Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia.

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.

The E3 ubiquitin ligase SPOP controls resolution of systemic inflammation by triggering MYD88 degradation.

The response to systemic infection and injury requires the rapid adaptation of hematopoietic stem cells (HSCs), which proliferate and divert their differentiation toward the myeloid lineage. Significant interest has emerged in understanding the signals that trigger the emergency hematopoietic program. However, the mechanisms that halt this response of HSCs, which is critical to restore homeostasis, remain unknown. Here we reveal that the E3 ubiquitin ligase Speckle-type BTB-POZ protein (SPOP) restrains the inflammatory activation of HSCs. In the absence of Spop, systemic inflammation proceeded in an unresolved manner, and the sustained response in the HSCs resulted in a lethal phenotype reminiscent of hyper-inflammatory syndrome or sepsis. Our proteomic studies decipher that SPOP restricted inflammation by ubiquitinating the innate signal transducer myeloid differentiation primary response protein 88 (MYD88). These findings unearth an HSC-intrinsic post-translational mechanism that is essential for reestablishing homeostasis after emergency hematopoiesis.

Amyloidogenic Oligomerization Transforms Drosophila Orb2 from a Translation Repressor to an Activator.

Memories are thought to be formed in response to transient experiences, in part through changes in local protein synthesis at synapses. In Drosophila, the amyloidogenic (prion-like) state of the RNA binding protein Orb2 has been implicated in long-term memory, but how conformational conversion of Orb2 promotes memory formation is unclear. Combining in vitro and in vivo studies, we find that the monomeric form of Orb2 represses translation and removes mRNA poly(A) tails, while the oligomeric form enhances translation and elongates the poly(A) tails and imparts its translational state to the monomer. The CG13928 protein, which binds only to monomeric Orb2, promotes deadenylation, whereas the putative poly(A) binding protein CG4612 promotes oligomeric Orb2-dependent translation. Our data support a model in which monomeric Orb2 keeps target mRNA in a translationally dormant state and experience-dependent conversion to the amyloidogenic state activates translation, resulting in persistent alteration of synaptic activity and stabilization of memory.

Proteome plasticity in response to persistent environmental change.

Temperature is a variable component of the environment, and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses, resulting in refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to reduction of temperature-sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find that many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and, importantly, cellular functions. We postulate that, in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.

A six-amino-acid motif is a major determinant in functional evolution of HOX1 proteins.

Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. We used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1-HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. Our findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.

Cyclin F-mediated degradation of ribonucleotide reductase M2 controls genome integrity and DNA repair.

F-box proteins are the substrate binding subunits of SCF (Skp1-Cul1-F-box protein) ubiquitin ligase complexes. Using affinity purifications and mass spectrometry, we identified RRM2 (the ribonucleotide reductase family member 2) as an interactor of the F-box protein cyclin F. Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides (dNTPs), which are necessary for both replicative and repair DNA synthesis. We found that, during G2, following CDK-mediated phosphorylation of Thr33, RRM2 is degraded via SCF(cyclin F) to maintain balanced dNTP pools and genome stability. After DNA damage, cyclin F is downregulated in an ATR-dependent manner to allow accumulation of RRM2. Defective elimination of cyclin F delays DNA repair and sensitizes cells to DNA damage, a phenotype that is reverted by expressing a nondegradable RRM2 mutant. In summary, we have identified a biochemical pathway that controls the abundance of dNTPs and ensures efficient DNA repair in response to genotoxic stress.

Human mediator subunit MED26 functions as a docking site for transcription elongation factors.

Promoter-proximal pausing by initiated RNA polymerase II (Pol II) and regulated release of paused polymerase into productive elongation has emerged as a major mechanism of transcription activation. Reactivation of paused Pol II correlates with recruitment of super-elongation complexes (SECs) containing ELL/EAF family members, P-TEFb, and other proteins, but the mechanism of their recruitment is an unanswered question. Here, we present evidence for a role of human Mediator subunit MED26 in this process. We identify in the conserved N-terminal domain of MED26 overlapping docking sites for SEC and a second ELL/EAF-containing complex, as well as general initiation factor TFIID. In addition, we present evidence consistent with the model that MED26 can function as a molecular switch that interacts first with TFIID in the Pol II initiation complex and then exchanges TFIID for complexes containing ELL/EAF and P-TEFb to facilitate transition of Pol II into the elongation stage of transcription.

Dynamic RNA acetylation revealed by quantitative cross-evolutionary mapping.

N4-acetylcytidine (ac4C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA1-3. However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac4C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac4C at single-nucleotide resolution. In human and yeast mRNAs, ac4C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac4C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac4C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac4C and its potential thermoadaptive role. Our studies quantitatively define the ac4C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease4-6.

Metabolic reprogramming underlies cavefish muscular endurance despite loss of muscle mass and contractility.

Physical inactivity is a scourge to human health, promoting metabolic disease and muscle wasting. Interestingly, multiple ecological niches have relaxed investment into physical activity, providing an evolutionary perspective into the effect of adaptive physical inactivity on tissue homeostasis. One such example, the Mexican cavefish Astyanax mexicanus, has lost moderate-to-vigorous activity following cave colonization, reaching basal swim speeds ~3.7-fold slower than their river-dwelling counterpart. This change in behavior is accompanied by a marked shift in body composition, decreasing total muscle mass and increasing fat mass. This shift persisted at the single muscle fiber level via increased lipid and sugar accumulation at the expense of myofibrillar volume. Transcriptomic analysis of laboratory-reared and wild-caught cavefish indicated that this shift is driven by increased expression of pparγ-the master regulator of adipogenesis-with a simultaneous decrease in fast myosin heavy chain expression. Ex vivo and in vivo analysis confirmed that these investment strategies come with a functional trade-off, decreasing cavefish muscle fiber shortening velocity, time to maximal force, and ultimately maximal swimming speed. Despite this, cavefish displayed a striking degree of muscular endurance, reaching maximal swim speeds ~3.5-fold faster than their basal swim speeds. Multi-omic analysis suggested metabolic reprogramming, specifically phosphorylation of Pgm1-Threonine 19, as a key component enhancing cavefish glycogen metabolism and sustained muscle contraction. Collectively, we reveal broad skeletal muscle changes following cave colonization, displaying an adaptive skeletal muscle phenotype reminiscent to mammalian disuse and high-fat models while simultaneously maintaining a unique capacity for sustained muscle contraction via enhanced glycogen metabolism.

The antiproliferative effect of FGF2 in K-Ras-driven tumor cells involves modulation of rRNA and the nucleolus.

The nucleolus is sensitive to stress and can orchestrate a chain of cellular events in response to stress signals. Despite being a growth factor, FGF2 has antiproliferative and tumor-suppressive functions in some cellular contexts. In this work, we investigated how the antiproliferative effect of FGF2 modulates chromatin-, nucleolus- and rDNA-associated proteins. The chromatin and nucleolar proteome indicated that FGF2 stimulation modulates proteins related to transcription, rRNA expression and chromatin-remodeling proteins. The global transcriptional rate and nucleolus area increased along with nucleolar disorganization upon 24 h of FGF2 stimulation. FGF2 stimulation induced immature rRNA accumulation by increasing rRNA transcription. The rDNA-associated protein analysis reinforced that FGF2 stimulus interferes with transcription and rRNA processing. RNA Pol I inhibition partially reversed the growth arrest induced by FGF2, indicating that changes in rRNA expression might be crucial for triggering the antiproliferative effect. Taken together, we demonstrate that the antiproliferative FGF2 stimulus triggers significant transcriptional changes and modulates the main cell transcription site, the nucleolus.

Paraspeckles interact with SWI/SNF subunit ARID1B to regulate transcription and splicing.

Paraspeckles are subnuclear RNA-protein structures that are implicated in important processes including cellular stress response, differentiation, and cancer progression. However, it is unclear how paraspeckles impart their physiological effect at the molecular level. Through biochemical analyses, we show that paraspeckles interact with the SWI/SNF chromatin-remodeling complex. This is specifically mediated by the direct interaction of the long-non-coding RNA NEAT1 of the paraspeckles with ARID1B of the cBAF-type SWI/SNF complex. Strikingly, ARID1B depletion, in addition to resulting in loss of interaction with the SWI/SNF complex, decreases the binding of paraspeckle proteins to chromatin modifiers, transcription factors, and histones. Functionally, the loss of ARID1B and NEAT1 influences the transcription and the alternative splicing of a common set of genes. Our findings reveal that dynamic granules such as the paraspeckles may leverage the specificity of epigenetic modifiers to impart their regulatory effect, thus providing a molecular basis for their function.

The ATAC acetyltransferase complex coordinates MAP kinases to regulate JNK target genes.

In response to extracellular cues, signal transduction activates downstream transcription factors like c-Jun to induce expression of target genes. We demonstrate that the ATAC (Ada two A containing) histone acetyltransferase (HAT) complex serves as a transcriptional cofactor for c-Jun at the Jun N-terminal kinase (JNK) target genes Jra and chickadee. ATAC subunits are required for c-Jun occupancy of these genes and for H4K16 acetylation at the Jra enhancer, promoter, and transcribed sequences. Under conditions of osmotic stress, ATAC colocalizes with c-Jun, recruits the upstream kinases Misshapen, MKK4, and JNK, and suppresses further activation of JNK. Relocalization of these MAPKs and suppression of JNK activation by ATAC are dependent on the CG10238 subunit of ATAC. Thus, ATAC governs the transcriptional response to MAP kinase signaling by serving as both a coactivator of transcription and as a suppressor of upstream signaling.

A Borrelia burgdorferi LptD homolog is required for flipping of surface lipoproteins through the spirochetal outer membrane.

Borrelia spirochetes are unique among diderm bacteria in their lack of lipopolysaccharide (LPS) in the outer membrane (OM) and their abundance of surface-exposed lipoproteins with major roles in transmission, virulence, and pathogenesis. Despite their importance, little is known about how surface lipoproteins are translocated through the periplasm and the OM. Here, we characterized Borrelia burgdorferi BB0838, a distant homolog of the OM LPS assembly protein LptD. Using a CRISPR interference approach, we showed that BB0838 is required for cell growth and envelope stability. Upon BB0838 knockdown, surface lipoprotein OspA was retained in the inner leaflet of the OM, as determined by its inaccessibility to in situ proteolysis but its presence in OM vesicles. The topology of the OM porin/adhesin P66 remained unaffected. Quantitative mass spectrometry of the B. burgdorferi membrane-associated proteome confirmed the selective periplasmic retention of surface lipoproteins under BB0838 knockdown conditions. Additional analysis identified a single in situ protease-accessible BB0838 peptide that mapped to a predicted ß-barrel surface loop. Alphafold Multimer modeled a B. burgdorferi LptB2 FGCAD complex spanning the periplasm. Together, this suggests that BB0838/LptDBb facilitates the essential terminal step in spirochetal surface lipoprotein secretion, using an orthologous OM component of a pathway that secretes LPS in proteobacteria.

An apical protein, Pcr2, is required for persistent movement by the human parasite Toxoplasma gondii.

The phylum Apicomplexa includes thousands of species of unicellular parasites that cause a wide range of human and animal diseases such as malaria and toxoplasmosis. To infect, the parasite must first initiate active movement to disseminate through tissue and invade into a host cell, and then cease moving once inside. The parasite moves by gliding on a surface, propelled by an internal cortical actomyosin-based motility apparatus. One of the most effective invaders in Apicomplexa is Toxoplasma gondii, which can infect any nucleated cell and any warm-blooded animal. During invasion, the parasite first makes contact with the host cell "head-on" with the apical complex, which features an elaborate cytoskeletal apparatus and associated structures. Here we report the identification and characterization of a new component of the apical complex, Preconoidal region protein 2 (Pcr2). Pcr2 knockout parasites replicate normally, but they are severely diminished in their capacity for host tissue destruction due to significantly impaired invasion and egress, two vital steps in the lytic cycle. When stimulated for calcium-induced egress, Pcr2 knockout parasites become active, and secrete effectors to lyse the host cell. Calcium-induced secretion of the major adhesin, MIC2, also appears to be normal. However, the movement of the Pcr2 knockout parasite is spasmodic, which drastically compromises egress. In addition to faulty motility, the ability of the Pcr2 knockout parasite to assemble the moving junction is impaired. Both defects likely contribute to the poor efficiency of invasion. Interestingly, actomyosin activity, as indicated by the motion of mEmerald tagged actin chromobody, appears to be largely unperturbed by the loss of Pcr2, raising the possibility that Pcr2 may act downstream of or in parallel with the actomyosin machinery.

DNA-PKcs-PIDDosome: a nuclear caspase-2-activating complex with role in G2/M checkpoint maintenance.

Caspase-2 is unique among all the mammalian caspases in that it is the only caspase that is present constitutively in the cell nucleus, in addition to other cellular compartments. However, the functional significance of this nuclear localization is unknown. Here we show that DNA damage induced by gamma-radiation triggers the phosphorylation of nuclear caspase-2 at the S122 site within its prodomain, leading to its cleavage and activation. This phosphorylation is carried out by the nuclear serine/threonine protein kinase DNA-PKcs and promoted by the p53-inducible death-domain-containing protein PIDD within a large nuclear protein complex consisting of DNA-PKcs, PIDD, and caspase-2, which we have named the DNA-PKcs-PIDDosome. This phosphorylation and the catalytic activity of caspase-2 are involved in the maintenance of a G2/M DNA damage checkpoint and DNA repair mediated by the nonhomologous end-joining (NHEJ) pathway. The DNA-PKcs-PIDDosome thus represents a protein complex that impacts mammalian G2/M DNA damage checkpoint and NHEJ.

SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.

RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins.

ATXN7L3 and ENY2 Coordinate Activity of Multiple H2B Deubiquitinases Important for Cellular Proliferation and Tumor Growth.

Histone H2B monoubiquitination (H2Bub1) is centrally involved in gene regulation. The deubiquitination module (DUBm) of the SAGA complex is a major regulator of global H2Bub1 levels, and components of this DUBm are linked to both neurodegenerative diseases and cancer. Unexpectedly, we find that ablation of USP22, the enzymatic center of the DUBm, leads to a reduction, rather than an increase, in global H2bub1 levels. In contrast, depletion of non-enzymatic components, ATXN7L3 or ENY2, results in increased H2Bub1. These observations led us to discover two H2Bub1 DUBs, USP27X and USP51, which function independently of SAGA and compete with USP22 for ATXN7L3 and ENY2 for activity. Like USP22, USP51 and USP27X are required for normal cell proliferation, and their depletion suppresses tumor growth. Our results reveal that ATXN7L3 and ENY2 orchestrate activities of multiple deubiquitinating enzymes and that imbalances in these activities likely potentiate human diseases including cancer.

Multiple roles for PARP1 in ALC1-dependent nucleosome remodeling.

The SNF2 family ATPase Amplified in Liver Cancer 1 (ALC1) is the only chromatin remodeling enzyme with a poly(ADP-ribose) (PAR) binding macrodomain. ALC1 functions together with poly(ADP-ribose) polymerase PARP1 to remodel nucleosomes. Activation of ALC1 cryptic ATPase activity and the subsequent nucleosome remodeling requires binding of its macrodomain to PAR chains synthesized by PARP1 and NAD+ A key question is whether PARP1 has a role(s) in ALC1-dependent nucleosome remodeling beyond simply synthesizing the PAR chains needed to activate the ALC1 ATPase. Here, we identify PARP1 separation-of-function mutants that activate ALC1 ATPase but do not support nucleosome remodeling by ALC1. Investigation of these mutants has revealed multiple functions for PARP1 in ALC1-dependent nucleosome remodeling and provides insights into its multifaceted role in chromatin remodeling.

The Enok acetyltransferase complex interacts with Elg1 and negatively regulates PCNA unloading to promote the G1/S transition.

KAT6 histone acetyltransferases (HATs) are highly conserved in eukaryotes and are involved in cell cycle regulation. However, information regarding their roles in regulating cell cycle progression is limited. Here, we report the identification of subunits of the Drosophila Enok complex and demonstrate that all subunits are important for its HAT activity. We further report a novel interaction between the Enok complex and the Elg1 proliferating cell nuclear antigen (PCNA)-unloader complex. Depletion of Enok in S2 cells resulted in a G1/S cell cycle block, and this block can be partially relieved by depleting Elg1. Furthermore, depletion of Enok reduced the chromatin-bound levels of PCNA in both S2 cells and early embryos, suggesting that the Enok complex may interact with the Elg1 complex and down-regulate its PCNA-unloading function to promote the G1/S transition. Supporting this hypothesis, depletion of Enok also partially rescued the endoreplication defects in Elg1-depleted nurse cells. Taken together, our study provides novel insights into the roles of KAT6 HATs in cell cycle regulation through modulating PCNA levels on chromatin.

Cytosolic proteostasis through importing of misfolded proteins into mitochondria.

Loss of proteostasis underlies ageing and neurodegeneration characterized by the accumulation of protein aggregates and mitochondrial dysfunction. Although many neurodegenerative-disease-associated proteins can be found in mitochondria, it remains unclear how mitochondrial dysfunction and protein aggregation could be related. In dividing yeast cells, protein aggregates that form under stress or during ageing are preferentially retained by the mother cell, in part through tethering to mitochondria, while the disaggregase Hsp104 helps to dissociate aggregates and thereby enables refolding or degradation of misfolded proteins. Here we show that, in yeast, cytosolic proteins prone to aggregation are imported into mitochondria for degradation. Protein aggregates that form under heat shock contain both cytosolic and mitochondrial proteins and interact with the mitochondrial import complex. Many aggregation-prone proteins enter the mitochondrial intermembrane space and matrix after heat shock, and some do so even without stress. Timely dissolution of cytosolic aggregates requires the mitochondrial import machinery and proteases. Blocking mitochondrial import but not proteasome activity causes a marked delay in the degradation of aggregated proteins. Defects in cytosolic Hsp70s leads to enhanced entry of misfolded proteins into mitochondria and elevated mitochondrial stress. We term this mitochondria-mediated proteostasis mechanism MAGIC (mitochondria as guardian in cytosol) and provide evidence that it may exist in human cells.