Histone H4 lysine 20 monomethylation promotes transcriptional repression by L3MBTL1

Abstract

Lethal 3 malignant brain tumor 1 (L3MBTL1), a homolog of the Drosophila polycomb tumor suppressor l(3)mbt, contains three tandem MBT repeats (3xMBT) that are critical for transcriptional repression. We recently reported that the 3xMBT repeats interact with mono- and dimethylated lysines in the amino termini of histones H4 and H1b to promote methylation-dependent chromatin compaction. Using a series of histone peptides, we now show that the recognition of mono- and dimethylated lysines in histones H3, H4 and H1.4 (but not their trimethylated or unmodified counterparts) by 3xMBT occurs in the context of a basic environment, requiring a conserved aspartic acid (D355) in the second MBT repeat. Despite the broad range of in vitro binding, the chromatin association of L3MBTL1 mirrors the progressive accumulation of H4K20 monomethylation during the cell cycle. Furthermore, transcriptional repression by L3MBTL1 is enhanced by the H4K20 monomethyltransferase PR-SET7 (to which it binds) but not SUV420H1 (an H4K20 trimethylase) or G9a (an H3K9 dimethylase) and knockdown of PR-SET7 decreases H4K20me1 levels and the chromatin association of L3MBTL1. Our studies identify the importance of H4K20 monomethylation and of PR-SET7 for L3MBTL1 function.

Figure 1

Chromatin association of L3MBTL1 (and its 3xMBT repeats) in vivo and in vitro. (a) Schematic representation of the domain architecture of the human L3MBTL1 protein. (b) Western blots show the progressive release of endogenous L3MBTL1 from a K562 chromatin pellet (RCP) (third panel) into the micrococcal nuclease (MNase) buffer supernatant (MBS) (second panel) with increasing MNase input. The concomitant release of a chromatin-associated factor, TATA box-binding protein (TBP), into the MBS served as control (top panel). (c) In vitro binding of recombinant GST (top) and GST-3xMBT (bottom) to post-translationally modified calf thymus histones (CTH) analysed in pull down experiments at indicated NaCl concentrations in binding and wash buffers. (d) Binding of individually purified HeLa core histones H3 and H4 (but not H2A or H2B) to GST-3xMBT (at 600mm NaCl).

Figure 2

3xMBT preferentially binds lower methylation states of lysine residues in histones H3 and H4 tails. Interaction of the 3xMBT region of L3MBTL1 with unmodified or differentially methylated (a) H4K20 and (b) H3K9 peptides was quantified using fluorescence polarization (FP). Binding curves represent averages from at least three independent experiments. Binding of 3xMBT proteins, with mutations in the ligand-binding pockets (D248N, D355N or D459N), to (c) H4K20me1 and (d) H3K9me1 peptides assayed using FP (K_D for binding to H3K9me1 peptide; Wt 11 µm, for D248N 15 µm, for D355N 163 µm, for D459N 11 µm).

Figure 3

The chromatin association of L3MBTL1 reflects the progressive accumulation of H4K20me1 during the cell cycle. (a) Confocal micrographs of K562 cells stained with anti-tubulin-α (green) and anti-L3MBTL1 (red) antibodies. DNA was counterstained with 4’-6-diamidino-2-phenylindole (blue). L3MBTL1 shows a diffuse nuclear distribution but is absent from mitotic cells (indicated by arrows). (b) Asynchronous cultures of K562 cells were size fractionated using centrifugal elutriation. Cell cycle status of each fraction was assessed by propidium iodide staining (indicated on top). Nuclear soluble and chromatin pellets isolated from each of the 10 fractions were analysed by immunoblotting using the indicated antibodies. Levels of L3MBTL1 and nucleophosmin (NPM; loading control) in the nuclear soluble fractions (panels 1 and 2) were assessed, as were the relative levels of L3MBTL1, H4K20 methylation (mono-, di- and tri-), H3K9 monomethylation and lamin B (loading control) in the chromatin pellet fractions (panels 3–8).

Figure 4

L3MBTL1 interacts with the H4K20 monomethyltransferase PR-SET7 in vivo and in vitro. (a) Reciprocal immunoprecipitations (anti-HA and anti-myc) from nuclear extracts of 293T cells transiently transfected with plasmids encoding myc-tagged PR-SET7 and HA-tagged L3MBTL1 were analysed by western blotting using anti-PR-SET7 and anti-L3MBTL1 antibodies. (b) K562 cells engineered to stably express HA-L3MBTL1 (K562-L3MBTL1) or empty vector (control) were used in immunoprecipitation (IP) experiments using anti-HA affinity beads. Immobilized protein complexes (HA-IP) as well as 10% of input nuclear extracts were analysed by western blotting, using the indicated antibodies. (c) Bacterially expressed His-tagged PR-SET7 (5 µg; lower panel) was bound to Ni affinity beads and incubated with ³⁵S-methionine-labeled in vitro-translated (IVTT) full-length L3MBTL1, the indicated L3MBTL1 deletional mutants or PR-SET7 (which forms homodimers). Ten percent of the IVTT input is shown (middle panel). Coomassie stained gels were dried and subjected to autoradiography (upper panels).

Figure 5

PR-SET7 promotes, and the 3xMBT repeats mediate, H4K20me1-targeted repression by L3MBTL1. (a) Schematic representation of the stably integrated luciferase reporter gene, downstream of multimeric GAL4-binding sites and a minimal thymidine kinase (TK) promoter, in the HEK293-TK22 cell line. (b–d) Plasmid constructs (amounts in ng) with or without fusion to the DNA-binding domain of GAL4 (DBD) were transfected into the reporter cell line. Luciferase activity in relation to the DBD alone control is shown (% activation). Averages from experiments performed in quadruplicate were calculated and plotted. The transcriptional repressor SMRT (DBD-SMRT) served as positive control (in panel b).

Figure 6

The chromatin association of L3MBTL1 is regulated by siRNA-mediated knockdown of PR-SET7. (a) Protein levels of G9a, PR-SET7, L3MBTL1 and α-tubulin (left side) in whole cell lysates of transiently transfected K562 cells (mock, siPR-SET7, or siG9a) were assessed by immunoblotting. Chromatin pellets were assessed for the levels of H3K9me2, H4K20me1, L3MBTL1 and total H3 by immunoblotting (right side). (b) Quantitative assessment of the impact of H4K20me1 status on the in vivo localization of L3MBTL1 to the cyclin E promoter by chromatin immunoprecipitation followed by quantitative-PCR using K562 cells transfected with siRNA directed against PR-SET7 or G9a (compared to mock-transfected cells). (c) Real-time PCR was used to quantify cyclin E mRNA levels in mock-, siPR-SET7- or siG9a-transfected K562 cells.