Week 7

Question 1

Central Dogma of Biology and its regulations

Answer 1

1. Transcription (on/off)
2. RNA processing (alternative splicing)
3. mRNA transport (stability, localization)
4. Translation (on/off, level)
5. active gene product

each step can be regulated

Question 2

• How does chromatin structure regulate gene expression
• How can chromatin structure be reversed

Answer 2

• Heterochromatin: closed, transcription off
• Euchromatin: Open, transcription ON in
  regions accessible to transcriptional regulators
• Chromatin structure can be reversed through the action of molecular switches

Question 3

• Factors that influence chromatin structure

Answer 3

1. Nucelosome remodelling
2. histone modification
3. DNA methylation

Question 4

• what modifications regulate gene expression and what are the 4 features of these modifications

Answer 4

• Epigenetic modifications regulate gene expression
• Features of Epigenetic modifications
  Ø Result from histone modifications or DNA methylation
  Ø Impact gene expression but do not alter DNA sequence
  Ø Heritable-are maintained after cell division
  Ø ensure that the repressed or active state of a gene is maintained after cell division

Question 5

DNA methylation

1. DNA in eukaryotes can be methylated at the – position of cytosine within —
2. CpG dinucleotides are not evenly distributed in the genome; most of them are present in regions called —
3. Approx. x% of CpG dinucleotides in CGIs are methylated in the mouse genome.
4. Approx.x% of CpGs in non-CGI regions are methylated in the mouse genome
5. DNA methylation does not affect -
6. DNA methylation of promoter regions of genes is mostly correlated
7. An epigenetic modification

Answer 5

1. NA in eukaryotes can be methylated at the C5 position of cytosine within cytosine-phosphate-guanine (5’-CpG-3’) dinucleotides i.e.Cytosine followed by a Guanine on the same DNA strand in 5’->3’ direction
2. CpG dinucleotides are not evenly distributed in the genome; most of them are present in regions called “CpG islands (CGI)
3. Approx. 2% of CpG dinucleotides in CGIs are methylated in the mouse genome
4. Approx. 60-80% of CpGs in non-CGI regions are methylated in the mouse genome
5. DNA methylation does not affect base pairing ->Guanine still pairs with methylated Cytosine
6. DNA methylation of promoter regions of genes is mostly correlated with silencing of expression (i.e. gene will be transcriptionally inactive
7. An epigenetic modification=> this form of transcriptional repression can be passed on to daughter cells over many rounds of cell division. In other words, DNA methylation pattern is preserved as DNA is replicated. This ensures a stable pattern of gene expression required to maintain the cellular identity.

Question 6

Methylation of Cytosine

• why s making access by enzymes in a double helix difficult
• how are cytosines accesed

Answer 6

• Bases are tightly packed in the double helix, making access by enzymes difficult
• DNA methyltransferases (green) use “base-flipping” to access cytosines
• A cytosine residue (red) is flipped out of the DNA double-helix (black)
• An amino acid is temporarily inserted to stack in the double-helix
• The cytosine is methylated and reinserted into the double-helix

Question 7

1. 3 places where DNA methylation occur
2. DNA is also methylated in x and x regions
3. DNA methylation is absent from x
   a) Approx. x% of human gene promoters are embedded in CGIs. The human genome contains ∼x CGIs. While CGIs associated with promoters nearly always remain x especially those in vicinity of x, many of the ∼9,000 CGIs become x during x and x

Answer 7

1. a) CpG island (CGI) promoters
   b) Imprinted genes
   c) Transposable elements
2. DNA is also methylated in intragenic and intergenic regions
3. DNA methylation is absent from most of the CpG islands (CGI)
   a) Approx. 70% of human gene promoters are embedded in CGIs. The human genome contains ∼30,000 CGIs. While CGIs associated with promoters nearly always remain unmethylated especially those in vicinity of housekeeping genes, many of the ∼9,000 CGIs become methylated during development and differentiation

Question 8

1. x is required for normal development
2. DNA Methylation at CpG island (CGI)=> x
3. Normal developmentally regulated CGI methylation:
   a) x become methylated during differentiation (e.g., pluripotency genes Oct4 and Nanog in ES cells) in a x
   b) Most CGIs on the inactive X chromosome of females are x Exception- x
   c) Genes that are expressed exclusively from either the maternal or the paternal allele (imprinted genes), have x. Note DNA methylation may silence only x
4. DNA methylation silences x
   a) Transposable elements also called “x” are x. DNA methylation silences these elements. Methylation of transposable elements is necessary to x and prevent x
5. x is observed in cancer cells
   a) CpG islands of cancer cells: x
   b) Transposable elements of cancer cells: x

Answer 8

1. DNA methylation of some CpG islands is required for normal development
2. DNA Methylation at CpG island (CGI)=> silencing of gene expression
3. Normal developmentally regulated CGI methylation:
   a) CGI promoters of certain genes become methylated during differentiation (e.g., pluripotency genes Oct4 and Nanog in ES cells) in a tissue-specific manner
   b) Most CGIs on the inactive X chromosome of females are densely methylated (X chromosome inactivation) Exception- few genes that escape X-inactivation have their CGIs unmethylated
   c) Genes that are expressed exclusively from either the maternal or the paternal allele (imprinted genes), have CGI methylation on one of the alleles (e.g., H19, IGF2 imprinted gene cluster)- Note DNA methylation may silence only some of the genes in an imprinted gene cluster
4. DNA methylation silences transposable elements
   a) ransposable elements also called “jumping genes” are mobile elements which when activated can insert themselves anywhere in the genome. DNA methylation silences these elements. Methylation of transposable elements is necessary to promote genomic stability and prevent dysregulation of gene expression.
5. Aberrant DNA methylation is observed in cancer cells
   a) CpG islands of cancer cells: Specific hypermethylation of gene promoters
   b) Transposable elements of cancer cells: Global hypomethylation of the genome

Question 9

1. Writers: x-3 types . All three catalyze x. x does not have catalytic activity, but it enhances the DNA methylation activity of x and x
   a) DNMT1: Methylates x in x position as the x and is x after cell division
   b) DNMT3a and DNMT3b: x methylation by targeting previously x in the x
   * Often recruited to genes for x
   *Play an important role during x
2. Erasers: x from x occurs in x. A direct mechanism to break the the x bond that connects the x to x does not appear to exist.
3. Readers: x is recognized by x families of x: i) MBD contain a x an e.g., MeCP2

Answer 9

1. Writers: DNA methyl transferases-DNMT1, DNMT3a, DNMT3b. All three catalyze addition of methyl groups to the DNA strand. Dnmt3L does not have catalytic activity, but it enhances the DNA methylation activity of DNMT3a and DNMT3b
   a) DNMT1: Methylates new DNA strand in the same position as the parent strand and is Pattern inherited after cell division
   b) DNMT3a and DNMT3b: De Novo methylation by targeting previously unmethylated positions in the
   genome
   * Often recruited to genes for promoting
   gene silencing
   *Play an important role during embryonic
   development
2. Erasers: Removal of methyl group from Cytosine occurs in multiple steps. A direct mechanism to break the the strong covalent carbon-carbon bond that connects the methyl group to cytosine does not appear to exist.
3. Readers: DNA methylation is recognized by 3 families of proteins: i) MBD contain a conserved methyl-CpG-binding domain
   , an e.g., MeCP2

Question 10

What are the mechanisms by which DNA methylation prevents transcription?

Answer 10

A) DNA methylation interferes with the binding of TFs. Several transcription
factors recognize CG-rich sequences.
B) MeCP2 recruits HDAC ( histone deacetylase) which removes acetyl groups from histone
tails- promotes conversion to closed form of chromatin
C) MeCP2 recruits HMT (histone methyltransferase) which adds repressive histone modifications

Question 11

5. Biological functions of DNA methylation

Answer 11

• Regulation of gene expression and cellular differentiation.
• Required for normal embryonic development. Dnmt1-/- and Dnmt3a-/- , Dnmt3b-/- mutants die embryonically.
• Required to promote genomic stability through silencing of transposable elements.
  Loss of Dnmt3b function in mice led to chromosomal aberrations such as chromosome fusion, breakage and
  aneuploidy (abnormal chromosome number).
• Required for the formation of heterochromatin e.g., in the case of X chromosome inactivation in females
• Regulation of expression of imprinted genes

Question 12

1. Imprinted genes:
2. gDMRs/ICRs: (
3. Gamete:
4. Primordial Germ Cells
5. Epigentic reprograming

Answer 12

1. Imprinted genes: genes expressed predominantly from one parental allele and whose allelic expression is controlled by gDMRs/ICRs.
2. gDMRs/ICRs: (gametic or germline differentially methylated regions / imprinting control regions)=> sequences with differences in DNA methylation between male and female chromosomes which control genomic imprinting.
3. Gamete: a sperm (male gamete) or an egg (female gamete)
4. Precursors to sperm and eggs
5. In mammals, epigenetic reprogramming refers to the remodelling of epigenetic marks following fertilisation in the early embryo and during germ-cell specification

Question 13

Passive versus active DNA demethylation pathways

Answer 13

Passive: Dependent on DNA replication and abscence of DNMT1 in the nucleus
Active: addition of hydroxyl to generate 5hmC

Question 14

1. Epigenetic reprogramming during early mammalian development
   a) Paternal DNA undergoes x (x process), before x. Requires x
   b) Maternal DNA undergoes x (x process)- During DNA replication, x do not get new x
   x is absent in the nucleus
   * Eventually, both copies of DNA have
   x
   * x maintain the methylation status
   * x levels increase=> x methylation
   by x and x; maintenance methylation by x
2. Epigenetic reprogramming in Primordial Germ cells (PGCs): 2nd wave of reprogramming in PGCs
   a) * DNA methylation marks are x at
   x for both male and female specific PGCs
   * Imprinted genes also lose x
   * New genome-wide DNA methylation
   occurs in a x manner,
   * Imprinted genes x
   * Slower in x than x since x

Answer 14

1. Epigenetic reprogramming during early mammalian development
   a) Paternal DNA undergoes rapid
   genome-wide demethylation (active process), before DNA replication begins. Requires TET proteins
   b) Maternal DNA undergoes slow demethylation (passive process)- During DNA replication, the daughter strands do not get new methylation marks
   DNMT1 is absent in the nucleus
   * Eventually, both copies of DNA have
   very low levels of methylation
   * Imprinted genes maintain the methylation status
   * Post-implantation total methylation
   levels increase=> de novo methylation
   by DNMT3a and 3b; maintenance
   methylation by DNMT1
2. Epigenetic reprogramming in Primordial Germ cells (PGCs): 2nd wave of reprogramming in PGCs
   a) * DNA methylation marks are removed at
   the same rate for both male and female
   specific PGCs
   * Imprinted genes also lose DNA methylation marks
   * New genome-wide DNA methylation
   occurs in a sex-specific manner,
   * Imprinted genes also get methylated
   * Slower in oocytes than sperms since
   oocytes mature a little later in development
   compared to the sperms

Question 15

The first wave of epigenetic reprogramming
* x is required to establish the x state of the zygote and the x state of the forming x.
* Takes places after x- the paternal and the maternal DNA lose x through an x
* x is maintained during this reprogramming event.
* The epiblast cells undergo x as x
The second wave of epigenetic reprogramming
* Takes place in x. DNA methylation is x at the same rate for both male and female germ cells
* x also lose DNA methylation marks
* De novo methylation takes place in a x manner, depending upon whether x

Answer 15

The first wave of epigenetic reprogramming
* Global demethylation is required to establish the totipotent state of the zygote and the pluripotent state of the
forming epiblast.
* Takes places after fertilization- the paternal and the maternal DNA lose methylation marks through an active and passive process, respectively.
* Methylation status of imprinted genes is maintained during this reprogramming event.
* The epiblast cells undergo genome wide de novo methylation as they begin the somatic cell differentiation program
The second wave of epigenetic reprogramming
* Takes place in PGCs. DNA methylation is erased at the same rate for both male and female germ cells
* Imprinted genes also lose DNA methylation marks
* De novo methylation takes place in a sex-specific manner, depending upon whether the PGCs will differentiate into
sperms or oocytes.

Question 16

How do we measure DNA methylation?

Answer 16

Bisulfite sequencing
:

Question 17

1. Bisulfite sequencing (BS-seq) or whole genome bisulfite sequencing (WGBS) is a technique used to detect x at x in the x
   a) 4 Key Steps:
2. Designing PCR primers-
   * Since the methylation status of CpG dinucleotides is x, the primer sequences should strictly avoid x-> design primers for regions that x, strictly avoid x
   * Non-CpG cytosines in the original DNA should be x
3. Quality control checks to determine efficiency of Bisulfite conversion:
   * x (positive control): to determine x
   Methylated genomic DNA standard:
   -> negative control for bisulfite reaction (x: ex

Answer 17

1. Bisulfite sequencing (BS-seq) or whole genome bisulfite sequencing (WGBS) is a technique used to detect methylated cytosines at single-base resolution in the genomic DNA.
   a) Key Steps:
   (i) Denatured DNA (DNA separated into single strands) is treated with sodium bisulfite. Denatured DNA is used since sodium bisulfite can react
   with cytosine only in single-stranded DNA (ii) This converts the unmethylated cytosine residues to Uracil. The methylated cytosines are not converted.
   (iii) The uracil residues are amplified
   as thymine in subsequent PCR reaction, whereas the methylated cytosine residues are amplified as cytosine.
   (iv) Comparison of the original sequence with the bisulfite converted sequence provides information of DNA methylation status of every CpG dinucleotide
2. Designing PCR primers-
   * Since the methylation status of CpG dinucleotides is unknown, the primer sequences should strictly avoid CpG dinucleotides-> design primers for regions that do not contain CpG, strictly avoid CpG islands
   * Non-CpG cytosines in the original DNA should be changed to uracils before designing the primers
3. Quality control checks to determine efficiency of Bisulfite conversion:
   * Non-methylated genomic DNA standard (positive control): to determine the conversion efficiency of bisulfite reaction.
   * Methylated genomic DNA standard:
   -> negative control for bisulfite reaction (error in bisulfite conversion: methyl cytosine can get converted to thymine, so treatment with sodium bisulfite should be carefully optimized