In this study, we describe a new mechanism for control of hypoxic gene expression in which oxygen controls DNA binding of the Sre1 transcription factor through Ofd1, a 2-OG-Fe(II)-dependent dioxygenase. Our model for this regulatory mechanism is outlined in . In the presence of oxygen, Ofd1N-REG is active, and Nro1 is unable to bind and inhibit Ofd1CTD. Consequently, Ofd1CTD binds Sre1N leading to both transcriptional inhibition and proteasomal degradation. In the absence of oxygen, Nro1 binds Ofd1CTD, and accumulating free Sre1N can activate expression of itself and other target genes. In this model, the Ofd1CTD binds Sre1N and prevents DNA binding, while the Ofd1N-REG dioxygenase domain is an oxygen sensor and confers oxygen-dependent regulation to this interaction. This is in contrast to the HIF system in which protein hydroxylation is essential for regulation by either the PHDs or FIH.
Combining two cooperative regulatory functions in Ofd1 ensures a coordinated response to changes in oxygen concentration. These two mechanisms act together with positive feedback regulation of the sre1N
promoter to rapidly increase the Sre1N transcription factor under low oxygen (Supplemental Fig S3A
, lanes 6-10). In addition, Ofd1 controls Sre1N DNA binding across a wide concentration range of transcription factor ( and ), consistent its ability to inhibit basal levels of Sre1N and accumulated Sre1N upon reintroduction of oxygen (Hughes and Espenshade, 2008
Multiple independent lines of evidence support this model (). First, Ofd1 regulates Sre1N levels in ubr1Δ cells where Sre1N degradation is blocked, demonstrating a second function for Ofd1 (). Second, in sre1N-MP ubr1Δ cells Ofd1-Nro1 control Sre1N target gene expression without affecting Sre1N protein level (, , and ). Third, Ofd1-Nro1 regulate Sre1N target gene expression by controlling its DNA binding ( and ). Fourth, under low oxygen formation of Ofd1-Nro1 protein complex triggers Sre1N DNA binding (). Fifth, an Ofd1 iron-binding mutant lacking hydroxylase activity suppresses DNA binding and Sre1N target gene expression (). Sixth, Ofd1CTD binds Sre1N to block DNA binding, and Nro1 competes with Sre1N for binding to Ofd1CTD (). In summary, these data demonstrate that the oxygen-dependent regulation of Sre1N DNA binding involves stoichiometric binding between Ofd1CTD and Sre1N, rather than acting through a catalytic mechanism. The oxygen-dependent activity of the Ofd1N-REG dioxygenase determines the binding partner for Ofd1CTD.
A non-catalytic mechanism analogous to that discovered here may exist in the HIF system to provide an additional layer of regulatory control. Recently, genetic analyses in C. elegans
showed that the prolyl hydroxylase EGL-9 can repress HIF-1 transcriptional activity through a VHL-independent pathway. In these experiments, catalytically deficient EGL-9 failed to destabilize HIF-1, but still repressed HIF-1 transcriptional activity (Shao et al., 2009
). Other studies suggest that mammalian PHD prolyl hydroxylases may regulate HIF-1α in a VHL-independent mechanism (Ozer et al., 2005
; To and Huang, 2005
). Whether these VHL-independent mechanisms, act by regulating HIF DNA binding is unknown.
Previously, we demonstrated that Ofd1 accelerates Sre1N degradation by the proteasome in the presence of oxygen (Hughes and Espenshade, 2008
; Lee et al., 2009
). Here, we identify additional components required for Sre1N degradation: the E2 ubiquitin-conjugating enzyme Rhp6 and the E3 ligase Ubr1. These two proteins also cooperate to target the nuclear envelope protein Cut8 for degradation in fission yeast (Takeda and Yanagida, 2005
). Ofd1 acts upstream of Ubr1 in the same pathway, because deletion of ofd1+
in sre1N-MP ubr1Δ
cells had no effect on Sre1N protein level (). Whether Ofd1 acts directly on Sre1N to accelerate degradation or whether, for example, Ofd1 affects Ubr1 activity is unknown. Ubr1 functions in the N-end rule degradation pathway in which N-terminal residues determine protein stability (Bartel et al., 1990
; Meinnel et al., 2006
). Indeed, the Sre1 N-terminus contains a tertiary destabilizing residue (Gln) that may affect protein stability. Alternatively, Sre1N may contain an internal degron, as is the case for the Ubr1 substrate Cup9 in S. cerevisiae
(Byrd et al., 1998
; Turner et al., 2000
). Future experiments will address whether Sre1N is a substrate for Ubr1 and the sequence requirements for degradation.
Ofd1 Now has two oxygen-dependent functions in the Sre1 pathway: control of Sre1N DNA binding and Sre1N degradation. Both functions require Nro1 and the Ofd1N-REG
dioxygenase for oxygen-dependent regulation. How the Ofd1N-REG
dioxygenase relays information regarding oxygen availability to Ofd1CTD
is currently unknown. Crystal structure data for the S. cerevisiae
Ofd1 homolog, Tpa1, indicate that Ofd1 is likely a prolyl hydroxylase (Henri et al., 2010
; Kim et al., 2010
). It remains to be determined whether Ofd1 hydroxylates itself or another protein, and what role hydroxylation plays in regulation of Sre1N DNA binding. Recently, the mammalian ortholog of Ofd1, named Ogfod1, was shown to be involved in the recovery of cells from stress and to decrease viability of cells under ischemic conditions (Saito et al., 2010
; Wehner et al., 2010
). Whether Ogfod1 also regulates hypoxic gene expression is unknown, but our results provide a mechanistic understanding of how such a pathway could be controlled. Current studies focus on these unanswered questions.