Comparing Gene Regulation in Prokaryotes and Eukaryotes
The lac operon provides an excellent example of how bacteria perform gene regulation in response to an environment that lacks glucose yet contains lactose. In the case of the lac operon, we learned that gene regulation involves an activator protein (CAP) and a repressor protein (lac repressor). Effector molecules (cAMP and allolactose) regulate CAP and the lac repressor binding to regulatory DNA sequences (CAP site, operator) near the promoter for the lac operon. Ultimately the binding of the CAP and the lac repressor proteins determine if the sigma (σ) factor protein and the RNA polymerase core enzyme can activate transcription.
Even though gene regulation in prokaryotes and eukaryotes is similar (e.g., both involve activator proteins, repressor proteins, and effector molecules), eukaryotic gene regulation is more complex. This complexity is needed to produce multicellular eukaryotic organisms with cells in each tissue having unique phenotypes. For example, a white blood cell (leukocyte) and a muscle cell have the same collection of structural genes; however, gene regulation ensures that a leukocyte expresses leukocyte-specific proteins, while a muscle cell expresses muscle-specific proteins. Further, many eukaryotic organisms progress from a fertilized egg through complex developmental stages to produce the mature adult. Gene regulation ensures that embryonic genes are expressed only during embryonic development, while other genes are expressed only in an adult.
Regulation of a typical eukaryotic gene involves combinatorial control. For example, a single eukaryotic gene can be regulated by a combination of:
- Activator proteins binding to enhancer DNA sequences.
- Repressor proteins binding to silencer DNA sequences.
- Regulation of activator and repressor protein function. This regulation of activator and repressor proteins involves effector molecules, covalent modification, and protein-protein interactions.
- Modifying the structure of chromatin to activate or repress transcription. Modifying chromatin involves chemically modifying histone proteins or altering the arrangement of nucleosomes near the core promoter of a gene.
- DNA methylation to silence transcription. The methylation of cytosine bases near the core promoter region of a gene inhibits transcription.
- What is meant by combinatorial control?
- What combinations of processes influence the transcription of a eukaryotic gene?
Core Promoter vs. Regulatory Promoter
We learned in Part 9 that transcription in eukaryotes involves several types of DNA sequences. The core promoter, for example, determines where RNA polymerase II will bind to the DNA and begin transcription. The core promoter includes the TATA box (-25 sequence), which serves as the binding site for the general transcription factor protein TFIID and the +1 site, the first base in the template DNA strand that is transcribed by RNA polymerase II. For transcription to occur, the TATA box and the +1 site must be present. If these two sequences are the only sequences present upstream of a gene, the gene will be transcribed at a low, yet constant rate (the so-called basal level of transcription).
In addition to the core promoter, many eukaryotic genes also include a regulatory promoter (see figure 14.1). The components of the regulatory promoter are required for transcription levels higher than the basal level provided by the core promoter. A common regulatory promoter component that is present in many eukaryotic genes is the CAAT box. The CAAT box is located at -80 and has the sequence 5’-GGCCAATCT-3’. Another common regulatory promoter component is a GC box (5’- GGGCGG – 3’) located at -100. The CAAT and GC boxes are the binding sites for certain activator proteins. Thus, the CAAT and GC boxes can be considered enhancers adjacent to many eukaryotic structural genes.
- What is meant by basal transcription?
- What is the function of the regulatory promoter?
- What are the names of two common DNA sequences found in the regulatory promoters of eukaryotic genes?
General and Regulatory Transcription Factors
Eukaryotic transcription factors are proteins that influence the ability of RNA polymerase II to bind to a eukaryotic core promoter. There are two categories of transcription factor proteins:
- General transcription factor proteins (GTFs). The general transcription factor proteins include the TFIID, TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH proteins described in Part 9. These proteins function to recruit RNA polymerase II to the core promoter to begin transcription. The general transcription factors are required for all transcription events. If these general transcription factors are the only proteins involved, the gene is transcribed at the basal level. The general transcription factors are also required for transcription rates above this basal level.
- Regulatory transcription factor proteins. Regulatory transcription factors function to regulate transcription by either increasing transcription above the basal level or decreasing transcription below the basal level. An activator protein increases the level of transcription above the basal level; a repressor protein decreases the level of transcription below the basal level. Many activator and repressor proteins are only expressed in certain tissues or at certain times during development, thus playing a critical role in tissue-specific or time-specific gene expression.
Transcription factors proteins are trans-acting factors (i.e., can regulate genes found throughout the genome) and bind to DNA sequences called cis-acting elements (i.e., the DNA binding sites for these transcription factors tend to be near the genes they control) (see figure 14.2). However, these cis-acting elements do not need to be immediately adjacent to the core promoter. Some transcription factor binding sites can be within the gene that they control or can be thousands of base pairs away.
Recall that the mediator protein complex communicates the signals from activator and repressor proteins to RNA polymerase II. Mediator thus serves as a link between transcription factors that bind to enhancer and silencer DNA sequences and RNA polymerase II, thereby determining the overall rate of transcription.
- Review the functions of TFIID, TFIIH, and mediator.
- Which transcription components are considered trans-acting factors?
- Which transcription components are considered cis-acting elements?
Enhancers and Silencers
Other regulatory DNA sequences assist the core promoter and regulatory promoter to regulate transcription by serving as the binding sites for transcription factor proteins. The binding of regulatory transcription factors to these DNA sequences may:
- Increase the rate of transcription. Transcription can increase 10 to 1000-fold when activator proteins bind to enhancer DNA sequences (up-regulation). Activator proteins and enhancer DNA sequences are generally responsible for tissue-specific expression of a gene.
- Decrease the rate of transcription. Transcription can decrease below the basal level when repressor proteins bind to silencer DNA sequences (down-regulation). Repressor proteins and silencer DNA sequences are generally responsible for tissue-specific repression of a gene.
A particular gene can be regulated by many transcription factors bound to different combinations of enhancers and silencers (see figure 14.2). The combination of the transcription factor proteins and regulatory DNA sequences involved determines the transcription pattern of the gene.
- Review the functions of activator proteins, repressor proteins, enhancer DNA sequences, and silencer DNA sequences (see Part 9).
Structural Features of Transcription Factors
Transcription factor proteins have been identified in many organisms, including viruses, bacteria, fungi, plants, and animals. Nearly all transcription factor proteins contain conserved structural features that are important in either binding to regulatory DNA sequences, effector molecules, or other transcription factor proteins. For example, most transcription factor proteins contain α-helices, a type of protein secondary structure. An α-helix is produced when certain amino acids in the polypeptide sequence interact through hydrogen bonding to produce a helical structure. An α-helix is the proper width to bind to the major groove in DNA. Thus, the α-helix is often used by transcription factors proteins to recognize specific base pair sequences located in the major groove of the DNA.
Four common structural motifs are found in transcription factor proteins. These structural motifs, based upon the α-helix structure described above, include (see figure 14.3):
- Helix-turn-helix (HTH) motif. The helix-turn-helix motif is found in both prokaryotic and eukaryotic transcription factor proteins. The HTH motif includes two α-helices separated by a “turn” of 3-4 amino acids. One α-helix is called the recognition helix, and functions to bind to specific base pair sequences in the DNA major groove. This recognition helix also includes basic (positively charged) amino acids that bind to the negatively charged DNA backbone. Many of the transcription factor proteins that we have discussed previously contain the HTH motif including sigma (σ) factor, the lac repressor protein, and the catabolite activator protein (CAP).
- Basic helix-loop-helix (bHLH) motif. The bHLH motif is similar to the helix-turn-helix motif, containing a recognition helix that binds to the DNA major groove. However, instead of a turn, bHLH transcription factors have a long amino acid loop to connect two α-helices. bHLH transcription factors play an important role in the differentiation of cells. For example, the MyoD and c-myc proteins are transcription factor proteins that contain the bHLH motif. MyoD activates muscle-specific genes, while c-myc activates genes involved in cell division.
- Zinc finger motif. The zinc finger motif is composed of a finger-like structure composed of an α-helix (i.e., the recognition helix) and two β-strands (another type of protein secondary structure). Electrostatic interactions between zinc ions (Zn2+) and negatively charged amino acid side chains within the transcription factor protein stabilize the zinc finger motif. Steroid hormone receptors, including the glucocorticoid receptor factor protein (see below), testosterone receptor protein, and the estrogen receptor protein contain zinc finger motifs.
- Leucine zipper motif. The leucine zipper motif not only contains a recognition helix, but also contains a second α-helix with many hydrophobic leucine amino acids. When the leucine-rich regions of two leucine zipper transcription factors interact, they form a coiled-coil to exclude water. The coiled-coil resembles a zipper with interlocking leucine amino acids. The DNA binding site is recognized by recognition helices that extend from the coiled-coil region of these two transcription factor proteins. The CREB protein (see below) contains a leucine zipper motif.
It is important to note that all four transcription factor motif structures described above permit transcription factor proteins to bind to each other. Two identical transcription factor proteins can interact to form a transcription factor homodimer, or two different transcription factor proteins can interact to form a heterodimer. For example, both the CAP protein and the lac repressor proteins are homodimers, composed of two identical transcription factor protein with HTH motifs. Higher order interactions (trimers, tetramers) are also possible when transcription factor proteins bind to each other.
- What are three examples of transcription factor proteins that contain the helix-turn-helix (HTH) motif?
- What are two examples of transcription factor proteins that contains the basic helix-loop-helix (bHLH) motif?
- What are three examples of transcription factor proteins that contains the zinc finger motif?
- What is an example of a transcription factor protein that contains the leucine zipper motif?
- What protein secondary structure is found in all transcription factor structural motifs?
- What is meant by a transcription factor homodimer or heterodimer?
Mechanisms to Regulate Transcription Factor Proteins
If an activator protein is present in a cell, it does not always bind to an enhancer DNA sequence and up-regulate transcription. Similarly, a repressor protein does not always bind to a silencer DNA sequence and repress transcription. The DNA-binding activities of activator and repressor proteins are regulated in three ways:
- Effector binding. Small effector molecules can bind to transcription factor proteins, change the conformation (shape) of the transcription factor, and influence the ability of the transcription factor protein to bind to enhancer or silencer DNA sequences. In animals, steroid hormones such as glucocorticoid, testosterone, and estrogen are effector molecules that regulate the functions of transcription factor proteins.
- Transcription factor dimerization. The formation of transcription factor homodimers or heterodimers influences binding to enhancer or silencer DNA sequences.
- Covalent modification. The addition of phosphate groups (phosphorylation) to activator or repressor proteins can stimulate binding to enhancer or silencer DNA sequences.
Note that for a particular gene, one or more of the above mechanisms may be involved in regulating gene expression. For example, the glucocorticoid receptor transcription factor protein (see below) is regulated by effector binding and dimerization, while the CREB transcription factor protein is regulated by dimerization and covalent modification.
- Describe the three ways that activator and repressor proteins can be regulated.
- What are three examples of eukaryotic effector molecules?
Regulating Transcription Through TFIID
Regulatory transcription factor proteins (activator and repressor proteins) influence the ability of RNA polymerase II to transcribe a gene. However, these regulatory transcription factor proteins do not typically bind to RNA polymerase II directly. Instead, transcription factor proteins communicate DNA binding indirectly to RNA polymerase II through other protein complexes. Eukaryotic regulatory transcription factors influence RNA polymerase II activity through TFIID, mediator, the enzymes involved in chromatin remodeling, and the enzymes involved in DNA methylation.
Consider the regulation of RNA polymerase II through the TFIID protein. Recall that TFIID is the general transcription factor protein that binds to the TATA box (the -25 sequence) within the core promoter. TFIID recruits the other five general transcription factors (TFIIA, TFIIB, TFIIF, TFIIH, and TFIIE) that bring RNA polymerase II to the +1 site and activate RNA polymerase II to begin transcription. Suppose an activator protein binds to an enhancer DNA sequence (see figure 14.4). This activator protein then encourages TFIID to bind to the TATA box, and TFIID then recruits the other general transcription factors and RNA polymerase II to the +1 site. As a result, transcription is up-regulated. Suppose instead that a repressor protein binds to a silencer DNA sequence adjacent to a gene. The repressor protein then prevents TFIID from binding to the TATA box. The absence of TFIID on the core promoter prevents the other general transcription factors and RNA polymerase II from binding to the core promoter. As a result, transcription is down-regulated.
- How do activator and repressor proteins influence TFIID?
Regulating Transcription Through Mediator
Mediator is a protein complex that mediates the interaction between the regulatory transcription factors (i.e., activator and repressor proteins) and RNA polymerase II. If mediator activates RNA polymerase II, transcription begins. Suppose an activator protein binds to an enhancer DNA sequence (see figure 14.5). The activator protein in turn activates mediator, and mediator then activates the general transcription factor protein TFIIH. Next, TFIIH acts as a helicase to separate the template and coding DNA strands. TFIIH also acts as a kinase, phosphorylating RNA polymerase II to begin transcription. Suppose a repressor protein binds to a silencer DNA sequence instead. The repressor protein then inhibits the activity of mediator. As a result, mediator fails to activate TFIIH, and TFIIH fails to separate the template and coding DNA strands. TFIIH also fails to phosphorylate RNA polymerase II, preventing the initiation of transcription. Note that the DNA between the enhancer/silencer DNA sequences and the core promoter can form a loop to permit the proteins described above to bind to each other.
- How do activator and repressor proteins influence the activity of mediator?
Transcription Activation Using the Glucocorticoid Receptor
Now let's apply what we have learned so far to two examples of gene regulation in the human body. The first example shows how steroid hormones produced by endocrine glands can activate the transcription of genes. For example, glucocorticoid hormones (GCs) are released by the adrenal glands in response to fasting, as well as physical activity. The GCs lead to an increase in glucose synthesis, an increase in protein metabolism, an increase in fat metabolism, and a decrease in inflammation. Glucocorticoid hormones can increase the transcription of a gene above the basal level as follows (see figure 14.6):
- The glucocorticoids are steroid hormones, which are nonpolar in structure. As a result, the glucocorticoids cross the cytoplasmic membrane and enter the cytoplasm of a target cell.
- Glucocorticoids act as effector molecules by binding to an activator protein called glucocorticoid receptor that is found in many cell types. Prior to glucocorticoid binding, the glucocorticoid receptor is bound to HSP90 proteins. HSP90 helps maintain the proper three-dimensional shape of the glucocorticoid receptor, so that glucocorticoid receptor can bind to glucocorticoid hormones produced by the adrenal glands. HSP90 is released when glucocorticoid hormone binds to glucocorticoid receptor.
- Glucocorticoid binding changes the conformation of glucocorticoid receptor, exposing a nuclear localization signal (NLS). The NLS is a polypeptide sequence that helps to target the glucortocoid receptor (with bound glucocorticoid) to the nucleus of the cell.
- Two glucocorticoid receptors with bound glucocorticoid hormones form a homodimer in the cytoplasm of the cell.
- The glucocorticoid receptor:glucocorticoid homodimer travels to the nucleus of the cell.
- The glucocorticoid receptor:glucocorticoid homodimer binds to two adjacent enhancer DNA sequences called glucocorticoid response elements (GREs). GREs are common enhancers found adjacent to many genes involved in metabolism.
- The glucocorticoid receptor: glucocorticoid bound to the GRE activates transcription.
Other steroid hormones, such as estrogen and testosterone, are effector molecules that activate transcription by binding to cytoplasmic transcription factor proteins.
- How does the production of glucocorticoid by an adrenal gland lead to transcriptional activation of a target gene?
Transcription Activation via CREB
Unlike glucocorticoid, many signaling molecules in the body, such as peptide hormones, growth factor proteins, and cytokine proteins, are not able to diffuse through the cytoplasmic membrane into the cytoplasm of the target cell. Instead, these signaling proteins bind to cell receptors on the surface of a target cell, and the receptor binding signal is then transmitted to the nucleus to activate transcription. Our second example of gene regulation demonstrates how transcription is up-regulated when receptor binding activates the transcription factor protein cAMP response element-binding protein (CREB). Transcription activation via CREB occurs when (see figure 14.7):
- A receptor protein embedded in the cytoplasmic membrane binds to a peptide hormone, growth factor, or cytokine protein.
- Receptor binding activates a G protein.
- The G protein activates adenylyl cyclase inside the cell, which converts ATP into cAMP.
- cAMP binds to and activates protein kinase A (PKA).
- PKA moves into the nucleus and phosphorylates an inactive CREB protein homodimer.
- The phosphorylated CREB protein homodimer binds to two adjacent enhancer sequences called cAMP response elements (CREs).
- The CREB homodimer bound to the CREs activates transcription.
- What is CREB and CRE?
- How does the binding of a signaling protein to a receptor lead to transcriptional activation of a target gene via the CREB pathway?
Chromosome Compaction and Transcription
The arrangement of nucleosomes on the DNA can also influence the transcription of a nearby gene (for a review of nucleosomes, refer to Part 2). For a gene to be transcribed, RNA polymerase II must be able to bind to the core promoter. If the core promoter region of a gene contains tightly packed nucleosomes (heterochromatin), RNA polymerase II struggles to find the core promoter. As a result, the heterochromatin form of DNA is said to be in a closed conformation and transcription is limited. Regions of the chromosome with loosely packed or absent nucleosomes are called euchromatin (open conformation). RNA polymerase II can better access a core promoter located in euchromatin, and as a result, transcription occurs more readily.
Recall that chromatin is a dynamic structure with a specific gene alternating between the closed and open conformations depending on the needs of the cell. When an activator protein binds to an enhancer DNA sequence, chromatin is converted to the open conformation. When a repressor protein binds to a silencer DNA sequence, chromatin is converted to the closed conformation.
- Review the structure of a nucleosome and the terms heterochromatin and euchromatin (see Part 2).
- What is the difference between the open conformation and the closed conformation?
Arrangement of Chromatin at the β-globin Gene
As an example of how chromatin structure can influence the transcription of a gene, consider the human β-globin gene (see figure 14.8). The β-globin gene, which encodes the β-globin protein components of hemoglobin, is not normally expressed in many cell types, including fibroblast cells. When the DNA region that encompasses the β-globin gene from fibroblasts was analyzed with respect to nucleosomes, scientists discovered that nucleosomes were found every 200 base pairs (bp) from the -3000 to +1500 region of the gene. Note that this closed conformation region from -3000 to +1500 includes the regulatory promoter, core promoter, and the beginning portion of the β-globin gene. Thus, the β-globin gene in fibroblasts is not accessible to the general transcription factors (GTFs) and RNA polymerase II. As a result, the β-globin gene is transcriptionally silent in fibroblasts.
The β-globin gene is expressed in erythroblasts (precursor red blood cells). When the nucleosome arrangement surrounding the β-globin gene was examined in erythroblasts, a different result was observed. Nucleosomes were displaced from the -500 to +200 region of the β-globin gene in erythroblasts. This open conformation (euchromatin) area includes the regulatory promoter, core promoter, and the beginning portion of the β-globin gene. Thus, the GTFs and RNA polymerase II can access the promoter region in erythroblasts, leading to the transcription of the β-globin gene.
- In terms of the core promoter for the β-globin gene, describe the difference between chromatin structure in fibroblasts and erythroblasts.
The results from fibroblasts and erythroblasts discussed above suggest that nucleosomes can be altered to influence transcription. Alterations in chromatin structure to promote transcription include the covalent modification of histone proteins and the rearrangement of nucleosomes by ATP-dependent chromatin remodeling (see figure 14.9).
Covalent modification includes the acetylation of histone proteins within nucleosomes. Enzymes called histone acetyltransferases (HATs) add acetyl groups to the tails of histone proteins. Acetylation neutralizes the positive charge on lysine amino acids within the histone tail, disrupting the interaction between the histone tail and the negatively charged DNA backbone. As a result, neutralization of the positive charges on the histone tails causes the histones to release from the DNA; the DNA is now accessible for transcription. When transcription needs to be turned off, the histones can be modified using histone deacetylase (HDAC) proteins. HDACs remove the acetyl groups from histones, restoring the positive charge on the histone tail. The histone tails once again bind to the negatively charged DNA backbone, and the chromatin is converted from the open to the closed conformation (heterochromatin), decreasing transcription of the gene.
Note that when an activator protein binds to an enhancer DNA sequence, the activator recruits HATs to the promoter, activating transcription. Alternatively, when repressor proteins bind to silencer DNA sequences, HDACs are recruited to the promoter, silencing transcription.
ATP-dependent Chromatin Remodeling
The ATP-dependent chromatin remodeling process uses the energy in ATP to alter the spacing of the nucleosomes near a gene (see figure 14.9). One example of an ATP-dependent chromatin remodeling enzyme is a multi-subunit protein complex called SWI/SNF. SWI/SNF performs at least two types of chromatin remodeling:
- SWI/SNF can change the distribution of nucleosomes along the DNA, creating larger gaps between adjacent nucleosomes. When these larger gaps between nucleosomes includes the core promoter region of a gene, transcription in activated.
- SWI/SNF can replace the standard histone proteins (H2A, H2B, H3, and H4) within a nucleosome with histone variant proteins. The presence of these histone variant proteins within the modified nucleosome increases transcription.
- When a HAT is active, what effect does this have on transcription?
- When a HDAC is active, what effect does this have on transcription?
- What is the function of the SWI/SNF complex?
Overview of DNA Methylation
Silencing of gene expression in many eukaryotes involves the methylation of DNA sequences near the core promoters of genes. The methyl group that is added to the DNA double helix blocks the major groove of the DNA, preventing the binding of activator proteins to enhancer sequences in the DNA. Cytosine bases within CG-rich sequences called CpG islands are typically targets for DNA methylation. Not surprisingly, many CpG islands are located near the core promoters of genes (see figure 14.10). Typical CpG islands are 1,000 – 2,000 base pair (bp) long sequences that contain multiple CpG sites (i.e., many 5’-CG-3’ dinucleotide sequences in a row). Within CpG islands, adding methyl groups to the cytosine bases on both DNA strands is called full methylation. Full methylation inhibits transcription.
Housekeeping genes encode proteins that are required for the maintenance of a cell. For example, the structural genes that produce the enzymes involved in glycolysis are housekeeping genes The promoters of these housekeeping genes are typically unmethylated and as a result, housekeeping genes are always transcribed. Tissue-specific genes are only expressed in certain cell types. In cell types in which these genes are not expressed, the CpG island near the promoter is fully methylated. In cell types in which the gene is expressed, the CpG island near the promoter is unmethylated. As a final example, the inactive X chromosome (Barr body) in female mammals contains methylated CpG islands adjacent to most structural genes.
- How does methylation alter the structure of DNA?
- Where are many CpG islands located?
- In terms of DNA methylation, what is the difference between a housekeeping gene and a tissue-specific gene?
Methylation Blocks Activator Proteins and Recruits HDACs
DNA methylation is thought to silence the transcription of a nearby gene in two general ways. First, methylation at a CpG island near the promoter of a gene can block an activator protein from binding to an enhancer DNA sequence (see figure 14.11). DNA methylation inhibits activator binding because the methyl group on cytosine prevents the activator protein from binding to the DNA major groove in the enhancer sequence. Second, methylated CpG islands near promoters serve as the binding sites for methyl-CpG-binding proteins. When a methyl-CpG-binding protein binds to a methylated CpG island, the methyl-CpG-binding proteins recruit histone deacetylase (HDAC). HDAC then removes the acetyl groups from the histone tails, converting the promoter region of the gene into heterochromatin. Transcription of the nearby gene is therefore inhibited.
- Describe the two ways that DNA methylation can inhibit transcription.
DNA Methylation is Preserved During Cell Division
The DNA methylation pattern in the cell is established by a process called de novo methylation (see figure 14.12). De novo methylation converts unmethylated DNA to fully methylated DNA (i.e., both DNA strands are methylated). De novo methylation is a highly regulated process that is thought to occur during embryonic development or the differentiation of cells to form tissues. Unfortunately, the details of de novo methylation are currently poorly understood.
The DNA methylation pattern established during de novo methylation is preserved during cell division; if a CpG island is fully methylated in a cell prior to mitosis, the same CpG island is fully methylated in the two daughter cells at the conclusion of mitosis. Maintenance methylation ensures that the daughter cells produced by mitosis maintain the same methylation pattern as the parental cell. As an example, suppose that fully methylated DNA is replicated. Because the DNA replication machinery does not methylate nitrogenous bases in the DNA during replication, the daughter DNA strands produced do not contain methylated cytosine bases. Thus, the daughter double-stranded DNA molecules are initially hemimethylated, with a methylated parental strand and an unmethylated daughter DNA strand. This hemimethylated DNA is recognized by DNA methyltransferase, which subsequently methylates the cytosine bases on the daughter DNA strands, thus preserving the DNA methylation pattern established in the parental cell.
Methylation of DNA explains a genetic phenomenon called genomic imprinting. In oogenesis (egg cell formation) or spermatogenesis (sperm cell formation), a specific gene can be methylated by de novo methylation. Following fertilization, the methylation pattern is maintained as the fertilized egg begins to divide. For example, if the maternal allele for a gene is fully methylated by genomic imprinting, that maternal allele remains fully methylated in the cells of the offspring. We will discuss genomic imprinting more in Part 15.
- What is the difference between de novo and maintenance methylation?
- Which enzyme is responsible for maintenance methylation?
- What is meant by genomic imprinting?
In eukaryotes, the processes (activators/repressor proteins, altering chromatin structure, and DNA methylation) that regulate the expression of one structural genedo not necessarily influence the regulation of an adjacent gene. Insulator DNA sequences define the boundaries between genes (see figure 14.13); an insulator DNA sequence ensures that the gene regulation processes that affect one gene do not affect nearby genes. Insulator DNA sequences:
- Serve as the binding sites for proteins that act as physical barriers for the HATs, HDACs and SWI/SNF protein complexes. For example, suppose a gene is flanked by two insulator DNA sequences, and HATs modify histone tails and activate transcription of the gene. Because the proteins bound to insulators serve as physical barriers to the HATs, genes beyond the insulator sequences are not activated.
- Serve as the binding sites for proteins that limit the effects of enhancer/silencer sequences. Suppose that Gene A has an adjacent enhancer DNA sequence. Gene B is also near the enhancer DNA sequence. A protein bound to the insulator DNA sequence between Genes A and B ensures that the enhancer only activates Gene A; the transcription of Gene B is unaffected. Insulators can limit the effects of silencer DNA sequences in a similar way.
- What is an insulator DNA sequence?
- How do insulators ensure that gene regulation is limited to a single gene?
Part 14 Review
Fill in the blank:
- The core promoter consists of two consensus DNA sequences located at position ______________ and ______________.
- The general transcription factor (GTF) proteins are ________________________________________________________________.
- Some examples of regulatory transcription factor proteins are ____________________, which increase transcription and _________________________, which decrease transcription below basal levels.
- Transcription factor proteins contain structural motifs. Two transcription factors with the _____________________ motif interact and form a coiled coil. Two alpha-helices are part of a ______________________ motif seen in proteins involved in muscle cell differentiation.
- The interaction of two identical transcription factor proteins to produce one molecule is called a ________________________.
- One example of a steroid hormone is ________________________________________.
- A glucocorticoid receptor is bound to _________________ until a glucocorticoid molecule comes along and binds to the receptor.
- CREB is a (protein OR DNA sequence; circle the correct answer), whereas CRE is a (protein OR DNA sequence; circle the correct answer).
- Upon its activation in the CREB system, protein kinase A (PKA) enters the nucleus and phosphorylates CREB which then leads to transcription being (turned ON or turned OFF; circle the correct answer).
- Histone acetyltransferases add acetyl groups to _______________ amino acids on the histone tail.
- Acetyl groups are removed from histone tails by enzymes called _____________________________________________.
- Methyl groups added to cytosine bases usually project into the (major OR minor; circle the correct answer) groove of the DNA.
- Housekeeping genes are usually (methylated OR unmethylated; circle the correct answer) while tissue-specific genes are (methylated or unmethylated; circle the correct answer) in cells that do not express the gene.
- _________________ methylation ensures that the methylation pattern continues in the daughter cells produced by mitosis.