1.1 The Genome and Epigenome

Question 1

What are the five major classes of non-coding DNA?

Answer 1

1. Promoter and enhancer regions
2. Binding sites for factors that maintain higher order chromatin structures
3. Non-coding regulatory RNA (e.g. microRNAs and long non-coding RNAs)
4. Mobile genetic elements (e.g. transposons)
5. Special structural regions of DNA (in particular telomeres and centromeres)

Question 2

What is the function of a promoter or enhancer region in DNA?

Answer 2

Provide binding sites for transcription factors. Promoters initiate gene transcription; they are on the same strand and upstream of their associated gene. Enhancers modulate gene expression by looping back onto promoters and recruiting additional factors that drive the expression of pre-messenger RNA species

Question 3

What are non-coding regulatory RNAs?

Answer 3

RNAs that are transcribed, but never translated. They regulate gene expression through a variety of mechanisms (e.g. microRNA/miRNA or long-non-coding RNA/lncRNA)

Question 4

What are mobile genetic elements?

Answer 4

“Jumping genes” (e.g. transposons) - genes which move around the genome during evolution, resulting in variable copy number and positioning. They are implicated in gene regulation and chromatin organisation, but their function is not well established.

Question 5

What is a major component of telomeres? Describe this component and the function that it serves.

Answer 5

Satellite DNA.

These are large repetitive sequences of DNA (from 5bp to 5kbp) that are associated with the spindle apparatus, but also maintain the dense, tightly packed organisation of heterochromatin

Question 6

What are the two most common forms of DNA polymorphisms (variation)?

Answer 6

1. Single nucleotide polymorphisms
2. Copy number variations

Question 7

What percentage of single nucleotide polymorphisms (SNPs) occur within coding regions?

What percentage of copy number variations (CNVs) occur within coding regions?

Answer 7

1% of single nucleotide polymorphisms occur within coding regions

50% of copy number variations occur within coding regions

Question 8

Define a single nucleotide polymorphism

Answer 8

Single nucleotide polymorphisms are a variation at a single nucleotide. They are almost always biallelic, occurring across the genome (within exons, introns, intergenic regions, and coding regions).

Question 9

Define copy number variations

Answer 9

Copy number variations are a form of genetic variation consisting of different numbers of large contiguous stretches of DNA.

Question 10

FILL IN THE BLANKS

1___________________ occur across the genome - within exons, introns, intergenic regions, and coding regions.

1___________________ occurring in 2___________________ can occur within genomic regulatory elements, thus altering 3_________________

Some 1__________________ are termed “4__________” variants and are thought to have 5__ effect on 6_____________ or 7________________________________

However, even 4____________ 1__________________ may be useful markers if they happen to be coinherited with a disease-associated polymorphism as a result of physical proximity. In other words, the 1_________________________ and the causative genetic factor are in 8___________________________________.

Answer 10

1. Single nucleotide polymorphisms
2. Non-coding regions
3. Gene expression
4. Neutral
5. No
6. Gene function
7. Individual phenotype
8. Linkage disequilibrium

Question 11

Fill in the blanks

Answer 11

1. Heterochromatin (dense, inactive)
2. Nucleolus
3. Nucleus
4. Euchromatin (sparse, active)
5. Cell
6. p arm
7. q arm
8. Telomeres
9. Centromere
10. Chromosome
11. Nucleosome
12. DNA
13. Transcription
14. Pre-mRNA
15. Promoter
16. Exon
17. Enhancer
18. Intron
19. Splicing
20. 5’ UTR
21. Open reading frame
22. 3’ UTR
23. Translation
24. Protein
25. mRNA

Question 12

What are copy number variations?

Answer 12

Copy number consist of different numbers of large contiguous stretches of DNA. They can be biallelic and simply duplicated, or alternatively deleted in some individuals. At other sites, there are complex rearrangements of the genomic material, with multiple variants in the human population.

Question 13

What percentage of single nucleotide polymorphisms involve gene coding areas?

Answer 13

1%

Question 14

What percentage of copy number variations involve gene coding areas?

Answer 14

50% (thus underlying a large portion of human phenotypic diversity)

Question 15

Describe the structure of chromatin

Answer 15

A segment of DNA 147 base pairs long is wrapped 1.8 times around an octameric low-molecular weight histone proteins (consistening of 8 subunit histone proteins - 2 x H2A, 2 x H2B, 2 x H3, and 2 x H4), resembling spools of thread. These complexes are called nucleosomes and joined together by 20-80 base nucleotide stretches of linker DNA and H1 histone proteins.

Histone units are positively charged, and DNA is negatively charged, thus allowing compaction.

This allows the genome to be packed into the nucleus. Chromatin is not wound uniformly; it can be dense and inactive (heterochromatin) or disperse and active (euchromatin).

Question 16

How does chromatin structure allow regulation of transcription and help define cellular identity and activity?

Answer 16

Generally only “unwound” regions of DNA (euchromatin) are available for transcription. Histones, which are highly dynamic structures, can be regulated by nuclear proteins, therefore allowing remodeling of chromatin structure and changing which genes available are for transcription

Question 17

What are the ways in which chromatin structure can be changed? What complexes are involved in these processes?

Answer 17

Chromatin remodeling complexes can reposition nucleosomes on DNA, exposing (or obscuring) gene regulatory elements such as promoters.

1. Chromatin writer complexes carry out over 70 different histone modifications (including methylation, acetylation and phosphorylation) generically donated as “marks”
2. Chromatin eraser complexes can reverse histone marks
3. Chromatin reader complexes bind histones that bear particular marks, thereby regulating gene expression

Question 18

What are the specific chemical processes by which gene expression is regulated?

Answer 18

1. Histone methylation
2. Histone acetylation
3. Histone phosphorylation
4. DNA methylation
5. Chromatin organizing factors

Question 19

What effect does histone methylation have on gene regulation?

Answer 19

Lysines and arginines can be methylated by specific writer enzymes. Lysine methylation leads to transcriptional activation or repression, depending on which histone residue is marked.

Question 20

What effect does histone acetylation have on gene regulation?

Answer 20

Lysine residues are acetylated by histone acetyltransferases (HATs), which tends to open up chromatin and increase transcription.

Similarly, these changes can be reversed by histone deacetylases (HDACs), leading to chromatin condensation

Question 21

What effect does histone phosphorylation have on gene regulation?

Answer 21

Serine residues can be modified by phosphorylation; depending on the specific residue, the DNA may be opened for transcription or condensed and inactive

Question 22

What effect does DNA methylation have on gene regulation?

Answer 22

High levels of DNA methylation typically lead to transcriptional silencing.

Question 23

What is DNA methylation regulated by?

Answer 23

1. Methyltransferases
2. Demethylating enzymes
3. Methylated-DNA-binding proteins

Question 24

How do chromatin organizing factors affect gene regulation?

Answer 24

They are believed to bind to non-coding regions and control long-range looping of DNA, thus regulating the spatial relationships between enhancers and promoters that control gene expression.

Question 25

Which of genetic changes (i.e. polymorphisms) or epigenetic alterations (e.g. histone acetylation and DNA methylation) are amenable to therapeutic intervention and why? What is an example of this?

Answer 25

Epigenetic alterations are more amenable to therapeutic intervention because they are reversible. An example of this is HDAC and DNA methylation inhibitors which are being tested in treatment of various forms of cancer.

Question 26

How does microRNA (miRNA) regulate gene expression? Describe and draw the stepwise fashion of this process

Answer 26

MicroRNA post-transcriptionally silence mRNA (messenger RNA). One miRNA can regulate multiple protein-coding genes.

1) Transcription of miRNA genes produces a primary transcript (pri-miRNA).
2) This is then processed into smaller segments within the nucleus to form pre-miRNA is a composed of a single strand of RNA with a secondary hairpin loop structure and stretches of double stranded RNA.
3) The pre-miRNA is exported into the cytoplasm by specific transporter proteins
4) It is then trimmed by cytoplasmic Dicer enzyme to generate mature double stranded miRNAs (21-30 nucleotides long)
5) The miRNA subsequently unwinds and the single strands are incorporated into multi-protein RNA-induced silencing complexes (RISC)
6) Base-pairing between the miRNA and the targeted messenger RNA (mRNA) directs RISC to either cleave or repress translation of the mRNA

Question 27

How many microRNA genes does the human genome encode?

Answer 27

~6000

Question 28

Describe an example by which the function of miRNA can be used therapeutically

Answer 28

siRNA (small interfering RNAs) are short RNA sequences that can be introduced experimentally into cells where they serve as substrates for Dicer and interact with RISC, thereby reproducing endogenous miRNA function. Synthetic siRNAs can be used to study gene function and as potential therapeutic agents to silence pathogenic genes (e.g. oncogenes)

Question 29

How do long non-coding RNAs module gene expression?

Answer 29

A) lncRNAs can facilitate transcription factor binding, promoting gene activation

B) lncRNAs can pre-emptively bind transcription factors and inhibit transcription

C) They can promote acetylases, methylases or deacetylases or demethylases that modify histones or DNA

D) They can act as scaffolds to stabilise other structures/complexes that influence chromatin structure and gene activity

E) They can bind to chromatin itself, restricting RNA polymerase from accessing coding genes within that region

Question 30

What is the best known example of a lncRNA binding chromatin, stopping RNA polymerase from accessing coding genes?

Answer 30

XIST (X-Inactive Specific Transcript) is a long non-coding RNA that is transcribed from the X-chromosome and binds chromatin on the X-chromosome, thus resulting in gene silencing of one X-chromosome in females.

Question 31

What is the relationship between enhancers and lncRNAs?

Answer 31

Many enhancers are the site of lncRNA synthesis and expand transcription from gene promoters.

Question 32

Describe the process of gene editing

Answer 32

Gene editing utilises a system found in prokaryotic cells (e.g. bacteria) which gives them acquired immunity against phages and plasmids.

1) DNA is sampled from infected agents

2) Portions of it are then integrated into bacterial or host cell genome as CRISPR (clustered regularly interspersed short palindromic repeats).

3) This is then transcribed into RNA (called guide RNA or gRNA). Each gRNA has approximately 20 bases.

4) The gRNA binds proteins (such as cas9 nuclease - CRISPR associated protein 9 nuclease). Every gRNA strand contains a variable portion and a homologous portion. The homologous portion binds to the cas protein while the variable portion binds to target DNA.

5) The cas9 protein then cleaves the DNA to produce a DNA break

6) The double stranded break is then healed by a process called nonhomologous end joining (NHEJ) which is an error prone mechanism that typically introduces disruptive insertions or deletions (mutations). Alternatively, in the presence of homologous “donor” DNA that spans the region target, cells can use homologous DNA recombination (HDR) to repair the break. This allows us to introduce precise changes in DNA sequence - “editing DNA”

Question 33

What are the practical uses of CRISPR technology?

Answer 33

1) Inserting specific mutations into cells and tissues to model cancers and other diseases
2) Rapidly generating transgenic animal models from edited embryonic stem cells
3) Selectively edit mutations that cause heritable disease, or “less desirable” traits.