David A. Wassarman, Ph. D Associate Professor Department of Pharmacology dawassarman@wisc.edu

Research Description:

We are interested in understanding how signal transduction pathways regulate gene expression and development.  There are currently three projects in the laboratory that explore this interest.

Project 1: Signal-dependent regulation of alternative splicing.
Alternative splicing is the process by which pre-mRNA exons are differentially ligated together to produce different mature mRNAs.  The coupling of signal transduction pathways to alternative splicing is predicted to be a major mechanism for regulating gene expression during development and in response to altered physiological or pathological conditions.  Recent estimates based on genomic analyses indicate that ~75% of human pre-mRNAs are alternatively spliced.  Alternative splicing can generate multiple, distinct mRNAs from a single gene and each distinct mRNA can potentially encode a functionally distinct protein.  Accordingly, alternative splicing affects basic cellular processes such as transcription and apoptosis, and mistakes in alternative splicing underlie human diseases such as autoimmune diseases and many types of cancer.  While documented examples of changes in splicing pattern in response to extracellular stimuli are plentiful, little is known about the mechanisms by which signaling events modulate the activity of the splicing machinery.

To learn about the mechanisms that underlie signal-dependent alternative splicing, we are using Drosophila as a model organism to understand how signals induced by DNA damage regulate alternative splicing of the TAF1 (TBP-associated factor 1) pre-mRNA (Figure 1).  To date, these studies have identified the ATM and ATR signaling pathways as transducers of the DNA damage signal to the splicing machinery that regulates TAF1 alternative splicing (Katzenberger et al. 2006).  Ongoing genetic and molecular studies are aimed at identifying cis-acting pre-mRNA sequences and trans-acting splicing regulatory proteins that are targets of these signals.

Figure 1.  Alternative splicing of the Drosophila TAF1 pre-mRNA.  The TAF1 pre-mRNA contains 14 constitutive exons (gray boxes) and two alternative exons (red or green box) separated by introns (lines).  Alternative splicing produces four TAF1 mRNA isoforms: TAF1-1, TAF1-2, TAF1-3, and TAF1-4.

Project 2: Gene-specific transcription initiation
The long-term goal of this project is to understand the mechanisms by which Transcription Factor IID (TFIID) regulates transcription initiation.  TFIID is an ~700 kDa complex composed of TATA-binding protein (TBP) and 8-14 TBP-associated factors (TAFs) that is required for transcription initiation at most RNA polymerase II (pol II) genes in eukaryotic organisms.  Binding of TFIID to a gene promoter constitutes a rate-limiting step in transcription initiation by RNA pol II, and TAFs play critical roles in this process.  These roles include binding core promoter DNA elements, binding transcription activator proteins, binding other components of the preinitiation complex (PIC), and post-translationally modifying histones.  Moreover, multicellular eukaryotic organisms express TAF isoforms that have restricted expression patterns and, through unknown mechanisms, direct transcription of specific genes.  Thus, it has been hypothesized that TFIID complexes containing different TAFs play mechanistically distinct roles during transcription initiation to specify developmental and stress response profiles of gene expression in multicellular eukaryotic organisms, analogous to sigma factors in bacteria.  However, evidence for structurally distinct TFIID complexes is limited.  This lack of knowledge has impeded progress in understanding the mechanisms underlying transcription regulation in normal and disease states.

To learn about the role of distinct TFIID complexes in gene-specific transcription initiation, we are using Drosophila as a model system to study the biochemical and biological roles of TAF1 protein isoforms, described in Project 1.  To date, these studies have shown that TAF1 isoforms have different DNA binding specificities (Metcalf and Wassarman 2007) and are differentially expressed during Drosophila development (Katzenberger et al. 2006, Metcalf and Wassarman 2006, 2007) (Figure 2).  Ongoing genetic and molecular studies are aimed at identifying transcriptional gene targets of TAF1 protein isoforms.

Figure 2.  Localization of TAF1 and other TFIID proteins during Drosophila spermatogenesis.  Immunoflurescence images reveal the localization of DNA (DAPI), TAF1, TBP, TAF4, and TAF9 at the tip of Drosophila testes.  The lack of co-localization of TAF1, TAF4, and TAF9 suggest that multiple distinct TFIID complexes function to regulate transcription during spermatogenesis (for more details, see Metcalf and Wassarman 2007).

Project 3: Neurodegeneration in the human disease Ataxia-telangiectasia
Ataxia-telangiectasia (A-T) is a recessive genetic disorder associated with progressive neurodegeneration.  The gene responsible for A-T, ATM (A-T mutated), encodes a protein kinase that plays a central role in the response to DNA damage in humans and other animals, including Drosophila.  Cells derived from A-T patients exhibit chromosomal instability and hypersensitivity to DNA damaging agents.  ATM responds to DNA damage by phosphorylation of proteins that regulate the DNA repair, cell cycle, and apoptosis machineries.  Despite these advances in our understanding of ATM activities, there is no convincing explanation for how ATM protects neurons from degeneration.  Furthermore, there is no whole animal system to study degeneration of ATM-deficient neurons.  These shortfalls have prevented the development of therapies for the most debilitating clinical manifestation of A-T, neuromotor dysfunction.

To learn about why ATM deficient neurons degenerate, we have generated a Drosophila model of A-T in which ATM levels were reduced in photoreceptor neurons of the eye by RNA interference.  To date, these studies suggest that ATM deficient neurons degenerate as a result of re-entering the cell cycle (Rimkus et al. 2008) (Figure 3). Ongoing genetic and molecular studies are aimed at understanding why ATM-deficient neurons re-enter the cell cycle and why cell cycle re-entry causes neurons to die rather than divide.

Figure 3.  Reducing the level of ATM in photoreceptor neurons of the eye causes neurodegeneration and a rough eye phenotype.  (A) A scanning electron micrograph (SEM) image of a wild type eye showing the ordered patterning of eye.  (B) An SEM image of an ATM knockdown eye showing the rough eye phenotype.  (C) An SEM image of a genetic suppressor of the ATM knockdown rough eye phenotype.

Selected Publications: Articles on PubMed

• Rimkus SA, Katzenberger RJ, Trinh AT, Dodson GE, Tibbetts RS, and Wassarman DA. (2008). Mutations in String/CDC25 inhibit cell cycle re-entry and neurodegeneration in a Drosophila model of Ataxia telangiectasia. Genes Dev. 22:1205-1220. PMID 18408079
  
  
• Metcalf CE and Wassarman DA. (2007). Nucleolar colocalization of TAF1 and testis-specific TAFs during Drosophila spermatogenesis. Dev Dyn. 236:2836-2843. PMID 17823958
  
  
• Katzenberger RJ, Marengo MS, and Wassarman DA. (2006). ATM and ATR Pathways Signal Alternative Splicing of Drosophila TAF1 Pre-mRNA in Response to DNA Damage. Mol Cell Biol. 26:9256-67. PDF PMID 17030624
  
  
• Metcalf CE and Wassarman DA. (2006). DNA binding properties of TAF1 isoforms with two AT-hooks. J Biol Chem. 281:30015-23. PDF PMID 16893881
  
  
• Maile T, Kwozynski S, Katzenberger RJ, Wassarman DA, and Sauer F. (2004). TAF1 activates transcription by phosphorylation of serine 33 in histone H2B. Science. 304:1014-1018. PMID 15143281
  
  
• Pile LA, Spellman PT, Katzenberger RJ, and Wassarman DA. (2003). The SIN3 deacetylase complex represses genes encoding mitochondrial proteins: Implications for the regulation of energy metabolism. J Biol Chem. 278:37840-37848. PMID 12865422
  
  
• Pile LA, Schlag EM, and Wassarman DA. (2002). The SIN3/RPD3 deacetylase complex is essential for G2 phase cell cycle progression and regulation of SMRTER corepressor levels. Mol Cell Biol. 22:4965-4976. PDF PMID 12077326
  
  
• Aoyagi N and Wassarman DA. (2001). Developmental and transcriptional consequences of mutations in Drosophila TAFII60. Mol Cell Biol. 21:6808-6819. PDF PMID 11564865
  
  
• Wassarman DA and Sauer F. (2001). TAFII250: a transcription toolbox. J Cell Sci. 114:2895-2902. PDF PMID 11686293
  
  
• Wassarman DA, Aoyagi N, Pile LA, and Schlag EM. (2000). TAF250 is required for multiple developmental events in Drosophila. PNAS. 97:1154-1159. PDF PMID 10655500
  

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