Research Interests
Transcriptional and Post-transcriptional Roles for Huntingtin, the Huntington’s Disease Protein
Defining the molecular mechanisms underlying the pathogenesis of a human disease is an ultimate challenge for researchers in the basic sciences. In collaboration with Dimitri Krainc, M.D., Ph.D. (Massachusetts General Hospital), we have made important discoveries that shed new insights into the pathogenesis of Huntington's disease (HD), an inherited neurodegenerative disorder. HD is caused by an expansion of CAG trinucleotide repeats encoding polyglutamines in huntingtin (Htt), a 300 kD protein of unknown function. Although the precise mechanisms by which mutant Htt causes neuropathologic damage remains to be determined, we have found that the site-specific transcription factor Sp1 and TAF4, a component of the basal transcription factor TFIID, interact with Htt leading to transcriptional deregulation of neuronal genes. Strikingly, over-expression of both Sp1 and TAF4 in cultured neuronal cells reversed mutant Htt-mediated transcriptional inhibition, and protected neurons from Htt-induced cellular toxicity. These experiments suggest that Htt physically associates with glutamine-rich transcription factors, thereby interfering with their ability to activate select target genes. Future experiments are aimed at defining the mechanisms of Htt-induced gene repression using biochemical and cell biological methods.
To gain further insight into Htt function, we recently purified epitope-tagged Htt from HeLa cells and identified Argonaute (Ago) proteins as Htt-interacting proteins. Agos have been shown in part to localize to discrete cytoplasmic foci known as P (processing)-bodies. Proteins involved in small RNA-mediated gene silencing, translational repression, mRNA surveillance, and mRNA degradation accumulate with their bound mRNAs at P-bodies, where their destiny is determined. Co-localization studies demonstrated Htt to be present in P-bodies. Further, depletion of Htt showed compromised RNAi-mediated gene silencing, indicating that normal Htt contributes to post-transcriptional regulation of gene expression. Analysis of mouse neuronal cells expressing endogenous mutant Htt showed fewer P-bodies and exhibited reduced gene silencing activity compared with their wild-type counterparts. These data suggest that transcriptional deregulation in HD may be attributed in part to mutant Htt’s role in post-transcriptional processes. Neuronal RNA granules have been described to be involved in transport and translational control of neuronal mRNAs. A recent study reported presence of P-body proteins in neuronal granules in Drosophila. It is tempting to speculate that Htt is involved in neuronal RNA granule function, which may be perturbed by mutant Htt, contributing to specificity for neurons in HD. We are investigating the role of wild-type and mutant Htt in post-transcriptional processes in order to uncover new pathogenic mechanisms that contribute to HD.
Regulation of gene expression by the chromatin remodeling complex SWI/SNF
Eukaryotic genomes require significant compaction in order to fit into the nucleus. This extraordinary degree of packing is achieved by wrapping chromosomal DNA around a core of histones to form nucleosomes, the building blocks of chromatin. Modulation of chromatin structure plays a fundamental role in gene expression because nucleosomes generally inhibit transcription. Genetic and biochemical approaches have led to the discovery of multiple protein complexes that activate or repress transcription by targeting histones or nucleosomes. We previously purified the Brahma chromatin remodeling complex to homogeneity from Drosophila cells and demonstrated that Osa, a protein previously shown to be required for photoreceptor differentiation and embryonic segmentation, is an integral component of the Drosophila SWI/SNF complex (in collaboration with Jessica Treisman, NYU School of Medicine). Our lab subsequently isolated cDNA clones encoding two distinct human orthologues of Osa and demonstrated their role in transcriptional activation by steroid hormone receptors. Cell lines induced to over-express hOsa protein exhibit growth and cell cycle defects. Similar to the observations made in flies, hOsa proteins may contribute to activation or repression of target genes in a pathway associated with tumor development. Ongoing research focuses on determining how hOsa regulates specific growth and cell cycle regulatory genes and contributes to the events leading to tumorigenesis. Our recent study has also focused on hOsa’s interaction with specific histones and its effect on the regulation of gene expression.