Mechanism of hsp70i gene bookmarking.

In contrast to most genomic DNA in mitotic cells, the promoter regions of some genes, such as the stress-inducible hsp70i gene that codes for a heat shock protein, remain uncompacted, a phenomenon called bookmarking. Here we show that hsp70i bookmarking is mediated by a transcription factor called HSF2, which binds this promoter in mitotic cells, recruits protein phosphatase 2A, and interacts with the CAP-G subunit of the condensin enzyme to promote efficient dephosphorylation and inactivation of condensin complexes in the vicinity, thereby preventing compaction at this site. Blocking HSF2-mediated bookmarking by HSF2 RNA interference decreases hsp70i induction and survival of stressed cells in the G1 phase, which demonstrates the biological importance of gene bookmarking.

Structural and topological studies on the lipid-mediated assembly of a membrane-associated lipomannan in Micrococcus luteus.

The biosynthesis of three mannolipids and the presence of a membrane-associated lipomannan in Micrococcus luteus (formerly Micrococcus lysodeikticus) were documented over 30 years ago. Structural and topological studies have been conducted to learn more about the possible role of the mannolipids in the assembly of the lipomannan. The major mannolipid has been purified and characterized as alpha-D-mannosyl-(1 --> 3)-alpha-D-mannosyl-(1 --> 3)-diacylglycerol (Man2-DAG) by negative-ion electrospray-ionization multistage mass spectrometry (ESI-MSn). Analysis of the fragmentation patterns indicates that the sn-1 position is predominantly acylated with a 12-methyltetradecanoyl group and the sn-2 position is acylated with a myristoyl group. The lipomannan is shown to be located on the exterior face of the cytoplasmic membrane, and not exposed on the surface of intact cells, by staining of intact protoplasts with fluorescein isothiocyanate (FITC)-linked concanavalin A (Con A). When cell homogenates of M. luteus are incubated with GDP-[3H]mannose (GDP-Man), [3H]mannosyl units are incorporated into Man1-2-DAG, mannosylphosphorylundecaprenol (Man-P-Undec) and the membrane-associated lipomannan. The addition of amphomycin, an inhibitor of Man-P-Undec synthesis, had no effect on the synthesis of Man1-2-DAG, but blocked the incorporation of [3H]mannose into Man-P-Undec and consequently the lipomannan. These results strongly indicate that GDP-Man is the direct mannosyl donor for the synthesis of Man1-2-DAG, and that the majority of the 50 mannosyl units in the lipomannan are derived from Man-P-Undec. Protease-sensitivity studies with intact and lysed protoplasts indicate that the active sites of the mannosyltransferases catalyzing the formation of Man1-2-DAG and Man-P-Undec are exposed on the inner face, and the Man-P-Undec-mediated reactions occur on the outer surface of the cytoplasmic membrane. Based on all of these results, a topological model is proposed for the lipid-mediated assembly of the membrane-bound lipomannan.

Use of a modified yeast one-hybrid screen to identify BAF60a interactions with the Vitamin D receptor heterodimer.

A modified yeast one-hybrid screen was used to isolate proteins capable of interacting with the Vitamin D receptor (VDR) heterodimer complex while driving expression from a repressor Vitamin D response element (VDRE). Four of nine independent colonies recovered in the screen coded for full-length BAF60a, a component of the mammalian SWI/SNF complex. Deletion studies in yeast were unable to localize a unique region of BAF60a responsible for interaction with the heterodimer complex, as only the full-length protein would support reporter gene expression. Pull-down analyses revealed that BAF60a displayed strong interactions with either the unliganded or liganded heterodimer complex, but neither individual receptor component alone. Transient transfection analysis in opossum kidney (OK) cells indicated that BAF60a decreased basal transcriptional activity from the negative VDRE, but had no effect on hormone-induced repression. Transcriptional activity from an enhancer VDRE also exhibited decreased basal transcriptional activity, but also augmented hormone-dependent enhancer activity, resulting in an overall increased sensitivity to hormone. In summary, BAF60a has been identified as a factor that specifically interacts with the VDR heterodimer complex using a modified yeast one-hybrid selection strategy. This suggests that BAF60a may be a link between mammalian SWI/SNF-like chromatin remodeling complexes and the VDR heterodimer.

Interaction between protein phosphatase 2A and members of the importin beta superfamily.

While performing a yeast two-hybrid library screen to uncover novel PP2A-interacting proteins, we discovered a specific interaction between a member of the importin beta/karyopherin beta superfamily, importin 9, and the A subunit of PP2A (PR65). This interaction between importin 9 and the A subunit was confirmed by in vitro pulldown, immunoprecipitation, and microcystin-Sepharose chromatography. We also found that another family member, importin beta, interacted specifically with the A subunit of PP2A. Finally, we showed that treatment of cells with a concentration of okadaic acid known to inhibit PP2A impeded the nuclear localization of an NLS-containing protein. These results provide evidence that these importins can exist in a native complex with endogenous PP2A and that this serine/threonine phosphatase plays a role in regulating the nuclear import of NLS-containing proteins in vivo.