Histone Analysis

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.

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