Uncoupling of X-linked gene silencing from XIST binding by DICER1 and chromatin modulation on human inactive X chromosome.

In mammals, X-inactivation process is achieved by the cis-spreading of long noncoding Xist RNA over one of the female X chromosomes. The Xist binding accumulates histones H3 methylation and H4 hypoacetylation required for X inactivation that leads to proper dosage compensation of the X-linked genes. Co-transcription of Tsix, an antisense copy of Xist, blocks the Xist coating on the Xi. In mice ES cells, an RNase III enzyme Dicer1 disrupts Xist binding and methylated H3K27me3 accumulation on the Xi. Later, multiple reports opposed these findings raising a question regarding the possible role of Dicer1 in murine X silencing. Here, we show that reduction of DICER1 in human female cells increases XIST transcripts without compromising the binding of the XIST and histone tail modifications on the Xi. Moreover, DICER1-depleted cells show differential upregulation of many human X-linked genes by binding different amounts of acetylated histone predominantly on their active promoter sites. Therefore, X-linked gene silencing, which is thought to be coupled with the accumulation of XIST and heterochromatin markers on Xi can be disrupted in DICER1 depleted human cells. These results suggest that DICER1 has no apparent effect on the recruitment of heterochromatic markers on the Xi but is required for inactivation of differentially regulated genes for the maintenance of proper dosage compensation in differentiated cells.

TIM family proteins promote the lysosomal degradation of the nuclear receptor NUR77.

T cell immunoglobulin and mucin domain (TIM) proteins are cell-surface signaling receptors in T cells and scavenger receptors in antigen-presenting cells and kidney tubular epithelia. Here, we demonstrated a function for TIM proteins in mediating the degradation of NUR77, a nuclear receptor implicated in apoptosis and cell survival. TIM proteins interacted with and mediated the lysosomal degradation of NUR77 in a phosphoinositide 3-kinase-dependent pathway. We also showed dynamic cycling of TIM-1 to and from the cell surface through clathrin-dependent constitutive endocytosis. Blocking this process or mutating the phosphatidylserine-binding pocket in TIM-1 abrogated TIM-1-mediated degradation of NUR77. In an in vitro model of kidney injury, silencing TIM-1 increased NUR77 abundance and decreased epithelial cell survival. These results show that TIM proteins may affect immune cell function and the response of the kidney to injury.

Cullin4B/E3-ubiquitin ligase negatively regulates beta-catenin.

Beta-catenin is the key transducer of Wingless-type MMTV integration site family member (Wnt) signalling, upregulation of which is the cause of cancer of the colon and other tissues. In the absence of Wnt signals, beta-catenin is targeted to ubiquitin-proteasome-mediated degradation. Here we present the functional characterization of E3-ubiquitin ligase encoded by cul4B. RNAi-mediated knock-down of Cul4B in a mouse cell line C3H T10 (1/2) results in an increase in beta-catenin levels. Loss-of-function mutation in Drosophila cul4 also shows increased beta-catenin/Armadillo levels in developing embryos and displays a characteristic naked-cuticle phenotype. Immunoprecipitation experiments suggest that Cul4B and beta-catenin are part of a signal complex in Drosophila, mouse and human. These preliminary results suggest a conserved role for Cul4B in the regulation of beta-catenin levels.

Cloning and characterization of mouse cullin4B/E3 ubiquitin ligase.

Heat induced differentiation of mouse embryonal carcinoma cells PCC4 has been reported earlier. We have further characterized the phenotype of the differentiated cells and by DD-RT-PCR identified several partial cDNAs that are differentially expressed during differentiation. Nucleotide homology search revealed that the genes corresponding to some of the up-regulated partial cDNAs are indeed part of differentiation pathway. 5'extension of an EST that has homology to one of the partial cDNAs led to the identification of mouse cullin4B. Cullin4B is coded by a separate gene and has a unique and longer amino-terminal end with a putative nuclear localization signal sequence (NLS). We have cloned, expressed and raised antibodies against the amino and carboxy-terminal halves of cullin4B. Immuno staining of differentiated PCC4 cells with N-terminal Cul4B antibody showed enhanced expression of Cul4B and its translocation into the nucleus upon differentiation. Transient transfection of a chimeric gene encoding the N-terminal part of Cul4B fused to green fluorescent protein into PCC4 cells revealed that the protein was localized in the nucleus confirming the functional significance of the putative NLS. Since cullins are involved in recognition of specific proteins for degradation, based on the evidence presented here, we hypothesize that cullin4B is probably involved in differentiation specific degradation/modification of nuclear proteins.