This ppt gives a general overview of how noncoding RNA can regulate epigenetics

1. Eddie McTaggart
Ammar Ahmedani
Advanced Topics in Molecular Genetics
Non-coding RNAs as regulators
in epigenetics
Ageneraloverview

2. The Central Dogma
“At this stage I must make four points about the formulation of the
central dogma which have occasionally produced misunderstandings…
(1) It says nothing about what the machinery of transfer is made of,
and in particular nothing about errors. (it was assumed that, in
general, the accuracy of transfer was high.)
(2) It says nothing about control mechanisms – that is, about the rate
at which the processes work.
(3) It was intended to apply only to present-day organisms, and not to
events in the remote past, such as the origin of life or the origin of the
code.
(4) It is not the same, as is commonly assumed, as the sequence
hypothesis, which was clearly distinguished from it in the same article.
In particular the sequence hypothesis was a positive statement, saying
that the (overall) transfer nucleic acid → protein did exist, whereas
the central dogma was a negative statement, saying that these
transfers from protein did not exist.
Francis Crick, 1970

3. The Central Dogma
“At this stage I must make four points about the formulation of the
central dogma which have occasionally produced misunderstandings…
(1) It says nothing about what the machinery of transfer is made of,
and in particular nothing about errors. (it was assumed that, in
general, the accuracy of transfer was high.)
(2) It says nothing about control mechanisms – that is, about the rate
at which the processes work.
(3) It was intended to apply only to present-day organisms, and not to
events in the remote past, such as the origin of life or the origin of the
code.
(4) It is not the same, as is commonly assumed, as the sequence
hypothesis, which was clearly distinguished from it in the same article.
In particular the sequence hypothesis was a positive statement, saying
that the (overall) transfer nucleic acid → protein did exist, whereas
the central dogma was a negative statement, saying that these
transfers from protein did not exist.
Francis Crick, 1970
“Stated in this way it is clear that the special transfers are
those about which there is the most uncertainty. It might
indeed have ‘profound implications for molecular biology’ if
any of these special transfers could be shown to be …
widely distributed.”

4. Non-coding RNAs
 Known for decades
 Functionality disputed
 Often labelled ‘junk’
 Increasingly shown to
have regulatory roles
Kevin V. Morris & John S. Mattick, 2014 , & Palazzo & Lee, 2015

5. Epigenetics
 Meaning “above or
around” genetics
 Precise meaning disputed
 Chemical or non-sequence
based changes
 Affecting gene expression
or activity
 Information carried on the
genome that is not coded by the
DNA
 Definitions: must it be heritable?

6. Histone Modifications
Tony Kouzarides, 2007

7. Histone Modifications - Acetylation
 Neutralises charge of basic
Lysine
 Charge change disrupts
higher order structure
 Activation of Transcription
Other cell cycle steps
 Persists for DNA repair
 Also associated with replication
 HBO1 (histone acetyltransferase
bound to ORC) associated with
replication.

8. Histone Modifications - Methylation
 May activate or repress
transcription
 Site specific
 Contradictory signals possible
 For example: H3K27 and H3K4
methylation
 Involved in silent and activated
chromatin, respectively
 Signals are complicated, and at
times contradictory:
 ES cells shown (Azuara et al
2006) to have H3K27me and
H3K4me

9. DNA Methylation
 Predominantly methylation of
5’Cytosine in CpG context
 CpG islands in 40% of promoters
 Methylation ≈ lack of expression
 Mutation hotspot – spontaneous
deamination to Thymine
 CpG underrepresented in genome

10. Effects of Epigenetic Modifications
Chromatin Structure and Remodelling
 Euchromatin = accessible
 Heterochromatin = inaccessible
Recruitment of Proteins
Result: Heritable and reversible
gene silencing
N. Sasai and P.A. Defossez, 2009

11. Effects of Epigenetic Modifications
Chromatin Structure and Remodelling
 Euchromatin = accessible
 Heterochromatin = inaccessible
Recruitment of Proteins
Result: Heritable and reversible
gene silencing
N. Sasai and P.A. Defossez, 2009

12. Studying DNA Modifications
Bisulfite Sequencing
 Cytosine to Uracil
 5-methylcytosine
unaffected
 Sequence as normal
Huang et al., 2010

13. Studying Histone Modifications
ChIP-seq
 Immunoprecipitation of
proteins
 Bound DNA sequenced
Ku et al., 2010

14. Epigenetics in Differentiation
 Differentiation is epigenetic
 Methylation “cleared” in
early development.
 Patterns of epigenetic
silencing created during
differentiation
Wolf Reik, 2007

15. Types of ncRNA
Losko M, et al., 2016

16. Chromatin-associated RNA

17. Chromatin-associated RNA
Li X & Fu X, 2017
Chromatin-associated RNA involved in
transcriptional activation or repression
 RNAs that are physically associated with
chromatin.
 Either retained in cis at their site of
transcription or recruited in trans to other
genomic regions.
 Chromatin-associated RNAs belong to
several classes including both short and long
ncRNAs
 RNAs that are physically associated with
chromatin.
 Either retained in cis at their site of
transcription or recruited in trans to other
genomic regions.
 Chromatin-associated RNAs belong to
several classes including both short and long
ncRNAs

18. Discovery of chromatin-associated RNA
Experimental strategies to identify chromatin-
associated RNAs and subsequent understanding
of their roles:
i. Chromatin-associated RNA sequencing
(ChAR-seq)
ii. Molecular and biochemical methods
iii. Epigenetics analysis
Sridhar, Bharat et al., 2017
Maps genome-wide RNA-to-DNA contacts

19. Discovery of chromatin-associated RNA
Experimental strategies to identify chromatin-
associated RNAs and subsequent
understanding of their roles:
i. Chromatin-associated RNA sequencing
(ChAR-seq)
ii. Molecular and biochemical methods
iii. Epigenetics analysis
Delás MJ, Hannon GJ, 2017
 Knockdown by siRNA, LNA, ASO and CRISPRi.
 Insertion of an early terminator sequence or
complete deletion of the locus or promoter.
Different tools for different questions

20. Discovery of chromatin-associated RNA
Experimental strategies to identify chromatin-
associated RNAs and subsequent
understanding of their roles:
i. Chromatin-associated RNA sequencing
(ChAR-seq)
ii. Molecular and biochemical methods
iii. Epigenetics analysis
Sridhar, Bharat et al., 2017
DNA in closed heterochromatin is resistant to
digestion or transposition. Conversely, DNA in
open euchromatin is vulnerable to nuclease and
transposase.
Endonuclease
Chromatin accessibility
DNase Seq
ATAC Seq

21. Discovery of chromatin-associated RNA
Experimental strategies to identify chromatin-
associated RNAs and subsequent
understanding of their roles:
i. Chromatin-associated RNA sequencing
(ChAR-seq)
ii. Molecular and biochemical methods
iii. Epigenetics analysis
@Pine Biotech
DNA Methylation Analysis
Bisulfite Sequencing

22. Discovery of chromatin-associated RNA
Experimental strategies to identify chromatin-
associated RNAs and subsequent
understanding of their roles:
i. Chromatin-associated RNA sequencing
(ChAR-seq)
ii. Molecular and biochemical methods
iii. Epigenetics analysis
© Copyright by Healthcare Business Today
RNA-Seq Analysis

23. Short ncRNA

24. Classical RNAi pathway
dolcera.com

25. RNAi and epigenetics
The scope of this review is to provide an overview of
the roles of small RNA and … in epigenetic inheritance
via heterochromatin formation
RNA interference (RNAi) is important in directing
heterochromatin assembly at centromeres in
fission yeast,…

26. RNAi-directed heterochromatin formation
RITS: RNA induced transcription
silencing complex

27. S. pombe
RNAi-directed heterochromatin formation
1
6
5
4
3
2
Ekwall K, 2007
1. Precursor siRNA (P-siRNA) is generated
2. P-siRNA serves as a template for Rdp1
to produce a long duplex RNA
3. Processed by Dicer into siRNA
4. Guides RITS into the target
5. Recruit Clr4 and HDAC
6. Reinforce the regulatory loop of siRNA
production

28. What do the jumping genes do?
Transposable elements
(jumping genes) Functional
Non
Functional
1
2
Center for Computational Biology and Bioinformatics @ Penn State
1. Landing inside a gene can result in disruption of
that gene.
2. Transposition can activate nearby genes.

29. PIWI-interacting RNAs: small RNAs with big functions
Transposable elements
(jumping genes)
 21–35 nucleotides in length.
 Transcribed from genomic loci known as piRNA
clusters.
 Silence transposable elements, regulate gene
expression and fight viral infection.
 In most animals, piRNAs defend the germline
genome against transposon mobilization
 How the piRNA pathway discriminates between self
transcripts and non-self transcripts remains a central
question in piRNA research.
Ozata DM, et al., 2018

30. Biogenesis of piRNAs
Transposable elements
(jumping genes)
Ernst C, , et al., 2017

31. Functions of piRNAs
Transposable elements
(jumping genes)
 piRNAs silence transposons transcriptionally and
post-transcriptionally.
 Nuclear PIWI proteins generate heterochromatin
via DNA or histone methylation.
 Cytoplasmic PIWI proteins slice target transcripts
Ozata DM, et al., 2018

32. Long ncRNAs

33.  Non-protein-coding RNA molecules that’re
>200nt.
 Resemble mRNA (transcribed by RNA pol II,
are often capped and polyadenylated).
 Exhibit cell, tissues, and developmental stage
specific expression pattern.
 Categorized as antisense, intronic,
bidirectional and intergenic.
 Reside in the nucleus or cytoplasm.
 Exploit biological functional diversity
LncRNAs
Chu C, et al., 2015

34. Epigenetic Regulation by Long Noncoding RNAs

35. Localizing particular chromatin modifying complexes to specific
chromatin targets.
Chu C, et al., 2015

36. Help in assembly of protein complexes that are involved in
chromatin remodeling
Chu C, et al., 2015

37. Bind to and titrate away chromatin modifying complexes
Chu C, et al., 2015

38. X-Chromosome
Inactivation
Chu C, et al., 2015 & Chen X, et al., 2018

39. Ayub ALP, et al 2019 & Gagnon KT, Corey DR, 2012 & Wing, J. & O'Connor, C, 2008
 The X-inactive-specific transcript (Xist).
 17- to 20-kb RNA that expressed only from
the inactive X chromosome (Xi) and coats the
X chromosome in cis.
 Xist RNA is expressed and initiates XCI only
when more than one X chromosome is
present.
 Through a conserved repeat motif (RepA),
Xist binds PRC2, targets PRC2 to the Xi for
H3K27me3.
XIST Exists to
Silence

40. Ayub ALP, et al 2019 & Gagnon KT, Corey DR, 2012 & Wing, J. & O'Connor, C, 2008
X-chromosomal “painting” by Xist RNA is
associated with the accumulation of (PRC2) on
the X chromosome and subsequent deposition
of H3K27me3 mark

41. Ayub ALP, et al 2019 & Gagnon KT, Corey DR, 2012 & Wing, J. & O'Connor, C, 2008
Xist is controlled by two other lncRNAs, one
acting negatively (Tsix), the other positively (Jpx)
LncRNA REGULATORS OF Xist

42. Leeb M, et al., 2009
Tsix keeps Xist Expression in Check
 Tsix mediated recruitment of Dnmt3a/b
 xiRNAs have been implicated in inhibition of
Xist expression.

43. Sun S, et al 2013
 Jpx RNA binds CTCF, and extricates CTCF from
one Xist allele.
POSITIVE Xist REGULATORS

44. IPSC and Epigenetic reprogramming
 Differentiation is epigenetic
 Differentiated cells can be turned
into stem cells (iPSCs) (Takahashi
and Yamanaka)
 ncRNA crucial to this process/ESC
 Lack of functional Dicer or DGCR8
causes delays and defects in
differentiation
 miRNAs promote differentiation or
the pluripotent state
 miR-134, miR-296, miR-470 target
mRNA from Nanog, Oct4, Sox2,
promoting differentiation
 Let-7 miRNAs promote loss of
pluripotency markers via hundreds
of target genes
 miR-290-295 miRNAs increase
Lin28, which interfere with Let-7
function

45. IPSC Generation – Enhancement through RNA
 miR-290-295 enhanced
iPSC generation by Oct4,
Sox2, and Klf4
 Similarly, inhibition of
let-7 miRNA increased
efficiency of a MEF/OSK
system.
 UTF1 and p53 siRNA increase
iPSC generation by 100x
Cell colonies created from 10^5 cells, using 4 gene mixture: OCT4, SOX2, KLF4, c-MYC. With or without additional non-coding RNAs
Zhao et al., 2008

46. IPSC Generation – non-coding RNA alone
 miRNA also sufficient to
generate iPSC
 Serial transfections of miR-
302s, miR-200c, mir-369s
generated iPSC from
adipose stromal cells.
 5 colonies generated from
50,000 cells
Teratomas formed by human mi-iPSCs implanted in
NOD/SCID mice. Bar = 100 µm.mi-IPS cell colonies. GFP reporter driven by Nanog promoter
Miyoshi et al., 2011

47. Advantages of RNA drugs
 Specificity: Tissue or even
disease
 Ease of off-target prediction and
avoidance
 Pharmacoevolution
 Can target any gene
 Lasting effects – six months per
dose with the right chemistry
 Hydrophobicity and stability can
be acheived chemically
Steven S. Dowdy, 2017

48. Uses – RNA Drugs on or near the Market
Effects of single dose subcutaneous Inclisiran, an siRNA targetting
Proprotein Convertase Subtilisin–Kexin Type 9 (PCSK9).
Fitzgerald et al. 2017
Inclisiran
 Phase 3 trials completed this year with over 3000 participants
 Further phase 3 trials ongoing 15,000 participants, to
examine cardiovascular outcomes
 Not yet approved by FDA or others
 Created by The Medicines Company – to be purchased by
Novartis for 9.7 billion USD
Nusinersen/Spinraza
 FDA approved
 Treatment of SMA
 Phosphothorothioate
 Causes alternate splicing of SMN2
 Prohibitively expensive
Eteplirsen/Exondys 51

49. Uses – RNA Drugs on or near the Market
Effects of single dose subcutaneous Inclisiran, an siRNA targetting
Proprotein Convertase Subtilisin–Kexin Type 9 (PCSK9).
Fitzgerald et al. 2017
Inclisiran
 Phase 3 trials completed this year with over 3000 participants
 Further phase 3 trials ongoing 15,000 participants, to
examine cardiovascular outcomes
 Not yet approved by FDA or others
 Created by The Medicines Company – to be purchased by
Novartis for 9.7 billion USD
Nusinersen/Spinraza
 FDA approved
 Treatment of SMA
 Phosphothorothioate
 Causes alternate splicing of SMN2
 Prohibitively expensive
Eteplirsen/Exondys 51

50. Uses – RNA Drugs on or near the Market
Effects of single dose subcutaneous Inclisiran, an siRNA targetting
Proprotein Convertase Subtilisin–Kexin Type 9 (PCSK9).
Fitzgerald et al. 2017
Inclisiran
 Phase 3 trials completed this year with over 3000 participants
 Further phase 3 trials ongoing 15,000 participants, to
examine cardiovascular outcomes
 Not yet approved by FDA or others
 Created by The Medicines Company – to be purchased by
Novartis for 9.7 billion USD
Nusinersen/Spinraza
 FDA approved
 Treatment of SMA
 Phosphothorothioate
 Causes alternate splicing of SMN2
 Prohibitively expensive
Eteplirsen/Exondys 51

51. References
• Losko M, Kotlinowski J, Jura J. Long Noncoding RNAs in Metabolic Syndrome Related Disorders.
Mediators Inflamm. 2016;2016:5365209. doi:10.1155/2016/5365209
• Lam JK, Chow MY, Zhang Y, Leung SW. siRNA Versus miRNA as Therapeutics for Gene Silencing. Mol
Ther Nucleic Acids. 2015;4(9):e252. Published 2015 Sep 15. doi:10.1038/mtna.2015.23
• Chen X, Sun Y, Cai R, Wang G, Shu X, Pang W. Long noncoding RNA: multiple players in gene
expression. BMB Rep. 2018;51(6):280–289. doi:10.5483/bmbrep.2018.51.6.025
• Ana Luisa Pedroso Ayub, Debora D’Angelo Papaiz, Roseli da Silva Soares and Miriam Galvonas
Jasiulionis (July 17th 2019). The Function of lncRNAs as Epigenetic Regulators [Online First],
IntechOpen, DOI: 10.5772/intechopen.88071. Available from: https://www.intechopen.com/online-
first/the-function-of-lncrnas-as-epigenetic-regulators
• Wing, J. & O'Connor, C. (2008) Sex chromosomes in mammals: X inactivation. Nature Education
1(1):221
• Talon I, Janiszewski A, Chappell J, Vanheer L, Pasque V. Recent Advances in Understanding the
Reversal of Gene Silencing During X Chromosome Reactivation. Front Cell Dev Biol. 2019;7:169.
Published 2019 Sep 3. doi:10.3389/fcell.2019.00169.

52. • Gagnon KT, Corey DR. Argonaute and the nuclear RNAs: new pathways for RNA-mediated control
of gene expression. Nucleic Acid Ther. 2012;22(1):3–16. doi:10.1089/nat.2011.0330
• Shim G, Kim D, Park GT, Jin H, Suh SK, Oh YK. Therapeutic gene editing: delivery and regulatory
perspectives. Acta Pharmacol Sin. 2017 Jun;38(6):738–753. doi: 10.1038/aps.2017.2. Epub 2017
Apr 10. under Creative Commons licence (CC BY-NC-ND 3.0)
• Lam JK, Chow MY, Zhang Y, Leung SW. siRNA Versus miRNA as Therapeutics for Gene
Silencing. Mol Ther Nucleic Acids. 2015;4(9):e252. Published 2015 Sep 15.
doi:10.1038/mtna.2015.23
• Delás MJ, Hannon GJ. lncRNAs in development and disease: from functions to mechanisms. Open
Biol. 2017;7(7):170121. doi:10.1098/rsob.170121