Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

Large-scale genetic perturbations reveal regulatory networks and an abundance of gene-specific repressors.

To understand regulatory systems, it would be useful to uniformly determine how different components contribute to the expression of all other genes. We therefore monitored mRNA expression genome-wide, for individual deletions of one-quarter of yeast genes, focusing on (putative) regulators. The resulting genetic perturbation signatures reflect many different properties. These include the architecture of protein complexes and pathways, identification of expression changes compatible with viability, and the varying responsiveness to genetic perturbation. The data are assembled into a genetic perturbation network that shows different connectivities for different classes of regulators. Four feed-forward loop (FFL) types are overrepresented, including incoherent type 2 FFLs that likely represent feedback. Systematic transcription factor classification shows a surprisingly high abundance of gene-specific repressors, suggesting that yeast chromatin is not as generally restrictive to transcription as is often assumed. The data set is useful for studying individual genes and for discovering properties of an entire regulatory system.

Single-cell atlas reveals meningeal leukocyte heterogeneity in the developing mouse brain.

The meninges are important for brain development and pathology. Using single-cell RNA sequencing, we have generated the first comprehensive transcriptional atlas of neonatal mouse meningeal leukocytes under normal conditions and after perinatal brain injury. We identified almost all known leukocyte subtypes and found differences between neonatal and adult border-associated macrophages, thus highlighting that neonatal border-associated macrophages are functionally immature with regards to immune responses compared with their adult counterparts. We also identified novel meningeal microglia-like cell populations that may participate in white matter development. Early after the hypoxic-ischemic insult, neutrophil numbers increased and they exhibited increased granulopoiesis, suggesting that the meninges are an important site of immune cell expansion with implications for the initiation of inflammatory cascades after neonatal brain injury. Our study provides a single-cell resolution view of the importance of meningeal leukocytes at the early stage of development in health and disease.

From structure to systems: high-resolution, quantitative genetic analysis of RNA polymerase II.

RNA polymerase II (RNAPII) lies at the core of dynamic control of gene expression. Using 53 RNAPII point mutants, we generated a point mutant epistatic miniarray profile (pE-MAP) comprising ∼60,000 quantitative genetic interactions in Saccharomyces cerevisiae. This analysis enabled functional assignment of RNAPII subdomains and uncovered connections between individual regions and other protein complexes. Using splicing microarrays and mutants that alter elongation rates in vitro, we found an inverse relationship between RNAPII speed and in vivo splicing efficiency. Furthermore, the pE-MAP classified fast and slow mutants that favor upstream and downstream start site selection, respectively. The striking coordination of polymerization rate with transcription initiation and splicing suggests that transcription rate is tuned to regulate multiple gene expression steps. The pE-MAP approach provides a powerful strategy to understand other multifunctional machines at amino acid resolution.

Sequence-Directed Action of RSC Remodeler and General Regulatory Factors Modulates +1 Nucleosome Position to Facilitate Transcription.

Accessible chromatin is important for RNA polymerase II recruitment and transcription initiation at eukaryotic promoters. We investigated the mechanistic links between promoter DNA sequence, nucleosome positioning, and transcription. Our results indicate that positioning of the transcription start site-associated +1 nucleosome in yeast is critical for efficient TBP binding and is driven by two key factors, the essential chromatin remodeler RSC and a small set of ubiquitous general regulatory factors (GRFs). Our findings indicate that the strength and directionality of RSC action on promoter nucleosomes depends on the arrangement and proximity of two specific DNA motifs. This, together with the effect on nucleosome position observed in double depletion experiments, suggests that, despite their widespread co-localization, RSC and GRFs predominantly act through independent signals to generate accessible chromatin. Our results provide mechanistic insight into how the promoter DNA sequence instructs trans-acting factors to control nucleosome architecture and stimulate transcription initiation.

Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast.

Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes, including yeast, and has been assigned multiple functions in addition to acting as building block of the lipid bilayer. Here, by isolating S. cerevisiae suppressor mutants that exhibit robust growth in the absence of PC, we show that PC essentiality is subject to cellular evolvability in yeast. The requirement for PC is suppressed by monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl-CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis in different ways, both decrease Acc1 activity, thereby reducing average acyl chain length. Consistently, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, feedback inhibition of Acc1 through acyl-CoA produced by fatty acid synthase (FAS) results from upregulation of lipid synthesis. The results show that budding yeast regulates acyl chain length by fine-tuning the activities of Acc1 and FAS and indicate that PC evolved by benefitting the maintenance of membrane fluidity.

Functional overlap and regulatory links shape genetic interactions between signaling pathways.

To understand relationships between phosphorylation-based signaling pathways, we analyzed 150 deletion mutants of protein kinases and phosphatases in S. cerevisiae using DNA microarrays. Downstream changes in gene expression were treated as a phenotypic readout. Double mutants with synthetic genetic interactions were included to investigate genetic buffering relationships such as redundancy. Three types of genetic buffering relationships are identified: mixed epistasis, complete redundancy, and quantitative redundancy. In mixed epistasis, the most common buffering relationship, different gene sets respond in different epistatic ways. Mixed epistasis arises from pairs of regulators that have only partial overlap in function and that are coupled by additional regulatory links such as repression of one by the other. Such regulatory modules confer the ability to control different combinations of processes depending on condition or context. These properties likely contribute to the evolutionary maintenance of paralogs and indicate a way in which signaling pathways connect for multiprocess control.

Functional organization of the S. cerevisiae phosphorylation network.

Reversible protein phosphorylation is a signaling mechanism involved in all cellular processes. To create a systems view of the signaling apparatus in budding yeast, we generated an epistatic miniarray profile (E-MAP) comprised of 100,000 pairwise, quantitative genetic interactions, including virtually all protein and small-molecule kinases and phosphatases as well as key cellular regulators. Quantitative genetic interaction mapping reveals factors working in compensatory pathways (negative genetic interactions) or those operating in linear pathways (positive genetic interactions). We found an enrichment of positive genetic interactions between kinases, phosphatases, and their substrates. In addition, we assembled a higher-order map from sets of three genes that display strong interactions with one another: triplets enriched for functional connectivity. The resulting network view provides insights into signaling pathway regulation and reveals a link between the cell-cycle kinase, Cak1, the Fus3 MAP kinase, and a pathway that regulates chromatin integrity during transcription by RNA polymerase II.

Single-cell atlas of developing murine adrenal gland reveals relation of Schwann cell precursor signature to neuroblastoma phenotype.

Neuroblastoma is the most common extracranial solid tumor and accounts for ∼10% of pediatric cancer-related deaths. The exact cell of origin has yet to be elucidated, but it is generally accepted that neuroblastoma derives from the neural crest and should thus be considered an embryonal malignancy. About 50% of primary neuroblastoma tumors arise in the adrenal gland. Here, we present an atlas of the developing mouse adrenal gland at a single-cell level. Five main cell cluster groups (medulla, cortex, endothelial, stroma, and immune) make up the mouse adrenal gland during fetal development. The medulla group, which is of neural crest origin, is further divided into seven clusters. Of interest is the Schwann cell precursor ("SCP") and the "neuroblast" cluster, a highly cycling cluster that shares markers with sympathoblasts. The signature of the medullary SCP cluster differentiates neuroblastoma patients based on disease phenotype: The SCP signature score anticorrelates with ALK and MYCN expression, two indicators of poor prognosis. Furthermore, a high SCP signature score is associated with better overall survival rates. This study provides an insight into the developing adrenal gland and introduces the SCP gene signature as being of interest for further research in understanding neuroblastoma phenotype.

Systematic discovery of gene fusions in pediatric cancer by integrating RNA-seq and WGS.

BACKGROUND: Gene fusions are important cancer drivers in pediatric cancer and their accurate detection is essential for diagnosis and treatment. Clinical decision-making requires high confidence and precision of detection. Recent developments show RNA sequencing (RNA-seq) is promising for genome-wide detection of fusion products but hindered by many false positives that require extensive manual curation and impede discovery of pathogenic fusions. METHODS: We developed Fusion-sq to overcome existing disadvantages of detecting gene fusions. Fusion-sq integrates and "fuses" evidence from RNA-seq and whole genome sequencing (WGS) using intron-exon gene structure to identify tumor-specific protein coding gene fusions. Fusion-sq was then applied to the data generated from a pediatric pan-cancer cohort of 128 patients by WGS and RNA sequencing. RESULTS: In a pediatric pan-cancer cohort of 128 patients, we identified 155 high confidence tumor-specific gene fusions and their underlying structural variants (SVs). This includes all clinically relevant fusions known to be present in this cohort (30 patients). Fusion-sq distinguishes healthy-occurring from tumor-specific fusions and resolves fusions in amplified regions and copy number unstable genomes. A high gene fusion burden is associated with copy number instability. We identified 27 potentially pathogenic fusions involving oncogenes or tumor-suppressor genes characterized by underlying SVs, in some cases leading to expression changes indicative of activating or disruptive effects. CONCLUSIONS: Our results indicate how clinically relevant and potentially pathogenic gene fusions can be identified and their functional effects investigated by combining WGS and RNA-seq. Integrating RNA fusion predictions with underlying SVs advances fusion detection beyond extensive manual filtering. Taken together, we developed a method for identifying candidate gene fusions that is suitable for precision oncology applications. Our method provides multi-omics evidence for assessing the pathogenicity of tumor-specific gene fusions for future clinical decision making.

Poised RNA polymerase II gives pause for thought.

Transcription by RNA polymerase II (Pol II) is thought to be predominantly regulated by recruitment of Pol II to promoters. Recent genome-wide analyses demonstrate that many genes are in fact regulated after recruitment of Pol II, by mechanisms such as pausing of Pol II proximal to promoters.

Suboptimal Global Transcriptional Response Increases the Harmful Effects of Loss-of-Function Mutations.

The fitness impact of loss-of-function mutations is generally assumed to reflect the loss of specific molecular functions associated with the perturbed gene. Here, we propose that rewiring of the transcriptome upon deleterious gene inactivation is frequently nonspecific and mimics stereotypic responses to external environmental change. Consequently, transcriptional response to gene deletion could be suboptimal and incur an extra fitness cost. Analysis of the transcriptomes of ∼1,500 single-gene deletion Saccharomyces cerevisiae strains supported this scenario. First, most transcriptomic changes are not specific to the deleted gene but are rather triggered by perturbations in functionally diverse genes. Second, gene deletions that alter the expression of dosage-sensitive genes are especially harmful. Third, by elevating the expression level of downregulated genes, we could experimentally mitigate the fitness defect of gene deletions. Our work shows that rewiring of genomic expression upon gene inactivation shapes the harmful effects of mutations.

CHETAH: a selective, hierarchical cell type identification method for single-cell RNA sequencing.

Cell type identification is essential for single-cell RNA sequencing (scRNA-seq) studies, currently transforming the life sciences. CHETAH (CHaracterization of cEll Types Aided by Hierarchical classification) is an accurate cell type identification algorithm that is rapid and selective, including the possibility of intermediate or unassigned categories. Evidence for assignment is based on a classification tree of previously available scRNA-seq reference data and includes a confidence score based on the variance in gene expression per cell type. For cell types represented in the reference data, CHETAH's accuracy is as good as existing methods. Its specificity is superior when cells of an unknown type are encountered, such as malignant cells in tumor samples which it pinpoints as intermediate or unassigned. Although designed for tumor samples in particular, the use of unassigned and intermediate types is also valuable in other exploratory studies. This is exemplified in pancreas datasets where CHETAH highlights cell populations not well represented in the reference dataset, including cells with profiles that lie on a continuum between that of acinar and ductal cell types. Having the possibility of unassigned and intermediate cell types is pivotal for preventing misclassification and can yield important biological information for previously unexplored tissues.

Regulation of Skn7-dependent, oxidative stress-induced genes by the RNA polymerase II-CTD phosphatase, Fcp1, and Mediator kinase subunit, Cdk8, in yeast.

Fcp1 is a protein phosphatase that facilitates transcription elongation and termination by dephosphorylating the C-terminal domain of RNA polymerase II. High-throughput genetic screening and gene expression profiling of fcp1 mutants revealed a novel connection to Cdk8, the Mediator complex kinase subunit, and Skn7, a key transcription factor in the oxidative stress response pathway. Briefly, Skn7 was enriched as a regulator of genes whose mRNA levels were altered in fcp1 and cdk8Δ mutants and was required for the suppression of fcp1 mutant growth defects by loss of CDK8 under oxidative stress conditions. Targeted analysis revealed that mutating FCP1 decreased Skn7 mRNA and protein levels as well as its association with target gene promoters but paradoxically increased the mRNA levels of Skn7-dependent oxidative stress-induced genes (TRX2 and TSA1) under basal and induced conditions. The latter was in part recapitulated via chemical inhibition of transcription in WT cells, suggesting that a combination of transcriptional and posttranscriptional effects underscored the increased mRNA levels of TRX2 and TSA1 observed in the fcp1 mutant. Interestingly, loss of CDK8 robustly normalized the mRNA levels of Skn7-dependent genes in the fcp1 mutant background and also increased Skn7 protein levels by preventing its turnover. As such, our work suggested that loss of CDK8 could overcome transcriptional and/or posttranscriptional alterations in the fcp1 mutant through its regulatory effect on Skn7. Furthermore, our work also implicated FCP1 and CDK8 in the broader response to environmental stressors in yeast.

The ability of transcription factors to differentially regulate gene expression is a crucial component of the mechanism underlying inversion, a frequently observed genetic interaction pattern.

Genetic interactions, a phenomenon whereby combinations of mutations lead to unexpected effects, reflect how cellular processes are wired and play an important role in complex genetic diseases. Understanding the molecular basis of genetic interactions is crucial for deciphering pathway organization as well as understanding the relationship between genetic variation and disease. Several hypothetical molecular mechanisms have been linked to different genetic interaction types. However, differences in genetic interaction patterns and their underlying mechanisms have not yet been compared systematically between different functional gene classes. Here, differences in the occurrence and types of genetic interactions are compared for two classes, gene-specific transcription factors (GSTFs) and signaling genes (kinases and phosphatases). Genome-wide gene expression data for 63 single and double deletion mutants in baker's yeast reveals that the two most common genetic interaction patterns are buffering and inversion. Buffering is typically associated with redundancy and is well understood. In inversion, genes show opposite behavior in the double mutant compared to the corresponding single mutants. The underlying mechanism is poorly understood. Although both classes show buffering and inversion patterns, the prevalence of inversion is much stronger in GSTFs. To decipher potential mechanisms, a Petri Net modeling approach was employed, where genes are represented as nodes and relationships between genes as edges. This allowed over 9 million possible three and four node models to be exhaustively enumerated. The models show that a quantitative difference in interaction strength is a strict requirement for obtaining inversion. In addition, this difference is frequently accompanied with a second gene that shows buffering. Taken together, these results provide a mechanistic explanation for inversion. Furthermore, the ability of transcription factors to differentially regulate expression of their targets provides a likely explanation why inversion is more prevalent for GSTFs compared to kinases and phosphatases.

H2B ubiquitylation controls the formation of export-competent mRNP.

Histone H2B ubiquitylation is a transcription-dependent modification that not only regulates nucleosome dynamics but also controls the trimethylation of histone H3 on lysine 4 by promoting ubiquitylation of Swd2, a component of both the histone methyltransferase COMPASS complex and the cleavage and polyadenylation factor(CPF). We show that preventing either H2B ubiquitylation or H2B-dependent modification of Swd2 results in nuclear accumulation of poly(A) RNA due to a defect in the integrity and stability of APT, a subcomplex of the CPF. Ubiquitin-regulated APT complex dynamics is required for the correct recruitment of the mRNA export receptor Mex67 to nuclear mRNPs. While H2B ubiquitylation controls the recruitment of the different Mex67 adaptors to mRNPs, the effect of Swd2 ubiquitylation is restricted to Yra1 and Nab2, which, in turn, controls poly(A) tail length. Modification of H2B thus participates in the crosstalk between cotranscriptional events and assembly of mRNPs linking nuclear processing and mRNA export.

Dot1 promotes H2B ubiquitination by a methyltransferase-independent mechanism.

The histone methyltransferase Dot1 is conserved from yeast to human and methylates lysine 79 of histone H3 (H3K79) on the core of the nucleosome. H3K79 methylation by Dot1 affects gene expression and the response to DNA damage, and is enhanced by monoubiquitination of the C-terminus of histone H2B (H2Bub1). To gain more insight into the functions of Dot1, we generated genetic interaction maps of increased-dosage alleles of DOT1. We identified a functional relationship between increased Dot1 dosage and loss of the DUB module of the SAGA co-activator complex, which deubiquitinates H2Bub1 and thereby negatively regulates H3K79 methylation. Increased Dot1 dosage was found to promote H2Bub1 in a dose-dependent manner and this was exacerbated by the loss of SAGA-DUB activity, which also caused a negative genetic interaction. The stimulatory effect on H2B ubiquitination was mediated by the N-terminus of Dot1, independent of methyltransferase activity. Our findings show that Dot1 and H2Bub1 are subject to bi-directional crosstalk and that Dot1 possesses chromatin regulatory functions that are independent of its methyltransferase activity.

EBV MicroRNA BART16 Suppresses Type I IFN Signaling.

Type I IFNs play critical roles in orchestrating the antiviral defense by inducing direct antiviral activities and shaping the adaptive immune response. Viruses have evolved numerous strategies to specifically interfere with IFN production or its downstream mediators, thereby allowing successful infection of the host to occur. The prototypic human gammaherpesvirus EBV, which is associated with infectious mononucleosis and malignant tumors, harbors many immune-evasion proteins that manipulate the adaptive and innate immune systems. In addition to proteins, the virus encodes >40 mature microRNAs for which the functions remain largely unknown. In this article, we identify EBV-encoded miR-BART16 as a novel viral immune-evasion factor that interferes with the type I IFN signaling pathway. miR-BART16 directly targets CREB-binding protein, a key transcriptional coactivator in IFN signaling, thereby inducing CREB-binding protein downregulation in EBV-transformed B cells and gastric carcinoma cells. miR-BART16 abrogates the production of IFN-stimulated genes in response to IFN-α stimulation and it inhibits the antiproliferative effect of IFN-α on latently infected BL cells. By obstructing the type I IFN-induced antiviral response, miR-BART16 provides a means to facilitate the establishment of latent EBV infection and enhance viral replication.

A central role for TFIID in the pluripotent transcription circuitry.

Embryonic stem (ES) cells are pluripotent and characterized by open chromatin and high transcription levels, achieved through auto-regulatory and feed-forward transcription factor loops. ES-cell identity is maintained by a core of factors including Oct4 (also known as Pou5f1), Sox2, Klf4, c-Myc (OSKM) and Nanog, and forced expression of the OSKM factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs) resembling ES cells. These gene-specific factors for RNA-polymerase-II-mediated transcription recruit transcriptional cofactors and chromatin regulators that control access to and activity of the basal transcription machinery on gene promoters. How the basal transcription machinery is involved in setting and maintaining the pluripotent state is unclear. Here we show that knockdown of the transcription factor IID (TFIID) complex affects the pluripotent circuitry in mouse ES cells and inhibits reprogramming of fibroblasts. TFIID subunits and the OSKM factors form a feed-forward loop to induce and maintain a stable transcription state. Notably, transient expression of TFIID subunits greatly enhanced reprogramming. These results show that TFIID is critical for transcription-factor-mediated reprogramming. We anticipate that, by creating plasticity in gene expression programs, transcription complexes such as TFIID assist reprogramming into different cellular states.

The specificity and topology of chromatin interaction pathways in yeast.

Packaging of DNA into chromatin has a profound impact on gene expression. To understand how changes in chromatin influence transcription, we analyzed 165 mutants of chromatin machinery components in Saccharomyces cerevisiae. mRNA expression patterns change in 80% of mutants, always with specific effects, even for loss of widespread histone marks. The data are assembled into a network of chromatin interaction pathways. The network is function based, has a branched, interconnected topology, and lacks strict one-to-one relationships between complexes. Chromatin pathways are not separate entities for different gene sets, but share many components. The study evaluates which interactions are important for which genes and predicts additional interactions, for example between Paf1C and Set3C, as well as a role for Mediator in subtelomeric silencing. The results indicate the presence of gene-dependent effects that go beyond context-dependent binding of chromatin factors and provide a framework for understanding how specificity is achieved through regulating chromatin.