2. • What is imprinting?
– Expand your vocabulary
• Inheritance
– Mendelian/Non-Mendelian
– Pattern of Imprinting
• Importance
• Mechanisms
– Big Picture
• Disease
– Detection of imprinted genes
• Disclaimer *Your results may vary*
OVERVIEW
3. • Epigenetics- “On genes,” modifications to DNA that
retain sequence fidelity yet alter gene expression
• Imprinting- Gamete-specific differential modification
– Unique to placental mammals, marsupials and flowering plants
– Typically marked with DNA methylation
– Often occur in clusters
– Gene is silenced
• Epigenetic memory- The set of epigenetic modifications
inherited in descendant cells
WHAT IS IMPRINTING?
4. • 2 broad categories of inheritance
– Mendelian
– Non-mendelian
• Law of Segregation- Each parent
contributes a single gamete
containing single alleles from
either maternal or parental
chromosomes
MENDELIAN GENETICS…. INHERITANCE
5. IMPRINTING INHERITANCE PATTERN (GAMETES)
http://hihg.med.miami.edu/code/http/modules/education/Design/Print.asp?CourseNum=2&LessonNum=2
• Imprinting
established
in gametes
6. IMPRINTING INHERITANCE PATTERN (POST
FERTILIZATION)
http://hihg.med.miami.edu/code/http/modules/education/Design/Print.asp?CourseNum=2&LessonNum=2
• Imprinting
maintained
during
embryogenesis
• Genome-wide
epigenetic
reprogramming
• Paternal X
inactivation
8. IMPRINTING INHERITANCE PATTERN ( BACK TO
GAMETES)
http://hihg.med.miami.edu/code/http/modules/education/Design/Print.asp?CourseNum=2&LessonNum=2
• Imprinting
erased and
modified
based on
sex
9. • Imprinted genes
– Are crucial for normal development
– Bypass epigenetic reprogramming
– Are vulnerable to epigenetic copying
machinery
• Roles in
– Growth
– Behavior
– Stem cells
– Disease
IMPORTANCE
10. • Paternal expression
– Placental development
– Enhance growth
– Large offspring (benefit for father)
• Maternal expression
– Suppress growth
– Limit expression of paternal genes
– Small offspring (benefit for mother)
• Resource use
IMPORTANCE
Moon, Y. S., Smas, C. M., Lee, K., Villena, J. A., Kim, K.-H., Yun, E. J., & Sul, H. S. (2002). Mice lacking paternally expressed
Pref-1/Dlk1 display growth retardation and accelerated adiposity. Molecular and Cellular Biology, 22, 5585–5592.
doi:10.1128/MCB.22.15.5585-5592.2002
12. MECHANISM- HOW DOES IT WORK?
Zakhari, S. (2013). Alcohol metabolism and epigenetics changes.
Alcohol Research : Current Reviews, 35, 6–16.
• DNA Methylation
– CpG- Cytosine-
phosphate-
guanine
– DNMT- DNA
methyltransferase
– 5’Methyl-cytosine
can easily be
deaminated to
form thymine
13. • CpG Islands- Regions with high
concentrations of Cpg motifs
– Promoter region
– ~70% of human promoter regions
contain CpG islands
• Imprinting Control Regions (ICR) &
Differentially Methylated Regions
(DMR)
– Cis-acting
– Control gene clusters ~3.0 Mb away
– Deletion of ICR leads to loss of
imprinting
DNA METHYLATION
http://biol10005.tumblr.com/post/31972466118
/epigenetics
14. DNA METHYLATION
Li, E. (2002). Chromatin modification and epigenetic reprogramming in mammalian development. Nature
Reviews. Genetics, 3, 662–673. doi:10.1038/nrg887
• Dnmt3 family
facilitates imprinting
in gametes
• Dnmt1 maintains
methylation states
post-embryogenesis
• 98% of genome 1
CpG/100bp- Me
• <2% of genome 1
CpG/10bp- Un-Me
15. • Chromatin condensation
• Gene imprinting on rare
occasions will modify
histones through
acetylation/methylation
DNA METHYLATION- QUICK STEP BACK
Duygu, B., Poels, E. M., & da Costa Martins, P. A. (2013). Genetics and epigenetics of arrhythmia and heart
failure. Frontiers in Genetics, 4, 219. doi:10.3389/fgene.2013.00219
16. • Acetylation generally
leads to increased
expression
• Methylation generally
leads to decreased
expression
• Ideas on why imprinting
occurs in clusters?
HISTONE MODIFICATION
Duygu, B., Poels, E. M., & da Costa Martins, P. A. (2013). Genetics and epigenetics of arrhythmia and heart
failure. Frontiers in Genetics, 4, 219. doi:10.3389/fgene.2013.00219
17. • Imprinted genes
contain regions of
ncRNAs
• Air paternally
expressed
• Repress flanking
protein-coding genes
in cis
LNC-RNA
Fatica, A., & Bozzoni, I. (2014). Long non-coding RNAs: new players in cell differentiation and development.
Nature Reviews. Genetics, 15, 7–21. doi:10.1038/nrg3606
18. • CTCF- 11-zinc finger
protein
• Binds Imprinting
Control Element
• Prevents methylation
of ICE and H19
• Ins2- Insulin 2
• H19- lnc-RNA,
negative regulator of
growth and
proliferation
INSULATOR MODEL
19. • “Random” X-chromosome inactivation
– Cell-to-cell basis
– Actually regulated by several cis-Elements (Choice elements)
• Xist- X-Inactive Specific Transcript- encodes lnc-RNA
• Tsix- Antisense to Xist- negative regulator of Xist
X CHROMOSOME INACTIVATION CO-
EVOLUTION?
Reik, W. & Lewis, A. Co-evolution of X-chromosome inactivation and imprinting in mammals. Nature Rev.
Genet. 6, 403–410 (2005).
20. • Xist
X CHROMOSOME INACTIVATION CO-
EVOLUTION?
• Xist lncRNA acts cis- coating chromosome
• Histone modification silences coated chromosome
• Very similar to imprinting
Reik, W. & Lewis, A. Co-evolution of X-chromosome inactivation and imprinting in mammals. Nature Rev.
Genet. 6, 403–410 (2005).
21. X CHROMOSOME INACTIVATION CO-
EVOLUTION?
• DNA methylation steps identical to imprinting
Reik, W. & Lewis, A. Co-evolution of X-chromosome inactivation and imprinting in mammals. Nature Rev.
Genet. 6, 403–410 (2005).
22. • Xist
X CHROMOSOME INACTIVATION CO-
EVOLUTION?
• Methylation is maintained in placenta
• Lost in embryonic tissue
Reik, W. & Lewis, A. Co-evolution of X-chromosome inactivation and imprinting in mammals. Nature Rev.
Genet. 6, 403–410 (2005).
23. • Paternal genome
undergoes rapid de-
methylation
• Maternal genome
de-methylates more
slowly
• Imprinted genes
escape methylome
reprogramming
UNDERSTANDING THE TIMELINE OF
METHYLATION
Vertino, P. (2011), "DNA methylation in cancer", in Issa, J. (ed.), DNA Methylation: Physiology, pathology and
disease, The Biomedical & Life Sciences Collection, Henry Stewart Talks Ltd, London
24. THE METHYLOME AND YOU (BIG PICTURE)
Vertino, P. (2011), "DNA methylation in cancer", in Issa, J. (ed.), DNA Methylation: Physiology, pathology and
disease, The Biomedical & Life Sciences Collection, Henry Stewart Talks Ltd, London
27. • Angelman Syndrome (AS)
– 1/20k births
– Severe mental retardation
– Lack of speech
– Ataxic gait
– Unnaturally happy disposition
• Random bouts of laughter
– Hand flapping
DISEASE
28. • Prader-Willi Syndrome (PWS)
– 1/25k births
– Mild mental retardation
– Chronic hunger- obesity
– Stunted height
– Most common syndromal cause of
human obesity
DISEASE
29. • Loss of function
• Chromosome 15
– Loss of expression in
15q11-13
• AS- Maternal
• PWS- Paternal
DISEASE
30. • Beckwith-Wiedemann
Syndrome
– Microcephaly- small head
– Macroglossia- enlarged tongue
– Visceromegaly- enlarged organs
– Macrosomia- large body size
– Umbilical hernia
• Loss of imprinting on
chromosome region 11p15.5
DISEASE
http://health.shorehealth.org/imagepages/17076.htm
31. • Cancer
– Wilms’ tumor
• Embryonic kidney cancer
– Loss of imprinting control on H19
– Hypermethylation of imprinting control
region
– Overexpression of IGF2
DISEASE
Martens-Uzunova ES, Bottcher R, Croce CM, Jenster G, Visakorpi T, Calin GA. Long noncoding RNA in prostate,
bladder, and kidney cancer. Eur Urol. 2013;65:1140–51. doi: 10.1016/j.eururo.2013.12.003.
32. DISEASE
• Cancer
– Bladder cancer
– Loss of imprinting on H19
– Hypomethylation of imprinting control
region
– Overexpression of IGF2
Martens-Uzunova ES, Bottcher R, Croce CM, Jenster G, Visakorpi T, Calin GA. Long noncoding RNA in prostate,
bladder, and kidney cancer. Eur Urol. 2013;65:1140–51. doi: 10.1016/j.eururo.2013.12.003.
33. • Bisulfite sequencing
– Cytosine converted to
uracil
– Methylated cytosine
unaffected
DETECTION OF IMPRINTED GENES
34. • Sequencing after treatment should
reveal no bands in lane C
• Bands in lane C represent methylated
cytosines
DETECTION OF IMPRINTED GENES
Herman, J G et al. “Methylation-Specific PCR: A Novel PCR Assay for Methylation Status of
CpG Islands.” Proc of Nat. Ac. of Sci. 93.18 (1996): 9821–9826.
35. • Hongerwinter 1944
– Dutch period of starvation during WWII
– Childen born were short, and
diagnosed with anemia, edema,
diabetes, and depression
– The women who were born of this era
were shown to have children that
mimicked the same symptoms as their
mothers.
– Hyopmethylation of IGF2 six decades
later
– Implication in environmental imprinting
YOU ARE WHAT YOU DON’T EAT?
Veenendaal M, Painter R, de Rooij S, Bossuyt P, van der Post J, Gluckman P, et al. Transgenerational effects of prenatal
exposure to the 1944-45 Dutch famine. BJOG (2013) 120(5):548–5410.1111/1471-0528.12136
36. • Duygu, B., Poels, E. M., & da Costa Martins, P. A. (2013). Genetics and epigenetics of arrhythmia and heart failure. Frontiers in
Genetics, 4, 219. doi:10.3389/fgene.2013.00219
• Fatica, A., & Bozzoni, I. (2014). Long non-coding RNAs: new players in cell differentiation and development. Nature Reviews.
Genetics, 15, 7–21. doi:10.1038/nrg3606
• Herman, J G et al. “Methylation-Specific PCR: A Novel PCR Assay for Methylation Status of CpG Islands.” Proceedings of the
National Academy of Sciences of the United States of America 93.18 (1996): 9821–9826.
• http://health.shorehealth.org/imagepages/17076.htm
• http://biol10005.tumblr.com/post/31972466118/epigenetics
• Li, E. (2002). Chromatin modification and epigenetic reprogramming in mammalian development. Nature Reviews. Genetics,
3, 662–673. doi:10.1038/nrg887
• Martens-Uzunova ES, Bottcher R, Croce CM, Jenster G, Visakorpi T, Calin GA. Long noncoding RNA in prostate, bladder, and
kidney cancer. Eur Urol. 2013;65:1140–51. doi: 10.1016/j.eururo.2013.12.003.
• Moon, Y. S., Smas, C. M., Lee, K., Villena, J. A., Kim, K.-H., Yun, E. J., & Sul, H. S. (2002). Mice lacking paternally expressed Pref-
1/Dlk1 display growth retardation and accelerated adiposity. Molecular and Cellular Biology, 22, 5585–5592.
doi:10.1128/MCB.22.15.5585-5592.2002
• Reik, W. & Lewis, A. Co-evolution of X-chromosome inactivation and imprinting in mammals. Nature Rev. Genet. 6, 403–410
(2005).
• Veenendaal M, Painter R, de Rooij S, Bossuyt P, van der Post J, Gluckman P, et al. Transgenerational effects of prenatal
exposure to the 1944-45 Dutch famine. BJOG (2013) 120(5):548–5410.1111/1471-0528.12136
• Vertino, P. (2011), "DNA methylation in cancer", in Issa, J. (ed.), DNA Methylation: Physiology, pathology and disease, The
Biomedical & Life Sciences Collection, Henry Stewart Talks Ltd, London
• Zakhari, S. (2013). Alcohol metabolism and epigenetics changes. Alcohol Research : Current Reviews, 35, 6–16.
REFERENCES
