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Epigenetics & Chromatin

Histone Analysis

Relevant Topics

Histone Overview

Chromatin, the material into which genomic DNA is packaged in eukaryotes, is a very dynamic structure. The smallest subunit of chromatin is the nucleosome, consisting of 147 base pairs of DNA wrapped around an octamer of core histone proteins. The histone octamer is composed of a central heterotetramer of histones H3 and H4, flanked by two heterodimers of histones H2A and H2B. Each nucleosome is separated by 10 to 60 bp of linker DNA. The resulting nucleosomal array constitutes a chromatin fiber of about 10 nm in diameter. This arrangement is folded into more condensed fibers (about 30 nm) that are stabilized by binding of a linker histone (Histone H1) to each nucleosome core. Such 30 nm fibers are then condensed in vivo to form thicker interphase fibers or the most highly compacted metaphase chromosome structures1.

But the role of histones and nucleosomes is not limited to the compaction of the chromatin. Indeed, over the past decade, evidence has accumulated indicating that chromatin structure is dynamic and plays an important role in biological processes such as transcription. For example, the discovery of nucleosome mobility has important biological implications. Indeed, nucleosomes positioned at promoters play a crucial role in the regulation of transcription. To allow access to the transcriptional apparatus, such nucleosomes have to be disrupted2,3.

Chromatin is subject to a variety of chemical modifications, including post-translational modifications of the N-terminal tails of histone and the methylation of cytosine residues in the DNA. Core histones are characterized by the presence of a histone fold domain and N-terminal tails of variable length that are the subject of extensive post-translational modifications. Reported histone modifications include acetylation, methylation, phosphorylation, ubiquitylation, glycosylation, ADP-ribosylation, carbonylation and SUMOylation. Many histones amino acids are modified. These include lysine residues that may be acetylated, methylated or coupled to ubiquitin; arginine residues that may be methylated; and serine or threonine residues that can be phosphorylated. Many of the modifications can affect the others, collectively constituting the “histone code”. They are positively or negatively correlated with specific transcriptional states or the specific organization of repressive or open chromatin.

Histone methylation is a post-translational modification of histones which takes place on the side chains of both lysine (K) and arginine (R) residues. Histone methylation is a reversible process which is catalysed by histone methyltransferases (HMT), such as PRMT1 or Suv39H whereas histone demethylation is catalyzed by histone demethylases, such as LSD1 or Jumanji domain-containing proteins. The regulational consequence of histone methylation on transcriptional state of a gene depends on the methylated residue and degree of methylation. Lysines can indeed be mono-, di- or tri-methylated4-7.

The modulation of chromatin condensation can be achieved via reversible acetylation on the lysine residues of histone tails. The acetylation reaction consists in the transfer of an acetyl group from acetyl coenzyme A (acetyl-coA) on the ε-amino group of the lysine residue, neutralizing the positive charge. This process results from a balance between the activity of two families of antagonistic enzymes: histone deacetylases (HDACs) and histone acetyltransferases (HATs), respectively removing or adding acetyl groups into core histone7,8.

Histone phosphorylation occurs on serine and threonine residues and influences transcription, chromosome condensation, DNA repair and apoptosis. For example, phosphorylation of serine 10 and serine 28 on the tail of histone H3 (H3 phospho Ser10 or H3 phospho Ser28) occurs early in mitosis when chromosome condensation induced during S-phase9.

Chromatin proteins are dynamic and a histone can be exchanged for a variant within its own class. Variants differ from canonical histones in their primary sequence, and their incorporation has functional consequences on the biophysical properties of the nucleosome core particle, altering accessibility of DNA to transcription factors and chromatin remodelers. Histone H2A has the largest number of identified variants. For example, histone H2A.Z (H2AZ, H2AFZ) is a histone H2A variant, a protein similar to canonical histone H2A but with different molecular identity and unique functions. H2A.Z is highly conserved during evolution. It plays an important role in basic cellular mechanisms such as gene activation, chromosome segregation, heterochromatic silencing and progression through the cell cycle. Histone H2AX (H2AX, H2A histone family member X) replaces conventional histone H2A in a subset of nucleosomes. Histone H2AX is required for checkpoint-mediated arrest of cell cycle progression in response to low doses of ionizing radiation, and for efficient repair of DNA double-strand breaks (DSBs), specifically when modified by C-terminal phosphorylation. Histone macroH2A (mH2A) is a histone variant that has a region that is similar to histone H2A but has a unique C-terminal domain (the macro domain, also called the non-histone domain (NHD)) in addition to the histone-like region. mH2A associates with condensed chromatin, including the inactive mammalian female X chromosome, senescence-associated heterochromatin foci, imprinted genetic loci, and regions of chromatin that are CpG methylated10,11.

The organization of genetic material in the nucleus has profound effects on all processes that require access to DNA. Much of the regulation of the genome involves the histone proteins that are the core of chromatin and chromosome structure. Changes to the epigenetic information within a cell play a significant role in a number of diseases such as cancer or developmental diseases.

Active Motif provides useful products to study histones and histone modifications.

Histone Antibodies

Active Motif provides high quality antibodies directed against histones and post-translationally modified histones or chromatin remodelers. Follow the link to our Histone Antibodies to get more information.

Histone Modification ELISAs

Histone Modification ELISAs provide a simple, sensitive method for detecting changes in histone modification levels from purified core histones or histones isolated by acid extraction. Histones can be extracted using our Histone Purification Kits. Kits are available to analyze acetylation, methylation or phosphorylation of histone H3, or total histone H3. For added convenience and a more quantitative interpretation of results, the Histone Modification ELISAs also include a site- and degree-specific methylated recombinant histone protein or acetylated recombinant histone protein for use as a reference standard curve. For complete details, follow our link to Histone Modification ELISAs.

Recombinant Histones

Histones with specific methylation states are an essential tool for investigating which methylation patterns are key to complex functional questions about chromatin-associated proteins, nucleosome remodeling, transcriptional regulation, replication and DNA repair.

In addition to methylation specific recombinant histones, Active Motif also offers histones with site-specific acetylation modifications. Histone acetylation is a post-translational modification that affects the nucleosome structure and therefore the ability of transcription factors to access the DNA and regulate gene expression.

Histone phosphorylation at serine and threonine residues can influence transcription, chromosome condensation, DNA repair and apoptosis. Active Motif offers Recombinant Histones to study site-specific histone phosphorylation.

To learn more, click on our link for information on our patented protein synthesis technologies and to get a complete list of our Recombinant Histones and Modified Histones.

Recombinant Histone Methyltransferases, Demethylases and Acetyltransferases

Active Motif offers a complete line of recombinant proteins to meet your research needs. Follow the link to get more information about our Recombinant Proteins.

Histone Peptide Array

With the increasing use of antibodies as research tools in techniques such as ChIP, ChIP-seq, ChIP-chip and IF, it is critical to assess antibody specificity to ensure accurate data analysis. Active Motif's MODified™ Histone Peptide Array offers a better way to screen antibodies for cross reactivity. Each peptide array contains 384 different histone modification combinations, inculding up to four separate modifications on the same peptide. This extensive coverage of histone modifications will enable you to study not only individual sites, but also to determine the effects of neighboring modifications on antibody recognition and binding. MODified Histone Peptide Arrays screen 59 acetylation, methylation, phosphorylation and citrullination modifications on the N-terminal tails of histones H2A, H2B, H3 and H4.

Array Labeling Kits are available which contain the buffers and ECL reagents needed for detection of the Histone Peptide Array (arrays are not included). The MODified™ Array Labeling Kit can be used to study antibody and enzyme interactions. A positive control antibody that recognizes the c-Myc spot on the array is included. The MODified™ Protein Domain Binding Kit is designed to interrogate the binding specificity of His-tagged protein reading domains. A positive control protein is included.

Luminex® Histone PTM Multiplex Assays

Active Motif has partnered with Luminex®, the industry leader in multiplexing technology, to develop the Histone H3 PTM Multiplex Assay, the first multiplex epigenetic assay for the study of histone PTMs. This novel bead-based sandwich ELISA assay is designed for use with Luminex instrumentation. The assay enables the use of low (nanogram) amounts of acid extracted cell lysates or purified histone samples to interrogate multiple histone modifications simultaneously within a single well. The 96-well format makes the assay adaptable to high throughput processing. The assay format also offers the ability to normalize PTM data against total histone H3 levels for evaluation of relative histone PTM levels.

Histone Purification Kits

Histones are known to be acid soluble and need an acid extraction to be isolated. Now you can easily purify histones and further separate the fractions of core histones from any cell culture or tissue sample while maintaining post-translational modifications like acetylation, methylation and phosphorylation states. The Histone Purification Kits enable you to isolate core histones from any cell culture or tissue sample. The core histones may be purified using the convenient spin column format as one total population containing H2A, H2B, H3 and H4 (Histone Purification Mini-kit), or further purified into separate fractions of H2A/H2B dimers and H3/H4 tetramers with the efficient gravity flow protocol (Histone Purification Kit). For high throughput applications, the Histone Purification Microplate Kit enables purification of core histones using very small amounts (as little as 0.9 x 105 cells) of starting material, and its microplate spin column format enables elution into a 96-well plate to easily transition into downstream high-throughput applications.


Chromatin Assembly Kit

The Chromatin Assembly Kit enables you to study your DNA sequence of interest in a native chromatin environment. The simple, easy-to-follow protocol generates assembled chromatin in hours with few manipulations, providing you with material that is ideal for downstream applications such as in vitro ChIP, transcription and histone modification assays.

Histone Acetyltransferase (HAT) Assay

Histone Acetylation is a reversible process resulting from a balance between the activity of two families of antagonistic enzymes: histone deacetylases (HDACs) and histone acetyltransferases (HATs), respectively removing or adding acetyl groups into core histone. The Histone Acetyltransferase (HAT) Assay Kit is a fluorescent method to assay samples for histone acetyltransferase (HAT) activity or to screen HAT inhibitors. The assay is easy to use, features a simple protocol that gives you results in 30 minutes and is ideal for high-throughput usage in 96-well plates.

Histone Deacetylase (HDAC) Assays

Histone Deacetylase (HDAC) Assay Kits are 96-well plate-based assays to determine the HDAC (Histone Deacetylase) activity in your extracts or purified samples. HDAC Assay Kits are available in both colorimetric and fluorescent formats to suit your needs.

Histone Demethylase Assays

The Histone Demethylase Assay (Fluorescent) Kits are designed to detect the formaldehyde released from the reaction of lysine specific demethylase 1 (LSD1, also known as KDM1) with a recombinant histone H3K4me2 substrate. The recombinant histone H3K4me2 substrate used in the assay mimics a native histone substrate, allowing for results that more closely resemble in vivo conditions. This fluorescent assay can be used to analyze overall histone conversion efficiencies or to screen compounds for changes in demethylation activity.

Mitotic Assay Kits

Two significant mitotic events include microtubule spindle formation and chromosome condensation. Histone H3 is phosphorylated on serine 28 during mitotic chromatin condensation before nuclear division occurs, making this event a reliable marker for cells undergoing mitosis. As mitosis is the most defining stage of cell growth, Active Motif's chemiluminescent and colorimetric Mitotic Assay Kits are an easy way to test the effect of various treatments on cellular division. The kits utilize the highly specific mitosis marker antibody, Histone H3 phospho Ser28 antibody (H3 phospho Ser28 pAb). Histone H3 phospho Ser28 ELISA (H3S28ph) kit provided by Active Motif allows also to screen for phosphorylated serine 28 on histone H3 in human, mouse, hamster and bovine systems.

Mitotic Index (Mitosis) Assay

The Mitotic Index Assay Kit offers you a simple, accurate method to determine the percentage of cells undergoing mitosis within a specified population. Because mitosis is the most defining stage of cell division, the ability to detect cells undergoing mitosis is a valuable way to characterize the effects of test compounds on cellular progression.


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