Practice Exam III: Q5

Progressive process

pluripotenet stem cells lose their developmental potency and commit to a fate through a series of genetically controlled steps

Fate specified, committing to a fate, different gene activity, transcription factors 

Process of Determination/Specification

Morphogenesis, Realization of fate, Maintenance of cell fate, Structural genes

Fully Differentiated Cell

pluripotent stem cells lose their developmental potency and commit to a fate through a series of genetically controlled steps

a. Describe how development is a progressive process. What are two key differences between a cell that is in the process of determination/specification, and a fully differentiated cell?

multiple enhancers can regulate the same gene, but at different times/in types of tissue

(Expression is governed by tissue-specific enhancers that are uniquely open depending on the tissue and/or only the TFs that would bind within that enhancer region are present in the tissue/cells)

b. How is the expression of a gene across tissues determined by regulatory enhancers?

Reporter Assays and ChIP-Seq

c. What are the two experimental methods that were learned in class to study enhancer dependency?

Actin enhancer fused to lacZ coding sequence and look for beta-gal expression pattern

Reporter assay

Chromatin Immunoprecipitation Sequencing (ChIP-Seq)

A technique used to map the exact physical binding locations of transcription factors (TFs) or histone modifications across the entire genome.

Fix cells, cross-link protiens to DNA and fragment through sonication

Pulldown, use specific antibodies to immunoprecipitate target protein-DNA complexes

Sequence & Map, Purify the DNA, sequence fragments and align them to a reference genome

Peak Analysis, identify high-intensity “peaks” to find exact binding sites relative to gene loci or TSS

• (F)ix cells

• (S)onicate to fragment DNA

• use (A)ntibodies specific to a histone mark or spcecific TF

• (P)ull-down / immunoprecipitate only the DNA fragments that had marks present/TFs bound release the Ab,

• (S)equence the fragments and map them all back to the genome

• look at the intensity/peak of where the most fragments aligned (software/peak-calling programs);

• compare (P)eaks to known gene loci/TSS