Information

How do CpG islands remain unmethylated?

How do CpG islands remain unmethylated?


We are searching data for your request:

Forums and discussions:
Manuals and reference books:
Data from registers:
Wait the end of the search in all databases.
Upon completion, a link will appear to access the found materials.

In most of the genome CpG sites are pretty much always methylated, but CpG islands are instead often unmethylated. This has been linked to the fact that they often are associated to transcripted genes.

What are the current theories on the mechanisms involved in this preferential demethylation?


Methylation is increasingly seen as a consequence of gene activity rather than a regulatory mechanism. There are cases where methylation is controlled because of gene regulatory control, especially at the famous H19/Igf2 locus[1]. Here is a generally good recent review[2], note they mention that DNA methylation does not cause transcriptional silencing, and likely methylated promoters are probably more active than unmethylated, they just create silencing RNAs when methylated (resulting in an apparent silencing). This may help explain some of the story[3], but note how old that paper is, yet generally I'd say few people know of its existance.

The exception seems to be transposable elements[4], but their control is probably also controlled by silencing RNAs.

References:

  1. Zampieri M, Guastafierro T, Calabrese R, Ciccarone F, Bacalini MG, Reale A, Perilli M, Passananti C, Caiafa P. 2012. ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites. The Biochemical journal 441: 645-52.

  2. Deaton AM, Bird A. 2011. CpG islands and the regulation of transcription. Genes & development 25: 1010-22.

  3. Rountree MR, Selker EU. 1997. DNA methylation inhibits elongation but not initiation of transcription in Neurospora crassa. Genes & Development 11: 2383-2395.

  4. Bourc'his D, Bestor TH. 2004. Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L. Nature 431: 96-9.


Human genes with CpG island promoters have a distinct transcription-associated chromatin organization

More than 50% of human genes initiate transcription from CpG dinucleotide-rich regions referred to as CpG islands. These genes show differences in their patterns of transcription initiation, and have been reported to have higher levels of some activation-associated chromatin modifications.

Results

Here we report that genes with CpG island promoters have a characteristic transcription-associated chromatin organization. This signature includes high levels of the transcription elongation-associated histone modifications H4K20me1, H2BK5me1 and H3K79me1/2/3 in the 5' end of the gene, depletion of the activation marks H2AK5ac, H3K14ac and H3K23ac immediately downstream of the transcription start site (TSS), and characteristic epigenetic asymmetries around the TSS. The chromosome organization factor CTCF may be bound upstream of RNA polymerase in most active CpG island promoters, and an unstable nucleosome at the TSS may be specifically marked by H4K20me3, the first example of such a modification. H3K36 monomethylation is only detected as enriched in the bodies of active genes that have CpG island promoters. Finally, as expression levels increase, peak modification levels of the histone methylations H3K9me1, H3K4me1, H3K4me2 and H3K27me1 shift further away from the TSS into the gene body.

Conclusions

These results suggest that active genes with CpG island promoters have a distinct step-like series of modified nucleosomes after the TSS. The identity, positioning, shape and relative ordering of transcription-associated histone modifications differ between genes with and without CpG island promoters. This supports a model where chromatin organization reflects not only transcription activity but also the type of promoter in which transcription initiates.


Abstract

DNA methylation is frequently described as a 'silencing' epigenetic mark, and indeed this function of 5-methylcytosine was originally proposed in the 1970s. Now, thanks to improved genome-scale mapping of methylation, we can evaluate DNA methylation in different genomic contexts: transcriptional start sites with or without CpG islands, in gene bodies, at regulatory elements and at repeat sequences. The emerging picture is that the function of DNA methylation seems to vary with context, and the relationship between DNA methylation and transcription is more nuanced than we realized at first. Improving our understanding of the functions of DNA methylation is necessary for interpreting changes in this mark that are observed in diseases such as cancer.


The TET proteins and the putative functions of 5hmC

The TET protein family members—TET1, TET2 and TET3—are 2-oxoglutarate and Fe(II)-dependent dioxygenases that all have the capacity to convert 5mC into 5hmC in vitro and in vivo (Fig 1A Ito et al, 2010 Ko et al, 2010 Tahiliani et al, 2009 ). In addition to the conserved iron-binding catalytic subunit, TET1 and TET3 also have a CXXC domain. This DNA-binding domain has previously been described as a CpG-binding motif, which could be involved in the recruitment of TET1 and TET3 to DNA. The TET proteins show tissue-specific differential expression with TET1 being mainly expressed in ESCs, whereas TET2 and TET3 are more ubiquitously expressed ( Szwagierczak et al, 2010 Tahiliani et al, 2009 ).

Interestingly, whereas levels of 5mC are relatively constant, 5hmC intensities vary significantly between tissues, with the highest levels reported for specific cell types of the brain ( Globisch et al, 2010 Kriaucionis & Heintz, 2009 ). ESCs also have relatively high levels of 5hmC, which decrease during differentiation ( Globisch et al, 2010 Koh et al, 2011 Szwagierczak et al, 2010 Tahiliani et al, 2009 ).

Several biological functions of TET-mediated conversion of 5mC into 5hmC can be envisioned (Fig 1B). The fact that de novo methyltransferase activity is low in differentiated tissues, taken together with the observation that 5hmC is found in relatively high levels in certain tissues, suggests that 5hmC in some tissues has a low turnover. In these situations, 5hmC might function by altering the local chromatin environment through the recruitment or displacement of proteins. Favouring this model, it has been reported that most 5mC-binding proteins do not recognize 5hmC ( Jin et al, 2010 Valinluck et al, 2004 ) and thereby presumably dissociate from DNA when 5mC is converted into 5hmC. However, it is also apparent that the generation of 5hmC could be involved in the demethylation of DNA. First, this could be through a passive mechanism in which the mark, in contrast to 5mC, would not be maintained through DNA replication. Passive DNA demethylation would be particularly relevant in rapidly dividing cells such as ESCs. The fact that brain cells, which are not rapidly dividing, have high levels of 5hmC, supports this model. Moreover, it has been shown that DNMT1 methylates 5hmC-containing DNA with a lower efficiency to 5mC hemi-methylated DNA ( Valinluck & Sowers, 2007 ).

Second, production of 5hmC could be an intermediate in an active demethylation pathway that ultimately replaces 5mC with cytosine in non-dividing cells. This has been proposed to involve specific DNA repair mechanisms such as deamination mediated by the AID/APOBEC family of cytidine deaminases, converting hmC into hmU followed by base excision repair (BER) or 5hmC glycosylation followed by BER (reviewed in Guo et al, 2011 ). In addition, the hypothesis that generation of 5hmC is an intermediate in an enzymatic pathway of active demethylation was recently supported by studies demonstrating the existence of formylcytosine and carboxylcytosine in mammalian DNA (Fig 1B He et al, 2011 Ito et al, 2011 Pfaffeneder et al, 2011 ). These new cytosine modifications can be generated by two successive oxidation reactions of 5hmC catalysed by the TET proteins ( He et al, 2011 Ito et al, 2011 ), raising the possibility that the TET proteins might be involved in several steps in converting 5mC to cytosine. As the TET proteins cannot convert carboxylcytosine to cytosine, a decarboxylase or a glycosylase might be involved in this step. In agreement with this, depletion of thymidine-DNA glycosylase (TDG) leads to accumulation of carboxylcytosine in mouse ESCs ( He et al, 2011 ), and other studies have shown that TDG is required for DNA demethylation ( Cortellino et al, 2011 ).


Results and Discussion

ZBTB2 binds at active CpG island promoters in mouse embryonic stem cells in vivo

To determine the genome-wide in vivo DNA binding sites of the putative unmethylated DNA reader ZBTB2, we performed chromatin immunoprecipitation followed by deep sequencing (ChIP-seq). To this end, we generated a mouse ESC line with a stably integrated bacterial artificial chromosome (BAC) transgene encoding the Zbtb2 gene fused to a C-terminal green fluorescent protein (GFP) tag under the control of its endogenous promoter 15 . Through ChIP-seq analysis with an antibody against GFP, we identified ± 4,000 ZBTB2-binding sites in ESCs. Genomic localisation of these peaks revealed that the majority of the ZBTB2-binding sites (81%) is located within 5 kb of a transcription start site (TSS) (Fig 1A). A more detailed analysis of the genomic distribution of these peaks confirmed the preferential binding of ZBTB2 to TSSs and, in addition, showed that most of these TSSs contain a CpG island (CGI) (Fig 1B). Motif enrichment analysis showed that a recently reported ZBTB2-binding motif 16 was not found to be enriched in our ZBTB2 peaks in ESCs compared to all CGIs (Fig EV1C), suggesting that in vitro and in vivo binding sites may differ. In addition, de novo motif searches in our ChIP-seq data set did not identify any clear consensus motif for binding of ZBTB2, but did indicate that one or multiple CpG dinucleotides are usually present. To substantiate this finding, we compared the number of CpG dinucleotides at ZBTB2-binding sites to a set of random promoters of the same average length and found that ZBTB2 peaks indeed contain significantly more CpGs (Fig EV1D). However, not all TSSs or even CGIs are bound by ZBTB2, suggesting that CpG dinucleotides are important but not sufficient for ZBTB2 recruitment (Figs 1C and EV1E). In order to identify other factors that determine ZBTB2 binding, we examined whether ZBTB2 peaks are enriched for particular histone marks. This analysis revealed that ZBTB2 primarily interacts with TSSs and CGIs that are marked by H3K4me3 and H3K27ac. Since these histone marks are associated with active promoters 17 18 (Figs 1C and EV1E), our data thus demonstrate that ZBTB2 preferentially binds to active CpG island promoters in ESCs in vivo.

Figure 1. Genomic localisation of ZBTB2 in mouse embryonic stem cells in vivo

  1. Histogram depicting the genomic localisation relative to a TSS of the called ZBTB2 ChIP-seq peaks in ESCs.
  2. Genomic distribution of the called ZBTB2 ChIP-seq peaks in ESCs.
  3. Heat map showing the ChIP-seq read density (normalised on RPKM) for ZBTB2, H3K4me3 43 and H3K27ac 44 in ESCs, centred on all mouse TSSs.

ZBTB2-binding dynamics anticorrelate with differential DNA methylation in vivo

Since most CGIs are hypomethylated 4 , our finding that ZBTB2 binding is enriched at CGIs in vivo is in line with ZBTB2 being a reader of unmethylated DNA. To examine whether ZBTB2 occupancy is influenced by DNA methylation in vivo, we altered global DNA methylation levels in two ways. Firstly, we induced global DNA hypomethylation in ZBTB2-GFP BAC ESCs by adding two small-molecule kinase inhibitors targeting MEK and GSK3β to the culture medium (2i) 19 . Secondly, we differentiated ZBTB2-GFP BAC ESCs into neural progenitor cells (NPCs), thereby changing the DNA methylation landscape in a cell-type-specific manner 20 . ChIP-seq analyses on these cell lines revealed that genome-wide ZBTB2 binding increases after addition of 2i and decreases upon differentiation into NPCs (Fig 2A). Importantly, ZBTB2 protein levels remain within the same range for all these conditions (Fig EV2A and B), even if mRNA levels seem to be more variable upon addition of 2i (Fig EV2C), indicating that these binding dynamics are not solely caused by changes in abundance. To investigate the extent to which the observed dynamics in ZBTB2 binding are related to changes in DNA methylation, we used publicly available methylation profiles from ESCs grown in serum/LIF or in complete 2i conditions 21 or ESCs and NPCs 20 as a proxy for genome-wide DNA methylation levels in our cell lines. We took the union of all ZBTB2 peaks that were called in either one of our ChIP-seq experiments, grouped them according to their genomic annotation, and then compared the difference in DNA methylation as predicted by the methylation profiles to the fold change in ZBTB2 binding as determined by ChIP-seq (Fig EV2D and E). These analyses suggest that on average, sites where the most dynamics in ZBTB2 binding occur also show the largest change in DNA methylation. To validate these findings, we selected a couple of loci containing a CGI and performed ChIP–qPCR and methylated DNA immunoprecipitation (MeDIP)–qPCR to measure ZBTB2 and DNA methylation levels, respectively (Figs 2B and C, and EV2F and G). Also in our cell lines, we observed an anticorrelation between ZBTB2 binding on the one hand and DNA methylation on the other, suggesting that ZBTB2-binding dynamics in vivo are sensitive to differential DNA methylation.

Figure 2. Comparison of ZBTB2 occupancy and differential DNA methylation in vivo

  • A. Heat map showing the ChIP-seq read density (normalised on RPKM) for ZBTB2 in ZBTB2-GFP BAC ESCs cultured in serum/LIF conditions (middle) or serum/LIF + 2i conditions (left), and in ZBTB2-GFP BAC NPCs (right), centred on the union of the ZBTB2 peaks in these three conditions.
  • B, C. Comparisons of ZBTB2 occupancy (assessed by ChIP–qPCR) and DNA methylation levels (measured by MeDIP–qPCR) at three loci (see Fig EV2F and G), between ZBTB2-GFP BAC ESCs in serum/LIF and serum/LIF + 2i conditions (B) or between ZBTB2-GFP BAC ESC and NPCs (C). Data are depicted as mean ± SEM of three biological replicates (P = as determined by Welch t-test).

Figure EV2. Comparison of ZBTB2 occupancy and differential DNA methylation (related to Fig 2 )

  • A, B. Western blot analyses of endogenous and GFP-tagged ZBTB2 levels in ZBTB2-GFP BAC ESCs in serum/LIF or serum/LIF + 2i conditions (A) or ZBTB2-GFP BAC ESCs or NPCs (B). Histone H3 was used as a loading control. NE = nuclear extract (nuclear soluble fraction), NP = nuclear pellet (chromatin-bound fraction).
  • C. qRT–PCR analysis of Zbtb2 levels in ZBTB2-GFP BAC ESCs cultured without or with 2i. Data are depicted as mean ± SEM of three biological replicates (*P = 0.02, Welch t-test).
  • D, E. Comparison of ZBTB2-binding dynamics and differential DNA methylation 2021 at the union of ZBTB2 peaks that were called in our three ChIP-seq experiments, presented as the average values for each genomic category. ZBTB2-binding dynamics (plotted on the x-axis) are calculated as the fold change in RPKM values obtained from ZBTB2-GFP ChIP-seq in +2i versus serum/LIF conditions (D), or in NPCs versus ESCs (E). Differential DNA methylation (plotted on the y-axis) is the difference in average methylation status between +2i and serum/LIF (D) or NPCs and ESCs (E).
  • F, G. UCSC genome browser views of genomic regions that were used for locus-specific analysis of ZBTB2 binding and DNA methylation (Fig 2B and C). All loci except the negative control locus contain a CGI. qPCR primers (Appendix Table S5) were designed to amplify a 100- to 200-bp region within the dashed lines. BS-seq = bisulphite sequencing 2145 .

Source data are available online for this figure.

ZBTB2 recruits a zinc finger module to unmethylated DNA

We next performed in vitro binding assays to further elucidate the mechanisms behind ZBTB2 binding to DNA. For this purpose, we selected a locus containing a CGI on the mouse genome where we saw a strong anticorrelation between ZBTB2 occupancy and DNA methylation in ESCs compared to NPCs, while DNase I hypersensitivity data suggest that this genomic region is accessible in both cell types in vivo (Figs 3A and EV3A). To test whether the DNA binding properties of ZBTB2 change upon differentiation into a different cell type, we performed DNA pull-downs with this genomic sequence containing either unmethylated or methylated CpGs, and using nuclear protein extracts from either ESCs or NPCs. These experiments revealed that in vitro, ZBTB2 is able to bind to the unmethylated sequence independent of the origin of the protein extract, suggesting that the inherent ability of ZBTB2 to bind to unmethylated DNA is unaltered in NPCs (Figs 3B and C and EV3B). We next investigated the possibility that ZBTB2 interacts with different proteins in ESCs and NPCs, which could also affect its recruitment to DNA. Therefore, we prepared nuclear protein extracts from ZBTB2-GFP BAC ESCs and NPCs and performed GFP pull-downs to identify ZBTB2 interaction partners. These experiments demonstrated that ZBTB2 interacts with two other zinc finger proteins, ZBTB25 and ZNF639, in both cell types (Fig 3D and E). We validated these protein–protein interactions in ESCs by reciprocal pull-downs and Western blot analysis (Fig EV3C–E). While the interaction partners of ZBTB2 thus do not change during differentiation, zinc finger proteins are known to have the capacity to bind to DNA 22 , giving rise to the possibility that recruitment of ZBTB2 to DNA is mediated by one or both of its interaction partners. We subsequently examined whether ZBTB2 would retain its DNA binding capability in the absence of ZBTB25 and ZNF639. For this purpose, we produced recombinant GST-fused ZBTB2 protein in Escherichia coli and used this for DNA pull-downs with generic unmethylated and methylated DNA probes 7 . Analysis by Western blot revealed that recombinant ZBTB2 binds to the unmethylated probe with high affinity, while almost no binding can be observed to the methylated probe (Fig 3F). A similar result was obtained when the genomic region indicated in Fig 3A was used (Fig EV3F). This confirms our findings that ZBTB2 preferentially binds to unmethylated DNA and shows that this binding is direct and independent of its interaction with ZBTB25 or ZNF639. In contrast, when we repeated the DNA pull-down with the genomic region that we described above using nuclear protein extract from Zbtb2 knockout (KO) ESCs, we found that ZBTB25 and ZNF639 are no longer able to bind to this region (Fig EV3G). Although we cannot exclude the possibility that protein levels of ZBTB25 and ZNF639 are affected upon knockout of ZBTB2, RNA-seq analysis comparing wild-type and Zbtb2 KO ESCs shows no significant difference in mRNA levels (Fig EV3H). Thus, we conclude that ZBTB2 forms a module with ZBTB25 and ZNF639 and that the capacity of this module to bind to unmethylated DNA is dependent on ZBTB2.

Figure 3. ZBTB2 reads unmethylated DNA directly and independently of its interaction partners

  • A. UCSC genome browser view of ZBTB2 binding, bisulphite sequencing data 45 and DNase I hypersensitivity tracks 46 in ESCs and NPCs. The region that was used for DNA pull-downs is located within the dashed lines.
  • B, C. Scatterplots of DNA pull-downs with the genomic region indicated in (A), using nuclear protein extract from wild-type ESCs (B) or NPCs (C). Proteins binding specifically to the unmethylated or methylated sequence are depicted in the lower left or upper right quadrant, respectively. Significant outliers were determined through box plot statistics. In (C), ZBTB2 and ZNF639 were just below the statistical cut-offs and have been indicated in dark red. All pull-downs were performed in duplicate and included a label-swap. Box: median (central line), first and third quartile (box limits) whiskers: 1.5 × interquartile range.
  • D, E. Volcano plots of label-free GFP pull-downs using nuclear extracts from ZBTB2-GFP BAC ESCs (D) or NPCs (E). Label-free quantification (LFQ) intensity of the experiment relative to the control [fold change (FC)] is plotted on the x-axis FDR-corrected t-test values are plotted on the y-axis. Grey lines indicate statistical cut-offs. All pull-downs were performed in triplicate.
  • F. Western blot analysis of DNA pull-downs using generic unmethylated or methylated DNA probes with unpurified (upper panel) or GST-purified (lower panel) recombinant GST-ZBTB2. Bound fraction consists of proteins that bound to the DNA probes, and unbound fraction is 5% of the remaining supernatant.

Source data are available online for this figure.

Figure EV3. ZBTB2 reads unmethylated DNA directly and independently of its interaction partners (related to Fig 3 )

  1. Comparison of ZBTB2 occupancy (assessed by ChIP–qPCR) and DNA methylation levels (measured by MeDIP–qPCR) between ZBTB2-GFP BAC ESC and NPCs, at the genomic locus depicted in Fig 3A. Data are depicted as mean ± SEM of three biological replicates (P = as determined by Welch t-test).
  2. Western blot validation of the DNA pull-downs depicted in Fig 3B and C. Bound fraction consists of proteins that bound to the DNA probes, and unbound fraction is 5% of the remaining supernatant.
  3. Western blot validation of the GFP pull-downs depicted in Fig 3D. GFP pull-down on ZBTB2-GFP yields the GFP-tagged (upper band) as well as the endogenous protein (lower band).
  4. Western blot analysis of endogenous ZBTB25 pull-downs with wild-type ESC nuclear protein extract. Pull-downs with IgG are used as a control.
  5. Western blot analysis of FLAG pull-downs with nuclear protein extract from ESCs transiently transfected with a ZNF639-FLAG construct. FLAG pull-downs with wild-type ESC nuclear extract are used as a control.
  6. Western blot analysis of DNA pull-downs using the unmethylated or methylated genomic region indicated in Fig 3A with unpurified (upper panel) or GST-purified (lower panel) recombinant GST-ZBTB2. Bound fraction consists of proteins that bound to the DNA probes, and unbound fraction is 5% of the remaining supernatant.
  7. Scatterplot of DNA pull-downs with the genomic region indicated in Fig 3A, using nuclear protein extract from Zbtb2 KO ESCs. All pull-downs were performed in duplicate and included a label-swap. Box: median (central line), first and third quartile (box limits) whiskers: 1.5 × interquartile range.
  8. RNA-seq analysis of Zbtb25 and Znf639 levels in Zbtb2 KO ESCs, relative to wild-type ESCs. Data are depicted as mean of two biological replicates.

Source data are available online for this figure.

ZBTB2 binding and expression and DNA methylation are intertwined

While the previous results suggest that DNA methylation can influence ZBTB2 binding, we wondered whether ZBTB2 levels can conversely also affect DNA methylation levels. To investigate this, we measured global levels of meC and hmC in wild-type ESCs, Zbtb2 KO ESCs and ZBTB2-GFP BAC ESCs. We found that global meC levels are lower in Zbtb2 KO ESCs and higher in ZBTB2-GFP BAC ESCs compared to wild-type ESCs, while the reverse is true for global hmC levels (Fig 4A). To gain insight into the underlying mechanism of this effect, we looked in our RNA-seq data comparing wild-type and Zbtb2 KO ESCs whether the expression of any known regulators of DNA methylation or demethylation is changed in Zbtb2 KO ESCs compared to wild-type cells. Indeed, we found the DNA methyltransferases Dnmt3A, Dnmt3B and Dnmt3L, as well as the demethylases Tet1 and Tet2, to be differentially expressed in Zbtb2 KO ESCs (Fig EV4A). Together, this suggests that the DNA methylation network is enhanced in Zbtb2 KO ESCs, resulting in a higher turnover of methylated DNA. We validated these results by qRT–PCR in wild-type and Zbtb2 KO ESCs and also showed that the expression levels can be restored towards wild-type levels in a transient overexpression of ZBTB2 in the Zbtb2 KO line (Fig 4B). In addition, we found that the changes in expression levels of TET1 are reflected in its occupancy at CpG islands (Fig EV4B), suggesting an active role for ZBTB2 in regulating meC and hmC levels at CGIs. Given our previous findings that ZBTB2 binding is sensitive to DNA methylation, this is particularly interesting, as it suggests that ZBTB2 is involved in a feedback loop to regulate its own recruitment to DNA. We thus speculate that ZBTB2 binding to unmethylated CGI promoters is regulated by a complex interplay between DNA methylation levels and the expression of DNA methylases and demethylases, in which ZBTB2 itself plays a key role.

Figure 4. Effect of ZBTB2 on genome-wide DNA methylation levels

  1. Global meC and hmC levels in wild-type, Zbtb2 knockout and ZBTB2-GFP BAC ESCs. Data are depicted as mean ± SEM of five biological replicates (*P = 0.01857, **P = 0.003181, ***P = 0.0007674, n.s. = not significant, Welch t-test).
  2. qRT–PCR analysis of gene expression levels in wild-type, Zbtb2 knockout (KO) and Zbtb2 KO ESCs transiently transfected with ZBTB2 [Zbtb2 rescue (RC)]. Data are depicted as mean ± SEM of three biological replicates (*P < 0.05 compared to WT, Welch t-test).

Figure EV4. Effect of ZBTB2 on DNA (de)methylases abundance and TET1 occupancy (related to Fig 4 )

  1. RNA-seq analysis of gene expression levels in Zbtb2 KO ESCs, relative to wild-type ESCs. Data are depicted as mean of two biological replicates.
  2. ChIP–qPCR analysis of TET1 binding at selected CpG islands in wild-type, Zbtb2 knockout (KO) and Zbtb2 KO ESCs transiently transfected with ZBTB2 [Zbtb2 rescue (RC)]. Data are depicted as mean ± SEM of three biological replicates (P > 0.05 for all pairwise comparisons, Welch t-test).

ZBTB2 is a gene activator and regulates the exit from pluripotency

To explore the biological significance of ZBTB2 occupancy at unmethylated CGI promoters in ESCs, we more deeply analysed our RNA-seq data of wild-type and Zbtb2 KO ESCs. Since DNA methylation has generally been associated with gene silencing, we expected that ZBTB2 would act as a gene activator rather than a repressor. Supporting this notion, we found that in Zbtb2 KO ESCs, ± 850 genes are differentially regulated compared to wild-type ESCs, with the majority becoming downregulated. To investigate whether these differentially expressed genes are direct ZBTB2 target genes, we integrated our RNA-seq and ChIP-seq data sets (Dataset EV2). This resulted in the identification of ± 300 genes that are bound by ZBTB2 in wild-type ESCs and whose expression is significantly changed in Zbtb2 KO ESCs, suggesting that ZBTB2 directly regulates the expression of these genes (Fig 5A). The majority of these directly regulated genes are downregulated in Zbtb2 KO compared to wild-type ESCs. Furthermore, when comparing the ratio of expressed to silence genes in the subset of ZBTB2-bound genes to the whole transcriptome, as determined by RNA-seq in wild-type ESCs, we found that this ratio is significantly higher for ZBTB2-bound genes (Fig EV5A). Altogether, these results propose a role for ZBTB2 as a gene activator, consistent with our understanding of DNA methylation as a repressive epigenetic mark.

Figure 5. ZBTB2 acts as a gene activator and stimulates cellular differentiation

  1. Venn diagram comparing genes that are bound by ZBTB2 in ESCs (as identified by ChIP-seq, green) and genes whose expression changes significantly in Zbtb2 KO ESCs (as identified by RNA-seq, orange).
  2. Top five hits of GO terms enrichment analysis of genes that were identified in (A) to be directly regulated by ZBTB2.
  3. Alkaline phosphatase staining of wild-type and Zbtb2 KO ESCs after culturing for 5 days in the absence of LIF to induce differentiation. Cells that were cultured for 5 days in the presence of 2i served as a reference for completely undifferentiated cells. Pictures are representative of eight biological replicates. Scale bars: 100 μm.
  4. qRT–PCR analysis of pluripotency and differentiation markers in wild-type and Zbtb2 KO ESCs after culturing for 5 days in the absence of LIF. Data are depicted as mean ± SEM of three biological replicates (*P < 0.05, Welch t-test).
  5. Proposed model of ZBTB2-binding dynamics. In ESCs, ZBTB2 recruits a zinc finger module to unmethylated CpG island promoters, thereby regulating genes that are important for differentiation. ZBTB2 influences its own binding by regulating TET enzymes and consequently the rate of DNA demethylation. Upon differentiation, binding of the zinc finger module is reduced by increased DNA methylation levels.

Figure EV5. ZBTB2 acts as a gene activator and stimulates cellular differentiation (related to Fig 5 )

  1. Percentages of expressed and not expressed genes in the total transcriptome of wild-type ESCs and the subset of ZBTB2-bound genes (*P = 8.348e-09, Fisher's exact test).
  2. RNA-seq analysis of gene expression levels in Zbtb2 KO ESCs, relative to wild-type ESCs. Data are depicted as mean of two biological replicates.
  3. Western blot analyses of protein levels in wild-type and Zbtb2 KO ESCs. Histone H3 was used as a loading control.
  4. qRT–PCR analysis of gene expression levels in wild-type, Zbtb2 knockout (KO) and Zbtb2 KO ESCs transiently transfected with ZBTB2 [Zbtb2 rescue (RC)]. Data are depicted as mean ± SEM of three biological replicates (*P < 0.05 compared to WT, Welch t-test).

Source data are available online for this figure.

Next, we performed gene ontology (GO) term enrichment analysis on genes that are directly regulated by ZBTB2 and found that one of the processes that these genes are primarily associated with is embryonic development (Fig 5B). In line with this, our RNA-seq data revealed that a number of pluripotency factors (such as ESRRB, KLF4 and NANOG) are significantly upregulated in Zbtb2 KO ESCs compared to wild-type cells (Fig EV5B), suggesting that the pluripotency network in Zbtb2 KO cells is perturbed. We validated this by Western blot and qRT–PCR in wild-type and Zbtb2 KO ESCs and also showed that a transient overexpression of ZBTB2 in the Zbtb2 KO line causes expression levels to decrease towards wild-type levels (Fig EV5C and D). To investigate whether a higher expression of these pluripotency factors induces delayed differentiation in Zbtb2 KO ESCs, we induced differentiation in these cells by culturing them in the absence of LIF and assessed their pluripotency by alkaline phosphatase staining and qRT–PCR. We found that after 5 days in medium without LIF, Zbtb2 KO ESCs display a less differentiated morphology compared to wild-type cells that were treated equally (Fig 5C). Furthermore, qRT–PCR analysis at day five after LIF withdrawal showed that several pluripotency factors remain more highly expressed in Zbtb2 KO ESCs compared to wild-type cells, while the opposite was observed for differentiation makers (Fig 5D). Taken together, these observations suggest that Zbtb2 KO ESCs differentiate more slowly and hence imply that ZBTB2 is involved in the regulation of genes that are important for ESC differentiation.

Concluding remarks

On the basis of our findings, we propose a model of ZBTB2-binding dynamics (Fig 5E) in which ZBTB2, together with ZBTB25 and ZNF639, binds to unmethylated CpG island promoters in ESCs, which is mostly positively correlated with expression of the associated genes. ZBTB2 influences its own binding by regulating the TET enzymes, which catalyse the first step of DNA demethylation by converting meC into hmC. ZBTB2 itself does not bind at the promoters of the TETs, indicating that this regulation is probably indirect. In differentiated cells, increased methylation levels lead to a decreased binding of the zinc finger module.

While this model recapitulates the findings we presented in this study, further research is needed to gain a more complete picture of the biological mechanisms that regulate ZBTB2 binding in the genome and its function. Although we have demonstrated that ZBTB2 binding is sensitive to DNA methylation, DNA methylation dynamics at dynamic ZBTB2-binding sites are not always very pronounced, suggesting that there may be mechanisms other than DNA methylation that can regulate ZBTB2 binding to genomic loci. Furthermore, it remains to be resolved why ESCs show a delay in differentiation upon Zbtbt2 knockout. The genes that are directly regulated by ZBTB2 include some well-known pluripotency factors, such as Klf4, Tbx3, Tfcp2l1 and Nr5a2 23 24 . However, these genes are all among the subset of genes whose expression goes up rather than down in the Zbtb2 KO, raising the question through which exact mechanisms ZBTB2 regulates gene expression. Previous studies have identified interactions between ZBTB2 and co-activator or co-repressor complexes containing histone acetyltransferase or deacetyltransferase activity, respectively, leading to gene activation or gene repression 25 26 27 . In addition, ZBTB2 has been reported to interact with numerous other proteins, such as OCT4 and PTIP 28 29 . However, we could not confirm any of these interactions in our GFP-ZBTB2 interaction proteomics data, suggesting that these interactions are either quite transient, substoichiometric or cell-type specific. Another putative mechanism of ZBTB2-regulated gene expression is through inhibition of binding of other transcription factors. Indeed, ZBTB2 has been suggested to compete with Sp1 for binding to G/C box motifs and to prevent p53 binding through a direct protein–protein interaction 26 . Comprehensive and integrative analysis of various genomics data sets will enable us to investigate to what extent these observations are representative of a general phenomenon.


The chromatin signature of CGIs

Unstable nucleosomes

There is evidence that nonmethylated CGIs are organized in a characteristic chromatin structure that predisposes them toward promoter activity. A study of chromatin at LPS-inducible genes in macrophages found that CGIs are relatively nucleosome-deficient (Ramirez-Carrozzi et al. 2009). Inducible “primary response genes” fall into two classes: those that require SWI/SNF chromatin remodeling complexes for their activation and those that do not. It was noticed that these groups corresponded with non-CGI and CGI promoters, respectively, suggesting that DNA in CGI chromatin is intrinsically accessible without the need for ATP-dependent nucleosome displacement (Ramirez-Carrozzi et al. 2009). In macrophages, the CGIs showed a reduced density of histone H3 even in the uninduced state. Accordingly, in vitro nucleosome assembly indicated that a set of these CGIs is significantly more reluctant to assemble into nucleosomes than other genomic DNA (Ramirez-Carrozzi et al. 2009). An attractive interpretation of the in vitro instability of CGI chromatin is that weakening of this barrier allows greater accessibility of the underlying DNA to transcriptional regulators in vivo. Other evidence has shown that nucleosome deficiency is a feature of CGI promoters in general. Early analysis of CGI chromatin detected abundant nonnucleosomal DNA that was absent in preparations of bulk chromatin (Tazi and Bird 1990). The same conclusion emerged from a re-examination of genome-wide nucleosome mapping data (Schones et al. 2008 Choi 2010). In addition to chromatin instability, nucleosome deficiency in vivo may also arise because CGI promoters, in common with all eukaryotic promoters, typically possess a nucleosome-free region surrounding the TSS (Schones et al. 2008). In other words, active promoters by definition may be nucleosome-deficient whether or not they are CGIs. It is not yet certain whether nucleosome deficiency at CGIs is due primarily to intrinsic chromatin instability or nucleosome exclusion due to the presence of the transcription initiation complex. It could, of course, be a mixture of both, and may even vary between individual CGIs.

Characteristic histone modifications

Early biochemical studies of isolated CGI chromatin showed high levels of histone H3 and H4 acetylation, which are characteristic of transcriptionally active chromatin. Histone H1, on the other hand, which is regarded as antagonistic to transcription, was depleted in this fraction (Tazi and Bird 1990). Genome-wide studies have confirmed this association at high resolution (Birney et al. 2007 Wang et al. 2008) and have revealed that H3K4me3 is a signature histone mark of CGI promoters, often persisting even when the associated gene is inactive (Guenther et al. 2007 Mikkelsen et al. 2007). Recent work has established a biochemical connection between the abundance of CpG in CGIs and H3K4me3, mediated by a CXXC domain protein that binds specifically to nonmethylated CpG (Voo et al. 2000). Cfp1 (CXXC finger protein 1 also known as CGBP) is an integral component of the Setd1 H3K4 methyltransferase complex (Lee and Skalnik 2005) and localizes to the vast majority of CGIs in the mouse genome, suggesting dependence of this histone modification on the DNA sequence (Thomson et al. 2010). In keeping with this model, depletion of Cfp1 reduces H3K4me3 at many CGIs. Importantly, insertion of an artificial CGI-like DNA sequence into the genome results in recruitment of Cfp1 and creates a novel peak of H3K4me3 in the absence of RNAPII (Thomson et al. 2010). Further support for a mechanistic link between unmethylated CpG residues, Cfp1, and H3K4me3 comes from the finding that CpG density in CGIs correlates positively with H3K4me3 levels (Illingworth et al. 2010). The ability of CpG density alone to directly influence the chromatin modification state (Thomson et al. 2010) is likely to be a key function of CGIs.

The presence of H3K4me3 appears to facilitate transcription in a number of ways. The H3K4me3 tail has been shown to interact with the NuRF chromatin remodeling complex (Li et al. 2006 Wysocka et al. 2006 Ruthenburg et al. 2007) as well as ING4-containing histone acetyltransferase complexes (Saksouk et al. 2009). Also, H3K4me3 interacts with the transcriptional machinery directly, as the core transcription factor TFIID has an affinity for the H3K4me3 mark (Vermeulen et al. 2007 van Ingen et al. 2008). Core transcriptional machinery can recruit H3K4 methyltransferases to chromatin (for review, see Ruthenburg et al. 2007), so it is likely that transcription also contributes to H3K4me3 at CGIs. The relative contributions of CpG-mediated and transcription-mediated H3K4me3 to CGI chromatin modification have yet to be determined.

Another distinctive feature of CGI chromatin is depletion of histone H3K36 dimethylation (H3K36me2) compared with non-CGI promoters and gene bodies (Blackledge et al. 2010). The H3K36me2 histone demethylase Kdm2a (Tsukada et al. 2006) is a CXXC domain protein that, like Cfp1, binds specifically to nonmethylated CpG. Accordingly, Kdm2a is bound in vivo at ∼90% of CGIs in mouse ES cells and mediates demethylation of H3K36me2 at these regions (Blackledge et al. 2010). Why H3K36me2 should be depleted at CGIs is uncertain, but this modification has been reported to inhibit transcriptional initiation through histone deacetylase (HDAC) recruitment in yeast (Strahl et al. 2002 Youdell et al. 2008 Li et al. 2009). H3K36me2 depletion may therefore contribute to a transcriptionally permissive state at CGIs (Fig. 4A). Both Cfp1 and Kdm2a have the characteristics of CGI-specific proteins that use CpG density to influence chromatin modification. It is likely that more factors of this kind are yet to be identified, for example, by a comprehensive characterization of the CGI proteome.

The chromatin state at CGIs. (A) CGIs usually exist in an unmethylated transcriptionally permissive state. They are marked by histone acetylation (H3/H4Ac) and H3K4me3, which is directed by Cfp1, and show Kdm2a-dependent H3K36me2 depletion. Nucleosome deficiency and constitutive binding of RNAPII may also contribute to this transcriptionally permissive state. (B) DNA methylation is associated with stable long-term silencing of CGI promoters. This can be mediated by MBD proteins, which recruit corepressor complexes associated with HDAC activity, or may be due to directed inhibition of transcription factor binding by DNA methylation. (C) CGIs can also be silenced by PcG proteins and may be key elements involved in polycomb recruitment. An unknown CGI-binding factor could be responsible for recruiting PRC2 to CGIs that then trimethylates H3K27. This H3K27me3 is recognized by PRC1 complexes that act to impede transcriptional elongation, thereby silencing genes. Note that the transcriptionally permissive and polycomb-repressed states can coexist at bivalent CGIs, predominantly in totipotent embryonic cells.


R-loop formation is a distinctive characteristic of unmethylated human CpG island promoters

CpG islands (CGIs) function as promoters for approximately 60% of human genes. Most of these elements remain protected from CpG methylation, a prevalent epigenetic modification associated with transcriptional silencing. Here, we report that methylation-resistant CGI promoters are characterized by significant strand asymmetry in the distribution of guanines and cytosines (GC skew) immediately downstream from their transcription start sites. Using innovative genomics methodologies, we show that transcription through regions of GC skew leads to the formation of long R loop structures. Furthermore, we show that GC skew and R loop formation potential is correlated with and predictive of the unmethylated state of CGIs. Finally, we provide evidence that R loop formation protects from DNMT3B1, the primary de novo DNA methyltransferase in early development. Altogether, these results suggest that protection from DNA methylation is a built-in characteristic of the DNA sequence of CGI promoters that is revealed by the cotranscriptional formation of R loop structures.

Copyright © 2012 Elsevier Inc. All rights reserved.

Figures

Figure 1. Co-oriented positive GC skew is…

Figure 1. Co-oriented positive GC skew is a common property of strong human CGI promoters

Figure 2. Formation of genomic R-loops at…

Figure 2. Formation of genomic R-loops at the endogenous SNRPN CGI promoter

Figure 3. R-loop formation at the endogenous…

Figure 3. R-loop formation at the endogenous APOE promoter

All symbols are as described in…

Figure 4. Formation of genomic R-loops at…

Figure 4. Formation of genomic R-loops at the endogenous mouse Airn CGI promoter

Figure 5. Widespread R-loop formation at human…

Figure 5. Widespread R-loop formation at human promoters

Figure 6. R-loop formation potential is predictive…

Figure 6. R-loop formation potential is predictive of the unmethylated status of CGI promoters


Эпигенетический контроль экспрессии генов

While the human genome sequence has transformed our understanding of human biology, it isn’t just the sequence of your DNA that matters, but also how you use it! How are some genes activated and others are silenced? How is this controlled? The answer is epigenetics. Epigenetics has been a hot topic for research over the past decade as it has become clear that aberrant epigenetic control contributes to disease (particularly to cancer). Epigenetic alterations are heritable through cell division, and in some instances are able to behave similarly to mutations in terms of their stability. Importantly, unlike genetic mutations, epigenetic modifications are reversible and therefore have the potential to be manipulated therapeutically. It has also become clear in recent years that epigenetic modifications are sensitive to the environment (for example diet), which has sparked a large amount of public debate and research. This course will give an introduction to the fundamentals of epigenetic control. We will examine epigenetic phenomena that are manifestations of epigenetic control in several organisms, with a focus on mammals. We will examine the interplay between epigenetic control and the environment and finally the role of aberrant epigenetic control in disease. All necessary information will be covered in the lectures, and recommended and required readings will be provided. There are no additional required texts for this course. For those interested, additional information can be obtained in the following textbook. Epigenetics. Allis, Jenuwein, Reinberg and Caparros. Cold Spring Harbour Laboratory Press. ISBN-13: 978-0879697242 | Edition: 1

Получаемые навыки

Cancer, Molecular Biology, Cancer Epigenetics, Dna Methylation

Рецензии

This is an excellent course that introduces you to fundamental knowledge of several hot topics in the field of epigenetics. Dr. Blewitt herself studies X inactivation, and I learned so much!

Amazingly structured and delivered course by the entire team. Including a few practical assays or how to read some of the assay results related to epigenetic techniques would be welcome.

An introduction to and definition of epigenetic control of gene expression, and its importance in normal development. We will learn what chromatin is, and how its composition and packaging can alter gene expression. We’ll also discuss the best-characterised epigenetic modification, DNA methylation, and how it is not only implicated in regulating gene expression, but also in maintaining genome stability.

Преподаватели

Dr. Marnie Blewitt

Текст видео

So now we are going to go through in the next series of lectures, each of the specific epigenetic modifications that are called epigenetic marks, and they have functional consequences for how the genes are expressed and how the chromatin is packaged. So while we'll go through each of these. This is the relevance of these, and how they specifically relate to the epigenetic phenomena, or the particular examples we are going to go through in the later lectures, we'll deal with that in later lectures and just have brief highlights of that here. But we need to go through and learn about how each of these work in order to really be able to understand at the molecular level how these processes are working in other instances. So we're going to start with DNA methylation. So DNA methylation is, as it sounds, the addition of a methyl group to the DNA, and this happens at cytosines. So here we're showing a cytosine single ring base but we can add on a 5- methyl group so its added on to the 5 carbon the fifth carbon here. And the methyl group is CHC group that's added directly onto that base. We know that, in mammals, DNA methylation occurs almost exclusively at Cs that are followed by Gs. So cytosines are followed by guanines. And this is called a CpG dinucleotide. And that p is just for the phosphate bond between the two. There's good reason that it's found almost exclusively at CpG dinucleotides. And that's because these dinucleotides are symmetrical when you look at the other side, the other strand of DNA, and this allows them to be maintained through cell division. And you remember this is one of those hallmark features of epigenetic modifications. So, how is it then that DNA methylation can be laid down and how can it be copied mitotically? So how is it that it can have this mitotic memory? So he pictured is a very simple picture of a DNA strand, shown by the black lines, and then in the middle this CpG dinucleotide. And in each case the cytosine is methylated. So, this methylation, because it's close by to one another. The cytosine on the other strand is very close by, and so it's methylated as well. What happens when the cell divides? Sorry, I’ll go back a step and say that the first thing that happens, is that DNA methyltransferases. So these are enzymes, in mammals they're know as DNMT3A and DNMT3B, these lay down the methylation. And they do this in a de novo fashion. In other words, they work on DNA that begins out being unmethylated. So they lay down these, these methyl-marks during development. Then how is it that it can be maintained? Well if we go through cell division, we have the parent strand in each case shown here in black. And the daughter strand shown in green. So, when the DNA is replicated, there's only going to be the parent strand that maintains the methyl group on that side within the CPG dinucleotide. And then the daughter strand, now, shown in green we have an unmethylated cytosine in each case. Now, what happens is we bring in DNMT1, so another DNA methyltransferase enzyme, and this stain of methyltransferase specifically recognises hemi-methylated DNA. It has a preference for hemi-methylated DNA, that is where one strand is methylated and the other is not. And this hemi-methylated DNA is then bound DNMT1. And DNMT1 the lays down methylation on the daughter strand and we have the restoration of a fully methlayted CPG dinucleotide. And this is how we know that DNA methylation can be a stable epigenetic mark. Because at every cell division, this DNA methylation will be copied by DNMT1 onto the new daughter strands of DNA. So we know I said that CpGs are where you, you mostly find methylation, or almost exclusively find methylation in mammals. So where are these CpG's found? Well many CpG’s are found in what are CPG islands. So this is where you find more CG dinucleotides than you would expect by chance and they tend to be found at the promoters of gene’s. So these, promoter are just to remind you is the region upstream of the start side of transcription of the gene and it's where the transcription factors bind. The general rule to remember is that CpG islands although they have many CpG’s there in fact tend to be protected from methylation, so methylation doesn't tend to occur at CpG islands. It tends to be that it occurs at other places in the genome. But if you do find methylation of CpG island. Then, this is almost universally synonymous with silencing of gene expression. So, DNA methylation is an inactive epigenetic mark. So, there are some CpG islands in the genome that are found to be methylated. So, while the general rule is that they are unmethylated There are some that are associated with gene silencing, and these are found at particular times and with dynamic methylation between different cell types. However, it’s mostly being studied, DNA methylation at CpG islands, for the inactive X chromosomes that I have mentioned a few times. So Iɽ like to go into a little bit more detail here about X inactivation. So, the reason for that is X inactivation really clearly demonstrates the mitotic heritability of DNA methylation. So we know that females have two X chromosomes, where as males have one X and one Y. So if we just consider the nucleus of these female and male cells, then the female cell will have twice the dose of all the genes that reside on the X chromosome, of which there are over 1000 genes. This in fact is not what ends up happening. What happens is that one of those two X chromosomes, either the one you inherited from your mom or the one you inherited from your dad, is chosen and its densely packed down in a cell as shown here. And this is called the inactive X chromosome. So while females have two X chromosomes they only ever use one. The second goes by unused. And is put in indeed is even put to the side in the nucleus literally put to the side. So what's interesting is this - when this X inactivation first occurs it occurs when there are only a few hundred cells in the embryo at gastrulation. And we know that when this choice is made it's a random choice. So each cell at that couple-of-hundred-cell stage can make the choice individually as to which X chromosome to inactivate - the one from your mom or the one from your dad. That choice is then mitotically heritable to all of the daughter cells. So, this X inactivation involves DNA methylation of the CpG islands. All thousand of them or so, or almost all thousand of them on the inactive X chromosome. And it's this mitotic heritability after the choice is made is partly ensured by the DNA methylation of those CpG islands. So, this is actually able to be observed in a visible way in the coat colour of cats. So, I'm going to show you a small movie here which is about Calico cats. What we can see, is that Calico cats, that are shown in this picture here, have genes that encode the coat colour genes are on the X chromosome. So, they can either have the ginger version or the black version. And then the choice is made early in development to have the ginger one being active or the black one being active. And the other one is silenced. The other chromosome is silenced. And so you end up with these cats that have a mottled appearance based on when that choice of which X to be inactivated was made early in development. So this could, is also the case not only in cats, of course, but is also true in humans. So in humans if we had a coat colour marker like this, which of course we don't, but if we had a coat colour marker like this, you would actually see these patches of green skin and patches of pink skin, based on when that choice was made early in development. Just as in female, humans just as you see in female cats they have this Calico appearance if they happen to have the right genetics. It's also interesting to note then, that you actually can't get male Calico cats. Male Calico cats, if they exist, have had a mutation where they actually have two copies an X chromosome but still have a Y chromosome. Otherwise they could not have this Calico appearance, traditional mottled appearance. So, how is then that DNA methylation which we've just discussed is heritable for many cell divisions and if you think about it in human mammals can can be heritable for maybe a 100 years. How is it that the DNA methylation actually is associated with gene silencing? There are probably at least a couple of mechanisms by which this can happen. So perhaps the primary mechanism is because the CpG, these methylated CPGs. Are associated with a condensation of the chromatin, and the way that this happens in the primary case is because the methylated CpG is bound by methylated CpG binding proteins, which are otherwise known as MeCP1 and MeCP2. So these MeCP1 and MeCP2.proteins bind to the methylated CpG dinucleotide and they themselves can alter transcription because they possess a transcriptional repression domain. Alternatively the MeCP2 or CeCP1 protein can itself bring in it's own protein partners and they can condense the chromatin. But for this primary mechanism it seems to be the binding of the methylated CPG by the methylated binding protein domain family. Probably the secondary mechanism, which doesn't seem to be is important, but can occasionally occur is that the methylated CpG will stop a transcription factor binding. So, transcription factors have particular binding sites. Say for example C,G,A,T. So, this binding site might be bound by a protein, a transcription factor, and it will then enable the transcription of a neighbouring gene, or the nearby gene. However, if this particular site now has the same sequence C,G,A,T, but its a methylated C,G,A,T, this will then block the binding of that transcription factor. So just that small addition of the methyl group will not allow the transcription factor to bind and therefore we don't have transcription in ensuing. So we don't think, although there are some specific examples of this occurring, for example for the transcription factor SP1 we don't think it's a generalisable mechanism and instead it seems to be just true For promoters that don't have so many CpGs. And so therefore, even single CpGs will have a large consequence. Rather we think that primary mechanism where the methylated CpG binding proteins bind to the methylated CpG is likely to be the most dominant mechanism within the nucleus.


DNA methylation establishment of CpG islands near maternally imprinted genes on chromosome 7 during mouse oocyte growth

Correspondence Qing-Yuan Sun, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 100101 Beijing, China.

Xiang-Hong Ou, Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, 510317 Guangzhou, China.

Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, China

Correspondence Qing-Yuan Sun, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 100101 Beijing, China.

Xiang-Hong Ou, Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, 510317 Guangzhou, China.

State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, China

Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, China

State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri

State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

Ferring Institute of Reproductive Biology, FIRM, Beijing, China

Correspondence Qing-Yuan Sun, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 100101 Beijing, China.

Xiang-Hong Ou, Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, 510317 Guangzhou, China.

Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, Guangzhou, China

Correspondence Qing-Yuan Sun, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 100101 Beijing, China.

Xiang-Hong Ou, Fertility Preservation Lab, Reproductive Medicine Center, Guangdong Second Provincial General Hospital, 510317 Guangzhou, China.

Abstract

The genome methylation is globally erased in early fetal germ cells, and it is gradually re-established during gametogenesis. The expression of some imprinted genes is regulated by the methylation status of CpG islands, while the exact time of DNA methylation establishment near maternal imprinted genes during oocyte growth is not well known. Here, growing oocytes were divided into three groups based on follicle diameters including the S-group (60–100 μm), M-group (100–140 μm), and L-group (140–180 μm). The fully grown germinal vesicle (GV)-stage and metaphase II (M2)-stage mature oocytes were also collected. These oocytes were used for single-cell bisulfite sequencing to detect the methylation status of CpG islands near imprinted genes on chromosome 7. The results showed that the CpG islands near Ndn, Magel2, Mkrn3, Peg12, and Igf2 were completely unmethylated, but those of Peg3, Snrpn, and Kcnq1ot1 were hypermethylated in MII-stage oocytes. The methylation of CpG islands near different maternal imprinted genes occurred asynchronously, being completed in later-stage growing oocytes, fully grown GV oocytes, and mature MII-stage oocytes, respectively. These results show that CpG islands near some maternally imprinted genes are not necessarily methylated, and that the establishment of methylation of other maternally imprinted genes is completed at different stages of oocyte growth, providing a novel understanding of the establishment of maternally imprinted genes in oocytes.


Epigenetics & Memory: How siRNA and Epimutations Help Yeast Cells Remember Their Past

They say that memory is the first thing to go as we get older. Memory research, and neuroscience in general, is one of the most fascinating areas of research. How do the different experiences we face every day shape our memories, and how does our brain know which memories to hold on to and which to let slip away?

It is difficult to study memory at a molecular level in humans and other mammals, so scientists often turn to model organisms like yeast to investigate the basic mechanisms involved behind making and remembering memories.

Lea Duempelmann and colleagues in Switzerland and the Netherlands recently described a phenomenon of transgenerational memory at the molecular level in the fission yeast, Schizosaccharomyces pombe. They were able to show that RNA-induced silencing of gene expression was mediated by epigenetic methods (trimethylation of histone H3 on lysine 9, H3K9me3) and that their yeast cells maintained a &ldquomemory&rdquo of this epigenetic silencing.

The team of researchers observed that the RNA-induced silent state of the gene encoding polymerase-associated factor 1 (Paf1) in S. pombe was stably maintained for at least 18 generations. When Paf1 was reintroduced to the yeast cells, the original silencing was lost. However, when Paf1 activity was impaired again later, the silencing was restored, even if the RNA-mediated silencing mechanism was no longer present.

While this study focused on a specific type of epigenetic memory in yeast, it's exciting to see scientists starting to unravel how organisms can remember their past experiences at a molecular level and we're looking forward to seeing what will come next.

Reference: Duempelmann, L. et al. Inheritance of a Phenotypically Neutral Epimutation Evokes Gene Silencing in Later Generations. Mol. Cell (2019)
Link



Comments:

  1. Pwyll

    So, will you open the topic to the end?

  2. Shakataxe

    It is interesting to read in theoretical terms.

  3. Merton

    you are not like the expert :)

  4. Shandon

    Granted, a very useful thing

  5. Manny

    Something so does not leave

  6. Mareo

    I consider, that you are mistaken. Let's discuss. Write to me in PM, we will communicate.

  7. Stefn

    Why not?



Write a message