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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 a characteristic of genes within lad calls?
What is a feature of the boundaries of lad calls?
What is the advantage of DamID over other assays?
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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?
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What is the result of cross-linking DNA in the high-C assay?
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What is a characteristic of lad calls?
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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)?
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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?
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What is the purpose of the DamID assay?
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What is the result of the fusion protein in the DamID assay?
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What is the main characteristic of chromosome territories?
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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.
