Research interests.

• A new mechanism of Myc-dependent gene regulation: control of mRNA cap methylation through Pol II CTD phosphorylation. Myc has been recognized as a transcription factor for over 20 years.  We have recently discovered a novel biochemical function of Myc: Myc induces mRNA cap methylation.  Methylation of the 5’mRNA cap is essential for mRNA to be translated and thus is a rate limiting step in gene regulation.  We have found that for a subset of genes, Myc can efficiently upregulate gene expression by simultaneously promoting mRNA transcription and cap methylation.  For another subset of genes, Myc induces cap methylation independently of transcription - this set of genes represents a novel set of Myc target genes.  To our knowledge, this is the first report that a transcription factor or oncogene can upregulate cap methylation.  mRNA cap methylation occurs co-transcriptionally and is dependent on TFIIH phosphorylation of the RNA pol II C-terminal domain.  Consistent with upregulation of cap methylation, we have demonstrated that Myc binds to TFIIH subunits, promotes TFIIH recruitment to specific promoters, and elevates RNA pol II phosphorylation. (More Information)

• Novel domains of the Myc transcription factor. The myc family of oncogenes is well conserved throughout evolution.  We have characterized a domain conserved in c-, N-, and L-Myc from fish to man, N-Myc317-337, designated Myc Box IV (MBIV).  A deletion of this domain leads to a defect in Myc-induced apoptosis and in some transformation assays but not in cell proliferation.  Unlike other Myc mutants Myc∆MBIV is not a simple loss-of-function mutation because it is hyperactive for G2 arrest in primary cells.  Microarray analysis of genes regulated by N-Myc∆MBIV reveals that it is weakened for transactivation and repression, but not nearly as defective as N-Myc∆MBII.  Although the mutated region is not part of the previously defined DNA-binding domain, we find that N-Myc∆MBIV has a significantly lower affinity for DNA than the wild-type protein in vitro.  Furthermore, chromatin immunoprecipitation shows reduced binding of N-Myc∆MBIV to some target genes in vivo which correlates with the defect in transactivation.  Thus, this conserved domain has an unexpected role in Myc DNA binding activity. This data also provides a novel separation of Myc functions linked to the modulation of DNA binding activity. (More Information)

• Mechanism of Myc-mediated Repression. Aberrant accumulation of the Myc oncoprotein propels proliferation and induces carcinogenesis.  In normal cells, however, an abundance of Myc protein represses transcription at the c-myc locus (Penn et al. 1990). Cancer cells often lose this autorepression (Grignani et al. 1990).  We examined the control of myc in Drosophila and show here that the Drosophila ortholog, dmyc, also undergoes autorepression.  We find that the developmental repressor Polycomb (Pc) is required for dmyc autorepression, and that this Pc-dMyc mediated repression spreads across an 875 kb region encompassing the dmyc gene.  To further investigate the relationship between Myc and Polycomb, we used microarrays to identify genes regulated by each, and identify a striking relationship between the two: a large set of dMyc activation targets are normally repressed by Pc, and 73% of dMyc repression targets require Pc for this repression.  Chromatin immunoprecipitation confirmed that many dMyc-Pc repressed loci have an epigenetic mark recognized by Pc.  Our results suggest a novel relationship between Myc and Polycomb, wherein Myc enhances Polycomb repression in order to repress targets, and Myc suppresses Polycomb repression in order to activate targets. (More Information)

• The ATM-related protein TRRAP.   TRRAP (Transformation/transactivation domain associated protein) was discovered as a nuclear cofactor that is essential for the oncogenic activity of c-Myc. (More Information) TRRAP is a component of both the SAGA and Nu4A chromatin remodeling complexes (Diagram), where it is associated with the histone acetyltransferases GCN5 and Esa1.  (More Information) This finding indicates that one function of c-Myc is to recruit chromatin modifying complexes to specific target sites. (Diagram; More Information). Recent studies show that the C-terminal domain that is related to ATM is required for the recruitment of histone acetyltransferase activity (More Information). However, since both the huge size (3750-3850 amino acids) and primary sequence of TRRAP protein are highly conserved in evolution, it suggests that TRRAP has an intrinsic function beyond any role as a scaffold in these complexes.   We are exploring the function of TRRAP through genetic studies in yeast and biochemical purification of tightly associated proteins in mammalian cells.

• Functional studies of the TIP49/TIP48/actin-related protein complex.  By expanding on our analysis of c-Myc function through affinity chromatography with the Myc transactivation domain, we identified two new nuclear cofactors as TIP49 and TIP48.  These cofactors have highly conserved ATPase/helicase motifs, and we established that the TIP49 ATPase is essential for Myc oncogenic function.  (Diagram; More Information)  Further analysis of this complex revealed that it contained two additional actin-related proteins, BAF53 and ß-actin itself (Wood et al., manuscript in review).  Targeted mutations in BAF53 inhibit oncogenic transformation by c-Myc.  Interestingly, the TIP complex appears to modulate the apoptotic activity of c-Myc, whereas the TRRAP complex does not.  Ongoing studies of TIP49/TIP48 parallel those of TRRAP, using both biochemical and genetic approaches to investigate the function of these highly conserved nuclear factors.

• The role of putative Myc-target genes in mediating cell proliferation.  The analysis of cell lines devoid of Myc function due to targeted gene knockout showed that virtually all proposed target genes of the c-Myc transcription factor were not misregulated in log phase cells, even though the myc knockout cells exhibit a stable, very slow growth phenotype.  (More Information) These studies indicate that as yet undefined targets must be responsible for the effect of c-Myc on the cell cycle and hence presumably on tumor cell growth.  (More Information)  We set up a functional complementation screen to isolate cDNAs from complex libraries that can rescue the slow growth phenotype of Myc-deficient cells. (Diagram) (More Information)  Remarkably, every fast growing cell had acquired a myc cDNA from the library, indicating that the Myc pathway is unique and not readily bypassed by other cellular factors.  By eliminating myc cDNAs from the library and repeating the screen with modified recipient cells, we have identified the serine hydroxymethyltransferase genes as critical and direct c-Myc target genes involved in growth control (More Information).