HIF-1 regulation by NF-κB family of transcription factors:

Hypoxia Inducible Factor (HIF) is a transcription factor that rapidly responds to changes in O2 levels. It is a heterodimer of α and β subunits. HIF-1β is also known as aryl hydrocarbon receptor nuclear translocator (ARNT). There are 3 HIF-α isoforms identified so far, HIF-1α, HIF-2α, HIF-3α see diagram). These share a number ofdomains but have non-redundant functions in the cell. The most well knownmechanism of HIF activation derives from protein stabilization of the HIF-α subunits in conditions of lack of O2, iron ions or 2-oxoglutarate. In these situations, the activity of a subset of enzymes is inhibited. These enzymes catalyse the hydroxylation of proline residues in the oxygen dependent degradation domain, which signals for binding for the E3 ubiquitin ligase complex, thetumour suppressor von Hippel Lindau (VHL). This results in K48-linked ubiquitination and proteasomal mediated degradation (see diagram).

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-κB. NF-κ is the collected name for a family of 5 genes that encode 7 proteins. These are RelA, RelB, c-Rel, NF-κB1 (p105/p50) and NF-κB2 (p100/p52). These form homo and heterodimers and are usually held inactive in the cytoplasm by a number of inhibitory molecules called Inhibitor of κB (IκBs) (see diagram).

We have found that all of NF-κB subunits are present at the HIF-1α promoter and are important for production of HIF-1α mRNA, both at basal and in situations of NF-κB activation. These include expose to cytokines such as TNF-α and hypoxia (see model).


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).

In collaboration with the Owen-Hughes lab, we are using imaging and genomic approaches to understand how chromatin responds to low oxygen, and in particular investigating the role SWI/SNF chromatin remodellers and the histone demethylases JmJCs.

The interplay between PHDs and the cell cycle

Until recently, the HIF system were the only known targets for the PHD enzymes. However, in the last few years novel targets for PHD3 and PHD1 have been discovered. Our work, in collaboration with the Swedlow and Lamond labs has focus on the role of PHDs in the control of the cell cycle. We have identified Cep192 as a novel target for PHD1. This provided the first mechanistic link between hypoxia a sensing and the cell cycle machinery. Furthermore, it was the first HIF-independent PHD1 target identified.

In addition, we are also investigating how PHDs are controlled by the cell cycle, to understand how target specificity is achieved in a coordinated manner.