1. Volume 53, Issue 2, Pages 193-208 (January 2014)
Cyclin-Dependent Kinase 6 Is a Chromatin-Bound Cofactor for NF-κB-Dependent Gene Expression 
Katja Handschick, Knut Beuerlein, Liane Jurida, Marek Bartkuhn, Helmut Müller, Johanna Soelch, Axel Weber, Oliver Dittrich-Breiholz, Heike Schneider, Maren Scharfe, Michael Jarek, Julia Stellzig, M. Lienhard Schmitz, Michael Kracht 
Molecular Cell 
Volume 53, Issue 2, Pages (January 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 53, 193-208DOI: (10.1016/j.molcel.2013.12.002)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
Modulation of Cytokine-Regulated Gene Expression by the Cell Cycle
(A) HeLa cells were subjected to cell-cycle arrest at G0/G1 by serum deprivation for 48 hr (arrest). Thereafter, cells were released for 6 hr by addition of 20% serum (G1 release). In addition, cells were treated with IL-1 for 30 min as indicated. Differentially expressed genes relative to the arrested state were determined by microarrays from four independent sets of samples.
(B–D) Venn diagrams were used to visualize overlapping sets of upregulated genes as identified in (A). G1-released plus IL-1-treated cells were compared with G1-released cells (B). Additionally, arrested cells treated with IL-1 were compared with G1-released cells (C) or G1-released plus IL-1-treated cells (D). Boxes highlight coregulated inflammatory genes.
(E) Quantitative changes of IL-1 response genes regulated by cell cycle. The left graph shows mean individual ratios of all 29 genes (gray lines) as well as the mean fold change across all genes (red line). The right graph shows box and whisker plots for the distribution of ratio values for IL-1, G1 release, and IL-1 + G1 release (n = 4; red line, mean; black line, median).
(F) Expression of a catalytically active mutant of CDK6 (CDK6 S178P) and of GFP driven by a bidirectional promoter (Figures S1C–S1E) was induced by doxycycline for 48 hr. Then, some cells were pretreated for 30 min with 10 μM of PD followed by IL-1 stimulation for 1 hr as indicated. IL8 mRNA expression relative to the untreated control was determined (mean ± SEM; n = 2).
(G) GFP-positive and GFP-negative cells from untreated, doxycycline-treated, IL-1-treated, or doxycycline plus IL-1-treated cultures were excised by LMD. Cells were pooled (20–50), and CDK6 and IL8 mRNA expression relative to the untreated controls was determined (mean ± SEM; n = 4).
(H) HeLa cells were treated as in (A), and enzymatic activity of CDK6 was analyzed by in vitro immune complex protein kinase assays using a fragment of the RB protein fused to glutathione S-transferase (GST) and 32P-labeled ATP as substrates. In parallel, aliquots of the immune complexes were analyzed with CDK6 and cyclin D3 antibodies. Numbers show relative CDK6 kinase activities compared to arrested cells.
(I) The same experiment was performed using cell extracts from unsynchronized HeLa cells left untreated or stimulated for 1 hr with IL-1. Immunoglobulin G (IgG) was used as a negative control for the IP:kinase assays. Numbers show relative CDK6 kinase activities compared to untreated unsynchronized cells. See also Figure S1 and Table S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Identification of CDK6- and CDK4-Dependent Cell Cycle- and IL-1-Induced Genes
(A and B) HeLa cells were stably transfected with pSuper (vector), pSuper-shCDK6 (shCDK6), or pGeneClip-shCDK4 (shCDK4). Vector control cells and cells showing strong CDK6 (A) or CDK4 (B) knockdown were subjected for 48 hr to cell-cycle arrest at G0/G1 by serum deprivation (arrest) followed by addition of 20% serum for 6 hr (G1 release). In addition, cells were treated with IL-1 for 30 min as indicated. Total cell lysates were analyzed by immunoblotting with antibodies directed against CDK6 and CDK4 to validate specific suppression, antibodies against cyclin D1 and cyclin D3 to control for cell cycle release, and anti-tubulin or anti-β-actin to validate equal loading.
(C) RNAs generated from CDK6 or CDK4 knockdown cells as described in (A) and (B) were analyzed for expression changes of inflammatory genes by RT-qPCR (mean ± SEM; n = 2).
(D and E) The same RNAs were used for microarray analyses. CDK6- or CDK4-dependent genes were defined as having log2 ratios below the mean log2 ratio minus two SD as calculated from four control experiments.
(D) Venn diagrams and numbers display cell cycle- or cytokine-induced genes, their overlap, and their dependency on CDK6, CDK4, or on both kinases.
(E) Overall numbers of CDK6- or CDK4-regulated genes extracted from this data set. Genes consistently showing mRNA expression levels in CDK knockdown cells 2-fold diminished or elevated (along with additional criteria as described in the Experimental Procedures) were counted, and numbers are summarized in the inserted table. Genes showing differential expression in at least one condition are additionally visualized as Venn diagrams to graphically display the overall CDK6 and CDK4 overlap.
(F) Unsynchronized vector control cells or CDK6 or CDK4 knockdown cell lines as described above were left untreated or were stimulated with IL-1 for 1 hr, and mRNA expression was determined (mean ± SEM; n = 2). In parallel, cell extracts were analyzed for expression of CDK6 and CDK4 by immunoblotting. See also Figure S2 and Table S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Interphase-Specific Requirement of CDK6 for TNF-α-Induced mRNA Expression of Specific Genes
(A) HeLa FUCCI cells were stably transfected with pSuper (vector) or pSuper-shCDK6 (shCDK6). Cells were serum deprived for 48 hr (arrest). Thereafter, cells were released for 6 hr with 20% serum followed by 30 min of TNF-α (G1 release + TNF). Cells in different cell cycle phases were identified by fluorescence of FUCCI proteins and separated by LMD. Examples of individual LMD-excised cells representative of each cell cycle state are shown.
(B) Expression of the indicated genes in the isolated cells was analyzed by RT-qPCR (mean ± SEM; n = 3; 20 cells per cell cycle phase). Colors indicate the following cell cycle stages: red, G1; yellow, G1/S; green, S/G2 or mitosis.
(C) Unsynchronized HeLa FUCCI cells were left untreated or were stimulated with TNF-α. A total of 50 individual cells were isolated by LMD, and mRNA expression of the indicated genes was determined (mean ± SEM; n = 2). Asterisks indicate significant changes of IL8 mRNA after suppression of CDK6. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Cell Cycle Release Promotes Nuclear Entry of CDK6, Where It Interacts with p65 and Modulates Stable Association of p65 with Chromatin
(A) Whole-cell extracts from HeLa cells transfected with the indicated expression constructs were used for immunoprecipitation of CDK6. IgG served as a negative control. Proteins in lysates and coprecipitating proteins were detected by immunoblotting.
(B) HeLa cells transfected to express a control plasmid or GFP-p65 were analyzed for subcellular localization of GFP-p65 and endogenous CDK6 by indirect immunofluorescence analysis. Enlarged areas show localization of CDK6 and GFP-p65 in nuclear domains. The scale bar is 10 μm.
(C) Cells transfected as in (A) were lysed; cytosolic (C), soluble nuclear proteins (N1), and insoluble nuclear proteins (N2) were prepared; and the subcellular distribution of p65 and CDK6 was analyzed by immunoblotting. Antibodies against tubulin, histone H3, and β-actin were used to assess purity and equal loading of subcellular fractions. The same fractions were also used for CDK6 immunoprecipitations and CDK6-interacting proteins were detected by immunoblotting using the indicated antibodies. Arrows indicate specific GFP-p65, HA-CDK6, CDK6, and cyclin D1 or D2 protein bands; n.s. indicates a nonspecifically detected protein.
(D) KB cells were left untreated or were stimulated for 1 hr with IL-1. Then, antibodies against p65 and CDK6 were used to determine intracellular interactions by PLAs. The upper panel shows representative images with p65:CDK6 immune complexes indicated in red; nuclei were stained with Hoechst dye (blue). White lines indicate cell borders. Scale bar = 10 μM. Left graph: numbers of p65:CDK6 complexes per cell (mean ± SEM; n = 150). Right graph: relative cytoplasmic (C) versus nuclear (N) distribution of positive spots before and after IL-1 treatment. Samples omitting both or individual primary antibodies served as negative controls. Additional images are shown in Figure S4.
(E) Vector controls or CDK6 knockdown cells were subjected to cell-cycle arrest (arrest, A), G1 release (R), and IL-1 treatment as described in Figure 2. Cytosolic (C), nucleosolic (N1), or chromatin (N2) fractions were prepared, and equal amounts of proteins were analyzed by immunoblotting for p65 and CDK6 subcellular distribution. Antibodies against phosphorylated RNA polymerase II (P-S2 Pol II) or tubulin were used to validate the purity of fractions.
(F) The experiment shown in E) was performed three times, and protein bands were quantified from comparable enhanced chemiluminescence (ECL) exposures. Shown are mean arbitrary units ± SEM. Asterisks indicate significant changes. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
High-Resolution Mapping Reveals IL-1-Inducible p65 and CDK6 Binding to Active Promoters
Chromatin from KB cells treated for 1 hr with IL-1 or from untreated cells was immunoprecipitated with antibodies recognizing p65 NF-κB, CDK6, RNA polymerase II (Pol II), histone H3K9 acetylation (H3K9Ac), or H3K9 trimethylation (H3K9me3), followed by deep sequencing of the retrieved DNA.
(A) Shown are the ChIP-seq profiles for six IL-1-induced genes (IL8, CXCL3, IL6, CCL20, PTGS2, ICAM1); the positions of TSSs and exons are indicated. Profiles for CCND1 and MMP1 are shown as examples for genes that are regulated by cell cycle transition but not by IL-1 (see Figure S2); y axis shows normalized read coverage.
(B) Venn diagrams indicate the total number of peaks across the human genome as well as their overlap using pairwise comparisons.
(C) DNA sequence reads obtained in the experiments described in (A) for all 34,450 RefSeq annotated promoters are shown as clustered heatmaps centered ± 5 kb around the TSS. Binding events are indicated with red.
(D) Average ChIP-seq binding profiles for 245 IL-1-inducible genes that had been compiled from microarray experiments were plotted to regions ± 3 kb next to the TSS; y axis shows normalized read coverage.
(E) We used 50 bp of sequence flanking the maximum of the top 500 IL-1-induced or top 500 noninduced binding events to search for DNA motif enrichments underlying p65 peaks. The three best-matching de novo motifs and the three most significantly correlating known motifs are shown (upper part of the graph). The same analysis was performed for CDK6, revealing only four de novo motifs, of which two match to PAX4 sites, whereas the other two are unknown. Numbers indicate p values.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
CDK6 Regulates Inducible Recruitment of NF-κB p65 and Transcription Initiation at the IL8 Promoter
(A) Scheme of the IL8 locus. Black lines indicate DNA regions examined by ChIP.
(B and C) HeLa cells were transfected with empty vector and with pCDNA3-CDK6 (B) or with pEBB-HA-p65 (C). After 24 hr, IL8 mRNA expression and ChIP analysis of the IL8 promoter was performed using the indicated antibodies. The left panels show immunoblots of whole-cell extracts probed with CDK6 or p65 antibodies. A vertical line indicates that intervening lanes of the same blot have been removed.
(D) Arrested or G1-released HeLa cells stably transfected with pSuper (vector, black bars) or with pSuper-shCDK6 (white bars) were treated with IL-1 for 30 min as indicated. ChIP analyses were performed on the IL8 promoter using the indicated antibodies. A nonregulatory genomic region 1 kb upstream of the IL8 promoter served as a negative control.
(E) After 48 hr of selection in puromycin, p65 knockdown or control HeLa cells were left untreated or IL-1 was added for 1 hr. Cells were used to ensure either efficient p65 knockdown by immunoblotting or IL-1-inducible expression of the IL8 mRNA (left panel). In parallel, the cells were analyzed for chromatin recruitment of p65, CDK6, c-FOS, Pol II, phosphorylated Pol II, or for histone modifications and histone H3 density at the IL8 promoter (right panels).
(F–H) HeLa cells were transiently transfected with expression vectors encoding shRNAs directed against TRIP6 (F), CDK7 (G), or cyclin D1 and cyclin D3 (H). After 48 hr selection in puromycin, cells were left untreated or stimulated with IL-1 for 1 hr. IL8 mRNA expression and recruitment of p65, Pol II, or CDK6 to the IL8 promoter were determined as described above. Efficient knockdown was controlled by immunoblotting. In all bar graph panels, mean values ± SEM for at least two independent experiments performed in duplicate are shown. See also Figures S5 and S6.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
NF-κB p65 and CDK6 Coassociate at Promoters of Inflammatory Genes in a TAK1-Dependent Manner
(A) KB cells were treated for 30 min with the TAK1 inhibitor 5Z-7-oxozeaenol (1 μM) followed by IL-1 stimulation for 1 hr. ChIP analyses were performed using the indicated antibodies and either primers covering the IL8 upstream region as a negative control (upper panel) or the IL8 promoter (lower panel). The right panel shows a Re-ChIP analysis of p65 ChIPs performed under identical conditions. Eluted protein DNA complexes were reimmunoprecipitated with anti-CDK6 or control antibodies (IgG) and analyzed for the presence of IL8 promoter DNA by qPCR.
(B) ChIP DNA isolated from cells treated as in (A) was analyzed by primer pairs covering the regulatory regions of the indicated inflammatory and cell cycle-regulated genes. Mean values ± SEM for at least two independent experiments performed in duplicate are shown. See also Figure S7.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions