Professor Sonia Rocha
Investigating the mechanisms controlling gene expression in hypoxia
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.
HIF and NF-kappaB Crosstalk
Research in the lab has lead to the identification of an extensive and intricate crosstalk between the HIF system and 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). NF-kappaB is a major regulator of the cellular response to inflammation.
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 exposure to cytokines such as TNF-alpha and hypoxia. Recently, we have been investigating how HIF controls the NF-kappaB pathway both in hypoxia and inflammation. We have uncouvered that HIF-1alpha keeps NF-kappaB under control. This has led to new insights on how these transcription factors are crosstalking in conditions of inflammation and hypoxia such as the ones seen in a great number of human diseases. Furthermore, using Drosophila melanogaster as a model organism, in collaboration with Dr. Arno Muller, we have been able to demonstrate that this crosstalk is evolutionary conserved and physiologically relevant.
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. In particular, we are interested in the action of the JmJC histone demethylases, as these are also oxygen regulated.
The interplay between PHDs and the cell cycle.
Oxygen sensitivity of the HIF system is conferred by a class of dioxygenases called Prolyl-hydroxylases (PHD1, PHD2 and PHD3). Until recently, HIF-alpha subunits were the only known and validated targets of PHDs. However, now it is becoming clear that PHDs have other targets in the cell, and control other important proteins. Our work, in collaboration with the Swedlow and Lamond labs, identified a novel target of PHD1, in the centrosomal protein Cep192. This finding linked oxygen sensing to the cell cycle machinery directly.
In addition, using a proteomic approach our collaborative efforts have identified additional hydroxylation targets. Validation and functional characterisation of these novel hydroxylation sites by PHDs will open the field to novel functional outcomes of selective targeting of these enzymes. In addition, we are also investigating how the cell cycle controls the activity of these enzymes.
- Lecturer in Level 3 Module-Gene Regulation and Expression
- Lecturer in Level 4 Module-Gene Regulation and Expression
- Lecturer in MRes Cancer Biology
- Tutor in MSci course
Ortmann, B., Bensaddek, D., Carvalhal, S., Moser, S. C., Mudie, S., Griffis, E. R., Swedlow, J.R., Lamond, A. I., and Rocha, S. (2016). CDK-dependent phosphorylation of PHD1 on serine 130 alters its substrate preference in cells. J. Cell Sci. 129, 191-205. View Paper
Moniz, S., Bandarra, D., Biddlestone, J., Campbell, K.J., Komander, D., Bremm, A., and Rocha, S. (2015). Cezanne regulates E2F1-dependent HIF-2alpha expression. J. Cell Sci. 128, 3082-3093. View Paper
Bandarra, D., Biddlestone, J., Mudie, S., Muller, H. A., and Rocha, S. (2015). NF-kappaB-dependent gene expression to control innate immunity signals. HIF-1alpha restricts Dis. Models and Mech. 8, 169-181. View Paper
Bremm, A., Moniz, S., Mader, J., Rocha, S., and Komander, D. (2014). Cezanne (OTUD7B) regulates HIF-1alpha homeostasis in a proteasome-independent manner. EMBO Rep. 15, 1268-1277 View Paper
Bandarra, D., Biddlestone, J., Mudie, S., Muller, H. A., and Rocha, S. (2014). Hypoxia activates IKK-NF-kappaB and the immune system in Drosophila melanogaster. Biosci. Rep. 34, e00127. View Paper
Shmakova, A., Batie, M., Druker, J., and Rocha, S. (2014). Chromatin and oxygen sensing in the context of JMJC histone demethylases. Biochem. J. 462, 385-395. View Paper
Moser S, Bensaddek D, Ortmann B, Maure JF, Mudie S, Blow JJ, Lamond AI, Swedlow JR, and Rocha S. (2013) PHD1 links Cell-Cycle progression to oxygen sensing through hydroxylation of the centrosomal protein Cep192. Dev. Cell. PMID: 23932902 View Paper
Yamada K, Ono M, Perkins ND, Rocha S, Lamond AI. (2013) Identification and functional characterization of FMN2, a regulator of the cyclin dependent kinase inhibitor p21. Mar 7; 49(5):922-933. Molecular Cell. PMID: 23375502 View Paper
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