The formation and maintenance of polarized distributions of membrane proteins in the cell membrane are key to the function of polarized cells. In polarized neurons, various membrane proteins are localized to the somatodendritic domain or the axon. Neurons control polarized delivery of membrane proteins to each domain, and in addition, they must also block diffusional mixing of proteins between these domains. However, the presence of a diffusion barrier in the cell membrane of the axonal initial segment (IS), which separates these two domains, has been controversial: it is difficult to conceive barrier mechanisms by which an even diffusion of phospholipids could be blocked. Here, by observing the dynamics of individual phospholipid molecules in the plasma membrane of developing hippocampal neurons in culture, we found that their diffusion was blocked in the IS membrane. We also found that the diffusion barrier is formed in neurons 7-10 days after birth through the accumulation of various transmembrane proteins that are anchored to the dense actin-based membrane skeleton meshes being formed under the IS membrane. We conclude that various membrane proteins anchored to the dense membrane skeleton function as rows of pickets, which even stop the overall diffusion of phospholipids, and may represent a universal mechanism for formation of diffusion barriers in the cell membrane.

Oct1 and Oct4 are homologous transcription factors with similar DNA-binding specificities. Here we show that Oct1 is dynamically phosphorylated in vivo following exposure of cells to oxidative and genotoxic stress. We further show that stress regulates the selectivity of both proteins for specific DNA sequences. Mutation of conserved phosphorylation target DNA-binding domain residues in Oct1, and Oct4 confirms their role in regulating binding selectivity. Using chromatin immunoprecipitation, we show that association of Oct4 and Oct1 with a distinct group of in vivo targets is inducible by stress, and that Oct1 is essential for a normal post-stress transcriptional response. Finally, using an unbiased Oct1 target screen we identify a large number of genes targeted by Oct1 specifically under conditions of stress, and show that several of these inducible Oct1 targets are also inducibly bound by Oct4 in embryonic stem cells following stress exposure. Oct1 and Oct4 (products of the Pou2f1 and Pou5f1 genes) are members of the POU (Pit-1, Oct1/2, Unc-86) domain transcription factor family (Herr et al. 1988;Ryan and Rosenfeld 1997). This family is defined by the presence of a bipartite DNA-binding domain in which two subdomains, covalently connected by a flexible linker, typically recognize DNA through major groove interactions on opposite sides of the helix (Klemm et al. 1994). The classical DNA recognition sequence is known as an octamer motif (59-ATGCAAAT-39, hereafter called a ''simple'' octamer). However, we demonstrated recently that native binding sites for Oct4 frequently exist in complex paired, overlapping, and nonconsensus configurations (Tantin et al. 2008).Oct4 is a master regulator of the stem cell state and has recently been shown to be one of three proteins sufficient to reprogram differentiated adult mouse and human cells to the embryonic stem (ES) cell lineage (Okita et al. 2007;Takahashi et al. 2007;Nakagawa et al. 2008). The biological function of Oct1 is more enigmatic. Oct1 is known to interact with regulatory sites in interleukin, immunoglobulin, and histone genes (Garrity et al. 1994;Zheng et al. 2003;Ushmorov et al. 2004;Murayama et al. 2006). Oct1 also moderately stimulates gene expression reporter constructs linked to target sequences in transient transfection assays (Sive et al. 1986;LeBowitz et al. 1988). However, we showed that Oct1 is nonessential for native H2B, IgH, and Igk expression (V.E. V.E.H. Wang et al. 2004). Oct1-deficient cells appear morphologically normal in light microscopy and divide at normal rates. Oct1-deficient mice die in mid-late gestation (embryonic days 12,5-18.5 [E12.5-E18.5]) (V.E.H. .We determined previously that Oct1 À/À mouse embryonic fibroblasts (MEFs) are hypersensitive to oxidative and genotoxic stress (Tantin et al. 2005). One explanation for this result is that constitutive products of Oct1-mediated transcription participate in stress response pathways. Support for an alternative hypothesis, namely that Oct1 directly senses cellular stress, comes from the f...

Transcription elongation factor DSIF/Spt4-Spt5 is capable of promoting and inhibiting RNA polymerase II elongation and is involved in the expression of various genes. While it has been known for many years that DSIF inhibits elongation in collaboration with the negative elongation factor NELF, how DSIF promotes elongation is largely unknown. Here, an activity-based biochemical approach was taken to understand the mechanism of elongation activation by DSIF. We show that the Paf1 complex (Paf1C) and Tat-SF1, two factors implicated previously in elongation control, collaborate with DSIF to facilitate efficient elongation. In human cells, these factors are recruited to the FOS gene in a temporally coordinated manner and contribute to its high-level expression. We also show that elongation activation by these factors depends on P-TEFb-mediated phosphorylation of the Spt5 C-terminal region. A clear conclusion emerging from this study is that a set of elongation factors plays nonredundant, cooperative roles in elongation. This study also shows unambiguously that Paf1C, which is generally thought to have chromatin-related functions, is involve directlyd in elongation control.

No abstract