Lsh, an epigenetic guardian of repetitive elements - PubMed (original) (raw)

. 2004 Sep 24;32(17):5019-28.

doi: 10.1093/nar/gkh821. Print 2004.

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Lsh, an epigenetic guardian of repetitive elements

Jiaqiang Huang et al. Nucleic Acids Res. 2004.

Abstract

The genome is burdened with repetitive sequences that are generally embedded in silenced chromatin. We have previously demonstrated that Lsh (lymphoid-specific helicase) is crucial for the control of heterochromatin at pericentromeric regions consisting of satellite repeats. In this study, we searched for additional genomic targets of Lsh by examining the effects of Lsh deletion on repeat regions and single copy gene sequences. We found that the absence of Lsh resulted in an increased association of acetylated histones with repeat sequences and transcriptional reactivation of their silenced state. In contrast, selected single copy genes displayed no change in histone acetylation levels, and their transcriptional rate was indistinguishable compared to Lsh-deficient cells and wild-type controls. Microarray analysis of total RNA derived from brain and liver tissues revealed that <0.4% of the 15 247 examined loci were abnormally expressed in Lsh-/-embryos and almost two-thirds of these deregulated sequences contained repeats, mainly retroviral LTR (long terminal repeat) elements. Chromatin immunoprecipitation analysis demonstrated a direct interaction of Lsh with repetitive sites in the genome. These data suggest that the repetitive sites are direct targets of Lsh action and that Lsh plays an important role as 'epigenetic guardian' of the genome to protect against deregulation of parasitic retroviral elements.

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Figures

Figure 1

Figure 1

Increased association of acetylated histones with repetitive elements in Lsh-deficient cells. (A) ChIP assay was performed from Lsh-deficient and wild-type chromatin extracts using indicated antibodies (normal rabbit IgG as control) to detect acetylation of histone 3 (Lys-9/14) or histone 4 (Lys-5/8/12/16). Precipitated DNA or chromatin input was diluted (1:3) for the detection of linear PCR amplification followed by Southern analysis. (B) Results of ChIPs were quantified using a PhosporImager, and the ratio of Lsh−/− to wild-type controls was calculated. The average values of four to five experiments are plotted in the bar graph.

Figure 2

Figure 2

Unchanged histone acetylation levels at single copy sequences. ChIP assay was performed using antibodies as indicated to detect acetylation of histone 3 (Lys-9/14) or histone 4 (Lys-5/8/12/16) in nuclear extracts derived from Lsh−/−, Lsh+/+ MEFs followed by PCR analysis. (A) Southern analysis. (B) Taqman-PCR analysis. The average values were calculated from four experiments.

Figure 3

Figure 3

Deregulation of transcripts with repetitive elements in the absence of Lsh. (A) Total RNA derived from Lsh−/− or Lsh+/+ MEF cells or Lsh−/− and Lsh+/+ embryos of day 13.5 of gestation was subjected to RT–PCR analysis. Repeat sequences were analyzed in dilutions of 1:3, single copy genes in dilutions of 1:10. Omission of reverse transcriptase (−RT) or treatment with RNase served as further controls. (B) Analysis of strand-specific transcripts using forward and reverse primers during reverse transcription. Oligo(dt) primed cDNA served as control for detection of β-actin and IAP transcripts.

Figure 4

Figure 4

Microarray analysis of gene expression after Lsh deletion. Total RNA derived from Lsh−/− or Lsh+/+ liver or brain tissue of day 17.5 gestation embryos were subjected to Microarray analysis. Results represent the average of three hybridizations performed for each tissue type. (A) Proportion of clones from the whole microarray that are abnormally up- or downregulated more than 2-fold above wild-type controls. (B) Proportion of deregulated genes that are either up- or downregulated in the brain or liver tissues. (C) Proportion of deregulated genes that are up- or downregulated and their degree of deregulation expressed as a fold above wild-type control.

Figure 5

Figure 5

Presence of repeat elements in deregulated sequences. (A) Proportion of deregulated clones in brain or liver tissues that contain a repeat element. (B) Proportion of clones that contain different types of repeat elements. Note that a few clones contain more than one type of repeat elements. About 9% of clones did contain neither LTR elements, SINE, LINE nor DNA elements, and only simple repeats were present. (C) Whole bars indicate the proportion of deregulated sequences that contain repeats in relation to their magnitude of deregulation. Black bars indicate the proportion of deregulated sequences containing an LTR.

Figure 6

Figure 6

Lsh targets to repetitive sequences. (A) Western analysis of Lsh expression in the embryonal carcinoma cell-line P19 and embryonal fibroblasts (MEFs) by using a specific antibody raised against the C-terminal portion of Lsh. (B) ChIPs were performed on P19 cells using the C-terminal antibody against Lsh or an IgG control. The precipitated material was examined by western analysis using the C-terminal anti-Lsh antibody for detection. (C) ChIPs were performed from P19 chromatin extracts using the C-terminal antibody against Lsh or an IgG control. Precipitated DNA or chromatin input was analyzed for the presence of the indicated repeat sequences. DNA was diluted (1:3) for the detection of linear PCR amplification.

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