Publications by authors named "Albert J Keung"

33 Publications

Human Pluripotent Stem Cell-Derived Medium Spiny Neuron-like Cells Exhibit Gene Desensitization.

Cells 2022 04 21;11(9). Epub 2022 Apr 21.

Chemical & Biomolecular Engineering Department, North Carolina State University, Campus Box 7905, Raleigh, NC 27695, USA.

Gene desensitization in response to a repeated stimulus is a complex phenotype important across homeostatic and disease processes, including addiction, learning, and memory. These complex phenotypes are being characterized and connected to important physiologically relevant functions in rodent systems but are difficult to capture in human models where even acute responses to important neurotransmitters are understudied. Here through transcriptomic analysis, we map the dynamic responses of human stem cell-derived medium spiny neuron-like cells (hMSN-like cells) to dopamine. Furthermore, we show that these human neurons can reflect and capture cellular desensitization to chronic versus acute administration of dopamine. These human cells are further able to capture complex receptor crosstalk in response to the pharmacological perturbations of distinct dopamine receptor subtypes. This study demonstrates the potential utility and remaining challenges of using human stem cell-derived neurons to capture and study the complex dynamic mechanisms of the brain.
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http://dx.doi.org/10.3390/cells11091411DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9100557PMC
April 2022

Yeast Display Guided Selection of pH-Dependent Binders.

Methods Mol Biol 2022 ;2491:293-311

Mythic Therapeutics, Waltham, MA, USA.

pH-dependent antigen binding has proven useful in engineering next-generation therapeutics specifically via antibody recycling technology. This technology allows for half-life extension, thereby lowering the amount and frequency of dosing of therapeutics. Cell sorting, coupled with display techniques, has been used extensively for the selection of high-affinity binders. Herein, we describe a cell sorting methodology utilizing yeast surface display for selection of binding proteins with strong binding at physiological pH and weak to no binding at acidic pH. This methodology can be readily applied to engineer proteins and/or antibodies that do not have pH-dependent binding or for selection of de novo pH-dependent binders using library-based methods.
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http://dx.doi.org/10.1007/978-1-0716-2285-8_16DOI Listing
January 2022

Modified Histone Peptides Linked to Magnetic Beads Reduce Binding Specificity.

Int J Mol Sci 2022 Feb 1;23(3). Epub 2022 Feb 1.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC 27606, USA.

Histone post-translational modifications are small chemical changes to the histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET-based systems. High-throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet, published studies on their use with histones have been noticeably sparse. Here, we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.
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http://dx.doi.org/10.3390/ijms23031691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8836101PMC
February 2022

Effects of early geometric confinement on the transcriptomic profile of human cerebral organoids.

BMC Biotechnol 2021 10 12;21(1):59. Epub 2021 Oct 12.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC, 27695-7905, USA.

Background: Human cerebral organoids (hCO) are attractive systems due to their ability to model important brain regions and transcriptomics of early in vivo brain development. To date, they have been used to understand the effects of genetics and soluble factors on neurodevelopment. Interestingly, one of the main advantages of hCOs are that they provide three dimensionality that better mimics the in vivo environment; yet, despite this central feature it remains unclear how spatial and mechanical properties regulate hCO and neurodevelopment. While biophysical factors such as shape and mechanical forces are known to play crucial roles in stem cell differentiation, embryogenesis and neurodevelopment, much of this work investigated two dimensional systems or relied on correlative observations of native developing tissues in three dimensions. Using hCOs to establish links between spatial factors and neurodevelopment will require the use of new approaches and could reveal fundamental principles of brain organogenesis as well as improve hCOs as an experimental model.

Results: Here, we investigated the effects of early geometric confinements on transcriptomic changes during hCO differentiation. Using a custom and tunable agarose microwell platform we generated embryoid bodies (EB) of diverse shapes mimicking several structures from embryogenesis and neurodevelopment and then further differentiated those EBs to whole brain hCOs. Our results showed that the microwells did not have negative gross impacts on the ability of the hCOs to differentiate towards neural fates, and there were clear shape dependent effects on neural lineage specification. In particular we observed that non-spherical shapes showed signs of altered neurodevelopmental kinetics and favored the development of medial ganglionic eminence-associated brain regions and cell types over cortical regions. Transcriptomic analysis suggests these mechanotransducive effects may be mediated by integrin and Wnt signaling.

Conclusions: The findings presented here suggest a role for spatial factors in brain region specification during hCO development. Understanding these spatial patterning factors will not only improve understanding of in vivo development and differentiation, but also provide important handles with which to advance and improve control over human model systems for in vitro applications.
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http://dx.doi.org/10.1186/s12896-021-00718-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8507123PMC
October 2021

Mapping the dynamic transfer functions of eukaryotic gene regulation.

Cell Syst 2021 11 31;12(11):1079-1093.e6. Epub 2021 Aug 31.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC 27606, USA. Electronic address:

Biological information can be encoded within the dynamics of signaling components, which has been implicated in a broad range of physiological processes including stress response, oncogenesis, and stem cell differentiation. To study the complexity of information transfer across the eukaryotic promoter, we screened 119 dynamic conditions-modulating the pulse frequency, amplitude, and pulse width of light-regulating the binding of an epigenome editor to a fluorescent reporter. This system revealed tunable gene expression and filtering behaviors and provided a quantification of the limit to the amount of information that can be reliably transferred across a single promoter as ∼1.7 bits. Using a library of over 100 orthogonal chromatin regulators, we further determined that chromatin state could be used to tune mutual information and expression levels, as well as completely alter the input-output transfer function of the promoter. This system unlocks the information-rich content of eukaryotic gene regulation.
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http://dx.doi.org/10.1016/j.cels.2021.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8602734PMC
November 2021

Mapping the residue specificities of epigenome enzymes by yeast surface display.

Cell Chem Biol 2021 12 28;28(12):1772-1779.e4. Epub 2021 Jun 28.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC 27606, USA. Electronic address:

Histone proteins are decorated with a combinatorially and numerically diverse set of biochemical modifications. Here, we describe a versatile and scalable approach which enables efficient characterization of histone modifications without the need for recombinant protein production. As proof-of-concept, we first use this system to rapidly profile the histone H3 and H4 residue writing specificities of the human histone acetyltransferase, p300. Subsequently, a large panel of commercially available anti-acetylation antibodies are screened for their specificities, identifying many suitable and unsuitable reagents. Furthermore, this approach enables efficient mapping of the large binary crosstalk space between acetylated residues on histones H3 and H4 and uncovers residue interdependencies affecting p300 activity. These results show that using yeast surface display to study histone modifications is a useful tool that can advance our understanding of chromatin biology by enabling efficient interrogation of the complexity of epigenome modifications.
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http://dx.doi.org/10.1016/j.chembiol.2021.05.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8665936PMC
December 2021

Promiscuous molecules for smarter file operations in DNA-based data storage.

Nat Commun 2021 06 10;12(1):3518. Epub 2021 Jun 10.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.

DNA holds significant promise as a data storage medium due to its density, longevity, and resource and energy conservation. These advantages arise from the inherent biomolecular structure of DNA which differentiates it from conventional storage media. The unique molecular architecture of DNA storage also prompts important discussions on how data should be organized, accessed, and manipulated and what practical functionalities may be possible. Here we leverage thermodynamic tuning of biomolecular interactions to implement useful data access and organizational features. Specific sets of environmental conditions including distinct DNA concentrations and temperatures were screened for their ability to switchably access either all DNA strands encoding full image files from a GB-sized background database or subsets of those strands encoding low resolution, File Preview, versions. We demonstrate File Preview with four JPEG images and provide an argument for the substantial and practical economic benefit of this generalizable strategy to organize data.
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http://dx.doi.org/10.1038/s41467-021-23669-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8192770PMC
June 2021

Evaluation of UBE3A antibodies in mice and human cerebral organoids.

Sci Rep 2021 03 18;11(1):6323. Epub 2021 Mar 18.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, 27695-7905, USA.

UBE3A is an E3 ubiquitin ligase encoded by the neurally imprinted UBE3A gene. The abundance and subcellular distribution of UBE3A has been the topic of many previous studies as its dosage and localization has been linked to neurodevelopmental disorders including Autism, Dup15q syndrome, and Angelman syndrome. While commercially available antibodies have been widely employed to determine UBE3A localization, an extensive analysis and comparison of the performance of different UBE3A antibodies has not been conducted. Here we evaluated the specificities of seven commercial UBE3A antibodies in two of the major experimental models used in UBE3A research, mouse and human pluripotent stem cell-derived neural cells and tissues. We tested these antibodies in their two most common assays, immunofluorescence and western blot. In addition, we also assessed the ability of these antibodies to capture dynamic spatiotemporal changes of UBE3A by utilizing human cerebral organoid models. Our results reveal that among the seven antibodies tested, three antibodies demonstrated substantial nonspecific immunoreactivity. While four antibodies show specific localization patterns in both mouse brain sections and human cerebral organoids, these antibodies varied significantly in background signals and staining patterns in undifferentiated human pluripotent stem cells.
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http://dx.doi.org/10.1038/s41598-021-85923-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973473PMC
March 2021

DNA stability: a central design consideration for DNA data storage systems.

Nat Commun 2021 03 1;12(1):1358. Epub 2021 Mar 1.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, USA.

Data storage in DNA is a rapidly evolving technology that could be a transformative solution for the rising energy, materials, and space needs of modern information storage. Given that the information medium is DNA itself, its stability under different storage and processing conditions will fundamentally impact and constrain design considerations and data system capabilities. Here we analyze the storage conditions, molecular mechanisms, and stabilization strategies influencing DNA stability and pose specific design configurations and scenarios for future systems that best leverage the considerable advantages of DNA storage.
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http://dx.doi.org/10.1038/s41467-021-21587-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7921107PMC
March 2021

Human Cerebral Organoids Reveal Early Spatiotemporal Dynamics and Pharmacological Responses of UBE3A.

Stem Cell Reports 2020 10 10;15(4):845-854. Epub 2020 Sep 10.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC 27606, USA. Electronic address:

Angelman syndrome is a complex neurodevelopmental disorder characterized by delayed development, intellectual disability, speech impairment, and ataxia. It results from the loss of UBE3A protein, an E3 ubiquitin ligase, in neurons of the brain. Despite the dynamic spatiotemporal expression of UBE3A observed in rodents and the potential clinical importance of when and where it is expressed, its expression pattern in humans remains unknown. This reflects a common challenge of studying human neurodevelopment: prenatal periods are hard to access experimentally. In this work, human cerebral organoids reveal a change from weak to strong UBE3A in neuronal nuclei within 3 weeks of culture. Angelman syndrome human induced pluripotent stem cell-derived organoids also exhibit early silencing of paternal UBE3A, with topoisomerase inhibitors partially rescuing UBE3A levels and calcium transient phenotypes. This work establishes human cerebral organoids as an important model for studying UBE3A and motivates their broader use in understanding complex neurodevelopmental disorders.
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http://dx.doi.org/10.1016/j.stemcr.2020.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7561513PMC
October 2020

Capturing complex epigenetic phenomena through human multicellular systems.

Curr Opin Biomed Eng 2020 Dec 7;16:34-41. Epub 2020 Jun 7.

North Carolina State University, Raleigh, NC 27606.

Epigenetic states inherently define a wide range of complex biological phenotypes and processes in development and disease. Accurate cellular modeling would ideally capture the epigenetic complexity of these processes as well as the accompanying molecular changes in chromatin biochemistry including in DNA and histone modifications. Here we highlight recent work that demonstrate how multicellular systems provide a natural approach to capture complex epigenetic phenomena. They accomplish this through more closely matching the environment and through the intrinsic nature of multicellular systems being able to generate and model multiple distinct cellular states, all within one system. We also discuss challenges and limitations of such systems, efforts to tune and modulate epigenetics directly in multicellular systems, and how molecular interventional approaches could advance and improve the utility of these models.
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http://dx.doi.org/10.1016/j.cobme.2020.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7467398PMC
December 2020

Dynamic and scalable DNA-based information storage.

Nat Commun 2020 06 12;11(1):2981. Epub 2020 Jun 12.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Campus Box 7905, Raleigh, NC, 27695-7905, USA.

The physical architectures of information storage systems often dictate how information is encoded, databases are organized, and files are accessed. Here we show that a simple architecture comprised of a T7 promoter and a single-stranded overhang domain (ss-dsDNA), can unlock dynamic DNA-based information storage with powerful capabilities and advantages. The overhang provides a physical address for accessing specific DNA strands as well as implementing a range of in-storage file operations. It increases theoretical storage densities and capacities by expanding the encodable sequence space and simplifies the computational burden in designing sets of orthogonal file addresses. Meanwhile, the T7 promoter enables repeatable information access by transcribing information from DNA without destroying it. Furthermore, saturation mutagenesis around the T7 promoter and systematic analyses of environmental conditions reveal design criteria that can be used to optimize information access. This simple but powerful ss-dsDNA architecture lays the foundation for information storage with versatile capabilities.
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http://dx.doi.org/10.1038/s41467-020-16797-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293219PMC
June 2020

Modular one-pot assembly of CRISPR arrays enables library generation and reveals factors influencing crRNA biogenesis.

Nat Commun 2019 07 3;10(1):2948. Epub 2019 Jul 3.

Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, NC, 27695, USA.

CRISPR-Cas systems inherently multiplex through CRISPR arrays-whether to defend against different invaders or mediate multi-target editing, regulation, imaging, or sensing. However, arrays remain difficult to generate due to their reoccurring repeat sequences. Here, we report a modular, one-pot scheme called CRATES to construct CRISPR arrays and array libraries. CRATES allows assembly of repeat-spacer subunits using defined assembly junctions within the trimmed portion of spacers. Using CRATES, we construct arrays for the single-effector nucleases Cas9, Cas12a, and Cas13a that mediated multiplexed DNA/RNA cleavage and gene regulation in cell-free systems, bacteria, and yeast. CRATES further allows the one-pot construction of array libraries and composite arrays utilized by multiple Cas nucleases. Finally, array characterization reveals processing of extraneous CRISPR RNAs from Cas12a terminal repeats and sequence- and context-dependent loss of RNA-directed nuclease activity via global RNA structure formation. CRATES thus can facilitate diverse multiplexing applications and help identify factors impacting crRNA biogenesis.
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http://dx.doi.org/10.1038/s41467-019-10747-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6610086PMC
July 2019

Driving the Scalability of DNA-Based Information Storage Systems.

ACS Synth Biol 2019 06 24;8(6):1241-1248. Epub 2019 May 24.

Department of Chemical and Biomolecular Engineering , North Carolina State University , Raleigh , North Carolina 27695 , United States.

The extreme density of DNA presents a compelling advantage over current storage media; however, to reach practical capacities, new systems for organizing and accessing information are needed. Here, we use chemical handles to selectively extract unique files from a complex database of DNA mimicking 5 TB of data and design and implement a nested file address system that increases the theoretical maximum capacity of DNA storage systems by five orders of magnitude. These advancements enable the development and future scaling of DNA-based data storage systems with modern capacities and file access capabilities.
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http://dx.doi.org/10.1021/acssynbio.9b00100DOI Listing
June 2019

Engineering Epigenetic Regulation Using Synthetic Read-Write Modules.

Cell 2019 Jan 6;176(1-2):227-238.e20. Epub 2018 Dec 6.

Biological Design Center, Boston University, Boston, MA 02215, USA; Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. Electronic address:

Chemical modifications to DNA and histone proteins are involved in epigenetic programs underlying cellular differentiation and development. Regulatory networks involving molecular writers and readers of chromatin marks are thought to control these programs. Guided by this common principle, we established an orthogonal epigenetic regulatory system in mammalian cells using N6-methyladenine (m6A), a DNA modification not commonly found in metazoan epigenomes. Our system utilizes synthetic factors that write and read m6A and consequently recruit transcriptional regulators to control reporter loci. Inspired by models of chromatin spreading and epigenetic inheritance, we used our system and mathematical models to construct regulatory circuits that induce m6A-dependent transcriptional states, promote their spatial propagation, and maintain epigenetic memory of the states. These minimal circuits were able to program epigenetic functions de novo, conceptually validating "read-write" architectures. This work provides a toolkit for investigating models of epigenetic regulation and encoding additional layers of epigenetic information in cells.
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http://dx.doi.org/10.1016/j.cell.2018.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6329643PMC
January 2019

Chromatin Immunoprecipitation in Human and Yeast Cells.

Methods Mol Biol 2018 ;1767:257-269

Chemical and Biomolecular Engineering Department, North Carolina State University, Raleigh, NC, USA.

Chromatin immunoprecipitation (ChIP) is an invaluable method to characterize interactions between proteins and genomic DNA, such as the genomic localization of transcription factors and posttranslational modification of histones. DNA and proteins are reversibly and covalently crosslinked using formaldehyde. Then the cells are lysed to release the chromatin. The chromatin is fragmented into smaller sizes either by micrococcal nuclease (MNase) or sonication and then purified from other cellular components. The protein-DNA complexes are enriched by immunoprecipitation (IP) with antibodies that target the epitope of interest. The DNA is released from the proteins by heat and protease treatment, followed by degradation of contaminating RNAs with RNase. The resulting DNA is analyzed using various methods, including PCR, qPCR, or sequencing. This protocol outlines each of these steps for both yeast and human cells.
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http://dx.doi.org/10.1007/978-1-4939-7774-1_14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987192PMC
February 2019

Designing Epigenome Editors: Considerations of Biochemical and Locus Specificities.

Methods Mol Biol 2018 ;1767:65-87

Chemical and Biomolecular Engineering Department, North Carolina State University, Raleigh, NC, USA.

The advent of locus-specific protein recruitment technologies has enabled a new class of studies in chromatin biology. Epigenome editors enable biochemical modifications of chromatin at almost any specific endogenous locus. Their locus specificity unlocks unique information including the functional roles of distinct modifications at specific genomic loci. Given the growing interest in using these tools for biological and translational studies, there are many specific design considerations depending on the scientific question or clinical need. Here we present and discuss important design considerations and challenges regarding the biochemical and locus specificities of epigenome editors. These include how to account for the complex biochemical diversity of chromatin; control for potential interdependency of epigenome editors and their resultant modifications; avoid sequestration effects; quantify the locus specificity of epigenome editors; and improve locus specificity by considering concentration, affinity, avidity, and sequestration effects.
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http://dx.doi.org/10.1007/978-1-4939-7774-1_3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972380PMC
February 2019

The epigenome: the next substrate for engineering.

Genome Biol 2016 08 31;17(1):183. Epub 2016 Aug 31.

Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, MA, 02215, USA.

We are entering an era of epigenome engineering. The precision manipulation of chromatin and epigenetic modifications provides new ways to interrogate their influence on genome and cell function and to harness these changes for applications. We review the design and state of epigenome editing tools, highlighting the unique regulatory properties afforded by these systems.
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http://dx.doi.org/10.1186/s13059-016-1046-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006378PMC
August 2016

MOLECULAR BIOLOGY. A unifying model of epigenetic regulation.

Science 2016 Feb;351(6274):661-2

Department of Biomedical Engineering and Biological Design Center, Boston University, Boston, MA, USA. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA.

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http://dx.doi.org/10.1126/science.aaf1647DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124315PMC
February 2016

Chromatin regulation at the frontier of synthetic biology.

Nat Rev Genet 2015 Mar 10;16(3):159-71. Epub 2015 Feb 10.

1] Institute for Medical Engineering &Science, Synthetic Biology Center and Department of Biological Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts 02139, USA. [2] Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, USA. [3] Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA.

As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including 'epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication.
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http://dx.doi.org/10.1038/nrg3900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846386PMC
March 2015

Using targeted chromatin regulators to engineer combinatorial and spatial transcriptional regulation.

Cell 2014 Jul;158(1):110-20

Department of Biomedical Engineering and Center of Synthetic Biology, Boston University, Boston, MA 02215, USA; Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA. Electronic address:

The transcription of genomic information in eukaryotes is regulated in large part by chromatin. How a diverse array of chromatin regulator (CR) proteins with different functions and genomic localization patterns coordinates chromatin activity to control transcription remains unclear. Here, we take a synthetic biology approach to decipher the complexity of chromatin regulation by studying emergent transcriptional behaviors from engineered combinatorial, spatial, and temporal patterns of individual CRs. We fuse 223 yeast CRs to programmable zinc finger proteins. Site-specific and combinatorial recruitment of CRs to distinct intralocus locations reveals a range of transcriptional logic and behaviors, including synergistic activation, long-range and spatial regulation, and gene expression memory. Comparing these transcriptional behaviors with annotated CR complex and function terms provides design principles for the engineering of transcriptional regulation. This work presents a bottom-up approach to investigating chromatin-mediated transcriptional regulation and introduces chromatin-based components and systems for synthetic biology and cellular engineering.
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http://dx.doi.org/10.1016/j.cell.2014.04.047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4110908PMC
July 2014

Pan-neuronal maturation but not neuronal subtype differentiation of adult neural stem cells is mechanosensitive.

Sci Rep 2013 ;3:1817

Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA.

Most past studies of the biophysical regulation of stem cell differentiation have focused on initial lineage commitment or proximal differentiation events. It would be valuable to understand whether biophysical inputs also influence distal endpoints more closely associated with physiological function, such as subtype specification in neuronal differentiation. To explore this question, we cultured adult neural stem cells (NSCs) on variable stiffness ECMs under conditions that promote neuronal fate commitment for extended time periods to allow neuronal subtype differentiation. We find that ECM stiffness does not modulate the expression of NeuroD1 and TrkA/B/C or the percentages of pan-neuronal, GABAergic, or glutamatergic neuronal subtypes. Interestingly, however, an ECM stiffness of 700 Pa maximizes expression of pan-neuronal markers. These results suggest that a wide range of stiffnesses fully permit pan-neuronal NSC differentiation, that an intermediate stiffness optimizes expression of pan-neuronal genes, and that stiffness does not impact commitment to particular neuronal subtypes.
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http://dx.doi.org/10.1038/srep01817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650663PMC
June 2014

Soft microenvironments promote the early neurogenic differentiation but not self-renewal of human pluripotent stem cells.

Integr Biol (Camb) 2012 Sep 2;4(9):1049-58. Epub 2012 Aug 2.

Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA.

Human pluripotent stem cells (hPSCs) are of great interest in biology and medicine due to their ability to self-renew and differentiate into any adult or fetal cell type. Important efforts have identified biochemical factors, signaling pathways, and transcriptional networks that regulate hPSC biology. However, recent work investigating the effect of biophysical cues on mammalian cells and adult stem cells suggests that the mechanical properties of the microenvironment, such as stiffness, may also regulate hPSC behavior. While several studies have explored this mechanoregulation in mouse embryonic stem cells (mESCs), it has been challenging to extrapolate these findings and thereby explore their biomedical implications in hPSCs. For example, it remains unclear whether hPSCs can be driven down a given tissue lineage by providing tissue-mimetic stiffness cues. Here we address this open question by investigating the regulation of hPSC neurogenesis by microenvironmental stiffness. We find that increasing extracellular matrix (ECM) stiffness in vitro increases hPSC cell and colony spread area but does not alter self-renewal, in contrast to past studies with mESCs. However, softer ECMs with stiffnesses similar to that of neural tissue promote the generation of early neural ectoderm. This mechanosensitive increase in neural ectoderm requires only a short 5-day soft stiffness "pulse", which translates into downstream increases in both total neurons as well as therapeutically relevant dopaminergic neurons. These findings further highlight important differences between mESCs and hPSCs and have implications for both the design of future biomaterials as well as our understanding of early embryonic development.
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http://dx.doi.org/10.1039/c2ib20083jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3459311PMC
September 2012

A genetic strategy for the dynamic and graded control of cell mechanics, motility, and matrix remodeling.

Biophys J 2012 Feb 7;102(3):434-42. Epub 2012 Feb 7.

Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California, USA.

Cellular mechanical properties have emerged as central regulators of many critical cell behaviors, including proliferation, motility, and differentiation. Although investigators have developed numerous techniques to influence these properties indirectly by engineering the extracellular matrix (ECM), relatively few tools are available to directly engineer the cells themselves. Here we present a genetic strategy for obtaining graded, dynamic control over cellular mechanical properties by regulating the expression of mutant mechanotransductive proteins from a single copy of a gene placed under a repressible promoter. With the use of constitutively active mutants of RhoA GTPase and myosin light chain kinase, we show that varying the expression level of either protein produces graded changes in stress fiber assembly, traction force generation, cellular stiffness, and migration speed. Using this approach, we demonstrate that soft ECMs render cells maximally sensitive to changes in RhoA activity, and that by modulating the ability of cells to engage and contract soft ECMs, we can dynamically control cell spreading, migration, and matrix remodeling. Thus, in addition to providing quantitative relationships between mechanotransductive signaling, cellular mechanical properties, and dynamic cell behaviors, this strategy enables us to control the physical interactions between cells and the ECM and thereby dictate how cells respond to matrix properties.
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http://dx.doi.org/10.1016/j.bpj.2011.12.048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3274793PMC
February 2012

Rho GTPases mediate the mechanosensitive lineage commitment of neural stem cells.

Stem Cells 2011 Nov;29(11):1886-97

Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720-3220, USA.

Adult neural stem cells (NSCs) play important roles in learning and memory and are negatively impacted by neurological disease. It is known that biochemical and genetic factors regulate self-renewal and differentiation, and it has recently been suggested that mechanical and solid-state cues, such as extracellular matrix (ECM) stiffness, can also regulate the functions of NSCs and other stem cell types. However, relatively little is known of the molecular mechanisms through which stem cells transduce mechanical inputs into fate decisions, the extent to which mechanical inputs instruct fate decisions versus select for or against lineage-committed blast populations, or the in vivo relevance of mechanotransductive signaling molecules in native stem cell niches. Here we demonstrate that ECM-derived mechanical signals act through Rho GTPases to activate the cellular contractility machinery in a key early window during differentiation to regulate NSC lineage commitment. Furthermore, culturing NSCs on increasingly stiff ECMs enhances RhoA and Cdc42 activation, increases NSC stiffness, and suppresses neurogenesis. Likewise, inhibiting RhoA and Cdc42 or downstream regulators of cellular contractility rescues NSCs from stiff matrix- and Rho GTPase-induced neurosuppression. Importantly, Rho GTPase expression and ECM stiffness do not alter proliferation or apoptosis rates indicating that an instructive rather than selective mechanism modulates lineage distributions. Finally, in the adult brain, RhoA activation in hippocampal progenitors suppresses neurogenesis, analogous to its effect in vitro. These results establish Rho GTPase-based mechanotransduction and cellular stiffness as biophysical regulators of NSC fate in vitro and RhoA as an important regulatory protein in the hippocampal stem cell niche.
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http://dx.doi.org/10.1002/stem.746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5990277PMC
November 2011

Progress and prospects for stem cell engineering.

Annu Rev Chem Biomol Eng 2011 ;2:479-502

Department of Chemical Engineering, University of California, Berkeley, CA 94720, USA.

Stem cells offer tremendous biomedical potential owing to their abilities to self-renew and differentiate into cell types of multiple adult tissues. Researchers and engineers have increasingly developed novel discovery technologies, theoretical approaches, and cell culture systems to investigate microenvironmental cues and cellular signaling events that control stem cell fate. Many of these technologies facilitate high-throughput investigation of microenvironmental signals and the intracellular signaling networks and machinery processing those signals into cell fate decisions. As our aggregate empirical knowledge of stem cell regulation grows, theoretical modeling with systems and computational biology methods has and will continue to be important for developing our ability to analyze and extract important conceptual features of stem cell regulation from complex data. Based on this body of knowledge, stem cell engineers will continue to develop technologies that predictably control stem cell fate with the ultimate goal of being able to accurately and economically scale up these systems for clinical-grade production of stem cell therapeutics.
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http://dx.doi.org/10.1146/annurev-chembioeng-061010-114105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991629PMC
May 2012

Akt increases sox2 expression in adult hippocampal neural progenitor cells, but increased sox2 does not promote proliferation.

Stem Cells Dev 2011 Jul 22;20(7):1153-61. Epub 2010 Dec 22.

Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720-3220, USA.

Multiple extracellular factors have been shown to modulate adult hippocampal neural progenitor cell (NPC) proliferation and self-renewal, and we have previously shown that Akt is an important mediator of the effects of these extracellular factors on NPC proliferation and differentiation. However, very little work has investigated how and whether Akt is involved in maintaining the multipotency of these cells. Here we demonstrate that Akt promotes expression of Sox2, a core transcription factor important for the self-renewal of NPCs. Retroviral-mediated overexpression of wild-type Akt increased Sox2 protein expression, particularly under conditions that promote cell differentiation, whereas Akt inhibition decreased Sox2. Similarly, quantitative reverse transcription (RT)-PCR in differentiating cultures indicated that Akt rescued Sox2 mRNA to levels present under conditions that promote cell proliferation. Additionally, pharmacological inhibition of Akt did not affect Sox2 protein levels in cells constitutively expressing Sox2 from a retroviral vector, indicating that Akt does not affect Sox2 protein stability. Further, in contrast to Akt overexpression, Sox2 overexpression does not increase NPC viable cell number or proliferation yet does inhibit differentiation. Collectively, these results indicate that Akt promotes cell proliferation and maintenance of a multipotent state via two downstream paths.
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http://dx.doi.org/10.1089/scd.2010.0130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3121932PMC
July 2011

Biophysics and dynamics of natural and engineered stem cell microenvironments.

Wiley Interdiscip Rev Syst Biol Med 2010 Jan-Feb;2(1):49-64

Department of Chemical Engineering, Department of Bioengineering, The Helen Wills Neuroscience Institute, University of California, Berkeley, CA, USA.

Stem cells are defined by their ability to self-renew and to differentiate into one or more mature lineages, and they reside within natural niches in many types of adult and embryonic tissues that present them with complex signals to regulate these two hallmark properties. The diverse nature of these in vivo microenvironments raises important questions about the microenvironmental cues regulating stem cell plasticity, and the stem cell field has built a strong foundation of knowledge on the biochemical identities and regulatory effects of the soluble, cellular, and extracellular matrix factors surrounding stem cells through the isolation and culture of stem cells in vitro within microenvironments that, in effect, emulate the properties of the natural niche. Recent work, however, has expanded the field's perspective to include biophysical and dynamic characteristics of the microenvironment. These include biomechanical characteristics such as elastic modulus, shear force, and cyclic strain; architectural properties such as geometry, topography, and dimensionality; and dynamic structures and ligand profiles. We will review how these microenvironmental characteristics have been shown to regulate stem cell fate and discuss future research directions that may help expand our current understanding of stem cell biology and aid its application to regenerative medicine.
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http://dx.doi.org/10.1002/wsbm.46DOI Listing
January 2011

Presentation counts: microenvironmental regulation of stem cells by biophysical and material cues.

Annu Rev Cell Dev Biol 2010 ;26:533-56

Department of Chemical Engineering, University of California, Berkeley, California, 94720, USA.

Stem cells reside in adult and embryonic tissues in a broad spectrum of developmental stages and lineages, and they are thus naturally exposed to diverse microenvironments or niches that modulate their hallmark behaviors of self-renewal and differentiation into one or more mature lineages. Within each such microenvironment, stem cells sense and process multiple biochemical and biophysical cues, which can exert redundant, competing, or orthogonal influences to collectively regulate cell fate and function. The proper presentation of these myriad regulatory signals is required for tissue development and homeostasis, and their improper appearance can potentially lead to disease. Whereas these complex regulatory cues can be challenging to dissect using traditional cell culture paradigms, recently developed engineered material systems offer advantages for investigating biochemical and biophysical cues, both static and dynamic, in a controlled, modular, and quantitative fashion. Advances in the development and use of such systems have helped elucidate novel regulatory mechanisms controlling stem cell behavior, particularly the importance of solid-phase mechanical and immobilized biochemical microenvironmental signals, with implications for basic stem cell biology, disease, and therapeutics.
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http://dx.doi.org/10.1146/annurev-cellbio-100109-104042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5989312PMC
October 2010

Extracellular matrix rigidity modulates neuroblastoma cell differentiation and N-myc expression.

Mol Cancer 2010 Feb 10;9:35. Epub 2010 Feb 10.

Department of Bioengineering, University of California, Berkeley, CA, USA.

Neuroblastoma is a pediatric malignancy characterized by tremendous clinical heterogeneity, in which some tumors are extremely aggressive while others spontaneously differentiate into benign forms. Because the degree of differentiation correlates with prognosis, and because differentiating agents such as retinoic acid (RA) have proven to decrease mortality, much effort has been devoted to identifying critical regulators of neuroblastoma differentiation in the cellular microenvironment, including cues encoded in the extracellular matrix (ECM). While signaling between tumor cells and the ECM is classically regarded to be based purely on biochemical recognition of ECM ligands by specific cellular receptors, a number of recent studies have made it increasingly clear that the biophysical properties of the ECM may also play an important role in this cross-talk. Given that RA-mediated neuroblastoma differentiation is accompanied by profound changes in cell morphology and neurite extension, both of which presumably rely upon mechanotransductive signaling systems, it occurred to us that mechanical cues from the ECM might also influence RA-mediated differentiation, which in turn might regulate clinically-relevant aspects of neuroblastoma biology. In this study, we tested this hypothesis by subjecting a series of neuroblastoma culture models to ECM microenvironments of varying mechanical stiffness and examined the regulatory role of ECM stiffness in proliferation, differentiation, and expression of tumor markers. We find that increasing ECM stiffness enhances neuritogenesis and suppresses cell proliferation. Remarkably, increasing ECM stiffness also reduces expression of N-Myc, a transcription factor involved in multiple aspects of oncogenic proliferation that is used for evaluating prognosis and clinical grading of neuroblastoma. Furthermore, the addition of RA enhances all of these effects for all ECM stiffnesses tested. Together, our data strongly support the notion that the mechanical signals from the cellular microenvironment influence neuroblastoma differentiation and do so synergistically with RA. These observations support further investigation of the role of microenvironmental mechanical signals in neuroblastoma proliferation and differentiation and suggest that pharmacological agents that modulate the underlying mechanotransductive signaling pathways may have a role in neuroblastoma therapy.
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http://dx.doi.org/10.1186/1476-4598-9-35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2831820PMC
February 2010
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