Lsh is involved in de novo methylation of DNA

Abstract

Deletion of Lsh perturbs DNA methylation patterns in mice yet it is unknown whether Lsh plays a direct role in the methylation process. Two types of methylation pathways have been distinguished: maintenance methylation by Dnmt1 occurring at the replication fork, and de novo methylation established by the methyltransferases Dnmt3a and Dnmt3b. Using an episomal vector in Lsh-/- embryonic fibroblasts, we demonstrate that the acquisition of DNA methylation depends on the presence of Lsh. In contrast, maintenance of previously methylated episomes does not require Lsh, implying a functional role for Lsh in the establishment of novel methylation patterns. Lsh affects Dnmt3a as well as Dnmt3b directed methylation suggesting that Lsh can cooperate with both enzymatic activities. Furthermore, we demonstrate that embryonic stem cells with reduced Lsh protein levels show a decreased ability to silence retroviral vector or to methylate endogenous genes. Finally, we demonstrate that Lsh associates with Dnmt3a or Dnmt3b but not with Dnmt1 in embryonic cells. These results suggest that the epigenetic regulator, Lsh, is directly involved in the control of de novo methylation of DNA.

Figure 1

Lsh is required for methylation of an episomal vector in MEF cells. (A) Map of the episomal vector pCEP4 illustrating the location of HpaII/MspI and DpnI sites as well as the position of the primers used for methylation-sensitive PCR analysis. Primer pairs P5/P6 are designed for detection of methylation, P3/P4 for detection of successful replication, and P1/P2 as an internal control. The length of the expected PCR fragment is indicated in base pairs (bp). (B) Western blot analysis using nuclear extracts derived from Lsh−/− and Lsh+/+ embryonal fibroblasts (MEF) and specific antibodies against Lsh, Dnmt3a, Dnmt3b and PCNA (serving as control). (C) Methylation-sensitive PCR (upper panel). Episomal DNA derived from stably transfected Lsh−/− and Lsh +/+ MEF cells was digested with DpnI and then either with the methylation-sensitive enzyme HpaII (H) or the methylation independent enzyme MspI (M) followed by PCR analysis with indicated primers. Replication of the episomal vector was confirmed using DpnI (lower panel). DpnI cuts only DNA that has been methylated in bacteria by the dam methylase. The replicated episomal DNA in mouse cells should be DpnI resistant. For adjustment of input undigested DNA (Un) was used before digestion. (D) Episomal DNA was derived as in C. and subjected to real time-PCR analysis using methylation-sensitive primer pair P5/P6 (upper panel) or the internal control P1/P2 (lower panel).

Figure 2

Lsh does not play a role in the maintenance of methylation in MEF cells. (A) Ethidium bromide stain of in vitro methylated episomal vector pCEP4 that has been digested with HpaII (H) and MspI (M). Resistance to HpaII digestion indicates that the episome has been fully methylated. (B) Methylation-sensitive PCR. Methylated episomal DNA was stably transfected into Lsh−/− and Lsh+/+ MEF cells and the recovered DNA was first digested with DpnI. Then the DNA was either digested with the methylation-sensitive enzyme HpaII (H) or the methylation independent enzyme MspI (M) followed by PCR analysis with the indicated primers as shown in Figure 1A. (C) Real-time PCR analysis. Episomal DNA was derived as described in (B). and subjected to real time-PCR analysis using the methylation-sensitive primer pair P5/P6 (upper panel) or the internal control primer pair P1/P2 (lower panel). (D) The relative copy numbers for real time-PCR products of HpaII digested episomal DNA were calculated based on the standard curve equation. Results of Figure 1 are represented as ‘de novo' and results from Figure 2 as ‘maintenance' methylation.

Figure 3

Lsh is required for silencing of Dnmt3a or Dnmt3b mediated silencing of a retroviral transgene. (A) Western blot analysis using nuclear extracts derived from Lsh−/− and Lsh+/+ mouse embryonal fibroblasts stably expressing Dnmt3a, Dnmt3b, Dnmt3a/Dnmt3b, or untransfected (Un) MEFs. For detection, specific antibodies were used against Dnmt3a, Dnmt3b, or PCNA as control. (B) Fluorescence analysis. Lsh+/+ and Lsh−/− MEFs that were stably expressing Dnmt3a, Dnmt3b, or Dnmt3a/Dnmt3b were infected with pMSCV-hGFP and examined after 8 days for GFP expression using a fluorescence microscope. (C) FACS analysis. The GFP intensity of Lsh+/+ and Lsh−/− MEF cells expressing Dnmt3a, Dnmt3b, Dnmt3a/Dnmt3b, or untransfected (Un) was measured by FACS analysis 8 days after retroviral infection. The difference in fluorescence intensity was expressed as GFP ratio of Lsh+/+ over Lsh−/−. (D) Map of the retroviral vector pMSCV-hGFP indicating the location of HpaII/MspI sites and the position of the primers used for methylation-sensitive PCR analysis. Primer pair P9/P10 detects methylation within the GFP region. Primer pair P7/P8 serves as internal control. Primer pair P11/P12 detects methylation in the 5′-LTR and the adjacent region. The length of the expected PCR fragments is indicated in base pairs (bp).

Figure 4

Lsh functionally cooperates with de novo methylation mediated by either Dnmt3a or Dnmt3b. (A) Methylation-sensitive PCR. At 8 days after the retroviral infection, genomic DNA from Lsh−/− and Lsh+/+ MEF cells stably expressing Dnmt3a, Dnmt3b, or Dnmt3a/Dnmt3b was extracted, digested with HpaII (H) or MspI (M) and subjected to PCR with the indicated primer pairs. For adjustment of DNA undigested DNA (Un) was used before digestion. (B–D). Real-time PCR analysis of HpaII digested DNA using primer pair P11/P12. Control primers P7/P8 are used in the lower panel of the graphs.

Figure 5

Silencing of Lsh in embryonic stem cells results in loss of de novo methylation. (A) Western analysis: nuclear extracts from two ES cell lines (#1 and #2 both received the same construct) stably expressing a silencing vector for Lsh (siLsh) were examined by Western analysis using specific antibodies against Lsh, Dnmt3a, Dnmt3b, Chd4 or PCNA as control. A scrambled sequence serves as the siRNA vector control. (B) Southern blot analysis: genomic DNA derived from two siLsh ES cell lines, an ES cell control, Lsh−/− MEFs, and Lsh+/+ MEF cells was digested with HpaII (H) or MspI (M), blotted, and probed for minor satellite sequence using the probe MR150. (C) RT–PCR analysis: siLsh ES cells were infected with the retroviral vector pMSCV-hGFP and after the indicated time points (24, 48, 72 h) RNA was extracted, reverse transcribed and analyzed by PCR for expression of GFP. β-Actin serves as a control. (D) FACS analysis: 72 h after retroviral infection of siLsh and control ES cells GFP expression was measured by FACS analysis. (E) Methylation-sensitive PCR: genomic DNA derived from the siLsh and control ES cells 3 days after retroviral infection was digested with HpaII or MspI and subjected to PCR analysis with the indicated primer pairs. (F) Real time-PCR analysis: as in (E), HpaII digested genomic DNA was subjected to real time-PCR using P11/P12. The right panel shows the internal control reaction with primers P7/P8.

Figure 6

Lsh is involved in de novo methylation at the Oct-4 gene. (A) Map of murine Oct4 gene and its promoter indicating the location of HpaII/MspI site (H in Site 1) and HpyCh4 IV (Hy in Site 2) and the position of the primers used for methylation-sensitive PCR analysis. Due to different recognition sequences of HpaII and HpyCh4 IV, site 1 and site 2 can be used as internal control for each other in methylation-sensitive PCR. (B) Methylation-sensitive PCR. Genomic DNA derived from the Lsh wild type and Lsh−/− MEF cells was digested with HpyCh4 IV and subjected to PCR analysis with the indicated primer pairs. Site 1 served as internal control for site 2. Undigested DNA (Un) was used for adjustment of DNA input before digestion. (C) Methylation-sensitive PCR. Genomic DNA derived from the siLsh and control ES cells at 0 day and 6 days after differentiation was digested with HpaCh4 IV and subjected to PCR analysis with the indicated primer pairs. Site 1 served as internal control for site 2. Undigested DNA (Un) was used for adjustment of DNA input before digestion. (D) Methylation-sensitive PCR. Genomic DNA derived from the siLsh and control ES cells at 0 day and 6 days after differentiation was digested with HpaII and MspI and subjected to PCR analysis with the indicated primer pairs. Site 2 served as internal control for site 1.

Figure 7

Lsh interacts with Dnmt3a and Dnmt3b. (A) Western analysis for detection of Dnmt3a, Dnmt3b or Dnmt1 after immunoprecipitation (IP) with Lsh-specific antibodies (Ab): Lsh#1 (C-terminal peptide, affinity purified) and Lsh#2 (recombinant protein, affinity purified) using P19 nuclear extracts. The blots are probed with a monoclonal anti-Dnmt3a, Dnmt3b and Dnmt1 antibodies. The negative controls are normal rabbit IgG or omission of antibodies (mock). Nuclear extracts before IP serve as positive controls. (B) Western analysis for detection of Lsh and Dnmt1 after IP with specific antibodies against Dnmt3a, Dnmt3b or Dnmt1 using P19 nuclear extracts. The following antibodies were used for Western analysis: anti-Lsh#2 (recombinant protein, affinity purified) Dnmt1. The negative controls are normal mouse IgG or omission of antibodies (mock). (C) Western analysis for detection of Myc-Dnmt3a and Myc-Dnmt3b fusion proteins after IP with anti-Lsh#1 (C-terminal peptide) and Lsh#2 (recombinant protein). The nuclear extracts were prepared from P19 cells stably expressing Myc-Dnmt3a (upper panel) or Myc-Dnmt3b (lower panel). The negative controls are normal rabbit IgG or omission of antibodies (mock). (D) Western analysis for detection of Lsh after IP with anti-Myc antibodies using nuclear extracts derived from P19 cells stably expressing Myc-Dnmt3a or Myc-Dnmt3b. Two different anti-myc antibodies were used for IP. The negative controls are normal mouse IgG (IgG#1), mouse IgG1 (IgG#2) or normal rabbit IgG (IgG#3).