Department of Biology

Michael Marr

Associate Professor of Biology

Research Description

Mechanisms Controlling Gene Expression

My lab is focused on investigating how a metazoan cell responds to developmental and environmental signals and how this response is manifest in changes in gene expression. The developmental, metabolic, and environmental signals a cell receives induce changes in both RNA and protein synthesis. Strict control of these pathways is required for integrity of homeostasis at both the cellular and the organismal level. Loss of this control leads to disease and developmental abnormalities. As a result, organisms and cells control these pathways at both the transcriptional and post transcriptional level. I utilize two model systems to investigate both levels of regulation of gene expression. For the investigation of a robust transcriptional response, I utilize the Drosophila metallothionein genes and the cellular response to heavy metal shock. For experiments investigating post-transcriptional regulation, I use the Drosophila insulin-like receptor pathway and the cellular response to insulin.

Transcriptional control of gene expression

The cellular response to heavy metal shock is mediated directly by a metal dependent transcription factor (MTF-1) and is primarily transcriptional. This provides an excellent model system for investigating the mechanisms governing transcriptional responses to cellular signals. I use a combination of RNA interference (RNAi), biochemical assays, and single cell immunofluorescence to investigate the role of many of the cofactors required for transcription activation. Some of the questions we are trying to address are: What steps are controlled in vivo by the various co-factors? Are the roles redundant? How many pathways are there to activation of transcription?

Post-transcriptional control of gene expression

The insulin receptor pathway is a classical signal transduction pathway that plays important roles in governing cell and organ size in metazoans and also metabolic homeostasis and even lifespan.  Interestingly, the gene for insulin receptor (dINR) is a direct transcriptional target of the pathway. A second important target gene is the global translation inhibitor eIF4E-binding protein (4E-BP). This discovery highlights one mechanism of insulin-mediated control of protein synthesis. This finding also exposes an apparent inconsistency. Under these conditions 4E-BP is repressing cap-dependent translation but the dINR protein continues to be synthesized. These findings were reconciled once we found that the dINR utilizes an internal ribosome entry site (IRES) that allows cap-independent translation initiation. This finding demonstrates a novel functional coupling of transcription and translation that allows amplification of the insulin receptor pathway specific target genes. The pathway activates 4E-BP and at the same time activates the transcription of genes that are resistant to 4E-BP. This leads to an effective switch of the cellular machinery to translation of targets of the pathway. Currently we are trying to understand how this cellular IRES engages the translational machinery. In addition we are trying to determine how often this type of functional coupling is utilized.

Some of the questions our lab is trying to answer. Do the co-activators communicate with one another? Are they redundant? What does it mean for an activator to communicate with the basal machinery?

Recruitment of coactivators to the transgenic locus.  In situ staining to determine protein recruitment to the transgenic MtnA cluster. The protein specified above the first column in each panel was detected with an FITC conjugated secondary antibody. In the middle column of each panel, MTF-1 was detected with a Rhodamin-X conjugated secondary antibody, and DNA was stained with DAPI. The third column shows the merge of these channels. In each panel, the top row shows a cell in the absence of copper; the bottom row shows a cell in the presence of copper for 2 or 4 hr. Scale bar, 2 µm.

Model of the role of IRES activity in production of Drosophila insulin receptor (dINR).  On the right, in conditions of high nutrients or insulin-like peptides, dFOXO and d4E-BP are phosphorylated and inactive. This results in a basal level of expression of the dINR and 4E-BP genes. The translational machinery is divided evenly between dINR and other cellular transcripts. On the left, in conditions of low nutrients or insulin-like peptides, dFOXO and d4E-BP are unphosphorylated. dFOXO can activate transcription of both the dINR and d4E-BP genes. The unphosphorylated d4E-BP is able to bind to 4E, preventing the translation of cellular cap-dependent translation. This results in an increase in the dINR transcript and an uneven distribution of the translation machinery amplifying expression of dINR at both the transcription and translation levels. This results in the accumulation of dINR in the cellular membrane, poising the cell for a rapid response to signals when nutrients are again available.

Models for translation initiation.

Under normal growth conditions both cap-dependent and cap-independent (IRES) mechanisms of translation initiation are utilized (top panel). In stress conditions such as low nutrients 4E-BP prevents cap-dependent initiation but transcripts containing an IRES are still actively translated (bottom panel). [Modified from Holcik & Sonenberg, Nat. Rev. Mol. Cell. Biol. 2005]