Genome Organization and Structure

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.