Nonmuscle Myosin Promotes Cytoplasmic Localization of PBX

In the absence of MEIS family proteins, two mechanisms are known to restrict the PBX family of homeodomain (HD) transcription factors to the cytoplasm. First, PBX is actively exported from the nucleus via a CRM1-dependent pathway. Second, nuclear localization signals (NLSs) within the PBX HD are masked by intramolecular contacts. In a screen to identify additional proteins directing PBX subcellular localization, we identified a fragment of murine nonmuscle myosin II heavy chain B (NMHCB). The interaction of NMHCB with PBX was verified by coimmunoprecipitation, and immunofluorescence staining revealed colocalization of NMHCB with cytoplasmic PBX in the mouse embryo distal limb bud. The interaction domain in PBX mapped to a conserved PBC-B region harboring a potential coiled-coil structure. In support of the cytoplasmic retention function, the NMHCB fragment competes with MEIS1A to redirect PBX, and the fly PBX homologue EXD, to the cytoplasm of mammalian and insect cells. Interestingly, MEIS1A also localizes to the cytoplasm in the presence of the NMHCB fragment. These activities are largely independent of nuclear export. We show further that the subcellular localization of EXD is deregulated in Drosophila zipper mutants that are depleted of nonmuscle myosin heavy chain. This study reveals a novel and evolutionarily conserved mechanism controlling the subcellular distribution of PBX and EXD proteins

Sequential Histone Modifications at Hoxd4 Regulatory Regions Distinguish Anterior from Posterior Embryonic Compartments

Hox genes are differentially expressed along the embryonic anteroposterior axis. We used chromatin immunoprecipitation to detect chromatin changes at the Hoxd4 locus during neurogenesis in P19 cells and embryonic day 8.0 (E8.0) and E10.5 mouse embryos. During Hoxd4 induction in both systems, we observed that histone modifications typical of transcriptionally active chromatin occurred first at the 3′ neural enhancer and then at the promoter. Moreover, the sequential distribution of histone modifications between E8.0 and E10.5 was consistent with a spreading of open chromatin, starting with the enhancer, followed by successively more 5′ intervening sequences, and culminating at the promoter. Neither RNA polymerase II (Pol II) nor CBP associated with the inactive gene. During Hoxd4 induction, CBP and RNA Pol II were recruited first to the enhancer and then to the promoter. Whereas the CBP association was transient, RNA Pol II remained associated with both regulatory regions. Histone modification and transcription factor recruitment occurred in posterior, Hox-expressing embryonic tissues, but never in anterior tissues, where such genes are inactive. Together, our observations demonstrate that the direction of histone modifications at Hoxd4 mirrors colinear gene activation across Hox clusters and that the establishment of anterior and posterior compartments is accompanied by the imposition of distinct chromatin states

RhoA Proteolysis Regulates the Actin Cytoskeleton in Response to Oxidative Stress.

The small GTPase RhoA regulates the actin cytoskeleton to affect multiple cellular processes including endocytosis, migration and adhesion. RhoA activity is tightly regulated through several mechanisms including GDP/GTP cycling, phosphorylation, glycosylation and prenylation. Previous reports have also reported that cleavage of the carboxy-terminus inactivates RhoA. Here, we describe a novel mechanism of RhoA proteolysis that generates a stable amino-terminal RhoA fragment (RhoA-NTF). RhoA-NTF is detectable in healthy cells and tissues and is upregulated following cell stress. Overexpression of either RhoA-NTF or the carboxy-terminal RhoA cleavage fragment (RhoA-CTF) induces the formation of disorganized actin stress fibres. RhoA-CTF also promotes the formation of disorganized actin stress fibres and nuclear actin rods. Both fragments disrupt the organization of actin stress fibres formed by endogenous RhoA. Together, our findings describe a novel RhoA regulatory mechanism

RhoA fragments and cleavage-resistant promote the formation of actin stress fibres.

<p><b>A)</b> Serum-starved Swiss 3T3 fibroblasts were transfected with Flag-tagged RhoA 1–56 and 57–193, which correspond to RhoA-NTF and -CTF respectively, as well as WT-, Q63L- and the cleavage-resistant L57A/Q63L-RhoA. Cells were stained with anti-Flag M2 antibody (green), Alexa-Fluor 546 phalloidin (red) and Hoechst (blue) to label RhoA, the actin stress fibres and the nucleus respectively. Scale bar, 20 μm. <b>B)</b> Classification of the actin phenotype in transfected Swiss 3T3 cells. Significance was determined by the Chi-square test. *, <i>p</i> < 0.05; ** <i>p</i> < 0.005; ***, <i>p</i> < 0.0005; ****, <i>p</i> < 0.0001. <i>n</i> > 40 cells from 7 independent experiments. <b>C)</b> Quantification of the ratio of actin-covered area divided by the total surface of the transfected cells represented as the mean +/- S.E.M. Significance was established by one-way ANOVA from <i>n</i> > 40 cells collected from 7 independent experiments. *, <i>p</i> < 0.05; ** <i>p</i> < 0.005; ***, <i>p</i> < 0.0005; ****, <i>p</i> < 0.0001. <b>D)</b> Representative side view of a z-stack of a Swiss 3T3 cell overexpressing RhoA-CTF showing the localization of nuclear actin rods (red) relative to the nucleus (blue) and RhoA 57–193 (green). Scale bar, 10 μm.</p

Identification of a 10 kDa amino-terminal RhoA fragment.

<p><b>A)</b> Western blot analysis of COS-7 cell lysates transfected with Flag-tagged WT-RhoA or with 2Xmyc-tagged WT-RhoA using an anti-Flag M2 or anti-c-myc antibody reveals the presence of FL-RhoA and RhoA-NTF bands. <b>B, C)</b> Western blot of cell lysates following treatment with the proteosome inhibitors MG132 <b>(B)</b> or epoxomicin <b>(C)</b>. <b>D)</b> Expression of wild-type (WT), constitutively active (Q63L and G14V), dominant-negative (T19N) as well as non-prenylated wild-type (C190A), active (Q63L/C190A) and inactive (T19N/C190A) RhoA constructs in COS-7 cells analyzed by western blot using the Flag M2 antibody. Western blot panels illustrating FL-RhoA only are exposed to evaluate loading of full-length protein while panels with FL-RhoA and RhoA-NTF are a longer exposure of the same blot to visualize RhoA-NTF.</p