Cover1 - Chromosome Structure2 - Chromosome Compaction3 - Chromosome Variation5 - Nucleic Acid Structure6 - DNA Replication7 - Mutations and DNA Repair8 - Polymerase Chain Reaction (PCR)9 - Transcription10 - RNA Modifications11 - Translation12 - Gene Cloning13 - The lac Operon14 - Gene Regulation in Eukaryotes15 - Epigenetics16 - Genome Editing

14 - Gene Regulation in Eukaryotes

Comparing Gene Regulation in Prokaryotes and Eukaryotes

The lac operon provided an excellent example of how bacteria perform gene regulation in response to an environment that lacked glucose yet contained 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 DNA sequences near the lac operon structural genes.  Ultimately the binding of the CAP and the lac repressor proteins determined if sigma (σ) factor protein and the RNA polymerase core enzyme could activate transcription.

Even though gene regulation in prokaryotes and eukaryotes is similar (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 organism.  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:

Key Questions

  • What is meant by combinatorial control?
  • What factors can influence the transcription of a eukaryotic gene?

Core Promoter vs. Regulatory Promoter

We learned earlier this semester 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 transcriptionThe 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 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.

Figure 14.1 Core and Regulatory Promoter --- Image created by SL

Key Questions

  • 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

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:

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 and regulatory promoters.  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.

Figure 14.2 Trans-acting factors binding to cis-acting elements.  In this case, mediator interprets three activation signals and two silencing signals.  Overall, transcription is increased above the basal level. --- Image created by SL.

Key Questions

  • 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:

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. 

Key Questions

  • Review the functions of activator proteins, repressor proteins, enhancer sequences, and silencer sequences.

Structural Features of Transcription Factors

Transcription factor proteins have been identified in many organisms, including 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.  These structural features are called structural motifs

The structural motifs found in transcription factors 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 nucleotide sequences in the major groove of DNA.

The four common structural motifs that are found in transcription factor proteins include (see figure 14.3):

It is important to note that all transcription factor motif structures allow transcription factors to bind to each other.  Two identical transcription factors can interact to form a transcription factor homodimer, or two different transcription factor proteins can interact to form a heterodimer.  Higher order interactions (trimers, tetramers) are also possible when transcription factor proteins bind to each other.


Figure 14.3 Transcription Factor Structural Motifs a) Helix-turn-helix motif b) Basic helix-loop-helix motif c) Zinc finger motif d) Leucine zipper motif --- Images created by SL

Key Questions

  • What are three examples of transcription factor proteins that contain the helix-turn-helix (HTH) motif?
  • What is an example of a transcription factor protein that contains the basic helix-loop-helix (bHLH) motif?
  • What is an example of a transcription factor protein 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 is regulated in three general ways:

Note that for a particular gene, one or more of the above mechanisms may be involved in regulating gene expression.

Key Questions

  • Describe the three ways that activator and repressor proteins can be regulated?
  • What is an example of a eukaryotic effector molecule?

Control of Transcription (TFIID)

We have seen that 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 transcription factors influence RNA polymerase II activity through TFIID, mediator, the enzymes involved in chromatin remodeling, and the enzymes involved in DNA methylation.

We will consider regulation of RNA polymerase II activity through TFIID first.  TFIID is a general transcription factor that binds to the TATA box (the -25 sequence) within the core promoter.  TFIID recruits the other five  general transcription factors (GTFs) that bring RNA polymerase II to the +1 site to initiate 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 GTFs 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.  The repressor protein then prevents TFIID from binding to the TATA box. The absence of TFIID on the core promoter prevents the other GTFs and RNA polymerase II from binding to the core promoter.  As a result, transcription is down regulated.

Figure 14.4 Regulating TFIID - Image created by SL

Key Questions

  • How do activator and repressor proteins influence TFIID?

Control of Transcription (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 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.  The repressor protein inhibits the activity of mediator.  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.

Figure 14.5 Regulating Mediator --- Image created by SL

Key Questions

  • How do activator and repressor proteins influence the activity of mediator?

An Example of Transcription Activation (glucocorticoid receptor)

Steroid hormones produced by endocrine glands can activate the transcription of many genes.  One example is a group of steroid hormones called glucocorticoid hormones (GCs) produced by the adrenal glands. Glucocorticoid hormones are produced in response to fasting as well as physical activity, leading to an increase in glucose synthesis, an increase in protein metabolism, an increase in fat metabolism, and a decrease in inflammation.  Other steroid hormones, such as estrogen and testosterone, influence the development of gonad tissue. 

Glucocorticoid hormones can increase the transcription of a gene above the basal level as follows (see figure 14.6):

  1. GCs are steroid hormones, which are nonpolar in structure. As a result, these nonpolar steroid hormones cross the cytoplasmic membrane and enter the cytoplasm of a target cell.
  2. GCs act as effector molecules by binding to an inactive activator protein called glucocorticoid receptor (GR) that is found in many cell types. Prior to GC binding, GR is bound to HSP90 proteins.  HSP90 helps maintain the proper three-dimensional shape of GR, so that GR can bind to GC when GCs are produced by the adrenal glands.
  3. GC binds to GR and HSP90 is released.
  4. GC binding changes the conformation of GR, exposing a nuclear localization signal (NLS). The NLS is a polypeptide sequence that helps to target the GR (with bound GC) to the nucleus of the cell.
  5. Two GRs (with bound GC hormones) from a homodimer in the cytoplasm of the cell.
  6. The GR homodimer travels to the nucleus of the cell.
  7. The GR homodimer binds to an enhancer DNA sequence called a glucocorticoid response element (GRE). GREs are common enhancers found adjacent to many genes involved in metabolism.
  8. GR bound to GRE activates transcription.
Figure 14.6 Transcription Regulation by Glucocorticoid --- Image created by SL

Key Questions

  • What is GC, GR, and GRE?
  • How does the production of GC by the adrenal gland lead to transcriptional activation of a target gene?

An Example of Transcription Activation (CREB)

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 binding of the signaling protein to the receptor is transmitted to the nucleus to activate transcription.

Consider how transcription is activated by an activator protein called cAMP response element-binding protein (CREB).  CREB activates transcription when (see figure 14.7):

  1. A receptor embedded in the cytoplasmic membrane binds to a peptide hormone, growth factor, or cytokine protein.
  2. The binding of the signaling protein to the receptor activates a G protein.
  3. The G protein activates adenylyl cyclase inside the cell, which converts ATP into cAMP.
  4. cAMP binds to and activates protein kinase A (PKA).
  5. PKA moves into the nucleus and phosphorylates the inactive CREB protein homodimer.
  6. The phosphorylated CREB protein homodimer binds to enhancer sequences called cAMP response elements (CREs).
  7. CREB bound to CRE activates transcription.
Figure 14.7 Transcriptional Regulation by CREB --- Image created by SL

Key Questions

  • What is CREB and CRE?
  • How does the binding of a peptide hormone to a receptor lead to transcriptional activation of a target gene via the CREB pathway?

Chromosome Compaction and Transcription

The arrangement of nucleosomes (see Part 2) can also influence the transcription of a eukaryotic gene.  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 is in a chromosomal region with tightly packed nucleosomes (heterochromatin), RNA polymerase II struggles to bind to 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 access a core promoter located in euchromatin, and as a result, transcription occurs.

Chromatin is a dynamic structure with a specific region of DNA 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.

Key Questions

  • 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 are found in regular intervals from -3000 to +1500.  Thus, the β-globin gene in fibroblasts is in the closed conformation (heterochromatin) and 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 cell).  When the nucleosome arrangement surrounding the β-globin gene was examined in erythroblasts, a different result was observed.  Nucleosomes are displaced from the -500 to +200 region of the gene.  This open conformation (euchromatin) area includes the regulatory and core promoters.  Thus, the GTFs and RNA polymerase II can access the promoter region, leading to the transcription of the β-globin gene in erythroblasts. 

Figure 14.8 Nucleosome arrangement on the B-globin gene --- Image created by SL

Key Questions

  • In terms of the core promoter for the β-globin gene, describe the difference between chromatin structure in fibroblasts and erythroblasts.

Histone Acetylation

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 involves the acetylation, methylation, and phosphorylation of histone proteins within nucleosomes.  Acetylation will serve as an example of the covalent modification of histones.  Enzymes called histone acetyltransferases (HATs) add acetyl groups to the tails of histone proteins.  Specifically, 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.  Neutralization of the positive charges on the histone tails cause 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.  As a result, the histone tails once again bind to the negatively charged DNA backbone, and the chromatin is converted 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 nucleosomes (see figure 14.9).  One example of an ATP-dependent chromatin remodeling enzyme is a multi-subunit complex called SWI/SNF.  SWI/SNF performs at least three types of chromatin remodeling:


Figure 14.9 Histone Acetylation and ATP-Dependent Chromatin Remodeling --- Images created by SL.

Key Questions

  • 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 genes.  The methyl group that is added to the DNA double helix blocks the major groove of the DNA, preventing the binding of activator protein to the DNA. Cytosine bases within CG-rich sequences called CpG islands are typically targets for 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’ 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. 

Figure 14.10 Overview of DNA Methylation.  CpG islands are the targets for DNA methylation to silence a gene. --- Image created by SL

Housekeeping genes encode proteins that are required for cell viability.  The promoters of these genes are 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.

Key Questions

  • 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 major groove in the enhancer region. 

Second, methylated CpG islands near promoters serve as the binding sites for a group of proteins called methyl-CpG-binding proteins.  When a methyl-CpG-binding protein binds to a methylated CpG island, the methyl-CpG-binding proteins can recruit a 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.

Figure 14.11 Methylation Inhibits Transcription --- Image created by SL

Key Questions

  • 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 full methylation (both DNA strands methylated).  De novo methylation is a highly regulated process that is thought to occur during embryonic and tissue development. 

The DNA methylation pattern established during de novo methylation is preserved during cell division.  For example, if a CpG island is fully methylated in a cell prior to mitosis, the same CpG island is fully methylated in the daughter cells after 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 bases during replication, the daughter DNA strands produced do not contain methylated cytosines.  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 phenomenon called genomic imprinting.  In oogenesis (egg cell formation) or spermatogenesis (sperm cell formation), a specific gene is 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, that maternal allele remains fully methylated in the cells of the offspring.  We will discuss genomic imprinting more in Part 15.

Figure 14.12 Preserving DNA Methylation During Cell Division --- image created by SL

Key Questions

  • What is the difference between de novo and maintenance methylation?
  • What is the name of the enzyme responsible for maintenance methylation?
  • What is meant by genomic imprinting?


In eukaryotes, the processes that regulate the expression of one gene (activators/repressor proteins bound to enhancer/silencer DNA sequences, altering chromatin structure, and DNA methylation) do not necessarily influence the regulation of an adjacent gene.  DNA sequences called insulators function to define the boundaries between genes (see figure 14.13); an insulator sequence ensures that the gene regulation processes that affect one gene do not affect nearby genes.

Insulator DNA sequences can:

Figure 14.13 Insulators --- Image created by SL

Key Questions

  • 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:

  1. The core promoter consists of two consensus DNA sequences located at position ______________ and ______________.
  2. The general transcription factor (GTF) proteins are ________________________________________________________________.
  3. Some examples of regulatory transcription factor proteins are ____________________, which increase transcription and _________________________, which decrease transcription below basal levels.
  4. 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.
  5. The interaction of two identical transcription factor proteins to produce one molecule is called a ________________________.
  6. One example of a steroid hormone is ________________________________________.
  7. A glucocorticoid receptor is bound to _________________ until a glucocorticoid molecule comes along and binds to the receptor thus liberating it.
  8. CREB is a (protein OR DNA sequence; circle the correct answer), whereas CRE is a (protein OR DNA sequence; circle the correct answer).
  9. 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).
  10. Histone acetyltransferases add acetyl groups to _______________ amino acids on the histone tail.
  11. Acetyl groups are removed from histone tails by enzymes called _____________________________________________.
  12. Methyl groups added to cytosine bases usually project into the (major OR minor; circle the correct answer) groove of the DNA.
  13. 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.
  14. _________________ methylation ensures that the methylation pattern continues in the daughter cells produced by mitosis.


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