STATs and ChIPs- Learning A Lesson Or Two About Transcriptional Activation

During my childhood, my family and I spent many a vacation in the Swiss Alps.  From the mountain tops I used to look out into the horizon as far as the eye could see with peak upon peak stretching out into the distance.  If I was lucky, I would have a map that allowed me to identify each peak, perhaps even distinguish the highest from the lowest and thus really get a sense that I understood the underlying topography.  However, I quickly realized how little I actually knew about the vast, undulating Swiss countryside.  What I had initially observed as a homogenous ‘mat’ of peaks stretching out into the horizon was in fact a rippling of deep valleys that would make an afternoon hike anything but a walk in the breeze. 

 
Looking back on these experiences I am struck by how closely they reflect the landscape of modern science— a broad mat of detailed knowledge with its own peaks of specialization.  I am reminded of the words of writer Bill Bryson who described science as “tens of thousands of people that do tiny, tiny things that all accrete into a larger body of knowledge” (1).  In fact one of the most captivating aspects of my work as a Technical Services Scientist at Promega is the opportunity that I get to talk over the phone with researchers at the forefront of their own specific areas of expertise.  Frequently these conversations lead to gold nuggets of information that compel me to investigate further. 

 
Working in the laboratoryJust recently for example one scientist drew my attention to a couple of papers about a family of mammalian transcription factors called STAT (short for Signal Transducers And Activators Of Transcription).  What I gleaned from these papers set off my sense of curiosity.   STATs are activated through a process known as cytokine-mediated phosphorylation (2).  Subsequently they are translocated to the nucleus where they bind to specific recognition sequences in the genome (2,3).  A team from the Dana Farber Cancer Institute, Harvard Medical School and Brigham and Women’s Hospital in Boston have speculated that the human genome might have as many as 200 binding sites for one of the STATs- STAT5 (although even this might be a conservative estimate; 2).
 

I have learned a lot since that initial conversation.  As Dana Farber molecular biologist Erik Nelson and his group have shown through their own experiments, STATs can bind to sequences tens of thousands of base pairs away from the gene promoters that they regulate (2).  In several cases, the downstream effects of STATs have been well characterized.  STAT5, for example, activates a gene called NCAM2 which appears to play an important part in ‘Natural Killer’ (NK) cell function during an immune response (2).  NCAM proteins are themselves cell adhesion molecules that facilitate cell-to-cell interactions and play a role in cellular differentiation and proliferation (2).

 
A key part of my technical services role involves deciphering where Promega’s products can fit into the large number of experimental schemes that I hear and talk about each day.  The Dual Luciferase Reporter Assay System features prominently in my discussions primarily because of the opportunities it offers customers to study promoter activation using highly sensitive luciferase reporters.  Indeed in their own work,  Nelson and colleagues used the Dual Luciferase Reporter Assay to show transcriptional activation of luciferase under the control of an NCAM2 intronic promoter sequence in cells expressing STAT5 (2). Moreover, they provided compelling evidence demonstrating the need for two STAT5 dimers to bind cooperatively on adjacent binding sites for such activation to occur (2).
 

The work of Nelson and his group highlighted other applications for which Promega offers key technologies.  Notably they used Chromatin Immunoprecipitation (ChIP) to confirm STAT5 binding to target DNA sequences (3).  ChIP is a technique that allows the characterization of a DNA binding protein’s target DNA sequence.  The resulting protein-DNA complex is then ‘pulled out’ using a protein-specific antibody.  Subsequent PCR amplification and sequencing of the bound DNA allows the identification of possible DNA-binding sites in the host cell genome (3).  Promega’s  HaloCHIP™ System  has been designed for doing ChIP assays without using antibodies.  By cloning the protein of interest as a fusion with the  HaloTag® protein, protein-DNA complexes can be pulled out using Promega’s HaloLink™ Resin.

 

Studies such as those undertaken on STATs have paved the way for future research into transcriptional regulation as we try to better understand the molecular basis of disease (3).  As I close up at the end of the day there is a sense of satisfaction that comes from knowing that my colleagues and I have in our own small way contributed to the greater cause of scientific advancement.  Indeed while science is truly a range of peaks made up of different areas of specialization, we can take comfort from knowing that there are threads of commonality such as the betterment of human health that unify them all.  

 

Literature Cited

  1. Jonathan Amos (2004) Bryson wins £10,000 science prize, 14 June, 2004, See full article at http://news.bbc.co.uk/1/hi/sci/tech/3806375.stm
  2. Nelson, E. A. et al. (2006) Identification of Human STAT5-dependent Gene Regulatory Elements Based on Interspecies Homology J. Biol. Chem. 281, 26216–24.
  3. Nelson, E. A. et al. (2004) Isolation Of Unique STAT5 Targets by Chromatin Immunoprecipitation-based Identification. J. Biol. Chem. 279, 54724–30.
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Robert Deyes

Robert has been a Technical Services Scientist at Promega for over 10 years. He also worked for two years as a Technical Advisor at the Paisley, Scotland facility of Life Technologies Inc. After earning his Masters in Medical Genetics from the University of Glasgow, he spent 18 months at the Université Louis Pasteur in Strasbourg, France where he did research into the molecular basis of the inherited disorder Spinal Muscular Atrophy. He also holds a BSc from the University of Portsmouth in England.

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