Position: CR-UK Senior Research Fellow and Lecturer
Division: Wellcome Trust Centre for Gene Regulation and Expression
Address: College of Life Sciences,
University of Dundee,
Dundee
Telephone: +44 1382 385792, int ext. 85792
Fax: +44 (0)1382 386375
Email: s.rocha@dundee.ac.uk
Website: Rocha Lab Website
Hypoxia (low levels of oxygen) is involved in a variety of patho- and physiological conditions such as cancer, ischemia (stroke and cardiac arrest), acute renal failure and intense muscle contraction during exercise. It also constitutes a great challenge to current cancer therapies, in particular in the treatment of solid tumours since many current therapies rely on the formation of reactive oxygen species (Figure 1). During hypoxia, gene expression is mainly controlled by the transcription factor hypoxia inducible factor-1 (HIF-1). HIF-1 is composed of HIF-1alpha and HIF-1beta subunits. While HIF-1beta is present at detectable levels, HIF-1alpha is rapidly degraded at normal oxygen tensions. Despite the identification of the crucial role for HIF-1 in hypoxia induced gene expression much is still unknown about the mechanisms of gene regulation and expression under such a physiologically relevant condition.
Despite most of the mechanistic studies on HIF regulation have focused on protein stabilization, we have been investigating additional levels of control over these important and vital factors.One project in the lab has lead to the identification of a direct control mechanism of HIF-1α expression by the transcription factor family NF-kappaB. NF-kappaB is the collected name for a family of 5 genes that encode 7 proteins. These are RelA, RelB, c-Rel, NF-kappaB1 (p105/p50) and NF-kappaB2 (p100/p52). These form homo and heterodimers and are usually held inactive in the cytoplasm by a number of inhibitory molecules called Inhibitor of kappaB (IkappaBs).
We have found that all of NF-kappaB subunits are present at the HIF-1alpha promoter and are important for production of HIF-1alpha mRNA, both at basal and in situations of NF-kappaB activation. These include expose to cytokines such as TNF-alpha and hypoxia.
Hypoxia induced NF-kappaB, mechanisms of activation and function.
The observation that hypoxia induces NF-kappaB has been made in the 1990s. However, what pathways are activated that result in nuclear accumulation of NF-kappaB have not been elucidated thus far. In addition, what subunits are involved and what is their function in the cellular response to hypoxia has not been investigated.
Using genetic knockout cells as well as stable and transient siRNA knockdown we are currently testing which molecules are involved in hypoxia induced NF-kappaB. In particular we are interested in identifying the sensor of low oxygen in the pathway leading to NF-kappaB activation. Furthermore, we are undertaking a quantitative proteomic approach to identify new components of the pathway in particular following hypoxia.
An additional and important question we are interested in is, which of the NF-kappaB subunits and targets are selectively activated by hypoxia and how do these contribute to the final response.
Hypoxia induced transcription, effects on chromatin
For transcription factors to access their target sequences in the DNA they must deal with chromatin structure. Chromatin is extremely compacted and this presents a great challenge for processes such as transcription and replication (see diagram). Thus, cells have developed several mechanisms of changing chromatin structure and compaction. These include histone modifications, replacement and/or loss of core histones from the nucleosome and movement of nucleosomes through the action of ATP-dependent enzymes, known as chromatin remodeling complexes.
Given that the hypoxia response relies heavily on transcription through HIF activation, are interested in identifying the mechanism behind hypoxia-induced transcription. In particular what are the changes in chromatin structure that happen through the course of hypoxia exposure. In addition, are ATP-dependent remodeller involved? This is particular interesting since in hypoxia, ATP production is solely dependent on glycolysis and hence not as efficient as respiration. Some of our results indicate global and temporal changes in chromatin markers such as histone modifications (see diagram).
Culver, C., Sunqvist, A., Mudie, S., Melvin, A., Xirodimas, D., and Rocha, S. (2010) Mechanism of Hypoxia Induced NF-kappaB. Mol. Cell Biol. PMID 20696840 View Paper
Kenneth, N. S., Mudie, S., and Rocha, S. (2010). IKK and NF-kappaB-mediated regulation of Claspin impacts on ATR checkpoint function. EMBO J. 29, 2966-2978. PMID 29657549 View Paper
Kenneth, N., Mudie, S., van Uden, P., and Rocha, S. (2009). SWI/SNF regulates the cellular response to hypoxia. J. Biol. Chem. 284, 4123-4131. PMID 19097995 View Paper
Kenneth, N. S., and Rocha, S. (2008). Regulation of Gene Expression by Hypoxia. Biochem. J. 414, 19-29. PMID 18651837 View Paper
Van Uden, P., Kenneth, N. S., and Rocha, S. (2008). Regulation of Hypoxia Inducible Factor-1alpha by NF-kappaB. Biochem. J. 412, 477-484. PMID 18393939 View Paper
Rocha, S. (2007) Gene Regulation Under Low Oxygen: holding your breath for transcription. TIBS 32, 389-397. PMID 17624786 View Paper
