UBR5 Is a Hect E3 Ubiquitin Ligase That Regulates Chromatin Bound Nuclear Hormone Receptor Stability

Acute promyelocytic leukemia (APML) is driven by the oncogenic PML-RARA fusion, which is unique in that it is degraded by two small molecules, all-trans retinoic acid (ATRA) and arsenic trioxide. ATRA-induced degradation of retinoic acid receptor (RARA) is necessary for sustained anti-tumor effect (Ablain et al, JEM, 2013). More generally, nuclear hormone receptors (NRs) are degraded through a ligand-dependent manner of an unknown mechanism. Here we demonstrate that the HECT E3 ubiquitin ligase UBR5 is a ligand-dependent regulator of RARA and other NRs, solve its structure, and show it binds to NRs on chromatin, regulating transcription by degradation.

We performed CRISPR-Cas9 screens on RARA and its partner, retinoid X receptor alpha (RXRA), to identify the ubiquitin ligase responsible for ATRA-dependent degradation. We found that UBR5 sgRNAs rescued ATRA-induced degradation. Moreover, we found that RARA and RXRA bind to UBR5 only in the presence of ATRA using co-immunoprecipitation experiments. NRs undergo a stereotypic structural change in the ligand binding domain (LBD) following ligand binding, revealing a conserved hydrophobic cleft that allows for nuclear co-activator (NCOA) binding through LxxLL peptide motifs, which are also present in UBR5. We performed mutagenesis screens on the RARA LBD and identified key residues within the cleft that, when altered, prevented ATRA-induced degradation and UBR5-RARA binding. Mutation of UBR5 LxxLL motifs also led to deficient RARA binding. We solved a 1.7Å crystal structure of RARA bound to a UBR5 LxxLL peptide, confirming ATRA dependent UBR5 binding in the cleft.

To understand UBR5/RARA interactions in the context of full-length UBR5, we solved its structure via cryo-electron microscopy and performed a detailed characterization of its functional domains and binding sites. We confirmed the HECT domain is needed for degradation through a UBR5 CRISPR tiling screen and showed specific loss of function of the HECT domain through targeted sgRNAs led to a rescue of ATRA induced RARA degradation.

RARA and PML-RARA are tightly chromatin associated, especially in the context of APML. To examine whether UBR5 binds RARA on chromatin, we performed chromatin-immunoprecipitation and sequencing (ChIP-seq) with anti-RARA and anti-UBR5 antibodies in an APML line (NB4) with and without ATRA treatment. Consistent with ATRA-induced degradation, we found significant loss of RARA peaks after ATRA treatment. RARA binding co-localized with UBR5 binding, suggesting UBR5 associates with RARA on chromatin. RNA-seq analysis of NB4 cells with or without UBR5 KD revealed significant alterations in transcript expression, implying that UBR5 regulates RARA-dependent transcription by degradation and therefore potentially downstream cellular behavior. To test this, we knocked down UBR5 in NB4s, treated with ATRA, and found that UBR5 KD led to increased ATRA resistance.

More broadly, we found that UBR5 KD rescues ligand-induced degradation of other NRs including the glucocorticoid (GR), estrogen, vitamin-D, progesterone receptors and liver-X-receptor alpha. Through similar experiments we showed that UBR5 binds to GR in the presence of dexamethasone and recognizes and competes with NCOAs for a similar degron. ChIP and RNA-seq in A549 cells following dexamethasone treatment revealed that UBR5 also co-localizes with GR and regulates its transcription, suggesting UBR5-mediated degradation may be a general regulator of multiple NRs and their transcriptional activities.

Our findings reveal a novel molecular mechanism regulating nuclear hormone receptors: ligand dependent E3 ligase binding on chromatin, leading to degradation and transcriptional regulation.