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Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering the environmental cause of COPD (e.g., cigarette smoke) and disease phenotypes, including stem-cell senescence and impaired differentiation, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) isolated from distal parenchyma of ex-smoker controls and COPD patients, with both mild and severe disease. The epigenetic landscape is markedly changed in lung fibroblasts across COPD stages, with DNA methylation changes occurring predominantly in regulatory regions, including promoters and enhancers. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Notably, we identified epigenetic and transcriptional dysregulation already in mild COPD patients, providing unique insights into early disease. Integration of profiling data identified 110 candidate regulators of disease phenotypes, including epigenetic factors. Using phenotypic screens, we verified the regulator capacity of multiple candidates and linked them to repair processes in the human lung.Our study provides first integrative high-resolution epigenetic and transcriptomic maps of human lung fibroblasts across stages of COPD. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic respiratory diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients.

Complexity of lung microenvironment and changes in cellular composition during disease make it exceptionally hard to understand molecular mechanisms driving development of chronic lung diseases. Although recent advances in cell type–resolved approaches hold great promise for studying complex diseases, their implementation relies on local access to fresh tissue, as traditional tissue storage methods do not allow viable cell isolation. To overcome these hurdles, we developed a versatile workflow that allows storage of lung tissue with high viability, permits thorough sample quality check before cell isolation, and befits sequencing-based profiling. We demonstrate that cryopreservation enables isolation of multiple cell types from both healthy and diseased lungs. Basal cells from cryopreserved airways retain their differentiation ability, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing and EPIC Array, we show that gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for omics profiling of lung cells. Moreover, we obtained high-quality single-cell RNA-sequencing data of cells from cryopreserved human lungs, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our workflow is well suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to viable human tissue.

Despite decades of intensive research, patients with COPD are still desperately waiting for curative treatments. For these to be developed, better understanding of molecular changes driving disease development is required. Based on key features and the environmental cause of COPD (cigarette smoke), we hypothesised that the disease will be triggered by altered epigenetic signalling in lung cells. Using cutting-edge, low-input whole genome bisulfite sequencing technology, we generated first unbiased DNA methylation maps of parenchymal fibroblasts isolated from lungs of smoker controls and COPD patients, both with early and established disease. We demonstrate that epigenetic landscape is severely changed in fibroblasts across COPD stages, with methylation changes occurring predominantly in regulatory regions, including promoters and enhancers. RNA-seq analysis of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair and extracellular matrix organisation. Excitingly, we identify differentially methylated regions and gene subsets dysregulated already in mild COPD patients, providing unique insight into early disease. Integration of profiling data, together with upstream regulator analysis identified 112 candidate regulators of COPD phenotypes. Using high-content phenotypic screens in primary lung fibroblasts from normal and COPD donors, we linked the function of multiple of these genes to key processes in COPD, establishing them as potential novel targets for therapy. Interestingly, many of these candidates are epigenetic factors, emphasising the critical role of epigenetic regulation in COPD.

The complexity of lung microenvironment together with changes in cellular composition during disease progression hampered deeper understanding of molecular mechanisms leading to development of lung pathologies. Although recent advances in cell type resolved sequencing and single cell -omics hold great promise for studying complex diseases, their implementation greatly relies on local access to fresh tissue, as traditional tissue storage formats do not allow viable cell isolation. To overcome these hurdles, we developed a versatile workflow that allows long term storage of lung tissue with high cell viability, permits thorough sample quality check before cell isolation, and is compatible with next generation sequencing-based profiling, including single cell approaches. We demonstrate that cryopreservation is suitable for isolation of multiple cell types from different lung locations and is applicable to both healthy and tumor tissue. Basal cells isolated from cryopreserved airways retain ability to differentiate, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNAseq and EPIC Array we show that genome-wide gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for -omics profiling of lung cells. In addition, we obtained high quality single-cell RNAseq from cryopreserved lung tissue, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our cryopreservation workflow is well-suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to tissue.

Protein kinase D3 (PKD3) is upregulated in triple-negative breast cancer (TNBC) and associated with cell proliferation and metastasis development but its precise pro-oncogenic function is unknown. Here we show that PKD3 is required for the maintenance of the TNBC stem cell population. The depletion of PKD3 in MDA-MB-231 cells reduced the cancer stem cell frequency in vitro and tumor initiation potential in vivo. We further provide evidence that the RhoGEF GEF-H1 is upstream of PKD3 activation in TNBC stem cells. Most importantly, pharmacological PKD inhibition in combination with paclitaxel synergistically decreased oncosphere and colony formation efficiency in vitro and tumor recurrence in vivo. Based on our results we propose that targeting the GEF-H1/PKD3 signaling pathway in combination with chemotherapy might provide an effective therapeutic option for TNBC.

COPD is a prevalent disease with complex phenotypes and pathogenesis. The contribution of epigenetic regulation in COPD has been recognised recently, but specific epigenetic signals that trigger and maintain COPD-specific gene expression patterns are largely unknown. The main goal of the project is to discover specific molecular and epigenetic changes associated with COPD development and progression in human lung fibroblasts and to identify epigenetic regulators of the disease. We obtained genome-wide maps of DNA methylation using whole genome bisulfite sequencing and gene expression using RNA-seq for parenchymal lung fibroblasts isolated from 3 smoker controls and 8 COPD patients, both with early and established disease. We identified 6279 differentially methylated regions in COPD and show that epigenetic changes occur already in mild COPD. Importantly, we discovered clusters of regions with methylation changes progressing with decline of lung function of patients. Differential gene expression analysis identified 949 genes dysregulated in COPD and enrichment analysis revealed that genes up-regulated in COPD are involved in proliferation, cell cycle regulation and DNA replication, whereas genes down-regulated in COPD are involved in extracellular matrix organization and extracellular matrix degradation. We have also derived gene expression signatures of disease progression and identified gene subsets that are already dysregulated in early stages of the disease. Identification of early gene expression changes associated with COPD development is of high clinical relevance, as it might offer a unique advantage for disease modifying therapy.

Mutations in the ATRX protein are associated with the alpha-thalassemia and mental retardation X-linked syndrome (ATR-X). Almost half of the disease-causing mutations occur in its ATRX-Dnmt3-Dnmt3L (ADD) domain. By employing peptide arrays, chromatin pull-down and peptide binding assays, we show specific binding of the ADD domain to H3 histone tail peptides containing H3K9me3. Peptide binding was disrupted by the presence of the H3K4me3 and H3K4me2 modification marks indicating that the ATRX-ADD domain has a combined readout of these two important marks (absence of H3K4me2 and H3K4me3 and presence of H3K9me3). Disease-causing mutations reduced ATRX-ADD binding to H3 tail peptides. ATRX variants, which fail in the H3K9me3 interaction, show a loss of heterochromatic localization in cells, which indicates the chromatin targeting function of the ADD domain of ATRX. Disruption of H3K9me3 binding may be a general pathogenicity pathway of ATRX mutations in the ADD domain which may explain the clustering of disease mutations in this part of the ATRX protein.

Huntingtin interacting protein 14 (HIP14), a membrane-bound palmitoyl transferase, palmitoylates a number of neuronal proteins (including Huntingtin) and affects the trafficking, stability, aggregation, and/or functional activity of substrate proteins. HIP14 contains an N-terminal ankyrin repeat domain that may function in its substrate recognition. Sequence analysis suggests that the HIP14 ankyrin repeats share approximately 50% identity with the ankyrin repeats of G9a and G9a-like protein (GLP) histone lysine methyltransferases. The crystal structure of the HIP14 ankyrin repeats reveals a surface aromatic cage, formed by two tryptophans, one tyrosine, and one methionine. The all-hydrophobic cage resembles the tri-methylated lysine binding pocket of the plant homeodomain (PHD) of human BPTF (bromodomain and PHD domain transcription factor) 1. HIP14, a Huntingtin interacting protein 2, is a 633-residue protein, including 7-8 ankyrin repeats in the N-terminal region followed by five predicted transmembrane helices. The protein contains a signature DHHC palmitoyl transferases motif located close to the predicted fourth transmembrane helix 3. Importantly, the ankyrin repeats (a protein-protein interaction domain that may function in substrate recognition) and the DHHC sequence (the hypothetical active site) are both predicted to reside on the cytoplasmic face of the lipid bilayer 4, presumably allowing the substrate recognition and the active site to interact with the same substrate. Posttranslational palmitoylation involves the attachment of the saturated C16 fatty acid palmitate to specific cysteines via a thioester linkage 5-7. HIP14 palmitoylates Huntingtin at cysteine 214 8. Other substrates of HIP14-mediated palmitoylation include SNAP-25 (synaptosome associated protein 25 kDa), PSD-95 (postsynaptic density 95 kDa), GAD-65 (glutamate decarboxylase 65 kDa), and Synaptotagmin I 9. Ankyrin repeats are known to mediate protein-protein interactions 10. Recently we showed that the ankyrin repeat domains of G9a and GLP, two euchromatin associated histone lysine methyltranferases, bind N-terminal histone H3 peptides containing mono- or di-methylated lysine 9 (H3K9me1, me2) via a partial aromatic cage with three tryptophans and one acidic residue 11. Besides the ankyrin repeats of G9a and GLP, other protein domains including the Chromodomain 12, plant homeodomain 1,13-15, and Tudor domain 16, also recognize methylated lysines (Review 17). The common mode of methyl-lysine interactions is via a surface aromatic cage consisting of 2-4 aromatic residues. These aromatic cages are highly selective for methyllysine. In a study of Chromodomain-methyllysine interaction, binding was driven primarily by cation-π interactions (i.e, a methyllysine carries a positive charge), and secondarily by the packing of methyl group(s) against the aromatic ring(s) of the cage, with the hydrophobic effect contributing the least to binding 18. This eliminates the possibility of such cages generically binding hydrophobic residues, and suggests that other methyllysine binding ankyrin repeats could be identified by the presence of a surface aromatic cage.

The unstructured N-terminal tails of histones protrude from the core nucleosome and harbor complex patterns of post-translational modifications (PTMs) (Kouzarides 2007; Margueron and Reinberg 2010; Bannister and Kouzarides 2011; Tan et al. 2011). These PTMs regulate a multitude of chromatin-templated transactions, play a central role in development, and are implicated in many diseases, such as cancer (Suva et al. 2013). Therefore, understanding the role of histone marks in chromatin-dependent processes is of paramount importance. So far, techniques based on the specific binding of antibodies to modified histone proteins have been the only method available for genome-wide analyses of histone modifications with locus-specific resolution. The central role of antibodies for the characterization of histone PTMs in chromatin research makes the quality and reliability of these reagents a very important scientific issue. In general, antibodies are very powerful and important reagents in biomolecular research, but the validation of commercial antibodies is not always sufficiently rigorous (Bordeaux et al. 2010). This is particularly important in the chromatin field, where specific recognition and discrimination of subtle epitopes defined only by the presence of distinct PTMs is required. Moreover, several important modifications occur in very similar amino acid sequence motifs, like the methylation of H3K9 and H3K27, which are both placed in the context of an ARKS sequence. In addition, histone tails are hypermodified, as exemplified by the H3 tail, where the adjacent R8, K9, and S10 amino acid side chains are known to be methylated, acetylated, or phosphorylated. This implies that secondary modifications often occur on the peptide segment contacted by the antibody in the immediate vicinity of the target PTM and sometimes prevent the binding of antibodies in spite of the presence of the target modification, yielding false negative results. When undocumented, the cross-reactivity with related or unrelated marks and the combinatorial effect of neighboring marks compromise the application of antibodies, as illustrated in Figure 1. Additionally, different antibodies show distinct profiles of false positive and false negative signals, and even antibodies with the same catalog numbers regularly show lot-to-lot fluctuations of properties (also illustrated in Fig. 1). This variability is not unexpected for polyclonal antibodies, where new batches are produced by immunization of a new animal, but changes of purification procedure may cause variance of properties of monoclonal antibodies as well. Occasionally some lots of commercial antibodies even prefer to bind to secondary targets (see Fig. 1 and H3K36me3 antibodies documented in Bock et al. 2011a). This necessitates a detailed quality control and documentation of each antibody and each lot in order to give the user all relevant information for correct data interpretation, which is often not sufficiently provided. The urgency for better quality assessment and documentation of antibodies used in chromatin research has been widely recognized in the field (Bock et al. 2011a; Egelhofer et al. 2011; Fuchs et al. 2011; Nishikori et al. 2012; Peach et al. 2012; Hattori et al. 2013; Heubach et al. 2013), and the ENCODE Project Consortium has set up quality criteria for histone PTM antibodies (Egelhofer et al. 2011; Landt et al. 2012). According to these guidelines, antibodies must specifically detect modified histones in Western blots and fulfill one or more of the following secondary criteria: (1) specific binding to modified peptides in dot blot assays; (2) mass spectrometric detection of the modification in precipitated chromatin; (3) loss of signal upon knockdown of the corresponding histone modifying enzyme; (4) reproducibility of ChIP-seq; (5) similarity of ChIP-seq results of two different antibodies directed against the same modification; or (6) overlap of ChIP-seq peaks with expected genomic annotations. Figure 1. Peptide array analyses showing lot-to-lot fluctuations, cross-reactivity, and effects of proximal marks on the binding of popular histone tail antibodies. Peptide spots are annotated on the left side of the glass slide. The color-coded boxes denote the ... To develop an alternative to antibodies for chromatin research, we assessed the applicative potential and utility of naturally occurring and engineered histone modification interacting domains (HMIDs). This approach has several distinct advantages over antibodies, such as the ease and cost-effectiveness of recombinant production of HMIDs in Escherichia coli, the amenability of HMIDs to protein engineering, and the possibility of producing them at constant quality, eliminating lot-to-lot variability. In support of this concept, affinity methods based on protein domains have been successfully employed for the enrichment of methylated or unmethylated CpG islands in the analysis of DNA methylation (Cross et al. 1994; Blackledge et al. 2012) or in proteome-wide analyses of non-histone lysine methylation (Liu et al. 2013; Moore et al. 2013). In this proof-of-principle study, we started by characterizing the specificity of several HMIDs and compared them with ENCODE-validated antibodies for the same PTM using peptide arrays. Specificities were further validated in Western blots by detecting histone tail PTMs using unmodified histones and histones specifically depleted with the target PTM as controls. Then, we investigated the applicative potential of HMIDs in ChIP-like experiments to enrich for chromatin with particular modifications, which represents one of the most important and commonly used applications of histone tail PTM antibodies.

PHF1 associates with the Polycomb repressive complex 2 and it was demonstrated to stimulate its H3K27-trimethylation activity. We studied the interaction of the PHF1 Tudor domain with modified histone peptides and found that it recognizes H3K36me3 and H3tK27me3 (on the histone variant H3t) and that it uses the same trimethyllysine binding pocket for the interaction with both peptides. Since both peptide sequences are very different, this result indicates that reading domains can have dual specificities. Sub-nuclear localization studies of full-length PHF1 in human HEK293 cells revealed that it co-localizes with K27me3, but not with K36me3, and that this co-localization depends on the trimethyllysine binding pocket indicating that K27me3 is an in vivo target for the PHF1 Tudor domain. Our data suggest that PHF1 binds to H3tK27me3 in human chromatin, and H3t has a more general role in Polycomb regulation.

Background Epigenetic reading domains are involved in the regulation of gene expression and chromatin state by interacting with histones in a post-translational modification specific manner. A detailed knowledge of the target modifications of reading domains, including enhancing and inhibiting secondary modifications, will lead to a better understanding of the biological signaling processes mediated by reading domains. Results We describe the application of Celluspots peptide arrays which contain 384 histone peptides carrying 59 post translational modifications in different combinations as an inexpensive, reliable and fast method for initial screening for specific interactions of reading domains with modified histone peptides. To validate the method, we tested the binding specificities of seven known epigenetic reading domains on Celluspots peptide arrays, viz. the HP1ß and MPP8 Chromo domains, JMJD2A and 53BP1 Tudor domains, Dnmt3a PWWP domain, Rag2 PHD domain and BRD2 Bromo domain. In general, the binding results agreed with literature data with respect to the primary specificity of the reading domains, but in almost all cases we obtained additional new information concerning the influence of secondary modifications surrounding the target modification. Conclusions We conclude that Celluspots peptide arrays are powerful screening tools for studying the specificity of putative reading domains binding to modified histone peptides.

The Dnmt3a DNA methyltransferase contains in its N-terminal part a PWWP domain that is involved in chromatin targeting. Here, we have investigated the interaction of the PWWP domain with modified histone tails using peptide arrays and show that it specifically recognizes the histone 3 lysine 36 trimethylation mark. H3K36me3 is known to be a repressive modification correlated with DNA methylation in mammals and heterochromatin in Schizosaccharomyces pombe. These results were confirmed by equilibrium peptide binding studies and pulldown experiments with native histones and purified native nucleosomes. The PWWP-H3K36me3 interaction is important for the subnuclear localization of enhanced yellow fluorescent protein-fused Dnmt3a. Furthermore, the PWWP-H3K36me3 interaction increases the activity of Dnmt3a for methylation of nucleosomal DNA as observed using native nucleosomes isolated from human cells after demethylation of the DNA with 5-aza-2′-deoxycytidine as substrate for methylation with Dnmt3a. These data suggest that the interaction of the PWWP domain with H3K36me3 is involved in targeting of Dnmt3a to chromatin carrying that mark, a model that is in agreement with several studies on the genome-wide distribution of DNA methylation and H3K36me3.

By methylation of peptide arrays, we determined the specificity profile of the protein methyltransferase G9a. We show that it mostly recognizes an Arg-Lys sequence and that its activity is inhibited by methylation of the arginine residue. Using the specificity profile, we identified new non-histone protein targets of G9a, including CDYL1, WIZ, ACINUS and G9a (automethylation), as well as peptides derived from CSB. We demonstrate potential downstream signaling pathways for methylation of non-histone proteins.