The transcription factor YY1 is a novel substrate for Aurora B kinase at G2/M transition of the cell cycle

Abstract

Yin Yang 1 (YY1) is a ubiquitously expressed and highly conserved multifunctional transcription factor that is involved in a variety of cellular processes. Many YY1-regulated genes have crucial roles in cell proliferation, differentiation, apoptosis, and cell cycle regulation. Numerous mechanisms have been shown to regulate the function of YY1, such as DNA binding affinity, subcellular localization, and posttranslational modification including phosphorylation. Polo-like kinase 1(Plk1) and Casein kinase 2α (CK2 α) were the first two kinases identified to phosphorylate YY1. In this study, we identify a third kinase. We report that YY1 is a novel substrate of the Aurora B kinase both in vitro and in vivo. Serine 184 phosphorylation of YY1 by Aurora B is cell cycle regulated and peaks at G2/M and is rapidly dephosphorylated, likely by protein phosphatase 1 (PP1) as the cells enter G1. Aurora A and Aurora C can also phosphorylate YY1 in vitro, but at serine/threonine residues other than serine 184. We present evidence that phosphorylation of YY1 in the central glycine/alanine (G/A)-rich region is important for DNA binding activity, with a potential phosphorylation/acetylation interplay regulating YY1 function. Given their importance in mitosis and overexpression in human cancers, Aurora kinases have been identified as promising therapeutic targets. Increasing our understanding of Aurora substrates will add to the understanding of their signaling pathways.

Figure 1. Phosphorylation of YY1 in nocodazole blocked extracts is detected by anti-phospho-S184 antibody.

(A) Diagram showing the different domains of the YY1 protein. Amino acid residues 177–191 are shown, which include serine 180 and 184, as indicated (left panel). Amino acid sequence alignment of residues 177–191 of human YY1 from different animal species, as indicated (right panel). (B) Dot blot assay of non-phosphorylated and serine 184 phosphorylated synthetic peptides of YY1 amino acid sequence 177 to 189 probed with anti-pS184. (C) Asynchronous, nocodazole treated (100 ng/ml for 18 hours) and thymidine treated (2.5 mM for 18 hours) HEK293 cell lysates were prepared, followed by Western blot. The blot was probed with anti-pS184 antibody with relative levels indicated below, then stripped and reprobed with anti-YY1 antibody (left panel). Western blot analysis of asynchronous and nocodazole treated HEK293 cells transiently transfected with Flag-YY1, probed with anti-pS184 antibody with relative levels indicated below, then stripped and reprobed with anti-YY1 antibody (middle panel). Flag-YY1 was immunoprecipitated from transiently transfected HEK293 cells treated with nocodazole. Flag-YY1 bound to anti-Flag mouse MAb cross-linked to resin beads was resuspended in phosphatase buffer, and incubated with (+) or without (-) λ-phosphatase at 30°C for 30 minutes, followed by a Western blot of the samples. The blot was probed with anti-pS184 antibody with relative levels indicated below, then stripped and reprobed with anti-YY1 (right panel) (D) Western blot analysis of nocodazole treated HEK293 cells transiently transfected with Flag- vector, Flag-YY1 wild type, Flag-YY1 S180A, Flag-YY1 S184A and Flag-YY1 S180,184A was probed with anti-pS184 antibody with relative levels indicated below, then stripped and reprobed with anti-YY1 antibody. (E) Asynchronous and nocodazole treated (Asy or Noc) HeLa, HEK293 and U2OS cell lysates were prepared, followed by Western blot analysis. The blot was probed with anti-pS184 antibody, then stripped and reprobed with anti-YY1 antibody followed by anti-cyclin B1 antibody to show proper mitotic synchrony (left panel). (F) Cold in vitro kinase assay reactions using Hela and HEK293 whole cell extracts (50 µg) as the kinase source. Both asynchronous (Asy) and nocodazole treated (Noc) extracts were used. Bacterially expressed GST-YY1 wild type bound to glutathione beads were used as substrate. The reactions were performed at 30°C for 45 minutes followed by Western blot analysis. The blot was probed with anti-pS184 antibody, then stripped and reprobed with anti-YY1 antibody to show equal GST-YY1 levels.

Figure 2. Serine 184 phosphorylation on YY1 peaks at the G2/M stage of the cell cycle.

(A) Cell cycle progression of HEK293 cells released after double-thymidine (2.5 mM) block was analyzed by fluorescence-activated cell sorting. Cells were stained with propidium iodide to analyze DNA content. (B) HEK293 cells were synchronized at early S phase by double-thymidine block and then released into fresh media. Western blot was performed on HEK293 cell lysates collected at the indicated time points. The blot was probed with anti-pS184 antibody with relative levels indicated below, then anti-YY1 antibody, followed by anti-cyclin B1 antibody. (C) HEK293 cells were also synchronized in mitosis by nocodazole block (100 ng/ml) for 18 hours and then released into fresh media. Western blot was performed on HEK293 cell lysates collected at the indicated time points after nocodazole block and release. The blot was probed with anti-pS184 antibody with relative levels indicated below, then anti-YY1 antibody followed by anti-cyclin B1.

Figure 3. Aurora B phosphorylates YY1 at serine 184 in vitro.

(A) Radioactive in vitro kinase assay using purified Aurora kinase isoforms and GST-YY1 as substrate. The kinase reactions include GST-YY1 only (no kinase), Aurora A, Aurora B and Aurora C only (no substrate) and GST-YY1 with each Aurora isoform. The reactions were performed at 30°C for 30 minutes. The reaction mixture was separated on a 10% SDS-PAGE gel and stained with Coomassie blue to visualize the protein bands and exposed to a phosphorimager screen. (B) Cold in vitro kinase assay reactions using purified Aurora A, Aurora B, Aurora C, Plk1 and PAK1 kinases and purified non-tagged YY1 as substrate. The reactions were performed at 30°C for 30 minutes followed by Western blot. The blot was probed with anti-pS184 antibody, then stripped and reprobed with anti-YY1 antibody. (C) Radioactive in vitro kinase assay using purified Aurora B kinase and GST-YY1 as substrate. The kinase reactions include GST-YY1 only (no kinase), Aurora B only (no substrate) and GSTY-YY1, GST-YY1 S180A, GST-YY1 S184A or GST-YY1 S180,184A with Aurora B. The reactions were performed as described for panel A. (D) Cold in vitro kinase assay reactions using purified Aurora B and GST-YY1 as substrate. The kinase reactions include GST-YY1 only (no kinase), Aurora B only (no substrate) and GST-YY1, GST-YY1 S180A, GST-YY1 S184A or GST-YY1 S180,184A with Aurora B. The reactions were performed at 30°C for 30 minutes followed by Western blot. The blot was probed with anti-pS184 antibody, then anti-YY1 antibody. (E) Cold in vitro kinase assay reactions using purified Aurora B and GST-YY1 as substrate. The kinase reactions were performed at 30°C for 30 minutes. After the reaction, GST-YY1 was washed with lysis buffer and resuspended in phosphatase buffer, and incubated with protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) or λ-phosphatase at 30°C for 30 minutes, followed by Western blot. The blot was probed with anti-pS184 antibody, then stripped and reprobed with anti-YY1.

Figure 4. Aurora B phosphorylates YY1 at serine 184 in vivo.

(A) HEK293 cells were synchronized in mitosis with nocodazole block for 17 hours. After the block, the cells were treated with the Aurora inhibitor VX-680, with the indicated concentrations for 15 minutes. Cell lysates were prepared, followed by Western blot. The blot was probed with anti-pS184 antibody with relative levels indicated below, then stripped and reprobed with anti-YY1 antibody followed by anti-cyclin B1antibody to show proper mitotic synchrony. (B) HEK293 cells were plated, cultured overnight, and then transfected with 20 nm control scrambled siRNA or Aurora B siRNA. After 48 hours of knockdown, the cells were lysed, and extracts were analyzed by Western blotting. The blot was probed with anti-pS184 antibody then stripped and reprobed with anti-YY1 antibody followed by anti-Aurora B antibody. (C) Co-immunoprecipitation of YY1 with Aurora A and Aurora B from HEK293 cells transiently transfected with HA-Aurora A, Flag-Aurora B and no transfection (control) followed by nocodazole block. Aurora A was immunoprecipitated using anti-HA antibody and Aurora B was immunoprecipitated using anti-Flag mouse MAb cross-linked to resin beads. Non-transfected cells were also immunprecipitated using anti-HA antibody and anti-Flag mouse MAb cross-linked to resin beads, which were used as a control for the specificity of the immunoprecipitation. Western blot analysis was performed on the immunoprecipitated samples and probed with anti-YY1 antibody. Input samples were probed with anti-YY1 antibody, anti-HA antibody and anti-Flag antibody.

Figure 5. YY1 S180,184D phospho-mutant exhibits an increased DNA binding affinity in vitro.

(A) Whole cell lysates were prepared from nococdazole treated HEK293 cells transiently transfected with Flag-vector, Flag-YY1 wild type, Flag-YY1 S180,184A and Flag-YY1 S180,184D followed by Western blot. The blot was probed with anti-pS184 antibody, then stripped and reprobed with anti-YY1 antibody. (B) Whole cell lysates were prepared from asynchronous HEK293 cells transiently transfected with Flag-vector, Flag-YY1 wild type, Flag-YY1 S180,184A and Flag-YY1 S180,184D. Cell lysates were used in an electrophoretic mobility shift assay (EMSA) using ³²P-labeled H3.2 α, P5-60 and p16 DNA oligonucleotides as probes. (C) The same lysates used in the EMSA were also used in a Western blot probed with anti-YY1 antibody to show equal Flag-YY1 levels.

Figure 6. The histone acetyltransferase p300 acetylates full length YY1 wild type in vitro.

(A) Cold in vitro acetylation assay reactions using purified p300-HAT domain and purified GST-YY1 (left panel) and non-tagged YY1 (right panel) as substrate. The reactions were performed at 30°C for 30 minutes followed by Western blot. The blot was probed with anti-acetyl-lysine antibody, then stripped and reprobed with anti-YY1 antibody. (B) Cold in vitro acetylation assay reactions using purified p300-HAT domain and purified GST-YY1 (1–200 a.a.) wild type, S180,184A and S180,184D deletion mutants. The reactions were performed at 30°C for 1 hour, followed by Western blot. The blot was probed with anti-acetyl-lysine antibody, then stripped and reprobed with anti-YY1 antibody.

Figure 7. Schematic model of the regulation of YY1 by Aurora B at G2/M.

YY1 is an Aurora B substrate at G2/M. Phosphorylation of YY1 in the regulatory domain prevents its association and acetylation by the histone acetyltransferase p300 and regulates YY1 DNA binding activity.