Molecular Cell, Volume 57 Supplemental Information Chromatin-wide Profiling of DYRK1A Reveals a Role as a Gene-Specific RNA Polymerase II CTD Kinase Chiara Di Vona, Daniela Bezdan, Abul B.M.M.K. Islam, Eulàlia Salichs, Nuria López-Bigas, Stephan Ossowski, and Susana de la Luna Figure S1 A 20 5 min 10 min 15 15 min 10 5 0 IP IgG-Control IP anti-DYRK1A 1 GAL4 κB * 130 130 95 95 72 GAL4 G4E1b-Luc GAL4 Luciferase 55 36 36 Wb: anti-G4-DBD 8 6 4 2 0 - DYRK1AWT DYRK1AKR G Wb: anti-G4-DBD G4E1b-Luc 140 Relative transcriptional activity (fold change) 10 8 G4E1b-Luc TATA Less 120 100 6 4 2 0 G4-DBD: - K1 YR A WT K1 AK R C D K9 W T C D K9 KM 12 G5HIV1-Luc D 14 G4-DBD: F YR G5HIV1-Luc D 16 Relative transcriptional activity (fold change) Relative transcriptional activity (fold change) E YR D K1A YR K D 1B YR D K2 YR D K3 YR K4 72 Luciferase TATA 55 G4E1b-Luc-TATAless - D KM K9 C D kDa Luciferase TATA G4-DBD fusion proteins R AK W T K9 K1 YR D YR - D kDa D His S/T K179R KR G5HIV1-Luc Reporters NLS2 PEST AW KD K1 NLS1 Sp G4DBD-DYRK1A G4-DBD N F- Effectors G4DBD-DYRK1AWT D G4-DBD fusion proteins T C B C kinase activity (cpmx103) 25 80 60 40 20 0 G4-DBD: - DYRK1AWT DYRK1AKR Figure S2 A Percentage 100 20 60 Promoters 61.3% (2.7e-77) 1.1% 1.8% >= 1000 bp B 62.5% (1.2e-67) Genome ChIP >= 2000 bp T98G cells 466 56 HeLa cells C 40 RBM39 ASXL1 19 RPS11 25 DYRK1A (T98G) 6.7 7 6 DYRK1A (HeLa) → chr20:34,330,000-34,323,000 → chr20:30,945,000-30,943,000 → chr19:49,999,000-50,003,000 C -2.0 0.0 +2.0 D Expression Scale -3.00 -7.50 ** -10.0 -3.75 *** 0.0 0 3.75 7.50 1 TSS XL 0.4 -4000 AS 4000 2 H3K27ac K1 DYRK1A target genes D Average profile 40 C 0.6 R 0 EN 80 D -4000 2 Average profile A 7L C B LU 11 PS R Relative mRNA expression 0 DYRK1A expression CD8_T cells Leukemia lymphoblastic_MOLT4 CD105_Endothelial CD71_Early erythroid Broquial epithelial cells Thymus CD19_B cells CD4_T cells BDCA4_Dendritic cells Colorectal adenocarcinoma X721_B lymphoblasts Testis_Germ cells Fetal Thyroid Uterus Leukemia promyelocytic_HL60 CD14_Monocytes CD56_NK cells Colon Leukemia chromic myelogenous Small intestine Lymp node CD34_Lymphatic endothelial cells Thyroid Pituitary Fetal lung Burkitt’s lymphoma_Daudi Pineal gland_Day Whole brain Tonsil Testis_Intersitial Prefrontal cortex Pineal gland_Night Amigdala CD33_Myeloid Whole blood Fetal liver Smooth muscle Fetal brain Testis_Seminiferous tubule Ovary Salivary gland Trachea Prostate Hypothalamus Pancreas Burkitt’s lymphoma_Raji Testis Olfatory bulb Occipital lobe Cerebellum Caudate nucleus Skin Globus pallidus Medulla oblongata Retina Parietal lobe Adrenal gland Testis_Leyding cell Cardiac Myocytes Placenta Pancreatic islet Dorsal root ganglion Appendix Bone marrow Thalamus Tongue Adipocyte Kidney Uterus_Corpus Temporal lobe Liver Pons Cerebellum peduncles Heart Lung Spinal Cord Skeletal muscle Cingulate cortex Superior cervial ganglion Subthalamic nucleus Adrenal cortex Atrioventricular node Trigeminal ganglion Ciliary ganglion Figure S3 Random genes 8 H4K20me1 6 4 2 0 0 Z-score DYRK1A targets expression 11.25 4000 1.2 1 *** 3.00 2.25 1.50 0.75 0 -0.75 -1.50 -2.25 15.00 Distance (bp) TSS shControl shDYRK1A.2 0.8 ** ** 0.2 0 Z-score Scale +10.0 DYRK1A expression DYRK1A targets Z-score Figure S4 A B average conservation score 0.5 DYRK1A random Nuclear extracts - cold competitor - t mu wt - 0.4 - Complex II - Complex I 0.3 0.2 0.1 -200 -100 0 100 - free probe 200 Distance to DYRK1A-peaks mid point (bp) kDa E (m) (r) K1AYRK1A R ) Y (m nti-D nti-D G (r) gG g HNE IP I IP a IP a IP I 95 2.5 kDa DYRK1A (m) 2.0 Wb: anti-DYRK1A (m) % of input 72 D IgG (m) (r) K1AYRK1A(r) R ) n Y i t ) D nti-D i-H3 (m oma gG G (r ntit Chr IP I IP Ig IP a IP a IP an DYRK1A (r) 1.5 1.0 0.5 DYRK1A 95 0.0 RBM39 RPS11 Histone H3 95 DENR ASXL G 0.06 IgG 0.05 DYRK1A (m) 0.01 0 6 2 0 1 2 9 1 R M3 PS1 DEN SXL C7L PL1 RPS _cov R A R 0 RB LU r2 ch TCTCGCGAGA 250 200 150 100 50 0 pGL2 Multimeric Luc 100 Relative transcriptional activity (fold change) 0.02 80 60 40 20 0 shControl 95 DYRK1A 80 KAISO 95 YR D Ig G IP H N E 130 IP IS O (m ) (r) (r) K1 A kDa IP YR D IP KA A (r) K1 Ig G IP YR D Ig G IP IP E (m ) (m ) (m ) H N RPS6 chr20_cov0 Multimeric reporter Relative transcriptional activity (fold change) 0.03 kDa RPL12 TATA 0.04 H % of input F LUC7L K1 A C p120-catenin DYRK1A shDYRK1A.1 shDYRK1A.2 Figure S5 B Not Promoters C Promoters 60 number of cells (x104) 80 60 40 20 0.8 0.6 0.4 0.2 0 sh 35 Cyclin D1 95 DYRK1A 54 Tubulin 30 20 10 1 2 3 4 days Se Prolierating cells Serum starved cells E DYRK1A mRNA 1.2 Relative expression levels 1.0 kDa A.1 A.2 l tro RK1 RK1 Y D hDY sh s n Co ru sp m s ec ta ifi rve c d on m om C Pr o sp lifer ec at ifi ing c DYRK1A protein 1.2 shDYRK1A.2 40 0 D shControl 50 0 Relative expression levels percentage of peaks 100 Proliferating cells Serum starved cells 1.0 0.8 0.6 0.4 0.2 0 F Cell cycle profiles 100 G1 S G2/M 80 * percentage of cells A 60 40 20 0 Proliferating Serum Starved Figure S6 A HA-DYRK1BWT B HA-DYRK1BKR ut 6) 6) inp G1 0) G1 0) IgG IgG (8W (N2 IgG IgG (8W I (N2 e e it it II I II II s s tes ou rabb POL POL tes ou rabb POL POL sa m sa m Ly IP IP Ly IP IP IP IP IP IP kDa IIo IIa 230 96 ST ST G G Wb: anti-HA D - GST: kDa 130 A RK1 DY E B RK1 Antibody DYRK1A DY anti-GST - 130 DYRK1B DYRK1BWT 43 GADPH DYRK1BKR 17 H3 + H5 (Ser2p) - + hyperP-CTD hypoP-CTD F hyperP-CTD A.1 A.2 l tro RK1 RK1 Y D hDY sh s n Co Ser5p kDa hypoP-CTD 95 sh 230 130 hyperP-CTD 95 hypoP-CTD 130 Ser7p DYRK1A 95 130 DYRK1B 54 95 IIo H5 (Ser2p) IIo H14 (Ser5p) IIo Thr4p 230 130 kDa N20 RNAPII 230 G IIo IIa 230 GST-CTD (anti-GST) 95 RNAPII 80 Ser2p hypoP-CTD IIo IIa 230 hyperP-CTD 95 C WT DYRK1AKR Wb: anti-RNAPII (N20) in ol leus omat c r Nu Ch s yto kDa DYRK1A * 75 C D CT 1B 1A IgG YRK YRK l o r D D tes Cont anti- antisa IP IP Ly IP * DYRK1A Tubulin B H kDa 95 1 G RK l Ig tro ti-DY n ut Co P an I inp IP ] DYRK1B DYRK1A 72 95 IgG 55 DYRK1B 72 J 3.5 IgG 3.0 DYRK1B 2.5 DYRK1A 2.0 1.5 1.0 IgG DYRK1B 0.03 0.02 0.01 0.5 0.0 0.05 0.04 % of input % of input I RBM39 RPS11 ASXL CDK12 DENR LUC7L RPL12 RPS6 0.00 RBM39 RPS11 ASXL CDK12 DENR LUC7L RPL12 RPS6 Figure S7 kDa B A.1 A.2 l tro RK1 RK1 n Co hDY hDY s sh s 3.0 % of input H3 17 17 H3-Ac 17 H3-Ser10p 17 H3-Ser28p 17 H3-Thr45p H4-Ac 95 DYRK1A 54 Tubulin normalized values (8WG16) DENR A 1.4 B C 0.6 *** ** *** *** ** 0.4 0.2 0.0 A B C RPL12 A 2.5 EIF4 CDK12 → B D DENR 10 kb C D Ser2p shControl Ser2p shDYRK1A Ser5p shControl Ser5p shDYRK1A E 2.0 1.5 1.0 * ** ** *** *** 0.5 A B C E RPS6 A 1.4 *** *** D → D 1.0 * CDK12 E 2 kb 1.2 0.8 1.0 3.0 0.0 → ChIP:N20 1.5 0 normalized values (8WG16) 14 2.0 0.5 C D IgG shControl shDYRK1A.1 shDYRK1A.2 2.5 normalized values (8WG16) A 1.5 kb B C D 1.2 1.0 0.8 ** *** 0.6 *** 0.4 *** ** *** 0.2 0.0 A B C D SUPPLEMENTAL FIGURE LEGENDS Figure S1. Related to Figure 1 Nuclear DYRK1A is an active nuclear kinase present in high molecular weight complexes that co-fractionates with RNAPII. (A) DYRK1A-immunocomplexes from HeLa nuclear extracts were used in in vitro kinase (IVK) assays using DYRKtide as the substrate to determine the DYRK1A kinase activity at the time points indicated. Immunocomplexes obtained with mouse IgGs were used as a control. DYRK1A activates transcription in a dose and kinase-dependent manner when tethered to promoter regions. (B) Schematic representation of the constructs used in the reporter assays in Figure 1 and panels E-G of this Figure. The effector plasmid G4DBDDYRK1AWT directs the expression of the full-length DYRK1A protein fused to the Gal4 DNA binding domain (DBD) while G4DBD-DYRK1AKR expresses a kinase-inactive DYRK1A through mutation of the ATP binding site. KD, kinase catalytic domain; NLS, nuclear localization signal; PEST region; His, stretch of 13 consecutive His residues; S/T, region enriched in Ser and Thr residues. The target plasmid G5HIV1-Luc contains 5 Gal4 binding sites (Gal4), the HIV-1 promoter, with NF-κB and Sp1 binding sites driving the expression of the luciferase reporter gene (Montanuy et al., 2008). The target plasmid G4E1b-Luc contains 5 Gal4 binding sites upstream of the E1b adenovirus minimal promoter (de la Luna et al., 1999). (C, D) Soluble extracts from HEK-293 cells expressing the indicated G4-DBD fusion plasmids were separated by SDS-PAGE and analyzed in Western blots probed with a G4-DBD specific antibody. The position of the marker proteins (in kDa) is indicated. The Western blots show that the G4-DBD/DYRK fusion proteins used in the reporter assays shown in Figure 1G and 1H were expressed at similar levels. (E) HEK-293 cells were co-transfected with the reporter G5HIV1-Luc together with increasing amounts of the expression plasmids encoding G4-DBD fusions to wild type DYRK1A (5 ng, 10 ng or 20 ng) or the kinase inactive DYRK1A (10 ng, 20 ng or 40 ng). A plasmid expressing unfused G4DBD (-) was used to measure the basal activity of the reporter. Luciferase activity was measured in triplicate plates and the values were corrected for transfection efficiency as measured by the Renilla activity. The graph represents transcriptional activity as the fold change compared to the control G4-DBD value, set as 1 (mean ± s.d.), from a representative experiment of 3 performed. (F) The efficiency of DYRK1A to activate transcription was compared with that of CDK9 on the G5HIV1-Luc reporter, at similar protein levels (as assessed in Western blots, in panel D). (G) The efficiency of DYRK1A to activate transcription was assessed with the G4E1b-Luc reporter or the G4E1b-Luc TATA-less reporter, which lacks the TATA-box of the minimal E1b promoter. The graph represents transcriptional activity as the fold change compared to the control G4-DBD value, set as 1, for each reporter. Note the strong reduction in the induction of luciferase expression by DYRK1A on the TATA-less reporter when compared with the G4E1b-luc reporter. Figure S2. Related to Figure 2 DYRK1A is recruited to promoters. (A) Bar plot showing the relative enrichment of promoters in the ChIP fragments with respect to the genome background. The light violet bars represent the percentage of annotated promoters in the genome and the dark bars the percentage in ChIP fragments. P-values for the significance (hypergeometric test) of the relative enrichment in ChIP fragments with respect to the genome background are shown in parentheses. (B) Venn diagram of the overlap between DYRK1A target chromatin loci of in T98G cells and HeLa cells. (C) DYRK1A signal maps are shown for representative target genes in T98G cells and HeLa cells. Figure S3. Related to Figure 3 (A) Profiles of the histone modifications indicated around the TSS for DYRK1A associated genes (in red) and for random Ensemble genes (in black). (B) DYRK1A protein levels were down-regulated in HeLa cells by lentiviral delivery of two different shRNAs targeting DYRK1A and the expression level of several DYRK1A target genes was determined by RT-qPCR. The data represent the mean and standard deviation of five independent experiments. (C) Correlation of the expression of DYRK1A and its targets in human tissues. Data from the GeneAtlas dataset (GEO GSE1133 dataset) was used. DYRK1A target genes in both HeLa and T98G cells were defined as genes with DYRK1A peaks between -300 to +1 of the TSS. The graphs show the absolute (log2) expression values for DYRK1A in different tissues and cell lines, plotted as a color-coded heatmap, where red indicates stronger expression and green indicates weaker expression (see "Expression scale"). The correlation analysis is represented as Z-score values for preferential expression and under-expression of DYRK1A target genes defined in a different colored heatmap, where red signifies over-expression of the targets and blue indicates under-expression of the targets. The samples are ordered according to the Z-score of preferential expression of the DYRK1A targets. (D) The scatter plot shows the correlation between DYRK1A expression and the Z-score of the expression of their targets (Pearson correlation coefficient 0.62). Figure S4. Related to Figure 4 The TCTCGCGAGA-sequence is a regulatory motif in DYRK1A target promoters. (A) Average conservation of the enriched motif in DYRK1A promoters (red line) compared to random sequences of equal length (green). On the X axis, 0 indicates the mid point of the DYRK1A peaks. Minus indicates upstream bases and plus values indicate bases downstream of the DYRK1A binding site sequence. (B) EMSA was performed with HeLa nuclear extracts and a 32P-labeled oligonucleotide probe containing the sequence motif from the RPS11 promoter region. A competition assay was performed with increasing amounts of the wild type cold probe (wt, 20-fold, 50-fold and 100-fold) or of a probe of the same size without the consensus sequence (mut, 20fold, 50-fold and 100-fold). The position of the shifted band corresponding to Complex I and II is indicated, as well as that of the free probe. (C) HeLa nuclear extracts were immunoprecipitated with the two antibodies against DYRK1A used in the EMSA experiments (m: mouse monoclonal 7D10 from Abnova; r: rabbit polyclonal from Abcam). Both the lysates and the immunoprecipitates were analyzed by Western blot with the anti-DYRK1A monoclonal antibody. IgGs from mouse and IgGs from rabbit were used as controls in the immunoprecipitation. (D) ChIP-Western from T98G cells with the two antibodies against DYRK1A used in the ChIP-qPCR assays included in panels E and F (m: mouse monoclonal 7D10 from Abnova; r: rabbit polyclonal from Abcam). The chromatin fraction and the immunoprecipitates were analyzed by Western blot with anti-DYRK1A and anti-histone H3 antibodies as indicated. Note that although both antibodies immunoprecipitate DYRK1A with similar efficiency, H3 is only detected in the rabbit anti-DYRK1A immunoprecipitates. (E, F) ChIP-qPCR assays on samples from T98G cells immunoprecipitated with the two anti-DYRK1A antibodies. ChIP signals were normalized with inputs. As control, IgG from mouse were used. In F, only the results with the mouse antibodies are shown to highlight the poor enrichment over the IgG control. (G) T98G cells were transfected with a pGL2-based plasmid in which five TCTCGCGAGA sequences (represented as white boxes in the scheme) were cloned in tandem upstream of the TATA-box of the adenovirus E1B promoter. In the left panel, the relative transcriptional activity of the multimeric reporter is compared with that of pGL2-basic. In the right panel, transfections were done in shRNA-control infected cells or in cells depleted of DYRK1A by lentiviral transduction of two different shRNAs; values are expressed as the fold change over that of a pGL2-basic reporter in each of the shRNA-treated cell pools. (H) HeLa nuclear extracts were immunoprecipitated with two different antibodies to DYRK1A (Abnova mouse monoclonal and Abcam rabbit polyclonal) or a rabbit polyclonal anti-KAISO antiserum. Normal mouse IgGs (mIgG) or normal rabbit IgGs (rIgG) were used as controls for specificity. Both the lysates (10%) and the immunocomplexes were analyzed by immunoblotting with antibodies against DYRK1A, KAISO and p120-catenin, as indicated in the Figure. Figure S5. Related to Figure 5 DYRK1A-peak enrichment T98G cells grown in proliferating or resting conditions. (A) Distribution of DYRK1A ChIP-Seq target regions as promoters or regions not associated to promoters in the overlapping peaks between T98G proliferating cells and T98G serum starved cells (common), in the peaks only detected in the proliferating ChIP-Seq or only in the T98G serum starved cells ChIP-Seq. DYRK1A depletion does not affect the cell proliferation. (B) Cumulative cell growth curve of T98G cells infected with a lentivirus expressing a shControl or shDYRK1A.2. The graph represents the total number of cells over the period indicated. (C) Total cells extracts prepared from T98G infected with lentiviruses expressing a shControl or two different shRNAs to DYRK1A were analyzed by Western blot with antibodies to detect cyclin D1. It has been shown that DYRK1A depletion in human fibroblast induces increased cyclin D1 accumulation with one subpopulation of cells arresting proliferation by co-stabilizing the CDK inhibitor p21 (Chen et al., 2013). No changes in cyclin D1 accumulation were detected in T98G upon DYRK1A depletion, which would be in agreement with a lack of effect in cell proliferation. (D) Protein levels of DYRK1A were determined by Western blot in proliferating and serum starved T98G cells. The graph shows the quantification of two independent experiments. No significant differences were found. (E) The expression levels of DYRK1A mRNA were determined by RTqPCR in proliferating and serum starved T98G cells. The data represent the mean and standard deviation of five independent experiments (p-value=0.00187). The reduction in DYRK1A mRNA levels could be the consequence of the existence of E2Fresponsive elements in one of the DYRK1A promoters (Maenz et al., 2008), which would be repressed in the serum starved cells. (F) The cell cycle profile of T98G proliferating and serum starved cells was determined by FACs analysis. The distribution of the G1, S and G2/M subpopulations is shown. Note that about 90% of the cells were arrested at G1 phase upon serum withdrawal. Figure S6. Related to Figure 6 DYRK1B interacts with the CTD of RNAPII. (A) Soluble cell extracts from HEK-293T cells expressing HA-DYRK1B wild-type (WT) or a kinase-inactive version (KR) were immunoprecipitated either with control IgGs (mouse and rabbit, as indicated) or with the anti-RNAPII antibodies N20 (rabbit) and 8WG16 (mouse). Both the input and the immunoprecipitates were analyzed with an anti-HA antibody and with the anti-RNAPII antibody N20. The position of the hypo- and the hyperphosphorylated forms of the RNAPII (IIa and IIo, respectively) is indicated. The asterisk points to a cross-reacting band. DYRK1B (wt and kinase-dead) is present in the N20 immunoprecipitates but no in the 8WG16 immunoprecipitates, similarly to what happens with its paralogue DYRK1A. (B) The proteins indicated were expressed in HEK-293T cells by transient transfection of the corresponding plasmids (Flag-tagged in the case of DYRK1A and HA-tagged in the case of DYRK1B). Soluble cell extracts were incubated with unfused GST or GST-CTD immobilized on gluthatione-Sepharose beads. The bound proteins were detected by Western blot with anti-Flag or anti-HA antibodies. Although DYRK1B is detected as present in the GST control pull-down, a clear increased is observed in the GST-CTD bound fraction for both the wt and the kinase inactive version. Therefore, DYRK1B interacts with the non-phosphorylated CTD independently of its kinase activity. Subcellular distribution of DYRK1B. (C) Panc-1 cells were fractionated into cytosolic, soluble nuclear and insoluble nuclear (chromatin) fractions. Equivalent aliquots of each fraction were analyzed by Western blot with antibodies to the indicated proteins. The purity of the fractions was assessed with the compartment-specific marker proteins GAPDH and histone H3. DYRK1B is mostly detected in the cytosolic fraction; however, a small pool of DYRK1B is present in the nuclear soluble and insoluble (chromatin) fractions. DYRK1B phosphorylates the CTD of RNAPII. (D) The in vitro kinase assays were performed at equal specific activities of DYRK1A and DYRK1B obtained from commercial sources (DYRK1A specific activity on DYRKtide peptide=2,880 nmol/mg; dosage= 6.4, 12.8 ng; DYRK1B specific activity on DYRKtide=2,210 nmol/mg; dosage=10, 20 ng) as the source of the enzyme and the fusion protein GST-CTD as the substrate (150 ng). An aliquot of the reaction was analyzed in immunoblots probed with antibodies to: i) GST, to detect the fusion GSTCTD (note that due to differences in amounts, the GST-DYRK fusions are not detected); ii) the CTD phosphoresidues indicated; iii) the DYRK proteins. Note that both kinases phosphorylate Ser2, Ser5 and Ser7 to similar extent. DYRK1A phosphorylates the RNAPII CTD at Ser2. (E) DYRK1A phosphorylates Ser2 within the CTD as detected by the H5 antibody. GST-CTD (150 ng) was incubated with GST (-) or GST-DYRK1A (20 ng) in and IVK assay. An aliquot of the reaction was analyzed in Western blots probed with antibodies to GST, to detect the fusion GST-CTD, and the H5 antibody to detect the CTD Ser2 phosphoresidue. Depletion of DYRK1A does not change global phosphorylation levels of the CTD of RNAPII. (F) Total cells extracts prepared from T98G infected with lentiviruses expressing a shControl or two different shRNAs to DYRK1A were analyzed by Western blot with antibodies to detect total RNAPII (N20) or the H5 and H14 antibodies, which recognize mostly Ser2 phosphorylated (in the context of phosphorylated neighboring Ser5 residues) or Ser5 phosphorylated (Chapman et al. 2007). The position of the hyper- (IIo) and hypo- (IIa) phosphorylated forms of RNAPII is indicated. The asterisk indicates a cross-reacting band for the DYRK1A antibody. Assessing the ability of DYRK1B to be recruited to DYRK1A target sites. (G) Soluble extracts of Panc-1 cells were immunoprecipitated with rabbit polyclonal antibodies to either DYRK1A or DYRK1B. Both the lysates and the immunoprecipitates were assayed by Western blot for the presence of the kinases. Rabbit IgGs were used for control immunoprecipitations. The results show that each antibody specifically immunoprecipitates the corresponding DYRK protein. (H) ChIPWestern blot on Panc-1 cells with the anti-DYRK1B antibody to assess the ability of this antibody to immunoprecipitate DYRK1B from formaldehyde-crosslinked cells. (I, J) ChIP-qPCR assays using samples from Panc-1 cells immunoprecipitated with anti- DYRK1A and anti-DYRK1B antibodies. As control, rabbit IgGs were used. In G, only the results with the DYRK1B antibody were shown to highlight the lack of enrichment over the IgG control. Figure S7. Related to Figure 7 DYRK1A depletion does not affect globally the acetylation levels of H3 or the phosphorylation of H3 at Ser10, Ser28 or Thr45. (A) Total cells extracts prepared from T98G infected with lentiviruses expressing a shControl or two different shRNAs to DYRK1A were analyzed by Western blot with antibodies to detect histone H3 (H3) or the following post-translational modifications: acetylated H3, phosphorylated H3 at residues Ser10, Ser28, Thr45, and acetylated H4. DYRK1A depletion reduces RNAPII occupancy at target promoters. (B) The presence of RNAPII at DYRK1A target promoters was assessed by ChIP-qPCR with the N20 antibody in cells infected with a shControl or two different shDYRK1A. Values are normalized with input. (C, D, F) DYRK1A depletion leads to a decreased in elongating RNAPII along gene body. The presence of RNAPII phosphorylated at Ser2 and Ser5 along the indicated DYRK1A target gene bodies was assessed by ChIP-qPCR with specific antibodies, in control cells and cells with reduced DYRK1A expression. For each gene, the scheme shows the basic gene structure and the position of the amplicons. ChIP signals were normalized with RNAPII (8WG16). Signals in shControl cells were set at 1 for each amplicon, and the values from the shDYRK1A cells were expressed relative to these signals. The graphs show the average of two independent experiments with the bars indicating standard deviations. Table S1. Related to Figure 2. DYRK1A ChIP-Seq results on T98G and HeLa cells. SUPPLEMENTAL EXPERIMENTAL PROCEDURES Plasmids The plasmids to express HA- or GST-tagged, wild type or kinase inactive DYRK1A (in which the ATP binding Lys179 was replaced by Arg), have been described previously (Alvarez et al., 2007; Alvarez et al., 2003). To express N-terminal fusions of the different human DYRKs (Accession Nº for DYRK1A: NP_569120; Acc. Nº for DYRK1B: NP_004705; Acc. Nº for DYRK2: NP_006473; Acc. Nº for DYRK3: NP_003573; Acc. Nº for DRYK4: NP_003836) to the yeast Gal4 DNA binding domain (DBD; amino acids 1-147 of the yeast transcription factor), EcoRI/XbaI fragments containing the corresponding open reading frames were ligated in-frame into the EcoRI/XbaI sites of pCG4-DBD (de la Luna et al., 1999). The DYRK1B open reading frame was cloned into pCDNA3-HA to express a HA-tagged version of the kinase. To generate a DYRK1B kinase inactive mutant, we substituted the codon for Lys140 in the ATP binding site by an Arg codon, and the loss of activity was confirmed in in vitro kinase assays. As reporter plasmids we used, pG5HIV-Luc (Montanuy et al., 2008), kindly provided by C. Suñe (Instituto de Parasitología y Biomedicina López Neyra, Granada, Spain), and pG4E1b-Luc (de la Luna et al., 1999). The pG4E1b-TATAless-Luc reporter plasmid was generated with the QuickChange Site-Directed Mutagenesis Kit (Invitrogen) on the pG4-E1b-Luc using the TATA-less primer (Supplemental Tables). The reporter plasmid in which luciferase expression is under the control of the promoter of the RPS11 gene was generated by PCR amplification of the corresponding genomic regions with primers RPS11-f and RPS11-r (Supplemental Tables), which was subcloned into pGL2-basic (Promega). The two consensus sequences within the promoter region were deleted with the QuickChange Site-Directed Mutagenesis Kit and primers RPS11-mut1 and RPS11-mut2 (Supplemental Tables). For the multimeric reporter, sense and antisense oligonucleotides with 5 repetitions of the TCTCGCGAGA sequence followed by the E1B adenovirus TATA box (AGGGTATATAATG) were annealed and cloned into pGL2-basic. For the pull-down assays, pGEX-CTD and pMyc-Cyclin T1 (Taube et al., 2002) were kindly provided by M. Peterlin (University of California, San Francisco, USA). For the expression of cyclin L2, the open reading frame of human cyclin L2 (Acc. Nº NP_112199) was cloned in-frame into pCDNA-3 with a Flag-tag at the N-terminus to generate pFlag-Cyclin L2. Cell culture and DNA transfection The HeLa, T98G, Panc-1, HEK-293T and HEK-293 cell lines were supplied by the American Type Culture Collection (http://www.atcc.org). All the cell lines were cultured at 37ºC and in 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen) with 10% fetal bovine serum (FBS; Invitrogen) and supplemented with antibiotics (100 u/ml penicillin and 100 µg/ml streptomycin; Invitrogen). The cells were transfected by the calcium phosphate precipitation method and processed 24-48 h after transfection. For serum starvation conditions, T98G cells were grown in DMEM without FBS for 48 h. For harmine treatment, T98G cells were treated with 10 µM harmine or with vehicle (0.02% DMSO) for 16 h. Lentivirus preparation and infection HEK-293 cells were transfected with pVSV-G (Stewart et al., 2003) and pCMV∆R8.91 (Zufferey et al., 1997), together with the pLKO.1-puro non-targeting vector (Sigma Mission clone SHC001) or pLKO.1-shRNA DYRK1A (Sigma Mission clones TRCN0000022999, TRCN0000199464 and TRCN0000199188), and the supernatant was harvested after 72 h. Viral particles were concentrated by centrifugation through a 4% sucrose cushion at 87,500 x g for 90 min at 4ºC. The cells were infected by adding the virus in DMEM with 5 µg/ml hexadimethrine bromide (Polybrene, Sigma). The medium was removed 24 h after infection and the cells processed 48 h later to quantify mRNA expression or for ChIP analysis. For selection, 1 µg/ml puromycin was added for 3 days and the cells were cultured for a further 24 h in the absence of puromycin. For the rescue experiments, the open reading frames of DYRK1Awt and the kinaseinactive version were cloned into the pWPXL lentiviral vector (Didier Trono's group; Addgene plasmid 12257). The expression of DYRK1A from these lentivectors is resistant to the effect of the DYRK1A shRNA expressed by the Mission clone TRCN0000199188 that targets the DYRK1A 3'-UTR. FACS analysis of cell cycle parameters Cells harvested in PBS-5 mM EDTA were fixed and permeabilized overnight with 100% ethanol. The cells were then resuspended in phosphate-buffered saline (PBS) containing 0.5 µg/ml Ribonuclease A (Sigma), 50 µg/ml propidium iodide (Sigma) and 3.8 mM sodium citrate, and analyzed by FACS flow cytometry with the Cell Quest software (Becton Dickinson). Cell lysate preparation, glycerol gradient sedimentation and size exclusion chromatography To prepare the total cell lysates, 1x106 cells were resuspended in 150 µl 2x sample loading buffer (100 mM Tris-HCl [pH 6.8], 200 mM dithiothreitol [DTT], 4% [w/v] sodium dodecyl sulfate [SDS], 20% [v/v] glycerol, 0.2% [w/v] bromophenol blue) and heated for 10 min at 98ºC. Soluble extracts were prepared by incubating cells for 15 min at 4ºC in lysis buffer A (50 mM HEPES [pH 7.4], 150 mM NaCl, 2 mM EDTA, 1% [v/v] Nonidet P40 [NP-40], protease inhibitor cocktail [PIC; Roche Diagnostics] and phosphatase inhibitors [2 mM sodium orthovanadate, 30 mM sodium pyrophosphate, and 25 mM sodium fluoride]), followed by centrifugation at 13,000xg for 30 min at 4ºC. Subcellular fractionation of cells was performed using NE-PER (Nuclear and Cytoplasmic Extraction Reagent; Pierce) according to the manufacturer’s protocol. Protein quantification was performed with the commercial BCA Protein Assay Kit (Pierce). For the subnuclear fractionation, 3 mg of HeLa nuclear extracts (HNE; CIL Biotech) was applied to a 10 ml 10–40% glycerol gradient in 20 mM HEPES [pH 7.9], 150 mM KCl, 0.2 mM EDTA, 0.1 % NP-40 and PIC. After centrifugation at 33,000 rpm in a SW41 Ti rotor (Beckman) for 20 h at 4°C, 24 fractions (500 µl each) were collected from the top of the gradient and a 30 µl aliquot of each fraction was analyzed in Western blots. Molecular mass standards (Gel Filtration High Molecular Weight Calibration Kit, Amersham) were used to identify the fractionation profiles within the gradient and the marker proteins were detected by silver staining of SDS-PAGE gels using standard protocols. Western blotting Cell lysates were resolved on SDS-polyacrylamide gels and the proteins transferred onto Hybond-ECL nitrocellulose membranes (Amersham Biosciences). Membranes were blocked for 30 min at room temperature (RT) with 10% (w/v) skimmed milk diluted in TBS (10 mM Tris-HCl [pH 7.4] and 100 mM NaCl)-0.1% Tween-20 (TBS-T), and then probed for 1 h at RT with primary antibodies diluted in 5% skimmed milk in TBS-T. After several washes, the membranes were incubated at RT for 45 min with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz) diluted in 5% skimmed milk in TBS-T. Antibody binding was detected by chemiluminiscence (SuperSignal West Pico Chemiluminescent Substrate, Pierce) on a LAS-3000 image analyzer (Fuji PhotoFilm). Primary antibodies are listed in the Supplemental Tables for Extended Experimental Procedures. Immunoprecipitation Soluble cell extracts were incubated overnight at 4°C with protein G-Magnetic beads (Invitrogen) pre-bound with a mouse monoclonal anti-DYRK1A antibody. Mouse IgGs or rabbit IgGs (Santa Cruz) were used as controls. The beads were washed four times with lysis buffer A, adding 0.1% NP-40 to the three initial washes, and they were finally resuspended in sample loading buffer. Samples were resolved by SDS-PAGE and analyzed by immunoblotting or used for in vitro kinase assays. The antibodies used in the immunoprecipitation experiments are listed in the Supplemental Tables for Extended Experimental Procedures. GST-fusion protein expression in bacteria and pull-down assays GST-fusion constructs were transformed into E. coli BL21(DE3)pLysS (Stratagene) and protein expression was induced with 0.1 mM isopropyl-β-D-thiogalactoside for 3 h at 37ºC for the GST-CTD fusion protein and for 8 h at 20ºC for GST-DYRK1A (WT or KR). Cells were lysed in lysis buffer B (10 mM Tris-HCl [pH 8], 100 mM NaCl, 1 mM EDTA, 0.5% NP-40 and a protease inhibitor cocktail), and the bacterial lysates were incubated with glutathione-Sepharose 4B beads (Amersham Biosciences) for 45 min at RT and then washed four times with lysis buffer B. For pull-down assays, prey proteins were transcribed and translated in vitro in the presence of [35S]-methionine using the TnT T7 Coupled Reticulocyte Lysate System (Promega), following manufacturer’s instructions. Equivalent amounts of synthesized proteins were incubated with unfused GST or GST-CTD immobilized on glutathioneSepharose beads that had been previously equilibrated in binding buffer (20 mM HEPES-KOH, 200 mM KCl, 0.1% Triton-X-100, 0.05% NP-40, 5 mM EDTA, 0.3% BSA and 5 mM DTT). After 3 h rolling at 4ºC, the beads were washed extensively with cold binding buffer containing 500 mM KCl, and the bound proteins were resolved on SDSPAGE gels that were then dried and exposed to a film. In vitro kinase assays For in vitro kinase (IVK) assays, we used GST-DYRK1A expressed in bacteria as a purified enzyme source (Alvarez et al., 2007). Both the substrate (GST-CTD) and the enzyme were eluted from the glutathione beads with 10 mM reduced glutathione (Sigma) in 50 mM Tris-HCl pH 8 and dialyzed against a buffer containing 50 mM HEPES pH 7.4, 150 mM NaCl and 2 mM EDTA. For the IVK assays, we used 100-150 ng of GST-CTD and 5, 10 or 20 ng of GST-DYRK1AWT or GST-DYRK1AKR. The proteins were incubated for 20 min at 30oC in 20 µl of phosphorylation buffer (50 mM HEPES [pH 7.4], 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT and 50 µM ATP]. For the comparative IVK assays of DYRK1A and DYRK1B, we used commercial proteins (Life Technologies). The IVK assays were performed at the same specific activity for each kinase as determined by the supplier on DYRKtide (DYRK1A: 2,880 nmol/mg; DYRK1B: 2,210 nmol/mg). For the IVKs, in which DYRK1A was purified by immunoprecipitation from cells, the immunocomplexes were incubated for 20 min at 30oC in 20 µl of phosphorylation buffer (50 mM HEPES [pH 7.4], 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT and 50 µM ATP [γ32P-ATP, 2.5x10-3 µCi/pmol] in the presence of the peptide substrate DYRKtide (Himpel et al., 2000) and 32P incorporation was determined as described (Alvarez et al., 2007). Harmine was used as a DYRK1A inhibitor (Bain et al., 2007) and in this case the immunoprecipitates were preincubated for 15 min with 10 µM harmine or with the vehicle alone (DMSO) prior to carrying out the IVK reaction in the presence of the inhibitor. Electrophoretic mobility-shift assay (EMSA) A [32P]-labeled double stranded oligonucleotide (0.03 pmol) spanning either the wt or the mutant DYRK1A motif (for details see Supplemental Experimental Procedures) was incubated with 5-10 µg of nuclear cell extract and 30 ng of poly-dI-dC (Roche Diagnostics) at 20°C for 20 min in 250 mM Tris-HCl [pH7.5], 250 mM NaCl, 5 mM MgCl2, 5 mM DTT, 1 mM EDTA and 50% glycerol. For the supershift assay, 2 µg of either mouse IgG (Santa Cruz), mouse monoclonal (Abnova H00001859) or rabbit polyclonal (Abcam ab69811) anti-DYRK1A antibodies were pre-incubated with the extracts for 15 min on ice before the addition of the DNA probe. Binding reactions were analyzed on non-denaturing 4% acrylamide gels. Reverse transcription (RT) and quantitative PCR (qPCR) Total RNA was isolated with the RNeasy extraction Kit (Qiagen), treated with DNase I (Ambion, 2 U/µl) and quantified with Nanodrop. Superscript II Reverse Transcriptase (RT: Invitrogen) was used to synthesize cDNA from the total RNA (500 ng) as recommended by the manufacturer’s instructions. RT-qPCR reactions were performed with SYBR Green (Fermentas) in 384 well plates using the Roche LC-480 cycler (Roche Applied Science). The template was denatured at 95º for 5 min, and subjected to 50 cycles of 15 s 95º, 20 s 60º, 20 s 72º. Each sample was assayed in triplicate. The Ct (threshold cycle) was calculated for each sample using the Relative Quantification 2nd Derivative Maximum method with the Lightcycler 480 1.2 software (Roche). No PCR products were observed in the absence of template and all the primer sets gave narrow single melting point curves that were checked at the end of each run. For RTqPCR, analysis was performed using a 1/10 dilution of cDNA as the template. A "noRT" control was included in each experiment to ensure that the PCR products were not amplified from contaminating DNA, and all results were normalized to GAPDH, β-actin or EF1α expression. Chromatin immunoprecipitation (ChIP) assay ChIP assays were performed from approximately 107 HeLa or T98G cells per experiment. Briefly, formaldehyde was added to the culture medium to a final concentration of 1% for 10 min at RT and the crosslinking was then quenched with 0.125 M glycine for 5 min. Crosslinked cells were washed twice with TBS, resuspended in 1 ml of Buffer I (5 mM PIPES [pH 8.0], 85 mM KCl, 0.5% NP-40 plus PIC) and incubated on ice for 10 min. Cells were centrifuged at 800 x g for 5 min, and the cell pellet was resuspended in 1.2 ml of Buffer II (1% SDS, 10 mM EDTA, 50 mM Tris-HCl [pH 8.0], 1 mM phenylmethylsulfonyl fluoride and PIC) and incubated for a further 10 min on ice. Samples were centrifuged and the cell pellet was resuspended in 1.2 ml of IP Buffer (100 mM Tris-HCl [pH 8.6], 0.3% SDS, 1.7% Triton X-100 and 5 mM EDTA) prior to sonicating the chromatin to an average size of 200-500 bp (Bioruptor, Diagenode). Extracts (600 µg of total protein) were diluted in 1 ml of IP Buffer and immunoprecipitated overnight at 4°C with the corresponding antibodies (listed in the Supplemental Tables for Extended Experimental Procedures) or control IgG antibodies. Immune complexes were recovered by incubating for 4 h at 4°C with protein G-Sepharose beads (40 µl), and the beads were then washed with three successive 1 ml washes in Low salt Buffer III (50 mM HEPES [pH 7.5], 140 mM NaCl, 1% Triton X-100 plus PIC), and one wash with High salt Buffer IV (50 mM HEPES [pH 7.5], 500 mM NaCl, 1% Triton X-100 plus PIC). DNA was eluted twice from the beads for 1 h at 65ºC in Elution Buffer (1% SDS, 100 mM NaHCO3) with constant agitation (1,000 rpm). The crosslinking was reverted by further incubating the DNA at 65ºC overnight at 1,000 rpm. The eluted material was phenol/chloroform extracted and ethanol-precipitated, and the ChIP DNA was resuspended in 50 µl of H2O for further analysis. For ChIP-qPCR, a 1/10 dilution of ChIP DNA was used as the template for the PCR reaction, and a 1/1000 dilution of input DNA was used as standard for normalization. For all antibodies, ChIP signals were normalized with inputs. In the case of antibodies detected post-translational modifications, the data was represented as the ratio to the non-modified protein: H3-T45p/H3, H3-Ac/H3, Ser2p/8WG16, Ser5p/8WG16. For standarization, values in T98G cells infected with a control lentivirus (shControl) were set at 1 and values from the cells infected with a lentivirus expressing a shRNA against DYRK1A (shDYRK1A), or other treatments, expressed relative to these signals. Statistical analysis All results are expressed as the mean ± SD of three independent experiments unless otherwise stated. Statistical analyses were performed with a two-tailed unpaired Student's t-test to determine statistical significance between the groups. P-values are represented in all Figures as follows: *, p-value ≤ 0.05; **, p-value ≤ 0.01; ***, p-value ≤ 0.001. Computational analysis Sequencing and read mapping ChIP-Seq libraries were sequenced to a length of 36bp on an Illumina GAIIx sequencer, and an average of 25,752,900 reads were obtained per library. The quality of the sequenced reads was controlled using FastQC (Trinklein et al., 2003). The reads were aligned to the hg19 human reference genome using bwa (Li and Durbin, 2009), allowing up to 3 mismatches and no gaps (whereby >97% of the reads were aligned). The resulting SAM files were sorted by position and transformed to BAM using samtools (Li et al., 2009). Picard (http://picard.sourceforge.net) was used to mark clonal duplicate reads in the BAM files (e.g. ~20% duplicate reads marked in TG98G) and ‘samtools flagstat’ to obtain quality information about the mapping. Peak identification and summary of the ChIP-Seq data The ChIP-Seq peak caller ‘shore peak’ (Moyroud et al., 2011; Pose et al., 2013) from the open source package SHORE (Ossowski et al., 2010) (http://sourceforge.net/projects/shore/) was used for the calling and visualization of peaks. BAM files were converted to the native SHORE format map.list using ‘shore convert’, and the peaks were predicted by the peak caller ‘shore peak’, using only uniquely aligned reads and allowing for up to 3 duplicate reads at a position. Peaks were accepted when: i) fold-change (FC, DYRK1A IgG versus control IgG) was ≥ 3; ii) the false discovery rate (FDR) was ≤ 0.05; iii) peak size was at least 80 bp; and iv) the x-shift (distance between forward and reverse peak) was at least 10 bp. The parameters of peak calling were: shore peak –i ChIP.map.list –c Control.map.list -S 10000 -V 10 -Q 0.5 -J 80 -B 3 -F 0 - n 3 --min-foldchange=3 -H 1,1 --rplot=500 –r hg19.index.shore T98G ChIP-Seq: 966 peaks were detected with a FDR≤10-2 (598 peaks with a FDR<10-3). However, only 604 peaks also showed a significant x-shift (x-shift≥80) and normalized FC scores (454 peaks for a FDR<10-3). Visual inspection indicated that the top 500 peaks mostly present an S-shaped coverage pattern. MACS (Zhang et al., 2008) was used, in no-model mode, assuming the fragment sizes estimated by SHORE, to produce wiggle files for visual inspection in IGV. MACS returned 713 peaks with a FDR<5%. More than 90% of the top 500 SHORE peaks were also detected with MACS. Visual inspection of the overlapping peaks as well as removal of peaks with a small x-shift (<80 bp) or forward-reverse coverage imbalance resulted in a final set of 539 high quality peaks. Serum-starved T98G ChIP-Seq: SHORE identified 477 peaks with a FDR≤0.05 (247 for a FDR≤10-2). Correction for x-shift resulted in 341 peaks (221 for a FDR≤10-2), of which 233 (199 for a FDR≤10-2), were included within the top 500 MACS results. Visual inspection and quality filtering by x-shift and fwd-rev coverage distribution resulted in a final selection of 337 high quality peaks. HeLa ChIP-Seq: Peak detection in HeLa ChIPs revealed substantially fewer peaks than the T98G ChIPs. To reduce false positives, we used SHORE’s ability to analyze two independent biological replicates together. We selected 73 peak predictions with pvalue < 0.05 (p<10-2: 64 peaks), of which 68 were found in the top 100 MACS results. 77 SHORE peaks showed a good x-shift (≥80), of which 67 (87%) were found in the top 100 MACS results. Peaks resulting from the three ChIP-Seq experiments were loaded into a MySQL database, and SQL commands were used to overlap the three peak sets using a 10% minimal overlap between peaks as threshold (Supplemental Table S1). Peak annotation An in-house gene and promoter annotation pipeline (ChIPanno) was used to annotate potential target genes and promoters. ChIPanno uses gene, transcript, promoter and transcription start site (TSS) annotation data from Ensembl, NCBI RefSeq, SwitchGear-TSS (Landolin et al., 2010; Trinklein et al., 2003) (http://www.switchgeargenomics.com/) and the UCSC genome browser database (Meyer et al., 2013), all obtained via the UCSC Table Browser (Karolchik et al., 2004). TSS from SwitchGear-TSS were filtered by setting confScore > 20 to decrease false positive TSS annotations (Landolin et al., 2010). We further used the promoter definition provided by the annotation tool HOMER (Heinz et al., 2010) and a custom set of promoters defined as SwitchGear-TSS 1 kb upstream to 100 bp downstream (called SW-TSS-1.1k from here on). All information was uploaded into the MySQL database to facilitate further motif, peak and functional enrichment analyses. Peak distribution at various genomic locations and peak density heatmaps The distribution of various genomic features in binding sites was assessed using CEAS software (Shin et al., 2009), and with in-house Python and Perl scripts. The distribution of the peaks within a -1 kb to +1 kb window centered on the TSS of each RefSeq transcript was calculated in 25 bp bins using scripts contained in the HOMER package (Heinz et al., 2010) and plotted using R. Expression of target genes according to public microarray data analysis Normalized mRNA expression (Affymetrix Human U133A/GNF1H Gene Atlas microarray chip) in 73 normal human tissues was downloaded from the BioGPS database (Wu et al., 2009). When more than one probe was present for the same gene, they were averaged. Based on the ‘‘gene-normalized’’ expression of the DYRK1A targets in the GeneAtlas data, we carried out Sample Level Enrichment Analysis (Gundem and Lopez-Bigas, 2012) using Gitools (www.gitools.org; (PerezLlamas and Lopez-Bigas, 2011)), which compares the median expression value of the genes in each module (DRKY1A targets) with a distribution of random modules of the same size drawn from the expression values for the sample. The result is a z-score, which is a measure of the difference between the observed median expression value compared with the expected value. Positive and negative z-scores indicate significantly higher or lower expression level of genes in the module under this condition, respectively. Similarly, DYRK1A-target expression was analyzed in brain samples from six Down syndrome individuals and compared to controls (GEO dataset GSE5390; (Lockstone et al., 2007), as well as in teratozoospermic samples with respect to the average level in samples from 14 normospermic individuals (GEO dataset GSE6872; (Platts et al., 2007). Conservation score We extracted sequences 50 bp upstream and 50 bp downstream of each peak's midpoint, and calculated the average conservation score of this region based on UCSC PhastCons conservation score data (Siepel et al., 2005) in placental mammals (hg19, phastCons46way for human). The calculation was done using the scripts from ChIPseeqer (Giannopoulou and Elemento, 2011). To plot the conservation score, the score was calculated for 250 bp upstream and downstream regions from the peak’s summit. We compared the conservation score of random peaks within 5 kb region of each DYRK1A peak and plotted an average conservation score of 10 bp window using R. De novo motif search MEME was used for de novo identification of motifs in peak regions and associated promoters (Bailey et al., 2009) (http://meme.nbcr.net/downloads/meme_4.6.0.tar.gz). Motifs were considered if they were found in at least 5 sites (-minsites 5), and MEME was requested to retrieve the 10 motifs with lowest p-value (-nmotif 10) and to search within a motif length range of 6-20 bp (-minw 6 -maxw 20). In separate executions, MEME was performed for a fixed motif length of 10 bp (-minw 10 -maxw 10) and 11 bp (-minw 11 -maxw 11) to optimize the prediction for the DYRK1A associated motif selected in the initial motif search. Gene ontology analysis and other analysis For Gene ontology analysis, the online software DAVID (Huang da et al., 2009) was used. The Venn diagrams were generated with the web application BioVenn (Hulsen et al., 2008). Antibodies used in this study Target Host Source Assay DYRK1A Mouse Abnova, #H000001859 Western blot Immunoprecipitation ChIP DYRK1A Rabbit Abcam, #ab69811 Immunoprecipitation ChIP DYRK1A Rabbit Sigma, #D1694 Western Blot DYRK1B Rabbit Abcam, #ab113968 Western blot Immunoprecipitation ChIP Histone H3 Rabbit Abcam, #ab1791 Western blot ChIP H3-Ser10p Rabbit Millipore, #04-817 Western blot H3-Ser28p Rabbit Millipore, #17-10269 Western blot H3-Thr45p Rabbit Abcam, #ab26127 Western blot ChIP H3-Ac Rabbit Millipore, #06-599 Western blot ChIP Histone H4-Ac Rabbit Millipore, #06-866 Western blot RPB1 (RNAPII) Rabbit N20, Santa Cruz Biotechnology, #sc-899 Western blot Immunoprecipitation ChIP RNAPII-Ser2p Rabbit Abcam, #ab5095 Western blot ChIP RNAPII-Ser5p Rabbit Abcam, #ab5131 Western blot ChIP RNAPII-IIa Mouse 8WG16, Covance, #MMS-126R Western blot Immunoprecipitation ChIP RNAPII-Ser2p Mouse H5, Covance, #MMS-129R Western blot RNAPII-Ser5p Mouse H14, Covance, #MMS-134R Western blot RNAPII-Tyr1p Rat Active Motif, #61383 Western blot RNAPII-Thr4p Rat Active Motif, #61361 Western blot RNAPII-Ser7p Rat Active Motif, #61087 Western blot Gal4-DBD Rabbit Santa Cruz Biotechnology, #sc-577 Western blot Lamin B1 Mouse Santa Cruz Biotechnology, #sc-20682 Western blot KAISO Rabbit Santa Cruz Biotechnology, #sc-23871 Western blot Immunoprecipitation CDK9 Rabbit Santa Cruz Biotechnology, #sc-484 Western blot Cyclin T1 Rabbit Santa Cruz Biotechnology, #sc-8127 Western blot p120-catenin Mouse BD-Bioscience, #610133 Western blot GAPDH Mouse Millipore, #374 Western blot α-tubulin Mouse Sigma, #T6199 Western blot Vinculin Mouse Sigma, #V9131 Western blot Flag Mouse M2, Sigma, #F1804 Western blot Immunoprecipitation HA Mouse Covance, #MMS-101R Western blot Immunoprecipitation Cyclin D1 Mouse Cell Signaling, #2926 Western blot GST Mouse Santa Cruz Biotechnology, #sc-138 Western blot Primers used for mutagenesis and cloning Name Sequence TATA-less GGCTCGCCTCTGAGACTAGACGCTAGATTC RPS11-f CCCGGGAACAAAGATGGCGACGCCGC RPS11-r AGATCTCTTCCCGGCCGCCTGAAAA RPS11-mut1 CAGGATGGACTCCGTACGACATACGGGCGGGCTGAAGGC RPS11-mut2 GGCGTTGTGGGGCCTAAGACCACCGTCTAGCACTTCCCGC Primers used for ChIP-qPCR Region (gene) Forward primer Reverse primer RPS11 (-2) ACGCCCGGCTAATTTTTGT TGAGGTCAGGGGTTCAACA RPS11 (-1) GCCACACATGCTCCTAGCAC GCTCTGTTCGTTCAGCCTTC RPS11 (0) GAAGGCTGAACGAACAGAGC CGTACGGAGTCCATCCTGTT RPS11 (+1) GGTTTAAGTGCAGCCTGTCAA ACGTAAGGAGAAACGCAGCA RPS11 (+2) GAGGCTCAAGCGTTTTAGGA CCGTTTCCACAGAATTACCC RBM39 AATTTGAGCGGCCGAAGTAT GAATGGGGGATGGGAATATC RPS11 GCTGAAGGCTGGTCACATCT GGGCACTGTGAAGGACTGAC ASXL1 AGCATCGCCTCCCAGAAT CACCGACCTCAGCTAGGAAC CDK12-A TGATAAGCAGGGGAATGAGG CTCCCTCACACAGACCCAGT CDK12-B GGAATGCCCAATTCAGAGAG CTTCGATACCAAGCGGTGAC CDK12-C TGGTTTGGTGCACTTTTCTG ATCCCGATGCAGGAAATTCT CDK12-D ATCCTGCCTTCAGCAGAACA GCTCTTGGGTTTTCATCAGC CDK12-E AAAACTCTATCGGGGGCCTA GAACTGGATGGAAGGATGGA DENR-A ACGCTCCGCAATTTTTCTC CTCCCGCGAGACAATGAG DENR-B GTTTGTGAAATGGCTGCTGA GGACTCGAAGTGGGTAATCG DENR-C TGGAAGGGGTCAAATAAAACA TGCAAGGCCACATACTCTTG DENR-D CGAAGATCTTGGAGAAGTAAAGAA TTTAAAAGGCCTCTCTCCCTTT LUC7L2 CCGGGAGGGAATGTAATGTA CTCCTCCCGCCCCTTTAC CDC5L CTCTGCCACTCGGTGACG CGCATGTCCAAAACAGAATG SMEK2 GGGAGTACTTCGGCGAGAC GGGAGATCGCGAGAACCT RPL12 GCGGACAAGCCAGATATAGG CTGCCCACAACAAACATGG RBM15 GAAAAAGGGGGTCGAAAGAG AAACCCGTCCTTTAAACCACTA RPS6-A CATCTTGAAGCAGCTGAACG CTCACTTCCGCTATCCCGTA RPS6-B CATGAAGCAGGGTGTCTTGA ACAATGCAACCACGAACTGA RPS6-C CTAGGACCAAAGCACCCAAG GCATATTCTGCAGCCTCTTCTT RPS6-D CGAAGAGACGCAGACTTTCC TCAGCAATGAAAAGTCAACAGA chr19_cov0 ATCTTGGTTCACCGCAACCT AATTAGCTGGGTGTGGTGGT chr20_cov0 GCTTGGCCAACAAGGTAAAA CTCTCTGCAACCTCCACCTC Primers used for RT-qPCR Gene Forward primer Reverse primer RPS11 GCTTCAAGACACCCAAGGAG TGACAATGGTCCTCTGCATC LUC7L2 TCGTCAACGAATCAAATTCAG ATCCGCTCTTAAAGCCAGGT ASXL1 TGCAGGTCATAGAGGCAGAA GAGCGTGAAAAGGCTGATTC CDC5L AGCTGCCCAAAGAGACAATG TGGCTTCAGAAAGCATCTCA DENR CGATTACCCACTTCGAGTCC CAGCTTCTTGTTTGGGTGAA RPS6 GCTAGCAGAATCCGCAAACT CTGCAGGACACGTGGAGTAA CDK12 TCCCACCCTTATTACCTGGA AGCTCTGGAGGGAGAGGAAG GADPH ACCCAGAAGACTGTGGATGG TTCAGCTCAGGGATGACCTT EF1α AGGTGATTATCCTGAACCATCC GAACGGCGATCAATCTTTTCC DNA-oligonucleotide probes used in EMSAs Name DYRK1A-WT DYRK1A-MUT Forward probe Reverse probe GGCCTAAGACTCTCGCGAGACACC GTCTAG GGCCTAAGACAGGTGTACAACACCG TCTAG CCGGATTCTGAGAGCGCTCTGTGGC AGATC CCGGATTCTGTCCACATGTTGTGGC AGATC SUPPLEMENTAL REFERENCES Alvarez, M., Altafaj, X., Aranda, S., and de la Luna, S. (2007). DYRK1A autophosphorylation on serine residue 520 modulates its kinase activity via 14-3-3 binding. Mol. Biol. Cell. 18, 1167-1178. Alvarez, M., Estivill, X., and de la Luna, S. (2003). DYRK1A accumulates in splicing speckles through a novel targeting signal and induces speckle disassembly. J. Cell Sci. 116, 3099-3107. Bailey, T.L., Boden, M., Buske, F.A., Frith, M., Grant, C.E., Clementi, L., Ren, J., Li, W.W., and Noble, W.S. (2009). MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res. 37, W202-208. Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C.J., McLauchlan, H., Klevernic, I., Arthur, J.S., Alessi, D.R., and Cohen, P. (2007). The selectivity of protein kinase inhibitors: a further update. Biochem J. 408, 297-315. Chapman, R.D., Heidemann, M., Albert, T.K., Mailhammer, R., Flatley, A., Meisterernst, M., Kremmer, E., and Eick, D. (2007).Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7. Science. 318, 1780–1782. Chen, J.Y., Lin, J.R., Tsai, F.C., Meyer, T. (2013). Dosage of Dyrk1a shifts cells within a p21-cyclin D1 signaling map to control the decision to enter the cell cycle. Mol Cell. 52, 87-100. de la Luna, S., Allen, K.E., Mason, S.L., and La Thangue, N.B. (1999). Integration of a growth-suppressing BTB/POZ domain protein with the DP component of the E2F transcription factor. EMBO J. 18, 212-228. Giannopoulou, E.G., and Elemento, O. (2011). An integrated ChIP-seq analysis platform with customizable workflows. BMC Bioinformatics 12, 277. Gundem, G., and Lopez-Bigas, N. (2012). Sample-level enrichment analysis unravels shared stress phenotypes among multiple cancer types. Genome medicine 4, 28. Heinz, S., Benner, C., Spann, N., Bertolino, E., Lin, Y.C., Laslo, P., Cheng, J.X., Murre, C., Singh, H., and Glass, C.K. (2010). Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576-589. Himpel, S., Tegge, W., Frank, R., Leder, S., Joost, H.G., and Becker, W. (2000). Specificity determinants of substrate recognition by the protein kinase DYRK1A. J. Biol. Chem. 275, 2431-2438. Huang da, W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44-57. Hulsen, T., de Vlieg, J., and Alkema, W. (2008). BioVenn - a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams. BMC Genomics 9, 488. Karolchik, D., Hinrichs, A.S., Furey, T.S., Roskin, K.M., Sugnet, C.W., Haussler, D., and Kent, W.J. (2004). The UCSC Table Browser data retrieval tool. Nucleic Acids Res. 32, D493-496. Landolin, J.M., Johnson, D.S., Trinklein, N.D., Aldred, S.F., Medina, C., Shulha, H., Weng, Z., and Myers, R.M. (2010). Sequence features that drive human promoter function and tissue specificity. Genome Res. 20, 890-898. Li, H., and Durbin, R. (2009). Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 25, 1754-1760. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., and Durbin, R. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078-2079. Lockstone, H.E., Harris, L.W., Swatton, J.E., Wayland, M.T., Holland, A.J., and Bahn, S. (2007). Gene expression profiling in the adult Down syndrome brain. Genomics 90, 647-660. Maenz, B., Hekerman, P., Vela, E.M., Galceran, J., and Becker, W. (2008). Characterization of the human DYRK1A promoter and its regulation by the transcription factor E2F1. BMC Mol. Biol. 9, 30. Meyer, L.R., Zweig, A.S., Hinrichs, A.S., Karolchik, D., Kuhn, R.M., Wong, M., Sloan, C.A., Rosenbloom, K.R., Roe, G., Rhead, B., et al. (2013). The UCSC Genome Browser database: extensions and updates 2013. Nucleic Acids Res. 41, D64-69. Montanuy, I., Torremocha, R., Hernandez-Munain, C., and Sune, C. (2008). Promoter influences transcription elongation: TATA-box element mediates the assembly of processive transcription complexes responsive to cyclin-dependent kinase 9. J. Biol. Chem. 283, 7368-7378. Moyroud, E., Minguet, E.G., Ott, F., Yant, L., Pose, D., Monniaux, M., Blanchet, S., Bastien, O., Thevenon, E., Weigel, D., et al. (2011). Prediction of regulatory interactions from genome sequences using a biophysical model for the Arabidopsis LEAFY transcription factor. Plant Cell 23, 1293-1306. Ossowski, S., Schneeberger, K., Lucas-Lledo, J.I., Warthmann, N., Clark, R.M., Shaw, R.G., Weigel, D., and Lynch, M. (2010). The rate and molecular spectrum of spontaneous mutations in Arabidopsis thaliana. Science 327, 92-94. Perez-Llamas, C., and Lopez-Bigas, N. (2011). Gitools: analysis and visualisation of genomic data using interactive heat-maps. PloS One 6, e19541. Platts, A.E., Dix, D.J., Chemes, H.E., Thompson, K.E., Goodrich, R., Rockett, J.C., Rawe, V.Y., Quintana, S., Diamond, M.P., Strader, L.F., et al. (2007). Success and failure in human spermatogenesis as revealed by teratozoospermic RNAs. Hum. Mol. Genet. 16, 763-773. Pose, D., Verhage, L., Ott, F., Yant, L., Mathieu, J., Angenent, G.C., Immink, R.G., and Schmid, M. (2013). Temperature-dependent regulation of flowering by antagonistic FLM variants. Nature 503, 414-417. Shin, H., Liu, T., Manrai, A.K., and Liu, X.S. (2009). CEAS: cis-regulatory element annotation system. Bioinformatics 25, 2605-2606. Siepel, A., Bejerano, G., Pedersen, J.S., Hinrichs, A.S., Hou, M., Rosenbloom, K., Clawson, H., Spieth, J., Hillier, L.W., Richards, S., et al. (2005). Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. Genome Res. 15, 1034-1050. Stewart, S.A., Dykxhoorn, D.M., Palliser, D., Mizuno, H., Yu, E.Y., An, D.S., Sabatini, D.M., Chen, I.S., Hahn, W.C., Sharp, P.A., et al. (2003). Lentivirus-delivered stable gene silencing by RNAi in primary cells. RNA 9, 493-501. Taube, R., Lin, X., Irwin, D., Fujinaga, K., and Peterlin, B.M. (2002). Interaction between P-TEFb and the C-terminal domain of RNA polymerase II activates transcriptional elongation from sites upstream or downstream of target genes. Mol. Cell. Biol. 22, 321-331. Trinklein, N.D., Aldred, S.J., Saldanha, A.J., and Myers, R.M. (2003). Identification and functional analysis of human transcriptional promoters. Genome Res. 13, 308-312. Wu, C., Orozco, C., Boyer, J., Leglise, M., Goodale, J., Batalov, S., Hodge, C.L., Haase, J., Janes, J., Huss, J.W., 3rd, et al. (2009). BioGPS: an extensible and customizable portal for querying and organizing gene annotation resources. Genome Biol. 10, R130. Zhang, Y., Liu, T., Meyer, C.A., Eeckhoute, J., Johnson, D.S., Bernstein, B.E., Nusbaum, C., Myers, R.M., Brown, M., Li, W., et al. (2008). Model-based analysis of ChIP-Seq (MACS). Genome Biol. 9, R137. Zufferey, R., Nagy, D., Mandel, R.J., Naldini, L., and Trono, D. (1997). Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat. Biotechnol. 15, 871-875.
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