Week 8

Question 1

Monoallelic expression:
Biallelic expression:
Long non-coding RNAs (lnc RNA):
Dosage compensation:
Autosome:

Answer 1

Monoallelic expression: Only one of the 2 copies of a gene is transcriptionally active while the second copy is silent.
Biallelic expression: Both copies of a gene are expressed. More common form of gene expression
Long non-coding RNAs (lnc RNA): RNA transcript longer than 200 nucleotides that is not translated into protein.
Dosage compensation: Dosage compensation refers to the equalization of most X-linked gene products between
males, which have one X chromosome and a single dose of X-linked genes, and females, which have two X’s and two
doses of such genes.
Autosome: any chromosome that is not a sex chromosome

Question 2

1. Why do we need to inactivate an X chromosome in female mammals?
2. which is the only chromosome that is globablly inactivated
3. what does x inactivation involve
4. what is the barr body

Answer 2

1. One of the X chromosomes is inactivated to equalize the expression of genes between males and females, also called Dosage compensation. This is necessary
   for development to proceed normally in females.
2. X chromosome is the only chromosome that is globally inactivated- epigenetic silencing
3. X-inactivation involves the action of lncRNAs
4. The Barr body=> inactive X chromosome

Question 3

1. X chromosome inactivation is the mechanism for **
   a) 1. Female mice need only ** active X chromosome. XO female mice develop **. ** is not seen in
   the cells of XO female mice
   b) X-inactivation occurs early in development during the **
   c) The choice of which X chromosome will be inactivated is **. The inactive X chromosome could be either ** in different cells of the
   same female animal.
   d) Once the decision to inactivate ** or ** has been made by a cell, its descendants will **
   e). Later, she and others showed that X-inactivation occurred ** as well
2. Every female is a mosaic of ** in which either ** or ** is active
   a) Because there are subtle differences in the sequences of the two X chromosomes, **
   b) Both X chromosomes are initially **
   c) Initially the X chromosome is randomly
   ** in the cells of the **
   d) Once the decision to inactivate ** or ** is made in a cell, its descendants will **
   e) Daughter cells remain **. As a result, female is a **

Answer 3

1. X chromosome inactivation is the mechanism for dosage compensation in female mammals
   a) 1. Female mice need only 1 active X chromosome. XO female mice develop normally. Barr body is not seen in
   the cells of XO female mice
   b) X-inactivation occurs early in development during the blastocyst stage.
   c) The choice of which X chromosome will be inactivated is random. The inactive X chromosome could be either paternal (Xp) or maternal (Xm) in different cells of the
   same female animal.
   d) Once the decision to inactivate Xp or Xm has been made by a cell, its descendants will continue to inactivate the same X chromosome.
   e). Later, she and others showed that X-inactivation occurred in other mammals and humans as well
2. Every female is a mosaic of clonal groups of cells in which either Xp or Xm is active
   a) Because there are subtle differences in the sequences of the two X chromosomes, the female cells will not be genetically identical with respect to active X-linked genes
   b) Both X chromosomes are initially active in the early embryo
   c) Initially the X chromosome is randomly
   inactivated in the cells of the epiblast lineage
   d) Once the decision to inactivate Xp or Xm
   is made in a cell, its descendants will maintain the same X chromosome
   in a silent state.
   e) Daughter cells remain close together
   during development. As a result, female is a mosaic of clones of cells in which Xp or Xm is active

Random X-inactivation occurs in the
cells of the epiblast lineage at the time
of implantation

Question 4

1. Rett syndrome
   a) what is it
   b) Affects X and is caused by X in one of the 2 copies of X gene that is present on the X
   c) 1 in 8,500 females are affected. Males die soon after birth.
   d) Females with Rett syndrome have a mixture of cells that X
2. X-inactivation in karyotypically abnormal cells
   a) Normal female: XX, ** autosomes =>XXX
   b) Normal male: XY, ** autosomes=> **
   c) Trisomy patients: **, ** autosomes=> **
   d) Klinefelter male patients: **, ** autosomes=> **
   e) Tetraploid cell: **, ** autosomes=> **

Answer 4

1. Rett syndrome
   a) Neurodevelopmental disorder.
   b) Affects females and is caused by mutations in one of the 2 copies of MeCP2 gene that is present on the X chromosome.
   c) 1 in 8,500 females are affected. Males die soon after birth.
   d) Females with Rett syndrome have a mixture of cells that express normal or mutant copy of MeCP2.
2. X-inactivation in karyotypically abnormal cells
   a) Normal female: XX, 44 autosomes => 1 X chromosome is inactivated
   b) Normal male: XY, 44 autosomes=> 0 X chromosomes are inactivated
   c) Trisomy patients: XXX, 44 autosomes=> 2 X chromosomes are inactivated
   d) Klinefelter male patients: XXY, 44 autosomes=> 1 X chromosome is inactivated
   e) Tetraploid cell: XXXX, 88 autosomes=> 2 X chromosomes are inactivated

Question 5

1. Mechanism of X chromosome inactivation in female placental mammals
   a) ** two X chromosomes sense each other and **
   b) ** is transcribed on one X chromosone; ** on the other
   c) **coats the chromosome from **
   d) Changes in ** inactivate most **e
2. Broad summary of X chromosome inactivation
   a) Counting of X chromosomes: ** is determined. A= **
   b) X-inactivation rule: **
   c) X chromosome inactivation (XCI) is initiated through **
   d) X-inactivation is mediated by **, a **, from ** chromosome. Xist RNA is transcribed only from **. Expt. Technique EMSA
   e) Tsix, a lncRNA, prevents **. Tsix RNA is transcribed only from **
   f) Xist RNA spreads throughout ** and recruits (i) **. Expt. Technique **
   g) Other proteins recruited to Xi include ** that ** (including CGIs) and ** that **
   h) The X chromosome undergoes **, becomes **, and is **
3. lncRNAs can ** and thus control **

Answer 5

1. Mechanism of X chromosome inactivation in female placental mammals
   a) XICs (X inactivation centre) on two X chromosomes sense each other and pair
   b) Xist (I-inactive specific transcript) is transcribed on one X chromosone; Tsix (lncRNA is an antisense transcript) on the other
   c) Xist RNA coats the chromosome from which it is transcribed
   d) Changes in chromatin in Xist-coated X chrmosome inactivate most transcription from the chromosome
2. Broad summary of X chromosome inactivation
   a) Counting of X chromosomes: X:A ratio is determined. A= autosome
   b) X-inactivation rule: Only one X chromosome per 44 autosomes is maintained in an active state. Remaining X
   chromosomes are inactivated.
   c) X chromosome inactivation (XCI) is initiated through transient pairing between the two X chromosomes.
   d) X-inactivation is mediated by expression of Xist, a lncRNA, from the future inactive X chromosome. Xist RNA is transcribed only from the inactive X chromosome. Expt. Technique EMSA
   e) Tsix, a lncRNA, prevents expression of Xist from the active X chromosome. Tsix RNA is transcribed only from
   the active X chromosome
   f) Xist RNA spreads throughout the future inactive X chromosome and recruits (i) histone modifying enzymes (e.g., PRC2) that deposit repressive histone modifications (H3K27me3). Expt. Technique CHART- seq
   g) Other proteins recruited to Xi include DNMTs that methylate DNA (including CGIs) and proteins that condense the X chromosome.
   h) The X chromosome undergoes 3D-conformational changes, becomes heterochromatic, and is transcriptionally
   silenced
3. lncRNAs can regulate chromatin states and thus control gene expression

Question 6

1. X-inactivation center (XIC) in mice
   a) The process of X-inactivation is driven **
   b) ** is sufficient to direct silencing, contains only **. 0.1% of mouse X
   c) Human XIC contains many of XCI
   d) ** act as a positive regulator of Xist expression (positive regulator => **)
   e) ** mediates pairing between the X chromosomes
2. How does Jpx regulate Xist expression?
   a) MALES:
   i) ** binds to **r and prevents **
   b) FEMALES:
   i) Prior to XCI: ** is constitutively bound to ** => prevents ** Thus, in epiblast cells of ** stage embryo both the X chromosomes are **
   ii) Post-implantation:- binds to ** and ** from the ** on **
   Xa= **
   Xi= **
   XCI= **

Answer 6

1. X-inactivation center (XIC) in mice
   a) The process of X-inactivation is driven by genes that lie in the XIC
   b) Core region of XIC (boxed region) ~ 100Kb is sufficient to direct silencing, contains only lnc RNAs. 0.1% of mouse X
   c) Genes in colour- established function in X chromosome inactivation (XCI)
   d) Human XIC contains many of these genes
   e) Jpx act as a positive regulator of Xist expression (positive regulator => enhances gene expression)
   f) ~15 kb long pairing center mediates pairing between the X chromosomes
2. How does Jpx regulate Xist expression?
   a) MALES:
   i) CTCF binds to Xist promoter and prevents synthesis of Xist RNA.
   b) FEMALES:
   i) Prior to XCI: CTCF is constitutively bound to Xist promoter => prevents
   synthesis of Xist RNA. Thus, in epiblast cells of pre-implantation stage embryo
   both the X chromosomes are maintained in an active state (Xa)
   ii) Post-implantation:-Jpx binds to CTCF and titrates it away from the Xist
   promoter on one of the chromosomes
   Xa= active X chromosome
   Xi= inactive X chromosome
   XCI= X chromosome inactivation

Question 7

1. EMSA for studying **
   a) Principle: RNA-protein complexes are ** and will move ** when subjected to ** compared to unbound RNA probe
   b) Specific binding of protein to the RNA
   results **. Due to this, ** resulting in an **
2. CTCF directly binds **

Answer 7

1. EMSA for studying direct interaction between RNA-protein interaction
   a) Principle: RNA-protein complexes are larger and will move more slowly when subjected to non-denaturing polyacrylamide gel electrophoresis compared to unbound RNA probe
   b) Specific binding of protein to the RNA
   results in the formation of protein-RNA
   complex. Due to this, RNA migrates slowly through the gel resulting in an upward shift in the band.
2. CTCF directly binds Jpx RNA

Question 8

1. CHART-seq: **
2. Principle: Affinity capture of ** by using **, followed by ** & **
3. Key steps:
   a. 1
   b. 2
   c. 3
   e.5
   f) 6
   g) Proteins can be identified **
4. CO:
   Oligo:

Answer 8

1. CHART-seq: Capture hybridization analysis of RNA targets with deep sequencing for investigating RNA-chromatin interactions
2. Principle: Affinity capture of target lncRNA:chromatin complex by using antisense-oligos, followed by sequencing of DNA & identification of bound protein
3. Key steps:
   a. Cross-linking (using formaldehyde) to form covalent bonds between lncRNA , chromatin and other proteins bound to lncRNA
   b. Chromatin shearing (by sonication) to fragment DNA into ~150bp-3kb long pieces
   c. Hybridize RNA to biotinylated capture oligonucleotides (CO) Negative control- biotinylated sense capture oligonucleotide
   d. Bind to Streptavidin beads to precipitate lncRNA with chromatin & proteins
   e. Elute CHART enriched material => lncRNA + chromatin + protein
   f) Reverse cross-links, isolate DNA and sequence (Illumina sequencing)
   g) Proteins can be identified via western blotting or Mass-spectrometry
4. CO: Capture oligo (antisense DNA strand in this case)
   Oligo: short single strand of synthetic DNA

Question 9

1. DNA methylation pattern of x is erased and rewritten in x
2. Two types of silencing mechanisms regulate gene expression from imprinted clusters
   a) The ** model
   i) In this case, the ** is defined as x. The x functions as the **.
   b)

Answer 9

1. DNA methylation pattern of gDMRs is erased and rewritten in PGCs
2. Two types of silencing mechanisms regulate gene expression from imprinted clusters
   a) The insulator model: Igf2/H19 cluster
   i) In this case, the insulator is defined as a regulatory sequence located between an enhancer and a promoter that blocks their interaction. The ICR/gDMR functions as the insulator.
   b) The ncRNA (non coding RNA) model: Igf2r/Airn cluster

Question 10

1. The insulator model: Igf2/H19 cluster
   a) Maternal: ** is not transcribed since ** is bound to the **. ** is
   transcriptionally active. H19 is a ** gene
   b) Paternal: DNA methylation of ** prevents binding of **. H19 gene is **
   since **. ** are brought
   in the vicinity of Igf2 promoter elements through **. Igf2 is ** gene.
2. CTCF participates in the formation of ** which prevents **
3. In absence on CTCF, chromatin ** which ** and this turns on **
4. DNA methylation of ICR results in **
5. ** acts as a methylation-sensitive switch to ** through **
6. Disruption of Igf2-H19 locus results in **
   a) WS is a ** with a prevalence of ** Children affected with BWS are at increased risk of ** (a form of kidney cancer) and ** (a form of liver cancer).
   b) One of the causes of BWS is **on the ** chromosome which cause **
   c) Higher incidence of BWS is observed in ** Embryonic culture media used in these procedures may result in **

Answer 10

1. The insulator model: Igf2/H19 cluster
   a) Maternal: Igf2 is not transcribed since CTCF is bound to the ICR. lncRNA H19 is
   transcriptionally active. H19 is a maternally expressed imprinted gene
   b) Paternal: DNA methylation of ICR prevents
   binding of CTCF. H19 gene is silenced
   since DNA methylation of the ICR
   region extends into the promoter
   region of H19. Enhancers are brought
   in the vicinity of Igf2 promoter elements through Chromatin looping. Igf2 is a paternally expressed imprinted
   gene.
2. CTCF participates in the formation of intrachromosomal loop which prevents the enhancers from interacting with Igf2
   promote
3. In absence on CTCF, chromatin adopts a different conformation which places the enhancers in the vicinity of Igf2 promoter and this turns on transcription of Igf2 gene
4. DNA methylation of ICR results in silencing of H19 gene and activation of Igf2 gene on the paternal chromosome
5. ICR acts as a methylation-sensitive switch to guide allele-specific expression through chromatin conformation
6. Disruption of Igf2-H19 locus results in Beckwith-Weidemann Syndrome
   a) WS is a pediatric overgrowth disorder with a prevalence of 1/13,700. Children affected with BWS are at increased risk of
   developing tumors Wilms tumor (a form of kidney cancer) and hepatoblastoma (a form of liver cancer).
   b) One of the causes of BWS is gain of methylation of ICR on the maternal chromosome which cause biallelic expression of Igf2.
   c) Higher incidence of BWS is observed in children born through use of assisted reproductive technologies-1 in 2,500 in children resulting from in vitro fertilization. Embryonic culture media used in these procedures may result in abnormal DNA methylation patterns