Modelo pedagógico social

To gain more insights into the role of Uhrf2 in regulating DNA methylation we performed transient knock downs of uhrf2 in wt and uhrf1-/-_{ESCs with two different siRNAs. Scrambled}

siRNA (control siRNA) was used to control for possible off- target effects. As currently no specific antibody against murine Uhrf2 is available, we monitored the knock down efficiency by qPCR (Fig. 30A, B). After two days of treatment, uhrf2 expression was already very efficiently down regulated by both specific siRNAs and transcript levels stayed low during consecutive rounds of siRNA treatment. As previous studies on RNAi- mediated knock down of dnmt1 in HCT116 cells have demonstrated that a dramatic effect on genomic DNA methylation could only be observed after at least 8 days of siRNA treatment (Spada et al., 2007), we chose to analyze DNA methylation levels after 10 days of repeated siRNA transfections. This prolonged treatment should also ensure a good knock down efficiency of uhrf2 and accumulation of potential errors in DNA methylation patterns. Hence, after 10 days

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of siRNA treatment, we isolated genomic DNA and analyzed the DNA methylation status at repetitive sequences like major satellites using bisulfite treatment followed by pyrosequencing (Fig. 30C, D). As expected, uhrf1-/- _{ESCs show a drastic reduction in DNA}

methylation at repetitive sequences in comparison to wt ESCs. However, DNA methylation levels were neither affected in wt nor in uhrf1-/- _{ESCs by the reduced uhrf2 mRNA level,}

suggesting that Uhrf2 might not play a role in the regulation of maintenance DNA methylation in ESCs.

Figure 30. Transient knock down of uhrf2 in ESCs does not affect DNA methylation levels at repetitive sequences.

(A and B) Wildtype (left side) and uhrf1-/- (right side) ESCs were treated with two different siRNAs targeting uhrf2

(siRNA- 1 and siRNA- 2). Cells were transfected with siRNA every second day for a total of 10 days and samples were taken for qPCR analysis to investigate the success of uhrf2 downregulation. Control siRNA was used to monitor possible off- targets caused by repeated transfections with siRNA. Untreated cells (not shown) were used for normalization and set to 1. Shown is the standard error of technical replicates of one biological replicate. (C and D). DNA methylation levels were analyzed at Major satellite sequences in ESCs treated with specific or control siRNA for 10 days using bisulfite treatment followed by pyrosequencing.

Since our first experiments of silencing uhrf2 expression by RNAi showed no effect on maintenance DNA methylation, we wondered whether the residual uhrf2 mRNA levels (about 20 % with respect to control treated cells) were sufficient to maintain relative high Uhrf2 protein levels in the cells. As we were unable to monitor the remaining levels of Uhrf2 protein after RNAi treatment due to the lack of a Uhrf2 specific antibody we acquired uhrf2-/+ _ESCs,

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Figure 31. RNAi- mediated silencing of uhrf2 in ESCs does not affect maintenance DNA methylation.

(A and B) Wildtype (left side) and uhrf2+/- (right side) ESCs were either transfected with control or specific siRNA (siRNA- 1) every second day and knock down efficiency was monitored by qPCR. Untreated wt cells (not shown) were used as reference and set to 1 so that levels in wt and uhrf2-/+ ESCs are directly comparable. Shown is the standard error of technical replicates of one biological replicate. (C–G). Pyrosequencing data at major satellite sequences (C, D), dnmt1o promoter (E, F) and skeletal α-actin promoter (G, H) in wt (left) and uhrf2-/+ _ESCs treated with specific or control siRNA for 8 days. For the skeletal α-actin promoter, only the results from the first 6 CpG sites are displayed.

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We hypothesized that RNAi treatment of these cells should achieve a higher knock down efficiency. Hence, we treated wt and uhrf2-/+ _{with control or specific siRNA for 8 days and}

monitored efficiency of uhrf2 silencing by qPCR. In wt ESCs, we obtained similar down regulation of uhrf2 to about 80 % compared to control treated cells. As expected, in ESCs heterozygous for uhrf2, mRNA levels of uhrf2 were reduced to about 95 % with respective to wt cells, demonstrating that the lower starting transcript levels indeed improved the total knock down efficiency. After 8 days, we then analyzed DNA methylation levels at repetitive sequences as well as single- copy genes in both cell lines (Fig. 31C-G). Although uhrf2 expression was drastically reduced after 8 days of siRNA treatment, neither an effect on DNA methylation level at repetitive sequences (i.e. major satellites) nor at single copy genes (i.e. promoter of dnmt1o or skeletal α- actin) was detected. These data, together with the previous RNAi mediated knock down experiment suggest that Uhrf2 does not play a role in maintenance DNA methylation in undifferentiated ESCs.

The observation that uhrf2 transcript levels increase during differentiation could imply a role of Uhrf2 in maintenance methylation during development. As uhrf2-/+ _{ESCs show reduced}

mRNA levels of uhrf2, we wondered whether these cells exhibit any defects in maintaining DNA methylation patterns during differentiation. Therefore, we differentiated wt and uhrf2-/+

ESCs to EBs and analyzed the expression of uhrf1 and uhrf2 in the undifferentiated state (day 0) as well as 4 and 16 days after differentiation (Fig. 32A,B). Whereas uhrf1 transcript levels were similarly down regulated independently of the genotype of the cell lines, uhrf2 expression in uhrf2-/+ _{ESCs was increased in both EBs during differentiation. However, in}

heterozygous EBs, uhrf2 mRNA levels reached on 50% of the transcript levels measured in

wt EBs. We then analyzed the level of DNA methylation in the ESCs state (d0) and at both time points during differentiation (d4 and d16) by bisulfite treatment followed by pyrosequencing (Fig. 32C-F). For both analyzed sequences, no difference in DNA methylation was observed in uhrf2-/+ _{ESCs and during EB differentiation, possibly because of}

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Figure 32. Reduced uhrf2 transcript levels do not alter DNA methylation during differentiation.

Wildtype (left) and uhrf2-/+ (right) ESCs were differentiated to EBs and samples for measuring expression levels as well as DNA methylation levels were taken and analyzed in the pluripotent state (d0) as well as 4 and 16 days after differentiation. (A and B) Expression analysis of uhrf1 and uhrf2 in wt and uhrf2-/+_{ESCs and EBs by qPCR.} Transcript levels of uhrf1 in undifferentiated wt cells were used as a reference (set to 1), so that mRNA levels between the two cell lines (wt and uhrf2-/+) are directly comparable. Error bars represent the standard error of three technical replicates. (C–F). DNA methylation of ESCs (d0) and 4 and 16 days old EBs at major satellite sequences (C, D) and the dnmt1o promoter (E, F) was measured by pyrosequencing.

In general, the residual levels of uhrf2 mRNA in uhrf2-/+ _{ESCs and during RNAi- mediated}

knock down could mask the real phenotype of Uhrf2 and hence might not be the best approach to analyze the function of Uhrf2, especially in light of a missing antibody to control for knock down efficiency at the protein level. Therefore, the best solution to analyze a possible function of Uhrf2 in maintenance DNA methylation, would be to generate uhrf2-/-

ESCs and then analyze DNA methylation pattern in the undifferentiated state as well as during differentiation and mouse development.

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