Knowledge of how proteins interact with DNA is essential for understanding gene regulation. Although DNA-binding specificities for thousands of transcription factors (TFs) have been determined, the specific amino acid-base interactions comprising their structural interfaces are largely unknown. This lack of resolution hampers attempts to leverage these data in order to predict specificities for uncharacterized TFs or TFs mutated in disease. Here we introduce recognition code learning via automated mapping of protein-DNA structural interfaces (rCLAMPS), a probabilistic approach that uses DNA-binding specificities for TFs from the same structural family to simultaneously infer both which nucleotide positions are contacted by particular amino acids within the TF as well as a recognition code that relates each base-contacting amino acid to nucleotide preferences at the DNA positions it contacts. We apply rCLAMPS to homeodomains, the second largest family of TFs in metazoans and show that it learns a highly effective recognition code that can predict de novo DNA-binding specificities for TFs. Furthermore, we show that the inferred amino acid-nucleotide contacts reveal whether and how nucleotide preferences at individual binding site positions are altered by mutations within TFs. Our approach is an important step toward automatically uncovering the determinants of protein-DNA specificity from large compendia of DNA-binding specificities and inferring the altered functionalities of TFs mutated in disease.

The Gram-positive bacterium Listeria monocytogenes occurs widespread in the environment and infects humans when ingested along with contaminated food. Such infections are particularly dangerous for risk group patients, for whom they represent a life-threatening disease. To invent novel strategies to control contamination and disease, it is important to identify those cellular processes that maintain pathogen growth inside and outside the host. Here, we have applied transposon insertion sequencing (Tn-Seq) to L. monocytogenes for the identification of such processes on a genome-wide scale. Our approach identified 394 open reading frames that are required for growth under standard laboratory conditions and 42 further genes, which become necessary during intracellular growth in macrophages. Most of these genes encode components of the translation machinery and act in chromosome-related processes, cell division, and biosynthesis of the cellular envelope. Several cofactor biosynthesis pathways and 29 genes with unknown functions are also required for growth, suggesting novel options for the development of antilisterial drugs. Among the genes specifically required during intracellular growth are known virulence factors, genes compensating intracellular auxotrophies, and several cell division genes. Our experiments also highlight the importance of PASTA kinase signaling for general viability and of glycine metabolism and chromosome segregation for efficient intracellular growth of L. monocytogenes.

Argonaute 2 (AGO2) is a ubiquitously expressed protein critical for regulation of mRNA translation and vital to animal development. AGO2 protein is found in both cytoplasmic and nuclear compartments, and although its cytoplasmic role is well studied, the biological relevance of nuclear AGO2 is unclear. Here, we address this problem in vivo using spermatogenic cells as a model. We find that AGO2 transiently binds both chromatin and nucleus-specific mRNA transcripts of hundreds of genes required for sperm production during male meiosis in mice, and that germline conditional knockout (cKO) of Ago2 causes depletion of the encoded proteins. Correspondingly, Ago2 cKO males show abnormal sperm head morphology and reduced sperm count, along with reduced postnatal viability of offspring. Together, our data reveal an unexpected nuclear role for AGO2 in enhancing expression of developmentally important genes during mammalian male reproduction.

The unicellular amoeba Acanthamoeba castellanii is ubiquitous in aquatic environments, where it preys on bacteria. The organism also hosts bacterial endosymbionts, some of which are parasitic, including human pathogens such as Chlamydia and Legionella spp. Here we report complete, high-quality genome sequences for two extensively studied A. castellanii strains, Neff and C3. Combining long- and short-read data with Hi-C, we generated near chromosome-level assemblies for both strains with 90% of the genome contained in 29 scaffolds for the Neff strain and 31 for the C3 strain. Comparative genomics revealed strain-specific functional enrichment, most notably genes related to signal transduction in the C3 strain and to viral replication in Neff. Furthermore, we characterized the spatial organization of the A. castellanii genome and showed that it is reorganized during infection by Legionella pneumophila Infection-dependent chromatin loops were found to be enriched in genes for signal transduction and phosphorylation processes. In genomic regions where chromatin organization changed during Legionella infection, we found functional enrichment for genes associated with metabolism, organelle assembly, and cytoskeleton organization. Given Legionella infection is known to alter its host's cell cycle, to exploit the host's organelles, and to modulate the host's metabolism in its favor, these changes in chromatin organization may partly be related to mechanisms of host control during Legionella infection.

Epigenetic regulation plays a critical role in many neurodevelopmental disorders (NDDs), including autism spectrum disorder (ASD). In particular, many such disorders are the result of mutations in genes that encode chromatin-modifying proteins. However, although these disorders share many features, it is unclear whether they also share gene expression disruptions resulting from the aberrant regulation of chromatin. We examined five chromatin modifiers that are all linked to ASD despite their different roles in regulating chromatin. Specifically, we depleted ASH1L, CHD8, CREBBP, EHMT1, and NSD1 in parallel in a highly controlled neuronal culture system. We then identified sets of shared genes, or transcriptional signatures, that are differentially expressed following loss of multiple ASD-linked chromatin modifiers. We examined the functions of genes within the transcriptional signatures and found an enrichment in many neurotransmitter transport genes and activity-dependent genes. In addition, these genes are enriched for specific chromatin features such as bivalent domains that allow for highly dynamic regulation of gene expression. The down-regulated transcriptional signature is also observed within multiple mouse models of NDDs that result in ASD, but not those only associated with intellectual disability. Finally, the down-regulated transcriptional signature can distinguish between control and idiopathic ASD patient iPSC-derived neurons as well as postmortem tissue, demonstrating that this gene set is relevant to the human disorder. This work identifies a transcriptional signature that is found within many neurodevelopmental syndromes, helping to elucidate the link between epigenetic regulation and the underlying cellular mechanisms that result in ASD.