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Epigenetic control of gene expressions

In eukaryotes, including plants, the genome is compacted into chromatin, which forms a physical barrier for gene transcription. Therefore, mechanisms that alter chromatin structure play an essential role in gene regulation. When changes in the chromatin states are inherited trough mitotic or meiotic cell division, the mechanisms responsible for these changes are defined as epigenetic.

Chromatin,nucleosome,histone
Chromatin is the combination of DNA and other proteins that make up the contents of the nucleus. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene expression and DNA replication. The primary protein components of chromatin are histones that compact the DNA. Chromatin is only found in eukaryotic cells: prokaryotic cells have a very different organization of their DNA which is referred to as genophore (not chromatin). Nucleosomes are the basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound around a histone protein core. This structure is often compared to thread wrapped around a spool The crystal structure of the nucleosome core particle consisting of H2A , H2B , H3 and H4 core histones, and DNA.

Histone Proteins -In biology, histones are highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes.They are the chief protein components of chromatin, acting as spools around which DNA winds, and play a role in gene regulation. Without histones, the unwound DNA in chromosomes would be very long

Histones "are highly conserved and can be grouped into five major classes: H1/H5, H2A, H2B, H3, and H4".

core histones H2A, H2B, H3 and H4 linker histones H1 and H5

The nucleosomes are loops of DNA, 147 base pairs long, wrapped tightly around a core made of eight protein molecules called histones. Each loop or knot of DNA is connected to the next by a stretch of unwrapped DNA (called linker DNA) that can be anything between 10 and 50 base pairs long. DNA in the nucleus, therefore, resembles a string of beads, each bead representing a nucleosome

Nucleosome positioning
Now the fascinating thing about this arrangement is that its physical structure affects the cell and the organisms. It has a profound effect on the readout of the DNA sequence. The very compact structure of DNA in the nucleosomes makes it difficult to physically access the DNA. So, genes or parts of genes which are located in a nucleosome are far less easily transcribed than genes located in linker DNA. Transcription factors and other regulatory factors will bind more readily to target sites located in linker DNA than in nucleosomes. Indeed not only do individual nuclesomes hide DNA from regulators and from transcription, but complexes of nucleosomes, packed tihghtly together to physically hide the DNA do the same job, but in spades. This enables the genome to physically hide non-functional binding sites from regulatory factors and moderate the expression of genes. The epigenetic code is hypothesised to be a defining code in every eukaryotic cell consisting of the specific epigenetic modification in each cell. It consists of histone modifications defined by the histone code and additional epigenetic modifications such as DNA methylation. The base for the epigenetic code is a system above the genetic code of a single cell. While in one individual the genetic code in each cell is the same, the epigenetic code is tissue and cell specific . The epigenetic code can be multidimensional in nature. It could include any of the three major cellular macromolecucles; namely, DNA (code independent), RNA, and/or protein.

Modern view of gene regulation


A modern view of gene regulation must take into account that eukaryotic DNA is tightly packaged around a core of structural proteins, the histones, to generate the chromatin nucleosome array . The nucleosome is composed of a histone octamer containing two copies of histones H2A, H2B, H3, and H4 and around which 147 bp of DNA is wrapped. The discovery that nucleosomes inhibit transcription in vitro that deletion of histones leads to a global increase in gene transcription in vivo. A negative correlation with gene expression was also reported for cytosine methylation (mC), which can repress transcription by altering chromatin structure and therefore, although basic principles of gene regulation are universal, the DNA packaging into chromatin determines a more sophisticated regulatory option.

Epigenetic phenomena
In biology, and specifically genetics, epigenetics is the study of inherited changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence, hence the name epi- (Greek: over, above) -genetics. These changes may remain through cell divisions for the remainder of the cell's life and may also last for multiple generations. However, there is no change in the underlying DNA sequence of the organism; instead, non-genetic factors cause the organism's genes to behave (or "express themselves") differently.

Historical basis of epigenetics


Epigenetics was coined by C. H. Waddington in 1942 .When Waddington coined the term the physical nature of genes and their role in heredity was not known; he used it as a conceptual model of how genes might interact with their surroundings to produce a phenotype. Robin Holliday defined epigenetics as "the study of the mechanisms of temporal and spatial control of gene activity during the development of complex organisms.Thus epigenetic can be used to describe anything other than DNA sequence that influences the development of an organism.

Epigenetic mechanisms

Molecular basis of epigenetics


The similarity of the word to "genetics" has generated many parallel usages. The "epigenome" is a parallel to the word "genome", and refers to the overall epigenetic state of a cell. The phrase "genetic code" has also been adaptedthe "epigenetic code" has been used to describe the set of epigenetic features that create different phenotypes in different cells. Taken to its extreme, the "epigenetic code" could represent the total state of the cell, with the position of each molecule accounted for in an epigenomic map, a diagrammatic representation of the gene expression, DNA methylation and histone modification status of a particular genomic region. More typically, the term is used in reference to systematic efforts to measure specific, relevant forms of epigenetic information such as the histone code or DNA methylation pattern. Cont.

Cont.
The molecular basis of epigenetics is complex. It involves modifications of the activation of certain genes, but not the basic structure of DNA. Additionally, the chromatin proteins associated with DNA may be activated or silenced. This accounts for why the differentiated cells in a multi-cellular organism express only the genes that are necessary for their own activity. Epigenetic changes are preserved when cells divide. Most epigenetic changes only occur within the course of one individual organism's lifetime, but, if a mutation in the DNA has been caused in sperm or egg cell that results in fertilization, then some epigenetic changes are inherited from one generation to the next

Applications of epigenetics
Epigenetic research uses a wide range of molecular biologic techniques to enhance our understanding of epigenetic phenomena, including chromatin immunoprecipitation (together with its large-scale variants ChIP-on-chip and ChIP-seq), fluorescent in situ hybridization, methylationsensitive restriction enzymes, DNA adenine methyltransferase identification (DamID) and bisulfite sequencing. Furthermore, the use of bioinformatic methods is playing an increasing role (computational epigenetics).

-Examples
Histone PTMs can be investigated performing ChIP with PTM specific antibodies. Genome-wide analyses were performed in Arabidopsis, rice, and maize by coupling ChIP to microarray hybridization (ChIP-chip) or to high-throughput sequencing (ChIP-seq). These studies showed that histone H3 acetylated at lysine 9 (H3K9ac) and histone H3 acetylated at lysine 27 (H3K27ac) are almost exclusively located within genes and invariably correlate with transcriptional activation, with both marks being biased towards 5-end of genes and peaking around TSS . A similar distribution was found for histone H3 acetylated at lysine 56 (H3K56), but unlike H3K9ac and H3K27ac, this modification is not correlated with active transcription and seems to be a mark of transcriptional competence.

Epigenetics in microorganism- Bacteria


Bacteria make widespread use of postreplicative DNA methylation for the epigenetic control of DNA-protein interactions. Bacteria make use of DNA adenine methylation (rather than DNA cytosine methylation) as an epigenetic signal. DNA adenine methylation is important in bacteria virulence in organisms such as Escherichia coli, Salmonella, Vibrio, Yersinia, Haemophilus, and Brucella. In Alphaproteobacteria, methylation of adenine regulates the cell cycle and couples gene transcription to DNA replication. In Gammaproteobacteria, adenine methylation provides signals for DNA replication, chromosome segregation, mismatch repair, packaging of bacteriophage, transposase activity and regulation of gene expression.

Prions

Prions are infectious forms of proteins. Proteins generally fold into discrete units which perform distinct cellular functions, but some proteins are also capable of forming an infectious conformational state known as a prion. Although often viewed in the context of infectious disease, prions are more loosely defined by their ability to catalytically convert other native state versions of the same protein to an infectious conformational state. It is in this latter sense that they can be viewed as epigenetic agents capable of inducing a phenotypic change without a modification of the genome. PSI+ and URE3, discovered in yeast in 1965 and 1971, are the two best studied of this type of prion. Prions can have a phenotypic effect through the sequestration of protein in aggregates, thereby reducing that protein's activity. In PSI+ cells, the loss of the Sup35 protein (which is involved in termination of translation) causes ribosomes to have a higher rate of read-through of stop codons.

- Fungus
The filamentous fungus Neurospora crassa is a prominent model system for understanding the control and function of cytosine methylation. In this organisms, DNA methylation is associated with relics of a genome defense system called RIP (repeatinduced point mutation) and silences gene expression by inhibiting transcription elongation.

- Yeast
The yeast prion PSI is generated by a conformational change of a translation termination factor, which is then inherited by daughter cells. This can provide a survival advantage under adverse conditions. This is an example of epigenetic regulation enabling unicellular organisms to respond rapidly to environmental stress. Prions can be viewed as epigenetic agents capable of inducing a phenotypic change without modification of the genome.

Review-Epigenetic control of gene regulation in plants


Biochemica et Biophysica Acta (BBA)-2011 by Massimiliano Lauria & vincenzo Rossi (Italy)

Epigenetic and their relationship with gene transcription


Mechanisms that regulate chromatin structure include: mC, factors affecting nucleosome positioning and composition, and post-translational modifications (PTMs) of histones; all of which are often interlaced with the small RNA (sRNA) pathway that acts in establishing and in maintaining specific chromatin states. Recent advances, which make it possible to couple methods for epigenetic marks analysis with microarrays, or next generation sequencing-based techniques, have provided us with a genome-wide picture of the epigenetic landscapes. Importantly, in most of these studies, changes in the mRNA and/or sRNA transcriptome were concomitantly investigated in order to associate transcriptome and epigenome alterations.

Cytosine methylation
In plants, mC occurs at cytosine bases in all sequence contexts: the symmetric CG and CHG contexts (where H is A, T, or C) and the asymmetric CHH context, with specific enzymes that establish and successively maintain mC patterns during DNA replication; removal of mC also take places by means of DNA glycosylase activity . Because CG methylation is coupled to DNA replication, it is a classical example of a stably inherited epigenetic mark and it was correlated with repression of gene transcription due to its ability to recruit, through interaction with methyl domain binding (MBD) proteins, chromatin remodeling complexes that establish a more condensed chromatin structure mC act as a signal for nucleosome targeting of histone modifiers.

Nucleosome positioning and composition


The work of Chodavarapu et al., not only provides important information regarding the possible mechanisms of interaction between mC and gene transcription, but also represents the first example of genome-wide mapping of nucleosome positioning performed in plants. Nucleosome positioning is controlled by both ATP-dependent chromatin remodeling complexes and the underlying DNA sequence, which either favors or disfavors nucleosome interactions.

Remodelers act by sliding, ejecting, or restructuring the nucleosome, thus directly modifying chromatin accessibility for RNAPII and TFs. In addition, some specialized complexes are involved in changing the nucleosome composition by replacing canonical histones with variants, and these changes can differentially modify the level of chromatin condensation . At the genome-wide level the nucleosome positioning can be analyzed using micrococcal nuclease I to obtain mononucleosomes or by chromatin immunoprecipitation (ChIP) with antibodies that recognize a conserved histone domain independently of the presence of PTMs (e.g. the Cterminal region of histones H3 or H4). ChIP with specific antibodies can also be used to analyze histone variant patterns. Similarly to methylome analysis, DNA from mononucleosome or ChIP samples can be employed for microarray hybridization or deep sequencing

Cont

Post-translational modification of histones


A great variety of histone PTMs, mainly but not exclusively occurring in the amino-terminal tail, have been described These include: acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and ADP ribosylation. Histone PTMs can affect chromatin structure in different ways. With the exception of methylation, they can cause a change in the net charge of nucleosomes, thus altering electrostatic or intranucleosomal contacts . However, an increasing body of evidence indicates that individual histone PTMs, or a specific combination of them, act as signals that are read by other proteins able to influence chromatin structure (the so called histone language) and,.since histone PTMs modulate chromatin structure, they are correlated with gene regulation .Histone PTMs can directly determine the formation of a transcriptionally active state (e.g. by favoring the recruitment of TFs to facilitate chromatin accessibility to RNAPII) or their deposition can be guided by transcription itself (e.g. by histone exchange occurring during elongation). Cont

The mechanism of propagation of a histone PTMs pattern during DNA replication remains unclear and it is believed that not all PTM types, or not in all circumstances, they are transmitted to daughter cells after that the signal provoking their depositions is removed .Nevertheless, examples of stably inherited histone PTMs have been provided and in plants they are well represented by the vernalization-induced histone signatures in the Arabidopsis floral repressor FLOWERING LOCUS C (FLC), which are associated with transcriptionally silenced chromatin, maintained through mitotic division even after exposure to cold has ended

Chromatin modifications in the transcription factor-mediated gene regulation


Gene regulation is often driven by DNA-binding TFs, which are transiently expressed in response to environmental or developmental cues. During transcription activation, these TFs act by recruiting general TFs (GTFs) and the RNAPII machinery to their targets. To do this they need to alter the physical barrier formed by chromatin by recruiting chromatin modifiers. During transcription repression, TFs again affect the chromatin environment, but in this case by modifying it in order to impede the RNAPII accessibility.

Transcription initiation and transition into elongation


Once an activator binds its promoter target site, it modifies the chromatin environment to allow pre-initiation complex (PIC) assembly and then triggers recruitment of additional epi-regulatory factors. The cascade of events following PIC formation appears to be conserved in eukaryotes.

A different array of histone PTMs characterizes the potentiated and activated state in O2 targets compared to the phas gene. Moreover, differences in chromatin state and co-activator requirement for distinct sub-sets of O2 targets during the activation phase suggest that different mechanisms drive transcription activation of targets regulated by the same TF . Furthermore, the same gene can be regulated by distinct epiregulators in different tissues or in response to different developmental cues. One example is the repression of the Arabidopsis floral organ identity gene AGAMOUS (AG) . In vegetative tissues AG is repressed through a PRC2 complex leading to H3K27me3 accumulation. Conversely, in early floral meristems, and in petals and sepals, AG is repressed by the combined action of the MADS-box APETALA 1 and SEPALLATA 3, which form a complex with LEUNIG (LUG) corepressor, resulting in a transcriptionally incompetent hypoacetylated chromatin state.

General model and specificities in transcription factor-mediated control of gene regulation through chromatin modifications

Epigenetic mechanisms in the transposon (or repeat)-mediated gene regulation


Genome sequencing has confirmed that large parts of eukaryotic genomes are constituted of transposable elements (TEs) and other repeat sequences. TEs and repeats represent an important threat to the stability of genomes. TEs can insert into the host genes or induce major chromosomal rearrangements such as translocations and inversions, whereas repeats can increase unfavourable recombination events . For these reasons epigenetic mechanisms, mainly those mediated by sRNA pathway, evolved to maintain these DNA elements in an inactive state by triggering cytosine hypermethylation and deposition of repressive histone PTMs, through a process called RNA-directed DNA methylation (RdDM). Although TEs and repeats tend to accumulate in heterochromatin (centromeric, telomeric, and pericentromeric regions), where they play a key role in shaping the epigenetic architecture of these regions , they are also scattered across euchromatin, among and within genes. This is particularly evident for large plant genomes, such as maize, where TE-related heterochromatin clusters separate genes or are tightly associated to them.

-Plants
Plants are sessile organisms continually exposed to environmental cues and, compared to animals, they have a developmental program that is not restricted to embryo formation, but, rather, extends to all stages of the life cycle. This implies that plants need strategies for relatively flexible and dynamic control of gene regulation. Since chromatin modifications are essential to modulate gene expression, it is very likely that plant-specific epigenetic regulatory patterns (e.g. different epi-regulatory proteins and different distribution of epigenetic marks) may have been shaped by, or selected for, during evolution. In this context, the large number of TEs found in plants, especially those with large genomes, suggests that TE-mediated epigenetic regulation of neighboring genes might be of particular relevance for plants compared to other eukaryotes.

Plant Peculiarities in the epigenetic control of gene regulation


Although mechanisms controlling gene regulation through modification of chromatin states are largely conserved among eukaryotes, some peculiarities that distinguish plants from other kingdoms have been observed. For example, plants have a specific DMT, the CHROMOMETHYLASE 3, which is required for maintenance of CHG methylation . DNA methylation occurring at non-CG sites (i.e. CHG and CHH) has been long considered as plant-specific. However, the recent analysis of the human methylome revealed that even mammals make some use of this . In mammals, non CG-methylation seems to be restricted to specific cell types. Cont

Cont
Plants also show peculiarities regarding the set of enzymes modulating histone PTMs, as well as distribution and correlation with transcription of these epigenetic marks. For example, plants have unique HDACs (the HD2-type) and specific mechanisms for regulation of HDAC activity . In addition, divergent distribution of H3K9me2 and H3K9me3 has been observed between plants and animals. In plants, H3K9me2 is a classical heterochromatin mark, while H3K9me3 is mainly localized within genes. Cont

Cont..
In plants, genome-wide analysis in different species showed that H3K27me3 forms small domains, less than 1 kb in length, which are localized to the coding region of single genes . Conversely, in flies and mammals, H3K27me3 chromosomal domains are large, up to 100 kb, and include multiple genes. Furthermore, in plants, H3K27me3 co-localizes with LHP1, while in mammals it is bound by the Polycomb repressive complex 1 (PRC1)

Future perspectives
The availability of full sequenced genomes and powerful genomic technologies has tremendously increased our knowledge about the rules that govern epigenetic gene regulation Another important challenge for plant epigenetics is to elucidate the role of chromatin in processing of pre-mRNAs. Observations from different eukaryotic systems indicate that pre-mRNA processing (i.e. 5-end capping, splicing, and 3-end processing) occurs co-transcriptionally and is regulated by transcription-related processes, including nucleosome positioning and histone PTMs . Moreover, recent findings from study of the FLC model show that the co-transcriptional processing of long non-coding antisense RNAs triggers chromatin modifications to repress transcription of sense RNA . Importantly, it was reported that synthesis of plant long non-coding RNAs is influenced by various abiotic factors . Therefore, to understand at a mechanistic level how the co-transcriptional processing of sense and antisense pre-RNAs interacts with chromatin modifications is expected to provide information about mechanisms linking plant phenotypic plasticity to environmental cues.