Liu Research
Zhe 'James' is interested in mechanisms of gene regulation that give
rise to such cell-type diversity during animal development. With a PhD
training on biochemistry and genomics, Zhe is now shifting his research
approach to live-cell microscopy. He will apply the fast-advancing
imaging techniques developed at Janelia to understand the missing links
between transcriptional regulation and cell-fate determination.
The model system
In vitro differentiation of embryonic stem cell provides us with a minimal system to isolate and study critical regulatory factors of cell-fate commitment. Zhe uses this system to understand how DNA information is selectively decoded to cell-type specificity during early embryonic lineage segregation. Zhe is collaborating with Transcription Imaging Consortium (Wu lab, Singer lab, and Tjian lab) and Keller lab to study: 1) enhancer-promoter communication; 2) dynamic protein-DNA interactions in the nucleus; and 3) the possible link between gene expression kinetics and cell-fate choices.
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.
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.
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) 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.
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.