The yeast two hybrid (Y2H) system, developed in 1989, is a technique for studying protein-protein interactions by utilizing specific protein domains to link transcription factors and activate reporter genes. While it offers advantages like simplicity and partner identification, it has limitations such as potential absence of interactions in yeast and possible interference from fusion proteins. Conversely, chromatin immunoprecipitation (ChIP) is a method to analyze protein-DNA interactions but faces challenges like distinguishing protein isoforms and difficulty with large-scale assays.

1. The Yeast Two Hybrid
System
The technique was pioneered by Stanley
Fields and Ok-kyu Song in 1989.
It is an important technique used to study
protein-protein interactions.
This procedure is based on the discovery that
gene expression in S. cerevisiae depends on
interactions between pairs of transcription
factors or proteins.

2. A protein is composed of domains, which
allow the same protein to perform different
functions.
The Y2H system uses two protein domains
that have specific functions.
A DNA binding domain(BD) capable of
binding to DNA
An activation domain(AD) capable of
activating transcription of DNA.

3. The assay relies on the expression of a
reporter gene (such as lacZ or GFP), which
is activated by the binding of a particular
transcription factor.
The query protein of interest fused with the
BD is known as the Bait, and the protein
library fused with the AD is referred to as
the Prey.
The successful interaction between the
proteins is linked to a change in the cell
phenotype.

4. In the absence of Bait-Prey interaction, the
AD domain is unable to localize to the
reporter gene to drive gene expression.
However, when Bait and Prey interact, the
BD domain binds to the DNA localizing the
AD domain upstream of the reporter gene,
leading to the expression of reporter gene.

5. .

6. RECOVERY OF
INFORMATION:
Once the selection has been performed, the
primary structure of the proteins which
display the appropriate characteristics must
be determined. This is achieved by retrieval
of the protein-encoding sequences (as
originally inserted) from the cells showing the
appropriate phenotype.

7. Advantages of Y2H
system:-
1. Its low tech, can be easily carried out in a
lab.
2. It helps in identification of interaction
partners.
3. Determination of crucial sequences for
interaction.
4.Determination of unknown protein function.

8. Limitations of Y2H system:-
1. A particular interaction may be absent in
yeast(like if a bacterial protein is tested, the
system may lack the chaperones important
for folding or interaction).
2. If test protein are not localized to the
nucleus, interacting proteins may show a
negative result.
3. Fusion proteins may inhibit the original
interaction.

9. Chromatin
Immunoprecipitation(ChI
P)
It is a type of immunoprecipitation
experimental technique used to
investigate the interaction between
proteins and DNA in the cell. It aims to
determine whether specific proteins are
associated with specific genomic regions,
such as transcription factors on promoters
or other DNA binding sites.

10. Procedure:
1) DNA and associated proteins on chromatin
in living cells or tissues are cross linked.
2) The DNA-protein complexes are then
sheared into ~500 bp DNA fragments by
sonication or nuclease digestion.
3) Cross-linked DNA fragments associated
with the protein(s) of interest are selectively
immunoprecipitated from the cell debris using
an appropriate protein-specific antibody.

11. 4)The associated DNA fragments
are purified and their sequence is
determined.
5)Enrichment of specific DNA
sequences represents regions on
the genome that the protein of
interest is associated with in vivo.

12. .

13. Types of ChIP :-
1. Cross-linked ChIP
Cross-linked ChIP is mainly suited for mapping the
DNA target of transcription factors or other
chromatin-associated proteins, and uses reversibly
cross-linked chromatin as starting material.
2. Native ChIP
Native ChIP is mainly suited for mapping the
DNA target of histone modifiers. Generally, native
chromatin is used as starting chromatin.

14. ChIP Limitations:
1)It cannot distinguish between transcription
factor isoforms.
2)Large scale assays are challenging, as
antibodies for each TF needs to be made.
3) ChIP does not distinguish between a single
protein and a complex.
4) The results from ChIP cannot be viewed or
analyzed directly, so further protocols such as
Polymerase Chain Reaction (PCR).