Epigenetic regulation of a murine retrotransposon by a dual histone modification mark

Abstract

Large fractions of eukaryotic genomes contain repetitive sequences of which the vast majority is derived from transposable elements (TEs). In order to inactivate those potentially harmful elements, host organisms silence TEs via methylation of transposon DNA and packaging into chromatin associated with repressive histone marks. The contribution of individual histone modifications in this process is not completely resolved. Therefore, we aimed to define the role of reversible histone acetylation, a modification commonly associated with transcriptional activity, in transcriptional regulation of murine TEs. We surveyed histone acetylation patterns and expression levels of ten different murine TEs in mouse fibroblasts with altered histone acetylation levels, which was achieved via chemical HDAC inhibition with trichostatin A (TSA), or genetic inactivation of the major deacetylase HDAC1. We found that one LTR retrotransposon family encompassing virus-like 30S elements (VL30) showed significant histone H3 hyperacetylation and strong transcriptional activation in response to TSA treatment. Analysis of VL30 transcripts revealed that increased VL30 transcription is due to enhanced expression of a limited number of genomic elements, with one locus being particularly responsive to HDAC inhibition. Importantly, transcriptional induction of VL30 was entirely dependent on the activation of MAP kinase pathways, resulting in serine 10 phosphorylation at histone H3. Stimulation of MAP kinase cascades together with HDAC inhibition led to simultaneous phosphorylation and acetylation (phosphoacetylation) of histone H3 at the VL30 regulatory region. The presence of the phosphoacetylation mark at VL30 LTRs was linked with full transcriptional activation of the mobile element. Our data indicate that the activity of different TEs is controlled by distinct chromatin modifications. We show that activation of a specific mobile element is linked to a dual epigenetic mark and propose a model whereby phosphoacetylation of histone H3 is crucial for full transcriptional activation of VL30 elements.

Figure 1. Effects of HDAC1 depletion, TSA treatment, and Aza-dC treatment on DNA–methylation and histone acetylation patterns in mouse fibroblasts.

Mouse wildtype fibroblasts were left untreated (HDAC1^+/+) or treated with TSA (166nM; 24h for (A) and (B); 3h for (C)) or Aza-dC (1µM for 24h; 24h recovery). In addition untreated HDAC1 deficient fibroblasts (HDAC1^−/−) were included in these experiments. (A) Western blot analysis of chromatin modifying enzymes. Proteins were extracted, separated by SDS-PAGE and analysed with antibodies specific for HDAC1, HDAC2 and DNMT1. Equal loading was controlled with an antibody raised against β-ACTIN. (B) DNA methylation of IAP retrotransposons in HDAC1^+/+ (black bars) and HDAC1^−/− (grey bars) fibroblasts. Both cell lines were treated with TSA or Aza-dC as described above. Average DNA methylation levels of three CpG sites within the LTR were determined employing the Ms-SNuPE technique. (C) Analysis of histone acetylation patterns. Histones were extracted and analysed on Western blots using H3ac and H4ac specific antibodies. Equal loading was monitored with an antibody recognising the C-terminus of histone H3 (c-H3).

Figure 2. Histone acetylation states of transposable elements upon HDAC1 depletion, TSA treatment, and Aza-dC treatment.

The histone acetylation state of chromatin associated with defined transposable elements was determined via ChIP technique using antibodies specifically recognising acetylated histone H3 (H3ac) and acetylated histone H4 (H4ac). Values were normalised to the nucleosome density as determined via ChIP using antibodies recognising the C-terminal region of histone H3 (c-H3) and are given relative to the acetylation levels in untreated fibroblasts. Control experiments employing unspecific IgG antibodies consistently led to negligible amounts of precipitated material (data not shown). (A) Histone H3 and H4 acetylation levels of a variety of transposable elements in HDAC1^−/− (green bars) and TSA (166nM; 24h; blue bars) treated fibroblasts as compared to untreated HDAC1^+/+ (black bars) fibroblasts. (p) promoter region ±500bp of transcriptional start site; (int) internal region. N = 3; ± SEM *p<0.05 (paired, one sided student's t-test). (B) Corresponding acetylation levels of transposable elements in Azd-dC treated fibroblasts (1µM; 24h, 24h recovery; red bars). N = 5; ± SEM *p<0.05.

Figure 3. Effect of TSA and Aza-dC treatment on the expression of transposable elements.

(A) Logarithmically growing fibroblasts show increased mRNA expression levels of VL30 LTR elements when treated with TSA. Expression levels were determined with qRT-PCR. Values are normalised to HPRT expression and are shown relative to the expression in untreated HDAC1^+/+ fibroblasts. Treatment as in Figure 2. N = 3; ± SD *p<0.05. (B) Aza-dC causes high level induction of VL30 and IAP LTR element expression. N = 3; ± SD *p<0.05.

Figure 4. Characterisation of transcribed VL30 elements reveals preferential expression of defined VL30 elements upon stimulation.

(A) Schematic view of a canonical VL30 retrotransposon. Two similar LTRs flank an internal region without functional open reading frame (upper part). Some elements have a hybrid structure and are flanked by LTRs belonging to different subtypes (lower part). Primers used to amplify fragments of genomic and transcribed VL30 elements from untreated, TSA and Aza-dC treated fibroblasts are indicated as black arrows. (B) To characterise the genomic VL30 elements identified as bona fide transcriptional sources, their 5′LTR sequences were aligned (using the Megalign program with the following settings: Clustal W, gap penalty 15, gap length penalty 6.66, delay divergent seqs 30%, DNA transition weight 0.5, DNA weight matrix IUB) and a phylogenetic tree was generated. The LTR sequences fall into three clades. The affiliation to one of the clades is determined by the structure of the highly polymorphic U3 region. While many VL30 elements identified in untreated fibroblasts are associated with a subtype III U3 region, TSA and Aza-dC treatment leads to preferential expression of elements with a type I U3 region. Coloured squares indicate the source, where the VL30 element has been isolated from (white, genomic DNA; black, untreated fibroblasts; blue, TSA treated fibroblasts (166nM; 24h); red, Aza-dC treated fibroblasts (1µM; 24h; recover 24h), numbers indicate the transcriptional activity of the respective elements (i.e. number of clones mapping best to this genomic locus). NVL3, BVL1, NVL1/2 serve as reference elements with known U3 regions and are described elsewhere –. (C) To survey the chromatin signature of individual VL30 elements, ChIP experiments in logarithmically growing fibroblasts untreated or treated for 12h with TSA (166nM) were performed. H3K4me3, H3ac and H4ac antibodies were used to survey the enrichment of active histone marks, H3K9me2 and K3K27me3 antibodies to measure levels of repressive marks. Values were normalised to local histone occupancy as determined with ChIP experiments using the c-H3 antibody and are given as percentage of Input. Primers targeting the upstream regions and 5′LTRs of individual elements allowed determining histone modification levels of five specific VL30 elements. NT_039207 4840bp is an element highly expressed upon TSA treatment. NT_039649 4919bp and NT_039341 4902bp are two additional elements found expressed in TSA treated fibroblasts. Those three elements are associated with a U3 subtype I LTR region. NT_039589 5224bp and NW_001030889 5248bp are equipped with subtype III and IV U3 regions, respectively. N = 2; ± SD.

Figure 5. VL30 expression correlates with the formation of a phosphoacetylation mark at chromatin associated with VL30 LTR sequences.

(A) Schematic view of a histone H3 tail carrying a so-called phosphoacetylation mark, i.e. concomitantly phosphorylated serine 10 (H3S10ph) and acetylated lysine 14 (H3K14ac). (B) Phosphoacetylation of histone H3 in response activation of the MAP kinase pathway and TSA treatment in quiescent Swiss 3T3 fibroblasts. Serum deprived Swiss 3T3 fibroblasts were left untreated (arr) or treated for 2 hours with 166nM TSA, 189nM anisomycin (Aniso) or 20% v/v fetal calf serum (FCS) alone or combinations of anisomycin and TSA or FCS and TSA. Western blot analysis was performed with antibodies directed against acetylated histone H4, H3S10pK14ac and an antibody against the C-terminus of histone H3 (c-H3) as loading control. H3S10pK14ac The H3S10pK14ac signals were determined by densitometric scanning, normalised to c-H3 signals and are shown relative to the value of untreated cells taken arbitrarily as 1. (C,D) The LTR region of VL30 elements is associated with histones carrying a phosphoacetylation mark only upon MAP kinase pathway activation together with HDAC inhibition. ChIP experiments using an H3S10pK14ac specific antibody reveals the presence of phosphoacetylated histones at VL30 LTR regions after anisomycin (189nM; 3h) or FCS (20%; 3h) and TSA (166nM; 3h) treatment (Aniso + TSA; FCS + TSA). (E–G) Combinatorial treatment efficiently activates VL30 element expression in quiescent Swiss 3T3 fibroblasts. Single treatment with TSA (166nM), FCS (20%) or anisomycin (189nM) (dark grey bars) leads to a modest induction compared to VL30 mRNA levels in untreated cells (light grey bars). In contrast, combinatorial treatment with anisomycin or FCS and TSA robustly increases VL30 expression after 6h (black bars). VL30 expression levels were determined with qRT-PCR; values are normalised to HPRT levels and given relative to VL30 levels in untreated cells. N = 3; ± SD *p<0.05.

Figure 6. Inhibition of H3S10 phosphorylation with the protein kinase inhibitor H89 precludes transcriptional activation of VL30 elements.

(A) Treatment of arrested Swiss 3T3 fibroblasts with the protein kinase inhibitor H89 (10µM), inhibiting H3S10 phosphorylation, prohibits the creation of a phosphoacetylation mark upon 1h of Aniso+TSA treatment. ChiP experiments were performed as in Figure 5C. (B) Precluding phosphoacetylation of VL30 elements with H89 also abolishes transcriptional induction upon Aniso + TSA treatment. Treatments as in (A); quantification of VL30 transcript levels were determined as in Figure 5E. N = 3; ± SD *p<0.05.

Figure 7. Model of VL30 element activation.

Full transcriptional activation of VL30 elements is triggered by a dual histone modification mark, i.e. phosphoacetylation of histone H3 at serine 10 and lysine 14. Phosphorylation of serine 10 (green circle) is conveyed by activation of the p38 stress (e.g. via anisomycin (Aniso)) or the ERK growth factor inducible MAPK pathway (e.g. via serum (FCS)) ultimately leading to the activation of the MSK1/2 kinase and H3S10 phosphorylation. Histone hyperacetylation (red circle) can be induced by HDAC inhibition (e.g. via TSA). Combined treatment results in the formation of phosphoacetylated histones at VL30 LTRs and full transcriptional activation.