N-terminal tails of histones are the most accessible regions for modifications. These post-translational modification (PTM) of histones is a crucial step in epigenetic regulation of a gene.
Dna methylation ppt
definition of Dna methylation ppt
discovery of Dna methylation ppt
types of Dna methylation ppt
history of Dna methylation ppt
process of Dna methylation ppt
mechanism of Dna methylation ppt
methylation in cancer
cytosine methylation
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It is the DNA located in the mitochondria.Mitochondrial DNA (mtDNA or mDNA) is the DNA located in the mitochondria.
They are double stranded circular DNA molecule.
It is only 16 kb in length – contains 16,600 bp.
It is haploid in nature.
It codes for 37 genes.
13 genes provide instructions for making enzymes involved in oxidative phosphorylation.
It is a process that uses oxygen and simple sugars to create ATP, the cells main energy source.
Cot curve dispersed repeated DNA or interspersed repeated DNA tandem repeated DNA Long interspersed repeat sequences (LINEs) Short interspersed nuclear elements (SINEs) satellite, minisatellite and microsatellite DNA Variable Number Tandem Repeat (or VNTR)
A detail ppt about Genome organization with focus on all levels of organization. Most recent research and findings about CT is also added in this ppt. Detail account of 30nm fiber and its ultra structure and types is also included.
Dna methylation ppt
definition of Dna methylation ppt
discovery of Dna methylation ppt
types of Dna methylation ppt
history of Dna methylation ppt
process of Dna methylation ppt
mechanism of Dna methylation ppt
methylation in cancer
cytosine methylation
genomic imprinting
It is the DNA located in the mitochondria.Mitochondrial DNA (mtDNA or mDNA) is the DNA located in the mitochondria.
They are double stranded circular DNA molecule.
It is only 16 kb in length – contains 16,600 bp.
It is haploid in nature.
It codes for 37 genes.
13 genes provide instructions for making enzymes involved in oxidative phosphorylation.
It is a process that uses oxygen and simple sugars to create ATP, the cells main energy source.
Cot curve dispersed repeated DNA or interspersed repeated DNA tandem repeated DNA Long interspersed repeat sequences (LINEs) Short interspersed nuclear elements (SINEs) satellite, minisatellite and microsatellite DNA Variable Number Tandem Repeat (or VNTR)
A detail ppt about Genome organization with focus on all levels of organization. Most recent research and findings about CT is also added in this ppt. Detail account of 30nm fiber and its ultra structure and types is also included.
presented by HAFIZ M WASEEM
university of education LAHORE Pakistan
i am from mailsi vehari and studied in lahore
bsc in science college multan
msc from lahore
Histone demethylase and it srole in cell biology reviewChristopher Wynder
This document provides a scientific review of the histone demethylase enzymes; particularly the H3K4 demethlases (KDM5 family) focusing on their role in cell biology. This review was written in 2014
Salt stressRole of Organelle Membranes in Salt Stress Sensing and Signalling ...Suresh Antre
Soil salinity is a major environmental constraint to crop production, affecting an estimated 45 million hectares of irrigated land, and is expected to increase due to global climate changes and as a consequence of many irrigation practices. The deleterious effects of salt stress on agricultural yield are significant, mainly because crops exhibit slower growth rates, reduced tillering and, over months, reproductive development is affected.
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The Nature and The Functional Complexity of Retrograde Signals. Retrograde signaling refers to the fact that chloroplasts and mitochondria utilize specific signaling molecules to convey information on their developmental and physiological states to the nucleus and modulate the expression of nuclear genes accordingly.
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As a consequence of global warming, plants have to face more severe and frequently occurring periods of high temperature stress. Crop production is highly sensitive to elevated temperatures. A rise of a few degrees above the optimum growing temperature can lead to a dramatic yield loss. While this affects the whole plant, development of the male gametophyte, the pollen, seems to be the most sensitive to adverse climatic environments and abiotic stress.
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Students, digital devices and success - Andreas Schleicher - 27 May 2024..pptxEduSkills OECD
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The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
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3. Histones are a group of basic protein that associate with
DNA and help the DNA to condense it into chromatin.
Histones contain a large proportion of the positively charged
(basic) amino acids, lyseine and arginine in their structure.
DNA is negatively charged due to the phosphate groups on its
backbone.
These result of these opposite charges is strong attraction and
therefore high binding affinity between histones and DNA
structure called nucleosome.
DNA wraps around histones, they also play a role in gene
regulation.
3Department of Plant Biotechnology
Luger et al., Nature 1997
Principles of Biochemistry, Lehninger 4th
Edition.
03/11/2016
IntroductionIntroduction
Histones Histones
4. The basic unit of chromatin is the nucleosome core particle, which contains 147 bp of DNA
wrapped nearly twice around an octamer of the core histones.
Each nucleosome is separated by 10–60 bp of ‘linker’ DNA, and the resulting nucleosomal
array constitutes a chromatin fiber of ~10 nm in diameter.
4Department of Plant Biotechnology03/11/2016
Genome Organization Genome Organization
Mol. Bio of Gene, Watson et al., 7th
edition
Luger et al., Nature 1997
5. Histone proteins are of two types:
Core Histones - H2A, H2B, H3, and H4
Linker Histones - H1
5Department of Plant Biotechnology
The eight histones in the core are
arranged into a (H3)2(H4)2 tetramer and a
pair of H2A–H2B dimers.
The tetramer and dimers come together
to form a left-handed superhelical ramp
around which the DNA wraps.
Hydrogen bonds between the DNA
backbone and the amide group on the main
chain of histone proteins
03/11/2016
Types of HistonesTypes of Histones
Mol. Bio of Gene, Watson et al., 7th
edition
6. Department of Plant Biotechnology 6
Histone H1 is not part of the nucleosome core particle.
Instead, it binds to the linker DNA and is referred to as a linker
histone.
H1 is half as abundant as the other histones, which is consistent
with the finding that only one molecule of H1 can associate with a
nucleosome.
Since H2A packages DNA molecules into chromatin, the
packaging process will effect gene expression.
Refers to a variety of closely related proteins that vary often by
only a few amino acids.
H2A plays a major role in determining the overall structure of
chromatin. Inadvertently, H2A has been found to regulate gene
expression.
H2B is also involved with the structure of the nucleosomes of the
'beads on a string' structure.
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H1 H1
H2A H2A
H2B H2B
http://www.actrec.gov.in/histome/histone_main.php
7. Department of Plant Biotechnology 7
H4 is a structural component of the nucleosome, and is subject
to covalent modification ,including acetylation and methylation,
which may alter expression of genes located on DNA associated
with its parent histone octamer.
Featuring a main globular domain and a long N-terminal tail.
Also an important protein in the emerging field of epigenetics,
where its sequence variants and variable modification states are
thought to play a role in the dynamic and long term regulation of
genes.
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H4H4
H3H3
http://www.actrec.gov.in/histome/histone_main.php
10. 10Department of Plant Biotechnology
Modifications in histone proteins affects the structure of chromatin.
Gene regulation
DNA damage and repair
Chromosome condensation
Heterochromatin
-Highly packed form
- Low gene expression
Euchromatin
- Loosely packed form
- High gene
expression
03/11/2016
11. N-terminal tails of all histones are particularly of interest since they protrude out of the compact
structure. These N-terminal tails are often subjected to a variety of post-translational modifications
such as,
11Department of Plant Biotechnology
Methyl Acetyl Phospho Ubiquitin SUMO
Bhaumik, Smith, and Shilatifard, 2007.
It has been proposed that these
modifications result in a ‘code’ which
can be read by proteins involved in
gene expression and other DNA
translations
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Types of Histone ModificationTypes of Histone Modification
12. Department of Plant Biotechnology 12
Amino acid
Residue
Modification
Type
Modiying
Enzyme
Lysine
Acetylation
Deacetylation
HAT
HDAC
Lysine
Methylation
Demethylation
HMT
HDM
Lysine
Ubiquitylation
Deubiquitylation
Ub ligase
Ub protease
Serine/Threonine
Phosphorylation
Dephosphorylatio
n
Kinase
Phosphatase
Arginine
Methylation
Demethylation
PRMT
Deiminase/De
methylase
Some Examples of Histone Modification and Modifiers
Others: Sumoylation (Lysine), ADP Ribosylation
(Glutamate)
Post-translational Modification of Histone N-terminal Tails
03/11/2016
13. 13Department of Plant Biotechnology
N-term tails reversible acetylated in Lys, particularly in H3+H4
While the globular core is involved in histone-histone packing and
DNA-contact, the N-terminal tails point outwards and is available for
interaction.
Acetylation also provides binding sites for a number of proteins
with an approximately 100-amino-acid sequence motif called a
bromodomain.
+
++
+
+
Luger et al., 1997
03/11/2016
1. Acetylation1. Acetylation
P.J. Barnes et al., Eur Respir J 2005
17. 17Department of Plant Biotechnology
It is the introduction of an Methyl functional group to only on Lysine or Arginine of the
histone tail.
These reactions are catalyzed by enzymes with "histone methyltransferases (HMTs)”
Histone lysine methyl transferases (HKMTs) Methylate lysine (K) residues
Protein argenin methyl transferases (PRMTs) Methylate arginine (R) residues
A role in both activation and repression
Arginines can be mono- or dimethylated whereas lysines can be mono-, di- or trimethylated .
Arg
Lys
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2. Methylation2. Methylation
Eric J. Richards., et al., 2002
18. Department of Plant Biotechnology 18
Methylation can result in activation or repression of expression .
Activation (H3K4, H3K36, H3K79)
Trimethylation of histone H3 at lysine 4 (H3K4) is an universal active mark for transcription.
Repression (H3K9, H3K27, H4K20)
Dimethylation of histone H3 at lysine 9 (H3K9) and at 27 (H3K27) are the universal signal for
transcriptional silencing.
Modification Histone Site Possible Function
Methylation H3 K4
Permissive euchromatin (di-Me), Active
euchromatin (tri-Me)
Transcriptional activation
K9
Transcriptional silencing (tri-Me), DNA
methylation (tri-Me), Heterochromatin formation
R17 Transcriptional activation
K27 Transcriptional silencing, X inactivation (tri-Me)
K36 Transcriptional elongation
K79 Euchromatin, Transcriptional elongation
H4 R3 Transcriptional activation
K20
Transcriptional silencing (mono-Me),
Heterochromatin (tri-Me).
K59 Transcriptional silencing
Sites of histone methylation with their function
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http://www.actrec.gov.in/histome/histones.php?histone=H3
19. Department of Plant Biotechnology 19
Model for Role of Methylation in Heterochromatin Formation
Condensation assisted by recruitment of
HMT (histone methyltransferase), where HP1
(heterochromatin protein 1) binds to H3K9-Me3
which methylates adjacent H3K9.
Chromatin condensed until a boundary
element is reached and turned into
heterochromatin
Methylation of histone tails long lasting
compared to acetylation.
Can be Inherited by daughter cells:
Responsible for X-inactivation in female.
Phillips, T. (2008) Nature Education
03/11/2016
20. 20Department of Plant Biotechnology
Phosphorylation is the addition of a phosphate group (PO4
3−
) to a molecule.
Phosphorylation is catalyzed by various specific protein kinases, whereas phosphatases
mediate removal of the phosphate group.
Histones can also get phosphorylated and the most studied sites of histone phosphorylation are
the serine 10 of histone H3 (H3S10) that is deposited by the Aurora-B kinase during mitosis.
Phosphorylation of histones, in particular phosphorylation
of H2AX, has a role in DNA damage response and DNA
repair.
Rapid phosphorylation of H2AX, at serine 129 (H2AX) by
the PI3K kinases at double strand break (DSB) sites, is one
of the first and most easily detectable DNA damage signaling
post-translational events.
Role of histone phosphorylation in DNA repair
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3. Phosphorylation3. Phosphorylation
Dorine Rossetto, et. al., Epigenetics 7:10 2012
21. Department of Plant Biotechnology 21
Modification Histone Site Possible Function
.Phosphorylation H2A S1
Mitosis, Transcriptional
repression, Chromatin
assembly
T119 Mitosis
S129 (S.
cerevisiae) DNA repair
S139
(mammalian
H2AX) DNA repair
H2B
S14
(vertebrates) Apoptosis
S33 (D.
melanogaster) Transcriptional activation
H3 T3 Mitosis
S10
Mitosis, meiosis,
Transcriptional activation
T11
(mammals) Mitosis
S28
(mammals) Mitosis
H4 S1 Mitosis
Role of histone phosphorylation in transcription regulation
The phosphorylation of H3S10 (H3S10P) was initially linked to chromosome relaxation and
segregation during mitosis and meiosis.
The role of H3S10P in chromatin condensation suggests that it should be involved in
transcriptional activation.
Transient derepression by histone H3 phosphorylation.
Mitogen-activated protein kinases (MAPK)
Signal
molecule
absent
Signal
molecule
present
Sites of histone phosphorylation with their function
03/11/2016 Anna Sawicka et al., Biochimie 94 (2012)
22. Ubiquitination (or ubiquitylation) refers to the post-translational modification of the amino group of a
lysine residue by the covalent attachment of one (monoubiquitination) or more (polyubiquitination)
ubiquitin monomers.
Ubiquitin is a 76 amino acid protein highly conserved in eukaryotes.
Histone ubiquitination alters chromatin structure and allows the access of enzymes involved in
transcription
Ubiquitination is carried out in three main steps: activation, conjugation, and ligation, performed by
ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases
(E3s), respectively.
22Department of Plant Biotechnology
Jian Cao et.al.,Frontiers in oncology
03/11/2016
4. Ubiquitination4. Ubiquitination
Modification Histone Site Possible Function
Ubiquitylation H2A
K119
(mammals) Spermatogenesis
H2B
K120
(mammals) Meiosis
K123 (S.
cerevisiae)
Transcriptional
activation
Sites of histone ubiquitination with their
function
23. Small Ubiquitin-like Modifier (or SUMO) proteins are a family of small proteins that are
attached to and detached from other proteins in cell to modify their function.
Sumoylation consists in the addition of a “Small Ubiquitin-related MOdifier protein”
(SUMO) of ~100 amino acids.
Histone sumoylation was first reported in 2003, when Shiio et al. found that H4 can be
modified by SUMO and they suggested that this modification leads to the repression of
transcriptional activity through the recruitment of HDACs and HP1 proteins
The putative sumoylation sites were identified as K6/7 and to a lesser extent K16/17
of H2B, K126 of H2A, and all four lysines in the N-terminal tail of H4.
Histone sumoylation has a role in transcription repression by opposing other active
marks such as acetylation, methylation, ubiquitination etc.
23Department of Plant Biotechnology
Shiio et. al., PNAS, 2003
03/11/2016
5. Sumoylation5. Sumoylation
http://www.pnas.org/content/100/23/13118/F1.expansion.html
24. ADP-ribosylation is the addition of an ADP-ribose moiety onto a protein using NAD+
as a substrate.
Mono(ADP-ribosyl)ation is mediated by ADP ribosyl transferases (ART) and the
enzymes responsible for the PARation (Poly-ADP-ribosylation) are the poly(ADP-
ribose) polymerases (PARPs).
PARP1 prefers to linker histone H1 while PARP2 prefers core histones.
24Department of Plant Biotechnology
Zdenko Herceg & Rabih Murr
Sascha Beneke* www.frontiersin.org, 2012
03/11/2016
6. ADP-Ribosylation6. ADP-Ribosylation
FIGURE 1 | Poly(ADP-ribose) polymerase1 in DNA repair
25. Department of Plant Biotechnology 25
H1 to nucleosomes increases chromatin compaction, ADP-ribosylation of H1 is suggested to alter
the chromatin structure and possibly the chromatin composition.
Messner and Michael , Trends in Cell
Biology September 201103/11/2016
1
2
3
ADP-ribosylation cycle of
histones in chromatin
Suggested biological
consequences of mono- or poly-
ADP-ribosylated histones in
chromatin.
26. 26Department of Plant Biotechnology
The histone code is a hypothesis that the transcription of genetic information encoded
in DNA is in part regulated by chemical modifications to histone proteins, primarily on their
unstructured ends .
This hypothesis predicts that-
•Distinct modifications of the histone tails will induce interaction affinities for chromatin-
associated proteins.
•Modifications may be interdependent generate various combinations
•Local concentrations and combinations of differently modified nucleosomes determine
qualities of higher order chromatin.
03/11/2016
Histone codeHistone code
27. Department of Plant Biotechnology 27
Readers-
Chromodomain
•CH3(Methyl)- recognition domain
•HP1 has a chromodomain
•Targets to Me-lys or H3K9me
•Promote packed “CLOSED” chromatin
Demethylation of Lys 9 in H3 tail
facilitates phosphorylation (P) of Ser 10
Acetylation (Ac) of Lys 9 and 14
leads to “OPEN” chromatin
Bromodomain
Binds to acetylated lysines “OPEN”
Wide range of histone modifications >>>
Writers: enzymes that add a mark
Readers: proteins that bind to and
“interpret” the mark
Erasers: enzymes that remove a mark
Tarakhovsky, A., Nature Immunology, 2010.
03/11/2016
Reading / translating the histone codeReading / translating the histone code
28. Department of Plant Biotechnology 28
The core histones are among the most conserved eukaryotic proteins; therefore, the
nucleosomes formed by these proteins are very similar in all eukaryotes. But there are numerous
histone variants found in eukaryotic cells.
Such unorthodox histones can replace one of the four standard histones to form alternate
nucleosomes and may serve to demarcate particular regions of chromosomes
Examples-
Moggs and Orphanides, oxicological Sciences, 2004.
Play role in DNA damage repair
When chromosomal DNA is broken, H2A.X located
adjacent to the break is phosphorylated at a serine
residue that is not present in H2A.
PhosphorylatedH2A.X is specifically recognized by
DNA repair enzymes leading to their localization at the
site of DNA damage.
03/11/2016
Histone VarientsHistone Varients
1) H2A.X is a variant of H2A1) H2A.X is a variant of H2A
29. Department of Plant Biotechnology 29
In the centromeric region of chromosome , CENP-A replaces the histone H3 subunits in
nucleosomes.
These nucleosomes are incorporated into the kinetochore that mediates attachment of the
chromosome to the mitotic spindle core structure of the nucleosome.
Consistent with this interaction being critical for kinetochore formation, loss of CENP-A
interferes with the association of kinetochore components with centromeric DNA.
Alteration of chromatin by incorporation of histone variants. incorporation of CENP-A in place of
histone H3 is proposed to act as a binding site for one or more protein components of the
kinetochore. Mol. Bio of Gene, Watson et al., 7th
edition
03/11/2016
2) CENP-A varient histone H32) CENP-A varient histone H3
30. Department of Plant Biotechnology 30
The decreased HAT activity of CBP is a key contributor to
the RSTS phenotype.
Features- Broad thumbs and toes, facial abnormalities,
congenital heart defects, and increased risk of tumor
formation.
Frequency- This condition is uncommon; it occurs in an
estimated 1 in 100,000 to 125,000 newborns.
Additional features of the disorder can include eye
abnormalities, heart and kidney defects, dental problems,
and obesity.
Infants born with this severe form of the disorder usually
survive only into early childhood.
RSTS - Facial features (A), left hand and
feet showing broad thumb and big toes (B,
C) and X-ray of both hands showing short
broad thumbs (D). (Limb Malformations &
Skeletal Dysplasia)
Huda Y. Zoghbi and Arthur L. Beaudet Cold Spring Harbor
Laboratory Press, 2016
Mutations in the CREBBP gene, the gene provides instructions for making CREB binding protein,
plays an essential role in controlling cell growth and division and prompting cells to mature
This mutation abolishes the histone acetyltransferase (HAT) activity of CBEP (Murata et al.
2001).
03/11/2016
Diseses associated with histone modificationDiseses associated with histone modification
1. Rubinstein–Taybi Syndrome (RSTS)1. Rubinstein–Taybi Syndrome (RSTS)
31. Department of Plant Biotechnology 31
The syndrome is caused by mutations in the RPS6KA3 gene (histone phosphorylation) and is
located on the short arm of the X chromosome. Males are usually more severely affected than
females.
Ribosomal protein S6 kinase, 90kDa, polypeptide 3, also known as RPS6KA3, is an enzyme
that in humans is encoded by the RPS6KA3 gene.
The protein RSK2 which is encoded by the RPS6KA3 gene is a kinase which phosphorylates
some substrates like CREB and histone H3.
A rare genetic disorder characterized by mental retardation and abnormalities of the head
and facial and other areas.
Cardiac abnormalities affect 15% of the patients.
Coffin-Lowry syndrome
03/11/2016
2. Coffin-Lowry syndrome2. Coffin-Lowry syndrome
CLSF- Coffin-Lowry Syndrome Foundation
Created in 1991 by Mary Hoffman in France.
CLS a general forum in which to exchange information, ideas and
advice and a great resource for families affected by CLS.
Huda Y. Zoghbi and Arthur L. Beaudet Cold Spring Harbor
Laboratory Press, 2016
32. Department of Plant Biotechnology 3203/11/2016
Techniques to Study Histone ModificationTechniques to Study Histone Modification
2) Mass Spectrometry (MS)
Other genome-wide techniques combined
with ChIPs
33. Department of Plant Biotechnology 33
ChIP is a technique whereby a protein of interest is selectively immunoprecipitated from a
chromatin preparation to determine the DNA sequences associated with it.
Chromatin immunoprecipitation (ChIP) has become the technique of choice to investigate
protein–DNA interactions inside the cell.
ChIP has been used for mapping the localization of post-translationally modified histones
and histone variants in the genome, and for mapping DNA target sites for transcription
factors and other chromosome-associated proteins.
There are mainly two types of ChIP, primarily differing in the starting chromatin preparation.
Philippe Collas, Mol Biotechnol (2010) 45:87–100
Thomas A. Methods in Molecular Biology, vol. 538
1. Cross-linked ChIP (XChIP)
Uses reversibly cross-linked chromatin as
a starting material then sheared by
sonication.
Mainly suited for mapping the DNA target
of transcription factors or other chromatin-
associated proteins,
2. Native ChIP (NChIP)
Chromatin sheared by
micrococcal nuclease digestion.
Mainly suited for mapping the DNA
target of histone modifiers.
03/11/2016
1. Chromatin Immunoprecipitation (ChIP)
34. Department of Plant Biotechnology 34
1. DNA and associated proteins reversibly
cross-linked with formaldehyde followed
by cell lysis.
2. The DNA-protein complexes (chromatin-
protein) are then sheared into ~500 bp DNA
fragments by sonication or nuclease
digestion.
3. Immunoprecipitation (IP) by using an
appropriate protein-specific antibody.
4. Reverse cross linking and sequence
determination
5. Enrichment of specific DNA sequences
represents regions on the genome that the
protein of interest is associated with in vivo.
Chromatin immunoprecipitation assay (ChIP) and various methods of
analysis
Philippe Collas, Mol Biotechnol (2010) 45:87–100
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Major Steps Involved in ChIP AssayMajor Steps Involved in ChIP Assay
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Chromatin Immunoprecipitation (ChIP) – Seq Assay
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ChIP-on-chip (also known as ChIP-chip) is a technology that combines chromatin
immunoprecipitaion ('ChIP') with DNA microarray ("chip").
Development of genomic DNA microarrays (chips) has, when combined with ChIP assays,
enabled the mapping of transcription factor binding sites and of histone modifications.
The most prominent representatives of this class are transcription factors, replication -related
proteins, like Origin Recognition Complex Protein (ORC), histones, their variants, and histone
modifications.
Philippe Collas, Mol Biotechnol (2010) 45:87–100
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ii) ChIP–on-chip
37. Department of Plant Biotechnology 37
Chromatin immunoprecipitation followed by sequencing (ChIP–seq) is a technique for
genome-wide profiling of DNA-binding proteins, histone modifications or nucleosomes.
1. Crosslinking
2. Chromatin is sheared by sonication (200–600 bp )
3. Immunoprecipitation
4. Reverse crosslinking DNA is assayed to determine
the sequences bound by the protein.
5. Construction of a sequencing library,
6. Sequencing of DNA.
Overview of a ChiP–seq experiment.
NATuRe RevIewS | Genetics
Peter J. Park 200903/11/2016
ii) ChIP–seq
Major StepsMajor Steps
In ChIP–seq, the DNA fragments of interest are
sequenced directly instead of being hybridized on
an array.
39. Department of Plant Biotechnology 39
Chromatin Immunoprecipitation (ChIP) coupled with quantitative PCR can be used to investigate
protein-DNA interaction at known genomic binding sites.
This technique is now used in a variety of life science disciplines including cellular differentiation, tumor
suppressor gene silencing, and the effect of histone modifications on gene expression.
1. Cell fixation (cross-linking)
2. Chromatin shearing: by Sonication (100-500 bp)
3. Chromatin IP: Using specific ChIP-grade
antibodies
4. Reverse cross-linking and DNA purification.
5. qPCR and analysis
EpiTect®
ChIP PCR System03/11/2016
Major StepsMajor Steps
iii) ChIP-qPCR
41. Department of Plant Biotechnology 41
Mass Spectrometry is an analytic technique that utilizes the degree of deflection of charged
particles by a magnetic field to find the relative masses of molecular ions and fragments.
A mass spectrum is a plot of the ion signal as a function of the mass-to-charge ratio (m/z)
The two primary methods for ionization of whole proteins,
i). Electrospray ionization(ESI)
ii). Matrix-assisted laser desorption/ionization (MALDI).
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2. Mass Spectrometry (MS)
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1.Extraction of histones.
2. Further separation by SDS-PAGE
3. Protease digestion
4. Trypsin is used to cleave proteins
5. Reversed phase chromatography.
6. Mass spectrometer analysis
Quantification and identification of the example peptide H3
27-40 by mass spectrometry
A. The areas of peptides with different modification states are
highlighted in grey. The presence of fragmentation spectra for the
monomethylated peptide is also displayed.
B. MS/MS spectrum -fragment ions b2+ and b3+ clearly demonstrate
methylation in K27 while y6+-y9+ show only propionylation in positions
K36 and K37.
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Major StepsMajor Steps
Ignasi Forné et.al., CIPSM,2012
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Recent Review on Histone Modification
A genetically encoded probe for live-cell imaging of H4K20 monomethylation
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(a) Schematic structure of H4K20-mintbody.
(b)The localization of H4K20me1-mintbodies in living cells.
H4K20me1-mintbody was concentrated in nuclei during
interphase and bound to chromosomes during mitosis.
VH- Blue colour, LH- Red colour, The amino acids that differ in
12C8 are indicated in yellow. .
Space filling models of two important residues (I95 and A99;
letters shown in red) are blown-up to the side. The oxygen and
nitrogen atoms arecolored in red and dark blue. Bars, 10 μm.
(c) The crystal structure of 15F11-scFv at 1.94 Å
resolution.
Yuko Sato, et. al., JMB, 2016
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Conventional techniques used to study regulation by histone modifications are limited to fixed (dead)
cells, thus preventing assessment of histone modification in single, living cells.
e.g. ChIP-chip, ChIP-seq , ChIP-qPCR etc.
The specificity of the H4K20me1-mintbody in living cells was verified using yeast mutants and
mammalian
cells in which this target modification was diminished.
In a roundworm Caenorhabditis elegans model, the H4K20me1-mintbody could be used to monitor
changes in H4K20me1 over the cell cycle and localization of dosage-compensated X chromosomes
without disrupting cell function.
Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K20me1 levels
during the cell cycle.
This research also identified key amino acids responsible for H4K20me1-mintbody conformational
stability, solubility, and consequently, functional performance using X-ray crystallography and genetic
analyses.
This research has future implications and can be used to monitor the dynamics of histone
modifications and genome integrity in single living cells without disturbing cellular functions.
In the future, development of additional mintbodies specific to diverse post-translational histone
modifications will facilitate the identification of regulatory mechanisms that control epigenetic modifications.
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Importance
Yuko Sato, et. al., JMB, 2016
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Histone proteins are most important for packaging and ordering of DNA.
N-terminal tails of histones are the most accessible for post translational modificatins.
Histone tail modification participate in regulation of many processes including,
– Transcription activation
– Transcription repression
– DNA damage and repair
– Chromatin assembly
– Domain bindings
– Cell cycle
– Long-range packaging (heterochromatin formation, silencing)
– Chromosome condensation
Post-translational modification (PTM) of histones play an impotanat role in epigenetic
regulation of a gene.
Strategy for localizing histone marks- ChIP-chip, ChIP –seq., ChIP-qPCR, Mass
Spectromery etc.
The H4K20me1-Mintbody could be used to monitor changes in H4K20me1 in living
cells.
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Conclusion Conclusion
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ReferencesReferences
Sato, Y., Kujirai, T., Arai, R., Asakawa, H., Ohtsuki, C., Horikoshi, N., Yamagata, K., Ueda, J.,
Nagase, T., Haraguchi, T., Hiraoka, Y., Kimura, A., Kurumizaka, H. & Kimura, H., A genetically
encoded probe for livecell imaging of H4K20 monomethylation, Journal of Molecular Biology
(2016).
Mechanisms of Histone Modifications, Zdenko Herceg and Rabih Murr
(download.bioon.com.cn/upload/201105/30165853_5528.pdf )
Shelley L Berger , Histone modifications in transcriptional regulation. Current Opinion in
Genetics & Development 2002, 12:142–148.
Sascha Beneke Regulation of chromatin structure by poly(ADP-ribosyl)ation .
www.frontiersin.org REVIEW ARTICLE September2012|Volume3|Article169 |
Peter J. Park ChIP–seq: advantages and challenges of a maturing technologyNATUREReviews
| Genetics vOlume 10 | OCTOBER 2009 |
Anna Sawicka, Christian Seiser. Histone H3 phosphorylation e A versatile chromatin
modification for different occasions. Biochimie 94 (2012) 2193e2201
Eric J. Richards and Sarah C.R. Elgin Epigenetic Codes for Review Heterochromatin Formation
and Silencing: Rounding up the Usual Suspects. Cell, Vol. 108, 489–500, February 22, 2002,
49. 03/11/2016 Department of Plant Biotechnology 49
EpiTect®
ChIP PCR System For reliable analysis of protein—DNA interactions
(www.sabiosciences.com/manuals/1073038_PP_GEF_Chip_0712_lr.pdf )
Huda Y. Zoghbi1,2 and Arthur L. Beaudet. Epigenetics and Human Disease Published by Cold
Spring Harbor Laboratory Press at University of Hong Kong Libraries on February 1, 2016
Vikki M. Weake and Jerry L. Workman, Histone Ubiquitination: Triggering Gene Activity , Molecular
Cell 29, March 28, 2008
Mol Biology of the Gene by Watson 7th
Edition
Principles of Biochemistry by Lehninger 4th
Edition
https://en.wikipedia.org/wiki/Histone
http://www.abcam.com/epigenetics/histone-modifications-a-guide
https://www.youtube.com/watch?v=fCd6B5HRaZ8
https://www.youtube.com/watch?v=4oFdS9EN9Pk
https://www.youtube.com/watch?v=eYrQ0EhVCYA