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Facioscapulohumeral muscular dystrophy (FSHD) is one of the most prevalent neuromuscular disorders. The disease is linked to copy number reduction and/or epigenetic alterations of the D4Z4 macrosatellite on chromosome 4q35 and associated with aberrant gain of expression of the transcription factor DUX4, which triggers a pro-apoptotic transcriptional program leading to muscle wasting. As today, no cure or therapeutic option is available to FSHD patients. Given its centrality in FSHD, blocking DUX4 expression with small molecule drugs is an attractive option. We previously showed that the long non protein-coding RNA DBE-T is required for aberrant DUX4 expression in FSHD. Using affinity purification followed by proteomics, here we identified the chromatin remodeling protein WDR5 as a novel DBE-T interactor and a key player required for the biological activity of the lncRNA. We found that WDR5 is required for the expression of DUX4 and its targets in primary FSHD muscle cells. Moreover, targeting WDR5 rescues both cell viability and myogenic differentiation of FSHD patient cells. Notably, comparable results were obtained by pharmacological inhibition of WDR5. Importantly, WDR5 targeting was safe to healthy donor muscle cells. Our results support a pivotal role of WDR5 in the activation of DUX4 expression identifying a druggable target for an innovative therapeutic approach for FSHD.

Supplementary Figure from HDAC2 Facilitates Pancreatic Cancer Metastasis

Supplementary Table from HDAC2 Facilitates Pancreatic Cancer Metastasis

Supplementary Data from HDAC2 Facilitates Pancreatic Cancer Metastasis

The mortality of patients with pancreatic ductal adenocarcinoma (PDAC) is strongly associated with metastasis, a multistep process that is incompletely understood in this disease. Although genetic drivers of PDAC metastasis have not been defined, transcriptional and epigenetic rewiring can contribute to the metastatic process. The epigenetic eraser histone deacetylase 2 (HDAC2) has been connected to less differentiated PDAC, but the function of HDAC2 in PDAC has not been comprehensively evaluated. Using genetically defined models, we show that HDAC2 is a cellular fitness factor that controls cell cycle in vitro and metastasis in vivo, particularly in undifferentiated, mesenchymal PDAC cells. Unbiased expression profiling detected a core set of HDAC2-regulated genes. HDAC2 controlled expression of several prosurvival receptor tyrosine kinases connected to mesenchymal PDAC, including PDGFRα, PDGFRβ, and EGFR. The HDAC2-maintained program disabled the tumor-suppressive arm of the TGFβ pathway, explaining impaired metastasis formation of HDAC2-deficient PDAC. These data identify HDAC2 as a tractable player in the PDAC metastatic cascade. The complexity of the function of epigenetic regulators like HDAC2 implicates that an increased understanding of these proteins is needed for implementation of effective epigenetic therapies.Significance:HDAC2 has a context-specific role in undifferentiated PDAC and the capacity to disseminate systemically, implicating HDAC2 as targetable protein to prevent metastasis.

The mortality of patients with pancreatic ductal adenocarcinoma (PDAC) is strongly associated with metastasis, a multistep process that is incompletely understood in this disease. Although genetic drivers of PDAC metastasis have not been defined, transcriptional and epigenetic rewiring can contribute to the metastatic process. The epigenetic eraser histone deacetylase 2 (HDAC2) has been connected to less differentiated PDAC, but the function of HDAC2 in PDAC has not been comprehensively evaluated. Using genetically defined models, we show that HDAC2 is a cellular fitness factor that controls cell cycle in vitro and metastasis in vivo, particularly in undifferentiated, mesenchymal PDAC cells. Unbiased expression profiling detected a core set of HDAC2-regulated genes. HDAC2 controlled expression of several prosurvival receptor tyrosine kinases connected to mesenchymal PDAC, including PDGFRα, PDGFRβ, and EGFR. The HDAC2-maintained program disabled the tumor-suppressive arm of the TGFβ pathway, explaining impaired metastasis formation of HDAC2-deficient PDAC. These data identify HDAC2 as a tractable player in the PDAC metastatic cascade. The complexity of the function of epigenetic regulators like HDAC2 implicates that an increased understanding of these proteins is needed for implementation of effective epigenetic therapies. Significance: HDAC2 has a context-specific role in undifferentiated PDAC and the capacity to disseminate systemically, implicating HDAC2 as targetable protein to prevent metastasis.

Well-differentiated liposarcoma (WDLPS) is a malignant neoplasia hard to diagnose and treat. Its main molecular signature is amplification of the MDM2-containing genomic region. The MDM2 oncogene is the master regulator of p53: its overexpression enhances p53 degradation and inhibits apoptosis, leading to the tumoral phenotype. Here, we show that the MDM2 inducible promoter G-rich region folds into stable G-quadruplexes both in vitro and in vivo and it is specifically recognized by cellular helicases. Cell treatment with G-quadruplex-ligands reduces MDM2 expression and p53 degradation, thus stimulating cancer cell cycle arrest and apoptosis. Structural characterization of the MDM2 G-quadruplex revealed an extraordinarily stable, unique four-tetrad antiparallel dynamic conformation, amenable to selective targeting. These data indicate the feasibility of an out-of-the-box G-quadruplex-targeting approach to defeat WDLPS and all tumours where restoration of wild-type p53 is sought. They also point to G-quadruplex-dependent genomic instability as possible cause of MDM2 expansion and WDLPS tumorigenesis., Graphical Abstract Graphical AbstractScheme representing how G4-ligand-induced stabilization of MDM2 P2 promoter G4 leads to accumulation of p53 and hnRNP-K proteins, resulting in stimulation of apoptosis and cell cycle arrest.

Glucocorticoids (such as Dexamethasone) are commonly used immunomodulatory drugs with potent anti-inflammatory effects, whose mechanisms of action remain incompletely understood. They bind to the Glucocorticoid Receptor (GR), a nuclear hormone receptor that acts as a transcription factor to directly control the expression of inflammatory genes. To elucidate the complex molecular mechanisms employed by GR during the suppression of innate immune responses, we have performed proteomics, ChIP-seq, ATAC-seq, RNA-seq and bioinformatics together with genetic and pharmacological loss of function studies in primary mouse macrophages. We found that GR interacts with the ATP-dependent SWI/SNF chromatin remodeling complex to regulate a specific subset of target genes. Here we show that the central catalytic subunit BRG1 is required not only for the transcriptional activation of classical GR target genes such as Fkbp5 or Klf9, but also for the transcriptional repression of cytokines and chemokines such as Ccl2, Cxcl10 or Il1a. We demonstrate that loss of BRG1 activity leads to reduced histone deacetylase (HDAC) function, and consequently increased histone acetylation, at these repressive GR binding sites. Altogether, our findings suggest that GR interacts with BRG1 to assemble a functional co-repressor complex at a defined fraction of macrophage cis-regulatory elements. These results may indicate additional non-classical, remodeling-independent functions of the SWI/SNF complex and may have implications for the development of future immunomodulatory therapies.GRAPHICAL ABSTRACTGraphical Abstract.In macrophages (mΦ) responding to bacterial LPS and Dexamethasone, the Glucocorticoid Receptor (GR) activates target genes like Klf9 or Fkbp5 via interaction with the BRG1-containing SWI/SNF complex, chromatin remodeling and Mediator recruitment. At the same time, GR represses the expression of inflammatory cytokines and chemokines such as Ccl2, Cxcl10, Il1a etc. by assembling a BRG1-containing co-repressor complex and de-acetylating surrounding histone tails. Loss of BRG1 activity affects both the transcriptional activation and repression of a subset of myeloid GR target genes via distinct mechanisms. (iTF: inflammatory transcription factor; Ac: histone acetylation) (Created with BioRender.com.)

GATA2 zinc-finger (ZF) mutations are associated with distinct entities of myeloid malignancies. The specific distribution of these mutations points toward different mechanisms of leukemogenesis depending on the ZF domain affected. In this study, we compared recurring somatic mutations in ZF1 and ZF2. All tested ZF mutants disrupted DNA binding in vitro. In transcription assays, co-expression of FOG1 counteracted GATA2-dependent transcriptional activation, while a variable response to FOG1-mediated repression was observed for individual GATA2 mutants. In primary murine bone marrow cells, GATA2 wild-type (WT) expression inhibited colony formation, while this effect was reduced for both mutants A318T (ZF1) and L359V (ZF2) with a shift toward granulopoiesis. In primary human CD34

Pioneer transcription factors (PTF) can recognize their binding sites on nucleosomal DNA and trigger chromatin opening for recruitment of other non-pioneer transcription factors. However, critical properties of PTFs are still poorly understood, such as how these transcription factors selectively recognize cell type-specific binding sites and under which conditions can they can initiate chromatin remodelling. Here we show that early endoderm binding sites of the paradigm PTF Foxa2 are epigenetically primed by low levels of active chromatin modifications in embryonic stem cells (ESC). Priming of these binding sites is supported by preferential recruitment of Foxa2 to endoderm binding sites compared to lineage-inappropriate binding sites, when ectopically expressed in ESCs. We further show that binding of Foxa2 is required for chromatin opening during endoderm differentiation. However, increased chromatin accessibility was only detected on binding sites which are synergistically bound with other endoderm transcription factors. Thus, our data suggest that binding site selection of PTFs is directed by the chromatin environment and that chromatin opening requires collaboration of PTFs with additional transcription factors.

Objective Hundreds of missense mutations in the coding region of PDX1 exist; however, if these mutations predispose to diabetes mellitus is unknown. Methods In this study, we screened a large cohort of subjects with increased risk for diabetes and identified two subjects with impaired glucose tolerance carrying common, heterozygous, missense mutations in the PDX1 coding region leading to single amino acid exchanges (P33T, C18R) in its transactivation domain. We generated iPSCs from patients with heterozygous PDX1P33T/+, PDX1C18R/+ mutations and engineered isogenic cell lines carrying homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations and a heterozygous PDX1 loss-of-function mutation (PDX1+/−). Results Using an in vitro β-cell differentiation protocol, we demonstrated that both, heterozygous PDX1P33T/+, PDX1C18R/+ and homozygous PDX1P33T/P33T, PDX1C18R/C18R mutations impair β-cell differentiation and function. Furthermore, PDX1+/− and PDX1P33T/P33T mutations reduced differentiation efficiency of pancreatic progenitors (PPs), due to downregulation of PDX1-bound genes, including transcription factors MNX1 and PDX1 as well as insulin resistance gene CES1. Additionally, both PDX1P33T/+ and PDX1P33T/P33T mutations in PPs reduced the expression of PDX1-bound genes including the long-noncoding RNA, MEG3 and the imprinted gene NNAT, both involved in insulin synthesis and secretion. Conclusions Our results reveal mechanistic details of how common coding mutations in PDX1 impair human pancreatic endocrine lineage formation and β-cell function and contribute to the predisposition for diabetes., Highlights • Missense mutations in the transactivation domain reduce PDX1 target gene expression. • Lack of PDX1 target gene activation impairs both β-cell development and function. • Common PDX1 coding mutations likely predispose for diabetes.

Background: Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second leading cause of cancer mortality within a decade with overall 5-year survival of 8% for all stages combined. Currently, it is well documented that mechanisms driving PDAC progression involve epigenetic and transcriptional rewiring. Here we combined assay for transposase-accessible chromatin using sequencing (ATAC-seq) and enrichment for H3K27 acetylation chromatin immunoprecipitation (H3K27ac ChIP-seq) measures to explore the epigenetic landscape of different mouse primary pancreatic tumor (PPT) cell lines. Methods: Kras-driven (n=36) and PI3K-driven PPT cell lines (n=9) were cultured in DMEM medium (Gibco). DNA was extracted using manufacture protocols (Qiagen, MinElute PCR Purification Kit) then DNA libraries and high-throughput sequencing were performed. Bioinformatics analysis (ROSE2 Python script) was conducted on H3k27ac ChIP-seq data to define super-enhancer (SEs) and SE-associated genes. Then, ATAC-seq data was explored using Coltron Python package to distinguish enriched Transcription Factor (TF) motifs into SEs. Transcriptomic data was used to slim down the list of potential cis-regulatory elements. We developed knockout (ko) PPT cell lines using CRISPR/CAS9 gene editing method to better characterize the role of Foxj1 as a novel potential master regulator in pancreatic cancer. Lastly, immunohistochemistry (IHC) staining for FOXJ1 was conducted on human PDAC cohort. Results: By k-means clustering, we identified 463 SE-associated genes. Many of them are associated with Kras-driven (epithelial or mesenchymal) or PI3K-driven cell lines exclusively. Surprisingly, we found Foxj1 as SE-associated TF exclusively in PI3K-driven PPT cell lines. Consistent with the epigenetic data, transcriptomic analysis confirmed higher expression of Foxj1 in PI3K-driven PPT cell lines. Then, RNA-seq data revealed downregulation of predicted Foxj1 target genes and enhanced EMT and Wnt/β-catenin signatures in Foxj1 ko cells. These data suggest that epithelial properties of PDAC cells are stabilized by Foxj1 activity. Consistent with these results we detect a higher potential of TGFβ treatment to induce mesenchymal features in Foxj1 ko cells. Furthermore, overexpression of β-catenin protein was confirmed by immunofluorescence. Enhanced Wnt/β-catenin signaling could be responsible for the higher proliferation of Foxj1 ko cells as revealed by proliferation assay. Finally, we investigated FOXJ1 protein level in our PDAC human cohort. Interestingly, we found high nuclear FOXJ1 expression in 23% of cases which is linked with better overall survival. Conclusions: In summary, our data revealed Foxj1 as a novel PDAC associated TF with the ability to reduce the cancer aggressiveness blocking epithelial to mesenchymal transition and β-catenin activity elucidating the better prognosis into the FOXJ1 high expressed patients. Citation Format: Andrea Terrasi, Swathi Subramanian, Christine Klement, Sruthi Ramesh, Heike Bollig, Chiara Falcomatà, Katja Steiger, Rupert Öllinger, Dieter Saur, Roland Rad, Maximilian Reichert, Günter Schneider, Gunnar Schotta. Foxj1 is a new master regulator of activated PI3K pathway pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2350.

It is generally accepted that epiblast cells ingress into the primitive streak by epithelial-to-mesenchymal transition (EMT) to give rise to the mesoderm; however, it is less clear how the endoderm acquires an epithelial fate. Here, we used embryonic stem cell and mouse embryo knock‐in reporter systems to combine time-resolved lineage labelling with high-resolution single-cell transcriptomics. This allowed us to resolve the morphogenetic programs that segregate the mesoderm from the endoderm germ layer. Strikingly, while the mesoderm is formed by classical EMT, the endoderm is formed independent of the key EMT transcription factor Snail1 by mechanisms of epithelial cell plasticity. Importantly, forkhead box transcription factor A2 (Foxa2) acts as an epithelial gatekeeper and EMT suppressor to shield the endoderm from undergoing a mesenchymal transition. Altogether, these results not only establish the morphogenetic details of germ layer formation, but also have broader implications for stem cell differentiation and cancer metastasis., Scheibner et al. demonstrate that, during gastrulation in the mouse, epithelial epiblast progenitors upregulate Foxa2 and form the definitive endoderm independently of a full EMT–MET cycle.

Epstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable, long-term latent infection in human B cells, its preferred host. Upon induction of EBV’s lytic phase, the latently infected cells turn into a virus factory, a process that is governed by EBV. In the lytic, productive phase, all herpes viruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105 binding sites with different sequence motifs in cellular chromatin in a concentration dependent manner implementing a binary molar switch probably to prevent noise-induced erroneous induction of EBV’s lytic phase. Concomitant with DNA binding of BZLF1, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBV’s lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficient de novo synthesis of this herpes virus.

The mammalian epigenome contains thousands of heterochromatin nanodomains (HNDs) marked by di- and trimethylation of histone H3 at lysine 9, which have a typical size of 3-10 nucleosomes. However, the (epi)genetic determinants of their location and boundaries are only partly understood. Here, we compare four HND types in mouse embryonic stem cells, that are defined by histone methylases SUV39H1/2 or GLP, transcription factor ADNP or chromatin remodeller ATRX. Based on a novel chromatin hierarchical lattice framework termed ChromHL, we are able to predict HND maps with singe-nucleotide resolution. We find that HND nucleation can be rationalized by DNA sequence specific protein binding to PAX3/9, ADNP and LINE1 repeats. Depending on type of microdomains, boundaries are determined either by CTCF binding sites or by nucleosome-nucleosome and nucleosome-HP1 interactions. Our new framework allows predicting how patterns of H3K9me2/3 and other chromatin nanodomains are established and changed in processes such as cell differentiation.

The mammalian epigenome contains thousands of heterochromatin nanodomains (HNDs) marked by di- and trimethylation of histone H3 at lysine 9 (H3K9me2/3), which have a typical size of 3-10 nucleosomes. However, what governs HND location and extension is only partly understood. Here, we address this issue by introducing the chromatin hierarchical lattice framework (ChromHL) that predicts chromatin state patterns with single-nucleotide resolution. ChromHL is applied to analyse four HND types in mouse embryonic stem cells that are defined by histone methylases SUV39H1/2 or GLP, transcription factor ADNP or chromatin remodeller ATRX. We find that HND patterns can be computed from PAX3/9, ADNP and LINE1 sequence motifs as nucleation sites and boundaries that are determined by DNA sequence (e.g. CTCF binding sites), cooperative interactions between nucleosomes as well as nucleosome-HP1 interactions. Thus, ChromHL rationalizes how patterns of H3K9me2/3 are established and changed via the activity of protein factors in processes like cell differentiation.

Endogenous retroviruses (ERVs) comprise a significant portion of mammalian genomes. Although specific ERV loci feature regulatory roles for host gene expression, most ERV integrations are transcriptionally repressed by Setdb1-mediated H3K9me3 and DNA methylation. However, the protein network which regulates the deposition of these chromatin modifications is still incompletely understood. Here, we perform a genome-wide single guide RNA (sgRNA) screen for genes involved in ERV silencing and identify the GHKL ATPase protein Morc3 as a top-scoring hit. Morc3 knock-out (ko) cells display de-repression, reduced H3K9me3, and increased chromatin accessibility of distinct ERV families. We find that the Morc3 ATPase cycle and Morc3 SUMOylation are important for ERV chromatin regulation. Proteomic analyses reveal that Morc3 mutant proteins fail to interact with the histone H3.3 chaperone Daxx. This interaction depends on Morc3 SUMOylation and Daxx SUMO binding. Notably, in Morc3 ko cells, we observe strongly reduced histone H3.3 on Morc3 binding sites. Thus, our data demonstrate Morc3 as a critical regulator of Daxx-mediated histone H3.3 incorporation to ERV regions., Endogenous retroviruses (ERVs) compose a significant portion of mammalian genomes; however, how ERVs are regulated is not well known. Here the authors performed a genome-wide sgRNA screen to identify Morc3 as a mediator of ERV silencing. They show Morc3 associates with the H3.3 chaperone Daxx, and that loss of Morc3 leads to reduced H3.3 at ERVs.

Endogenous retroviruses (ERVs) comprise a significant portion of mammalian genomes. Although, specific ERV loci feature regulatory roles for host gene expression, most ERV integrations are transcriptionally repressed by Setdb1 mediated H3K9me3 and DNA methylation. However, the protein network which regulates deposition of these chromatin modifications is still incompletely understood. Here, we performed a genome-wide sgRNA screen for genes involved in ERV silencing and identified the GHKL ATPase protein Morc3 as top scoring hit. Morc3 knock-out cells display de-repression, reduced H3K9me3 and increased chromatin accessibility of distinct ERV classes. We found that the GHKL ATPase domain of Morc3 is critical for ERV silencing, since mutants which cannot bind ATP, or which are defective in ATP hydrolysis cannot rescue the Morc3 ko phenotype. Proteomic analysis revealed that Morc3 mutant protein which cannot bind ATP fails to interact with the H3.3 chaperone Daxx. This interaction depends on Morc3 SUMOylation, as Daxx lacking the SUMO interaction domain shows reduced association with Morc3. Notably, in Morc3 ko cells, we observed strongly reduced H3.3 on Morc3 binding sites. Thus, our data demonstrate Morc3 as critical regulator of Daxx-mediated H3.3 incorporation to ERV regions.

Although endogenous retroviruses (ERVs) are known to harbor cis-regulatory elements, their role in modulating cellular immune responses remains poorly understood. Using an RNA-seq approach, we show that several members of the ERV9 lineage, particularly LTR12C elements, are activated upon HIV-1 infection of primary CD4+ T cells. Intriguingly, HIV-1-induced ERVs harboring transcription start sites are primarily found in the vicinity of immunity genes. For example, HIV-1 infection activates LTR12C elements upstream of the interferon-inducible genes GBP2 and GBP5 that encode for broad-spectrum antiviral factors. Reporter assays demonstrated that these LTR12C elements drive gene expression in primary CD4+ T cells. In line with this, HIV-1 infection triggered the expression of a unique GBP2 transcript variant by activating a cryptic transcription start site within LTR12C. Furthermore, stimulation with HIV-1-induced cytokines increased GBP2 and GBP5 expression in human cells, but not in macaque cells that naturally lack the GBP5 gene and the LTR12C element upstream of GBP2. Finally, our findings suggest that GBP2 and GBP5 have already been active against ancient viral pathogens as they suppress the maturation of the extinct retrovirus HERV-K (HML-2). In summary, our findings uncover how human cells can exploit remnants of once-infectious retroviruses to regulate antiviral gene expression.

Summary The spatial organization of chromatin is known to be highly dynamic in response to environmental stress. However, it remains unknown how chromatin dynamics contributes to or modulates disease pathogenesis. Here, we show that upon influenza virus infection, the H4K20me3 methyltransferase Suv4-20h2 binds the viral protein NP, which results in the inactivation of Suv4-20h2 and the dissociation of cohesin from Suv4-20h2. Inactivation of Suv4-20h2 by viral infection or genetic deletion allows the formation of an active chromatin loop at the HoxC8-HoxC6 loci coincident with cohesin loading. HoxC8 and HoxC6 proteins in turn enhance viral replication by inhibiting the Wnt-β-catenin mediated interferon response. Importantly, loss of Suv4-20h2 augments the pathology of influenza infection in vivo. Thus, Suv4-20h2 acts as a safeguard against influenza virus infection by suppressing cohesin-mediated loop formation., Graphical abstract, Highlights • H4K20me3 methyltransferase Suv4-20h2 suppresses influenza viral replication • Influenza virus NP protein binds to Suv4-20h2 and causes dissociation from cohesin • Suv4-20h2 inactivation generates cohesion-mediated loop formation at HoxC8 -HoxC6 • HoxC8-HoxC6 enhance viral replication by suppressing Wnt/β-catenin signaling, Molecular biology; Epigenetics; Virology ; Omics

Cell identity is maintained by activation of cell-specific gene programs, regulated by epigenetic marks, transcription factors and chromatin organization. DNA G-quadruplex (G4)-folded regions in cells were reported to be associated with either increased or decreased transcriptional activity. By G4-ChIP-seq/RNA-seq analysis on liposarcoma cells we confirmed that G4s in promoters are invariably associated with high transcription levels in open chromatin. Comparing G4 presence, location and transcript levels in liposarcoma cells to available data on keratinocytes, we showed that the same promoter sequences of the same genes in the two cell lines had different G4-folding state: high transcript levels consistently associated with G4-folding. Transcription factors AP-1 and SP1, whose binding sites were the most significantly represented in G4-folded sequences, coimmunoprecipitated with their G4-folded promoters. Thus, G4s and their associated transcription factors cooperate to determine cell-specific transcriptional programs, making G4s to strongly emerge as new epigenetic regulators of the transcription machinery., G-quadruplex (G4) structures play complex roles linked to transcription regulation. Here the authors, by comparing G4 location and transcript levels in liposarcoma and keratinocyte cells, reveal that G4s cooperate with transcription factors to determine cell-specific transcriptional programs.

Objective The intestinal epithelium is a rapidly renewing tissue which plays central roles in nutrient uptake, barrier function and the prevention of intestinal inflammation. Control of epithelial differentiation is essential to these processes and is dependent on cell type-specific activity of transcription factors which bind to accessible chromatin. Here, we studied the role of SET Domain Bifurcated Histone Lysine Methyltransferase 1, also known as ESET (SETDB1), a histone H3K9 methyltransferase, in intestinal epithelial homeostasis and IBD. Design We investigated mice with constitutive and inducible intestinal epithelial deletion of Setdb1, studied the expression of SETDB1 in patients with IBD and mouse models of IBD, and investigated the abundance of SETDB1 variants in healthy individuals and patients with IBD. Results Deletion of intestinal epithelial Setdb1 in mice was associated with defects in intestinal epithelial differentiation, barrier disruption, inflammation and mortality. Mechanistic studies showed that loss of SETDB1 leads to de-silencing of endogenous retroviruses, DNA damage and intestinal epithelial cell death. Predicted loss-of-function variants in human SETDB1 were considerably less frequently observed than expected, consistent with a critical role of SETDB1 in human biology. While the vast majority of patients with IBD showed unimpaired mucosal SETDB1 expression, comparison of IBD and non-IBD exomes revealed over-representation of individual rare missense variants in SETDB1 in IBD, some of which are predicted to be associated with loss of function and may contribute to the pathogenesis of intestinal inflammation. Conclusion SETDB1 plays an essential role in intestinal epithelial homeostasis. Future work is required to investigate whether rare variants in SETDB1 contribute to the pathogenesis of IBD.

Despite important advances in the treatment of breast cancer, the 5-year survival rate for patients with distant metastasis remains less than 30%. Metastasis is a complex, multi-step process beginning with local invasion and ending with the outgrowth of systemically disseminated cells into actively proliferating metastases that ultimately cause the destruction of vital organs. It is this last step that limits patient survival and, at the same time, remains the least understood mechanistically. Here, we focus on understanding determinants of metastatic outgrowth using metastatic effusion biopsies from stage IV breast cancer patients. By modelling metastatic outgrowth through xenograft transplantation, we show that tumour initiation potential of patient-derived metastatic breast cancer cells across breast cancer subtypes is strongly linked to high levels of EPCAM expression. Breast cancer cells with high EPCAM levels are highly plastic and, upon induction of epithelial-mesenchymal transition (EMT), readily adopt mesenchymal traits while maintaining epithelial identity. In contrast, low EPCAM levels are caused by the irreversible reprogramming to a mesenchymal state with concomitant suppression of metastatic outgrowth. The ability of breast cancer cells to retain epithelial traits is tied to a global epigenetic program that limits the actions of EMT-transcription factor ZEB1, a suppressor of epithelial genes. Our results provide direct evidence that maintenance of epithelial identity is required for metastatic outgrowth while concomitant expression of mesenchymal markers enables plasticity. In contrast, loss of epithelial traits is characteristic of an irreversible mesenchymal reprogramming associated to a deficiency for metastatic outgrowth. Collectively, our data provide a framework for the intricate intercalation of mesenchymal and epithelial traits in metastatic growth.

BACKGROUND: Mononuclear phagocytes (MPs), including macrophages, monocytes, and dendritic cells (DCs), are phagocytic cells with important roles in immunity. The developmental origin of kidney DCs has been highly debated because of the large phenotypic overlap between macrophages and DCs in this tissue. METHODS: We used fate mapping, RNA sequencing, flow cytometry, confocal microscopy, and histo-cytometry to assess the origin and phenotypic and functional properties of renal DCs in healthy kidney and of DCs after cisplatin and ischemia reperfusion–induced kidney injury. RESULTS: Adult kidney contains at least four subsets of MPs with prominent Clec9a-expression history indicating a DC origin. We demonstrate that these populations are phenotypically, functionally, and transcriptionally distinct from each other. We also show these kidney MPs exhibit unique age-dependent developmental heterogeneity. Kidneys from newborn mice contain a prominent population of embryonic-derived MHCII(neg)F4/80(hi)CD11b(low) macrophages that express T cell Ig and mucin domain containing 4 (TIM-4) and MER receptor tyrosine kinase (MERTK). These macrophages are replaced within a few weeks after birth by phenotypically similar cells that express MHCII but lack TIM-4 and MERTK. MHCII(+)F4/80(hi) cells exhibit prominent Clec9a-expression history in adulthood but not early life, indicating additional age-dependent developmental heterogeneity. In AKI, MHCII(neg)F4/80(hi) cells reappear in adult kidneys as a result of MHCII downregulation by resident MHCII(+)F4/80(hi) cells, possibly in response to prostaglandin E2 (PGE2). RNA sequencing further suggests MHCII(+)F4/80(hi) cells help coordinate the recruitment of inflammatory cells during renal injury. CONCLUSIONS: Distinct developmental programs contribute to renal DC and macrophage populations throughout life, which could have important implications for therapies targeting these cells.

Novel immunotherapeutic strategies like BiTE ® (bispecific T cell engager) constructs aim to eradicate neoplastic cells by TCR-independent T-cell activation, and therefore rely on the function of autologous T cells. Currently, their efficacy is also evaluated in heavily pre-treated patients with relapsed/refractory acute myeloid leukemia (AML). Previous data demonstrated dysfunction in CD8 + T cells of AML patients (Knaus et al 2018). Thus, we aimed to characterize the progressive modulation of T-cell activity over the course of AML progression to improve the optimal application of T-cell based immunotherapeutic approaches. Bone marrow mononuclear cells (BMMCs) from AML patients at time of initial diagnosis (ID), complete remission (CR), relapse (RL), as well as of age-matched healthy donors (HD) were analyzed for T-cell subset distribution and expression of exhaustion markers by flow cytometry. Additionally, T-cell function was assessed after stimulation with 1) CD3/CD28 beads; 2) AMG 330, a CD33/CD3 specific BiTE ® construct, after incubation with OCI-AML3 target cells; or 3) AMG 330 in an autologous ex vivo long-term culture system after incubation with primary AML cells (pAML). After 6 days, T cell proliferation, expression of effector molecules and cytokines, and AMG 330-mediated T-cell cytotoxicity were assessed by flow cytometry. Lastly, we performed longitudinal bulk RNA-sequencing on 5000 sorted T cells from 7 matched ID-RL primary AML samples. Immunophenotypic analysis of BM T-cell subsets revealed a shift from T NAIVE toward central/effector memory subsets during AML progression. We observed lower percentages of T NAIVE in RL (n=3) compared to CR (n=3) CD8 + T cells(11.8 vs. 45.2%, p=0.07; RL vs. CR). Conversely, RL patients showed increased percentages of CD8 + memory T cells (T CM: 23.4 vs. 6.7%; T EM: 29.4 vs. 20.2%; T EMRA: 35.3 vs. 27.8%; RL vs. CR). Further characterization of exhaustion markers exhibited a significantly higher percentage of both CD4 + and CD8 + T cells expressing 2B4 (CD244) in ID (n=19) and RL (n=13) compared to HD (n=10, both p < 0.001). A higher percentage of PD-1 + CD8 + and TIM-3 + CD4 + T cells was detected in both ID and RL relative to HD (all p < 0.05). However, a significantly increased percentage of CD8 + T cells expressing TIM-3 and CD160 was detected in ID relative to HD (p < 0.05). Intriguingly, RL CD4 + T cells demonstrated a significantly higher level of LAG3 compared to ID (p < 0.01). In line with phenotypic exhaustion features, ID (n=4) and RL (n=5) CD8 + T cells showed reduced proliferation compared to HD (n=4) CD8 + T cells after CD3/CD28 bead stimulation (both p < 0.01). Correspondingly, we observed a marked reduction in the expression of Granzyme B (GZMB) by CD8 + T cells (both p < 0.05). Interestingly, when compared to ID, RL CD4 + T cells showed decreased TNF-α secretion (p < 0.05). In contrast to these findings, AMG 330-mediated T cell cytotoxicity against OCI-AML3 target cells was superior with RL T cells compared to ID T cells (p < 0.001). The percentage of GZMB + CD8 + T cells strikingly enhanced in RL relative to ID (p < 0.01). In an autologous setting with pAML samples, T cells from RL patients (n=6) showed higher AMG 330-mediated cytotoxicity compared to ID (n=9) T cells (67.7 vs. 35.2; RL vs. ID). In our longitudinal RNA-sequencing, differentially expressed genes analysis detected 61 up- and 30 downregulated genes (log2 FC > 1 or < -1; p < 0.01) in RL T cells compared to their matched ID counterparts. Among the significantly upregulated genes in RL, we identified genes associated with memory T cell function (TP53INP2, DUSP4) and exhaustion (NR4A1, TOX2). Moreover, Gene set enrichment analysis showed significant enrichment of gene signatures associated to memory and exhausted T cells (normalized enrichment score (NES)=1.2 and 1.3; p-value= 0.026 and 0.008, respectively), depletion of oxidative phosphorylation (NES=-2.05; p adj < 0.0001) and protein secretion (NES=-1.49; p adj < 0.05) gene signatures in RL vs. ID T cells. Taken together, our data show that patient T cells acquire an activated/exhausted phenotype upon AML progression. However, this is not reflected in the T-cell effector functions upon AMG 330 stimulation, in contrast to bead stimulation. These observations may highlight the significant role of the AML target cells in shaping a T-cell response. To this end, we will further analyze the longitudinal communication between T cells and their corresponding AML blasts. Disclosures Brauchle: Adivo: Current Employment. Kischel: Amgen GmbH Munich: Current Employment. Buecklein: BMS/Celgene: Consultancy, Research Funding; Amgen: Consultancy, Honoraria; Kite/Gilead: Consultancy, Honoraria, Other: Congress and travel support, Research Funding; Miltenyi: Research Funding; Novartis: Consultancy, Other: congress and travel support, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau. Subklewe: Novartis: Consultancy, Research Funding, Speakers Bureau; MorphoSys: Research Funding; Roche: Research Funding; Miltenyi: Research Funding; Seattle Genetics: Consultancy, Research Funding; Gilead: Consultancy, Research Funding, Speakers Bureau; BMS/Celgene: Consultancy, Research Funding, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Klinikum der Universität München: Current Employment.