Emerging Approaches to Profile Accessible Chromatin from Formalin-Fixed Paraffin-Embedded Sections.

Nucleosomes are non-uniformly distributed across eukaryotic genomes, with stretches of 'open' chromatin strongly associated with transcriptionally active promoters and enhancers. Understanding chromatin accessibility patterns in normal tissue and how they are altered in pathologies can provide critical insights to development and disease. With the advent of high-throughput sequencing, a variety of strategies have been devised to identify open regions across the genome, including DNase-seq, MNase-seq, FAIRE-seq, ATAC-seq, and NicE-seq.

Epigenetics of Genes Preferentially Expressed in Dissimilar Cell Populations: Myoblasts and Cerebellum.

While studying myoblast methylomes and transcriptomes, we found that had a remarkable preference for expression in both myoblasts and cerebellum. To understand how widespread such a relationship was and its epigenetic and biological correlates, we systematically looked for genes with similar transcription profiles and analyzed their DNA methylation and chromatin state and accessibility profiles in many different cell populations. Twenty genes were expressed preferentially in myoblasts and cerebellum (Myob/Cbl genes).

NicE-viewSeq: An Integrative Visualization and Genomics Method to Detect Accessible Chromatin in Fixed Cells.

A novel genome-wide accessible chromatin visualization, quantitation, and sequencing method is described, which allows in situ fluorescence visualization and sequencing of the accessible chromatin in the mammalian cell. The cells are fixed by formaldehyde crosslinking, and processed using a modified nick translation method, where a nicking enzyme nicks one strand of DNA, and DNA polymerase incorporates biotin-conjugated dCTP, 5-methyl-dCTP, Fluorescein-12-dATP or Texas Red-5-dATP, dGTP, and dTTP. This allows accessible chromatin DNA to be labeled for visualization and on bead NGS library preparation.

Measuring Inaccessible Chromatin Genome-Wide Using Protect-seq.

Chromatin accessibility has been an immensely powerful metric for identifying and understanding regulatory elements in the genome. Many important regulatory elements, such as enhancers and transcriptional start sites, are characterized by "open" or nucleosome-free regions. Understanding the areas of the genome that are not considered open chromatin has been more difficult.

Universal NicE-Seq: A Simple and Quick Method for Accessible Chromatin Detection in Fixed Cells.

Genome-wide accessible chromatin sequencing and identification has enabled deciphering the epigenetic information encoded in chromatin, revealing accessible promoters, enhancers, nucleosome positioning, transcription factor occupancy, and other chromosomal protein binding. The starting biological materials are often fixed using formaldehyde crosslinking. Here, we describe accessible chromatin library preparation from low numbers of formaldehyde-crosslinked cells using a modified nick translation method, where a nicking enzyme nicks one strand of DNA and DNA polymerase incorporates biotin-conjugated dATP, dCTP, and methyl-dCTP.

Diverse silent chromatin states modulate genome compartmentalization and loop extrusion barriers.

The relationships between chromosomal compartmentalization, chromatin state and function are poorly understood. Here by profiling long-range contact frequencies in HCT116 colon cancer cells, we distinguish three silent chromatin states, comprising two types of heterochromatin and a state enriched for H3K9me2 and H2A.Z that exhibits neutral three-dimensional interaction preferences and which, to our knowledge, has not previously been characterized.

Promoter-Adjacent DNA Hypermethylation Can Downmodulate Gene Expression: in the Muscle Lineage.

TBX15, which encodes a differentiation-related transcription factor, displays promoter-adjacent DNA hypermethylation in myoblasts and skeletal muscle (psoas) that is absent from non-expressing cells in other lineages. By whole-genome bisulfite sequencing (WGBS) and enzymatic methyl-seq (EM-seq), these hypermethylated regions were found to border both sides of a constitutively unmethylated promoter. To understand the functionality of this DNA hypermethylation, we cloned the differentially methylated sequences (DMRs) in CpG-free reporter vectors and tested them for promoter or enhancer activity upon transient transfection.

Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation in mammalian nuclear genome.

In mammalian cells, SET8 mediated Histone H4 Lys 20 monomethylation (H4K20me1) has been implicated in regulating mitotic condensation, DNA replication, DNA damage response, and gene expression. Here we show SET8, the only known enzyme for H4K20me1 is post-translationally poly ADP-ribosylated by PARP1 on lysine residues. PARP1 interacts with SET8 in a cell cycle-dependent manner.

Polyglutamylation: biology and analysis.

Polyglutamylation is a posttranslational modification (PTM) that adds several glutamates on glutamate residues in the form of conjugated peptide chains by a family of enzymes known as polyglutamylases. Polyglutamylation is well documented in microtubules. Polyglutamylated microtubules consist of different α- and β-tubulin subunits with varied number of added glutamate residues.

One-pot universal NicE-seq: all enzymatic downstream processing of 4% formaldehyde crosslinked cells for chromatin accessibility genomics.

Background: Accessible chromatin landscape allows binding of transcription factors, and remodeling of promoter and enhancer elements during development. Chromatin accessibility along with integrated multiomics approaches have been used for determining molecular subtypes of cancer in patient samples.

Results: One-pot Universal NicE-seq (One-pot UniNicE-seq) is an improved accessible chromatin profiling method that negate DNA purification and incorporate sonication free enzymatic fragmentation before library preparation and is suited to a variety of mammalian cells.

Aba-Seq: High-Resolution Enzymatic Mapping of Genomic 5-Hydroxymethylcytosine.

Aba-Seq (DNA modification-dependent restriction endonuclease AbaSI coupled with sequencing) provides a cost-effective and non-chemical based method for the high-resolution mapping of genomic 5-hydroxymethylcytosine (5hmC). The high specificity of the AbaSI enzyme allows sensitive detection of 5hmC in the genome. Here, we describe the Aba-Seq technique that was used for the high-resolution mapping of 5hmC in the genome of mouse embryonic stem cells (E14).

Nondestructive enzymatic deamination enables single-molecule long-read amplicon sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution.

The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA, leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite-treated DNA, we applied a new enzymatic deamination approach, termed enzymatic methyl-seq (EM-seq), to long-range sequencing technologies.

Universal NicE-seq for high-resolution accessible chromatin profiling for formaldehyde-fixed and FFPE tissues.

Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negates the optimization step and is suited to a variety of mammalian cells and tissues.

Visualization and Sequencing of Accessible Chromatin Reveals Cell Cycle and Post-HDAC inhibitor Treatment Dynamics.

Chromatin accessibility is a predictor of gene expression, cell division, and cell type specificity. NicE-viewSeq (Nicking Enzyme-assisted viewing and Sequencing) allows accessible chromatin visualization and sequencing with overall lower mitochondrial DNA and duplicated sequences interference relative to ATAC-see. Using NicE-viewSeq, we interrogated the accessibility of chromatin in a cell cycle (G1, S, and G2/M)-specific manner using mammalian cells.

Heterochromatin establishment during early mammalian development is regulated by pericentromeric RNA and characterized by non-repressive H3K9me3.

Following fertilization in mammals, the gametes are reprogrammed to create a totipotent zygote, a process that involves de novo establishment of chromatin domains. A major feature occurring during preimplantation development is the dramatic remodelling of constitutive heterochromatin, although the functional relevance of this is unknown. Here, we show that heterochromatin establishment relies on the stepwise expression and regulated activity of SUV39H enzymes.

Mapping of polyglutamylation in tubulins using nanoLC-ESI-MS/MS.

Tubulin polyglutamylation is a polymeric modification that extends from the carboxyl-terminus of tubulins. Molecular description of amino acids and their branching polyglutamyls is a hallmark of tubulin in microtubules. There are different chemical approaches for detecting these polymeric structures, mostly reported prior to development of nESI peptide analysis.

The microtubule-associated histone methyltransferase SET8, facilitated by transcription factor LSF, methylates α-tubulin.

Microtubules are cytoskeletal structures critical for mitosis, cell motility, and protein and organelle transport and are a validated target for anticancer drugs. However, how tubulins are regulated and recruited to support these distinct cellular processes is incompletely understood. Posttranslational modifications of tubulins are proposed to regulate microtubule function and dynamics.

Protect-seq: genome-wide profiling of nuclease inaccessible domains reveals physical properties of chromatin.

In metazoan cell nuclei, heterochromatin constitutes large chromatin domains that are in close contact with the nuclear lamina. These heterochromatin/lamina-associated domains (LADs) domains are difficult to profile and warrants a simpler and direct method. Here we report a new method, Protect-seq, aimed at identifying regions of heterochromatin via resistance to nuclease degradation followed by next-generation sequencing (NGS).

Mechanisms of DNA Methyltransferase Recruitment in Mammals.

DNA methylation is an essential epigenetic mark in mammals. The proper distribution of this mark depends on accurate deposition and maintenance mechanisms, and underpins its functional role. This, in turn, depends on the precise recruitment and activation of de novo and maintenance DNA methyltransferases (DNMTs).

Levels of DNA cytosine methylation in the genome.

Insects provide an accessible system to study the contribution of DNA methylation to complex epigenetic phenotypes created to regulate gene expression, chromatin states, imprinting and dosage compensation. The members of genus have been used as a model system to study aspects of biology like development, behaviour and genetics. Despite the popularity of as a genetic and epigenetic model organism, DNA methylation studies are limited due to low levels of genomic 5-methylcytosine.

Targeting the SET and RING-associated (SRA) domain of ubiquitin-like, PHD and ring finger-containing 1 (UHRF1) for anti-cancer drug development.

Ubiquitin-like containing PHD Ring Finger 1 (UHRF1) is a multi-domain protein with a methyl-DNA binding SRA (SET and RING-associated) domain, required for maintenance DNA methylation mediated by DNMT1. Primarily expressed in proliferating cells, UHRF1 is a cell-cycle regulated protein that is required for S phase entry. Furthermore, UHRF1 participates in transcriptional gene regulation by connecting DNA methylation to histone modifications.

S-adenosylhomocysteine Hydrolase Participates in DNA Methylation Inheritance.

DNA (cytosine-5) methyltransferase 1 (DNMT1) is essential for mammalian development and maintenance of DNA methylation following DNA replication in cells. The DNA methylation process generates S-adenosyl-l-homocysteine, a strong inhibitor of DNMT1. Here we report that S-adenosylhomocysteine hydrolase (SAHH/AHCY), the only mammalian enzyme capable of hydrolyzing S-adenosyl-l-homocysteine binds to DNMT1 during DNA replication.