The WDR11 complex is a receptor for acidic-cluster-containing cargo proteins.

Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e., "cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.

Efficient embryonic homozygous gene conversion via RAD51-enhanced interhomolog repair.

Searching for factors to improve knockin efficiency for therapeutic applications, biotechnology, and generation of non-human primate models of disease, we found that the strand exchange protein RAD51 can significantly increase Cas9-mediated homozygous knockin in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is a hallmark of meiosis but only occurs at low frequencies in somatic cells, and its occurrence in zygotes is controversial. Using multiple approaches, we provide evidence for an endogenous IHR mechanism in the early embryo that can be enhanced by RAD51. This process can be harnessed to generate homozygotes from wild-type zygotes using exogenous donors and to convert heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we identify additional IHR-promoting factors and describe features of IHR events. Together, our findings show conclusive evidence for IHR in mouse embryos and describe an efficient method for enhanced gene conversion.

Non-canonical substrate recognition by the human WDR26-CTLH E3 ligase regulates prodrug metabolism.

The yeast glucose-induced degradation-deficient (GID) E3 ubiquitin ligase forms a suite of complexes with interchangeable receptors that selectively recruit N-terminal degron motifs of metabolic enzyme substrates. The orthologous higher eukaryotic C-terminal to LisH (CTLH) E3 complex has been proposed to also recognize substrates through an alternative subunit, WDR26, which promotes the formation of supramolecular CTLH E3 assemblies. Here, we discover that human WDR26 binds the metabolic enzyme nicotinamide/nicotinic-acid-mononucleotide-adenylyltransferase 1 (NMNAT1) and mediates its CTLH E3-dependent ubiquitylation independently of canonical GID/CTLH E3-family substrate receptors. The CTLH subunit YPEL5 inhibits NMNAT1 ubiquitylation and cellular turnover by WDR26-CTLH E3, thereby affecting NMNAT1-mediated metabolic activation and cytotoxicity of the prodrug tiazofurin. Cryoelectron microscopy (cryo-EM) structures of NMNAT1- and YPEL5-bound WDR26-CTLH E3 complexes reveal an internal basic degron motif of NMNAT1 essential for targeting by WDR26-CTLH E3 and degron mimicry by YPEL5's N terminus antagonizing substrate binding. Thus, our data provide a mechanistic understanding of how YPEL5-WDR26-CTLH E3 acts as a modulator of NMNAT1-dependent metabolism.

Assembly mechanism of Integrator's RNA cleavage module.

The modular Integrator complex is a transcription regulator that is essential for embryonic development. It attenuates coding gene expression via premature transcription termination and performs 3'-processing of non-coding RNAs. For both activities, Integrator requires endonuclease activity that is harbored by an RNA cleavage module consisting of INTS4-9-11. How correct assembly of Integrator modules is achieved remains unknown. Here, we show that BRAT1 and WDR73 are critical biogenesis factors for the human cleavage module. They maintain INTS9-11 inactive during maturation by physically blocking the endonuclease active site and prevent premature INTS4 association. Furthermore, BRAT1 facilitates import of INTS9-11 into the nucleus, where it is joined by INTS4. Final BRAT1 release requires locking of the mature cleavage module conformation by inositol hexaphosphate (IP6). Our data explain several neurodevelopmental disorders caused by BRAT1, WDR73, and INTS11 mutations as Integrator assembly defects and reveal that IP6 is an essential co-factor for cleavage module maturation.

Transcription elongation defects link oncogenic SF3B1 mutations to targetable alterations in chromatin landscape.

Transcription and splicing of pre-messenger RNA are closely coordinated, but how this functional coupling is disrupted in human diseases remains unexplored. Using isogenic cell lines, patient samples, and a mutant mouse model, we investigated how cancer-associated mutations in SF3B1 alter transcription. We found that these mutations reduce the elongation rate of RNA polymerase II (RNAPII) along gene bodies and its density at promoters. The elongation defect results from disrupted pre-spliceosome assembly due to impaired protein-protein interactions of mutant SF3B1. The decreased promoter-proximal RNAPII density reduces both chromatin accessibility and H3K4me3 marks at promoters. Through an unbiased screen, we identified epigenetic factors in the Sin3/HDAC/H3K4me pathway, which, when modulated, reverse both transcription and chromatin changes. Our findings reveal how splicing factor mutant states behave functionally as epigenetic disorders through impaired transcription-related changes to the chromatin landscape. We also present a rationale for targeting the Sin3/HDAC complex as a therapeutic strategy.

Ring domains are essential for GATOR2-dependent mTORC1 activation.

The GATOR2-GATOR1 signaling axis is essential for amino-acid-dependent mTORC1 activation. However, the molecular function of the GATOR2 complex remains unknown. Here, we report that disruption of the Ring domains of Mios, WDR24, or WDR59 completely impedes amino-acid-mediated mTORC1 activation. Mechanistically, via interacting with Ring domains of WDR59 and WDR24, the Ring domain of Mios acts as a hub to maintain GATOR2 integrity, disruption of which leads to self-ubiquitination of WDR24. Physiologically, leucine stimulation dissociates Sestrin2 from the Ring domain of WDR24 and confers its availability to UBE2D3 and subsequent ubiquitination of NPRL2, contributing to GATOR2-mediated GATOR1 inactivation. As such, WDR24 ablation or Ring deletion prevents mTORC1 activation, leading to severe growth defects and embryonic lethality at E10.5 in mice. Hence, our findings demonstrate that Ring domains are essential for GATOR2 to transmit amino acid availability to mTORC1 and further reveal the essentiality of nutrient sensing during embryonic development.

Co-transcriptional genome surveillance by HUSH is coupled to termination machinery.

The HUSH complex recognizes and silences foreign DNA such as viruses, transposons, and transgenes without prior exposure to its targets. Here, we show that endogenous targets of the HUSH complex fall into two distinct classes based on the presence or absence of H3K9me3. These classes are further distinguished by their transposon content and differential response to the loss of HUSH. A de novo genomic rearrangement at the Sox2 locus induces a switch from H3K9me3-independent to H3K9me3-associated HUSH targeting, resulting in silencing. We further demonstrate that HUSH interacts with the termination factor WDR82 and-via its component MPP8-with nascent RNA. HUSH accumulates at sites of high RNAPII occupancy including long exons and transcription termination sites in a manner dependent on WDR82 and CPSF. Together, our results uncover the functional diversity of HUSH targets and show that this vertebrate-specific complex exploits evolutionarily ancient transcription termination machinery for co-transcriptional chromatin targeting and genome surveillance.

A restrictor complex of ZC3H4, WDR82, and ARS2 integrates with PNUTS to control unproductive transcription.

The transcriptional termination of unstable non-coding RNAs (ncRNAs) is poorly understood compared to coding transcripts. We recently identified ZC3H4-WDR82 ("restrictor") as restricting human ncRNA transcription, but how it does this is unknown. Here, we show that ZC3H4 additionally associates with ARS2 and the nuclear exosome targeting complex. The domains of ZC3H4 that contact ARS2 and WDR82 are required for ncRNA restriction, suggesting their presence in a functional complex. Consistently, ZC3H4, WDR82, and ARS2 co-transcriptionally control an overlapping population of ncRNAs. ZC3H4 is proximal to the negative elongation factor, PNUTS, which we show enables restrictor function and is required to terminate the transcription of all major RNA polymerase II transcript classes. In contrast to short ncRNAs, longer protein-coding transcription is supported by U1 snRNA, which shields transcripts from restrictor and PNUTS at hundreds of genes. These data provide important insights into the mechanism and control of transcription by restrictor and PNUTS.

RNA Targets Ribogenesis Factor WDR43 to Chromatin for Transcription and Pluripotency Control.

Transcription regulation underlies stem cell function and development. Here, we elucidate an unexpected role of an essential ribogenesis factor, WDR43, as a chromatin-associated RNA-binding protein (RBP) and release factor in modulating the polymerase (Pol) II activity for pluripotency regulation. WDR43 binds prominently to promoter-associated noncoding/nascent RNAs, occupies thousands of gene promoters and enhancers, and interacts with the Pol II machinery in embryonic stem cells (ESCs). Nascent transcripts and transcription recruit WDR43 to active promoters, where WDR43 facilitates releases of the elongation factor P-TEFb and paused Pol II. Knockdown of WDR43 causes genome-wide defects in Pol II release and pluripotency-associated gene expression. Importantly, auxin-mediated rapid degradation of WDR43 drastically reduces Pol II activity, precluding indirect consequences. These results reveal an RNA-mediated recruitment and feedforward regulation on transcription and demonstrate an unforeseen role of an RBP in promoting Pol II elongation and coordinating high-level transcription and translation in ESC pluripotency.

Expanded profiling of WD repeat domain 5 inhibitors reveals actionable strategies for the treatment of hematologic malignancies.

WD40 Repeat Domain 5 (WDR5) is a highly conserved nuclear protein that recruits MYC oncoprotein transcription factors to chromatin to stimulate ribosomal protein gene expression. WDR5 is tethered to chromatin via an arginine-binding cavity known as the "WIN" site. Multiple pharmacological inhibitors of the WDR5-interaction site of WDR5 (WINi) have been described, including those with picomolar affinity and oral bioavailability in mice. Thus far, however, WINi have only been shown to be effective against a number of rare cancer types retaining wild-type p53. To explore the full potential of WINi for cancer therapy, we systematically profiled WINi across a panel of cancer cells, alone and in combination with other agents. We report that WINi are unexpectedly active against cells derived from both solid and blood-borne cancers, including those with mutant p53. Among hematologic malignancies, we find that WINi are effective as a single agent against leukemia and diffuse large B cell lymphoma xenograft models, and can be combined with the approved drug venetoclax to suppress disseminated acute myeloid leukemia in vivo. These studies reveal actionable strategies for the application of WINi to treat blood-borne cancers and forecast expanded utility of WINi against other cancer types.

WDR20 prevents hepatocellular carcinoma senescence by orchestrating the simultaneous USP12/46-mediated deubiquitination of c-Myc.

The dysfunction of the ubiquitin-proteasome system (UPS) facilitates the malignant progression of hepatocellular carcinoma (HCC). While targeting the UPS for HCC therapy has been proposed, identifying effective targets has been challenging. In this study, we conducted a focused screen of siRNA libraries targeting UPS-related WD40 repeat (WDR) proteins and found that silencing WDR20, a deubiquitinating enzyme activating factor, selectively inhibited the proliferation of HCC cells without affecting normal hepatocytes. Moreover, the downregulation of WDR20 expression induced HCC cellular senescence and suppressed tumor progression in xenograft, sleeping beauty transposon/transposase, and hydrodynamic tail vein injection-induced HCC models, and Alb-Cre+/MYC+ HCC transgenic mouse models. Mechanistically, we found that WDR20 silencing disturbed the protein stability of c-Myc, orchestrating the simultaneous USP12/46-mediated deubiquitination of c-Myc, thereby promoting the transcriptional activation of CDKN1A. Further investigation revealed a positive coexpression of WDR20 and c-Myc in a tissue microarray with 88 HCC clinical samples. By employing three patient-derived organoids from individuals with HCC, we have validated the decrease in c-Myc expression and the significant induction of senescence and growth inhibition following silencing of WDR20. This study not only uncovers the biological function of WDR20 and elucidates the molecular mechanism underlying its negative regulation of HCC cellular senescence but also highlight the potential of WDR20 as a promising target for HCC therapy.

Mettl1/Wdr4-Mediated m7G tRNA Methylome Is Required for Normal mRNA Translation and Embryonic Stem Cell Self-Renewal and Differentiation.

tRNAs are subject to numerous modifications, including methylation. Mutations in the human N7-methylguanosine (m7G) methyltransferase complex METTL1/WDR4 cause primordial dwarfism and brain malformation, yet the molecular and cellular function in mammals is not well understood. We developed m7G methylated tRNA immunoprecipitation sequencing (MeRIP-seq) and tRNA reduction and cleavage sequencing (TRAC-seq) to reveal the m7G tRNA methylome in mouse embryonic stem cells (mESCs). A subset of 22 tRNAs is modified at a "RAGGU" motif within the variable loop. We observe increased ribosome occupancy at the corresponding codons in Mettl1 knockout mESCs, implying widespread effects on tRNA function, ribosome pausing, and mRNA translation. Translation of cell cycle genes and those associated with brain abnormalities is particularly affected. Mettl1 or Wdr4 knockout mESCs display defective self-renewal and neural differentiation. Our study uncovers the complexity of the mammalian m7G tRNA methylome and highlights its essential role in ESCs with links to human disease.

Wdr59 promotes or inhibits TORC1 activity depending on cellular context.

Target of Rapamycin Complex I (TORC1) is a central regulator of metabolism in eukaryotes that responds to a wide array of negative and positive inputs. The GTPase-activating protein toward Rags (GATOR) signaling pathway acts upstream of TORC1 and is comprised of two subcomplexes. The trimeric GATOR1 complex inhibits TORC1 activity in response to amino acid limitation by serving as a GTPase-activating protein (GAP) for the TORC1 activator RagA/B, a component of the lysosomally located Rag GTPase. The multi-protein GATOR2 complex inhibits the activity of GATOR1 and thus promotes TORC1 activation. Here we report that Wdr59, originally assigned to the GATOR2 complex based on studies performed in tissue culture cells, unexpectedly has a dual function in TORC1 regulation in Drosophila. We find that in the ovary and the eye imaginal disc brain complex, Wdr59 inhibits TORC1 activity by opposing the GATOR2-dependent inhibition of GATOR1. Conversely, in the Drosophila fat body, Wdr59 promotes the accumulation of the GATOR2 component Mio and is required for TORC1 activation. Similarly, in mammalian HeLa cells, Wdr59 prevents the proteolytic destruction of GATOR2 proteins Mio and Wdr24. Consistent with the reduced levels of the TORC1-activating GATOR2 complex, Wdr59KOs HeLa cells have reduced TORC1 activity which is restored along with GATOR2 protein levels upon proteasome inhibition. Taken together, our data support the model that the Wdr59 component of the GATOR2 complex functions to promote or inhibit TORC1 activity depending on cellular context.

Structure-based discovery of potent WD repeat domain 5 inhibitors that demonstrate efficacy and safety in preclinical animal models.

WD repeat domain 5 (WDR5) is a core scaffolding component of many multiprotein complexes that perform a variety of critical chromatin-centric processes in the nucleus. WDR5 is a component of the mixed lineage leukemia MLL/SET complex and localizes MYC to chromatin at tumor-critical target genes. As a part of these complexes, WDR5 plays a role in sustaining oncogenesis in a variety of human cancers that are often associated with poor prognoses. Thus, WDR5 has been recognized as an attractive therapeutic target for treating both solid and hematological tumors. Previously, small-molecule inhibitors of the WDR5-interaction (WIN) site and WDR5 degraders have demonstrated robust in vitro cellular efficacy in cancer cell lines and established the therapeutic potential of WDR5. However, these agents have not demonstrated significant in vivo efficacy at pharmacologically relevant doses by oral administration in animal disease models. We have discovered WDR5 WIN-site inhibitors that feature bicyclic heteroaryl P7 units through structure-based design and address the limitations of our previous series of small-molecule inhibitors. Importantly, our lead compounds exhibit enhanced on-target potency, excellent oral pharmacokinetic (PK) profiles, and potent dose-dependent in vivo efficacy in a mouse MV4:11 subcutaneous xenograft model by oral dosing. Furthermore, these in vivo probes show excellent tolerability under a repeated high-dose regimen in rodents to demonstrate the safety of the WDR5 WIN-site inhibition mechanism. Collectively, our results provide strong support for WDR5 WIN-site inhibitors to be utilized as potential anticancer therapeutics.

Structural studies of WDR5 in complex with MBD3C WIN motif reveal a unique binding mode.

The nucleosome remodeling and deacetylase (NuRD) complex plays a pivotal role in chromatin regulation and transcriptional repression. In mice, methyl-CpG binding domain 3 isoform C (MBD3C) interacts specifically with the histone H3 binding protein WD repeat-containing protein 5 (WDR5) and forms the WDR5-MBD3C/Norde complex. Despite the functional significance of this interaction on embryonic stem cell gene regulation, the molecular mechanism underlying MBD3C recognition by WDR5 remains elusive. Here, we determined the crystal structure of WDR5 in complex with the peptide (residues 40-51) derived from the MBD3C protein at a resolution of 1.9 Å. Structural analysis revealed that MBD3C utilizes a unique binding mode to interact with WDR5, wherein MBD3C Arg43 and Phe47 are involved in recognizing the WDR5-interacting (WIN) site and Tyr191-related B site on the small surface of WDR5, respectively. Notably, the binding induces a ∼91° rotation of WDR5 Tyr191, generating the hydrophobic B site. Furthermore, mutation experiments combined with isothermal titration calorimetry (ITC) assays confirmed the importance of both Arg43 and Phe47 in mediating WDR5 binding affinity. By determining structures of various peptides bound to WDR5, we demonstrated that the WDR5 WIN site and B site can be concurrently recognized by WIN motif peptides containing ''Arg-Cies/Ser-Arg-Val-Phe'' consensus sequence. Overall, this study reveals the structural basis for the formation of the WDR5-MBD3C subcomplex and provides new insights into the recognition mode of WDR5 for the WIN motif. Moreover, these findings shed light on structural-based designs of WDR5-targeted anti-cancer small molecule inhibitors or peptide-mimic drugs.

Unraveling MLL1-fusion leukemia: Epigenetic revelations from an iPS cell point mutation.

Our understanding of acute leukemia pathology is heavily dependent on 11q23 chromosomal translocations involving the mixed lineage leukemia-1 (MLL1) gene, a key player in histone H3 lysine 4 (H3K4) methylation. These translocations result in MLL1-fusion (MLL1F) proteins that are thought to drive leukemogenesis. However, the mechanism behind increased H3K4 trimethylation in MLL1F-leukemic stem cells (MLL1F-LSCs), following loss of the catalytic SET domain of MLL1 (known for H3K4 monomethylation and dimethylation) remains unclear. In our investigation, we introduced a homozygous loss-of-function point mutation in MLL1 within human-induced pluripotent stem cells. This mutation mimics the histone methylation, gene expression, and epithelial-mesenchymal transition phenotypes of MLL1F-LSCs-without requiring a translocation or functional WT MLL1. The mutation caused a genome-wide redistribution of the H3K4 trimethyl mark and upregulated LSC-maintenance genes like HoxA9-A13, Meis1, and the HOTTIP long noncoding RNA. Epithelial-mesenchymal transition markers such as ZEB1, SNAI2, and HIC-5 were also increased leading to enhanced cellular migration and invasiveness. These observations underscore the essential role of MLL1's enzymatic activity in restraining the cascade of epigenetic changes associated with the gene-activating H3K4 trimethylation mark, which we show may be catalyzed by mislocalized SETd1a H3K4 trimethyltransferase in the absence of MLL1's enzymatic activity. Challenging existing models, our findings imply that MLL1F-induced leukemias arise from a dominant-negative impact on MLL1's histone methyltransferase activity. We propose targeting SETd1a in precision medicine as a new therapeutic approach for MLL1-associated leukemias.

Calcium flux control by Pacs1-Wdr37 promotes lymphocyte quiescence and lymphoproliferative diseases.

Endoplasmic reticulum (ER) calcium (Ca2+ ) stores are critical to proteostasis, intracellular signaling, and cellular bioenergetics. Through forward genetic screening in mice, we identified two members of a new complex, Pacs1 and Wdr37, which are required for normal ER Ca2+ handling in lymphocytes. Deletion of Pacs1 or Wdr37 caused peripheral lymphopenia that was linked to blunted Ca2+ release from the ER after antigen receptor stimulation. Pacs1-deficient cells showed diminished inositol triphosphate receptor expression together with increased ER and oxidative stress. Mature Pacs1-/- B cells proliferated and died in vivo under lymphocyte replete conditions, indicating spontaneous loss of cellular quiescence. Disruption of Pacs1-Wdr37 did not diminish adaptive immune responses, but potently suppressed lymphoproliferative disease models by forcing loss of quiescence. Thus, Pacs1-Wdr37 plays a critical role in stabilizing lymphocyte populations through ER Ca2+ handling and presents a new target for lymphoproliferative disease therapy.

Disease-associated mutations in WDR34 lead to diverse impacts on the assembly and function of dynein-2.

The primary cilium is a sensory organelle, receiving signals from the external environment and relaying them into the cell. Mutations in proteins required for transport in the primary cilium result in ciliopathies, a group of genetic disorders that commonly lead to the malformation of organs such as the kidney, liver and eyes and skeletal dysplasias. The motor proteins dynein-2 and kinesin-2 mediate retrograde and anterograde transport, respectively, in the cilium. WDR34 (also known as DYNC2I2), a dynein-2 intermediate chain, is required for the maintenance of cilia function. Here, we investigated WDR34 mutations identified in Jeune syndrome, short-rib polydactyly syndrome and asphyxiating thoracic dysplasia patients. There is a poor correlation between genotype and phenotype in these cases, making diagnosis and treatment highly complex. We set out to define the biological impacts on cilia formation and function of WDR34 mutations by stably expressing the mutant proteins in WDR34-knockout cells. WDR34 mutations led to different spectrums of phenotypes. Quantitative proteomics demonstrated changes in dynein-2 assembly, whereas initiation and extension of the axoneme, localization of intraflagellar transport complex-B proteins, transition zone integrity and Hedgehog signalling were also affected.

Host WD repeat-containing protein 5 inhibits protein kinase R-mediated integrated stress response during measles virus infection.

Some negative-sense RNA viruses, including measles virus (MeV), share the characteristic that during their infection cycle, cytoplasmic inclusion bodies (IBs) are formed where components of the viral replication machinery are concentrated. As a foci of viral replication, how IBs act to enhance the efficiency of infection by affecting virus-host interactions remains an important topic of investigation. We previously established that upon MeV infection, the epigenetic host protein, WD repeat-containing protein 5 (WDR5), translocates to cytoplasmic viral IBs and facilitates MeV replication. We now show that WDR5 is recruited to IBs by forming a complex with IB-associated MeV phosphoprotein via a conserved binding motif located on the surface of WDR5. Furthermore, we provide evidence that WDR5 promotes viral replication by suppressing a major innate immune response pathway, the double-stranded RNA-mediated activation of protein kinase R and integrated stress response. IMPORTANCE: MeV is a pathogen that remains a global concern, with an estimated 9 million measles cases and 128,000 measles deaths in 2022 according to the World Health Organization. A large population of the world still has inadequate access to the effective vaccine against the exceptionally transmissible MeV. Measles disease is characterized by a high morbidity in children and in immunocompromised individuals. An important area of research for negative-sense RNA viruses, including MeV, is the characterization of the complex interactome between virus and host occurring at cytoplasmic IBs where viral replication occurs. Despite the progress made in understanding IB structures, little is known regarding the virus-host interactions within IBs and the role of these interactions in promoting viral replication and antagonizing host innate immunity. Herein we provide evidence suggesting a model by which MeV IBs utilize the host protein WDR5 to suppress the protein kinase R-integrated stress response pathway.

Nup98 regulation of histone methylation promotes normal gene expression and may drive leukemogenesis.

Nuclear pore proteins (Nups) interact with chromosomes to regulate gene expression and chromatin structure. A new study by Franks and colleagues (pp. 2222-2234) provides new mechanistic insight into the molecular basis by which Nup98 promotes gene activation in normal hematopoietic cells and how that process is altered by translocations to cause excess expression of developmental genes in leukemia.