Histone Modification Profiles Research Articles

The CUT&RUN suspect list of problematic regions of the genome

BackgroundCleavage Under Targets and Release Using Nuclease (CUT&RUN) is an increasingly popular technique to map genome-wide binding profiles of histone modifications, transcription factors, and co-factors. The ENCODE project and others have compiled blacklists for ChIP-seq which have been widely adopted: these lists contain regions of high and unstructured signal, regardless of cell type or protein target, indicating that these are false positives. While CUT&RUN obtains similar results to ChIP-seq, its biochemistry and subsequent data analyses are different. We found that this results in a CUT&RUN-specific set of undesired high-signal regions.ResultsWe compile suspect lists based on CUT&RUN data for the human and mouse genomes, identifying regions consistently called as peaks in negative controls. Using published CUT&RUN data from our and other labs, we show that the CUT&RUN suspect regions can persist even when peak calling is performed with SEACR or MACS2 against a negative control and after ENCODE blacklist removal. Moreover, we experimentally validate the CUT&RUN suspect lists by performing reiterative negative control experiments in which no specific protein is targeted, showing that they capture more than 80% of the peaks identified.ConclusionsWe propose that removing these problematic regions can substantially improve peak calling in CUT&RUN experiments, resulting in more reliable datasets.

Targeting BRPF3 moderately reverses olaparib resistance in high grade serous ovarian carcinoma.

PARP inhibitors (PARPi) kill cancer cells by stalling DNA replication and preventing DNA repair, resulting in a critical accumulation of DNA damage. Resistance to PARPi is a growing clinical problem in the treatment of high grade serous ovarian carcinoma (HGSOC). Acetylation of histone H3 lysine 14 (H3K14ac) and associated histone acetyltransferases (HATs) and epigenetic readers have known functions in DNA repair and replication. Our objectives are to examine their expression and activities in the context of PARPi-resistant HGSOC, and to determine if targeting H3K14ac or associated proteins has therapeutic potential. Using mass spectrometry profiling of histone modifications, we observed increased H3K14ac enrichment in PARPi-resistant HGSOC cells relative to isogenic PARPi-sensitive lines. By reverse-transcriptase quantitative PCRand RNA-seq, we also observed altered expression of numerous HATs in PARPi-resistant HGSOC cells and a PARPi-resistant PDX model. Knockdown of HATs only modestly altered PARPi response, although knockdown and inhibition of PCAF significantly increased resistance. Pharmacologic inhibition of HBO1 depleted H3K14ac but did not affect PARPi response. However, knockdown and inhibition of BRPF3, a bromodomain and PHD-finger containing protein that is known to interact in a complex with HBO1, did reduce PARPi resistance. This study demonstrates that depletion of H3K14ac does not affect PARPi response in HGSOC. Our data suggest that the bromodomain function of HAT proteins, such as PCAF, or accessory proteins, such as BRPF3, may play a more direct role compared to direct HATsfunction in PARPi response.

High-throughput identification of regulatory elements and functional assays to uncover susceptibility genes for nasopharyngeal carcinoma

Large-scale genetic association studies have identified multiple susceptibility loci for nasopharyngeal carcinoma (NPC), but the underlying biological mechanisms remain to be explored. To gain insights into the genetic etiology of NPC, we conducted a follow-up study encompassing 6,907 cases and 10,472 controls and identified two additional NPC susceptibility loci, 9q22.33 (rs1867277; OR= 0.74, 95% CI= 0.68-0.81, p= 3.08×10-11) and 17q12 (rs226241; OR= 1.42, 95% CI= 1.26-1.60, p= 1.62×10-8). The two additional loci, together with two previously reported genome-wide significant loci, 5p15.33 and 9p21.3, were investigated by high-throughput sequencing for chromatin accessibility, histone modification, and promoter capture Hi-C (PCHi-C) profiling. Using luciferase reporter assays and CRISPR interference (CRISPRi) to validate the functional profiling, we identified PHF2 at locus 9q22.33 as a susceptibility gene. PHF2 encodes a histone demethylase and acts as a tumor suppressor. The risk alleles of the functional SNPs reduced the expression of the target gene PHF2 by inhibiting the enhancer activity of its long-range (4.3 Mb) cis-regulatory element, which promoted proliferation of NPC cells. In addition, we identified CDKN2B-AS1 as a susceptibility gene at locus 9p21.3, and the NPC risk allele of the functional SNP rs2069418 promoted the expression of CDKN2B-AS1 by increasing its enhancer activity. The overexpression of CDKN2B-AS1 facilitated proliferation of NPC cells. In summary, we identified functional SNPs and NPC susceptibility genes, which provides additional explanations for the genetic association signals and helps to uncover the underlying genetic etiology of NPC development.

Histone Modification of Osteogenesis Related Genes Triggered by Substrate Topography Promotes Human Mesenchymal Stem Cell Differentiation.

The clinical success of orthopedic implants is closely related to their integration in the bone tissue promoted by rough device surfaces. The biological response of precursor cells to their artificial microenvironments plays a critical role in this process. In this study, we elucidated the relation between cell instructivity and surface microstructure of polycarbonate (PC)-based model substrates. The rough surface structure (hPC) with an average peak spacing (Sm) similar to the trabecular spacing of trabecular bone improved osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), as compared to the smooth surface (sPC) and the surface with a moderate Sm value (mPC). The hPC substrate promoted the cell adhesion and assembling of F-actin and enhanced cell contractile force by upregulating phosphorylated myosin light chain (pMLC) expression. The increased cell contractile force led to YAP nuclear translocation and the elongation of cell nuclei, presenting higher levels of active form of Lamin A/C. The nuclear deformation alternated the histone modification profile, particularly the decrease of H3K27me3 and increase of H3K9ac on the promoter region of osteogenesis related genes (ALPL, RUNX2, and OCN). Mechanism study using inhibitors and siRNAs elucidated the role of YAP, integrin, F-actin, myosin, and nuclear membrane proteins in such a regulatory process of surface topography on stem cell fate. These mechanistical insights on the epigenetic level give a new perspective in understanding of the interaction of substrate and stem cells as well as provide valuable criteria for designing bioinstructive orthopedic implants.

Abstract 4725: The epigenetic determinants of BTKi efficacy in chronic lymphocytic leukemia

Abstract Introduction: The introduction of Bruton’s tyrosine kinase (BTK) inhibitors (BTKi) to target B cell receptor (BCR) pathways in chronic lymphocytic leukemia (CLL) has significantly improved clinical responses. However, the effectiveness of BTKi is limited by the development of adaptive drug resistance. Therefore, there is an urgent need to identify critical biologic features that determine BTKi efficacy in order to develop more effective therapies. Epigenetic aberrations play an important role in tumor initiation, progression, and drug resistance. However, it is currently unknown if BCR signaling is also dependent on epigenetic mechanisms to support oncogenic gene expression and survival in CLL B cells. Methods and Results: To gain new insights into the role of epigenetic regulation of BTKi treatment in CLL B cells, we analyzed the genome wide chromatin accessibility (ATAC-seq) and histone modification (H3K4me1, H3K4me3, H3K27ac, H3K27me3) profiles (CUT&Tag) of leukemic cells from 4 CLL patients on ibrutinib treatment in a sequential fashion (i.e., baseline, on ibrutinib treatment, and at relapse). We also performed the same analysis in other cohorts at two stages of ibrutinib treatment (baseline and while on treatment, n=20). Our studies show that suppressing BCR signaling by BTKi treatment leads to an alteration of a pro-survival epigenetic signature (defined by histone modifications and chromatin accessibility landscapes) in CLL and that the genes regulated by these chromatin changes are enriched in pathways associated with malignant B cell survival (i.e., BCR signaling and apoptosis pathways). Importantly, disruption of this pro-survival epigenetic signature is necessary for effective BTKi treatment. We further find that the transcription factor (TF) Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) couples the BCR signaling to control this pro-survival epigenetic program that supports CLL B cell survival. In specific, we show that nuclear depletion of NFATc1 is required for BTKi induced epigenetic alteration and effective BTKi treatment. Depletion of NFATc1 disrupts the pro-survival epigenetic signature and inhibits CLL B cells survival, which improves the efficacy of BTKi treatment in CLL. Conclusion: Here using leukemic cells from CLL patients with BTKi treatment, we demonstrate that epigenetic regulators are exploited by the oncogenic signaling pathway to support malignant cell survival. Specifically, NFATc1 is utilized by the BCR signaling pathway to control an epigenetic signature that supports CLL cell survival. This BCR regulated epigenetic machinery can be targeted to maintain and enhance BTKi treatment efficacy in CLL. Citation Format: Zhiquan Wang, Huihuang Yan, Justin C. Boysen, Esteban Braggio, Sameer A. Parikh, Neil E. Kay. The epigenetic determinants of BTKi efficacy in chronic lymphocytic leukemia. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4725.

Integration of epigenetic and genetic profiles identifies multiple sclerosis disease-critical cell types and genes

Genome-wide association studies (GWAS) successfully identified multiple sclerosis (MS) susceptibility variants. Despite this notable progress, understanding the biological context of these associations remains challenging, due in part to the complexity of linking GWAS results to causative genes and cell types. Here, we aimed to address this gap by integrating GWAS data with single-cell and bulk chromatin accessibility data and histone modification profiles from immune and nervous systems. MS-GWAS associations are significantly enriched in regulatory regions of microglia and peripheral immune cell subtypes, especially B cells and monocytes. Cell-specific polygenic risk scores were developed to examine the cumulative impact of the susceptibility genes on MS risk and clinical phenotypes, showing significant associations with risk and brain white matter volume. The findings reveal enrichment of GWAS signals in B cell and monocyte/microglial cell-types, consistent with the known pathology and presumed targets of effective MS therapeutics.

Linking chromatin acylation mark-defined proteome and genome in living cells.

A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed distinct crotonylation (e.g., H3K56cr) and β-hydroxybutyrylation (e.g., H3K56bhb) upon short chain fatty acids stimulation and established linkages for chromatin acylation mark-defined proteome, genome, and functions. This led to the identification of GLYR1 as a distinct interacting protein in modulating H3K56cr's gene body localization as well as the discovery of an elevated super-enhancer repertoire underlying bhb-mediated chromatin modulations. SiTomics offers a platform technology for elucidating the "metabolites-modification-regulation" axis, which is widely applicable for multi-omics profiling and functional dissection of modifications beyond acylations and proteins beyond histones.

Peaksat: an R package for ChIP-seq peak saturation analysis

BackgroundEpigenomic profiling assays such as ChIP-seq have been widely used to map the genome-wide enrichment profiles of chromatin-associated proteins and posttranslational histone modifications. Sequencing depth is a key parameter in experimental design and quality control. However, due to variable sequencing depth requirements across experimental conditions, it can be challenging to determine optimal sequencing depth, particularly for projects involving multiple targets or cell types.ResultsWe developed the peaksat R package to provide target read depth estimates for epigenomic experiments based on the analysis of peak saturation curves. We applied peaksat to establish the distinctive read depth requirements for ChIP-seq studies of histone modifications in different cell lines. Using peaksat, we were able to estimate the target read depth required per library to obtain high-quality peak calls for downstream analysis. In addition, peaksat was applied to other sequence-enrichment methods including CUT&RUN and ATAC-seq.Conclusionpeaksat addresses a need for researchers to make informed decisions about whether their sequencing data has been generated to an adequate depth and subsequently sufficient meaningful peaks, and failing that, how many more reads would be required per library. peaksat is applicable to other sequence-based methods that include calling peaks in their analysis.

Simultaneous profiling of histone modifications and DNA methylation via nanopore sequencing

The interplay between histone modifications and DNA methylation drives the establishment and maintenance of the cellular epigenomic landscape, but it remains challenging to investigate the complex relationship between these epigenetic marks across the genome. Here we describe a nanopore-sequencing-based-method, nanoHiMe-seq, for interrogating the genome-wide localization of histone modifications and DNA methylation from single DNA molecules. nanoHiMe-seq leverages a nonspecific methyltransferase to exogenously label adenine bases proximal to antibody-targeted modified nucleosomes in situ. The labelled adenines and the endogenous methylated CpG sites are simultaneously detected on individual nanopore reads using a hidden Markov model, which is implemented in the nanoHiMe software package. We demonstrate the utility, robustness and sensitivity of nanoHiMe-seq by jointly profiling DNA methylation and histone modifications at low coverage depths, concurrently determining phased patterns of DNA methylation and histone modifications, and probing the intrinsic connectivity between these epigenetic marks across the genome.

The Chromatin Structure at the MECP2 Gene and In Silico Prediction of Potential Coding and Non-Coding MECP2 Splice Variants.

Methyl CpG binding protein 2 (MeCP2) is an epigenetic reader that binds to methylated CpG dinucleotides and regulates gene transcription. Mecp2/MECP2 gene has 4 exons, encoding for protein isoforms MeCP2E1 and MeCP2E2. MeCP2 plays key roles in neurodevelopment, therefore, its gain- and loss-of-function mutations lead to neurodevelopmental disorders including Rett Syndrome. Here, we describe the structure, functional domains, and evidence support for potential additional alternatively spliced MECP2 transcripts and protein isoforms. We conclude that NCBI MeCP2 isoforms 3 and 4 contain certain MeCP2 functional domains. Our in silico analysis led to identification of histone modification and accessibility profiles at the MECP2 gene and its cis-regulatory elements. We conclude that the human MECP2 gene associated histone post-translational modifications exhibit high similarity between males and females. Between brain regions, histone modifications were found to be less conserved and enriched within larger genomic segments named as "S1-S11". We also identified highly conserved DNA accessibility regions in different tissues and brain regions, named as "A1-A9" and "B1-B9". DNA methylation profile was similar between mid-frontal gyrus of donors 35 days-25 years of age. Based on ATAC-seq data, the identified hypomethylated regions "H1-H8" intersected with most regions of the accessible chromatin (A regions).

Genome-wide profiling of histone modifications in Plasmodium falciparum using CUT&RUN.

We recently adapted a CUT&RUN protocol for genome-wide profiling of chromatin modifications in the human malaria parasite Plasmodium Using the step-by-step protocol described below, we were able to generate high-quality profiles of multiple histone modifications using only a small fraction of the cells required for ChIP-seq. Using antibodies against two commonly profiled histone modifications, H3K4me3 and H3K9me3, we show here that CUT&RUN profiling is highly reproducible and closely recapitulates previously published ChIP-seq-based abundance profiles of histone marks. Finally, we show that CUT&RUN requires substantially lower sequencing coverage for accurate profiling compared with ChIP-seq.

2cChIP-seq and 2cMeDIP-seq: The Carrier-Assisted Methods for Epigenomic Profiling of Small Cell Numbers or Single Cells.

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) can profile genome-wide epigenetic marks associated with regulatory genomic elements. However, conventional ChIP-seq is challenging when examining limited numbers of cells. Here, we developed a new technique by supplementing carrier materials of both chemically modified mimics with epigenetic marks and dUTP-containing DNA fragments during conventional ChIP procedures (hereafter referred to as 2cChIP-seq), thus dramatically improving immunoprecipitation efficiency and reducing DNA loss of low-input ChIP-seq samples. Using this strategy, we generated high-quality epigenomic profiles of histone modifications or DNA methylation in 10-1000 cells. By introducing Tn5 transposase-assisted fragmentation, 2cChIP-seq reliably captured genomic regions with histone modification at the single-cell level in about 100 cells. Moreover, we characterized the methylome of 100 differentiated female germline stem cells (FGSCs) and observed a particular DNA methylation signature potentially involved in the differentiation of mouse germline stem cells. Hence, we provided a reliable and robust epigenomic profiling approach for small cell numbers and single cells.

H3K18 lactylation marks tissue-specific active enhancers

BackgroundHistone lactylation has been recently described as a novel histone post-translational modification linking cellular metabolism to epigenetic regulation.ResultsGiven the expected relevance of this modification and current limited knowledge of its function, we generate genome-wide datasets of H3K18la distribution in various in vitro and in vivo samples, including mouse embryonic stem cells, macrophages, adipocytes, and mouse and human skeletal muscle. We compare them to profiles of well-established histone modifications and gene expression patterns. Supervised and unsupervised bioinformatics analysis shows that global H3K18la distribution resembles H3K27ac, although we also find notable differences. H3K18la marks active CpG island-containing promoters of highly expressed genes across most tissues assessed, including many housekeeping genes, and positively correlates with H3K27ac and H3K4me3 as well as with gene expression. In addition, H3K18la is enriched at active enhancers that lie in proximity to genes that are functionally important for the respective tissue.ConclusionsOverall, our data suggests that H3K18la is not only a marker for active promoters, but also a mark of tissue specific active enhancers.

Nucleosome sliding can influence the spreading of histone modifications.

Nucleosomes are the fundamental building blocks of chromatin that not only help in the folding of chromatin, but also in carrying epigenetic information. It is known that nucleosome sliding is responsible for dynamically organizing chromatin structure and the resulting gene regulation. Since sliding can move two neighboring nucleosomes physically close or away, can it play a role in the spreading of histone modifications? We investigate this by simulating a stochastic model that couples nucleosome dynamics with the kinetics of histone modifications. We show that the sliding of nucleosomes can affect the modification pattern as well as the time it takes to modify a given region of chromatin. Exploring different nucleosome densities and modification kinetic parameters, we show that nucleosome sliding can be important for creating histone modification domains. Our model predicts that nucleosome density coupled with sliding dynamics can create an asymmetric histone modification profile around regulatory regions. We also compute the probability distribution of modified nucleosomes and relaxation kinetics of modifications. Our predictions are comparable with known experimental results.

Genome-wide profiling of histone H3K4me3 and H3K27me3 modifications in individual blastocysts by CUT&Tag without a solid support (NON-TiE-UP CUT&Tag)

Individual analysis of the epigenome of preimplantation embryos is useful for characterizing each embryo and for investigating the effects of environmental factors on their epigenome. However, it is difficult to analyze genome-wide epigenetic modifications, especially histone modifications, in a large number of single embryos due to the small number of cells and the complexity of the analysis methods. To solve this problem, we further modified the CUT&Tag method, which can analyze histone modifications in a small number of cells, such that the embryo is handled as a cell mass in the reaction solutions in the absence of the solid-phase magnetic beads that are used for antibody and enzyme reactions in the conventional method (NON-TiE-UP CUT&Tag; NTU-CAT). By using bovine blastocysts as a model, we showed that genome-wide profiles of representative histone modifications, H3K4me3 and H3K27me3, could be obtained by NTU-CAT that are in overall agreement with the conventional chromatin immunoprecipitation-sequencing (ChIP-seq) method, even from single embryos. However, this new approach has limitations that require attention, including false positive and negative peaks and lower resolution for broad modifications. Despite these limitations, we consider NTU-CAT a promising replacement for ChIP-seq with the great advantage of being able to analyze individual embryos.

HOTAIR interacts with PRC2 complex regulating the regional preadipocyte transcriptome and human fat distribution.

Mechanisms governing regional human adipose tissue (AT) development remain undefined. Here, we show that the long non-coding RNA HOTAIR (HOX transcript antisense RNA) is exclusively expressed in gluteofemoral AT, where it is essential for adipocyte development. We find that HOTAIR interacts with polycomb repressive complex 2 (PRC2) and we identify core HOTAIR-PRC2 target genes involved in adipocyte lineage determination. Repression of target genes coincides with PRC2 promoter occupancy and H3K27 trimethylation. HOTAIR is also involved in modifying the gluteal adipocyte transcriptome through alternative splicing. Gluteal-specific expression of HOTAIR is maintained by defined regions of open chromatin across the HOTAIR promoter. HOTAIR expression levels can be modified by hormonal (estrogen, glucocorticoids) and genetic variation (rs1443512 is a HOTAIR eQTL associated with reduced gynoid fat mass). These data identify HOTAIR as a dynamic regulator of the gluteal adipocyte transcriptome and epigenome with functional importance for human regional AT development.

Abstract 2967: ACTR5 maintains hepatocellular carcinoma via H3K9 epigenetic silencing of CDKN2A

Abstract Hepatocellular carcinoma (HCC) is one of the most common cancers (>800,000 new cases each year worldwide) and a leading cause of cancer mortality (~700,000 deaths per year globally), with an average 5-year survival rate of 20~30%. The prevalence of HCC and the unmet clinical needs emphasize the urge for novel regimens for these malignancies. In this study, we performed an epigenetics-focused CRISPR interference (CRISPRi) library screen in the HepG2-dCas9-Krab cells (a total of 3,669 sgRNAs targeting 728 chromatin regulator genes at their transcription start sites) and identified ACTR5 as a novel essential gene in HCC. CRISPR depletion of ACTR5 led to arrested cell cycle and attenuated HCC tumor progression in vivo. Transcriptomic analysis revealed a significantly increased CDKN2A expression in the sgACTR5 transduced HCC cells, which led to the activation of Rb and repression of CDK6 and E2F signaling. Meanwhile, histone modification profiling identified loss of H3K9me2 and Hek9me3 at the CDKN2A promoters upon ACTR5 depletion, indicating the involvement of the epigenetic silencing of tumor suppressors (including CDKN2A) underlying the ACTR5-driven HCC progression. Mechanistically, we found that CRISPR targeting the ACTR5’s interacting protein IES6 (also known as INO80C) recapitulated the sgACTR5 phenotypes, highlighting the collaborative roles between these two partners. We then conducted a high-density CRISPR gene tiling scan using a sgRNA library that targets every NGG protospacer adjacent motif (PAM) within the ACTR5 (284 sgRNAs) and IES6 (90 sgRNAs) coding exons. This saturating mutagenesis screen revealed the requirement of 2 regions (G502-S519 and I552-C569) within ACTR5’s 2nd Actin-fold domain for IES6 interaction. Reciprocally, our unbiased screen identified a previously uncharacterized domain (T163-V187) in IES6’s C-terminal region to mediate the ACTR5-IES6 complex formation. To investigate the clinical impact of ACTR5, we examined the response of HCC cells to various inhibitors upon ACTR5 depletion. While sgACTR5 did not affect the cellular response to sorafenib (an FDA-approved RAF and VEGF inhibitor for HCC patients), we found that suppression of ACTR5 sensitized the HCC cells to ribociclib (a CDK6 inhibitor) and UNC0642 (an H3K9 methyltransferase G9a and GLP inhibitor). Based on these observations, we rationalized a combination treatment of ribociclib/UNC0642 that synergistically suppresses the proliferation of the HCC cells. In summary, our study revealed a pivotal role of ACTR5 (and its complex member IES6) in supporting the CDK6-driven cell cycle progression via an H3K9me2 H3K9me3-mediated silencing of CDKN2A. These notions can be exploited to develop novel combinational therapies against the difficult to cure liver cancers. Citation Format: Xiaobao Xu, Lu Yang, Anthony Chan, Sheela Pangeni Pokharel, Mingli Li, Qiao Liu, Nicole Mattson, Chun-wei Chen. ACTR5 maintains hepatocellular carcinoma via H3K9 epigenetic silencing of CDKN2A [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 2967.

Time makes histone H3 modifications drift in mouse liver.

To detect the epigenetic drift of time passing, we determined the genome-wide distributions of mono- and tri-methylated lysine 4 and acetylated and tri-methylated lysine 27 of histone H3 in the livers of healthy 3, 6 and 12 months old C57BL/6 mice. The comparison of different age profiles of histone H3 marks revealed global redistribution of histone H3 modifications with time, in particular in intergenic regions and near transcription start sites, as well as altered correlation between the profiles of different histone modifications. Moreover, feeding mice with caloric restriction diet, a treatment known to retard aging, reduced the extent of changes occurring during the first year of life in these genomic regions.

Graph-Based Integration of Histone Modification Profiles

In this work, we introduce a similarity-network-based approach to explore the role of interacting single-cell histone modification signals in haematopoiesis—the process of differentiation of blood cells. Histones are proteins that provide structural support to chromosomes. They are subject to chemical modifications—acetylation or methylation—that affect the degree of accessibility of genes and, in turn, the formation of different phenotypes. The concentration of histone modifications can be modelled as a continuous signal, which can be used to build single-cell profiles. In the present work, the profiles of cell types involved in haematopoiesis are built based on all the major histone modifications (i.e., H3K27ac, H3K27me3, H3K36me3, H3K4me1, H3K4me3, H3K9me3) by counting the number of peaks in the modification signals; then, the profiles are used to compute modification-specific similarity networks among the considered phenotypes. As histone modifications come as interacting signals, we applied a similarity network fusion technique to integrate these networks in a unique graph, with the aim of studying the simultaneous effect of all the modifications for the determination of different phenotypes. The networks permit defining of a graph-cut-based separation score for evaluating the homogeneity of subgroups of cell types corresponding to the myeloid and lymphoid phenotypes in the classical representation of the haematopoietic tree. Resulting scores show that separation into myeloid and lymphoid phenotypes reflects the actual process of haematopoiesis.

Modified Adenosines Sensitize Glioblastoma Cells to Temozolomide by Affecting DNA Methyltransferases.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor, and due to its unique features, its management is certainly one of the most challenging ones among all cancers. N6-isopentenyladenosine (IPA) and its analog N6-benzyladenosine (N6-BA) are modified nucleosides endowed with potent antitumor activity on different types of human cancers, including GBM. Corroborating our previous finding, we demonstrated that IPA and N6-BA affect GBM cell line proliferation by modulating the expression of the F-box WD repeat domain-containing-7 (FBXW7), a tumor suppressor with a crucial role in the turnover of many proteins, such as SREBPs and Mcl1, involved in malignant progression and chemoresistance. Luciferase assay revealed that IPA-mediated upregulation of FBXW7 translates in transcriptional inactivation of its oncogenic substrates (Myc, NFkB, or HIF-1α). Moreover, downregulating MGMT expression, IPA strongly enhances the killing effect of temozolomide (TMZ), producing a favorable sensitizing effect starting from a concentration range much lower than TMZ EC50. Through DNA methyltransferase (DNMT) activity assay, analysis of the global DNA methylation, and the histone modification profiles, we demonstrated that the modified adenosines behave similar to 5-AZA-dC, known DNMT inhibitor. Overall, our results provide new perspectives for the first time, suggesting the modified adenosines as epigenetic tools able to improve chemo- and radiotherapy efficacy in glioblastoma and potentially other cancers.