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. 2009;4(3):e4750.
doi: 10.1371/journal.pone.0004750. Epub 2009 Mar 9.

Functional conservation of Asxl2, a murine homolog for the Drosophila enhancer of trithorax and polycomb group gene Asx

Heather A Baskind  1 , Lucy Na, Quanhong Ma, Mayur P Patel, David L Geenen, Q Tian Wang
Affiliations

Functional conservation of Asxl2, a murine homolog for the Drosophila enhancer of trithorax and polycomb group gene Asx

Heather A Baskind et al. PLoS One. 2009.

Abstract

Background: Polycomb-group (PcG) and trithorax-group (trxG) proteins regulate histone methylation to establish repressive and active chromatin configurations at target loci, respectively. These chromatin configurations are passed on from mother to daughter cells, thereby causing heritable changes in gene expression. The activities of PcG and trxG proteins are regulated by a special class of proteins known as Enhancers of trithorax and Polycomb (ETP). The Drosophila gene Additional sex combs (Asx) encodes an ETP protein and mutations in Asx enhance both PcG and trxG mutant phenotypes. The mouse and human genomes each contain three Asx homologues, Asx-like 1, 2, and 3. In order to understand the functions of mammalian Asx-like (Asxl) proteins, we generated an Asxl2 mutant mouse from a gene-trap ES cell line.

Methodology/principal findings: We show that the Asxl2 gene trap is expressed at high levels in specific tissues including the heart, the axial skeleton, the neocortex, the retina, spermatogonia and developing oocytes. The gene trap mutation is partially embryonic lethal and approximately half of homozygous animals die before birth. Homozygotes that survive embryogenesis are significantly smaller than controls and have a shortened life span. Asxl2(-/-) mice display both posterior transformations and anterior transformation in the axial skeleton, suggesting that the loss of Asxl2 disrupts the activities of both PcG and trxG proteins. The PcG-associated histone modification, trimethylation of histone H3 lysine 27, is reduced in Asxl2(-/-) heart. Necropsy and histological analysis show that mutant mice have enlarged hearts and may have impaired heart function.

Conclusions/significance: Our results suggest that murine Asxl2 has conserved ETP function and plays dual roles in the promotion of PcG and trxG activity. We have also revealed an unexpected role for Asxl2 in the heart, suggesting that the PcG/trxG system may be involved in the regulation of cardiac function.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Generation of Asxl2 mutant mice.
(A) Schematic representation of the gene trap allele of murine Asxl2 (not drawn to scale). Exon 1 contains the 5'UTR (the narrow portion of the red box) and coding sequence for the first 19 amino acids of Asxl2. The gene trap cassette (blue rectangle) is inserted 5,016 bp downstream of exon 1. P1, P2 and P3: primers used in genotyping PCRs. R1, R2 and R3: primers used in RT-PCR analysis of the wild-type and gene trapped transcripts. (B) Conservation between mouse Asxl2 intron 1 and corresponding sequences in human ASXL2 and rat Asxl2 loci. The mouse, human and rat sequences were compared by SLAGAN. The 15,261-bp intron 1 of mouse Asxl2 was used as the base sequence. The top two panels show distribution of conserved sequence modules within the entire length of mouse Asxl2 intron 1. The bottom two panels show close-up views of 500 bp upstream and 500 bp downstream of the gene trap insertion site, which is indicated by red arrows. (C) RT-PCR analysis of the AQ0356 gene trap ES cell line. Transcripts from the wild-type (+) and mutant (−) alleles are detected with primer sets R1/R2 and R1/R3, respectively. The RT-PCR product in the mutant lane reflects splicing event that joins exon 1 with β-geo. (D) Schematic representation of the domain structures of Drosophila Asx, vertebrate Asxl1, Asxl2, Asxl3 and the predicted protein product from the Asxl2− allele. The ASXH (green box) and PHD (red box) domains are conserved from flies to mammals. The ASXL1 boxes 1 and 2 (blue and orange boxes) are conserved in the three mammalian Asx-like proteins but are not present in Asx. The mutant protein contains the first 19 amino acids of Asxl2 joined to β-geo. None of the conserved domains is present in the mutant protein. (E) PCR genotyping of genomic DNA from Asxl2 wild-type (+/+), heterozygous (+/−) and mutant (−/−) mice. P1 and P2 generate a 250-bp product from the wild-type allele. P1 and P3 generate a 480-bp product from the mutant allele. (F) Real-time RT-PCR quantification of wild-type Asxl2 transcripts in Asxl2 wild-type (+/+), heterozygous (+/−) and mutant (−/−) hearts. The transcript levels in heterozygous and mutant hearts were 52.1% and 3.0% of that in wild-type hearts, respectively.
Figure 2
Figure 2. The expression pattern of the Asxl2 gene trap in developing embryos and adult organs.
Developing embryos and postnatal organs were stained with X-gal. (A) A whole-mount E9.5 embryo. (B–D) Cryo-sections of whole-mount stained E10.5 (B), E12.5 (C) and E14.5 (D) embryos. (E) Top view of a whole-mount stained postnatal heart showing strong X-gal staining in the heart but not in the aorta. (F) Cryo-section of a whole-mount stained postnatal heart. (G) Cryo-section of a piece of whole-mount stained skeletal muscle. The skeletal muscle in (G) and the heart in (F) were taken from the same animal and stained together. There was no detectable X-gal activity in the skeletal muscle. (H) Cryo-section of the retina in a whole-mount stained eye. Arrows point to stained cells in the ganglion cell layer. (I) Cross-section of a seminiferous tubule in a whole-mount stained testis showing expression in the primary spermatocytes. (J) Cross-section of an early tertiary follicle in a whole-mount stained ovary, showing expression in the oocyte (arrow). The blue color around the periphery of the follicle (arrowhead) was non-specific and also detected in wild-type samples. Phr: 1st pharyngeal arch; A: common atrial chamber; AS: aorta sac; SV: sinus venosus; Som: somites; V: common ventricular chamber; TA: truncus arteriosus; H: heart; AS: axial skeleton; NC: neocortex; RV: right ventricle; LV: left ventricle; ONL: outer nuclear layer.
Figure 3
Figure 3. Growth defect in Asxl2−/− animals.
(A) Comparison of body weights at weaning (3 weeks) for wild-type (blue columns), heterozygous (green columns), runt mutant (red columns) and non-runt mutant (black columns) mice. Error bars stand for standard deviation. M: males. F: females. (B) Growth of wild-type (blue line), heterozygous (green line) and non-runt mutant (black line) animals. Body weights were measured every two weeks after weaning and plotted against time. Animals of all three genotypes gained weight over time, but the weight gap between non-runt mutants and wild-type or heterozygous animals widens from 3 to 5 weeks and remains wide throughout the time course of measurements. The growth curve of runt mutants was not generated because very few runt mutants were born and all died early.
Figure 4
Figure 4. The loss of Asxl2 disrupts axial skeleton patterning.
(A, B) Ventral view of the sixth lumbar vertebra (L6) and the sacral vertebrae 1–4 (S1–4). (A) A wild-type skeleton. The wing-shaped lateral processes of S1, S2 and S3 fuse at the tip (arrowheads). The lateral processes of S4 do not fuse with those of S3. (B) A mutant skeleton. The lateral processes of S1, S2 and S3 fail to fuse. Arrowheads point to the sites at which fusion should happen. The lateral processes of mutant S2 resemble those of wild-type S3; those of mutant S3 resemble those of wild-type S4. One of the lateral processes of mutant L6 resembles that of S1 (arrow). (C, D) Caudal view of isolated T6 and T7 vertebrae, showing anterior transformation in the mutant. The wild-type T6 and T7 differ in the length of the neural spine (arrows) and the arch of the attached ribs (C). The mutant T7 resembles T6 in both the neural spine and the arch of ribs. (E, F) Caudal view of the first thoracic vertebrae (T1) in a heterozygous skeleton (E) and a mutant skeleton (F). The mutant T1 is split at the dorsal midline (asterisk).
Figure 5
Figure 5. Cardiac phenotypes of Asxl2−/− animals.
(A, B) H&E stainings of heart sections. The myocardial fibers, composed of cardiomyocytes, are well organized in the wild-type heart (A). In contrast, the heart of a moribund 5-month-old Asxl2−/− mouse shows cardiomyocyte disarray (B). (C, D) Examination of fibrosis in heart sections by Mason's trichrome (MTC) staining. Blue staining marks regions of fibrosis. The mutant (D) but not the wild-type (C) heart displays extensive interstitial fibrosis. (E) The heart/body weight ratios of mutant and control animals at 1 month, 2 months and 3 months after birth. The table below the chart shows the average ratio + standard deviation for each genotype and the p value for the time point. Error bars in the chart represent standard deviations. (F, G) Western blot analysis of the levels of H3K27me3 and H3K4me3 in wild-type and mutant hearts. As a control for equal loading, the blots were stripped and re-probed with an antibody against pan histone H3. The mutant heart has decreased level of H3K27me3 (F). There is no obvious change in the level of H3K4me3 (G).
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