ANDA: an open-source tool for automated image analysis of in vitro neuronal cells.

BACKGROUND: Imaging of in vitro neuronal differentiation and measurements of cell morphologies have led to novel insights into neuronal development. Live-cell imaging techniques and large datasets of images have increased the demand for automated pipelines for quantitative analysis of neuronal morphological metrics. RESULTS: ANDA is an analysis workflow that quantifies various aspects of neuronal morphology from high-throughput live-cell imaging screens of in vitro neuronal cell types. This tool automates the analysis of neuronal cell numbers, neurite lengths and neurite attachment points. We used chicken, rat, mouse, and human in vitro models for neuronal differentiation and have demonstrated the accuracy, versatility, and efficiency of the tool. CONCLUSIONS: ANDA is an open-source tool that is easy to use and capable of automated processing from time-course measurements of neuronal cells. The strength of this pipeline is the capability to analyse high-throughput imaging screens.

Multi-omics approach reveals dysregulated genes during hESCs neuronal differentiation exposure to paracetamol.

Prenatal paracetamol exposure has been associated with neurodevelopmental outcomes in childhood. Pharmacoepigenetic studies show differences in cord blood DNA methylation between unexposed and paracetamol-exposed neonates, however, causality and impact of long-term prenatal paracetamol exposure on brain development remain unclear. Using a multi-omics approach, we investigated the effects of paracetamol on an in vitro model of early human neurodevelopment. We exposed human embryonic stem cells undergoing neuronal differentiation with paracetamol concentrations corresponding to maternal therapeutic doses. Single-cell RNA-seq and ATAC-seq integration identified paracetamol-induced chromatin opening changes linked to gene expression. Differentially methylated and/or expressed genes were involved in neurotransmission and cell fate determination trajectories. Some genes involved in neuronal injury and development-specific pathways, such as KCNE3, overlapped with differentially methylated genes previously identified in cord blood associated with prenatal paracetamol exposure. Our data suggest that paracetamol may play a causal role in impaired neurodevelopment.

MYB regulates the SUMO protease SENP1 and its novel interaction partner UXT, modulating MYB target genes and the SUMO landscape.

SUMOylation is a post-translational modification frequently found on nuclear proteins, including transcription factors (TFs) and coactivators. By controlling the activity of several TFs, SUMOylation may have far-reaching effects. MYB is an example of a developmental TF subjected to SUMO-mediated regulation, through both SUMO conjugation and SUMO binding. How SUMO affects MYB target genes is unknown. Here, we explored the global effect of reduced SUMOylation of MYB on its downstream gene programs. RNA-Seq in K562 cells after MYB knockdown and rescue with mutants having an altered SUMO status revealed a number of differentially regulated genes and distinct gene ontology term enrichments. Clearly, the SUMO status of MYB both quantitatively and qualitatively affects its regulome. The transcriptome data further revealed that MYB upregulates the SUMO protease SENP1, a key enzyme that removes SUMO conjugation from SUMOylated proteins. Given this role of SENP1 in the MYB regulome, we expanded the analysis, mapped interaction partners of SENP1, and identified UXT as a novel player affecting the SUMO system by acting as a repressor of SENP1. MYB inhibits the expression of UXT suggesting that MYB is able not only to control a specific gene program directly but also indirectly by affecting the SUMO landscape through SENP1 and UXT. These findings suggest an autoactivation loop whereby MYB, through enhancing SENP1 and reducing UXT, is itself being activated by a reduced level of repressive SUMOylation. We propose that overexpressed MYB, seen in multiple cancers, may drive this autoactivation loop and contribute to oncogenic activation of MYB.

ANDA: An open-source tool for automated image analysis of neuronal differentiation.

Background: Imaging of in vitro neuronal differentiation and measurements of cell morphologies has led to novel insights into neuronal development. Live-cell imaging techniques and large datasets of images has increased the demand for automated pipelines for quantitative analysis of neuronal morphological metrics. Results: We present ANDA, an analysis workflow for quantification of various aspects of neuronal morphology from high-throughput live-cell imaging screens. This tool automates the analysis of neuronal cell numbers, neurite lengths and neurite attachment points. We used rat, chicken and human in vitro models for neuronal differentiation and have demonstrated the accuracy, versatility, and efficiency of the tool. Conclusions: ANDA is an open-source tool that is easy to use and capable of automated processing from time-course measurements of neuronal cells. The strength of this pipeline is the capability to analyse high-throughput imaging screens.

A multi-omics approach to visualize early neuronal differentiation from hESCs in 4D.

Neuronal differentiation of pluripotent stem cells is an established method to study physiology, disease, and medication safety. However, the sequence of events in human neuronal differentiation and the ability of in vitro models to recapitulate early brain development are poorly understood. We developed a protocol optimized for the study of early human brain development and neuropharmacological applications. We comprehensively characterized gene expression and epigenetic profiles at four timepoints, because the cells differentiate from embryonic stem cells towards a heterogeneous population of progenitors, immature and mature neurons bearing telencephalic signatures. A multi-omics roadmap of neuronal differentiation, combined with searchable interactive gene analysis tools, allows for extensive exploration of early neuronal development and the effect of medications.

Robust neuronal differentiation of human embryonic stem cells for neurotoxicology.

Here, we describe a protocol for rapid neuronal differentiation from human embryonic stem cells (hESCs) toward a heterogenous population of telencephalic progenitors, immature and mature neurons, for drug-screening and early-brain differentiation studies. hESC neuronal differentiation depends on adhesion and minimal cell-passaging to avert monolayer cross-connectivity rupture. In this protocol, we detail optimized cell-seeding densities and coating conditions with high cell viability suitable for neurotoxicology and high-resolution single-cell omics studies. Daily media changes reduce compound instability and degradation for optimal screening. For complete details on the use and execution of this protocol, please refer to Samara et al. (2022).

Paracetamol perturbs neuronal arborization and disrupts the cytoskeletal proteins SPTBN1 and TUBB3 in both human and chicken in vitro models.

Epidemiological studies have linked long-term/high-dose usage of paracetamol (N-acetyl-para-aminophenol, APAP) during pregnancy to adverse neuropsychiatric outcomes, primarily attention-deficit hyperactive disorder (ADHD), in the offspring. Though variable, ADHD has been associated with phenotypic alterations characterized by reductions in grey matter densities and aberrations in structural connectivity, effects which are thought to originate in neurodevelopment. We used embryonic chicken cerebellar granule neurons (CGNs) and neuronally differentiating human NTERA2 cells (NT2Ns) to investigate the in vitro effects of APAP on cell viability, migration, neuritogenesis, and the intracellular levels of various proteins involved in neurodevelopment as well as in the maintenance of the structure and function of neurites. Exposure to APAP ranging from 100 to 1600 µM yielded concentration- and time-dependent reductions in cell viability and levels of neurite arborization, as well as reductions in the levels of the cytoskeletal protein ß2-spectrin, with the highest APAP concentration resulting in between 50 and 75% reductions in the aforementioned metrics over the course of 72 h. Exposure to APAP also reduced migration in the NT2Ns but not CGNs. Moreover, we found concentration- and time-dependent increases in punctate aggregation of the cytoskeletal protein ß3-tubulin following exposure to APAP in both cell model systems, with the highest APAP concentration approximately doubling the number of aggregates over 72-120 h. Our findings demonstrate that APAP negatively perturbs neurite arborization degree, with concurrent reductions in the protein levels of ß2-spectrin and disruption of the integrity of ß3-tubulin, both proteins of which play important roles in neuronal structure and function.

Pioneer transcription factors are associated with the modulation of DNA methylation patterns across cancers.

Methylation of cytosines on DNA is a prominent modification associated with gene expression regulation. Aberrant DNA methylation patterns have recurrently been linked to dysregulation of the regulatory program in cancer cells. To shed light on the underlying molecular mechanism driving this process, we hypothesised that aberrant methylation patterns could be controlled by the binding of specific transcription factors (TFs) across cancer types. By combining DNA methylation arrays and gene expression data with TF binding sites (TFBSs), we explored the interplay between TF binding and DNA methylation in 19 cancer types. We performed emQTL (expression-methylation quantitative trait loci) analyses independently in each cancer type and identified 13 TFs whose expression levels are correlated with local DNA methylation patterns around their binding sites in at least 2 cancer types. The 13 TFs are mainly associated with local demethylation and are enriched for pioneer function, suggesting a specific role for these TFs in modulating chromatin structure and transcription in cancer patients. Furthermore, we confirmed that de novo methylation is precluded across cancers at CpGs lying in genomic regions enriched for TF binding signatures associated with SP1, CTCF, NRF1, GABPA, KLF9, and/or YY1. The modulation of DNA methylation associated with TF binding was observed at cis-regulatory regions controlling immune- and cancer-associated pathways, corroborating that the emQTL signals were derived from both cancer and tumor-infiltrating cells. As a case example, we experimentally confirmed that FOXA1 knock-down is associated with higher methylation in regions bound by FOXA1 in breast cancer MCF-7 cells. Finally, we reported physical interactions between FOXA1 with TET1 and TET2 both in an in vitro setup and in vivo at physiological levels in MCF-7 cells, adding further support for FOXA1 attracting TET1 and TET2 to induce local demethylation in cancer cells.

ShinyArchR.UiO: user-friendly,integrative and open-source tool for visualization of single-cell ATAC-seq data using ArchR.

MOTIVATION: Mapping of chromatin accessibility landscapes in single-cells and the integration with gene expression enables a better understanding of gene regulatory mechanisms defining cell identities and cell-fate determination in development and disease. Generally, raw data generated from single-cell Assay for Transposase-Accessible Chromatin sequencing (scATAC-seq) are deposited in repositories that are generally inaccessible due to lack of in-depth knowledge of computational programming. RESULTS: We have developed ShinyArchR.UiO, an R-based shiny app, that facilitates scATAC-seq data accessibility and visualization in a user-friendly, interactive and open-source web interface. ShinyArchR.UiO is an application that can streamline collaborative efforts for interpretation of massive chromatin accessibility datasets and allow for open access data sharing for wider audiences. AVAILABILITY AND IMPLEMENTATION: https://Github.com/EskelandLab/ShinyArchRUiO and a demo server with a hematopoietic tutorial dataset https://cancell.medisin.uio.no/ShinyArchR.UiO. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Chromatin occupancy and target genes of the haematopoietic master transcription factor MYB.

The transcription factor MYB is a master regulator in haematopoietic progenitor cells and a pioneer factor affecting differentiation and proliferation of these cells. Leukaemic transformation may be promoted by high MYB levels. Despite much accumulated molecular knowledge of MYB, we still lack a comprehensive understanding of its target genes and its chromatin action. In the present work, we performed a ChIP-seq analysis of MYB in K562 cells accompanied by detailed bioinformatics analyses. We found that MYB occupies both promoters and enhancers. Five clusters (C1-C5) were found when we classified MYB peaks according to epigenetic profiles. C1 was enriched for promoters and C2 dominated by enhancers. C2-linked genes were connected to hematopoietic specific functions and had GATA factor motifs as second in frequency. C1 had in addition to MYB-motifs a significant frequency of ETS-related motifs. Combining ChIP-seq data with RNA-seq data allowed us to identify direct MYB target genes. We also compared ChIP-seq data with digital genomic footprinting. MYB is occupying nearly a third of the super-enhancers in K562. Finally, we concluded that MYB cooperates with a subset of the other highly expressed TFs in this cell line, as expected for a master regulator.

The SUMO protease SENP1 and the chromatin remodeler CHD3 interact and jointly affect chromatin accessibility and gene expression.

The small ubiquitin-like modifier (SUMO) post-translationally modifies lysine residues of transcription factors and co-regulators and thereby contributes to an important layer of control of the activities of these transcriptional regulators. Likewise, deSUMOylation of these factors by the sentrin-specific proteases (SENPs) also plays a role in gene regulation, but whether SENPs functionally interact with other regulatory factors that control gene expression is unclear. In the present work, we focused on SENP1, specifically, on its role in activation of gene expression investigated through analysis of the SENP1 interactome, which revealed that SENP1 physically interacts with the chromatin remodeler chromodomain helicase DNA-binding protein 3 (CHD3). Using several additional methods, including GST pulldown and co-immunoprecipitation assays, we validated and mapped this interaction, and using CRISPR-Cas9-generated CHD3- and SENP1-KO cells (in the haploid HAP1 cell line), we investigated whether these two proteins are functionally linked in regulating chromatin remodeling and gene expression. Genome-wide ATAC-Seq analysis of the CHD3- and SENP1-KO cells revealed a large degree of overlap in differential chromatin openness between these two mutant cell lines. Moreover, motif analysis and comparison with ChIP-Seq profiles in K562 cells pointed to an association of CHD3 and SENP1 with CCCTC-binding factor (CTCF) and SUMOylated chromatin-associated factors. Lastly, genome-wide RNA-Seq also indicated that these two proteins co-regulate the expression of several genes. We propose that the functional link between chromatin remodeling by CHD3 and deSUMOylation by SENP1 uncovered here provides another level of control of gene expression.

The pioneer factor activity of c-Myb involves recruitment of p300 and induction of histone acetylation followed by acetylation-induced chromatin dissociation.

BACKGROUND: The concept of pioneer transcription factors is emerging as an essential part of the epigenetic regulation, taking place during cell development and differentiation. However, the precise molecular mechanism underlying pioneer factor activity remains poorly understood. We recently reported that the transcription factor c-Myb acts as a pioneer factor in haematopoiesis, and a point mutation in its DNA binding domain, D152V, is able to abrogate this function. RESULTS: Here, we show that specific histone modifications, including H3K27ac, prevent binding of c-Myb to histone tails, representing a novel effect of histone modifications, namely restricting binding of a pioneer factor to chromatin. Furthermore, we have taken advantage of the pioneer-defect D152V mutant to investigate mechanisms of c-Myb's pioneer factor activity. We show that c-Myb-dependent transcriptional activation of a gene in inaccessible chromatin relies on c-Myb binding to histones, as well as on c-Myb interacting with the histone acetyltransferase p300. ChIP assays show that both wild type and the D152V mutant of c-Myb bind to a selected target gene at its promoter and enhancer, but only wild-type c-Myb causes opening and activation of the locus. Enhancement of histone acetylation restores activation of the same gene in the absence of c-Myb, suggesting that facilitating histone acetylation is a crucial part of the pioneer factor function of c-Myb. CONCLUSIONS: We suggest a pioneer factor model in which c-Myb binds to regions of closed chromatin and then recruits histone acetyltransferases. By binding to histones, c-Myb facilitates histone acetylation, acting as a cofactor for p300 at c-Myb bound sites. The resulting H3K27ac leads to chromatin opening and detachment of c-Myb from the acetylated chromatin. We propose that the latter phenomenon, acetylation-induced chromatin dissociation, represents a mechanism for controlling the dynamics of pioneer factor binding to chromatin.

DNA methylation at enhancers identifies distinct breast cancer lineages.

Breast cancers exhibit genome-wide aberrant DNA methylation patterns. To investigate how these affect the transcriptome and which changes are linked to transformation or progression, we apply genome-wide expression-methylation quantitative trait loci (emQTL) analysis between DNA methylation and gene expression. On a whole genome scale, in cis and in trans, DNA methylation and gene expression have remarkably and reproducibly conserved patterns of association in three breast cancer cohorts (n = 104, n = 253 and n = 277). The expression-methylation quantitative trait loci associations form two main clusters; one relates to tumor infiltrating immune cell signatures and the other to estrogen receptor signaling. In the estrogen related cluster, using ChromHMM segmentation and transcription factor chromatin immunoprecipitation sequencing data, we identify transcriptional networks regulated in a cell lineage-specific manner by DNA methylation at enhancers. These networks are strongly dominated by ERα, FOXA1 or GATA3 and their targets were functionally validated using knockdown by small interfering RNA or GRO-seq analysis after transcriptional stimulation with estrogen.

Mobilization of LINE-1 retrotransposons is restricted by Tex19.1 in mouse embryonic stem cells.

Mobilization of retrotransposons to new genomic locations is a significant driver of mammalian genome evolution, but these mutagenic events can also cause genetic disorders. In humans, retrotransposon mobilization is mediated primarily by proteins encoded by LINE-1 (L1) retrotransposons, which mobilize in pluripotent cells early in development. Here we show that TEX19.1, which is induced by developmentally programmed DNA hypomethylation, can directly interact with the L1-encoded protein L1-ORF1p, stimulate its polyubiquitylation and degradation, and restrict L1 mobilization. We also show that TEX19.1 likely acts, at least in part, through promoting the activity of the E3 ubiquitin ligase UBR2 towards L1-ORF1p. Moreover, loss of Tex19.1 increases L1-ORF1p levels and L1 mobilization in pluripotent mouse embryonic stem cells, implying that Tex19.1 prevents de novo retrotransposition in the pluripotent phase of the germline cycle. These data show that post-translational regulation of L1 retrotransposons plays a key role in maintaining trans-generational genome stability in mammals.

A c-Myb mutant causes deregulated differentiation due to impaired histone binding and abrogated pioneer factor function.

The transcription factor c-Myb is involved in early differentiation and proliferation of haematopoietic cells, where it operates as a regulator of self-renewal and multi-lineage differentiation. Deregulated c-Myb plays critical roles in leukaemias and other human cancers. Due to its role as a master regulator, we hypothesized it might function as a pioneer transcription factor. Our approach to test this was to analyse a mutant of c-Myb, D152V, previously reported to cause haematopoietic defects in mice by an unknown mechanism. Our transcriptome data from K562 cells indicates that this mutation specifically affects c-Myb's ability to regulate genes involved in differentiation, causing failure in c-Myb's ability to block differentiation. Furthermore, we see a major effect of this mutation in assays where chromatin opening is involved. We show that each repeat in the minimal DNA-binding domain of c-Myb binds to histones and that D152V disrupts histone binding of the third repeat. ATAC-seq data indicates this mutation impairs the ability of c-Myb to cause chromatin opening at specific sites. Taken together, our findings support that c-Myb acts as a pioneer factor and show that D152V impairs this function. The D152V mutant is the first mutant of a transcription factor specifically destroying pioneer factor function.

GSuite HyperBrowser: integrative analysis of dataset collections across the genome and epigenome.

Background: Recent large-scale undertakings such as ENCODE and Roadmap Epigenomics have generated experimental data mapped to the human reference genome (as genomic tracks) representing a variety of functional elements across a large number of cell types. Despite the high potential value of these publicly available data for a broad variety of investigations, little attention has been given to the analytical methodology necessary for their widespread utilisation. Findings: We here present a first principled treatment of the analysis of collections of genomic tracks. We have developed novel computational and statistical methodology to permit comparative and confirmatory analyses across multiple and disparate data sources. We delineate a set of generic questions that are useful across a broad range of investigations and discuss the implications of choosing different statistical measures and null models. Examples include contrasting analyses across different tissues or diseases. The methodology has been implemented in a comprehensive open-source software system, the GSuite HyperBrowser. To make the functionality accessible to biologists, and to facilitate reproducible analysis, we have also developed a web-based interface providing an expertly guided and customizable way of utilizing the methodology. With this system, many novel biological questions can flexibly be posed and rapidly answered. Conclusions: Through a combination of streamlined data acquisition, interoperable representation of dataset collections, and customizable statistical analysis with guided setup and interpretation, the GSuite HyperBrowser represents a first comprehensive solution for integrative analysis of track collections across the genome and epigenome. The software is available at: https://hyperbrowser.uio.no.

PIAS1 binds p300 and behaves as a coactivator or corepressor of the transcription factor c-Myb dependent on SUMO-status.

The PIAS proteins (Protein Inhibitor of Activated STATs) constitute a family of multifunctional nuclear proteins operating as SUMO E3 ligases and being involved in a multitude of interactions. They participate in a range of biological processes, also beyond their well-established role in the immune system and cytokine signalling. They act both as transcriptional corepressors and coactivators depending on the context. In the present work, we investigated mechanisms by which PIAS1 causes activation or repression of c-Myb dependent target genes. Analysis of global expression data shows that c-Myb and PIAS1 knockdowns affect a subset of common targets, but with a dual outcome consistent with a role of PIAS1 as either a corepressor or coactivator. Our mechanistic studies show that PIAS1 engages in a novel interaction with the acetyltransferase and coactivator p300. Interaction and ChIP analysis suggest a bridging function where PIAS1 enhances p300 recruitment to c-Myb-bound sites through interaction with both proteins. In addition, the E3 activity of PIAS1 enhances further its coactivation. Remarkably, the SUMO status of c-Myb had a decisive role, indicating a SUMO-dependent switch in the way PIAS1 affects c-Myb, either as a coactivator or corepressor. Removal of the two major SUMO-conjugation sites in c-Myb (2KR mutant), which enhances its activity significantly, turned PIAS1 into a corepressor. Also, p300 was less efficiently recruited to chromatin by c-Myb-2KR. We propose that PIAS1 acts as a "protein inhibitor of activated c-Myb" in the absence of SUMOylation while, in its presence, PIAS behaves as a "protein activator of repressed c-Myb".

In the loop: promoter-enhancer interactions and bioinformatics.

Enhancer-promoter regulation is a fundamental mechanism underlying differential transcriptional regulation. Spatial chromatin organization brings remote enhancers in contact with target promoters in cis to regulate gene expression. There is considerable evidence for promoter-enhancer interactions (PEIs). In the recent years, genome-wide analyses have identified signatures and mapped novel enhancers; however, being able to precisely identify their target gene(s) requires massive biological and bioinformatics efforts. In this review, we give a short overview of the chromatin landscape and transcriptional regulation. We discuss some key concepts and problems related to chromatin interaction detection technologies, and emerging knowledge from genome-wide chromatin interaction data sets. Then, we critically review different types of bioinformatics analysis methods and tools related to representation and visualization of PEI data, raw data processing and PEI prediction. Lastly, we provide specific examples of how PEIs have been used to elucidate a functional role of non-coding single-nucleotide polymorphisms. The topic is at the forefront of epigenetic research, and by highlighting some future bioinformatics challenges in the field, this review provides a comprehensive background for future PEI studies.

c-Myb Binding Sites in Haematopoietic Chromatin Landscapes.

Strict control of tissue-specific gene expression plays a pivotal role during lineage commitment. The transcription factor c-Myb has an essential role in adult haematopoiesis and functions as an oncogene when rearranged in human cancers. Here we have exploited digital genomic footprinting analysis to obtain a global picture of c-Myb occupancy in the genome of six different haematopoietic cell-types. We have biologically validated several c-Myb footprints using c-Myb knockdown data, reporter assays and DamID analysis. We show that our predicted conserved c-Myb footprints are highly dependent on the haematopoietic cell type, but that there is a group of gene targets common to all cell-types analysed. Furthermore, we find that c-Myb footprints co-localise with active histone mark H3K4me3 and are significantly enriched at exons. We analysed co-localisation of c-Myb footprints with 104 chromatin regulatory factors in K562 cells, and identified nine proteins that are enriched together with c-Myb footprints on genes positively regulated by c-Myb and one protein enriched on negatively regulated genes. Our data suggest that c-Myb is a transcription factor with multifaceted target regulation depending on cell type.

Spatial genome organization: contrasting views from chromosome conformation capture and fluorescence in situ hybridization.

Although important for gene regulation, most studies of genome organization use either fluorescence in situ hybridization (FISH) or chromosome conformation capture (3C) methods. FISH directly visualizes the spatial relationship of sequences but is usually applied to a few loci at a time. The frequency at which sequences are ligated together by formaldehyde cross-linking can be measured genome-wide by 3C methods, with higher frequencies thought to reflect shorter distances. FISH and 3C should therefore give the same views of genome organization, but this has not been tested extensively. We investigated the murine HoxD locus with 3C carbon copy (5C) and FISH in different developmental and activity states and in the presence or absence of epigenetic regulators. We identified situations in which the two data sets are concordant but found other conditions under which chromatin topographies extrapolated from 5C or FISH data are not compatible. We suggest that products captured by 3C do not always reflect spatial proximity, with ligation occurring between sequences located hundreds of nanometers apart, influenced by nuclear environment and chromatin composition. We conclude that results obtained at high resolution with either 3C methods or FISH alone must be interpreted with caution and that views about genome organization should be validated by independent methods.