Publications by authors named "D V Krishna Pantakani"

18 Publications

  • Page 1 of 1

MPA Modulates Tight Junctions' Permeability via Midkine/PI3K Pathway in Caco-2 Cells: A Possible Mechanism of Leak-Flux Diarrhea in Organ Transplanted Patients.

Front Physiol 2017 26;8:438. Epub 2017 Jun 26.

Institute for Clinical Chemistry/UMG-Laboratories, University Medical CenterGoettingen, Germany.

Mycophenolic acid (MPA) is prescribed to prevent allograft rejection in organ transplanted patients. However, its use is sporadically linked to leak flux diarrhea and other gastrointestinal (GI) disturbances in around 75% of patients through yet unknown mechanisms. Recently, we identified Midkine as a modulator of tight junctions (TJs) permeability in MPA treated Caco-2 monolayer. In the present study, we investigated the possible involvement of Midkine dependent PI3K pathway in alteration of TJs under MPA treatment. Caco-2 cells were grown as monolayer to develop TJs and were treated for 72 h with DMSO (control) or MPA in presence and absence of Midkine inhibitor (iMDK) or PI3K inhibitors (LY/AMG). Caco-2 monolayer integrity was assessed by transepithelial electrical resistance (TEER) and FITC-dextran assays. Our functional assays showed that PI3K inhibitors (LY/AMG) can significantly inhibit the compromised TJs integrity of MPA-treated Caco-2 cells monolayer. Chromatin immunoprecipitation analyses showed a significant epigenetic activation of M, and genes and epigenetic repression of gene after MPA treatment. The MPA-induced epigenetic alterations were further confirmed by mRNA and protein expression analysis. Collectively, our data shows that PI3K pathway as the downstream target of Midkine which in turn modulates p38MAPK and pAKT signaling to alter TJs permeability in Caco-2 cell monolayers treated with MPA. These results highlight the possible use of either Midkine or PI3K inhibitors as therapeutic agents to prevent MPA induced GI disturbances.
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http://dx.doi.org/10.3389/fphys.2017.00438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5483464PMC
June 2017

Active and Repressive Chromatin-Associated Proteome after MPA Treatment and the Role of Midkine in Epithelial Monolayer Permeability.

Int J Mol Sci 2016 Apr 20;17(4). Epub 2016 Apr 20.

Institute for Clinical Chemistry/UMG-Laboratories, University Medical Center, Robert-Koch-Str. 40, 37075 Göttingen, Germany.

Unlabelled: Mycophenolic acid (MPA) is prescribed to maintain allografts in organ-transplanted patients. However, gastrointestinal (GI) complications, particularly diarrhea, are frequently observed as a side effect following MPA therapy. We recently reported that MPA altered the tight junction (TJ)-mediated barrier function in a Caco-2 cell monolayer model system. This study investigates whether MPA induces epigenetic changes which lead to GI complications, especially diarrhea.

Methods: We employed a Chromatin Immunoprecipitation-O-Proteomics (ChIP-O-Proteomics) approach to identify proteins associated with active (H3K4me3) as well as repressive (H3K27me3) chromatin histone modifications in MPA-treated cells, and further characterized the role of midkine, a H3K4me3-associated protein, in the context of epithelial monolayer permeability.

Results: We identified a total of 333 and 306 proteins associated with active and repressive histone modification marks, respectively. Among them, 241 proteins were common both in active and repressive chromatin, 92 proteins were associated exclusively with the active histone modification mark, while 65 proteins remained specific to repressive chromatin. Our results show that 45 proteins which bind to the active and seven proteins which bind to the repressive chromatin region exhibited significantly altered abundance in MPA-treated cells as compared to DMSO control cells. A number of novel proteins whose function is not known in bowel barrier regulation were among the identified proteins, including midkine. Our functional integrity assays on the Caco-2 cell monolayer showed that the inhibition of midkine expression prior to MPA treatment could completely block the MPA-mediated increase in barrier permeability.

Conclusions: The ChIP-O-Proteomics approach delivered a number of novel proteins with potential implications in MPA toxicity. Consequently, it can be proposed that midkine inhibition could be a potent therapeutic approach to prevent the MPA-mediated increase in TJ permeability and leak flux diarrhea in organ transplant patients.
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http://dx.doi.org/10.3390/ijms17040597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4849051PMC
April 2016

Immunosuppressant MPA Modulates Tight Junction through Epigenetic Activation of MLCK/MLC-2 Pathway via p38MAPK.

Front Physiol 2015 22;6:381. Epub 2015 Dec 22.

Proteomics Group, Institute for Clinical Chemistry/UMG-Laboratories, University Medical Centre Goettingen, Germany.

Background: Mycophenolic acid (MPA) is an important immunosuppressive drug (ISD) prescribed to prevent graft rejection in the organ transplanted patients, however, its use is also associated with adverse side effects like sporadic gastrointestinal (GI) disturbances. Recently, we reported the MPA induced tight junctions (TJs) deregulation which involves MLCK/MLC-2 pathway. Here, we investigated the global histone acetylation as well as gene-specific chromatin signature of several genes associated with TJs regulation in Caco-2 cells after MPA treatment.

Results: The epigenetic analysis shows that MPA treatment increases the global histone acetylation levels as well as the enrichment for transcriptional active histone modification mark (H3K4me3) at promoter regions of p38MAPK, ATF-2, MLCK, and MLC-2. In contrast, the promoter region of occludin was enriched for transcriptional repressive histone modification mark (H3K27me3) after MPA treatment. In line with the chromatin status, MPA treatment increased the expression of p38MAPK, ATF-2, MLCK, and MLC-2 both at transcriptional and translational level, while occludin expression was negatively influenced. Interestingly, the MPA induced gene expression changes and functional properties of Caco-2 cells could be blocked by the inhibition of p38MAPK using a chemical inhibitor (SB203580).

Conclusions: Collectively, our results highlight that MPA disrupts the structure of TJs via p38MAPK-dependent activation of MLCK/MLC-2 pathway that results in decreased integrity of Caco-2 monolayer. These results led us to suggest that p38MAPK-mediated lose integrity of epithelial monolayer could be the possible cause of GI disturbance (barrier dysfunction) in the intestine, leading to leaky style diarrhea observed in the organ-transplanted patients treated with MPA.
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http://dx.doi.org/10.3389/fphys.2015.00381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4687409PMC
January 2016

Dppa3 expression is critical for generation of fully reprogrammed iPS cells and maintenance of Dlk1-Dio3 imprinting.

Nat Commun 2015 Jan 23;6:6008. Epub 2015 Jan 23.

Institute of Human Genetics, University of Goettingen, Heinrich-Dueker-Weg 12, 37073 Goettingen, Germany.

Reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs) often generates partially reprogrammed iPSCs (pre-iPSCs), low-grade chimera forming iPSCs (lg-iPSCs) and fully reprogrammed, high-grade chimera production competent iPSCs (hg-iPSCs). Lg-iPSC transcriptome analysis revealed misregulated Dlk1-Dio3 cluster gene expression and subsequently the imprinting defect at the Dlk1-Dio3 locus. Here, we show that germ-cell marker Dppa3 is present only in lg-iPSCs and hg-iPSCs, and that induction with exogenous Dppa3 enhances reprogramming kinetics, generating all hg-iPSCs, similar to vitamin C (Vc). Conversely, Dppa3-null fibroblasts show reprogramming block at pre-iPSCs state and Dlk1-Dio3 imprinting defect. At the molecular level, we show that Dppa3 is associated with Dlk1-Dio3 locus and identify that Dppa3 maintains imprinting by antagonizing Dnmt3a binding. Our results further show molecular parallels between Dppa3 and Vc in Dlk1-Dio3 imprinting maintenance and suggest that early activation of Dppa3 is one of the cascades through which Vc facilitates the generation of fully reprogrammed iPSCs.
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http://dx.doi.org/10.1038/ncomms7008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354275PMC
January 2015

Pelota regulates the development of extraembryonic endoderm through activation of bone morphogenetic protein (BMP) signaling.

Stem Cell Res 2014 Jul 26;13(1):61-74. Epub 2014 Apr 26.

Institute of Human Genetics, University of Göttingen, D-37073 Göttingen, Germany. Electronic address:

Pelota (Pelo) is ubiquitously expressed, and its genetic deletion in mice leads to embryonic lethality at an early post-implantation stage. In the present study, we conditionally deleted Pelo and showed that PELO deficiency did not markedly affect the self-renewal of embryonic stem cells (ESCs) or their capacity to differentiate in teratoma assays. However, their differentiation into extraembryonic endoderm (ExEn) in embryoid bodies (EBs) was severely compromised. Conversely, forced expression of Pelo in ESCs resulted in spontaneous differentiation toward the ExEn lineage. Failure of Pelo-deficient ESCs to differentiate into ExEn was accompanied by the retained expression of pluripotency-related genes and alterations in expression of components of the bone morphogenetic protein (BMP) signaling pathway. Further experiments have also revealed that attenuated activity of BMP signaling is responsible for the impaired development of ExEn. The recovery of ExEn and down-regulation of pluripotent genes in BMP4-treated Pelo-null EBs indicate that the failure of mutant cells to down-regulate pluripotency-related genes in EBs is not a result of autonomous defect, but rather to failed signals from surrounding ExEn lineage that induce the differentiation program. In vivo studies showed the presence of ExEn in Pelo-null embryos at E6.5, yet embryonic lethality at E7.5, suggesting that PELO is not required for the induction of ExEn development, but rather for ExEn maintenance or for terminal differentiation toward functional visceral endoderm which provides the embryos with growth factors required for further development. Moreover, Pelo-null fibroblasts failed to reprogram toward induced pluripotent stem cells (iPSCs) due to inactivation of BMP signaling and impaired mesenchymal-to-epithelial transition. Thus, our results indicate that PELO plays an important role in the establishment of pluripotency and differentiation of ESCs into ExEn lineage through activation of BMP signaling.
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http://dx.doi.org/10.1016/j.scr.2014.04.011DOI Listing
July 2014

The roles of DAZL in RNA biology and development.

Wiley Interdiscip Rev RNA 2014 Jul-Aug;5(4):527-35. Epub 2014 Apr 8.

Institute of Human Genetics, University of Goettingen, Goettingen, Germany.

RNA-binding proteins play an important role in the regulation of gene expression by modulating translation and localization of specific messenger RNAs (mRNAs) during early development and gametogenesis. The DAZ (Deleted in Azoospermia) family of proteins, which includes DAZ, DAZL, and BOULE, are germ cell-specific RNA-binding proteins that are implicated in translational regulation of several transcripts. Of particular importance is DAZL, which is present in vertebrates and arose from the duplication of the ancestral BOULE during evolution. Identification of DAZL target mRNAs and characterization of the RNA-binding sequence through in vitro binding assays and crystallographic studies revealed that DAZL binds to GUU triplets in the 3' untranslated region of target mRNAs. Although there is compelling evidence for the role of DAZL in translation stimulation of target mRNAs, recent studies indicate that DAZL can also function in translational repression and transport of specific mRNAs. Furthermore, apart from the well-characterized function of DAZL in gametogenesis, recent data suggest its role in early embryonic development and differentiation of pluripotent stem cells toward functional gametes. In light of the mounting evidence for the role of DAZL in various cellular and developmental processes, we summarize the currently characterized biological functions of DAZL in RNA biology and development.
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http://dx.doi.org/10.1002/wrna.1228DOI Listing
March 2015

Zfp819, a novel KRAB-zinc finger protein, interacts with KAP1 and functions in genomic integrity maintenance of mouse embryonic stem cells.

Stem Cell Res 2013 Nov 30;11(3):1045-59. Epub 2013 Jul 30.

Institute of Human Genetics, University of Goettingen, Heinrich-Dueker-Weg 12, 37073 Goettingen, Germany.

Pluripotency is maintained by both known and unknown transcriptional regulatory networks. In the present study, we have identified Zfp819, a KRAB-zinc finger protein, as a novel pluripotency-related factor and characterized its role in pluripotent stem cells. We show that Zfp819 is expressed highly in various types of pluripotent stem cells but not in their differentiated counterparts. We identified the presence of non-canonical nuclear localization signals in particular zinc finger motifs and identified them as responsible for the nuclear localization of Zfp819. Analysis of the Zfp819 promoter region revealed the presence of a transcriptionally active chromatin signature. Moreover, we confirmed the binding of pluripotency-related factors, Oct4, Sox2, and Nanog to the distal promoter region of Zfp819, indicating that the expression of this gene is regulated by a pluripotency transcription factor network. We found that the expression of endogenous retroviral elements (ERVs) such as Intracisternal A Particle (IAP) retrotransposons, Long Interspersed Nuclear Elements (LINE1), and Short Interspersed Nuclear Elements (SINE B1) is significantly upregulated in Zfp819-knockdown (Zfp819_KD) cells. In line with the activation of ERVs, we observed the occurrence of spontaneous DNA damage in Zfp819_KD cells. Furthermore, we tested whether Zfp819 can interact with KAP1, a KRAB-associated protein with a transcriptional repression function, and found the interaction between these two proteins in both in vitro and in vivo experiments. The challenging of Zfp819_KD cells with DNA damaging agent revealed that these cells are inefficient in repairing the damaged DNA, as cells showed presence of γH2A.X foci for a prolonged time. Collectively, our study identified Zfp819 as a novel pluripotency-related factor and unveiled its function in genomic integrity maintenance mechanisms of mouse embryonic stem cells.
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http://dx.doi.org/10.1016/j.scr.2013.07.006DOI Listing
November 2013

Mouse Dazl and its novel splice variant functions in translational repression of target mRNAs in embryonic stem cells.

Biochim Biophys Acta 2013 May 6;1829(5):425-35. Epub 2013 Jan 6.

University of Goettingen, Goettingen, Germany.

Dazl (deleted in azoospermia-like) is an RNA binding protein that is important for germ cell differentiation in vertebrates. In the present study, we report the identification of a novel Dazl isoform (Dazl_Δ8) that results from alternative splicing of exon8 of mouse Dazl. We observed the expression of Dazl_Δ8 in various pluripotent cell types, but not in somatic cells. Furthermore, the Dazl_Δ8 splice variant was expressed along with the full-length isoform of Dazl (Dazl_FL) throughout male germ-cell development and in the ovary. Sub-cellular localization studies of Dazl_Δ8 revealed a diffused cytoplasmic and large granular pattern, which is similar to the localization patterns of Dazl_FL protein. In contrast to the well documented translation stimulation function in germ cells, overexpression and downregulation studies of Dazl isoforms (Dazl_FL and Dazl_Δ8) revealed a role for Dazl in the negative translational regulation of Mvh, a known target of Dazl, as well as Oct3/4 and Sox2 in embryonic stem cells (ESCs). In line with these observations, a luciferase reporter assay with the 3'UTRs of Oct3/4 and Mvh confirmed the translational repressive role of Dazl isoforms in ESCs but not in germ cells derived cell line GC-1. Further, we identified several putative target mRNAs of Dazl_FL and Dazl_Δ8 in ESCs through RNA-binding immunoprecipitation followed by whole genome transcriptome analysis. Collectively, our results show a translation repression function of Dazl in pluripotent stem cells.
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http://dx.doi.org/10.1016/j.bbagrm.2012.12.010DOI Listing
May 2013

Apoptosis-related gene expression profiles of mouse ESCs and maGSCs: role of Fgf4 and Mnda in pluripotent cell responses to genotoxicity.

PLoS One 2012 7;7(11):e48869. Epub 2012 Nov 7.

Institute of Human Genetics, University of Goettingen, Goettingen, Germany.

Stem cells in the developing embryo proliferate and differentiate while maintaining genomic integrity, failure of which may lead to accumulation of mutations and subsequent damage to the embryo. Embryonic stem cells (ESCs), the in vitro counterpart of embryo stem cells are highly sensitive to genotoxic stress. Defective ESCs undergo either efficient DNA damage repair or apoptosis, thus maintaining genomic integrity. However, the genotoxicity- and apoptosis-related processes in germ-line derived pluripotent cells, multipotent adult germ-line stem cells (maGSCs), are currently unknown. Here, we analyzed the expression of apoptosis-related genes using OligoGEArray in undifferentiated maGSCs and ESCs and identified a similar set of genes expressed in both cell types. We detected the expression of intrinsic, but not extrinsic, apoptotic pathway genes in both cell types. Further, we found that apoptosis-related gene expression patterns of differentiated ESCs and maGSCs are identical to each other. Comparative analysis revealed that several pro- and anti-apoptotic genes are expressed specifically in pluripotent cells, but markedly downregulated in the differentiated counterparts of these cells. Activation of the intrinsic apoptotic pathway cause approximately ∼35% of both ESCs and maGSCs to adopt an early-apoptotic phenotype. Moreover, we performed transcriptome studies using early-apoptotic cells to identify novel pluripotency- and apoptosis-related genes. From these transcriptome studies, we selected Fgf4 (Fibroblast growth factor 4) and Mnda (Myeloid cell nuclear differentiating antigen), which are highly downregulated in early-apoptotic cells, as novel candidates and analyzed their roles in apoptosis and genotoxicity responses in ESCs. Collectively, our results show the existence of common molecular mechanisms for maintaining the pristine stem cell pool of both ESCs and maGSCs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048869PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492253PMC
May 2013

MicroRNA signature in various cell types of mouse spermatogenesis: evidence for stage-specifically expressed miRNA-221, -203 and -34b-5p mediated spermatogenesis regulation.

Biol Cell 2012 Nov 24;104(11):677-92. Epub 2012 Sep 24.

Institute of Human Genetics, University of Goettingen, Goettingen 37073, Germany.

Background Information: Recently, it became apparent that microRNAs (miRNAs) can regulate gene expression post-transcriptionally. Despite the advances in identifying the testis-expressed miRNAs and their role in spermatogenesis, only few data are available showing the spatiotemporal expression of miRNAs during this process.

Results: To understand how different miRNAs can regulate germ cells differentiation, we generated a transgenic mouse model and purified pure populations of premeiotic (PrM) cells and primary spermatocytes (meiotic cells). We also established spermatogonial stem cell (SSC) culture using relatively simple and robust culture conditions. Comparison of global miRNA expression in these germ cell populations revealed 17 SSC-, 11 PrM- and 13 meiotic-specific miRNAs. We identified nine miRNAs as specific for both SSC and PrM cells and another nine miRNAs as specific for PrM and meiotic cells. Additionally, 45 miRNAs were identified as commonly expressed in all three cell types. Several of PrM- and meiotic-specific miRNAs were identified as exclusively/preferentially expressed in testis. We were able to identify the relevant target genes for many of these miRNAs. The luciferase reporter assays with SSC (miR-221)-, PrM (miR-203)- and meiotic (miR-34b-5p)-specific miRNAs and 3'-untranslated region constructs of their targets, c-Kit, Rbm44 and Cdk6, respectively, showed an approximately 30%-40% decrease in reporter activity. Moreover, we observed a reduced expression of endogenous proteins, c-Kit and Cdk6, when the testis-derived cell lines, GC-1 and GC-4, were transfected with miRNA mimics for miR-221 and miR-34b-5p, respectively.

Conclusions: Taken together, we established the miRNA signature of SSC, PrM and meiotic cells and show evidence for their functional relevance during the process of spermatogenesis by target prediction and validation. Through our observations, we propose a working model in which the stage-specific miRNAs such as miR-221, -203 and -34b-5p coordinate the regulation of spermatogenesis.
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http://dx.doi.org/10.1111/boc.201200014DOI Listing
November 2012

Generation and characterization of yeast two-hybrid cDNA libraries derived from two distinct mouse pluripotent cell types.

Mol Biotechnol 2013 Jun;54(2):228-37

Institute of Human Genetics, University of Goettingen, Goettingen, Germany.

Pluripotent stem cells have the therapeutic potential in future regenerative medicine applications. Therefore, it is highly important to understand the molecular mechanisms governing the pluripotency and differentiation potential of these cells. Our current knowledge of pluripotent cells is largely limited owing to the candidate gene/protein approach rather than studying the complex interactions of the proteins. Experimentally, yeast two-hybrid system (Y2H) is by far the most useful and widely used method to detect the protein-protein interactions in high-throughput screenings. Unfortunately, currently there is no GAL4-based pluripotent stem cell-specific cDNA library available for screening the interaction proteins impeding the large-scale studies. In this study, we report the construction of Y2H cDNA libraries derived from mouse pluripotent embryonic stem cells (ESCs) and multipotent adult germ-line stem cells (maGSCs) in GAL4-based Y2H vector system with very high transformation efficiency. Furthermore, we have constructed two different baits and screened for interaction partners in an effort to characterize the libraries and also as a part of our ongoing studies. Consequently, many putative interaction proteins were identified in both cases and their interaction was further validated by direct-Y2H. The observed interactions between bait proteins and their respective analyzed putative interaction proteins were further confirmed using two independent approaches in mammalian cells, thus highlighting the biological significance of the identified interactor (s). Finally, we would like to make these cDNA libraries as a resource that can be distributed to the research community.
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http://dx.doi.org/10.1007/s12033-012-9561-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636440PMC
June 2013

Oligomerization of ZFYVE27 (Protrudin) is necessary to promote neurite extension.

PLoS One 2011 28;6(12):e29584. Epub 2011 Dec 28.

Institute of Human Genetics, University of Goettingen, Goettingen, Germany.

ZFYVE27 (Protrudin) was originally identified as an interacting partner of spastin, which is most frequently mutated in hereditary spastic paraplegia. ZFYVE27 is a novel member of FYVE family, which is implicated in the formation of neurite extensions by promoting directional membrane trafficking in neurons. Now, through a yeast two-hybrid screen, we have identified that ZFYVE27 interacts with itself and the core interaction region resides within the third hydrophobic region (HR3) of the protein. We confirmed the ZFYVE27's self-interaction in the mammalian cells by co-immunoprecipitation and co-localization studies. To decipher the oligomeric nature of ZFYVE27, we performed sucrose gradient centrifugation and showed that ZFYVE27 oligomerizes into dimer/tetramer forms. Sub-cellular fractionation and Triton X-114 membrane phase separation analysis indicated that ZFYVE27 is a peripheral membrane protein. Furthermore, ZFYVE27 also binds to phosphatidylinositol 3-phosphate lipid moiety. Interestingly, cells expressing ZFYVE27(ΔHR3) failed to produce protrusions instead caused swelling of cell soma. When ZFYVE27(ΔHR3) was co-expressed with wild-type ZFYVE27 (ZFYVE27(WT)), it exerted a dominant negative effect on ZFYVE27(WT) as the cells co-expressing both proteins were also unable to induce protrusions and showed cytoplasmic swelling. Altogether, it is evident that a functionally active form of oligomer is crucial for ZFYVE27 ability to promote neurite extensions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0029584PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3247280PMC
May 2012

Stage-specific germ-cell marker genes are expressed in all mouse pluripotent cell types and emerge early during induced pluripotency.

PLoS One 2011 25;6(7):e22413. Epub 2011 Jul 25.

Institute of Human Genetics, University of Goettingen, Goettingen, Germany.

Embryonic stem cells (ESCs) generated from the in-vitro culture of blastocyst stage embryos are known as equivalent to blastocyst inner cell mass (ICM) in-vivo. Though several reports have shown the expression of germ cell/pre-meiotic (GC/PrM) markers in ESCs, their functional relevance for the pluripotency and germ line commitment are largely unknown. In the present study, we used mouse as a model system and systematically analyzed the RNA and protein expression of GC/PrM markers in ESCs and found them to be comparable to the expression of cultured pluripotent cells originated from the germ line. Further, siRNA knockdown experiments have demonstrated the parallel maintenance and independence of pluripotent and GC/PrM networks in ESCs. Through chromatin immunoprecipitation experiments, we observed that pluripotent cells exhibit active chromatin states at GC marker genes and a bivalent chromatin structure at PrM marker genes. Moreover, gene expression analysis during the time course of iPS cells generation revealed that the expression of GC markers precedes pluripotency markers. Collectively, through our observations we hypothesize that the chromatin state and the expression of GC/PrM markers might indicate molecular parallels between in-vivo germ cell specification and pluripotent stem cell generation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0022413PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3143132PMC
December 2011

Global and gene-specific histone modification profiles of mouse multipotent adult germline stem cells.

Mol Hum Reprod 2011 Mar 8;17(3):166-74. Epub 2010 Oct 8.

Institute of Human Genetics, Georg-August-University Goettingen, Heinrich-Dueker-Weg 12, 37073 Goettingen, Germany.

We previously reported the generation of multipotent adult germline stem cells (maGSCs) from spermatogonial stem cells (SSCs) isolated from adult mouse testis. In a later study, we substantiated the pluripotency of maGSCs by demonstrating their close similarity to pluripotent male embryonic stem cells (ESCs) at the epigenetic level of global and gene-specific DNA methylation. Here, we extended the comparative epigenetic analysis of maGSCs and male ESCs by investigating the second main epigenetic modification in mammals, i.e. global and gene-specific modifications of histones (H3K4 trimethylation, H3K9 acetylation, H3K9 trimethylation and H3K27 trimethylation). Using immunofluorescence staining, flow cytometry and western blot analysis, we show that maGSCs are very similar to male ESCs with regard to global levels and nuclear distribution patterns of these modifications. Chromatin immunoprecipitation real-time PCR analysis of these modifications at the gene-specific level further revealed modification patterns of the pluripotency marker genes Oct4, Sox2 and Nanog in maGSCs that are nearly identical to those of male ESCs. These genes were enriched for activating histone modifications including H3K4me3 and H3K9ac and depleted of repressive histone modifications including H3K27me3 and H3K9me3. In addition, Hoxa11, a key regulator of early embryonic development showed the ESC-typical bivalent chromatin conformation with enrichment of both the activating H3K4me3 and the repressive H3K27me3 modification also in maGSCs. Collectively, our results demonstrate that maGSCs also closely resemble ESCs with regard to their chromatin state and further evidence their pluripotent nature.
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http://dx.doi.org/10.1093/molehr/gaq085DOI Listing
March 2011

Evaluating the effect of spastin splice mutations by quantitative allele-specific expression assay.

Eur J Neurol 2011 Jan;18(1):99-105

Department of Neurology, University Medical Center, Johannes Gutenberg University, Mainz, Germany.

Background: mutations in the SPG4/SPAST gene are the most common cause for hereditary spastic paraplegia (HSP). The splice-site mutations make a significant contribution to HSP and account for 17.4% of all types of mutations and 30.8% of point mutations in the SPAST gene. However, only few studies with limited molecular approach were conducted to investigate and decipher the role of SPAST splice-site mutations in HSP.

Methods: a reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and quantitative allele-specific expression assay were performed.

Results: we have characterized the consequence of two novel splice-site mutations (c.1493 + 1G>A and c.1414-1G>A) in the SPAST gene in two different families with pure HSP. The RT-PCR analysis revealed that both spastin mutations are indeed splice-site mutations and cause skipping of exon 12. Furthermore, RT-PCR data suggested that these splice-site mutations may cause leaky splicing. By means of a quantitative allele-specific expression assay, we could confirm that both splice-site mutations cause leaky splicing, as the relative expression of the exon 12-skipped transcript was reduced (21.1 ± 3.6 compared to expected 50%).

Conclusions: our finding supports a "threshold-effect-model" for functional spastin in HSP. A higher level (78.8 ± 3.9%) of functional spastin than the expected ratio of 50% owing to leaky splicing might cause late age at onset of HSP. Remarkably, we could show that a quantitative allele-specific expression assay is a simple and effective tool to evaluate the role of most types of spastin splice-site mutations in HSP.
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http://dx.doi.org/10.1111/j.1468-1331.2010.03079.xDOI Listing
January 2011

Expansion of mutation spectrum, determination of mutation cluster regions and predictive structural classification of SPAST mutations in hereditary spastic paraplegia.

Eur J Hum Genet 2009 Feb 13;17(2):187-94. Epub 2008 Aug 13.

Institute of Human Genetics, University of Goettingen, Goettingen, Germany.

The SPAST gene encoding for spastin plays a central role in the genetically heterogeneous group of diseases termed hereditary spastic paraplegia (HSP). In this study, we attempted to expand and refine the genetic and phenotypic characteristics of SPAST associated HSP by examining a large cohort of HSP patients/families. Screening of 200 unrelated HSP cases for mutations in the SPAST gene led to detection of 57 mutations (28.5%), of which 47 were distinct and 29 were novel mutations. The distribution analysis of known SPAST mutations over the structural domains of spastin led to the identification of several regions where the mutations were clustered. Mainly, the clustering was observed in the AAA (ATPases associated with diverse cellular activities) domain; however, significant clustering was also observed in the MIT (microtubule interacting and trafficking), MTBD (microtubule-binding domain) and an N-terminal region (228-269 residues). Furthermore, we used a previously generated structural model of spastin as a framework to classify the missense mutations in the AAA domain from the HSP patients into different structural/functional groups. Our data also suggest a tentative genotype-phenotype correlation and indicate that the missense mutations could cause an earlier onset of the disease.
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http://dx.doi.org/10.1038/ejhg.2008.147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2986068PMC
February 2009

Spastin oligomerizes into a hexamer and the mutant spastin (E442Q) redistribute the wild-type spastin into filamentous microtubule.

J Neurochem 2008 Jul 12;106(2):613-24. Epub 2008 Apr 12.

Institute of Human Genetics, University of Goettingen, Goettingen, Germany.

Spastin, a member of the ATPases associated with various cellular activities (AAA) family of proteins, is the most frequently mutated in hereditary spastic paraplegia. The defining feature of the AAA proteins is a structurally conserved AAA domain which assembles into an oligomer. By chemical cross-linking and gel filtration chromatography, we show that spastin oligomerizes into a hexamer. Furthermore, to gain a comprehensive overview of the oligomeric structure of spastin, we generated a structural model of the AAA domain of spastin using template structure of VPS4B and p97/VCP. The generated model of spastin provided us with a framework to classify the identified missense mutations in the AAA domain from hereditary spastic paraplegia patients into different structural/functional groups. Finally, through co-localization studies in mammalian cells, we show that E442Q mutant spastin acts in a dominant negative fashion and causes redistribution of both wild-type spastin monomer and spastin interacting protein, RTN1 into filamentous microtubule bundles.
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http://dx.doi.org/10.1111/j.1471-4159.2008.05414.xDOI Listing
July 2008

Compound heterozygosity in the SPG4 gene causes hereditary spastic paraplegia.

Clin Genet 2008 Mar 9;73(3):268-72. Epub 2007 Jan 9.

Institute of Human Genetics, University of Goettingen, Goettingen, Germany.

The SPG4 gene is frequently mutated in autosomal dominant form of hereditary spastic paraplegia (HSP). We report that the compound heterozygous sequence variants S44L, a known polymorphism, and c.1687G>A, a novel mutation in SPG4 cause a severe form of HSP in a patient. The family members carrying solely c.1687G>A mutation are asymptomatic for HSP. The reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the c.1687G>A mutation is a splice site mutation and causes skipping of the exon 15 of spastin. Furthermore, quantification of RT-PCR products by sequencing and quantification of allele-specific expression by pyrosequencing assay revealed that c.1687G>A is a leaky or hypomorphic splice site mutation. At the protein level, c.1687G>A mutation in SPG4 leads to E563K substitution. In ex vivo study, about 10% of cells expressing E563K mutant spastin showed filamentous expression pattern, suggesting a hypomorphic effect at the protein level. Collectively, our results suggest that S44L in association with c.1687G>A (E563K) drops the functional level of spastin below a threshold limit sufficient to manifest HSP.
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http://dx.doi.org/10.1111/j.1399-0004.2007.00953.xDOI Listing
March 2008