CHROMATIN AND THE INTERPHASE NUCLEUS
Functional States of Chromatin, Alterations in Chromatin Organization, and Structural and Functional Organization of the Interphase Nucleus
Prepared by
Dr Bhabesh Nath
Assistant Professor
Department of Zoology
B N College Autonomous, Dhubri
1. Introduction
Each human diploid cell carries about 2 metres of DNA that must fit inside a nucleus only 5–10 µm across, and yet remain accessible for transcription, replication and repair. This is achieved by packaging DNA with histone and non-histone proteins into chromatin. Chromatin is not a uniform, static structure. Different regions exist in different functional states, from open and transcriptionally active to tightly compacted and silent, and these states are dynamically altered during development, differentiation, the cell cycle and disease. In the interphase nucleus the chromatin is further organised in space, together with the nuclear envelope, nucleolus and nuclear bodies, so that nuclear architecture itself influences gene expression.
1.1 Levels of chromatin packaging (a quick recall)
• Nucleosome (10 nm fibre, 'beads on a string'): about 147 bp of DNA wrapped in 1.65 left-handed superhelical turns around a histone octamer (two each of H2A, H2B, H3 and H4). Linker DNA (about 20–80 bp) joins nucleosomes. Linker histone H1 binds the DNA entering and leaving the nucleosome (Luger et al., 1997).
• 30 nm fibre: formed by further coiling (solenoid or zigzag models) and stabilised by H1; its existence in living cells is debated, and many regions appear to be irregular 10 nm fibre clusters.
• Loops and higher-order domains: 30–100 kb loops anchored on a protein scaffold, then miniband and chromonema fibres, reaching the maximally condensed metaphase chromosome (about 10,000-fold compaction).
2. Functional States of Chromatin
On the basis of staining and function, chromatin is divided into two classical forms, euchromatin and heterochromatin, a distinction made by Emil Heitz in 1928 from cytological observations in mosses and liverworts. Modern epigenomic mapping has refined this into several more specific chromatin states.
2.1 Euchromatin
• Lightly stained, decondensed, located mainly in the nuclear interior; contains most of the gene-rich DNA and is generally transcriptionally active or competent.
• Replicates early in S phase, is highly sensitive to DNase I, and shows an open nucleosome arrangement at promoters and enhancers.
• Marked by histone acetylation (H3K9ac, H3K27ac), H3K4 methylation (H3K4me3 at promoters, H3K4me1 at enhancers), H3K36me3 along the transcribed region, and the histone variants H2A.Z and H3.3.
• Low levels of DNA methylation, particularly at CpG islands of promoters.
2.2 Heterochromatin
Heterochromatin remains condensed throughout the cell cycle (except during replication), stains darkly, is gene-poor, replicates late in S phase and is usually transcriptionally repressed. It is of two types.
Feature | Constitutive heterochromatin | Facultative heterochromatin |
Nature | Permanently condensed in all cells | Condensed only in some cells or stages; can revert to euchromatin |
DNA content | Repetitive, satellite DNA, transposons; few or no genes | Contains genes that are silenced in a developmental or cell-specific way |
Location | Centromeres, pericentromeric regions, telomeres, Y chromosome long arm, secondary constrictions | Inactive X chromosome (Barr body), imprinted loci, developmentally regulated genes (e.g. Hox) |
Histone marks | H3K9me2/3, bound by HP1; H4K20me3 | H3K27me3 (PRC2/EZH2), H2AK119ub (PRC1); macroH2A |
DNA methylation | High | Variable |
Key proteins | SUV39H1/H2, HP1, DNMTs | Polycomb repressive complexes, Xist RNA |
Examples | Centromeric and telomeric chromatin | X inactivation in female mammals |
2.3 X-chromosome inactivation (a classic example of facultative heterochromatin)
• Barr and Bertram (1949) observed a dark body, the Barr body, in nuclei of female cats; Mary Lyon (1961) proposed the Lyon hypothesis: in each somatic cell of a female mammal one X chromosome is randomly inactivated early in embryonic development, and this state is clonally inherited.
• Number of Barr bodies = number of X chromosomes − 1 (normal female 1, normal male 0, XXY Klinefelter 1, XXX 2).
• Inactivation is initiated at the X-inactivation centre (XIC), from which the long non-coding RNA Xist is transcribed; Xist coats the future inactive X in cis, recruits Polycomb complexes (H3K27me3, H2AK119ub), histone deacetylation, macroH2A and finally DNA methylation of CpG islands to lock the silent state.
• About 15–25% of genes (such as those in the pseudoautosomal regions) escape inactivation. The mouse also shows imprinted (paternal) X inactivation in extra-embryonic tissue.
2.4 Chromatin states defined by histone modifications
Genome-wide mapping (ChIP-seq, ChromHMM) has defined distinct functional states, which can be summarised as follows.
State | Typical marks | Function |
Active promoter | H3K4me3, H3K9ac, H3K27ac, H2A.Z | Transcription initiation |
Active enhancer | H3K4me1, H3K27ac, p300/CBP | Gene activation at a distance |
Transcribed gene body | H3K36me3, H3K79me2 | Elongation, prevents cryptic initiation |
Poised/bivalent | H3K4me3 together with H3K27me3 | Genes silent but ready for activation, e.g. in embryonic stem cells |
Polycomb-repressed | H3K27me3, H2AK119ub | Reversible silencing of developmental genes |
Constitutive heterochromatin | H3K9me3, HP1, H4K20me3, DNA methylation | Stable silencing of repeats; centromere and telomere function |
Quiescent / nuclear-lamina-associated | Low or no marks; H3K9me2 at LADs | Gene-poor, inactive regions |
2.5 The histone code, writers, readers and erasers
The histone code hypothesis (Strahl and Allis, 2000) states that combinations of covalent modifications of the histone N-terminal tails are 'read' by effector proteins and determine chromatin function. Modifications include acetylation, methylation, phosphorylation, ubiquitination and SUMOylation.
Class | Examples | Role |
Writers | HATs (p300/CBP, GCN5); HMTs (SUV39H1, EZH2, SET1/MLL); kinases (Aurora B) | Add acetyl, methyl, phosphate groups |
Erasers | HDACs; histone demethylases (LSD1, JmjC family); phosphatases | Remove the marks |
Readers | Bromodomain (acetyl-lysine); chromodomain of HP1 and PHD fingers (methyl-lysine) | Recognise marks and recruit other complexes |
• Acetylation neutralises the positive charge of lysine, weakening histone–DNA contacts and opening chromatin (associated with activation); deacetylation by HDACs restores compaction.
• Methylation does not change charge; its effect depends on the residue and the degree: H3K4me3, H3K36me3 and H3K79me are activating, whereas H3K9me3 and H3K27me3 are repressive.
• Phosphorylation of H3 at Ser10 by Aurora B is linked with chromosome condensation in mitosis; phosphorylation of H2A.X at Ser139 (γH2AX) marks DNA double-strand breaks.
3. Alterations in Chromatin Organization
The chromatin state of a locus can be changed by several interconnected mechanisms. These changes are epigenetic, as they alter gene expression without changing the DNA sequence, and they can be inherited through cell division.
3.1 Covalent histone modifications
Described in section 2.5. Addition or removal of marks by writers and erasers switches a region between open and closed states, and is the commonest regulatory mechanism.
3.2 ATP-dependent chromatin remodelling
Multi-subunit remodelling complexes use the energy of ATP hydrolysis to slide, eject, restructure or exchange nucleosomes, exposing or hiding regulatory DNA sequences.
Family | Example complexes | Main action |
SWI/SNF | BAF, PBAF (mammals); SWI/SNF (yeast) | Nucleosome sliding and eviction; transcription activation; frequently mutated in cancers (ARID1A, SMARCA4) |
ISWI | NURF, ACF, CHRAC | Nucleosome spacing and assembly; both repression and activation |
CHD | Mi-2/NuRD, CHD1 | Nucleosome spacing; NuRD combines remodelling with HDAC activity (repression) |
INO80/SWR1 | INO80, SWR1 | Histone variant exchange (H2A.Z) and DNA repair |
3.3 DNA methylation
• Methylation of cytosine at C-5 in CpG dinucleotides (5-methylcytosine) by DNA methyltransferases: DNMT1 copies the pattern on the new strand after replication (maintenance), while DNMT3A/3B establish new patterns (de novo).
• Methylated CpG recruits methyl-binding proteins (MeCP2, MBD1–4) that bring HDACs and chromatin-remodelling complexes, causing silencing. Hypermethylation of CpG islands in promoters silences the gene.
• Demethylation occurs passively through replication or actively through TET enzymes, which oxidise 5mC to 5-hydroxymethylcytosine (5hmC) and onwards.
• Roles: genomic imprinting, X inactivation, silencing of transposons, tissue-specific gene expression.
3.4 Histone variants
Variant | Role |
H2A.Z | Enriched at promoters and enhancers; transcription regulation and boundary function |
H2A.X | Phosphorylated (γH2AX) at sites of DNA double-strand breaks; recruits repair factors |
macroH2A | Associated with the inactive X chromosome and transcriptional repression |
H3.3 | Deposited independently of replication in active genes, enhancers and telomeres |
CENP-A | Centromere-specific H3 variant; epigenetic mark that specifies the kinetochore site |
3.5 Non-coding RNAs, insulators and nuclear positioning
• Long non-coding RNAs (Xist, HOTAIR) and small RNAs (siRNA pathway in fission yeast, piRNAs) guide histone-modifying complexes to specific loci.
• Insulators (bound by CTCF) block enhancer–promoter communication and prevent spreading of heterochromatin; locus control regions such as the β-globin LCR open large domains.
• Movement of a gene towards the nuclear periphery or the lamina generally correlates with silencing.
3.6 Chromatin changes in physiological processes
• Cell cycle: chromatin condenses (H3Ser10 phosphorylation, condensins) for mitosis and decondenses again in telophase and G1.
• Differentiation: pluripotent stem cells have more open chromatin and bivalent domains; differentiation brings progressive heterochromatin formation.
• Spermatogenesis: histones are replaced by protamines for extreme compaction of sperm chromatin.
• Apoptosis: chromatin condensation (pyknosis) and fragmentation of DNA into 180-bp multiples (ladder pattern).
• Senescence and ageing: formation of senescence-associated heterochromatin foci (SAHF) and loss of peripheral heterochromatin.
3.7 Position effect variegation (PEV)
In Drosophila, when a gene such as white (eye colour) is moved by an inversion or translocation next to centromeric heterochromatin (H. J. Muller, 1930), it is silenced in some cells but not others, giving a mosaic (variegated) eye. The spreading of heterochromatin into the neighbouring euchromatic gene is clonally inherited, demonstrating that gene expression depends on the chromatin context. Suppressors and enhancers of PEV include HP1 and Su(var)3-9.
3.8 Altered chromatin and disease
Condition | Chromatin alteration |
Cancer | Global hypomethylation with promoter hypermethylation of tumour suppressor genes (e.g. p16, BRCA1, MLH1); mutations in EZH2, SWI/SNF subunits, histone genes (H3K27M) and IDH (changing demethylase activity) |
Rett syndrome | Mutations in MeCP2, a methyl-CpG-binding protein |
Rubinstein–Taybi syndrome | Mutation in CBP/p300 histone acetyltransferase |
ICF syndrome | Mutations in DNMT3B leading to hypomethylation of pericentromeric repeats |
Fragile X syndrome | CGG repeat expansion in FMR1, with hypermethylation and silencing |
Prader–Willi and Angelman syndromes | Loss of imprinted expression at 15q11–q13 |
Laminopathies (e.g. Hutchinson–Gilford progeria) | LMNA mutations disrupt the nuclear lamina and peripheral heterochromatin |
Because epigenetic changes are reversible, drugs such as HDAC inhibitors (vorinostat) and DNA methyltransferase inhibitors (azacitidine, decitabine) are used in cancer treatment.
4. Structural and Functional Organization of the Interphase Nucleus
The interphase nucleus is the site of DNA replication, transcription and RNA processing, and is far from a random bag of chromatin. Typical dimensions are 5–10 µm (about 10% of the cell volume). Its major structural components are the nuclear envelope, nuclear lamina, nuclear pore complexes, chromatin, nucleolus, nuclear bodies and the nucleoplasm/nuclear matrix.
Figure 1. Schematic organisation of the interphase nucleus (not to scale).
4.1 Nuclear envelope
• A double membrane (outer and inner nuclear membranes) separated by a perinuclear space of about 20–40 nm. The outer membrane is continuous with the rough endoplasmic reticulum and may carry ribosomes; the perinuclear space is continuous with the ER lumen.
• The inner membrane carries integral proteins (lamin B receptor, emerin, LAP2, SUN proteins) that bind lamins and chromatin; the SUN–KASH (LINC) complexes connect the nucleus to the cytoskeleton.
• The two membranes fuse at the nuclear pores. The envelope breaks down in prometaphase and reassembles in telophase in higher eukaryotes (open mitosis).
4.2 Nuclear lamina
• A thin meshwork (about 10–30 nm) beneath the inner membrane, made of lamins A/C, B1 and B2, which are type V intermediate filament proteins.
• Functions: gives mechanical strength and shape to the nucleus, anchors chromatin (lamina-associated domains, LADs) and nuclear pores, helps organise heterochromatin at the periphery, and takes part in DNA replication, transcription regulation and nuclear reassembly.
• Phosphorylation of lamins by CDK1 at the onset of mitosis disassembles the lamina; dephosphorylation allows reassembly. Mutations in LMNA cause laminopathies such as progeria, Emery–Dreifuss muscular dystrophy and some cardiomyopathies.
4.3 Nuclear pore complex (NPC) and nucleocytoplasmic transport
• A large (about 120 MDa in vertebrates) octagonal, eight-fold symmetrical channel built from about 30 different proteins called nucleoporins, with cytoplasmic filaments and a nuclear basket. A mammalian nucleus has a few thousand pores.
• Small molecules and proteins up to about 40 kDa diffuse passively; larger macromolecules are transported actively, signal-mediated and energy-dependent.
• Proteins with a nuclear localisation signal (NLS) (short stretches of basic residues, e.g. PKKKRKV of SV40 large T antigen) bind importins (karyopherins); proteins with a nuclear export signal (NES) bind exportins. Directionality is provided by the Ran GTPase gradient: Ran-GTP is high in the nucleus (made by the nuclear RCC1 GEF) and Ran-GDP is high in the cytoplasm (made by RanGAP).
• RNA is exported as ribonucleoprotein particles (mRNA through NXF1/TAP; rRNA and tRNA through exportins).
4.4 Chromosome territories
Each interphase chromosome occupies a discrete, non-overlapping region of the nucleus called a chromosome territory, rather than being mixed with others. Theodor Boveri predicted this in 1909 and it was demonstrated by Thomas and Christoph Cremer using laser UV micro-irradiation, and later by chromosome painting (FISH).
• Territory positioning is non-random: gene-rich chromosomes (human chr 19) tend to lie toward the nuclear interior, while gene-poor chromosomes (chr 18) lie near the periphery.
• Between territories lies an interchromatin compartment (channel network) where splicing factors and transcription machinery operate; loops from adjacent territories can intermingle at their borders.
4.5 Higher-order genome organization
Chromosome conformation capture methods (3C, 4C, Hi-C) have revealed several levels of folding.
Level | Description |
A and B compartments (Lieberman-Aiden et al., 2009) | Megabase-scale; A compartment is gene-rich, open, active (euchromatin, interior); B compartment is gene-poor, compact, inactive (heterochromatin, periphery) |
Topologically associating domains (TADs) (Dixon et al., 2012) | Self-interacting domains of about 0.2–1 Mb with sharp boundaries enriched for CTCF; restrict enhancer–promoter interactions |
Loops | Formed by loop extrusion by cohesin, halted by convergent CTCF sites; link enhancers and promoters |
Lamina-associated domains (LADs) (Guelen et al., 2008) | 0.1–10 Mb regions contacting the lamina; gene-poor, low expression, H3K9me2/3 |
Nucleolus-associated domains (NADs) | Chromatin contacting the nucleolus; often repressed, including rDNA-adjacent regions |
• Inverted nuclei: in rod photoreceptors of nocturnal mammals heterochromatin occupies the centre and euchromatin the periphery, acting as a lens that reduces light scattering (Solovei et al., 2009).
4.6 Nucleolus
• The largest nuclear body, not membrane bound, formed around nucleolar organiser regions (NORs) carrying tandem rDNA repeats. In humans the NORs are on the short arms of acrocentric chromosomes 13, 14, 15, 21 and 22.
• Three zones seen by electron microscopy: fibrillar centres (FC) containing rDNA and RNA polymerase I; dense fibrillar component (DFC), the site of pre-rRNA transcription and early processing; and the granular component (GC), where ribosomal subunits are assembled.
• Functions: synthesis of 45S pre-rRNA by Pol I (processed into 18S, 5.8S and 28S rRNA), 5S rRNA import, assembly of ribosomal subunits with ribosomal proteins, and also stress sensing (p53 regulation) and assembly of some RNPs. The nucleolus disperses in mitosis and reforms at telophase.
4.7 Nuclear bodies and nuclear matrix
Structure | Marker / content | Function |
Nuclear speckles (interchromatin granule clusters) | SC35 (SRSF2), snRNPs, splicing factors | Storage and assembly of splicing factors; supply active genes nearby |
Cajal bodies | Coilin, SMN, snRNPs, snoRNPs | Maturation of snRNPs and snoRNPs; histone mRNA processing; telomerase assembly |
PML bodies | PML protein, SUMO | Transcription regulation, DNA repair, antiviral defence, apoptosis |
Paraspeckles | NEAT1 lncRNA, PSPC1 | Retention of edited RNAs in the nucleus |
Histone locus bodies | NPAT | Transcription and processing of histone genes |
Transcription factories | RNA Pol II clusters | Sites where many active genes are transcribed together |
The nuclear matrix (nuclear scaffold) is the insoluble fibro-granular network that remains after extraction of DNA, histones and soluble proteins with nuclease and high salt. It consists of lamins, hnRNP proteins and others. DNA loops are attached at scaffold/matrix attachment regions (S/MARs), AT-rich sequences, and matrix proteins such as topoisomerase II. It is believed to organise DNA replication, transcription and splicing, though its in vivo structure remains debated.
4.8 Functional organization: replication, transcription and repair
• Transcription: active genes loop out of their territories to reach transcription factories and speckles. The nucleolus is the factory for Pol I transcription, whereas Pol II and Pol III transcription takes place in the nucleoplasm.
• Replication: replication occurs at discrete foci (replication factories). Early-replicating euchromatin lies in the interior and late-replicating heterochromatin lies at the periphery and around the nucleolus; the pattern of foci changes through S phase.
• RNA processing: splicing, 5′ capping and 3′ polyadenylation are coupled to transcription and concentrated around speckles.
• DNA repair: breaks recruit γH2AX, 53BP1 and repair factors in foci; PML bodies and the nuclear lamina also contribute.
• Nuclear transport and signalling: NPCs regulate access of transcription factors and mRNA export, and mechanical forces from the cytoskeleton via the lamina can alter chromatin and gene expression.
5. Quick Summary
• Euchromatin: open, early replicating, gene-rich, H3K4me3/acetyl marks. Heterochromatin: condensed, late replicating, gene-poor; constitutive (H3K9me3, HP1, centromeres) vs facultative (H3K27me3, Barr body).
• Alterations come from histone modifications (writers, readers, erasers), ATP-dependent remodelling (SWI/SNF, ISWI, CHD, INO80), DNA methylation (DNMT1, DNMT3A/B, TET), histone variants and non-coding RNAs; PEV and many diseases show their importance.
• The nucleus has an envelope with pores, lamina, chromosome territories, A/B compartments, TADs and LADs, a nucleolus that makes ribosomes, and nuclear bodies (speckles, Cajal bodies, PML bodies) that compartmentalise function.
6. Multiple Choice Questions (10)
Q1. Constitutive heterochromatin is typically marked by:
(a) H3K4me3 and H3K27ac
(b) H3K9me3 bound by HP1
(c) H3K36me3
(d) H2A.Z
Q2. The Barr body represents:
(a) An active X chromosome
(b) The Y chromosome
(c) An inactivated X chromosome
(d) The nucleolus
Q3. How many Barr bodies are present in the nucleus of a person with the karyotype 47,XXY?
(a) 0
(b) 1
(c) 2
(d) 3
Q4. The long non-coding RNA that initiates X-chromosome inactivation is:
(a) HOTAIR
(b) Xist
(c) NEAT1
(d) 7SK
Q5. Which enzyme maintains DNA methylation patterns after replication?
(a) DNMT3A
(b) DNMT3B
(c) DNMT1
(d) TET1
Q6. Which of the following is an ATP-dependent chromatin remodelling complex?
(a) HDAC1
(b) SWI/SNF
(c) SUV39H1
(d) p300
Q7. Position effect variegation in Drosophila is explained by:
(a) Point mutation of the white gene
(b) Spread of heterochromatin into a gene moved near it
(c) Loss of the gene
(d) DNA methylation of rRNA genes
Q8. The nuclear lamina is composed of:
(a) Actin filaments
(b) Microtubules
(c) Lamins (type V intermediate filaments)
(d) Keratins
Q9. The nucleolar organiser regions in humans are located on chromosomes:
(a) 1, 9 and 16
(b) 13, 14, 15, 21 and 22
(c) 6, 7 and 8
(d) X and Y
Q10. Topologically associating domains (TADs) have boundaries enriched for which protein?
(a) CTCF
(b) HP1
(c) Coilin
(d) Lamin B1
Answer Key
Q1 | Q2 | Q3 | Q4 | Q5 | Q6 |
(b) | (c) | (b) | (b) | (c) | (b) |
Q7 | Q8 | Q9 | Q10 |
(b) | (c) | (b) | (a) |
7. Exam-Oriented Questions
A. One-Mark Questions (with answers)
1. Who coined the terms euchromatin and heterochromatin?
Ans: Emil Heitz (1928).
2. Name the histone mark characteristic of facultative heterochromatin.
Ans: H3K27me3 (deposited by PRC2/EZH2).
3. What is a Barr body?
Ans: The condensed, inactivated X chromosome seen at the nuclear periphery in female somatic cells.
4. State the Lyon hypothesis in one line.
Ans: One of the two X chromosomes in each female mammalian somatic cell is randomly inactivated early in development.
5. Which histone variant marks the centromere?
Ans: CENP-A.
6. Name the enzyme family that removes acetyl groups from histones.
Ans: Histone deacetylases (HDACs).
7. What is the thickness of the nuclear lamina?
Ans: About 10–30 nm.
8. Name the GTPase that gives directionality to nuclear transport.
Ans: Ran.
9. What are LADs?
Ans: Lamina-associated domains, gene-poor chromatin regions anchored to the nuclear lamina.
10. Where is rRNA synthesised in the cell?
Ans: In the nucleolus, by RNA polymerase I.
B. Two-Mark Questions (with answers)
1. Distinguish between euchromatin and heterochromatin.
Ans: Euchromatin is light staining, decondensed, gene-rich, early replicating and transcriptionally active (acetylated histones, H3K4me3). Heterochromatin is dark staining, condensed, gene-poor, late replicating and mostly inactive (H3K9me3/H3K27me3, high DNA methylation).
2. What is the difference between constitutive and facultative heterochromatin?
Ans: Constitutive heterochromatin is permanently condensed in all cells and contains repetitive DNA (centromeres, telomeres). Facultative heterochromatin is condensed only in certain cells or times and can revert to euchromatin (inactive X).
3. What is the histone code hypothesis?
Ans: Combinations of covalent modifications on histone tails (acetylation, methylation, phosphorylation) are read by specific proteins and together specify chromatin structure and gene activity (Strahl and Allis, 2000).
4. Write a short note on chromatin remodelling complexes.
Ans: ATP-dependent multi-subunit complexes (SWI/SNF, ISWI, CHD, INO80) that slide, eject or exchange nucleosomes, thereby exposing or hiding regulatory DNA sequences.
5. What is position effect variegation?
Ans: Variegated expression of a gene placed next to heterochromatin by rearrangement, because heterochromatin spreads into it in some cells; shown by the white gene in Drosophila.
6. Mention the functions of the nuclear lamina.
Ans: Mechanical support and shape of the nucleus, anchoring of chromatin (LADs) and pores, organisation of peripheral heterochromatin, and roles in replication, transcription and nuclear reassembly.
7. What are chromosome territories?
Ans: Discrete, non-overlapping nuclear regions occupied by individual interphase chromosomes, with gene-rich chromosomes in the interior and gene-poor ones near the periphery.
8. What are TADs?
Ans: Topologically associating domains: self-interacting chromatin domains of about 0.2–1 Mb with CTCF-rich boundaries that limit enhancer–promoter contacts.
C. Five-Mark Questions (answer outlines)
1. Describe the functional states of chromatin with suitable examples.
Key points for answer:
– Define chromatin; classical division by Heitz (1928).
– Euchromatin: features, marks (H3K4me3, acetylation), early replication.
– Heterochromatin: constitutive (H3K9me3, HP1, centromeres) vs facultative (H3K27me3, Barr body); give a comparison table.
– X inactivation: Lyon hypothesis, Xist RNA, Barr body count.
– Modern states: active promoters, enhancers, poised/bivalent, Polycomb-repressed.
2. Explain how chromatin organization is altered by epigenetic mechanisms.
Key points for answer:
– Histone modifications: writers, erasers, readers; histone code.
– ATP-dependent remodelling: SWI/SNF, ISWI, CHD, INO80.
– DNA methylation: DNMT1, DNMT3A/B, CpG islands, MeCP2, TET enzymes.
– Histone variants (H2A.Z, H2A.X, CENP-A, H3.3) and non-coding RNAs.
– Consequences: PEV, imprinting, cancer and other diseases.
3. Describe the structure and functions of the nuclear envelope, lamina and nuclear pore complex.
Key points for answer:
– Double membrane, perinuclear space, continuity with ER.
– Lamina: lamins A/C, B1, B2; functions and laminopathies.
– NPC: 8-fold symmetry, nucleoporins, transport routes.
– NLS/NES, importins/exportins and the Ran cycle.
– Breakdown and reassembly during mitosis.
4. Discuss the organization of chromatin in the interphase nucleus.
Key points for answer:
– Chromosome territories and interchromatin compartment.
– A/B compartments, TADs, loop extrusion by cohesin and CTCF.
– LADs and nucleolus-associated domains; peripheral heterochromatin.
– Replication timing and transcription factories.
– Inverted nuclei of rod cells; labelled diagram.
5. Write an account of the nucleolus and other nuclear bodies.
Key points for answer:
– Nucleolus: NORs on chromosomes 13, 14, 15, 21, 22; FC, DFC and GC zones.
– Pol I transcription of 45S pre-rRNA; ribosome subunit assembly.
– Speckles (splicing factors), Cajal bodies (snRNP maturation), PML bodies, paraspeckles.
– Nuclear matrix and S/MARs.
– Dynamic behaviour in cell cycle and stress.
D. Additional Practice Questions
1. Explain X-chromosome inactivation with the role of Xist RNA.
2. Compare the histone modifications associated with active and repressed chromatin.
3. What is position effect variegation? Explain with reference to Drosophila.
4. Describe the three zones of the nucleolus and their functions.
5. How do laminopathies illustrate the importance of nuclear architecture?
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