Mammalian polycomb-mediated repression of Hox genes requires the essential spliceosomal protein Sf3b1

Abstract

Polycomb group (PcG) proteins are responsible for the stable repression of homeotic (Hox) genes by forming multimeric protein complexes. We show (1) physical interaction between components of the U2 small nuclear ribonucleoprotein particle (U2 snRNP), including Sf3b1 and PcG proteins Zfp144 and Rnf2; and (2) that Sf3b1 heterozygous mice exhibit skeletal transformations concomitant with ectopic Hox expressions. These alterations are enhanced by Zfp144 mutation but repressed by Mll mutation (a trithorax-group gene). Importantly, the levels of Sf3b1 in PcG complexes were decreased in Sf3b1-heterozygous embryos. These findings suggest that Sf3b1-PcG protein interaction is essential for true PcG-mediated repression of Hox genes.

Figure 1.

Physical interaction between Sf3b1 and PcG proteins. (A) Primary structure of Sf3b1 (Isono et al. 2001). Sf3b1 has WD-40 like repeats and PP2A repeats (usually termed HEAT motifs) originally identified in the PR65 subunit of protein phosphatase 2A. The N-terminal region (amino acids 1-489) and the internal region (amino acids 312-777), respectively, were associated with Rnf2 and Zfp144 in the yeast two-hybrid system. (B) GST pull-down assays probed with the Sf3b1-specific antibody. Nuclear extracts from ES cells were incubated with GST alone (lane 2) and GST fusions with Zfp144 (lane 3), Rnf2 (lane 4), and the N-terminal (lane 5) or C-terminal (lane 6) regions of Rnf2. In lane 1, one-fifteenth volume of extract sample used in a reaction was applied. (C) Coimmunoprecipitation with whole-cell lysates. Lysate of a mouse embryo at 10.75-11.25 dpc was incubated with or without antibodies to Zfp144, Rnf2, or TMG. Each precipitant was divided into two, and each was used in immunoblotting with Sf3b1 and Rnf2 or Sp1 and Sf3b4 antibodies. Anti-Sp1 was used as a negative control. In each “extract” lane, one-fortieth volume of extract sample used in a reaction was applied. In addition, lysates treated with ethidium bromide (EtBr) were also used to exclude DNA. (D) Coimmunoprecipitation with chromatin-rich lysates. Insoluble nuclear extract from an embryo at 10.75-11.25 dpc was prepared by osmotic shock, solubilized by sonication, and then incubated with antibodies. Phc1 is a mouse homolog to the fly PcG protein, Polyhomeotic (the fly protein homologous to mouse Phc1). B2.4-4 is a polyclonal antibody against the Xenopus Sf3b1, which has an epitope different from the monoclonal antibody (αSf3b1). In the “INE” lane, one-fortieth volume of the insoluble extract used in a reaction was applied.

Figure 2.

Skeletal abnormalities and ectopic Hox expressions in Sf3b1^+/- mice. Cervical-thoracic (lateral view) and thoracic-sacral (ventral view) regions of wild-type (A,D) and two independent Sf3b1^+/- (B,C,E,F) newborns are shown. Numbering of vertebrae of mutants makes them consistent with those of wild type. In wild type, C6 and T2 vertebrae have characteristic ventral processes as indicated by arrowheads (orange) and a prominent spinous process (yellow circle), respectively. An arrow indicates the disappearance of the T13 rib in the Sf3b1^+/- mutant. (G,H) the lateral view of cervical-thoracic region and the ventral view of rib cage of the Sf3b1^+/-Zfp144^+/- compound mutant. Ectopic ribs with C7 are represented by black arrowheads. An asterisk indicates an additional ossification center. Ribs numbered as 1-6 associate with T1-T6 vertebrae, respectively. The right ectopic rib with C7 separates from the rib with T1 on the sternum, as shown by green arrowheads. (I-Q) RNA in situ hybridization of Hox genes in embryos at 11.5 dpc. Boundaries of prevertebrae in lateral view are indicated by lines and the prevertebrae are numbered from prospective C1. Vertebral arteries are indicated by arrowheads and anterior boundaries of expressions are indicated by arrows. In Sf3b1^+/- embryos, anterior boundaries of Hoxb6, Hoxb8, and Hoxc6 expressions are shown to shift by sixth, seventh, and sixth prevertebrae, respectively. (R) Summary of Hoxa5, Hoxb3, Hoxb4, Hoxb6, Hoxb8, Hoxc6, and Hoxd4 expressions in the paraxial mesoderm is depicted schematically. Each black (wild type) or gray (Sf3b1^+/-) bar represents expressional regions and a number of bars show individual embryos. Segment numbers are counted from the prospective C1.

Figure 3.

Comparison of wild-type and Sf3b1^+/- embryos. Genotyping of embryos was performed with total DNA from yolk sacs. (A) In vitro splicing assay. The soluble fraction of a nuclear extract was prepared from wild-type or Sf3b1^+/- embryos at 10.5 dpc. Equal amounts (60 μg) of total protein were confirmed by Western blotting with anti-Sf3b1 and anti-Rnf2 antibodies (upper panels) and subjected to a splicing assay (lower panel). One-step RT-PCR revealed that unspliced forms consisting of two exons and an intron (upper bands) as the RNA substrates were converted into spliced forms (lower bands) by the splicing reaction. Band intensities were measured by the image-processing program Image J and the ratios of Sf3b1^+/- to wild type were obtained.(B) Northern analysis of total RNAs (15 μg each) from independent embryos at 12.5 dpc. Ethidium-bromide-stained 28S rRNAs prior to transfer to membranes are displayed below their blots. Intensity of each detected band was normalized by their rRNAs and then the ratios of Sf3b1^+/- to wild type were calculated (indicated below their panels). (C) Immunoprecipitation assay with embryos at 11.5 dpc. Three independent embryos with the same genotype were sonicated individually, and the soluble lysates were mixed and then divided into four. Each was immunoprecipitated with a specific antibody. (D) ChIP assay with ES cells. Cross-linked chromatins were immunoprecipitated with or without indicated antibodies and subjected to amplification of Hox and Hprt regions (260-480 bp) by PCR. The amplified regions were detected in 1.5% agarose gels.