Integration of circadian and hypoxia signaling via non‐canonical heterodimerization

Abstract

CLOCK, BMAL1, and HIFs are basic helix‐loop‐helix and Per‐Arnt‐Sim domain (bHLH‐PAS) proteins, which function as transcription factors. bHLH‐PAS proteins are designated in two classes. Many class I proteins are regulated by environmental signals via their PAS domains, but such signals have not been identified for all. Class II (ARNTs and BMALs) are partners for Class I and can be regulated by synthetic PAS ligands. Previous studies suggested restricted dimerization for bHLH‐PAS proteins. BMAL1 and BMAL2 were believed to dimerize only with CLOCK and NPAS2, while ARNT was thought to dimerize with most Class I proteins except for CLOCK and NPAS2. The logic underlying these assumptions was flawed, and evidence supports dimerization of both HIF1α and HIF2α with BMAL1.

CLOCK, BMAL1, and HIFs are basic helix‐loop‐helix and Per‐Arnt‐Sim domain (bHLH‐PAS) proteins, which function as transcription factors. bHLH‐PAS proteins are designated in two classes. Many class I proteins are regulated by environmental signals via their PAS domains, but such signals have not been identified for all. Class II (ARNTs and BMALs) are partners for Class I and can be regulated by synthetic PAS ligands. Previous studies suggested restricted dimerization for bHLH‐PAS proteins. BMAL1 and BMAL2 were believed to dimerize only with CLOCK and NPAS2, while ARNT was thought to dimerize with most Class I proteins except for CLOCK and NPAS2. The logic underlying these assumptions was flawed, and evidence supports dimerization of both HIF1α and HIF2α with BMAL1.

Abbreviations

ARNT

aryl hydrocarbon receptor nuclear translocator

bHLH‐PAS

basic helix‐loop‐helix and PER‐ARNT‐SIM domains

BMAL1

brain and muscle ARNT‐like protein 1

CBP

CREB binding lysine acetyltransferase

CLOCK

circadian locomotor output cycles kaput

CRY

cryptochrome circadian regulator

DNA

deoxyribonucleic acid

HAS

hypoxia accessory sequence

HIF

hypoxia‐inducible factor

HRE

hypoxia response element

NPAS2

neuronal PAS domain protein 2

P300

EP300 lysine acetyltransferase

PER

period circadian regulator

SCF

Skp‐Cullin‐Fbox

TAD

transcriptional activation domain

TTFL

transcription‐translation feedback loop

VHL

von Hippel‐Lindau tumor suppressor

Circadian‐hypoxia crosstalk

Mammalian circadian clocks are centered on a transcription‐translation feedback loop (TTFL), in which circadian locomotor output cycle kaput (CLOCK), brain and muscle aryl hydrocarbon receptor nuclear translocator‐like protein 1 (BMAL1), periods (PERs), and cryptochromes (CRYs) control rhythmic gene expression. Non‐overlapping gene sets, representing 3‐16% of the transcriptome, exhibit rhythmicity in mammalian organs [1], and circadian disruption leads to dysregulation of biological processes and increased risks of disease and mortality [2, 3, 4, 5, 6].

Partial pressure of oxygen in blood and tissues ranges from 0.5% to 21% [7] and exhibits circadian rhythmicity [8]. Pathological conditions like cancer or sleep apnea cause local or widespread hypoxia. Hypoxia‐inducible factors (HIFs) are heterodimers of alpha (HIF1α, HIF2α, or HIF3α) and beta (HIF1β or HIF2β, also known as ARNT or ARNT2) subunits. When oxygen is plentiful, HIFα subunits are destabilized by oxygen‐dependent post‐translational modifications [9]. Under hypoxia, HIFs stimulate transcription in an isoform‐ and tissue‐specific manner [10]. HIFs promote genes critical for oxygen‐replenishing processes like angiogenesis, glycolysis, respiration, and erythropoiesis [10].

Crosstalk between circadian clocks and HIFs is increasingly recognized [11, 12, 13, 14, 15, 16, 17]. HIFs exhibit circadian expression, and hypoxia shifts clock time [8, 15]. Responses to hypoxia depend on time of day [15, 16, 17, 18, 19]. BMAL1 is extensively co‐localized with HIF1α and HIF2α in mammalian genomes [17, 20], and depletion of BMAL1 alters genomic localization of HIF2α [20]. Intriguingly, ARNT and BMAL1 are required for expression of distinct HIF target genes, including some that require both, suggesting ARNT and BMAL1 cooperatively regulate some targets [20].

BMAL1‐CLOCK, ARNT‐HIF2α, and BMAL1‐HIF2α

CLOCK, BMAL1, and HIFs are basic helix‐loop‐helix and Per‐Arnt‐Sim domain (bHLH‐PAS) transcription factors [21], which are designated in two classes. Many class I proteins are regulated by environmental signals via their PAS domains [22], but such signals have not been identified for all. Class II (ARNTs and BMALs) are partners for class I and can be regulated by synthetic PAS ligands [23]. Previous studies suggested that BMALs dimerize only with CLOCK and NPAS2, while ARNT was thought to dimerize with most other class I proteins [21]. New evidence supports dimerization of both HIF1α [13, 14, 17, 24, 25] and HIF2α [18, 20] with BMAL1. HIF3α may interact with BMAL1 but that has not been investigated. BMAL2 is associated with high expression of HIF target genes in pancreatic cancer [26] and may interact with HIFs.

In the BMAL1‐CLOCK intertwined heterodimer, interactions between corresponding bHLH and PAS domains are stabilizing [27]. In ARNT‐HIF2α, ARNT domains wrap around HIF2α and do not form intramolecular interactions [21]. As in BMAL1‐CLOCK, interfaces between corresponding domains contribute to stability. Unlike CLOCK‐BMAL1, ARNT PAS‐A interacts with HIF2α PAS‐B. Despite reports that BMAL1 interacts with HIF1α and HIF2α, these findings remained controversial until BMAL1 was found to adopt an ARNT‐like fold in BMAL1‐HIF2α [18]. Notably, most bHLH‐PAS heterodimers resemble ARNT‐HIF2α, highlighting the uniqueness of BMAL1‐CLOCK [22]. BMAL1 adopting an ARNT‐like fold suggests it may interact with additional class I partners. Flexibility in BMAL1‐CLOCK enables interaction with nucleosomes in two distinct positions [28], likely facilitating pioneer activity [28, 29]. BMAL1‐containing heterodimers may assist other bHLH‐PAS complexes by establishing open chromatin, which could explain the requirement for both BMAL1 and ARNT for some transcripts [20].

Target genes and response elements

CLOCK‐BMAL1 activates transcription through E‐boxes [30, 31]. HIFs recognize a similar motif, the hypoxia response element (HRE) [32]. Hypoxia accessory sequences (HASes) enhance activation by HIFs [33]. ARNT and HIFα subunits form dimers of heterodimers when combined with DNA containing both HRE and HAS sequences [34]; such multimerization enhances transcription [35]. BMAL1‐HIFα heterodimers activate E‐boxes and HREs and their target gene networks overlap with those of CLOCK‐BMAL1 and classical HIFs [17, 20]. It is unknown whether BMAL1‐containing heterodimers form larger multimers or bind HASes. Mixed dimers of ARNT‐HIFα and BMAL1‐HIFα may contribute to the reliance of some target genes on both ARNT and BMAL1 [20]. Research integrating genetic, genomic, and transcriptomic approaches in a range of cell and tissue types under diverse circadian and oxygen exposures is needed to clarify overlapping and distinct roles of various HIF heterodimers.

Transcriptional coregulators

PERs and CRYs repress CLOCK‐BMAL1 [36]. One mechanism by which CRYs suppress CLOCK‐BMAL1 involves displacing CBP/p300 from the BMAL1 TAD [37, 38]. A peptide containing part of the CRY1 C‐terminal tail displaces co‐activators from BMAL1 [38]. CRYs interact with HIF1α and HIF2α and repress BMAL1‐HIFα heterodimers [24, 39]. In cells, CRY1‐HIF1α interaction requires the CRY1 tail and the HIF1α bHLH domain [39]. CRY2‐HIF1α interaction is suppressed by mutations that prevent BMAL1‐HIF1α interaction [24]. These findings indicate that CRYs interact with HIF1α primarily in BMAL1‐containing heterodimers and that CRY C‐terminal tails compete with co‐activators to bind the BMAL1 TAD. In CLOCK‐BMAL1, W362 in the CLOCK PAS‐B domain is essential for interaction with CRY1 [40]. HIF1/2α do not contain tryptophan in the analogous loop, so HIF interactions with CRYs may involve a distinct mechanism. Finally, CRY1 and CRY2 destabilize HIF1α via recruitment to SCF^FBXL3 [24]. Additional work is needed to understand how CRYs regulate HIFs and whether PERs repress non‐canonical heterodimers.

PAS domain ligands

PAS domains are attractive drug targets [41]. Belzutifan, which disrupts HIF2α heterodimerization through allosteric regulation of the PAS‐B domain [42], is used to treat VHL‐deficient ccRCC [43]. In cells, BMAL1‐HIF2α is more sensitive than ARNT‐HIF2α to disruption by PT2399, a HIF2α antagonist related to belzutifan [20]. Furthermore, PT2399 most effectively reduced growth of ccRCC xenograft tumors when delivered at the peak of BMAL1 expression [20]. Belzutifan and related drugs disrupt HIF2α heterodimers in vivo but cannot disrupt pre‐formed HIF2α heterodimers in vitro [34]. Additional research is needed to elucidate how diverse HIF2α antagonists impact distinct heterodimers and larger protein complexes. HIF‐2 agonists [44, 45, 46] may be useful to enhance HIF‐2 activity in conditions like anemia, recurrent pregnancy loss, and myocardial injury. A more detailed understanding of how diverse heterodimers respond to ligands for HIF‐2 and other PAS domains [23, 47] will support the development of chronotherapy in these and related conditions.