How do we know that promoter and enhancer sequences control the initiation of transcription in eukaryotes?

How do we know that the orientation of promoters relative to the transcription start site is important while enhancers are orientation independent?

How do we know that eukaryotic transcription factors bind to DNA sequences at or near promoter regions?

How do we know that there is an association between disease susceptibility in humans and regulatory DNA sequences?

Write a short essay describing how cis-acting regulatory elements, activators, and chromatin modifiers are all coordinately involved in regulating transcription initiation.

What features of eukaryotes provide additional opportunities for the regulation of gene expression compared to bacteria?

Provide a definition of chromatin remodeling, and give two examples of this phenomenon.

Describe the organization of the interphase nucleus. Include in your presentation a description of chromosome territories, interchromatin compartments, and transcription factories.

A number of experiments have demonstrated that areas of the genome that are transcriptionally inactive are also resistant to DNase I digestion. However, transcriptionally active areas are DNase I sensitive. Describe how DNase I resistance or sensitivity might indicate transcriptional activity.

Provide a brief description of two different types of histone modification and how they impact transcription.

Present an overview of the manner in which chromatin can be remodeled. Describe the manner in which these remodeling processes influence transcription.

Chromatin remodeling by the SWI/SNF complex requires hydrolysis of ATP. What purpose does this serve?

Explain how the addition of acetyl groups to histones leads to a weaker association of DNA in nucleosomes.

Distinguish between the cis-acting regulatory elements referred to as promoters and enhancers.

Enhancers can influence the transcription of genes far away on the same chromosome. How are the effects of enhancers restricted so that they do not exert inappropriate transcriptional activation of non-target genes?

Describe the manner in which activators and repressors influence the rate of transcription initiation. How might chromatin structure be involved in such regulation?

Compare the control of gene regulation in eukaryotes and bacteria at the level of initiation of transcription. How do the regulatory mechanisms work? What are the similarities and differences in these two types of organisms in terms of the specific components of the regulatory mechanisms?

Many promoter regions contain CAAT boxes containing consensus sequences CAAT or CCAAT approximately 70 to 80 bases upstream from the transcription start site. How might one determine the influence of CAAT boxes on the transcription rate of a given gene?

Research indicates that promoters may fall into one of two classes: focused or dispersed. How do these classes differ, and which genes tend to be associated with each?

Explain the features of the Initiator (Inr) elements, BREs, DPEs, and MTEs of focused promoters.

Many transcriptional activators are proteins with a DNA-binding domain (DBD) and an activation domain (AD). Explain how each domain contributes to transcriptional initiation. Would you expect repressors to also have each of these domains?

How do the ENCODE data vastly help determine which enhancers regulate which genes?

DNA supercoiling, which occurs when coiling tension is generated ahead of the replication fork, is relieved by DNA gyrase. Supercoiling may also be involved in transcription regulation. Researchers discovered that enhancers operating over a long distance (2500 bp) are dependent on DNA supercoiling, while enhancers operating over shorter distances (110 bp) are not so dependent [Liu et al. (2001). Proc. Natl. Acad. Sci. USA 98:14,883–14,888]. Using a diagram, suggest a way in which supercoiling may positively influence enhancer activity over long distances.

Because the degree of DNA methylation appears to be a relatively reliable genetic marker for some forms of cancer, researchers have explored the possibility of altering DNA methylation as a form of cancer therapy. Initial studies indicate that while hypomethylation suppresses the formation of some tumors, other tumors thrive. Why would one expect different cancers to respond differently to either hypomethylation or hypermethylation therapies?

Explain how the following mutations would affect transcription of the yeast GAL1 gene in the presence of galactose.

A deletion within the GAL4 gene that removes the region encoding amino acids 1 to 100.

Explain how the following mutations would affect transcription of the yeast GAL1 gene in the presence of galactose.

A deletion of the entire GAL3 gene.

Explain how the following mutations would affect transcription of the yeast GAL1 gene in the presence of galactose.

A mutation within the GAL80 gene that blocks the ability of Gal80 protein to interact with Gal3p.

Explain how the following mutations would affect transcription of the yeast GAL1 gene in the presence of galactose.

A deletion of one of the four UAS_G elements upstream from the GAL1 gene.

Explain how the following mutations would affect transcription of the yeast GAL1 gene in the presence of galactose.

A point mutation in the GAL1 core promoter that alters the sequence of the TATA box.

The interphase nucleus is a highly structured organelle with chromosome territories, interchromatin compartments, and transcription factories. In cultured human cells, researchers have identified approximately 8000 transcription factories per cell, each containing an average of eight tightly associated RNAP II molecules actively transcribing RNA. If each RNAP II molecule is transcribing a different gene, how might such a transcription factory appear? Provide a simple diagram that shows eight different genes being transcribed in a transcription factory and include the promoters, structural genes, and nascent transcripts in your presentation.