Chromatin Structure and Nuclear Organization

Question 1

How is 2 meters of DNA compacted to fit into the nucleus of a cell?

Answer 1

DNA is compacted by forming nucleosomes, where it wraps around histone octamers, creating a 10 nm chromatin fiber. Strings of nucleosomes, connected by linker DNA, further compact DNA to fit inside the nucleus.

Question 2

What is a nucleosome, and what proteins are involved in its structure?

Answer 2

A nucleosome is a unit of chromatin where DNA wraps around a core histone octamer consisting of two copies each of H2A, H2B, H3, and H4 histone proteins. The DNA wraps ~1.6 turns around this protein core.

Question 3

What is the role of histone tails in gene expression?

Answer 3

Histone tails are flexible, unstructured domains that extend from the nucleosome. They can be post-translationally modified to alter gene expression by making the DNA more or less accessible for transcription.

Question 4

How does the positive charge of histone proteins influence their interaction with DNA?

Answer 4

Histone proteins are positively charged, which neutralizes the negatively charged DNA phosphate backbone. This electrostatic interaction helps package DNA tightly around histones, forming nucleosomes.

Question 5

Why are 30 nm chromatin fibers considered an in vitro artifact, and how is chromatin organized in vivo?

Answer 5

30 nm fibers are an artifact of in vitro purification. In vivo, chromatin is organized as 10 nm fibers, which are packaged into mitotic chromosomes without transitioning to 30 nm fibers.

Question 6

How do chromatin and naked DNA differ in their biophysical properties?

Answer 6

Chromatin is stabilized by the positive charges of histones, reducing steric repulsion and enabling flexibility. Naked DNA, with its periodic negatively charged phosphate backbone, requires metal ions or small molecules to neutralize the charge and remain flexible.

Question 7

How is tissue-specific gene expression regulated in multicellular organisms?

Answer 7

Tissue-specific gene expression is regulated by post-translational modifications of histone proteins and DNA, which either restrict or activate gene expression. These modifications include histone tail modifications and DNA methylation.

Question 8

What role does DNA methylation play in gene expression?

Answer 8

DNA methylation, particularly of cytosine at CpG sites, can influence gene expression by either repressing or activating specific genes. Methylation impacts the recruitment of transcription factors and overall DNA metabolism.

Question 9

Which DNA bases are most frequently methylated, and what enzyme is responsible for this process?

Answer 9

Adenine (A) and cytosine (C) are the most frequently methylated bases. DNA methylases use S-adenosylmethionine (SAM) as a methyl group donor for this process.

Question 10

How do histone tail modifications affect nucleosome packaging and gene expression?

Answer 10

Histone tail modifications can change nucleosome packaging, leading to either a closed conformation (gene silencing) or an open conformation (gene activation). They also recruit protein complexes that regulate gene expression in combination with DNA methylation.

Question 11

How can the length and staining density of genomic regions be assessed in human cells during mitosis?

Answer 11

The length and staining density of genomic regions can be assessed using light microscopy during mitosis when chromosomes compact and pair, forming an “X” shape, which is helpful for karyotyping.

Question 12

What kind of genomic changes are often associated with disease states like cancer?

Answer 12

Disease states, especially cancer, are often associated with large-scale genomic changes such as duplications, deletions, and inversions of chromosome segments.

Question 13

What is the purpose of FISH (Fluorescence In Situ Hybridization) experiments?

Answer 13

FISH experiments are used to visualize or detect specific DNA sequences using fluorescent probes that anneal to specific regions of the genome, allowing researchers to assess genomic stability and gene expression.

Question 14

How are chromosomal regions visualized in FISH experiments?

Answer 14

Fluorescent probes are annealed to denatured chromatin after the DNA is chemically denatured and heated, and the specific binding is visualized using fluorescence microscopy.

Question 15

What are chromosome territories, and when do they form?

Answer 15

Chromosome territories are distinct domains formed by chromatin from individual chromosomes during interphase. Each chromosome occupies its own domain rather than mixing with others.

Question 16

How is chromatin organized during interphase?

Answer 16

During interphase, chromatin is organized into discrete structures known as chromosome territories, composed of 10-nm chromatin fibers made of nucleosomes connected by linker DNA sequences.

Question 17

What role does nuclear organization play in cellular functions?

Answer 17

The nucleus is highly compartmentalized, with sub-nuclear bodies like the nucleolus responsible for ribosome production. These bodies, lacking membranes, compartmentalize and concentrate molecules to increase efficiency of processes like transcription and splicing.

Question 18

How do nuclear bodies, such as the nucleolus, hold together if they lack membranes?

Answer 18

Nuclear bodies are held together through protein phase separation. Proteins and RNA self-organize into phase-separated compartments due to high concentrations or post-translational modifications, allowing them to remain intact in an aqueous environment.

Question 19

What 2 mechanisms allow proteins to form phase-separated nuclear bodies?

Answer 19

Two mechanisms allow proteins to form phase-separated nuclear bodies: (1) interlocking via multivalent domains, and (2) pi-stacking via disordered, low-complexity regions like phenylalanine residues.

Question 20

How are biological processes like transcription sub-compartmentalized in the nucleus?

Answer 20

Processes such as transcription are sub-compartmentalized in nuclear bodies called transcription factories, where RNA polymerase clusters to transcribe active genes. Genomic regions move to these factories when active transcription is required.

Question 21

How is fluorescence microscopy used to detect specific proteins within cells?

Answer 21

luorescence microscopy uses antibodies raised to detect a specific protein of interest, often tagged with a fluorescent protein. Cells are fixed and permeabilized, and the tagged antibody binds to the target protein, allowing its visualization.

Question 22

What are DNA counterstains like DAPI or Hoescht used for in fluorescence microscopy experiments?

Answer 22

DNA counterstains such as DAPI or Hoescht are used to define the nucleus, allowing researchers to visualize regions of more or less DNA, which helps in identifying compacted heterochromatin versus less compacted regions.

Question 23

What genetic mutation causes Hutchinson-Gilford Progeria Syndrome?

Answer 23

A de novo C->T point mutation in exon 11 of the lamin A gene, which creates a cryptic splice site and results in abnormal splicing of the gene.

Question 24

How does the C->T mutation in exon 11 of the lamin A gene affect splicing?

Answer 24

The mutation causes splicing to start at the beginning of exon 11 instead of the normal splice junction between exon 11 and exon 12, resulting in the removal of part of exon 11 and the adjoining intron.

Question 25

How can the RNA from the lamin A gene be visualized in affected individuals versus their parents?

Answer 25

RT-PCR analysis shows that parents have only the full-length lamin A mRNA, while the affected individual has both a full-length and a shortened version of lamin A mRNA.

Question 26

How does the cryptic splicing of the lamin A gene result in two protein products?

Answer 26

Western blot analysis reveals that the affected individual has both the wild-type lamin A protein and a smaller, alternatively spliced version, while the parents have only the wild-type protein.

Question 27

What differences in nuclear organization are seen in cells affected by progeria compared to normal cells?

Answer 27

In progeria cells, the nucleus has an irregular, reticulated structure with lamin spread throughout the nucleoplasm, while normal cells have a smooth oval nucleus with lamin mostly localized at the nuclear periphery.

Question 28

How is lamin dynamics altered in progeria cells, as shown by fluorescence recovery after photobleaching (FRAP)?

Answer 28

Lamin A in progeria cells shows a much slower recovery of fluorescence compared to wild-type lamin A, indicating that the mutated lamin is less dynamic and takes longer to return to the nuclear envelope.

Question 29

What effect does treating progeria cells with a small oligo have on the aberrant lamin protein?

Answer 29

Treatment with a small oligo reduces the amount of alternatively spliced lamin A mRNA and decreases the production of the aberrant lamin protein in progeria cells.

Question 30

What technique is used to measure the dynamic movement of lamin proteins in cells, and how does it work?

Answer 30

Fluorescence recovery after photobleaching (FRAP) is used to monitor the recovery of GFP-labeled lamin proteins. After a region is bleached, the return of fluorescence indicates the movement of unbleached lamin proteins into the area.

Question 31

How does the lamin A mutation in progeria affect the wild-type lamin protein?

Answer 31

The lamin A mutation in progeria affects the dynamics of even the wild-type lamin protein, slowing its movement and recovery at the nuclear envelope.

Question 32

What is the potential therapeutic approach for Progeria involving oligos, and how does it work?

Answer 32

Treating progeria cells with a small oligo can suppress the production of the alternatively spliced lamin A mRNA, reducing the amount of the aberrant protein, although the exact mechanism is still unknown.