hCAF1, a new regulator of PRMT1-dependent arginine methylation.

Protein arginine methylation is an emergent post-translational modification involved in a growing number of cellular processes, including transcriptional regulation, cell signaling, RNA processing and DNA repair. Although protein arginine methyltransferase 1 (PRMT1) is the major arginine methyltransferase in mammals, little is known about the regulation of its activity, except for the regulation induced by interaction with the antiproliferative protein BTG1 (B-cell translocation gene 1). Since the protein hCAF1 (CCR4-associated factor 1) was described to interact with BTG1, we investigated a functional link between hCAF1 and PRMT1. By co-immunoprecipitation and immunofluorescence experiments we demonstrated that endogenous hCAF1 and PRMT1 interact in vivo and colocalize in nuclear speckles, a sub-nuclear compartment enriched in small nuclear ribonucleoproteins and splicing factors. In vitro methylation assays indicated that hCAF1 is not a substrate for PRMT1-mediated methylation, but it regulates PRMT1 activity in a substrate-dependent manner. Moreover, small interfering RNA (siRNA)-mediated silencing of hCAF1 in MCF-7 cells significantly modulates the methylation of endogenous PRMT1 substrates. Finally, we demonstrated that in vitro and in the cellular context, hCAF1 regulates the methylation of Sam68 and histone H4, two PRMT1 substrates. Since hCAF1 and PRMT1 have been involved in the regulation of transcription and RNA metabolism, we speculate that hCAF1 and PRMT1 could contribute to the crosstalk between transcription and RNA processing.

Sumoylation of the estrogen receptor alpha hinge region regulates its transcriptional activity.

The steroid hormone 17beta-estradiol (estrogen) plays a significant role in the normal physiology of the mammary gland and breast cancer development primarily through binding to its receptor, the estrogen receptor alpha (ERalpha). ERalpha is a nuclear transcription factor undergoing different types of posttranslational modifications, i.e. phosphorylation, acetylation, and ubiquitination, which regulate its transcriptional activation and/or stability. Here we identify ERalpha as a new target for small ubiquitin-like modifier (SUMO)-1 modification in intact cells and in vitro. Moreover, ERalpha sumoylation occurs strictly in the presence of hormone. SUMO-1 appears to regulate ERalpha-dependent transcription. Using a series of mutants, we demonstrated that ERalpha is sumoylated at conserved lysine residues within the hinge region. Mutations that prevented SUMO modification impaired ERalpha-induced transcription without influencing ERalpha cellular localization. In addition to identifying protein inhibitor of activated signal transducer and activator of transcription (PIAS)1 and PIAS3 as E3 ligases for ERalpha, we also found that PIAS1 and PIAS3, as well as Ubc9, modulated ERalpha-dependent transcription independently from their SUMO-1 conjugation activity. These findings identify sumoylation as a new mechanism modulating ERalpha-dependent cellular response and provide a link between the SUMO and estrogen pathways.

BTG2 antiproliferative protein interacts with the human CCR4 complex existing in vivo in three cell-cycle-regulated forms.

The yeast CCR4-NOT complex exists in two forms (1.0 and 1.9 MDa) that share several common subunits, including yCCR4, yCAF1 and five NOT proteins (NOT1-5). Here, we report that different complexes containing mammalian homologs of CCR4-NOT subunits exist in mammalian cells, with estimated sizes of approximately 1.9 MDa, approximately 1 MDa and approximately 650 kDa, and that BTG2, a member of a protein family with antiproliferative functions, can associate with these complexes. Immunoprecipitation and gel filtration experiments established that BTG2 interacts in vivo with hCCR4 protein via hCAF1 and hPOP2. Moreover, we show that hCCR4, as well as hCAF1 and BTG2, modulate the transcription regulation mediated by ERalpha. Finally, we demonstrate that the cellular localization of hCAF1 and the cell content in hCAF1-containing complexes change as cells progress from quiescence to S phase. These findings suggest that the different regulatory pathways in which hCAF1 is involved, notably transcription regulation and mRNA turnover, may occur through distinct CCR4 complexes in the course of cell-cycle progression.