VL 10: Chromosomal Inactivation II

Question 1

X-chromosomal dosage compensation in drosophila

Answer 1

Mechanism: Male X-linked genes are turned up (hyperactivated) to match female X-linked gene expression.

Transcription rate of male X-linked genes is higher than that of autosomal or female X-linked genes.

average transcription rate (per unit DNA)
female X = female autosomes = male autosomes < male X

Question 2

Consequences of Mutations in Dosage Compensation Machinery

Answer 2

Male-Specific Lethal (MSL): Mutations disrupting MSL complex cause male-specific lethality due to lack of X chromosome hyperactivation.

Phenotypes:
Female (XX): No X hyperactivation.
Male (XY): Required X hyperactivation.

Question 3

Sex determination of drosophila

Answer 3

• X counting mechanism is linked to sex
  determination
• X:autosome ratio determines both
• X/A ratio =1 → female diff.
• X/A ratio =0.5 → male diff. + active MSL
  complex

Question 4

MSL Complex

Answer 4

The MSL (Male-Specific Lethal) complex is a multi-protein complex responsible for the dosage compensation mechanism in male Drosophila. It upregulates the transcription of X-linked genes in males to ensure that their expression levels are equivalent to those in females, who have two X chromosomes.

MSL complex
* MSL1 - Scaffold protein eesential for the assemlbe of MSL complex
* MSL2 – ubiquitin ligase, key for targeting. Target of the Sex-lethal (Sxl) protein, which prevents its expression in females.
* MSL3 – chromodomain, binds H3K36me
* MOF – acetylates H4K16
* MLE – ATPase RNA/DNA helicase
* roX RNA - Facilitate assembly and targeting

Function
* Transcriptional Upregulation: Increases X-linked gene transcription in males.
* Targeting/Localization: Enriched at 3’ ends of active genes.
* Chromatin Modification: Acetylates H4K16 for chromatin relaxation.
* Nucleation/Spreading: Binds at HASs and spreads along X chromosome.

Question 5

roX ncRNAS

Answer 5

Facilitate assembly and targeting of MSL complex on X chromosome.

• 2 roX lncRNAs = RNA on X
• fully redundant
• Double mutant males die with mislocalization of MSL complex, single mutants no phenotype
• Different sequence and length (3.7 kb vs. 0.5-1.4 kb)
• Common sequence motif: repeated roX box forms stem loop structure
• RoXs act in cis (at site of production) and in trans

Question 6

MSL Complex Localization

Answer 6

• Enriched at: 3’ end of active genes.
• Nucleation Sites: MRE (MSL recognition elements) enriched in specific sequence motifs.
• Spreading: High H4K16ac follows nucleation.

Question 7

Targeting MSL Complex to X Chromosome

Answer 7

• DNA sequence elements
• ncRNAs
• Active chromatin marks

–> all factors contribute

Question 8

Dosage compensation in c.elegans

Answer 8

Hermaphrodite X chromosomes have transcription turned down; male X chromosome is twice as active.

Genes: Hermaphrodite-specific lethal genes encode a protein complex that reduces transcription on hermaphrodite X chromosomes.

Question 9

Sex determinationa nd dosage compensation in c.elegans

Answer 9

Sex determination and dosage compensation are linked in worms

• XOL XO lethal = master switch
  The gene xol-1 acts as a master switch for sex determination.
  xol-1 Expression: High xol-1 expression in XO animals leads to male development, while low xol-1 expression in XX animals leads to hermaphrodite development.
• SDC-2 critical for assembly of DCC
  Hermaphrodites: (XX)
  Both X chromosomes are active, but their transcriptional activity is reduced by half to balance gene expression with males.
  The dosage compensation complex (DCC) is recruited to both X chromosomes to achieve this reduction.
  Males (XO)
  The single X chromosome in males is not subject to dosage compensation and remains fully active.
• sdc-2: A critical gene for the assembly of the DCC.
  Function: sdc-2 expression in XX embryos leads to the recruitment of the DCC to the X chromosomes.
• Ectopic SDC-2 expression in XO embryos sufficient to cause assembly of DCC and trigger dosage compensation

Question 10

Chromosome Condensation is caused by condensins

Answer 10

Condensins = protein complexes that play a crucial role in chromosome condensation, ensuring that chromosomes are properly compacted and organized during cell division.

Components
1. SMC Protein (Structural Maintenance of Chromosomes):
These are ATPase Proteins that that include condensins and cohesins.
2. Non-SMC Subunits

Condensins typically consist of heterodimers of two SMC proteins, such as SMC2 and SMC4.

Function
* Condensins cause chromatin to be more tightly coiled by introducing positive supercoils into DNA.
* Condensins are responsible for condensing chromosomes at mitosis.
* Chromosome-specific condensins are responsible for condensing inactive X chromosomes in C. elegans.

Mechanism
* ATP hydrolysis for DNA supercoiling.
* Bending and looping DNA.

Roles in c elegans
* DCC resembles condensin.
* Compacts X chromosomes in hermaphrodites.
* Reduces transcriptional activity for dosage compensation.

(A) The basic architecture of condensin and cohesin complexes.

Question 11

How is DCC recruited to the X?

Answer 11

In C. elegans, the Dosage Compensation Complex (DCC) resembles the condensin complex and is involved in reducing the transcriptional activity of both X chromosomes in hermaphrodites.

• Recruitment Elements:
• 100-300 specific DNA sequences on the X chromosomes.
• Key Protein:
  SDC-2 binds to recruitment elements and facilitates DCC assembly.
• High-Affinity Sites (HASs): Enriched in specific sequence motifs; initial binding sites for the DCC.
• Spreading Mechanism: DCC spreads from high-affinity sites to lower-affinity sites along the X chromosomes.
  Factors Contributing to Recruitment:
• DNA sequence elements.
• Non-coding RNAs.
• Chromatin state.

Question 12

Overvie Dosage Compensation Complexes

Answer 12

• Drosophila: MSL complex (histone acetylation).
• Mammals: Xist, PRC, H3K9me.
• C. elegans: DCC complex (condensin-like).

https://www.youtube.com/watch?v=CtgZAIAVtGs