World Scientific Lecture Notes in Complex Systems, 2012
ABSTRACT Synthetic biology is a new branch of interdisciplinary science that has been developed i... more ABSTRACT Synthetic biology is a new branch of interdisciplinary science that has been developed in recent years. The main purpose of synthetic biology is to apply successful principles that have been developed in electronic and chemical engineering to develop basic biological functional modules, and through rational design, develop man-made biological systems that have predicted useful functions. Here, we discuss an important principle in rational design of functional biological circuits: the reverse engineering design. We will use a research project that was conducted at Peking University for the International Genetic Engineering Machine Competition (iGEM) to illustrate the principle: synthesis a cell which has a semi-log dose-response to the environment. Through this work we try to demonstrate the potential application of network engineering in synthetic biology.
A central goal of synthetic biology is to apply successful principles that have been developed in... more A central goal of synthetic biology is to apply successful principles that have been developed in electronic and chemical engineering to construct basic biological functional modules, and through rational design, to build synthetic biological systems with predetermined functions. Here, we apply the reverse engineering design principle of biological networks to synthesize a gene circuit that executes semi-log dose-response, a logarithmically linear sensing function, in Escherichia coli cells. We first mathematically define the object function semi-log dose-response, and then search for tri-node network topologies that can most robustly execute the object function. The simplest topology, transcriptional coherent feed-forward loop (TCFL), among the searching results is mathematically analyzed; we find that, in TCFL topology, the semi-log dose-response function arises from the additive effect of logarithmical linearity intervals of Hill functions. TCFL is then genetically implemented in E. coli as a logarithmically linear sensing biosensor for heavy metal ions [mercury (II)]. Functional characterization shows that this rationally designed biosensor circuit works as expected. Through this study we demonstrated the potential application of biological network reverse engineering to broaden the computational power of synthetic biology.
Living cells respond to changing environments by regulating their genes and activities. In unicel... more Living cells respond to changing environments by regulating their genes and activities. In unicellular organisms such as yeasts, the cell division cycle is coupled to the nutrient availability. However, it is unclear how tight this coupling is and how the intrinsic time scales of the different cell cycle processes respond to varying nutrient conditions. Here we study the cell cycle behavior of the budding yeast Saccharomyces cerevisiae in response to periodically modulated nutrient availability, using a microfluidic platform which allows for longtime cultivation, programmed medium switching, and automated time-lapse image acquisition. We observe that the division cycle of the yeast cells can follow a periodically modulated medium so that the whole population can be driven into synchrony. When the period of the nutrient modulation is optimized, as many as 80% of the cells in a population are continuously synchronized. The degree of synchronization as a function of the nutrient modulation period can be qualitatively captured by a stochastic phenomenological model. Our work may shed light on the coupling between the cell growth and cell division as well as provide a nontoxic and non-invasive method to continuously synchronize the cell cycle. † Electronic supplementary information (ESI) available. See
Microsatellite instability (MSI) is characterized by the expansion or contraction of DNA repeat t... more Microsatellite instability (MSI) is characterized by the expansion or contraction of DNA repeat tracts as a consequence of DNA mismatch repair deficiency (MMRD). Accurate detection of MSI in cancer cells is important since MSI is associated with several cancer subtypes and can help inform therapeutic decisions. Although experimental assays have been developed to detect MSI, they typically depend on a small number of known microsatellite loci or mismatch repair genes and have limited reliability. Here, we report a novel genome-wide approach for MSI detection based on the global detection of insertions and deletions (indels) in microsatellites found in expressed genes. Our large-scale analyses of 20 cancer cell lines and 123 normal individuals revealed striking indel features associated with MSI: there is a significant increase of short microsatellite deletions in MSI samples compared to microsatellite stable (MSS) ones, suggesting a mechanistic bias of repair efficiency between insertions and deletions in normal human cells. By incorporating this observation into our MSI scoring metric, we show that our approach can correctly distinguish between MSI and MSS cancer cell lines. Moreover, when we applied this approach to primal tumor samples, our metric is also well consistent with diagnosed MSI status. Thus, our study offers new insight into DNA mismatch repair system, and also provides a novel MSI diagnosis method for clinical oncology with better reliability.
RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood... more RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578 leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-mRNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte di...
MicroRNA (miRNA)-dependent regulation of gene expression confers robustness to cellular phenotype... more MicroRNA (miRNA)-dependent regulation of gene expression confers robustness to cellular phenotypes and controls responses to extracellular stimuli. Although a single miRNA can regulate expression of hundreds of target genes, it is unclear whether any of its distinct biological functions can be due to the regulation of a single target. To explore in vivo the function of a single miRNA-mRNA interaction, we mutated the 3' UTR of a major miR-155 target (SOCS1) to specifically disrupt its regulation by miR-155. We found that under physiologic conditions and during autoimmune inflammation or viral infection, some immunological functions of miR-155 were fully or largely attributable to the regulation of SOCS1, whereas others could be accounted only partially or not at all by this interaction. Our data suggest that the role of a single miRNA-mRNA interaction is dependent on cell type and biological context.
Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therap... more Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. Leukemia cells exhibit a dysregulated developmental program as the result of genetic and epigenetic alterations. Overexpression of the RNA-binding protein Musashi2 (MSI2) has been previously shown to predict poor survival in leukemia. Here, we demonstrated that conditional deletion of Msi2 in the hematopoietic compartment results in delayed leukemogenesis, reduced disease burden, and a loss of LSC function in a murine leukemia model. Gene expression profiling of these Msi2-deficient animals revealed a loss of the hematopoietic/leukemic stem cell self-renewal program and an increase in the differentiation program. In acute myeloid leukemia patients, the presence of a gene signature that was similar to that observed in Msi2-deficent murine LSCs correlated with improved survival. We determined that MSI2 directly maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting with and retaining efficient translation of Hoxa9, Myc, and Ikzf2 mRNAs. Moreover, depletion of MLL target Ikzf2 in LSCs reduced colony formation, decreased proliferation, and increased apoptosis. Our data provide evidence that MSI2 controls efficient translation of the oncogenic LSC self-renewal program and suggest MSI2 as a potential therapeutic target for myeloid leukemia.
Hematopoietic stem cells (HSCs) are maintained through the regulation of symmetric and asymmetric... more Hematopoietic stem cells (HSCs) are maintained through the regulation of symmetric and asymmetric cell division. We report that conditional ablation of the RNA-binding protein Msi2 results in a failure of HSC maintenance and engraftment caused by a loss of quiescence and increased commitment divisions. Contrary to previous studies, we found that these phenotypes were independent of Numb. Global transcriptome profiling and RNA target analysis uncovered Msi2 interactions at multiple nodes within pathways that govern RNA translation, stem cell function, and TGF- signaling. Msi2-null HSCs are insensitive to TGF--mediated expansion and have decreased signaling output, resulting in a loss of myeloid-restricted HSCs and myeloid reconstitution. Thus, Msi2 is an important regulator of the HSC translatome and balances HSC homeostasis and lineage bias.
World Scientific Lecture Notes in Complex Systems, 2012
ABSTRACT Synthetic biology is a new branch of interdisciplinary science that has been developed i... more ABSTRACT Synthetic biology is a new branch of interdisciplinary science that has been developed in recent years. The main purpose of synthetic biology is to apply successful principles that have been developed in electronic and chemical engineering to develop basic biological functional modules, and through rational design, develop man-made biological systems that have predicted useful functions. Here, we discuss an important principle in rational design of functional biological circuits: the reverse engineering design. We will use a research project that was conducted at Peking University for the International Genetic Engineering Machine Competition (iGEM) to illustrate the principle: synthesis a cell which has a semi-log dose-response to the environment. Through this work we try to demonstrate the potential application of network engineering in synthetic biology.
A central goal of synthetic biology is to apply successful principles that have been developed in... more A central goal of synthetic biology is to apply successful principles that have been developed in electronic and chemical engineering to construct basic biological functional modules, and through rational design, to build synthetic biological systems with predetermined functions. Here, we apply the reverse engineering design principle of biological networks to synthesize a gene circuit that executes semi-log dose-response, a logarithmically linear sensing function, in Escherichia coli cells. We first mathematically define the object function semi-log dose-response, and then search for tri-node network topologies that can most robustly execute the object function. The simplest topology, transcriptional coherent feed-forward loop (TCFL), among the searching results is mathematically analyzed; we find that, in TCFL topology, the semi-log dose-response function arises from the additive effect of logarithmical linearity intervals of Hill functions. TCFL is then genetically implemented in E. coli as a logarithmically linear sensing biosensor for heavy metal ions [mercury (II)]. Functional characterization shows that this rationally designed biosensor circuit works as expected. Through this study we demonstrated the potential application of biological network reverse engineering to broaden the computational power of synthetic biology.
Living cells respond to changing environments by regulating their genes and activities. In unicel... more Living cells respond to changing environments by regulating their genes and activities. In unicellular organisms such as yeasts, the cell division cycle is coupled to the nutrient availability. However, it is unclear how tight this coupling is and how the intrinsic time scales of the different cell cycle processes respond to varying nutrient conditions. Here we study the cell cycle behavior of the budding yeast Saccharomyces cerevisiae in response to periodically modulated nutrient availability, using a microfluidic platform which allows for longtime cultivation, programmed medium switching, and automated time-lapse image acquisition. We observe that the division cycle of the yeast cells can follow a periodically modulated medium so that the whole population can be driven into synchrony. When the period of the nutrient modulation is optimized, as many as 80% of the cells in a population are continuously synchronized. The degree of synchronization as a function of the nutrient modulation period can be qualitatively captured by a stochastic phenomenological model. Our work may shed light on the coupling between the cell growth and cell division as well as provide a nontoxic and non-invasive method to continuously synchronize the cell cycle. † Electronic supplementary information (ESI) available. See
Microsatellite instability (MSI) is characterized by the expansion or contraction of DNA repeat t... more Microsatellite instability (MSI) is characterized by the expansion or contraction of DNA repeat tracts as a consequence of DNA mismatch repair deficiency (MMRD). Accurate detection of MSI in cancer cells is important since MSI is associated with several cancer subtypes and can help inform therapeutic decisions. Although experimental assays have been developed to detect MSI, they typically depend on a small number of known microsatellite loci or mismatch repair genes and have limited reliability. Here, we report a novel genome-wide approach for MSI detection based on the global detection of insertions and deletions (indels) in microsatellites found in expressed genes. Our large-scale analyses of 20 cancer cell lines and 123 normal individuals revealed striking indel features associated with MSI: there is a significant increase of short microsatellite deletions in MSI samples compared to microsatellite stable (MSS) ones, suggesting a mechanistic bias of repair efficiency between insertions and deletions in normal human cells. By incorporating this observation into our MSI scoring metric, we show that our approach can correctly distinguish between MSI and MSS cancer cell lines. Moreover, when we applied this approach to primal tumor samples, our metric is also well consistent with diagnosed MSI status. Thus, our study offers new insight into DNA mismatch repair system, and also provides a novel MSI diagnosis method for clinical oncology with better reliability.
RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood... more RBM15, an RNA binding protein, determines cell-fate specification of many tissues including blood. We demonstrate that RBM15 is methylated by protein arginine methyltransferase 1 (PRMT1) at residue R578 leading to its degradation via ubiquitylation by an E3 ligase (CNOT4). Overexpression of PRMT1 in acute megakaryocytic leukemia cell lines blocks megakaryocyte terminal differentiation by downregulation of RBM15 protein level. Restoring RBM15 protein level rescues megakaryocyte terminal differentiation blocked by PRMT1 overexpression. At the molecular level, RBM15 binds to pre-mRNA intronic regions of genes important for megakaryopoiesis such as GATA1, RUNX1, TAL1 and c-MPL. Furthermore, preferential binding of RBM15 to specific intronic regions recruits the splicing factor SF3B1 to the same sites for alternative splicing. Therefore, PRMT1 regulates alternative RNA splicing via reducing RBM15 protein concentration. Targeting PRMT1 may be a curative therapy to restore megakaryocyte di...
MicroRNA (miRNA)-dependent regulation of gene expression confers robustness to cellular phenotype... more MicroRNA (miRNA)-dependent regulation of gene expression confers robustness to cellular phenotypes and controls responses to extracellular stimuli. Although a single miRNA can regulate expression of hundreds of target genes, it is unclear whether any of its distinct biological functions can be due to the regulation of a single target. To explore in vivo the function of a single miRNA-mRNA interaction, we mutated the 3' UTR of a major miR-155 target (SOCS1) to specifically disrupt its regulation by miR-155. We found that under physiologic conditions and during autoimmune inflammation or viral infection, some immunological functions of miR-155 were fully or largely attributable to the regulation of SOCS1, whereas others could be accounted only partially or not at all by this interaction. Our data suggest that the role of a single miRNA-mRNA interaction is dependent on cell type and biological context.
Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therap... more Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. Leukemia cells exhibit a dysregulated developmental program as the result of genetic and epigenetic alterations. Overexpression of the RNA-binding protein Musashi2 (MSI2) has been previously shown to predict poor survival in leukemia. Here, we demonstrated that conditional deletion of Msi2 in the hematopoietic compartment results in delayed leukemogenesis, reduced disease burden, and a loss of LSC function in a murine leukemia model. Gene expression profiling of these Msi2-deficient animals revealed a loss of the hematopoietic/leukemic stem cell self-renewal program and an increase in the differentiation program. In acute myeloid leukemia patients, the presence of a gene signature that was similar to that observed in Msi2-deficent murine LSCs correlated with improved survival. We determined that MSI2 directly maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting with and retaining efficient translation of Hoxa9, Myc, and Ikzf2 mRNAs. Moreover, depletion of MLL target Ikzf2 in LSCs reduced colony formation, decreased proliferation, and increased apoptosis. Our data provide evidence that MSI2 controls efficient translation of the oncogenic LSC self-renewal program and suggest MSI2 as a potential therapeutic target for myeloid leukemia.
Hematopoietic stem cells (HSCs) are maintained through the regulation of symmetric and asymmetric... more Hematopoietic stem cells (HSCs) are maintained through the regulation of symmetric and asymmetric cell division. We report that conditional ablation of the RNA-binding protein Msi2 results in a failure of HSC maintenance and engraftment caused by a loss of quiescence and increased commitment divisions. Contrary to previous studies, we found that these phenotypes were independent of Numb. Global transcriptome profiling and RNA target analysis uncovered Msi2 interactions at multiple nodes within pathways that govern RNA translation, stem cell function, and TGF- signaling. Msi2-null HSCs are insensitive to TGF--mediated expansion and have decreased signaling output, resulting in a loss of myeloid-restricted HSCs and myeloid reconstitution. Thus, Msi2 is an important regulator of the HSC translatome and balances HSC homeostasis and lineage bias.
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