MARCO - open accessible chromatin

Question 1

Transcription factors:

Answer 1

• Open, accessible chromatin allows for binding of transcription factors
• Proteins that bind DNA in a sequence specific manner (only binds to specific DNA motifs of 5-6 nucleotides) & up/down regulate gene expression by either stabilizing or blocking the binding of RNA polymerase to DNA
• Some recognize short sequences while some recognizes dimers of sequences
• Any single point mutation in TF motif will prevent TF recognition

o Ex of important TF binding motif: the dimer sequences upstream of p53, a tumor suppressor gene. Binding of TF to the motif leading to p53 activation is crucial to prevent cancer. Any mutations to the TF motif will result in cancer.

Question 2

To identify TF motifs, can identify open, accessible chromatin regions using:

Answer 2

• ChIP-seq
• DNase-seq
• ATAC-seq
• MNase-seq
• FAIRE-seq

Question 3

ChIP-seq: (method)

Answer 3

Chromatin ImmunoPrecipitation sequencing
1. Chromatin (with bound transcription factors) are
cross-linked, using formaldehyde to prevent detachment of transcription factors, and then sheared by sonification
2. The DNA fragments bound by a specific TF are separated by immunoprecipitation using antibody known to bind the specific TF
3. The precipitated fragments are purified by magnetic beads that recognize the antibodies
4. Proteases are added to degrade transcription factors to only obtain DNA fragments
5. Those DNA fragments are sequenced with next-gen sequencing to obtain reads
6. The reads are mapped back to the genome to identify region bound by the specific TF

ChIP-seq result shows transcription factor binding peakà

Question 4

ChIP-seq can also be used to study histone modifications

Answer 4

• Utilize antibody that binds to modified histone instead (ex. recognize methylated/ acetylated histones)
  1. Fractionate DNA
  2. Use antibody that binds to modified histone to separate them by immunoprecipitation
  3. Sequence the isolated fraction using a next generation sequencing
  4. Map sequence back onto genome to identify regions with modified histones
• Can be used to identify chromatin states since active chromatin will have different sets of histone modifications from inactive chromatin

Question 5

DNase-seq:

Answer 5

utilize DNase1
* Easily accessible regions are highly sensitive to cleavage by DNase1 – thus called hypersensitive sites (HS).
* However, regions bound by TF in those HS are not cleaved by DNase 1

Question 6

DNase-seq: (method)

Answer 6

1. Treat DNA with DNase1 to cut the chromatin where it is accessible
2. Fragments are sequenced by next-gen sequencing and mapped back onto the genome – identify easily accessible regions nearby of specific gene
3. Downstream analyses (ex. gel electrophoresis) is required in order to observe DNA footprints - region of HS that is not cut by DNase1 due to TF binding - providing the exact sequence of TF motif upstream of a certain gene

Obtained sequences of open, accessible chromatin/TF motif by DNase-seq can then be computationally analyzed based on known TF motifs –> enable us to deduce which TF is responsible for which gene based on their binding patterns.
* Can then be validated with ChIP-seq (because now know the specific TF & can use antibody specific to the TF to perform ChIP-seq)

Question 7

DNase-seq advantage over ChIP-seq

Answer 7

• Does not require prior knowledge of a specific TF. Chip-seq requires prior knowledge of specific TF to select for an antibody for immunoprecipitation

DNase-seq = identify regions bound by TF & use computational analysis to deduce which TF binds to those regions
ChIP seq = have a specific TF in mind & want to identify region bound by that TF

Question 8

FAIRE-seq

Answer 8

an alternative method to DNase-seq

• FAIRE-Seq has higher coverage at enhancer regions over promoter regions
• DNase-Seq has a higher sensitivity towards promoter regions

Disadvantages of DNase-seq and FAIRE-seq:
* Require a lot of starting materials

Question 9

FAIRE-seq (method)

Answer 9

1. Perform formaldehyde cross-linking on nucleosome-bound DNA (inactive/closed conformation) & sheared by sonification
2. Perform Phenol/ Chloroform extraction to separate out the cross-linked inactive region which will be in the organic phase from the aqueous, nucleosome-depleted, non- crosslinked active region
3. The extracted active region is then sequenced & reads are mapped back onto the reference genome to identify open chromatin regions
4. Like DNase-seq, computational analysis based on known TF binding motif can be used to deduce which TF = responsible for regulation of which gene

Question 10

ATAC-seq: (method)

Answer 10

Assay for Transposase-Accessible Chromatin
1. Treat sample with mutated hyperactive transposase Tn5, an enzyme derived from transposons which its only function is cutting exposed chromatin
2. The fragments are then isolated, amplified, sequenced, and mapped – can identify open, accessible chromatin

Question 11

ATAC-seq: advantages

Answer 11

• Requires smaller sample than DNase-seq which require 1000-fold more cells (50k vs millions of cells) – which is good for when working with primary cells
• MNase-seq extracted from actual tissues ex. neurons, bone marrow cells, hepatocytes
• Very fast (completed in 3 hours)

Question 12

MNase-seq:

Answer 12

Micrococcal nuclease sequencing
1. Treat sample (either native or cross-linked) with MNase (Micrococcal nuclease) which cleave the linker DNA between nucleosomes in nucleosome-rich inactive chromatin. This generates a pool of mononucleosomes
2. Sequencing of mono-nucleosomal DNA yields nucleosomal maps – allow precise determination of nucleosomes positions, identifying closed, inactive chromatin regions

Question 13

comparison of methods

Answer 13

Chip-seq = requires antibody which gives result specific to TF of interest
MNase-seq = profile all closed, inactive chromatin regions of the whole genome
Other methods = profile all open, active chromatin regions of the whole genome

Question 14

Hi

Answer 14

Hi