Assistant Professor of Biology
B.Sc. (Hon.), University of British Columbia
Ph.D, Stanford University
Member of: Institute of Molecular Biology
Office: Streisinger 233D
Lab: Streisinger 233
Our laboratory pursues innovative and multidisciplinary research to understand how epigenetic information is regulated to allow complex organs such as the heart to form. Epigenetics refers to the inheritance of traits or features outside that provided by DNA sequence alone. We focus on the roles of chromatin and its regulators in providing a molecular basis for epigenetic information and its control. The developing heart provides both an ideal model for studying chromatin contributions to organogenesis and is itself of tremendous and understudied medical importance. Congenital heart defects are the most common class of birth defects and a major contributor to heart disease. Studies of normal heart development will reveal new insights into human congenital diseases and provide clues towards recapitulating developmental regulatory networks for regenerative purposes.
Chromatin Dynamics & Heart Development
Like all organs, the heart is crafted through an ordered series of differentiation and morphogenetic events beginning from tiny clusters of pluripotent cells. This sequential elaboration of pattern depends upon cellular memories to produce specific responses at each stage of development. Memories are stored in epigenetically inherited open or closed chromatin states of genetic loci that determine unique patterns of gene activation in response to recycled signals. Chromatin remodeling complexes, including the Brg1-associated factor (BAF) complex, regulate chromatin structure by repositioning nucleosomes in response to cellular signals. This activity modifies whether and how a loci will respond to the present combination of active transcription factors. In cooperation with chromatin remodelers, chromatin-modifying enzymes provide an additional level of information by covalently altering the histone subunits of nucleosomes. While it is understood that chromatin modifiers are universally important throughout development, the extent and dynamics of their in vivo roles remain largely unknown. The mammalian heart provides a medically relevant and accessible organ for a combination of genetic, biochemical, and systems approaches to these questions.
The BAF Complex & Heart Development
Early in mammalian development, the heart contains two tissue layers, the myocardium and endocardium, that are separated by a thick extracellular matrix called the cardiac jelly. In the ventricles, the myocardium develops a network of projections, called trabeculae, into the cardiac jelly. As the cardiac jelly dissipates, trabeculae compact to form the thick muscle that characterizes a mature heart. We found that mice with endocardial-specific deletion of Brg1 fail to undergo trabeculation (1). Brg1 is the core subunit of the BAF complex, an essential chromatin remodeling complex. Strikingly, the BAF complex does not have general roles in regulating transcription or differentiation during trabeculation. Instead, chemical rescue studies demonstrate a remarkably specific function in fine tuning morphogenesis by directly repressing transcription of the matrix protease, ADAMTS1. This allows the correct microenvironment to form that supports morphogenesis of the neighboring myocardial cells. ADAMTS1 later becomes derepressed to prevent excessive trabeculation. We are currently studying the mechanism of BAF complex recruitment to ADAMTS1 and how it induces changes in chromatin structure that support transcriptional repression. In addition to their relevance to heart development, further studies on ADAMTS1 will provide a “template” locus to understand how changes in chromatin structure provide transcriptional switches during mammalian organogenesis. Additional conditional Brg1-knockout mice we have produced are revealing further roles for endocardial BAF complexes during valve development, cardiac septation, and maturation of the heart ventricles.
Chemical Genetic Approaches to Developmental Biology
Genetic or RNA-based loss-of-function studies are compromised by slow, unpredictable, and irreversible inhibition of targeted products. In response to the universal need for alternative approaches, we developed a chemical genetic method for regulating the stability of any protein using a polypeptide tag and generic small molecules (4,7). This method was used to determine temporal requirements for GSK-3β in palatogenesis and sternal development, the first time a mouse gene has been regulated in this manner (5). In collaboration with external labs, we have developed a new technology that allows a gene-targeted nuclear protein to be rapidly and reversibly exported from the nucleus, and therefore inactivated, by induced dimerization to a second protein fused to nuclear export sequences (6). We continue to optimize these and related technologies and intend to apply such approaches to our studies on chromatin and mammalian heart development. Synergies will be pursued with next-generation sequencing methods to reveal at a systems-level how widespread and how dynamic chromatin modifications are during organogenesis.
Our research program will yield specific insights into the molecular genetics of heart development and broader conclusions on how regulatory networks use epigenetic information during organogenesis. For example, our molecular studies on BAF complex control of ADAMTS1 during heart muscle formation will reveal new candidate genes to underlie congenital heart defects and will generally serve as a model for chromatin control of gene expression in a developing organ. Refined genetic approaches are uncovering additional roles for the BAF complex in heart formation, opening up a range of new projects to broaden our insights. Finally, improvements of our chemical genetic technologies will provide completely new ways for researchers to regulate a chosen gene activity at the protein level. Trainees in the lab can pursue these or related projects in a dynamic, interactive, and interdisciplinary environment.
1. Stankunas, K., Hang, C. T., Tsun, Z. Y., Chen, H., Lee, N. V., Wu, J. I., Shang, C., Bayle, J. H., Shou, W., Iruela-Arispe, M. L. et al. (2008). Endocardial Brg1 represses ADAMTS1 to maintain the microenvironment for myocardial morphogenesis. Dev Cell 14, 298-311.
2. Chang, C. P.*, Stankunas, K.*, Shang, C., Kao, S. C., Twu, K. Y. and Cleary, M. L. (2008). Pbx1 functions in distinct regulatory networks to pattern the great arteries and cardiac outflow tract. Development 135, 3577-86.
3. Stankunas, K.*, Shang, C.*, Twu, K. Y., Kao, S. C., Jenkins, N. A., Copeland, N. G., Sanyal, M., Selleri, L., Cleary, M. L. and Chang, C. P. (2008). Pbx/Meis deficiencies demonstrate multigenetic origins of congenital heart disease. Circ Res 103, 702-9.
4. Stankunas, K., Bayle, J. H., Havranek, J. J., Wandless, T. J., Baker, D., Crabtree, G. R. and Gestwicki, J. E. (2007). Rescue of Degradation-Prone Mutants of the FK506-Rapamycin Binding (FRB) Protein with Chemical Ligands. Chembiochem. 8, 1162-1169.
5. Liu, K. J., Arron, J. R., Stankunas, K., Crabtree, G. R. and Longaker, M. T. (2007). Chemical rescue of cleft palate and midline defects in conditional GSK-3beta mice. Nature 446, 79-82.
6. Bayle, J. H., Grimley, J. S., Stankunas, K., Gestwicki, J. E., Wandless, T. J. and Crabtree, G. R. (2006). Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chem Biol 13, 99-107.
7. Stankunas, K., Bayle, J. H., Gestwicki, J. E., Lin, Y. M., Wandless, T. J. and Crabtree, G. R. (2003). Conditional protein alleles using knockin mice and a chemical inducer of dimerization. Mol Cell 12, 1615-24.
* Equal Contributions