Question

Briefly describe how post-translational modifications of histones affect gene expression.

Briefly explain how restriction enzymes can be used for the generation of recombinant DNA.

Answer 1

A histone modification is a covalent post-translational modification (PTM) to histone proteins which includes methylation, phosphorylation, acetylation, ubiquitylation, and sumoylation. The PTMs made to histones can impact gene expression by altering chromatin structure or recruiting histone modifiers. Histone proteins act to package DNA, which wraps around the eight histones, into chromosomes. Histone modifications act in diverse biological processes such as transcriptional activation/inactivation, chromosome packaging, and DNA damage/repair. In most species, histone H3 is primarily acetylated at lysines 9, 14, 18, 23, and 56, methylated at arginine 2 and lysines 4, 9, 27, 36, and 79, and phosphorylated at ser10, ser28, Thr3, and Thr11. Histone H4 is primarily acetylated at lysines 5, 8, 12 and 16, methylated at arginine 3 and lysine 20, and phosphorylated at serine 1. Thus, quantitative detection of various histone modifications would provide useful information for a better understanding of epigenetic regulation of cellular processes and the development of histone modifying enzyme-targeted drugs.

Histone Acetylation/Deacetylation

Histone acetylation occurs by the enzymatic addition of an acetyl group (COCH3) from acetyl coenzyme A. The process of histone acetylation is tightly involved in the regulation of many cellular processes including chromatin dynamics and transcription, gene silencing, cell cycle progression, apoptosis, differentiation, DNA replication, DNA repair, nuclear import, and neuronal repression. The modifying enzymes involved in histone acetylation are called histone acetyltransferases (HATs) and they play a critical role in controlling histone H3 and H4 acetylation.

Histone deacetylaces (HDACs) catalyze the hydrolytic removal of acetyl groups from histone lysine residues. An imbalance in the equilibrium of histone acetylation has been associated with tumorigenesis and cancer progression. Detecting whether histone H3 is acetylated at its lysine residues would provide useful information for further characterization of acetylation patterns or sites, thereby leading to a better understanding of epigenetic regulation of gene activation as well as the development of HAT-targeted drugs. Similar to HATs, HDACs play a critical role in various cellular processes involving histone H3 and H4.

Histone Methylation/Demethylation

Histone methylation is defined as the transfer of one, two, or three methyl groups from S-adenosyl-L-methionine to lysine or arginine residues of histone proteins by histone methyltransferases (HMTs). HMTs control or regulate DNA methylation through chromatin-dependent transcriptional repression or activation. In the cell nucleus, when histone methylation occurs, specific genes within the DNA complexed with the histone may be activated or silenced. Several different histone methyltransferases exist that are specific for the lysine or arginine residue which they modify.

On the other hand, arginine methylation of histones H3 and H4 promotes transcriptional activation and is mediated by a family of protein arginine methyltransferases (PRMTs). There are 9 types of PRMTs found in humans but only 7 members are reported to methylate histones. They can mediate mono or dimethylation of arginine residues. Histone demethylation is the removal of methyl groups in modified histone proteins via histone demethylases. These demethylases have been found to have potential oncogenic functions and involvement in other pathological processes. The discovery of histone demethylases demonstrates that histone methylation is not a permanent modification but rather a more dynamic process. Two major families of demethylases have been discovered: Lysine specific demethylase 1 (LSD1) and Jumonji domain containing (JmjC domain) histone demethylases (JMJD2, JMJD3/UTX and JARIDs).

•             A restriction enzyme is a DNA-cutting enzyme that recognizes specific sites in DNA. Many restriction enzymes make staggered cuts at or near their recognition sites, producing ends with a single-stranded overhang.

•             If two DNA molecules have matching ends, they can be joined by the enzyme DNA ligase. DNA ligase seals the gap between the molecules, forming a single piece of DNA.

Restriction enzymes and DNA ligase are often used to insert genes and other pieces of DNA into plasmids during DNA cloning.

Restriction enzymes are found in bacteria (and other prokaryotes). They recognize and bind to specific sequences of DNA, called restriction sites. Each restriction enzyme recognizes just one or a few restriction sites. When it finds its target sequence, a restriction enzyme will make a double-stranded cut in the DNA molecule. Typically, the cut is at or near the restriction site and occurs in a tidy, predictable pattern.

(i) Selection and isolation of DNA insert:

First step in rec DNA technology is the selection of a DNA segment of interest which is to be cloned. This desired DNA segment is then isolated enzymatically. This DNA segment of interest is termed as DNA insert or foreign DNA or target DNA or cloned DNA.

(ii) Selection of suitable cloning vector:

A cloning vector is a self-replicating DNA molecule, into which the DNA insert is to be integrated. A suitable cloning vector is selected in the next step of rec DNA technology. Most commonly used vectors are plasmids and bacteriophages.

(iii) Introduction of DNA-insert into vector to form recDNA molecule:

The target DNA or the DNA insert which has been extracted and cleaved enzymatically by the selective restriction endonuclease enzymes [in step (i)] are now ligated (joined) by the enzyme ligase to vector DNA to form a rec DNA molecule which is often called as cloning-vector-insert DNA construct.

(iv) rec DNA molecule is introduced into a suitable host:

Suitable host cells are selected and the rec DNA molecule so formed [in step (iii)] is introduced into these host cells. This process of entry of rec DNA into the host cell is called transformation. Usually selected hosts are bacterial cells like E. coli, however yeast, fungi may also be utilized.

(v) Selection of transformed host cells:

Transformed cells (or recombinant cells) are those host cells which have taken up the recDNA molecule. In this step the transformed cells are separated from the non-transformed cells by using various methods making use of marker genes.

(vi) Expression and Multiplication of DNA insert in the host:

Finally, it is to be ensured that the foreign DNA inserted into the vector DNA is expressing the desired character in the host cells. Also, the transformed host cells are multiplied to obtain sufficient number of copies. If needed, such genes may also be transferred and expressed into another organism.

Suppose we have a target gene, flanked with EcoRI recognition sites, and a plasmid, containing a single EcoRI site:

Our goal is to use the enzyme EcoRI to insert the gene into the plasmid. First, we separately digest (cut) the gene fragment and the plasmid with EcoRI. This step produces fragments with sticky ends:

Next, we take the gene fragment and the linearized (opened-up) plasmid and combine them along with DNA ligase. The sticky ends of the two fragments stick together by complementary base pairing: Once they are joined by ligase, the fragments become a single piece of unbroken DNA. The target gene has now been inserted into the plasmid, making a recombinant plasmid.