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Agrobacterium tumefaciens, a pathogenic bacterium capable of transforming plants through horizontal gene transfer, is nowadays the preferred vector for plant genetic engineering. The vehicle for transfer is the T-strand, a single-stranded DNA molecule bound by the bacterial protein VirD2, which guides the T-DNA into the plant's nucleus where it integrates. How VirD2 is removed from T-DNA, and which mechanism acts to attach the liberated end to the plant genome is currently unknown. Here, using newly developed technology that yields hundreds of T-DNA integrations in somatic tissue of Arabidopsis thaliana, we uncover two redundant mechanisms for the genomic capture of the T-DNA 5' end. Different from capture of the 3' end of the T-DNA, which is the exclusive action of polymerase theta-mediated end joining (TMEJ), 5' attachment is accomplished either by TMEJ or by canonical non-homologous end joining (cNHEJ). We further find that TMEJ needs MRE11, whereas cNHEJ requires TDP2 to remove the 5' end-blocking protein VirD2. As a consequence, T-DNA integration is severely impaired in plants deficient for both MRE11 and TDP2 (or other cNHEJ factors). In support of MRE11 and cNHEJ specifically acting on the 5' end, we demonstrate rescue of the integration defect of double-deficient plants by using T-DNAs that are capable of forming telomeres upon 3' capture. Our study provides a mechanistic model for how Agrobacterium exploits the plant's own DNA repair machineries to transform it.

Pharmacogenomics (PGx)-guided drug treatment is one of the cornerstones of personalized medicine. However, the genes involved in drug response are highly complex and known to carry many (rare) variants. Current technologies (short-read sequencing and SNP panels) are limited in their ability to resolve these genes and characterize all variants. Moreover, these technologies cannot always phase variants to their allele of origin. Recent advance in long-read sequencing technologies have shown promise in resolving these problems. Here we present a long-read sequencing panel-based approach for PGx using PacBio HiFi sequencing.A capture based approach was developed using a custom panel of clinically-relevant pharmacogenes including up- and downstream regions. A total of 27 samples were sequenced and panel accuracy was determined using benchmarking variant calls for 3 Genome in a Bottle samples and GeT-RM star(*)-allele calls for 21 samples..The coverage was uniform for all samples with an average of 94% of bases covered at >30×. When compared to benchmarking results, accuracy was high with an average F1 score of 0.89 for INDELs and 0.98 for SNPs. Phasing was good with an average of 68% the target region phased (compared to ~20% for short-reads) and an average phased haploblock size of 6.6kbp. Using Aldy 4, we compared our variant calls to GeT-RM data for 8 genes (CYP2B6, CYP2C19, CYP2C9, CYP2D6, CYP3A4, CYP3A5, SLCO1B1, TPMT), and observed highly accurate star(*)-allele calling with 98.2% concordance (165/168 calls), with only one discordance inCYP2C9leading to a different predicted phenotype.We have shown that our long-read panel-based approach results in high accuracy and target phasing for SNVs as well as for clinical star(*)-alleles.

In mammalian embryos, proper zygotic genome activation (ZGA) underlies totipotent development. Double homeobox (DUX)-family factors participate in ZGA, and mouse Dux is required for forming cultured two-cell (2C)-like cells. Remarkably, in mouse embryonic stem cells, Dux is activated by the tumor suppressor p53, and Dux expression promotes differentiation into expanded-fate cell types. Long-read sequencing and assembly of the mouse Dux locus reveals its complex chromatin regulation including putative positive and negative feedback loops. We show that the p53–DUX/DUX4 regulatory axis is conserved in humans. Furthermore, we demonstrate that cells derived from patients with facioscapulohumeral muscular dystrophy (FSHD) activate human DUX4 during p53 signaling via a p53-binding site in a primate-specific subtelomeric long terminal repeat (LTR)10C element. In summary, our work shows that p53 activation convergently evolved to couple p53 to Dux/DUX4 activation in embryonic stem cells, embryos and cells from patients with FSHD, potentially uniting the developmental and disease regulation of DUX-family factors and identifying evidence-based therapeutic opportunities for FSHD. p53 activates Dux in mouse embryos and embryonic stem cells, as well as DUX4 in human facioscapulohumeral muscular dystrophy cell models.

We report a patient case with pseudomembranous colitis associated with a mono-toxin producingClostridioides difficilebelonging to the very rarely diagnosed PCR ribotype (RT) 151. The infection was difficult to diagnose, since the isolate and the feces sample tested negative for toxin-encoding genes using a routine commercial test. This prompted us to sequence n = 11 RT151s from various geographical regions to study their genomic characteristics and relatedness. By including whole genome sequence data from other sources, we could further place these isolates into the phylogenetic tree ofC. difficileand assign them to their respective clades. These analyses revealed that 1) RT151s are polyphyletic with isolates falling into clades 1, and cryptic clades C-I and C-II 2) RT151 contains both non-toxigenic and toxigenic isolates and 3) RT151 C-II isolates contained mono-toxin pathogenicity loci (PaLoc). Additional analysis with PacBio circular consensus sequencing revealed that the isolate from our patient case report contains a novel PaLoc insertion site, lackedtcdAand a had significantly divergenttcdBsequence that might explain the failure of the diagnostic test. The study is noteworthy as 1) RT151 encompasses both typical and cryptic clades and 2) conclusive evidence for CDI due to clade C-II isolates was hitherto lacking.

A subset of clinical isolates of Clostridioides difficile contains one or more plasmids and these plasmids can harbor virulence and antimicrobial resistance determinants. Despite their potential importance, C. difficile plasmids remain poorly characterized. Here, we describe a human clinical isolate that carries three plasmids from three different plasmid families that are therefore compatible. For two of these, we identify a region capable of sustaining plasmid replication in C. difficile. Together, our data advance our understanding of C. difficile plasmid biology.HighlightsThe complete circular genome sequence is provided for a C. difficile isolate harboring three plasmidsThese three plasmids (pJMR5-1, pJMR5-4 and pJRM5-W) are therefore compatible in a single strainSequence analysis suggest a modular nature of plasmid families to which the pJMR-plasmids belongA functional replicon was cloned from pJMR5-1 (pCD-ECE1 family) and pJMR5-W (pCD-WTSI1 family) and plasmids carrying this replicon are compatible with plasmid pCD630

We report a patient case of pseudomembranous colitis associated with a monotoxin-producing Clostridioides difficile belonging to the very rarely diagnosed polymerase chain reaction (PCR) ribotype (RT) 151. To understand why this isolate was not identified using a routine commercial test, we performed a genomic analysis of RT151.Illumina short-read sequencing was performed on n = 11 RT151s from various geographical regions to study their genomic characteristics and relatedness. Subsequently, we used PacBio circular consensus sequencing to determine the complete genome sequence of isolates belonging to cryptic clades C-I and C-II, which includes the peatient isolate.We found that 1) RT151s are polyphyletic with isolates falling into clades 1 and cryptic clades C-I and C-II; 2) RT151 contains both nontoxigenic and toxigenic isolates and 3) RT151 C-II isolates contained monotoxin pathogenicity loci. The isolate from our patient case report contains a novel-pathogenicity loci insertion site, lacked tcdA and had a divergent tcdB sequence that might explain the failure of the diagnostic test.This study shows that RT151 encompasses both typical and cryptic clades and provides conclusive evidence for C. difficile infection due to clade C-II isolates that was hitherto lacking. Vigilance towards C. difficile infection as a result of cryptic clade isolates is warranted.

The evolution of a placenta is predicted to be accompanied by rapid evolution of genes involved in processes that regulate mother–offspring interactions during pregnancy, such as placenta formation, embryonic development, and nutrient transfer to offspring. However, these predictions have only been tested in mammalian species, where only a single instance of placenta evolution has occurred. In this light, the genus Poeciliopsis is a particularly interesting model for placenta evolution, because in this genus a placenta has evolved independently from the mammalian placenta. Here, we present and compare genome assemblies of two species of the livebearing fish genus Poeciliopsis (family Poeciliidae) that differ in their reproductive strategy: Poeciliopsis retropinna which has a well-developed complex placenta and P. turrubarensis which lacks a placenta. We applied different assembly strategies for each species: PacBio sequencing for P. retropinna (622-Mb assembly, scaffold N50 of 21.6 Mb) and 10× Genomics Chromium technology for P. turrubarensis (597-Mb assembly, scaffold N50 of 4.2 Mb). Using the high contiguity of these genome assemblies and near-completeness of gene annotations to our advantage, we searched for gene duplications and performed a genome-wide scan for genes evolving under positive selection. We find rapid evolution in major parts of several molecular pathways involved in parent–offspring interaction in P. retropinna, both in the form of gene duplications as well as positive selection. We conclude that the evolution of the placenta in the genus Poeciliopsis is accompanied by rapid evolution of genes involved in similar genomic pathways as found in mammals.

Inserting large DNA payloads (>10 kb) into specific genomic sites of mammalian cells remains challenging. Applications ranging from synthetic biology to evaluating the pathogenicity of disease-associated variants for precision medicine initiatives would greatly benefit from tools that facilitate this process. Here, we merge the strengths of different classes of site-specific recombinases and combine these with CRISPR-Cas9-mediated homologous recombination to develop a strategy for stringent site-specific replacement of genomic fragments at least 50 kb in size in human induced pluripotent stem cells (hiPSCs). We demonstrate the versatility of STRAIGHT-IN (serine and tyrosine recombinase-assisted integration of genes for high-throughput investigation) by (1) inserting various combinations of fluorescent reporters into hiPSCs to assess the excitation-contraction coupling cascade in derivative cardiomyocytes and (2) simultaneously targeting multiple variants associated with inherited cardiac arrhythmic disorders into a pool of hiPSCs. STRAIGHT-IN offers a precise approach to generate genetically matched panels of hiPSC lines efficiently and cost effectively.

Agrobacterium tumefaciens, a pathogenic bacterium capable of transforming plants through horizontal gene transfer, is nowadays the preferred vector for plant genetic engineering. The vehicle for transfer is the T-strand, a single-stranded DNA molecule bound by the bacterial protein VirD2, which guides the T-DNA into the plant's nucleus where it integrates. How VirD2 is removed from T-DNA, and which mechanism acts to attach the liberated end to the plant genome is currently unknown. Here, using newly developed technology that yields hundreds of T-DNA integrations in somatic tissue of Arabidopsis thaliana, we uncover two redundant mechanisms for the genomic capture of the T-DNA 5' end. Different from capture of the 3' end of the T-DNA, which is the exclusive action of polymerase theta-mediated end joining (TMEJ), 5' attachment is accomplished either by TMEJ or by canonical non-homologous end joining (cNHEJ). We further find that TMEJ needs MRE11, whereas cNHEJ requires TDP2 to remove the 5' end-blocking protein VirD2. As a consequence, T-DNA integration is severely impaired in plants deficient for both MRE11 and TDP2 (or other cNHEJ factors). In support of MRE11 and cNHEJ specifically acting on the 5' end, we demonstrate rescue of the integration defect of double-deficient plants by using T-DNAs that are capable of forming telomeres upon 3' capture. Our study provides a mechanistic model for how Agrobacterium exploits the plant's own DNA repair machineries to transform it.

Immune homeostasis is essential to protect mucosal airway surfaces from unnecessary and damaging inflammation against inhaled harmless environmental antigens, such as allergens. However, in allergic individuals this protective homeostatic response seems to be absent. Innate cells that are part of the mononuclear phagocytic system (MPS) play an important role in these processes. Most of our knowledge on allergic immune responses comes from animal models or from peripheral blood immune responses in humans. Information on tissue-specific responses in humans is scarce, however allergen-specific immune responses are initiated locally and this information is therefore crucial for the development of novel therapies. In this study we performed mass-cytometry proteomics and single cell RNA sequencing on immune cells from nasal biopsies of allergic rhinitis subjects and non-allergic controls, before and three days after repeated nasal challenge with House Dust Mite allergen. Following challenge, patients displayed a clinical nasal response together with enhanced eosinophilia, a cardinal feature of allergic inflammation. Although clinically silent, we observed a distinct, local, innate immune response to allergen in non-allergic individuals, characterized by infiltration of HLA-DRlowCD14+ monocytes expressing anti-microbial genes (S100A8, S100A9, S100A12) as well as transcriptional activation in cDC2, including several inhibitory/tolerogenic genes (NR4A1, IL4I1, ANXA2, TIMP1). The innate response in allergic individuals indicated an inflammatory role for infiltrating HLA-DRhi CD14+ monocytes, CD16+ monocytes, and CD1A+ cDC2 (ALOX15, CD1A, CCL17), in the development/maintenance of an allergic response. Future therapies should address those innate MPS populations, either enhancing or reducing their activity for the treatment of inflammatory airway disease.

Background Drawing genotype-to-phenotype maps in tumors is of paramount importance for understanding tumor heterogeneity. Assignment of single cells to their tumor clones of origin can be approached by matching the genotypes of the clones to the mutations found in RNA sequencing of the cells. The confidence of the cell-to-clone mapping can be increased by accounting for additional measurements. Follicular lymphoma, a malignancy of mature B cells that continuously acquire mutations in parallel in the exome and in B cell receptor loci, presents a unique opportunity to join exome-derived mutations with B cell receptor sequences as independent sources of evidence for clonal evolution. Methods Here, we propose CACTUS, a probabilistic model that leverages the information from an independent genomic clustering of cells and exploits the scarce single cell RNA sequencing data to map single cells to given imperfect genotypes of tumor clones. Results We apply CACTUS to two follicular lymphoma patient samples, integrating three measurements: whole exome, single-cell RNA, and B cell receptor sequencing. CACTUS outperforms a predecessor model by confidently assigning cells and B cell receptor-based clusters to the tumor clones. Conclusions The integration of independent measurements increases model certainty and is the key to improving model performance in the challenging task of charting the genotype-to-phenotype maps in tumors. CACTUS opens the avenue to study the functional implications of tumor heterogeneity, and origins of resistance to targeted therapies. CACTUS is written in R and source code, along with all supporting files, are available on GitHub (https://github.com/LUMC/CACTUS).

Differentiation of mammalian pluripotent cells involves large-scale changes in transcription and, among the molecules that orchestrate these changes, chromatin remodellers are essential to initiate, establish and maintain a new gene regulatory network. The NuRD complex is a highly conserved chromatin remodeller which fine-tunes gene expression in embryonic stem cells. While the function of NuRD in mouse pluripotent cells has been well defined, no study yet has defined NuRD function in human pluripotent cells. We investigated the structure and function of NuRD in human induced pluripotent stem cells (hiPSCs). Using immunoprecipitation followed by mass-spectrometry in hiPSCs and in naive or primed mouse pluripotent stem cells, we find that NuRD structure and biochemical interactors are generally conserved. Using RNA sequencing, we find that, whereas in mouse primed stem cells and in mouse naïve ES cells, NuRD is required for an appropriate level of transcriptional response to differentiation signals, hiPSCs require NuRD to initiate these responses. This difference indicates that mouse and human cells interpret and respond to induction of differentiation differently.Graphical AbstractNuRD acts like a conductor in an orchestra.A. In the presence of NuRD (pink blob figure, centre) differentiation occurs in an ordered fashion in both mouse (left) and human (right) ES cells. Gene expression changes in both cell types are tightly controlled with down-regulation of pluripotency genes and up-regulation of lineage appropriate genes. This is akin to a group of musicians producing musical notes in the right order and at the right amplitude to create a coherent piece of music. B. Loss of “the conductor” NuRD results in increased transcriptional noise in both systems, indicated here as a low-level blanket of sound in both systems. Consequences of MBD3/NuRD loss differs between human and mouse ES cells. In mouse ES cells, differentiation cues lead to some down-regulation of pluripotency genes and incomplete progression along a lineage appropriate pathway. This is like musicians who know that they should be making music but who lose their way without a conductor’s influence. In human iPS cells the background level of noise without NuRD results in a lack of order to gene expression changes in response to differentiation. The noise from these “musicians” would be truly awful.

Endocrine therapy is important for management of patients with estrogen receptor (ER)–positive breast cancer; however, positive ER staining does not reliably predict therapy response. We assessed the potential to improve prediction of response to endocrine treatment of a novel test that quantifies functional ER pathway activity from mRNA levels of ER pathway–specific target genes. ER pathway activity was assessed on datasets from three neoadjuvant-treated ER-positive breast cancer patient cohorts: Edinburgh: 3-month letrozole, 55 pre-/2-week/posttreatment matched samples; TEAM IIa: 3- to 6-month exemestane, 49 pre-/28 posttreatment paired samples; and NEWEST: 16-week fulvestrant, 39 pretreatment samples. ER target gene mRNA levels were measured in fresh-frozen tissue (Edinburgh, NEWEST) with Affymetrix microarrays, and in formalin-fixed paraffin-embedded samples (TEAM IIa) with qRT-PCR. Approximately one third of ER-positive patients had a functionally inactive ER pathway activity score (ERPAS), which was associated with a nonresponding status. Quantitative ERPAS decreased significantly upon therapy (P < 0.001 Edinburgh and TEAM IIa). Responders had a higher pretreatment ERPAS and a larger 2-week decrease in activity (P = 0.02 Edinburgh). Progressive disease was associated with low baseline ERPAS (P = 0.03 TEAM IIa; P = 0.02 NEWEST), which did not decrease further during treatment (P = 0.003 TEAM IIa). In contrast, the staining-based ER Allred score was not significantly associated with therapy response (P = 0.2). The ERPAS identified a subgroup of ER-positive patients with a functionally inactive ER pathway associated with primary endocrine resistance. Results confirm the potential of measuring functional ER pathway activity to improve prediction of response and resistance to endocrine therapy.

The nucleosome remodeling and deacetylase (NuRD) complex plays an important role in gene expression regulation, stem cell self-renewal, and lineage commitment. However, little is known about the dynamics of NuRD during cellular differentiation. Here, we study these dynamics using genome-wide profiling and quantitative interaction proteomics in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We find that the genomic targets of NuRD are highly dynamic during differentiation, with most binding occurring at cell-type specific promoters and enhancers. We identify ZFP296 as an ESC-specific NuRD interactor that also interacts with the SIN3A complex. ChIP-sequencing in Zfp296 knockout (KO) ESCs reveals decreased NuRD binding both genome-wide and at ZFP296 binding sites, although this has little effect on the transcriptome. Nevertheless, Zfp296 KO ESCs exhibit delayed induction of lineage-specific markers upon differentiation to embryoid bodies. In summary, we identify an ESC-specific NuRD-interacting protein which regulates genome-wide NuRD binding and cellular differentiation., The NuRD complex plays an important role in regulating lineage commitment and cell fate during early embryonic development. Here the authors present an integrative analysis of MBD3/NuRD composition and binding in mouse embryonic stem cells and neural progenitor cells, providing a molecular basis for genome-wide NuRD localization

Introduction Follicular Lymphoma (FL) is one of the most prevalent B-cell neoplasms and despite recent advances remains incurable in most cases. FL cells are malignant counterparts of normal germinal center B-cells. At molecular level FL are characterized by the t(14;18) which results in the overexpression of the BCL-2. These apoptosis resistant cells accumulate somatic mutations associated with tumorigenesis and progression. Whereas several mutagenic mechanisms shape the FL genomic landscape, up to 22% of the mutations may be attributed to activation-induced cytidine deaminase (AID) activity. Under physiological conditions AID is responsible for somatic hypermutation (SHM) in immunoglobulin genes (IG) of germinal center B-cells. In fact, it is accepted that ongoing SHM still occurs at relatively high rates in FL cells as compared with other germinal center lymphoid neoplasms. Moreover, we recently demonstrated the direct effect of AID overexpression inducing somatic mutations driving murine and human B-cell neoplasm progression in vivo. Therefore, unveiling the molecular basis of AID activity in lymphoma cells remains essential to understand lymphomagenesis and to develop novel targeted therapies. The advent of single-cell high-throughput sequencing has enabled the analysis of molecular events at an unprecedented resolution. Therefore, we designed a study to capture AID-induced somatic hypermutation at the single cell level in FL. Methods Tumor samples derived from 14 FL patients were analyzed, as controls, we chose a B-cell malignancy with lower SHM rates and included 5 samples derived from chronic lymphocytic leukemia (CLL) and 2 from monoclonal B lymphocytosis (MBL). Single cell whole cDNA libraries were obtained by 10X Genomics. Immunoglobulin gene single cell libraries were prepared by enrichment with seminested amplification using 3 ′ constant domain primers followed by 10X Genomics prep. Both single-cell libraries were sequenced in paired-end mode (2 × 150 bp) on an Illumina Hiseq platform. We developed an immunoglobulin alignment tool to enable the analysis of highly mutated IG sequences derived from FL. A consensus sequence was obtained by transcript associated with a unique molecular identifier (UMI) for every cell. Then, the presence of variants in the IG by position in a particular cell was annotated. These observations were filtered using quality parameters (read depth => 25 , frequency of the event => 20%, UMIs supporting every variant => 5). Results We analyzed an average of 926 cells per case. Our data confirms at the single cell level previous reports on high clonal heterogeneity and hypermutation rate (mean 18.7%) in FL. When analyzing intracellular heterogeneity we observed single FL cells expressing simultaneously transcripts derived from the same immunoglobulin VDJ rearrangement but displaying high confidence single nucleotide variants. After applying strict filtering strategies to account for potential technical issues we defined the occurrence of "SHM snapshot events'' when a single cell displayed a set of transcripts from a particular VDJ rearrangement with and without the occurring single nucleotide variant. Such events were detected in 8 of the 14 FL analyzed samples. In those 8 samples, 114 events were observed in which two different transcripts of one specific IG were found within a single cell. AID-related motifs (WRCY, WA and RCG) were found in 45% of the SHM snapshot events. SHM snapshot events were undetectable in CLL (5 samples) and 3 SHM snapshot events were detected in MBL (2 samples). In addition, the occurrence of SHM snapshot events was significantly associated with AID expression (Fisher exact test, p-value < 0.001). On the other hand, AID expression was not detectable on CLL and MBL samples. Conclusion Here we report for the first time the occurrence of ongoing somatic hypermutation at a single cell level in FL. The simultaneous detection of both the pre and post mutation IG mRNA transcripts within a single cell may be indicative of SHM occurring more recently than the lifespan of mRNA transcript. The detection of this phenomenon in 57% of FL samples and its association with AID expression suggests that AID-induced mutagenesis may be acting at a much higher rate than expected. This work highlights the role of AID in shaping the tumor heterogeneity in FL and the need to further understand the role of this enzyme in lymphomagenesis and tumor progression. Disclosures No relevant conflicts of interest to declare.

International audience; The pluripotent ground state is defined as a basal state free of epigenetic restrictions, which influence lineage specification. While naive embryonic stem cells (ESCs) can be maintained in a hypomethylated state with open chromatin when grown using two small-molecule inhibitors (2i)/leukemia inhibitory factor (LIF), in contrast to serum/LIF-grown ESCs that resemble early post-implantation embryos, broader features of the ground-state pluripotent epigenome are not well understood. We identified epigenetic features of mouse ESCs cultured using 2i/LIF or serum/LIF by proteomic profiling of chromatin-associated complexes and histone modifications. Polycomb-repressive complex 2 (PRC2) and its product H3K27me3 are highly abundant in 2i/LIF ESCs, and H3K27me3 is distributed genome-wide in a CpG-dependent fashion. Consistently, PRC2-deficient ESCs showed increased DNA methylation at sites normally occupied by H3K27me3 and increased H4 acetylation. Inhibiting DNA methylation in PRC2-deficient ESCs did not affect their viability or transcriptome. Our findings suggest a unique H3K27me3 configuration protects naive ESCs from lineage priming, and they reveal widespread epigenetic crosstalk in ground-state pluripotency.

Multiprotein chromatin remodelling complexes show remarkable conservation of function amongst metazoans, even though components present in invertebrates are often found as multiple paralogous proteins in vertebrate complexes. In some cases, these paralogues specify distinct biochemical and/or functional activities in vertebrate cells. Here, we set out to define the biochemical and functional diversity encoded by one such group of proteins within the mammalian Nucleosome Remodelling and Deacetylation (NuRD) complex: Mta1, Mta2 and Mta3. We find that, in contrast to what has been described in somatic cells, MTA proteins are not mutually exclusive within embryonic stem (ES) cell NuRD and, despite subtle differences in chromatin binding and biochemical interactions, serve largely redundant functions. ES cells lacking all three MTA proteins exhibit complete NuRD loss of function and are viable, allowing us to identify a previously unreported function for NuRD in reducing transcriptional noise, which is essential for maintaining a proper differentiation trajectory during early stages of lineage commitment.

Item does not contain fulltext Facioscapulohumeral muscular dystrophy (FSHD) is characterized by sporadic de-repression of the transcription factor DUX4 in skeletal muscle. DUX4 activates a cascade of muscle disrupting events, eventually leading to muscle atrophy and apoptosis. Yet, how sporadic DUX4 expression leads to the generalized muscle wasting remains unclear. Transcriptome analyses have systematically been challenged by the majority of nuclei being DUX4neg, weakening the DUX4 transcriptome signature. Moreover, DUX4 has been shown to be expressed in a highly dynamic burst-like manner, likely resulting in the detection of the downstream cascade of events long after DUX4 expression itself has faded. Identifying the FSHD transcriptome in individual cells and unraveling the cascade of events leading to FSHD development may therefore provide important insights in the disease process. We employed single-cell RNA sequencing, combined with pseudotime trajectory modeling, to study FSHD disease etiology and cellular progression in human primary myocytes. We identified a small FSHD-specific cell population in all tested patient-derived cultures and detected new genes associated with DUX4 de-repression. We furthermore generated an FSHD cellular progression model, reflecting both the early burst-like DUX4 expression as well as the downstream activation of various FSHD-associated pathways, which allowed us to correlate DUX4 expression signature dynamics with that of regulatory complexes, thereby facilitating the prioritization of epigenetic targets for DUX4 silencing. Single-cell transcriptomics combined with pseudotime modeling thus holds valuable information on FSHD disease etiology and progression that can potentially guide biomarker and target selection for therapy.

Although the core subunits of Polycomb group (PcG) complexes are well characterized, little is known about the dynamics of these protein complexes during cellular differentiation. We used quantitative interaction proteomics and genome-wide profiling to study PcG proteins in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We found that the stoichiometry and genome-wide binding of PRC1 and PRC2 were highly dynamic during neural differentiation. Intriguingly, we observed a downregulation and loss of PRC2 from chromatin marked with trimethylated histone H3 K27 (H3K27me3) during differentiation, whereas PRC1 was retained at these sites. Additionally, we found PRC1 at enhancer and promoter regions independently of PRC2 binding and H3K27me3. Finally, overexpression of NPC-specific PRC1 interactors in ESCs led to increased Ring1b binding to, and decreased expression of, NPC-enriched Ring1b-target genes. In summary, our integrative analyses uncovered dynamic PcG subcomplexes and their widespread colocalization with active chromatin marks during differentiation.

Graphical abstract, Differentiation of mammalian pluripotent cells involves large-scale changes in transcription and, among the molecules that orchestrate these changes, chromatin remodellers are essential to initiate, establish and maintain a new gene regulatory network. The Nucleosome Remodelling and Deacetylation (NuRD) complex is a highly conserved chromatin remodeller which fine-tunes gene expression in embryonic stem cells. While the function of NuRD in mouse pluripotent cells has been well defined, no study yet has defined NuRD function in human pluripotent cells. Here we find that while NuRD activity is required for lineage commitment from primed pluripotency in both human and mouse cells, the nature of this requirement is surprisingly different. While mouse embryonic stem cells (mESC) and epiblast stem cells (mEpiSC) require NuRD to maintain an appropriate differentiation trajectory as judged by gene expression profiling, human induced pluripotent stem cells (hiPSC) lacking NuRD fail to even initiate these trajectories. Further, while NuRD activity is dispensable for self-renewal of mESCs and mEpiSCs, hiPSCs require NuRD to maintain a stable self-renewing state. These studies reveal that failure to properly fine-tune gene expression and/or to reduce transcriptional noise through the action of a highly conserved chromatin remodeller can have different consequences in human and mouse pluripotent stem cells.

H3K27ac is associated with regulatory active enhancers, but its exact role in enhancer function remains elusive. Using mass spectrometry-based interaction proteomics, we identified the Super Elongation Complex (SEC) and GBAF, a non-canonical GLTSCR1L- and BRD9-containing SWI/SNF chromatin remodeling complex, to be major interactors of H3K27ac. We systematically characterized the composition of GBAF and the conserved GLTSCR1/1L ‘GiBAF’-domain, which we found to be responsible for GBAF complex formation and GLTSCR1L nuclear localization. Inhibition of the bromodomain of BRD9 revealed interaction between GLTSCR1L and H3K27ac to be BRD9-dependent and led to GLTSCR1L dislocation from its preferred binding sites at H3K27ac-associated enhancers. GLTSCR1L disassociation from chromatin resulted in genome-wide downregulation of enhancer transcription while leaving most mRNA expression levels unchanged, except for reduced mRNA levels from loci topologically linked to affected enhancers. Our results indicate that GBAF is an enhancer-associated chromatin remodeler important for transcriptional and regulatory activity of enhancers.Graphical abstract

Multiprotein chromatin remodelling complexes show remarkable conservation of function amongst metazoans, even though components present in invertebrates are often present as multiple paralogous proteins in vertebrate complexes. In some cases these paralogues specify distinct biochemical and/or functional activities in vertebrate cells. Here we set out to define the biochemical and functional diversity encoded by one such group of proteins within the mammalian Nucleosome Remodelling and Deacetylation (NuRD) complex: Mta1, Mta2 and Mta3. We find that, in contrast to what has been described in somatic cells, MTA proteins are not mutually exclusive within ES cell NuRD and, despite subtle differences in chromatin binding and biochemical interactions, serve largely redundant functions. Nevertheless, ES cells lacking all three MTA proteins represent a complete NuRD null and are viable, allowing us to identify a previously undetected function for NuRD in maintaining differentiation trajectory during early stages of lineage commitment.

Objectives: Follicular lymphoma (FL) typically originates from premalignant mature B cells that carry the founder t(14;18) BCL2 translocation. Mutations in epigenetic modifiers and acquisition of N-glycosylation sites in CDR regions of the B-cell receptor (BCR) are recurrent secondary events in FL pathogenesis. Despite these oncogenic drivers, FL can remain indolent and clinically stable for years. The molecular events driving subclonal evolution into symptomatic progression and eventual transformation to aggressive lymphoma are insufficiently understood. FL cells are frozen in their B-cell development at the germinal center stage and undergo continuous somatic hypermutation mediated by expression of activation-induced deaminase (AID). We aim to identify crucial drivers of subclonal FL evolution by high-throughput mapping at single-cell resolution. Methods: Viable FL cells were isolated and cryopreserved from 23 histologically or immunocytologically confirmed FL samples from 13 patients with informed consent. Full-length VDJ/VJ transcripts were isolated by unbiased template-switching ARTISAN PCR and massive parallel NGS sequencing on the PacBio platform. The clonal primordial FL BCR (pBCR) was reconstructed from unmutated IGV/IGJ sequences with the CDR3 of the least mutated BCR. Since the IgTree program was unable to process the obtained numbers of BCR sequences, we developed the WILLOW algorithm for analysis of BCR evolution based on the principle of maximum parsimony and on distance from the pBCR. Intraclonal BCR variability was quantified by Shannon's diversity index. 5' single cell transcriptomics and VDJ/VJ sequencing was performed on 2 pools of highly purified FL cells from 5 lymph node biopsies on the 10x Genomics platform. Data were deconvoluted based on expressed variants by the Single Cell Sample Matcher (SCSM) algorithm. Clustering based on gene expression profiles was performed by shared nearest neighbour (SNN) modularity optimization within the R Seurat package. Genes whose expression differed significantly (adjusted p Results: ARTISAN PCR/PacBio NGS yielded a median of 743 full-length VDJ and VJ sequences (range 62-12782) per BCR chain with expected high intraclonal diversity (median 200 subclones, range 15-3301). WILLOW revealed dominant FL subclones with a subclonal hierarchy wherein multiple routes converged to offspring nodes with identical additional mutations rather than tree-like branching (Figure). In serial samples of 4 patients, lymph node biopsies had only marginally higher subclonal diversity than blood or bone marrow samples (p=0,055; Wilcoxon's matched-pairs signed rank test). Overall BCR mutational burden increased over time in sequential biopsies. Two cases of histological FL transformation were dominated by a single subclone (65% and 80% of all VDJ/VJ sequences, respectively) that was rare in the preceding FL BCR network (0.2% and 1.8%). Pooled transcriptomics data from 6050-6500 cells were assigned to individual samples by SCSM and revealed up to seven transcriptional clusters per FL. In 9 of 10 FL, genes assigned to immune function strongly contributed to separation into one or more clusters. Single cell VDJ/VJ sequencing yielded combined heavy and light chain BCR sequences for a median of 502 FL cells per biopsy (range 22 - 1919) that permitted mapping of subclonal evolution by WILLOW based on complete BCR information. Transcriptome clusters were not distributed evenly throughout the WILLOW FL BCR networks but rather statistically associated with distinct major FL subclones. Vice versa, major FL subclones within the same biopsy were distinguished by particular gene expression profiles. Conclusions: WILLOW facilitates mapping of subclonal FL evolution based on high-throughput BCR sequencing. FL evolution proceeds in networks rather than tree-like branching, whereby acquisition of certain combinations of several BCR mutations can occur in parallel in different trajectories. Transcriptomic profiling of single FL cells identifies distinct clusters within a single biopsy. Mapping of these clusters to the FL cell position in the subclonal FL evolutionary network identifies putative mechanisms that are associated with subclonal progression. These mechanisms involve physiological B-cell signalling pathways. Figure Disclosures No relevant conflicts of interest to declare.

Background: The NuRD complex controls gene expression through altering chromatin structure. Results: The MTA1-RbAp48 structure shows how the RbAp46/p48 histone chaperones are recruited to NuRD. Conclusion: The MTA subunits act as scaffolds for NuRD complex assembly. Significance: The MTA/RbAp48 interaction prevents binding of histone H4, which is crucial for understanding the role of the RbAp46/p48 chaperones in the complex., The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex.

The largest transcription factor IID (TFIID) subunit, TBP-associated factor 1 (TAF1), possesses protein kinase and histone acetyltransferase (HAT) activities. Both enzymatic activities are essential for transcription from a subset of genes and G(1) progression in mammalian cells. TAF7, another TFIID subunit, binds TAF1 and inhibits TAF1 HAT activity. Here we present data demonstrating that disruption of the TAF1/TAF7 interaction within TFIID by protein phosphorylation leads to activation of TAF1 HAT activity and stimulation of cyclin D1 and cyclin A gene transcription. Overexpression and small interfering RNA knockdown experiments confirmed that TAF7 functions as a transcriptional repressor at these promoters. Release of TAF7 from TFIID by TAF1 phosphorylation of TAF7 increased TAF1 HAT activity and elevated histone H3 acetylation levels at the cyclin D1 and cyclin A promoters. Serine-264 of TAF7 was identified as a substrate for TAF1 kinase activity. Using TAF7 S264A and S264D phosphomutants, we determined that the phosphorylation state of TAF7 at S264 influences the levels of cyclin D1 and cyclin A gene transcription and promoter histone H3 acetylation. Our studies have uncovered a novel function for the TFIID subunit TAF7 as a phosphorylation-dependent regulator of TAF1-catalyzed histone H3 acetylation at the cyclin D1 and cyclin A promoters.

Background Forward genetic screens in mice provide an unbiased means to identify genes and other functional genetic elements in the genome. Previously, a large scale ENU mutagenesis screen was conducted to query the functional content of a ~50 Mb region of the mouse genome on proximal Chr 5. The majority of phenotypic mutants recovered were embryonic lethals. Results We report the high resolution genetic mapping, complementation analyses, and positional cloning of mutations in the target region. The collection of identified alleles include several with known or presumed functions for which no mutant models have been reported (Tbc1d14, Nol14, Tyms, Cad, Fbxl5, Haus3), and mutations in genes we or others previously reported (Tapt1, Rest, Ugdh, Paxip1, Hmx1, Otoe, Nsun7). We also confirmed the causative nature of a homeotic mutation with a targeted allele, mapped a lethal mutation to a large gene desert, and localized a spermiogenesis mutation to a region in which no annotated genes have coding mutations. The mutation in Tbc1d14 provides the first implication of a critical developmental role for RAB-GAP-mediated protein transport in early embryogenesis. Conclusion This collection of alleles contributes to the goal of assigning biological functions to all known genes, as well as identifying novel functional elements that would be missed by reverse genetic approaches.