1. (Long) Non-Coding RNAs
Genetics and Epigenetics of lncRNAs in
Normal Development and Complex Diseases
R. Michael Sheetz, PhD
Center for Computational Sciences

2.  Tumorigenesis and metastatic progression  Cardiovascular diseases
Outline of presentation
 Characteristics of lncRNAs
 Involvement of lncRNAs in:
  Normal development
 X chromosome inactivation
 Genomic imprinting
 Normal development
- Complex diseases
 Neurodevelopmental disorders
 Tumorigenesis and metastatic progression
 Cardiovascular diseases

3. < 2% of the human genome codes for proteins (~20K protein-coding genes)
Much of the remaining genome (once considered to be “ancestral noise” or “junk”
DNA) encodes regulatory information (~90% of the genome)

4. The functions of these non-coding regions of the genome include:
< 2% of the human genome codes for proteins (~20K protein-coding genes)
Much of the remaining genome (once considered to be “ancestral noise” or “junk”
DNA) encodes regulatory information (~90% of the genome)
The functions of these non-coding regions of the genome include:
 substrates for DNA-binding proteins that regulate both gene expression
and the 3D architecture of the genome
 template for non-coding RNA (ncRNA) transcription

5. The functions of these non-coding regions of the genome include:
< 2% of the human genome codes for proteins (~20K protein-coding genes)
Much of the remaining genome (once considered to be “ancestral noise” or “junk”
DNA) encodes regulatory information (~90% of the genome)
The functions of these non-coding regions of the genome include:
 substrates for DNA-binding proteins that regulate both gene expression
and the 3D architecture of the genome
 template for non-coding RNA (ncRNA) transcription
Noncoding RNAs fall into three major groups based on transcript size:
 small ncRNAs < 200 nt ( e.g., rRNA, tRNA, miRNA (~ 22 nt), piRNA (24-30 nt),
snRNA, siRNA, shRNA )
 mid-size ncRNAs (e.g., snoRNA ( nt) )

6. Biogenesis and functional mechanisms of miRNAs, piRNAs and snoRNAs
Esteller, M. Nature Reviews Genetics 12, (2011)

7. The functions of these non-coding regions of the genome include:
< 2% of the human genome codes for proteins.
Much of the remaining genome (once considered to be “junk” DNA) encodes
regulatory information.
The functions of these non-coding regions of the genome include:
 substrates for DNA-binding proteins that regulate both gene expression
and the 3-D architecture of the genome
 template for non-coding RNA (ncRNA) transcription
Noncoding RNAs fall into three major groups based on transcript size:
 small ncRNAs < 200 nt ( e.g., rRNA, tRNA, miRNA (~ 22 nt), piRNA (24-30 nt),
snRNA, siRNA, shRNA )
 mid-size ncRNAs (e.g., snoRNA ( nt) )
 long non-coding RNAs (lncRNAs) > 200 nt exhibiting “no apparent ORFs”
(although they often are polyadenylated, transcribed by RNA pol II (in both sense and antisense orientation to protein-coding genes), and exhibit
complex splicing patterns)
The estimated number of lncRNAs > the number of protein-coding RNAs

8. Categories of lncRNA action
Sana, J. et al. Journal of Translational Medicine 10, (2012)

9. lncRNAs allow the cell to fine-tune gene expression in response to its environment or
to up-regulate, down-regulate, or completely silence a gene to ensure proper tissue
patterning during programmed embryonic development.

10. lncRNAs allow the cell to fine-tune gene expression in response to its environment or
to up-regulate, down-regulate, or completely silence a gene to ensure proper tissue
patterning during programmed embryonic development. This includes:
 control of cell division, development, cell metabolism, human diseases
(including cancer progression and metastasis)
 regulation of transcription (can act in either cis or trans)
 regulation of RNA splicing, RNA transport, translation, mRNA processing and
stability
 chromatin remodeling (can act in either cis or trans) and 3D architecture of
the genome, nuclear organization
 scaffolding for assembly of RNA-protein complexes, ligands for proteins
involved in gene regulation and in base-pairing interactions capable of guiding
lncRNA-containing ribonucleoprotein complexes to specific target sites on
DNA or RNA, decoys for sequestering of regulatory proteins from their target
DNA sequences, localization and/or activity of proteins

11. lncRNAs allow the cell to fine-tune gene expression in response to its environment or
to up-regulate, down-regulate, or completely silence a gene to ensure proper tissue
patterning during programmed embryonic development. This includes:
 control of cell division, development, cell metabolism, human diseases
(including cancer progression and metastasis)
 regulation of transcription (can act in either cis or trans)
 regulation of RNA splicing, RNA transport, translation, mRNA processing and
stability
 chromatin remodeling (can act in either cis or trans) and 3D architecture of
the genome, nuclear organization
 scaffolding for assembly of RNA-protein complexes, ligands for proteins
involved in gene regulation and in base-pairing interactions capable of guiding
lncRNA-containing ribonucleoprotein complexes to specific target sites on
DNA or RNA, decoys for sequestering of regulatory proteins from their target
DNA sequences, localization and/or activity of proteins
Many lncRNAs are differentially expressed during individual stages of differentiation
and their expression and activity often exhibit tissue specificity.

12. relative to protein-coding genes:
lncRNAs can be grouped into 4 separate subclassifications based on their location
relative to protein-coding genes:
 exonic - at least one of its exons intersects a protein-coding exon
 intronic – completely contained within a protein coding intron (sense/antisense)
 overlapping - contains a coding gene within a lncRNA intron (sense/antisense)
 intergenic – do not intersect with any protein-coding loci
Overview of lncRNA populations
Categories of lncRNAs based on where they are found relative to nearby protein-coding genes.
(A) Proportion of lncRNA subgroups (B) Location of each type of lncRNA
Lee, C. and N. Kikyo. Cell Biosci. 2, (2012)

13. Models of nuclear lncRNA function
Sana, J. et al. Journal of Translational Medicine 10, (2012)

14. Models of cytoplasmic lncRNA function
A common recognition mechanism is through base pairing of complementary
regions between the lncRNA and their target RNA sequence
Fatica, A. and I. Bozzoni. Nature Reviews Genetics 15, 7-21 (2014)

15. X chromosome inactivation
In mammals females carry two X chromosomes whereas males carry only a single X giving a double dosage of X-linked genes in XX females relative to XY males.
To balance X-linked gene expression levels in males and females, female cells invoke a mechanism (X chromosome inactivation, XCI) to randomly silence one of the maternal X chromosomes (Xm) during early embryogenesis.  
Prior to cell differentiation: both X chromosomes are active (Xa)

16. X chromosome inactivation
In mammals females carry two X chromosomes whereas males carry only a single X giving a double dosage of X-linked genes in XX females relative to XY males.
To balance X-linked gene expression levels in males and females, female cells invoke a mechanism (X chromosome inactivation, XCI) to randomly silence one of the maternal X chromosomes (Xm) during early embryogenesis.  
Prior to cell differentiation: both X chromosomes are active (Xa)
During cell differentiation:
   each cell counts its number of X chromosomes
 one X chromosome is randomly selected for inactivation
 the lncRNA, Xist, is up-regulated on the future inactive X (Xi)
 a gradual chromosome-wide silencing process is then initiated on Xi
Once established, this silent state is stably transmitted through each round of cell
division in a heritable manner.

17. X chromosome inactivation
During development females undergo two forms of XCI - imprinted and random.
imprinted XCI – occurs during early embryogenesis - the paternal X chromosome (Xp) is
preferentially silenced (maintained in extra-embryonic tissues throughout
development)
random XCI – all imprinted epigenetic marks on Xp are erased in cells of epiblast lineage (these
form the future embryo) - a second round of XCI is then initiated where either Xp
or the maternal X chromosome (Xm) is randomly silenced (this randomness in
chromosome selection is directly observable in the coloration pattern of female
calico cats)
XIC is initiated from the X inactivation center (Xic) which encodes five ncRNAs of known function: Xist, Tsix, Xite, RepA, and Jpx.
Xist (X-inactive specific transcript) is up-regulated only after transient pairing of the two X chromosomes (pairing is the way in which the cell counts the number of X chromosomes.
Depletion of any of the factors involved in pairing (e.g., CTCF and OCT4) blocks pairing  cells
with an aberrant number of active (2 Xa) or inactive (2 Xi) X chromosomes.
Xist expression is regulated by Tsix ( – ), Xite ( – ), Jpx (+), and RepA (+)

18. Random X chromosome inactivation in mouse embryonic stem cells upon differentiation
Jeon et al. Curr Opin Genet & Dev. 22, (2012)1 (2012)

19. CTCF (CCCTC-binding factor / 11-zinc finger protein)
A primary role of CTCF is in regulating the 3D structure of chromatin. CTCF binds together strands of DNA to form chromatin loops facilitating direct interaction between remote sites on the chromosome.
Models of enhancer and insulator function
Role of CTCF in facilitating long range
interaction between an enhancer and promoter
Krivega, I. and A. Dean. Curr Opin Genet & Dev. 22, (2012)1

20. Genomic Imprinting
In certain cases the phenotype conferred by an allele depends on whether that allele is inherited from the mother or the father. The basis of this ‘parent-of‑origin’ phenotypic variation that is most well characterized is genomic imprinting.

21. Genomic Imprinting
In certain cases the phenotype conferred by an allele depends on whether that allele is inherited from the mother or the father. The basis of this ‘parent-of‑origin’ phenotypic variation that is most well characterized is genomic imprinting.
Essential roles of imprinted genes include:
 growth and development of the fetus
 post-natal behavior and metabolism.

22. Genomic Imprinting
In certain cases the phenotype conferred by an allele depends on whether that allele is inherited from the mother or the father. The basis of this ‘parent-of‑origin’ phenotypic variation that is most well characterized is genomic imprinting.
Essential roles of imprinted genes include:
 growth and development of the fetus
 post-natal behavior and metabolism.
Imprinted genes may be either ubiquitously imprinted or exhibit tissue-specific and/or temporal-specific imprinting patterns. They are located throughout the genome in approximately 1 Mb clusters (typically) that are:
 expressed exclusively from the maternally or paternally inherited chromosomes
 under the control of a discrete imprinting control region (ICR)

23. Genomic Imprinting
In certain cases the phenotype conferred by an allele depends on whether that allele is inherited from the mother or the father. The basis of this ‘parent-of‑origin’ phenotypic variation that is most well characterized is genomic imprinting.
Essential roles of imprinted genes include:
 growth and development of the fetus
 post-natal behavior and metabolism.
Imprinted genes may be either ubiquitously imprinted or exhibit tissue-specific and/or temporal-specific imprinting patterns. They are located throughout the genome in approximately 1 Mb clusters (typically) that are:
 expressed exclusively from the maternally or paternally inherited chromosomes
 under the control of a discrete imprinting control region (ICR)
Imprinted genes must be marked with their parental origin so that the correct allele-specific expression patterns are observed in somatic tissues.

24. Genomic Imprinting
In certain cases the phenotype conferred by an allele depends on whether that allele is inherited from the mother or the father. The basis of this ‘parent-of‑origin’ phenotypic variation that is most well characterized is genomic imprinting.
Essential roles of imprinted genes include:
 growth and development of the fetus
 post-natal behavior and metabolism.
Imprinted genes may be either ubiquitously imprinted or exhibit tissue-specific and/or temporal-specific imprinting patterns. They are located throughout the genome in approximately 1 Mb clusters (typically) that are:
 expressed exclusively from the maternally or paternally inherited chromosomes
 under the control of a discrete imprinting control region (ICR)
Imprinted genes must be marked with their parental origin so that the correct allele-specific expression patterns are observed in somatic tissues.
These parental-specific marks must be:
 stable and heritable so that imprinting is maintained throughout development
 erasable so that imprints can be reset when embryonic germ cells are being reprogrammed
during germ cell migration and differentiation

25. Genomic Imprinting
Two dominating mechanisms have been described for mediating imprinting in clusters:
(1) insulator model (an evolutionarily ancient but least utilized mechanism) that is employed
by the H19/Igf2 imprinted locus
(2) lncRNA model (a recently evolved and more commonly utilized mechanism) that is
employed for ncRNA-mediated imprinting at the Igf2r locus

26. Insulator and lncRNA mediated imprinting
Genomic Imprinting
Two dominating mechanisms have been described for mediating imprinting in clusters:
(a) insulator model (evolutionarily ancient but least utilized mechanism) employed by the
H19/Igf2 imprinted locus
(b) lncRNA model (recently evolved and more commonly utilized mechanism) employed
for ncRNA-mediated imprinting at the Igf2r locus (differentially methylated IRC)
Insulator and lncRNA mediated imprinting
Abramowitz, L. K. and M. S. Bartolomei. Curr Opin Genet & Dev. 22, (2012)1

27. Involvement of (long) noncoding RNAs
in development

28. One set of TFs critical in embryonic development is encoded by genes within the Hox clusters
Dasen, J. S. Cell Reports 5, 1-2 (2013) ; Delpretti, S. et al . Cell Reports 5, (2013)
HOX genes - a group of related genes that control the body plan of the embryo along the anterior-posterior (head-tail) axis. Following formation of the embryonic segments, expressed Hox proteins determine the type of segment structures (e.g. legs, antennae, and wings in fruit flies; different types of vertebrae in humans) that will form on a given segment (why snakes have no legs!).
During development Hox genes are regulated (through modifications of histones and chromosome structure) by lncRNAs encoded by genes within the Hox clusters.
The Hox clusters encode the lncRNA genes HOTAIR (HOX antisense intergenic RNA) , HOTTIP (HOXA transcript at the distal tip), and HOTAIRM1 (HOXA transcript antisense RNA, myeloid-specific 1).
The lncRNAs Hog (Hotdog) and Tog (Twin of Hotdog) are encoded by two genes within a gene desert near the HoxD locus.

29. LSD1 encodes a flavin -dependent monoamine
HOTAIR (HOX antisense intergenic RNA), transcribed within the HOXC cluster, regulates Hox genes in trans by repressing genes within the HoxD cluster by recruitment of LSD1 and PRC2 (similar to the activity of Xist in XCI).
LSD1 encodes a flavin -dependent monoamine
oxidase that demethylase s both K4 (H3K4me)
and K9 (H3K9me) of histone H3
Hox genes are required for development of the cecum, a critical organ required for the metabolism of cellulose by herbivorous and omnivorous mammals.
Hog and Tog are expressed only in the cecum, and is
required for regulation of the profile of HoxD gene
expression during cecum budding.
This regulation requires the physical contact between
the shared start site of Hog and Tog transcription and
the expressed HoxD genes.
Dasen, J. S. Cell Reports 5, 1-2 (2013) ; Delpretti, S. et al . Cell Reports 5, (2013)

30. Homeosis Resulting From HoxD Gene Derepression in Hotair - /- mice
In the mouse, targeted deletion of Hotair causes derepression HoxD genes along with several imprinted loci and results in both homeotic transformation of the spine and malformation of metacarpal-carpal bones in mice homozygous for this deletion.
Li, L. et al. Cell Reports 5, 3-12 (2013)

31. Homeosis Resulting From HoxD Gene Derepression in Hotair - /- mice
Homeotic transformation of the lumber (L)
vertebrae results in loss of the sixth
lumbar and structurally deformed first sacral (S) vertebrae (arrow) in Hotair KO mice (L6  S1 transformation).
Alizarin red-Alcian blue staining shows the deformed wrist bones in KO mice. Note the fusion of carpal elements c-3 and 1-2-c (circled area), and missing radius (asterisk) in KOs.
Note that carpal elements 4/5
are always naturally fused in
WT wrist.
Li, L. et al. Cell Reports 5, 3-12 (2013)

32. ncRNA regulation of muscle differentiation
Fatica, A. and I. Bozzoni. Nature Reviews Genetics 15, 7-21 (2014)

33. lncRNAs Involved in Retinal Development
Vax2os1 contains a binding site for CRX (Cone-Rod Homeobox - a TF in cone and rod maturation involved in regulating the expression of a number of photoreceptor-specific genes). Vax2os1 is thought to play a role in the specification of the ventral rod photoreceptors by acting as a cell cycle regulator of the retinal progenitors and in the maintenance of the adult photoreceptor cells.
Six3OS (transcribed from the homeodomain transcription factor Six3 gene) is involved in
regulating retinal cell specification. Six3OS regulates Six3 activity in developing retina by binding
directly to Ezh2 and Eya family and acting as a molecular scaffold to recruit histone modification
enzymes to Six3 target genes.
The eya (eyes absent) gene (and its mammalian homologues) encode protein tyrosine
phosphatases that function as transcriptional coregulators controlling eye field specification.
RNCR2 (a.k.a. MIAT (myocardial infarction associated transcript) ) - negatively regulates the differentiation of amacrine interneurons and Müller glia (but does not affect development of other neuronal subtypes such as bipolar interneurons).
Probable a target of TF Oct4 in mESCs maintaining Oct4 expression in a feedback loop to help maintain pluripotency.
Variants confer susceptibility to myocardial infarction, possibly through altering the RNA's protein
binding properties.

34. Involvement of lncRNAs in complex diseases
 General Overview of ncRNAs in complex diseases
 lncRNAs in neurodevelopmental disorders
 lncRNAs in tumorigenesis and metastatic progression
 lncRNAs in CAD

35. Involvement of selected noncoding RNAs in complex diseases
Esteller, M. Nature Reviews Genetics 12, (2011)

36. ncRNAs identified in neurodevelopmental disorders
PWS (Prader–Willi syndrome), AS (Angelmann syndrome), FXS (fragile X syndrome)
DS (down syndrome), MCOPS3 (micropthalmia syndrome 3) ASD, (autism spectrum disorder)
van deVondervoort, Ilse I. G. M. et al. Frontiers in Molecular Neuroscience Reports 6, 1-9 (2013)

37. Only a very limited number of cancers are the result of inheritable genetic mutations, which typically involve non-synonymous mutations in protein-coding genes.
The majority of cancers result from somatic mutations that involve a complex combination of both genetic and environmental factors. Moreover, the majority
of cancer-associated SNPs are located outside of protein-coding genes (either within the introns of protein-coding genes or intergenic regions.
Cheetham, S.W. et al. British Journal of Cancer 108, (2013)

38. Mechanisms of lncRNA-induced cancer progression
Up-regulation of the maternally imprinted lncRNA H19 due to loss of imprinting occurs in a wide range of metastatic tumors. The exact mechanism of metastatic regulation varies with tumor identity.
Bladder cancer - transcription regulation
binding of H19 with EZH2 (enhancer of Zeste homolog 2), the histone methyl-transferase of
PRC2, recruits PRC2 to the E-cadherin (epithelial calcium-dependent adhesion) promoter
 suppression of E-cadherin expression  epithelial-to-mesechymal transition (EMT)
Colon cancer - post-transcriptional regulation
H19 serves as a precursor for miRNA-675 which
targets the tumor suppressor Rb
Up-regulation of miRNA-675   Rb  increase
in colony-forming ability in soft agar (phenotype
associated with acquisition of anchorage-independent
growth)
Suzanne J. Baker, S. J. & P. J. McKinnon Nature Reviews Cancer 4, (2004)

39. Mechanisms of lncRNA-induced cancer progression
HOTAIR
Over-expression leads to targeting of PRC2 and LSD1 repressive complexes to anti-metastatic loci
(in trans – as in the regulation of HOXD gene cluster) resulting in histone H3K27 trimethylation and H3K4 demethylation at the loci. Evidence that HOTAIR may also directly control DNA methylation (depletion of HOTAIR leads to a decrease in PTEN (phosphatase and tensin homologue) promotor methylation)
ZEB2-AS1 (ZEB2 antisense RNA 1)
Overlaps 5’-UTR intron of ZEB2 gene which contains an internal ribosomal binding site required for translation of ZEB2 mRNA, which is prevented by splicing of the 5’ intron. Over-expression of ZEB2-AS1 in epithelial cells prevents 5’-UTR splicing   ZEB2 protein (ZEB2 directly inhibits E-cadherin expression)   E-cadherin levels  EMT.

40. Mechanisms of lncRNA-induced cancer progression
KCNQ1OT1 (KCNQ1 overlapping transcript 1)
Particularly interesting in that the KCNQ1 gene exhibits tissue- or stage-specific imprinting
The Kcnq1 imprinted domain exhibits complex tissue-specific expression patterns co-existing with a domain-wide cis-acting control element. Transcription of the paternally expressed antisense non-coding RNA Kcnq1ot1 silences some neighboring genes in the embryo, while others are unaffected.
Kcnq1 (a critical gene for normal heart development and function) is imprinted in early cardiac development but becomes biallelic after midgestation
A recent study has identified regulatory mechanisms within the Kcnq1 imprinted domain that operate on Kcnq1 exclusively in the heart.
Down-regulation of Kcnq1ot1 occurs in colorectal cancer causing an over-expression of Kcnq1 in later cardiac development. This leads to an aberrant 3D structure of the chromatin allowing the Kcnq1 promoter to establish abnormal contact with enhancers activating aberrant transcription of multiple target genes resulting in cell proliferation.

41. Mechanisms of lncRNA-induced cancer progression
TERRA (telomeric repeat-containing RNA)
Telomeres are repetitive DNA sequences that protect the ends of chromosomes from deterioration or fusion with neighboring chromosomes. Telomeres are progressively shorten during cell division and trigger either cell death or senescense when reaching a critical length. Most cancer cells express telomerase, which prevents this shortening by adding telomeric repeats to the 3- end of the chromosome.
TERRA (a lncRNA transcribed from telomeric ends), which binds telomerase inhibiting its activity, is down-regulated in many cancer cells  increase in cancer cell longevity
Cheetham, S.W. et al. British Journal of Cancer 108, (2013)

42. Mechanisms of lncRNA-induced cancer progression
PTENP1 (PTEN pseudogene 1)
Aberrant activation of the PI3K/AKT pathway in melanoma is known to be caused by genomic deletion, promoter methylation, and loss-of-function mutations of the tumor suppressor gene
PTEN (phosphatase and tensin homolog).
Levels of PTEN tumor suppressor protein is also regulated the post-transcriptionally by a complex microRNA network involving the lncRNA PTENP1. PTENP1 behaves as a pseudogene of PTEN (or “PTEN decoy”) by competetive binding of miRNAs that down-regulate PTEN expression
 cell will maintain levels of tumor suppressor protein sufficient to restrict cell proliferation.
Many human cancers (e.g. melanoma) exhibit a loss of PTENP1 lncRNA resulting in decrease in PTEN expression and a loss in levels of PTEN tumor suppressor protein sufficient to allow unrestricted cell proliferation.
Cheetham, S.W. et al. British Journal of Cancer 108, (2013)

43. Mechanisms of lncRNA-induced cancer progression
MALAT1 (metastasis associated lung adenocarcinoma transcript 1; a.k.a NEAT2 (noncoding nuclear-enriched abundant transcript 2))
Overexpression linked to increase in cell proliferation in colorectal and lung cancer. MALAT1 localizes to nuclear speckles and acts post-transcriptionally by regulating levels of phosphorylated serine/arginine (SR) splicing factors.
MALAT1 interacts with CBX4 (E3 SUMO (small ubiquitin-like modifier)-protein ligase CBX4 , a.k.a. chromobox protein homolog 4), a component of a PRC1-like complex required to maintain the transcriptionally repressive state of many genes by mediating monoubiquitination of histone H2AK119. MALAT1 regulates subnuclear shuttling of CBX4 between polycomb bodies and interchromatin granules.
mascRNA (MALAT1-associated small cytoplasmic RNA) ???
Proposed that MALAT1 also may encode a tRNA-like structure generated by RNase P (although how this sncRNA functions is currently not known. It has been proposed that (i) mascRNA may act as a "sponge" for proteins, preventing them from reaching their natural destinations within the cell or (ii) may simply alert the cell that the MALAT1 is "available" in the nucleus for other cellular duties (Wilusz, J. E. et al. Cell 135, (2008)).

44. Mechanisms of lncRNA-induced cancer progression
Xist
Random XCI, initiated by Xist, occurs only once during development during embryonic days after which time the same X chromosome is maintained as Xi during all future cell divisions. Since Xi maintenance does not depend on Xist, it was always assumed that Xist should serve no purpose subsequent to initiation of XCI.

45. Mechanisms of lncRNA-induced cancer progression
Xist
Random XCI, initiated by Xist, occurs only once during development during embryonic days after which time the same X chromosome is maintained as Xi during all future cell divisions. Since Xi maintenance does not depend on Xist, it was always assumed that Xist should serve no purpose subsequent to initiation of XCI.
However, the fact that Xist is continuously expressed for the entire life of the female suggests that Xist serves some additional function(s) after XCI is established in the early embryo. A recent study in mice has shown that X reactivation resulting from Xist deletion leads to development of a highly aggressive, lethal blood cancer (mixed MPN/MDS) in females with 100% penetrance.
 up-regulation of X-linked genes resulting from the deletion of Xist leads to changes in
homeostatic pathways leading to cancer.

46. Mechanisms of lncRNA-induced cancer progression
Xist
Random XCI, initiated by Xist, occurs only once during development during embryonic days after which time the same X chromosome is maintained as Xi during all future cell divisions. Since Xi maintenance does not depend on Xist, it was always assumed that Xist should serve no purpose subsequent to initiation of XCI.
However, the fact that Xist is continuously expressed for the entire life of the female suggests that Xist serves some additional function(s) after XCI is established in the early embryo. A recent study in mice has shown that X reactivation resulting from Xist deletion leads to development of a highly aggressive, lethal blood cancer (mixed MPN/MDS) in females with 100% penetrance.
 up-regulation of X-linked genes resulting from the deletion of Xist leads to changes in
homeostatic pathways leading to cancer (Yildirim, E. Cell 152, (2013))
Consistent with the known association of supernumerary X chromosomes and human cancers:
 breast and ovarian cancer cells frequently show loss of Barr body and duplicate Xa
 XXY males exhibit a x increased risk of breast cancer
 testicular germ cell tumors often acquire supernumary X chromosomes
 In addition to its role in XCI Xist lncRNA plays a role in suppressing cancer.

47. ANRIL (antisense noncoding RNA in the INK4 locus)
Genomic layout of the p15/CDKN2B-p16/CDKN2A-p14/ARF-ANRIL
gene cluster at the 9p21.3 locus
Pasmant, E. et al. FASEB J. 25, (2011)
ANRIL (a.k.a. CDKN2BAS) contains 19 exons, spans a region of kb and is transcribed in an antisense direction relative to the p15/CDKN2B-p16/CDKN2A-p14/ARF gene cluster.
ANRIL intron 1 overlaps the two exons of p15/CDKN2B. The 5′ end of the first exon is located ∼300 bp upstream of the transcription start site of the p14/ARF gene.
Co-clustering of ANRIL with p14/ARF across normal human tissues and human tumors and the mapping of CTCF-binding sites map within the CpG island overlapping the ANRIL-p14/ARF promoters suggests the transcription of these two genes are co-regulated.
CTCF (CCCTC-binding factor), a major zinc-finger protein with insulator and chromatin barrier activity, is critical for transcription of the p15/CDKN2B-p16/CDKN2A-p14/ARF locus.

48. ANRIL (antisense noncoding RNA in the INK4 locus)
ANRIL is located in a GWAS ‘hot spot’ linked to many complex diseases, including type-2 diabetes, coronary artery disease and, recently, cancer. ANRIL interacts with PcG group proteins and may add repressive histone marks to the p15/CDKN2B-p16/CDKN2A-p14/ARF locus, suppressing cell proliferation.
Disease-associated SNPs in ANRIL cluster by disorder; vascular conditions are associated with the 3’-end of the transcript, while cancer susceptibility SNPs map to the 5’-region . Polymorphisms in different regions of the ANRIL RNA may affect the RNA–DNA or RNA–protein interactions necessary for ANRIL–induced gene silencing. The region mutated may cause changes in site selectivity of epigenetic programming, with some interactions required for vascular function while other interactions are required for cell cycle maintenance.
Cheetham, S.W. et al. British Journal of Cancer 108, (2013)