Epigenetics is the study of acquired modifications in chromatin structure that emerge independently of any changes in the underlying DNA nucleotide sequence. Epigenetic alterations – comprising acetylation, methylation, phosphorylation, and ubiquitination, among others – change the accessibility of DNA to transcription machinery and thus affect gene expression.
Epigenetic mechanisms integrate environmental alterations at the cellular level, thus facilitating cellular plasticity. Consequently, they have been implicated in multiple diseases comprising cancer, metabolic disorders, and inflammation. Proteins carrying out and interpreting these epigenetic changes are classifiable as either erasers, readers, or writers.
Epigenetic changes arise most typically on either the actual DNA or on the histone octamer around which DNA is coiled. The complex of DNA and the histone octamer is defined as ‘chromatin’, while the basic repeating unit of chromatin is known as the ‘nucleosome’. While DNA needs to be tightly compacted to enable it to fit into the nucleus, it needs to also be temporarily accessible during DNA transcription. To enable this, mechanisms of chromatin uncoiling and recoiling also need to exist.
The addition and removal of specific chemical groups, which are termed ‘epigenetic marks’, is believed to modulate chromatin architecture and thus impacts on gene expression. Tocris offers a broad array of pharmacological solutions for the study of such epigenetic targets, an assortment of which are featured in each section. A full product summary can be found at www.tocris.com/epigenetics.
Tocris Product Areas: Epigenetic Writers
Poly(ADP-ribose) Polymerase (PARP)
Epigenetic writers catalyze the addition of chemical groups onto either histone tails or the actual DNA. These alterations are termed epigenetic marks and are fundamental to gene expression and silencing. One such category of epigenetic writers is histone methyltransferases, comprising enzymes that catalyze the transfer of a methyl group onto a lysine or arginine residue on histone tails. As well as methyl marks, histone lysine residues can also be acetylated via the activity of histone acetyltransferases.
The transfer of an acetyl group from the cofactor acetyl-CoA to lysine residues on histone tails causes neutralization of the positive charge of lysine, thus enfeebling the affinity of the histone tail for the DNA and engendering a reduction of chromatin condensation. Because a more open chromatin architecture facilitates the recruitment of transcription factors and polymerases, the promotion of gene expression is caused by histone acetylation.
Moreover, enzymes catalyzing the phosphorylation of histone tails are critical epigenetic writers. For instance, phosphorylation of histone H3 (H3Y41) by JAK2 disrupts the binding of the heterochromatin protein HP1α to chromatin, engendering enhanced DNA accessibility and the transcription of the oncogene lmo2. Additional kinases, comprising ATM/ATR kinases, Aurora B Kinase, Haspin, Pim-1, and PKC, have also been implicated in the phosphorylation of histone proteins and subsequent alteration of gene expression.
Other epigenetic marks modifying gene expression comprise ADP-ribosyltation and ubiquitination. Lysine residues on histone proteins H2A and H2B can be monoubiquitinated via the concerted actions of E2 ubiquitin conjugases and E3 ubiquitin ligases, while activated PARP interacts with, and ADP-ribosylates, some histones to cause destabilization of chromatin structure, thus inducing transcription. DNA can also be methylated via divergent mechanisms.
The addition of a methyl group to a nucleotide by DNA methyltransferases (DNMTs) transpires at the major groove of the DNA double helix, preventing transcription by inhibiting the binding of transcription factors and polymerases.
There are two known classes of DNA methylation – maintenance and de novo methylation. The latter, which is primarily implemented by DNA methyltransferases DNMT3A and DNMT3B, catalyzes the addition of methyl groups onto cytosine nucleotides. Because cell replication does not preserve such methylation, maintenance methylation duplicates these marks from the parent DNA onto the daughter DNA strands. The high affinity of DNMT1 for hemimethylated DNA in vitro indicates that this enzyme is principally responsible for maintaining DNA methylation in vivo.
Cole (2008) Chemical probes for histone-modifying enzymes. Nat.Chem.Biol. 4 590
Spannhoff et al (2009) The emerging therapeutic potential of histone methyltransferase and demethylase inhibitors. Chem.Med.Chem. 4 1568
Featured Products for Epigenetic Writers
Histone Methyltransferase Inhibitor
UNC 0642 is a potent and selective G9a and GLP histone lysine methyltransferase inhibitor (IC50 < 2.5 nM). The compound exhibits >2,000-fold selectivity for G9a and GLP over PRC2-EZH2 and >20,000-fold selectivity over other methyltransferases.
UNC 0642 reduces H3K9 dimethylation levels in MDA-MB-231 cells (IC50 = 110 nM) and displays modest brain penetration in vivo. (Supplied in conjunction with the Structural Genomics Consortium)
DNA Methyltransferase Inhibitor
SGI 1027 is a DNMT3B, DNMT3A, and DNMT1 DNA methyltransferase inhibitor (IC50 values are 7.5, 8, and 12.5 μM respectively with Poly(dl-dC) as the substrate). The compound reactivates silenced tumor suppressor genes by reducing CpG island hypermethylation.
Tocris Product Areas: Epigenetic Readers
Epigenetic reader domains can be categorized as effector proteins that are capable of recognizing specific marks on histones or nucleotides, to which they are recruited. Enzymes that write or erase epigenetic marks might also comprise such reader domains, engendering the coordination of ‘read-write’ or ‘read-erase’ epigenetic processes. The structure of reader domains usually supplies a surface groove or cavity within which a specific epigenetic mark can be accommodated.
Proteins comprising reader domains are broadly classifiable into four categories: chromatin architectural proteins; chromatin remodeling enzymes; chromatin modifiers; and adaptor proteins which recruit other machinery implicated in gene expression. The first category, chromatin architectural proteins, tethers to nucleosomes, either directly inducing chromatin compaction or alternatively functioning as a shield to inhibit the binding of proteins implicated in RNA transcription.
Unlike chromatin architectural proteins, chromatin remodeling enzymes generate a more open chromatin architecture. The increased accessibility of chromatin enables DNA transcription, which promotes gene expression.
A number of additional proteins containing reader domains are unable to directly influence chromatin architecture but instead function by recruiting secondary chromatin modifiers to make further modifications to chromatin or by reversing an existing chromatin alteration. For example, the yeast chromatin remodeling enzyme complex, RSC, which comprises a tandem bromodomain within its Rsc4 subunit, recruits the complex to acetylated lysine residues on histone H3 (H3K14). RSC is implicated in multiple cellular processes.
One such example is nucleosome remodeling, which facilitates the promotion of RNA polymerase II recruitment to the underlying DNA, triggering gene transcription. Adaptor proteins represent the final category of reader domain-containing proteins: the primary role of these domains is to recruit factors that are connected to DNA metabolism processes, comprising DNA damage repair, DNA recombination, DNA replication, RNA processing and transcription.
Belkina and Davis (2012) BET domain co-regulators in obesity, inflammation, and cancer. Nat.Rev.Cancer 12 465
Musselman et al (2012) Perceiving the epigenetic landscape through histone readers.
Nat.Struct.Mol.Biol. 19 1218
Featured Products for Epigenetic Readers
BET Bromodomain Inhibitor
I-BET 151 is a BET bromodomain inhibitor that blocks the recruitment of BET to chromatin. The compound induces apoptosis and G0/G1 cell cycle arrest in MLL-fusion leukemic cell lines in vitro (IC50 values are 15, 26, 120 and 192 nM for NOMO1, MV4;11, MOLM13 and RS4;11 cell lines respectively). I-BET 151 hydrochloride reduces BCL2 expression in NOMO1 cells and improves survival in two rodent models of MLL-fusion leukemia in vivo. (Sold for research purposes under agreement from GlaxoSmithKline)
CBP/p300 Bromodomain Inhibitor
I-CBP 112 is a selective CBP/p300 bromodomain inhibitor (IC50 values are 142-170 and 625 nM respectively). The compound is selective for CBP/p300 over ATAD2, BAZ2B, BRD2(2), BRD4(1), PB1(5), PCAF, PHIP(2) and TIF1α bromodomains. (Supplied in conjunction with the Structural Genomics Consortium)
SMARCA2/4 and Polybromo 1 Inhibitor
PFI 3 is a potent and selective polybromo 1 (PB1) and SMARCA4 inhibitor (Kd values are 48 and 89 nM respectively). The compound also inhibits SMARCA2. PFI 3 displays selectivity for PB1 and SMARCA2/4 over other bromodomains. (Supplied in conjunction with the Structural Genomics Consortium)
Tocris Product Areas: Epigenetic Erasers
Protein Serine/Threonine Phosphatases
Protein Tyrosine Phosphatases
The alterations caused by epigenetic marks are not necessarily permanent; rather, they can be removed to alter gene expression, by a category of enzymes termed epigenetic ‘erasers’. There is more than one category of epigenetic erasers targetting histones, such as histone deubiquitinases, histone lysine/arginine demethylases, histone deacetylases, and histone serine/threonine/tyrosine phosphatases. The removal of acetyl groups via the actions of histone deacetylases (HDACs) is a critical mechanism for multiplying chromatin condensation and thus suppressing gene transcription.
HDACs are divisible into class I and class II HDACs. Protein phosphatases are able to target either threonine, phosphorylated serine, or tyrosine residues on histone proteins. PP1, PP2A, and PP4, among others, have been observed to cause dephosphorylation of histone proteins. For instance, the catalytic subunit of PP2A co-localizes with phosphorylated H2AX, a phosphorylated sequence variant of histone protein H2A, which is rapidly concentrated within chromatin domains flanking DNA double-strand breaks. Phosphorylated H2AX functions as a docking site for DNA repair proteins, and is released from chromatin as soon as double-strand breaks have been rejoined; this mechanism is believed to implicate PP2A.
Proteases catalyze the removal of ubiquitin groups from histone lysine residues termed deubiquitinating enzymes (DUBs). These proteins target histones H2A and H2B, where they modulate cell cycle progression, DNA repair, gene expression, and transcription. In comparison to alternative histone alterations, knowledge regarding the functions of histone ubiquitination is less comprehensive, although emerging research is suggesting a critical role for this epigenetic change in the DNA damage response.
Lysine-specific demethylase 1 (LSD1), also known as KDM1A, was the first histone lysine demethylase to be identified. LSD1 comprises an amino oxidase domain that tethers the cofactor, flavin adenine dinucleotide (FAD), which is essential for demethylation.
Another family of lysine demethylases has been discovered more recently. These are known as Jumonji C domain-containing demethylases (JMJD). JMJD does not need FAD as a cofactor but alternatively, relies on Fe2+/2-oxoglutarate (2-OG) for catalysis.
Up until now, two enzymes, which can remove methyl groups from arginine residues, have been determined: histone demethylase JMJD6 and peptidyl arginine deiminases. The process of DNA demethylation can be either active or passive. Passive demethylation comprises the loss of methyl groups from DNA during DNA replication. This necessitates the absence of DNA methylation machinery which would otherwise maintain DNA methylation.
The mechanism of active demethylation is less fully understood. One suggested mechanism comprises the ten-eleven translocation (TET) enzyme family and thymine DNA glycosylase (TDG), which are implicated in DNA base excision repair. TET enzymes are capable of oxidizing methylated cytosine residues, generating intermediates which, subsequently, can be excised from DNA by TDG, engendering DNA demethylation. Additional research is required to establish further DNA modifying enzymes implicated in the physiological modulation of the DNA demethylation pathway.
Arrowsmith et al (2012) Epigenetic protein families: a new frontier for drug discovery. Nat.Rev.Drug Discov. 11 384
Kooistra and Helin (2012) Molecular mechanisms and potential functions of histone demethylases. Nat.Rev.Mol.Cell.Biol. 13 297
Featured Products for Epigenetic Erasers
Histone Deacetylase Inhibitor
SAHA is a Class I and II histone deacetylase (HDAC) inhibitor that induces accumulation of acetylated histones H2A, H2B, H3 and H4 in transformed cultured cells. The compound suppresses cell growth in a range of cancer cell lines and induces apoptosis in cutaneous T-cell lymphoma cells in vitro.
Histone Demethylase Inhibitor
GSK J4 is a histone lysine demethylase (KDM) inhibitor that blocks GSK demethylation of histone H3K27. The compound attenuates lipopolysaccharide-induced proinflammatory cytokine production in primary human macrophages (IC50 = 9 μM for the inhibition of TNF-α release). GSK J4 is cell-permeable and is an ethyl ester derivative of GSK J1. (Supplied in conjunction with the Structural Genomics Consortium)
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