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Epigenetics
1. REGULATORY AND EPIGENETIC
LANDSCAPES OF MAMMALIAN
GENOME
PRESENTED TO:
Dr. MANISHA SACHAN
PRESENTED BY:
SHRADHA SUYAL (2015BT01)
MEGHNA SUHAG (2015BT04)
2. INTRODUCTION
•“Epigenetics” refers to modification of DNA, protein, or RNA, resulting in
changes to the function and/or regulation of these molecules, without altering
their primary sequences.
•The Greek prefix “epi” means “on top of” or “over”, so the term
“Epigenetics” literally describes regulation at a level above, or in addition to,
those of genetic mechanisms.
•Changes in gene transcription through modulation of chromatin which is not
brought about by changes in the DNA sequences.
•Three independent criteria should determine whether a certain molecular
signal is indeed epigenetic:
1. mechanism for propagation, i.e. pathways that explain how the molecular
signature is faithfully reproduced after DNA replication/cell division;
2. evidence of transmission, i.e epigenetic modifications are stable and
passed on to future generations, but they may change in response to
environmental stimuli
3. effect on gene expression, i.e. an epigenetic signal should be sufficient to
cause a transcriptional outcome.
3.
4. EPIGENETIC MECHANISMS
1. DNA methylation
2. Covalent Histone modifications
3. Chromatin remodelling or non covalent modifications
4. Non coding RNA
5. DNA METHYLATION
•plays an important role in regulating gene expression .
•Methylation of DNA is direct chemical modification of a cytosine nucleotides within
a continuous stretch of DNA, specifically at the 5-position of the pyrimidine ring.
• Not all cytosines can be methylated; cytosines must be immediately followed by a
guanine in order to be methylated i.e CpG dinucleotide.
CpG dinucleotides
Intragenically
Rest is
unmethylated “CpG
Islands”
70% methylated
Upstream of
Transcription start site
(promoter)
97% intra and
intergenic i,.e
centromeric and
retrotransposons
<3% in 5’ UTR
6. • DNA methylation patterns are generated and maintained by the cooperative
activity of DNMTs that transfer methyl groups.
DNMTs
(DNA Methyl
transferases)
De novo DNMT’s
•DNMT 3a & 3b isoforms.
•Methylate previously
unmethylated CpG sites in
DNA.
•Place new methylation marks
in DNA.
•Lay down initial patterns of
methylation when cell fate is
determined.
Maintenance DNMT
•DNMT 1
•Methylates hemi methylated
DNA.
•Causes methylation marks
after cell division.
•Propagates epigenetic
marks through cell
generations.
7. Functional consequences of DNA methylation
• Methylation of DNA is associated with suppression of gene transcription
and. Methylation is a process whereby a gene can be shut off functionally.
•Some CpG island promoters become methylated during development, which
results in long-term transcriptional silencing for example: X chromosome
inactivation and imprinted genes.
•Effect of cytosine methylation is highly dependent on the location of the
methylated CpGs.
1. Only when the methylated sites are located in promoter regions (i.e.
non-coding regions upstream from transcriptional start sites) then that
gene would be suppressed.
2. Methylation of CpGs located within gene bodies is associated with the
opposite effect – an increase in transcriptional activity. The precise
molecular processes through which this occurs are complex and an area
of intense investigation at present.
•DNA methylation can lead to gene silencing by either preventing or
promoting the recruitment of regulatory proteins to DNA.
8.
9. COVALENT HISTONE MODIFICATIONS
•Histones are highly basic proteins whose function is to organize DNA within
the nucleus.
•Unlike DNA methylation, histone modifications can lead to either activation
or repression depending upon which residues are modified and the type of
modifications present.
For example, lysine acetylation correlates with transcriptional activation
whereas lysine methylation leads to transcriptional activation or repression
depending upon which residue is modified and the degree of methylation.
•These modifications serve as epigenetic tags or marks.
•Histone proteins have tails that can have a number of post-translational
modifications including acetylation, methylation, phosphorylation,
ubiquitylation, sumoylation, ADP-ribosylation, glycosylation etc.
10.
11. Types of Histone Modifications
1.Acetylation:
•occurs at lysine residues, specifically on their side-chain amino group.
•Histone acetyltransferases (HATs) catalyze the direct transfer of an acetyl
group from acetyl-CoA to the ε-NH+ group of the lysine residues within a
histone.
•is a reversible process, and the enzymes that catalyze the reversal of histone
acetylation are known as HDACs.
•Creates a docking site for the binding of other proteins.
•Proteins that bind to acetylated lysines in histiones contain a domain called
bromodomain.
2.Ubiquitination
•Ubiquitin is usually, but not always, attached to proteins as a signal for
degradation by the proteasome.
•Like other proteins, histones are ubiquitinated through attachment of a
ubiquitin to the ε-NH+ group of a lysine.
•Ubiquitination of histones H2A, H2B, H3 and H1 has been observed.
•Most histones appear to be mono-ubiquitinated, although there is evidence
for poly-ubiquitination.
12. 3.Methylation
•Lys and Arg at the N-ter of histone undergo methylation.
•It may be mono-, di- or trimethyl for Lys and mono- or dimethyl for Arg.
•Catalyzed by methyl transferases.
•Unlike acetylation, methylation of lysines preserves their positive charge.
•Methylation of different parts of the N-ter tails of H3 & H4 histiones is
associated with both repressed & active chromatin, depending on the
particular amino acid that is modified.
Ex: methylation of H3K9 is associated with gene silencing whereas
methylation of H3K4 is associated with transcription activation.
•Proteins that bind to methylated histones contain a domain called
chromodomain.
4.Phosphorylation
•Limited information.
•Phosphorylation of histones H1 and H3 was first observed in the context
of chromosome condensation during mitosis. H3 was the first histone
whose phosphorylation was characterized in response to activation of
mitogenic signaling pathways.
•Phosphatases remove phosphate groups from histones.
13. CHROMATIN REMODELING
•For the creation of nucleosome free regions
of DNA for gene activation, nucleosome
remodeling complex is recruited.
•The complex slides the histone along the
DNA, thus expose sites for DNA binding
proteins, or evict the histone or may also bring
in variant of histone protein.
•Thus, the nucleosome is dynamic, i.e their
positions can shift.
•Nucleosomes may be irregularly packed or
fold into higher-order structures resulting in
euchromatin & heterochromatin regions.
14. 1.Sliding over 2. Histone eviction
3. Replacement with
variant histones
TYPES OF CHROMATIN
REMODELLING
15. HISTONE PROTEINS
HISTONE VARIANTS
•Are replacement histone proteins.
•Synthesis is not confined to S phase
only.
•Replication independent.
•mRNA with polyA tail.
•Ex: H3.3, H2A.Z
CONVENTIONAL HISTONES
•Are simple histone proteins.
•Synthesized during S phase.
•Replication dependent.
•mRNA formed without polyA tail.
•Ex: H1, H2A, H2B, H3, H4.
16. NON CODING RNA (ncRNA)
Regulatory ncRNAs includes short interfering RNAs (siRNAs), microRNAs
(miRNAs), and long non coding RNAs (lncRNAs) .
ncRNA play important roles in gene expression regulation at post-transcriptional
level by mRNA degradation, during splicing.
miRNAs
•A micro RNA is a small non-coding RNA molecule.
•about 22 nucleotides long.
•functions in RNA silencing and post-transcriptional regulation of gene expression.
•Endogenous in origin.
siRNA
•Short interfering RNA or silencing RNA.
• double-stranded RNA molecules, 20-25 base pairs long.
•Role in the RNA interference (RNAi) pathway.
•Can be endogenous or exogenous in origin.
lncRNAs
•Long non-coding RNAs (long ncRNAs, lncRNA)
•longer than 200 nucleotides.
•LincRNAs, a subset of lncRNA, exhibit high conservation across different species.
•LincRNA play an important role in developmental processes such as X-chromosome
inactivation and genomic imprinting.
18. 1. RNA Interference
RNA interference (RNAi) is a mechanism whereby the expression of genes is disrupted
through the action of double-stranded RNA molecules.
19. •Females silence one of their two X-chromosomes through a process referred
to as X-chromosome inactivation, to compensate gene dosage disparities.
•Embryos containing more than one X-chromosome (XX, XXX females and
XXY males) undergo random X-chromosome inactivation at the blastocyst
stage in early embryogenesis.
•It is initiated from XIC (X inactivation Centre) containing several genes of
which two, i.e XIST & TSIX, play an important role.
•Both these genes code for non coding RNA’s which are antagonistic in
nature.
2. X Inactivation (LYONIZATION)
20. 3. Genomic Imprinting
•Phenomenon by which certain genes are expressed in a parentof-origin-
specific manner. If the allele inherited from the father is imprinted, it is
thereby silenced, only the allele from the mother is expressed and vice
versa.
•is an inheritance process independent of the classical Mendelian
inheritance.
•It is rare in mammals, but recent studies have shown that <1% of genes
are imprinted.
•The expressed allele is dependent upon its parental origin. For example,
the gene encoding insulinlike growth factor 2 (IGF2) is only expressed
from the allele inherited from the father.
•Human diseases involving genomic imprinting include Angelman
syndrome and Prader–Willi syndrome.
22. EPIGENETICS IN DEVELOPMENT & DISEASE
•Although the initial establishment of the epigenome takes place during embryonic
development, the maintenance of the epigenetic state is important throughout life for
the production of differentiated cells from adult stem cells and proper gene expression
in specific cell types.
•The epigenomic state is dynamic and tightly regulated, and misregulation of
epigenetic patterns are observed in many human diseases and multiple types of
cancers.
•Changes in the epigenome are also correlated with the aging process. For example,
gene promoters become hypermethylated as an individual ages, whereas the CpG
sequences of non-coding centromeric repeat regions become hypomethylated.
•In a variety of cancers, tumor suppressor genes exhibit hypermethylation in the CpG
islands upstream of the coding regions, repressing their expression. Conversely, some
other genes are usually methylated and repressed, but are hypomethylated and
expressed in some cancers.
•Some diseases have epigenetic causes like Prader-Willi syndrome, Angelman
syndrome are all the result of uniparental disomy (UPD), a condition in which a person
inherits both homologous chromosomes from the same parent. UPD can be the result
of gene deletion, translocation, or a defect in imprinting.
23. EPIGENETICS RESEARCH TECHNIQUES
•The epigenetic modifications of DNA, histones, and RNA can often be distinguished
from their unmodified counterparts using both standard molecular biology techniques
and Next-Gen whole-genome approaches.
1. sodium bisulfite deaminates cytosine into uracil, but 5-methylcytosine is resistant
to this conversion, meaning a base change following bisulfite treatment can be
used to experimentally determine DNA methylation status.
2. There are several methylation-sensitive restriction endonucleases whose ability
to cut DNA at specific sequences is dependent upon the DNA methylation state of
the sequence.
3. Methylated DNA immunoprecipitation (MeDIP) assays utilize an antibody that
specifically recognizes methylated DNA, and the immunoprecipitated methylated
DNA can be identified by different methods.
4. Histone modifications can also be studied using antibodies specific for particular
histone modifications using chromatin immunoprecipitation (ChIP) assays.
These features, when coupled with other common molecular biological methods, such
as DNA sequencing, PCR, real-time PCR, Southern blotting, primer extension, HPLC,
and MALDI-TOF MS, whole genome sequencing provides variety of tools to
investigate epigenetic processes.
24. CONCLUSION
•Of great importance for the future are the integration of sequencing
technologies and the means to maintain and manipulate the large amount of
data produced by sequencing epigenomes.
•As more epigenetic marks are associated with specific diseases, tools can be
developed to diagnose patients and measure the severity of disease.
•In therapeutic epigenetics several drugs, such as DNA methyltransferase
inhibitors and histone deacetylase inhibitors, are already used in cancer
treatment. There are issues with specificity and efficacy of these drugs, so
further research into their mechanisms is needed to develop better therapeutic
agents.
•Likewise, better understanding of the various epigenetic diseases and
syndromes may lead to effective drugs designed to overcome epigenetic
defects.
•New data and knowledge relating to epigenetics obtained in recent years
paves a way for future epigenetics research.
25. REFERENCES
“Basic concepts of epigenetics”, Michal Inbar-Feigenberg, Sanaa Choufani,
Darci T. Butcher, Maian Roifman and Rosanna Weksberg.
“Overview and Concepts”, C. David Allis,Thomas Jenuwein, and Danny
Reinberg.
“Epigenetics in cancer”, Shikhar Sharma, Theresa K. Kelly and Peter A. Jones,
“An Overview of the Molecular Basis of Epigenetics”, J. David Sweatt, Eric J.
Nestler, Michael J. Meaney and Schahram Akbarian
https://en.wikipedia.org/wiki/Genomic_imprinting
https://en.wikipedia.org/wiki/Xinactivation
http://www.zymoresearch.com/learning-center/epigenetics/epigenetics-research-
techniques
http://www.zymoresearch.com/learning-center/epigenetics/epigenetics-research-
techniques
https://en.wikipedia.org/wiki/Epigenetics
Hinweis der Redaktion
Term coined by Waddington in 1942.
While most of the CpG sites in the genome are methylated, the majority of CpG islands usually remain unmethylated during development and in differentiated tissues (11).
The rest of the unmethylated CpG dinucleotides occur in small clusters, known as “CpG islands” that can occur both near gene transcription start sites and intragenically. Thus, CpGs in regions of the genome that actively regulate gene transcription, such as promoters, are largely unmethylated.
CpG dinucleotides are not evenly distributed across the human genome but are instead concentrated in short CpG-rich DNA stretches called ‘CpG islands’ and regions of large repetitive sequences (e.g. centromeric repeats, retrotransposon elements, rDNA etc.)
the repetitive genomic sequences scattered all over the human genome are heavily methylated, which prevents chromosomal instability by silencing non-coding DNA and transposable DNA elements.
CpG islands are preferentially located at the 5′ end of genes and occupy∼60% of human gene promoters. some CpG island promoters become methylated during development, which results in long-term transcriptional silencing for example: Xchromosome inactivation and imprinted genes.
DNA methylation can lead to genesilencing by either preventing or promoting the recruitment of regulatoryproteins to DNA.
through interactions with histone deacetylases (HDACs)
methylation is a process whereby a gene can be shut off functionally.
The repressive effect of DNA methylation in gene promoters is better understood
One simplified model for how methyl-DNA binding proteins might suppress transcription.
methylation of cytosines at CpG dinucleotides recruits methyl-DNA binding proteins at specific sites in the genome.Proteins that bind to methylated DNA have both a methyl-DNA binding domain (MBD) and a transcription-regulatory domain (TRD). The TRD recruits adapter/scaffolding proteins which,in turn, recruit histone deacetylases (HDACs) to the site.
suppression. Thus, through this complex and highly regulated biochemical machinery, methylation of DNA triggers localized regulation of the three-dimensionalstructure of DNA and its associated histone proteins, resulting in a higher-affinity interaction between DNA and the histone core, and transcriptional repression by allosteric means.
Histone modification can occur as a consequence of DNA methylation, or can be mediated by mechanisms that are independent of DNA methylation and controlled by intracellular signaling.
The basic unit of chromatin is the nucleosome, which is composed of an octomer of histoneproteins (containing two copies each of histones 2A, 2B, 3, and 4) around which is wrapped –like a rope on a windlass – the DNA double helix. The degree to which nucleosomes are condensed or packed is a critical determinant of the transcriptional activity of the associated DNAand this is mediated in part by chemical modifications of the N-terminal tails of histone proteinsStructural studies indicate that these N-terminal tails protrude from the nucleosomeand are extensively modified post-translationally.24
Classical isoforms of HDACs catalyze the removal of acetyl groups from lysine residuesthrough a Zn2+-dependent charge-relay system.
Histones in heterochromatin are generally deacetylated while those in euchromatin are acetylated.
There are a total of eleven HDAC isoforms broadly divided into two classes. HDACs 1, 2, 3, and 8 are ClassI HDACs, while Class II encompasses HDAC isoforms 4, 5, 6, 7, 9, 10, and 11. The newlycharacterized SIR2 family of HDACs (the “Sirtuins”), termed Class III, operate through anNAD+-dependent mechanism
Phosphorylation, through the addition of net negative charge,can generate “charge patches” (Dou and Gorovsky 2000)that are believed to alter nucleosome packaging or toexpose histone amino termini by altering the higher-orderfolded state of the chromatin polymer
The individual histone variants appear to differ in the capacity to support specific modifications, with somelikely more associated with increased epigenetic “plasticity” and others more closely allied toepigenetically stable genomic regions.
Unlike the major histone subtypes whose synthesis and incorporation is coupled to DNA replication in S phase, these variants are synthesized and incorporated into chromatin throughout the cell cycle.
The incorporation of histone variants, e.g. H3.3and H2A.Z, into nucleosomes also influences gene activity: H3.3 and H2A.Z are preferentially enriched at promoters of active genes or genes ready for activation.
They can mediate gene activation by altering the stability of nucleosomes. H2A.Z incorporation may also contribute to gene activation by protecting genes against DNA methylation.
Like canonical histones, histone variants undergo posttranslational modifications, which determine their nuclear localization and function.
SiRNAs are double-stranded RNAs (dsRNA) that mediate post-transcriptional silencing, in part by inducing heterochromatin to recruit histone deacetylase complexes (30). MiRNAs comprise a novel class of endogenous, small (18–24 nucleotides in length); single-stranded RNAs generated from precursor RNA cleaved by two RNA polymerase III enzymes DROSHA and DICER to produce mature miRNA. These miRNAs can control gene expression by targetingspecific mRNAs for degradation and/or translational repression (31, 32). They can also control gene expression by recruiting chromatin-modifying complexes to DNA through binding to DNA regulatory regions, thereby altering chromatin conformation (33, 34). Expression of miRNA in human blastocysts correlates with maintenance of pluripotency in embryo development (35). LincRNAs, a subset of lncRNA, exhibit high conservation across different species. They have been shown to guide chromatin-modifying complexes to specific genomic loci, thereby participating in the establishment of cell type–specificepigenetic states (36, 37). In embryonic development, expression of lncRNAs, regulated by the pluripotent transcription factors OCT4 and NANOG, facilitates cell lineage–specific gene expression (38). LincRNAs also play an important role in developmental processes such as X-chromosome inactivation and genomic imprinting.
Genetic disruption of RNAi pathways leads to relaxation of heterochromatin around centromeres, which causes erroneous expression of normally silent genic regions and a decrease in the repressive methylation of histone H3.
X-chromosome inactivation is regulatedby a master switch locus, the X inactivation center (XIC),which regulates in cis the expression of the lincRNA geneXIST (X-inactive specific transcript) and its antisensetranscription unit TSIX.
Prior to inactivation, both X chromosomes weakly express Xist RNA from the Xist gene. During theinactivation process, the future Xa ceases to express Xist, whereas the future Xi dramatically increasesXist RNA production. On the future Xi, the Xist RNA progressively coats the chromosome, spreadingout from the XIC;[19] the Xist RNA does not localize to the Xa. The silencing of genes along the Xioccurs soon after coating by Xist RNA.X chromosomeslacking Tsix expression (and thus having high levels of Xist transcription) are inactivated much morefrequently than normal chromosomes.
In germline cells the imprint is erased and then reestablished according to the sex of the individual, i.e. in the developing sperm (during spermatogenesis), a paternal imprint is established, whereas in developing oocytes (oogenesis), a maternal imprint is established
Epigenetics is a prominent theme in the study of human development from fertilization through aging and to death3. Some epigenetic programming events that occur during embryonic development are erasure and re-establishment of DNA methylation marks, genetic imprinting, X-chromosome inactivation, the development of pluripotent stem cells, and the differentiation of somatic cells
ChIP assays are also very useful to determine whether other non-histone proteins, such as chromatin-remodeling and histone-modifying factors are associated with chromatin. The chromatin immunoprecipitated in ChIP assays can be identified by performing PCR for specific genes of interest, or genome-wide by hybridization to a microarray (ChIP-chip), or by direct Next-Gen sequencing of the immunoprecipitated chromatin (ChIP-Seq).
Recent advances in embryology have posed more questions related to epigenetics, particularly to the mechanics of genome demethylation and the re-establishment of methylation in early embryonic development3. The epigenetic marks associated with the production of pluripotent embryonic stem cells is also of high interest for its relevance in reprogramming differentiated cells to make induced pluripotent stem cells2. Beyond embryonic development, phenomena relating to the acquisition of epigenetic marks during an organism’s life span and their passage to offspring is a tantalizing area of research with many questions to be answered regarding mechanisms, and environmental influences.