Charles R. Bradshaw

Systems survey of endocytosis by multiparametric image analysis

Endocytosis is a complex process fulfilling many cellular and developmental functions. Understanding how it is regulated and integrated with other cellular processes requires a comprehensive analysis of its molecular constituents and general design principles. Here, we developed a new strategy to phenotypically profile the human genome with respect to transferrin (TF) and epidermal growth factor (EGF) endocytosis by combining RNA interference, automated high-resolution confocal microscopy, quantitative multiparametric image analysis and high-performance computing. We identified several novel components of endocytic trafficking, including genes implicated in human diseases. We found that signalling pathways such as Wnt, integrin/cell adhesion, transforming growth factor (TGF)-β and Notch regulate the endocytic system, and identified new genes involved in cargo sorting to a subset of signalling endosomes. A systems analysis by Bayesian networks further showed that the number, size, concentration of cargo and intracellular position of endosomes are not determined randomly but are subject to specific regulation, thus uncovering novel properties of the endocytic system.

Small-molecule–induced DNA damage identifies alternative DNA structures in human genes

Guanine-rich DNA sequences that can adopt non-Watson-Crick structures in vitro are prevalent in the human genome. Whether such structures normally exist in mammalian cells has, however, been the subject of active research for decades. Here, we show that the G-quadruplex interacting drug pyridostatin promoted growth arrest in human cancer cells via inducing replication- and transcription-dependent DNA damage. Chromatin immunoprecipitation sequence (ChIP-Seq) analysis of the DNA damage marker γH2AX provided the genome-wide distribution of pyridostatin-induced sites of damage, and revealed that pyridostatin targets gene bodies containing clusters of sequences with a propensity for G-quadruplex formation. As a result, pyridostatin modulated the expression of these genes, including the proto-oncogene SRC. We observed that pyridostatin reduced SRC protein levels and SRC-dependent cellular motility in human breast cancer cells, validating SRC as a target. Our unbiased approach to define genomic sites of action for a drug establishes a framework for discovering functional DNA-drug interactions.

Familial dementia caused by polymerization of mutant neuroserpin

Aberrant protein processing with tissue deposition is associated with many common neurodegenerative disorders; however, the complex interplay of genetic and environmental factors has made it difficult to decipher the sequence of events linking protein aggregation with clinical disease. Substantial progress has been made toward understanding the pathophysiology of prototypical conformational diseases and protein polymerization in the superfamily of serine proteinase inhibitors (serpins). Here we describe a new disease, familial encephalopathy with neuroserpin inclusion bodies, characterized clinically as an autosomal dominantly inherited dementia, histologically by unique neuronal inclusion bodies and biochemically by polymers of the neuron-specific serpin, neuroserpin. We report the cosegregation of point mutations in the neuroserpin gene (PI12) with the disease in two families. The significance of one mutation, S49P, is evident from its homology to a previously described serpin mutation, whereas that of the other, S52R, is predicted by modelling of the serpin template. Our findings provide a molecular mechanism for a familial dementia and imply that inhibitors of protein polymerization may be effective therapies for this disorder and perhaps for other more common neurodegenerative diseases.

A Unique Gene Regulatory Network Resets the Human Germline Epigenome for Development

Summary Resetting of the epigenome in human primordial germ cells (hPGCs) is critical for development. We show that the transcriptional program of hPGCs is distinct from that in mice, with co-expression of somatic specifiers and naive pluripotency genes TFCP2L1 and KLF4. This unique gene regulatory network, established by SOX17 and BLIMP1, drives comprehensive germline DNA demethylation by repressing DNA methylation pathways and activating TET-mediated hydroxymethylation. Base-resolution methylome analysis reveals progressive DNA demethylation to basal levels in week 5–7 in vivo hPGCs. Concurrently, hPGCs undergo chromatin reorganization, X reactivation, and imprint erasure. Despite global hypomethylation, evolutionarily young and potentially hazardous retroelements, like SVA, remain methylated. Remarkably, some loci associated with metabolic and neurological disorders are also resistant to DNA demethylation, revealing potential for transgenerational epigenetic inheritance that may have phenotypic consequences. We provide comprehensive insight on early human germline transcriptional network and epigenetic reprogramming that subsequently impacts human development and disease.

Replication stress induces 53BP1-containing OPT domains in G1 cells

53BP1-OPT domains, nuclear bodies that arise in G1 cells at sites of DNA damage induced by incomplete DNA replication, preferentially localize to chromosomal common fragile sites.

Titration of four replication factors is essential for the Xenopus laevis midblastula transition.

Regulating the MBT It has been known for more than 30 years that a defined number of cell divisions in the frog embryo precede a crucial developmental event called the midblastula transition (MBT). Collart et al. (p. 893, published online 1 August) now elucidate a mechanism involved in the control of the MBT. DNA replication initiation factors are titrated out during early cell divisions, which controls the elongation of the cell cycle and the onset of zygotic transcription during the MBT. Increasing numbers of nuclei compared with the cytoplasmic volume promotes a key developmental step in frog embryos. The rapid, reductive early divisions of many metazoan embryos are followed by the midblastula transition (MBT), during which the cell cycle elongates and zygotic transcription begins. It has been proposed that the increasing nuclear to cytoplasmic (N/C) ratio is critical for controlling the events of the MBT. We show that four DNA replication factors—Cut5, RecQ4, Treslin, and Drf1—are limiting for replication initiation at increasing N/C ratios in vitro and in vivo in Xenopus laevis. The levels of these factors regulate multiple events of the MBT, including the slowing of the cell cycle, the onset of zygotic transcription, and the developmental activation of the kinase Chk1. This work provides a mechanism for how the N/C ratio controls the MBT and shows that the regulation of replication initiation is fundamental for normal embryogenesis.

Identification of methylated deoxyadenosines in vertebrates reveals diversity in DNA modifications

Methylation of cytosine deoxynucleotides generates 5-methylcytosine (m5dC), a well-established epigenetic mark. However, in higher eukaryotes much less is known about modifications affecting other deoxynucleotides. Here, we report the detection of N6-methyldeoxyadenosine (m6dA) in vertebrate DNA, specifically in Xenopus laevis but also in other species including mouse and human. Our methylome analysis reveals that m6dA is widely distributed across the eukaryotic genome and is present in different cell types but is commonly depleted from gene exons. Thus, direct DNA modifications might be more widespread than previously thought.

Human primary liver cancer–derived organoid cultures for disease modeling and drug screening

Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

Nuclear Wave1 Is Required for Reprogramming Transcription in Oocytes and for Normal Development

Egg WAVE1 Eggs not only activate sperm nuclei for normal development but also reprogram transplanted somatic nuclei. In addition to its well-established cytoplasmic role in actin organization, Miyamoto et al. (p. 1002) discovered that the Wiskott-Aldrich syndrome protein family member 1 in oocytes cooperates with transcriptional machineries in the nucleus to activate previously silenced genes. A cytoskeletal protein associates with the transcription machinery and is required for nuclear reprogramming. Eggs and oocytes have a remarkable ability to induce transcription of sperm after normal fertilization and in somatic nuclei after somatic cell nuclear transfer. This ability of eggs and oocytes is essential for normal development. Nuclear actin and actin-binding proteins have been shown to contribute to transcription, although their mode of action is elusive. Here, we find that Xenopus Wave1, previously characterized as a protein involved in actin cytoskeleton organization, is present in the oocyte nucleus and is required for efficient transcriptional reprogramming. Moreover, Wave1 knockdown in embryos results in abnormal development and defective hox gene activation. Nuclear Wave1 binds by its WHD domain to active transcription components, and this binding contributes to the action of RNA polymerase II. We identify Wave1 as a maternal reprogramming factor that also has a necessary role in gene activation in development.

Conformational Adaptability of Redβ during DNA Annealing and Implications for Its Structural Relationship with Rad52

Single-strand annealing proteins, such as Redbeta from lambda phage or eukaryotic Rad52, play roles in homologous recombination. Here, we use atomic force microscopy to examine Redbeta quaternary structure and Redbeta-DNA complexes. In the absence of DNA, Redbeta forms a shallow right-handed helix. The presence of single-stranded DNA (ssDNA) disrupts this structure. Upon addition of a second complementary ssDNA, annealing generates a left-handed helix that incorporates 14 Redbeta monomers per helical turn, with each Redbeta monomer annealing approximately 11 bp of DNA. The smallest stable annealing intermediate requires 20 bp DNA and two Redbeta monomers. Hence, we propose that Redbeta promotes base pairing by first increasing the number of transient interactions between ssDNAs. Then, annealing is promoted by the binding of a second Redbeta monomer, which nucleates the formation of a stable annealing intermediate. Using threading, we identify sequence similarities between the RecT/Redbeta and the Rad52 families, which strengthens previous suggestions, based on similarities of their quaternary structures, that they share a common mode of action. Hence, our findings have implications for a common mechanism of DNA annealing mediated by single-strand annealing proteins including Rad52.