Histone H2AX in the Study

Histone H2AX

In the study of biology, histones are the main, large and organic compounds made of amino acids that are considered as among the most important elements of chromatin. Chromatin is the compound and compact form of deoxyribonucleic acid (DNA) in the nucleus that makes up chromosomes. Let us focus our study to one of the major and core histones, the DNA that wraps the nucleosome around two copies each of histone proteins, the H2AX. Histone H2AX is characterized by having a long terminal tail on one end of the amino acid structure. This feature tells its main difference from H2A.

Once the DNA is damaged and its physiology is disturbed in normal healthy individual, the p53 protein or TP53 is activated and can start a cell cycle arrest. The tail of H2XA, also known as the carboxy terminus, rapidly becomes labeled with phosphate groups that generate species called gamma-H2AX. This protein functions as a tumor suppressor and protects the genes as it is closely controlled by phosphorylation. The phosphorylated histone H2AX cooperates in repairing the genetic damage. It preserves the stability of the cells and prevents the onset of tumors. In DNA repair, chromatic reorganization plays an important role. Chromatin is responsible in packaging the DNA into a smaller volume for it to fit in the cell. This process aims to strengthen the DNA to allow mitosis and meiosis, where the cells divides and separates into two identical sets. This is also vital in apoptosis, cell cycle checkpoints, and serves as a mechanism to control inheritable information from a gene. The H2AX histone phosphorylation kinetics correlated well with the kinetics of DNA-adducts removal at earlier recovery time points. The chromatin reorganization is composed of proteins that are involved in the process. Among these proteins is TIP60 histone acetyltransferase, which plays an important role in DNA repair and apoptosis.

Conversely, its function to regulate the chromatin reorganization in the response of human cells to DNA damage is not commonly known. Based on microscopic studies, gamma-H2AX is generated in the chromatic edging a DNA double-strand break, and signaling proteins are recruited to the sites, known as "foci." TIP60 normalizes the ubiquitination of H2AX by means of the ubiquitin-conjugating enzyme UBC13, which is largely induced by DNA damage. To continue the process, this ubiquitination of H2AX requires its prior acetylation. Researches show that aceylation-dependent ubiquitination by the TIP60-UBC13 complex leads to the release of H2AX from damaged chromatin.

It was concluded that the sequential acetylating and ubiquitination of H2AX by TIP60-UBC13 promote enhanced histone dynamics, which in turn stimulate a DNA damage response (ESR Nexus Medical Research Community). Please note that the gamma-H2AX is not that essential in the recruitment of the repair factors, but indeed in the later formation of the foci. If the DNS damage proves to be irreparable, p53 can initiate apoptosis or a programmed cell morphology and death. The p53 status of the cell determines the decision between life or death after genotoxic stress.

Tornaletti et al. conducted a detailed investigation of how nucleosomes are formed and arranged on the DNA sequence in order to better understand the molecular mechanism if DNA-dependent processes such as transcription, replication, DNA repair, and mutagenesis. In their report, they analyzed the chromatin structure of exons 5-8 of the p53 gene in human fibroblasts. They also mapped at the nucleotide level the positions of DNase I and microccocal nuclease cleavage sites in pemeabilized cells. Areas of clear DNAse I protection, which would be indicative of the binding of sequence-specific proteins, were not detected. Instead, the micrococcial nuclease and DNase digestion patterns suggested that the region was covered by nucleosomes and that two areas spanning exons 5 and 6 are occupied preferentially. These nucleosomes could influence DNA damage distribution, repair of certain lesions, and other aspects of the mutagenesis process in p53 sequences.

Now, let us take a look at histone, as a gene or protein in the aquatic environment. Aquatic plants are very diverse and encompass the kingdoms Bacteria, Archaea, Protista, Fungi, and Plantae. The cells of cyanobacteria are typically small and composed of single DNA and circular molecule lacking histones as basic proteins. They do not have membrane bound-organelles. Eukaryotic algae have not much larger cells. Their DNAs are organized into one or more chromosomes, including histones. There are various membrane-bound organelles in nucleus. Histone H2A is a component of eukaryotic chromatin whose expression has not been studied in plants. To identify the cell-specific expression of plant histone to H2A genes, Koning et al. isolated and characterized a tomato and a pea cDNA encoding histone H2A. They found that in tomato H2A is encoded by a small gene family and that both the pea and the tomato mRNAs are polyadenylated. Tomato H2A has 82 amino acid residue identity to pea H2A, 83% to wheat, and 65% to human and yeast H2A. Plant H2As differ from fungal and animal H2As in their amno-terminal and carboxy-terminal regions. Carboxy-terminal plant H2A regions contain the motif SPKK, a peptide implicated in binding of a/T-rich DNA regions. By using RNA gel blot analysis, the authors determined that the steady-state mRNA level of these genes was abundant in apices and early developing fruit and very low in mature tissues. They also found that in situ RNA hybridization showed strong spatial regulation because the mRNA was abundant in some cells and not detectable in others. In tomato root tips, H2A-expressing cells were distributed irregularly in or near meristems. In tomato or pea root tips, expressing cells were concentrated near the apex, and their distribution was consistent with that expected of cycling cells. The authors also found other H2A transcripts in nondividing cortical cells that are known to undergo endoduplication during late maturation phase of primary development (1991)

In PubMed Central's article, Busslinger et al. talked about the histone DNA clone h19 of the sea urchin Psammechinus miliaris. It was found that in clone h19 of polarity of transcription and the relative arrangement of the histone genes is identical to that in clone h22 of the same species. The histone proteins encoded by h19 DNA differ in their primary structure from those encoded by clone h22 and have been compared to histone protein sequences of other sea urchin species a well as other eukaryotes.

The transcription of the sea urchin early histone genes occurs transiently during early cleavage, reaching the maximum at the morula stage and declining to an undetectable level at the gastrula stage. To identify the regulatory elements responsible for the timing and the levels of transcription of the H2A gene, promoter binding studies in nuclear extracts and microinjection of a CAT transgene driven y the early H2A promoter was used. It was found out that morula and gastrula nuclear proteins produced indistinguishable NDase I footprint patterns on the H2A promoter. There were two sites of interactions, centered on the modulator/enhancer and on the CCAAT box respectively, were detected. Deletion of the modulator or coinjection of an excess of modulator sequences severely affected the expression of two transgenes driven by the enhancer-less and modulator-containing H2A promoter. Finally, a DNA fragment containing 3' coding and post-H2A spacer sequences, where upon silencing three micrococcal nuclease hypersensitive sites were previously mapped, specifically repressed at the gastrula stage the expression of the transgene driven by the H2A promoter. These results indicate that the modulator for the expression of early H2A gene and that sequences for downregulation are localized near the 3' end of the H2A gene (Palla et al.: 1999).

The aquatic environment exposes both human and animal life to the presence of bacteria and other forms of parasites like entamoeba. Entamoeba belongs to the genus of amoebozoa that is usually found as internal parasites of animals. Some of its species are found in humans; and species of E. moshkovskii are found from river and lake sediments. The pathogen that is responsible for amoebiasis is called Entamoeba histolytica.

During the 5th Annual Congress on Tropical Medicine and International Health, among the topics discussed was the silencing of gene expression. One of their purposes is to study the role of histone acetylation in the regulation of E. histolytica. This was done through methylated cytosine that recruits methylated CpG binding proteins that interact with histonedeacetylase to alter the chromatin. In most eukararyotes such as animals, plants, and fungi whose cells are organized into complex structures enclosed within membranes, the cytosines are methylated by DNA methyltransferases that belong to the DNMT1 and DNMT3 families. In the parasite Entamoeba histolytica, the formation of methylated cystosine is catalyzed by an unexpected DNA methyltransferase from the DNMT2 family. The targets of methylated comprise LINE retrotransposon, rDNA and S/MAR element which suggests that methylation plays a role in the control of repetitive elements. In their study, they recently identified a protein, EhMLBP, that binds with high affinity to methylated INE retrotransposon and rDNA. EhMLBP lacks homology with methyl-CpG binding proteins and it shares a weak sequence identity with several prokaryotic histone. To further their study about recombinational DNa repair, they surveyed the parasite genome and identified a core set of highly conserved predicted proteins involved in DNA double-strand breaks (DSBs) repair by homologous combination. These proteins include homologous members of yeast. The presences of these proteins suggest that E. histolytica is skilled to perform homologous recombination, which is the same as in other organisms. DNA damage was evaluated by TUNEL assay. In yeast and in human cells, histone H2AX becomes rapidly phosphorylated when DSBs are introduced into chromatin (Lavi et al.).

Studies show that histone as a protein plays a significant role in the transition between the expression of a fetal gene and that of the adult gene. The adult gene's metabolism becomes oxidative in order to adapt to air and to weight, as it generates methylated transmitters and creatine phosphate. The muscles get used to life on the ground as compared to the fetal life which takes place in an aquatic environment. Regulated proteins allow the muscles to respond in a more adequate manner to this environment.

Now, let us see how histone and histone-like proteins work in dinoflagellates, most of which are marine planktons that commonly live in fresh water and belongs to a large group of flagellate protists. When they reproduce, they generate red tide that contains toxins. According to BioMed Central, a significant finding of the study of histone and histone-llike proteins in dinoflagellates is the identification of two rare expressed sequence tags (ESTs) that encode a partial histone H2AX. The longest cDNA isolated form the library using PCR was predicted to encode a protein of 169 amino acids that share high sequence identity to eukaryotic histone H2AX. This clone putatively lacked only the start codon at the N-terminus. The divergent N-terminus of a. tamarense H2AX is somewhat longer than in other homologs but the remainder of the sequence is conserved. Several functional residues from the known crystal structure are also present in a. tamarense H2AX including the lysine at the trypsine cleavage site, the arginines in the loops that interact with the DNA ?-helix, and the lysine ubiquitination site (Luger: 1997). Here, H2AX functions in the recognition and repair of double-strand DNA breaks by non-homologous end-joining. Various studies have proven that H2AX can arise independently and in many times during eukaryotic evolution.

Crissman et al. made a cytochemical analyses of the nuclear histone composition of hepatic tissue from acid-exposed Brook trout. The liver sections were stained fro histones by the eosin-fast green method which has been used for detecting histone changes n other vertebrate species. Three spectrally distinct cell types were determined on the basis of differential affinities for eosin and fast green. They exposed the fish to low acidity or pH for four days and they found out that the exposure did not affect the relative proportions of the three cell types. It only indicates that the aspect of liver function is not influenced by short exposure to low pH (2001: 49-52).

Another study of the activity and cytological effects of histone-like proteins on the parasitic dinoflagellate Amyloodinium ocellatum were made by Noga et al. The authors isolated from the tissues of rainbow trout Oncorhynchus mykiss and hybrid striped bass some histone-like proteins (HLPs) that are broad spectrum that endogenously produced antibiotics. They found that the histone-like proteins, which have a high sequence homology to H2B, equally inhibited both young and mature trophonts of the important ectoparasite Amyloodinium ocellatum. They also found that the aside from direct killing of Amyloodinium trophonts, there was an evident that HLP-1 from both rainbow trout and hybrid striped bass caused severe developmental abnormalities, which include delayed development in both the parasitic trophont stage as well as reproductive tomont stage. Parasites of normal appearance would die later in development and this called the "delayed mortality" effect of HLP-1. They also have seen the same effect with calf histone H2B and unrelated peptide antibiotic magainin. The antibiotic activity in mucus vs. epidermis compartments of the skin of hybrid striped bass suggested that the majority of antibiotic, including HLP-1 activity, resided in the epidermis, although some activities were present in the mucus. With this, they concluded that normal, non-immune fish skin contains potent defenses against protozoan ectoparasites and that the effects of these defenses may extend beyond their transient interactions with the parasites, which has important implications for this host-parasite relationship (2002: 207-215). Further evidence that HLPs are important defensive molecules in fish is provided by the fact that the relatively mild extraction techniques which the authors used to obtain HLP-1 and HLP-2 from fish tissues are not strong enough to release histone bound to nuclei acid (Moehs et al. 1992). It was also found that the activity was just as strong when they extracted tissues in water (Robinette et al. 1998; E. Noga and Z. Fan, unpublished data). The most important thing in their study, was that they have found that HLP-1 and HLP-2 possess potent activity against important fish pathogens that include bacteria and water moulds (Robinette et al. 1998). As a result, HLPs and other endobiotics may prove to be a very rich source of anti-parasitic strategies in the future.

As a then PhD in Pharmacology student in MUSC, Severine Patat and her group made a research about shrimp innate immunity to identity antimicrobial peptides and proteins in shrimp hemolymp. Preliminary data showed that hemocytes or shrimp blood cells contain high levels of histone proteins. In their study, it was found that in various vertebrates, histones have been shown to have antimicrobial activity. Since hemocytes are immune effector cells in the shrimp, they hypothesized that hemocytes contained high levels of histones to fight microorganisms. Shrimp histones H2A, H2B/H4 and H1 fragment were purified and inhibited growth of the Gram positive bacteria M. luteus.

A research headed by Robinette et al. was made about "Antimicrobial activity in the skin of the channel catfish Ictalurus punctatus: characterization of broad-spectrum histone-like antimicrobial proteins." It is widely known that parasites are the major cause of morbidity and mortality in fish. (Schnick et al. 1997). Channel catfish skin contains three proteins with antimicrobial activity. This activity is acid -- and heat-stable, and highly basic in nature. They isolated three antibacterial proteins from acid extracts of channel catfish skin. Histone-like proteins (HLPs) from channel catfish which exhibit broad-expectrum activity against pathogenic bacteria and water moulds were isolated. The objective of the study was to determine if similar defences occurred in unrelated fish species and whether such antibiotics had activity against protozoan ectoparasites.

In their initial study, they obtained from the local farms some clinically normal rainbow trout and sunshine bass that range from thirty to forty five centimeters in length and about two to four kilograms in weight. These were processed on site. The fish were euthanized by a blow to the head and decapitation and then they were maintained on ice through the process. They scraped the epidermis from the dermis with a scalpel. The scrapings were placed into ice-cold 1% acetic acid. They also dissected from each fish the gill and spleen and applied the same process. Afterwards, they heated the suspension until it boiled, and then they allowed it to cool at room temperature. The material was then homogenized and they extracted the acid. laboratory results indicated that skin extracts from channel catfish had significant antibacterial activity. The process was made by cation exchange chromatography. They isolated the purity and apparent molecular mass of antibacterial proteins from RP-HPLC and were assessed using SDS-polyacrylamide gel electrophoresis. The molecular masses of the purified antibacterial proteins were determined by matrix-assisted laser desorption mass spectrometry and were measured at 15.5, 15.5 and 30 kD. The most abundant antibacterial protein was assayed for antibacterial activity using a spectrophotometric assay. It was confirmed that antimicrobial protein was accomplished using a modification of a gel overlay assay. Then they examined the antiparasitic activity with the use of standard in vitro assay. Since the previous data indicated that HLPs were highly basic molecules (Robinette et al. 1998), the authors produced a large amount of tissue extract in 1% acetic acid for further purification and then they exploited a method that was similar to that used in purifying histones (Moehs et al. 1992); Johns, 1977). Based on the gathered data on mass spectrometry, amino acid composition and amino acid sequence, the authors found that the sequence differed from the major antibacterial protein only by the substitution of lysine for glycine, with an approximately 82% homology with rainbow trout histone H2B. Because of their similarity to histones, the catfish antibacterial proteins that they isolated were designated histone-like proteins (HLP) 1,2, and 3 in decreasing order of predominance in RP-HPLC. HLP-1 is closely realated, if not identical, to histone H2B. The H2B-like protein was inhibitory to Aeromonas hydrohila and Saprolegnia spp., which are important bacterial and fungal pathogens of fish. Considering these findings, histones may be important defensive molecules in fish and functions in fish immunity. The authors also gathered other evidence that is suggestive of possible immunological roles for histones.

The above study was supported by other evidence that is suggestive of possible immunological roles for histones. Nondividing mouse liver cells and exponentially dividing Friend erythrleukaemia cells contain sizeable cytoplasmic pools of histone H1 (Zlatanova et al.: 1990). Histone H2B, H2A and H4 have been isolated from bovine milk and serum (Waga et al.: 1987), and N-terminal fragments of histone H2B are found in human wound and blister fluid, along with defensins, lysozyme and the peptide antibiotic FALL-39 (Frohm et al.: 1996). There are also several reports that suggest that histones are not confined to the nucleus after synthesis. They may have other roles to their classical involvement in chromatin formation. A good example is histone H2A that inhibits the binding of gonadotropin-releasing hormone (Aten et al.: 1989). Researches also show that the mahor constituents of homeostatic thymus hormones are histones H2A and H2B (Aten et al.: 1989).

With the introduction of beta and gamma radiation and subnuclear localization, there have been a number of reported effects of proliferative radiation-induced lesions that appeared to be rather specific to ionizing radiations. Because the wavelength of gamma radiation is so short, a single incident photon can impart significant damage to a living cell. This property of gamma radiation is used to kill living organisms in the process of irradiation. This application is used in sterilizing medical equipment, removing decay-causing bacteria from the food; or preventing the fruit and vegetables from sprouting to maintain freshness and flavor. Though gamma rays/X-rays are used in medical practice such as in CT Scans and radio therapy because they contain a tissue penetrating property, as a form of ionizing radiation, they also have the ability to effect molecular changes that give them the potential to cause cancer when DNA is affected. X-rays and gamma rays can ionize almost any molecule or atom but if used improperly, overuse can be hazardous to human health.. (Wikipedia).

In the research made by Korgaonkar et al. entitled Monolayer Technique to Study Effects of Ionizng Radiation on Histone, the authors adapted the monoloyer technique developed by Langmuir for interphase and thin film studies. They studied the effectsof beta-gamma radiations on the protein histone in dilute aqueous solutions, prior to and after irradiation from radon glass capillaries. They found monolayer changes in the surface properties of histone. From the observed changes, it was concluded that irradiation produces a degree of fragmentation and some modifications in the histone molecules, which cause chrophlilic groups to go into the aqueous under phase and results in lowering of the residual charges carried by the molecules in the monolayer (2003: 6225-35). The clustered lesions or locally damaged sites give rise to cell killing and lethal effects. They may have some mutagenic potential in conjunction with other lesions (Cadet, Averbeck). According to Rothkamm et Lbrich, the reaction of living cells to IR is different at low doses/rates and high doses/rates. At very low dose (1mG,), cells are going to die because no DNA signaling and no initiation of DNA repair of DSBs with oxidized endings. At slightly higher doses (5-20 mGy), DNA repair is initiated. At medium doses (200 mGy) DNA repair starts to b counteracted by apoptosis. Extrapolations from high dose effects to low dose effects do not respond to the actual reaction of living cells to IR (PNAS 2003; 100:5057-5062). This means that there is an absence of repair at a very low dose. As explained by Belyakov, the non-targeted effects of ionizing radiation may have positive consequences. It might be interrelated and possibly have a protective role under in vivo conditions. These effects might related to adaptive response because of increased non-targeted differentiation in irradiated samples. Based on these experimental data the authors proposed a theory that the main function of the non-targeted effects is to decrease the risk of carcinogenesis in a multicellular organism exposed to oxidative damage, including radiation induced. DNA can be damaged by agents such as toxic chemicals and radiation. With that, DNA strands are broken and the "ataxia telangiectasia mutated" or ATM comes to the rescue. This ATM gene coordinates with DNA repair by activating enzymes that fix the broken strands. Another function of the ATM gene is to provide instructions for making a protein in the nucleus of the cell, where it helps to control cell growth and cell division. Recent studies show that the lysine tail of histone proteins undergoes phosphorylation in cells exposed to ionizing radiation.

Aside from gamma rays, there is another radiation, known as beta rays which consist of high-energy and high-speed electrons. Since it is composed of charged particles, beta radiation is more strongly ionizing than gamma radiation. Therefore, it also has an effect on histone proteins. Just like gamma radiation, beta particles are able to penetrate living matter and can result to damage resulting to a sever cancer and even death. They have much lighter energy particles.