Enhanced gene trapping in mouse embryonic stem cells.

Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.

Proteasomal degradation of the multifunctional regulator YB-1 is mediated by an F-Box protein induced during programmed cell death.

F-Box proteins (FBPs) are variable adaptor proteins that earmark protein substrates for ubiquination and destruction by the proteasome. Through their N-terminal F-box motif, they couple specific protein substrates to a catalytic machinery known as SCF (Skp-1/Cul1/F-Box) E3-ubiquitin ligase. Typical FBPs bind the specific substrates in a phosphorylation dependent manner via their C-termini using either leucine rich repeats (LRR) or tryptophan-aspartic acid (WD40) domains for substrate recognition. By using a gene trap strategy that selects for genes induced during programmed cell death, we have isolated the mouse homolog of the hypothetical human F-Box protein 33 (FBX33). Here we identify FBX33 as a component of an SCF E3-ubiquitin ligase that targets the multifunctional regulator Y-box binding protein 1 (YB-1)/dbpB/p50 for polyubiquitination and destruction by the proteasome. By targeting YB-1 for proteasomal degradation, FBX33 negatively interferes with YB-1 mediated functions. In contrast to typical FBPs, FBX33 has no C-terminal LRR or WD40 domains and associates with YB-1 via its N-terminus. The present study confirms the existence of a formerly hypothetical F-Box protein in living cells and describes one of its substrates.

Thyroid hormone-regulated enhancer blocking: cooperation of CTCF and thyroid hormone receptor.

The highly conserved, ubiquitously expressed, zinc finger protein CTCF is involved in enhancer blocking, a mechanism crucial for shielding genes from illegitimate enhancer effects. Interestingly, CTCF-binding sites are often flanked by thyroid hormone response elements (TREs), as at the chicken lysozyme upstream silencer. Here we identify a similar composite site positioned upstream of the human c-myc gene. For both elements, we demonstrate that thyroid hormone abrogates enhancer blocking. Relief of enhancer blocking occurs even though CTCF remains bound to the lysozyme chromatin. Furthermore, chromatin immunoprecipitation analysis of the lysozyme upstream region revealed that histone H4 is acetylated at the CTCF-binding site. Loss of enhancer blocking by the addition of T3 led to increased histone acetylation, not only at the CTCF site, but also at the enhancer and the promoter. Thus, when TREs are adjacent to CTCF-binding sites, thyroid hormone can regulate enhancer blocking, thereby providing a new property for what was previously thought to be constitutive enhancer shielding by CTCF.