Systematic assessment of ISWI subunits shows that NURF creates local accessibility for CTCF.

Catalytic activity of the imitation switch (ISWI) family of remodelers is critical for nucleosomal organization and DNA binding of certain transcription factors, including the insulator protein CTCF. Here we define the contribution of individual subcomplexes by deriving a panel of isogenic mouse stem cell lines, each lacking one of six ISWI accessory subunits. Individual deletions of subunits of either CERF, RSF, ACF, WICH or NoRC subcomplexes only moderately affect the chromatin landscape, while removal of the NURF-specific subunit BPTF leads to a strong reduction in chromatin accessibility and SNF2H ATPase localization around CTCF sites. This affects adjacent nucleosome occupancy and CTCF binding. At a group of sites with reduced chromatin accessibility, CTCF binding persists but cohesin occupancy is reduced, resulting in decreased insulation. These results suggest that CTCF binding can be separated from its function as an insulator in nuclear organization and identify a specific role for NURF in mediating SNF2H localization and chromatin opening at bound CTCF sites.

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.

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.

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.

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.

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.

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.

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.

Cell cycle-resolved chromatin proteomics reveals the extent of mitotic preservation of the genomic regulatory landscape.

Regulation of transcription, replication, and cell division relies on differential protein binding to DNA and chromatin, yet it is unclear which regulatory components remain bound to compacted mitotic chromosomes. By utilizing the buoyant density of DNA-protein complexes after cross-linking, we here develop a mass spectrometry-based approach to quantify the chromatin-associated proteome at separate stages of the cell cycle. While epigenetic modifiers that promote transcription are lost from mitotic chromatin, repressive modifiers generally remain associated. Furthermore, while proteins involved in transcriptional elongation are evicted, most identified transcription factors are retained on mitotic chromatin to varying degrees, including core promoter binding proteins. This predicts conservation of the regulatory landscape on mitotic chromosomes, which we confirm by genome-wide measurements of chromatin accessibility. In summary, this work establishes an approach to study chromatin, provides a comprehensive catalog of chromatin changes during the cell cycle, and reveals the degree to which the genomic regulatory landscape is maintained through mitosis.

Binding of high mobility group A proteins to the mammalian genome occurs as a function of AT-content.

Genomic location can inform on potential function and recruitment signals for chromatin-associated proteins. High mobility group (Hmg) proteins are of similar size as histones with Hmga1 and Hmga2 being particularly abundant in replicating normal tissues and in cancerous cells. While several roles for Hmga proteins have been proposed we lack a comprehensive description of their genomic location as a function of chromatin, DNA sequence and functional domains. Here we report such a characterization in mouse embryonic stem cells in which we introduce biotin-tagged constructs of wild-type and DNA-binding domain mutants. Comparative analysis of the genome-wide distribution of Hmga proteins reveals pervasive binding, a feature that critically depends on a functional DNA-binding domain and which is shared by both Hmga proteins. Assessment of the underlying queues instructive for this binding modality identifies AT richness, defined as high frequency of A or T bases, as the major criterion for local binding. Additionally, we show that other chromatin states such as those linked to cis-regulatory regions have little impact on Hmga binding both in stem and differentiated cells. As a consequence, Hmga proteins are preferentially found at AT-rich regions such as constitutively heterochromatic regions but are absent from enhancers and promoters arguing for a limited role in regulating individual genes. In line with this model, we show that genetic deletion of Hmga proteins in stem cells causes limited transcriptional effects and that binding is conserved in neuronal progenitors. Overall our comparative study describing the in vivo binding modality of Hmga1 and Hmga2 identifies the proteins' preference for AT-rich DNA genome-wide and argues against a suggested function of Hmga at regulatory regions. Instead we discover pervasive binding with enrichment at regions of higher AT content irrespective of local variation in chromatin modifications.

Evidence for Converging DNA Methylation Pathways in Placenta and Cancer.

CpG island promoters are generally devoid of DNA methylation in somatic cells but are frequently methylated during tumorigenesis. Reporting recently in Nature, Smith et al. (2017) show that the signaling-induced methylome in early extraembryonic tissues resembles that of many cancers, suggesting that placental nuclear programming might be co-opted in tumorigenesis.

Multidimensional pooled shRNA screens in human THP-1 cells identify candidate modulators of macrophage polarization.

Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the 'classical' (M1) and 'alternative' (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.

Impact of cytosine methylation on DNA binding specificities of human transcription factors.

The majority of CpG dinucleotides in the human genome are methylated at cytosine bases. However, active gene regulatory elements are generally hypomethylated relative to their flanking regions, and the binding of some transcription factors (TFs) is diminished by methylation of their target sequences. By analysis of 542 human TFs with methylation-sensitive SELEX (systematic evolution of ligands by exponential enrichment), we found that there are also many TFs that prefer CpG-methylated sequences. Most of these are in the extended homeodomain family. Structural analysis showed that homeodomain specificity for methylcytosine depends on direct hydrophobic interactions with the methylcytosine 5-methyl group. This study provides a systematic examination of the effect of an epigenetic DNA modification on human TF binding specificity and reveals that many developmentally important proteins display preference for mCpG-containing sequences.

YAP1 Exerts Its Transcriptional Control via TEAD-Mediated Activation of Enhancers.

YAP1 is a major effector of the Hippo pathway and a well-established oncogene. Elevated YAP1 activity due to mutations in Hippo pathway components or YAP1 amplification is observed in several types of human cancers. Here we investigated its genomic binding landscape in YAP1-activated cancer cells, as well as in non-transformed cells. We demonstrate that TEAD transcription factors mediate YAP1 chromatin-binding genome-wide, further explaining their dominant role as primary mediators of YAP1-transcriptional activity. Moreover, we show that YAP1 largely exerts its transcriptional control via distal enhancers that are marked by H3K27 acetylation and that YAP1 is necessary for this chromatin mark at bound enhancers and the activity of the associated genes. This work establishes YAP1-mediated transcriptional regulation at distal enhancers and provides an expanded set of target genes resulting in a fundamental source to study YAP1 function in a normal and cancer setting.

Short sequences can efficiently recruit histone H3 lysine 27 trimethylation in the absence of enhancer activity and DNA methylation.

Trimethylation of histone H3 at lysine 27 (H3K27me3) is a chromatin mark associated with Polycomb-mediated gene repression. Despite its critical role in development, it remains largely unclear how this mark is targeted to defined loci in mammalian cells. Here, we use iterative genome editing to identify small DNA sequences capable of autonomously recruiting Polycomb. We inserted 28 DNA elements at a defined chromosomal position in mouse embryonic stem cells and assessed their ability to promote H3K27me3 deposition. Combined with deletion analysis, we identified DNA elements as short as 220 nucleotides that correctly recapitulate endogenous H3K27me3 patterns. Functional Polycomb recruiter sequences are invariably CpG-rich but require protection against DNA methylation. Alternatively, their activity can be blocked by placement of an active promoter-enhancer pair in cis. Taken together, these data support the model whereby PRC2 recruitment at specific targets in mammals is positively regulated by local CpG density yet obstructed by transcriptional activity or DNA methylation.

DNA methylation is required for the control of stem cell differentiation in the small intestine.

The mammalian intestinal epithelium has a unique organization in which crypts harboring stem cells produce progenitors and finally clonal populations of differentiated cells. Remarkably, the epithelium is replaced every 3-5 d throughout adult life. Disrupted maintenance of the intricate balance of proliferation and differentiation leads to loss of epithelial integrity or barrier function or to cancer. There is a tight correlation between the epigenetic status of genes and expression changes during differentiation; however, the mechanism of how changes in DNA methylation direct gene expression and the progression from stem cells to their differentiated descendants is unclear. Using conditional gene ablation of the maintenance methyltransferase Dnmt1, we demonstrate that reducing DNA methylation causes intestinal crypt expansion in vivo. Determination of the base-resolution DNA methylome in intestinal stem cells and their differentiated descendants shows that DNA methylation is dynamic at enhancers, which are often associated with genes important for both stem cell maintenance and differentiation. We establish that the loss of DNA methylation at intestinal stem cell gene enhancers causes inappropriate gene expression and delayed differentiation.

Gene regulation and priming by topoisomerase IIα in embryonic stem cells.

Topoisomerases resolve torsional stress, while their function in gene regulation, especially during cellular differentiation, remains unknown. Here we find that the expression of topo II isoforms, topoisomerase IIα and topoisomerase IIß, is the characteristic of dividing and postmitotic tissues, respectively. In embryonic stem cells, topoisomerase IIα preferentially occupies active gene promoters. Topoisomerase IIα inhibition compromises genomic integrity, which results in epigenetic changes, altered kinetics of RNA Pol II at target promoters and misregulated gene expression. Common targets of topoisomerase IIα and topoisomerase IIß are housekeeping genes, while unique targets are involved in proliferation/pluripotency and neurogenesis, respectively. Topoisomerase IIα targets exhibiting bivalent chromatin resolve upon differentiation, concomitant with their activation and occupancy by topoisomerase IIß, features further observed for long genes. These long silent genes display accessible chromatin in embryonic stem cells that relies on topoisomerase IIα activity. These findings suggest that topoisomerase IIα not only contributes to stem-cell transcriptome regulation but also primes developmental genes for subsequent activation upon differentiation.