Our main research interest involves deciphering the means by which the genetic information stored in DNA molecules is retrieved in a controlled and orderly fashion during transcription, the biochemical process that leads to the production of specific proteins in animal cells. We have used both in vitro and in vivo systems in a multipronged approach to the problem of gene control. For example, we devised various means of isolating the individual components of the cell responsible for transcription and of reconstructing this complex reaction in the test tube. In this way, we can study the detailed molecular mechanisms that direct the exquisitely sensitive turning up and down of gene expression in human and animal regulatory systems. We have also used cells and whole organisms (e.g., flies and mice) to interface our biochemical studies with physiologically relevant situations that require the switching on and off of specific genes during cell growth, development, and differentiation in metazoan organisms. In recent years, much of the lab has focused on studies of key transcription events in embryonic stem cells (ESCs) as well as various terminally differentiated cell types, including adipocytes, hepatocytes, myotubes, and motor neurons.
Proteins That Regulate Gene Expression
Among the most important families of proteins in the nucleus are the sequence-specific transcription factors that bind to DNA at select sites and regulate the expression of genes in a tissue-specific and developmentally regulated fashion. We previously devised biochemical methods to purify these rare and fragile proteins, which in turn allowed us to isolate the genes that encode these regulatory factors. These sequence-specific transcription factors not only regulate the temporal cascade of gene expression during development of multicellular organisms but also are often good candidates for genes that directly or indirectly play a role in disease. For example, many of these transcription factors turn out to be either oncogenes (Jun/Fos) or tumor-suppressor genes (p53, Rb) directly implicated in cancer. At the same time, a growing body of evidence links specific transcription factors (such as FOXO, PPARγ, NFκB) to various metabolic diseases and inflammation.
A fundamental question that remains poorly understood concerns how sequence-specific DNA-binding proteins actually work to regulate transcription. To address this critical issue, our lab fractionated and isolated the multiple components (more than 85 proteins) necessary to reconstitute activator-responsive transcription in the test tube. In the process of dissecting the general transcriptional apparatus, we discovered previously undetected components that serve as the functional bridge between upstream regulatory proteins, such as the human transcription factor Sp1, and the initiation complex that contains RNA polymerase. These novel factors, which we have called coactivators, appear to be part of the missing link that directs promoter-selective transcription in animal cells and are likely to represent a diverse and essential class of regulatory proteins.
The first class of coactivators we identified consists of the TATA-binding protein (TBP)-associated factors (TAFIIs), which make up the core transcription complex called TFIID. Biochemical characterization of these factors revealed that transcriptional activators can bind select TAFII subunits of the TFIID complex. The specificity and function of individual TAFIIs mediating transcriptional activation remains to be determined, however, and we continue to work toward this goal. To this end, we have used both x-ray crystallography and electron microscopy (EM) analysis to determine the three-dimensional structure of transcription proteins and large multicomponent complexes. These structure/function studies have revealed that TAFs can bind core promoter DNA, interact with selective activators, catalyze various enzymatic reactions (e.g., kinase, acetyltransferase), and target binding of TFIID to specifically acetylated nucleosomal templates.
Our biochemical studies also identified additional human cofactor complexes, CRSP/Med and ARC, that together with the TAFIIs help mediate transcriptional activation. Structural determination by EM and biochemical assays revealed that ARC-L and CRSP are substantially different in size and shape and display differential coactivator functions. Moreover, EM analysis of independently derived CRSP complexes reveals distinct conformations induced by different activators. These results suggest that CRSP may potentiate transcription via specific activator-induced conformational changes.
Identification of TBP-Related Factors and Cell-Type-Specific TAFIIs
Eukaryotic cells were originally thought to contain a single TBP that directs transcription by all cellular RNA polymerases. However, we later identified two TBP-related factors, TRF-1 and TRF-2, that interact with the basal transcription factors TFIIA and TFIIB to initiate gene-selective RNA synthesis. In addition to multiple TRFs, we have also discovered tissue- or cell-type-specific TAFIIs. In vitro biochemical and in vivo genetic analyses reveal that TAF II 4b is selectively expressed in the granulosa cells of the mouse ovary. Deletion of this gene by homologous recombination results in infertile female mice with defects in ovarian development due to down-regulated genes. More recently, we have uncovered the functions of several other cell-type-specific TAFs, including TAF7L, which serves a critical function during adipogenesis; TAF3, which plays a key role in the formation of the endoderm as well as in muscle differentiation; and TAF9B, which helps direct neuronal differentiation. The discovery of multiple TFIID-like complexes, as well as cell-specific transcriptional functions of TAFs operating by other mechanisms and not as integral subunits of TFIID, confirms that the core molecular machinery in metazoans has substantially diversified to accommodate complex patterns of transcription during cellular differentiation.
Transcriptional Mechanisms Regulating Stem Cell Self-Renewal and Differentiation
A new area of research we initiated targets the identification of cell-type-specific transcription factors responsible for directing human stem cell maintenance as well as changes to the core promoter recognition apparatus during differentiation. In particular, we have recently isolated novel stem cell–specific cofactor complexes (SCCs) that are required to mediate the transcriptional activation of the NANOG gene by OCT4/SOX2 in human embryonic stem cells. After an extended in vitro biochemical study of putative ESC-specific coactivators, we identified, purified, and characterized three distinct, previously unknown transcription cofactors (SCC-A, SCC-A', SCC-B) that play an important role in potentiating the activation of pluripotent gene expression networks under the regulation of the core factors OCT4 and SOX2. These findings open a new chapter in defining the transcriptional apparatuses in ESCs that are critical for the pluripotency and self-renewal capacity of stem cells.
We have also carried out genome-wide gene expression studies and transcription factor occupancy studies tracking the activities of various cell-type-specific coactivators and TAFs in ESCs, myotubes, hepatocytes, adipocytes, and motor neurons. Each of these studies has uncovered new functions and associations of core promoter components (TAFs, SCCs) and gene regulatory mechanisms.
Developing Single-Cell and Single-Molecule Assays to Study Transcription
Although our attempts to dissect the elaborate process of transcription have traditionally relied on a combination of in vitro biochemistry and in vivo reverse genetics, it has become evident that a significant gap remains between in vitro mechanisms and in vivo physiology. Thus, we have begun to generate a battery of reagents and novel assays to bridge this gap. One effort has been to develop assays at the single-cell level so that we can locate, track, and study transcription in individual cells. Using a combination of fluorescence microscopy, antibody staining, GFP (green fluorescent protein) tracking, ChIPs (chromatin immunoprecipitation), FISH (fluorescence in situ hybridization), and genomic arrays, we have begun to assemble tools to analyze the in vivo mechanisms that regulate transcription at single-cell resolution. In particular, my group, together with the Transcription Imaging Consortium at the Janelia Farm Research Campus, has begun to tackle the challenging problem of dissecting and studying the dynamics of long-distance enhancer/promoter DNA looping induced by sequence-specific transcriptional activators by super-resolution light microscopy. Our recent studies of the endogenous histone gene cluster in Drosophila cells revealed a number of unexpected findings regarding both the composition and the dynamics of His gene transcription during the S phase of the cell cycle. At the same time, our lab has been developing a suite of single-molecule assays to measure the assembly and kinetics of RNA Pol II transcription by visualizing the complex reactions of initiation and elongation in purified reconstituted systems using TIRF (total internal reflection fluorescence) microscopy. We are also adapting single-molecule techniques such as FRET (fluorescence resonance energy transfer) and cryo-EM (cryo-electron microscopy) to complement our functional studies of key molecular transactions during the regulation of transcription.
Transcriptional Control and Disease
Our studies of activators and coactivators have also provided insights into the molecular mechanisms underlying various human diseases. For example, we found that type II diabetes is regulated by a class of insulin sensitizers that modulate the interactions between specific coactivators and the ligand-binding domain of the PPARγ nuclear receptor. We also found that transcription factor FOXO regulates the insulin receptor in a feedback mechanism controlled by insulin and nutrient deprivation. This finding provides a potential link between FOXO and insulin-resistant diabetes. Recently we discovered that TAF7L is a key molecular regulator of adipocyte formation, and this finding may have implications for obesity and metabolic diseases. Most recently, our studies of TAF9B suggest a role in motor neuron or neuron differentiation with potential implication for neurodegenerative diseases such as ALS. As we learn more about the molecular mechanisms that have evolved to regulate transcription and disease mechanisms, opportunities for new diagnostic and therapeutic strategies may emerge.
A grant from the National Institutes of Health provided partial support for the work on Sp1, CRSP, ARC, and other human enhancer and basal transcription factors.
As of April 03, 2013