Combination Of Histone Marks Research Articles

Histone FRET reports the spatial heterogeneity in nanoscale chromatin architecture that is imparted by the epigenetic landscape at the level of single foci in an intact cell nucleus

Genome sequencing has identified hundreds of histone post-translational modifications (PTMs) that define an open or compact chromatin nanostructure at the level of nucleosome proximity, and therefore serve as activators or repressors of gene expression. Direct observation of this epigenetic mode of transcriptional regulation in an intact single nucleus, is however, a complex task. This is because despite the development of fluorescent probes that enable observation of specific histone PTMs and chromatin density, the changes in nucleosome proximity regulating gene expression occur on a spatial scale well below the diffraction limit of optical microscopy. In recent work, to address this research gap, we demonstrated that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) between fluorescently labelled histones core to the nucleosome, is a readout of chromatin nanostructure that can be multiplexed with immunofluorescence (IF) against specific histone PTMs. Here from application of this methodology to gold standard gene activators (H3K4Me3 and H3K9Ac) versus repressors (e.g., H3K9Me3 and H3K27Me), we find that while on average these histone marks do impart an open versus compact chromatin nanostructure, at the level of single chromatin foci, there is significant spatial heterogeneity. Collectively this study illustrates the importance of studying the epigenetic landscape as a function of space within intact nuclear architecture and opens the door for the study of chromatin foci sub-populations defined by combinations of histone marks, as is seen in the context of bivalent chromatin.

Abstract 1378: Identification of genomic regions associated with temozolomide resistance in glioblastoma through analysis of histone marks on chromatin

Abstract Glioblastoma (GBM) is an aggressive and fatal primary brain tumor. Temozolomide (TMZ) is a critical component of the standard care of newly diagnosed GBM patients, but unfortunately preexisting primary resistance and rapid emergence of secondary resistance invariably limits the therapeutic benefit of TMZ in GBM. Prior studies have identified genetic and epigenetic alterations that can modulate TMZ sensitivity and treatment outcome. However, comprehensive analysis of histone marks and knowledge of epigenetic regulation of genes associated with TMZ sensitivity or resistance is lacking. To identify epigenetic states associated with TMZ resistance, we performed an epigenetic profiling of eight different histone marks in GBM xenografts. Using chromatin immunoprecipitation combined with high throughput sequencing (ChIP-seq), distribution of H3K4me1, H3K4me3, H3K9ac, H3K9me2, H3K9me3, H3K27ac, H3K27me3 and H3K36me3 histone marks was compared in a panel of GBM patient-derived xenograft sub-lines derived by treating TMZ sensitive GBM12 tumors with placebo (n=2 sublines) or temozolomide (n=6 sub-lines) and then propagating resulting recurrent tumors. Our analysis revealed that H3K4me1 and H3K27ac modification patterns varied globally across individual sub-lines, while distribution of H3K4me3, H3K36me3 histone marks was specifically altered in discrete genomic regions in resistant sub-lines depending on the mechanisms of resistance. To find how epigenetic modifications affect TMZ sensitivity, we analyzed ChIP-Seq data using Hidden Markov Model to test if one or a combination of histone marks relates to TMZ resistance. The effect of histone modifications on transcription was simultaneously determined by RNA sequencing. These analyses helped identify specific histone modifications which could be functionally related to TMZ resistance. Through these analyses we have identified 1142 genomic regions governed by a specific epigenetic pattern. We subsequently analyzed a candidate genomic region on top of the list. By using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated nucleases 9 system (CRISPR/Cas9 system), we have generated clones from an intrinsically TMZ resistant SKMG3 cell line with deletion up to four-kilo base genomic region. Clonogenic growth assays showed that deletion of this genomic region enhanced TMZ sensitivity, reducing the IC50 from 186 μM to less than 60μM, p<0.05. This finding indicates that this genomic region is functionally related with TMZ sensitivity. Taken together, our study reveals epigenetic modifications related to TMZ resistance in GBM cells and a specific genomic region involved in regulating TMZ sensitivity. The analysis of epigenetic state at this genomic region could potentially be useful in predicting treatment response and may help in designing TMZ sensitizing therapy in GBM. Citation Format: Xiaoyue Chen, Haiyun Gan, Jeong Heong Lee, Dong Fang, Gaspar Kitange, Jann Sarkaria, Zhiguo Zhang. Identification of genomic regions associated with temozolomide resistance in glioblastoma through analysis of histone marks on chromatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1378. doi:10.1158/1538-7445.AM2017-1378

The structural basis of modified nucleosome recognition by 53BP1.

DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 Å resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.

Analysis of replacing DNase-seq data with histone marks in computational dimer prediction

Background Open chromatin regions and their findings are great pointers to genome-wide revelations of transcription factor activities, which in-turn defines the level of gene participation and their roles in metabolic well-being or disease condition in human body. Chromatin openness [1] is highly influenced by the presence of histone markers which are biomarkers that define the selective modification of some amino acid at specific positions. Worthy of note is that DNase-seq useful in detecting transcription [2] activity is scarcely available in most species compared to more abundant ChIP-seq data across several species. Our interest is to investigate an optimal combination of histone marks that could replace the DNase-seq data in transcription factor dimer prediction algorithm.

The Correlation Between Histone Modifications and Gene Expression

In the nuclei of eukaryotic cells, DNA wraps around the octamer of histone proteins to form the nucleosome, in a structure like ‘beads on a string’, which makes up the basic unit of chromatin. Chromatin further folds into higherlevel structures, loosely or tightly, which helps to determine the accessibility of the DNA. For instance, actively transcribed regions tend to be in looser chromatin structures so that transcription factors and RNA polymerases can access the genes. Chromatin structure can be altered by various post-translational modifications of the N-terminal tail residues of histone proteins. For example, acetylation of a lysine residue can neutralize its positive charge and weaken the binding between the histone and the negatively charged DNA, which exposes the DNA to regulatory proteins. Methylation is another common type of histone modification; for example, the lysine at the fourth position of the H3 histone can be mono-, dior tri-methylated (denoted as H3K4me1, H3K4me2 and H3K4me3, respectively). By examining histone modification patterns at highly conserved noncoding regions in mouse embryonic stem cells, Bernstein et al. found ‘bivalent domains’ of histone modifications (i.e., harboring both the repressive mark H3K27me3 and the active mark H3K4me3) near genes with poised transcription [1]. When embryonic stem cells differentiate into more specialized cells (e.g., neural precursor cells), a subset of the bivalent domains are resolved (i.e., H3K27me3 becomes weaker, while H3K4me3 becomes stronger, and these loci coincide with genes that are actively transcribed in neural precursor cells). Thus, combinations of histone marks are indicative of transcriptional states. Barski et al. mapped 20 histone methylations of lysine and arginine residues in human CD4 T cells using chromatin immunoprecipitation followed by sequencing (ChIP-seq) [2]. They found that monomethylated H3K27, H3K9, H4K20, H3K79 and H2BK5 were linked to gene activation, while trimethylated H3K27, H3K9 and H3K79 were linked to gene repression. In a later study, the group profiled 39 additional histone modifications in human CD4 T cells [3]. They identified more than 3000 genes that were highly expressed in these cells and the promoters of these genes showed high levels of 17 histone modifications (called a histone modification module). Other studies also investigated the correlation between individual histone marks and gene expression, although not in a quantitative way [4,5].

Recruitment of TIF1γ to Chromatin via Its PHD Finger-Bromodomain Activates Its Ubiquitin Ligase and Transcriptional Repressor Activities

The interplay between sequence-specific DNA-binding transcription factors, histone-modifying enzymes, and chromatin-remodeling enzymes underpins transcriptional regulation. Although it is known how single domains of chromatin "readers" bind specific histone modifications, how combinations of histone marks are recognized and decoded is poorly understood. Moreover, the role of histone binding in regulating the enzymatic activity of chromatin readers is not known. Here we focus on the TGF-β superfamily transcriptional repressor TIF1γ/TRIM33/Ectodermin and demonstrate that its PHD finger-bromodomain constitutes a multivalent histone-binding module that specifically binds histone H3 tails unmethylated at K4 and R2 and acetylated at two key lysines. TIF1γ's ability to ubiquitinate its substrate Smad4 requires its PHD finger-bromodomain, as does its transcriptional repressor activity. Most importantly, TIF1γ's E3 ubiquitin ligase activity is induced by histone binding. We propose a model of TIF1γ activity in which it dictates the residence time of activated Smad complexes at promoters of TGF-β superfamily target genes.

Recognition of a Mononucleosomal Histone Modification Pattern by BPTF via Multivalent Interactions

Little is known about how combinations of histone marks are interpreted at the level of nucleosomes. The second PHD finger of human BPTF is known to specifically recognize histone H3 when methylated on lysine 4 (H3K4me2/3). Here, we examine how additional heterotypic modifications influence BPTF binding. Using peptide surrogates, three acetyllysine ligands are indentified for a PHD-adjacent bromodomain in BPTF via systematic screening and biophysical characterization. Although the bromodomain displays limited discrimination among the three possible acetyllysines at the peptide level, marked selectivity is observed for only one of these sites, H4K16ac, in combination with H3K4me3 at the mononucleosome level. In support, these two histone marks constitute a unique trans-histone modification pattern that unambiguously resides within a single nucleosomal unit in human cells, and this module colocalizes with these marks in the genome. Together, our data call attention to nucleosomal patterning of covalent marks in dictating critical chromatin associations.

Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression of aberrantly silenced genes

Epigenetic chromatin modification is a major regulator of eukaryotic gene expression, and aberrant epigenetic silencing of gene expression contributes to tumorigenesis. Histone modifications include acetylation, phosphorylation, and methylation, resulting in a combination of histone marks known collectively as the histone code. The chromatin marks at a given promoter determine, in part, whether specific promoters are in an open/active conformation or closed/repressed conformation. Dimethyl-lysine 4 histone H3 (H3K4me2) is a transcription-activating chromatin mark at gene promoters, and demethylation of this mark by the lysine-specific demethylase 1 (LSD1), a homologue of polyamine oxidases, may broadly repress gene expression. We now report that novel biguanide and bisguanidine polyamine analogues are potent inhibitors of LSD1. These analogues inhibit LSD1 in human colon carcinoma cells and affect a reexpression of multiple, aberrantly silenced genes important in the development of colon cancer, including members of the secreted frizzle-related proteins (SFRPs) and the GATA family of transcription factors. Furthermore, we demonstrate by chromatin immunoprecipitation analysis that the reexpression is concurrent with increased H3K4me2 and acetyl-H3K9 marks, decreased H3K9me1 and H3K9me2 repressive marks. We thus define important new agents for reversing aberrant repression of gene transcription.