D. Mohamed Kamal Breast Cancer Research Team
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Lecture NO.5: Chromatin structure and Function IV (DNA Methylation)
Lecture No: 5 Chromatin structure and Function IV (DNA Methylation)
In this lecture we will discuss one of the most important regulatory processes of gene regulation; DNA methylation.
DNA Methylation
Although, DNA methylation does not relate to chromatin structure and function, it controls gene expression at the transcriptional level.
DNA methylation, not to be confused with histone methylation, is an archetypal epigenetic mark. It is borne by the genetic material but does not influence its sequence. It can regulate genomic activities, and can be maintained through mitosis and meiosis.
DNA methylation is essential in mammals: its loss leads to growth arrest or apoptosis in normal cells as well as in cancer lines. The presence of DNA methylation is absolutely required for embryonic development in mouse. The key role of DNA methylation is to control gene expression, and methylated sequences undergo transcriptional repression.
The DNA of mammals can be methylated on cytosines within the CpG dinucleotides (Fig. 1). The added methyl groups protrude in the major groove of DNA. When the DNA is symmetrically methylated, both methyls face the same direction and are close to one another. The addition of methyl groups changes the biophysical characteristics of the DNA and has two effects: it inhibits the recognition of DNA by some proteins and permits the binding of others (Vaissie` re, et al., 2008).
Fig 1: DNA methylation
DNA methyl-transferases (DNMTs)
The modification is brought about by enzymes called DNA methyltransferases (DNMTs). There are three such enzymes in mammals: DNMT1, DNMT3a, and DNMT3b. DNMT3L is structurally related, but is catalytically inactive and serves as a cofactor for DNMT3a and DNMT3b. The protein DNMT2 also has sequence similarity to these enzymes, but its function is quite different; it will not be discussed further here. Extensive enzymology studies have yielded important insight into the function of these enzymes. Notably, it was found that DNMT1 has preferential activity for hemi-methylated DNA over unmethylated DNA. It seems likely that, most of the time, DNMT3a and DNMT3b, aided by DNMT3L, set up the new imprints on previously naked DNA. For this they are called “de novo” methyltransferases. After DNA replication, methylated DNA becomes hemi-methylated, and DNMT1 would be the main player in making it fully methylated again. It is thus called the “maintenance” enzyme.
Table 1. The proteins involved in setting up and interpreting the methylation mark.
De novo Maintenance Cofactor
DNMT3a DNMT1 DNMT3L
DNMT3b
DNA methylation binding proteins
MBD Zinc finger SRA
MeCP2 Kaiso UHRF1
MBD1 ZBTB4 UHRF2
MBD2 ZBTB38
MBD4
Top panel: the enzymes that methylate DNA in mammals. Bottom panel: the three families of proteins that bind methylated DNA in mammals.
The methyl mark is translated into transcriptional repression by the action of proteins that recognize methylated DNA and inhibit gene expression by creating a repressive chromatin structure. Three families of proteins specifically recognize methylated DNA (Table 1). The first family contains MBD1, MBD2, MBD4, and MeCP2; these proteins share a related DNA binding domain called Methyl-binding Domain (MBD). The second family contains the Zinc-finger proteins Kaiso, ZBTB4, and ZBTB38 . These proteins are bifunctional: they bind methylated DNA, but also some non-methylated consensus sequences. Finally, the third family comprises UHRF1 and UHRF2 (also known as ICBP90 and NIRF), which bind methylated DNA through their SET-and-RING-Finger-Associated (SRA) domain.
An important question, discussed at length in an excellent recent review is that of the redundancy between methyl-binding proteins. Their degree of sequence specificity is poorly characterized, and it is unclear whether they can all bind the same target loci, or whether they have distinct targets. Even if the proteins do share some targets, they could be functionally different for other reasons. For instance, they could have different DNA-binding affinities. Also, the different proteins could be expressed at different times or places. Finally they could have different protein or nucleotide interactors that could possibly recruit them to different compartments of the nucleus (Vaissie` re, et al., 2008).
Targets of DNA methylation differ in normal and cancer cells
In normal cells, three main types of targets are repressed by DNA methylation. First: parentally imprinted genes, i.e. genes that are expressed differentially from the maternal and the paternal chromosome. They are key regulators of embryonic development and adult life. In most cases the inactive allele is marked by DNA methylation, and monoallelic expression is lost in the absence of methylation. As an aside, recent data indicates that many genes may be expressed monoallelicaly in somatic cells, but it is yet unclear if this depends at all on DNA methylation. Second: the transposons and other repeated sequences that constitute a large fraction of the mammalian genome. Third: a number of genes are methylated in a tissue-specific manner. An interesting subset of those is the Cancer/Testis (C/T) antigens, which are unmethylated and expressed in the testis, and methylated and repressed in all other tissues.
DNA methylation is deregulated in cancer. Tumor cells often have an abnormal pattern of DNA methylation where some tumor suppressor genes are methylated and inactive. Conversely, some normally methylated sequences, such as repeated DNA, imprinted genes, and C/T antigens, can become demethylated. Abnormal DNA methylation is an early causal event during cellular transformation. Demethylating agents can re-establish the expression of silenced tumor suppressor genes and have been approved for clinical use against some leukemias.
In this lecture we will discuss one of the most important regulatory processes of gene regulation; DNA methylation.
DNA Methylation
Although, DNA methylation does not relate to chromatin structure and function, it controls gene expression at the transcriptional level.
DNA methylation, not to be confused with histone methylation, is an archetypal epigenetic mark. It is borne by the genetic material but does not influence its sequence. It can regulate genomic activities, and can be maintained through mitosis and meiosis.
DNA methylation is essential in mammals: its loss leads to growth arrest or apoptosis in normal cells as well as in cancer lines. The presence of DNA methylation is absolutely required for embryonic development in mouse. The key role of DNA methylation is to control gene expression, and methylated sequences undergo transcriptional repression.
The DNA of mammals can be methylated on cytosines within the CpG dinucleotides (Fig. 1). The added methyl groups protrude in the major groove of DNA. When the DNA is symmetrically methylated, both methyls face the same direction and are close to one another. The addition of methyl groups changes the biophysical characteristics of the DNA and has two effects: it inhibits the recognition of DNA by some proteins and permits the binding of others (Vaissie` re, et al., 2008).
Fig 1: DNA methylation
DNA methyl-transferases (DNMTs)
The modification is brought about by enzymes called DNA methyltransferases (DNMTs). There are three such enzymes in mammals: DNMT1, DNMT3a, and DNMT3b. DNMT3L is structurally related, but is catalytically inactive and serves as a cofactor for DNMT3a and DNMT3b. The protein DNMT2 also has sequence similarity to these enzymes, but its function is quite different; it will not be discussed further here. Extensive enzymology studies have yielded important insight into the function of these enzymes. Notably, it was found that DNMT1 has preferential activity for hemi-methylated DNA over unmethylated DNA. It seems likely that, most of the time, DNMT3a and DNMT3b, aided by DNMT3L, set up the new imprints on previously naked DNA. For this they are called “de novo” methyltransferases. After DNA replication, methylated DNA becomes hemi-methylated, and DNMT1 would be the main player in making it fully methylated again. It is thus called the “maintenance” enzyme.
Table 1. The proteins involved in setting up and interpreting the methylation mark.
De novo Maintenance Cofactor
DNMT3a DNMT1 DNMT3L
DNMT3b
DNA methylation binding proteins
MBD Zinc finger SRA
MeCP2 Kaiso UHRF1
MBD1 ZBTB4 UHRF2
MBD2 ZBTB38
MBD4
Top panel: the enzymes that methylate DNA in mammals. Bottom panel: the three families of proteins that bind methylated DNA in mammals.
The methyl mark is translated into transcriptional repression by the action of proteins that recognize methylated DNA and inhibit gene expression by creating a repressive chromatin structure. Three families of proteins specifically recognize methylated DNA (Table 1). The first family contains MBD1, MBD2, MBD4, and MeCP2; these proteins share a related DNA binding domain called Methyl-binding Domain (MBD). The second family contains the Zinc-finger proteins Kaiso, ZBTB4, and ZBTB38 . These proteins are bifunctional: they bind methylated DNA, but also some non-methylated consensus sequences. Finally, the third family comprises UHRF1 and UHRF2 (also known as ICBP90 and NIRF), which bind methylated DNA through their SET-and-RING-Finger-Associated (SRA) domain.
An important question, discussed at length in an excellent recent review is that of the redundancy between methyl-binding proteins. Their degree of sequence specificity is poorly characterized, and it is unclear whether they can all bind the same target loci, or whether they have distinct targets. Even if the proteins do share some targets, they could be functionally different for other reasons. For instance, they could have different DNA-binding affinities. Also, the different proteins could be expressed at different times or places. Finally they could have different protein or nucleotide interactors that could possibly recruit them to different compartments of the nucleus (Vaissie` re, et al., 2008).
Targets of DNA methylation differ in normal and cancer cells
In normal cells, three main types of targets are repressed by DNA methylation. First: parentally imprinted genes, i.e. genes that are expressed differentially from the maternal and the paternal chromosome. They are key regulators of embryonic development and adult life. In most cases the inactive allele is marked by DNA methylation, and monoallelic expression is lost in the absence of methylation. As an aside, recent data indicates that many genes may be expressed monoallelicaly in somatic cells, but it is yet unclear if this depends at all on DNA methylation. Second: the transposons and other repeated sequences that constitute a large fraction of the mammalian genome. Third: a number of genes are methylated in a tissue-specific manner. An interesting subset of those is the Cancer/Testis (C/T) antigens, which are unmethylated and expressed in the testis, and methylated and repressed in all other tissues.
DNA methylation is deregulated in cancer. Tumor cells often have an abnormal pattern of DNA methylation where some tumor suppressor genes are methylated and inactive. Conversely, some normally methylated sequences, such as repeated DNA, imprinted genes, and C/T antigens, can become demethylated. Abnormal DNA methylation is an early causal event during cellular transformation. Demethylating agents can re-establish the expression of silenced tumor suppressor genes and have been approved for clinical use against some leukemias.