Lecture 9 (Kaufmann) Flashcards
Techniques to Study DNA Binding
EMSA (Electrophoretic Mobility Shift Assay)
EMSA (Electrophoretic Mobility Shift Assay)
Purpose
- EMSA is used to study sequence-specific DNA-binding proteins by detecting protein-DNA interactions.
Principle
- When a protein binds to DNA, the protein-DNA complex moves more slowly during gel electrophoresis compared to free DNA.
Steps:
- A labeled DNA probe (e.g., radiolabeled or fluorescently tagged) is mixed with the protein of interest.
- The mixture is run on a non-denaturing polyacrylamide gel.
- Protein-DNA complexes are visualized as shifted bands relative to the free DNA probe.
- Possibility to add an Antibody to identify the Protein added ⟶ causes ‘Supershift’
Gel Setup (4 slots):
- Only DNA.
- Protein and DNA.
- Mutated DNA (control sequence) and protein to check non-specific binding.
- Competitor (unlabeled) DNA and protein to verify affinity of binding.
Applications:
- Confirming DNA-binding specificity.
- Comparing binding affinities of different proteins to the same DNA sequence.
- Studying the impact of mutations on protein-DNA interactions.
Advantages
- Simple, sensitive, and allows direct observation of protein-DNA complexes.
Protein Binding Microarrays
Protein Binding Microarrays
Purpose
- Identify DNA-binding preferences of transcription factors (TFs).
Process:
- Hybridization: Tagged transcription factors (TFs) are introduced to an array with a variety of DNA sequences.
- Wash Step: Unbound TFs are washed away, leaving only TFs that bind specifically to the DNA sequences on the array.
- Detection: Bound TFs are identified using a fluorophore-tagged antibody specific to the tag on the TF.
- Scanning: The microarray is scanned to visualize the binding interactions and determine the DNA sequence preferences of the TF.
Advantages
- High-throughput method for studying protein-DNA interactions.
- Provides a detailed map of DNA sequence preferences.
SELEX-Seq
SELEX-Seq
Purpose
- Systematic Evolution of Ligands by Exponential Enrichment (SELEX) identifies DNA sequences that a protein of interest binds to with high affinity.
Process
- DNA Library Preparation: A pool of randomized DNA sequences (free-floating). Ends of the DNA are specific to allow primer binding for amplification.
- Incubation: The DNA library is incubated with the protein of interest, and the protein selectively binds to specific DNA sequences.
- Binding and Immunoprecipitation: Antibody-tagged beads capture the protein-DNA complexes. Free DNA and unbound proteins are washed out.
- Elution: Bound DNA is released (eluted) from the protein.
- Amplification & Recycling: The eluted DNA is amplified using primers and recycled into subsequent SELEX rounds to enrich high-affinity sequences.
- NGS (Next-Generation Sequencing): After several rounds, DNA is sequenced to identify the enriched sequences that bind the protein of interest.
Key Feature
- The protein selectively binds to DNA during each round, and repeated cycles increase the frequency of sequences with the highest binding affinity.
Quantitative Multiple Fluorescence Relative Affinity (QuMFRA)
Quantitative Multiple Fluorescence Relative Affinity (QuMFRA)
Purpose
- Quantify and compare the binding affinities of a protein for different DNA sequences.
Components
- Uses fluorescently labeled DNA (e.g., test and reference sequences).
- Combines EMSA to visualize binding and fluorescence intensity to measure affinity.
Process
- Incubate the protein with labeled test and reference DNA sequences.
- Separate protein-DNA complexes by electrophoresis.
- Measure fluorescence to determine relative binding affinities.
Application
- Validates SELEX results by confirming both specificity and affinity for enriched DNA sequences.
Yeast One Hybrid
Yeast One Hybrid
Purpose
- Identify DNA sequences bound by a specific transcription factor (TF) in a living system.
Process
- Test Transcription Factor (TF): The TF is fused to a transcription activation domain.
- Plasmid Setup: A plasmid contains a randomized DNA region (potential binding sites) upstream of a reporter gene (e.g., an auxotrophic or fluorescent marker).
- Binding Detection: If the TF binds the DNA on the plasmid, the fusion protein activates transcription of the reporter gene.
- Activation of the reporter gene confirms the TF-DNA interaction.
Key Features
- Performed in vivo using yeast as a host organism.
- Directly measures whether the TF can bind and activate transcription at a given DNA sequence.
ChIP (Chromatin Immunoprecipitation)
ChIP (Chromatin Immunoprecipitation)
Purpose
- Identify the specific DNA sequences bound by a protein in a living cell or tissue.
Process
- Crosslinking: Use formaldehyde to form covalent bonds between proteins and DNA they are bound to in the native chromatin context.
- Chromatin Isolation and Shearing: Extract the chromatin from cells. Shear the chromosomes into small DNA fragments using sonication or enzymatic digestion.
- Immunoprecipitation: Use a specific antibody to target and bind the protein of interest. Capture the protein-DNA complexes using beads bound to the antibody.
- DNA Purification: Reverse the crosslinks to separate the DNA and protein. Purify the DNA for analysis (e.g., qPCR, microarray, or sequencing).
ATAC-Seq
ATAC-Seq
- Process: Isolate nuclei and treat with transposase enzyme (e.g., Tn5), which cuts accessible chromatin and inserts sequencing adapters. Purify DNA, amplify fragments via PCR, and sequence
- Outcome: Sequencing identifies open chromatin regions as peaks in the genome.
- Applications: Detect regulatory elements (enhancers, promoters) and study chromatin accessibility dynamics genome-wide.
What are Homeotic Genes?
Homeotic Genes
- Control the development and identity of body segments or structures during embryogenesis.
- Function: Regulate the expression of other genes to ensure proper formation of body parts in specific locations.
- Mutations can cause homeotic transformations, where one body part is replaced by another (e.g., legs instead of antennae in fruit flies).
- Example: Hox genes, which are homeotic genes encoding transcription factors with conserved homeobox domains.
Homeodomain transcription factors
Homeodomain transcription factors
- Homeodomain is a DNA-binding domain of 60 amino acids that has 3 alpha helices
- C-terminal α-helix 3 binds the major groove of the DNA
- N-terminal arm binds the minor groove of the DNA
- Homeodomain TFs can either activate or repress transcription
- Hox TFs only a small subgroup of Homeodomain TFs (~39 in mammals)
- Recognize AT-rich DNA sites, potential binding sites are at high frequency in the genome, but only some a occupied
SELEX-Seq of Hox TF’s
SELEX-seq for HOX TFs
Process:
- Prepare a randomized DNA library.
- Incubate HOX proteins with cofactors (e.g., Exd/Pbx) to reveal full binding specificity.
- Isolate protein-DNA complexes, elute bound DNA, and amplify via PCR.
- Repeat cycles to enrich specific sequences and sequence via NGS.
Key Features:
- Cofactor Dependency: Includes cofactors to uncover latent specificity.
- Clustered Binding: Focuses on low-affinity, clustered sites.
- Distinct Preferences: Enriched motifs reflect anterior, central, or posterior HOX protein specificity.
Integration
- Combine SELEX results with in vivo data (e.g., ChIP-seq) for gene regulation insights.
HOX Binding (Involved Factors)
- Concept of “Latent Specifity” in HOX TFs
- Modes of Binding
Concept of “Latent Specifity” in HOX TFs
- Alone, HOX proteins have limited DNA-binding specificity, recognizing simple motifs like “TWAYnn.”
- Dimerization with the cofactor Exd (Extradenticle) reveals latent specificity, allowing HOX proteins to bind more complex motifs (e.g., “TGAYNNAYnn”).
- Exd-HOX heterodimers group into three specificity classes, linked to anterior, central, and posterior HOX proteins.
- Anterior and posterior HOX proteins prefer binding sites with distinct DNA shapes, reflecting their different regulatory roles.
- Exd-HOX binding sites (e.g., Exd-Ubx sites) are overrepresented in DNA fragments bound in vivo, showing their biological significance.
Widespread Binding Model
- Low-affinity binding at many clustered sites determines specificity through their collective presence.
Cooperative Binding Model
- High-affinity binding at few bipartite sites, aided by cofactors, ensures precise specificity.
Homeotic Genes in Plants ⟶ ABC(E) Model
Homeotic Genes in Plants ⟶ ABC(E) Model
- Describes the genetic basis of floral organ identity in plants, primarily studied in Arabidopsis.
- Highlights the combinatorial role of MADS-box transcription factors in floral development
Model Overview
- A-Class Genes: Specify sepals and contribute to petal development.
- B-Class Genes: Work with A-class to form petals and with C-class to form stamens.
- C-Class Genes: Determine stamens and carpels, and repress A-class activity in reproductive organs.
- E-Class Genes: Act as cofactors required for all organ development, stabilizing the ABC interactions.
Functionality:
- Combination of these gene classes determines the type of floral organ formed in each whorl of the flower.
- Mutations in these genes can cause organ identity transformations (e.g., stamens replaced by petals).
- Example: Double flowers often result from mutations in C-class genes like AGAMOUS, leading to repeated petal-like structures instead of reproductive organs.
Floral Quartets Model
Floral Quartets Model
Transcription Factor Interactions:
- Specific MADS-box transcription factors interact to form tetrameric complexes (quartets).
DNA Binding:
- These quartets bind to specific CArG-box motifs in the DNA.
Activation of Class Genes:
- The quartets act as transcriptional activators (or sometimes repressors) to regulate the expression of floral organ-specific genes, determining organ identity in each whorl (sepals, petals, stamens, carpels).
What are the determinants of molecular specificity of organspecific MADS-box protein complexes?
- DNA-Binding Specificity: The interaction of MADS-box proteins with specific CArG-box motifs (cis-regulation) determines their target genes.
- Protein-Protein Interactions: Cooperative binding with cofactors (e.g., SEP proteins, chromatin remodeling factors) enhances specificity (trans-activity).
–> SELEX-Seq can be used to determine DNA-binding specificities
Distinguish between “base readout” and “shape readout” in TF
binding site selection
Base Readout vs Shape Readout
Transcription factors often combine base readout and shape readout to achieve specificity in DNA binding
Base Readout:
- Direct interactions between amino acids of the transcription factor (TF) and the functional groups of the DNA bases.
- Relies on base-specific hydrogen bond donors, acceptors, and hydrophobic groups in the major groove.
- Example: Recognizing a specific DNA sequence by reading its chemical properties.
Shape Readout:
- Interprets the global and local shape of the DNA (e.g., bending, groove width, or electrostatic potential).
- Involves interactions with the minor groove, where the DNA shape is less sequence-specific.
- Example: Binding to structural features like a narrow minor groove or bent DNA helix.
