Using advanced optical and genomic tools, we devise new strategies to quantitatively understand such cell-type diversity at the molecular level.
How does one genome give rise to so many cell types during animal development? Even more mysteriously, how is the spatiotemporal choreography of development genetically encoded? Our lab undertakes a quantitative analysis of gene regulatory mechanisms at the single-cell, single-molecule level to discover how stereotypical gene control during normal development as well as altered states in diseases emerge from seemingly stochastic molecular transactions. Specifically, we use advanced imaging and genomic tools to understand how dynamic molecular regulations operating in the 3D nucleus of a live cell influence gene expression kinetics; and how this network of regulatory transactions eventually drives elaborate cellular behaviors during animal development. We are also keen to apply these new research approaches to answer outstanding questions in neuroscience.
We are generally interested in candidates who are passionate about the inter-discipline research that lies at the interface between quantitative imaging, cell biology, developmental biology, and genomics. We don’t expect you have a complete skill set. Instead, we would like to help you pick up new research skills during the training period.
Specifically, in the beginning, to synergize with existing projects, we give emphasis to candidates who have strong background in neuron cell biology, developmental biology, mouse genetics, or quantitative imaging analysis.
Applications (CV) should be sent to: firstname.lastname@example.org
In close collaboration with pioneer imaging tool builders on campus, we use both primary cell culture and early developing mouse embryos as complementary and parallel models systems to 1) discover novel gene networks that control cell-fate choices and 2) dissect the molecular and cellular dynamics of gene control mechanisms underlying complex developmental pathways.
Imaging Gene Regulation and Genome Organization in single live cells
Critical lineage commitment events during animal development must be exquisitely timed and orchestrated by dynamic molecular transactions of multiple transcription factors in the nucleus. In past couple years, by working closely with Transcription Imaging Consortium TIC (consisting of the labs of Carl Wu, Robert Singer, and Robert Tjian) and labs of Eric Betzig and Luke Lavis, we first developed a imaging method capable of discriminating specific DNA binding events at endogenous diploid allele sites from non-specific macro-molecular interactions in single-living embryonic stem cells; our single-molecule measurements also describe several key kinetic features (3D diffusion periods, number of trials, search times, residence times etc.) associated with in vivo TF dynamics. Recently, we developed a lattice light-sheet microscopy based single-molecule imaging strategy to map and functionally probe TF binding site spatial organization in single, living stem cells. In the next step, we plan to team up with multiple TIC labs in a highly collaborative fashion to further optimize new molecular imaging systems to study mechanisms linking TF dynamics, genome organization and transcription kinetics. Furthermore, with the help from labs specialized in imaging instrumentation on campus, we would like to further devise fast multicolor imaging strategies to study the 4D Nucleome (architecture and dynamics) in single live cells.
Linking Gene Regulation and Developmental Dynamics
A hallmark of nervous system development across bilateral animals is the internalization of neural precursor cells to form ventral or dorsal nerve cord along the anterior-posterior (AP) axis. Specifically, in mammals, the initial formation of neural tube requires extensive interplay between neuro-ectoderm and a transient structure of the axial mesoderm – the notochord. Currently, the transcription circuitry that drives the coordinated cellular dynamics and lineage diversification during neural tube and notochord development is still largely unknown. As a long-term project aimed at quantitatively elucidating gene regulation mechanisms underlying mammalian nerve system/notochord development, we have systematically characterized the developmental pathway mediated by a key developmental regulator – Brachyury during early mouse embryogenesis. Brachyury is a classic enhancer binding TF critical for mesoderm lineage specification and notochord formation. In the next step, we would like to work with labs of Philipp Keller and Tzumin Lee and use a novel combination of cutting-edge developmental imaging and single-cell genomics to characterize the dynamic interplays between neural tube induction and notochord development. Eventually, by correlating the imaging dataset to systematic genome-wide datasets, we would learn significant mechanistic insights into the molecular signatures associated with certain types of cellular behaviors identified in the whole-embryo imaging experiments.
Interfacing with Neurosciences
Gene regulation defects underlie neural disorders such as Huntington’s disease and Rett syndrome that deteriorate brain function of the patients. However, it remains largely unknown how genetic mutations eventually alter molecular dynamics to influence gene regulation in specific neuron populations and what are the downstream effector genes responsible for disease phenotypes. We are currently working with both inside and outside collaborators to characterize molecular dynamics associated with these genetic mutations. We plan to establish a robust microscopy platform for imaging neurons in both primary culture and acute brain slices.
Finally, we envision heterogeneous molecular distribution and dynamics also actively contribute to large-scale neural computation by influencing the basic biophysical properties of single neurons. We are looking forward to working with our colleagues at Janelia to test this hypothesis in the future.
Combinatorial cis-regulatory networks encoded in animal genomes represent the foundational gene expression mechanism for directing cell-fate commitment and maintenance of cell identity by transcription factors (TFs). However, the 3D spatial organization of cis-elements and how such sub-nuclear structures influence TF activity remain poorly understood. Here, we combine lattice light-sheet imaging, single-molecule tracking, numerical simulations, and ChIP-exo mapping to localize and functionally probe Sox2 enhancer-organization in living embryonic stem cells. Sox2 enhancers form 3D-clusters that are segregated from heterochromatin but overlap with a subset of Pol II enriched regions. Sox2 searches for specific binding targets via a 3D-diffusion dominant mode when shuttling long-distances between clusters while chromatin-bound states predominate within individual clusters. Thus, enhancer clustering may reduce global search efficiency but enables rapid local fine-tuning of TF search parameters. Our results suggest an integrated model linking cis-element 3D spatial distribution to local-versus-global target search modalities essential for regulating eukaryotic gene transcription.
Enhancer-binding pluripotency regulators (Sox2 and Oct4) play a seminal role in embryonic stem (ES) cell-specific gene regulation. Here, we combine in vivo and in vitro single-molecule imaging, transcription factor (TF) mutagenesis, and ChIP-exo mapping to determine how TFs dynamically search for and assemble on their cognate DNA target sites. We find that enhanceosome assembly is hierarchically ordered with kinetically favored Sox2 engaging the target DNA first, followed by assisted binding of Oct4. Sox2/Oct4 follow a trial-and-error sampling mechanism involving 84-97 events of 3D diffusion (3.3-3.7 s) interspersed with brief nonspecific collisions (0.75-0.9 s) before acquiring and dwelling at specific target DNA (12.0-14.6 s). Sox2 employs a 3D diffusion-dominated search mode facilitated by 1D sliding along open DNA to efficiently locate targets. Our findings also reveal fundamental aspects of gene and developmental regulation by fine-tuning TF dynamics and influence of the epigenome on target search parameters.
Charting Brachyury-mediated developmental pathways during early mouse embryogenesis.Proceedings of the National Academy of Sciences of the United States of America 2014
M. Lolas, P. D T. Valenzuela, R. Tjian, and Z. Liu Proceedings of the National Academy of Sciences of the United States of America, (2014)
To gain insights into coordinated lineage-specification and morphogenetic processes during early embryogenesis, here we report a systematic identification of transcriptional programs mediated by a key developmental regulator-Brachyury. High-resolution chromosomal localization mapping of Brachyury by ChIP sequencing and ChIP-exonuclease revealed distinct sequence signatures enriched in Brachyury-bound enhancers. A combination of genome-wide in vitro and in vivo perturbation analysis and cross-species evolutionary comparison unveiled a detailed Brachyury-dependent gene-regulatory network that directly links the function of Brachyury to diverse developmental pathways and cellular housekeeping programs. We also show that Brachyury functions primarily as a transcriptional activator genome-wide and that an unexpected gene-regulatory feedback loop consisting of Brachyury, Foxa2, and Sox17 directs proper stem-cell lineage commitment during streak formation. Target gene and mRNA-sequencing correlation analysis of the T(c) mouse model supports a crucial role of Brachyury in up-regulating multiple key hematopoietic and muscle-fate regulators. Our results thus chart a comprehensive map of the Brachyury-mediated gene-regulatory network and how it influences in vivo developmental homeostasis and coordination.
Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.Science (New York, N.Y.) 2014
B. Chen, W. R. Legant, K. Wang, L. Shao, D. E. Milkie, M. W. Davidson, C. Janetopoulos, X. S. Wu, J. A. Hammer, Z. Liu, B. P. English, Y. Mimori-Kiyosue, D. P. Romero, A. T. Ritter, J. Lippincott-Schwartz, L. Fritz-Laylin, D. R. Mullins, D. M. Mitchell, J. N. Bembenek, A. Reymann, R. Böhme, S. W. Grill, J. T. Wang, G. Seydoux, S. U. Tulu, D. P. Kiehart, and E. Betzig Science (New York, N.Y.), 346:1257998 (2014)
Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.
Prior Publications (5)
Deciphering the molecular basis of pluripotency is fundamental to our understanding of development and embryonic stem cell function. Here, we report that TAF3, a TBP-associated core promoter factor, is highly enriched in ES cells. In this context, TAF3 is required for endoderm lineage differentiation and prevents premature specification of neuroectoderm and mesoderm. In addition to its role in the core promoter recognition complex TFIID, genome-wide binding studies reveal that TAF3 localizes to a subset of chromosomal regions bound by CTCF/cohesin that are selectively associated with genes upregulated by TAF3. Notably, CTCF directly recruits TAF3 to promoter distal sites and TAF3-dependent DNA looping is observed between the promoter distal sites and core promoters occupied by TAF3/CTCF/cohesin. Together, our findings support a new role of TAF3 in mediating long-range chromatin regulatory interactions that safeguard the finely-balanced transcriptional programs underlying pluripotency.
Purification, characterization and crystallization of pyrroline-5-carboxylate reductase from the hyperthermophilic archeon Sulfolobus Solfataricus.Protein Expression and Purification 2009
Z. Meng, Z. Liu, Z. Lou, X. Gong, Y. Cao, M. Bartlam, K. Zhang, and Z. Rao Protein Expression and Purification, 64:125-30 (2009)
The gene SSO0495 (proC), which encodes pyrroline-5-carboxylate reductase (P5CR) from the thermoacidophilic archeon Sulfolobus solfataricus P2 (Ss-P5CR), was cloned and expressed. The purified recombinant enzyme catalyzes the thioproline dehydrogenase with concomitant oxidation of NAD(P)H to NAD(P)+. This archeal enzyme has an optimal alkaline pH in this reversible reaction and is thermostable with a half-life of approximately 30 min at 80 degrees C. At pH 9.0, the reverse activation rate is nearly 3-fold higher than at pH 7.0. The homopolymer was characterized by cross-linking and size exclusion gel filtration chromatography. Ss-P5CR was crystallized by the hanging-drop vapor-diffusion method at 37 degrees C. Diffraction data were obtained to a resolution of 3.5A and were suitable for X-ray structure determination.
Remarkably high activities of testicular cytochrome c in destroying reactive oxygen species and in triggering apoptosis.Proceedings of the National Academy of Sciences of the United States of America 2006
Z. Liu, H. Lin, S. Ye, Q. Liu, Z. Meng, C. Zhang, Y. Xia, E. Margoliash, Z. Rao, and X. Liu Proceedings of the National Academy of Sciences of the United States of America, 103:8965-70 (2006)
Hydrogen peroxide (H(2)O(2)) is the major reactive oxygen species (ROS) produced in sperm. High concentrations of H(2)O(2) in sperm induce nuclear DNA fragmentation and lipid peroxidation and result in cell death. The respiratory chain of the mitochondrion is one of the most productive ROS generating systems in sperm, and thus the destruction of ROS in mitochondria is critical for the cell. It was recently reported that H(2)O(2) generated by the respiratory chain of the mitochondrion can be efficiently destroyed by the cytochrome c-mediated electron-leak pathway where the electron of ferrocytochrome c migrates directly to H(2)O(2) instead of to cytochrome c oxidase. In our studies, we found that mouse testis-specific cytochrome c (T-Cc) can catalyze the reduction of H(2)O(2) three times faster than its counterpart in somatic cells (S-Cc) and that the T-Cc heme has the greater resistance to being degraded by H(2)O(2). Together, these findings strongly imply that T-Cc can protect sperm from the damages caused by H(2)O(2). Moreover, the apoptotic activity of T-Cc is three to five times greater than that of S-Cc in a well established apoptosis measurement system using Xenopus egg extract. The dramatically stronger apoptotic activity of T-Cc might be important for the suicide of male germ cells, considered a physiological mechanism that regulates the number of sperm produced and eliminates those with damaged DNA. Thus, it is very likely that T-Cc has evolved to guarantee the biological integrity of sperm produced in mammalian testis.
Pyrroline-5-carboxylate reductase (P5CR) catalyzes the reduction of Delta1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)(+). The enzymatic cycle between P5C and proline is very important in many physiological and pathological processes. Human P5CR was over-expressed in Escherichia coli and purified to homogeneity by chromatography. Enzymatic assays of the wild-type protein were carried out using 3,4-dehydro-L-proline as substrate and NAD(+) as cofactor. The homopolymer was characterized by cross-linking and size exclusion gel filtration chromatography. Human P5CR was crystallized by the hanging-drop vapor-diffusion method at 37 degrees C. Diffraction data were obtained to a resolution of 2.8A and were suitable for high resolution X-ray structure determination.
Pyrroline-5-carboxylate reductase (P5CR) is a universal housekeeping enzyme that catalyzes the reduction of Delta(1)-pyrroline-5-carboxylate (P5C) to proline using NAD(P)H as the cofactor. The enzymatic cycle between P5C and proline is very important for the regulation of amino acid metabolism, intracellular redox potential, and apoptosis. Here, we present the 2.8 Angstroms resolution structure of the P5CR apo enzyme, its 3.1 Angstroms resolution ternary complex with NAD(P)H and substrate-analog. The refined structures demonstrate a decameric architecture with five homodimer subunits and ten catalytic sites arranged around a peripheral circular groove. Mutagenesis and kinetic studies reveal the pivotal roles of the dinucleotide-binding Rossmann motif and residue Glu221 in the human enzyme. Human P5CR is thermostable and the crystals were grown at 37 degrees C. The enzyme is implicated in oxidation of the anti-tumor drug thioproline.