E2f6 and Bmi1 cooperate in axial skeletal development

Abstract

Bmi1 is a Polycomb Group protein that functions as a component of Polycomb Repressive Complex 1 (PRC1) to control axial skeleton development through Hox gene repression. Bmi1 also represses transcription of the Ink4a-Arf locus and is consequently required to maintain the proliferative and self-renewal properties of hematopoietic and neural stem cells. Previously, one E2F family member, E2F6, has been shown to interact with Bmi1 and other known PRC1 components. However, the biological relevance of this interaction is unknown. In this study, we use mouse models to investigate the interplay between E2F6 and Bmi1. This analysis shows that E2f6 and Bmi1 cooperate in the regulation of Hox genes, and consequently axial skeleton development, but not in the repression of the Ink4a-Arf locus. These findings underscore the significance of the E2F6-Bmi1 interaction in vivo and suggest that the Hox and Ink4a-Arf loci are regulated by somewhat different mechanisms.

Figure 1. E2f6 mutant mice are fully viable and display axial skeletal transformations

(A) Generation of the E2f6^−/− mice. The E2f6 genomic locus comprises 8 exons that include non-coding sequences (black boxes) coding sequences (gray boxes) and an alternatively spliced exon 2 (white box). DBD, DNA binding domain; DIM, dimerization domain; MB, marked box domain. The E2F6 mutant allele was generated by replacing coding sequences of exons 4 through 8 with a PGK-neo cassette that includes a STOP codon at the beginning. PGK-neo, neomycin resistance gene under the regulation of the PGK promoter for positive selection. Disruption of the E2f6 locus was confirmed by (B) Western of lysate from Wild-type and E2f6^−/− MEFs. (C) Survival curve of E2f6 wild-type, heterozygote and mutant mice followed for over 500 days. (D) Ventral view of axial skeletons of newborn E2f^+/+ and E2f6^−/− mice stained with alcian blue (cartilage) and alzarin red (bone). E2f6^−/− mice display two axial skeletal transformations, the thirteenth thoracic vertebra (T13) is transformed into the first lumbar vertebra (L1) as shown by the degeneration of the thirteenth ribs and the sixth lumbar vertebra (L6) is transformed into the first sacral vertebra (S1) as evidenced by the formation of the sacraliliac joints.

Figure 2. E2F6-loss does not modulate the hematopoietic defect within Bmi1 mutant mice

(A) Cell counts of single cell preparations made from the bone marrow, spleen, and thymus of eight week old mice. E2f6^+/+;Bmi1^+/+ n=2; E2f6^−/−;Bmi1^+/+ n=2; E2f6^+/+;Bmi1^−/− n=3; E2f6^+/−;Bmi1^−/− n=1; E2f6^−/−;Bmi1^−/− n=3. FACS analysis of single cell preparations made from the thymus (B) and bone marrow (C) and immunostained with the indicated antibodies. Results are presented as percentage of cells. E2f6^+/+;Bmi1^+/+ n=2; E2f6^−/−;Bmi1^+/+ n=2 for part B, n=3 for part C; E2f6^+/+;Bmi1^−/− n=3; E2f6^+/−;Bmi1^−/− n=1 for part B, n=2 for part C; E2f6^−/−;Bmi1^−/− n=3.

Figure 3. E2f6^−/−;Bmi1^−/− and Bmi1^−/− mice display defects in gross cerebellar structure

Hematoxylin and eosin staining of cerebellum sections from the midline of 8 week old wild-type, Bmi1 mutant, and E2f6;Bmi1 compound mutant mice.

Figure 4. Cell cycle properties of E2f6 and Bmi1 mutant MEFs

Mouse embryonic fibroblasts of wild-type (υ), Bmi1 mutant (σ, 5), and E2f6;Bmi1 compound mutant (Σ, λ) mice were assayed for (A) asynchronous proliferation rate, (B) senescence properties, (C) S-phase re-entry following serum withdrawal and re-addition, and (D) p16^INK4a and p19^ARF expression by RT-PCR from serum-starved cells.

Figure 5. E2f6 and Bmi1 show a genetic interaction in the development of the axial skeleton

(A) Alcian blue (cartilage) and alzarin red (bone) stainings of wild-type, Bmi1 mutant and E2f6;Bmi1 compound mutant mice at E18.5. The following skeletal transformations are depicted in the Bmi1 and E2f6:Bmi1 compound mutant mice: An extra piece of bone anterior to the first cervical vertebra (C1); the C1 vertebra is transformed into the second cervical vertebra (C2) as evidenced by the altered morphology; the seventh cervical vertebra (C7) is transformed into the first thoracic vertebra (T1) as shown by the presence of vertebrosternal ribs. The right panel shows the novel C5 to C6 transformation present in the E2f6 mutant and E2f6;Bmi1 compound mutant mice. This is evidenced by the presence of a piece of cartlidge on C5 instead of on C6. (B) Penetrance of axial skeletal transformations in E2f6;Bmi1 compound mutant embryos.

Figure 6. E2f6 and Bmi1 co-regulate Hox genes

(A) Real-time PCR analysis of Hox genes in MEFs. (B) Chromatin immunoprecipitation analysis of mouse embryonic stem cells. Sonicated, cross-linked chromatin was immunoprecipitated with a Bmi1, E2F6, or control IgG antibody and the purified DNA was analyzed by PCR with primers specific for the promoter of HoxA7, HoxA10, HoxA11, Arf, or a control sequence (1kb upstream of the E2F1 promoter).