The Experts below are selected from a list of 5595 Experts worldwide ranked by ideXlab platform
Mitsuo Oshimura - One of the best experts on this subject based on the ideXlab platform.
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An efficient protein production system via gene amplification on a Human Artificial Chromosome and the Chromosome transfer to CHO cells.
Scientific reports, 2019Co-Authors: Takahito Ohira, Yasuhiro Kazuki, Mitsuo Oshimura, Narumi Uno, Koichi Miyauchi, Noriaki Shimizu, Hiroyuki KugohAbstract:Gene amplification methods play a crucial role in establishment of cells that produce high levels of recombinant protein. However, the stability of such cell lines and the level of recombinant protein produced continue to be suboptimal. Here, we used a combination of a Human Artificial Chromosome (HAC) vector and initiation region (IR)/matrix attachment region (MAR) gene amplification method to establish stable cells that produce high levels of recombinant protein. Amplification of Enhanced green fluorescent protein (EGFP) was induced on a HAC carrying EGFP gene and IR/MAR sequences (EGFP MAR-HAC) in CHO DG44 cells. The expression level of EGFP increased approximately 6-fold compared to the original HAC without IR/MAR sequences. Additionally, anti-vascular endothelial growth factor (VEGF) antibody on a HAC (VEGF MAR-HAC) was also amplified by utilization of this IR/MAR-HAC system, and anti-VEGF antibody levels were approximately 2-fold higher compared with levels in control cells without IR/MAR. Furthermore, the expression of anti-VEGF antibody with VEGF MAR-HAC in CHO-K1 cells increased 2.3-fold compared with that of CHO DG44 cells. Taken together, the IR/MAR-HAC system facilitated amplification of a gene of interest on the HAC vector, and could be used to establish a novel cell line that stably produced protein from mammalian cells.
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identification of candidate progenitor populations which causes transient abnormal myelopoiesis
Blood, 2017Co-Authors: Yoko Nishinakaarai, Yasuhiro Kazuki, Mitsuo Oshimura, Yuwna Yakura, Akira Niwa, Shiori Matsuo, Tatsutoshi Nakahata, Megumu K SaitoAbstract:Transient abnormal myelopoiesis (TAM) is a temporal preleukemic status specific to neonates of Down syndrome (DS). TAM is characterized by leucocytosis and/or thrombocytopenia with the trisomy of Chromosome 21 and somatic GATA1 mutation. Morphologically, most blasts found in TAM are myeloblasts, and some other types of blasts are similar to those typically found in DS-related acute megakaryoblastic leukemia (DS-AMKL). Although TAM patients usually experience spontaneous remission within 6 months, 20 to 30 % of develop DS-AMKL. The cell population that causes TAM phenotypes in vitro or in vivo mouse and Human models has not yet been determined. Recently, the hematopoietic differentiation systems from Human pluripotent stem cells (PSCs) have revealed the pathogenesis in various diseases including TAM. Previous TAM models have succeeded in recapitulating the phenotypes, such as arrest and/or skewness of differentiation from immature hematopoietic progenitor cells (HPCs). However, identification of the responsible cell population for TAM at a precise subpopulation level based on functional evaluations has not yet been accomplished. Therefore, we applied our 2D-hematopoietic differentiation system for identifying the responsible cell population through the progenitor assay. A comprehensive understanding of TAM pathogenesis will enable us to predict and eventually prevent the disease progression from TAM into life-threatening DS-AMKL. First, we differentiated Artificial trisomy 21 embryonic stem cells (Ts21-ESCs) generated by transfer of Human Artificial Chromosome (HAC) 21 in our 2D-differentiation system. Five subpopulations of HPCs were harvested at day6 and day9, and cells were subjected to expression analysis of hematopoiesis-related genes. Principal component analysis (PCA) successfully mapped each HPC subpopulation corresponding to the hematopoietic developmental pathways, proving that our system can be used for isolating functional HPC subpopulations. Next, we used genome edition to establish strictly controlled isogenic ES/induced pluripotent stem cell (iPSC) pairs derived from TAM, non-TAM/DS patients and the Ts21-ESCs. Then we differentiated ES/iPSCs into each hematopoietic lineage to identify the abnormal phenotypes in vitro . GATA1 -mutated clones (G1s-clones) showed obviously different phenotypes, compared to GATA1-WT clones (WT-clones). In particular, completely restricted erythroid lineage cells (CD42b-CD71+CD235a+) and increased myeloid lineage cells (CD34-CD235a-CD41-CD42b-CD43+CD45+) were observed in G1s-clones. Moreover, in megakaryocytic lineage, the frequency of immature (CD235a-CD41+CD42b dim ) and mature (CD235a-CD41+CD42b bright ) cells differed between G1s- and WT-clones. Notably, the proportion of immature megakaryoblasts increased in G1s-clones. These data indicated that we have succeeded in recapitulation the TAM phenotype with our in vitro differentiation system. Then, we performed progenitor assay based on in vitro TAM phenotypes. We differentiated 5 isolated HPCs subpopulation into erythrocyte, megakaryocyte and myeloid cells with a suspension culture and colony forming assay. As a result, we found strong correlations between the increased or decreased specific cell fractions and abnormal phenotypes. Correlation pattern is slightly different between clones. In conclusion, we have identified putative responsible cell populations that contribute to the pathological phenotype in TAM. These populations were located on a series of spectra in the differentiation stage. Ubiquitous expression of GATA1 during the differentiation process and wide heterogeneity of the differentiation systems in general may be the reason why precise identification of the single responsible population was not possible. However, our model of TAM using iPSCs enabled us to narrow down the candidate cell populations which have the potential to be a source of DS-AMKL. The identification of responsible progenitor cells is an important first step to predict the transformation from TAM into DS-AMKL and to prevent its progression. Moreover, the method used in this study, such as 2D-differentiation system and progenitor assay, is a promising way to identify the responsible cell population of various types of leukemia other than TAM. Disclosures No relevant conflicts of interest to declare.
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Effects of duration of electric pulse on in vitro development of cloned cat embryos with Human Artificial Chromosome vector.
Reproduction in domestic animals = Zuchthygiene, 2016Co-Authors: Manita Wittayarat, Kanako Kazuki, Yasuhiro Kazuki, T. Terazono, Yoko Sato, Masayasu Taniguchi, Fuminori Tanihara, Tatsuya Takemoto, Mitsuo OshimuraAbstract:Contents The current applications for cat cloning include production of models for the study of Human and animal diseases. This study was conducted to investigate the optimal fusion protocol on in vitro development of transgenic cloned cat embryos by comparing duration of electric pulse. Cat fibroblast cells containing a Human Artificial Chromosome (HAC) vector were used as genetically modified nuclear donor cells. Couplets were fused and activated simultaneously with a single DC pulse of 3.0 kV/cm for either 30 or 60 μs. Low rates of fusion and embryo development to the blastocyst stage were observed in the reconstructed HAC-transchromosomic embryos, when the duration of fusion was prolonged to 60 μs. In contrast, the prolongation of electric pulse duration improved the embryo development and quality in the reconstructed control embryos without HAC vector. Our results suggested that the optimal parameters of electric pulses for fusion in cat somatic cell nuclear transfer vary among the types used for donor cells.
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Potential Usage of Human Artificial Chromosome for Regenerative Medicine
Gene Therapy and Cell Therapy Through the Liver, 2016Co-Authors: Narumi Uno, Yasuhiro Kazuki, Mitsuo OshimuraAbstract:Human Artificial Chromosome (HAC) vectors can carry a gene or genes of interest. HACs have been generated mainly by either a “top-down approach” (engineered creation) or a “bottom-up approach” (de novo creation). HACs with one or more acceptor sites exhibit several characteristics required by an ideal gene delivery vector, including stable episomal maintenance and the capacity to carry large genomic loci plus their regulatory elements, thus allowing the physiological regulation of the introduced gene in a manner similar to that of native Chromosomes. This mini-review introduces characteristics of engineered HAC and their ability to drive exogenous gene expression in cultured cells via microcell-mediated Chromosome transfer (MMCT). A new avenue for regenerative medicine in the future is also proposed.
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Use of a Human Artificial Chromosome for Delivering Trophic Factors in a Rodent Model of Amyotrophic Lateral Sclerosis
Molecular therapy. Nucleic acids, 2015Co-Authors: Yasuhiro Watanabe, Kanako Kazuki, Yasuhiro Kazuki, Mitsutaka Ebiki, Mami Nakanishi, Kazuomi Nakamura, Miho Yamakawa, Hiroyuki Hosokawa, Tetsuya Ohbayashi, Mitsuo OshimuraAbstract:A Human Artificial Chromosome (HAC) is maintained as an episome within a cell and avoids random integration into the host genome. It can transfer multiple and/or large transgenes along with their regulatory elements thereby resembling native Chromosomes. Using this HAC system, we established mesenchymal stem cells (MSCs) that simultaneously expressed hepatocyte growth factor, glial cell line-derived neurotrophic factor, and insulin-like growth factor 1, termed HAC-MSCs. This cell line provides an opportunity for stable transplantation and thorough analyses. We then introduced the cells for the treatment of a neurodegenerative disorder, amyotrophic lateral sclerosis. The HAC-MSCs were transplanted via the fourth cerebral ventricle (CV) or intravenous (i.v.) infusion at various ages of recipient mice. Littermate- and sex-matched mice underwent a sham procedure. Compared to the controls, there was an encouraging trend of increased life span via CV transplantation and delayed onset in i.v. infusion 60 days after transplantation. Further, we confirmed a statistically significant increase in life span via CV transplantation at 100 days. This effect was not seen in mice transplanted with MSCs lacking the HAC. We successfully enhanced the trophic potential of the MSCs using the HAC. This strategy could be a promising direction for the treatment of neurodegenerative disorders.
Vladimir Larionov - One of the best experts on this subject based on the ideXlab platform.
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Human Artificial Chromosome (HAC) for measuring Chromosome instability (CIN) and identification of genes required for proper Chromosome transmission
Experimental Cell Research, 2020Co-Authors: Natalay Kouprina, M A Liskovykh, Nikolai Petrov, Vladimir LarionovAbstract:Chromosomal instability (CIN) is one of the characteristics of cancer inherent for tumor initiation and progression, which is defined as a persistent, high rate of gain/loss of whole Chromosomes. In the vast majority of Human tumors the molecular basis of CIN remains unknown. The development of a conceptually simple colony color sectoring assay that measures yeast Artificial Chromosome (YAC) loss provided a powerful genetic tool to assess the rate of Chromosome mis-segregation and also identified 937 yeast genes involved in this process. Similarly, a Human Artificial Chromosome (HAC)-based assay has been recently developed and applied to quantify Chromosome mis-segregation events in Human cells. This assay allowed identification of novel Human CIN genes in the library of protein kinases. Among them are PINK1, TRIO, IRAK1, PNCK, and TAOK1. The HAC-based assay may be applied to screen siRNA, shRNA and CRISPR-based libraries to identify the complete spectrum of CIN genes. This will reveal new insights into mechanisms of Chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.
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h3k9me3 maintenance on a Human Artificial Chromosome is required for segregation but not centromere epigenetic memory
Journal of Cell Science, 2020Co-Authors: Nuno Martins, Vladimir Larionov, Fernanda Cisnerossoberanis, Elisa Pesenti, Natalia Y Kochanova, Weihao Shang, Tetsuya Hori, Takahiro Nagase, Hiroshi Kimura, Hiroshi MasumotoAbstract:Most eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize de novo centromere formation and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoidtetO Human Artificial Chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase, we removed heterochromatin mark H3K9me3 specifically from the HAC centromere. This caused no short-term defects, but long-term tethering reduced HAC centromere protein levels and triggered HAC mis-segregation. Yet, centromeric CENP-A was maintained at a reduced level. Furthermore, HAC centromere function was compatible with an alternative low-H3K9me3, high-H3K27me3 chromatin signature, as long as residual levels of H3K9me3 remained. When JMJD2D was released from the HAC, H3K9me3 levels recovered over several days back to initial levels along with CENP-A/-C and mitotic segregation fidelity. Our results suggest that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory, and that a homeostatic pathway maintains heterochromatin at centromeres.
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Novel screen for anti-cancer drugs that elevate Chromosome instability (CIN) using Human Artificial Chromosome (HAC).
Oncotarget, 2018Co-Authors: Natalay Kouprina, Yves Pommier, Vladimir LarionovAbstract:Human Artificial Chromosomes (HACs) bearing functional kinetochores have been exploited as promising systems for gene delivery and expression and in studies of different epigenetic modifications on kinetochore structure and function. The HAC-based technology has been also used to develop drug screening and assessment strategies to manipulate the CIN (Chromosome instability) phenotype in cancer cells. More recently, we designed a new protocol for systematic analysis of compounds specifically targeting telomeres and telomerase. This approach used two isogenic cell lines containing a circular HAC (lacking telomeres) and a linear HAC (containing telomeres): compounds that target telomerase or telomeres should preferentially induce loss of the linear HAC but not the circular HAC. This platform enables identification and ranking of compounds that greatly increase Chromosome mis-segregation rates as a result of telomere dysfunction and may expedite the development of new therapeutic strategies for cancer treatment.
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Human Artificial Chromosome with Regulated Centromere: A Tool for Genome and Cancer Studies.
ACS synthetic biology, 2018Co-Authors: Natalay Kouprina, William C Earnshaw, Hiroshi Masumoto, M A Liskovykh, Nikolai Petrov, Elisa Pesenti, Jun-ichirou Ohzeki, Oscar Molina, Vladimir LarionovAbstract:Since their description in the late 1990s, Human Artificial Chromosomes (HACs) bearing functional kinetochores have been considered as promising systems for gene delivery and expression. More recently a HAC assembled from a synthetic alphoid DNA array has been exploited in studies of centromeric chromatin and in assessing the impact of different epigenetic modifications on kinetochore structure and function in Human cells. This HAC was termed the alphoidtetO-HAC, as the synthetic monomers each contained a tetO sequence in place of the CENP-B box that can be targeted specifically with tetR-fusion proteins. Studies in which the kinetochore chromatin of the alphoidtetO-HAC was specifically modified, revealed that heterochromatin is incompatible with centromere function and that centromeric transcription is important for centromere assembly and maintenance. In addition, the alphoidtetO-HAC was modified to carry large gene inserts that are expressed in target cells under conditions that recapitulate the physio...
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Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications
MDPI AG, 2018Co-Authors: Sergey A. Sinenko, M A Liskovykh, Vladimir Larionov, Elena V. Skvortsova, Sergey V. Ponomartsev, Andrey A. Kuzmin, Aleksandr A. Khudiakov, Anna B. Malashicheva, Natalia Alenina, Natalay KouprinaAbstract:AlphoidtetO-type Human Artificial Chromosome (HAC) has been recently synthetized as a novel class of gene delivery vectors for induced pluripotent stem cell (iPSC)-based tissue replacement therapeutic approach. This HAC vector was designed to deliver copies of genes into patients with genetic diseases caused by the loss of a particular gene function. The alphoidtetO-HAC vector has been successfully transferred into murine embryonic stem cells (ESCs) and maintained stably as an independent Chromosome during the proliferation and differentiation of these cells. Human ESCs and iPSCs have significant differences in culturing conditions and pluripotency state in comparison with the murine naïve-type ESCs and iPSCs. To date, transferring alphoidtetO-HAC vector into Human iPSCs (hiPSCs) remains a challenging task. In this study, we performed the microcell-mediated Chromosome transfer (MMCT) of alphoidtetO-HAC expressing the green fluorescent protein into newly generated hiPSCs. We used a recently modified MMCT method that employs an envelope protein of amphotropic murine leukemia virus as a targeting cell fusion agent. Our data provide evidence that a totally Artificial vector, alphoidtetO-HAC, can be transferred and maintained in Human iPSCs as an independent autonomous Chromosome without affecting pluripotent properties of the cells. These data also open new perspectives for implementing alphoidtetO-HAC as a gene therapy tool in future biomedical applications
Natalay Kouprina - One of the best experts on this subject based on the ideXlab platform.
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Human Artificial Chromosome (HAC) for measuring Chromosome instability (CIN) and identification of genes required for proper Chromosome transmission
Experimental Cell Research, 2020Co-Authors: Natalay Kouprina, M A Liskovykh, Nikolai Petrov, Vladimir LarionovAbstract:Chromosomal instability (CIN) is one of the characteristics of cancer inherent for tumor initiation and progression, which is defined as a persistent, high rate of gain/loss of whole Chromosomes. In the vast majority of Human tumors the molecular basis of CIN remains unknown. The development of a conceptually simple colony color sectoring assay that measures yeast Artificial Chromosome (YAC) loss provided a powerful genetic tool to assess the rate of Chromosome mis-segregation and also identified 937 yeast genes involved in this process. Similarly, a Human Artificial Chromosome (HAC)-based assay has been recently developed and applied to quantify Chromosome mis-segregation events in Human cells. This assay allowed identification of novel Human CIN genes in the library of protein kinases. Among them are PINK1, TRIO, IRAK1, PNCK, and TAOK1. The HAC-based assay may be applied to screen siRNA, shRNA and CRISPR-based libraries to identify the complete spectrum of CIN genes. This will reveal new insights into mechanisms of Chromosome segregation and may expedite the development of novel therapeutic strategies to target the CIN phenotype in cancer cells.
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Novel screen for anti-cancer drugs that elevate Chromosome instability (CIN) using Human Artificial Chromosome (HAC).
Oncotarget, 2018Co-Authors: Natalay Kouprina, Yves Pommier, Vladimir LarionovAbstract:Human Artificial Chromosomes (HACs) bearing functional kinetochores have been exploited as promising systems for gene delivery and expression and in studies of different epigenetic modifications on kinetochore structure and function. The HAC-based technology has been also used to develop drug screening and assessment strategies to manipulate the CIN (Chromosome instability) phenotype in cancer cells. More recently, we designed a new protocol for systematic analysis of compounds specifically targeting telomeres and telomerase. This approach used two isogenic cell lines containing a circular HAC (lacking telomeres) and a linear HAC (containing telomeres): compounds that target telomerase or telomeres should preferentially induce loss of the linear HAC but not the circular HAC. This platform enables identification and ranking of compounds that greatly increase Chromosome mis-segregation rates as a result of telomere dysfunction and may expedite the development of new therapeutic strategies for cancer treatment.
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Human Artificial Chromosome with Regulated Centromere: A Tool for Genome and Cancer Studies.
ACS synthetic biology, 2018Co-Authors: Natalay Kouprina, William C Earnshaw, Hiroshi Masumoto, M A Liskovykh, Nikolai Petrov, Elisa Pesenti, Jun-ichirou Ohzeki, Oscar Molina, Vladimir LarionovAbstract:Since their description in the late 1990s, Human Artificial Chromosomes (HACs) bearing functional kinetochores have been considered as promising systems for gene delivery and expression. More recently a HAC assembled from a synthetic alphoid DNA array has been exploited in studies of centromeric chromatin and in assessing the impact of different epigenetic modifications on kinetochore structure and function in Human cells. This HAC was termed the alphoidtetO-HAC, as the synthetic monomers each contained a tetO sequence in place of the CENP-B box that can be targeted specifically with tetR-fusion proteins. Studies in which the kinetochore chromatin of the alphoidtetO-HAC was specifically modified, revealed that heterochromatin is incompatible with centromere function and that centromeric transcription is important for centromere assembly and maintenance. In addition, the alphoidtetO-HAC was modified to carry large gene inserts that are expressed in target cells under conditions that recapitulate the physio...
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Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies
2018Co-Authors: Elisa Pesenti, William C Earnshaw, Natalay Kouprina, Hiroshi Masumoto, M A Liskovykh, Vladimir Larionov, Joan Aurich-costa, Oscar MolinaAbstract:It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native Chromosomes. To address this problem, in our previous studies we generated a Human Artificial Chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoidtetO-HAC. The presence of tetO sequences allows the specific targeting of the centromeric region in the HAC with different chromatin modifiers fused to the tetracycline repressor. The alphoidtetO-HAC has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin to maintain a functional kinetochore. In this study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO, lacO and gal4, the alphoidhybrid-HAC. This new HAC can be used for detailed epigenetic engineering studies because its kinetochore can be simultaneously or independently targeted by different chromatin modifiers and other fusion proteins
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Transfer of Synthetic Human Chromosome into Human Induced Pluripotent Stem Cells for Biomedical Applications
MDPI AG, 2018Co-Authors: Sergey A. Sinenko, M A Liskovykh, Vladimir Larionov, Elena V. Skvortsova, Sergey V. Ponomartsev, Andrey A. Kuzmin, Aleksandr A. Khudiakov, Anna B. Malashicheva, Natalia Alenina, Natalay KouprinaAbstract:AlphoidtetO-type Human Artificial Chromosome (HAC) has been recently synthetized as a novel class of gene delivery vectors for induced pluripotent stem cell (iPSC)-based tissue replacement therapeutic approach. This HAC vector was designed to deliver copies of genes into patients with genetic diseases caused by the loss of a particular gene function. The alphoidtetO-HAC vector has been successfully transferred into murine embryonic stem cells (ESCs) and maintained stably as an independent Chromosome during the proliferation and differentiation of these cells. Human ESCs and iPSCs have significant differences in culturing conditions and pluripotency state in comparison with the murine naïve-type ESCs and iPSCs. To date, transferring alphoidtetO-HAC vector into Human iPSCs (hiPSCs) remains a challenging task. In this study, we performed the microcell-mediated Chromosome transfer (MMCT) of alphoidtetO-HAC expressing the green fluorescent protein into newly generated hiPSCs. We used a recently modified MMCT method that employs an envelope protein of amphotropic murine leukemia virus as a targeting cell fusion agent. Our data provide evidence that a totally Artificial vector, alphoidtetO-HAC, can be transferred and maintained in Human iPSCs as an independent autonomous Chromosome without affecting pluripotent properties of the cells. These data also open new perspectives for implementing alphoidtetO-HAC as a gene therapy tool in future biomedical applications
Hiroshi Masumoto - One of the best experts on this subject based on the ideXlab platform.
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Human Artificial Chromosome: Chromatin assembly mechanisms and CENP-B.
Experimental cell research, 2020Co-Authors: Jun-ichirou Ohzeki, Koichiro Otake, Hiroshi MasumotoAbstract:The centromere is a specialized chromosomal locus required for accurate Chromosome segregation. Heterochromatin also assembles around centromere chromatin and forms a base that supports sister chromatid cohesion until anaphase begins. Both centromere chromatin and heterochromatin assemble on a centromeric DNA sequence, a highly repetitive sequence called alphoid DNA (α-satellite DNA) in Humans. Alphoid DNA can form a de novo centromere and subsequent Human Artificial Chromosome (HAC) when introduced into the Human culture cells HT1080. HAC is maintained stably as a single Chromosome independent of other Human Chromosomes. For de novo centromere assembly and HAC formation, the centromere protein CENP-B and its binding sites, CENP-B boxes, are required in the repeating units of alphoid DNA. CENP-B has multiple roles in de novo centromere chromatin assembly and stabilization and in heterochromatin formation upon alphoid DNA introduction into the cells. Here we review recent progress in Human Artificial Chromosome construction and centromere/heterochromatin assembly and maintenance, focusing on the involvement of Human centromere DNA and CENP-B protein.
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h3k9me3 maintenance on a Human Artificial Chromosome is required for segregation but not centromere epigenetic memory
Journal of Cell Science, 2020Co-Authors: Nuno Martins, Vladimir Larionov, Fernanda Cisnerossoberanis, Elisa Pesenti, Natalia Y Kochanova, Weihao Shang, Tetsuya Hori, Takahiro Nagase, Hiroshi Kimura, Hiroshi MasumotoAbstract:Most eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize de novo centromere formation and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoidtetO Human Artificial Chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase, we removed heterochromatin mark H3K9me3 specifically from the HAC centromere. This caused no short-term defects, but long-term tethering reduced HAC centromere protein levels and triggered HAC mis-segregation. Yet, centromeric CENP-A was maintained at a reduced level. Furthermore, HAC centromere function was compatible with an alternative low-H3K9me3, high-H3K27me3 chromatin signature, as long as residual levels of H3K9me3 remained. When JMJD2D was released from the HAC, H3K9me3 levels recovered over several days back to initial levels along with CENP-A/-C and mitotic segregation fidelity. Our results suggest that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory, and that a homeostatic pathway maintains heterochromatin at centromeres.
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Human Artificial Chromosome with Regulated Centromere: A Tool for Genome and Cancer Studies.
ACS synthetic biology, 2018Co-Authors: Natalay Kouprina, William C Earnshaw, Hiroshi Masumoto, M A Liskovykh, Nikolai Petrov, Elisa Pesenti, Jun-ichirou Ohzeki, Oscar Molina, Vladimir LarionovAbstract:Since their description in the late 1990s, Human Artificial Chromosomes (HACs) bearing functional kinetochores have been considered as promising systems for gene delivery and expression. More recently a HAC assembled from a synthetic alphoid DNA array has been exploited in studies of centromeric chromatin and in assessing the impact of different epigenetic modifications on kinetochore structure and function in Human cells. This HAC was termed the alphoidtetO-HAC, as the synthetic monomers each contained a tetO sequence in place of the CENP-B box that can be targeted specifically with tetR-fusion proteins. Studies in which the kinetochore chromatin of the alphoidtetO-HAC was specifically modified, revealed that heterochromatin is incompatible with centromere function and that centromeric transcription is important for centromere assembly and maintenance. In addition, the alphoidtetO-HAC was modified to carry large gene inserts that are expressed in target cells under conditions that recapitulate the physio...
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Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies
2018Co-Authors: Elisa Pesenti, William C Earnshaw, Natalay Kouprina, Hiroshi Masumoto, M A Liskovykh, Vladimir Larionov, Joan Aurich-costa, Oscar MolinaAbstract:It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native Chromosomes. To address this problem, in our previous studies we generated a Human Artificial Chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoidtetO-HAC. The presence of tetO sequences allows the specific targeting of the centromeric region in the HAC with different chromatin modifiers fused to the tetracycline repressor. The alphoidtetO-HAC has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin to maintain a functional kinetochore. In this study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO, lacO and gal4, the alphoidhybrid-HAC. This new HAC can be used for detailed epigenetic engineering studies because its kinetochore can be simultaneously or independently targeted by different chromatin modifiers and other fusion proteins
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Method to Assemble Genomic DNA Fragments or Genes on Human Artificial Chromosome with Regulated Kinetochore Using a Multi-Integrase System
ACS synthetic biology, 2017Co-Authors: Nicholas C.o. Lee, William C Earnshaw, Hiroshi Masumoto, Vladimir Larionov, Nikolai Petrov, Jung-hyun Kim, Hee-sheung Lee, Natalay KouprinaAbstract:The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: (i) cloning of large genomic segments; (ii) insertion of multiple DNA blocks at a precise location and (iii) the capability to eliminate the assembled region from cells. In this study, we designed the iterative integration system (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with a Human Artificial Chromosome (HAC) possessing a regulated kinetochore (alphoidtetO-HAC). We have demonstrated that the IIS-alphoidtetO-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoidtetO-HAC has several notable advantages over other Artificial Chromosome-based systems. This includes the potential to assemble an unlimited number of genomic DNA segments; a DNA assembly process that leaves only a small insertion (
Huntington F Willard - One of the best experts on this subject based on the ideXlab platform.
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Human Artificial Chromosome assembly by transposon-based retrofitting of genomic BACs with synthetic alpha-satellite arrays.
Current protocols in human genetics, 2007Co-Authors: Joydeep Basu, Huntington F Willard, Gregory StrombergAbstract:The development of methodologies for the rapid assembly of synthetic alpha-satellite arrays recapitulating the higher-order periodic organization of native Human centromeres permits the systematic investigation of the significance of primary sequence and sequence organization in centromere function. Synthetic arrays with defined mutations affecting sequence and/or organization may be evaluated in a de novo Human Artificial Chromosome assay. This unit describes strategies for the assembly of custom built alpha-satellite arrays containing any desired mutation as well as strategies for the construction and manipulation of alpha satellite-based transposons. Transposons permit the rapid and reliable retrofitting of any genomic bacterial Artificial Chromosome (BAC) with synthetic alpha-satellite arrays and other functional components, thereby facilitating conversion into BAC-based Human Artificial Chromosome vectors. These techniques permit identification and optimization of the critical parameters underlying the unique ability of alpha-satellite DNA to facilitate de novo centromere assembly, and they will establish the foundation for the next generation of Human Artificial Chromosome vectors.
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Current Protocols in Human Genetics - Human Artificial Chromosome Assembly by Transposon‐Based Retrofitting of Genomic BACs with Synthetic Alpha‐Satellite Arrays
Current Protocols in Human Genetics, 2007Co-Authors: Joydeep Basu, Huntington F Willard, Gregory StrombergAbstract:The development of methodologies for the rapid assembly of synthetic alpha-satellite arrays recapitulating the higher-order periodic organization of native Human centromeres permits the systematic investigation of the significance of primary sequence and sequence organization in centromere function. Synthetic arrays with defined mutations affecting sequence and/or organization may be evaluated in a de novo Human Artificial Chromosome assay. This unit describes strategies for the assembly of custom built alpha-satellite arrays containing any desired mutation as well as strategies for the construction and manipulation of alpha satellite–based transposons. Transposons permit the rapid and reliable retrofitting of any genomic bacterial Artificial Chromosome (BAC) with synthetic alpha-satellite arrays and other functional components, thereby facilitating conversion into BAC-based Human Artificial Chromosome vectors. These techniques permit identification and optimization of the critical parameters underlying the unique ability of alpha-satellite DNA to facilitate de novo centromere assembly, and they will establish the foundation for the next generation of Human Artificial Chromosome vectors. Keywords: Transposon; alpha satellite; centromere; BAC; Human Artificial Chromosome
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Human Artificial Chromosomes: potential applications and clinical considerations.
Pediatric Clinics of North America, 2006Co-Authors: Joydeep Basu, Huntington F WillardAbstract:Human Artificial Chromosomes demonstrate promise as a novel class of nonintegrative gene therapy vectors. The authors outline current developments in Human Artificial Chromosome technology and examine their potential for clinical application.
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Rapid creation of BAC-based Human Artificial Chromosome vectors by transposition with synthetic alpha-satellite arrays.
Nucleic acids research, 2005Co-Authors: Joydeep Basu, Huntington F Willard, Gregory Stromberg, George Compitello, Gil B. Van BokkelenAbstract:Efficient construction of BAC-based Human Artificial Chromosomes (HACs) requires optimization of each key functional unit as well as development of techniques for the rapid and reliable manipulation of high-molecular weight BAC vectors. Here, we have created synthetic Chromosome 17-derived alpha-satellite arrays, based on the 16-monomer repeat length typical of natural D17Z1 arrays, in which the consensus CENP-B box elements are either completely absent (0/16 monomers) or increased in density (16/16 monomers) compared to D17Z1 alpha-satellite (5/16 monomers). Using these vectors, we show that the presence of CENP-B box elements is a requirement for efficient de novo centromere formation and that increasing the density of CENP-B box elements may enhance the efficiency of de novo centromere formation. Furthermore, we have developed a novel, high-throughput methodology that permits the rapid conversion of any genomic BAC target into a HAC vector by transposon-mediated modification with synthetic alpha-satellite arrays and other key functional units. Taken together, these approaches offer the potential to significantly advance the utility of BAC-based HACs for functional annotation of the genome and for applications in gene transfer.
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α satellite dna and vector composition influence rates of Human Artificial Chromosome formation
Molecular Therapy, 2002Co-Authors: Brenda R Grimes, Angela A Rhoades, Huntington F WillardAbstract:Human Artificial Chromosomes (HACs) have been proposed as a new class of potential gene transfer and gene therapy vector. HACs can be formed when bacterial cloning vectors containing α-satellite DNA are transfected into cultured Human cells. We have compared the HAC-forming potential of different sequences to identify features critical to the efficiency of the process. Chromosome 17 or 21 α-satellite arrays are highly competent HAC-forming substrates in this assay. In contrast, a Y-Chromosome-derived α-satellite sequence is inefficient, suggesting that centromere specification is at least partly dependent on DNA sequence. The length of the input array is also an important determinant, as reduction of the Chromosome-17-based array from 80 kb to 35 kb reduced the frequency of HAC formation. In addition to the α-satellite component, vector composition also influenced HAC formation rates, size, and copy number. The data presented here have a significant impact on the design of future HAC vectors that have potential to be developed for therapeutic applications and as tools for investigating Human Chromosome structure and function.