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Gavin Schnitzler, Ph.D. Assistant Professor, Tufts Department of Biochemistry
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Links:ResearchLab MembersRecent Publications |
Research Summary:The Schnitzler lab is interested in fundamental mechanisms of transcriptional regulation in the context of chromatin. Human DNA is coated with histone proteins to form nucleosomes and chromatin, which blocks transcription factor access. The human SWI/SNF complex is an ATP-dependent enzyme that is recruited to promoters by over two dozen transcription factors, including steroid receptors, p53, c-myc and Rb, to activate (or sometimes repress) transcription. It is required for mammalian development, and is a tumor suppressor complex that limits aberrant cell division. We are working to understand how hSWI/SNF regulates transcription, by measuring how it alters nucleosome positions and structures on the promoters of its target genes. Chromatin is a sufficiently complex molecule that a wide range of approaches is needed to understand functional changes in its structure and characteristics. We employ a variety of techniques including cell culture expression studies, chromatin immunoprecipitation, nuclease footprint assays, ultracentrifugation and single molecule imaging using Atomic Force Microscopy. |
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The assembly of DNA and histones into nucleosomes inhibits the binding of most transcription factors and the movement of RNA polymerase II. Within living cells however, despite the association of histones with cellular DNA to form chromatin, transcription can be initiated and elongation is rapid. Thus, cells must have mechanisms to combat the repressive effects or chromatin and render the underlying DNA accessible. Understanding these mechanisms will give us a better general picture of eukaryotic gene control which may aid, for instance, the development of gene therapy vectors which are not rapidly inactivated. Recently, genetic and functional studies have identified an evolutionarily conserved family of ATP-dependent chromatin remodeling machines that are capable of facilitating factor binding and transcription. In humans different members of this family are implicated in transcriptional activation, repression through facilitating histone deacetylation, recombination, DNA repair and growth control. Biochemical studies suggest that these complexes may act through a shared mechanism for altering nucleosome structure that is targeted or modified by associated factors to make DNA more accessible for specific functions such as the activation of a class of genes or histone deacetylation.
The human SWI/SNF complex (hSWI/SNF) is a tumor suppressor found mutated in several human cancers. The mouse homologue of hSWI/SNF is essential for embryonic development. hSWI/SNF is important for transcriptional coactivation by over two dozen human transcriptional activators (including almost all steroid receptors, p53, SP1 and MyoD), as well as corepression though the retinoblastoma protein and other transcriptional repressors. Despite its critical regulatory importance, very little is known about the mechanism by which hSIW/SNF coactivates or corepresses transcription. The major goal of our research is to understand how the specific biochemical products of hSWI/SNF accomplish this regulation.
We have shown that hSWI/SNF acts enzymatically to produce an stable non-covalent dimer from single mononucleosomes, which is more accessible to transcription factors and endonucleases (Schnitzler et al., 1998a). More recently, we have shown that hSWI/SNF generates abundant structurally-altered dinucleosomes, termed “altosomes” on polynucleosomal templates (Ulyanova et al, 2005). These results suggest that histones have evolved to allow the formation of alternative forms of the nucleosome, and that hSWI/SNF may function by converting nucleosomes to these altered forms.
In addition to altered nucleosome formation, hSWI/SNF moves nucleosomes relative to underlying DNA sequence. This was seen when hSWI/SNF-remodeled arrays were imaged by Atomic Force Microscopy (in collaboration with Dr. Charles Lieber at Harvard, Schnitzler et al., 2001). More recently, we explored the specific rules governing nucleosome movement by hSWI/SNF, using short mononucleosome templates (Ramachandran et al., 2003). These studies suggested some sequence specificity to repositioning, although this effect was largely eclipsed by movement towards DNA ends. Intriguingly, the linker histone H1 (a major component of mammalian chromatin) altered the effect of hSWI/SNF, resulting in movement away from DNA ends. These results suggest that hSWI/SNF action could regulate transcription by causing defined promoter nucleosome movements guided by the local DNA sequence and linker histone environment.
These results have laid the groundwork for ongoing studies into the regulatory functions of hSWI/SNF products on genes that it regulates, using a combination of biochemical, molecular imaging and cell biological techniques. For instance, we are applying novel mapping techniques to examine the distribution of nucleosomes and altosomes after hSWI/SNF action on target gene promoter chromatin. We are using a variety of biochemical techniques to further detail the structure and possible regulatory functions of altossomes. We are also developing new techniques to measure the regulatory importance of altosome formation at known hSWI/SNF target genes and genomewide. Finally, we are developing model cell culture systems which are expected to allow the mechanism of hSWI/SNF-dependent transcriptional regulation to be understood in much greater detail. Together, these studies are expected to provide answers to several outstanding general questions about remodeling complex function: 1) what role do altered nucleosomes play in transcriptional activation and attenuation?, 2) when hSWI/SNF is recruited to a specific site on chromatin, how far and how rapidly do its effects spread?, and 3) what is the role of promoter DNA sequence in determining the outcome of hSWI/SNF action? These studies will also provide important insights into the regulation of hSWI/SNF target genes involved in cancer control (including Cyclin D1, c-myc, and p21) as well as transcriptional regulation by nuclear hormone receptors.
For more details, please see the publications below.
