lecture 5-How transcription factors are activated Flashcards
(36 cards)
Learning Objectives
➢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
DNA binding proteins can move to the nucleus after “activation”
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)
DNA binding proteins are not always in the nucleus- So how do they get there?
DNA binding proteins are not always in the nucleusSo how do they get there? Part 2
DNA binding proteins can move to the nucleus after “activation
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
Homeobox (HOX) genes contain homeodomains
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)
p53: A β-sheet recognition protein
*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
Functions of p53
Zinc fingered nuclear hormone receptors
*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
Nuclear Receptors (which include steroid hormone receptors)
➢Contain two Zn-binding domains, one interacts with DNA, the other enables dimerisation.
➢Where the first “finger” determines sequence specificity
Fos: A Leucine Zipper that regulates bone
*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)
Fos Activation
Mycand its role in cancer
*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
Effects of multi-protein complexes
-Two are always better than one!
➢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
How dimer make up affects transcription
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:
- 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. - 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. - 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. - 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. - 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. - 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. - 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.
The significance of a mediator complex
Changes to structure outside the DNA binding domain can enhance DNA binding
how can Contents of the mediator complex can influence the rate of transcription
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.
Nuclear receptors as co-activators and co-repressors
Same response element, same DNA binding protein, different mediator proteins, different outcomes
Summary
➢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
Further reading
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
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
- 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.
- 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.
- 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.
- 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.
- 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.
Transcriptional activators
✓Promotes regulator binding
✓Recruit RNA polymerase II
✓Releases RNA polymerase II either to begin transcription OR from a paused state
Types of promoter
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
What does the term epigenetics mean?
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:
- DNA Methylation:
The addition of methyl groups to cytosine residues in DNA (typically at CpG sites), often leading to gene repression.
- 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.
- 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.
- 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.
