mitosis. NIMA, and related kinases, have an N-terminal kinase domain and a C-terminal extension. Deletion of the C-terminus does not completely inactivate NIMA kinase activity but does prevent functional complementation of a temperature sensitive mutation of nimA, showing it to be essential for function. Partial C-terminal deletion of NIMA generates a highly toxic kinase although the kinase domain alone is not toxic. Transient induction experiments demonstrate that the partially truncated NIMA is far more stable than the full length NIMA protein which likely accounts for its toxicity. Unlike full length NIMA, the truncated NIMA is not degraded during mitosis and this affects normal mitotic progression. Cells arrested in mitosis with non-degradable NIMA are able to destroy cyclin B, demonstrating that the arrest is not due to stabilization of p34cdc2/cyclin B activity. The data establish that NIMA degradation during mitosis is required for correct mitotic progression in A. nidulans.     

Dwarfism in homozygous Agc1CreERT mice is associated with decreased expression of aggrecan

Aggrecan (Acan), a large proteoglycan is abundantly expressed in cartilage tissue. Disruption of Acan gene causes dwarfism and perinatal lethality of homozygous mice. Because of sustained expression of Acan in the growth plate and articular cartilage, Agc^Cre model has been developed for the regulated ablation of target gene in chondrocytes. In this model, the IRES‐CreERT‐Neo‐pgk transgene is knocked‐in the 3′UTR of the Acan gene. We consistently noticed variable weight and size among the Agc^Cre littermates, prompting us to examine the cause of this phenotype. Wild‐type, Cre‐heterozygous (Agc^+/Cre), and Cre‐homozygous (Agc^Cre/Cre) littermates were indistinguishable at birth. However, by 1‐month, Agc^Cre/Cre mice showed a significant reduction in body weight (18–27%) and body length (19–22%). Low body weight and dwarfism was sustained through adulthood and occurred in both genders. Compared with wild‐type and Agc^+/Cre littermates, long bones and vertebrae were shorter in Agc^Cre/Cre mice. Histological analysis of Agc^Cre/Cre mice revealed a significant reduction in the length of the growth plate and the thickness of articular cartilage. The amount of proteoglycan deposited in the cartilage of Agc^Cre/Cre mice was nearly half of the WT littermates. Analysis of gene expression indicates impaired differentiation of chondrocyte in hyaline cartilage of Agc^Cre/Cre mice. Notably, both Acan mRNA and protein was reduced by 50% in Agc^Cre/Cre mice. A strong correlation was noted between the level of Acan mRNA and the body length. Importantly, Agc^+/Cre mice showed no overt skeletal phenotype. Thus to avoid misinterpretation of data, only the Agc^+/Cre mice should be used for conditional deletion of a target gene in the cartilage tissue.