A genome-wide screen identifies silencers with distinct chromatin properties and mechanisms of repression.

Differential gene transcription enables development and homeostasis in all animals and is regulated by two major classes of distal cis-regulatory DNA elements (CREs): enhancers and silencers. Although enhancers have been thoroughly characterized, the properties and mechanisms of silencers remain largely unknown. By an unbiased genome-wide functional screen in Drosophila melanogaster S2 cells, we discover a class of silencers that bind one of three transcription factors (TFs) and are generally not included in chromatin-defined CRE catalogs as they mostly lack detectable DNA accessibility.

A conserved fertilization complex bridges sperm and egg in vertebrates.

Fertilization, the basis for sexual reproduction, culminates in the binding and fusion of sperm and egg. Although several proteins are known to be crucial for this process in vertebrates, the molecular mechanisms remain poorly understood. Using an AlphaFold-Multimer screen, we identified the protein Tmem81 as part of a conserved trimeric sperm complex with the essential fertilization factors Izumo1 and Spaca6.

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 PIWI-interacting RNA (piRNA) pathway, is largely defined by a DNA sequence-specific CH zinc finger protein named Kipferl (Baumgartner et al.

Sororin is an evolutionary conserved antagonist of WAPL.

Cohesin mediates sister chromatid cohesion to enable chromosome segregation and DNA damage repair. To perform these functions, cohesin needs to be protected from WAPL, which otherwise releases cohesin from DNA. It has been proposed that cohesin is protected from WAPL by SORORIN.

Cohesin mediates DNA loop extrusion and sister chromatid cohesion by distinct mechanisms.

Cohesin connects CTCF-binding sites and other genomic loci in cis to form chromatin loops and replicated DNA molecules in trans to mediate sister chromatid cohesion. Whether cohesin uses distinct or related mechanisms to perform these functions is unknown. Here, we describe a cohesin hinge mutant that can extrude DNA into loops but is unable to mediate cohesion in human cells.

Cryo-EM structure of the chain-elongating E3 ubiquitin ligase UBR5.

UBR5 is a nuclear E3 ligase that ubiquitinates a vast range of substrates for proteasomal degradation. This HECT domain-containing ubiquitin ligase has recently been identified as an important regulator of oncogenes, e.g.

A phylogenetic profiling approach identifies novel ciliogenesis genes in Drosophila and C. elegans.

Cilia are cellular projections that perform sensory and motile functions in eukaryotic cells. A defining feature of cilia is that they are evolutionarily ancient, yet not universally conserved. In this study, we have used the resulting presence and absence pattern in the genomes of diverse eukaryotes to identify a set of 386 human genes associated with cilium assembly or motility.

Divergent molecular signatures in fish Bouncer proteins define cross-fertilization boundaries.

Molecular compatibility between gametes is a prerequisite for successful fertilization. As long as a sperm and egg can recognize and bind each other via their surface proteins, gamete fusion may occur even between members of separate species, resulting in hybrids that can impact speciation. The egg membrane protein Bouncer confers species specificity to gamete interactions between medaka and zebrafish, preventing their cross-fertilization.

SPOC domain proteins in health and disease.

Since it was first described >20 yr ago, the SPOC domain (Spen paralog and ortholog C-terminal domain) has been identified in many proteins all across eukaryotic species. SPOC-containing proteins regulate gene expression on various levels ranging from transcription to RNA processing, modification, export, and stability, as well as X-chromosome inactivation. Their manifold roles in controlling transcriptional output implicate them in a plethora of developmental processes, and their misregulation is often associated with cancer.

Systematic identification and characterization of repressive domains in Drosophila transcription factors.

All multicellular life relies on differential gene expression, determined by regulatory DNA elements and DNA-binding transcription factors that mediate activation and repression via cofactor recruitment. While activators have been extensively characterized, repressors are less well studied: the identities and properties of their repressive domains (RDs) are typically unknown and the specific co-repressors (CoRs) they recruit have not been determined. Here, we develop a high-throughput, next-generation sequencing-based screening method, repressive-domain (RD)-seq, to systematically identify RDs in complex DNA-fragment libraries.

Homologous chromosomes are stably conjoined for Drosophila male meiosis I by SUM, a multimerized protein assembly with modules for DNA-binding and for separase-mediated dissociation co-opted from cohesin.

For meiosis I, homologous chromosomes must be paired into bivalents. Maintenance of homolog conjunction in bivalents until anaphase I depends on crossovers in canonical meiosis. However, instead of crossovers, an alternative system achieves homolog conjunction during the achiasmate male meiosis of Drosophila melanogaster.

Human Papillomavirus 42 Drives Digital Papillary Adenocarcinoma and Elicits a Germ Cell-like Program Conserved in HPV-Positive Cancers.

Unlabelled: The skin is exposed to viral pathogens, but whether they contribute to the oncogenesis of skin cancers has not been systematically explored. Here we investigated 19 skin tumor types by analyzing off-target reads from commonly available next-generation sequencing data for viral pathogens. We identified human papillomavirus 42 (HPV42) in 96% (n = 45/47) of digital papillary adenocarcinoma (DPA), an aggressive cancer occurring on the fingers and toes.

Lysosomal enzyme trafficking factor LYSET enables nutritional usage of extracellular proteins.

Mammalian cells can generate amino acids through macropinocytosis and lysosomal breakdown of extracellular proteins, which is exploited by cancer cells to grow in nutrient-poor tumors. Through genetic screens in defined nutrient conditions, we characterized LYSET, a transmembrane protein (TMEM251) selectively required when cells consume extracellular proteins. LYSET was found to associate in the Golgi with GlcNAc-1-phosphotransferase, which targets catabolic enzymes to lysosomes through mannose-6-phosphate modification.

Cryo-EM structure of the plant 26S proteasome.

Targeted proteolysis is a hallmark of life. It is especially important in long-lived cells that can be found in higher eukaryotes, like plants. This task is mainly fulfilled by the ubiquitin-proteasome system.

Cornelia de Lange syndrome mutations in NIPBL can impair cohesin-mediated DNA loop extrusion.

Cornelia de Lange syndrome (CdLS) is a developmental multisystem disorder frequently associated with mutations in NIPBL. CdLS is thought to arise from developmental gene regulation defects, but how NIPBL mutations cause these is unknown. Here we show that several NIPBL mutations impair the DNA loop extrusion activity of cohesin.

The Sperm Protein Spaca6 is Essential for Fertilization in Zebrafish.

Fertilization is a key process in all sexually reproducing species, yet the molecular mechanisms that underlie this event remain unclear. To date, only a few proteins have been shown to be essential for sperm-egg binding and fusion in mice, and only some are conserved across vertebrates. One of these conserved, testis-expressed factors is SPACA6, yet its function has not been investigated outside of mammals.

AKIRIN2 controls the nuclear import of proteasomes in vertebrates.

Protein expression and turnover are controlled through a complex interplay of transcriptional, post-transcriptional and post-translational mechanisms to enable spatial and temporal regulation of cellular processes. To systematically elucidate such gene regulatory networks, we developed a CRISPR screening assay based on time-controlled Cas9 mutagenesis, intracellular immunostaining and fluorescence-activated cell sorting that enables the identification of regulatory factors independent of their effects on cellular fitness. We pioneered this approach by systematically probing the regulation of the transcription factor MYC, a master regulator of cell growth.

HUWE1 employs a giant substrate-binding ring to feed and regulate its HECT E3 domain.

HUWE1 is a universal quality-control E3 ligase that marks diverse client proteins for proteasomal degradation. Although the giant HECT enzyme is an essential component of the ubiquitin-proteasome system closely linked with severe human diseases, its molecular mechanism is little understood. Here, we present the crystal structure of Nematocida HUWE1, revealing how a single E3 enzyme has specificity for a multitude of unrelated substrates.

Caenorhabditis elegans RMI2 functional homolog-2 (RMIF-2) and RMI1 (RMH-1) have both overlapping and distinct meiotic functions within the BTR complex.

Homologous recombination is a high-fidelity repair pathway for DNA double-strand breaks employed during both mitotic and meiotic cell divisions. Such repair can lead to genetic exchange, originating from crossover (CO) generation. In mitosis, COs are suppressed to prevent sister chromatid exchange.

The oxidoreductase PYROXD1 uses NAD(P) as an antioxidant to sustain tRNA ligase activity in pre-tRNA splicing and unfolded protein response.

The tRNA ligase complex (tRNA-LC) splices precursor tRNAs (pre-tRNA), and Xbp1-mRNA during the unfolded protein response (UPR). In aerobic conditions, a cysteine residue bound to two metal ions in its ancient, catalytic subunit RTCB could make the tRNA-LC susceptible to oxidative inactivation. Here, we confirm this hypothesis and reveal a co-evolutionary association between the tRNA-LC and PYROXD1, a conserved and essential oxidoreductase.

Zebrafish Ski7 tunes RNA levels during the oocyte-to-embryo transition.

Post-transcriptional regulation of gene expression is crucial during the oocyte-to-embryo transition, a highly dynamic process characterized by the absence of nuclear transcription. Thus, changes to the RNA content are solely dependent on RNA degradation. Although several mechanisms that promote RNA decay during embryogenesis have been identified, it remains unclear which machineries contribute to remodeling the maternal transcriptome.

Site-specific ubiquitination of the E3 ligase HOIP regulates apoptosis and immune signaling.

HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), is a critical regulator of inflammation. However, how HOIP itself is regulated to control inflammatory responses is unclear. Here, we discover that site-specific ubiquitination of K784 within human HOIP promotes tumor necrosis factor (TNF)-induced inflammatory signaling.

LSD1 inhibition induces differentiation and cell death in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a highly aggressive, neuroendocrine skin cancer that lacks actionable mutations, which could be utilized for targeted therapies. Epigenetic regulators governing cell identity may represent unexplored therapeutic entry points. Here, we targeted epigenetic regulators in a pharmacological screen and discovered that the lysine-specific histone demethylase 1A (LSD1/KDM1A) is required for MCC growth in vitro and in vivo.

ANGEL2 is a member of the CCR4 family of deadenylases with 2',3'-cyclic phosphatase activity.

RNA molecules are frequently modified with a terminal 2',3'-cyclic phosphate group as a result of endonuclease cleavage, exonuclease trimming, or de novo synthesis. During pre-transfer RNA (tRNA) and unconventional messenger RNA (mRNA) splicing, 2',3'-cyclic phosphates are substrates of the tRNA ligase complex, and their removal is critical for recycling of tRNAs upon ribosome stalling. We identified the predicted deadenylase angel homolog 2 (ANGEL2) as a human phosphatase that converts 2',3'-cyclic phosphates into 2',3'-OH nucleotides.

Moyamoya disease factor RNF213 is a giant E3 ligase with a dynein-like core and a distinct ubiquitin-transfer mechanism.

RNF213 is the major susceptibility factor for Moyamoya disease, a progressive cerebrovascular disorder that often leads to brain stroke in adults and children. Characterization of disease-associated mutations has been complicated by the enormous size of RNF213. Here, we present the cryo-EM structure of mouse RNF213.