(Received for publication, May 15, 1995; and in revised form, August 2, 1995)
From the
To test the influence of cytosine methylation on homologous recombination and the rejoining of DNA double strand breaks in mammalian cells, we developed a sensitive and quantitative assay system using extrachromosomal substrates. First, methylation was introduced into substrates in vitro with the prokaryotic SssI methylase, which specifically methylates the C-5 position of cytosine bases within CpG dinucleotides, mimicking the mammalian DNA methyltransferase. Next, methylated substrates were incubated in mammalian cells for a sufficient length of time to recombine or rejoin prior to substrate recovery. Results from bacterial transformation of the substrates and from direct Southern analysis demonstrate that cytosine methylation has no detectable effect on either DNA end-joining or homologous recombination. Thus, the components of the protein machinery involved in these complex processes are unaffected by the major DNA modification in mammalian cells. These results leave open the possibility that methylation may modulate the accessibility of these components to chromosomal DNA by altering local chromatin structure.
Two key processes in the maintenance of genomic integrity in mammalian cells are DNA end-joining, a nonhomologous process in which DNA breaks are rejoined, and homologous recombination. Both of these processes are mobilized in the repair of double strand breaks in chromosomal DNA (1) and in transfected extrachromosomal DNA (reviewed in (2) and (3) ). Homologous recombination of transfected DNA substrates is postulated to occur primarily by the nonconservative single strand annealing pathway(4, 5) . In this pathway, the two substrates contain double strand breaks at or near their homologous regions on which an exonuclease (or helicase) acts to produce single strands. The homologous single strands anneal and subsequent processing steps result in the completion of the recombination event.
DNA end-joining has also been extensively studied by transfection of substrates into mammalian cells(2) . Surprisingly, many different combinations of DNA ends can be rejoined efficiently in vivo. In addition to the precise ligation of compatible ends, noncompatible ends are also rejoined. For example, blunt-ended DNA ends can be rejoined to either 3` or 5` overhangs. The rejoining of the noncompatible ends frequently occurs within very short homologies near the DNA ends(2) . Mechanistically, this type of end-joining may be similar to the single strand annealing pathway of homologous recombination, the key distinction being the length of the homology. Although the protein machinery involved in either recombination or DNA end-joining is not well characterized, a number of components, including exo- and endonucleases, DNA polymerase, ligase, and strand annealing proteins, can be expected to participate.
The major base modification of mammalian DNA, cytosine methylation, occurs on the C-5 position of cytosines within the context of CpG dinucleotides (reviewed in (6) ). Cytosine methylation has several demonstrated biophysical consequences on DNA and, thus, it may modulate the enzymatic activity of some of the components involved in recombination or end-joining. For example, methylation increases the melting temperature of naked DNA(7) . It also has other effects, such as influencing the extrusion of DNA cruciforms(8) , as well as the transition of DNA from the B form to Z form(9) . However, it appears to have almost no influence on the intrinsic flexibility of DNA(10) .
Functionally, methylation has a critical role in transcriptional regulation, and it has been implicated in both the establishment and maintenance of X chromosome inactivation and genomic imprinting patterns(6) . The consequences of cytosine methylation in gene regulation may be the result of directly or indirectly altering the binding or activity of transcription factors and chromatin proteins (11) . Although Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila have no detectable methylation, the importance of methylation in mammals has been underscored by gene knockout experiments in which it was found that mice defective in the DNA methyltransferase die during embryonic development(12) . Methylation may also play a role in modulating the timing of replication. For example, the inactive X chromosome is replicated later than the active X chromosome in female cells(13, 14) .
At present, it is unknown whether methylation affects homologous recombination in either mitotic or meiotic mammalian cells. However, it has been observed that recombination rates differ at identical chromosomal regions between males and females. Overall, females have a higher rate of recombination than males, although some chromosomal regions recombine more frequently in males than in females(15) . Methylation patterns also differ between male and female gametes, with spermatogenic cells having an overall higher level of methylation than oogenic cells(16, 17) . Thus, methylation could potentially directly or indirectly suppress recombination rates. Related to this, it has been demonstrated that methylation decreases the site-specific recombination of antigen receptor genes, at least in some contexts. In this report, we have begun to address whether methylation has a direct affect on either homologous recombination or DNA end-joining in mammalian cells.
Recombination
substrates were constructed based on the parental Mneo plasmid. Mneo
has an intact Tn5 neo gene and confers kanamycin resistance
(Kan) to bacteria (Fig. 1). The Tn5 neo gene expresses only in bacteria, so as to bypass any potential
transcriptional effects on recombination and end-joining in mammalian
cells. This plasmid and its derivatives do not contain a mammalian
replication origin. The two intermolecular recombination substrates,
M5neo and M3neo, contain 3`- and 5`-truncated Tn5 neo genes,
respectively, and have an overlap of 352 bp of homologous sequences
within the neo gene. Recombination within this homology
restores a functional neo gene. To stimulate recombination,
the plasmids are cleaved at the end of the homology region prior to
transfection. M5neo is cleaved with SphI (M5neo/S), and M3neo
is cleaved with PstI and AatII (M3neo/AP).
Restriction with each of these enzymes produces 4-base 3` overhangs,
although the sequence of the overhang differs for each.
Figure 1: Structure of the recombination substrates. The parental plasmid Mneo contains a functional Tn5neo gene, whereas the recombination substrates M5neo and M3neo contain 3`- and 5`-truncated neo genes, respectively. Base pair positions of relevant restriction sites are indicated for Mneo.
The
experimental design is shown in Fig. 2. M5neo/S and M3neo/AP are
methylated in vitro with a cytosine methylase and then
electroporated into mammalian cells. Cells are incubated for 4 h, after
which time plasmid DNA is recovered. We chose 4 h, since previous
results have shown that recovery of recombined DNA is optimal at this
time point. ()The recovered DNA is electroporated into E. coli, and colonies are selected on both ampicillin (Amp)
and kanamycin (Kan) plates. The design is such that kanamycin selection
should allow us to measure recombination, whereas ampicillin selection
should allow us to measure end-joining.
Figure 2: Experimental design to determine the effect of CpG methylation on extrachromosomal homologous recombination in mammalian cells. M5neo/S and M3neo/AP can recombine within the 352-bp neo gene homology region (black shading) to generate a functional neo gene. Recombination can also occur downstream of the neo gene, as indicated. The downstream homology (thick line) is within pUC19 sequences and is 487 bp. EP, electroporation.
Figure 3:
Agarose gel electrophoresis to demonstrate
the resistance of in vitro methylated DNA to HpaII
digestion. CpG, DNA methylated
with methylase SssI. AP, AatII/PstI; S, SphI; H, HindIII.
Prior to transfection of DNA into mammalian cells, we monitored the
transformation efficiency of the methylated plasmid DNAs in E.
coli. DNA was electroporated into E. coli strain DH12S,
which is defective in the restriction of methylcytosine containing DNA.
Transformation of the parental Mneo plasmid resulted in almost 2
10
colonies on either Amp or Kan plates, for an
overall transformation efficiency of approximately 4
10
colonies/µg (Table 1). The efficiency was the same
whether or not the DNA was methylated.
Transformation of M5neo/S,
either methylated or unmethylated, resulted in an approximately
500-fold reduction in the number of Amp transformants. This
is a result of powerful exonucleolytic activities present in bacterial
cells, which degrades incoming linear DNA. The resulting transformants
are likely due to a low level of repair of the broken DNA molecules by
the bacterial cells. No Kan
colonies are obtained with
M5neo/S, since the neo gene contains a 3` truncation.
The
other recombination substrate, M3neo/AP, transforms bacteria to
Amp to an even lower level than M5neo/S, possibly due to
the close proximity of the AatII site to the start of the amp gene. Thus, M3neo/AP plasmids which have recircularized
may have defective amp genes. Alternatively, differences in
the overhangs produced by restriction digestion may contribute to the
lower transformation efficiency of M3neo/AP. As with M5neo/S, M3neo/AP
does not give rise to Kan
colonies due to truncation of the neo gene.
M5neo/S and M3neo/AP were also mixed prior to
transformation into bacteria. Although Amp colonies were
obtained at approximately the same level as that seen for M5neo/S
alone, no Kan
colonies were obtained, indicating that
recombination between the two substrates is very inefficient during
bacterial transformation. These results indicate that the bacterial
transformation assay will be highly sensitive for the detection of
recombination products and that cytosine methylation will not affect
the outcome of the assay in bacteria.
Figure 4:
Southern blot analysis of DNA recovered
from transfected COS1 cells. DNA was methylated in vitro (indicated by CpG),
transfected into COS1 cells, and recovered after 4-h incubation. A
portion of the DNA was subjected to HpaII digestion, as
indicated. The Mneo plasmid was used as the
probe.
Retention of methylation was monitored by HpaII digestion. Substrates that were methylated in vitro retained their methylation, as evidenced by their resistance to HpaII (Fig. 4). By contrast, unmethylated substrates were completely digested by HpaII to smaller fragments.
No Kan colonies were obtained from
transfecting M5neo/S or M3neo/AP separately into COS1 cells. In
addition, premixing the M5neo/S and M3neo/AP which had been separately
transfected upon electroporation into bacteria did not result in any
Kan
colonies, such that Kan
was dependent upon
cotransfection of both of the DNAs into COS1 cells. These results
demonstrate that Kan
is a result of homologous
recombination between M5neo and M3neo in COS1 cells and, therefore,
that extrachromosomal recombination is not affected by CpG methylation.
The number of Amp colonies provides a measure of DNA
end-joining in COS1 cells. Transfection of unmethylated and methylated
M5neo/S results in 1.7
10
and 2
10
colonies, respectively. Transfection of unmethylated and
methylated M3neo/AP results in 5.1
10
and 4.3
10
colonies, respectively. These are roughly 2%
(M5neo/S) and 6% (M3neo/AP) the number of colonies obtained with Mneo.
These figures are substantially higher than what was seen with direct
bacterial transformation of the plasmids (Table 1). For M5neo it
is approximately 14-fold higher, whereas for M3neo it is more than
7000-fold higher. These results indicate that the COS1 cells are able
to rejoin the broken DNA ends more efficiently than the bacterial cells
and that this end-joining is unaffected by CpG methylation of the
plasmid DNA.
The DNA that was transfected into COS1 cells was first cleaved by the restriction endonucleases and then methylated. Since some of the CpG dinucleotides are located within or adjacent to the restriction sites, it is possible that these sites may remain unmethylated. To rule this out, we also performed experiments in which the methylation was performed prior to the restriction digestion. Nearly identical results were obtained in this experiment to those shown in Table 2(data not shown), proving that the methylation at the ends does not reduce end-joining efficiencies.
As mentioned
above, the difference between the M5neo/S and M3neo/AP plasmids in
end-joining efficiencies in bacteria is likely due to the close
proximity of the AatII site to the amp gene in M3neo.
Alternatively, the particular restriction enzyme cleavage for the two
plasmids may play a role. However, following COS1 cell transfection
there is little difference between the two plasmids in the generation
of Amp colonies. These results indicate that the joining of
ends differs mechanistically between mammalian cells and E.
coli.
Individual recombination and end-joining products were
analyzed by preparing plasmid DNA from bacterial colonies and
subjecting them to restriction analysis. For the recombination
products, plasmids were prepared from 30 Kan colonies
derived from COS1 cotransfection of M5neo/S and M3neo/AP for both the
methylated and unmethylated samples (Fig. 5). Similar results
were obtained. Most plasmids were identical to the positive Mneo
control (R), indicating that recombination had occurred within
both the neo gene and plasmid backbone sequences. A small
number of plasmids (RE), 1 or 2 out of 30, had undergone
recombination only within the neo gene. DNA end-joining had
occurred to join the molecules within the plasmid backbone.
Figure 5:
Structure of recombination and end-joining
products. DNA recovered from cotransfection of COS1 cells with M5neo/S
and M3neo/AP was electroporated into bacteria. Plasmid DNA was prepared
from either Kan or Kan
(Amp
)
colonies and its structure determined by restriction enzyme
digestion.
To
examine end-joining products, plasmids were prepared from 10
Kan/Amp
colonies from the cotransfection. One
class of end-joining products (5E) had precisely rejoined the
cohesive SphI ends of M5neo/S. This class consisted of three
plasmids derived from the unmethylated sample and one plasmid from the
methylated sample. The more numerous class of end-joining products (3E2) was derived from DNA end-joining of the AatII/PstI plasmid backbone fragment of M3neo/AP.
This class consisted of seven plasmids from the unmethylated sample and
nine plasmids from the methylated sample. Based on restriction
analysis, most of the plasmids in this class contained only a limited
modification of the AatII/PstI ends. AatII
has a 3` ACGT overhang and PstI has a 3` TGCA overhang. The
rejoining of these two heterologous ends is not unexpected, given the
promiscuity of end-joining in mammalian cells (2) .
Figure 6: Southern blot analysis of DNA recovered from transfected COS1 cells. A, structure of recombination and end-joining products, as described in the text. Both intermolecular (R, RE, ER, EE, and E) and intramolecular (3E1, 3E2, and 5E) products are shown. Only intermolecular products which have recircularized are diagrammed. B, Southern analysis. DNA was cleaved with HindIII/BamHI prior to gel electrophoresis. The positions of these sites are indicated in Fig. 1. T lanes: DNA recovered from transfected COS1 cells; C lanes: DNA prior to transfection into COS1 cells. The HindIII/BamHI neo fragment was used as the probe.
For Southern analysis, DNA is cleaved with HindIII and BamHI (Fig. 1) and probed with a neo gene fragment (Fig. 6B). No recombination or end-joining products are detected without prior transfection of DNA into mammalian cells (Fig. 6B, ``C'' lanes). However, a variety of products are detected upon transfection of either the methylated or unmethylated DNAs into COS1 cells (Fig. 6B, ``T'' lanes). Cotransfection of M5neo/S and M3neo/AP results in formation of both recombination and end-joining products. Recombination occurred within the neo gene (R and RE products) for about 5-10% of the input substrates and is similar in both the methylated and unmethylated samples. The Southern assay gives an apparently higher level of recombination than does the bacterial transformation assay. This difference is due to the nature of the two assays. Southern analysis directly examines the product of recombination within the neo gene. Bacterial transformation to kanamycin resistance requires a second event (recombination or end-joining) within the plasmid sequences, in addition to recombination within the neo gene, so as to generate a closed circular product. This requirement for a second event lowers the measured level of recombination in the bacterial transformation assay.
As with the recombination product, end-joining products are also detected at similar levels in both the methylated and unmethylated samples. The intermolecular products (ER/EE and E) are only detected upon cotransfection of M5neo/S and M3neo/AP, whereas the intramolecular product 3E1 is detected in both the cotransfection and the transfection of M3neo/AP alone. The intramolecular product derived from M5neo/S is not separated from the input linearized M5neo/S in the HindIII/BamHI digest shown in Fig. 6B. However, this product is detected using other restriction digests (data not shown).
We demonstrate that mammalian cytosine methylation has little
or no effect on extrachromosomal recombination in tissue culture cells.
End-joining processes also are unaffected by CpG methylation. These
results were obtained using a highly sensitive bacterial transformation
assay and were confirmed by direct Southern analysis of DNA recovered
from transfected COS1 cells. These results are not unique to COS1
cells, since experiments performed in mouse embryonic stem cells have
yielded similar results. ()As with mammalian cells,
transfection studies of plant protoplasts have demonstrated that CpG
methylation does not affect extrachromosomal
recombination(23) .
Our substrates consist of bacterial plasmids carrying portions of the Tn5 neo gene. As with most bacterial genes, Tn5 neo is G/C-rich, rendering it a good model system in which to examine the effect of cytosine methylation. The CpG content is 9% of dinucleotides in the entire neo gene as well as 9% of dinucleotides in the 352 bp neo homology region. Even though the CpG content in mammalian genomic DNA is in general much lower (e.g. 1% of dinucleotides in human DNA), CpG islands have a higher CpG content. For example, 14% of dinucleotides in the 5` region of the human PGK1 gene are CpG(24) . Therefore, it is reasonable to use Tn5 neo as a model system to study the effect of cytosine methylation on homologous recombination in mammalian cells.
As with extrachromosomal recombination, no effect of cytosine methylation is detected on DNA end-joining frequencies or products. CpG dinucleotides are located at two of the ends, as well as further in from the ends (Fig. 7). For M3neo/AP, there is a CpG dinucleotide within the AatII overhang itself, as well as 15-bp upstream of the cleavage site. The PstI end of M3neo/AP also has a CpG dinucleotide located 15 bp upstream. For M5neo/S, there are two CpG dinucleotides directly adjacent to the SphI site on one end. Both ends also have other CpG dinucleotides located further upstream (Fig. 7). The major end-joining products detected by the bacterial transformation assay involves recircularization of the M3neo/AP and M5neo/S plasmids (3E2 and 5E). As shown in Table 2, they are not affected by methylation. Thus, methylation within an overhang or adjacent to an overhang does not interfere with end-joining. We cannot rule out, however, that there are subtle differences between the methylated and unmethylated end-joining products from the M3neo/AP transformation. However, the M5neo/S end-joining appears to be identical whether or not the substrate is methylated, since the SphI cleavage site is restored. Thus, end-joining appears unaffected by general methylation of the substrates or methylation directly at the ends of a broken DNA molecule. The biochemical details of end-joining have not been fully elucidated. However, proteins such as the Ku autoantigen and its associated DNA-activated protein kinase are likely involved, since this complex binds to DNA ends(25) . It may be that methylation does not affect binding of this complex or subsequent processing and ligation steps postulated to be involved in end-joining.
Figure 7: Sequences at the ends of the end-joining substrates. The methylated residues are indicated by an asterisk. For M3neo/AP, only the two ends that form in the intramolecular product 3E2 are shown.
Our results have
demonstrated that extrachromosomal homologous recombination is
unaffected by CpG methylation. Recombination in these substrates likely
occurs via the single strand annealing pathway(4, 5) .
This pathway has the requirement that both recombination substrates be
cut at or near the regions of homology(26) . The DNA ends may
then provide an entry site for an exonuclease such that single strands
are exposed for annealing. Mechanistically, our results suggest that
CpG methylation has little or no effect on the exonuclease and strand
annealing activities. A second pathway for homologous recombination,
double strand break repair (27) has also been shown to be
operational in mammalian cells(28, 29, 30) .
In this pathway, a DNA substrate containing a double strand break is
repaired from an unbroken homologous DNA template after strand
invasion, conserving both partners of the recombination event. We
cannot rule out that CpG methylation may have an effect on this
pathway. Experiments that we have performed in which only one of the
recombination substrates is broken and the other is introduced as a
supercoiled plasmid have shown no difference between methylated and
unmethylated substrates. However, considering the
nonconservative nature of most recombination events between transfected
DNAs(31) , these results are inconclusive regarding the double
strand break repair pathway.
Previous work has shown that CpG methylation and the accessibility of loci to site-specific recombinases are interrelated. A transgene locus that can undergo V(D)J recombination has been identified that is refractory to recombination when methylated yet accessible for recombination when unmethylated(32) . CpG methylated minichromosomes are also inaccessible for V(D)J recombination(33) . Since the inhibition of V(D)J recombination is apparent only after replication of the minichromosomes, the interpretation of these results is that CpG methylation does not directly affect recombination, but rather results in an altered chromatin structure upon replication which inhibits V(D)J recombination(33) . In addition, demethylation of endogenous T cell receptor and immunoglobulin gene loci has been shown to occur upon activation of lymphocytes which leads to T cell receptor recombination and Ig class switching, another site-specific recombination event(34) . Thus, although we find that methylation has no effect on the homologous recombination of extrachromosomal DNA, methylation may yet to be found to exert effects on chromosomal DNA by altering the accessibility of DNA to recombination machinery.