Imprinting regulator DNMT3L is a transcriptional repressor associated with histone deacetylase activity

Abstract

DNMT3L is a regulator of imprint establishment of normally methylated maternal genomic sequences. DNMT3L shows high similarity to the de novo DNA methyltransferases, DNMT3A and DNMT3B, however, the amino acid residues needed for DNA cytosine methyltransferase activity have been lost from the DNMT3L protein sequence. Apart from methyltransferase activity, Dnmt3a and Dnmt3b serve as transcriptional repressors associating with histone deacetylase (HDAC) activity. Here we show that DNMT3L can also repress transcription by binding directly to HDAC1 protein. We have identified the PHD-like zinc finger of the ATRX domain as a main repression motif of DNMT3L, through which DNMT3L recruits the HDAC activity needed for transcriptional silencing. Furthermore, we show that DNMT3L protein contains an active nuclear localisation signal at amino acids 156-159. These results describe DNMT3L as a co-repressor protein and suggest that a transcriptionally repressed chromatin organisation through HDAC activity is needed for establishment of genomic imprints.

Figure 1

DNMT3L represses transcription in a TSA-sensitive manner. (A) Schematic representation of the DNMT3L fragments used for transfection experiments. Numbers indicate DNMT3L amino acids. (B) DNMT3L represses transcription when fused to the Gal4DB. RD cells were co-transfected with five different pMDNMT3L constructs fused to Gal4DB and a CAT repoter gene that contained five Gal4-binding sites upstream from a thymidine kinase promoter. CAT activity from cells transfected only with Gal4DB (pM), without a fusion partner, was set at 100%. (C) DNMT3L repression is dose-dependent. RD cells were transfected with the CAT reporter together with increasing amounts (200 ng or 1 µg) of the DNMT3L 1–195 construct. (D) DNMT3L repression is inhibited by TSA, a specific inhibitor of HDAC activity. RD cells were co-transfected with a CAT reporter gene and the N-terminal pM-DNMT3L 1–195 construct. After 22 h, cells were treated or left untreated with 100 nM TSA for 24 h.

Figure 2

DNMT3L interacts with the histone deacetylase HDAC1. (A) GST pull-down assay using in vitro translated (IVT) full-length HDAC1 and GST-DNMT3L 1–195. (B) GST pull-down assay using IVT pcDNMT3L 1–195 and full-length GST–HDAC1. (C) GST pull-down from RD cell lysate. Total lysate of RD cells transfected with pcHDAC1 was used for a GST pull-down assay performed with GST–DNMT3L 1–195, full-length GST–DNMT3L and GST alone. Input proteins are indicated by arrows on the left and molecular weights are indicated on the right.

Figure 3

DNMT3L associates with HDAC activity. Seven different GST–DNMT3L fusion proteins were used to pull-down the HDAC activity from THP-1 monocyte total protein lysates. α-HDAC1 and α-GAL4 antibodies were used as positive and negative controls, respectively. Deacetylase activity was detected as radioactivity released from ³H-labelled acetylated histone peptide substrate. To show that the repressional effect was mediated through HDAC1, 100 mM sodium butyrate (NaBu) was added to the reactions with PHD finger-containing constructs to inhibit the deacetylation reaction.

Figure 4

DNMT3L has a functional NLS. (A) DNMT3L has a functional NLS, RRRK, at amino acids 156–159. DNMT3L amino acids 142–173 were cloned into GFP vector pEGFP-C3 and transiently transfected into Cos-7 cells. (Upper) GFP fluorescence; (lower) DAPI staining of the same cell. (B) Full-length GFP–DNMT3L and (C) GFP vector alone are distributed in both the cytoplasm and nucleus.