BMAL1-HIF2α heterodimers contribute to ccRCC.

Circadian disruption enhances cancer risk, and many tumors exhibit disordered circadian gene expression. We show rhythmic gene expression is unexpectedly robust in clear cell renal cell carcinoma (ccRCC). Furthermore, the clock gene BMAL1 is higher in ccRCC than in healthy kidneys, unlike in other tumor types. BMAL1 is closely related to ARNT, and we show that BMAL1-HIF2α regulates a subset of HIF2α target genes in ccRCC cells. Depletion of BMAL1 reprograms HIF2α chromatin association and target gene expression and reduces ccRCC growth in culture and in xenografts. Analysis of pre-existing data reveals higher BMAL1 in patient-derived xenografts that are sensitive to growth suppression by a HIF2α antagonist (PT2399). We show that BMAL1-HIF2α is more sensitive than ARNT-HIF2α to suppression by PT2399, and increasing BMAL1 sensitizes 786O cells to growth inhibition by PT2399. Together, these findings indicate that an alternate HIF2α heterodimer containing the circadian partner BMAL1 contributes to HIF2α activity, growth, and sensitivity to HIF2α antagonist drugs in ccRCC cells.

Pioneer factors: Emerging rules of engagement for transcription factors on chromatinized DNA.

Pioneering transcription factors (TFs) can drive cell fate changes by binding their DNA motifs in a repressive chromatin environment. Recent structures illustrate emerging rules for nucleosome engagement: TFs distort the nucleosomal DNA to gain access or employ alternative DNA-binding modes with smaller footprints, they preferentially access solvent-exposed motifs near the entry/exit sites, and frequently interact with histones. The extent of TF-histone interactions, in turn, depends on the motif location on the nucleosome, the type of DNA-binding fold, and adjacent domains present. TF-histone interactions can phase TF motifs relative to nucleosomes, and we discuss how these complex and surprisingly diverse interactions between nucleosomes and TFs contribute to function.

BMAL1-HIF2α heterodimers contribute to ccRCC.

Circadian disruption enhances cancer risk, and many tumors exhibit disordered circadian gene expression. We show rhythmic gene expression is unexpectedly robust in clear cell renal cell carcinoma (ccRCC). Furthermore, the clock gene BMAL1 is higher in ccRCC than in healthy kidneys, unlike in other tumor types. BMAL1 is closely related to ARNT, and we show that BMAL1-HIF2α regulates a subset of HIF2α target genes in ccRCC cells. Depletion of BMAL1 reprograms HIF2α chromatin association and target gene expression and reduces ccRCC growth in culture and in xenografts. Analysis of pre-existing data reveals higher BMAL1 in patient-derived xenografts that are sensitive to growth suppression by a HIF2α antagonist (PT2399). We show that BMAL1-HIF2α is more sensitive than ARNT-HIF2α to suppression by PT2399, and increasing BMAL1 sensitizes 786O cells to growth inhibition by PT2399. Together, these findings indicate that an alternate HIF2α heterodimer containing the circadian partner BMAL1 contributes to HIF2α activity, growth, and sensitivity to HIF2α antagonist drugs in ccRCC cells.

DCAF1-based PROTACs with activity against clinically validated targets overcoming intrinsic- and acquired-degrader resistance.

Targeted protein degradation (TPD) mediates protein level through small molecule induced redirection of E3 ligases to ubiquitinate neo-substrates and mark them for proteasomal degradation. TPD has recently emerged as a key modality in drug discovery. So far only a few ligases have been utilized for TPD. Interestingly, the workhorse ligase CRBN has been observed to be downregulated in settings of resistance to immunomodulatory inhibitory drugs (IMiDs). Here we show that the essential E3 ligase receptor DCAF1 can be harnessed for TPD utilizing a selective, non-covalent DCAF1 binder. We confirm that this binder can be functionalized into an efficient DCAF1-BRD9 PROTAC. Chemical and genetic rescue experiments validate specific degradation via the CRL4DCAF1 E3 ligase. Additionally, a dasatinib-based DCAF1 PROTAC successfully degrades cytosolic and membrane-bound tyrosine kinases. A potent and selective DCAF1-BTK-PROTAC (DBt-10) degrades BTK in cells with acquired resistance to CRBN-BTK-PROTACs while the DCAF1-BRD9 PROTAC (DBr-1) provides an alternative strategy to tackle intrinsic resistance to VHL-degrader, highlighting DCAF1-PROTACS as a promising strategy to overcome ligase mediated resistance in clinical settings.

Design principles for cyclin K molecular glue degraders.

Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. Here, to investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.

From Thalidomide to Rational Molecular Glue Design for Targeted Protein Degradation.

Thalidomide and its derivatives are powerful cancer therapeutics that are among the best-understood molecular glue degraders (MGDs). These drugs selectively reprogram the E3 ubiquitin ligase cereblon (CRBN) to commit target proteins for degradation by the ubiquitin-proteasome system. MGDs create novel recognition interfaces on the surface of the E3 ligase that engage in induced protein-protein interactions with neosubstrates. Molecular insight into their mechanism of action opens exciting opportunities to engage a plethora of targets through a specific recognition motif, the G-loop. Our analysis shows that current CRBN-based MGDs can in principle recognize over 2,500 proteins in the human proteome that contain a G-loop. We review recent advances in tuning the specificity between CRBN and its MGD-induced neosubstrates and deduce a set of simple rules that govern these interactions. We conclude that rational MGD design efforts will enable selective degradation of many more proteins, expanding this therapeutic modality to more disease areas.

Cooperation between bHLH transcription factors and histones for DNA access.

The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2,3). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry-exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A-H2B acidic patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.

UBR5 forms ligand-dependent complexes on chromatin to regulate nuclear hormone receptor stability.

Nuclear hormone receptors (NRs) are ligand-binding transcription factors that are widely targeted therapeutically. Agonist binding triggers NR activation and subsequent degradation by unknown ligand-dependent ubiquitin ligase machinery. NR degradation is critical for therapeutic efficacy in malignancies that are driven by retinoic acid and estrogen receptors. Here, we demonstrate the ubiquitin ligase UBR5 drives degradation of multiple agonist-bound NRs, including the retinoic acid receptor alpha (RARA), retinoid x receptor alpha (RXRA), glucocorticoid, estrogen, liver-X, progesterone, and vitamin D receptors. We present the high-resolution cryo-EMstructure of full-length human UBR5 and a negative stain model representing its interaction with RARA/RXRA. Agonist ligands induce sequential, mutually exclusive recruitment of nuclear coactivators (NCOAs) and UBR5 to chromatin to regulate transcriptional networks. Other pharmacological ligands such as selective estrogen receptor degraders (SERDs) degrade their receptors through differential recruitment of UBR5 or RNF111. We establish the UBR5 transcriptional regulatory hub as a common mediator and regulator of NR-induced transcription.

Orphan quality control shapes network dynamics and gene expression.

All eukaryotes require intricate protein networks to translate developmental signals into accurate cell fate decisions. Mutations that disturb interactions between network components often result in disease, but how the composition and dynamics of complex networks are established remains poorly understood. Here, we identify the E3 ligase UBR5 as a signaling hub that helps degrade unpaired subunits of multiple transcriptional regulators that act within a network centered on the c-Myc oncoprotein. Biochemical and structural analyses show that UBR5 binds motifs that only become available upon complex dissociation. By rapidly turning over unpaired transcription factor subunits, UBR5 establishes dynamic interactions between transcriptional regulators that allow cells to effectively execute gene expression while remaining receptive to environmental signals. We conclude that orphan quality control plays an essential role in establishing dynamic protein networks, which may explain the conserved need for protein degradation during transcription and offers opportunities to modulate gene expression in disease.

Readout of histone methylation by Trim24 locally restricts chromatin opening by p53.

The genomic binding sites of the transcription factor (TF) and tumor suppressor p53 are unusually diverse with regard to their chromatin features, including histone modifications, raising the possibility that the local chromatin environment can contextualize p53 regulation. Here, we show that epigenetic characteristics of closed chromatin, such as DNA methylation, do not influence the binding of p53 across the genome. Instead, the ability of p53 to open chromatin and activate its target genes is locally restricted by its cofactor Trim24. Trim24 binds to both p53 and unmethylated histone 3 lysine 4 (H3K4), thereby preferentially localizing to those p53 sites that reside in closed chromatin, whereas it is deterred from accessible chromatin by H3K4 methylation. The presence of Trim24 increases cell viability upon stress and enables p53 to affect gene expression as a function of the local chromatin state. These findings link H3K4 methylation to p53 function and illustrate how specificity in chromatin can be achieved, not by TF-intrinsic sensitivity to histone modifications, but by employing chromatin-sensitive cofactors that locally modulate TF function.

A deep dive into degrader-induced protein-protein interfaces.

Targeted protein degradation (TPD) relies on a comprehensive understanding of interfaces between hijacked E3 ligases and their substrates. In vitro techniques often do not capture the interaction dynamics. Recently, Hanzl et al. introduced deep mutational scanning (DMS) in combination with structural and biochemical approaches to identify residues crucial for degrader activity.

Recognition of the CCT5 di-Glu degron by CRL4DCAF12 is dependent on TRiC assembly.

Assembly Quality Control (AQC) E3 ubiquitin ligases target incomplete or incorrectly assembled protein complexes for degradation. The CUL4-RBX1-DDB1-DCAF12 (CRL4DCAF12 ) E3 ligase preferentially ubiquitinates proteins that carry a C-terminal double glutamate (di-Glu) motif. Reported CRL4DCAF12 di-Glu-containing substrates include CCT5, a subunit of the TRiC chaperonin. How DCAF12 engages its substrates and the functional relationship between CRL4DCAF12 and CCT5/TRiC is currently unknown. Here, we present the cryo-EM structure of the DDB1-DCAF12-CCT5 complex at 2.8 Å resolution. DCAF12 serves as a canonical WD40 DCAF substrate receptor and uses a positively charged pocket at the center of the ß-propeller to bind the C-terminus of CCT5. DCAF12 specifically reads out the CCT5 di-Glu side chains, and contacts other visible degron amino acids through Van der Waals interactions. The CCT5 C-terminus is inaccessible in an assembled TRiC complex, and functional assays demonstrate that DCAF12 binds and ubiquitinates monomeric CCT5, but not CCT5 assembled into TRiC. Our biochemical and structural results suggest a previously unknown role for the CRL4DCAF12 E3 ligase in overseeing the assembly of a key cellular complex.

Quality control of protein complex assembly by the ubiquitin-proteasome system.

The majority of human proteins operate as multimeric complexes with defined compositions and distinct architectures. How the assembly of these complexes is surveyed and how defective complexes are recognized is just beginning to emerge. In eukaryotes, over 600 E3 ubiquitin ligases form part of the ubiquitin-proteasome system (UPS) which detects structural characteristics in its target proteins and selectively induces their degradation. The UPS has recently been shown to oversee key quality control steps during the assembly of protein complexes. We review recent findings on how E3 ubiquitin ligases regulate protein complex assembly and highlight unanswered questions relating to their mechanism of action.

RIF1 acts in DNA repair through phosphopeptide recognition of 53BP1.

The chromatin-binding protein 53BP1 promotes DNA repair by orchestrating the recruitment of downstream effectors including PTIP, RIF1, and shieldin to DNA double-strand break sites. While we know how PTIP recognizes 53BP1, the molecular details of RIF1 recruitment to DNA-damage sites remains undefined. Here, we report that RIF1 is a phosphopeptide-binding protein that directly interacts with three phosphorylated 53BP1 epitopes. The RIF1-binding sites on 53BP1 share an essential LxL motif followed by two closely apposed phosphorylated residues. Simultaneous mutation of these sites on 53BP1 abrogates RIF1 accumulation into ionizing-radiation-induced foci, but surprisingly, only fully compromises 53BP1-dependent DNA repair when an alternative mode of shieldin recruitment to DNA-damage sites is also disabled. Intriguingly, this alternative mode of recruitment still depends on RIF1 but does not require its interaction with 53BP1. RIF1 therefore employs phosphopeptide recognition to promote DNA repair but also modifies shieldin action independently of 53BP1 binding.

Targeted protein degradation: from small molecules to complex organelles-a Keystone Symposia report.

Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome-lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. On June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium "Targeting protein degradation: from small molecules to complex organelles." The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.

The CRL4DCAF1 cullin-RING ubiquitin ligase is activated following a switch in oligomerization state.

The cullin-4-based RING-type (CRL4) family of E3 ubiquitin ligases functions together with dedicated substrate receptors. Out of the ˜29 CRL4 substrate receptors reported, the DDB1- and CUL4-associated factor 1 (DCAF1) is essential for cellular survival and growth, and its deregulation has been implicated in tumorigenesis. We carried out biochemical and structural studies to examine the structure and mechanism of the CRL4DCAF1 ligase. In the 8.4 Å cryo-EM map of CRL4DCAF1 , four CUL4-RBX1-DDB1-DCAF1 protomers are organized into two dimeric sub-assemblies. In this arrangement, the WD40 domain of DCAF1 mediates binding with the cullin C-terminal domain (CTD) and the RBX1 subunit of a neighboring CRL4DCAF1 protomer. This renders RBX1, the catalytic subunit of the ligase, inaccessible to the E2 ubiquitin-conjugating enzymes. Upon CRL4DCAF1 activation by neddylation, the interaction between the cullin CTD and the neighboring DCAF1 protomer is broken, and the complex assumes an active dimeric conformation. Accordingly, a tetramerization-deficient CRL4DCAF1 mutant has higher ubiquitin ligase activity compared to the wild-type. This study identifies a novel mechanism by which unneddylated and substrate-free CUL4 ligases can be maintained in an inactive state.

Reading the chromatinized genome.

Eukaryotic DNA-binding proteins operate in the context of chromatin, where nucleosomes are the elementary building blocks. Nucleosomal DNA is wrapped around a histone core, thereby rendering a large fraction of the DNA surface inaccessible to DNA-binding proteins. Nevertheless, first responders in DNA repair and sequence-specific transcription factors bind DNA target sites obstructed by chromatin. While early studies examined protein binding to histone-free DNA, it is only now beginning to emerge how DNA sequences are interrogated on nucleosomes. These readout strategies range from the release of nucleosomal DNA from histones, to rotational/translation register shifts of the DNA motif, and nucleosome-specific DNA binding modes that differ from those observed on naked DNA. Since DNA motif engagement on nucleosomes strongly depends on position and orientation, we argue that motif location and nucleosome positioning co-determine protein access to DNA in transcription and DNA repair.