6.3.5 Regulation of Gene Expression by DNA Methylation
DNA methylation ( 22 CH 3 groups attached to DNA of the promoter or coding region) is a
major factor in regulating gene expression. There appears to be an inverse relationship
between percent methylation and the degree of expression.Hypomethylation is associated
with higher gene expression, whereashypermethylation is associated with greater gene
silencing. The most common methylated base in eukaryotic genomic DNA is 5-methyl-
cytosine (m^5 C).
Plants generally have higher levels of DNA methylation than do mammals. Also, in
plants methylation occurs mainly in transposable elements and other repeat sequences. If
a transposon is methylated, it will be inactive and not hop around the genome, but it can
be activated if the methylation is removed. However, as in mammals, methylation of the
cytosine on both strands of the CpG dinucleotide (linear sequence of cytosine followed
by a guanine separated by a phosphate, to be distinguished from a cytosine base-paired
to a guanine) is common in plants and is carried out by DNA methyltransferases such as
MET1 inArabidopsis. This enzyme is responsible for maintenance of global genomic
methylation. Plants mutant for MET1 have significantly lower levels of methylation and
show late flowering phenotypes (Kankel et al. 2003). Also, transgenes that are genetically
engineered into plants and become highly methylated are not expressed. However, if these
plants have a defective MET1, these transgenes will no longer be silenced. Plants also have
methylation of CpNpG trinucleotides (“N” can be any of the four DNA bases) and asym-
metric CpNpN trinucleotide sites that are performed by specific enzymes that are unique to
plants such aschromomethylases(CMT) anddomain-rearranged methylases(DRM). The
CMTs appear to be involved in maintaining methylation of sites that are heavily methylated
to keep them silenced. The DRMs function in RNA-directed DNA methylation by
somehow recognizingsmall interference RNA(siRNA—these RNAs are usually 20–25
nucleotides long and inhibit expression of specific genes) and then methylating the appro-
priate DNA sequences. Additionally, it has been shown that chromatin-remodeling factors,
as described above, can be necessary for maintaining methylation.
6.3.6 Processing to Produce Mature mRNA
Controlling transcription is one of the most important ways to alter gene expression for
biotechnology applications. Many of the mechanisms that plants possess to regulate the
transcription of DNA to mRNA have been introduced above. Promoters, transcription
factors, chromatin remodeling, and DNA methylation are all crucial for transcriptional
control. However, transcription is only the first step in gene regulation. The mRNA that
is made through transcription is not mature and is termed apre-mRNAor aheterogeneous
nuclear RNA(hnRNA). Before a gene transcript is transported out of the nucleus and into
the cytoplasm where it will ultimately be translated into protein, it must be processed in
several ways: 50 capping, 3^0 polyadenylation(polyA tail), andsplicingout ofintrons
and putting together ofexons(Fig. 6.10). The first processing step, occurring when approxi-
mately 20–30 ribonucleotides of the hnRNA have been made, is the addition of a 7-methyl-
guanosine to the 5^0 end of the transcript. This cap structure may play a role in mRNA
stability by physically protecting the mRNA from 5^0! 30 exonucleases, types of
RNAses, once it is in the cytoplasm. Most eukaryotic gene protein-coding regions are
interrupted by non-protein-coding sequences (introns) that are removed from the hnRNA,
so they are not found in mature mRNA. The hnRNA has consensus sequences at the
146 MOLECULAR GENETICS OF GENE EXPRESSION