SGN-B6A: A New Vedotin Antibody-Drug Conjugate Directed to Integrin Beta-6 for Multiple Carcinoma Indications.

Integrin beta-6, a component of the heterodimeric adhesion receptor alpha-v/beta-6, is overexpressed in numerous solid tumors. Its expression has been shown by multiple investigators to be a negative prognostic indicator in diverse cancers including colorectal, non-small cell lung, gastric, and cervical. We developed SGN-B6A as an antibody-drug conjugate (ADC) directed to integrin beta-6 to deliver the clinically validated payload monomethyl auristatin E (MMAE) to cancer cells. The antibody component of SGN-B6A is specific for integrin beta-6 and does not bind other alpha-v family members. In preclinical studies, this ADC has demonstrated activity in vivo in models derived from non-small cell lung, pancreatic, pharyngeal, and bladder carcinomas spanning a range of antigen expression levels. In nonclinical toxicology studies in cynomolgus monkeys, doses of up to 5 mg/kg weekly for four doses or 6 mg/kg every 3 weeks for two doses were tolerated. Hematologic toxicities typical of MMAE ADCs were dose limiting, and no significant target-mediated toxicity was observed. A phase I first-in-human study is in progress to evaluate the safety and antitumor activity of SGN-B6A in a variety of solid tumors known to express integrin beta-6 (NCT04389632).

Somatic PDGFRB Activating Variants in Fusiform Cerebral Aneurysms.

The role of somatic genetic variants in the pathogenesis of intracranial-aneurysm formation is unknown. We identified a 23-year-old man with progressive, right-sided intracranial aneurysms, ipsilateral to an impressive cutaneous phenotype. The index individual underwent a series of genetic evaluations for known connective-tissue disorders, but the evaluations were unrevealing. Paired-sample exome sequencing between blood and fibroblasts derived from the diseased areas detected a single novel variant predicted to cause a p.Tyr562Cys (g.149505130T>C [GRCh37/hg19]; c.1685A>G) change within the platelet-derived growth factor receptor ß gene (PDGFRB), a juxtamembrane-coding region. Variant-allele fractions ranged from 18.75% to 53.33% within histologically abnormal tissue, suggesting post-zygotic or somatic mosaicism. In an independent cohort of aneurysm specimens, we detected somatic-activating PDGFRB variants in the juxtamembrane domain or the kinase activation loop in 4/6 fusiform aneurysms (and 0/38 saccular aneurysms; Fisher's exact test, p < 0.001). PDGFRB-variant, but not wild-type, patient cells were found to have overactive auto-phosphorylation with downstream activation of ERK, SRC, and AKT. The expression of discovered variants demonstrated non-ligand-dependent auto-phosphorylation, responsive to the kinase inhibitor sunitinib. Somatic gain-of-function variants in PDGFRB are a novel mechanism in the pathophysiology of fusiform cerebral aneurysms and suggest a potential role for targeted therapy with kinase inhibitors.

Mutations in TRAPPC12 Manifest in Progressive Childhood Encephalopathy and Golgi Dysfunction.

Progressive childhood encephalopathy is an etiologically heterogeneous condition characterized by progressive central nervous system dysfunction in association with a broad range of morbidity and mortality. The causes of encephalopathy can be either non-genetic or genetic. Identifying the genetic causes and dissecting the underlying mechanisms are critical to understanding brain development and improving treatments. Here, we report that variants in TRAPPC12 result in progressive childhood encephalopathy. Three individuals from two unrelated families have either a homozygous deleterious variant (c.145delG [p.Glu49Argfs∗14]) or compound-heterozygous variants (c.360dupC [p.Glu121Argfs∗7] and c.1880C>T [p. Ala627Val]). The clinical phenotypes of the three individuals are strikingly similar: severe disability, microcephaly, hearing loss, spasticity, and characteristic brain imaging findings. Fibroblasts derived from all three individuals showed a fragmented Golgi that could be rescued by expression of wild-type TRAPPC12. Protein transport from the endoplasmic reticulum to and through the Golgi was delayed. TRAPPC12 is a member of the TRAPP protein complex, which functions in membrane trafficking. Variants in several other genes encoding members of the TRAPP complex have been associated with overlapping clinical presentations, indicating shared and distinct functions for each complex member. Detailed understanding of the TRAPP-opathies will illuminate the role of membrane protein transport in human disease.

Large-scale heterochromatin remodeling linked to overreplication-associated DNA damage.

Previously, we have shown that loss of the histone 3 lysine 27 (H3K27) monomethyltransferases ARABIDOPSIS TRITHORAX-RELATED 5 (ATXR5) and ATXR6 (ATXR6) results in the overreplication of heterochromatin. Here we show that the overreplication results in DNA damage and extensive chromocenter remodeling into unique structures we have named "overreplication-associated centers" (RACs). RACs have a highly ordered structure with an outer layer of condensed heterochromatin, an inner layer enriched in the histone variant H2AX, and a low-density core containing foci of phosphorylated H2AX (a marker of double-strand breaks) and the DNA-repair enzyme RAD51. atxr5,6 mutants are strongly affected by mutations in DNA repair, such as ATM and ATR. Because of its dense packaging and repetitive DNA sequence, heterochromatin is a challenging environment in which to repair DNA damage. Previous work in animals has shown that heterochromatic breaks are translocated out of the heterochromatic domain for repair. Our results show that atxr5,6 mutants use a variation on this strategy for repairing heterochromatic DNA damage. Rather than being moved to adjacent euchromatic regions, as in animals, heterochromatin undergoes large-scale remodeling to create a compartment with low chromatin density.

Evidence for ARGONAUTE4-DNA interactions in RNA-directed DNA methylation in plants.

RNA polymerase V (Pol V) long noncoding RNAs (lncRNAs) have been proposed to guide ARGONAUTE4 (AGO4) to chromatin in RNA-directed DNA methylation (RdDM) in plants. Here, we provide evidence, based on laser UV-assisted zero-length cross-linking, for functionally relevant AGO4-DNA interaction at RdDM targets. We further demonstrate that Pol V lncRNAs or the act of their transcription are required to lock Pol V holoenzyme into a stable DNA-bound state that allows AGO4 recruitment via redundant glycine-tryptophan/tryptophan-glycine AGO hook motifs present on both Pol V and its associated factor, SPT5L. We propose a model in which AGO4-DNA interaction could be responsible for the unique specificities of RdDM.

Periodontal Ehlers-Danlos Syndrome Is Caused by Mutations in C1R and C1S, which Encode Subcomponents C1r and C1s of Complement.

Periodontal Ehlers-Danlos syndrome (pEDS) is an autosomal-dominant disorder characterized by early-onset periodontitis leading to premature loss of teeth, joint hypermobility, and mild skin findings. A locus was mapped to an approximately 5.8 Mb region at 12p13.1 but no candidate gene was identified. In an international consortium we recruited 19 independent families comprising 107 individuals with pEDS to identify the locus, characterize the clinical details in those with defined genetic causes, and try to understand the physiological basis of the condition. In 17 of these families, we identified heterozygous missense or in-frame insertion/deletion mutations in C1R (15 families) or C1S (2 families), contiguous genes in the mapped locus that encode subunits C1r and C1s of the first component of the classical complement pathway. These two proteins form a heterotetramer that then combines with six C1q subunits. Pathogenic variants involve the subunit interfaces or inter-domain hinges of C1r and C1s and are associated with intracellular retention and mild endoplasmic reticulum enlargement. Clinical features of affected individuals in these families include rapidly progressing periodontitis with onset in the teens or childhood, a previously unrecognized lack of attached gingiva, pretibial hyperpigmentation, skin and vascular fragility, easy bruising, and variable musculoskeletal symptoms. Our findings open a connection between the inflammatory classical complement pathway and connective tissue homeostasis.

Identification of Multiple Proteins Coupling Transcriptional Gene Silencing to Genome Stability in Arabidopsis thaliana.

Eukaryotic genomes are regulated by epigenetic marks that act to modulate transcriptional control as well as to regulate DNA replication and repair. In Arabidopsis thaliana, mutation of the ATXR5 and ATXR6 histone methyltransferases causes reduction in histone H3 lysine 27 monomethylation, transcriptional upregulation of transposons, and a genome instability defect in which there is an accumulation of excess DNA corresponding to pericentromeric heterochromatin. We designed a forward genetic screen to identify suppressors of the atxr5/6 phenotype that uncovered loss-of-function mutations in two components of the TREX-2 complex (AtTHP1, AtSAC3B), a SUMO-interacting E3 ubiquitin ligase (AtSTUbL2) and a methyl-binding domain protein (AtMBD9). Additionally, using a reverse genetic approach, we show that a mutation in a plant homolog of the tumor suppressor gene BRCA1 enhances the atxr5/6 phenotype. Through characterization of these mutations, our results suggest models for the production atxr5 atxr6-induced extra DNA involving conflicts between the replicative and transcriptional processes in the cell, and suggest that the atxr5 atxr6 transcriptional defects may be the cause of the genome instability defects in the mutants. These findings highlight the critical intersection of transcriptional silencing and DNA replication in the maintenance of genome stability of heterochromatin.

MTHFD1 controls DNA methylation in Arabidopsis.

DNA methylation is an epigenetic mechanism that has important functions in transcriptional silencing and is associated with repressive histone methylation (H3K9me). To further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. Here, we identify the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Decreased levels of oxidized tetrahydrofolates in mthfd1-1 and lethality of loss-of-function demonstrate the essential enzymatic role of MTHFD1 in Arabidopsis. Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1. Comparative analysis of DNA methylation revealed that the CMT3 and CMT2 pathways involving positive feedback with H3K9me are mostly affected. Our work highlights the sensitivity of epigenetic networks to one-carbon metabolism due to their common S-adenosylmethionine-dependent transmethylation and has implications for human MTHFD1-associated diseases.

Domains rearranged methyltransferase3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis.

DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase domains rearranged methylase 2 (DRM2) controls RNA-directed DNA methylation in a pathway that also involves the plant-specific RNA Polymerase V (Pol V). Additionally, the Arabidopsis genome encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase, DRM3. Here, we show that DRM3 has moderate effects on global DNA methylation and small RNA abundance and that DRM3 physically interacts with Pol V. In Arabidopsis drm3 mutants, we observe a lower level of Pol V-dependent noncoding RNA transcripts even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts. This work sheds further light on the mechanism by which long noncoding RNAs facilitate RNA-directed DNA methylation.

Mechanism of DNA methylation-directed histone methylation by KRYPTONITE.

In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide, and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the ε-ammonium of K9 positioned adjacent to bound SAH. These structural insights, complemented by functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation.

Molecular mechanism of action of plant DRM de novo DNA methyltransferases.

DNA methylation is a conserved epigenetic gene-regulation mechanism. DOMAINS REARRANGED METHYLTRANSFERASE (DRM) is a key de novo methyltransferase in plants, but how DRM acts mechanistically is poorly understood. Here, we report the crystal structure of the methyltransferase domain of tobacco DRM (NtDRM) and reveal a molecular basis for its rearranged structure. NtDRM forms a functional homodimer critical for catalytic activity. We also show that Arabidopsis DRM2 exists in complex with the small interfering RNA (siRNA) effector ARGONAUTE4 (AGO4) and preferentially methylates one DNA strand, likely the strand acting as the template for RNA polymerase V-mediated noncoding RNA transcripts. This strand-biased DNA methylation is also positively correlated with strand-biased siRNA accumulation. These data suggest a model in which DRM2 is guided to target loci by AGO4-siRNA and involves base-pairing of associated siRNAs with nascent RNA transcripts.

Transcriptional gene silencing by Arabidopsis microrchidia homologues involves the formation of heteromers.

Epigenetic gene silencing is of central importance to maintain genome integrity and is mediated by an elaborate interplay between DNA methylation, histone posttranslational modifications, and chromatin remodeling complexes. DNA methylation and repressive histone marks usually correlate with transcriptionally silent heterochromatin, however there are exceptions to this relationship. In Arabidopsis, mutation of Morpheus Molecule 1 (MOM1) causes transcriptional derepression of heterochromatin independently of changes in DNA methylation. More recently, two Arabidopsis homologues of mouse microrchidia (MORC) genes have also been implicated in gene silencing and heterochromatin condensation without altering genome-wide DNA methylation patterns. In this study, we show that Arabidopsis microrchidia (AtMORC6) physically interacts with AtMORC1 and with its close homologue, AtMORC2, in two mutually exclusive protein complexes. RNA-sequencing analyses of high-order mutants indicate that AtMORC1 and AtMORC2 act redundantly to repress a common set of loci. We also examined genetic interactions between AtMORC6 and MOM1 pathways. Although AtMORC6 and MOM1 control the silencing of a very similar set of genomic loci, we observed synergistic transcriptional regulation in the mom1/atmorc6 double mutant, suggesting that these epigenetic regulators act mainly by different silencing mechanisms.

SRA- and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation.

RNA-directed DNA methylation in Arabidopsis thaliana depends on the upstream synthesis of 24-nucleotide small interfering RNAs (siRNAs) by RNA POLYMERASE IV (Pol IV) and downstream synthesis of non-coding transcripts by Pol V. Pol V transcripts are thought to interact with siRNAs which then recruit DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to methylate DNA. The SU(VAR)3-9 homologues SUVH2 and SUVH9 act in this downstream step but the mechanism of their action is unknown. Here we show that genome-wide Pol V association with chromatin redundantly requires SUVH2 and SUVH9. Although SUVH2 and SUVH9 resemble histone methyltransferases, a crystal structure reveals that SUVH9 lacks a peptide-substrate binding cleft and lacks a properly formed S-adenosyl methionine (SAM)-binding pocket necessary for normal catalysis, consistent with a lack of methyltransferase activity for these proteins. SUVH2 and SUVH9 both contain SRA (SET- and RING-ASSOCIATED) domains capable of binding methylated DNA, suggesting that they function to recruit Pol V through DNA methylation. Consistent with this model, mutation of DNA METHYLTRANSFERASE 1 (MET1) causes loss of DNA methylation, a nearly complete loss of Pol V at its normal locations, and redistribution of Pol V to sites that become hypermethylated. Furthermore, tethering SUVH9 [corrected] with a zinc finger to an unmethylated site is sufficient to recruit Pol V and establish DNA methylation and gene silencing. These results indicate that Pol V is recruited to DNA methylation through the methyl-DNA binding SUVH2 and SUVH9 proteins, and our mechanistic findings suggest a means for selectively targeting regions of plant genomes for epigenetic silencing.

Interplay between active chromatin marks and RNA-directed DNA methylation in Arabidopsis thaliana.

DNA methylation is an epigenetic mark that is associated with transcriptional repression of transposable elements and protein-coding genes. Conversely, transcriptionally active regulatory regions are strongly correlated with histone 3 lysine 4 di- and trimethylation (H3K4m2/m3). We previously showed that Arabidopsis thaliana plants with mutations in the H3K4m2/m3 demethylase JUMONJI 14 (JMJ14) exhibit a mild reduction in RNA-directed DNA methylation (RdDM) that is associated with an increase in H3K4m2/m3 levels. To determine whether this incomplete RdDM reduction was the result of redundancy with other demethylases, we examined the genetic interaction of JMJ14 with another class of H3K4 demethylases: lysine-specific demethylase 1-like 1 and lysine-specific demethylase 1-like 2 (LDL1 and LDL2). Genome-wide DNA methylation analyses reveal that both families cooperate to maintain RdDM patterns. ChIP-seq experiments show that regions that exhibit an observable DNA methylation decrease are co-incidental with increases in H3K4m2/m3. Interestingly, the impact on DNA methylation was stronger at DNA-methylated regions adjacent to H3K4m2/m3-marked protein-coding genes, suggesting that the activity of H3K4 demethylases may be particularly crucial to prevent spreading of active epigenetic marks. Finally, RNA sequencing analyses indicate that at RdDM targets, the increase of H3K4m2/m3 is not generally associated with transcriptional de-repression. This suggests that the histone mark itself--not transcription--impacts the extent of RdDM.

Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1.

DNA methylation is an epigenetic modification that has critical roles in gene silencing, development and genome integrity. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and targeted by 24-nucleotide small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM). This pathway requires two plant-specific RNA polymerases: Pol-IV, which functions to initiate siRNA biogenesis, and Pol-V, which functions to generate scaffold transcripts that recruit downstream RdDM factors. To understand the mechanisms controlling Pol-IV targeting we investigated the function of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1), a Pol-IV-interacting protein. Here we show that SHH1 acts upstream in the RdDM pathway to enable siRNA production from a large subset of the most active RdDM targets, and that SHH1 is required for Pol-IV occupancy at these same loci. We also show that the SHH1 SAWADEE domain is a novel chromatin-binding module that adopts a unique tandem Tudor-like fold and functions as a dual lysine reader, probing for both unmethylated K4 and methylated K9 modifications on the histone 3 (H3) tail. Finally, we show that key residues within both lysine-binding pockets of SHH1 are required in vivo to maintain siRNA and DNA methylation levels as well as Pol-IV occupancy at RdDM targets, demonstrating a central role for methylated H3K9 binding in SHH1 function and providing the first insights into the mechanism of Pol-IV targeting. Given the parallels between methylation systems in plants and mammals, a further understanding of this early targeting step may aid our ability to control the expression of endogenous and newly introduced genes, which has broad implications for agriculture and gene therapy.

DDR complex facilitates global association of RNA polymerase V to promoters and evolutionarily young transposons.

The plant-specific DNA-dependent RNA polymerase V (Pol V) evolved from Pol II to function in an RNA-directed DNA methylation pathway. Here, we have identified targets of Pol V in Arabidopsis thaliana on a genome-wide scale using ChIP-seq of NRPE1, the largest catalytic subunit of Pol V. We found that Pol V is enriched at promoters and evolutionarily recent transposons. This localization pattern is highly correlated with Pol V-dependent DNA methylation and small RNA accumulation. We also show that genome-wide chromatin association of Pol V is dependent on all members of a putative chromatin-remodeling complex termed DDR. Our study presents a genome-wide view of Pol V occupancy and sheds light on the mechanistic basis of Pol V localization. Furthermore, these findings suggest a role for Pol V and RNA-directed DNA methylation in genome surveillance and in responding to genome evolution.

DNA methyltransferases are required to induce heterochromatic re-replication in Arabidopsis.

The relationship between epigenetic marks on chromatin and the regulation of DNA replication is poorly understood. Mutations of the H3K27 methyltransferase genes, Arabidopsis trithorax-related protein5 (ATXR5) and ATXR6, result in re-replication (repeated origin firing within the same cell cycle). Here we show that mutations that reduce DNA methylation act to suppress the re-replication phenotype of atxr5 atxr6 mutants. This suggests that DNA methylation, a mark enriched at the same heterochromatic regions that re-replicate in atxr5/6 mutants, is required for aberrant re-replication. In contrast, RNA sequencing analyses suggest that ATXR5/6 and DNA methylation cooperatively transcriptionally silence transposable elements (TEs). Hence our results suggest a complex relationship between ATXR5/6 and DNA methylation in the regulation of DNA replication and transcription of TEs.

INVOLVED IN DE NOVO 2-containing complex involved in RNA-directed DNA methylation in Arabidopsis.

At least three pathways control maintenance of DNA cytosine methylation in Arabidopsis thaliana. However, the RNA-directed DNA methylation (RdDM) pathway is solely responsible for establishment of this silencing mark. We previously described INVOLVED IN DE NOVO 2 (IDN2) as being an RNA-binding RdDM component that is required for DNA methylation establishment. In this study, we describe the discovery of two partially redundant proteins that are paralogous to IDN2 and that form a stable complex with IDN2 in vivo. Null mutations in both genes, termed IDN2-LIKE 1 and IDN2-LIKE 2 (IDNL1 and IDNL2), result in a phenotype that mirrors, but does not further enhance, the idn2 mutant phenotype. Genetic analysis suggests that this complex acts in a step in the downstream portion of the RdDM pathway. We also have performed structural analysis showing that the IDN2 XS domain adopts an RNA recognition motif (RRM) fold. Finally, genome-wide DNA methylation and expression analysis confirms the placement of the IDN proteins in an RdDM pathway that affects DNA methylation and transcriptional control at many sites in the genome. Results from this study identify and describe two unique components of the RdDM machinery, adding to our understanding of DNA methylation control in the Arabidopsis genome.

MORC family ATPases required for heterochromatin condensation and gene silencing.

Transposable elements (TEs) and DNA repeats are commonly targeted by DNA and histone methylation to achieve epigenetic gene silencing. We isolated mutations in two Arabidopsis genes, AtMORC1 and AtMORC6, which cause derepression of DNA-methylated genes and TEs but no losses of DNA or histone methylation. AtMORC1 and AtMORC6 are members of the conserved Microrchidia (MORC) adenosine triphosphatase (ATPase) family, which are predicted to catalyze alterations in chromosome superstructure. The atmorc1 and atmorc6 mutants show decondensation of pericentromeric heterochromatin, increased interaction of pericentromeric regions with the rest of the genome, and transcriptional defects that are largely restricted to loci residing in pericentromeric regions. Knockdown of the single MORC homolog in Caenorhabditis elegans also impairs transgene silencing. We propose that the MORC ATPases are conserved regulators of gene silencing in eukaryotes.