A lethal mitonuclear incompatibility in complex I of natural hybrids.

The evolution of reproductive barriers is the first step in the formation of new species and can help us understand the diversification of life on Earth. These reproductive barriers often take the form of hybrid incompatibilities, in which alleles derived from two different species no longer interact properly in hybrids1-3. Theory predicts that hybrid incompatibilities may be more likely to arise at rapidly evolving genes4-6 and that incompatibilities involving multiple genes should be common7,8, but there has been sparse empirical data to evaluate these predictions. Here we describe a mitonuclear incompatibility involving three genes whose protein products are in physical contact within respiratory complex I of naturally hybridizing swordtail fish species. Individuals homozygous for mismatched protein combinations do not complete embryonic development or die as juveniles, whereas those heterozygous for the incompatibility have reduced complex I function and unbalanced representation of parental alleles in the mitochondrial proteome. We find that the effects of different genetic interactions on survival are non-additive, highlighting subtle complexity in the genetic architecture of hybrid incompatibilities. Finally, we document the evolutionary history of the genes involved, showing signals of accelerated evolution and evidence that an incompatibility has been transferred between species via hybridization.

RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis.

Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.

Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.

Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development. However, the mechanisms by which these complexes recognize their target sites and function together to form repressive chromatin domains remain poorly understood. Recruitment of PRC1 to target sites has been proposed to occur through a hierarchical process, dependent on prior nucleation of PRC2 and placement of H3K27me3. Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain. This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development. These observations provide a surprising PRC1-dependent logic for PRC2 occupancy at target sites in vivo.

RIPK1 blocks early postnatal lethality mediated by caspase-8 and RIPK3.

Receptor-interacting protein kinase (RIPK)-1 is involved in RIPK3-dependent and -independent signaling pathways leading to cell death and/or inflammation. Genetic ablation of ripk1 causes postnatal lethality, which was not prevented by deletion of ripk3, caspase-8, or fadd. However, animals that lack RIPK1, RIPK3, and either caspase-8 or FADD survived weaning and matured normally. RIPK1 functions in vitro to limit caspase-8-dependent, TNFR-induced apoptosis, and animals lacking RIPK1, RIPK3, and TNFR1 survive to adulthood. The role of RIPK3 in promoting lethality in ripk1(-/-) mice suggests that RIPK3 activation is inhibited by RIPK1 postbirth. Whereas TNFR-induced RIPK3-dependent necroptosis requires RIPK1, cells lacking RIPK1 were sensitized to necroptosis triggered by poly I:C or interferons. Disruption of TLR (TRIF) or type I interferon (IFNAR) signaling delayed lethality in ripk1(-/-)tnfr1(-/-) mice. These results clarify the complex roles for RIPK1 in postnatal life and provide insights into the regulation of FADD-caspase-8 and RIPK3-MLKL signaling by RIPK1.

Revealing protein-protein interactions at the transcriptome scale by sequencing.

We describe PROPER-seq (protein-protein interaction sequencing) to map protein-protein interactions (PPIs) en masse. PROPER-seq first converts transcriptomes of input cells into RNA-barcoded protein libraries, in which all interacting protein pairs are captured through nucleotide barcode ligation, recorded as chimeric DNA sequences, and decoded at once by sequencing and mapping. We applied PROPER-seq to human embryonic kidney cells, T lymphocytes, and endothelial cells and identified 210,518 human PPIs (collected in the PROPER v.1.0 database). Among these, 1,365 and 2,480 PPIs are supported by published co-immunoprecipitation (coIP) and affinity purification-mass spectrometry (AP-MS) data, 17,638 PPIs are predicted by the prePPI algorithm without previous experimental validation, and 100 PPIs overlap human synthetic lethal gene pairs. In addition, four previously uncharacterized interaction partners with poly(ADP-ribose) polymerase 1 (PARP1) (a critical protein in DNA repair) known as XPO1, MATR3, IPO5, and LEO1 are validated in vivo. PROPER-seq presents a time-effective technology to map PPIs at the transcriptome scale, and PROPER v.1.0 provides a rich resource for studying PPIs.

Xist RNA is a potent suppressor of hematologic cancer in mice.

X chromosome aneuploidies have long been associated with human cancers, but causality has not been established. In mammals, X chromosome inactivation (XCI) is triggered by Xist RNA to equalize gene expression between the sexes. Here we delete Xist in the blood compartment of mice and demonstrate that mutant females develop a highly aggressive myeloproliferative neoplasm and myelodysplastic syndrome (mixed MPN/MDS) with 100% penetrance. Significant disease components include primary myelofibrosis, leukemia, histiocytic sarcoma, and vasculitis. Xist-deficient hematopoietic stem cells (HSCs) show aberrant maturation and age-dependent loss. Reconstitution experiments indicate that MPN/MDS and myelofibrosis are of hematopoietic rather than stromal origin. We propose that Xist loss results in X reactivation and consequent genome-wide changes that lead to cancer, thereby causally linking the X chromosome to cancer in mice. Thus, Xist RNA not only is required to maintain XCI but also suppresses cancer in vivo.

Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53-null tumors.

Here, we show that a subset of breast cancers express high levels of the type 2 phosphatidylinositol-5-phosphate 4-kinases α and/or ß (PI5P4Kα and ß) and provide evidence that these kinases are essential for growth in the absence of p53. Knocking down PI5P4Kα and ß in a breast cancer cell line bearing an amplification of the gene encoding PI5P4K ß and deficient for p53 impaired growth on plastic and in xenografts. This growth phenotype was accompanied by enhanced levels of reactive oxygen species (ROS) leading to senescence. Mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly however, PIP4K2A(-/-), PIP4K2B(+/-), and TP53(-/-) mice were viable and had a dramatic reduction in tumor formation compared to TP53(-/-) littermates. These results indicate that inhibitors of PI5P4Ks could be effective in preventing or treating cancers with mutations in TP53.

A small UTX stabilization domain of Trr is conserved within mammalian MLL3-4/COMPASS and is sufficient to rescue loss of viability in null animals.

Catalytic-inactivating mutations within the Drosophila enhancer H3K4 mono-methyltransferase Trr and its mammalian homologs, MLL3/4, cause only minor changes in gene expression compared with whole-gene deletions for these COMPASS members. To identify essential histone methyltransferase-independent functions of Trr, we screened to identify a minimal Trr domain sufficient to rescue Trr-null lethality and demonstrate that this domain binds and stabilizes Utx in vivo. Using the homologous MLL3/MLL4 human sequences, we mapped a short ∼80-amino-acid UTX stabilization domain (USD) that promotes UTX stability in the absence of the rest of MLL3/4. Nuclear UTX stability is enhanced when the USD is fused with the MLL4 HMG-box. Thus, COMPASS-dependent UTX stabilization is an essential noncatalytic function of Trr/MLL3/MLL4, suggesting that stabilizing UTX could be a therapeutic strategy for cancers with MLL3/4 loss-of-function mutations.

HDAC3 ensures stepwise epidermal stratification via NCoR/SMRT-reliant mechanisms independent of its histone deacetylase activity.

Chromatin modifiers play critical roles in epidermal development, but the functions of histone deacetylases in this context are poorly understood. The class I HDAC, HDAC3, is of particular interest because it plays divergent roles in different tissues by partnering with tissue-specific transcription factors. We found that HDAC3 is expressed broadly in embryonic epidermis and is required for its orderly stepwise stratification. HDAC3 protein stability in vivo relies on NCoR and SMRT, which function redundantly in epidermal development. However, point mutations in the NCoR and SMRT deacetylase-activating domains, which are required for HDAC3's enzymatic function, permit normal stratification, indicating that HDAC3's roles in this context are largely independent of its histone deacetylase activity. HDAC3-bound sites are significantly enriched for predicted binding motifs for critical epidermal transcription factors including AP1, GRHL, and KLF family members. Our results suggest that among these, HDAC3 operates in conjunction with KLF4 to repress inappropriate expression of Tgm1, Krt16, and Aqp3 In parallel, HDAC3 suppresses expression of inflammatory cytokines through a Rela-dependent mechanism. These data identify HDAC3 as a hub coordinating multiple aspects of epidermal barrier acquisition.

Microcephaly gene links trithorax and REST/NRSF to control neural stem cell proliferation and differentiation.

Microcephaly is a neurodevelopmental disorder causing significantly reduced cerebral cortex size. Many known microcephaly gene products localize to centrosomes, regulating cell fate and proliferation. Here, we identify and characterize a nuclear zinc finger protein, ZNF335/NIF-1, as a causative gene for severe microcephaly, small somatic size, and neonatal death. Znf335 null mice are embryonically lethal, and conditional knockout leads to severely reduced cortical size. RNA-interference and postmortem human studies show that ZNF335 is essential for neural progenitor self-renewal, neurogenesis, and neuronal differentiation. ZNF335 is a component of a vertebrate-specific, trithorax H3K4-methylation complex, directly regulating REST/NRSF, a master regulator of neural gene expression and cell fate, as well as other essential neural-specific genes. Our results reveal ZNF335 as an essential link between H3K4 complexes and REST/NRSF and provide the first direct genetic evidence that this pathway regulates human neurogenesis and neuronal differentiation.

Whole-genome doubling confers unique genetic vulnerabilities on tumour cells.

Whole-genome doubling (WGD) is common in human cancers, occurring early in tumorigenesis and generating genetically unstable tetraploid cells that fuel tumour development1,2. Cells that undergo WGD (WGD+ cells) must adapt to accommodate their abnormal tetraploid state; however, the nature of these adaptations, and whether they confer vulnerabilities that can be exploited therapeutically, is unclear. Here, using sequencing data from roughly 10,000 primary human cancer samples and essentiality data from approximately 600 cancer cell lines, we show that WGD gives rise to common genetic traits that are accompanied by unique vulnerabilities. We reveal that WGD+ cells are more dependent than WGD- cells on signalling from the spindle-assembly checkpoint, DNA-replication factors and proteasome function. We also identify KIF18A, which encodes a mitotic kinesin protein, as being specifically required for the viability of WGD+ cells. Although KIF18A is largely dispensable for accurate chromosome segregation during mitosis in WGD- cells, its loss induces notable mitotic errors in WGD+ cells, ultimately impairing cell viability. Collectively, our results suggest new strategies for specifically targeting WGD+ cancer cells while sparing the normal, non-transformed WGD- cells that comprise human tissue.

Aneuploidy renders cancer cells vulnerable to mitotic checkpoint inhibition.

Selective targeting of aneuploid cells is an attractive strategy for cancer treatment1. However, it is unclear whether aneuploidy generates any clinically relevant vulnerabilities in cancer cells. Here we mapped the aneuploidy landscapes of about 1,000 human cancer cell lines, and analysed genetic and chemical perturbation screens2-9 to identify cellular vulnerabilities associated with aneuploidy. We found that aneuploid cancer cells show increased sensitivity to genetic perturbation of core components of the spindle assembly checkpoint (SAC), which ensures the proper segregation of chromosomes during mitosis10. Unexpectedly, we also found that aneuploid cancer cells were less sensitive than diploid cells to short-term exposure to multiple SAC inhibitors. Indeed, aneuploid cancer cells became increasingly sensitive to inhibition of SAC over time. Aneuploid cells exhibited aberrant spindle geometry and dynamics, and kept dividing when the SAC was inhibited, resulting in the accumulation of mitotic defects, and in unstable and less-fit karyotypes. Therefore, although aneuploid cancer cells could overcome inhibition of SAC more readily than diploid cells, their long-term proliferation was jeopardized. We identified a specific mitotic kinesin, KIF18A, whose activity was perturbed in aneuploid cancer cells. Aneuploid cancer cells were particularly vulnerable to depletion of KIF18A, and KIF18A overexpression restored their response to SAC inhibition. Our results identify a therapeutically relevant, synthetic lethal interaction between aneuploidy and the SAC.

Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets.

BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5' flap endonuclease but not the 5'-3' exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency.

An Adversarial DNA N6-Methyladenine-Sensor Network Preserves Polycomb Silencing.

Adenine N6 methylation in DNA (6mA) is widespread among bacteria and phage and is detected in mammalian genomes, where its function is largely unexplored. Here we show that 6mA deposition and removal are catalyzed by the Mettl4 methyltransferase and Alkbh4 dioxygenase, respectively, and that 6mA accumulation in genic elements corresponds with transcriptional silencing. Inactivation of murine Mettl4 depletes 6mA and causes sublethality and craniofacial dysmorphism in incross progeny. We identify distinct 6mA sensor domains of prokaryotic origin within the MPND deubiquitinase and ASXL1, a component of the Polycomb repressive deubiquitinase (PR-DUB) complex, both of which act to remove monoubiquitin from histone H2A (H2A-K119Ub), a repressive mark. Deposition of 6mA by Mettl4 triggers the proteolytic destruction of both sensor proteins, preserving genome-wide H2A-K119Ub levels. Expression of the bacterial 6mA methyltransferase Dam, in contrast, fails to destroy either sensor. These findings uncover a native, adversarial 6mA network architecture that preserves Polycomb silencing.

HP6/Umbrea is dispensable for viability and fertility, suggesting essentiality of newly evolved genes is rare.

The newly evolved gene Heterochromatin Protein 6 (HP6), which has been previously classified as essential, challenged the dogma that functions required for viability are only seen in genes with a long evolutionary history. Based on previous RNA-sequencing analysis in Drosophila germ cells, we asked whether HP6 might play a role in germline development. Surprisingly, we found that CRISPR-generated HP6 mutants are viable and fertile. Using previously generated mutants, we identified an independent lethal allele and an RNAi off-target effect that prevented accurate interpretation of HP6 essentiality. By reviewing existing data, we found that the vast majority of young genes that were previously classified as essential were indeed viable when tested with orthologous methods. Together, our data call into question the frequency with which newly evolved genes gain essential functions and suggest that using multiple independent genetic methods is essential when probing the functions of young genes.

Reticulons 1 and 3 are essential for axonal growth and synaptic maintenance associated with intellectual development.

Reticulon (RTN) proteins are a family of proteins biochemically identified for shaping tubular endoplasmic reticulum, a subcellular structure important for vesicular transport and cell-to-cell communication. In our recent study of mice with knockout of both reticulon 1 (Rtn1) and Rtn3, we discovered that Rtn1-/-;Rtn3-/- (brief as R1R3dKO) mice exhibited neonatal lethality, despite the fact that mice deficient in either RTN1 or RTN3 alone exhibit no discernible phenotypes. This has been the first case to find early lethality in animals with deletion of partial members of RTN proteins. The complete penetrance for neonatal lethality can be attributed to multiple defects including the impaired neuromuscular junction found in the diaphragm. We also observed significantly impaired axonal growth in a regional-specific manner, detected by immunohistochemical staining with antibodies to neurofilament light chain and neurofilament medium chain. Ultrastructural examination by electron microscopy revealed a significant reduction in synaptic active zone length in the hippocampus. Mechanistic exploration by unbiased proteomic assays revealed reduction of proteins such as FMR1, Staufen2, Cyfip1, Cullin-4B and PDE2a, which are known components in the fragile X mental retardation pathway. Together, our results reveal that RTN1 and RTN3 are required to orchestrate neurofilament organization and intact synaptic structure of the central nervous system.

Human TrkAR649W mutation impairs nociception, sweating and cognitive abilities: a mouse model of HSAN IV.

A functional nerve growth factor NGF-Tropomyosin Receptor kinase A (TrkA) system is an essential requisite for the generation and maintenance of long-lasting thermal and mechanical hyperalgesia in adult mammals. Indeed, mutations in the gene encoding for TrkA are responsible for a rare condition, named Hereditary Sensory and Autonomic Neuropathy type IV (HSAN IV), characterized by the loss of response to noxious stimuli, anhidrosis and cognitive impairment. However, to date, there is no available mouse model to properly understand how the NGF-TrkA system can lead to pathological phenotypes that are distinctive of HSAN IV. Here, we report the generation of a knock-in mouse line carrying the HSAN IV TrkAR649W mutation. First, by in vitro biochemical and biophysical analyses, we show that the pathological R649W mutation leads to kinase-inactive TrkA also affecting its membrane dynamics and trafficking. In agreement with the HSAN IV human phenotype, TrkAR649W/m mice display a lower response to thermal and chemical noxious stimuli, correlating with reduced skin innervation, in addition to decreased sweating in comparison to TrkAh/m controls. Moreover, the R649W mutation decreases anxiety-like behavior and compromises cognitive abilities, by impairing spatial-working and social memory. Our results further uncover unexplored roles of TrkA in thermoregulation and sociability. In addition to accurately recapitulating the clinical manifestations of HSAN IV patients, our findings contribute to clarifying the involvement of the NGF-TrkA system in pain sensation.

The long noncoding RNA, Jpx, is a molecular switch for X chromosome inactivation.

Once protein-coding, the X-inactivation center (Xic) is now dominated by large noncoding RNAs (ncRNA). X chromosome inactivation (XCI) equalizes gene expression between mammalian males and females by inactivating one X in female cells. XCI requires Xist, an ncRNA that coats the X and recruits Polycomb proteins. How Xist is controlled remains unclear but likely involves negative and positive regulators. For the active X, the antisense Tsix RNA is an established Xist repressor. For the inactive X, here, we identify Xic-encoded Jpx as an Xist activator. Jpx is developmentally regulated and accumulates during XCI. Deleting Jpx blocks XCI and is female lethal. Posttranscriptional Jpx knockdown recapitulates the knockout, and supplying Jpx in trans rescues lethality. Thus, Jpx is trans-acting and functions as ncRNA. Furthermore, ΔJpx is rescued by truncating Tsix, indicating an antagonistic relationship between the ncRNAs. We conclude that Xist is controlled by two RNA-based switches: Tsix for Xa and Jpx for Xi.

Mitochondrial fusion is required for mtDNA stability in skeletal muscle and tolerance of mtDNA mutations.

Mitochondria are highly mobile and dynamic organelles that continually fuse and divide. These processes allow mitochondria to exchange contents, including mitochondrial DNA (mtDNA). Here we examine the functions of mitochondrial fusion in differentiated skeletal muscle through conditional deletion of the mitofusins Mfn1 and Mfn2, mitochondrial GTPases essential for fusion. Loss of the mitofusins causes severe mitochondrial dysfunction, compensatory mitochondrial proliferation, and muscle atrophy. Mutant mice have severe mtDNA depletion in muscle that precedes physiological abnormalities. Moreover, the mitochondrial genomes of the mutant muscle rapidly accumulate point mutations and deletions. In a related experiment, we find that disruption of mitochondrial fusion strongly increases mitochondrial dysfunction and lethality in a mouse model with high levels of mtDNA mutations. With its dual function in safeguarding mtDNA integrity and preserving mtDNA function in the face of mutations, mitochondrial fusion is likely to be a protective factor in human disorders associated with mtDNA mutations.

The Role of N-α-acetyltransferase 10 Protein in DNA Methylation and Genomic Imprinting.

Genomic imprinting is an allelic gene expression phenomenon primarily controlled by allele-specific DNA methylation at the imprinting control region (ICR), but the underlying mechanism remains largely unclear. N-α-acetyltransferase 10 protein (Naa10p) catalyzes N-α-acetylation of nascent proteins, and mutation of human Naa10p is linked to severe developmental delays. Here we report that Naa10-null mice display partial embryonic lethality, growth retardation, brain disorders, and maternal effect lethality, phenotypes commonly observed in defective genomic imprinting. Genome-wide analyses further revealed global DNA hypomethylation and enriched dysregulation of imprinted genes in Naa10p-knockout embryos and embryonic stem cells. Mechanistically, Naa10p facilitates binding of DNA methyltransferase 1 (Dnmt1) to DNA substrates, including the ICRs of the imprinted allele during S phase. Moreover, the lethal Ogden syndrome-associated mutation of human Naa10p disrupts its binding to the ICR of H19 and Dnmt1 recruitment. Our study thus links Naa10p mutation-associated Ogden syndrome to defective DNA methylation and genomic imprinting.