lecture 5-How transcription factors are activated

Question 1

Learning Objectives

Answer 1

➢Explain the principles governing interactions between DNA and protein that take place at the promoter or enhancer.

➢Describe the differences between DNA binding domains, using an example protein of each to help you explain their functional differences.

➢Explain the effect of multicomponent complexes on DNA binding, including those recruited to the mediator complex

Question 2

DNA binding proteins can move to the nucleus after “activation”

Answer 2

1.Movement to the nucleus may require a shape change or dissociation from another protein.

2.The change reveals the nuclear localisation signal (NLS).

3.This then allows the protein to bind to importins to allow it to move through the nuclear pore complex and into the nucleus.

4.Once in the nucleus it can dimerise with other proteins and DNA.

5.(…and therefore affect gene expression by controlling initiation of transcription)

Question 3

DNA binding proteins are not always in the nucleus- So how do they get there?

Answer 3

Question 4

DNA binding proteins are not always in the nucleusSo how do they get there? Part 2

Answer 4

Question 5

DNA binding proteins can move to the nucleus after “activation

Answer 5

Revealing the NLS can be achieved by:
*Ligand binding
*Post translational modification (covalent modification)
*Addition of subunits (also known as dimerization)
*Dissociation from an inhibitor through covalent modification (unmasking) or naturally separating (stimulation of nuclear entry)
*Proteins being released from the plasma membrane

Question 6

Homeobox (HOX) genes contain homeodomains

Answer 6

Homeobox (HOX) proteins control body patterning during development. e.g. HOX9 is involved in limb development

Homeodomains contain 3 α helices which are packed closely together by hydrophobic interactions, one (red) touches the major grove of the DNA (left)

Question 7

p53: A β-sheet recognition protein

Answer 7

*Typical tumour suppressor gene.
*Contains a DNA binding domain made of two β-sheets which “sandwich” the DNA.
*Forms multimers through its oligomerisation domain (OD) which can modify its DNA sequence specificity

Question 8

Functions of p53

Answer 8

Question 9

Zinc fingered nuclear hormone receptors

Answer 9

*Strangely they do not always bind to zinc, some bind other metals, others nothing.

*They all have the finger like domains that interact with the DNA.

*Many different types.

*Regulate processes like bile acid detoxification.

*Contents of the dimer are key to sequence specificity and effect

Question 10

Nuclear Receptors (which include steroid hormone receptors)

Answer 10

➢Contain two Zn-binding domains, one interacts with DNA, the other enables dimerisation.

➢Where the first “finger” determines sequence specificity

Question 11

Fos: A Leucine Zipper that regulates bone

Answer 11

*Always bind to DNA as dimers.

*Dimer formation is enabled by hydrophobic interactions between alpha helices (mainly by leucine residues).

*Sometimes have a globular domain with basic properties, called a basic leucine zipper (bZIP).

*Activated by phosphorylation (by Mitogen Activated [MAP] Kinase)

Question 12

Fos Activation

Answer 12

Question 13

Mycand its role in cancer

Answer 13

*Mycis often upregulated in cancer
*Components of the heterodimer control the outcome of the signalling pathways.

*Mycis associated with
-cell cycle progression
-apoptosis
-proliferation
-metabolism

Question 14

Effects of multi-protein complexes
-Two are always better than one!

Answer 14

➢Some DNA binding domains only fold completely in the presence of DNA.
➢Usually this is because the DNA binding domain is partially unstructured and dimerisation enables better folding and support.
➢The composition of the dimer affects sequence recognition and therefore range of targets affected

Question 15

How dimer make up affects transcription

Answer 15

Greater DNA binding by monomers can reduce transcription rates.

Dimerization—the process where two molecules (often proteins) bind together to form a dimer—plays a crucial role in regulating transcription. Many transcription factors (TFs) function as dimers, and the formation of these dimers directly impacts their ability to regulate gene expression.

Here’s how dimer formation can affect transcription:

1. Stability and DNA Binding Affinity
   Increased Stability: When transcription factors dimerize, the dimer often becomes more stable than the monomer form. This stability enhances the ability of the TFs to stay bound to DNA, allowing them to regulate gene expression more effectively.
   Increased DNA Binding Affinity: Many TFs bind more strongly to DNA when in dimer form. Dimerization allows for greater surface area interaction between the TFs and the DNA, increasing the strength and specificity of binding to target sequences in the genome.
   Example: The basic leucine zipper (bZIP) transcription factors, such as AP-1, must dimerize to effectively bind DNA and regulate target genes.
2. Specificity of DNA Binding
   Heterodimer vs. Homodimer: Transcription factors can form either homodimers (two identical proteins) or heterodimers (two different proteins). The combination of different proteins in a heterodimer can change the specificity for which DNA sequences are recognized.
   Homodimer: Binds to specific consensus sequences that match the binding preferences of the individual protein.
   Heterodimer: The DNA-binding specificity may change because the two proteins in the dimer interact differently with the DNA. This allows cells to regulate different sets of genes depending on which transcription factor pair is formed.
   Example: In the case of bHLH (basic helix-loop-helix) proteins, different combinations of heterodimers can bind to different DNA sequences, modulating the regulation of various genes. The transcription factors Myc and Max form heterodimers to regulate genes involved in cell growth and division.
3. Allosteric Regulation
   Conformational Changes: Dimerization can induce conformational changes in transcription factors, altering their activity. This can affect whether they can interact with other proteins, such as coactivators or corepressors, and thus influence transcription.
   Example: The dimerization of nuclear hormone receptors like the estrogen receptor (which forms homodimers) is critical for binding to hormone response elements on DNA. The binding of the hormone (ligand) triggers dimerization, which then activates the transcription factor and initiates transcription.
4. Transcriptional Activation or Repression
   Cooperative Binding: In many cases, dimerized transcription factors work together more efficiently than monomers to recruit coactivators or corepressors that modify chromatin and help activate or repress transcription. This cooperativity often depends on dimer formation.
   Repression: Some transcription factors, when they form dimers, can act as repressors. By dimerizing, they may mask DNA binding domains or recruit corepressors that inhibit gene expression.
   Activation: In other cases, dimerization may be required for transcriptional activation. Some transcription factors can only recruit the necessary coactivators to start transcription when they dimerize.
   Example: NF-κB, a key transcription factor involved in immune responses, forms dimers (often p50-p65) that can bind DNA and activate or repress target genes depending on which coactivators or corepressors are recruited.
5. Response to Signaling Pathways
   Signal-Dependent Dimerization: Some transcription factors only dimerize in response to specific signaling events, such as phosphorylation or ligand binding. Dimerization acts as a switch that controls whether the transcription factor can activate or repress genes.
   Example: In the STAT (Signal Transducer and Activator of Transcription) proteins, phosphorylation induces dimerization. Once dimerized, STAT proteins can enter the nucleus and activate transcription of target genes involved in immune responses and cell growth.
6. Functional Diversity
   Expanded Regulatory Potential: By forming dimers, especially heterodimers, transcription factors gain functional diversity. This means a single transcription factor can participate in regulating multiple genes depending on its dimerization partner.
   Example: Fos and Jun proteins, which form the AP-1 transcription factor, create different responses based on whether they form heterodimers or homodimers. This variation affects which genes are turned on or off, impacting processes like cell differentiation and apoptosis.
7. Inhibition via Dominant-Negative Dimers
   Inactive Dimers: Sometimes, dimerization can result in a non-functional complex. A dominant-negative dimer is when one partner in the dimer lacks the ability to bind DNA or activate transcription but still forms a dimer with a functional partner, blocking the activity of the transcription factor.
   Example: In the Myc-Max system, the Mad-Max heterodimer competes with Myc-Max to bind DNA but represses transcription rather than activating it. This competition between dimers helps control the balance between cell growth and differentiation.

Conclusion:
Dimerization profoundly affects transcription by regulating the stability, specificity, and activity of transcription factors. Whether through homodimers or heterodimers, the process allows transcription factors to fine-tune gene expression in response to various cellular signals and conditions. Dimerization expands the range of gene regulation, offering more complexity and flexibility in controlling biological processes.

Question 16

The significance of a mediator complex

Answer 16

Changes to structure outside the DNA binding domain can enhance DNA binding

Question 17

how can Contents of the mediator complex can influence the rate of transcription

Answer 17

Contents of the mediator complex can influence the rate of transcription by influencing recruitment to the promoter and through changes to shape of the DNA binding proteins.

Question 18

Nuclear receptors as co-activators and co-repressors

Answer 18

Same response element, same DNA binding protein, different mediator proteins, different outcomes

Question 19

Summary

Answer 19

➢DNA binding proteins use a wide range of mechanisms to bind specifically to binding sites.

➢The three-dimensional structure of the binding site must be taken into consideration when understanding binding specificity.

➢The main readout mechanisms are*the recognition of bases *the recognition of DNA shape.

➢Any one DNA binding protein is likely to use a combination of readout mechanisms.

➢The formation of higher-order protein-DNA complexes may depend on sequence-dependent DNA structures that are optimized to promote assembl

Question 20

Further reading

Answer 20

Patricia J. Wittkopp, P.J., & Kalay, G. (2012) Cis-regulatory elements: molecular mechanisms and evolutionary processes underlying divergence. Nature Reviews in Genetics 13: 59-69.

Terrence RJ Lappin, David G Grier, Alexander Thompson, and Henry L Halliday. HOX GENES: Seductive Science, Mysterious Mechanisms Ulster Med J. 2006 Jan; 75(1): 23–31.

Fischer, M. (2017) Census and evaluation of p53 target genes. Oncogene. 2017 Jul 13; 36(28): 3943–3956.

Joerger, A.C.,andFersht, A.R. (2010) The TumorSuppressor p53: From Structures to Drug Discovery. Cold Spring HarbPerspectBiol.2(6): a000919.

Brázda, V., and Fojta, M. (2019) The Rich World of p53 DNA Binding Targets: The Role of DNA Structure. Int J Mol Sci.20(22): 5605.

Cassandri,M., Smirnov, A., Novelli, F., Pitolli, C., Agostini, M., Malewicz, M., Melino, G.,& Raschellà, G.(2017) Zinc-finger proteins in health and disease. Cell Death Discovery3:17071

Chanu SI and Sarkar S. (2014) Myc: Master Regulator of Global Genomic Expression. Austin J Genet Genomic Res. 1(1): 5.

Rohs, R., Jin, X., West, S.M, Joshi, R., Honig, B.,& Mann, R.S. (2010) Origins of specificity in protein-DNA recognition. Annu Rev Biochem. 79: 233–269.

Hiebl, V., Ladurner, A., Latkolik, S., & Dirsch, V.M. (2018) Natural products as modulators of the nuclear receptors and metabolic sensors LXR, FXR and RXR. Biotechnology Advances 36(6):1657-1698

Question 21

Learning outcomes on epigenetics

• *Describe factors that affect the affinity of general transcription factors for DNA and explain how this affects transcription rates.

*Identify the mechanisms of epigenetic control of gene expression and be able to apply this knowledge to explain its effects on the control of gene expression.

*Explain the basis of experimental methods used for epigenetic analysis.

*Integrate knowledge of diseases with that of methods used to detect epigenetic changes and be able to explain the link to epigenetic mechanisms.

*Use knowledge of the changes in chromatin packaging to explain how the above affect transcription rates

Answer 21

1. Factors That Affect the Affinity of General Transcription Factors for DNA and Their Effect on Transcription Rates
   General transcription factors (GTFs) are proteins essential for the initiation of transcription in eukaryotic cells. They bind to specific sequences in the promoter region of genes and recruit RNA polymerase II to the transcription start site.

Factors Affecting the Affinity of GTFs for DNA:
DNA Sequence:

The core promoter elements (e.g., TATA box, INR, BRE) are recognized by transcription factors such as TATA-binding protein (TBP). The exact sequence of these elements affects the strength of GTF binding.
Mutations in promoter regions can reduce or enhance the affinity of GTFs, influencing transcription rates.
Chromatin Structure:

Chromatin compaction affects the accessibility of DNA to transcription factors.
Heterochromatin (tightly packed chromatin) inhibits GTF binding, reducing transcription rates, while euchromatin (loosely packed chromatin) promotes GTF binding and increases transcription.
Post-translational Modifications of GTFs:

Phosphorylation, acetylation, and other modifications can either enhance or reduce the affinity of GTFs for DNA.
For instance, phosphorylation of TFIIB can affect its ability to recruit RNA polymerase II, impacting the initiation of transcription.
Co-activators and Co-repressors:

Co-activators (such as Mediator complex) and co-repressors (such as NCoR) can alter the conformation of GTFs, enhancing or inhibiting their binding to DNA.
This modulation in turn affects the transcription rate.
Effects on Transcription Rates:
Higher affinity of GTFs for DNA promotes more efficient recruitment of RNA polymerase II, leading to increased transcription.
Lower affinity results in weaker assembly of the transcription initiation complex and lower transcription rates.

2. Mechanisms of Epigenetic Control of Gene Expression
   Epigenetic modifications control gene expression without altering the DNA sequence. These modifications include:

DNA Methylation:

Methylation of cytosine residues (at CpG sites) typically represses gene expression by preventing the binding of transcription factors or recruiting methyl-binding proteins that promote chromatin compaction.
Example: Hypermethylation of tumor suppressor gene promoters can silence them, contributing to cancer.
Histone Modifications:

Acetylation of histones (H3 and H4) by histone acetyltransferases (HATs) reduces the positive charge on histones, leading to a relaxed chromatin state (euchromatin) and promoting transcription.
Deacetylation by histone deacetylases (HDACs) results in chromatin condensation (heterochromatin) and gene repression.
Other histone modifications include methylation, phosphorylation, and ubiquitination, each affecting gene expression in different ways.
Chromatin Remodeling Complexes:

Complexes such as SWI/SNF can reposition nucleosomes, making specific DNA regions more or less accessible to transcription factors.
Non-coding RNAs:

miRNAs and lncRNAs can regulate gene expression post-transcriptionally by degrading mRNA or blocking its translation.
Effects on Gene Expression:
Methylation typically represses genes, while acetylation promotes gene activation.
Epigenetic changes can be heritable and respond to environmental factors, linking them to long-term regulation of gene expression.

3. Experimental Methods for Epigenetic Analysis
   Bisulfite Sequencing:

Purpose: To analyze DNA methylation.
Method: Bisulfite treatment converts unmethylated cytosines to uracil, while methylated cytosines remain unchanged. Sequencing then reveals which cytosines were methylated.
ChIP-Seq (Chromatin Immunoprecipitation followed by Sequencing):

Purpose: To detect histone modifications or transcription factor binding.
Method: Specific antibodies target modified histones or transcription factors, allowing DNA associated with these proteins to be isolated and sequenced.
ATAC-Seq:

Purpose: To assess chromatin accessibility.
Method: Transposase cuts open regions of the genome, which are more likely to be transcriptionally active. Sequencing these regions identifies accessible chromatin regions.
RNA Interference (RNAi):

Purpose: To study the role of non-coding RNAs.
Method: Small interfering RNAs (siRNAs) are used to degrade target mRNA, allowing researchers to observe changes in gene expression.

4. Link Between Epigenetic Mechanisms and Diseases
   Epigenetic alterations are implicated in many diseases, particularly cancer, neurodevelopmental disorders, and metabolic diseases.

Example: Cancer
DNA Hypermethylation:
In cancers, hypermethylation of tumor suppressor gene promoters (e.g., p16, BRCA1) silences their expression, allowing uncontrolled cell growth.
Histone Modifications:
Loss of histone acetylation can lead to the repression of genes involved in cell cycle control and apoptosis. Drugs like HDAC inhibitors are used to restore histone acetylation and reactivate tumor suppressor genes.
Detection of Epigenetic Changes in Disease:
Methylation-Specific PCR (MSP) can be used to detect hypermethylation in the promoters of tumor suppressor genes, aiding in cancer diagnosis.
ChIP-Seq can identify changes in histone modifications associated with cancer progression.

5. Chromatin Packaging and Its Effect on Transcription Rates
   Euchromatin:

Loosely packed chromatin that is accessible to transcription machinery.
Active genes are generally found in euchromatin, as the open structure allows transcription factors and RNA polymerase to bind and initiate transcription.
Heterochromatin:

Tightly packed chromatin that is transcriptionally silent.
Genes located in heterochromatin are generally repressed due to the inaccessibility of the DNA.
Role of Histone Modifications:

Histone acetylation promotes the formation of euchromatin, increasing transcription rates.
Histone methylation can either activate or repress genes, depending on the specific amino acid residue and the number of methyl groups added.
Chromatin Remodeling:

Chromatin remodeling complexes (e.g., SWI/SNF) alter nucleosome positioning, making specific genes more accessible or less accessible, thus modulating transcription rates.

Summary:
Epigenetic modifications, such as DNA methylation and histone modifications, play a crucial role in regulating gene expression.
Techniques like ChIP-Seq and bisulfite sequencing help analyze these changes, which are often linked to diseases like cancer.
Changes in chromatin structure, controlled by epigenetic mechanisms, can either promote or inhibit transcription, depending on the degree of chromatin compaction.

Question 22

Transcriptional activators

Answer 22

✓Promotes regulator binding

✓Recruit RNA polymerase II

✓Releases RNA polymerase II either to begin transcription OR from a paused state

Question 23

Types of promoter

Answer 23

Broad promoters require assembly of multiple independent protein complexes to form across Kbpof DNA.

Sharp promoters are controlled by the binding of fewer protein complexes, located over a shorter span or non-coding DNA

A few things to remember that affect the strength of a promoter:
▪There can be more than one TSS.
▪There does not have to be a TATA box.
▪Chromatin structure can override all of this

Question 24

What does the term epigenetics mean?

Answer 24

Epigenetics refers to the study of heritable changes in gene expression or cellular phenotype that occur without changes to the underlying DNA sequence. These changes affect how genes are turned “on” or “off” and are influenced by factors such as the environment, lifestyle, and developmental stages.

Key Features of Epigenetics:

1. DNA Methylation:

The addition of methyl groups to cytosine residues in DNA (typically at CpG sites), often leading to gene repression.

2. Histone Modifications:

Chemical changes to histone proteins (such as acetylation, methylation, phosphorylation) that affect how tightly or loosely DNA is wrapped around histones, influencing gene accessibility for transcription.

3. Non-coding RNAs:

RNA molecules (such as miRNAs and lncRNAs) that do not code for proteins but can regulate gene expression post-transcriptionally by degrading mRNA or inhibiting translation.

4. Chromatin Remodeling:

The dynamic modification of chromatin structure, which affects the accessibility of the DNA to transcription machinery.

Importance of Epigenetics:
- Epigenetic changes are reversible and can be influenced by external factors such as diet, stress, toxins, and even social experiences.
- They play a crucial role in development and differentiation of cells, as well as in disease processes like cancer, where aberrant epigenetic modifications can lead to abnormal gene silencing or activation.

In summary, epigenetics controls how genes are expressed based on external factors, without altering the actual genetic code.

Question 25

What are the components of chromatin?

Answer 25

Chromatin is a complex of DNA and proteins that packages genetic material within the nucleus of eukaryotic cells. Its structure is dynamic, allowing it to regulate access to DNA for processes such as transcription, replication, and repair. The major components of chromatin are:

1. DNA
   Genetic Material: Chromatin contains the organism’s DNA, which carries the genetic information in the form of a double helix.
   Structure: The DNA is wrapped around histone proteins to form the basic structural unit of chromatin, the nucleosome.
2. Histones
   Core Histones: There are four main histone proteins—H2A, H2B, H3, and H4—which assemble into an octamer (a complex of eight proteins). DNA wraps around this histone octamer, forming the nucleosome.
   Linker Histone (H1): This histone binds to the nucleosome and helps in further packaging by linking nucleosomes together, contributing to higher-order chromatin structure.
3. Non-histone Proteins
   Chromatin Remodeling Complexes: These proteins help rearrange the structure of chromatin, making DNA more or less accessible to other proteins involved in transcription, replication, and repair.
   Transcription Factors: These proteins regulate gene expression by binding to specific DNA sequences within chromatin and influencing the recruitment of RNA polymerase and other transcription machinery.
   Scaffold Proteins: These provide structural support and help organize chromatin within the nucleus.
4. Non-coding RNAs
   Regulatory RNAs: Small and long non-coding RNAs (such as miRNAs and lncRNAs) play a role in regulating chromatin structure and function, often influencing gene expression and chromatin compaction.
5. Other Chemical Modifications
   DNA Methylation: Addition of methyl groups to the DNA (typically at cytosine bases) alters chromatin structure, leading to gene repression.
   Histone Modifications: Histone proteins can undergo post-translational modifications, including:
   Acetylation (often associated with active gene expression)
   Methylation (associated with both gene activation and repression, depending on the site)
   Phosphorylation
   Ubiquitination

These components combine to create a flexible and dynamic structure that regulates access to DNA and controls gene expression, replication, and repair processes within the cell.

Question 26

How does chromatin control gene expression?

Answer 26

Chromatin controls gene expression by regulating the accessibility of DNA to transcription machinery. The way DNA is packaged in chromatin can either facilitate or block the binding of transcription factors and RNA polymerase to specific genes, thus turning them “on” or “off.”

Key Mechanisms by Which Chromatin Controls Gene Expression:

1. Chromatin Structure: Euchromatin vs. Heterochromatin
   Euchromatin:
   Loosely packed chromatin, where the DNA is more accessible to transcription factors and RNA polymerase.
   Genes in euchromatin are usually active or transcriptionally “on” because the less compact structure allows transcription machinery to bind.
   Heterochromatin:
   Tightly packed chromatin, where the DNA is not easily accessible.
   Genes in heterochromatin are typically repressed or transcriptionally “off” due to the dense packaging of DNA, which prevents transcription factors from accessing the gene promoters.
2. Histone Modifications
   Histones, the proteins around which DNA is wrapped, can undergo various chemical modifications that change the structure of chromatin and affect gene expression. Some of the key modifications include:

Acetylation:
Acetylation of histones by enzymes like histone acetyltransferases (HATs) reduces the positive charge on histones, weakening their interaction with the negatively charged DNA. This results in a more open chromatin structure (euchromatin), promoting gene activation.
Histone deacetylation by histone deacetylases (HDACs) causes chromatin to become more compact, leading to gene repression.
Methylation:
Methylation of histone proteins can either activate or repress gene expression, depending on the specific amino acids modified and the number of methyl groups added.
For example, H3K4 methylation is often associated with active transcription, while H3K9 or H3K27 methylation is linked to gene repression.

3. DNA Methylation
   Methylation of DNA (usually at cytosine bases in CpG islands) is a major epigenetic mechanism that silences gene expression.
   When DNA is methylated at gene promoters, it prevents the binding of transcription factors and recruits proteins (like methyl-CpG binding proteins) that promote a repressive chromatin state, thus silencing the gene.
   DNA methylation is essential for processes like X-chromosome inactivation, genomic imprinting, and tissue-specific gene expression.
4. Chromatin Remodeling Complexes
   Chromatin remodeling complexes (e.g., SWI/SNF, ISWI) can reposition or evict nucleosomes (the basic units of chromatin) to expose or hide specific DNA regions.
   By moving nucleosomes away from a promoter, these complexes make it easier for transcription factors and RNA polymerase to bind, activating gene expression.
   Alternatively, by positioning nucleosomes over promoters, they can repress gene expression by blocking access to the DNA.
5. Non-coding RNAs (ncRNAs)
   MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) can influence chromatin structure and gene expression by interacting with chromatin-modifying proteins or recruiting complexes that modify histones.
   For example, lncRNAs can recruit histone-modifying enzymes to specific loci, leading to the activation or repression of nearby genes.
6. Topological Changes and Higher-Order Chromatin Structure
   The 3D organization of the genome within the nucleus also plays a role in gene regulation. Certain regions of chromatin can be brought into proximity with enhancers or repressive regions through chromatin looping, affecting the expression of genes in these regions.
   Topologically associated domains (TADs) are large regions of the genome that interact more frequently with themselves than with other regions, creating boundaries that help regulate gene expression.

Summary:
Chromatin controls gene expression by making DNA either more accessible (euchromatin) or less accessible (heterochromatin) to the transcription machinery. Through histone modifications, DNA methylation, chromatin remodeling, and higher-order chromatin organization, chromatin dynamically regulates the transcriptional activity of genes, playing a crucial role in processes such as development, differentiation, and disease.

Question 27

EUCHROMATIN and HETEROCHROMATIN

Answer 27

Relaxation of the structure:
*Replication
*Transcription
*DNA Repair

Condensation of the structure:
*Inhibit transcription
*Cell division

Question 28

Nucleosomes prevent promoter access by general transcription factors and RNA pol II

Answer 28

Transcriptional activators recruit coactivators including:
➢histone modification enzymes
➢ATP-dependent chromatin remodelling complexes
➢histone chaperones

Question 29

How does chromatin structure change?

Answer 29

4 main mechanisms:
✓Nucleosome sliding (Lecture 1)
✓Nucleosome eviction
✓Histone variant exchange
✓Histone tail modification & DNA methylation (Lecture 2)

Question 30

Basic principles of DNA : Protein interactions

Answer 30

Sugar-phosphate backbone is negatively charged so interactions between the histones and the DNA requires positively charged amino acid contacts

Question 31

Location and shape of histones are key

Answer 31

Remember that shape changes in DNA and protein change affinity for each other.

Generally regulators bind DNA in nucleosomes with lower affinity than to naked DNA because:

-Cis-regulatory sequence facing inwards

-Changes to the shape of the binding site due to associated protein binding

Question 32

How does chromatin structure change?

Answer 32

Modification by the components of the mediator complex or coactivator determine the longevity of the changes.

Question 33

Controlling gene expression: Summary

Answer 33

Broad promoters contain multipipe cis-elements and attract transcription factors that influence transcription in a variety of ways.

Question 34

Controlling gene expression: Summary

Answer 34

Question 35

Other Epigenetic mechanisms:

Answer 35

DNA methylation –direct modification of DNA bases

Interactions between DNA modification and protein modification

Question 36

Glossary

Answer 36

Epigenetics: the study of changes in organisms caused by modification of gene expression rather than alteration of the genetic code itself.

Promoter: Promoter sequences are DNA sequences that define where transcription of a gene by RNA polymerase begins. Promoter sequences are typically located directly upstream or at the 5’ end of the transcription initiation site.

Enhancer: Enhancer sequences are regulatory DNA sequences that, when bound by specific proteins called transcription factors, enhance the transcription of an associated gene.

Methylation: a process by which methyl groups (CH3) are added to the DNA molecule.

CpG island: CpG islands are defined as stretches of DNA 500–1500 bp long with a CG: GC ratio of more than 0.6, and they are normally found at promoters and contain the 5′ end of the transcript.

Euchromatin:a form of chromatin that is lightly packed. The presence of chromatin usually reflects that cells are transcriptionally active, i.e. they are actively transcribing DNA to mRNA.

Heterochromatin: densely packed chromatin found in the nucleus of eukaryotic cells. (opposite of Euchromatin)