The role of RNA in the maintenance of chromatin domains as revealed by antibody-mediated proximity labelling coupled to mass spectrometry.

Eukaryotic chromatin is organized into functional domains, that are characterized by distinct proteomic compositions and specific nuclear positions. In contrast to cellular organelles surrounded by lipid membranes, the composition of distinct chromatin domains is rather ill described and highly dynamic. To gain molecular insight into these domains and explore their composition, we developed an antibody-based proximity biotinylation method targeting the RNA and proteins constituents. The method that we termed antibody-mediated proximity labelling coupled to mass spectrometry (AMPL-MS) does not require the expression of fusion proteins and therefore constitutes a versatile and very sensitive method to characterize the composition of chromatin domains based on specific signature proteins or histone modifications. To demonstrate the utility of our approach we used AMPL-MS to characterize the molecular features of the chromocenter as well as the chromosome territory containing the hyperactive X chromosome in Drosophila. This analysis identified a number of known RNA-binding proteins in proximity of the hyperactive X and the centromere, supporting the accuracy of our method. In addition, it enabled us to characterize the role of RNA in the formation of these nuclear bodies. Furthermore, our method identified a new set of RNA molecules associated with the Drosophila centromere. Characterization of these novel molecules suggested the formation of R-loops in centromeres, which we validated using a novel probe for R-loops in Drosophila. Taken together, AMPL-MS improves the selectivity and specificity of proximity ligation allowing for novel discoveries of weak protein-RNA interactions in biologically diverse domains.

Alterations in the hepatocyte epigenetic landscape in steatosis.

Fatty liver disease or the accumulation of fat in the liver, has been reported to affect the global population. This comes with an increased risk for the development of fibrosis, cirrhosis, and hepatocellular carcinoma. Yet, little is known about the effects of a diet containing high fat and alcohol towards epigenetic aging, with respect to changes in transcriptional and epigenomic profiles. In this study, we took up a multi-omics approach and integrated gene expression, methylation signals, and chromatin signals to study the epigenomic effects of a high-fat and alcohol-containing diet on mouse hepatocytes. We identified four relevant gene network clusters that were associated with relevant pathways that promote steatosis. Using a machine learning approach, we predict specific transcription factors that might be responsible to modulate the functionally relevant clusters. Finally, we discover four additional CpG loci and validate aging-related differential CpG methylation. Differential CpG methylation linked to aging showed minimal overlap with altered methylation in steatosis.

CRUP: a comprehensive framework to predict condition-specific regulatory units.

We present the software Condition-specific Regulatory Units Prediction (CRUP) to infer from epigenetic marks a list of regulatory units consisting of dynamically changing enhancers with their target genes. The workflow consists of a novel pre-trained enhancer predictor that can be reliably applied across cell types and species, solely based on histone modification ChIP-seq data. Enhancers are subsequently assigned to different conditions and correlated with gene expression to derive regulatory units. We thoroughly test and then apply CRUP to a rheumatoid arthritis model, identifying enhancer-gene pairs comprising known disease genes as well as new candidate genes.

PHF13: A new player involved in RNA polymerase II transcriptional regulation and co-transcriptional splicing.

We recently identified PHF13 as an H3K4me2/3 chromatin reader and transcriptional co-regulator. We found that PHF13 interacts with RNAPIIS5P and PRC2 stabilizing their association with active and bivalent promoters. Furthermore, mass spectrometry analysis identified ∼50 spliceosomal proteins in PHF13s interactome. Here, we will discuss the potential role of PHF13 in RNAPII pausing and co-transcriptional splicing.

Epigenomic Profiling of Human CD4+ T Cells Supports a Linear Differentiation Model and Highlights Molecular Regulators of Memory Development.

The impact of epigenetics on the differentiation of memory T (Tmem) cells is poorly defined. We generated deep epigenomes comprising genome-wide profiles of DNA methylation, histone modifications, DNA accessibility, and coding and non-coding RNA expression in naive, central-, effector-, and terminally differentiated CD45RA+ CD4+ Tmem cells from blood and CD69+ Tmem cells from bone marrow (BM-Tmem). We observed a progressive and proliferation-associated global loss of DNA methylation in heterochromatic parts of the genome during Tmem cell differentiation. Furthermore, distinct gradually changing signatures in the epigenome and the transcriptome supported a linear model of memory development in circulating T cells, while tissue-resident BM-Tmem branched off with a unique epigenetic profile. Integrative analyses identified candidate master regulators of Tmem cell differentiation, including the transcription factor FOXP1. This study highlights the importance of epigenomic changes for Tmem cell biology and demonstrates the value of epigenetic data for the identification of lineage regulators.

reChIP-seq reveals widespread bivalency of H3K4me3 and H3K27me3 in CD4(+) memory T cells.

The combinatorial action of co-localizing chromatin modifications and regulators determines chromatin structure and function. However, identifying co-localizing chromatin features in a high-throughput manner remains a technical challenge. Here we describe a novel reChIP-seq approach and tailored bioinformatic analysis tool, normR that allows for the sequential enrichment and detection of co-localizing DNA-associated proteins in an unbiased and genome-wide manner. We illustrate the utility of the reChIP-seq method and normR by identifying H3K4me3 or H3K27me3 bivalently modified nucleosomes in primary human CD4(+) memory T cells. We unravel widespread bivalency at hypomethylated CpG-islands coinciding with inactive promoters of developmental regulators. reChIP-seq additionally uncovered heterogeneous bivalency in the population, which was undetectable by intersecting H3K4me3 and H3K27me3 ChIP-seq tracks. Finally, we provide evidence that bivalency is established and stabilized by an interplay between the genome and epigenome. Our reChIP-seq approach augments conventional ChIP-seq and is broadly applicable to unravel combinatorial modes of action.

PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3.

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.

Inference of interactions between chromatin modifiers and histone modifications: from ChIP-Seq data to chromatin-signaling.

Chromatin modifiers and histone modifications are components of a chromatin-signaling network involved in transcription and its regulation. The interactions between chromatin modifiers and histone modifications are often unknown, are based on the analysis of few genes or are studied in vitro. Here, we apply computational methods to recover interactions between chromatin modifiers and histone modifications from genome-wide ChIP-Seq data. These interactions provide a high-confidence backbone of the chromatin-signaling network. Many recovered interactions have literature support; others provide hypotheses about yet unknown interactions. We experimentally verified two of these predicted interactions, leading to a link between H4K20me1 and members of the Polycomb Repressive Complexes 1 and 2. Our results suggest that our computationally derived interactions are likely to lead to novel biological insights required to establish the connectivity of the chromatin-signaling network involved in transcription and its regulation.

SPOC1-mediated antiviral host cell response is antagonized early in human adenovirus type 5 infection.

Little is known about immediate phases after viral infection and how an incoming viral genome complex counteracts host cell defenses, before the start of viral gene expression. Adenovirus (Ad) serves as an ideal model, since entry and onset of gene expression are rapid and highly efficient, and mechanisms used 24-48 hours post infection to counteract host antiviral and DNA repair factors (e.g. p53, Mre11, Daxx) are well studied. Here, we identify an even earlier host cell target for Ad, the chromatin-associated factor and epigenetic reader, SPOC1, recently found recruited to double strand breaks, and playing a role in DNA damage response. SPOC1 co-localized with viral replication centers in the host cell nucleus, interacted with Ad DNA, and repressed viral gene expression at the transcriptional level. We discovered that this SPOC1-mediated restriction imposed upon Ad growth is relieved by its functional association with the Ad major core protein pVII that enters with the viral genome, followed by E1B-55K/E4orf6-dependent proteasomal degradation of SPOC1. Mimicking removal of SPOC1 in the cell, knock down of this cellular restriction factor using RNAi techniques resulted in significantly increased Ad replication, including enhanced viral gene expression. However, depletion of SPOC1 also reduced the efficiency of E1B-55K transcriptional repression of cellular promoters, with possible implications for viral transformation. Intriguingly, not exclusive to Ad infection, other human pathogenic viruses (HSV-1, HSV-2, HIV-1, and HCV) also depleted SPOC1 in infected cells. Our findings provide a general model for how pathogenic human viruses antagonize intrinsic SPOC1-mediated antiviral responses in their host cells. A better understanding of viral entry and early restrictive functions in host cells should provide new perspectives for developing antiviral agents and therapies. Conversely, for Ad vectors used in gene therapy, counteracting mechanisms eradicating incoming viral DNA would increase Ad vector efficacy and safety for the patient.

Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes.

Death domain-associated protein (Daxx) cooperates with X-linked α-thalassaemia retardation syndrome protein (ATRX), a putative member of the sucrose non-fermentable 2 family of ATP-dependent chromatin-remodelling proteins, acting as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription is regulated by cellular chromatin remodelling to allow efficient virus gene expression. Here, we focus on the repressive role of the Daxx/ATRX complex during Ad5 replication, which depends on intact protein-protein interaction, as negative regulation could be relieved with a Daxx mutant that is unable to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance and diversity of viral factors antagonizing Daxx/ATRX-mediated repression of viral gene expression and shed new light on the modulation of cellular chromatin remodelling factors by Ad5. We show for the first time that cellular Daxx/ATRX chromatin remodelling complexes play essential roles in Ad gene expression and illustrate the importance of early viral proteins to counteract cellular chromatin remodelling.

SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response.

Survival time-associated plant homeodomain (PHD) finger protein in Ovarian Cancer 1 (SPOC1, also known as PHF13) is known to modulate chromatin structure and is essential for testicular stem-cell differentiation. Here we show that SPOC1 is recruited to DNA double-strand breaks (DSBs) in an ATM-dependent manner. Moreover, SPOC1 localizes at endogenous repair foci, including OPT domains and accumulates at large DSB repair foci characteristic for delayed repair at heterochromatic sites. SPOC1 depletion enhances the kinetics of ionizing radiation-induced foci (IRIF) formation after γ-irradiation (γ-IR), non-homologous end-joining (NHEJ) repair activity, and cellular radioresistance, but impairs homologous recombination (HR) repair. Conversely, SPOC1 overexpression delays IRIF formation and γH2AX expansion, reduces NHEJ repair activity and enhances cellular radiosensitivity. SPOC1 mediates dose-dependent changes in chromatin association of DNA compaction factors KAP-1, HP1-α and H3K9 methyltransferases (KMT) GLP, G9A and SETDB1. In addition, SPOC1 interacts with KAP-1 and H3K9 KMTs, inhibits KAP-1 phosphorylation and enhances H3K9 trimethylation. These findings provide the first evidence for a function of SPOC1 in DNA damage response (DDR) and repair. SPOC1 acts as a modulator of repair kinetics and choice of pathways. This involves its dose-dependent effects on DNA damage sensors, repair mediators and key regulators of chromatin structure.

SPOC1 (PHF13) is required for spermatogonial stem cell differentiation and sustained spermatogenesis.

SPOC1 (PHF13) is a recently identified protein that has been shown to dynamically associate with somatic chromatin, to modulate chromatin compaction and to be important for proper cell division. Here, we report on the expression of SPOC1 in promyelocytic leukaemia zinc finger (PLZF)-positive undifferentiated spermatogonial stem cells (SSCs) of the mouse testis. To investigate further the biological function of SPOC1 in germ cells we generated Spoc1 mutant mice from a gene-trap embryonic stem cell clone. Postpubertal homozygous Spoc1(-/-) animals displayed a pronounced progressive loss of germ cells from an initially normal germ epithelium of the testis tubules leading to testis hypoplasia. This loss first affected non-SSC stages of germ cells and then, at a later time point, the undifferentiated spermatogonia. Remarkably, successive loss of all germ cells (at >20 weeks of age) was preceded by a transient increase in the number of undifferentiated A(aligned) (A(al)) spermatogonia in younger mice (at >10 weeks of age). The number of primary Spoc1(-/-) gonocytes, the proliferation of germ cells, and the initiation and progression of meiosis was normal, but we noted a significantly elevated level of apoptosis in the Spoc1(-/-) testis. Taken together, the data argue that SPOC1 is indispensable for stem cell differentiation in the testis and for sustained spermatogenesis.

SPOC1: a novel PHD-containing protein modulating chromatin structure and mitotic chromosome condensation.

In this study, we characterize the molecular and functional features of a novel protein called SPOC1. SPOC1 RNA expression was previously reported to be highest in highly proliferating tissues and increased in a subset of ovarian carcinoma patients, which statistically correlated with poor prognosis and residual disease. These observations implied that SPOC1 might play a role in cellular proliferation and oncogenesis. Here we show that the endogenous SPOC1 protein is labile, primarily chromatin associated and its expression as well as localization are regulated throughout the cell cycle. SPOC1 is dynamically regulated during mitosis with increased expression levels and biphasic localization to mitotic chromosomes indicating a functional role of SPOC1 in mitotic processes. Consistent with this postulate, SPOC1 siRNA knockdown experiments resulted in defects in mitotic chromosome condensation, alignment and aberrant sister chromatid segregation. Finally, we have been able to show, using micrococcal nuclease (MNase) chromatin-digestion assays that SPOC1 expression levels proportionally influence the degree of chromatin compaction. Collectively, our findings show that SPOC1 modulates chromatin structure and that tight regulation of its expression levels and subcellular localization during mitosis are crucial for proper chromosome condensation and cell division.

The path of murine serum amyloid A through peritoneal macrophages.

Serum amyloid A (SAA) is a family of proteins encoded by four related genes. Of the four, isoforms 1.1 and 2.1 are acute phase proteins synthesized by the liver. They become major components of the HDL plasma fraction during acute tissue injury and the HDL/SAA complex is readily taken up by macrophages. Herein we investigated the path SAA follows when presented to macrophages as HDL/SAA or in liposomes. Using antibodies specific to SAA and confocal microscopy, or EM autoradiography where only SAA is radio-labeled, we show that HDL/SAA is taken up rapidly by macrophages and within 30 min SAA, or fragments thereof, proceeds through the cytoplasm to the peri-nuclear region and then the nucleus. Within 45-60 min SAA, or fragments thereof, is found back in the cytoplasm and at the plasma membrane where it is subsequently extruded. The observation that SAA, or fragments thereof, traverse the nucleus is a novel finding and may implicate SAA in macrophage gene regulation. It also raises questions by what mechanism SAA enters and leaves the nucleus. We further investigated if both SAA isoforms traffic through the macrophage in a similar manner. Isoform differences were observed. Both isoforms bind well to the plasma membrane of macrophages at 4 degrees C, but at 37 degrees C only SAA2.1 is taken up by the cell in significant quantity, and is observed in the nucleus, suggesting that the two isoforms are handled differently and that they may have discrete physiological roles.