High Affinity Heme Binding to a Heme Regulatory Motif on the Nuclear Receptor Rev-erbβ Leads to Its Degradation and Indirectly Regulates Its Interaction with Nuclear Receptor Corepressor

Abstract

Rev-erb␣ and Rev-erb␤ are heme-binding nuclear receptors (NR) that repress the transcription of genes involved in regulating metabolism, inflammation, and the circadian clock. Previous gene expression and co-immunoprecipitation studies led to a model in which heme binding to Rev-erb␣ recruits nuclear receptor corepressor 1 (NCoR1) into an active repressor complex. However, in contradiction, biochemical and crystallographic studies have shown that heme decreases the affinity of the ligand-binding domain of Rev-erb NRs for NCoR1 peptides. One explanation for this discrepancy is that the ligand-binding domain and NCoR1 peptides used for in vitro studies cannot replicate the key features of the full-length proteins used in cellular studies. However, the combined in vitro and cellular results described here demonstrate that heme does not directly promote interactions between full-length Rev-erb␤ (FLRev-erb␤) and an NCoR1 construct encompassing all three NR interaction domains. NCoR1 tightly binds both apo-and heme-replete FLRev-erb␤⅐DNA complexes; furthermore, heme, at high concentrations, destabilizes the FLRev-erb␤⅐NCoR1 complex. The interaction between FLRev-erb␤ and NCoR1 as well as Reverb␤ repression at the Bmal1 promoter appear to be modulated by another cellular factor(s), at least one of which is related to the ubiquitin-proteasome pathway. Our studies suggest that heme is involved in regulating the degradation of Rev-erb␤ in a manner consistent with its role in circadian rhythm maintenance. Finally, the very slow rate constant (10 ؊6 s ؊1 ) of heme dissociation from Rev-erb␤ rules out a prior proposal that Reverb␤ acts as an intracellular heme sensor.Nuclear receptors (NRs) 2 are eukaryotic transcription factors that activate or repress gene expression in response to binding small signaling molecules such as steroid hormones, lipophilic vitamins, and fatty acids (1). Genes regulated by NRs are involved in a myriad of cellular processes ranging from metabolic homeostasis to growth and development. The subjects of this article, Rev-erb␣ and Rev-erb␤, are NRs that have overlapping functions and tissue expression patterns (2-9) and are under circadian control (10 -12). Rev-erb NRs repress the transcription of key genes, e.g. Bmal1, CLOCK, and Rev-erb␣ itself, involved in regulating the molecular clock (13)(14)(15). This is accomplished in part through the formation of a heterodimeric Bmal1-CLOCK complex that activates the transcription of clock-controlled genes including Rev-erb␣/␤, completing a critical feedback loop required for circadian rhythm maintenance (6, 7). Rev-erb␣ and Rev-erb␤ also regulate the expression of genes involved in gluconeogenesis, lipid homeostasis, and immune responses, ultimately synchronizing these processes to the diurnal cycle (16 -21).The multiple functions of NRs (DNA, ligand, and coregulator binding) are accomplished through their modular structure (22, 23). The N-terminal A/B domain is hypervariable, is involved in ligand-independent transcriptional regulation, and is subject to ...

Macrophages and Iron Metabolism

“…Heme-responsive transcription factors include the nuclear receptors RevErbα (Raghuram et al, 2007; Yin et al, 2007) and Rev-Erbβ (Carter et al, 2015), encoded by the nuclear receptor subfamily 1 group D member 1 (NR1D1) and 2 (NR1D2), respectively (Yin et al, 2007), as well as the circadian factor period 2 (Per2) (Yang et al, 2008). Upon heme binding, Rev-Erbα (Raghuram et al, 2007; Yin et al, 2007) and Rev-Erbβ (Carter et al, 2015) inhibit the transcription of a broad range of genes in macrophages (Lam et al, 2013) (Figure 2).…”

“…Heme-responsive transcription factors include the nuclear receptors RevErbα (Raghuram et al, 2007; Yin et al, 2007) and Rev-Erbβ (Carter et al, 2015), encoded by the nuclear receptor subfamily 1 group D member 1 (NR1D1) and 2 (NR1D2), respectively (Yin et al, 2007), as well as the circadian factor period 2 (Per2) (Yang et al, 2008). Upon heme binding, Rev-Erbα (Raghuram et al, 2007; Yin et al, 2007) and Rev-Erbβ (Carter et al, 2015) inhibit the transcription of a broad range of genes in macrophages (Lam et al, 2013) (Figure 2). These include the circadian rhythm regulators Aryl hydrocarbon receptor nuclear translocator-like (ARNTL), also known as BMAL1, the metabolic regulators peroxisome proliferator-activated receptor gamma co-activator 1-alpha (PGC-1α) and the first enzyme in heme synthesis (5′-aminolevulinate synthase 1; ALAS1) (Raghuram et al, 2007), the chemokine (C-C motif) ligand 2 (CCL2 or MCP1), IL-6 and IL-10 (Curtis et al, 2014).…”

“…When released from damaged RBC or muscle cells, extracellular hemoglobin and myoglobin are prone to oxidation, releasing their heme prosthetic groups. The resulting labile heme can be sensed in macrophages via Bach1 (Ogawa et al, 2001), Rev-Erbα (Yin et al, 2007) and Rev-Erbβ (Carter et al, 2015). In addition, heme is also sensed in macrophages via PRR, including TLR4 (Figueiredo et al, 2007; Fortes et al, 2012) and NLRP3 (Dutra et al, 2014).…”

“…Iron activates PHD2 and as such reducing macrophage heme-iron content, represses PHD2 activity and induces HIF1α activation under normoxic conditions. ( D ) Upon heme binding, RevErbα (Raghuram et al, 2007; Yin et al, 2007) and Rev-Erbβ (Carter et al, 2015) recruit the nuclear receptor co-repressor 1 (NCoR1) and the histone deacetylase 3 (HDAC3), forming protein complexes that inhibit the transcription of a broad range of genes in macrophages (Lam et al, 2013). Transcription repression occurs via a mechanism that involves the macrophage lineage-specific transcription PU.1, which renders gene enhancers available for Rev-Erbα and Rev-Erbβ targeting (Lam et al, 2013).…”

The Heme Connection: Linking Erythrocytes and Macrophage Biology

“…Importantly, exogenous administration of the heme scavenger hemopexin was found to counteract the induction of this inflammatory phenotype in macrophages of a mouse model of sickle cell disease. Heme is also a natural ligand for nuclear receptors RevErbα and RevErbβ (52–54). Heme binding to RevErbα has been shown to repress IL10 transcription in human monocytes and macrophages (55).…”