W9L1 lncRNAs and X-chromosome inactivation

Question 1

Features of long non-coding RNAs (lncRNAs)

Answer 1

§ >200nt in length, polyadenylated and lack an ORF
§ Tend to be expressed at low levels + tissue specific pattern
§ Interact with proteins -ribonucleoprotein complexes
§ Guide complexes to DNA/RNA targets via base pairing
§ Found in both plants and animals
Vast majority of lncRNAs have no known function

Question 2

The encode project found what about lncRNA

Answer 2

-In humans ~90% genome is transcribed, only 1.5% translated into proteins
Genome encodes 60K long non-coding genes (lncRNA)~80% were previously unannotated
-more than 50% are lncRNA, out number protein coding gene in human

Question 3

lncRNAs are functionally diverse

Answer 3

• Involve in post-transcriptional regulation by modulating alt splicing pattern, generate endo-siRNA, small RNA precursor
  -regulation of protein activity - modulation of protein activity
  -formation of assembly platforms- large ribonucleoprotein bodies
  -transcriptional regulation in cis and trans (transcriptional interference, induced chromatin remodeling and histone modification)

Question 4

lncRNA that interfere with transcription

Answer 4

§ Arise from enhancers/promoters of a gene
§ Regulatory activity is mediated by transcription of lncRNA (no sequence-specific function)
§ Reduces/prevents transcription factors/RNA pol II from binding to the promoter
-Fuction to tune gene expression

Question 5

lncRNA that function as tethers

Answer 5

§ lncRNAs remain tethered to the encoding locus during transcription
§ Acts as a scaffold for the assembly of chromatin modifying enzymes
§ Modify localized chromatin structures and/or expression of nearby genes (cis-regulation)

Question 6

lncRNA that function as guides

Answer 6

§ lncRNAs released from site of transcription
§ Act as a scaffold for the assembly of chromatin modifying enzymes
§ Sequence complementarity may be required for binding to specific chromosomal sites but not always
§ lncRNAs guide the associated proteins to sites on the same or different chromosomes (cis or trans regulation)

Question 7

Example 1: Hox gene regulation

Answer 7

Hox genes regulate segmental/regional identity along the embryonic A-P axis
-co linearity in location of hox gene and the location it affect
-multiple lncRNA in the Hoxcuster
-in HoxC have Hotair, HoxD have Hog and Tog
-HoxA have Hottip and HOTAIEM1

Question 8

lncRNA involved in Hox gene regulation in mammals

Answer 8

-Hottip and HOTAIRM1 function as tethers – cis regulation
HOX transcript antisense intergenic RNA (HOTAIR) – 2.2kb transcript arises from antisense strand of HoxC cluster
Not found in non-mammalian vertebrates or invertebrates
Displays spatially distinct expression pattern along the A-P axis

Question 9

Role of Hotair in Hox gene regulation

Answer 9

Homeotic conversion seen in Hotair mutant associated with anteriorly expanded Hoxd10/11 expression
* Hotair represses many genes (~200) – induces repressive chromatin marks on gene target
* Binds to (GA)n DNA motif in target loci
* Ectopic Hotair is detected in some human cancers – associated with metastasis/progression
Repressive chromatin is lost around the transcriptional start site of upregulated genes

Question 10

How does hotair work

Answer 10

-Hotair recruit PRC2
Polycomb repressive complex 2 – adds H3K27 tri-methyl marks (gene silencing)
Lysine-specific histone demethylase – removes H3K4 methyl marks (gene activating)
Hotair functions as a guide to promote a repressive chromatin state – trans regulation

Question 11

X chromosome inactivation (XCI)

Answer 11

§ X inactivation is an epigenetic dosage compensation mechanism in mammals
§ Transcriptional silencing of one X chromosome in females
§ Mitotically stable

Question 12

Two forms of XCI in mammals:

Answer 12

Random XCI
§ Inactive X can be either the maternal or paternal copy, but once established is maintained in daughter cells
Imprinted XCI (marsupials/mammalian placenta tissue)
§ Paternal X chromosome is selectively silenced

Question 13

Two waves of XCI in mammals

Answer 13

-In pre-implantation embryo, both X chromosome are inactive. Xp is then activated for the embryo and inactivated in the placenta
-Random XCI established and maintained in subsequent mitotic divisions
-In X chromosome for female, there will be patches of mosaic

Question 14

Mosaic example IRL

Answer 14

Question 15

XCI is associated with heterochromatin formation

Answer 15

§ XCI occurs progressively over developmental time
§ XCI results in the Xi forming a Barr body which migrates to the nuclear periphery – only present in female nuclei
§ XCI associated with:
transcriptional silencing, chromosome compaction, change in histone marks and recruitment of histone variants (e.g.MacroH2A)
Progressive accumulation of epigenetic marks leading to heterochromatin formation

Question 16

X-inactivation centre (XIC)

Answer 16

XIC is a <500 kb region that is the source of seven different lncRNAs – Xist, Tsix, Xite + small ncRNAs
>7 lncRNAs ensure orderly progression through XCI
Tsix, DXPas34, Xite and Tsx directly/indirectly repress Xist
Ftx and Jpx activate Xist
But many protein coding factors also regulate Xist
Focus on Xist and Tsix functions in mouse

Question 17

Xist – X-inactive specific transcript

Answer 17

§ 17 kb, spliced and polyadenylated non-coding transcript
§ Retained to the nucleus
§ Initially expressed at low levels from both X chromosomes in undifferentiated female mouse embryonic cells
§ Elevated expression in one X indicate future inactivation is the first detectable event in XCI
§ Xist RNA coats the future Xi in cis – cloud of RNA
§ Xist expression is absent from Xa
§ Xi drawn into a silent nuclear compartment
§ Depletion of active chromatin marks on promoters and enhancers
§ Deposition of silencing marks, gene silencing and heterochromatin formation

Question 18

Xist functions as a cis guide

Answer 18

• Xist lncRNA has a series of conserved repeat motifs at the 5’ end of the transcript that bind to proteins
  -interact with SPEN which cause Active histone marks (H3K27ac) marks removed by histone deacetylase (HDAC)
• PRC1/PRC2 recruited to Xi by Xist RNA via heterogeneous nuclear ribonucleoprotein K (hnRNP K)
• Deposit silencing histone marks
  H2AK119Ub (PRC1)
  H3K27me3 (PRC2)

Question 19

Xist interaction with PRC1 and PRC2

Answer 19

§ Histone marks act as nucleation sites for PRC1/2
§ Propagate silencing marks along Xi in cis
• Further PRC1/2 recruitment enhances Xist RNA binding to the X-chromosome
Promote better contact between RNA and chromatin
Polycomb-mediated changes to chromatin structure enhance Xist interactions
Positive feedback enables the spread of Xist binding along the Xi chromosome

Question 20

Controls Xist activity by Tsix

Answer 20

Expression of the 40-kb antisense Tsix transcript is associated with Xist regulation in cis
Prior to cell differentiation-High-level transcriptional activity of Tsix
interferes with Xist activation. Acts as tether
Onset of cell differentiation-XCI establishment is associated with transcriptionally inactivation of Tsix
Later in cell differentiation-Xist binds to CMF and deposits localized repressive histone marks

Question 21

X-X Pairing

Answer 21

First event in XCI involves transient interchromosomal pairing between X chromosomes with a protein to form a protein-RNA bridge
§ Involves Tsix lncRNA physically interacting Paired X chromosomes exchange bound pluripotency factors:
§ Bound factors accumulate on just one X chromosome (future Xa)
Distribution of bound factors determines Tsix expression
§ Chromosome lacking bound factors (future Xi) – Tsix no longer expressed, increase in repressive H3K27me marks, Xist expression elevated

Question 22

Summary – early stages of XCI

Answer 22

§ Xist expression at low levels prior to XCI – Tsix acting as a cis repressor
§ X-X pairing – Tsix transcript binds to proteins to form a bridge
§ Transcriptional silencing of Tsix leads to increased Xist expression from Xi
§ Progressive accumulation of repressive epigenetic marks on the Xi – Xist acting as a cis tether then guide

Question 23

Secondary chromatin modification

Answer 23

XCI requires Xist to coat Xi
1o silencing factors bind to Xist Chromosome wide silencing

2o silencing factors recruited Stabilisation of chromosome silencing

Maintenance of heterochromatic state through cell divisions

Xist expression is maintained throughout the life of the cell
Xist only has a minor role in maintaining Xi