The CCR4-NOT complex (1 mDa in size), consisting of the proteins CCR4, CAF1, and NOT1 to NOT5, regulates gene expression both positively and negatively and is distinct from other large transcriptional complexes in Saccharomyces cerevisiae such as SNF/SWI, TFIID, SAGA, and RNA polymerase II holoenzyme. The physical and genetic interactions between the components of the CCR4-NOT complex were investigated in order to gain insight into how this complex affects the expression of diverse genes and processes. The CAF1 protein was found to be absolutely required for CCR4 association with the NOT proteins, and CCR4 and CAF1, in turn, physically interacted with NOT1 through its central amino acid region from positions 667 to 1152. The NOT3, NOT4, and NOT5 proteins had no significant effect on the association of CCR4, CAF1, and NOT1 with each other. In contrast, the NOT2, NOT4, and NOT5 interacted with the C-terminal region (residues 1490 to 2108) of NOT1 in which NOT2 and NOT5 physically associated in the absence of CAF1, NOT3, and NOT4. These and other data indicate that the physical ordering of these proteins in the complex is CCR4-CAF1-NOT1-(NOT2, NOT5), with NOT4 and NOT3 more peripheral to NOT2 and NOT5. The physical separation of CCR4 and CAF1 from other components of the CCR4-NOT complex correlated with genetic analysis indicating partially separate functions for these two groups of proteins. ccr4 or caf1 deletion suppressed the increased 3-aminotriazole resistance phenotype conferred by not mutations, resulted in opposite effects on gene expression as compared to several not mutations, and resulted in a number of synthetic phenotypes in combination with not mutations. These results define the CCR4-NOT complex as consisting of at least two physically and functionally separated groups of proteins.

Protein ubiquitination has been implicated in the intracellular biochemical events transduced by TGF-beta receptor via different mechanisms including the degradation of Smads or their binding proteins. Here we show that loss of Itch E3 ligase in mouse embryonic fibroblasts (MEFs) results in reduced susceptibility of TGF-beta-induced cell growth arrest and decreased phosphorylation of Smad2, without apparent alteration in protein levels for Smad2, Smad4, and Smad7 in Itch-/- MEFs. Itch promotes ubiquitination of Smad2 and augments Smad2 phosphorylation that requires an intact ligase activity of Itch. Moreover, Itch facilitates complex formation between TGF-beta receptor and Smad2 and enhances TGF-beta-induced transcription. This study reveals a previously unrecognized positive TGF-beta signaling pathway via proteolysis-independent ubiquitination.

Estrogen receptor (ER)alpha and -beta interact with a variety of coactivator proteins, most notably members of the steroid receptor coactivator (SRC) family, and these interactions have been shown to be regulated by estrogenic ligands and growth factor signaling. Here, using fluorescence resonance energy transfer (FRET), the selectivity of different stimulants on ERalpha and -beta interactions with coactivator receptor interaction domains (RIDs) were examined in living cells. We first show that ERalpha and ERbeta homo- and heterodimers form in vivo independently of the presence of 17beta-estradiol (E2) or antiestrogens. We then demonstrate that E2 enhances interactions between ERalpha and the RIDs of SRC-1 and SRC-3, whereas the interaction between ERalpha with the SRC-2 RID is ligand independent. The transcriptionally inactive mutant ERalphaL539A showed no interaction with all three SRC RIDs. Similarly, treatment with the antagonists 4-hydroxytamoxifen and EM-652 abolished all interactions between ERalpha and the SRC RIDs. FRET data also demonstrate that, in contrast to ERalpha, ERbeta interacts with all three SRC RIDs in a ligand-independent manner. However, these interactions were further enhanced or stabilized by E2, whereas the antiestrogen EM-652 abolished all interactions. In the presence of both ERalpha and ERbeta, E2 treatment led to the recruitment of SRC RIDs to the nuclei. Finally, expression of the oncogenic activated ErbB-2/Neu protein specifically enhanced ERalpha but not ERbeta interactions with SRC RIDs to an extent similar to E2-stimulated interactions. In summary, using FRET, we demonstrated preferential interactions between ER isoforms and coactivators upon hormonal treatment and activation of a growth factor signal transduction pathway in living cells.

Forward genetic screens with ENU (N-ethyl-N-nitrosourea) mutagenesis can facilitate gene discovery, but mutation identification is often difficult. We present the first study in which an ENUinduced mutation was identified by massively parallel DNA sequencing. This mutation causes heterotaxy and complex congenital heart defects and was mapped to a 2.2-Mb interval on mouse chromosome 7. Massively parallel sequencing of the entire 2.2-Mb interval identified 2 single-base substitutions, one in an intergenic region and a second causing replacement of a highly conserved cysteine with arginine (C193R) in the gene Megf8. Megf8 is evolutionarily conserved from human to fruit fly, and is observed to be ubiquitously expressed. Morpholino knockdown of Megf8 in zebrafish embryos resulted in a high incidence of heterotaxy, indicating a conserved role in laterality specification. Megf8 C193R mouse mutants show normal breaking of symmetry at the node, but Nodal signaling failed to be propagated to the left lateral plate mesoderm. Videomicroscopy showed nodal cilia motility, which is required for left-right patterning, is unaffected. Although this protein is predicted to have receptor function based on its amino acid sequence, surprisingly confocal imaging showed it is translocated into the nucleus, where it is colocalized with Gfi1b and Baf60C, two proteins involved in chromatin remodeling. Overall, through the recovery of an ENU-induced mutation, we uncovered Megf8 as an essential regulator of left-right patterning.cardiogenesis ͉ left-right ͉ nodal

Werner Syndrome (WS) is an autosomal recessive disease characterized by premature aging and chromosome instability. The protein involved in WS, WRN, is a RecQ-type helicase that also has exonuclease activity. WRN has been demonstrated to bind to a variety of other proteins, including RPA, DNA-PKcs, and TRF2, suggesting that WRN is involved in DNA replication, repair, recombination, and telomere maintenance. In culture, WS cells show premature senescence, which can be overcome by transfection with an expression vector containing the gene for the catalytic subunit of telomerase. However, telomerase expression does not eliminate chromosome instability in WS cells, which led to the proposal that telomere loss is not the cause of the high rate of chromosome rearrangements in WS cells. In the present study, we have investigated how a WRN protein containing a dominant-negative mutation (K577M-WRN) influences the stability of telomeres in a human tumor cell line expressing telomerase. The results demonstrate an increased rate of telomere loss and chromosome fusion in cells expressing K577M-WRN. Expression of K577M-WRN results in reduced levels of telomerase activity, however, the absence of detectable changes in average telomere length demonstrates that WRN-associated telomere loss results from stochastic events involving complete telomere loss or loss of telomere capping function. Thus, telomere loss can contribute to chromosome instability in cells deficient in WRN regardless of the expression of telomerase activity.