Inhibition of S/G(2) phase CDK4 reduces mitotic fidelity

Cyclin-dependent kinase 4 (CDK4)/cyclin D has a key role in regulating progression through late G(1) into S phase of the cell cycle. CDK4-cyclin D complexes then persist through the latter phases of the cell cycle, although little is known about their potential roles. We have developed small molecule inhibitors that are highly selective for CDK4 and have used these to define a role for CDK4-cyclin D in G(2) phase. The addition of the CDK4 inhibitor or small interfering RNA knockdown of cyclin D3, the cyclin D partner, delayed progression through G(2) phase and mitosis. The G(2) phase delay was independent of ATM/ATR and p38 MAPK but associated with elevated Wee1. The mitotic delay was because of failure of chromosomes to migrate to the metaphase plate. However, cells eventually exited mitosis, with a resultant increase in cells with multiple or micronuclei. Inhibiting CDK4 delayed the expression of the chromosomal passenger proteins survivin and borealin, although this was unlikely to account for the mitotic phenotype. These data provide evidence for a novel function for CDK4-cyclin D3 activity in S and G(2) phase that is critical for G(2)/M progression and the fidelity of mitosis

The Yeast FACT Complex Has a Role in Transcriptional Initiation

A crucial step in eukaryotic transcriptional initiation is recognition of the promoter TATA by the TATA-binding protein (TBP), which then allows TFIIA and TFIIB to be recruited. However, nucleosomes block the interaction between TBP and DNA. We show that the yeast FACT complex (yFACT) promotes TBP binding to a TATA box in chromatin both in vivo and in vitro. The SPT16 gene encodes a subunit of yFACT, and we show that certain spt16 mutations are synthetically lethal with TBP mutants. Some of these genetic defects can be suppressed by TFIIA overexpression, strongly suggesting a role for yFACT in TBP-TFIIA complex formation in vivo. Mutations in the TOA2 subunit of TFIIA that disrupt TBP-TFIIA complex formation in vitro are also synthetically lethal with spt16. In some cases this spt16 toa2 lethality is suppressed by overexpression of TBP or the Nhp6 architectural transcription factor that is also a component of yFACT. The Spt3 protein in the SAGA complex has been shown to regulate TBP binding at certain promoters, and we show that some spt16 phenotypes can be suppressed by spt3 mutations. Chromatin immunoprecipitations show TBP binding to promoters is reduced in single spt16 and spt3 mutants but increases in the spt16 spt3 double mutant, reflecting the mutual suppression seen in the genetic assays. Finally, in vitro studies show that yFACT promotes TBP binding to a TATA sequence within a reconstituted nucleosome in a TFIIA-dependent manner. Thus, yFACT functions in establishing transcription initiation complexes in addition to the previously described role in elongation

KIR3DL01 upregulation on gut natural killer cells in response to SIV infection of KIR- and MHC class I-defined rhesus macaques

<div><p>Natural killer cells provide an important early defense against viral pathogens and are regulated in part by interactions between highly polymorphic killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their MHC class I ligands on target cells. We previously identified MHC class I ligands for two rhesus macaque KIRs: KIR3DL01 recognizes Mamu-Bw4 molecules and KIR3DL05 recognizes Mamu-A1*002. To determine how these interactions influence NK cell responses, we infected KIR3DL01⁺ and KIR3DL05⁺ macaques with and without defined ligands for these receptors with SIV_mac239, and monitored NK cell responses in peripheral blood and lymphoid tissues. NK cell responses in blood were broadly stimulated, as indicated by rapid increases in the CD16⁺ population during acute infection and sustained increases in the CD16⁺ and CD16^-CD56^- populations during chronic infection. Markers of proliferation (Ki-67), activation (CD69 & HLA-DR) and antiviral activity (CD107a & TNFα) were also widely expressed, but began to diverge during chronic infection, as reflected by sustained CD107a and TNFα upregulation by KIR3DL01⁺, but not by KIR3DL05⁺ NK cells. Significant increases in the frequency of KIR3DL01⁺ (but not KIR3DL05⁺) NK cells were also observed in tissues, particularly in the gut-associated lymphoid tissues, where this receptor was preferentially upregulated on CD56⁺ and CD16^-CD56^- subsets. These results reveal broad NK cell activation and dynamic changes in the phenotypic properties of NK cells in response to SIV infection, including the enrichment of KIR3DL01⁺ NK cells in tissues that support high levels of virus replication.</p></div

KIR3DL01 upregulation on NK cells of the gastrointestinal mucosa.

<p>Representative KIR3DL01 versus KIR3DL05 staining on NK cells isolated from colorectal biopsies is shown for two different rhesus macaques before (week -4) and after (week 2) SIV infection. CD16 versus CD56 staining is shown for the KIR3DL01⁺ population at week 2 post-infection (right panels).</p

Longitudinal changes in NK and T cell counts in peripheral blood in response to SIV infection.

<p>Absolute counts for total NK cells (A), CD8⁺ T cells (B), CD4⁺ T cells (C), KIR3DL01⁺ NK cells (D), KIR3DL05⁺ NK cells (E), KIR3DL01^-05^- NK cells (F), KIR3DL01⁺05⁺ NK cells (G), CD16⁺ NK cells (H), CD56⁺ NK cells (I), and CD16^-CD56^- NK cells (J) were monitored by staining whole blood and PBMCs as described in the methods. Individual (K) and geometric mean (L) SIV RNA loads in plasma are shown for Mamu-A1*002⁺ (blue) and–A1*002^- (red) animals. Gating strategies for determining absolute lymphocyte counts in blood and the percentages of PBMCs expressing CD16, CD56, KIR3DL01 and KIR3DL05 are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006506#ppat.1006506.s001" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006506#ppat.1006506.s004" target="_blank">S4</a> Figs. Viral loads were measured using a qRT-PCR assay with a detection threshold of 30 copies/ml (dotted line) and error bars indicate 95% CI for geometric mean values. Statistics were calculated using a mixed effects model by comparing results from acute (week 1–4) and chronic (weeks 6–24) infection to pre-infection (week 0) (p<0.05 *, p<0.01**, p< 0.005*** & p<0.001****).</p

NK cell degranulation and cytokine release in response to SIV infection.

<p>Longitudinal changes in the upregulation of CD107a (A & C) and TNFα (B & D) are shown for KIR3DL01⁺, KIR3DL05⁺ and KIR3DL01^-05^- NK cells in response to stimulation with 721.221 cells (A & B) or without 721.221 cell stimulation (C & D). PBMCs were incubated overnight with or without 721.221 cells in the presence of an antibody to CD107a, and stained the following day with antibodies to differentiate KIR3DL01⁺, KIR3DL05⁺ and KIR3DL01^-05^- NK cells and for intracellular accumulation of TNFα. Representative gating for CD107a and TNFα staining is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006506#ppat.1006506.s006" target="_blank">S6 Fig</a>. The mean and standard error (error bars) are plotted for the each NK cell population. Significance values for acute (weeks 1–4) and chronic (weeks 6–24) infection compared to pre-infection (week 0) are indicated with asterisks color-coded to the corresponding cell population (p<0.05*, p<0.01**, p< 0.005*** & p<0.001****, mixed effects models).</p

NK cell proliferation and activation in response to SIV infection.

<p>Longitudinal changes in the expression of Ki-67 (A & B), CD69 (C & D), HLA-DR (E & F), and α4β7 (G & H) were monitored for CD16⁺, CD56⁺ and CD16^-CD56^- NK cells (left panels: A, C, E & G) and for KIR3DL01⁺, KIR3DL05⁺ and KIR3DL01^-05^- NK cells (right panels: B, D, F & H). Absolute counts were calculated as a percentage of total NK cell counts by staining PBMCs with antibodies to CD3, CD8, NKG2A, CD16, CD56, KIR3DL01 and KIR3DL05 (tetramer), and to markers of proliferation (Ki-67), activation (CD69 & HLA-DR) and mucosal homing (α4β7). Representative gating for the proliferation and activation panel is shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006506#ppat.1006506.s004" target="_blank">S4 Fig</a>. The mean and standard error (error bars) are shown for each NK cell subset. Significance values for acute (weeks 1–4) and chronic (weeks 6–24) infection compared to pre-infection (week 0) are indicated with asterisks color-coded to the corresponding cell population (p<0.05*, p<0.01**, p< 0.005*** & p<0.001****, mixed effects models).</p

Comparison of the frequencies of KIR3DL01⁺ and KIR3DL05⁺ NK cells in lymph nodes and gut-associated lymphoid tissues.

<p>The frequencies of KIR3DL05⁺ (A), KIR3DL01⁺ (B) and KIR3DL01^-05^- (C) NK cells in PBMCs, lymph nodes (LN) and gut-associated lymphoid tissues (GALT) were compared prior to SIV infection (week -4) and at weeks 2 and 8 after SIV infection. The frequencies of CD16⁺, CD56⁺ and CD16^-CD56^- NK cells in lymph nodes (D & E) and gut-associated lymphoid tissues (F & G) expressing KIR3DL05 (D & F) or KIR3DL01 (E & G) were also compared at weeks -4, 2 and 8 pre- and post-infection. Representative gating for differentiating KIR3DL01⁺ versus KIR3DL05⁺ NK cells in lymphocytes isolated from colorectal biopsies is illustrated in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006506#ppat.1006506.s007" target="_blank">S7 Fig</a>. Significant differences are indicated with asterisks (p<0.05*, p<0.01**, p< 0.005*** & p<0.001****, Mann-Whitney <i>U</i>-test). Samples with less than 30 events per gate were excluded from the analysis.</p

Frequency and intensity of KIR expression as a function of the presence of MHC class I ligands.

<p>Comparison of the frequency (A) and the mean fluorescence intensity (B) of KIR3DL01 staining on NK cells prior to SIV infection versus the number of MHC class I alleles predicted to encode Mamu-Bw4 ligands for this receptor by linear regression analysis. Comparison of the frequency (C) and the mean fluorescence intensity (D) of KIR3DL05 staining on NK cells for Mamu-A1*002^-, -A3*13^- (open circle), Mamu-A1*002^-, -A3*13⁺ (red dot), Mamu-A1*002⁺, -A3*13^- (black dot), and Mamu-A1*002⁺, -A3*13⁺ (black circle, red dot) animals prior to SIV infection by linear regression analysis. Comparison of the frequency (E) and the mean fluorescence intensity (F) of KIR3DL05 staining on NK cells for Mamu-A1*002⁺ (blue) versus -A1*002^- (red) animals at the indicated time points before and after SIV infection. Significant differences are indicated with asterisks (p<0.05* & p<0.01**, Mann-Whitney <i>U</i>-test).</p

Changes in the frequency of CD16⁺, CD56⁺ and CD16^-CD56^- NK cells expressing KIR3DL01 and KIR3DL05.

<p>The percentages of CD16⁺, CD56⁺ and CD16^-CD56^- NK cells that are KIR3DL01⁺ (A), KIR3DL05⁺ (B) or KIR3DL01^-05^- (C) were monitored in peripheral blood. The mean and standard error (error bars) are shown for each subset. Significance values were calculated using a mixed effects model by comparing the results from acute (weeks 1–4) and chronic (weeks 6–24) infection to pre-infection (week 0) and are indicated with asterisks color-coded according to the corresponding NK cell population (p<0.05*, p<0.01**, p< 0.005*** & p<0.001****).</p