Diego Pasini

The PR-DUB complex is responsible for erasing the repressive histone modification, H2AK119ub1. ASXL1-3 proteins are mutually exclusive catalytic partners of BAP1 in the PR-DUB complex. Somatic heterozygous ASXL1-3 variants are associated with cancer, including myeloid malignancies, while de novo germline variants cause neurodevelopmental disorders such as Bohring-Opitz syndrome. These pathogenic variants are almost exclusively nonsense and frameshift and have been proposed to act as gain-of-function. However, the precise catalytic impact and mechanism of variant ASXL1-3 remains elusive. Using an isogenic embryonic stem cell model we have discovered that ASXL1 BOS variants drive reductions, but not global ablations, in H2AK119ub1, consistent with gain-of-function. This catalytic change occurs through the production of a truncated ASXL1 protein with enhanced stability. Hyper-stabilised ASXL1 drives a stoichiometric shift in PR-DUB assembly away from ASXL2 complexes. The drop in H2AK119ub1 levels ultimately reduces PRC2 binding and H3K27me3 deposition. Surprisingly, this phenotype is shared across PR-DUB loss-of-function models and indeed is emerging as a common phenotype across genetically and mechanistically distinct Polycomb-related chromatinopathies.

In multicellular organisms, the execution of developmental and homeostatic programs often relies on asymmetric cell divisions. These divisions require the alignment of the mitotic spindle axis to cortical polarity cues, and the unequal partitioning of cellular components between progeny cells. Asymmetric divisions are orchestrated by signals from the niche frequently presented in a directional manner, such as Wnt signals. Here we employ bioengineered Wnt-niches to demonstrate that in metaphase NuMA/dynein microtubule motors form a complex with activated LRP6 and β-catenin at the cortical sites of Wnt activation to orient cell division perpendicularly. We show that engagement of LRP6 co-receptors by Wnt ligands locally stabilizes actomyosin contractility through the accumulation of myosin1C. Additionally, we describe a proteomic-based approach to identify mitotic protein complexes enriched at the Wnt-contact site, revealing that mitochondria polarize toward localized Wnt3a sources and are asymmetrically apportioned to the Wnt-proximal daughter cell during Wnt-mediated asymmetric cell division of embryonic stem cells. Mechanistically, we show that CENP-F is required for mitochondria polarization towards localized sites of Wnt3a activation, and that deletion of the Wnt-co-receptor LRP6 impairs the asymmetric apportioning of mitochondria. Our findings enhance the understanding of mitotic Wnt-signaling and elucidate fundamental principles underlying Wnt-dependent mitochondrial polarization.

Heterozygous missense mutations in EZH2 cause Weaver syndrome (WS), a developmental disorder characterized by intellectual disability and overgrowth. EZH2 encodes the enzymatic subunit of Polycomb repressive complex 2 (PRC2), which mediates monomethylation, dimethylation, and trimethylation of histone H3 lysine 27 (H3K27me1/2/3). Most WS-associated EZH2 variants lack functional characterization but are presumed loss-of-function. However, the lack of early truncating mutations in EZH2 led us to hypothesize a dominant-negative mechanism for WS, which was supported by our structural analysis of all known WS-associated EZH2 variants. We isogenically modeled 10 representative variants in embryonic stem cells and showed that they reduce global H3K27me2/3 with concomitant increases in H3K27ac and chromatin decompaction. Notably, the pattern of H3K27me2/3 reductions indicated dominant-negative interference on PRC2 activity even when WS variants were expressed at low levels. RNA-seq identified weakly Polycomb-bound genes that lose canonical PRC1 (cPRC1) occupancy and become derepressed, including several phenotypically relevant growth control genes. Comparative analysis of a gain-of-function EZH2 variant causing growth restriction revealed reciprocal chromatin and transcriptional changes compared with WS-associated variants. Taken together, our findings support a model in which EZH2 variants associated with opposing developmental growth syndromes affect not only H3K27me3 but also intergenic H3K27me2, chromatin architecture, and cPRC1 recruitment.

Oncogenic mutations accumulating in many chromatin-associated proteins have been identified in different tumor types. With a mutation rate from 10 to 57%, ARID1A has been widely considered a tumor suppressor gene. However, whether this role is mainly due to its transcriptional-related activities or its ability to preserve genome integrity is still a matter of intense debate. Here, we show that ARID1A is largely dispensable for preserving enhancer-dependent transcriptional regulation, being ARID1B sufficient and required to compensate for ARID1A loss. We provide in vivo evidence that ARID1A is mainly required to preserve genomic integrity in adult tissues. ARID1A loss primarily results in DNA damage accumulation, interferon type I response activation, and chronic inflammation leading to tumor formation. Our data suggest that in healthy tissues, the increased genomic instability that follows ARID1A mutations and the selective pressure imposed by the microenvironment might result in the emergence of aggressive, possibly immune-resistant, tumors.

Histone H2A lysine 119 (H2AK119Ub) is monoubiquitinated by Polycomb repressive complex 1 and deubiquitinated by Polycomb repressive deubiquitinase complex (PR-DUB). PR-DUB cleaves H2AK119Ub to restrict focal H2AK119Ub at Polycomb target sites and to protect active genes from aberrant silencing. The PR-DUB subunits (BAP1 and ASXL1) are among the most frequently mutated epigenetic factors in human cancers. How PR-DUB establishes specificity for H2AK119Ub over other nucleosomal ubiquitination sites and how disease-associated mutations of the enzyme affect activity are unclear. Here, we determine a cryo-EM structure of human BAP1 and the ASXL1 DEUBAD in complex with a H2AK119Ub nucleosome. Our structural, biochemical, and cellular data reveal the molecular interactions of BAP1 and ASXL1 with histones and DNA that are critical for restructuring the nucleosome and thus establishing specificity for H2AK119Ub. These results further provide a molecular explanation for how >50 mutations in BAP1 and ASXL1 found in cancer can dysregulate H2AK119Ub deubiquitination, providing insight into understanding cancer etiology.

Heterozygous missense mutations in EZH2 cause Weaver syndrome (WS), a developmental disorder characterized by intellectual disability and overgrowth. EZH2 encodes the enzymatic subunit of Polycomb Repressive Complex 2 (PRC2), which mediates mono-, di-, and tri-methylation of histone H3 lysine residue 27 (H3K27me1/2/3). Although the functional characterization of most WS-associated EZH2 variants is lacking, they are presumed loss of function. However, the dearth of reported early truncating mutations in EZH2 led us to hypothesise that a dominant negative mutational mechanism may underlie the development of WS. To test this, we performed a detailed structural analysis of all known WS-associated EZH2 variants, which provided initial support that they are dominant negative. Next, we isogenically modelled 10 representative WS-associated EZH2 variants in embryonic stem cells and showed they induce global reductions in H3K27me2 and H3K27me3 with concomitant global increases in H3K27me1, H3K27ac, and chromatin decompaction. Importantly, the reductions in H3K27me2/3 methylation revealed a pattern of dominant-negative interference to PRC2 activity. Comparative analysis of a gain-of-function EZH2 variant causing growth restriction highlighted the reciprocal nature of the chromatin changes in these opposing growth syndromes. Our findings detail the molecular effects of developmental-syndrome-associated EZH2 variants in cells and implicate imbalanced landscapes of H3K27 modification in their pathology.

As wildfires burn millions of acres each year around the globe and have become more severe due to climate change, wildfire prevention is more important now than ever. Existing wildfire surveying techniques such as hotspotting and cold trailing require human interventions that can lead to dangerous situations or satellite imagery which does not provide real-time data on hotspots. To address this problem, we propose a low-cost and effective integrated system of robots composed of an unmanned aerial vehicle (UAV, or drone) and unmanned ground vehicle (UGV, or rover) that autonomously cooperate and pathfind to detect and investigate hotspots. The UAV monitors a post-forest fire area from the air and uses aerial footage to create long-term direction for the UGV to inspect specific suspected hotspots. The UGV then follows the path to investigate each hotspot with centimeter-level accuracy. Testing of the pathfinding system with satellite imagery yielded highly accurate and consistent results necessary for high-precision autonomous navigation when investigating hotspots in dynamic environments.