METHODS FOR STUDYING EPIGENETIC MODIFICATIONS

DNA Methylation:
• Bisulfite sequencing

Histone Modification:
• Chromatin immunoprecipitation (ChIP)
• DNA adenosine methylation identification (DamID)

siRNA Production:
• Deep Sequencing

Analysis of DNA Methylation by Bisulfite Sequencing

DNA treated with bisulfite
Cytosines deaminated: converted to uracil BUT methylated cytosines are
conserved
To determine methylation region: comparing of treated and untreated samples
In treated sample: unmethylated cytosines – thymines (u t olarak okunur)
In untreated sample: unmethylated cytosine

Methylation-Sensitive Nucleases
Restriction enzymes- sensitive to DNA methylation
Comparing: Digestion of genomic DNA with methylation-sensitive restriction
enzymes AND DNA digested by their methylation-insensitive isoschizomers
Endonucleases: digest specifically methylated DNA – detecting methylated
regions.
AFTER DIGESTION
Further proceed
Characterization of specific regions- PCR
Genome wide analysis- microarray hybridization and DNA sequencing
Endonuclease digestion (methylation sensitive) → PCR amplification→ in
methylated regions there are products.

Affinity Purification of Methylated DNA

Antibodies→ recognize methylated cytosine
Antibodies→ can be used to isolate methylated DNA by affinity purification or
Immunoprecipitation → these methods for genomic maps and methylcytosine
rich regions BUT NOT EXACT METHYLATED SITES

Conventional DNA Sequencing

Requirement:
• 4 standard dNTPs
• Primer
• Template strand
• DNA polymerase
DNA polymerase incorporates unlabeled dNTPs (that allows normal extension
or labeled ddNPTs (do not allow the strand to extend),
resulting in a mixture of differently sized labeled molecules
Separate DNA molecules by size (electrophoresis)

Next Generation DNA Sequencing Methods

Classical DNA sequencing= one molecule at a time
Next generation= one millions molecules at a time

NGS: Pyrosequencing
no labelled nucleotides or ddNTPs
After nucleotide addition pyrophosphate release
Pyrophosphate+ Adenosine 5’ phosphate sulphate → by ATP Sulfurylase = ATP
ATP + Luciferin → by luciferase → LIGHT PRODUCTION
Light intensity= added bases → dNTPs added and additions monitored by light
production → determine results by flowgram

NGS: Illumina Sequencing

DNA methylation on single gene → bisulfite conversion →microarray

OR directly Methyl sensitive cut counting
Results → indicating which cytosines are methylated and which are
unmethylated / The degree of methylation at each cytosine position can be
quantified – level of DNA methylation SORU

DNA methylation on whole genome→ bisulfite treatment → pcr

→pyrosequencing
Results: A comprehensive methylation profile of the entire genome, indicating
the methylation status of individual cytosines / Quantification of the overall
methylation levels across the genome / Identification of Differentially
Methylated Regions /Analysis of methylation in CpG islands, which are often
associated with gene regulation. SORU
 Methods for Studying Histone Modification
Chromatin Immunoprecipitation (ChiP)
Modified histone → Cross link DNA to histone → Shear DNA to smaller
pieces→ add specific antibody to modified histone → affinity purify antibody
→bound histone/DNA complex → remove cross link → purify DNA→ examine
by PCR, sequencing or microarray
antibody-based technology used to selectively enrich specific DNA-binding proteins along with their DNA targets.
ChIP is used to investigate a particular protein-DNA interaction, several protein-DNA interactions, or interactions
across the whole genome or a subset of genes.
Microarray: A microarray is a laboratory tool used to detect the expression of
thousands of genes at the same time. DNA microarrays are microscope slides
that are printed with thousands of tiny spots in defined positions, with each spot
containing a known DNA sequence or gene. Often, these slides are referred to as
gene chips or DNA chips. The DNA molecules attached to each slide act as probes
to detect gene expression, which is also known as the transcriptome or the set
of messenger RNA (mRNA) transcripts expressed by a group of genes.

qPCR: In qPCR vs PCR, the key differences are quantification, speed and
resolution. End point PCR enables qualitative or semi-quantitative analysis at the
end of all PCR cycles via an agarose gel or microchip. qPCR relies on fluorescent
dyes or probes and calibration curves to deliver quantitative data in real-time.

ChIP on chip → immunoprecipitation and microarray

Chip-Seq→ immunoprecipitation and sequencing
Chip-qPCR→ immunoprecipitation and qPCR
 SORU Microarray Advantages and Disadvantages:
Advantages:
simultaneous • simultaneous analysis of thousands of genes or genomic regions in a single
analyze
experiment, providing a high-throughput platform.
comparative • Multiple samples can be analysed in parallel, enabling comparative studies
studies
of gene expression, DNA copy number variations, or DNA methylation
different cond.
patterns.
cost effective • gene expression profiling, helping researchers understand how genes are

small amo.sample regulated under different conditions.

• Microarrays are cost-effective for analyzing a large number of genes

simultaneously compared to individual gene assays.
• Well-established protocols and standardized arrays contribute to data
reproducibility, allowing comparisons across different studies.
• Microarrays often require smaller amounts of sample material compared
to other genomic techniques. - sample conservation
Disadvantages:
limited dynamic • Microarrays may have a limited dynamic range, making it challenging to
cross hybrid.
not nonmodel
accurately quantify extreme expression levels.
fixed content • Cross-hybridization can occur, leading to non-specific binding and
potential misinterpretation of results.
• Designing specific and effective probes can be challenging, particularly
for species with incomplete genomic information.
• Microarray results are dependent on the accuracy and completeness of
genome annotations, limiting their utility for non-model organisms.
• Once manufactured, microarrays have a fixed content, making it
impossible to update probe sets with new genomic information.
• provide limited information on epigenetic modifications compared to
newer technologies like bisulfite sequencing.
 Next-Generation Sequencing (NGS):
Advantages:
no need pre- • NGS is highly versatile and can be applied to various genomic analyses,
existing genomic
inf.- non model including whole-genome sequencing, RNA-Seq, ChIP-Seq, and more.
• NGS does not rely on pre-existing genomic information, making it suitable
high resolution
for non-model organisms or studies in the absence of a reference genome.
detailed inf. meth• NGS provides high resolution and accuracy, enabling the detection of rare
variants and precise quantification of gene expression.
• NGS can detect novel sequences, offering insights into previously unknown
genes or genomic features.
• NGS methods, such as bisulfite sequencing, can provide detailed
information on DNA methylation patterns.

Disadvantages:
expensive • NGS can be more expensive, especially for whole-genome sequencing or
advanced compu.
complex sample large-scale projects.
preparation
• NGS generates large datasets, requiring advanced computational tools and
bioinformatics expertise for data analysis.
• NGS often requires more extensive and complex sample preparation
compared to microarrays.
• The turnaround time for NGS can be longer, especially for projects with
extensive data analysis requirements.
• The initial investment in NGS instrumentation can be high.

model organism sample preparation

Mass Spectrometry: Protein mixtures should be separated into less complex

mixtures containing fewer components.
Firstly separating proteins → digestion to peptides → MS analysis→MS data →
Identification
This method based on this hierarchy: amino acid→peptides→protein
DNA adenine methylation ID (DamID):
identify genomic regions that interact with a specific protein in living cells. It
allows researchers to map the binding sites of a protein of interest on genomic
DNA.
• Expression of Fusion Protein:
A fusion protein is created by attaching the protein of interest to the Escherichia
coli DNA adenine methyltransferase (Dam). This fusion protein is expressed in
the cells of interest.
• Methylation of Adjacent DNA:
Dam methyltransferase methylates adenine residues in the GATC sequence. As
the fusion protein moves along the genomic DNA in living cells, it methylates
adenine bases in GATC sites that are in proximity to its binding sites.
• Genomic DNA Extraction:
Genomic DNA is extracted from the cells expressing the Dam-fusion protein. The
methylation patterns on DNA reflect the protein's binding sites.
• Methyl-Sensitive Restriction Enzymes:
Methyl-sensitive restriction enzymes, such as DpnI, are used to digest the
genomic DNA. These enzymes cleave at GATC sites unless the adenine is
methylated.
• DNA Amplification by PCR and Microarray/Sequencing:
The remaining DNA fragments, containing the methylated GATC sites, are
amplified. The amplified DNA can be analysed using microarrays or high-
throughput sequencing to determine the genomic locations where the protein of
interest is bound.
Applications of DamID:
• Protein-DNA Interaction Mapping:
DamID is used to map the genomic regions that interact with specific proteins,
such as transcription factors or chromatin-associated proteins.
• Functional Genomics:
It provides insights into the regulatory networks and functions of DNA-binding
proteins.
• Epigenetic Studies:
DamID can be applied to study changes in chromatin structure and protein
binding during development or in response to environmental cues.
• Identification of Transcriptional Regulatory Elements:
It helps identify enhancers, promoters, and other regulatory elements in the
genome.
• Comparative Genomics:
Comparative DamID can be employed to study protein-DNA interactions across
different cell types or species.
ChIP-SEQ VS DamID:
ChIP-Seq (Chromatin Immunoprecipitation Sequencing):
Principle: protein binding to chromatin studying histone modifications

ChIP-Seq captures protein-DNA interactions by immunoprecipitating chromatin

fragments associated with a specific protein, followed by high-throughput
sequencing.
mapping transcription factor binding sites
DNA Methylation:
ChIP-Seq does not provide information on DNA methylation status. It focuses
on protein binding to chromatin.
Resolution:
Typically has higher resolution, allowing precise mapping of protein binding
sites.
Signal Specificity:
Provides direct signals of protein binding.
Protein Modification Studies:
Suitable for studying protein modifications like histone modifications.
Applications:
Widely used for mapping transcription factor binding sites, histone
modifications, and other chromatin-associated proteins.
DamID (DNA Adenine Methylation Identification):
Principle: studying dna methylation
DamID maps protein-DNA interactions by methylating adenine bases near
binding sites of a DNA-binding protein of interest, followed by DNA analysis.
DNA Methylation:
Provides information on DNA methylation patterns associated with the protein.
Resolution:
May have slightly lower resolution compared to ChIP-Seq.
Signal Specificity:
The signal indicates regions in proximity to the binding sites rather than direct
binding.
Protein Modification Studies:
Limited in studying protein modifications; mainly focuses on protein-DNA
interactions.
Applications:
Useful for mapping genomic regions interacting with DNA-binding proteins,
offering insights into the functional genomics of these proteins.
Considerations:
DNA Methylation Information:
DamID provides information on DNA methylation, which is not a focus of ChIP-
Seq.
Resolution:
ChIP-Seq often has higher resolution in pinpointing exact protein binding sites.
Signal Specificity:
ChIP-Seq provides a more direct signal of protein binding, while DamID
indicates regions in proximity to binding sites.
Applications:
ChIP-Seq is versatile, used for a wide range of chromatin-associated proteins,
while DamID is particularly suitable for studying DNA-binding proteins and their
interactions.

SORU
Histone modification on whole genome→ ChIP-Seq
ChIP-seq is a versatile technique that can be applied to study various histone
modifications simultaneously or sequentially.It is not limited to a specific
histone mark. It has high resolution. Cost-effective
Results→ ChIP-seq provides a genome-wide distribution of the histone
modification of interest, showing where on the genome the modification
occurs.
The intensity of peaks indicates the relative abundance or strength of the
histone modification.
Peaks can be annotated to specific genomic features such as promoters,
enhancers, or gene bodies.
SORU
Histone modification on single gene→ChIP-qPCR
Targeted Analysis: ChIP-qPCR allows researchers to focus on specific genomic
regions, such as individual genes or regulatory elements. This targeted
approach is beneficial when the area of interest is known, and a more focused
analysis is required.
Researchers can design qPCR primers to target specific regions within the gene,
allowing for customization of the analysis based on the research question.
Results→ Quantitative information about the presence or enrichment of the
histone modification at specific sites on the single gene.