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How Do Histone Modifications Regulate Gene Expression?

Arun Kumar
Tags: Epigenetics

Eukaryotic DNA is packaged and wrapped around proteins known as histones which protect and regulate gene expression. The structure of DNA wrapped around histone octamers is known as chromatin. Chromatin at the first level of its organization appears as a linear array of uniform structural units, nucleosomes. Nucleosomes are formed by 150–210 base pairs (bp) of double stranded (ds) DNA and an octamer of highly conserved histone proteins. The histone proteins have tails that project from the nucleosome and many residues in these tails can be post-translationally modified, influencing chromatin compaction and transcription. Modifications in the core regions of the histones had remained unknown until recently. The ability of eukaryotic cells to maintain distinct phenotype, although containing identical genetic constituents is ensured by various chemical modifications occurring on DNA and histones. The blueprint is accessed during development and differentiation in distinct cells is regulated by epigenetics. Chromatin in the nucleus of a cell exists in two different states, a closed chromatin known as heterochromatin, which is associated with transcriptional repression, and an open chromatin known as euchromatin favorable towards transcription.

The key players associated with these modifications have been deciphered progressively with our growing knowledge of epigenetics. These are: writers - a host of enzymes capable of modifying nucleotide base and specific amino acid residues on histones, erasers - a group of enzymes proficient in removing these marks, and readers - a diverse range of proteins that possess specialized domains recognizing specific epigenetic marks in a locus. These enzymes and protein domains together constitute the epigenetics tools as shown Table 1.

Writers Readers Erasers
HKMTs Bromodomain HKDMs
PRMTs Chromodomain

Table 1: Epigenetics tools. These enzymes and protein domains carry out most of the epigenetic modifications on DNA and histone tails.

Recent research has suggested that altered regulation of these epigenetics tools plays a key role in tumorigenesis. Since the modifications effected by these enzymes are reversible, therapeutic approach targeting these alterations is one of the current areas of research in academia and industry in terms of cancer drug discovery. Writers, enzymes that add PTMs to histones, are divided into classes based on the specific PTM they effect. Similarly, erasers, enzymes which remove specific PTMs from histone substrates, are divided into PTM-specific classes. Finally, readers are dedicated protein factors that recognize either specific post-translational marks on histones or a combination of marks and histone variants to direct a particular transcriptional outcome

Epigenetic Writers

Modifications of DNA and histone proteins occur through the addition of various chemical groups utilizing numerous enzymes. Although a variety of modifications operate, the two most widely studied epigenetic alterations viz., methylation and acetylation. Both DNA and histone proteins are prone to methylation, while acetylation is associated only with histones. These two modifications frequently govern the gene expression pattern in a cell by altering between transcriptional activation and repression.
The epigenetic writers are DNA methyltransferases, histone lysine methyltransferases and histone acetyltransferases. Enzo’s Acetyltransferase and Methyltransferase activity kits are homogeneous mix-and-read fluorescent assays for the determination of any acetyl-CoA dependent acetyltransferase activity or any S-adenosyl-L-methionine (AdoMet) dependent methyltransferase activity, respectively. They are high throughput amenable and suitable for end-point or kinetic assay protocols.

Methyltransferase activity kit

Figure 2: Methyltransferase activity kit is a homogeneous mix-and-read fluorescent assay for the determination of any AdoMet dependent methyltransferase activity. It is suitable for end-point or kinetic read options, which are ideal for determining mechanism of action, kinetics, and screening candidate compounds. The assay is amendable to HTS and miniaturization.

Histone Lysine Methyltransferase

Methylation of histones is a unique post-translational modification since it can add up to three methyl groups on the single lysine (K) residues resulting in mono (me1), di (me2) and tri-methylated (me3) states. These modifications are associated with transcriptional activation or repression based on the location of the lysine residues. Histone lysine methyltransferase (KMTs) catalyzes the transfer of methyl group from AdoMet, producing three methylated products, and adenosylhomocysteine. Based on the cellular location HATs are classified into two broad categories. HATs found in the nucleus are known as Type A, and are responsible for acetylating histones associated with chromatin, whereas Type B HATs are present in the cytoplasm, and acetylate newly translated histone. p300/CBP is the most studied HAT since it is associated with acetylating histones along with numerous other proteins.

Histone Acetyltransferase

Histone acetylation status is regulated by two groups of enzymes exerting opposite effects, histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs catalyze the transfer of an acetyl group from acetyl-CoA to an amino acid group of the target lysine residues in the histone tails, which leads to the removal of a positive charge on the histones, weakening the interaction between histones and (negatively charged phosphate groups of) DNA. This in turn makes the chromatin less compact and thus more accessible to the transcriptional machinery. HDACs remove acetyl groups from histone tail lysine residues and thereby work as repressors of gene expression.

Chloramphenicol acetyltransferase (CAT) titration

Figure 3: Chloramphenicol acetyltransferase (CAT) was tittered in the assay using 100 uM of the substrate chloramphenicol.

Epigenetic Readers

Just as histone PTMs are accomplished by “writers” and “erasers”, their actions to govern DNA transcription are mediated by “readers.” Identification of these proteins was originally driven by use of modified histone peptides to identify proteins that recognize histone PTMs. a number of domains have been identified that have high affinity for sites of histone methylation (e.g. PhD [plant homeodomain], chromo [chromatin organization modifier], MBT [Malignant Brain Tumor]) or acetylation (e.g. Bromo). Bromodomains are the most widely studied histone acetylation readers. Their involvement in various forms of cancer have made them an attractive target for new drug development. Most of the developments surrounding bromodomain inhibitors target the BET family of bromodomains. Recent advances in the field of histone modification and their involvement in health and diseases, will move closer to developing better therapies to fight diseases.

Epigenetic Erasers

The epigenetic marks laid down in the form of post-translational modifications on histones and co-valent modifications on DNA are not permanent. These marks can be removed depending on the requirement of the cell to modify the expression states of the locus. To implement this, a group of enzymes known as erasers are available that oppose the activity of the writers. The erasers catalyze the removal of epigenetic marks, which relieves its effect on transcription, resulting in the modulation of gene expression.

Histone Deacetylases

Deacetylation of histone proteins is carried out by histone deacetylases (HDAC), which remove the ε-amino acetyl group from lysine residues on histones, resulting in a compact transcriptionally repressive chromatin organization. The mammalian genome encodes two distinct class of HDAC enzymes based on their enzymatic property. The classical zinc- dependent deacetylases consist of 11 proteins, and are divided into four distinct classes (I, IIa, IIb and IV) based on their homology with yeast proteins. The members of class I HDAC family consist of HDAC1, 2, 3 and 8 with ubiquitous tissue expression and predominant nuclear localization. Class III HDACs are referred to as Sir2 (silent information regulator 2) proteins or Sirtuins (SIRT) which are NAD+ dependent deacetylases, consisting of seven family members (SIRT1–7). SIRT1 is predominantly localized in the nucleus. The catalytic domain of SIRT2 acts both as NAD+- dependent deacetylase (DAC) and mono-ADP-ribosyl transferase (ART). SIRT3 is present in the mitochondria and its catalytic domain functions both as DAC and ART. SIRT4 resides in the mitochondria and thought to function as ADP-ribosyltransferase.
Kinetics of CHEMILUM DE LYS® Substrate Deacetylation by HeLa HDAC Activity

Figure 4: Kinetics of CHEMILUM DE LYS® Substrate Deacetylation by HeLa HDAC Activity. The HDAC/SIRT Chemiluminescent Drug Discovery Kit is a complete assay system designed to measure histone deacetylase (HDAC) and sirtuin activity in cell or nuclear extracts, immunoprecipitates or purified enzymes.

Histone Deacetylase Inhibitors

Numerous reports have suggested that histone deacetylase govern the expression and function of various proteins that are associated with tumorigenesis. Trichostatin A, a naturally occurring derivative of dienohydroxamic acid obtained from the genus Streptomyces inhibits the zinc dependent HDACs. It was the first HDAC inhibitor (HDACi) to be identified and subsequent research led to the development of numerous hydroxamic acid derivatives among which Vorinostat was the first pan-HDACi approved by US-FDA for the treatment of advanced primary cutaneous T-cell lymphoma (CTCL). The NAD+ dependent HDAC (Sirtuins), which belong to class III HDACs, is another potential target for cancer drug development. Studies have demonstrated that Sirtinol which inhibits SIRT1/2 induces apoptosis in breast cancer cells. Enzo Life Sciences provides a comprehensive portfolio of widely cited active HDACs, inhibitors and Sirtuin proteins, HATS, methyltransferases and demethylases.
Trichostatin A inhibition of FLUOR DE LYS® Substrate Deacetylation by HeLa Nuclear Extract

Figure 5: Trichostatin A inhibition of FLUOR DE LYS® Substrate Deacetylation by HeLa Nuclear Extract.

Enzo’s FLUOR DE LYS® HDAC & Sirtuin Assays platform has revolutionized assessment of HDAC & Sirtuin enzyme activity, freeing researchers from cumbersome protocols required with radiolabeled or other modified histone-based methods. Our high-quality assays utilize patented substrate/developer chemistry in combination with high-activity, high-purity enzymes, to deliver more high-quality hits. Broad-class HDAC/Sirtuin screening assays are available in chemiluminescent, fluorescent, and colorimetric formats, and backed by a panel of epigenetic modulators and PTM-specific antibodies.

In addition to our HDAC & Sirtuin drug discovery kits, our SCREEN-WELL® Epigenetics library contains 43 compounds with defined activity against enzymes which carry out epigenetic modification of lysine. It also includes DNA methylation inhibitors. The library is a useful tool for chemical genomics, assay development and other pharmacological applications. The library contains inhibitors of these important enzymes: HDACs, SIRTs, Lysine demethylases, HATs, Histone methyl transferases, DNA methyltransferases as well as SIRT activators. A variety of structurally and mechanistically different compound classes are included for your research needs.

Inhibitor validation data

Figure 6: Inhibitor validation data. Inhibition curves for all compounds with HDAC3, 6, 8, and 10.

Enzo Life Sciences offers a complete portfolio of reagents and antibodies for the detection of key epigenetics-regulating enzymes and substrates, including modification-specific antibodies for acetylated, methylated, phosphorylated or ubiquitinated epitopes. Further details can be found on our Epigenetics platform page or our complete toolbox guide. Please contact our Technical Support Team for further assistance and check out our TechNotes, an easy way for you to see how Enzo is living up to our motto: Scientists Enabling Scientists™.

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