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Huifei Liu,1
Maite Huarte,1
Su Wu,1
Tucker Collins,2
En Li,3,
and
Yang Shi1*
Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115,1 Department of Pathology, Boston Children's Hospital, Boston, Massachusetts 02115,2 Cardiovascular Research Center, Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Charlestown, Massachusetts 021293
Received 13 October 2005/ Returned for modification 28 November 2005/ Accepted 11 January 2006
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ABSTRACT |
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75%, 50%, and 25% of the normal complement of YY1 identified a dosage-dependent requirement for YY1 during late embryogenesis. Indeed, reduction of YY1 levels impairs embryonic growth and viability in a dose-dependent manner. Analysis of the corresponding mouse embryonic fibroblast cells also revealed a tight correlation between YY1 dosage and cell proliferation, with a complete ablation of YY1 inducing cytokinesis failure and cell cycle arrest. Consistently, RNA interference-mediated inhibition of YY1 in HeLa cells prevents cytokinesis, causes proliferative arrest, and increases cellular sensitivity to various apoptotic agents. Genome-wide expression profiling identified a plethora of YY1 target genes that have been implicated in cell growth, proliferation, cytokinesis, apoptosis, development, and differentiation, suggesting that YY1 coordinates multiple essential biological processes through a complex transcriptional network. These data not only shed new light on the molecular basis for YY1 developmental roles and cellular functions, but also provide insight into the general mechanisms controlling eukaryotic cell proliferation, apoptosis, and differentiation. |
INTRODUCTION |
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Yin Yang 1 (YY1; also called delta, NF-E1, and UCRBP) is a ubiquitously expressed GLI-Krüppel zinc finger-containing transcription factor (23, 28, 44, 60). It is highly conserved from Xenopus to humans and has been shown to be the vertebrate homolog of the Drosophila melanogaster polycomb group protein Pleiohomeotic (2, 16, 64). YY1 is a multifunctional protein which can act as a transcriptional repressor or activator through combinatorial interactions with various other transcription factors, coactivators, and corepressors as well as chromatin-remodeling complexes displaying opposite functions, including the histone acetyltransferase p300/CBP, the arginine methyltransferase PRMT1, and the histone deacetylases HDAC1 and HDAC2 (4, 9-12, 26, 34, 35, 45, 47, 57, 62, 65, 80, 82, 83).
Since its original isolation, YY1 has been shown to control an ever-growing number of viral and cellular genes, among which are the human immunodeficiency virus type 1 and human papillomavirus oncogenes E6 and E7, several proto-oncogenes (c-myc, c-fos, and errb2), cdc-6 (cell division cycle 6 homolog), the DNA replication-dependent histone H3.2 gene, as well as various others (53, 59, 68, 76). With a few exceptions, many YY1 target genes have been characterized based on transient transfection (overexpression experiments and reporter gene assays) and/or in vitro approaches, and most of them have yet to be validated as bona fide YY1 target genes in vivo. Nevertheless, these studies suggest important roles for YY1 in the control of cell growth, proliferation, apoptosis, oncogenic transformation, and differentiation.
Consistent with this hypothesis, YY1 has been shown to functionally interact with c-Myc and E1A oncoproteins (3, 34, 36, 48), as well as to control the stability of the tumor suppressor p53 via a mechanism independent of its transcriptional activity (5, 26, 65). Further supporting the potential involvement of YY1 in tumorigenesis, recent studies suggest that elevated YY1 expression and/or transcriptional activity might contribute to tumor formation and/or progression (11, 14, 21, 56). Altogether, these findings highlight the complex nature of YY1 functions and possible mechanisms of action, but the molecular basis for YY1's biological activities remains largely unknown.
Several lines of evidence suggest a critical requirement for YY1 in embryonic development, morphogenesis, and organogenesis. Genetic and biochemical studies have attributed important roles to the Drosophila counterpart of YY1, pleiohomeotic (pho), in embryonic patterning; mutations of pho result in homeotic transformations associated with misexpression (i.e., derepression) of homeotic genes (15, 24, 25). YY1 is also essential for neural induction and patterning in Xenopus laevis (33, 51). Further emphasizing its critical requirement in embryonic development, we previously reported that constitutive ablation of YY1 in mice results in peri-implantation lethality (20). Heterozygous mice (yy1+/–) display a mild developmental delay, and a subset of these animals exhibit neurulation defects and exencephaly (20). While these observations strongly suggest that YY1 might play important additional roles during late embryogenesis, the early embryonic lethality caused by constitutive loss of function precluded investigation of YY1 requirement at later developmental stages.
In this study, we report the generation of conditional knockout mice bearing a yy1flox hypomorphic allele. Genetic crosses with heterozygous mice carrying a wild-type allele and a null allele of YY1 allowed us to generate mutant mice expressing 75%, 50%, and 25% of the normal YY1 level. Phenotypic analysis of these mice and corresponding embryonic fibroblasts revealed a critical, dosage-dependent requirement for YY1 in late embryonic development and cell proliferation. Significantly, complete ablation of YY1 induced cytokinesis failure and cell cycle arrest. Consistent with these findings, genomewide gene expression analysis revealed that YY1 regulates various genes involved in cytokinesis, cell proliferation, and differentiation as well as apoptosis, oncogenic transformation, and DNA repair. Taken together, these data shed new light on YY1 developmental roles, cellular functions, and target genes and set the stage for further investigation of the molecular mechanisms underlying its biological activities.
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MATERIALS AND METHODS |
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J1 embryonic stem (ES) cell clones bearing the targeted yy1flox conditional allele and chimeric mice were obtained as previously described (20, 37). Chimeric animals were bred to 129/Sv mice, and heterozygous yy1flox/+ offspring were intercrossed to obtain homozygous yy1flox/flox mice. Insertion of a neomycin resistance gene cassette in an intron by gene targeting has previously been reported to alter gene expression by virtue of the potential cryptic splice sites and polyadenylation signals it contains (39). In order to delete the selection cassette, yy1flox/+ mice were mated with transgenic "flipper" animals ubiquitously expressing Flp recombinase (22). While homologous recombination events targeting the yy1 locus were analyzed by Southern blot using external (i.e., derived from a genomic sequence that is not present in the targeting vector) and internal probes (20), Flp- and/or Cre-mediated recombination of the yy1flox and yy1f conditional alleles was monitored using a PCR-based approach. The sequences of the PCR primers and Southern blot probes will be made available upon request.
Histological analysis of hypomorphic mice. Newborn mice were fixed in Bouin solution, dehydrated in ethanol, incubated in xylene, and embedded in paraffin. Lung sections stained with eosin and hematoxylin were observed by light microscopy.
Isolation of MEFs. Mouse embryonic fibroblasts (MEFs) were derived from embryos 13.5 days postcoitum (dpc). Briefly, after removal of the head and internal organs, embryo carcasses were digested in trypsin solution for 30 min at 37°C. After addition of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% of fetal bovine serum (FBS), the remaining tissues were disrupted by pipetting. The resulting cell suspension was plated and cultured in DMEM supplemented with 10% FBS and antibiotics. Infection with adenovirus-Cre (Gene Transfer Vector Core, University of Iowa) was performed 2 days postisolation. Cells were maintained in the presence of viruses in DMEM containing 5% of FBS for 16 h before being plated for various assays.
RNAi. HeLa cells were transfected with a control or yy1 RNA interference (RNAi) plasmid (65) using Lipofectamine 2000 (Invitrogen Life Technologies). In some cases (see below), RNAi vectors were mixed with a puromycin resistance-encoding vector, and transfected cells were selected by adding puromycin (1.5 µg/ml; Sigma-Aldrich) to the culture medium for 2 days (65). Cells were then trypsinized and plated for various assays.
Analysis of growth properties. For the colony formation assay, equal numbers (5,000 to 20,000) of MEFs were seeded in six-well plates and cultured for 10 to 12 days. Cell colonies were then stained with a solution containing 30 mg/ml crystal violet, 9 mg/ml ammonium oxalate, and 20% ethanol. The same protocol was used for HeLa cells, but the colony formation assay was performed 14 days after puromycin selection of the transfected cells.
For the MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich] assay, equal numbers (10,000 to 30,000) of MEFs were seeded in six-well plates. After 2 to 10 days of culture, cells were incubated in the presence of 400 µg/ml of MTT in DMEM for 30 min at 37°C, allowing metabolic transformation of the MTT by the mitochondria and subsequent formation of an intracellular purple formazan (41). After solubilization of this precipitate in dimethyl sulfoxide, the absorbance was measured at 490 nm.
Analysis of cell viability and apoptotic cell death. Puromycin-selected HeLa cells were treated with various apoptotic agents 4 days after transfection of the RNAi vectors. Briefly, cells were treated with 200 nM of staurosporine (Sigma-Aldrich), 100 µM of etoposide (Sigma-Aldrich), 100 ng/ml of Fas ligand (Alexis Corporation), or 10 µg/ml of tumor necrosis factor alpha (R&D Systems) and harvested at various time points posttreatment. While viable cells were trypsinized and counted, apoptotic cell death was quantified by Western blot analysis of poly(ADP-ribose) polymerase (PARP) cleavage.
Flow cytometry analysis. The DNA content of MEF cells and puromycin-selected HeLa cells was analyzed essentially according to Shah et al. (58). Briefly, cells were harvested by trypsinization 4 days postinfection by adenovirus-Cre or posttransfection of the RNAi vectors and fixed with 50% ethanol. After one wash with phosphate-buffered saline (PBS), cells were treated with RNase A (100 µg/ml; Sigma-Aldrich) for 30 min at 37°C, stained with 50 µg/ml propidium iodide (Sigma-Aldrich), and analyzed with a FACSCalibur machine and Cellquest software (Becton Dickinson).
Bromodeoxyuridine incorporation and indirect immunofluorescence. MEFs and unselected RNAi-treated HeLa cells were cultured on coverslips and incubated with 10 µM bromodeoxyuridine (5-bromo-2'-deoxyuridine; Sigma-Aldrich) for 4 h prior to fixation in a 3% paraformaldehyde solution for 20 min. Cells were then permeabilized with 0.5% of NP-40 in PBS for 20 min and washed with PBS containing 0.1% NP-40. The cells were further treated with 4 M HCl for 30 min, followed by one wash with PBS and another wash with 100 mM borax solution. After an additional wash in PBS containing 0.1% NP-40, cells were incubated in blocking solution (PBS containing 0.1% NP-40 and 10% of FBS) and costained with a mouse monoclonal antibromodeoxyuridine (BU33; Sigma-Aldrich) and a rabbit polyclonal anti-YY1 (H414; Santa Cruz Biotechnology, Inc.).
Expression of various cell cycle markers was analyzed by immunostaining of HeLa cells, using antibodies directed against PCNA (PC10; Santa Cruz Biotechnology, Inc.), cyclin B1 (GNS1; Santa Cruz Biotechnology, Inc.), cyclin E (HE12; Santa Cruz Biotechnology, Inc.), phosphorylated (Ser10) histone H3 (06-570; Upstate Cell Signaling Solutions), 
-tubulin (B512; Sigma-Aldrich), or YY1 (H10; Santa Cruz Biotechnology, Inc.). With the exception of cyclin E immunodetection, which required methanol fixation, all immunofluorescence experiments were conducted on paraformaldehyde-fixed HeLa cells, as described previously (66).
Western blot analysis. Total cell extracts were prepared in lysis buffer (50 mM Tris-HCl, pH 7.3; 5 mM EDTA; 50 mM KCl; 0.1% NP-40; 1 mM phenylmethylsulfonyl fluoride; 1 mM dithiothreitol) supplemented with a complete antiprotease cocktail (Roche Diagnostics), and protein concentration was assessed by Bradford assay. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and peroxidase-based chemiluminescence detection were conducted according to standard laboratory procedures. Anti-YY1 (H414 and H10) and anti-PARP (F2) were from Santa Cruz (Santa Cruz Biotechnology, Inc.), the anti-lamin A and C antibody (2032) was from Cell Signaling (Cell Signaling Technology, Inc.), and the antiactin antibody (MAB1501) was from Chemicon International Inc. p53 induction was analyzed using an anti-p53 antibody (PAB240; Santa Cruz Biotechnology, Inc.), adriamycin treatment (2 µg/ml for 10 h) being used as a positive control. Densitometry analysis of YY1 protein levels in tissues and MEFs was performed using a digital camera coupled with band quantification software (Quantity One; Bio-Rad).
Microarray. MEFs were isolated from three wild-type (yy1+/+) embryos and compound heterozygous mutant (yy1flox/–) littermates, and RNA samples were prepared 4 days postisolation. Briefly, RNAs were prepared using Trizol reagent (Life Technologies) and the RNeasy kit (QIAGEN). cDNAs were generated from total RNAs with the Superscript Choice system (Gibco BRL Life Technologies) and T7-oligo(dT)24 primers. Subsequently, biotin-labeled cRNAs were synthesized using a BioArray high-yield RNA transcript labeling kit (Enzo Diagnostic, Inc.). After cleanup, fragmentation, and quality controls, the fragmented cRNAs were subjected to hybridization with a DNA-Chip mouse genome 430A array (Affymetrix) containing over 22,600 probe sets corresponding to transcripts and variants from approximately 15,000 well-characterized mouse genes. Data analysis was performed using DNA-Chip expression analysis software. Briefly, after determining the presence call and average differentiation parameters of each gene, gene expression levels from yy1+/+ and yy1flox/– MEFs were subjected to comparative analysis.
RT-PCR analysis. Reverse transcription (RT)-PCR analysis was performed to compare the mRNA expression levels of putative YY1 target genes in yy1+/+ and yy1flox/– MEFs 4 days postisolation, as well as in Cre-treated yy1+/+ and yy1f/f MEFs 6 days postinfection. Reverse transcription was carried out with 2 µg of total RNA (prepared as described above) using the Superscript III reverse transcriptase and oligo(dT)12-18 primers (Invitrogen Life Technologies). Once the semiquantitative conditions were set up, equivalent amounts of yy1+/+, yy1f/f and yy1flox/– RT products were submitted to amplification with various sets of PCR primers, and the PCR products were analyzed on ethidium bromide-stained agarose gels. The sequences of the PCR primers will be made available upon request.
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RESULTS |
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25% of the yy1flox/– animals died before day 14.5 dpc (Table 1). The discrepancies in the frequency of yy1flox/– embryos expected and obtained became even more pronounced at later stages, indicating that a fraction of embryos died between 13.5 and 14.5 dpc and 16.5 and 19.5 dpc, while another subpopulation died between 16.5 and 19.5 dpc and birth (Table 1). Examination of the morphological appearance of the yy1flox/– mice before birth (17.5 dpc) revealed that a significant subset of yy1flox/– embryos were severely growth retarded and displayed other developmental defects with various degrees of severity (Fig. 3A).
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To gain further insights into the cause of perinatal lethality of the yy1flox/– mice, embryos were isolated and examined at 19.5 dpc. As expected, most of the wild-type embryos isolated at this stage were able to breathe autonomously. In contrast, the yy1flox/– animals that were alive and reacted to mechanical stimulation at the time of isolation appeared unable to breathe despite frequent gasping and died within an hour postisolation. Postmortem analysis of lungs isolated from newborn yy1flox/– pups indicated that they were not filled with air. Consistently, histological examination revealed collapsed alveoli (Fig. 3D), confirming that the lungs failed to inflate at birth. Together, these results demonstrate an essential, dosage-dependent requirement for YY1 in late embryonic development.
Inhibition of YY1 function impairs cell proliferation in primary cells.
As mentioned earlier, generation of the yy1flox hypomorphic allele allowed us to produce mouse strains expressing different levels of YY1. As shown in Fig. 4A and B, Western blotting and densitometry analysis of YY1 expression in yyflox/+, yy1+/–, and yy1flox/– MEFs demonstrated that these cells expressed 75%, 50%, and 25% of the normal YY1 level, respectively. In contrast, MEFs isolated from embryos carrying two copies of the conditional yy1f allele (which lacks the PGK-Neo cassette [see above and Fig. S1 in the supplemental material]) expressed YY1 levels indistinguishable from that detected in their wild-type counterparts (Fig. 4C and Fig. S1H in the supplemental material). Time course analysis revealed that infection of yy1flox/– and yy1f/f MEFs with adenovirus-Cre led to a gradual decrease in YY1 protein level, YY1 expression becoming undetectable 2 to 3 days postinfection (Fig. 4B and C; see also Fig. S1 in the supplemental material). To gain insights into YY1 cellular functions, we took advantage of these conditional MEFs and examined the phenotype induced by partial and complete inhibition of YY1.
We first analyzed the viability and growth properties of MEFs of various genotypes in the absence and presence of adenovirus-Cre by a colony-forming assay. As shown in Fig. 4D, untreated yy1flox/+ MEFs expressing 75% of the normal YY1 complement displayed growth properties very similar to those of wild-type cells. While infection of the wild-type cells by adenovirus-Cre did not significantly affect their growth properties, reduction of the YY1 level to 50% in Cre-treated yy1flox/+ MEFs led to a marked decrease in cell proliferation (Fig. 4D). The growth-suppressive effect induced by depletion of YY1 became even more pronounced in untreated yy1flox/– cells expressing 25% of normal YY1, with complete depletion of YY1 in Cre-treated yy1flox/– MEFs further increasing the severity of this proliferation defect (Fig. 4D). Interestingly, while reduction of YY1 expression to 25% of the normal complement in untreated yy1flox/– MEFs affected mostly the size of the colonies formed by these cells, a marked decrease in both colony size and colony number was observed after complete depletion of YY1 in yy1flox/– MEFs infected with adenovirus-Cre (Fig. 4D).
Consistent with their ability to express wild-type levels of YY1 (Fig. 4C), no significant differences in cell colony formation were observed when comparing the untreated and yy1f/f MEFs with their wild-type counterparts (Fig. 4E). As expected based on our previous results (Fig. 4D), infection of the yy1f/f MEFs with adenovirus-Cre led to a dramatic decrease in cell colony size and cell colony number (Fig. 4E). Immunofluorescence experiments revealed that the few cell colonies formed by the Cre-treated yy1f/f MEFs were mainly attributable to the presence of YY1-positive cells (i.e., cells that had not been infected by adenovirus-Cre and therefore displayed normal YY1 expression levels and proliferation rates; data not shown). Taken together, these results demonstrate that inhibition of YY1 function in primary cells impairs cell viability and/or proliferation in a dose-dependent manner.
The dosage-dependent effect of YY1 on cell viability and/or proliferation was confirmed by the MTT assay, performed on yy1+/+, yy1flox/+, yy1+/–, and yy1flox/– MEFs expressing 100%, 75%, 50%, and 25%, respectively, of the normal amount of YY1 (Fig. 4F). Using a similar approach, we analyzed the growth kinetics of untreated and Cre-treated yy1+/+, yy1flox/–, and yy1f/f MEFs (Fig. 4G and data not shown). Consistent with our previous observations (Fig. 4D), the MTT assay confirmed that yy1flox/– MEFs expressing 25% of normal YY1 proliferate at a much lower rate than wild-type cells (Fig. 4G). Despite their impaired growth properties, untreated yy1flox/– MEFs were found to proliferate steadily throughout the course of the experiment (Fig. 4G). In contrast, complete depletion of YY1 in both Cre-treated yy1flox/– MEFs (Fig. 4G) and Cre-treated yy1f/f MEFs (data not shown) abolished cell proliferation.
The decrease in viable cell number caused by depletion of YY1 could result from an increase in cell death, a decrease in cell proliferation, or both. Microscopic observation of the untreated and Cre-treated yy1flox/– and yy1f/f MEFs demonstrated that these cells did not detach and/or die (data not shown). Consistently, these cells did not display any characteristics of apoptotic cells, including nuclear fragmentation or cleavage of lamins A and C, and no significant accumulation of apoptotic (sub-G0) cells was detected by fluorescence-activated cell sorting (FACS) analysis in either untreated or Cre-treated yy1flox/– and yy1f/f cell populations (Fig. 4H and data not shown).
Besides the appearance of a small subpopulation of cells having an 8N DNA content (see below), the major alteration of cell cycle profile observed upon treatment of the yy1f/f cells with Cre was an accumulation of cells with a 4N DNA content (Fig. 4H). Indeed, 4 days posttreatment, the tetraploid cell population in the yy1f/f MEFs treated with Cre was 62%, while the percentage of cells with a 4N DNA content in the wild-type (treated or untreated) and untreated yy1f/f MEFs ranged from 26 to 39% (Fig. 4H). Based on these observations, we conclude that inhibition of YY1 function in primary cells does not trigger apoptosis but impairs cellular proliferation in a dosage-dependent manner. Strikingly, complete depletion of YY1 leads to cell cycle arrest and accumulation of tetraploid cells.
YY1 is required for cytokinesis in primary cells. The accumulation of tetraploid cells observed after inhibition of YY1 function could be due to the inability of the cells to progress from G2 phase to mitosis (blockage at the G2/M transition), to successfully complete mitosis (mitotic arrest, failure of chromosome segregation), to initiate or complete cytokinesis, or to enter G1 phase. To characterize the proliferative arrest caused by depletion of YY1 in more detail, we examined the cellular and nuclear morphology of the YY1-depleted cells by phase-contrast light microscopy and fluorescence microscopy after 4',6'-diamidino-2-phenylindole (DAPI) staining. Most cells had nuclei with uncondensed chromosomes, arguing against mitotic arrest (data not shown). However, depletion of YY1 resulted in the gradual accumulation of binucleated cells (Fig. 4I). No sign of DNA condensation or cell cleavage was observed in these binucleated cells (data not shown), strongly suggesting that inhibition of YY1 function results in cytokinesis failure.
Identification of YY1 target genes in primary cells.
While a substantial body of studies established a role for YY1 in transcriptional regulation, most YY1-responsive genes identified so far have yet to be validated as bona fide YY1 target genes in vivo. To gain mechanistic insights into YY1 developmental and cellular functions, we used a genomewide approach to identify YY1 target genes in mouse embryonic fibroblasts. Microarray analysis performed on yy1+/+ and yy1flox/– MEFs (expressing 25% of the normal complement of YY1) allowed us to identify 524 genes whose expression was affected by a reduction of the YY1 level. The complete list of YY1-responsive genes is provided in Table S1 in the supplemental material, and the target genes whose functions are the most relevant to the phenotypes described in this study are presented in Table 2. It is worth mentioning that approximately 90% of the genes affected by partial depletion of YY1 were found to display similar (i.e., comparable or more pronounced) changes in their expression profiles after complete depletion of YY1 in Cre-treated yy1flox/– MEFs (data not shown).
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To confirm the microarray data, the expression of 22 putative YY1 target genes was analyzed by RT-PCR in untreated yy1+/+ and yy1flox/– MEFs (Fig. 5A). A similar approach was used to investigate the effects of complete depletion of YY1 on the expression of 21 of these putative targets in Cre-treated yy1+/+ and yy1f/f MEFs (Fig. 5B). Among the candidates analyzed were the genes for cell cycle regulators p21, p57, cdc46/mcm5, pol1, cyclin B1 and cyclin B2, known regulators of mitosis and cytokinesis (Incenp, CenpA, Plk1, Birc-5/Survivin, and Cap-d2), and epigenetic regulators of mammalian development (Suv39h2 and Dnmt3a). The p53 target genes Mdm2, Sip27, Gadd45
, Dda3, and Perp were selected due to their involvement in p53-dependent regulation of the cell cycle and apoptosis. Several genes involved in the regulation of cell survival, growth, and differentiation during development (e.g., Bdnf, Ntf3, Nsg1, Tgfß2, and Btg2), as well as a few other genes (BM88, Hmgb3, Nisnap1, and Sfxn1) regulating diverse biological processes, were also studied. The expression of all candidate target genes tested was found to be affected by partial (Fig. 5A) and/or complete (Fig. 5B) depletion of YY1, as predicted based on the microarray results (Table 2; see also Table S1 in the supplemental material).
Depletion of YY1 does not lead to p53 induction in primary cells. We and others have previously shown that, in some cellular contexts, ablation of YY1 results in elevated levels of the tumor suppressor p53 and subsequent activation of p53-dependent pathways controlling cell growth and/or apoptosis (5, 26, 65). Strikingly, analysis of the microarray data revealed that approximately 40 YY1-responsive genes have previously been identified as p53 target genes (18, 27, 31, 40, 42, 74), their deregulation upon depletion of YY1 mimicking the effects of p53 induction (see Table 2; see also Table S1 in the supplemental material). Taken together, these results raise the possibility that the cellular phenotype (i.e., cell cycle arrest) and changes in gene expression profiles induced by depletion of YY1 in mouse embryonic fibroblasts might be mediated, at least in part, via an increase in p53 expression and/or stability.
To investigate this hypothesis, we analyzed the effects of YY1 depletion on the cellular levels of p53 mRNA and protein (Fig. 6). Consistent with our microarray data (see Table S1 in the supplemental material, and data not shown), we did not observe any significant effect of partial (i.e., untreated yy1flox/– MEFs) or complete (i.e., Cre-treated yy1flox/– MEFs) depletion of YY1 on p53 mRNA levels (Fig. 6A). While a strong increase in p53 protein level was observed in adriamycin-treated wild-type MEFs (used as a positive control for p53 induction), Western blot analysis showed that YY1 depletion did not affect the steady-state level of p53 protein in yy1flox/– MEFs (Fig. 6B). Consistently, no significant fluctuations of p53 mRNA and protein levels were detected after complete depletion of YY1 in yy1f/f MEFs (data not shown).Together, these results strongly suggest that the cellular phenotype and molecular targets induced by depletion of YY1 in MEFs are unlikely to result from induction of p53.
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The effects of YY1 depletion on cell viability and proliferation were assessed by a colony-forming assay (Fig. 7B) and FACS analysis (Fig. 7C), performed after transfection with a control or YY1 RNAi vector and subsequent selection of the transfected cells. Under these conditions, the YY1 protein level was dramatically reduced upon transfection of the YY1 RNAi vector (Fig. 7A). We found that the number and size of cell colonies formed by the RNAi-depleted cells were greatly reduced compared to those of mock-transfected cells (Fig. 7B). This is consistent with the phenotype resulting from complete depletion of YY1 in mouse primary cells (Fig. 4D and E, Cre-treated yy1flox/– and yy1f/f MEFs) and reinforces the idea that YY1 is required for cell survival and/or cell proliferation.
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As shown in Fig. 7E, the majority of YY1-negative cells (i.e., in which YY1 nuclear staining was extremely low or undetectable) containing more than one nucleus were binucleated and showed no detectable sign of DNA condensation or cell cleavage. Only a very small percentage of YY1-negative cells accumulated more than two nuclei (Fig. 7E and data not shown). The YY1-RNAi treated cells also displayed other nuclear anomalies, including deformed or bilobed nuclei, micronuclei, and DNA bridges, with a high frequency (Fig. 7D and F). Besides these alterations of their nuclear morphology, we found that most cells affected by the YY1 RNAi were abnormally flat and large compared to the control cells, and the size of their nucleus was also increased (Fig. 7E and F, and data not shown). Thus, the phenotype induced by depletion of YY1 in HeLa cells is essentially identical to that observed in mouse primary cells (Fig. 4) and strongly suggests that inhibition of YY1 induces cytokinesis failure.
To gain further insights into the molecular mechanisms underlying the role of YY1 in the regulation of cell cycle progression and cell ploidy, we examined the expression of various markers specific to the different phases of the cell cycle (Fig. S2 and S3 in the supplemental material). Based on these stainings, the percentages of cells in G1, S, G2/early M (i.e., prophase-end of metaphase), and late M/cytokinesis (i.e., anaphase-telophase) phases in the control and YY1 RNAi cell populations were determined (Table 3).
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35% of the YY1-depleted cells) were undergoing DNA replication. Considering exclusively YY1-negative cells containing more than one nucleus, we determined that 44% ± 3% of such cells contained PCNA-positive DNA replication foci. Consistently, a significant number of binucleated YY1-negative cells (24.3% ± 1%) were found to be in G2 phase (see also Fig. S2 in the supplemental material). Based on these immunostaining experiments, we conclude that (i) the proliferation defects and accumulation of tetraploid cells induced by depletion of YY1 do not result from a blockage at the G2/M transition, (ii) depletion of YY1 impairs cytokinesis initiation, leading to the accumulation of cells containing two nuclei, and (iii) binucleated cells do not undergo cell cycle arrest immediately after cytokinesis failure, but instead progress through the next G1 phase and subsequently undergo at least one round of illegitimate DNA replication leading to polyploidization. The small percentage of YY1-negative cells displaying more than two nuclei strongly suggests that most binucleated cells cannot proceed to the next round of mitosis, but rather undergo cell cycle arrest. Supporting this view, the percentage of YY1-negative cells initiating mitosis (i.e., cells in early M phase) decreased slowly but gradually throughout the course of the experiment, while the percentage of control cells undergoing mitosis was not significantly affected (data not shown).
Taken together, these results demonstrate that complete inhibition of YY1 function results in cytokinesis failure and induces severe cell proliferation defects, uncovering new p53-independent functions of YY1-mediated regulation of cell cycle progression.
Depletion of YY1 sensitizes HeLa cells to various apoptosis-inducing agents. Inhibition of YY1 function has previously been shown to modulate the cellular sensitivity to p53-mediated apoptosis in response to DNA damage (26). In the present study, we report that depletion of YY1 leads to a deregulation of several apoptosis-related genes without any significant effect on p53 levels (Table 2 and Fig. 6). These observations led us to investigate the potential direct involvement of YY1 in the regulation of apoptotic programs. Therefore, we analyzed the effects of YY1 depletion on the cellular response of HeLa cells, which lack functional p53, to various DNA-damaging and nondamaging apoptotic inducers.
As shown in Fig. 8, puromycin-selected HeLa cells transfected with the control or YY1 RNAi were treated with known activators of mitochondrion-dependent apoptotic pathways (i.e., staurosporine and etoposide) and of the death receptor-dependent pathways (i.e., tumor necrosis factor and Fas ligand). After ensuring efficient depletion of YY1 in the YY1 RNAi-treated cells (Fig. 8B), we quantified cell viability (Fig. 8A) and cell death (using PARP cleavage as a marker of apoptotic cell death; Fig. 8B) in the untreated and treated control and YY1 RNAi cell populations. Consistent with our previous observations (see above), RNAi-mediated inhibition of YY1 in HeLa cells did not trigger apoptosis in the absence of any apoptotic agents (Fig. 8B, and data not shown). However, depletion of YY1 dramatically increased the cellular sensitivity to all apoptotic agents tested. Indeed, treatment with staurosporine, etoposide, tumor necrosis factor, and Fas ligand led to a much greater decrease in the number of viable cells in the YY1 RNAi cell population than in the control cell population (Fig. 8A).
Consistently, treatment of the YY1-depleted cells with staurosporine, etoposide, and Fas ligand led to a marked increase in PARP cleavage, this effect being much more pronounced than that observed upon treatment of the control cells (Fig. 8B). Taken together with previous results showing that YY1 inhibits genotoxic stress-induced activation of p53 (26), our data strongly suggest that YY1 plays important roles in the regulation of multiple p53-dependent as well as p53-independent pathways mediating programmed cell death in response to a broad spectrum of apoptotic agents.
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DISCUSSION |
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75% of the normal level of YY1 are viable and do not display any obvious developmental defects, reduction of the YY1 protein level by 50% induces lethality in a small subset of mice and affects embryonic growth. Further diminution of the YY1 level significantly increases the penetrance of the growth retardation and embryo-lethal phenotype. Approximately half of the mice expressing 25% of normal YY1 levels died during late embryogenesis, a developmental period during which active morphogenesis and organogenesis are occurring. Thus, the phenotype of mice expressing 25% of normal YY1 levels differs dramatically from that of yy1 null embryos, which die around the time of implantation, a stage characterized by intense cell proliferation (20). These observations suggest that 25% of the normal YY1 level can support cellular proliferation, at least to some extent, while complete depletion of YY1 cannot, which is consistent with the dose-dependent growth suppressor effect of YY1 observed in cultured embryonic fibroblasts (see below).
Approximately half of the mice expressing 25% of the normal complement of YY1 developed and survived until birth but died shortly thereafter from respiratory failure. While histological examination demonstrated that the lung alveoli were collapsed, electronic microscopy did not reveal any major ultrastructural defects. In particular, alveolar type II cells, which are responsible for the synthesis of lung surfactant, appeared normal (data not shown), suggesting that the respiratory failure induced by depletion of YY1 results from developmental defects affecting organs other than the lung. Interestingly, inactivation of several of the novel YY1 target genes identified here (including lamin B1, Bdnf, the transcription factor FoxM1, and the cell cycle regulator p57) impairs the development of various tissues and causes respiratory failure (30, 32, 67, 73, 81).
Functional defects of the respiratory musculature, of its motor innervation, as well as of the central control center (i.e., respiratory rhythm-generating neurons) have been shown to cause respiratory failure. In this regard, it is worth pointing out that a large number of muscle-specific genes were derepressed after depletion of YY1 in MEFs (including various myosin-, procollagen-, troponin-, and actin-encoding genes [Table 2; see also Table S1 in the supplemental material]). Importantly, YY1 was recently shown to recruit HDAC1 as well as the polycomb group protein EZH2 (enhancer of zeste homolog 2), a histone H3 methyltransferase whose activity is associated with stable transcriptional repression, to genomic regions of silent muscle-specific loci in undifferentiated myoblasts (17). Polycomb group proteins play important roles in long-term, inheritable gene silencing required for maintenance of cell identity during development and differentiation. However, the mechanisms underlying the recruitment of polycomb group protein complexes to their molecular targets as well as the identity of these targets remain largely unknown. It would be extremely interesting to investigate the possibility that YY1 might target HDACs and/or polycomb group protein-containing complexes to specific DNA recognition sites within the regulatory regions of its target genes and induce stable transcriptional repression of muscle-specific genes in undifferentiated muscle progenitor cells and differentiated nonmuscle cells.
Strikingly, depletion of YY1 in embryonic fibroblasts also led to the activation of various genes whose expression and function are normally restricted to neuronal cells, including the neuronal survival and plasticity factors neurotrophin 3 (Ntf3) and brain-derived neurotrophic factor (Bdnf), the imprinted gene neuronatin, and the brain-specific helix-loop-helix proteins encoded by Neurod6 (neurogenic differentiation 6), BM88, and several others. Although YY1 might directly repress some neuron-specific genes, the observation that YY1 depletion dramatically inhibits the expression of REST (repressor element 1 silencing transcription factor [Table 2]) in MEFs suggests alternative regulatory mechanisms.
Several studies demonstrated that the transcriptional repressor REST plays essential roles in restricting expression of neuronal genes to neurons by mediating active repression as well as long-term epigenetic silencing of these genes in differentiated nonneuronal cells (6, 8, 19, 29, 38, 43, 49, 50, 54, 55). Importantly, REST is highly expressed in pluripotent cells, in which it also represses neuron-specific gene expression, but is dramatically down-regulated during the transition to lineage-restricted neural progenitors, completely disappearing as progenitors exit the cell cycle and differentiate into mature neurons (7, 54). It is thus conceivable that YY1 might, directly or via the modulation of REST expression, participate in the maintenance of nonneuronal cell identity by contributing to neuron-specific gene silencing in such cells and/or play a role in the acquisition of neuronal traits in differentiating neuronal precursors. It would be extremely interesting to study YY1 expression during neuronal cell specification and differentiation and take advantage of our conditional knockout mice to investigate its role during neurogenesis.
While YY1 target genes are likely to play crucial roles in its developmental functions, various YY1-binding partners could also contribute to its biological activities. In this regard, it is interesting that abrogation of the acetyltransferase activity of p300, which acetylates YY1 and modulates its activity (34, 80), also causes neonatal lethality due to respiratory failure in mice (61). In addition, constitutive inactivation of p300 in mice reveals important dosage-dependent effects on cell proliferation, embryonic growth, and viability, as well as a partially penetrant neural tube closure defect similar to that displayed by a subset of heterozygous (yy1+/–) and compound heterozygous (yy1flox/–) mutant mice (20, 79) (data not shown). These phenotypic similarities suggest that YY1-p300 functional interplay could play an important role during development. Further analysis will be required to decipher the mechanisms underlying YY1 developmental functions and determine the contributions of its target genes and interacting partners.
Analysis of the cellular phenotypes induced by partial and complete depletion of YY1 in MEF cells (Fig. 4) allowed us to demonstrate that YY1 regulates cell proliferation in a dose-dependent manner. Interestingly, while the growth properties of MEFs expressing 50% and 25% of the normal complement of YY1 were indisputably impaired, these cells kept proliferating at a slow but steady rate. In contrast, complete depletion of YY1 in primary and tumor cells induced proliferative arrest (Fig. 4 and 7). In addition, loss of YY1 function resulted in cytokinesis failure in both embryonic fibroblasts (Fig. 4) and HeLa cells (Fig. 7), but no significant accumulation of binucleated cells was observed in untreated yy1flox/– MEFs expressing 25% of the normal complement of YY1 (data not shown). Taken together, theses results strongly suggest that partial depletion of YY1 impairs cell proliferation by inducing an extension of the cell cycle phases, while complete loss of YY1 blocks cell cycle progression and leads to cell cycle arrest.
Consistently, genome-wide gene expression analysis revealed that various cell cycle-related genes were deregulated upon depletion of YY1 (Table 2; see also Table S1 in the supplemental material). Interestingly, a large number of YY1-responsive genes involved in cell cycle control have previously been shown to be regulated by E2F transcription factors (13, 46). Among these YY1- and E2F-regulated genes are p21, cyclin A2, mcmd4-7, pol1, polo-like kinase, and aurora kinase B, as well as various other checkpoint regulators (including Bub1, Bub1b, Mad2, and CenpE). Supporting the hypothesis that YY1 might share common functions and/or molecular targets with E2Fs, coincident binding of YY1 and E2F2 or E2F3 on adjacent recognition sites within the cdc6 promoter results in synergistic activation of this promoter (53). As several other YY1 and/or E2F target genes contain binding sites for both transcription factors (Y. Shi, unpublished observations) (53), this mechanism might provide a molecular basis for the specificity of E2F functions as well as for YY1 in cell cycle control.
Consistent with the novel and unanticipated function for YY1 in cytokinesis uncovered in this study, several known key regulators of mitosis and cytokinesis, such as survivin/Birc5, polo-like kinase, inner centromere protein, aurora kinases A and B, and various others (Table 2), were found to be deregulated by depletion of YY1. Interestingly, RNAi-mediated inhibition of survivin, the expression of which is significantly down-regulated upon depletion of YY1, impairs cytokinesis and cell proliferation (78). Inactivation of survivin was shown to induce p53 up-regulation and subsequent activation of a p53- and p21-dependent checkpoint, resulting in cell cycle arrest in G1 phase (78). Despite contradictory reports, other studies support the hypothesis that cytokinesis failure activates a tetraploidy checkpoint response blocking cell cycle progression in G1 phase (1, 63, 69, 70, 75).
Several lines of evidence argue against the cell cycle arrest induced by complete depletion of YY1 being the result of the activation of such a checkpoint response. Indeed, we did not detect any significant effect of YY1 depletion on p53 levels in MEFs (Fig. 6), which, together with the previous demonstration that YY1 negatively regulates p53 stability in other cellular contexts (5, 26, 65), suggests that YY1-mediated regulation of p53 might be cell type and/or species specific. Consistent with the observation that depletion of YY1 does not affect steady-state p53 levels in MEFs, RNAi-mediated inhibition of YY1 function was found to induce proliferative arrest in p53 null mouse embryonic fibroblasts (Y. Shi, unpublished observation). In addition, inhibition of YY1 expression in HeLa cells, in which p53 is inactivated by the papillomavirus oncoprotein E6, also resulted in cell cycle arrest and cytokinesis failure.
Immunostaining of HeLa cells allowed us to demonstrate that binucleated cells did not undergo cell cycle arrest immediately after cytokinesis failure, but progressed through the next G1 phase and underwent at least one round of DNA endoreplication before exiting the cell cycle (see Fig. S2 and S3 in the supplemental material). Similarly, a significant fraction of YY1-depleted binucleated MEFs were found to undergo DNA replication, as indicated by their ability to incorporate bromodeoxyuridine (data not shown). Further strengthening the idea that YY1 ablation does not trigger activation of a p53- and p21-dependent tetraploidy checkpoint response, genetic rescue experiments demonstrated that YY1 depletion induced similar defects regardless of the status of p21. Indeed, no significant differences in growth properties or binucleated cell numbers were observed when comparing the effects of YY1 depletion in p21+/+ versus p21 null MEFs (see Fig. S4 in the supplemental material; also data not shown). Together, these data demonstrate that the effects of YY1 on cell cycle progression and cell proliferation are essentially p53 and p21 independent.
Despite the strong evidence that the effects of YY1 depletion on cell cycle progression and cell proliferation reported here are essentially p53 independent, we identified approximately 40 YY1-responsive genes that are known p53 targets (18, 27, 31, 40, 42, 74), suggesting that YY1 and p53 might directly regulate a common subset of related target genes in an antagonistic manner. While further analysis will be required to validate this hypothesis, it is supported by the recent finding that YY1 inhibits p53-mediated activation of p21 and Gadd45, through a mechanism involving competitive binding for overlapping DNA recognition sites within these promoters (77). Together with the transcription-independent cross talk between these two transcription factors (26, 65), such convergence of YY1 and p53 on shared molecular targets could contribute to their antagonistic effects on cell growth and apoptosis.
Interestingly, depletion of YY1 results in the derepression of several proapoptotic genes (Table 2), including known p53 target genes (18, 27, 31, 40, 42, 74). YY1 has previously been shown to modulate p53-dependent apoptotic programs induced by genotoxic stress (26). Here, we report that depletion of YY1 increases the sensitivity of cells lacking p53 activity to various DNA-damaging and nondamaging apoptosis inducers. Indeed, inhibition of YY1 was found to sensitize tumor cells to etoposide, a chemotherapeutic agent used to treat a wide spectrum of human cancers, and to staurosporine; both of these activate mitochondrion-dependent apoptotic pathways. In addition, YY1-depleted cells were also more sensitive to tumor necrosis factor and Fas ligand-induced apoptosis, mediated through death receptor-dependent pathways. This finding is consistent with the observation that expression of Fas receptor (Fas) is derepressed upon inhibition of YY1 (Table 2).
While this work was in preparation, Vega et al. reported that rituximab, a chimeric anti-CD20 antibody used in the treatment of B non-Hodgkin's lymphoma, up-regulates Fas expression and sensitizes Fas-resistant tumor cells to Fas ligand-induced apoptosis by modulating YY1 expression and activity (71, 72). Therefore, through its ability to modulate the sensitivity of various tumor cell types to a broad spectrum of apoptotic agents inducing p53-dependent as well as p53-independent pathways, YY1 might be an attractive therapeutic target for cancer.
Together, our data uncovered important dosage-dependent functions of the transcription factor YY1 in embryonic growth and development, cell proliferation, cytokinesis, and apoptosis. Moreover, genome-wide analysis allowed us to identify over 500 putative YY1 target genes. The large number of YY1-responsive genes identified in this study is not surprising, as it reflects the effects of YY1 removal on its direct targets as well as cascades of secondary events. Kinetic analysi
