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. 2010 Jul 2;329(5987):78-82.
doi: 10.1126/science.1187945.

Genome-wide reprogramming in the mouse germ line entails the base excision repair pathway

Petra Hajkova #  1   2 , Sean J Jeffries #  1 , Caroline Lee  1 , Nigel Miller  3 , Stephen P Jackson  1 , M Azim Surani  1
Affiliations

Genome-wide reprogramming in the mouse germ line entails the base excision repair pathway

Petra Hajkova et al. Science. .

Abstract

Genome-wide active DNA demethylation in primordial germ cells (PGCs), which reprograms the epigenome for totipotency, is linked to changes in nuclear architecture, loss of histone modifications, and widespread histone replacement. Here, we show that DNA demethylation in the mouse PGCs is mechanistically linked to the appearance of single-stranded DNA (ssDNA) breaks and the activation of the base excision repair (BER) pathway, as is the case in the zygote where the paternal pronucleus undergoes active DNA demethylation shortly after fertilization. Whereas BER might be triggered by deamination of a methylcytosine (5mC), cumulative evidence indicates other mechanisms in germ cells. We demonstrate that DNA repair through BER represents a core component of genome-wide DNA demethylation in vivo and provides a mechanistic link to the extensive chromatin remodeling in developing PGCs.

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Figures

Figure 1
Figure 1. Activation of BER during DNA demethylation in gonadal PGCs
a. qPCR analysis of PGC and neighbouring somatic cells at E11.5; note high expression of BER but not NER factors specifically in PGCs b. Chromatin bound XRCC1 detected by immunofluorescence in gonadal PGCs (arrowheads) at E11.5 at the time of DNA demethylation.. Single cell suspensions of embryonic genital ridges were pre-extracted with detergent prior to fixation (see Materials and Methods). c. Kinetics of BER activation with respect to chromatin changes in PGCs compared with neighbouring somatic cells at E11.25-E11.5 (Samples were prepared as described: see Supplementary Fig.1c). Immunofluorescence analysis of single cell suspension (upper panel), or after pre-extraction to detect chromatin bound XRCC1 (bottom panel). Note a transient subpopulation of H1 positive PGCs, which also display PAR and bound XRCC1 signals. There is than a progressive loss of H1 during between E11.25-E11.5, while the XRCC1 and of PAR signals persist in PGC population lacking H1. The kinetics of the process is depicted at the bottom with respect to the loss of 5mC. (Scale bar 10μM).
Figure 2
Figure 2. Chromatin bound XRCC1 in zygotes depicting ssDNA breaks in male pronucleus
a. Detection of total XRCC1 in male and female pronuclei. b. Chromatin bound XRCC1 specifically in the male pronucleus visible from PN3. ♀ – maternal pronucleus, ♂ – paternal pronucleus, pb – polar body. (Scale bar 10μM).
Figure 3
Figure 3. Inhibition of BER and the consequences for DNA demethylation in zygotes
a,c Small molecule PARP inhibitor 3AB and, APE1 inhibitor (CRT0044876) impede progression of DNA demethylation as detected by 5mC staining. b,d. Quantification of 5mC staining. The values represent a ratio between 5mC signal from paternal pronuclei relative to the signal from maternal pronuclei. Statistical analysis was by Student’s t-test. e. Bisulphite analysis of Line1 repetitive elements. The values show the percentage of methylated CpGs. Each line represents a unique DNA clone. Filled and open circles represent methylated and unmethylated CpGs, respectively. Scale bar 10μM. ♀ – maternal pronucleus, ♂ – paternal pronucleus, pb – polar body.
Figure 4
Figure 4. Molecular pathways for DNA demethylation and chromatin remodelling in mouse germ cells
The appearance of ssDNA breaks involves the BER pathway components (APE1, XRCC1 and PARP1) in the process of DNA demethylation. Several molecular pathways can lead to the activation of BER in the course of DNA demethylation.. 5mC could be subject to a chemical modification such as cytosine deamination although we did not detect expression of known candidate enzyme. Alternatively, 5mC may be converted to 5hmC by tet1 or other such enzymes that are expressed in PGCs, which would trigger a response from glycosylases. Alternatively, 5mC specific glycosylases, which have been identified in plants may have equivalents in mammals but none have yet been found. Note the temporal connections between DNA demethylation, appearance of ssDNA breaks and the activation of BER and the chromatin remodelling observed in PGCs.
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References

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