C) Repetitive elements
7. Conclusion
The genome is subject to many regulatory mechanisms, of which methylation is only one. This work serves to show that significant differences exist between closely related organisms, and that sex is one of the determining factors in every organisms particular methylation pattern. We also see that same genes are differently methylated in different organs. Organs are collections of many different cell populations, and greater exactness could be achieved by isolating homogeneous cell populations, which is technically challenging. We also know at this point that DNA methylation pattern of an individual changes during its life. To determine these dynamics, a work of much larger scale is required, involving greater numbers of individuals, DNA from more than one time point in their lives and, as said before, more exact isolation of cell populations. This shall also require methods capable of analyzing multiple CpG sites rapidly and from lesser DNA amounts. The complexity of the problem is further shown by the fact that sex specific methylation results could not be reproduced in another mice population – but were still reproducible in the tissue samples used in the original work. This shows that methylation patterns are possibly dependent on the environmental influences, seasonal variability and other factors. The first group of mice were raised in August/September, the second in December/January; though conditions were close, they may have not been fully identical. The exact CO2 concentration used to kill the mice to harvest tissue samples was not measured; the pathological anaerobic metabolism prior to stop of vital functions may have been different in both groups; exposure to CO2 results in a decrease in alpha-ketoglutarate, which is a cofactor in transforming methylcytosine into 5-carboxylcytosine; between these are the oxidation products which are the intermediate steps in demethylation process. Thus, different levels of CO2 could possibly have caused different methylation product concentration in tissues with active circulation, such as spleen and brain. The SIRPH technique used was very accurate and sensitive, but covered only few methylated spots in the genome. A technique capable of broader analysis and a larger cohort of experimental animals would be needed to visualize broader and general patterns of methylation.
101
Tables
1. Tab. 1: Bisulfite amplification primers... 47
2. Tab. 2: SNuPE reaction primers... 50
3. Tab. 3: HPLC elution gradient and temperature conditions... 52
4. Tab. 4: Primers used for RNA expression analysis... 54
5. Tab. 5: Level of methylation in spleen, lung and skin tissue samples.. 62
6. Tab. 6: Level of methylation in brain, bone marrow, and testicular
tissue samples ……….. 63
7. Tab. 7: Level of methylation in tongue, skeletal muscle, and heart
muscular tissue samples ………. 64
8. Tab. 8: Alpha actin methylation data………. 108-116
9 Tab. 9: Snrpn D1 methylation data……… 117-125
10 Tab. 10: IAP methylation data………. 126-134
11 Tab. 11: Line 1 methylation data………. 135-143
12 Tab. 12: Long intronic transcript methylation data……… 144-152
13. Tab. 13: Myosin methylation data……… 153-161
14. Tab. 14: Paternally expressed gene 3 methylation data………….. 162-170
102
15 Tab 15 and 16: Methylation correlation between different loci within one tissue, male bone marrow... 172
16. Tab. 17 and 18: Methylation correlation between different loci within one tissue,
female bone marrow………... 173
17 Tab.19 and 20: Methylation correlation between different loci within one tissue,
male brain……… 174
18 Tab. 21 and 22: Methylation correlation between different loci within one tissue,
female brain………. 175
19 Tab. 23 and 24: Methylation correlation between different loci within one tissue,
male heart……… 176
20 Tab. 25 and 26: Methylation correlation between different loci within one tissue,
female heart………. 177
21 Tab. 27 and 28: Methylation correlation between different loci within one tissue,
male lungs……… 178
22 Tab. 29 and 30: Methylation correlation between different loci within one tissue ,
female lungs………. 179
23 Tab. 31 and 32: Methylation correlation between different loci within one tissue,
male skeletal muscle……….. 180
24 Tab. 32 and 34: Methylation correlation between different loci within one tissue, female skeletal muscle………... 181
25 Tab. 35 and 36: Methylation correlation between different loci within one tissue,
male skin……….. 182
103
26 Tab. 37 and 38: Methylation correlation between different loci within one tissue,
female skin……… 183
27 Tab. 39 and 40: Methylation correlation between different loci within one tissue,
male spleen……… 184
28 Tab. 41 and 42: Methylation correlation between different loci within one tissue,
female spleen……… 185
28 Tab. 43 and 44: Methylation correlation between different loci within one tissue,
male tongue……… 186
29 Tab. 45 and 46: Methylation correlation between different loci within one tissue,
female tongue………. 187
30 Tab. 47 and 48: Methylation correlation between different loci within one tissue,
testes……… 188
104
Figures
1. Fig. 1: Adenosylmethionine synthesis scheme... 14
2. Fig. 2: DNA bases, minor base methylated cytosine and its overall
quantity in the genome………..……….... 18
3. Fig. 3: Mechanism of DNA methylation pattern inheritance... 20
4. Fig. 4: DNA rendered expressionally inactive by methylation... 22
5. Fig. 5: Myosin light chain exon 1, sequence before and after bisulfite
processing……….. 30
6. Fig. 6: Fragment of alpha actin gene, sequence before and after
bisulfite processing………... 32
7. Fig. 7: Fragment of paternally expressed gene 3, sequence before
and after bisulfite processing……….……… 34
8. Fig. 8: Snrpn fragment, sequence before and after bisulfite processing… 36
9. Fig. 9: Mouse partial Kcnq gene intron 1, sequence before and
after bisulfite processing………... 37
10. Fig. 10: Mouse LINE-1 repetitive element, sequence before and after
bisulfite processing……… 39
11. Fig. 11: Intracisternal A particle, sequence before and after
bisulfite processing………. 41
105
12. Fig. 12: Bisulfite conversion scheme………. 44
13. Fig. 13: Scheme of the SNuPE product quantification
using HPLC………. 53
14 Fig. 14: Scheme of the SNuPE product quantification using
HPLC with two primers………. 53
15 Fig. 15: Sample of genomic DNA analysis, electrophoresis on
agarose based gel ... 56
16. Fig. 16: Gel analysis of bisulfite amplification product ... 57
17. Fig. 17: HPLC chromatogram samples... 59
18. Fig. 18: Methylation variability at specific loci, alpha actin………… 65 19. Fig. 19: Methylation variability at specific loci, myosin light chain… 66 20. Fig. 20: Methylation variability at specific loci, paternally
expressed gene 3………. 67
21. Fig. 21: Methylation variability at specific loci, Snrpn-D1………… 68
22. Fig. 22: Methylation variability at specific loci, LIT1………. 69
23. Fig. 23: Methylation variability at specific loci, IAP……… 70
24. Fig. 24: Methylation variability at specific loci, LINE-1………. 71
25. Fig. 25: Relative RNA expression intensity in a given tissue for
alpha actin………... 72
106
26. Fig. 26: Relative RNA expression intensity in a given tissue
for myosin light chain... 74
27. Figures 27-36, correlation scatter plots……… 76-80
28 Fig. 37: Location of LIT1 within KvLQT1... 93
107