Heterochromatin production limitations

Heterochromatin production limitations

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Currently playing with some ideas for a project and needed some guidance. I am wondering, both in Drosophila melanogaster and in general, is the amount of heterochromatin a cell/nucleus can produce limited/consistent or variable?

Does one cell in a D. melanogaster produce a similar amount of heterochromatin as another cell in the same fly or another fly (assuming same conditions - environment etc.) or is the heterochromatin production quite variable? How is heterochromatin production regulated?

Heterochromatin profile is of course different in different cells but I am not sure if absolute heterochromatin content will vary greatly. This DNAse hypersensitivity region data is for human cells but same principles apply to all organisms. If I have to take a guess then I would say that quiescent cells are likely to have more heterochromatin. Heterochromatin production is regulated by different epigenetic mechanisms but I cannot comment (I feel it is unlikely) if there is a mechanism to control absolute heterochromatin content.

Generally speaking, heterochromatin or euchromatin structures mark specific regions to regulate the transcriptional activity and these marks carry the signature of developmental processes as they differ in different tissues (or cell types).

Therefore, we should not ignore the developmental processes if we want to understand how such epigenetic marks are formed. But first, we should reconsider the question as it assumes that the heterochromatin is produced. The assumption is somewhat valid, there are many enzymes responsible to shut the chromosome off thru the histone modifications. On the other hand, heterochromatin strucure comes as a default (or as the factory settings). Before the mid-blastula transition, where no (or negligable) transcription happens, all the genome is heterochromatin due to the excess amount of histones. After certain amount of division, the amount of histone per cell reduces but the genome per cell remains constant which then titrates the histones and some transcriptional activity kicks in.

Then HATs, methyl transferases etc. help to clear up the necessary genomic regions depending on the extracellular developmental signaling. Then for many somatic cells, the ratio of heterochromatin and euchromatin regions more or less remain similar.

Somatic Embryogenesis: Process and Applications | Plants

In this article we will discuss about: 1. Process of Somatic Embryogenesis 2. Embryo Maturation and Synchronisation 3. Cultural Conditions 4. Recurrent Embryogenesis and Mass Production 5. Applications.

After fertilization, zygote is transformed into adult status through a series of embryogenic processes. Despite the same genetic constituents, somatic cells on the other hand, do not reorient towards embryo production. However, isolated somatic cells under in vitro conditions have the potential to develop into embryo under the influence of growth factors.

Somatic cells are able to develop into whole plant through the stages of embryogenesis without gametic fusion. Therefore, somatic embyros are non-zygotic embryos originated from sporophytic cells. Somatic embryo production is either direct or indirect in vitro. Somatic embryos may be direct when embryonic cells develop directly from the explants’ cells or indirect when developed through the callus.

Plant cells undergoing somatic embryogenesis are either pro-embryonic determined cells (PEDC) or induced embryogenic determined cells (IEDC). There have been reports on the induction of somatic embryos frequently from various tissues like seedlings, shoot meristem, young inflorescence and zygotic embryos. In addition, other tissues such as root, nucellus has also yielded somatic embryos.

The favorable responses of a few of the above tissues actually contain proembryogenic determinant cells (PEDC) or these cells may require minor reprogramming to enter embryogenic state. The first report on the production of somatic embryos in carrot suspension cells was published by Steward and co-workers in 1958. Thereafter reports were flooded on the production of somatic embryos in plants.

Process of Somatic Embryogenesis:

Significance of Auxin:

Reprogramming of somatic cells and its entry into the embryogenic status requires ex­tensive proliferation through unorganized callus cycle and exposure to high doses of synthetic auxin such as 2, 4-D or picloram. Somatic embryo induction can also be accomplished by plasmolysis of explant cells.

The significance of auxin for embryo induction status from vegetative cells and tissue was recognized as the prime controlling factor. This is based on a critical assessment in species like Daucus carota, Atropa belladona and Ranunculus sceleratus. Transformation of embyrogenic cells into the callus system due to the differentiation of single cell is followed by the appearance of dense cytoplasm, prominent nucleus and high profiles of organelles.

These groups of small densely packed cytoplasmic cells arise by internal division. These groups of cells constitute pro-embyros which can develop into globular embryos. The formation of a mature embyro and plantlet via heart and torpedo shaped stages may proceed undisturbed even when exogenous auxin remains present at lowest concentration in the later development.

The later process in the prevalent media condition however, may disturb further establishment of embryogenesis unless auxin is completely omitted. It was even evidenced that embryogenic process may be completely arrested during transition of embryo to plantlet. Therefore auxin is reduced or entirely withdrawn once such anomaly appears during culture.

In date palm tissue culture, liquid media enriched with low amount of plant growth regulator resulted in the differentiation of large number of somatic embryos. High concentration of auxin may not encourage embryo formation. Therefore, two distinct conclusions can be drawn by the role of auxin in entire embryogenic episode.

First, induction of cells with reprogrammed embryogenic competence under the influence of auxin. Second one is directing embryogenic cells to undergo complete development by withdrawing auxin from the media. Low level of endogenous auxin can equally determine embryo induction.

Auxin deprivation acts as a development switch from nonpolar embryogenic units to induce somatic embryogenesis in maize. This developmental switch is accompanied by cytoskeletal rearrangements in embryogenic cells. Whole somatic embryogenetic process may derail the establishment of polarity if exogenous auxin is supplied.

One of the negative factors implicated in somatic embryogenesis is the pro­duction of ethylene in presence of auxin for a considerable period of time in the culture media. Production of ethylene in turn elevates the activity of enzymes, probably, cellulase and pectinase which degrade pectin compounds and consequently disturb establishment of polarity by reduc­ing cell to cell interaction and contact due to separation.

Role of 2, 4-D in particular, for the induction of somatic embryogenesis is exemplary. Literature survey has shown that this synthetic auxin is very often suitable in inducing somatic embryogenesis in most of the species. Another synthetic auxin NAA has been found to be suit­able for somatic embryo induction.

However, the role of phytohormones in somatic embryo induction is highly a complex process and varies depending on plant species as well as its en­dogenous concentration. Under no circumstances, gibberellic acid is useful for somatic embryo induction. But its role has been implicated in the maturation of somatic embryos.

Role of Reduced Nitrogen:

The embryogenic competent cells seem to have preference for high salt strength and specific nitrogen source. This was considered to be a second pre-requisite for somatic embryo induction after auxin. The reduced form of nitrogen, ammonia, provides triggering factors for embryogenesis.

Similarly, nitrogen in the form of casein hydrolysate can equally contribute in the stimulation of somatic embryos and has been critically assessed in carrot as a model plant. Presence of proline and serine, capable of stimulating somatic embryo induction was reported in carrot plant.

Addition of reduced nitrogen, ammonium ion (NH4 + salt) or amino acids into the media is conductive for embryogenesis after shifting callus from auxin to auxin free media. It is however, concentration of auxin and nitrogen rather than critical concentration of reduced ni­trogen which is crucial in empowering embryogenesis.

High frequency of somatic embryogenesis was achieved in cucumber plant. Addition of diazuron and sucrose treatment (3-6%) exerted positive effect on the relative position of somatic embryo induction. Addition of copper sulphate in the media induces high frequency somatic embryo induction.

Similarly, thiadiazuron when supplemented in the medium induced shoot organogenesis at low concentration and somatic embryogenesis at high concentration. Enhanced somatic embryo production and maturation into normal plants in cotton was achieved when calli cultured on half strength MS media.

A thorough examination of the role of reduced nitrogen ammonia shows that embryo formation is promoted when as little as 0.1 mM ammonium chloride is supplied to nitrate me­dia. Embryogenesis is promoted by 40 mM potassium nitrate and 30 mM ammonium chloride as optimum concentration.

Glutamine and alanine can serve as sole nitrogen source for the growth and embryo formation. Although nitrate is required for embryogenesis on several in­stances, ammonium alone can produce embryo in carrot suspension culture, provided pH of the medium containing 10mM ammonium chloride and 20 mM potassium chloride was controlled at pH 5.4.

Level of dissolved oxygen has some role to play in somatic embryogenesis at least in carrot plant where embryogenesis takes place only below critical level of dissolved oxygen (i.e., above 1.5 ppm). Higher level favors rhizogenesis. Addition of activated charcoal into the culture media can promote embryo induction by adsorbing inhibiting substances produced by tissue.

Embryo Maturation and Synchronisation:

Studies on embryo germination process shows that embryo development completes without any anomalies in the absence of auxin in the media. However, any abnormalities due to endogenous hormones can be avoided by supplementing balanced concentrations of abscisic acid (ABA), zeatin, and GA3. Addition of charcoal may increase the maturation of somatic embryo.

Presence of charcoal in the media reduces the level of auxin like IAA due to its binding effect. Somatic embryo maturation can be enhanced by subjecting to osmotic desiccation. Sucrose is generally used at different concentrations to achieve embryonic growth and maturation. This is achieved by providing sucrose concentration between 4 and 6%.

In certain species, progressive increase in sucrose concentration upto 4% is required for maturation, which consequently produces vigorous plantlets. Similarly, imposition of temporary desiccation before embryo germination facilitates conversion to plantlets. Imposition of desiccation can be progressed by placing somatic embryos in empty petridish and incubated at desiccated condition for 2-3 weeks and some plants upto several weeks.

Somatic embryos, when shriveled to 50% of their original volume rapidly imbibe water when rehydrated by transfer to media. The whole exercise of desiccation in embryo is to influence metabolic process for germination. Somatic embryos when subjected to show desiccation, it stimulates the production of high frequency of shoot regeneration.

Imposition of desiccation improves conversion to plantlets several times the frequency of non-desiccated embryos. In Alfalfa culture, somatic embryos have been trained to withstand desiccation by treating them with ABA at the torpedo stage. ABA treatment can promote the development of cotyledons and block the production of embryo clusters.

Cultural Conditions of Somatic Embryogenesis:

High light intensity can influence the process of somatic embryogenesis. However, cul­tures were incubated under both light and dark periods. Early maturation takes place more predominantly under complete dark conditions.

Reports on the influence of temperature on somatic embryogenesis are scarce. In citrus nucellus culture, embryogenic potential drops when the temperature was reduced from 27°C to 12°C. Similarly, conditioning of somatic embryos by cold treatment can escape dormancy and facilitate development.

Recurrent Embryogenesis and Mass Production of Somatic Embryogenesis:

The primary somatic embryo when fails to undergo maturation may enter continuous successive cycles of embryos. Certain specific superficial cells of the hypocotyl or cotyledon exhibit this tendency in provoking successive cycles of embryos or in other words continuous production of supernumerary embryos from somatic embryos itself.

This phenomenon is also known as secondary embryogenesis, recurrent embryogenesis, repetitive or accessory embryogenesis (Fig. 8.1). Recurrent embryogenic cycle can be maintained in culture by the removal of growth regulators and cycles can be spontaneous as this was evidenced in Alfalfa (Medicago sativa).

Recurrent embryogenesis cycle can be made spontaneous by locking the development of so­matic embryos particularly at proembryogenic status, beyond which they cannot proceed to develop. This can be accomplished by initial exposure to very high concentration of 2, 4-D upto 40 mg/L for brief period followed by exposure to a lowest concentration (3-5 mg/L).

This high concentration of auxin treatment may be involved in reprogramming of cells and reinduce embryogenic competence. Repetitive embryogenesis may be a serious problem during spontane­ous cycles of somatic embryo production when germination and further development is required.

Gene Expression Programme in Somatic Embryogenesis:

One of the most striking features of somatic embryogenesis is the successful crackdown on RNA expression in embryogenic and nonembryogenic tissues. Several striking similarities were cited in the gene products expressed in embryogenic and nonembryogenic cultures. The tissue culture conditions are typically defined as nonembryogenic. The pattern of gene expres­sion between embryogenic and nonembryogenic systems exhibit least diversity about RNA ex­pression profiles.

Limited number of changes has been recorded in protein expression pattern during somatic embryogenesis. Similarly, changes in mRNA populations take place during tran­sition from nonembryogenic to various embryo stages. Removal of auxin from the media during embryo induction triggers new profiles of gene expression that are eventually coupled to ob­serve morphogenetic events.

Applications of Somatic Embryogenesis:

Micro-Propagation Industries:

One of the most promising applications of somatic embryogenesis is large scale propagation of somatic embryos, which shows several advantages such as innumerable number of embryo production (60,000-70,000 embryos per litre of media), presence of both root and shoots meristems, easy to scale up and convert them into seedlings efficiently as far as commercial significance is considered. Somatic embryos are genetically well programmed to make a complete plant. Thus, unlike other micro-propagation systems, somatic embryogenesis avoids certain stages of micro-propagation particularly, the rooting stage.

Synthetic Seed Production:

Synthetic seeds or artificial seeds are the somatic embryos encapsulated by gel entrap­ment solution. Artificial seeds are generally produced in plant species which exhibit seed steril­ity and difficulty or slow phase of vegetative propagation.

This can be prepared by placing somatic embryos in alginate slurry (2%) as gel entrapment matrix and subsequently trans­ferred to calcium chloride (100 mM) solution to form beads in which embryos get entrapped. Artificial seeds can be stored at 4°C for a considerable period of time and used as efficient system for germplasm conservation. For regeneration, seeds can be placed in culture media or in sterile soil to facilitate germination and seedling development (Fig. 8.2).

Repetitive embryogenesis often provides innumerable number of somatic embryos, which in turn is useful in the mass production of plant propagules. Several embryo specific metabolites like seed storage proteins and lipids of industrial value can be recovered. Lack of seed tissue surrounding somatic embryos proves significant advantage for certain lipids such as α-linolenic acid present at high level in Borage seeds.

This lipid is of high commercial significance in the treatment of atopic eczema. Surprisingly, somatic embryos as an analogue of zygotic embryo also synthesize the same amount of α-linolenic acid. Similarly, jojoba plant contains high qual­ity industrial lubricant in their seeds.

Somatic embryos obtained from zygotic embryos as the explant possesses waxes identical to that of zygotic embryos. In addition, novel metabolites can be produced in somatic embryos throughout the season.

Somatic Embryos in Gene Transfer:

Somatic embryos are an ideal system for gene transfer process. This particular approach can avoid protoplast mediated regeneration of transformed plants which generally requires additional care. Moreover, protoplast mediated regenerated plants can exhibit genetic variation. Since somatic embryos maintain genetic stability, regenerated plants are not susceptible for somaclonal variation.

Somatic embryos can be transformed by incubating them in Agrobacterium solution or subjected for particle bombardment. Embryo cloning by recurrent approach is well suited for direct gene transfer to the mass of somatic embryos. The stably transformed somatic embryos can be farther subjected for recurrent embryogenic cycle to procure millions of transgenic plants.

RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production

In fission yeast, factors involved in the RNA interference (RNAi) pathway including Argonaute, Dicer, and RNA-dependent RNA polymerase are required for heterochromatin assembly at centromeric repeats and the silent mating-type region. Previously, we have shown that RNA-induced initiation of transcriptional gene silencing (RITS) complex containing the Argonaute protein and small interfering RNAs (siRNAs) localizes to heterochromatic loci and collaborates with heterochromatin assembly factors via a self-enforcing RNAi loop mechanism to couple siRNA generation with heterochromatin formation. Here, we investigate the role of RNA-dependent RNA polymerase (Rdp1) and its polymerase activity in the assembly of heterochromatin. We find that Rdp1, similar to RITS, localizes to all known heterochromatic loci, and its localization at centromeric repeats depends on components of RITS and Dicer as well as heterochromatin assembly factors including Clr4/Suv39h and Swi6/HP1 proteins. We show that a point mutation within the catalytic domain of Rdp1 abolished its RNA-dependent RNA polymerase activity and resulted in the loss of transcriptional silencing and heterochromatin at centromeres, together with defects in mitotic chromosome segregation and telomere clustering. Moreover, the RITS complex in the rdp1 mutant does not contain siRNAs, and is delocalized from centromeres. These results not only implicate Rdp1 as an essential component of a self-enforcing RNAi loop but also ascribe a critical role for its RNA-dependent RNA polymerase activity in siRNA production necessary for heterochromatin formation.


Rdp1 localization at centromeres depends…

Rdp1 localization at centromeres depends on RITS, Dcr1, and heterochromatin assembly factors. (…

Mutation of RdRP domain of…

Mutation of RdRP domain of Rdp1 abolishes silencing and heterochromatin formation at centromeres.…

Rdp1 possesses RdRP activity that…

Rdp1 possesses RdRP activity that is essential for generation of RITS-associated siRNAs. (…

RdRP activity is necessary for…

RdRP activity is necessary for RITS and Rdp1 localization at centromeres. ( A…

Rdp1 is an essential component…

Rdp1 is an essential component of a self-enforcing RNAi loop mediating heterochromatin formation.…

Why and Where to Include Limitations in My Research Paper

Every research has certain limitations, and it’s completely normal, but you need to minimize their range of scope in the process. Provide your acknowledgment of them in the conclusion. Identify and understand potential shortcomings in your work.

When discussing limitations in research, explain how they impact your findings because creating their short list or description isn’t enough. Your research may have many limitations. Your basic goal is to discuss the ones that relate to the problems that you choose for a specific academic assignment.

Limitations of your qualitative research can become clear to your readers even before they start to read your study. Sometimes, people can see the limitations only when they have viewed the whole document. You have to present your study limitations clearly in the Discussion paragraph. This is the final part of your work where it’s logical to place the limitations section. You should write the limitations at the very beginning of this paragraph, just after you have highlighted the strong sides of the research methodology. When you discuss the limitations before the findings are analyzed, it will help to see how to qualify and apply these findings in future research.

Biological functions

Protein aggregation has been classically viewed as an aberrant process with pathological consequences. Indeed, cellular aggregates are associated with a large number of human diseases, including neurodegeneration [98], type 2 diabetes [39], and aging [99], and extensive work has been dedicated to elucidating their role in these and other diseases (reviewed elsewhere [42, 43]). But in part due to the development of new techniques to study protein aggregates (Table 2), we are gaining a new appreciation and understanding of their non-pathological roles. Protein aggregates play positive functions in a variety of cellular processes, including gene regulation [126, 127], signaling [76, 128, 129], memory storage [56, 130, 131], DNA repair [132], cell fate decisions [130, 133,134,135], and even evolution [2]. These examples should serve as inspiration for synthetic biologists aiming to purposefully manipulate information flow in living systems (Fig. 2).

Protein assemblies play important roles in a variety of critical cellular processes. a In eukaryotic transcription, co-activators and transcription (txn.) factors form highly dynamic protein condensates that recruit RNA polymerase II (RNA pol II) and drive robust gene activation. b RNA-binding proteins (RBPs) and RNAs coalesce to form RNP granules, which serve different RNA processing functions, such as mRNA storage and degradation, ribosome biogenesis, and localized translation. In one intriguing example, prion-like aggregation of CPEB3 promotes translation in activated synapses to potentiate long-term memory. c Higher-order assemblies play key roles in innate immunity. For example, prion-like polymerization of the MAVS adaptor protein in response to viral infection leads to amplification and stabilization of the antiviral response. d In yeast, stochastic switching between [prion − ] and [PRION + ] states in a population of cells enables phenotypic diversification and may promote survival in uncertain environments. Figure adapted from Fig. 1B in [136]. In prion nomenclature, brackets denote non-Mendalian inheritance and capital letters denote dominance in crosses

Gene regulation

Many components controlling aspects of gene expression form dynamic protein assemblies that contribute to their regulatory mechanism. Strikingly, different steps of eukaryotic gene transcription appear to utilize regulated phase separation mechanisms [137]. A first step in transcription is the binding of transcription factors (TFs) to enhancer regions. Phase separation was found to be important in this process at super-enhancers, which are clusters of enhancers driving robust transcription of cell identity genes. In particular, certain TFs were shown to phase separate via their IDRs into liquid-like condensates that help to compartmentalize the transcriptional apparatus [138]. A following step in transcription involves Mediator, a complex that connects signals from TFs to RNA polymerase (Pol) II. Mediator has been shown to form phase-separated clusters both with TFs [127] and with Pol II [126] at active sites of transcription. Finally, the process of transcription elongation relies on phosphorylation of the C-terminal domain (CTD) of Pol II. This is accomplished in part by the enzyme complex positive transcription elongation factor b (P-TEFb). To ensure hyper-phosphorylation of the CTD and efficient elongation, P-TEFb undergoes phase separation into nuclear speckles capable of recruiting Pol II [139]. Interestingly, protein phase separation has also been implicated in gene silencing through recent work demonstrating that HP1α proteins, key factors involved in the formation of heterochromatin domains, have the ability to form liquid droplets in a regulated fashion [140,141,142].

Proteins regulating the RNA life cycle, downstream of transcription, are among the most prominent examples of molecules that undergo phase separation. RNA-binding proteins (RBPs) are particularly rich in disordered, low-complexity sequences. Many RBPs possess IDRs that have been shown to undergo liquid–liquid phase transition in cells [20, 22, 23], thus driving the formation of membraneless organelles important in RNA metabolism (these include nucleoli, stress granules, P-bodies, and Cajal bodies) [143]. As one example, condensation of components of the human miRISC complex facilitates recruitment of deadenylation factors that promote degradation and silencing of mRNAs [144]. RBPs have gained recent attention because, on the one hand, their aggregation can drive the formation of these functional membraneless RNP bodies, yet on the other hand, mutations in their low-complexity sequences are causal factors in neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and multisystem proteinopathy (MSP) [145]. Interestingly, nucleotide repeat expansions, one class of mutation associated with neurotoxicity, which can cause gain or loss of function of genes encoding RBPs, have also been shown to alter the properties of RNAs. Specifically, repeat-containing RNAs have been shown to form gels in vitro by creating opportunities for multivalent base-pairing, and can accumulate in aberrant and potentially toxic nuclear foci in cells that sequester RBPs [146].


The innate immune system is an ancient and rapid first-line defense that higher organisms deploy to defend against invading pathogens. This system consists of interconnected signaling pathways that activate inflammatory responses in an effort to eliminate the pathogen, as well as regulate different types of cell death, such as apoptosis and necroptosis (programmed necrosis). These pathways must be able to be rapidly deployed, but also tightly controlled and balanced in order to prevent excessive inflammatory responses or cell death. Many signaling components involved in these pathways are capable of oligomerization to form higher-order assemblies, sometimes generically referred to as signalosomes [147]. This capacity for oligomerization is an important mechanism for increasing specificity of and amplifying signal transduction [76].

One prominent example is the RIP1/RIP3 necrosome mediating programmed necrosis, a form of cell death (distinct from apoptosis) that represents an important host defense mechanism [128]. Here, the RIP1 and RIP3 kinases form a functional amyloid-based signaling complex to trigger programmed necrosis. Importantly, RIP1 and RIP3 kinase activation is required for this amyloid complex formation, which in turn can further enhance kinase activation through phosphorylation, thereby amplifying/propagating the pronecrotic signal.

Another key host defense mechanism is activation of inflammatory responses. This is carried out by the inflammasome complex, which translates pathogen and cellular danger signals recognized by sensors, such as NLRP3, into inflammatory responses through the adaptor protein ASC. Intriguingly, ASC was shown to be a bona fide prion in yeast [76, 77, 129]. Thus, in response to upstream sensors, initial oligomerization of ASC can result in prion-like nucleation this in turn enables the templating of other ASC molecules to form large polymers capable of robustly recruiting caspase-1 molecules to induce their activation and propagate the inflammatory signal. Similarly, viral infection triggers the prion-like aggregation of the mitochondrial antiviral-signaling (MAVS) adaptor protein into fibrillar structures, which in turn recruit other soluble MAVS proteins, amplifying and stabilizing the antiviral response [76, 148]. These examples highlight how prion-like polymerization may provide a (evolutionarily conserved) mechanism for highly sensitive and robust response to cellular signals.


One of the most fundamental and remarkable aspects of organismal behavior is the ability to make memories of past events, and to subsequently modify behavior by learning. Cells have multiple mechanisms for making molecular memories that outlast the half-life of proteins. One mechanism is prion-like aggregation [130, 149]. In animals, the cytoplasmic polyadenylation element binding protein 3 is a highly conserved RBP (CPEB in Aplysia, Orb2 in Drosophila, and CPEB3 in mice) that plays a role in the formation of new memories [56, 150, 151]. Specifically, prion-like aggregation of CPEB3 in the synapses of stimulated neurons leads to the formation of RNA granules that bind and drive translation of mRNAs involved in synaptic plasticity and growth [57]. CPEB3, and possibly also other RBPs, represents a fascinating example of how conformational changes at the molecular scale can produce macroscopic changes in animal behavior, linking molecular self-replication, cellular memory, and neuronal memory.


Since their initial discovery, we have come to understand prions not only as causative agents of disease, but also as sources of new and sometimes adaptive cellular functions [47, 66, 88, 121, 136, 152,153,154,155,156,157]. This has been most apparent in yeast, where several central regulators of information flow and metabolism have been determined to be prion proteins (Table 1). A canonical example is the S. cerevisiae prion [PSI + ], formed by the translation termination factor Sup35 [48]. At a low frequency, Sup35 converts from a soluble, functional conformation to a self-templating prion. This allows ribosomes to read through stop codons, uncovering previously silent genetic variation on a genome-wide level, and thus producing diverse and heritable phenotypes that are often disadvantageous but that can provide advantages in particular environments [158, 159]. This has led to the provocative hypothesis that yeast prions may serve as adaptive ‘bet-hedging’ elements to promote cellular survival in stressful environments [160]. In support of this was the discovery that hundreds of wild yeast strains contain heritable prion states, which frequently confer beneficial phenotypes under selective conditions [88].

Other notable examples of yeast prions that enable epigenetic switching to endow cells with beneficial phenotypes in specific metabolic and environmental conditions include [URE3] [47], [MOT3 + ] [88], and [GAR + ] [161]. In particular, the [SWI + ] prion formed by the yeast Swi1 protein represents an intriguing potential example of bet-hedging. As the main subunit of the SWI/SNF chromatin remodeling complex, Swi1 serves as a global transcriptional regulator. When Swi1 adopts its prion form, a variety of phenotypes can be induced, such as growth phenotypes on alternative carbon sources, sensitivity to antifungal agents, and, importantly, abolished adhesion to other cells or substrates [162]. By maintaining a small population of [SWI + ] cells, an isogenic population can effectively safeguard against unpredictable environments via these diverse and potentially beneficial phenotypes, for example, by providing an opportunity for non-adherent [SWI + ] cells to disperse to new locations to ensure re-population and survival [160].

Recently, the first bacterial protein capable of prion formation (the Clostridium botulinum global transcriptional terminator Rho [59]) and the first viral protein exhibiting prion-like self-propagating activity (formed by the baculovirus LEF-10 protein [78]) were discovered, suggesting that prion-based mechanisms for phenotypic diversification may be more pervasive than originally thought. We expect that more of these elements will be discovered with the development of new genetic tools to characterize and validate putative prions from other organisms [117, 163].

Plant Sex Chromosomes

Although individuals in most flowering plant species, and in many haploid plants, have both sex functions, dioecious species—in which individuals have either male or female functions only—are scattered across many taxonomic groups, and many species have genetic sex determination. Among these, some have visibly heteromorphic sex chromosomes, and molecular genetic studies are starting to uncover sex-linked markers in others, showing that they too have fully sex-linked regions that are either too small or are located in chromosomes that are too small to be cytologically detectable from lack of pairing, lack of visible crossovers, or accumulation of heterochromatin. Detailed study is revealing that, like animal sex chromosomes, plant sex-linked regions show evidence for accumulation of repetitive sequences and genetic degeneration. Estimating when recombination stopped confirms the view that many plants have young sex-linked regions, making plants of great interest for studying the timescale of these changes.


Analysis of SU(VAR)3-9 identified the key function of H3K9 methylation in heterochromatic gene silencing (Tschiersch et al. 1994). The protein contains a SET domain that enzymatically functions to methylate histone H3K9. That this protein is a histone methyltransferase (HKMT) targeting H3K9 was first shown by characterization of the human SUV39H1 homolog (Rea et al. 2000). In Drosophila, SU(VAR)3-9 is a major, but not the only, H3K9 HKMT (Schotta et al. 2002 Ebert et al. 2004). SU(VAR)3-9 contributes to di- and trimethylation of H3K9 (H3K9me2 and me3) in the bulk of the pericentromeric heterochromatin, but not in the majority of the fourth chromosome, telomeres, or euchromatic sites. The bulk of the dimethylation of these latter regions is independent of SU(VAR)3-9, as is monomethylation of H3K9 in pericentromeric heterochromatin (Ebert et al. 2004). dSETDB1 (“eggless”) plays a major role in H3K9 methylation on the fourth chromosome (Seum et al. 2007 Tzeng et al. 2007 Brower-Toland et al. 2009 Riddle et al. 2012) G9a and potentially other HKMTs could also contribute, but the specifics are still unknown. The importance of H3K9 dimethylation in heterochromatic gene silencing is shown by the strong dosage-dependent effect of SU(VAR)3-9 on the PEV phenotype, as well as by the finding that suppression of gene silencing by Su(var)3-9 mutations correlates with HKMT activity. The enzymatically hyperactive Su(var)3-9 ptn mutation is a strong enhancer of PEV and causes elevated H3K9 di- and trimethylation (H3K9me2 and H3K9me3) at the chromocenter, as well as generating prominent H3K9me2 and me3 signals at many euchromatic sites (ectopic heterochromatin) (Ebert et al. 2004). S-adenosylmethionine functions as the methyl donor for all of these methylation reactions consequently, mutations in the gene encoding S-adenosylmethionine synthase, Su(z)5, are dominant suppressors of PEV (Larsson et al. 1996).

Studies using mutations in SU(VAR) genes have begun to reveal the sequence of molecular reactions required to establish heterochromatic domains. SU(VAR)3-9 binding at heterochromatic sequences depends on both its chromo and its SET domains (see Patel 2014 for details of protein structure Schotta et al. 2002). How SU(VAR)3-9 binding is controlled is not yet understood. The act of methylating H3K9 by SU(VAR)3-9 establishes binding sites for HP1a. The HP1a chromo domain specifically binds H3K9me2 and H3K9me3 (Jacobs et al. 2001). That SU(VAR)3-9 also binds HP1a has been shown by yeast two-hybrid tests and by immunoprecipitation (Schotta et al. 2002). In fact, the region of SU(VAR)3-9 amino-terminal to its chromodomain interacts with the chromoshadow domain of HP1a, and this interaction stabilizes HP1a binding to H3K9me2/3 (Fig. 4A) (Eskeland et al. 2007). This region of SU(VAR)3-9 also interacts with the carboxy-terminal domain of SU(VAR)3-7. The SU(VAR)3-7 protein interacts at three different sites with the chromoshadow domain of HP1a (Delattre et al. 2000). This pattern of interactions suggests that the three proteins—HP1a, SU(VAR)3-7, and SU(VAR)3-9—physically associate in multimeric heterochromatin protein complexes.

Interaction of SU(VAR)3-9 and HP1a in setting the distribution pattern of H3K9 methylation. (A) HP1a interacts with H3K9me2/3 through its chromodomain, and with SU(VAR)3-9 through its chromoshadow domain. By recognizing both the histone modification and the enzyme responsible for that modification, HP1a provides a mechanism for heterochromatin spreading and epigenetic inheritance. (B) SU(VAR)3-9 is responsible for much of the dimethylation of H3K9 (H3K9me2) loss of the enzyme results in loss of this modification in the pericentric heterochromatin, as shown by loss of antibody staining of the polytene chromosomes (compare middle panel with top panel). Loss of HP1a results in a loss of targeting of SU(VAR)3-9 high levels of H3K9me are consequently now seen throughout the chromosome arms (bottom panel).

Association of SU(VAR)3-9 and HP1a with pericentric heterochromatin is interdependent (Schotta et al. 2002). SU(VAR)3-9 causes H3K9 di- and trimethylation, which are specifically recognized by the chromodomain of HP1a (Jacobs et al. 2011). Consequently, in Su(var)3-9 null larvae, HP1a binding to pericentric heterochromatin is impaired. As H3K9 dimethylation does not depend exclusively on SU(VAR)3-9 in the inner chromocenter, the fourth chromosome, telomeres and euchromatic sites, HP1a continues to be found at all of these sites in the mutant lines. Thus although SU(VAR)3-9 associates with these sites in wild-type cells, it appears to be relatively inactive.

Conversely, if HP1a is not present (having been depleted by mutations), SU(VAR)3-9 is no longer associated primarily with the pericentric heterochromatin, but is found along the euchromatic chromosome arms. It is now seen at almost all bands, where it causes ectopic mono- and dimethylation of H3K9 (H3K9me1 and H3K9me2) (Fig. 4B). Thus HP1a is essential for the restricted binding of SU(VAR)3-9 to pericentric heterochromatin. These data suggest a sequence of reactions starting with SU(VAR)3-9 association with heterochromatic domains and consequent generation of H3K9me2/3. This mark is recognized by the chromodomain of HP1a binding of SU(VAR)3-9 to the HP1a chromoshadow domain ensures its association with heterochromatin (Fig. 4A). Interestingly, a chimeric HP1a-Pc protein has been generated in which the chromodomain of HP1a is replaced with the chromodomain of the Pc protein (Platero et al. 1996). The chromodomain of Pc binds strongly to H3K27me3 (Fischle et al. 2003), and the HP1a-Pc chimeric protein binds these sites in the euchromatic arms. In the presence of such a chimeric HP1a-Pc protein, the SU(VAR)3-9 protein is also found at Pc binding sites, demonstrating its strong association with the chromoshadow domain of HP1a (Schotta et al. 2002).

In SU(VAR)3-9 null cells another heterochromatin-specific methylation mark, H4K20 trimethylation (H4K20me3), is strongly reduced. The interdependence between H3K9 dimethylation and H4K20 trimethylation in heterochromatin has been shown to reflect an interaction between the SU(VAR)3-9, HP1a, and SUV4-20 proteins. SUV4-20 is a histone lysine methyl transferase (HKMT) that controls H4K20 methylation in heterochromatin. This heterochromatin-specific methylation mark is also strongly impaired in HP1a null cells, suggesting association of SU(VAR)3-9, HP1a, and SUV4-20 in a mutually dependent protein complex, although such a complex has not yet been isolated from flies. Mutations in the Suv4-20 gene cause suppression of PEV-induced gene silencing, indicating that the H4K20me3 mark is required for this process (Schotta et al. 2004).

Taken together, the evidence argues that the HP1a protein has a central function in pericentric heterochromatin formation and associated gene silencing it binds H3K9me2 and H3K9me3, and interacts directly with SU(VAR)3-9 (one of the H3K9 HKMTs) as well as several other key chromosomal proteins. The resulting complexes probably include several additional heterochromatin-specific proteins. Variations on this theme apply to other heterochromatic domains, such as the fourth chromosome (Riddle et al. 2012). However, given the number of identified Su(var) loci, the model is certain to become more complex!

In mammals and plants, histone H3K9 methylation and DNA methylation represent interrelated marks of repressed chromatin (Martienssen and Colot 2001 Bird 2002). Whether or not DNA methylation occurs at all in Drosophila has been a point of contention for many years. Recent reports showing low levels of DNA methylation in the early embryo have renewed this discussion (reviewed in Krauss and Reuter 2011). In Drosophila the only recognizable DNA methyltransferase present is Dnmt2. Mutations in this gene have a significant impact on retrotransposon silencing in somatic cells (Phalke et al. 2009). However, many inbred laboratory strains show only a very low level of Dnmt2 expression variation of this sort could explain conflicting results concerning DNA methylation in Drosophila (O Nickel, C Nickel, and G Reuter, unpubl.).

Top 5 Techniques of Chromosome Banding

The technique of C-banding originated after the work of Pardue and Gall who reported that constitutive heterochromatin can be stained specifically by Giemsa-solution. Each chromosome possesses a different degree of constitutive heterochromatin which enables the identification of individual chromosomes.

Constitutive heterochromatin is located near the centromere, at telomeres and in the nucleolar organizer regions it is composed of highly repetitive DNA. C-banding represents the constitutive heterochromatin, and the banding is caused by differential staining reactions of the DNA of heterochromatin and euchromatin.

The banding method is a complex technique that involves several treatments with acid, alkali or increased temperature. Denaturation of DNA is caused by these treatments. Subsequently, DNA renaturation occurs in treatments with sodium-citrate at 60°C.

By these treatments, the repetitive DNA (heterochromatin) re-natures but low repetitive and unique DNAs do not re-nature. This results in differential staining of the specific chromosome regions. Giemsa-C-banding technique has been used to identify chromosomes of various plant and animal species including human. The Y chromosome of mammals is mostly heterochromatic and therefore, the technique of C-banding is quite useful for its identification.

In barley chromosomes, Linde-Laursen in 1978, divided the C-bands into the following classes based on their position:

(i) Centromeric bands situated at one or both sides of the centromere,

(iv) Bands beside the secondary constriction in the short arm of satellited chromosomes.

He observed polymorphism in C-banding pattern in different barley lines, Giemsa-C-banding patterns may also be used to identify the extra-chromosomes of trisomies and telotrisomics.

Chromosome Banding: Technique # 2. G-Bands:

The technique of G-banding involves Giemsa staining following pretreatment with weak trypsin solution, urea or protease. It provides greater detail than C-banding. It was first used for human chromosomes by Summer et al. in 1971. G-bands may reflect a stronger chromatin condensation. However, this technique is not suitable for plant chromosomes.

Chromosome Banding: Technique # 3. Q-Bands:

The method of Q-banding was developed by Caspersson et al. in 1968. The chromosomes stained with Quinacrine mustard show bright and dark zones under UV light. This technique is used to identify human and mice chromosomes.

Chromosome Banding: Technique # 4. N-Bands:

The technique of N-banding was originally described by Matsui and Sasaki in 1973. Briefly, air-dried chromosomes slides are stained for 90 minutes with Giemsa (diluted 1 : 10 in 1/15 M phosphate buffer at pH 7.0) following extraction with 5% trichloroacetic acid at 95°C for 30 minutes and then 0.1 NHCl at 60″C for 30 minutes.

The N-bands are generally located at the secondary constriction, satellites, centromeres, telomeres and heterochromatic segments. It is suggested that the N-bands represent certain structural non-histone proteins specifically linked to the nucleolar organizer region of the eukaryotic chromosomes.

The N- banding patterns have been used for the location of nucleolar regions in the different organisms, such as, mammals, birds, amphibians, fishes, insects and plants. N-banding patterns differ in the chromosomes of different species.

In 1980, Islam used this method to identify the barley chromosomes from those of wheat in the reciprocal wheat-barely F, hybrids, and to detect translocations between the wheat and barley chromosomes. He also used this technique to isolate lines possessing a pair of barely chromosomes substituted for particular pair of wheat chromosomes.

A modified Giemsa-N-banding technique was developed by Singh and Tsuchiya in 1982 for the identification of barley chromosomes. This method is a combination of acetocarmine staining and Giemsa-N-banding. After processing according to this method, the centromeric region looks like a “diamond-shaped” structure this is not seen in other techniques.

Early metaphase or prometaphase chromosomes are more suitable for this staining as they show better banding pattern than the chromosomes at mid-metaphase in somatic cells.

Chromosome Banding: Technique # 5. Other Techniques of Chromosome Banding:

Besides the above, there are other techniques for chromosome banding, e.g., R-banding (Reverse Giemsa banding). H-banding, and T-banding (Terminal banding). Chromosome banding patterns can be used not only for the identification of individual chromosomes of an organism but also to establish evolutionary relationships between different species.

Banding patterns in human, chimpanzee, gorilla and orangutan have indicated that the evolutionary relationship between human and chimpanzee is closer than that between human and gorilla. It has further indicated that humans have a more distant evolutionary relationship with orangutans.

Heterochromatin, histone modifications, and nuclear architecture in disease vectors

Heterochromatin contains essential genes and performs important biological functions.

Finished genome assemblies must include heterochromatic sequences.

Histone modifications regulate immunity, development, and reproduction.

Nuclear architecture affects gene expression and longevity.

Interactions between a pathogen and a vector are plastic and dynamic. Such interactions can be more rapidly accommodated by epigenetic changes than by genetic mutations. Gene expression can be affected by the proximity to the heterochromatin, by local histone modifications, and by the three-dimensional position within the nucleus. Recent studies of disease vectors indicate that gene regulation by these factors can be important for susceptibility to pathogens, reproduction, immunity, development, and longevity. Knowledge about heterochromatin, histone modifications, and nuclear architecture will help our understanding of epigenetic mechanisms that control gene function at traits related to vectorial capacity.


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