Chromatin remodeling of histone H3 variants by DDM1 underlies epigenetic inheritance of DNA methylation.

Nucleosomes block access to DNA methyltransferase, unless they are remodeled by DECREASE in DNA METHYLATION 1 (DDM1LSH/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 promotes replacement of histone variant H3.3 by H3.1. In ddm1 mutants, DNA methylation is partly restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals engagement with histone H3.3 near residues required for assembly and with the unmodified H4 tail. An N-terminal autoinhibitory domain inhibits activity, while a disulfide bond in the helicase domain supports activity. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1, but is blocked by H4K16 acetylation. The male germline H3.3 variant MGH3/HTR10 is resistant to remodeling by DDM1 and acts as a placeholder nucleosome in sperm cells for epigenetic inheritance.

Manipulating PTPRD function with ectodomain antibodies.

Protein tyrosine phosphatases (PTPs) are critical regulators of signal transduction but have yet to be exploited fully for drug development. Receptor protein tyrosine phosphatase δ (RPTPδ/PTPRD) has been shown to elicit tumor-promoting functions, including elevating SRC activity and promoting metastasis in certain cell contexts. Dimerization has been implicated in the inhibition of receptor protein tyrosine phosphatases (RPTPs). We have generated antibodies targeting PTPRD ectodomains with the goal of manipulating their dimerization status ectopically, thereby regulating intracellular signaling. We have validated antibody binding to endogenous PTPRD in a metastatic breast cancer cell line, CAL51, and demonstrated that a monoclonal antibody, RD-43, inhibited phosphatase activity and induced the degradation of PTPRD. Similar effects were observed following chemically induced dimerization of its phosphatase domain. Mechanistically, RD-43 triggered the formation of PTPRD dimers in which the phosphatase activity was impaired. Subsequently, the mAb-PTPRD dimer complex was degraded through lysosomal and proteasomal pathways, independently of secretase cleavage. Consequently, treatment with RD-43 inhibited SRC signaling and suppressed PTPRD-dependent cell invasion. Together, these findings demonstrate that manipulating RPTP function via antibodies to the extracellular segments has therapeutic potential.

OCA-T1 and OCA-T2 are coactivators of POU2F3 in the tuft cell lineage.

Tuft cells are a rare chemosensory lineage that coordinates immune and neural responses to foreign pathogens in mucosal tissues1. Recent studies have also revealed tuft-cell-like human tumours2,3, particularly as a variant of small-cell lung cancer. Both normal and neoplastic tuft cells share a genetic requirement for the transcription factor POU2F3 (refs. 2,4), although the transcriptional mechanisms that generate this cell type are poorly understood. Here we show that binding of POU2F3 to the uncharacterized proteins C11orf53 and COLCA2 (renamed here OCA-T1/POU2AF2 and OCA-T2/POU2AF3, respectively) is critical in the tuft cell lineage. OCA-T1 and OCA-T2 are paralogues of the B-cell-specific coactivator OCA-B; all three proteins are encoded in a gene cluster and contain a conserved peptide that binds to class II POU transcription factors and a DNA octamer motif in a bivalent manner. We demonstrate that binding between POU2F3 and OCA-T1 or OCA-T2 is essential in tuft-cell-like small-cell lung cancer. Moreover, we generated OCA-T1-deficient mice, which are viable but lack tuft cells in several mucosal tissues. These findings reveal that the POU2F3-OCA-T complex is the master regulator of tuft cell identity and a molecular vulnerability of tuft-cell-like small-cell lung cancer.

NSD3-Short Is an Adaptor Protein that Couples BRD4 to the CHD8 Chromatin Remodeler.

The bromodomain and extraterminal (BET) protein BRD4 is a therapeutic target in acute myeloid leukemia (AML). Here, we demonstrate that the AML maintenance function of BRD4 requires its interaction with NSD3, which belongs to a subfamily of H3K36 methyltransferases. Unexpectedly, AML cells were found to only require a short isoform of NSD3 that lacks the methyltransferase domain. We show that NSD3-short is an adaptor protein that sustains leukemia by linking BRD4 to the CHD8 chromatin remodeler, by using a PWWP chromatin reader module, and by employing an acidic transactivation domain. Genetic targeting of NSD3 or CHD8 mimics the phenotypic and transcriptional effects of BRD4 inhibition. Furthermore, BRD4, NSD3, and CHD8 colocalize across the AML genome, and each is released from super-enhancer regions upon chemical inhibition of BET bromodomains. These findings suggest that BET inhibitors exert therapeutic effects in leukemia by evicting BRD4-NSD3-CHD8 complexes from chromatin to suppress transcription.

Tryp-N: A Thermostable Protease for the Production of N-terminal Argininyl and Lysinyl Peptides.

Bottom-up proteomics is a mainstay in protein identification and analysis. These studies typically employ proteolytic treatment of biological samples to generate suitably sized peptides for tandem mass spectrometric (MS) analysis. In MS, fragmentation of peptides is largely driven by charge localization. Consequently, peptides with basic centers exclusively on their N-termini produce mainly b-ions. Thus, it was long ago realized that proteases that yield such peptides would be valuable proteomic tools for achieving simplified peptide fragmentation patterns and peptide assignment. Work by several groups has identified such proteases, however, structural analysis of these suggested that enzymatic optimization was possible. We therefore endeavored to find enzymes that could provide enhanced activity and versatility while maintaining specificity. Using these previously described proteases as informatic search templates, we discovered and then characterized a thermophilic metalloprotease with N-terminal specificity for arginine and lysine. This enzyme, dubbed Tryp-N, affords many advantages including improved thermostability, solvent and detergent tolerance, and rapid digestion time.

The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis.

PIWI-family proteins and their associated small RNAs (piRNAs) act in an evolutionarily conserved innate immune mechanism to provide essential protection for germ-cell genomes against the activity of mobile genetic elements. piRNA populations comprise a molecular definition of transposons, which permits them to distinguish transposons from host genes and selectively silence them. piRNAs can be generated in two distinct ways, forming either primary or secondary piRNAs. Primary piRNAs come from discrete genomic loci, termed piRNA clusters, and seem to be derived from long, single-stranded precursors. The biogenesis of primary piRNAs involves at least two nucleolytic steps. An unknown enzyme cleaves piRNA cluster transcripts to generate monophosphorylated piRNA 5' ends. piRNA 3' ends are probably formed by exonucleolytic trimming, after a piRNA precursor is loaded into its PIWI partner. Secondary piRNAs arise during the adaptive 'ping-pong' cycle, with their 5' termini being formed by the activity of PIWIs themselves. A number of proteins have been implicated genetically in primary piRNA biogenesis. One of these, Drosophila melanogaster Zucchini, is a member of the phospholipase-D family of phosphodiesterases, which includes both phospholipases and nucleases. Here we produced a dimeric, soluble fragment of the mouse Zucchini homologue (mZuc; also known as PLD6) and show that it possesses single-strand-specific nuclease activity. A crystal structure of mZuc at 1.75 Å resolution indicates greater architectural similarity to phospholipase-D family nucleases than to phospholipases. Together, our data suggest that the Zucchini proteins act in primary piRNA biogenesis as nucleases, perhaps generating the 5' ends of primary piRNAs.

Evolutionary adaptation of an HP1-protein chromodomain integrates chromatin and DNA sequence signals.

Members of the diverse heterochromatin protein 1 (HP1) family play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin-binding patterns, likely due to interactions with various specificity factors. Previously, we showed that the chromatin-binding pattern of the HP1 protein Rhino, a crucial factor of the Drosophila PIWI-interacting RNA (piRNA) pathway, is largely defined by a DNA sequence-specific C2H2 zinc finger protein named Kipferl (Baumgartner et al., 2022). Here, we elucidate the molecular basis of the interaction between Rhino and its guidance factor Kipferl. Through phylogenetic analyses, structure prediction, and in vivo genetics, we identify a single amino acid change within Rhino's chromodomain, G31D, that does not affect H3K9me2/3 binding but disrupts the interaction between Rhino and Kipferl. Flies carrying the rhinoG31D mutation phenocopy kipferl mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of rhinoG31D flies. Thus, Rhino's chromodomain functions as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.

Allostery Illuminated: Harnessing AI and Machine Learning for Drug Discovery.

In the past several years there has been rapid adoption of artificial intelligence (AI) and machine learning (ML) tools for drug discovery. In this Microperspective, we comment on recent AI/ML applications to the discovery of allosteric modulators, focusing on breakthroughs with AlphaFold, structure-based drug discovery (SBDD), and medicinal chemistry applications. We discuss how these technologies are facilitating drug discovery and the remaining challenges to identify allosteric binding sites and ligands.

ETV6 dependency in Ewing sarcoma by antagonism of EWS-FLI1-mediated enhancer activation.

The EWS-FLI1 fusion oncoprotein deregulates transcription to initiate the paediatric cancer Ewing sarcoma. Here we used a domain-focused CRISPR screen to implicate the transcriptional repressor ETV6 as a unique dependency in this tumour. Using biochemical assays and epigenomics, we show that ETV6 competes with EWS-FLI1 for binding to select DNA elements enriched for short GGAA repeat sequences. Upon inactivating ETV6, EWS-FLI1 overtakes and hyper-activates these cis-elements to promote mesenchymal differentiation, with SOX11 being a key downstream target. We show that squelching of ETV6 with a dominant-interfering peptide phenocopies these effects and suppresses Ewing sarcoma growth in vivo. These findings reveal targeting of ETV6 as a strategy for neutralizing the EWS-FLI1 oncoprotein by reprogramming of genomic occupancy.

Chromatin remodeling of histone H3 variants underlies epigenetic inheritance of DNA methylation.

Epigenetic inheritance refers to the faithful replication of DNA methylation and histone modification independent of DNA sequence. Nucleosomes block access to DNA methyltransferases, unless they are remodeled by DECREASE IN DNA METHYLATION1 (DDM1 Lsh/HELLS ), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 activity results in replacement of the transcriptional histone variant H3.3 for the replicative variant H3.1 during the cell cycle. In ddm1 mutants, DNA methylation can be restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals direct engagement at SHL2 with histone H3.3 at or near variant residues required for assembly, as well as with the deacetylated H4 tail. An N-terminal autoinhibitory domain binds H2A variants to allow remodeling, while a disulfide bond in the helicase domain is essential for activity in vivo and in vitro . We show that differential remodeling of H3 and H2A variants in vitro reflects preferential deposition in vivo . DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1 Dnmt1 . DDM1 localization to the chromosome is blocked by H4K16 acetylation, which accumulates at DDM1 targets in ddm1 mutants, as does the sperm cell specific H3.3 variant MGH3 in pollen, which acts as a placeholder nucleosome in the germline and contributes to epigenetic inheritance.