QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 15 November 2006
doi: 10.1242/dev.02677

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, S. Y. Articles by Schumacher, A. Search for Related Content PubMed PubMed Citation Articles by Kim, S. Y. Articles by Schumacher, A. Social Bookmarking                         What's this?

Juxtaposed Polycomb complexes co-regulate vertebral identity

¹ Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA.
² Department of Genetics, University of North Carolina, Chapel Hill, North Carolina 27599, USA.

View larger version (97K): [in this window] [in a new window]   Fig. 1. Coexpression of eed and Bmi1 in wild-type embryos. Representative images of flat-mount E8.5 embryos (A,E) and sectioned E12.5 embryos (B,F) mRNA in situ hybridization with eed (A,B) and Bmi1 antisense cRNA probes (E,F). Note coexpression of eed and Bmi1 mRNA in somites (arrowheads in A,E) and prevertebrae (arrowheads in B,F), as well as neuroectoderm (arrows in A,B,E,F). Control hybridization with eed and Bmi1 sense cRNA probes revealed no specific signal on sectioned E12.5 embryos (C,G). Immunohistochemistry detected nuclear expression of EED and BMI1 in E12.5 prevertebrae (D,H). The images were captured at 20x (B,C,F,G), 50x (A,E), and 1000x (D,H).

View larger version (35K): [in this window] [in a new window]   Fig. 2. Homeotic transformations and ectopic Hox gene expression in eedxBmi1 mutants. (A) The chart summarizes the penetrance of posterior homeotic transformations in P0 skeletons. The inset depicts the color-coding of the five transformations assessed. The columns represent the percentage of skeletons exhibiting the cervical (C), thoracic (T), lumbar (L) or sacral (S) transformations. Unilateral and bilateral transformations were combined. See the legend for Fig. 3 for a description of the transformations. The genotypes and the number (n) of skeletons analyzed per genotype are shown at the bottom of the figure. (B) The chart summarizes the penetrance of ectopic Hox gene expression in prevertebrae of E12.5 embryos. The inset depicts the color-coding of the four Hox genes tested (Hoxa5, b6, c8 and b4). The columns indicate the percentage of embryos exhibiting ectopic Hox gene expression. The genotypes and the number (n) of embryos analyzed per genotype and Hox probe are shown below the columns.

View larger version (84K): [in this window] [in a new window]   Fig. 3. Ectopic Hox gene expression and skeletal analysis in eedxBmi1 mutants. Genotypes are indicated above the panels. (A,B) Representative images of sectioned wild-type and mutant embryos following mRNA in situ hybridization with Hoxa5 (A) and Hoxb4 (B) antisense probes. Arrows denote the anterior Hox gene expression boundary and the first prevertebra. All images were captured at 50 x magnification. (C-G) Homeotic transformations and vertebral abnormalities in cervical (C-E), thoracic (F) and lumbar regions (G). (C) Two ectopic ossification centers (arrowheads) and broadening of the neural arch (asterisk) constitute Bmi1-specific defects (van der Lugt et al., 1994). (D) The first cervical vertebra reveals a significantly broader ventral arch in eed and Bmi1 mutant skeletons (arrow), which is likely to result from an incomplete regression of the vertebral body during embryogenesis (Verbout, 1985). As all vertebrae posterior to C1 contain a body, incomplete regression of the body in C1 represents a posterior homeotic transformation (C1C2). (D,E) The rudimentary vertebral body also broadens the anterior arch, which is visible in both rostral and lateral views of C1 (arrowheads). (E) The presence of ribs transforms the seventh cervical vertebra toward the identity of the first thoracic vertebra (C7T1*). (F) Posterior transformation of the seventh thoracic vertebra is evident from lack of sternal fusion of the ribs (T7T8). (G) Fusion with the ilial bones represents a homeotic transformation of the sixth lumbar vertebra toward a sacral identity (L6S1*). Images were captured at 20x (C,D), 16x (E), 12x (F) and 18x magnification (G). pv, prevertebra.

View larger version (57K): [in this window] [in a new window]   Fig. 4. Normal Hox gene expression in E8.5 eed mutant embryos. Representative flat-mount images of wild-type (A,C) and l7Rn51989SB/1989SB mutant embryos (B,D) following mRNA in situ hybridization with Hoxa5 (A,B) and Hoxb6 (C,D) antisense cRNA probes. Note that wild-type and l7Rn51989SB/1989SB mutant embryos present the same anterior Hox gene expression boundaries in somites (s). Arrows denote the first somite and the anterior Hox gene expression boundary. All images were captured at 50x magnification.

View larger version (93K): [in this window] [in a new window]   Fig. 5. EED and BMI1 engage in separate protein complexes. Immunoprecipitation of EED (left column) from E12.5 trunk identified three isoforms of approximately 50 and 75 kDA. Note the absence of the EED isoforms in the input lane, indicating low levels of EED expression. EED co-immunoprecipitated with EZH2 and dimethylated H3-K27, which presented a molecular weight of approximately 100 and 17 kDA, respectively. Immunoprecipitation of BMI1 (right column) from E12.5 trunk revealed three isoforms in the 39-41 kDA range. A fourth band, slightly larger than the triplet, was occasionally detected in mock immunoprecipitation without the BMI1 antibody and, hence, could not be confirmed as a BMI1 isoform. RING1B co-immunoprecipitated with BMI1 as a band of approximately 38 kDa. Note that reciprocal co-immunoprecipitation did not detect EED and BMI1 in a common protein complex, and trimethylated H3-K27 did not pull down with either complex. YY1, as a band of 49 kDA, co-immunoprecipitated with both EED and BMI1. IP, immunoprecipitation; beads, mock immunoprecipitation without antibody; input, 2 µg protein lysate; H3M2K27, dimethylated histone 3-K27, H3M3K27, trimethylated histone 3-K27.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Juxtaposition of EED and BMI1 complexes at Hox target loci. (A) A schematic representation of the Hoxc8 upstream region is shown. The blue arrow depicts the Hoxc8 transcription start site, and the green lines demark two upstream regions identified by ChIP located immediately upstream (a) and 1.5 kb upstream (b) of the Hoxc8 transcription start site. Red asterisks indicate putative YY1-binding sites. (B) ChIP using antibodies against EED, BMI1, YY1 and trimethylated H3-K27 detected the proteins at the two upstream regions (a and b) of the Hoxc8 locus. While dimethylated H3-K27 localized to region b of the Hoxc8 locus, results for region a were variable and, hence, inconclusive. EED, BMI1, YY1 and trimethylated H3-K27 were also detected 1.5 kb upstream of the Hoxa5 transcription start site. ChIP using an antibody against Fpn1 served as a negative control. (C) ChIP detected differential binding of EED and BMI1 binding to Hox regulatory regions in dissected anterior and posterior regions of E12.5 trunk. In all cases, input encompassed 1% of the chromatin used for ChIP, and mock ChIP without antibody served as additional negative controls. As a negative control, EED and BMI1 did not associate with the ß-actin promoter.

View larger version (13K): [in this window] [in a new window]   Fig. 7. Model of PcG complex assembly at Hox target loci. Nucleosomes and methylated H3-K27 are depicted as blue ovals and orange diamonds, respectively. Clustered YY1 binding sites are indicated in red. The purple line represents potential cooperative interactions between RNA molecules and PcG proteins. Based on previous PcG and trxG mutant analysis (Yu et al., 1995; Yu et al., 1998; Deschamps et al., 1999; Tomotsune et al., 2000), the transition from initiation to maintenance of Hox gene expression should occur between E9 and E10 of mouse development, herein referred to as `Early Maintenance' phase. Complex assembly during the `Late Maintenance' phase was ascertained from E12.5 embryos.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter