Chromatin architecture (TADs, loops), phase separation in nuclear organization, DNA damage response & repair pathway choice

This PPT contan inforation about the DNA, its TADs, loop, liquid-liquid Pase seperation, Hi-C, gene regulation and DNA repair

1. Chromatin architecture (TADs, loops), phase separation in nuclear organization, DNA damage response & repair pathway choice DR. AKBAR

2. Hierarchy and definitions

3. Chromatin Architecture Chromatin Loops Within and across TADs, loops bring together distal regulatory elements (e.g., enhancers and promoters). Proteins like CTCF (CCCTC-binding factor) and cohesin are key in loop extrusion and stabilization.

4. Mechanisms It’s a mechanical model that explains how DNA forms loops inside the nucleus — helping organize chromatin into TADs (Topologically Associating Domains) . Boundary formation : enriched with CTCF, active transcription, tRNA genes, and chromatin modifications; can be reinforced by transcriptional activity and by certain chromatin remodelers . Compartmentalization via phase-separation–like mechanisms: similarity to LLPS: homotypic interactions among chromatin regions bearing similar histone marks (e.g., H3K27me3 clusters) can drive self-association, distinct from loop extrusion. Think of it like thread being pulled through a ring — the ring slides along the thread, forming a loop in between Component Role Cohesin Acts like a sliding ring that grabs DNA and extrudes (pushes) it into a loop. CTCF Acts like a stop sign — it stops cohesin from sliding further when bound in a specific direction. DNA The “thread” being looped out. The Key Players

5. Step-by-Step (Simple View) Cohesin loads onto DNA A protein complex called NIPBL helps cohesin attach to DNA. Cohesin starts extruding DNA It reels in DNA from both sides, forming a growing loop between its two arms. Imagine pulling rope through a ring — the middle forms a loop. CTCF stops the loop When cohesin reaches CTCF binding sites (facing toward each other), it stops . These two CTCF sites mark the boundary of a loop or TAD . Stable loop forms The region inside the loop can now have enhancers and promoters close together — making gene activation easier . Why It’s Important Organizes the genome into functional neighborhoods (TADs) . Ensures enhancers only talk to their correct promoters — prevents misexpression. Helps control DNA replication and repair regions efficiently. Step Description 1 Cohesin loads onto DNA Cohesin extrudes DNA and forms a growing loop CTCF blocks cohesin movement at boundaries Loop is stabilized (forms TAD)

6. CTCF (CCCTC-binding factor) A highly conserved zinc finger DNA-binding protein plays a central role in 3D genome organization and gene regulation . It acts as an insulator, transcriptional regulator, and architectural protein that helps form TAD (Topologically Associating Domain) boundaries. Known as the “master insulator protein” of the genome. Plays a central role in shaping chromatin architecture. Functions : Boundary element for TADs CTCF binds at TAD boundaries . Often works with cohesin to halt loop extrusion → defining where one TAD ends and another begins. Insulator activity Prevents an enhancer from inappropriately activating a gene outside its domain. Chromatin looping CTCF binding sites in convergent orientation (arrows pointing toward each other) create stable DNA loops . Gene regulation Organizes promoters, enhancers, and silencers into proper spatial neighborhoods. Contains 11 zinc finger domains, allowing flexible DNA-binding patterns. Has N-terminal and C-terminal domains that interact with other chromatin-associated proteins like cohesin and YY1.

7. Mechanism with Cohesin (Loop Extrusion) Disease relevance : CTCF mutations → observed in cancers (e.g., breast, endometrial). Boundary disruptions → cause enhancer hijacking (misexpression of oncogenes like MYC ). Developmental disorders → linked to CTCF haploinsufficiency . Boundaries: Usually enriched for CTCF binding sites, cohesin, and active housekeeping genes. Function: They act as regulatory units, keeping enhancers and promoters in the same "zone" so that gene regulation stays specific and insulated.

8. What is “Phase Separation”? Think of what happens when you mix oil and water they separate into two phases . Inside cells, something very similar happens but instead of oil and water, it’s biomolecules (proteins, RNA, DNA). Certain proteins and RNAs “stick together” to form droplet-like condensates , separating from the surrounding solution (cytoplasm or nucleoplasm). This process is called Liquid–Liquid Phase Separation (LLPS) . Why It Happens Many nuclear proteins have special regions called Intrinsically Disordered Regions (IDRs) — flexible parts that don’t fold into fixed structures . These regions can make weak, multivalent interactions (many low-strength bonds) with other molecules. When enough of these interactions occur, the molecules condense into droplets — dense, liquid-like assemblies that: form quickly, are dynamic (molecules can move in/out), and dissolve when not needed.

9. Nuclear Body Formed by Phase Separation? Main Function Nucleolus ✅ Yes rRNA transcription and ribosome assembly Cajal Bodies ✅ Yes snRNP and snoRNP maturation Nuclear Speckles ✅ Yes mRNA splicing factor storage PML Bodies ✅ Yes DNA repair, transcription regulation DNA Damage Foci (e.g., 53BP1) ✅ Yes Organize repair machinery Heterochromatin Condensates (HP1 α) ✅ Yes Gene silencing phase separation organizes the nucleus without membranes!

10. Role in Gene Regulation Phase-separated condensates are not just “blobs” — they control how genes are read and repaired : Transcriptional Condensates Mediated by transcription factors (e.g., MED1 , BRD4 , Pol II ). Form at super-enhancers to boost gene expression. Function like “molecular factories” concentrating transcription machinery. Chromatin Compartmentalization HP1 α can phase-separate heterochromatin regions, forming compact, silenced domains. CTCF and cohesin organize TADs and loops , while LLPS reinforces boundaries. DNA Damage Response (DDR) Proteins like 53BP1 , FUS , and BRCA1 phase-separate at DNA break sites, forming repair foci . Molecule Type Feature Enabling LLPS Example Proteins Intrinsically disordered regions (IDRs), low-complexity domains FUS, TDP-43, MED1, HP1 α RNA Multivalent negative charges attract proteins snRNA, mRNA Post-translational modifications Regulate interaction strength Phosphorylation, methylation, acetylation Molecular Drivers of LLPS

11. Hi-C (High-throughput Chromosome Conformation Capture) Hi-C is a genome-wide 3D mapping method that detects which DNA sequences are physically close together in the nucleus. It’s an extension of 3C (Chromosome Conformation Capture) → but scaled up to the whole genome with sequencing. How Hi-C Works (basic workflow) Crosslinking Cells are treated with formaldehyde to “freeze” physical DNA–protein–DNA interactions in place. Restriction digestion Chromatin is cut with a restriction enzyme (e.g., HindIII , MboI ). Fill-in & biotin labeling The ends are filled in with nucleotides, one of which is biotin- labeled . Ligation DNA ends that are crosslinked and nearby are ligated → producing chimeric fragments (two originally distant DNA regions now ligated). Purification & sequencing Crosslinks are reversed, DNA is purified, ligation products pulled down (biotin), and then sequenced. Mapping Reads are mapped back to the reference genome. A contact matrix is built, showing how often two loci interact. Variants of Hi-C Micro-C → uses micrococcal nuclease, gives nucleosome-resolution maps. HiChIP / PLAC- seq show how certain proteins (such as cohesin) help form DNA loops. Single-cell Hi-C → captures heterogeneity. Capture Hi-C → enriches for selected regions of interest. Applications Studying enhancer-promoter contacts. Identifying structural variants in cancer (e.g., translocations). Understanding developmental gene regulation. Investigating how DNA repair foci and γ H2AX domains spread in 3D.

12. Method Captures Focus Resolution Hi-C All DNA–DNA interactions Global 3D map Moderate ChIP-seq Protein–DNA binding 1D linear view High HiChIP Protein-mediated DNA–DNA loops 3D, protein-specific High

13. Dynamics and Plasticity TADs and loops keep changing: The 3D structure of DNA inside the nucleus isn’t fixed—it moves and changes over time. Cells are not all the same: Even though we can find an average pattern, every single cell has small differences in its DNA folding. Single-cell studies show variation: Using special tools (like single-cell Hi-C and live-cell imaging), scientists see that each cell’s DNA structure can be different, but overall gene activity stays consistent. During the cell cycle: When a cell divides, DNA becomes tightly packed (TADs disappear). After division, in the G1 phase, the structure forms again. During development or differentiation: When cells change their type (like from stem cell to nerve cell), parts of DNA can open or close. This allows enhancers (DNA switches) to turn genes on or off, changing the DNA loops and boundaries.

14. Functional consequences

15. LLPS and Diseases

16. What is DNA Damage Response (DDR)?

17. The DDR System Has Three Major Steps Step What Happens Key Players 1. Detection (Sensors) Detect damage or abnormal DNA structure MRN complex (MRE11–RAD50–NBS1), RPA, Ku70/80 2. Signal Transduction (Transducers) Activate kinases to spread the alarm ATM, ATR, DNA-PK 3. Response (Effectors) Repair DNA, stop the cell cycle, or induce apoptosis p53, CHK1/CHK2, BRCA1, 53BP1, Rad51

18. Types of DNA Damage Type of Damage Common Cause Repair Mechanism Single-strand break (SSB) Oxidation, alkylation Base excision repair (BER) Bulky adducts / thymine dimers UV light Nucleotide excision repair (NER) Base mismatch DNA replication errors Mismatch repair (MMR) Double-strand break (DSB) Ionizing radiation, replication collapse Non-homologous end joining (NHEJ) or Homologous recombination (HR) Cells experience different types of DNA damage, and each type needs a specific repair pathway.

19. Two Major Pathways for Double-Strand Break (DSB) Repair When both DNA strands break, it’s the most dangerous kind of damage. The cell must choose how to repair it — quickly or accurately. A. Non-Homologous End Joining (NHEJ) “Quick but Risky” Active in: All cell cycle phases (especially G1) Mechanism: Directly joins broken DNA ends no template. Key Proteins: Ku70/Ku80, DNA- PKcs , XRCC4, Ligase IV, XLF Advantage: Fast Disadvantage: Error-prone (can cause small insertions/deletions)

20. Homologous Recombination (HR) “Slow but Accurate

21. How Does the Cell Choose Between HR and NHEJ? Condition Preferred Pathway Key Regulators G1 phase NHEJ 53BP1 promotes end protection S/G2 phase HR BRCA1 promotes end resection High chromatin compaction NHEJ Less access for HR machinery Open chromatin / replication HR More access to sister chromatid The “pathway choice” depends on cell cycle phase , chromatin state , and protein competition . The “pathway choice” depends on cell cycle phase , chromatin state , and protein competition . The balance between 53BP1 and BRCA1 determines the pathway: 53BP1 → blocks DNA end resection → NHEJ BRCA1 → promotes resection → HR

22. DDR Signaling Cascade (Simplified) Step-by-step: Damage detection: MRN binds DSB → recruits ATM Signal spread: ATM phosphorylates H2AX → γ H2AX foci Recruitment: MDC1, 53BP1, BRCA1, and repair proteins gather at damage site Pathway choice: HR or NHEJ activated Checkpoint activation: CHK1/CHK2 → stop cell cycle for repair Outcome: DNA repaired → resume cycle or trigger apoptosis if damage is irreparable

23. DDR and Diseases When DDR or repair pathway choice fails: Cancer: BRCA1/2 mutations → defective HR → genomic instability Neurodegeneration: Persistent DDR activation (e.g., in ALS, FTD) Aging: Accumulated DNA damage leads to cellular senescence Therapeutics: PARP inhibitors target HR-deficient cancers (synthetic lethality)

24. Formation Mechanism

25. Foci (plural: foci; singular: focus) in the context of cell biology and molecular genetics —particularly when discussing DNA damage, repair, or chromatin organization refers to distinct, localized accumulations of proteins or protein–DNA complexes that form visible spots in the nucleus (or cytoplasm) under a fluorescence microscope. However, if by “ fOCI ” you mean “foci” in the DNA damage response (DDR) context (for example, γ H2AX foci , 53BP1 foci