O6–methylgunine-DNA methyltransferace (MGMT) is a DNA binding protein that is involved in repairing mutations. MGMT gene - a tumor suppressor gene that codes MGMT (O6-methylguanine DNA methyltransferase) protein. The MGMT protein removes mutagenic methyl groups from guanines through the methyltransferase activity.

1. Epigenetic silencing of the
MGMT gene in cancer

2. Arman Mahmud Khan
3rd year, 1st term.
Biotechnology & Genetic Engineering Discipline,
Khulna University.

3. Contents
• Gene Silencing
• Epigenetic gene silencing
• MGMT gene
• MGMT gene product
• Epigenetic gene silencing mechanisms
• Clinical implications of MGMT silencing

4. What is Gene Silencing?
• Gene silencing is a technique that aims to reduce or eliminate the
production of a protein from it’s corresponding gene.
• It generally describe the “switching off” of a gene by a mechanism other
than genetic modification.
• That is, a gene which would be expressed (“turned on”) under normal
circumstances is switched off by machinery in the cell.
• It occurs when RNA is unable to make a protein during translation.
• Gene silencing is same as gene knock down but is totally different from
gene knock out.
So, we can say-
Gene silencing is the regulation of gene expression in a cell to prevent the
expression of a certain gene.

5. Epigenetic gene silencing
Epigenetic gene silencing indicates heritable changes in gene expression that
do not involve changes in the DNA sequence.
(Silenced)
This type of gene silencing aims to nonmutational gene inactivation that can
be faithfully propagated from precursor cells to clones of daughter cells. The
addition of methyl groups to cytosine residues in CpG dinucleotides in DNA
(a biochemical modification) that meets this requirement.
…A T T G G G T…
…T A A C C C A…
…A T T G G G T…
…T A A T C C A…
methylation

7. Epigenetic regulation of gene expression plays a critical role in development
of tumors as well as cancers.
The genetic aberrations(mutations, deletions, or genomic rearrangements)
cause tumor suppressor genes to lose their function. Therefore, the tumor
suppressor protein can’t act appropriately to its specific site of action.
However, a number of those suppressor genes are known to be inactivated by
an epigenetic mechanism, such as promoter hypermethylation, in human
cancer.
Epigenetic gene silencing associated with aberrant promoter
hypermethylation is an alternative mechanism to genetic aberration to
inactivate tumor suppressor genes, but it can have an effect similar to genetic
aberration, leading to a loss of gene function.

8. MGMT gene
MGMT gene - a tumor suppressor gene that codes MGMT (O6-
methylguanine DNA methyltransferase) protein.
This gene product (MGMT protein) is one of the critical key elements to
DNA repair that removes methyl group from mutagenic O6-
methylguanine.

9. MGMT gene
• MGMT is a large gene spanning approximately 300 kb, including 5 exons
at chromosomal region 10q26.
• Transcribed mRNA from this gene is 866 bp long, encoding 207 amino
acids.
• The MGMT gene has a GC-rich promoter containing a CpG island, a short
stretch of GC-rich sequences associated with promoter region. The CpG
island in the MGMT promoter region is approximately 780 bp long,
including 97 CpG dinucleotides.

10. MGMT gene
• The promoter with maximal activity lies approximately 1 kb upstream
of the transcription initiation site to 200 bp downstream of it.
• It consists of minimal promoter, enhancer, glucocorticoid responsive
elements, and several transcription factor-binding sites, such as SP1,
AP1, and AP2.

12. MGMT gene product
O6–methylgunine-DNA methyltransferace (MGMT) is a DNA binding
protein that is involved in repairing mutations.
The MGMT protein removes mutagenic methyl groups from guanines
through the methyltransferase activity.
…………A T C G G C C G T……….
…………T A G C T G G C A………
m
MGMT
…………A T C G G C C G T……….
…………T A G C C G G C A………
( methyltransferase activity)
methyl group is removed from the
O6-mehylguanine

13. • MGMT adds a methyl group onto a specific cysteine
residue, Cys146 , by breaking an ether bond connecting the
methyl group of O6 m-guanine within the DNA chain.
• MGMT binds the methyl to the sulfur molecule in its cystein
residue located within the C-terminal end.
• Performing a nucleophilic attack by the thiolate, thereby
breaking the O6 guanine’s ether bond to the DNA and binding it
to the thiolate.
How MGMT Works?

14. MGMT
Cys
G
m
MGMT
Cys
m
G
..A..A..A..A…
CH3
..A..A..A..A…
Cysteine
O6- methylguanine

15. MGMT gene product
• Loss of MGMT function causes mutations leading to tumor induction.
• MGMT deficient cells are hypersensitive to alkylating agents, leading
to tumorigenesis.
• Aberrant hypermethylation of the MGMT promoter region is
correlated with lack of mRNA and (or) protein, as well as with loss of
its function in various human primary tumors.
• An experiment in which MGMT transgenic mice were treated with
alkylating agent revealed that O6-mG was crucial for tumor initiation
and that MGMT prevents initiation.

16. Epigenetic gene silencing mechanisms in general
• In recent studies there have been focused on two molecular mechanism for gene
silencing
• 1) DNA methylation
• 2) Histone modification
• DNA methylation:
a. Methylation of cytosine in CpG dinucleotides,
• localized mainly in the CpG islands,
• is the major modification in mammals.
• b. The CpG islands are normally unmethylated,
• but some of them are aberrantly hyper methylated
• in cancer cells, leading to gene silencing.

17. c. There are three biologically active DNA methyltransferases (DNMTs) present in mammals.
They are DNMT1, DNMT3a, and DNMT3b. DNMT1 works mainly on maintenance of
methylation, whereas DNMT3a and DNMT3b work on de novo methylation.

18. Histone modification:
Histone: Any group of basic proteins found in chromation.
Histone modification is related with chromatin because of the presence of histone in
chromation that is associated with epigenetic gene silencing.
i. The DNA of all eukaryotes is packed into chromatin consisting of a repetitive fundamental
unit, the nucleosome, which contains a highly conserved histone octamer wrapped twice with
147 bp of DNA.
ii. Chromatin structures are divided into 2 types of domain,
1. Heterochromatin and
2. Euchromatin.

19. iii. Heterochromatin is transcriptionally silenced, showing tightly compacted, inaccessibility
DNA-binding factors, and late replication. In contrast, euchromatin is transcriptionally
active, accessibility of transcriptional factors, and early replication .
iv. The histone amino termini are susceptible to a variety of covalent modifications, such as
methylation, acetylation, phosphorylation, ubiquitylation, and ADP ribosylation.
v. Among them methylation and acetylation of lysine residues, which are modified by
histone methyltransferase (HMT) and histone acetyltransferase (HAT), respectively ,are
most prominent.
vi. As to methylation at the lysine residue, 3 patterns based on the number of added
methyl groups are known: monomethylation, dimethylation, and trimethylation.

21. vii. The silencing process occurring through DNA methylation was initially thought to be due
simply to physical interference between transcription factors and their binding sequences.
viii. However, it seems that DNA methylation does not by itself lead to gene silencing.
Recruitment of proteins to methylated DNA is required for the formation of
heterochromatin to silence genes.
ix. Binding of methyl-CpG binding proteins (MBDs) to methylated CpG nucleotides recruits a
co repressor complex containing histone deacetylases (HDACs), resulting in highly
deacetylated nucleosomes. MBDs themselves also function as transcriptional repressors in
vitro.
x. MeCP2 (MBD protein) is associated with methyltransferase activity on H3K9 in vivo,
though the identity of the H3K9 methyltransferase is unknown.
xi. Histone methyltransferase SUV39H1 and its interacting partner HP1β associate with
DNMTs.
xii. Recently, a nuclear amine oxidase LSD1 has been reported as a demethylase,may be
involved in heterochromatin formation leading to gene silencing.

23. Epigenetic mechanism of MGMT silencing
• As mentioned before, MGMT is silenced in various cancers and cancer cell lines, and it is
believed that the silencing is associated with DNA hypermethylation at a discrete region
of the promoter CpG island.
• In more than 20 cancer cell lines in which MGMT is transcriptionally silenced, the precise
methylation status of each CpG in the island was reported.
• The reports showed that the island was actually methylated and was divided into 2
hypermethylated regions.
• However, among the cell lines, DNA methylation status varied: some showed heavily and
entirely methylated CpG islands, but others showed hypomethylation, regardless of gene
silencing.

24. Is there any common silencing factor other than DNA methylation?
• Analyses of histone modification in several cancer cell lines, in which DNA
methylation at the promoter CpG island varied from hypermethylation to
hypomethylation, showed that dimethylation of H3K9 was always associated
with MGMT silencing .
• In contrast, histones were highly acetylated on H3 and H4 and dimethylated
on H3K4 were seen in some MGMT-expressing cells.
• Interestingly, neither mono nor trimethylation of H3K9 was associated with
the silencing, that is, apparent mono and trimethylation of H3K9 in MGMT-
expressing cells.
• The evidence suggests that histone deacetylation and demethylation of
H3K4 are not critical for the silencing of MGMT.

25. • Furthermore, among MBD proteins such as MeCP2, MBD1, MBD2, and MBD3, only
MeCP2 binding to the CpG island was associated with MGMT silencing in cell lines .
• These findings suggest that H3K9 dimethylation and MeCP2 binding of the CpG island
are common and are more important for MGMT silencing in these cells than DNA
hypermethylation, histone deacetylation, or demethylation of H3K4.

27. Clinical implications of MGMT silencing
i. Levels of MGMT protein and mRNA are lower in human tumors than in their normal tissue
counterparts.
ii. Although H3K9 dimethylation and MBD binding is critical for the silencing, DNA methylation at
the promoter CpG island would occur earlier.
iii. Thus, DNA methylation is thought to be a good marker for the silencing.
iv. The methylated DNA fragment of MGMT has been investigated in biological fluid. Aberrant
methylation of MGMT was detected in serum, sputum, and broncho alveolar lavage from lung
cancer patients.
v. Methylation analysis, combined with other tumor suppressor genes in body fluid, increased the
detection rate and seems to be a useful epigenetic tumor marker for early diagnosis or
detection of groups at high risk of cancer or for monitoring disease recurrence.
vi. MGMT silencing is a poor prognostic factor for several cancers, such as hepatocellular, biliary
tract, gastric, and breast cancers .
vii. MGMT silencing is a good predictive marker for chemotherapy of alkylating agents.
viii. MGMT silencing is a poor prognostic factor, but it is also a good predictive marker for
chemotherapy using alkylating agents. MGMT is an important molecule in both basic and
clinical fields of cancer research and should be studied further to extend our understanding of
cancer biology.

28. THANKS TO ALL

29. Reference
• https://www.nrcresearchpress.com/doi/abs/10.1139/o05-
140#.XKcSB5gzZPY