Research proposal

M.SC. Research proposal: Epigenetic modification of AVT during the critical

Student Name: Benny (Ben) Ami Goldenberg
Instructor:
Department: Animal science - volcani
Faculty: Agricultural faculty in rehovot
Lab Tel:

Subject of the Research

Stress response is a learned entity. Exposure to stress during the critical period of
chick's postnatal development induces stress tolerance later in life. Although the
phenotype of stress tolerance is clear the molecular mechanism underlying these
phenomena is unknown. The working hypothesis which will be investigated in this
proposal is that there is a printing of an epigenetic code that induces long term
adaptation to extreme climatic conditions. To this end I will evaluate the epigenetic
regulation of the stress related hormone argentine-vasotocine (AVT) during heat
exposure and heat-response-memory, concentrating on the methylation pattern of
itsTo elucidate the role of epigenetic modification on the promoter and on the post
translation modifications of histones of the AVT gene during conditioning of chicks
to extreme temperature rangeat the AVT promoter. It is speculated that during
conditioning in the critical period of a chick's life there is a printing of an epigenetic
code that induce long term adaptation to postnatal climatic conditions. We will try to
asses if this is true for the AVT gene and to speculate on the mechanism of action.

Major goals of research project

1. Evaluate difference in AVT mRNA levels in heat conditioned chicks
compared to unconditioned chicksage matched counterparts.
2.
3. Asses the epigenetic difference in the AVT promoter and histones
between those two groups in their basal levels and during heat challenge. Pay
close attention to TF binding sites like CREB and AP-1.
4.

Specific objectives:
1. To determine the mRNA levels of the stress hormone AVT during
thermal control establishmentCondition chicks in day 3 post hatch to extreme
temperatures in different time frames challenge those chicks in day 10. Chicks
are then , during heat stress and during heat challenge of chicks that were
previously heat conditioned. (This stage will be performed using real time
PCR)scarified and brain tissue kept in 'RNA later'.
2. Extract DNA and RNA from brain tissue. Determine levels of mRNA
between 10 day chicks and 3 day chicks using Real-Time-PCR both basal
levels of mRNA and levels of mRNA during challenge between conditioned
and unconditioned chicks.
3. To asses the methylation levels of CpG dinucleotide at the promoter of
AVT in the frontal hypothalamus during heat control establishment and
memory using the bisulphate technology.We will react DNA with Bisulfate
reagent, this reaction turn every un methylated cytosine to uracil, DNA then
 goes thru sequencing or MSP (use two sets of primers to determine
methylation).
4.
5. To asses epigenetic modification of on Lysine 14 of histones histone 3 we will
useat the promoter of AVT during heat termotolerance establishment and memory,
using the Chromatin Immunoprecipitation (CHIP) methodologyduring which
chromatin goes thru sonication
and precipitated using antibodies that react with the modified histones.
6. Using Real-Time-PCR we will quantify the levels of the gene.

Relevance to Biotechnology
The growing demand for improvements in poultry animal welfare on one hand and
poultry production on the other requires an understanding of the mechanisms
governing stress responses. One of the major stressors affecting animal welfare and
hence, the poultry industry is thermal responses. This project aims on understanding
the epigenetic mechanisms underlying thermal stress responses, relating alteration in
the epigenetic code with direct effects on poultry physiology.

Loss of birds due to heat stress is a major loss to the industry. Birds are homeotherms
(after hatch) that regulate their temperature and grow best in temperatures between
18C-20C. To insure productivity farmers use to cool enclosure or, more recently, try
to condition chicks to higher temperature during their critical period. Those methods
are not cost affective to the farmers. Understanding the mechanism that governs brain
.plasticity in thermoregulation will help finding better solutions
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