Mouse cofactor of BRCA1 (Cobra1) is required for early embryogenesis

Abstract

Background: Negative elongation factor (NELF) is a four-subunit protein complex conserved from Drosophila to humans. In vitro biochemical and tissue culture-based studies have demonstrated an important role of NELF in controlling RNA polymerase II (Pol II) pausing in transcription. However, the physiological significance of NELF function is not clear due to the lack of any genetic systems for studying NELF.

Principal findings: Here we show that disruption of the mouse B subunit of NELF (NELF-B), also known as cofactor of BRCA1 (Cobra1), causes inner cell mass (ICM) deficiency and embryonic lethality at the time of implantation. Consistent with the phenotype of the Cobra1 knockout (KO) embryos, knockdown of Cobra1 in mouse embryonic stem cells (ESCs) reduces the efficiency of colony formation and increases spontaneous differentiation. Cobra1-depleted ESCs maintain normal levels of Oct4, Nanog, and Sox2, master regulators of pluripotency in ESCs. However, knockdown of Cobra1 leads to precocious expression of developmental regulators including lymphoid enhancer-binding factor 1 (Lef1). Chromatin immunoprecipitation (ChIP) indicates that Cobra1 binds to the Lef1 promoter and modulates the abundance of promoter-bound RNA polymerase.

Conclusions: Cobra1 is essential for early embryogenesis. Our findings also indicate that Cobra1 helps maintain the undifferentiated state of mESCs by preventing unscheduled expression of developmental genes.

Figure 1. Cobra1 is essential for early embryonic development.

A. Illustration of the wild type Cobra1 locus (top) and a portion of the targeting construct (bottom). The short (left) and long (right) homology arms encompass genomic regions 1.1 kb upstream of exon 1 and ∼8.4 kb downstream of exon 4, respectively. Also indicated are loxP (red arrows), FRT sites (crescents), exons (open bars), BamH1 sites (B), promoter (solid arrow), 5′ probe (blue bar), and PCR primers (block arrows). B. Southern blot for the BamH1-digested genomic DNA from 3 wk old pups of Cobra1^+/− intercross. C. Summary of the genotypes from the Cobra1^+/− intercrosses. D and E. Phenotype of Cobra1^+/+ (D) and Cobra1^−/− (E) embryos retrieved at E8 of embryonic development. The block arrows point to embryo proper.

Figure 2. Impaired outgrowth of Cobra1^−/− blastocysts.

A. In vitro developed blastocysts (Cobra1^+/+ and Cobra1^−/−) from two-cell stage embryos. B. Cobra1 deletion resulted in elevated incidence of outgrowths with defective or no ICM derivatives. C–D. Normal (Cobra^+/+; C) and defective (Cobra^−/−; D) blastocyst outgrowth four days after in vitro culture. The ICM derivatives (*) and trophoblast giant cells (TGC) are indicated.

Figure 3. Cobra1 knockdown in mouse ESCs results in reduced colony formation and increased spontaneous differentiation.

A. Knockdown of Cobra1 by siRNA does not affect protein levels of Oct4, Nanog, or Sox2. B–E. Cobra1 knockdown reduces the colony formation capability of mESCs. F. Quantitation of the colony formation efficiency in B–E. The value for the mock-transfected cells is set at 1. G–H. Representatives of AP-stained undifferentiated (G) and differentiated (H) ESC colonies. I. Quantitation of the percentage of differentiated colonies.

Figure 4. Cobra1 is required for transcriptional repression of Lef1 in ESCs.

A. Real-time RT-PCR of Cobra1 mRNA in control and Cobra1-knockdown cells 3 and 6 days after siRNA transfection. B. Lef1 mRNA in control and Cobra1-knockdown cells. C. Cobra1 ChIP at the promoter and exon 1 of the Lef1 gene in parental ESCs. Preimmune antiserum was used as the negative control. D. Pol II ChIP at the promoter and exon 1 of the Lef1 locus in control and Cobra1-knockdown ESCs six days after transfection.