Epigenetics3: Histone Modification and ChIP-seq

Summary

This course delves into epigenetics, focusing on how histone modifications regulate gene expression without altering DNA sequences. It explains the role of histones in chromatin structure and the impact of various post-translational modifications on gene transcription. The script introduces Chromatin Immunoprecipitation (ChIP) and its sequencing (ChIP-seq) as tools for studying these modifications genome-wide. It also covers the analytical challenges and methods for processing and interpreting ChIP-seq data, using Engelmann syndrome as a case study to illustrate the practical application of these techniques in understanding neurodegenerative diseases.

Takeaways

• 🧬 Epigenetics is the study of gene expression changes not encoded in the DNA sequence, including DNA methylation, histone modification, and non-coding RNA activity.
• 🌟 Histone modification is a significant part of epigenetic regulation, affecting how DNA is packed around histones and influencing gene transcription.
• 🔬 Nucleosomes, made up of histones, are the basic repeating units of chromatin, with DNA wrapped around core histones H3, H4, H2A, and H2B.
• 📐 Histone modifications can alter DNA packing density and include various types such as acetylation, phosphorylation, ubiquitination, and methylation.
• 🔑 Histone modifications can either activate or repress transcription, with certain modifications linked to active transcription and others to gene silencing.
• 🔍 Chromatin Immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) is a powerful tool for identifying genome-wide profiles of histone modifications and transcription factor binding sites.
• 🧲 The specificity of antibodies used in ChIP is crucial for high-quality data, as they bind to specific histone modifications or proteins attached to DNA.
• 📈 ChIP-seq data analysis involves identifying enriched genome fragments, which can indicate transcription factor binding sites, chromatin remodeling, or gene transcription events.
• 🛠️ Accurate peak detection in ChIP-seq is challenging and involves comparing signal enrichment against a control to determine the significance of binding sites.
• 🧬 Histone modifications are associated with specific widths and profiles of peaks in ChIP-seq data, which can be studied in the context of diseases like Angelman syndrome.
• 📚 The course encourages further exploration of epigenomics data analysis, suggesting deeper dives into projects related to Angelman syndrome and asthma for practical experience.

Q & A

What is the main focus of the course 'Epigenetics Three: Histone Modification and Chromatin Immunoprecipitation'?

The course focuses on understanding how gene expression is controlled by histones and how histone modifications can be studied using specialized protocols like chromatin immunoprecipitation (ChIP).

What are the mechanisms included in epigenetic regulation mentioned in the script?

Epigenetic regulation includes DNA methylation, histone modification, the activity of non-coding RNAs such as microRNAs, and the regulatory function of non-coding repeating regions found in the DNA.

What is the significance of histones in epigenetic regulation?

Histones are proteins around which double-stranded DNA is wrapped. Their modification plays a major role in gene expression by influencing how densely the histones are grouped together or spread apart, affecting the accessibility of the DNA and thus gene transcription.

What is a nucleosome, and what is its composition?

A nucleosome is the basic repeating unit of chromatin where 146 base pairs of DNA are wrapped around an octamer of core histones, consisting of pairs of H3, H4, H2A, and H2B. The N-terminal tails of these histones protrude out and are subject to various post-translational modifications.

What types of post-translational modifications can histones undergo?

Histones can undergo modifications such as acetylation, phosphorylation, ubiquitination, and methylation of lysine and arginine.

What is the role of histone modifications in gene transcription?

Histone modifications can either activate or repress gene transcription. For example, acetylation was the first modification linked with active transcription, while some histone methylation events have been associated with transcription activation or gene silencing.

What is the purpose of Chromatin Immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq)?

ChIP-seq is a powerful tool used to identify genome-wide profiles of transcription factor binding sites, histone modifications, and nucleosome positioning, providing insights into the regulatory mechanisms of gene expression.

How does the specificity of an antibody impact ChIP-seq data quality?

The specificity of the antibody, whether it is monoclonal or polyclonal and the organism it is specific for, is crucial in generating high-quality ChIP-seq data, as it ensures that the correct protein-DNA complexes are selected for sequencing.

What is the main challenge in analyzing ChIP-seq data?

The main challenge in ChIP-seq data analysis is accurately detecting enriched genome fragments, known as peaks, which represent regions where the protein of interest is bound to the DNA. This requires proper peak calling and accounting for sequencing and mapping errors.

How does the script relate ChIP-seq analysis to the study of diseases like Angelman syndrome?

The script uses Angelman syndrome as an example to illustrate how ChIP-seq analysis can help understand the epigenetic deregulation responsible for the disorder, by studying histone modifications and their impact on gene expression in affected individuals.

What are some of the techniques used in the ChIP-seq workflow for peak detection and peak shift?

Techniques such as MACS (Model-based Analysis of ChIP-Seq), BinH's peak calling method, and the Penn algorithm are used in the ChIP-seq workflow for accurate peak detection and determining the position of protein-DNA binding sites.

How can researchers use ChIP-seq data to study the regulatory mechanisms of specific genes?

Researchers can analyze the enrichment of reads in specific regions of the genome, which can correspond to genes of interest. By comparing ChIP-seq profiles of different samples, they can identify differential profiles of histone modifications and understand their regulatory roles in specific conditions or diseases.

Outlines

00:00

🧬 Epigenetics and Histone Modification Overview

This paragraph introduces the concept of epigenetics, focusing on how gene expression is controlled by histones and their modifications. It explains that epigenetics involves mechanisms that alter gene expression without changing the DNA sequence, including DNA methylation, histone modification, and non-coding RNA activity. The paragraph delves into the role of histones as proteins around which DNA is wrapped, forming nucleosomes. It describes how histone modifications, such as acetylation and methylation, can affect the compaction of DNA and, consequently, gene expression. The importance of specific histone modifications in transcription activation or gene silencing is highlighted, emphasizing the complexity of this regulatory machinery.

05:02

🔬 Chromatin Immunoprecipitation (ChIP) and Sequencing

This section discusses the technique of Chromatin Immunoprecipitation (ChIP) followed by high-throughput sequencing, known as ChIP-seq, as a method to study histone modifications genome-wide. It details the ChIP protocol, starting with cross-linking to bind proteins to DNA, followed by DNA fragmentation and the use of antibodies to extract DNA fragments with proteins of interest. The paragraph explains the process of sequencing these fragments and analyzing the data to identify binding sites of the proteins. It also touches on the importance of antibody specificity and the challenges of peak detection and analysis in ChIP-seq data.

10:03

🧠 Engelmann Syndrome and ChIP-seq Analysis

The third paragraph focuses on applying ChIP-seq analysis to study Engelmann syndrome, a neurodegenerative disease linked to epigenetic deregulation. It describes the process of preparing and analyzing ChIP-seq data, including the use of control samples to identify differential profiles of histone modifications. The paragraph provides a hands-on approach to analyzing ChIP-seq data, mentioning the use of specific software and algorithms for alignment, peak detection, and segmentation. It also discusses the significance of identifying enriched regions and the potential insights these can provide into the epigenetic regulation of diseases like Engelmann syndrome.

15:04

📊 Analyzing ChIP-seq Data and Interpreting Results

The final paragraph discusses the analysis of ChIP-seq data, emphasizing the importance of accurate peak detection and the interpretation of results. It describes a simple pipeline for ChIP-seq data analysis, including alignment, segmentation, and the use of various algorithms to identify enriched regions. The paragraph also mentions the use of gene ontology terms for enrichment analysis to understand the biological significance of the identified regions. It concludes with an invitation to explore the data further and provides resources for deeper study, including guides for accessing and analyzing multi-omics data.

Keywords

💡Epigenetics

Epigenetics is the study of changes in gene expression that do not involve alterations to the underlying DNA sequence. It is a key theme of the video, as it sets the stage for understanding how gene expression can be influenced by mechanisms other than genetic mutation. In the script, epigenetic regulation is discussed in the context of histone modification, DNA methylation, and the role of non-coding RNAs.

💡Histone Modification

Histone modification refers to chemical changes to histone proteins around which DNA is wound. These modifications play a crucial role in the regulation of gene expression by affecting how tightly or loosely the DNA is packaged. The script emphasizes the significance of histone modification as a major aspect of epigenetic regulation, with various types of modifications impacting gene transcription differently.

💡Chromatin Immunoprecipitation (ChIP)

ChIP is a technique used to study protein-DNA interactions, such as where a specific protein binds to DNA. It is central to the video's discussion on how histone modifications can be studied. The script describes the ChIP process in detail, including the use of antibodies to precipitate DNA fragments with specific histone modifications.

💡Nucleosome

A nucleosome is the basic unit of chromatin, consisting of a segment of DNA wound around a core of histone proteins. The script explains that the accessibility of DNA to transcription factors is influenced by how the nucleosomes are positioned and modified, which in turn affects gene expression.

💡Post-translational Modifications

Post-translational modifications are changes made to proteins after their translation from mRNA, such as acetylation, phosphorylation, and methylation. In the context of the video, these modifications occur on the N-terminal tails of histones and can influence the compaction state of chromatin and gene expression.

💡Transcription

Transcription is the process by which the genetic information in DNA is copied into RNA, specifically mRNA, which can then be translated into proteins. The video discusses how histone modifications can either activate or repress transcription, thereby controlling gene expression.

💡ChIP-Seq

ChIP-Seq is a method that combines chromatin immunoprecipitation with high-throughput DNA sequencing to identify the genome-wide distribution of proteins that bind to DNA. The script describes the ChIP-Seq process as a powerful tool for studying histone modifications and their impact on gene regulation.

💡Peak Calling

Peak calling in ChIP-Seq analysis refers to the identification of regions in the genome where there is a high concentration of reads, indicating where the protein of interest is bound to the DNA. The script discusses the importance of accurate peak calling for determining the exact positions of protein binding sites.

💡Engelmann Syndrome

Engelmann Syndrome, also known as AS, is a neurodegenerative disorder discussed in the script as an example of how epigenetic deregulation can lead to disease. The video uses this syndrome to illustrate the practical application of ChIP-Seq in understanding the epigenetic mechanisms behind specific conditions.

💡Gene Ontology (GO) Enrichment

GO enrichment is a method used to determine which biological processes, molecular functions, or cellular components are overrepresented among a list of genes. In the script, GO enrichment is mentioned as a way to gauge the significance of the regions identified through ChIP-Seq analysis, particularly in the context of Engelmann Syndrome.

💡KLHL17

KLHL17 is a gene mentioned in the script that has been implicated in neural development and is associated with actin-based neuronal function. The video uses KLHL17 as an example of a gene identified through ChIP-Seq analysis, demonstrating how such analysis can reveal important regulatory genes.

Highlights

Epigenetics involves mechanisms that alter gene expression without changing the DNA sequence, including DNA methylation, histone modification, and non-coding RNA activity.

Histone modification is a key aspect of epigenetic regulation, influencing how DNA is packed around histones and affecting gene transcription.

Nucleosomes, composed of histones, are the basic repeating units of chromatin, with DNA wrapped around them.

Post-translational modifications of histone tails, such as acetylation and methylation, can either activate or repress gene transcription.

Histone modifications can have a combinatorial effect on processes like transcription, DNA repair, and apoptosis.

Chromatin Immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) is a powerful tool for identifying genome-wide profiles of histone modifications.

The specificity of antibodies used in ChIP is crucial for generating high-quality data, with factors like monoclonal vs. polyclonal and organism specificity being important.

ChIP-seq data analysis involves cleaning raw reads, aligning them to the reference genome, and identifying enriched regions indicative of protein binding.

Peak calling in ChIP-seq experiments is essential for accurately identifying binding sites, with algorithms like MACS used for this purpose.

Engelmann syndrome, a neurodegenerative disorder, is linked to epigenetic deregulation affecting neuron differentiation.

Hands-on analysis of ChIP-seq data can help understand the epigenetic mechanisms behind diseases like Engelmann syndrome.

Different histone modifications are associated with specific widths and profiles of peaks in ChIP-seq data, influencing gene expression levels.

Gene ontology terms can be used to assess the significance of enriched regions identified in ChIP-seq analysis.

KLHL17, a gene involved in neural development, was identified through ChIP-seq as having enrichment of reads, indicating its potential role in neuronal function.

ChIP-seq analysis can reveal differential profiles of histone modifications, aiding in understanding condition-specific epigenetic regulation.

The course provides practical guides for further exploration of epigenomics data analysis, including projects on Engelmann syndrome and asthma.

Data integration from multiple sources, such as ChIP-seq, methyl-seq, and RNA-seq, is crucial for a comprehensive understanding of epigenetic regulation.

Transcripts

welcome to epigenetics three histone

modification and chromatin

immunoprecipitation in this course we

will explore how gene expression is

controlled by histones and how histone

modification can be studied using

specialized protocols like chromatin

immunoprecipitation we will also discuss

analytical approaches to study genome

wide histone modification data or chip

seek and specific challenges these

methods are designed to address before

we jump in let's first do a quick

overview of epigenetic regulation and

discuss the significance of histone

modification as you remember from

previous courses epigenetics is the

study of mechanisms that cause changes

in gene expression but that are not

encoded in the DNA sequence itself these

mechanisms include DNA methylation

histone modification activity of

non-coding RNAs such as micro RNAs and

the regulatory function of non-coding

repeating regions found in the DNA

histone modification is a major aspect

of epigenetic regulation histones are

proteins that the double-stranded DNA is

wrapped around on these images you can

see how the DNA string has small bumps

or beads on it these visible beads are

nucleosomes that are made up of histones

when these beads were originally

discovered scientists confused them with

jeans until was eventually proven that

the string like structure is wrapped

around and not to beat themselves

actually contain the genetic code

histones can be either grouped together

forming lumps of DNA or as we can see in

the picture on the right more relaxed

and spread out it turns out that the

modification of histones plays a major

role in how densely the histones are

grouped together or spread apart

moreover additional modifications of

histone tails can have other effects on

gene transcription one important aspect

of histones is that they can be changed

to alter how much packing the DNA is

capable of there are several key

modifications that take place as a

result of changes to groups of atoms

that are at the ends of histones these

changes can be of several types and will

have a positive or negative charge that

either attract them closer together or

force them apart as a result the DNA

region that is wrapped around the head

stones can be more or less accessible

causing variation in gene expression the

nucleosome is considered to be the basic

repeating unit of chromatin in which 146

base pairs of DNA are wrapped around an

octamer of core histones consisting of

pairs of h3 h4 h2a and h2b n terminal

tails of these systems protrude out of

the nucleosome and are subject to a

variety of post translational

modifications such as acetylation

phosphorylation ubiquitination

and lysine and arginine methylation a

set elation was the first of these

modifications to be linked with active

transcription and subsequently

phosphorylation of histone h3 was found

to cooperate with a substation in

transcriptional activation some histone

methylation events have also been

associated with transcription activation

and others with gene silencing histone

modifications can function either

individually or combinatorially to

govern such processes as transcription

replication DNA repair and apoptosis it

has been proposed that methyl group of

atoms increases packing and a central

group decreases packing also phosphoryl

group can be attached to the histones

and causes a decrease in packing these

modifications and their impact on

transcription and other processes are

being actively studied and as our

understanding grows researchers

appreciate how complex this machinery

works in real life as we mentioned

histones are organized in october's

forming nucleosomes the four histones

are called h-2a h-2b h3 and h4 DNA is

wrapped around with structure of four

times with histone h1 holding everything

together the most studied modification

of histones are methylation in a set

elation of the core histone tails as you

can see modification to the tails of

histones is linked to the length of the

tail itself when a specific type of the

modification is studied it will be

referred to as h3k4me3 or h3k27 me3 the

name simply refers to the position on

histone tail and the type of

modification it is

here you can see a table of histone

modifications and their reported impact

on transcription for example you might

read about h3k4 methylation free signal

this means that h3 histone tail is being

modified by removal of methyl group

items different signals act as different

regulatory mechanisms and sometimes work

together in the complex patterns the

function of specific histone

modifications is an active field of

research and many of these modifications

are not yet known on the right you can

see how these modifications and appear

in chip seek data against the control

sample to understand this better let's

discuss chromatin immunoprecipitation

for studying histone modification

chromatin immunoprecipitation followed

by high-throughput sequencing for chip

seek is a powerful tool to identify

genome-wide profiles of transcription

factor binding sites histone

modifications and nucleosome positioning

to understand the way this data is

generated we have to look at the process

in greater detail and appreciate the

biological elements involved in various

steps of the chip seek protocol

chromatin immunoprecipitation uses

antibodies designed to bind to specific

proteins of interest for pris

these can be histones or other complexes

attached to the DNA first cross-linking

ensures the proteins are tightly bound

to DNA so that DNA shattering does not

remove them

DNA is shattered leaving fragments of

DNA with or without protein of interest

on them special antibodies bind to

fragments of DNA with proteins of

interest the antibodies are typically

attached to magnetic beads so that it

can be used to extract fragments of DNA

with protein antibody complexes the

complexes can then be removed and

libraries for sequencing are prepared

the sequence reads will contain tags

that are used to denote the position

next to where the protein was bound to

DNA as a result peaks of reads will

accumulate next to positions of DNA

bound to protein in the analysis that

these positions are identified Peaks

quantified and position of the protein

binding site is determined accumulation

of reads with tags is analyzed for

accurate detection of binding sites to

detect accumulation properly a second

library called I DNA is prepared as a

control abundance of reads vs. control

provides assessment of abundant

significance then reads align to

positive and negative strands are

analyzed for overlap helping select the

exact position of PRI binding sites a

key step for chromatin

immunoprecipitation is the design of an

antibody that binds to a specific

protein for example you might have a kid

for histone h3 or even more specific to

h3k27 or h3k4 specificity of the

antibody whether it is monoclonal or

polyclonal and the organism it is

specific for are all key factors in

generating high quality data the whole

process of data preparation looks like

this proteins of interest like histones

are bound to DNA DNA is fragmented

resulting in DNA fragments that have

protein of interest on them and others

that do not they might have other

proteins on them or nothing at all only

DNA with proteins of interest bound to

them or event

they're selected with specially designed

antibodies the DNA is then separated

from the protein and the DNA fragments

sequenced as a result reads that were in

the region bound to the protein of

interest can be aligned to the reference

genome and regions with abundance of

reads are analyzed when we deal with

chip sig data we are analyzing sequences

of short reads that came from DNA

fragments that were attached to protein

of interest we selected after we

sequence the raw reads they need to be

cleaned from adapter sequences and PCR

duplicates then - they're cleaned reads

are aligned to the positive and negative

strands of the DNA separately this is

done because when the DNA is wrapped

around the protein of interest and

fragmented there will be a slight shift

to the left or right left from the

positive strand and right from the

negative string that needs to be

accounted for then the challenge is to

accurately identify Peaks what is

commonly referred to as peak Collin peak

calling is performed on the two strands

separately which leads to the next step

determining the position of the p/y

Center by shifting the positive and

negative strand reads and identifying

the intersecting segments finally the

intersecting segments are annotated and

marked as positions where the DNA was

wrapped around a py the major objective

of chip seek analysis to detect genome

fragments that are enriched with

upregulated signals these fragments

could be transcription factor binding

sites chromatin remodeling or gene

transcription events the main

algorithmic challenge is to accurately

detect enriched genome fragments

generating a whole genome landscape for

the signal to do that let's review

several techniques related to peak

detection and peak shift that are part

of the chip seek workflow probably the

most discussed issue and chip seek

experiments is the best method to find

true peaks in the data and precisely

identify a binding site a peak is a site

where multiple reads have been mapped to

and produce a pilot to accurately assess

the pilot significance a control library

is often used also referred to as I DNA

chip sequencing is most often performed

with single and reads and chip fragments

per sequence from the five prime ends

only this creates two disty

pekes one on each strand with the

binding site fallen in the middle of

these Peaks the distance from the middle

of the peaks to the binding site is

often referred to as the shift as the

DNA is wrapped around the histones and

can be more or less accessible various

portions of the DNA are affected these

include the promoter region

transcription start sites and the

introns and exons of genes other

regulatory regions like enhancers and

micro rna's are encoded in introns or in

proximity to coding regions each one of

these elements can have a significant

effect on the level of gene expression

alternative splicing as well as other

downstream regulation of pathways the

size of the region and its location are

both important

researchers are reporting that different

histone modifications are associated

with specific widths and profiles of

Peaks that can be detected in chip seek

data often times specific regions like

enhancers and transcription factors or

micro RNAs are being studied in cancer

neurodegenerative diseases and other

conditions researchers are studying one

or multiple types of modifications and

the elements they regulate by comparing

chip seek profiles of groups of samples

accurate identification of differential

profiles of histone modification can

lead to the understanding of epigenetic

regulation specific to a condition now

that we understand the way chip seek

data is prepared and analyzed let us try

to perform an analysis ourselves in this

hands-on section we will build a simple

pipeline to analyze the data and discuss

methods that were used in pipeline

results Before we jump in to building

our pipeline let's review the options we

have for analysis the two options are

for analysis of Peaks in a given sample

or for detecting differences between two

conditions in our case we will use the I

DNA option to identify areas that have

histone modifications by comparing a

condition versus control the condition

we will study is Engelmann syndrome an

inherited neurodegenerative disease

Engelmann syndrome also known as a s is

a neurogenic disorder caused by deletion

of the maternally inherited ube 3a

allele and is characterized by the

development delay

intellectual disability ataxia seizures

and a happy effect it is believed to be

caused by epigenetic deregulation of a

region that is responsible for proper

neuron differentiation we will use a

pair of samples from the study to try a

hands-on analysis of chip seek data the

full article is available here let's

navigate to server dot T - bio info and

open the areas of analysis here you will

see a dedicated section for analysis of

chip on chip and chip seek data when you

are in select single and reads as e and

Homo sapiens grch38 for reference genome

we already have to fast queue files in

SRA format archive of sequence reads

uploaded to the server so we don't have

to wait for lengthy uploads over various

connection speeds SR are five nine nine

zero eight eight five is a healthy

control sample and SR are five nine nine

zero eight eight two is a cell line from

a patient with angleman syndrome we can

place the healthy and sample group and

sick and I DNA essentially we will get a

contrast between the two so it does not

matter which one goes where our first

pipeline will be very simple for now

let's skip the pre-processing steps and

discuss two central processing steps

alignment and segmentation alignment or

mapping as it is also called can be done

with several methods the main goal is to

align reads to the reference genome we

selected Homo sapiens grch38 both i2g is

a newer version of the bowtie algorithm

that was published in 2012 the second

main step in the analysis of map reads

is accurate detection of the rich

regions for this purpose we will use

Isaac a method that uses a Bayesian

hidden Ising model for chip seek data to

ignore falsely enriched regions caused

by sequencing and/or mapping errors

another more complete pipeline is shown

here the main change is with including

PCR clean to remove PCR duplicates and

using multiple methods for segmentation

these include Binh s a segmentation

method developed by the Taliban from

Alex

research center and max to the popular

method for analysis of chip seek data

penis algorithm is based on a method

developed by broadsky Adele in 2010

called the binary search approach to

whole genome data analysis this dividing

conquer type algorithm detects a series

of non-interest in fragments of various

lengths with locally optimal scores

original version of blacks - was

published by Ying chieh and Cao Lu Lu

from the lab of extra Li Lu at the

dana-farber Cancer Institute Boston here

we will use version two developed and

maintained by taolu as a result of the

second pipeline we can use mapping

statistics as well as a table of each

chromosome

let's select from some one to see what

the output looks like in the output

table you will see a number of columns

corresponding to outputs by each method

bin s I seek and max - the most reliable

results will be when we each method

gives an enrichment score significantly

greater than zero for example these

positions highlighted between nine

hundred sixty one thousand two hundred

and 960 1800 on chromosome one since the

output is already annotated we can see

this segment corresponds with NS G zero

zero zero zero zero one eight seven nine

six one this is KLH seventeen catch like

family member 17 let's look at this gene

in greater detail the protein encoded by

this gene is expressed in neurons of

most regions of the brain it contains an

n-terminal BTB domain which mediates

dimerization of the protein na seed

terminal Kelch domain which mediates

binding to f-actin this protein may play

a key role in the regulation of actin

based neuronal function KHL 17 has been

reported to be involved in neural

development for example in their study

of infantile spasms I assess pack yakov

ski at I'll link a leech l7 theme to

expansion of epilepsy phenotypes as you

can see in this chart where enrichment

scores from I seek

were plotted KHL 17 is only one of the

genes identified by enrichment of reeds

as you remember these reeds mark a

protein of interest attached to the DNA

reading deeper into the method section

of the paper we can find what antibody

they used for preparing their chip seek

data the protein of interest what was

h3k4me3 if we plot the scores on the

scatterplot with y-axis and the position

on the chromosome we will see that KHL

17 is at the start of the chromosome one

of the first and rich regions identified

by all three algorithms but that is not

the only region that we can see highly

enriched in this type of methylation of

h3 histone we also see another region

around the 150 million position that

stands out one of the ways we can gauge

the significance of the whole region is

to use David for enrichment with gene

ontology terms now that you have this

data you are welcome to play around with

these enriched regions to see what you

can discover thank you for taking this

course and we hope that you enjoyed

learning about epigenomics data analysis

if you want to take things further

we recommend diving deeper into the

Engelmann project or this other project

focused on asthma to help you get

started with these datasets we've

prepared guides on accessing all of the

data that include chip seek my sulphate

seek and RNA sequence that's in the

project you will find data guides and

explanations of methods you can rely on

analyzing and integrating this multi

onyx data