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Questions and Answers
What is the purpose of comparing the distribution of methylated adenines between the foreground and control experiments in DamID?
What is the purpose of comparing the distribution of methylated adenines between the foreground and control experiments in DamID?
What is a characteristic of genes within lad calls?
What is a characteristic of genes within lad calls?
What is a feature of the boundaries of lad calls?
What is a feature of the boundaries of lad calls?
What is the advantage of DamID over other assays?
What is the advantage of DamID over other assays?
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What is the purpose of the high-C assay?
What is the purpose of the high-C assay?
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What is a possible reason why different genomic loci may be in close physical proximity?
What is a possible reason why different genomic loci may be in close physical proximity?
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What is the result of cross-linking DNA in the high-C assay?
What is the result of cross-linking DNA in the high-C assay?
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What is a characteristic of lad calls?
What is a characteristic of lad calls?
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What is the primary challenge of DNA organization in the nucleus?
What is the primary challenge of DNA organization in the nucleus?
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What is the main characteristic of a Topologically Associated Domain (TAD)?
What is the main characteristic of a Topologically Associated Domain (TAD)?
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What is the purpose of grouping TADs together?
What is the purpose of grouping TADs together?
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What is the main difference between the two categories of experimental assays used to measure genome interactions?
What is the main difference between the two categories of experimental assays used to measure genome interactions?
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What is the purpose of the DamID assay?
What is the purpose of the DamID assay?
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What is the result of the fusion protein in the DamID assay?
What is the result of the fusion protein in the DamID assay?
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What is the main characteristic of chromosome territories?
What is the main characteristic of chromosome territories?
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What is the main difference between TADs and chromosome territories?
What is the main difference between TADs and chromosome territories?
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Study Notes
Nuclear Organization of the Genome
- The nucleus has to pack approximately 2 meters worth of DNA into a 6 micrometer diameter.
- To achieve this packing, the genome is organized at different scales of resolution.
- The fundamental level is the linear DNA sequence, which is wrapped up into nucleosomes, and not all regions of the genome are wrapped up into nucleosomes.
Topologically Associated Domains (TADs)
- A TAD is a contiguous segment of a chromosome from which you tend to find a lot of physical interactions between loci within the TAD.
- TADs are somewhat loosely compartmentalized sections of the chromosome for which you find a lot of interactions within that TAD.
- TADs can consist of regions of the genome that tend to be highly associated with transcriptionally active elements or regions that are transcriptionally inactive.
Higher Level Organization
- TADs can group together and interact with each other, forming what is called the A component (transcriptionally active regions) and the B component (transcriptionally inactive regions).
- Chromosomes as a whole don't randomly distribute themselves around the nucleus, with certain chromosomes having preferences for location within the nucleus, forming what is known as chromosome territories.
Measuring Genome Interactions
- Experimental assays can be categorized into two groups: those that measure interactions with relatively fixed nuclear landmarks (e.g. nuclear lamina) and those that measure interactions between genomic loci.
- Techniques like ChIP-seq, DamID, and high C are used to measure these interactions.
DamID
- DamID is an assay that measures protein-genome interactions.
- It involves creating a fusion protein between Dam (a protein domain that can methylate adenine in the GATC motif) and a protein of interest (e.g. lamin).
- The fusion protein will methylate adenines in the GATC motif near regions of the genome that are associated with the protein of interest.
- By comparing the distribution of methylated adenines between the foreground and control experiments, you can identify regions of the genome that are associated with the protein of interest.
Lad Calls
- Lad calls are regions of the genome that are associated with the nuclear lamina.
- To identify lad calls, you can plot the distribution of reads across the genome and look for regions with a high log2 ratio of reads mapping to particular genomic locations in the foreground vs. control experiment.
- Lad calls are generally made where you see a whole region of a chromosome with values above zero.
Characteristics of Lads
- Regions inside lads are generally gene poor.
- Genes within lads tend to be more poorly expressed than genes outside of lads.
- Lads are associated with epigenetic marks of inactive transcription.
- Boundaries of lads tend to have a lot of CTCF binding sites or CpG islands and even promoters of genes that are facing outwards with respect to the lad boundary.
Flexibility of DamID
- DamID can be used for a wide range of assays.
- You can fuse Dam to any transcription factor that binds DNA and identify potential binding sites of that transcription factor.
- You can also use DamID to identify open chromatin regions or RNA-DNA interactions.
High-C Assay
- The high-C assay is used to measure interactions between genomic loci.
- It involves cross-linking DNA such that genomic loci that are physically proximal to each other get cross-linked together.
- The cross-linking results in the formation of chimeric DNA fragments that contain sequences from both genomic loci.
- The mapping of these reads to the genome tells you which genomic loci were in close physical proximity.
Reasons for Close Proximity
- There are several reasons why different genomic loci may be in close physical proximity, including:
- Protein-protein interactions (e.g. enhancer-promoter interactions)
- Bystander interactions (non-functional interactions between genomic loci that are physically close)
- Chromatin looping
- Interactions with fixed nuclear landmarks (e.g. lamina)
3C Technologies
- 3C technologies are based on sequencing to detect interactions between genomic loci.
- They involve cross-linking, restriction enzyme digestion, ligation, and purification to generate chimeric DNA fragments.
- High-C is a type of 3C technology that is used to measure interactions between genomic loci.Here are the study notes in detailed bullet points, focusing on key facts with context:
- 3C Technology*
- 3C (Chromatin Conformation Capture) is a laboratory technique used to analyze the spatial organization of chromatin in the nucleus
- It involves cross-linking, sonication, and ligation to create chimeric reads that represent interactions between different genomic regions
- 3C is a one-versus-one approach, where interactions are measured between a specific locus and other genomic regions
- 3C Variants*
- 4C (Circular 3C): a variation of 3C that uses a circularization step to create chimeric reads that represent interactions between a specific locus and other genomic regions
- High-C: a high-throughput version of 3C that uses deep sequencing to analyze interactions between all possible pairs of genomic regions
- Chromatin Looping*
- Chromatin looping refers to the formation of loops between distant genomic regions
- Loops are formed through the interaction of cohesion complexes and CTCF (CCCTC-binding factor) proteins
- Loops are important for regulating gene expression and maintaining genomic stability
- Chromatin Organization*
- Chromatin is organized into Topologically Associated Domains (TADs), which are self-interacting genomic regions
- TADs are separated by boundary elements, which are enriched with CTCF binding sites
- TADs are further organized into A and B compartments, which correspond to active and inactive chromatin regions, respectively
- Hi-C Maps*
- Hi-C maps are visual representations of chromatin organization, showing interactions between genomic regions
- Hi-C maps can be used to identify TADs, compartments, and topological features of chromatin organization
- Corner dots on Hi-C maps represent tight interactions between genomic regions, indicating looping events
- Loop Extrusion Model*
- The loop extrusion model is a hypothesis for how loops form in chromatin
- According to this model, cohesion complexes extrude chromatin fibers to form loops, which are stabilized by CTCF binding
- The loop extrusion model is still a topic of ongoing research and debate.### Cohesin and CTCF Binding
- Cohesin is loaded onto chromosomes through complexes like NIPBL
- Cohesin helps to facilitate chromosome looping, which is the process of bringing together distal loci
- CTCF binding sites are frequently found at the boundaries of TADs (topologically associated domains)
- CTCF binding sites physically interact with cohesion to prevent further looping
Loop Extrusion Model
- The loop extrusion model proposes that cohesin is loaded onto chromosomes, and then loops form as the chromosome is fed through
- Looping continues until it reaches a CTCF binding site, which prevents further looping
- Cohesion is not a permanent event and can be released from the chromosome through the activity of regulators like WAPL
Effects of Perturbing Components of the Loop Extrusion Model
- If CTCF is knocked down, the corner dots in a high-C heatmap (representing interactions between distal loci) disappear
- Without CTCF, subtad structures start to disappear, and interactions between distal loci are lost
- Depleting cohesin results in the loss of TAD structure and a loss of looping
High-C Heat Maps
- High-C heat maps show the 3D structure of chromatin and can be used to study gene regulation
- Changes in the high-C heat map can indicate changes in gene regulation
- High-C heat maps can be used to identify the molecular effects of genetic variation on gene regulation
3C-Based Assays
- 3C-based assays can be used to identify the molecular effects of genetic variation on gene regulation
- 4C (circular chromosome conformation capture) is a type of 3C-based assay that can be used to study the effects of genetic variation on gene regulation
Comparing 3C-Based Assays to Hi-C
- Hi-C has lower resolution than 3C-based assays
- Hi-C measures all possible pairwise interactions between genomic loci, which can make it difficult to get enough coverage
- 3C-based assays are more suitable for studying promoter-enhancer interactions for a single promoter or locus
Library Complexity and Coverage
- Library complexity refers to the number of unique molecules in a library
- Library complexity is affected by the amount of input material
- Low library complexity can lead to poor coverage of interactions, even with deep sequencing
- Approximately 100 million mapped reads is usually enough to get sufficient coverage of the human genome for a high-C interaction, assuming a library complexity of 40 KB bin size.
Nuclear Organization of the Genome
- The nucleus packs 2 meters of DNA into a 6 micrometer diameter by organizing the genome at different scales of resolution.
- The fundamental level is the linear DNA sequence, which is wrapped up into nucleosomes, but not all regions are wrapped up into nucleosomes.
Topologically Associated Domains (TADs)
- A TAD is a contiguous segment of a chromosome with many physical interactions between loci within the TAD.
- TADs are loosely compartmentalized sections of the chromosome with interactions within the TAD.
- TADs consist of regions with highly associated transcriptionally active elements or transcriptionally inactive regions.
Higher Level Organization
- TADs group together and interact, forming the A component (transcriptionally active regions) and the B component (transcriptionally inactive regions).
- Chromosomes don't randomly distribute themselves around the nucleus, with certain chromosomes having preferences for location within the nucleus, forming chromosome territories.
Measuring Genome Interactions
- Experimental assays measure interactions with relatively fixed nuclear landmarks (e.g. nuclear lamina) or interactions between genomic loci.
- Techniques like ChIP-seq, DamID, and high-C are used to measure these interactions.
DamID
- DamID measures protein-genome interactions by creating a fusion protein between Dam and a protein of interest (e.g. lamin).
- The fusion protein methylates adenines in the GATC motif near regions associated with the protein of interest.
Lad Calls
- Lad calls are regions associated with the nuclear lamina, identified by plotting the distribution of reads across the genome.
- Lad calls are made where a whole region of a chromosome has values above zero.
Characteristics of Lads
- Regions inside lads are generally gene poor and have poorly expressed genes.
- Lads are associated with epigenetic marks of inactive transcription.
- Boundaries of lads have CTCF binding sites, CpG islands, and promoters of genes facing outwards.
Flexibility of DamID
- DamID is used for a wide range of assays, including identifying potential binding sites of transcription factors, open chromatin regions, or RNA-DNA interactions.
High-C Assay
- The high-C assay measures interactions between genomic loci by cross-linking DNA.
- Chimeric DNA fragments are formed, containing sequences from both genomic loci, indicating physical proximity.
Reasons for Close Proximity
- Genomic loci may be in close physical proximity due to protein-protein interactions, bystander interactions, or chromatin looping.
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Description
Learn about the organization of the genome at different scales, from linear DNA sequence to topologically associated domains (TADs) and how the nucleus packs DNA into a small space.
