A particular type of anemia in humans, called β-thalassemia, results from a severe reduction or absence of the normal β-globin chain of hemoglobin. However, the γ-globin chain, normally only expressed during fetal development, can functionally substitute for β-globin. A variety of studies have explored the use of the nucleoside 5-azacytidine for the expression of γ-globin in adult patients with β-thalassemia.

How might 5-azacytidine lead to expression of γ-globin in adult patients?

A particular type of anemia in humans, called β-thalassemia, results from a severe reduction or absence of the normal β-globin chain of hemoglobin. However, the γ-globin chain, normally only expressed during fetal development, can functionally substitute for β-globin. A variety of studies have explored the use of the nucleoside 5-azacytidine for the expression of γ-globin in adult patients with β-thalassemia.

Explain why this drug may also have some adverse side effects.

Regulation of the lac operon in E. coli and regulation of the GAL system in yeast are analogous in that they both serve to adapt cells to growth on different carbon sources. However, the transcriptional changes are accomplished very differently. Consider the conceptual similarities and differences as you address the following.

Compare and contrast the roles of the lac operon inducer in bacteria and Gal3p in eukaryotes in the regulation of their respective systems.

Regulation of the lac operon in E. coli and regulation of the GAL system in yeast are analogous in that they both serve to adapt cells to growth on different carbon sources. However, the transcriptional changes are accomplished very differently. Consider the conceptual similarities and differences as you address the following.

Compare and contrast the cis-regulatory elements of the lac operon and GAL gene system.

Regulation of the lac operon in E. coli and regulation of the GAL system in yeast are analogous in that they both serve to adapt cells to growth on different carbon sources. However, the transcriptional changes are accomplished very differently. Consider the conceptual similarities and differences as you address the following.

Compare and contrast how these two systems are negatively regulated such that they are downregulated in the presence of glucose.

DNA methylation is commonly associated with a reduction of transcription. The following data come from a study of the impact of the location and extent of DNA methylation on gene activity in eukaryotic cells. A bacterial gene, luciferase, was inserted into plasmids next to eukaryotic promoter fragments. CpG sequences, either within the promoter and coding sequence (transcription unit) or outside of the transcription unit, were methylated to various degrees, in vitro. The chimeric plasmids were then introduced into cultured cells, and luciferase activity was assayed. These data compare the degree of expression of luciferase with differences in the location of DNA methylation [Irvine et al. (2002). Mol. and Cell. Biol. 22:6689–6696]. What general conclusions can be drawn from these data? 

During an examination of the genomic sequences surrounding the human β-globin gene, you discover a region of DNA that bears sequence resemblance to the glucocorticoid response element (GRE) of the human metallothionein IIA (hMTIIA) gene. Describe experiments that you would design to test

(1) whether this sequence was necessary for accurate β-globin gene expression and

(2) whether this sequence acted in the same way as the hMTIIA gene's GRE.

Marine stickleback fish have pelvic fins with long spines that provide protection from larger predatory fish. Some stickleback fish were trapped in lakes and have adapted to life in a different environment. Many lake populations of stickleback fish lack pelvic fins. Shapiro et al. (2004) (Nature 428:717.723) mapped the mutation associated with the loss of pelvic fins to the Pitx1 locus, a gene expressed in pelvic fins, the pituitary gland, and the jaw. However, the coding sequence of the Pitx1 gene is identical in marine and lake stickleback [Chan et al. (2010). Science 327:5963,302–305]. Moreover, when the Pitx1 coding region is deleted, the fish die with defects in the pituitary gland and the jaw, and they lack pelvic fins. Explain how a mutation near, but outside of, the coding region of Pitx1 may cause a loss of pelvic fins without pleiotropic effects on the pituitary gland and jaw.

Although a single activator may bind many enhancers in the genome to control several target genes, in many cases, the enhancers have some sequence conservation but are not all identical. Keeping this in mind, consider the following hypothetical example:

- Undifferentiated cells adopt different fates depending on the concentration of activator protein, Act1.

- A high concentration of Act1 leads to cell fate 1, an intermediate level leads to cell fate 2, and low levels to cell fate 3.

- Research shows that Act1 regulates the expression of three different target genes (A, B, and C) with each having an enhancer recognized by Act1 but a slightly different sequence that alters the affinity of Act1 for the enhancer. Act1 has a high affinity for binding the enhancer for gene A, a low affinity for the gene B enhancer, and an intermediate affinity for the gene C enhancer.

From these data, speculate on how Act1 concentrations can specify different cell fates through these three target genes? Furthermore, which target genes specify which fates?

Hereditary spherocytosis (HS) is a disorder characterized by sphere-shaped red blood cells, anemia, and other abnormal traits. Ankyrin-1 (ANK1) is a protein that links membrane proteins to the cytoskeleton. Loss of this activity is associated biochemically to HS. However, Gallagher et al. (2010) (J. Clin. Invest. 120:4453–4465) show that HS can also be caused by mutations within a region from -282 to -101 relative to the transcriptional start site, which lead to constitutive transcriptional repression in erythroid cells due to local chromatin condensation. Propose a hypothesis for the function of the -282 to -101 region of the ANK1 gene.

Transcription factors play key roles in the regulation of gene expression, but to do so, they must act within the nucleus. Like most proteins, however, transcription factors are translated in the cytoplasm. To enter the nucleus, transcription factors contain nuclear localization signals, which in some cases can work only when bound to some other molecule such as a steroid hormone. After entering the nucleus, transcription factors must bind to appropriate DNA sites and must interact with other transcription proteins at promoters, enhancers, and silencers. Transcription factors then activate or repress transcription through their activation or repression domains. Many drug therapies target transcription factors. Based on the information provided above, suggest three specific mechanisms through which a successful drug therapy, targeted to a transcription factor, might work.