@article {48280, title = {A variant Sp1 (R218Q) transcription factor might enhance HbF expression in β(0) -thalassaemia homozygotes.}, journal = {British Journal of Haematology}, volume = {180}, year = {2017}, month = {2017 Feb 27}, pages = {755-7}, issn = {1365-2141}, doi = {10.1111/bjh.14445}, author = {Jiang, Zhihua and Luo, Hong-Yuan and Farrell, John J and Zhang, Zhengjian and Schulz, Vincent P and Albarawi, Dilan and Steinberg, Martin H and Al-Allawi, Nasir A S and Gallagher, Patrick G and Forget, Bernard G and Chui, David H K} } @article {47938, title = {Rapid dynamics of general transcription factor TFIIB binding during preinitiation complex assembly revealed by single-molecule analysis.}, journal = {Genes and Development}, volume = {30}, year = {2016}, month = {2016 Sep 15}, pages = {2106-18}, abstract = {

Transcription of protein-encoding genes in eukaryotic cells requires the coordinated action of multiple general transcription factors (GTFs) and RNA polymerase II (Pol II). A {\textquotedblleft}step-wise{\textquotedblright} preinitiation complex (PIC) assembly model has been suggested based on conventional ensemble biochemical measurements, in which protein factors bind stably to the promoter DNA sequentially to build a functional PIC. However, recent dynamic measurements in live cells suggest that transcription factors mostly interact with chromatin DNA rather transiently. To gain a clearer dynamic picture of PIC assembly, we established an integrated in vitro single-molecule transcription platform reconstituted from highly purified human transcription factors and complemented it by live-cell imaging. Here we performed real-time measurements of the hierarchal promoter-specific binding of TFIID, TFIIA, and TFIIB. Surprisingly, we found that while promoter binding of TFIID and TFIIA is stable, promoter binding by TFIIB is highly transient and dynamic (with an average residence time of 1.5 sec). Stable TFIIB{\textendash}promoter association and progression beyond this apparent PIC assembly checkpoint control occurs only in the presence of Pol II{\textendash}TFIIF. This transient-to-stable transition of TFIIB-binding dynamics has gone undetected previously and underscores the advantages of single-molecule assays for revealing the dynamic nature of complex biological reactions.

}, doi = {10.1101/gad.285395.116}, author = {Zhang, Zhengjian and English, Brian P and Grimm, Jonathan B and Kazane, Stephanie A and Hu, Wenxin and Tsai, Albert and Inouye, Carla and You, Changjiang and Piehler, Jacob and Schultz, Peter G and Lavis, Luke D and Revyakin, Andrey and Tjian, Robert} } @article {47512, title = {Real-time quantification of single RNA translation dynamics in living cells.}, journal = {Science}, volume = {352}, year = {2016}, month = {2016 May 05}, pages = {1425-9}, abstract = {

Although mRNA translation is a fundamental biological process, it has never been imaged in real-time with single molecule precision in vivo. To achieve this, we developed Nascent Chain Tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify single mRNA protein synthesis dynamics. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 s. Polysomes contain ~1 ribosome every 200-900 nucleotides and are globular rather than elongated in shape. By developing multi-color probes, we show most polysomes act independently; however, a small fraction (~5\%) form complexes in which two distinct mRNAs can be translated simultaneously. The sensitivity and versatility of NCT make it a powerful new tool for quantifying mRNA translation kinetics.

}, doi = {10.1126/science.aaf0899}, author = {Morisaki, Tatsuya and Lyon, Kenneth and DeLuca, Keith F and DeLuca, Jennifer G and English, Brian P and Zhang, Zhengjian and Lavis, Luke D and Grimm, Jonathan B and Viswanathan, Sarada and Looger, Loren L} } @article {47034, title = {Chemical perturbation of an intrinsically disordered region of TFIID distinguishes two modes of transcription initiation.}, journal = {eLife}, volume = {4}, year = {2015}, month = {2015 Aug 28}, abstract = {

Intrinsically disordered protein regions (IDRs) are peptide segments that fail to form stable 3-dimensional structures in the absence of partner proteins. They are abundant in eukaryotic proteomes and are often associated with human diseases, but their biological functions have been elusive to study. Here we report the identification of a tin(IV) oxochloride-derived cluster that binds an evolutionarily conserved IDR within the metazoan TFIID transcription complex. Binding arrests an isomerization of promoter-bound TFIID that is required for the engagement of Pol II during the first (de novo) round of transcription initiation. However, the specific chemical probe does not affect reinitiation, which requires the re-entry of Pol II, thus, mechanistically distinguishing these two modes of transcription initiation. This work also suggests a new avenue for targeting the elusive IDRs by harnessing certain features of metal-based complexes for mechanistic studies, and for the development of novel pharmaceutical interventions.

}, issn = {2050-084X}, doi = {10.7554/eLife.07777}, author = {Zhang, Zhengjian and Boskovic, Zarko and Hussain, Mahmud M and Hu, Wenxin and Inouye, Carla and Kim, Han-Je and Abole, A Katherine and Doud, Mary K and Lewis, Timothy A and Koehler, Angela N and Schreiber, Stuart L and Tjian, Robert} } @article {46267, title = {A general method to improve fluorophores for live-cell and single-molecule microscopy.}, journal = {Nature Methods}, volume = {12}, year = {2015}, month = {2015 Jan 19}, pages = {244-50}, abstract = {

Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range.

}, issn = {1548-7105}, doi = {10.1038/nmeth.3256}, author = {Grimm, Jonathan B and English, Brian P and Chen, Jiji and Slaughter, Joel P and Zhang, Zhengjian and Revyakin, Andrey and Patel, Ronak and Macklin, John J and Normanno, Davide and Singer, Robert H and Lionnet, Timoth{\'e}e and Lavis, Luke D} } @article {6919, title = {Single-molecule dynamics of enhanceosome assembly in embryonic stem cells.}, journal = {Cell}, volume = {156}, year = {2014}, month = {2014 Mar 13}, pages = {1274-85}, abstract = {

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.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2014.01.062}, author = {Chen, Jiji and Zhang, Zhengjian and Li Li and Chen, Bi-Chang and Revyakin, Andrey and Hajj, Bassam and Legant, Wesley and Dahan, Maxime and Lionnet, Timoth{\'e}e and Betzig, Eric and Tjian, Robert and Liu, Zhe} } @article {6845, title = {Single-molecule tracking of the transcription cycle by sub-second RNA detection.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Jan 28}, pages = {e01775}, abstract = {

Transcription is an inherently stochastic, noisy, and multi-step process, in which fluctuations at every step can cause variations in RNA synthesis, and affect physiology and differentiation decisions in otherwise identical cells. However, it has been an experimental challenge to directly link the stochastic events at the promoter to transcript production. Here we established a fast fluorescence in situ hybridization (fastFISH) method that takes advantage of intrinsically unstructured nucleic acid sequences to achieve exceptionally fast rates of specific hybridization (\~{}10e7 M(-1)s(-1)), and allows deterministic detection of single nascent transcripts. Using a prototypical RNA polymerase, we demonstrated the use of fastFISH to measure the kinetic rates of promoter escape, elongation, and termination in one assay at the single-molecule level, at sub-second temporal resolution. The principles of fastFISH design can be used to study stochasticity in gene regulation, to select targets for gene silencing, and to design nucleic acid nanostructures. DOI: http://dx.doi.org/10.7554/eLife.01775.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.01775}, author = {Zhang, Zhengjian and Revyakin, Andrey and Grimm, Jonathan B and Lavis, Luke D and Tjian, Robert} } @article {6846, title = {Dual functions of TAF7L in adipocyte differentiation.}, journal = {eLife}, volume = {2}, year = {2013}, month = {2013 Jan 8}, pages = {e00170}, abstract = {

The diverse transcriptional mechanisms governing cellular differentiation and development of mammalian tissue remains poorly understood. Here we report that TAF7L, a paralogue of TFIID subunit TAF7, is enriched in adipocytes and white fat tissue (WAT) in mouse. Depletion of TAF7L reduced adipocyte-specific gene expression, compromised adipocyte differentiation, and WAT development as well. Ectopic expression of TAF7L in myoblasts reprograms these muscle precursors into adipocytes upon induction. Genome-wide mRNA-seq expression profiling and ChIP-seq binding studies confirmed that TAF7L is required for activating adipocyte-specific genes via a dual mechanism wherein it interacts with PPARγ at enhancers and TBP/Pol II at core promoters. In vitro binding studies confirmed that TAF7L forms complexes with both TBP and PPARγ. These findings suggest that TAF7L plays an integral role in adipocyte gene expression by targeting enhancers as a cofactor for PPARγ and promoters as a component of the core transcriptional machinery.DOI:http://dx.doi.org/10.7554/eLife.00170.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.00170}, author = {Zhou, Haiying and Kaplan, Tommy and Li, Yan and Grubisic, Ivan and Zhang, Zhengjian and Wang, P Jeremy and Eisen, Michael B and Tjian, Robert} } @article {6518, title = {Transcription initiation by human RNA polymerase II visualized at single-molecule resolution.}, journal = {Genes \& Development}, volume = {26}, year = {2012}, month = {2012 Aug 1}, pages = {1691-702}, abstract = {

Forty years of classical biochemical analysis have identified the molecular players involved in initiation of transcription by eukaryotic RNA polymerase II (Pol II) and largely assigned their functions. However, a dynamic picture of Pol II transcription initiation and an understanding of the mechanisms of its regulation have remained elusive due in part to inherent limitations of conventional ensemble biochemistry. Here we have begun to dissect promoter-specific transcription initiation directed by a reconstituted human Pol II system at single-molecule resolution using fluorescence video-microscopy. We detected several stochastic rounds of human Pol II transcription from individual DNA templates, observed attenuation of transcription by promoter mutations, observed enhancement of transcription by activator Sp1, and correlated the transcription signals with real-time interactions of holo-TFIID molecules at individual DNA templates. This integrated single-molecule methodology should be applicable to studying other complex biological processes.

}, keywords = {Fluorescence, Genetic, Humans, Microscopy, Molecular Imaging, Mutation, Promoter Regions, RNA Polymerase II, Sp1 Transcription Factor, Transcription, Transcription Factor TFIID, Video}, issn = {1549-5477}, doi = {10.1101/gad.194936.112}, author = {Revyakin, Andrey and Zhang, Zhengjian and Coleman, Robert A and Li, Yan and Inouye, Carla and Lucas, Julian K and Park, Sang-Ryul and Chu, Steven and Tjian, Robert} } @article {2849, title = {A novel mechanism of antagonism between ATP-dependent chromatin remodeling complexes regulates RNR3 expression.}, journal = {Molecular and Cellular Biology}, volume = {29}, year = {2009}, month = {2009 Jun}, pages = {3255-65}, abstract = {

Gene expression depends upon the antagonistic actions of chromatin remodeling complexes. While this has been studied extensively for the enzymes that covalently modify the tails of histones, the mechanism of how ATP-dependent remodeling complexes antagonize each other to maintain the proper level of gene activity is not known. The gene encoding a large subunit of ribonucleotide reductase, RNR3, is regulated by ISW2 and SWI/SNF, complexes that repress and activate transcription, respectively. Here, we studied the functional interactions of these two complexes at RNR3. Deletion of ISW2 causes constitutive recruitment of SWI/SNF, and conditional reexpression of ISW2 causes the repositioning of nucleosomes and reduced SWI/SNF occupancy at RNR3. Thus, ISW2 is required for restriction of access of SWI/SNF to the RNR3 promoter under the uninduced condition. Interestingly, the binding of sequence-specific DNA binding factors and the general transcription machinery are unaffected by the status of ISW2, suggesting that disruption of nucleosome positioning does not cause a nonspecific increase in cross-linking of all factors to RNR3. We provide evidence that ISW2 does not act on SWI/SNF directly but excludes its occupancy by positioning nucleosomes over the promoter. Genetic disruption of nucleosome positioning by other means led to a similar phenotype, linking repressed chromatin structure to SWI/SNF exclusion. Thus, incorporation of promoters into a repressive chromatin structure is essential for prevention of the opportunistic actions of nucleosome-disrupting activities in vivo, providing a novel mechanism for maintaining tight control of gene expression.

}, keywords = {Adenosine Triphosphatases, Adenosine Triphosphate, Base Sequence, Chromatin, Chromatin Assembly and Disassembly, DNA, Fungal, Gene Expression, Genes, Genetic, Multiprotein Complexes, Mutation, Nucleosomes, Promoter Regions, Ribonucleoside Diphosphate Reductase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors}, issn = {1098-5549}, doi = {10.1128/MCB.01741-08}, author = {Tomar, Raghuvir S and Psathas, James N and Zhang, Hesheng and Zhang, Zhengjian and Reese, Joseph C} } @article {2850, title = {Isolation of highly purified yeast nuclei for nuclease mapping of chromatin structure.}, journal = {Methods in Molecular Biology}, volume = {463}, year = {2008}, month = {2008}, pages = {43-53}, abstract = {

Probing chromatin structure with nucleases is a well-established method for determining the accessibility of DNA to gene regulatory proteins and measuring competency for transcription. A hallmark of many silent genes is the presence of translationally positioned nucleosomes over their promoter regions, which can be inferred by the sensitivity of the underlying DNA to nucleases, particularly micrococcal nuclease. The quality of this data is highly dependent upon the nuclear preparation, especially if the digestion products are analyzed by high-resolution detection methods such as reiterative primer extension. Here we describe a method to isolate highly purified nuclei from the budding yeast Saccharomyces cerevisiae and the use of micrococcal nuclease to map the positions of nucleosomes at the RNR3 gene. Nuclei isolated by this procedure are competent for many of the commonly used chromatin mapping and detection procedures.

}, keywords = {Cell Nucleus, Chromatin, DNA, Fungal, Genetic, Micrococcal Nuclease, Mycology, Promoter Regions, Ribonucleoside Diphosphate Reductase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins}, issn = {1940-6029}, doi = {10.1007/978-1-59745-406-3_3}, author = {Reese, Joseph C and Zhang, Hesheng and Zhang, Zhengjian} } @article {2855, title = {Isolation of yeast nuclei and micrococcal nuclease mapping of nucleosome positioning.}, journal = {Methods in Molecular Biology }, volume = {313}, year = {2006}, month = {2006}, pages = {245-55}, abstract = {

Chromatin structure and nucleosome positioning play a crucial role in gene expression regulation. Nucleosome positioning is often inferred by the protection of underlying DNA to nucleases. Because nucleases are excluded by plasma membranes, chromatin mapping requires isolating nuclei from cells and digesting the chromatin in situ with nucleases. The quality of this data is highly dependent on the nuclei preparation. Here we describe a method to isolate nuclei from the budding yeast Saccharomyces cerevisiae and the use of micrococcal nuclease to map the chromatin structure at the RNR3 gene. Nuclei isolated by this procedure are competent for many of the common chromatin mapping and detection procedures.

}, keywords = {Cell Fractionation, Cell Nucleus, Chromatin, DNA, Fungal, Gene Expression Regulation, Genes, Micrococcal Nuclease, Mycology, Nucleosomes, Saccharomyces cerevisiae}, issn = {1064-3745}, author = {Zhang, Zhengjian and Reese, Joseph C} } @article {2851, title = {Molecular genetic analysis of the yeast repressor Rfx1/Crt1 reveals a novel two-step regulatory mechanism.}, journal = {Molecular and Cellular Biology}, volume = {25}, year = {2005}, month = {2005 Sep}, pages = {7399-411}, abstract = {

In Saccharomyces cerevisiae, the repressor Crt1 and the global corepressor Ssn6-Tup1 repress the DNA damage-inducible ribonucleotide reductase (RNR) genes. Initiation of DNA damage signals causes the release of Crt1 and Ssn6-Tup1 from the promoter, coactivator recruitment, and derepression of transcription, indicating that Crt1 plays a crucial role in the switch between gene repression and activation. Here we have mapped the functional domains of Crt1 and identified two independent repression domains and a region required for gene activation. The N terminus of Crt1 is the major repression domain, it directly binds to the Ssn6-Tup1 complex, and its repression activities are dependent upon Ssn6-Tup1 and histone deacetylases (HDACs). In addition, we identified a C-terminal repression domain, which is independent of Ssn6-Tup1 and HDACs and functions at native genes in vivo. Furthermore, we show that TFIID and SWI/SNF bind to a region within the N terminus of Crt1, overlapping with but distinct from the Ssn6-Tup1 binding and repression domain, suggesting that Crt1 may have activator functions. Crt1 mutants were constructed to dissect its activator and repressor functions. All of the mutants were competent for repression of the DNA damage-inducible genes, but a majority were "derepression-defective" mutants. Further characterization of these mutants indicated that they are capable of receiving DNA damage signals and releasing the Ssn6-Tup1 complex from the promoter but are selectively impaired for TFIID and SWI/SNF recruitment. These results imply a two-step activation model of the DNA damage-inducible genes and that Crt1 functions as a signal-dependent dual-transcription activator and repressor that acts in a transient manner.

}, keywords = {Chromatin, DNA Damage, DNA-Binding Proteins, Epistasis, Fungal, Gene Expression Regulation, Genetic, Histone Deacetylases, Mutation, Nuclear Proteins, Protein Binding, Protein Structure, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Structure-Activity Relationship, Tertiary, Transcriptional Activation}, issn = {0270-7306}, doi = {10.1128/MCB.25.17.7399-7411.2005}, author = {Zhang, Zhengjian and Reese, Joseph C} } @article {2852, title = {Redundant mechanisms are used by Ssn6-Tup1 in repressing chromosomal gene transcription in Saccharomyces cerevisiae.}, journal = {The Journal of Biological Chemistry}, volume = {279}, year = {2004}, month = {2004 Sep 17}, pages = {39240-50}, abstract = {

The Ssn6-Tup1 corepressor complex regulates many genes in Saccharomyces cerevisiae. Three mechanisms have been proposed to explain its repression functions: 1) nucleosome positioning by binding histone tails; 2) recruitment of histone deacetylases; and 3) direct interference with the general transcription machinery or activators. It is unclear if Ssn6-Tup1 utilizes each of these mechanisms at a single gene in a redundant manner or each individually at different loci. A systematic analysis of the contribution of each mechanism at a native promoter has not been reported. Here we employed a genetic strategy to analyze the contributions of nucleosome positioning, histone deacetylation, and Mediator interference in the repression of chromosomal Tup1 target genes in vivo. We exploited the fact that Ssn6-Tup1 requires the ISW2 chromatin remodeling complex to establish nucleosome positioning in vivo to disrupt chromatin structure without affecting other Tup1 repression functions. Deleting ISW2, the histone deacetylase gene HDA1, or genes encoding Mediator subunits individually caused slight or no derepression of RNR3 and HUG1. However, when Mediator mutations were combined with Deltaisw2 or Deltahda1 mutations, enhanced transcription was observed, and the strongest level of derepression was observed in triple Deltaisw2/Deltahda1/Mediator mutants. The increased transcription in the mutants was not due to the loss of Tup1 at the promoter and correlated with increased TBP cross-linking to promoters. Thus, Tup1 utilizes multiple redundant mechanisms to repress transcription of native genes, which may be important for it to act as a global corepressor at a wide variety of promoters.

}, keywords = {Acetylation, Adenosine Triphosphatases, Chromatin, Chromosomes, DNA-Binding Proteins, Fungal, Fungal Proteins, Gene Expression Regulation, Genetic, Histone Deacetylases, Histones, Nuclear Proteins, Nucleosomes, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Transcription Factors}, issn = {0021-9258}, doi = {10.1074/jbc.M407159200}, author = {Zhang, Zhengjian and Reese, Joseph C} } @article {2853, title = {Ssn6-Tup1 requires the ISW2 complex to position nucleosomes in Saccharomyces cerevisiae.}, journal = {The EMBO Journal}, volume = {23}, year = {2004}, month = {2004 Jun 2}, pages = {2246-57}, abstract = {

The Imitation SWItch (ISWI) chromatin remodeling factors have been implicated in nucleosome positioning. In vitro, they can mobilize nucleosomes bi-directionally, making it difficult to envision how they can establish precise translational positioning of nucleosomes in vivo. It has been proposed that they require other cellular factors to do so, but none has been identified thus far. Here, we demonstrate that both ISW2 and TUP1 are required to position nucleosomes across the entire coding sequence of the DNA damage-inducible gene RNR3. The chromatin structure downstream of the URS is indistinguishable in Deltaisw2 and Deltatup1 mutants, and the crosslinking of Tup1 and Isw2 to RNR3 is independent of each other, indicating that both complexes are required to maintain repressive chromatin structure. Furthermore, Tup1 repressed RNR3 and blocked preinitiation complex formation in the Deltaisw2 mutant, even though nucleosome positioning was completely disrupted over the promoter and ORF. Our study has revealed a novel collaboration between two nucleosome-positioning activities in vivo, and suggests that disruption of nucleosome positioning is insufficient to cause a high level of transcription.

}, keywords = {Adenosine Triphosphatases, Cell Nucleus, Chromatin, Chromatin Immunoprecipitation, DNA-Binding Proteins, Genetic, Nuclear Proteins, Nucleosomes, Promoter Regions, Repressor Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription Factors}, issn = {0261-4189}, doi = {10.1038/sj.emboj.7600227}, author = {Zhang, Zhengjian and Reese, Joseph C} }