Swi1 associates with chromatin through the DDT domain and recruits Swi3 to preserve genomic integrity.

Swi1 and Swi3 form the replication fork protection complex and play critical roles in proper activation of the replication checkpoint and stabilization of replication forks in the fission yeast Schizosaccharomyces pombe. However, the mechanisms by which the Swi1-Swi3 complex regulates these processes are not well understood. Here, we report functional analyses of the Swi1-Swi3 complex in fission yeast. Swi1 possesses the DDT domain, a putative DNA binding domain found in a variety of chromatin remodeling factors. Consistently, the DDT domain-containing region of Swi1 interacts with DNA in vitro, and mutations in the DDT domain eliminate the association of Swi1 with chromatin in S. pombe cells. DDT domain mutations also render cells highly sensitive to S-phase stressing agents and induce strong accumulation of Rad22-DNA repair foci, indicating that the DDT domain is involved in the activity of the Swi1-Swi3 complex. Interestingly, DDT domain mutations also abolish Swi1's ability to interact with Swi3 in cells. Furthermore, we show that Swi1 is required for efficient chromatin association of Swi3 and that the Swi1 C-terminal domain directly interacts with Swi3. These results indicate that Swi1 associates with chromatin through its DDT domain and recruits Swi3 to function together as the replication fork protection complex

Plasmids used in this study.

*<p>The <i>S. pombe nmt1</i> promoter for overpexpression of GST- or FLAG-fused proteins.</p

Domains of Swi1 required for Swi1-Swi3 complex formation.

<p>The indicated Swi1 truncations mutants fused to FLAG were expressed in and purified from <i>swi3</i>Δ cells (top panel). Anti-FLAG agarose beads bound to the indicated Swi1 truncation mutant were incubated with recombinant His₆-Swi3. The beads were washed and analyzed by Western blotting using the anti-FLAG or His₆ antibody (middle panel). Asterisks indicate non-specific bands. Quantification of His₆-Swi3 bands was performed using EZQuant, normalizing the values to the amounts of Swi1 truncations used in the reactions. Swi3 binding activity of Swi1 (1–971) was set to 1. Representative image of repeat experiments are shown.</p

TBK1 at the Crossroads of Inflammation and Energy Homeostasis in Adipose Tissue

The noncanonical IKK family member TANK-binding kinase 1 (TBK1) is activated by pro-inflammatory cytokines, but its role in controlling metabolism remains unclear. Here, we report that the kinase uniquely controls energy metabolism. Tbk1 expression is increased in adipocytes of HFD-fed mice. Adipocyte-specific TBK1 knockout (ATKO) attenuates HFD-induced obesity by increasing energy expenditure; further studies show that TBK1 directly inhibits AMPK to repress respiration and increase energy storage. Conversely, activation of AMPK under catabolic conditions can increase TBK1 activity through phosphorylation, mediated by AMPK's downstream target ULK1. Surprisingly, ATKO also exaggerates adipose tissue inflammation and insulin resistance. TBK1 suppresses inflammation by phosphorylating and inducing the degradation of the IKK kinase NIK, thus attenuating NF-κB activity. Moreover, TBK1 mediates the negative impact of AMPK activity on NF-κB activation. These data implicate a&nbsp;unique role for TBK1 in mediating bidirectional crosstalk between energy sensing and inflammatory signaling pathways in both over- and undernutrition

DDT domain is essential for Swi1’s functions.

<p>(A) Five-fold serial dilutions of the indicated cells were incubated on YES agar medium supplemented with the indicated drugs for 2 to 4 days at 32°C. Representative images of repeat experiments are shown. (B) Cells of indicated <i>swi1</i> mutants were engineered to express Rad22-YFP and grown in YES medium at 25°C until midlog phase. The percentages of nuclei with at least one Rad22-YFP focus are shown (left panel). At least 200 cells were counted for each strain. Error bars correspond to standard deviations obtained from at least three independent experiments. Quantification of Rad22-YFP foci according to the cell cycle stages was also performed by analyzing cell length, nuclei number and position, and the presence of a division plate, as described in our previous publications (right panel) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043988#pone.0043988-Noguchi1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043988#pone.0043988-Noguchi2" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043988#pone.0043988-Ansbach1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043988#pone.0043988-Noguchi4" target="_blank">[43]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043988#pone.0043988-Noguchi6" target="_blank">[55]</a>. Schematic drawing for nuclear and morphological changes during the <i>S. pombe</i> cell cycle is shown (right panel). <i>swi1-h1</i> and <i>swi1</i>^Δ<i>DDT</i> cells displayed strong accumulation of Rad22-YFP foci during S and early G2 phases.</p

Swi1 facilitates the recruitment of Swi3 to chromatin.

<p>(A, B) ChIP assays of the indicated Swi1 truncated mutants were performed. Swi1 full-length (1–971), Swi1 (250–550), and Swi1 (250–550) precipitate the <i>ori2004</i> regions (<i>ori2004</i> and two positions 14- and 30-kb away from <i>ori2004</i>) in wild-type (WT, in A, middle panels) and <i>swi3</i>Δ mutants (in B, middle panels). Western blotting with anti-FLAG antibodies shows that all FLAG-fused Swi1 truncations mutants were similarly expressed (top panels). Fold increase in chromatin association over background (FLAG, set to 1) was calculated for each band. The average fold increase in the association of Swi1 truncations with the three positions (<i>ori200</i>4, −14 kb, and −30 kb) is shown, and error bars represent standard deviations obtained from the three positions (bottom panels). Representative results of repeat experiments are shown. WCE, whole cell extract; WB, Western blotting. (C) ChIP assay of GST-Swi3 was performed in wild-type or <i>swi1</i>Δ cells expressing the indicated proteins. GST-Swi3 strongly associates with the <i>ori2004</i> region in wild-type (WT), while GST-Swi3 has weak chromatin association in the absence of Swi1. Western blotting with anti-GST antibodies shows that GST-Swi3 was expressed similarly in wild-type and <i>swi1</i>Δ cells. Quantification of PCR bands was performed as described above. Fold increase in chromatin association over background (GST, set to 1) in each cell line (WT or <i>swi1</i>Δ) is shown. Representative results of repeat experiments are shown. (D) In vitro DNA binding assays of Swi1 (235–564) and Swi3 purified from the indicated <i>S. pombe</i> cells (top panel). The indicated proteins were mixed with radiolabeled plasmid pUC28, and associated DNA was analyzed by agarose gel electrophoresis (middle panel). Quantification of bound DNA was performed as described in Materials and Method. The values of bound DNA were normalized to the amount of proteins used in the reactions, and relative levels of bound DNA over background (GST) are shown. Representative results of repeat experiments are shown. C, input radiolabeled DNA.</p

The structure of the <i>S. pombe</i> Swi1 protein.

<p>(A) The Swi1 polypeptide was divided into 9 putative functional sub-domains. Hatched boxes indicate the regions with amino acid sequences that are conserved throughout evolution. Swi1 contains the Timeless domain (22–279 aa), NLS (304–314 aa), the DDT domain (323–378 aa) and the Timeless-C domain (595–817 aa). h1, h2 and h3 in the DDT domain indicate alpha-helix regions. Swi1 also has stretches of acidic amino acids at 535–542, 858–867, and 916–924 aa regions. The four truncated versions of Swi1 constructed in this study are shown. aa, amino acid. (B) Multiple sequence alignment of DDT domains of various transcription factors, chromatin-remodeling proteins, human Tim1 (Timeless) and <i>S. pombe</i> Swi1. Conserved aromatic and hydrophobic residues are shown in red. The predicted helices are boxed. Asterisks indicate mutated amino acids in <i>swi1</i> mutants constructed in this study.</p

<i>S. pombe</i> strains used in this study.

*<p>All strains are <i>leu1-32</i> and <i>ura4-D18</i>.</p

RalA controls glucose homeostasis by regulating glucose uptake in brown fat.

Insulin increases glucose uptake into adipose tissue and muscle by increasing trafficking of the glucose transporter Glut4. In cultured adipocytes, the exocytosis of Glut4 relies on activation of the small G protein RalA by insulin, via inhibition of its GTPase activating complex RalGAP. Here, we evaluate the role of RalA in glucose uptake in vivo with specific chemical inhibitors and by generation of mice with adipocyte-specific knockout of RalGAPB. RalA was profoundly activated in brown adipose tissue after feeding, and its inhibition prevented Glut4 exocytosis. RalGAPB knockout mice with diet-induced obesity were protected from the development of metabolic disease due to increased glucose uptake into brown fat. Thus, RalA plays a crucial role in glucose transport in adipose tissue in vivo