Lysine Methylation Marks Research Articles

Histone Methylation Mark Analyses Distinguish Carts Manufactured from Distinct Sources and Uncover Novel Transcription Factors Associated with In Vivo Function of Carts after Infusion into DLBCL Patients That Are Not Identified By RNA-Seq

Chimeric antigen receptor-modified T cell (CART) therapy has heralded a revolution in cancer immunotherapy. Yet, understanding the qualities that govern effects on in vivo function of CART infusion products (IP) has been challenging. RNA sequencing (RNA-seq), albeit high dimensional in scope, is limited by the dynamic nature of transcription, especially in T cells and CARTs where activation may hinder identification of transiently or minimally expressed genes. We investigated whether epigenomic analyses of histone 3 lysine methylation marks (H3Kme) might uncover genes associated with the potential of starting T cell subsets and CART IP that could not be identified by RNA-seq. We used Cleavage Under Targets and Restriction Using Nuclease (CUT&RUN) to assess whole genome transcriptionally permissive H3K4me2 and repressive H3K27me3 marks in naïve (N), central memory (CM) and effector memory (EM) CD8+ T cells and in CARTs of different potencies and manufactured from different sources. We identify, for the first time, epigenetic predictors of CART cell expansion in a clinical trial (NCT01865617). In preclinical models, CARTs derived from less differentiated N and CM T cells control tumor more efficiently than their EM-derived counterparts. We conducted parallel RNA-seq and histone mark analyses on N, CM and EM CD8+ T cells. We found superior distinction between T cell subsets by individual histone mark analyses compared to RNA-seq. H3K4me2 marks identified 2.7- to 4.5-fold more differentially enriched transcription factors (TFs) than RNA-seq when comparing subsets. Statistical noise in high transcript abundance genes and transcript sparsity in low transcript abundance genes complicates the discriminative capacity of RNA-seq. To further elucidate factors governing T cell qualities we assessed combinatorial patterns of change in histone marks between CD8+ T cell subsets in both high and low transcript abundance genes that were not identified as differentially expressed by RNA-seq. Patterns of histone mark changes between subsets identified novel genes associated with T cell differentiation not seen by RNA-seq or differential enrichment of individual histone marks. We then determined whether histone mark analyses could distinguish CARTs manufactured from similar, yet distinct CD8 + T cell sources. When comparing CM- and EM-derived CART cells from healthy donors (HD) we found that analyses of histone marks identified 33- and 44-fold more genes, respectively, than RNA-seq, including TFs associated with T cell differentiation (KLF7, ZEB2, LEF1) that were not identified by RNA-seq (Fig. 1). By assessing transitional histone mark patterns (H3K4me2+H3K27me3+ and H3K4me2-H3K27me3-), we identified genesets associated with T cell differentiation, cellular stress response and metabolism, as well as master regulators associated with T cell differentiation, exhaustion, and lipid metabolism (POU2F1, RELA, NFKB, MYC, STAT1, SREBF1) that were not identified by RNA-seq (Fig. 2). Similarly, histone mark analyses distinguished and identified increased differentiation in CARTs manufactured from phenotypically CM cells isolated from DLBCL patients on a phase 1/2 clinical trial (NCT01865617) from CARTs manufactured from CM cells from healthy donors. Identification of genetic programming in CARTs associated with in vivo function in clinical trials of CART immunotherapy has been exceptionally challenging. We analysed CART IP from NCT01865617 and found that histone mark analyses, but not RNA-seq, identified differences between IP that associated with the magnitude of CART counts in patient blood after infusion. We demonstrated that the proliferation-augmenting TF, KLF7, and its epigenomic regulome, were associated with in vivo CART cell accumulation. Histone mark analyses enable identification of novel parameters of T cell and CART quality that could be modified to improve outcomes of CART immunotherapy.

Targeting LSD1 in tumor immunotherapy: rationale, challenges and potential.

Lysine-specific demethylase 1 (LSD1) is an enzyme that removes lysine methylation marks from nucleosome histone tails and plays an important role in cancer initiation, progression, metastasis, and recurrence. Recent research shows that LSD1 regulates tumor cells and immune cells through multiple upstream and downstream pathways, enabling tumor cells to adapt to the tumor microenvironment (TME). As a potential anti-tumor treatment strategy, immunotherapy has developed rapidly in the past few years. However, most patients have a low response rate to available immune checkpoint inhibitors (ICIs), including anti-PD-(L)1 therapy and CAR-T cell therapy, due to a broad array of immunosuppressive mechanisms. Notably, inhibition of LSD1 turns "cold tumors" into "hot tumors" and subsequently enhances tumor cell sensitivity to ICIs. This review focuses on recent advances in LSD1 and tumor immunity and discusses a potential therapeutic strategy for combining LSD1 inhibition with immunotherapy.

Sulforaphane and iberin are potent epigenetic modulators of histone acetylation and methylation in malignant melanoma.

Growing evidence supports that isothiocyanates exert a wide range of bioactivities amongst of which is their capacity to interact with the epigenetic machinery in various cancers including melanoma. Our aim was to characterise the effect of sulforaphane and iberin on histone acetylation and methylation as a potential anti-melanoma strategy. We have utilised an in vitro model of malignant melanoma [consisting of human (A375, Hs294T, VMM1) and murine (B16F-10) melanoma cell lines as well as a non-melanoma (A431) and a non-tumorigenic immortalised keratinocyte (HaCaT) cell line] exposed to sulforaphane or iberin. Cell viability was evaluated by the Alamar blue assay whilst total histone deacetylases and acetyltransferases activities were determined by the Epigenase HDAC Activity/Inhibition and EpiQuik HAT Activity/Inhibition assay kits, respectively. The expression levels of specific histone deacetylases and acetyltransferases together with those of lysine acetylation and methylation marks were obtained by western immunoblotting. Overall, both sulforaphane and iberin were able to (1) reduce cell viability, (2) decrease total histone deacetylase activity and (3) modulate the expression levels of various histone deacetylases as well as acetyl and methyl transferases thus modulating the acetylation and methylation status of specific lysine residues on histones 3 and 4 in malignant melanoma cells. Our findings highlight novel insights as to how sulforaphane and iberin differentially regulate the epigenetic response in ways compatible with their anticancer action in malignant melanoma.

Allyl isothiocyanate regulates lysine acetylation and methylation marks in an experimental model of malignant melanoma

Objective(s)Isothiocyanates (ITCs) are biologically active plant secondary metabolites capable of mediating various biological effects including modulation of the epigenome. Our aim was to characterize the effect of allyl isothiocyanate (AITC) on lysine acetylation and methylation marks as a potential epigenetic-induced anti-melanoma strategy.MethodsOur malignant melanoma model consisted of (1) human (A375) and murine (B16-F10) malignant melanoma as well as of human; (2) brain (VMM1) and lymph node (Hs 294T) metastatic melanoma; (3) non-melanoma epidermoid carcinoma (A431) and (4) immortalized keratinocyte (HaCaT) cells subjected to AITC. Cell viability, histone deacetylases (HDACs) and acetyltransferases (HATs) activities were evaluated by the Alamar blue, Epigenase HDAC Activity/Inhibition and EpiQuik HAT Activity/Inhibition assay kits, respectively, while their expression levels together with those of lysine acetylation and methylation marks by western immunoblotting. Finally, apoptotic gene expression was assessed by an RT-PCR-based gene expression profiling methodology.ResultsAITC reduces cell viability, decreases HDACs and HATs activities and causes changes in protein expression levels of various HDACs, HATs, and histone methyl transferases (HMTs) all of which have a profound effect on specific lysine acetylation and methylation marks. Moreover, AITC regulates the expression of a number of genes participating in various apoptotic cascades thus indicating its involvement in apoptotic induction.ConclusionsAITC exerts a potent epigenetic effect suggesting its potential involvement as a promising epigenetic-induced bioactive for the treatment of malignant melanoma.

In Vitro Enzyme Assays for JmjC-Domain-Containing Lysine Histone Demethylases (JmjC-KDMs).

Histone modifications, including lysine methylation marks on histone tails, modulate the accessibility of genes for transcription. Changes in histone tail methylation patterns can cause transcriptional activation or repression. The dynamic regulation of lysine methylation patterns is enabled by two distinct groups of enzymes: histone methyltransferases (KMTs) and demethylases (KDMs). The Jumonji C (JmjC) domain-containing lysine histone demethylases (JmjC-KDMs) alter the methylation levels of histone tails by removing tri-, di-, or mono-methylation marks. Because JmjC-KDMs activities are dysfunctional in cancer and other clinical conditions, they are targets for drug discovery. Efforts are underway to develop high-throughput assays capable of identifying selective, small-molecule inhibitors of KDMs. Detailed in this unit are protocols for mass spectrometry-based and formaldehyde dehydrogenase-coupled enzyme-based assays that can be used to identify inhibitors of JmjC-KDMs. © 2018 by John Wiley & Sons, Inc.

KDM4B-mediated reduction of H3K9me3 and H3K36me3 levels improves somatic cell reprogramming into pluripotency

Correct reprogramming of epigenetic marks is essential for somatic cells to regain pluripotency. Repressive histone (H) lysine (K) methylation marks are known to be stable and difficult to reprogram. In this study, we generated transgenic mice and mouse embryonic fibroblasts (MEFs) for the inducible expression of KDM4B, a demethylase that removes H3 K9 and H3K36 trimethylation (me3) marks (H3K9/36me3). Upon inducing Kdm4b, H3K9/36me3 levels significantly decreased compared to non-induced controls. Concurrently, H3K9me1 levels significantly increased, while H3K9me2 and H3K27me3 remained unchanged. The global transcriptional impact of Kdm4b-mediated reduction in H3K9/36me3 levels was examined by comparative microarray analysis and mRNA-sequencing of three independent transgenic MEF lines. We identified several commonly up-regulated targets, including the heterochromatin-associated zinc finger protein 37 and full-length endogenous retrovirus repeat elements. Following optimized zona-free somatic nuclear transfer, reduced H3K9/36me3 levels were restored within hours. Nevertheless, hypo-methylated Kdm4b MEF donors reprogrammed six-fold better into cloned blastocysts than non-induced donors. They also reprogrammed nine-fold better into induced pluripotent stem cells that gave rise to teratomas and chimeras. In summary, we firmly established H3K9/36me3 as a major roadblock to somatic cell reprogramming and identified transcriptional targets of derestricted chromatin that could contribute towards improving this process in mouse.

Traceless semisynthesis of a set of histone 3 species bearing specific lysine methylation marks.

Considerable mechanistic insight into the function of histone post-translational modifications and the enzymes that install and remove them derives from in vitro experiments with modified histones, often embedded in nucleosomes. We report the first semisyntheses of native-like histone 3 (H3) bearing tri- and dimethyllysines at position 79 and trimethyllysine at position 36, as well as more facile and traceless semisyntheses of K9 and K27 trimethylated species. These semisyntheses are practical on a multi-milligram scale and can also generate H3 with combinations of marks. Each of these modifications has distinct functional consequences, although the pathways by which H3K36me3 and H3K79me2/3 act have not been entirely mapped. To this end, we demonstrated that our semisynthetic histones, when reconstituted into nucleosomes, are valuable affinity reagents for unbiased binding partner discovery and compare them to their methyllysine analogue (MLA) counterparts at the nucleosome level.

Mechanisms of epigenetic memory and addiction.

Epigenetic regulation of cellular identity and function is at least partly achieved through changes in covalent modifications on DNA and histones. Much progress has been made in recent years to understand how these covalent modifications affect cell identity and function. Despite the advances, whether and how epigenetic factors contribute to memory formation is still poorly understood. In this review, we discuss recent progress in elucidating epigenetic mechanisms of learning and memory, primarily at the DNA level, and look ahead to discuss their potential implications in reward memory and development of drug addiction.

CpG Island Chromatin Is Shaped by Recruitment of ZF-CxxC Proteins

Most mammalian gene promoters are embedded within genomic regions called CpG islands, characterized by elevated levels of nonmethylated CpG dinucleotides. Here, we describe recent work demonstrating that CpG islands act as specific nucleation sites for the zinc finger CxxC domain-containing proteins CFP1 and KDM2A. Importantly, both CFP1 and KDM2A are associated with enzymatic activities that modulate specific histone lysine methylation marks. The action of these zinc finger CxxC domain proteins therefore imposes a defined chromatin architecture on CpG islands that distinguishes these important regulatory elements from the surrounding genome. The functional consequence of this CpG island-directed chromatin environment is discussed.

Transient JMJD2B-Mediated Reduction of H3K9me3 Levels Improves Reprogramming of Embryonic Stem Cells into Cloned Embryos

Correct reprogramming of epigenetic marks in the donor nuclei is crucial for successful cloning by nuclear transfer. Specific epigenetic modifications, such as repressive histone lysine methylation marks, are known to be very stable and difficult to reprogram. The discovery of histone lysine demethylases has opened up opportunities to study the effects of removing repressive histone lysine methylation marks in donor cells prior to nuclear transfer. In this study, we generated mouse embryonic stem (ES) cells for the inducible expression of JMJD2B (also known as KDM4B), a demethylase that primarily removes the histone-3 lysine-9 trimethylation (H3K9me3) mark. Induction of jmjd2b in the ES cells decreased total levels of H3K9me3 by 63%. When these cells were used for nuclear transfer, H3K9me3 levels were normalized within minutes following fusion with an enucleated oocyte. This transient reduction of H3K9me3 levels improved in vitro development into cloned embryos by 30%.

Structural Biology of Methylation‐mediated Epigenetic Regulation

The lecture will present our research on recognition events controlling epigenetic regulation mediated by methylation marks on histone tails and at CpG sites on DNA.Specific topics will include recognition of methyllyisne marks by reader elements in ADDATRX (Haitao Li), TRIM24/33 (Zhanxin Wang) and MLL1 (Zhanxin Wang and Jikui Song), erasure of lysine methylation marks by the KMD2A jumonji family lysine demethylases (Zhongjun Cheng), and DNMT1‐mediated active and autoinhibitory mechanisms of maintenance DNA methylation (Jikui Song).Collaborators: Yang Shi (Harvard) and Haitao Li (Tsinghua) on ADDATRX, C. David Allis (Rockefeller) on MLL1, Michelle Barton (M. D. Anderson) on TRIM24, Joan Massague (Sloan‐Kettering) on TRIM33, and Or Gozani (UCSF) on KMD2A.

CHD3 protein recognizes and regulates methylated histone H3 lysines 4 and 27 over a subset of targets in the rice genome

Histone lysine methylation is an important component of the epigenetic system demarcating transcriptionally active and inactive chromatin domains. It is of primary importance in understanding how different histone lysine methylation marks and a specific combination of them are read and interpreted by chromatin proteins to regulate gene expression. In this paper, we report that the rice CHD3 protein CHR729 that was required for many aspects of plant development can interact with dimethylated histone H3 lysine 4 (H3K4me2, a mark associated with moderately expressed or repressed genes) and with trimethylated histone H3 lysine 27 (H3K27me3, a mark associated with repressed genes), respectively, through the chromodomains and the plant homeodomain (PHD) finger of the protein. A mutation or down-regulation of the gene provoked a decrease of H3K27me3 and H3K4me3 (a mark associated with active genes). Genome-wide analysis revealed that H3K27me3 and H3K4me3, respectively, were lost from about 56 and 23% of marked loci, which correspond mostly to under-expressed or repressed genes. In the mutant, a higher-than-expected proportion of down-regulated genes lost H3K4me3, among which many encode DNA-binding transcription factors. These results suggest that the rice CHD3 protein is a bifunctional chromatin regulator able to recognize and modulate H3K4 and H3K27 methylation over repressed or tissue-specific genes, which may be associated with regulation of a gene transcription program of plant development.

L3MBTL2 Protein Acts in Concert with PcG Protein-Mediated Monoubiquitination of H2A to Establish a Repressive Chromatin Structure

We have identified human MBT domain-containing protein L3MBTL2 as an integral component of a protein complex that we termed Polycomb repressive complex 1 (PRC1)-like 4 (PRC1L4), given the copresence of PcG proteins RING1, RING2, and PCGF6/MBLR. PRC1L4 also contained E2F6 and CBX3/HP1γ, known to function in transcriptional repression. PRC1L4-mediated repression necessitated L3MBTL2 that compacted chromatin ina histone modification-independent manner. Genome-wide location analyses identified several hundred genes simultaneously bound by L3MBTL2 and E2F6, preferentially around transcriptional start sites that exhibited little overlap with those targeted by other E2Fs or by L3MBTL1, another MBT domain-containing protein that interacts with RB1. L3MBTL2-specific RNAi resulted in increased expression of target genesthat exhibited a significant reduction in H2A lysine 119 monoubiquitination. Our findings highlight a PcG/MBT collaboration that attains repressive chromatin without entailing histone lysine methylation marks.

Systematic Identification of Methyllysine-Driven Interactions for Histone and Nonhistone Targets

An important issue in epigenetic research is to understand how the numerous methylation marks associated with histone and certain nonhistone proteins are recognized and interpreted by the hundreds of chromatin-binding modules (CBMs) in a cell to control chromatin state, gene expression, and other cellular functions. We have assembled a peptide chip that represents known and putative lysine methylation marks on histones and p53 and probed the chip for binding to a group of CBMs to obtain a comprehensive interaction network mediated by lysine methylation. Interactions revealed by the peptide array screening were validated by in-solution binding assays. This study not only recapitulated known interactions but also uncovered new ones. A novel heterochromatin protein 1 beta (HP1β) chromodomain-binding site on histone H3, H3K23me, was discovered from the peptide array screen and subsequently verified by mass spectrometry. Data from peptide pull-down and colocalization in cells suggest that, besides the H3K9me mark, H3K23me may play a role in facilitating the recruitment of HP1β to the heterochromatin. Extending the peptide array and mass spectrometric approach presented here to more histone marks and CBMs would eventually afford a comprehensive specificity and interaction map to aid epigenetic studies.

Recognition of Histone H3K4 Trimethylation by the Plant Homeodomain of PHF2 Modulates Histone Demethylation

Distinct lysine methylation marks on histones create dynamic signatures deciphered by the "effector" modules, although the underlying mechanisms remain unclear. We identified the plant homeodomain- and Jumonji C domain-containing protein PHF2 as a novel histone H3K9 demethylase. We show in biochemical and crystallographic analyses that PHF2 recognizes histone H3K4 trimethylation through its plant homeodomain finger and that this interaction is essential for PHF2 occupancy and H3K9 demethylation at rDNA promoters. Our study provides molecular insights into the mechanism by which distinct effector domains within a protein cooperatively modulate the "cross-talk" of histone modifications.

Histone Lysine Methylation: An Epigenetic Modification?

Histone lysine methylation regulates a variety of nuclear processes including transcription and maintenance of genome integrity. Many attributes of the histone lysine methylation system suggest that it might also contribute to epigenetic memory. Here, we examine the recent advances in our understanding of three intensely-studied histone lysine methylation marks, focusing on the potential mechanisms by which these marks may be maintained during cell proliferation and adhere to the principles of epigenetics.

Global levels of histone modifications predict prostate cancer recurrence

Epigenetic alterations such as DNA methylation and histone modifications play important roles in carcinogenesis. It was reported that global histone modification patterns are predictors of cancer recurrence in various tumor entities. Our study was performed to evaluate histone lysine (H(x)K(y)) and histone acetyl (H(x)Ac) modifications in prostate tissue. A tissue microarray with 113 prostate cancer (PCA), 23 non-malignant prostate tissues was stained with antibodies against H3K4 mono-(H3K4me1), di-(H3K4me2), tri-(H3K4me3) methylation, H3K9me1, H3K9me2, H3K9me3, H3 and H4 pan-acetylation (H3Ac, H4Ac). We also analyzed H3K4 methylation in patients with advanced PCA (hormone-refractory PCA-HRPC, n = 34; hormone-dependent PCA, n = 30). Sections were scored according the staining intensity and the proportion of epithelial cells showing nuclear staining. H3K4me1, H3K9me2, H3K9me3, H3Ac, and H4Ac were significantly reduced in PCA compared to non-malignant prostate tissue. H3Ac and H3K9me2 levels allowed discrimination of PCA and non-malignant prostate tissue highly specifically (>91%) and sensitively (>78%) as determined via ROC analyses (AUC >0.91). Histone lysine methylation and histone acetylation marks were correlated with clinical-pathological parameters (i.e., digital rectal examination, preoperative PSA, pT-stage, lymph node metastasis, Gleason score). In addition, H3K4me1 was a significant predictor of PSA recurrence following radical prostatectomy. H3K4me1, H3K4me2, and H3K4me3 levels were significantly increased in HRPC. Global histone modification levels may help to identify patients with adverse prognosis, and represent a target for the future therapy of PCA.

Impaired recruitment of the histone methyltransferase DOT1L contributes to the incomplete reactivation of tumor suppressor genes upon DNA demethylation

Understanding the mechanisms that link changes in DNA methylation with histone modifications is particularly relevant in the case of tumor suppressor genes that undergo transcriptional silencing in cancer cells in association with promoter CpG island hypermethylation. In this study, we show that two histone lysine methylation marks associated with active transcription, dimethylation of H3K79 (H3K79me2) and trimethylation of H3K4 (H3K4me3), are present in all the unmethylated promoters analysed, and both of them are lost when these promoters become hypermethylated. Most importantly, pharmacological and genetic interventions that cause DNA demethylation and partial recovery of gene transcription, result in the restoration of H3K4me3, but not of H3K79me2. We also show that DOT1L, the major H3K79 histone methyltransferase, is no longer recruited to the promoters that are demethylated after 5-aza-deoxycytidine treatment or genetic deletion of DNA methyltransferases. Knock-down and transfection experiments for DOT1L show that this enzyme has a direct role in maintaining the euchromatic and active status of these genes when unmethylated. These findings suggest that DNA demethylating interventions alone are not able to restore a complete euchromatic status and a full transcriptional reactivation of the epigenetically silenced tumor suppressor genes, and reinforce the necessity of targeting multiple elements of the epigenetics machinery for a successful treatment of malignancies.

L3MBTL1, a Histone-Methylation-Dependent Chromatin Lock

Distinct histone lysine methylation marks are involved in transcriptional repression linked to the formation and maintenance of facultative heterochromatin, although the underlying mechanisms remain unclear. We demonstrate that the malignant-brain-tumor (MBT) protein L3MBTL1 is in a complex with core histones, histone H1b, HP1gamma, and Rb. The MBT domain is structurally related to protein domains that directly bind methylated histone residues. Consistent with this, we found that the L3MBTL1 MBT domains compact nucleosomal arrays dependent on mono- and dimethylation of histone H4 lysine 20 and of histone H1b lysine 26. The MBT domains bind at least two nucleosomes simultaneously, linking repression of transcription to recognition of different histone marks by L3MBTL1. Consistently, L3MBTL1 was found to negatively regulate the expression of a subset of genes regulated by E2F, a factor that interacts with Rb.

PLU-1 Is an H3K4 Demethylase Involved in Transcriptional Repression and Breast Cancer Cell Proliferation

Posttranslational modification of chromatin by histone methylation has wide-ranging effects on nuclear function, including transcriptional regulation, maintenance of genome integrity, and epigenetic inheritance. The enzymes utilized to place histone methylation marks are well characterized, but the identity of a histone demethylation system remained elusive until recently. The discovery of histone demethylase enzymes capable of directly removing methyl groups from modified lysine residues has demonstrated that histone methylation is a dynamic modification. The most extensive family of histone demethylase enzymes identified so far contains a JmjC domain and catalyzes demethylation through a hydroxylation reaction. Here, we identify PLU-1, a transcriptional repressor implicated in breast cancer, as a histone demethylase enzyme that has the ability to reverse the trimethyl H3K4 modification state. Furthermore, we reveal that PLU-1-mediated H3K4 demethylase activity plays an important role in the proliferative capacity of breast cancer cells through repression of tumor suppressor genes, including BRCA1.