Question

Exercise A student interested in the regulation of facultative heterochomatin needs to optimize the experimental conditions of her chromatin immunoprecipitation (Chip) assay using heart tissue. For this, she crosslinks mouse heart tissue with 1% formaldehyde for either 10 or 30 minutes. The student then performs Chip with the RING1B and SUZ12 antibodies. RING1B and SUZ12 belong to the Polycomb Repressor complex 1 (PRC1), which maintains genes silenced in specific tissues. The enriched DNA is quantified by real-time PCR using primers to the indicated genes. The amount of immunoprecipitated DNA in each sample is represented as normalized signal to the negative locus GAPDH, which is equal to 1. 010 30 Input Normalized Perce FUNGIB 22 p Rato mb 5804 SUZ12 ore pe Rabbimib 3737 Questions: A. Why does the student consider GAPDH a negative locus, i.e. where RING1B and SUZ12 do not bind? Ans: B. What is the transcriptional status of the HoxD10 and HoxA1 genes in the heart? In one sentence explain your reasoning Ans: C. If you were this student, which fixation condition (10 or 30 min) would you use? In one sentence explain your reasoning Ans:

Answer 1

1) The GAPDH is capable of binding to the DNA telomeric sequences, offering them protection and regulation. The locus does not correspond to DNA binding domain of RING1 and SUZ12 genes that often functional as a transcriptional repressor.

GAPDH is stably expressed in almost all the human tissues and cells and hence it is considered as a housekeeping gene or an endogenous gene.

Being a housekeeping gene the expression of GAPDH would remain constant throughout the cell cycle that gives a constant normalized signal in during Chromatin immunoprecipitation.

All these factors make it suitable for being used as a negative locus for the experiment.

2) HoxD10 and HoxA1 genes consist of DNA binding domain (homeobox). Besides possessing a strong DNA binding affinity, the increased normalized percent input at 30 minutes in comparison to the 10 minute cross-linking duration show that HoxD10 and HoxA1 genes are weakly transcribed in heart tissues of mouse.

3)In general, the cross-linking should be done only for short duration (~ 10 minutes), as longer duration causes decreased availability antigen and reduces the efficiency of chromatin-shearing step. Prolonged cross-linking is only required in case of proteins with weak DNA binding affinity.  

If I were a student I will use 10minutes fixation condition since it will increase specificity and accuracy as HoxD10 and Hox A1 are strong DNA  binding proteins.