Center for Proteomics Research at UT Southwestern

Transcription Factor Related Research

Transcription factors (TFs) are key regulators of almost all biological processes. Indeed, a significant percentage of molecular biologists in the world spend much of their time: 1) trying to delineate which gene-specific TFs regulate the expression of their favorite genes and 2) then try to deduce how these regulators are regulated. We propose to develop proteomics tools to address these critical problems using orexin-regulated gene expression in cultured cells as a sleep-relevant model system.

This area is an excellent case where genomics is of limited utility and proteomics methods are desperately needed. This is because TF levels (either of the transcript or the protein), while important, provide an incomplete picture because TF activity is usually determined by post-transcriptional events. For example, phosphorylation, ubiquitination and proteolysis of the negative factor, I-kB, releases NF-kB and allows it to move into the nucleus. Glucocorticoid receptor is freed from an inhibitory cytoplasmic Hsp90-containing complex upon steroid binding. SREBP-1, a membrane-anchoring sequence on the protein is cleaved site-specifically, allowing the TF to move into the nucleus. On the other hand, the yeast Gal4 regulatory protein is present on the promoters of genes it controls even before induction of transcription. GAL genes are activated by dissociation or remodeling of the Gal4 activation domain-Gal80 repressor complex. Another constitutively bound factor is mammalian MEF2, which is activated by phosphorylation-induced dissociation of an associated histone deacetylase.

There are many other examples in which post-transcriptional events, particularly ones that influence the protein interactions and/or intracellular localization of TFs, play central roles in determining activity. Thus, tools for monitoring TF activity would ideally address the following issues:

Are specific transcription factors present in the cells/tissues of interest at particular times and how much of each protein is present? Ideally, we would like to know what TFs are present in all relevant cell types.
Are the factors present capable of binding the relevant DNA sites, or even more to the point, is the protein on specific sites on the DNA in vivo?
What other factors are associated with each TF? As mentioned above, TF activity is often dominated by the nature of the associated proteins.
Which TFs co-occupy a given promoter/enhancer region? For most mammalian genes the level of transcription or even the on/off state of a gene is determined by the combination of regulatory factors on the promoter.
What is the PTM state of each TF? There are increasing numbers of examples where modification of the regulatory protein itself controls one of its crucial functions. To date most examples involve phosphorylation. However, there are examples of other forms of modification including ubiquitination acetylation and probably many more to come.
What is the state of the chromatin on the TF-regulated genes? The histones on most active genes exhibit characteristic PTM states, which would help distinguish between active and inactive promoter-bound TFs.
What are the half-lives of TFs under different conditions. Very little is known about this aspect of regulatory proteins, or for that matter any other quantitative aspect of these proteins. However, from first-principles it seems that changing the half-life of a regulatory protein could be a central regulatory step.

In all of these efforts, it will be important to develop sensitive detection methods, since many TFs are low abundance proteins. For example, the Gal4 protein is present at a level of 60 dimers per cell (unpublished results) which translates into 0.00005% of the total cellular protein. While this is a somewhat extreme case, it highlights the need for sensitive techniques. Indeed, in mammalian cells the Sp1 protein which is fairly abundant still only represents 0.0002% of total cellular protein.