Chromatin protein L3MBTL1 is dispensable for development and tumor suppression in mice

Abstract

L3MBTL1, a paralogue of Drosophila tumor suppressor lethal(3)malignant brain tumor (l(3)mbt), binds histones in a methylation state-dependent manner and contributes to higher order chromatin structure and transcriptional repression. It is the founding member of a family of MBT domain-containing proteins that has three members in Drosophila and nine in mice and humans. Knockdown experiments in cell lines suggested that L3MBTL1 has non-redundant roles in the suppression of oncogene expression. We generated a mutant mouse strain that lacks exons 13-20 of L3mbtl1. Markedly reduced levels of a mutant mRNA with an out-of-frame fusion of exons 12 and 21 were expressed, but a mutant protein was undetectable by Western blot analysis. L3MBTL1(-/-) mice developed and reproduced normally. The highest expression of L3MBTL1 was detected in the brain, but its disruption did not affect brain development, spontaneous movement, and motor coordination. Despite previous implications of L3mbtl1 in the biology of hematopoietic transcriptional regulators, lack of L3MBTL1 did not result in deficiencies in lymphopoiesis or hematopoiesis. In contrast with its demonstrated biochemical activities, embryonic stem (ES) cells lacking L3MBTL1 displayed no abnormalities in H4 lysine 20 (H4K20) mono-, di-, or trimethylation; had normal global chromatin density as assessed by micrococcal nuclease digests; and expressed normal levels of c-myc. Embryonic fibroblasts lacking L3MBTL1 displayed unaltered cell cycle arrest and down-regulation of cyclin E expression after irradiation. In cohorts of mice followed for more than 2 years, lack of L3MBTL1 did not alter normal lifespan or survival with or without sublethal irradiation.

FIGURE 1.

Disruption of L3mbtl1 in mice. a and b, L3mbtl1 expression levels in wild type mice vary among different tissues. S. muscle, skeletal muscle. a, Northern blot analysis demonstrates expression in testes, eyes, and ES cells and most abundantly in the brain. b, expression analysis by RT-PCR reveals widespread, but weaker, expression in other tissues. c and d, strategy for L3mbtl1 disruption (for additional details, see supplemental Fig. S2). d, the knock-out allele lacks the coding region for two MBT domains as well as the zinc finger (amino acids 413 through 708). Additionally, fusion of exon 12 to exon 21 predicts a frameshift that generates several stop codons, so any residual message would not code for the SAM domain but instead express 60 alternative amino acids. e, Southern blot analysis of SpeI-digested DNA from ES cells after targeting and/or Cre-mediated excision of the loxP-flanked regions. As expected, the targeted allele results in a lower band (7.3 kb) compared with the wild type allele (20 kb) using a probe upstream of the targeting vector (5′ probe) (left panel). Correct insertion of the 3′ end was confirmed with a probe downstream of the targeted region (3′ probe; expected bands: wild type, 20 kb; and targeted, 13 kb) (right panel). Excision of the floxed region after transient expression of Cre in vitro results in a 10-kb band (right panel, left lane). f, germ line transmission of the targeted allele documented by Southern blot analysis of tail DNA. g, Northern blot analysis from brains using the entire coding region as a probe demonstrates abundant message in the wild type but no signal in L3mbtl1 knock-out mice. h, RT-PCR analysis for L3mbtl1 using primers upstream and downstream of the deleted region reveals a faint band in the mutant consistent with a shorter message as predicted. i and j, sequence analysis of the PCR product from the mutant in h confirms deletion of exons 13–20 after exon 12 resulting in 60 abnormal residues followed by five stop codons. k, Western blot analysis using an antiserum against a His-tagged fusion protein corresponding to the N-terminal 215 amino acid residues of L3MBTL1 demonstrates absence of the protein in knock-out cells and tissues. Note that a theoretically possible truncated protein (expected size, ∼55 kDa) is not detectable.

FIGURE 2.

Disruption of L3mbtl1 is compatible with ostensibly normal CNS development and function. a, normal brain morphology and cytoarchitecture in L3mbtl1^−/− mice. Representative 15–20-μm-thick Nissl-stained coronal sections display normal morphology and cytoarchitecture in forebrain (upper panels) and hindbrain (lower panels) of 6–8-week-old mutants (right) and littermate controls (left). Scale bar, 0.5 mm. b, unaltered body weight after disruption of L3mbtl1 (mean ± standard error (S.E.), n = 8–15 animals/group; black bars, control; white bars, mutants). c and d, behavioral testing. c, locomotor activity is not perturbed in the absence of L3MBTL1. Shown is the total ambulation over 17 h tracked with a photobeam (5 p.m. to 10 a.m.; summed ambulation, means ± S.E., n = 4–6 animals/group). d, rotarod motor coordination performance is not perturbed in L3mbtl1^−/− mice. The bar graph shows latency to failure in seconds (see “Experimental Procedures”; day 2, three-trial mean ± S.E., n = 5).

FIGURE 3.

Disruption of L3mbtl1 does not perturb hematopoiesis or lymphopoiesis. a, normal blood cell counts in the absence of L3MBTL1 (n = 5, mean and standard deviation (S.D.)). b, total bone marrow cell counts (number per two femurs, tibiae, and fibulae) and immuno-flow cytometric analysis of major stem cell and myeloid progenitor population demonstrate normal hematopoiesis in L3mbtl1^−/− mice. Hematopoietic stem cells (HSC) were identified as lineage marker⁻ (L⁻), cKit⁺ (K⁺), Sca1⁺ (S⁺) cells with or without additional SLAM markers CD48 (48⁻) and CD150 (150⁺). Frequencies of common myeloid progenitor (CMP; L⁻K⁺S⁻CD34⁺FCγR⁻), granulocyte-monocyte progenitors (GMP; L⁻K⁺S⁻CD34⁺FCγR⁺), and megakaryocyte-erythrocyte progenitors (MEP; L⁻K⁺S⁻CD34⁻FCγR⁻) are shown. b and c, no major abnormalities in lymphopoiesis in the absence of L3MBTL1. c, frequencies (%) of B cells and their progenitors. Pro-B cells (IgM⁻, B220⁺, CD43⁺), pre-B cells (IgM⁻, B220⁺,CD43⁻), and IgM⁺ bone marrow cells as well as mature spleen B cells (IgM^low, IgD^hi) were determined as shown previously. d, frequencies of T cell precursors in the thymus and mature T cells in the spleen were determined as described. Bar graphs represent averages and S.D. Black bars, wild type littermates (n = 3); white bars, L3mbtl1^−/− mice (n = 3). No differences were significant (<0.05) by two-tailed Student t test.

FIGURE 4.

Impact of disruption of L3mbtl1 on ES cells. a, biallelic disruption of L3mbtl1 in ES cells was achieved after selection of clones that had duplicated the targeted allele by crossing over in the presence of increased neomycin concentration (third lane). After confirmation of a normal karyotype (not shown), both alleles of L3mbtl1 were disrupted by transient expression of Cre (right lane). b, no major differences in cell cycle profiles (G₁, S, and G₂/M) of L3mbtl1^−/− ES cells compared with control ES cells. Analysis of cell cycle distribution after staining DNA content with propidium iodide (PI) is shown. c, the G₂/M checkpoint is not altered in L3mbtl1^−/− ES cells. Simultaneous staining for DNA content and serine 28-phosporylated histone H3 (H3pS28) was used to resolve G₂ and M phases of the cell cycle. d and e, analysis of global chromatin structure using limited micrococcal nuclease (MNase) digests of wild type or L3mbtl1^−/− ES cells. Micrococcal nuclease cuts DNA that is not occupied by nucleosomes. In dense chromatin or with limited amounts of enzyme, digestion within the internucleosomal spaces is inefficient, resulting in the generation of oligo- and multinucleosomes with distinct sizes (the lowest band represents mononucleosome; higher bands represent oligonucleosome (dimers, trimers, etc.)). In loose (relaxed) chromatin and with high amounts of micrococcal nuclease, fragments representing DNA protected by a single nucleosome are generated preferentially. Note that an equal abundance of the various bands suggests that there is no marked global difference in compaction of chromatin after disruption (e); an equal size of fragments demonstrates that nucleosome spacing is not affected by loss of L3MBTL1. Each panel shows one representative of three independent experiments. DNA markers are shown with sizes of prominent bands labeled. f, global levels of H4K20 mono-, di-, or trimethylation are not affected by the absence of L3MBTL1 in ES cells.

FIGURE 5.

MEFs lacking L3MBTL1 display unaltered cell cycle arrest in response to irradiation. a, MEFs from L3mbtl^−/− embryos lack L3MBTL1 protein. b, scheme of experiment. c, RT-PCR before and after irradiation demonstrates dose-dependent, diminished expression of cyclin E after irradiation in the presence or absence of L3MBTL1. d, cell cycle analysis reveals dose-dependent arrest in the cell cycle irrespective of L3mbtl1 gene status. Gy, grays; PI, propidium iodide.

FIGURE 6.

Disruption of L3mbtl1 does not affect lifespan with or without sublethal dose irradiation. a, Kaplan-Meyer survival curves of L3mbtl1^−/− (n = 10) and littermate L3mbtl1^+/+ control mice (n = 10) reveal no significant difference in lifespan. b, single dose (6 grays (Gy)), non-lethal total body irradiation of cohorts of L3mbtl1^−/− (n = 10) and littermate L3mbtl1^+/+ control mice (n = 9) at 3 months of age results in no significant difference in survival; p values were calculated by log rank test. c, no alteration of global c-myc expression levels in L3mbtl1^−/− brains or ES cells as analyzed by Northern blot.