MARCO - chromatin + RNA-protein

Question 1

Chromatin

Answer 1

very dynamic – require interaction of TF at diff. enhancers, promotors, and many regulatory factors over long distance, forming 3D interaction of chromatin

• The interactions are limited to specific regions that form the same TAD (transcription active domains)
• The components in 1 TAD do not interact with other TADs. Enhancers and promotors can only regulate genes inside the same TAD (with very few exceptions)
• DNase-seq, etc identify regulatory regions but not the distant interactions

Question 1

ChIA-PET: Chromatin Interaction Analysis with Paired-End Tag

Answer 1

*Identification of 3D Chromatin interaction technique:

• Based on the assumption that DNA fragments that are in close proximity due to 3D chromosome interaction are more likely to re-ligate with each other. (Not naturally close to each other but since interacting, end up being near each other)
  *Other similar techniques: 3C, 4C, HiSEQ

Question 2

ChIA-PET: (method)

Answer 2

1. Cross-linking of TF bound chromatin which also has long-distance interaction with other GSTF on distal enhancers, then shearing
2. Immunoprecipitation of region bound by a selected TF to also pull down the distal DNA region associated with the TF
3. Proximity ligation of interacting regions forming paired-end sequences
4. De-crosslinking of the proteins from DNAs, PCR amplification, and purification
5. Next-generation sequencing obtaining reads of paired-end sequences
6. Mapping back to genome to identify distal interacting regions

Question 3

RIP-seq:

Answer 3

RNA ImmunoPrecipitation

• Identification of RNA-protein/ RNA-DNA interactions
  ex. long non-coding RNA, proteins regulating RNA post transcription

Question 4

RIP-seq: method

Answer 4

1. Immunoprecipitation of a specific RNA-protein complex using antibody that recognizes a specific RNA binding protein (RBP)
2. RNase digestion of unbound regions & degradation of RBP
3. Extraction of bound RNA
4. Reverse transcription into cDNA
5. Next-gen sequencing on cDNA & mapping to reference genome

Question 5

RIP-seq: problem

Answer 5

cannot perform cross-linking to keep the RBP associated with RNA because it would be too damaging to the RNA - results in low stringency & specificity

Question 6

CLIP-seq:

Answer 6

Cross-linking ImmunoPrecipitation
Relies on UV cross-linking: treating RNA-protein complex with UV light to immobilize the interaction for an amount of time - results in increased stringency during immunoprecipitation

Question 7

CLIP-seq method

Answer 7

1. UV cross-linking
2. Immunoprecipitation
3. RNase digestion of unbound regions & degradation of cross-linked RBP
4. Reverse transcription into cDNA
5. Next-gen sequencing on cDNA & mapping to reference genome
   * The process of degrading the cross- linked proteins utilize proteinase K which is not able to digest the whole protein (leaves a peptide at the binding site)
   * During reverse transcription, the peptide that is left behind will induce mutations, resulting in cross-linking induced mutation sites – CIMS
   * Therefore, the cDNA will not be identical to the original DNA sequence that coded for the bound RNA.
   * When mapped back to the reference genome, the CIMS can be identified to indicate protein-RNA binding sites

Question 8

Clip-seq variants

Answer 8

• PAR-CLIP - improves crosslinking with photoreactive RNA nucleotides
• iCLIP - uses reverse transcriptase stalling to map individual nucleotide-protein
  interactions
• miCLIP - modifies an RNA methylase to map its binding sites

Question 9

-seq method key features

Answer 9

• identify region of interest (protein bidning site, open chromatin, etc)
• isolate sequence eg. fragmentation and immunoprecipitation, phenol/chloroform, etc)
  *sequence fragment and map back to genome

Question 10

ENCODE – Encyclopedia of DNA elements

Answer 10

aims to identify all functional elements in the human genome involved in gene regulation across ~50-60 cell types using these -seq methods

Question 11

ENCODE controversy

Answer 11

Identified millions of enhancers/ 100s, 1000s of promotors/ millions of transcription factor binding sites across genome which correspond to a big proportion of genome – cause controversy during the earlier times (now known to be true)

Question 12

Limitations of ENCODE

Answer 12

Most of the work was conducted in immortalized cell lines – cell lines derived from human cancers (ex. HeLa cells):
* cancer cells model tissue of origin BUT are not the exact same as the primary cell type
* Identification of those regions from a primary cell type (closer to natural state) is
preferred – partially accomplished by a recent consortium called ROADMAP – identified histone modifications across 25 human tissues

both ENCODE and ROADMAP are ongoing which keep adding information to our knowledge of these regulatory regions