LIN54 is an essential core subunit of the DREAM / LINC complex that binds to the cdc2 promoter in a sequence-specific manner Fabienne Schmit, Sarah Cremer and Stefan Gaubatz Department of Physiological Chemistry I, Biocenter, University of Wuerzburg, Germany Keywords cell cycle; CXC; DNA binding; LIN54; LINC/DREAM Correspondence S Gaubatz, Department of Physiological Chemistry I, Biocenter, University of Wuerzburg, 97074 Wuerzburg, Germany Fax: +49 931 3184150 Tel: +49 931 3184138 E-mail: stefan.gaubatz@biozentrum uni-wuerzburg.de (Received June 2009, revised 23 July 2009, accepted August 2009) doi:10.1111/j.1742-4658.2009.07261.x Recently, the conserved human LINC ⁄ DREAM complex has been described as an important regulator of cell cycle genes LINC consists of a core module that dynamically associates with E2F transcription factors, p130 and the B-MYB transcription factor in a cell cycle-dependent manner In this study, we analyzed the evolutionary conserved LIN54 subunit of LINC We found that LIN54 is required for cell cycle progression Protein interaction studies demonstrated that a predicted helix–coil–helix motif is required for the interaction of LIN54 with p130 and B-MYB In addition, we found that the cysteine-rich CXC domain of LIN54 is a novel DNAbinding domain that binds to the cdc2 promoter in a sequence-specific manner We identified two binding sites for LIN54 in the cdc2 promoter, one of which overlaps with the cell cycle homology region at the transcriptional start site Gel shift assays suggested that, in quiescent cells, the binding of LIN54 at the cell cycle homology region is stabilized by the binding of E2F4 to the adjacent cell cycle-dependent element Our data demonstrate that LIN54 is an important and integral subunit of LINC Structured digital abstract l MINT-7239362: LIN54 (uniprotkb:Q6MZP7) physically interacts (MI:0915) with p130 (uniprotkb:Q08999) by anti tag coimmunoprecipitation (MI:0007) l MINT-7239376: LIN54 (uniprotkb:Q6MZP7) physically interacts (MI:0915) with B-Myb (uniprotkb:P10244) by anti tag coimmunoprecipitation (MI:0007) Introduction The retinoblastoma tumor suppressor protein (pRB) and the related pocket proteins (p107 and p130) function together with E2F transcription factors to regulate the cell cycle, differentiation and development [1] The activity of the pocket proteins is regulated by cyclindependent kinases which phosphorylate the pocket proteins and thereby release E2F, which then further transcriptionally activates cyclins and other cell cycledependent genes [2] We and others have recently identified a new E2F– pocket protein complex in mammalian cells that plays an important role in gene repression in quiescent cells and in the activation of mitotic genes [3–5] This complex, called LINC or human DREAM, consists of a five-protein core module that dynamically associates with p130 and the E2F4 and B-MYB transcription factors in a cell cycle-dependent manner LINC has been highly conserved throughout evolution A similar Abbreviations BrdU, bromodeoxyuridine; CDE, cell cycle-dependent element; CHR, cell cycle homology region; pRB, retinoblastoma tumor-suppressor protein; shRNA, small hairpin RNA FEBS Journal 276 (2009) 5703–5716 ª 2009 The Authors Journal compilation ª 2009 FEBS 5703 LIN54 is an essential core subunit of the DREAM ⁄ LINC complex F Schmit et al complex, called DREAM or MMB, was first identified in Drosophila [6,7] The Caenorhabditis elegans homologs of DREAM and LINC form the highly related DRM complex that mainly acts in the repression of developmental genes [8] Unlike the Drosophila and C elegans complexes, the composition of human LINC is regulated during the cell cycle Specifically, in quiescent cells, LINC interacts with p130 and E2F4 and contributes to the repression of E2F target genes [3,5,9] The binding of LINC with E2F4 ⁄ p130 is disrupted in the S phase At this time, B-MYB, a member of the vertebrate MYB family, is incorporated into the complex LINC–B-MYB binds to and activates the promoters of G2 ⁄ M genes [4,10,11] One particular well-studied subunit of LINC is the LIN9 protein LIN9 depletion in human and mouse cells leads to reduced activation of G2 ⁄ M target genes and cell cycle inhibition [9] LIN9 also plays a role in zebrafish development and in transcriptional regulation in response to DNA damage [12,13] Another conserved core subunit of LINC is LIN54 LIN54 homologs are present in many species, including plants (Arabidopsis thaliana), invertebrates (C elegans and Drosophila melanogaster) and humans, who, in addition to LIN54, express a testis-specific homolog, tesmin [14–18] LIN54 homologs contain dual cysteinerich domains, termed CXC, with the consensus sequence CXCX4CX3YCXCX6CX3CXCX2C The two CXC domains are separated by a short spacer It is possible, yet unproven, that the dual CXC domain of LIN54 functions as a DNA-binding domain In this study, we investigated the function of LIN54 We found that LIN54 is required for the proliferation of human cells Using LIN54 deletion mutant constructs, we demonstrated that a conserved helix–coil– helix (HCH) region is essential for the binding of LIN54 to p130 and B-MYB We also found that the dual CXC domain of LIN54 is a DNA-binding domain that binds to the cdc2 promoter in a sequencespecific manner We identified two binding sites for LIN54 in the cdc2 promoter, one of which overlaps with the cell cycle homology region (CHR) at the transcriptional start site Our data suggest that, in quiescent cells, the binding of LIN54 at CHR is stabilized by E2F4 bound to the adjacent cell cycle-dependent element (CDE) Taken together, these data establish LIN54 as an essential member of the LINC ⁄ DREAM complex delayed entry into mitosis [9] To address whether this is an isolated function of LIN9 or whether it is mediated by LINC, we examined another subunit of LINC, LIN54 Immortalized BJ cells containing the ecotrophic receptor (BJ-ET) were infected with a retrovirus encoding a small hairpin RNA (shRNA) against LIN54, or with a control retrovirus The efficiency of LIN54 depletion was tested at the mRNA and protein level LIN54 mRNA was reduced by 70% and a significant reduction in the protein level was detected in immunoblots in cells infected with the LIN54-specific shRNA (Fig 1A, B) Next, the proliferation of LIN54-depleted cells compared with control cells was analyzed When LIN54depleted cells were monitored for 12 days in culture, we found that they grew significantly more slowly than control cells, indicating that LIN54 is required for the proliferation of human cells (Fig 1C) To better characterize the cell cycle defects, flow cytometry profiles of control cells and LIN54-depleted BJ-ET cells were compared Cells were labeled with bromodeoxyuridine (BrdU) to identify the population of cells in S phase As shown in Fig 2A, LIN54 depletion results in an accumulation of cells in G2 ⁄ M and in a reduction in the fraction of cells in S phase Staining with an antibody against phosphorylated histone H3 revealed a decrease in the percentage of mitotic cells in LIN54depleted cells compared with control cells (Fig 2B) Together with the fluorescence-activated cell sorting data, this indicates that cell cycle inhibition occurs in G2 before entry into mitosis To address whether LIN54-depleted cells are completely blocked in G2, the kinetics of cell cycle progression were analyzed in more detail LIN54-depleted cells were labeled with BrdU for h BrdU was washed away and, h later, the cells were analyzed by flow cytometry; 11.6% of the control cells that were in S phase during labeling had progressed to the next G1 phase at h after labeling (Fig 2C); in contrast, only 4.5% of the LIN54depleted S-phase cells had re-entered into the next G1 phase at h after labeling This shows that the G2 phase of LIN54-depleted cells is prolonged, but not blocked completely Taken together, these results indicate that LIN54 is required for entry into mitosis of human cells These results are consistent with the recent identification of LIN54 in an RNA interference (RNAi)-based screen for cell division defects in human cells [19] LIN54 is an evolutionarily conserved protein that contains a conserved sequence in the C-terminus that is predicted to form an HCH secondary structure (Fig 3A) The corresponding region of tombola, a testis-specific D melanogaster homolog of LIN54, is Results We have shown previously that the depletion of LIN9 in human cells inhibits proliferation and results in 5704 FEBS Journal 276 (2009) 5703–5716 ª 2009 The Authors Journal compilation ª 2009 FEBS LIN54 is an essential core subunit of the DREAM ⁄ LINC complex F Schmit et al B LIN54 expression (normalized to GAPDH) A 0.6 LIN54 0.4 β-tubulin 0.2 Ctrl BrdU-FITC BrdU positive cells in G1: 11.6 % shRNA Ctrl 20 15 10 LIN54 kd 0 12 Days B LIN54 kd G1: 59.4 % S: 17.8 % G2/M: 10.3 % Control LIN54 25 G1: 66.3 % 2.7 % S: G2/M: 22.0 % DNA content (PI intensity) C LIN54 LIN54 kd BrdU positive cells in G1: 4.5 % Control LIN54 kd Nuclei Control Ctrl 0.8 Anti-PH3 BrdU-FITC A C Cumulative growth number of cells (×1×105) Fig Depletion of LIN54 leads to growth defects hTert immortalized BJ fibroblasts (BJ-ET) were infected with retroviral shRNA against LIN54 LIN54 mRNA (A) and protein (B) levels after shRNA depletion compared with the levels in control infected cells (C) Infected and selected BJ-ET cells were counted and reseeded in triplicate in each experiment Cell numbers were analyzed and the cumulative growth was plotted against time The experiment was repeated three times One representative experiment is shown shRNA 14.8 % +/– 2.6 % 5.8 % +/– 1.8 % Phospho-H3 positive cells DNA content (PI intensity) Fig Depletion of LIN54 leads to defects in the G2 ⁄ M transition (A) LIN54-depleted and control-depleted BJ-ET cells were labeled with BrdU and analyzed by flow cytometry to determine the percentage of cells in different phases of the cell cycle (B) Control and LIN54depleted cells were stained with an antibody against phosphorylated histone H3 (PH3) Nuclei were counterstained with Hoechst 33258 At least 300 cells were counted in triplicate in each experiment and the percentage of PH3-positive cells was determined Representative regions of one of three independent experiments are shown (C) To determine the number of cells re-entering the next cell cycle, control-depleted and LIN54-depleted BJ-ET cells were pulse labeled with BrdU for h and grown for an additional h without BrdU The percentage of BrdU-positive cells in G1 was determined by flow cytometry required for binding to the interaction partner Aly [14] It is possible that the HCH region of human LIN54 is also involved in protein–protein interaction To address this possibility, we generated a set of LIN54 deletion mutants (Fig 3B) Flag-tagged LIN54 wild-type or mutants were transfected into 293 cells HA-tagged FEBS Journal 276 (2009) 5703–5716 ª 2009 The Authors Journal compilation ª 2009 FEBS 5705 LIN54 is an essential core subunit of the DREAM ⁄ LINC complex A F Schmit et al 749 H.s LIN54 509 H.s Tesmin 950 D.m mip120 243 D.m tombola 435 C.e LIN-54 896 G.m CPP1 695 A.t TSO1 B CXC Helix-Coil-Helix LIN54 LIN54 ΔN LIN54 CXC LIN54 HCH LIN54 ΔHCH LIN54 ΔCXC Input flag-LIN54 10% gel * * * * Input flag-LIN54 15% gel * * * * * Input flag-LIN54 10% gel * * * Bound HA-p130 (IP: flag > WB: HA) pCDNA Input HA-B-MYB Input HA-p130 * LIN54-HCH LIN54-CXC LIN54-ΔHCH LIN54-ΔN LIN54 LIN54 pCDNA LIN54-ΔCXC HA-B-MYB D LIN54-HCH LIN54-ΔHCH LIN54-CXC LIN54-ΔN LIN54 LIN54 LIN54-ΔCXC HA-p130 C * Input flag-LIN54 15% gel Bound HA-B-MYB (IP: flag > WB: HA) Fig The putative helix–coil–helix (HCH) region of LIN54 is required for binding to p130 and B-MYB (A) Schematic alignment of LIN54 proteins A.t., Arabidopsis thaliana; C.e., Caenorhabditis elegans; D.m., Drosophila melanogaster; G.m., Glycine max; H.s., Homo sapiens (B) Scheme of the flag-tagged LIN54 deletion mutants to analyze the function of the conserved CXC and HCH domains (C, D) 293T cells were cotransfected with the indicated flag-tagged LIN54 constructs and HA-p130 (C) or HA-B-MYB (D) Whole-cell lysates were immunoprecipitated with anti-flag agarose Input levels of full-length LIN54 and mutants were detected by immunoblotting with flag antibodies (indicated by asterisks) Because of their small size, 15% SDS gels were used to detect LIN54 CXC and HCH mutants Bound HA-p130 and HA-B-MYB were detected by immunoblotting p130 was coexpressed The binding of LIN54 mutants to p130 was analyzed by immunoprecipitation of LIN54 from lysates with anti-flag IgG and the detection of bound p130 with anti-HA IgG Compared with the other deletion mutants, we found that it was very difficult to overexpress full-length LIN54, DHCH and DN mutants in a panel of different cell lines Although difficult to overexpress, distinct bands corresponding to LIN54 and the DHCH and DN mutants could be detected (marked by asterisks) Fluorescence-activated 5706 cell sorting studies did not suggest that the expression of LIN54, DHCH and DN was toxic to the cells (data not shown) Therefore, it is possible that full-length LIN54, DHCH and DN are unstable Although some LIN54 constructs were difficult to express, coimmunoprecipitation experiments showed unequivocally that p130 bound to full-length LIN54 and to the DN, DCXC and HCH mutants, all of which contained an intact HCH region (Fig 3C) In contrast, mutants that lacked the HCH region (DHCH and CXC) showed no binding FEBS Journal 276 (2009) 5703–5716 ª 2009 The Authors Journal compilation ª 2009 FEBS F Schmit et al LIN54 is an essential core subunit of the DREAM ⁄ LINC complex to p130 Importantly, these two mutants were both strongly overexpressed, indicating that a lack of binding of these mutants to p130 was not a result of weak expression In similar experiments, binding of B-MYB could also be confined to HCH of the LIN54 region (Fig 3D) These data indicate that the LIN54 HCH domain is required for the interaction of LIN54 with p130 and B-MYB Chromatin immunoprecipitation assays have shown that LINC subunits, including LIN54, bind to the promoters of mitotic genes, such as cdc2 [4] As described above, LIN54 proteins contain two cysteinerich CXC domains that could function as a DNAbinding domain (Fig 3A) In this study, therefore, we wanted to address whether purified LIN54 can interact directly with DNA in a sequence-specific manner Because it was not possible to express full-length LIN54 in heterologous bacterial expression systems, we decided to focus on the CXC domains The dual CXC domain of LIN54 was fused to GST (GSTCXC), expressed in bacteria and affinity purified (Fig 4A) Next, we performed gel shift experiments with GST-CXC and with a  400 bp fragment of the human cdc2 promoter, a known LINC target gene [4,9] As shown in Fig 4B, binding of GST-CXC to the cdc2 promoter was readily detected Binding was competed with the unlabeled cdc2 promoter, indicating that the binding was specific Next, to test whether the conserved cysteines in the dual CXC domain were required for binding to the cdc2 pro- moter, we mutated two conserved cysteine residues in the first CXC domain (C525 and C527) to tyrosine Mutation of the residue equivalent to C525 of Arabidopsis TSO1 disrupts flower development and cell division [20] We found that mutation of C525 and C527 abolished the binding of GST-CXC to the cdc2 promoter (Fig 4B) Thus, the conserved cysteine residues are essential for the binding of LIN54 to DNA Previous studies have indicated that multiple positively and negatively acting elements are involved in cell cycle-dependent transcription of the human cdc2 promoter [21,22] For example, a CDE–CHR element overlapping the transcription start site mediates repression in quiescent cells and an E2F site is responsible for activation by E2F1–3 [22,23] In addition, a MYB binding site upstream of the E2F site and CCAAT elements between the E2F sites are involved in cdc2 promoter regulation [23] Examination of the cdc2 promoter sequence identified four additional putative MYB elements and a sequence related to a CHR element adjacent to MYB binding site (Fig 5A) To address which elements are involved in the binding of LIN54 to the cdc2 promoter, we generated mutated cdc2 promoter fragments and used them in competition experiments (Fig 5) In the first set of experiments, we analyzed the CDE-CHR region at the transcription start site, the potential MYB binding sites and the upstream region with CHR homology (CHR-up) (Fig 5C) As expected, the binding of GST-CXC to the cdc2 promoter was competed with the wild-type cdc2 promoter A Fig The dual CXC domain of LIN54 binds to the cdc2 promoter (A) Recombinant GST, GST-CXC (comprising the two CXC domains of human LIN54) and GST-CXCC525 ⁄ 527Y (with two C to Y point mutations in the first CXC domain) were expressed in bacteria and purified A Coomassie-stained gel of the purified proteins is shown The asterisks indicate the position of GST and the GST-CXC fusion proteins (B) Recombinant GST-CXC and GST-CXCC525 ⁄ 527Y were incubated with a [32P]labeled fragment of the cdc2 promoter and separated on a non-denaturing acrylamide gel; 250 ng of unlabeled cdc2 promoter fragment was used to compete for the binding of the GST protein to the labeled probe (+) Labeled DNA was detected by autoradiography The positions of the free probe and the shifted GST-CXC protein are indicated GSTCXCGST- C525/ CXC 527Y GST M (kDa) 100 72 B Competitor GST-CXC + – GST-CXCC525/527Y + – 55 40 33 24 * GSTCXC * * FEBS Journal 276 (2009) 5703–5716 ª 2009 The Authors Journal compilation ª 2009 FEBS Free probe 5707 LIN54 is an essential core subunit of the DREAM ⁄ LINC complex A CHRup MYB1 MYB2 MYB3 MYB4 MYB5 F Schmit et al CAAT CAAT E2F CDE CHR wt CDE mut CHR mut CHR-up mut MYB1 mut MYB4 mut MYB5 mut 5′ del B Wt sequence Mut sequence CDE CHR CHR-up MYB1 TAGCGCGGT AGTTTGAAAC ATTTGAA TAGCGCtGT AGTagctAAC ATccGAA GAACTGTG GAAtcGTG MYB4 MYB5 AGAAACAGT CAGTTGGCG AGAggaAGT CAGcctGCG C MYB5 mut MYB4 mut MYB1 mut CHRup mut CHR mut CDE mut Wt Competitor: – GST-CXC GSTCXC Free probe Fig Binding of LIN54 to the CHR element of the cdc2 promoter Scheme (A) and sequences (B) of the mutated cdc2 promoter mutants used for competition in electrophoretic motility shift assay experiments (C) Purified GST-CXC was incubated with a [32P]-labeled fragment of the cdc2 promoter Competition was performed with 100 ng of the unlabeled wild-type cdc2 promoter or the indicated promoter mutants cdc2 promoter fragments in which the CDE region, the upstream CHR region or potential MYB binding sites 1, or were mutated (MYB1-mut, MYB4-mut, MYB5-mut) also specifically competed for binding This indicates that these regions are not essential for the binding of LIN54 to the cdc2 promoter In contrast, the same amount of a promoter fragment with a mutated CHR region (CHR-mut) did not compete efficiently, indicating that the CHR region is important 5708 for the binding of LIN54 to the cdc2 promoter (Fig 5C) We next asked whether the same element was involved in the binding of a LIN54-containing complex from cellular lysates To address this possibility, we performed gel shift assays with nuclear extracts from T98G cells and an oligonucleotide encompassing the CDE-CHR element of the cdc2 promoter as a probe Because the CDE-CHR element is occupied by FEBS Journal 276 (2009) 5703–5716 ª 2009 The Authors Journal compilation ª 2009 FEBS LIN54 is an essential core subunit of the DREAM ⁄ LINC complex NE – + – B NE – + Comp.: – – G0-compl + + + CHR Comp CDE A WT F Schmit et al G0-compl * * Free probe + + Antibody: – – – – LIN54 + + + Comp.: – – wt E2F – – – Ab: – Ab: – LIN9 + E2F4 G0-compl D E NE α-LIN54 (µg): – – Peptide (µg): – – – 1.25 – 2.5 25 Comp.: – + – – – – 20 0.25 0.25 0.5 G0-compl repressor complexes in G0, we prepared nuclear extracts from serum-starved cells In gel shift reactions, a specific band shift was observed that was competed with a 100-fold excess of unlabeled oligo- 0.5 Relative activity Fig Binding of LINC from nuclear extracts of serum-starved T98G cells to the cdc2 CDE-CHR element (A) Gel shift analysis with nuclear extracts from serumstarved T98G cells and the CDE-CHR element of the cdc2 promoter Competition was performed with a 100-fold excess of the unlabeled oligonucleotide (B) Gel shift assay as described in (A) Competition with a 100-fold excess of wild-type CDE-CHR or with oligonucleotides with mutation in the CDE or CHR region (C) A gel shift assay was performed as described in (A) Competitor oligonucleotides or polyclonal antibodies were added as indicated E2F, competition with the canonical E2F-binding site from the DHFR promoter is an additional demonstration that the G0-specific complex contains E2F (D) The LIN54 antibody was preincubated with the indicated amount of peptide against which it was raised A gel shift assay was performed as described in (A) NE, nuclear extract; *, nonspecific band (E) The activities of the indicated cdc2 promoter constructs (wild-type, CDE mutant or CHR mutant) were compared in a luciferase reporter assay – E2F1 NE p130 C 15 10 wt CDE CHR nucleotide, suggesting that the binding is specific (Fig 6A) To identify which part of the CDE-CHR is required for this binding activity, we performed competition experiments with oligonucleotides in which FEBS Journal 276 (2009) 5703–5716 ª 2009 The Authors Journal compilation ª 2009 FEBS 5709 LIN54 is an essential core subunit of the DREAM ⁄ LINC complex F Schmit et al either the CDE or CHR element was mutated As shown in Fig 6B, the mutated oligonucleotides were unable to compete for binding, indicating that both elements are necessary for the binding of the complex in serum-starved cells Next, we asked whether the G0 binding activity contains members of the LINC complex To address this possibility, we performed gel shift experiments with antibodies directed at LINC subunits As a control, we used an antibody directed at E2F1, which is not a component of LINC and which did not interfere with the formation of the G0 phase-specific band shift (Fig 6C, middle panel) In contrast, polyclonal antibodies against p130, E2F4, LIN9 and LIN54 abolished binding (Fig 6C) To verify that the inhibition of binding by the LIN54 antibody is a specific effect, we preincubated LIN54 antiserum with the peptide against which it was raised After blocking with the peptide, the antibody failed to affect complex formation, verifying the specificity of the LIN54 antibody (Fig 6D) Together, these data demonstrate that a complex containing p130, E2F4, LIN9 and LIN54 binds to the CDECHR element of the cdc2 promoter in serum-starved cells, and that both parts of the CDE-CHR element are required for binding of the complex These findings are consistent with previous studies that reported the binding of E2F4 and p130 to the CDE element in G0 [22] Binding of LIN54 to the adjacent CHR element could therefore stabilize the binding of E2F4 ⁄ p130 at CDE To address whether the CDECHR element is required for repression, we performed luciferase reporter assays using the same CDE and CHR mutations as were employed in the gel retardation experiments Consistent with previous studies, the activity of the cdc2 reporter constructs that contained mutations in either the CDE or CHR element was strongly increased when compared with the wild-type promoter (Fig 6E) Thus, the loss of DNA binding by the LINC complex correlates with increased activity of the cdc2 promoter Because chromatin immunoprecipitation experiments indicate that LINC also binds to the cdc2 promoter in the S phase [9], we next asked whether LINC also binds to the CDE-CHR element during this phase of the cell cycle In S-phase extracts, however, the G0-specific band was no longer present, indicating that LINC dissociates from CDE-CHR after cell cycle re-entry (data not shown) Because LINC is no longer present at CDE-CHR in the S phase, it should bind to other elements in the cdc2 promoter at this time in the cell cycle This binding could be mediated by additional LIN54-binding sites in the cdc2 promoter, or may be independent of LIN54 To address the possibility of additional LIN54-binding sites in the cdc2 promoter, we performed additional gel shift experiments with purified GST-CXC and the cdc2 promoter In competition experiments in which the CHR region was mutated, little competition was observed with small amounts of this competitor, as shown previously However, partial competition was observed with larger amounts This indicates that the binding of GST-CXC is reduced, but not completely abolished, when the proximal CHR element is mutated (Fig 7A) To further confirm that LIN54 can still bind to the cdc2 promoter with a mutated CHR element, the reverse experiment was performed and CHR-mut was used as a probe in gel shift experiments (Fig 7A) GST-CXC was able to shift this mutated promoter construct, confirming that it can indeed still bind to a promoter in which the CHR element is mutated Thus, it appears that there are additional binding sites for LIN54 in the cdc2 promoter Further evidence for the presence of additional binding sites came from the observation that although a 5¢ cdc2 promoter deletion (5¢ del) competed for binding it required higher concentrations than the longer cdc2 promoter to fully compete for binding (Fig 7B,C) Thus, a second binding site for LIN54 appears to be located in or close to the upstream region that is missing in this deletion Further support for this concept came from the observation that a cdc2 promoter fragment that contained a deletion at the 5¢ region and a mutated downstream CHR site (CHRmut + del) did not compete for binding (Fig 8B, lane 4) To define more precisely the upstream binding site for LIN54, we generated a set of point mutants Each of the constructs contained a three-base-pair mutation within the upstream LIN54-binding region, in addition to a mutation in the downstream CHR element (mutants A–I, Fig 8A) The downstream CHR region was mutated in these constructs, because we wanted to analyze binding to the upstream region independently from binding to the downstream CHR element Mutated cdc2 promoter fragments were used as competitors in gel shift experiments As shown in Fig 8B, the ability of mutant E (lane 10) to compete was reduced slightly, whereas the two mutants G and I (lanes 12 and 14) completely lost their ability to compete for LIN54 binding Thus, in addition to the CHR region, the cdc2 promoter regions corresponding to mutants G and I are required for the binding of LIN54 Taken together, these data indicate that LIN54 interacts with the cdc2 promoter at two different sites Although not addressed in this study, it is possible that the in vivo binding of LIN54 to the upstream element is stabilized by the binding of 5710 FEBS Journal 276 (2009) 5703–5716 ª 2009 The Authors Journal compilation ª 2009 FEBS LIN54 is an essential core subunit of the DREAM ⁄ LINC complex F Schmit et al GST-CXC 50 100 200 – GSTCXC ut GSTCXC Free probe Probe: wt CHRmut Free probe GST-CXC C 5′ del (ng) – wt 5′ del H – Comp GST-CXC C 50 100 200 R CHR-mut t Comp (ng) -m wt B GST-CXC w A – 50 100 150 200 GSTCXC Fig Evidence for additional binding sites for LIN54 in the cdc2 promoter (A) Purified GST-CXC was incubated with a [32P]-labeled fragment of the cdc2 promoter or a cdc2 promoter carrying a mutation in the CHR region Competition was performed with the indicated amounts of wild-type cdc2 promoter or CHR mutant promoter fragment (B) GST-CXC was incubated with a [32P]-labeled fragment of the cdc2 promoter Bound labeled probe was competed with 150 ng of wild-type or deleted cdc2 promoter (C) Gel shift assay with GST-CXC and the cdc2 promoter Competition experiments were performed with increasing amounts of 5¢ del B-MYB to adjacent low-affinity sites Our results provide the basis for a further investigation of the regulation of the cdc2 promoter by LINC in different phases of the cell cycle Discussion The primary goal of this study was to investigate LIN54, a conserved subunit of the human DREAM ⁄ LINC complex We found that RNAi-mediated depletion of LIN54 in primary human cells results in cell cycle arrest and delayed entry into mitosis A similar phenotype has been found previously on depletion of human LIN9 and B-MYB [9–11] This suggests that the ability to promote cell cycle progression and mitotic entry is not an isolated function of LIN9 and B-MYB, but that it is mediated by the LINC complex Our study demonstrates that LIN54 is an integral and essential subunit of this complex To analyze the function of LIN54, we created a set of deletion mutants Using these mutants, we found that a region of LIN54 that is predicted to form an HCH secondary structure is required and sufficient for binding to p130 and B-MYB Because the corresponding region of tombola, a testis-specific Drosophila homolog of LIN54, is involved in binding to the LIN9 homolog Aly [14], it is possible that the LIN54 HCH domain does not interact directly with p130 and B-MYB, but is required for the formation of the LINC complex, with which p130 and B-MYB interact LIN54 is an evolutionarily conserved protein of the tesmin ⁄ TSO1 family [14,24] Members of this family FEBS Journal 276 (2009) 5703–5716 ª 2009 The Authors Journal compilation ª 2009 FEBS 5711 LIN54 is an essential core subunit of the DREAM ⁄ LINC complex A MYB4 CHRup MYB1 MYB2 MYB3 CHR MYB1 A B C D MYB5 E F E2F G H F Schmit et al CAAT CAAT CDE CHR I AT T T G A A C T G T G C C A AT G C T G G G A G A A A A A AT T TA A G AT C T AT T T G A A C T A G A C C A AT G C T G G G A G A A A A A AT T TA A G AT C T Mut A AT T T G A A C T G T G A A G AT G C T G G G A G A A A A A AT T TA A G AT C T Mut B AT T T G A A C T G T G C C A GGAC T G G G A G A A A A A AT T TA A G AT C T Mut C AT T T G A A C T G T G C C A AT G A G A G G A G A A A A A AT T TA A G AT C T Mut D AT T T G A A C T G T G C C A AT G C T G A A G G A A A A A AT T TA A G AT C T Mut E AT T T G A A C T G T G C C A AT G C T G G G A A G G A A A AT T TA A G AT C T M u t F AT T T G A A C T G T G C C A AT G C T G G G A G A A G G G AT T TA A G AT C T M u t G AT T T G A A C T G T G C C A AT G C T G G G A G A A A A A GGGTA A G AT C T M u t H AT T T G A A C T G T G C C A AT G C T G G G A G A A A A A AT T GGGG AT C T M u t I B GST-CXC CHR mut + Comp – wt del wt A B C D E F G H I Fig Identification of the 5¢-binding region for LIN54 in the cdc2 promoter (A) Scheme of the mutants in the 5¢ region of the cdc2 promoter used for competition in gel shift experiments (B) GST-CXC was incubated with a [32P]-labeled fragment of the cdc2 promoter Bound labeled probe was competed with the indicated fragments containing a mutated triplet in the 5¢ region and a mutated CHR region Del, construct contains deletion of the 5¢ region in addition to a mutated CHR region GSTCXC Free probe 10 11 12 13 14 contain one or two related cysteine-rich domains, termed the CXC domain CXC proteins have been found in plants and animals, but not in yeast The founding family member TSO1 is essential for flower development [17,18] Mutations in TSO1 cause defects in mitosis and cytokinesis Interestingly, all tso1 mutants that have been described harbor point mutations in the CXC domain, indicating that this domain is critical for the function of this protein A function for the CXC domain is suggested by the observation that soybean CPP1, a member of the family, binds to the promoter of the leghemoglobin gene Gmlbc3 through the cysteine-rich domains [25] Moreover, Mip120, the Drosophila LIN54 protein of the DREAM complex, and C elegans Lin-54 also bind to DNA [15,16] Therefore, it has been suggested that the CXC domain functions as a sequence-specific DNA-binding domain To test this concept directly, we performed gel shift experiments with the dual CXC domain of human LIN54 We found that the CXC domain binds to the cdc2 promoter, an in vivo target of LINC Binding was critically dependent on the conserved cysteines, as mutation of two cysteine residues in the first CXC domain completely abolished binding to the cdc2 promoter Interestingly, our data indicate that there are two LIN54-binding sites in the cdc2 promoter The 5712 first binding site overlaps with the CHR at the transcriptional start site of the cdc2 promoter The second binding site is found further upstream in the cdc2 promoter, and is located between two potential MYBbinding sites The CHR element, which is required for the binding of LINC subunits in quiescent cells, is typically found in cell cycle-regulated promoters adjacent to a second element, termed the ‘cell cycle-dependent element’ or CDE [26] Mutation in either the CDE or CHR element abolishes repression of cell cycle genes in G0 ⁄ G1 [27–30] Previous studies have reported the binding of E2F4 and p130 to the CDE part of the composite element [22,31] Because LIN54 binds to CHR, interaction of E2F4 ⁄ p130 with CDE could be stabilized by binding of LIN54 to the adjacent CHR element Two different unidentified in vitro binding activities that interact with CHR have been described [32–34] The first, CDF-1, interacts with CHR elements of multiple promoters in quiescent cells [32,33] However, unlike LINC, CDF-1 does not interact with E2Fs or pocket proteins, suggesting that CDF-1 is unrelated to LIN54 Because the second known CHR-interacting activity, termed CHF, more selectively interacts with the CHR element found in the cyclin A promoter, it is also unlikely that it corresponds to LIN54 [34] Although FEBS Journal 276 (2009) 5703–5716 ª 2009 The Authors Journal compilation ª 2009 FEBS F Schmit et al LIN54 is an essential core subunit of the DREAM ⁄ LINC complex LIN54 binds to the CHR element, its RNAi-mediated depletion does not result in a dramatic upregulation of genes with CDE-CHR elements in their promoters, most probably as a result of compensation by other repressive complexes [4] LINC not only binds to target genes in quiescent cells when they are repressed, but also binds to mitotic promoters in the S phase and mediates their activation [4,5] The composition of the S-phase complex differs from that of the G0 complex Specifically, in the S phase, the interaction of LINC with E2F4 ⁄ p130 is lost and LINC interacts with B-MYB [4,5] Consistent with the dissociation of E2F4 ⁄ p130, LINC cannot be detected at the CDE-CHR element in the S phase The second LIN54binding site in the cdc2 promoter, which is distinct from CHR, could be involved in the recruitment of the complex in the S phase Because LINC associates with B-MYB in the S phase, it is possible that LINC binding to the cdc2 promoter is stabilized by simultaneous binding of B-MYB to neighboring sites In this model, LIN54 would increase the binding affinity of the complex to otherwise low-affinity sites and thus would allow a more stringent selection of target genes Although the primary focus of this study was to characterize LINC binding in quiescent cells, our results provide the basis to further investigate the regulation of the cdc2 promoter by LINC in different phases of the cell cycle Although, so far, only the cdc2 promoter has been analyzed, the promoters of many other LINC target genes also carry E2F- and B-MYB-binding sites, as well as CDE and CHR elements Although the arrangement of these elements is different in every promoter, it is possible that they are regulated in a similar manner counted and · 105 cells were reseeded The mean values of the cumulative cell numbers were calculated Experimental procedures BrdU pulse labeling and flow cytometry For flow cytometry, cells were labeled with 10 lm BrdU for h, harvested by trypsinization and fixed with 80% ethanol at )20 °C overnight The DNA was denatured with m HCl–0.5% Triton-X-100 for 30 The mixture was neutralized with 0.1 m Na2B4O7 Cells (1 · 106) were resuspended in 50 lL NaCl ⁄ Pi–0.5% Tween-20–1% BSA and 20 lL of an a-BrdU–fluorescein isothiocyanate antibody After incubation for h, the cells were washed with 50 lL NaCl ⁄ Pi–0.5% Tween-20–1% BSA and resuspended with 500 lL of 38 mm sodium citrate–25 lL RNase A (10 mgỈmL)1) The cells were incubated for 1–2 h at 37 °C, stained with 30 lL of propidium iodide (1 mgỈmL)1) and measured by flow cytometry Immunofluorescence Cells were plated and grown on coverslips for 24 h Cells were fixed with NaCl ⁄ Pi–3% paraformaldehyde–2% sucrose, permeabilized with NaCl ⁄ Pi–0.2% Triton-X-100 and washed with NaCl ⁄ Pi–0.1% Triton-X-100 Nonspecific staining was minimized by blocking for 15 with NaCl ⁄ Pi–0.1% Triton-X-100–5% goat serum Cells were washed three times in NaCl ⁄ Pi–5 mm MgSO4 and incubated with the primary antibody directed against phosphorylated histone H3 (Upstate 06-570), diluted in NaCl ⁄ Pi–5 mm MgSO4 for h The coverslips were washed three times with NaCl ⁄ Pi–0.1% Triton-X-100 and incubated with a rhodamine-labeled secondary antibody (Jackson Immuno-Research, West Grove, PA, USA) in NaCl ⁄ Pi–0.1% Triton-X-100 for 15 Nuclei were stained with Hoechst 33258 (Sigma, St Louis, MO, USA) To analyze the mitotic index, the percentage of phospho-H3-stained cells was determined In each experiment, 300 cells were counted in triplicate Tissue culture, constructs and infection 293T, BJ-ET, T98G and Phoenix cells were cultured in DMEM supplemented with 10% fetal bovine serum Retroviruses were produced in the Phoenix retroviral packaging cell line and BJ-ET cells were infected as described previously [9] with the following shRNA target sequence directed at LIN54: 5¢-GCAGTTACAGGACAGACAA-3¢; 48 h after infection, the cells were selected with blasticidin (final concentration, gỈm)1) for days T98G cells were made quiescent by incubation for 72 h in serum-free medium Growth curves The infected and selected BJ-ET cells(1 · 105) were seeded onto a six-well plate in triplicate Every days, they were Transfection and SDS-PAGE Expression plasmids for HA-p130 [35], HA-B-MYB [36] and flag-LIN-54 [4] have been described previously LIN54 deletion constructs were generated by PCR using the following primers: 5¢-GGCGGATCCAAGCCAGTGGTTGTTAAT AC-3¢ and 5¢-GCCTCGAGAATCAAGTGTCCCTGCACC T-3¢ (LIN-54-DN); 5¢-GCGGATCCGAGGTGGTGCCAG CTGAG-3¢, 5¢-GCTCTAGAGAATGGAAGCCGTGCCT G-3¢, 5¢-GCTCTAGATTGGCAGATGCAGCTGAAGTA3¢ and 5¢-GCCTCGAGAATCAAGTGTCCCTGCACCT-3¢ (LIN-54-DCXC); 5¢-GCGGATCCGAGGTGGTGCCAGC TGAG-3¢ and 5¢-GCCTCGAGTTAAACTTCGTCTTGGC TGC-3¢ (LIN-54-DHCH); 5Â-ATGGATCCCCATCAGAGT FEBS Journal 276 (2009) 57035716 ê 2009 The Authors Journal compilation ª 2009 FEBS 5713 LIN54 is an essential core subunit of the DREAM ⁄ LINC complex F Schmit et al CGGCCAGT-3¢ and 5¢-CGCTCGAGCATCTGCCAAAT GCATCA-3¢ (LIN-54-CXC); 5¢-GCGGATCCAGGGTAC AGCAACAAACAGC-3¢ and 5¢-GCCTCGAGAATCAAG TGTCCCTGCACCT-3¢ (LIN-54-HCH) 293T cells were transfected with the calcium phosphate method; 48 h after transfection, cells were lysed in TNN buffer (50 mm Tris ⁄ HCl pH 7.5, 120 mm NaCl, mm EDTA, 0.5% NP-40, 10 mm Na4P2O7, mm Na3VO4, 100 mm NaF, protease inhibitors) and immunoprecipitated with flag-M2-agarose (Sigma) overnight Precipitated lysate was resolved on a denaturing SDS gel and immunoblotted with anti-flag-M2 (Sigma) or anti-HA (Covance, Princeton, NJ, USA) antibodies temperature with the labeled probe Reactions were separated by nondenaturing gel electrophoresis with 6% acrylamide ⁄ bisacrylamide (75 : 1) in 0.5 · TAE (20 mm Tris-acetate, 0.5 mm EDTA, pH 8.3) For antibody experiments, gel shift reactions were preincubated for 10 on ice with the indicated antibodies before the addition of the probe The a-LIN9 antiserum has been described previously [12] The LIN54 antiserum was raised in rabbits against the peptide AAKTKLSSQISDLLTR The LIN54 serum was affinity purified Other antibodies used were a-E2F4 (C-108), a-p130 (C-20) and a-E2F1 (C-20) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA) Reporter assays Electrophoretic motility shift assay pGex-4T2-CXC (amino acids 514–646 of LIN54) and pGex4T2-CXC-C525 ⁄ 527Y constructs were used for recombinant expression in bacteria For fusion protein recovery, bacterial cultures were lysed with bacterial lysis buffer (20 mm Hepes pH 7.5, 120 mm NaCl, mm EDTA, 10% glycerol, mm dithiothreitol, protease inhibitors) Sarcosyl was added to a final concentration of 1.5% and the lysate was sonicated and centrifuged The supernatant was incubated with 2% Triton-X-100 and 250 lL of glutathione-sepharose beads The recombinant protein was eluted from the beads with elution buffer (100 mm Hepes pH 8.0, 100 mm NaCl, 0.1% Triton-X-100, 0.5 mm dithiothreitol, 15 mgỈmL)1 glutathione) As probe, a cdc2 promoter fragment (427–24 bp upstream of the transcriptional start site) was prepared by PCR from ng of the wild-type or mutant cdc2 promoter constructs using a-[32P]-dCTP The PCR products were purified through Sephadex G-50 spin columns (ProbeQuant G-50, GE Healthcare Europe, Freiburg, Germany); 10 000 counts were used in each gel shift reaction DNA fragments for competition were prepared by PCR and gel purified; 50–300 ng of PCR fragment were used for competition The following primers were used for amplification of the competitor: full-length fragment, 5¢-GGGGATCCATTTGAACTGTGCCAATGC-3¢ (sense) and 5¢-GTCAAGCTTCACTGTACCCGGCTTATTATT-3¢ (antisense) The fragment with a deletion from the 5¢ end was prepared using the following forward primer: 5¢-GCTGGGAGAAAAAATTTAAAAGAAGAACG-3¢ For gel shifts with nuclear extracts, an oligonucleotide encompassing the CDE-CHR element of the human cdc2 promoter was used as a probe: 5¢-CCCTTTAGCGCGGT GAGTTTGAAACTGCTC-3¢ The sequences of the mutated derivatives are shown in Fig Gel shift binding reactions were prepared on ice using gel shift binding buffer (65 mm Hepes pH 7.9, 65% glycerol, 320 mm KCl, 0.65 mm EDTA, 2.5 mm MgCl2) with 0.05 lL of salmon sperm DNA (10 mgỈmL)1) and lL of BSA (20 mgỈmL)1) 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G A G A A A A A AT T TA A G AT C T AT T T G A A C T A G A C C A AT G C T G G G A G A A A A A AT T TA A G AT C T Mut A AT T T G A A C T G T G A A G AT G C T G G G A G A A A A A AT T TA A G AT... together, these data establish LIN54 as an essential member of the LINC ⁄ DREAM complex delayed entry into mitosis [9] To address whether this is an isolated function of LIN9 or whether it is. .. integral and essential subunit of this complex To analyze the function of LIN54, we created a set of deletion mutants Using these mutants, we found that a region of LIN54 that is predicted to form an