Analysis Of Epigenetic Marks Research Articles

Abstract B004: Analysis of epigenetic marks and mechanisms in disease: Your guide to a successful assay

Abstract Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets & Release Using Nuclease is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell. The assay can be combined with downstream qPCR or NG-seq to analyze histone modifications and binding of transcription factors, DNA replication factors, or DNA repair proteins at specific target genes or across the entire genome. It provides a rapid, robust, and true low cell number assay for detection of protein-DNA interactions in the cell. Unlike the ChIP assay, it is free from formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation. Previously, we have shown that, compared to ChIP, it requires fewer starting cells (100K), has a much faster protocol (one day from cells to DNA), generates lower background signal (requires less sequencing depth), and offers spike-in control DNA for effective normalization of signal between samples and between experiments. We recently updated the CST Assay Kit for use with 5,000-20,000 cells and added protocols for fixed cells and tissue. I will discuss the basics of the assay and important factors to consider when setting up your experiment. In addition, I will provide data showing the versatility of this assay for mapping various histone modifications, transcription factor, and transcription cofactor binding across multiple sample types. Finally, I will discuss how the general protocol is optimized for greater signal to noise ratio, reduced number of starting cells, and provide an alternative digestion method to prepare the input DNA as a critical control of the experiment. Citation Format: Christopher C. Comeau, Angela H. Guo, Fang Chen, Christopher J. Fry. Analysis of epigenetic marks and mechanisms in disease: Your guide to a successful assay. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr B004.

PAPST, a User Friendly and Powerful Java Platform for ChIP-Seq Peak Co-Localization Analysis and Beyond.

Comparative co-localization analysis of transcription factors (TFs) and epigenetic marks (EMs) in specific biological contexts is one of the most critical areas of ChIP-Seq data analysis beyond peak calling. Yet there is a significant lack of user-friendly and powerful tools geared towards co-localization analysis based exploratory research. Most tools currently used for co-localization analysis are command line only and require extensive installation procedures and Linux expertise. Online tools partially address the usability issues of command line tools, but slow response times and few customization features make them unsuitable for rapid data-driven interactive exploratory research. We have developed PAPST: Peak Assignment and Profile Search Tool, a user-friendly yet powerful platform with a unique design, which integrates both gene-centric and peak-centric co-localization analysis into a single package. Most of PAPST’s functions can be completed in less than five seconds, allowing quick cycles of data-driven hypothesis generation and testing. With PAPST, a researcher with or without computational expertise can perform sophisticated co-localization pattern analysis of multiple TFs and EMs, either against all known genes or a set of genomic regions obtained from public repositories or prior analysis. PAPST is a versatile, efficient, and customizable tool for genome-wide data-driven exploratory research. Creatively used, PAPST can be quickly applied to any genomic data analysis that involves a comparison of two or more sets of genomic coordinate intervals, making it a powerful tool for a wide range of exploratory genomic research. We first present PAPST’s general purpose features then apply it to several public ChIP-Seq data sets to demonstrate its rapid execution and potential for cutting-edge research with a case study in enhancer analysis. To our knowledge, PAPST is the first software of its kind to provide efficient and sophisticated post peak-calling ChIP-Seq data analysis as an easy-to-use interactive application. PAPST is available at https://github.com/paulbible/papst and is a public domain work.

Large replication skew domains delimit GC-poor gene deserts in human

Besides their large-scale organization in isochores, mammalian genomes display megabase-sized regions, spanning both genes and intergenes, where the strand nucleotide composition asymmetry decreases linearly, possibly due to replication activity. These so-called skew-N domains cover about a third of the human genome and are bordered by two skew upward jumps that were hypothesized to compose a subset of “master” replication origins active in the germline. Skew-N domains were shown to exhibit a particular gene organization. Genes with CpG-rich promoters likely expressed in the germline are over represented near the master replication origins, with large genes being co-oriented with replication fork progression, which suggests some coordination of replication and transcription. In this study, we describe another skew structure that covers ∼13% of the human genome and that is bordered by putative master replication origins similar to the ones flanking skew-N domains. These skew-split-N domains have a shape reminiscent of a N, but split in half, leaving in the center a region of null skew whose length increases with domain size. These central regions (median size ∼860kb) have a homogeneous composition, i.e. both a null and constant skew and a constant and low GC content. They correspond to heterochromatin gene deserts found in low-GC isochores with an average gene density of 0.81promoters/Mb as compared to 7.73promoters/Mb genome wide. The analysis of epigenetic marks and replication timing data confirms that, in these late replicating heterochomatic regions, the initiation of replication is likely to be random. This contrasts with the transcriptionally active euchromatin state found around the bordering well positioned master replication origins. Altogether skew-N domains and skew-split-N domains cover about 50% of the human genome.

Dynamic epigenetic states of ribosomal RNA promoters during the cell cycle

It has previously been shown that the ribosomal RNA promoter is regulated through epigenetic mechanisms. It is unclear however whether epigenetic marks are stable in somatic cells or whether and how they vary with cell cycle dynamics. Here we present an analysis of epigenetic marks in cells positioned at different phases of the cell cycle following synchronization using a double thymidine block. We show that the levels of acetylated histone 4 are highest in early S phase, coinciding with the peak of binding of the transcriptional activators UBF and MBD3 to the ribosomal RNA promoter. Additionally, binding of the DNA methyltransferase DNMT1 is highest during mid-S phase, while DNMT3B binding peaks later in G2. Bisulfite mapping of the ribosomal RNA promoter reveals that the DNA methylation state varies during the cell cycle being lowest during early and late S phase. Interestingly, although the interaction of RNA polymerase I with the promoter and its progress along the gene coincides with epigenetic activation, the burst in levels of rRNA transcript did not occur until after DNA synthesis was complete. This suggests that although the rRNA promoter is poised for transcription early in the cell cycle, the accumulation of rRNA transcripts requires additional signals later in the cell cycle. This data is consistent with the idea that epigenetic states are dynamic in somatic cells and might participate in physiological cellular responses.