EU-LIFE charter of independent life science research institutes.

The diverse range of organizations contributing to the global research ecosystem is believed to enhance the overall quality and resilience of its output. Mid-sized autonomous research institutes, distinct from universities, play a crucial role in this landscape. They often lead the way in new research fields and experimental methods, including those in social and organizational domains, which are vital for driving innovation. The EU-LIFE alliance was established with the goal of fostering excellence by developing and disseminating best practices among European biomedical research institutes. As directors of the 15 EU-LIFE institutes, we have spent a decade comparing and refining our processes. Now, we are eager to share the insights we've gained. To this end, we have crafted this Charter, outlining 10 principles we deem essential for research institutes to flourish and achieve ground-breaking discoveries. These principles, detailed in the Charter, encompass excellence, independence, training, internationality and inclusivity, mission focus, technological advancement, administrative innovation, cooperation, societal impact, and public engagement. Our aim is to inspire the establishment of new institutes that adhere to these principles and to raise awareness about their significance. We are convinced that they should be viewed a crucial component of any national and international innovation strategies.

Cancer lineage-specific regulation of YAP responsive elements revealed through large-scale functional epigenomic screens.

YAP is a key transcriptional co-activator of TEADs, it regulates cell growth and is frequently activated in cancer. In Malignant Pleural Mesothelioma (MPM), YAP is activated by loss-of-function mutations in upstream components of the Hippo pathway, while, in Uveal Melanoma (UM), YAP is activated in a Hippo-independent manner. To date, it is unclear if and how the different oncogenic lesions activating YAP impact its oncogenic program, which is particularly relevant for designing selective anti-cancer therapies. Here we show that, despite YAP being essential in both MPM and UM, its interaction with TEAD is unexpectedly dispensable in UM, limiting the applicability of TEAD inhibitors in this cancer type. Systematic functional interrogation of YAP regulatory elements in both cancer types reveals convergent regulation of broad oncogenic drivers in both MPM and UM, but also strikingly selective programs. Our work reveals unanticipated lineage-specific features of the YAP regulatory network that provide important insights to guide the design of tailored therapeutic strategies to inhibit YAP signaling across different cancer types.

Cooperation between bHLH transcription factors and histones for DNA access.

The basic helix-loop-helix (bHLH) family of transcription factors recognizes DNA motifs known as E-boxes (CANNTG) and includes 108 members1. Here we investigate how chromatinized E-boxes are engaged by two structurally diverse bHLH proteins: the proto-oncogene MYC-MAX and the circadian transcription factor CLOCK-BMAL1 (refs. 2,3). Both transcription factors bind to E-boxes preferentially near the nucleosomal entry-exit sites. Structural studies with engineered or native nucleosome sequences show that MYC-MAX or CLOCK-BMAL1 triggers the release of DNA from histones to gain access. Atop the H2A-H2B acidic patch4, the CLOCK-BMAL1 Per-Arnt-Sim (PAS) dimerization domains engage the histone octamer disc. Binding of tandem E-boxes5-7 at endogenous DNA sequences occurs through direct interactions between two CLOCK-BMAL1 protomers and histones and is important for circadian cycling. At internal E-boxes, the MYC-MAX leucine zipper can also interact with histones H2B and H3, and its binding is indirectly enhanced by OCT4 elsewhere on the nucleosome. The nucleosomal E-box position and the type of bHLH dimerization domain jointly determine the histone contact, the affinity and the degree of competition and cooperativity with other nucleosome-bound factors.

Readout of histone methylation by Trim24 locally restricts chromatin opening by p53.

The genomic binding sites of the transcription factor (TF) and tumor suppressor p53 are unusually diverse with regard to their chromatin features, including histone modifications, raising the possibility that the local chromatin environment can contextualize p53 regulation. Here, we show that epigenetic characteristics of closed chromatin, such as DNA methylation, do not influence the binding of p53 across the genome. Instead, the ability of p53 to open chromatin and activate its target genes is locally restricted by its cofactor Trim24. Trim24 binds to both p53 and unmethylated histone 3 lysine 4 (H3K4), thereby preferentially localizing to those p53 sites that reside in closed chromatin, whereas it is deterred from accessible chromatin by H3K4 methylation. The presence of Trim24 increases cell viability upon stress and enables p53 to affect gene expression as a function of the local chromatin state. These findings link H3K4 methylation to p53 function and illustrate how specificity in chromatin can be achieved, not by TF-intrinsic sensitivity to histone modifications, but by employing chromatin-sensitive cofactors that locally modulate TF function.

ZFP462 safeguards neural lineage specification by targeting G9A/GLP-mediated heterochromatin to silence enhancers.

ZNF462 haploinsufficiency is linked to Weiss-Kruszka syndrome, a genetic disorder characterized by neurodevelopmental defects, including autism. Though conserved in vertebrates and essential for embryonic development, the molecular functions of ZNF462 remain unclear. We identified its murine homologue ZFP462 in a screen for mediators of epigenetic gene silencing. Here we show that ZFP462 safeguards neural lineage specification of mouse embryonic stem cells (ESCs) by targeting the H3K9-specific histone methyltransferase complex G9A/GLP to silence meso-endodermal genes. ZFP462 binds to transposable elements that are potential enhancers harbouring pluripotency and meso-endoderm transcription factor binding sites. Recruiting G9A/GLP, ZFP462 seeds heterochromatin, restricting transcription factor binding. Loss of ZFP462 in ESCs results in increased chromatin accessibility at target sites and ectopic expression of meso-endodermal genes. Taken together, ZFP462 confers lineage and locus specificity to the broadly expressed epigenetic regulator G9A/GLP. Our results suggest that aberrant activation of lineage non-specific genes in the neuronal lineage underlies ZNF462-associated neurodevelopmental pathology.

Evidence that direct inhibition of transcription factor binding is the prevailing mode of gene and repeat repression by DNA methylation.

Cytosine methylation efficiently silences CpG-rich regulatory regions of genes and repeats in mammalian genomes. To what extent this entails direct inhibition of transcription factor (TF) binding versus indirect inhibition via recruitment of methyl-CpG-binding domain (MBD) proteins is unclear. Here we show that combinatorial genetic deletions of all four proteins with functional MBDs in mouse embryonic stem cells, derived neurons or a human cell line do not reactivate genes or repeats with methylated promoters. These do, however, become activated by methylation-restricted TFs if DNA methylation is removed. We identify several causal TFs in neurons, including ONECUT1, which is methylation sensitive only at a motif variant. Rampantly upregulated retrotransposons in methylation-free neurons feature a CRE motif, which activates them in the absence of DNA methylation via methylation-sensitive binding of CREB1. Our study reveals methylation-sensitive TFs in vivo and argues that direct inhibition, rather than indirect repression by the tested MBD proteins, is the prevailing mechanism of methylation-mediated repression at regulatory regions and repeats.

DNA sequence and chromatin modifiers cooperate to confer epigenetic bistability at imprinting control regions.

Genomic imprinting is regulated by parental-specific DNA methylation of imprinting control regions (ICRs). Despite an identical DNA sequence, ICRs can exist in two distinct epigenetic states that are memorized throughout unlimited cell divisions and reset during germline formation. Here, we systematically study the genetic and epigenetic determinants of this epigenetic bistability. By iterative integration of ICRs and related DNA sequences to an ectopic location in the mouse genome, we first identify the DNA sequence features required for maintenance of epigenetic states in embryonic stem cells. The autonomous regulatory properties of ICRs further enabled us to create DNA-methylation-sensitive reporters and to screen for key components involved in regulating their epigenetic memory. Besides DNMT1, UHRF1 and ZFP57, we identify factors that prevent switching from methylated to unmethylated states and show that two of these candidates, ATF7IP and ZMYM2, are important for the stability of DNA and H3K9 methylation at ICRs in embryonic stem cells.

Generating specificity in genome regulation through transcription factor sensitivity to chromatin.

Cell type-specific gene expression relies on transcription factors (TFs) binding DNA sequence motifs embedded in chromatin. Understanding how motifs are accessed in chromatin is crucial to comprehend differential transcriptional responses and the phenotypic impact of sequence variation. Chromatin obstacles to TF binding range from DNA methylation to restriction of DNA access by nucleosomes depending on their position, composition and modification. In vivo and in vitro approaches now enable the study of TF binding in chromatin at unprecedented resolution. Emerging insights suggest that TFs vary in their ability to navigate chromatin states. However, it remains challenging to link binding and transcriptional outcomes to molecular characteristics of TFs or the local chromatin substrate. Here, we discuss our current understanding of how TFs access DNA in chromatin and novel techniques and directions towards a better understanding of this critical step in genome regulation.

monaLisa: an R/Bioconductor package for identifying regulatory motifs.

SUMMARY: Proteins binding to specific nucleotide sequences, such as transcription factors, play key roles in the regulation of gene expression. Their binding can be indirectly observed via associated changes in transcription, chromatin accessibility, DNA methylation and histone modifications. Identifying candidate factors that are responsible for these observed experimental changes is critical to understand the underlying biological processes. Here, we present monaLisa, an R/Bioconductor package that implements approaches to identify relevant transcription factors from experimental data. The package can be easily integrated with other Bioconductor packages and enables seamless motif analyses without any software dependencies outside of R. AVAILABILITY AND IMPLEMENTATION: monaLisa is implemented in R and available on Bioconductor at https://bioconductor.org/packages/monaLisa with the development version hosted on GitHub at https://github.com/fmicompbio/monaLisa. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

BANP opens chromatin and activates CpG-island-regulated genes.

The majority of gene transcripts generated by RNA polymerase II in mammalian genomes initiate at CpG island (CGI) promoters1,2, yet our understanding of their regulation remains limited. This is in part due to the incomplete information that we have on transcription factors, their DNA-binding motifs and which genomic binding sites are functional in any given cell type3-5. In addition, there are orphan motifs without known binders, such as the CGCG element, which is associated with highly expressed genes across human tissues and enriched near the transcription start site of a subset of CGI promoters6-8. Here we combine single-molecule footprinting with interaction proteomics to identify BTG3-associated nuclear protein (BANP) as the transcription factor that binds this element in the mouse and human genome. We show that BANP is a strong CGI activator that controls essential metabolic genes in pluripotent stem and terminally differentiated neuronal cells. BANP binding is repelled by DNA methylation of its motif in vitro and in vivo, which epigenetically restricts most binding to CGIs and accounts for differential binding at aberrantly methylated CGI promoters in cancer cells. Upon binding to an unmethylated motif, BANP opens chromatin and phases nucleosomes. These findings establish BANP as a critical activator of a set of essential genes and suggest a model in which the activity of CGI promoters relies on methylation-sensitive transcription factors that are capable of chromatin opening.

Systematic dissection of transcriptional regulatory networks by genome-scale and single-cell CRISPR screens.

Millions of putative transcriptional regulatory elements (TREs) have been cataloged in the human genome, yet their functional relevance in specific pathophysiological settings remains to be determined. This is critical to understand how oncogenic transcription factors (TFs) engage specific TREs to impose transcriptional programs underlying malignant phenotypes. Here, we combine cutting edge CRISPR screens and epigenomic profiling to functionally survey ≈15,000 TREs engaged by estrogen receptor (ER). We show that ER exerts its oncogenic role in breast cancer by engaging TREs enriched in GATA3, TFAP2C, and H3K27Ac signal. These TREs control critical downstream TFs, among which TFAP2C plays an essential role in ER-driven cell proliferation. Together, our work reveals novel insights into a critical oncogenic transcription program and provides a framework to map regulatory networks, enabling to dissect the function of the noncoding genome of cancer cells.

PAX8 and MECOM are interaction partners driving ovarian cancer.

The transcription factor PAX8 is critical for the development of the thyroid and urogenital system. Comprehensive genomic screens furthermore indicate an additional oncogenic role for PAX8 in renal and ovarian cancers. While a plethora of PAX8-regulated genes in different contexts have been proposed, we still lack a mechanistic understanding of how PAX8 engages molecular complexes to drive disease-relevant oncogenic transcriptional programs. Here we show that protein isoforms originating from the MECOM locus form a complex with PAX8. These include MDS1-EVI1 (also called PRDM3) for which we map its interaction with PAX8 in vitro and in vivo. We show that PAX8 binds a large number of genomic sites and forms transcriptional hubs. At a subset of these, PAX8 together with PRDM3 regulates a specific gene expression module involved in adhesion and extracellular matrix. This gene module correlates with PAX8 and MECOM expression in large scale profiling of cell lines, patient-derived xenografts (PDXs) and clinical cases and stratifies gynecological cancer cases with worse prognosis. PRDM3 is amplified in ovarian cancers and we show that the MECOM locus and PAX8 sustain in vivo tumor growth, further supporting that the identified function of the MECOM locus underlies PAX8-driven oncogenic functions in ovarian cancer.

Mammalian SWI/SNF continuously restores local accessibility to chromatin.

Chromatin accessibility is a hallmark of regulatory regions, entails transcription factor (TF) binding and requires nucleosomal reorganization. However, it remains unclear how dynamic this process is. In the present study, we use small-molecule inhibition of the catalytic subunit of the mouse SWI/SNF remodeler complex to show that accessibility and reduced nucleosome presence at TF-binding sites rely on persistent activity of nucleosome remodelers. Within minutes of remodeler inhibition, accessibility and TF binding decrease. Although this is irrespective of TF function, we show that the activating TF OCT4 (POU5F1) exhibits a faster response than the repressive TF REST. Accessibility, nucleosome depletion and gene expression are rapidly restored on inhibitor removal, suggesting that accessible chromatin is regenerated continuously and in a largely cell-autonomous fashion. We postulate that TF binding to chromatin and remodeler-mediated nucleosomal removal do not represent a stable situation, but instead accessible chromatin reflects an average of a dynamic process under continued renewal.

Molecular Co-occupancy Identifies Transcription Factor Binding Cooperativity In Vivo.

Gene activation requires the cooperative activity of multiple transcription factors at cis-regulatory elements (CREs). Yet, most transcription factors have short residence time, questioning the requirement of their physical co-occupancy on DNA to achieve cooperativity. Here, we present a DNA footprinting method that detects individual molecular interactions of transcription factors and nucleosomes with DNA in vivo. We apply this strategy to quantify the simultaneous binding of multiple transcription factors on single DNA molecules at mouse CREs. Analysis of the binary occupancy patterns at thousands of motif combinations reveals that high DNA co-occupancy occurs for most types of transcription factors, in the absence of direct physical interaction, at sites of competition with nucleosomes. Perturbation of pairwise interactions demonstrates the function of molecular co-occupancy in binding cooperativity. Our results reveal the interactions regulating CREs at molecular resolution and identify DNA co-occupancy as a widespread cooperativity mechanism used by transcription factors to remodel chromatin.

A genome-scale map of DNA methylation turnover identifies site-specific dependencies of DNMT and TET activity.

DNA methylation is considered a stable epigenetic mark, yet methylation patterns can vary during differentiation and in diseases such as cancer. Local levels of DNA methylation result from opposing enzymatic activities, the rates of which remain largely unknown. Here we developed a theoretical and experimental framework enabling us to infer methylation and demethylation rates at 860,404 CpGs in mouse embryonic stem cells. We find that enzymatic rates can vary as much as two orders of magnitude between CpGs with identical steady-state DNA methylation. Unexpectedly, de novo and maintenance methylation activity is reduced at transcription factor binding sites, while methylation turnover is elevated in transcribed gene bodies. Furthermore, we show that TET activity contributes substantially more than passive demethylation to establishing low methylation levels at distal enhancers. Taken together, our work unveils a genome-scale map of methylation kinetics, revealing highly variable and context-specific activity for the DNA methylation machinery.

Mechanisms of OCT4-SOX2 motif readout on nucleosomes.

Transcription factors (TFs) regulate gene expression through chromatin where nucleosomes restrict DNA access. To study how TFs bind nucleosome-occupied motifs, we focused on the reprogramming factors OCT4 and SOX2 in mouse embryonic stem cells. We determined TF engagement throughout a nucleosome at base-pair resolution in vitro, enabling structure determination by cryo-electron microscopy at two preferred positions. Depending on motif location, OCT4 and SOX2 differentially distort nucleosomal DNA. At one position, OCT4-SOX2 removes DNA from histone H2A and histone H3; however, at an inverted motif, the TFs only induce local DNA distortions. OCT4 uses one of its two DNA-binding domains to engage DNA in both structures, reading out a partial motif. These findings explain site-specific nucleosome engagement by the pluripotency factors OCT4 and SOX2, and they reveal how TFs distort nucleosomes to access chromatinized motifs.

Targeting neuronal and glial cell types with synthetic promoter AAVs in mice, non-human primates and humans.

Targeting genes to specific neuronal or glial cell types is valuable for both understanding and repairing brain circuits. Adeno-associated viruses (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is an unsolved problem. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that a number of these AAVs specifically target expression to neuronal and glial cell types in the mouse and non-human primate retina in vivo and in the human retina in vitro. We demonstrate applications for recording and stimulation, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.

Publisher Correction: DNA damage detection in nucleosomes involves DNA register shifting.

In this Article, in Fig. 1a, the 5' and 3' labels were reversed in the DNA sequence, and Fig. 4 was missing panel labels a-e. These errors have been corrected online.

DNA damage detection in nucleosomes involves DNA register shifting.

Access to DNA packaged in nucleosomes is critical for gene regulation, DNA replication and DNA repair. In humans, the UV-damaged DNA-binding protein (UV-DDB) complex detects UV-light-induced pyrimidine dimers throughout the genome; however, it remains unknown how these lesions are recognized in chromatin, in which nucleosomes restrict access to DNA. Here we report cryo-electron microscopy structures of UV-DDB bound to nucleosomes bearing a 6-4 pyrimidine-pyrimidone dimer or a DNA-damage mimic in various positions. We find that UV-DDB binds UV-damaged nucleosomes at lesions located in the solvent-facing minor groove without affecting the overall nucleosome architecture. In the case of buried lesions that face the histone core, UV-DDB changes the predominant translational register of the nucleosome and selectively binds the lesion in an accessible, exposed position. Our findings explain how UV-DDB detects occluded lesions in strongly positioned nucleosomes, and identify slide-assisted site exposure as a mechanism by which high-affinity DNA-binding proteins can access otherwise occluded sites in nucleosomal DNA.

Mammalian ISWI and SWI/SNF selectively mediate binding of distinct transcription factors.

Chromatin remodelling complexes evict, slide, insert or replace nucleosomes, which represent an intrinsic barrier for access to DNA. These remodellers function in most aspects of genome utilization including transcription-factor binding, DNA replication and repair1,2. Although they are frequently mutated in cancer3, it remains largely unclear how the four mammalian remodeller families (SWI/SNF, ISWI, CHD and INO80) orchestrate the global organization of nucleosomes. Here we generated viable embryonic stem cells that lack SNF2H, the ATPase of ISWI complexes, enabling study of SNF2H cellular function, and contrast it to BRG1, the ATPase of SWI/SNF. Loss of SNF2H decreases nucleosomal phasing and increases linker lengths, providing in vivo evidence for an ISWI function in ruling nucleosomal spacing in mammals. Systematic analysis of transcription-factor binding reveals that these remodelling activities have specific effects on binding of different transcription factors. One group critically depends on BRG1 and contains the transcriptional repressor REST, whereas a non-overlapping set of transcription factors, including the insulator protein CTCF, relies on SNF2H. This selectivity readily explains why chromosomal folding and insulation of topologically associated domains requires SNF2H, but not BRG1. Collectively, this study shows that mammalian ISWI is critical for nucleosomal periodicity and nuclear organization and that transcription factors rely on specific remodelling pathways for correct genomic binding.