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Department of Pathology, New York University School of Medicine and New York University Cancer Institute, New York, New York,1 Division of Molecular Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands,2 Molecular Medicine Program, Ottawa Health Research Institute, Ottawa, Ontario, Canada K1H 8L6,3 Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, Massachusetts 021424
Received 4 April 2005/ Returned for modification 26 May 2005/ Accepted 27 June 2005
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ABSTRACT |
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INTRODUCTION |
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Although p107, p130, and pRB are closely related members of the same family, they have different affinities for E2F family members, and they exhibit distinct temporal regulation during the cell cycle. While E2F4/p130 complexes are the most abundant in quiescent cells, E2F4/p107 and E2F4/pRB complexes accumulate in G1 cells (31). In addition, while p130 and p107 are bound to a number of promoters in asynchronously growing cells, only p130 is recruited to promoters in quiescent or serum-restimulated human cells (43).
These and other observations suggest unique properties among pRB and E2F family members. Indeed, a recent study revealed a distinct novel function for p107 as a negative regulator of stem cell development in the adult brain (47). Furthermore, knockout studies implicate both p130 and p107 in embryonic development and cell cycle regulation, though the mechanisms are likely different (23, 24, 47). Mice lacking either p107 or p130 in a mixed genetic background are viable and fertile, and exhibit no overt phenotypes, and mutant embryonic fibroblasts exhibit normal cell cycle kinetics (10, 21, 25). However, ablation of the p130 gene in BALB/cJ mice results in death between embryonic days E11 and E13, whereas in the same genetic background, p107-null mice display impaired growth but are viable and fertile (23, 24).
E2F cell cycle control mechanisms have been studied primarily by analyzing quiescent cells stimulated by mitogens. However, recent evidence suggests that cells reentering the cell cycle from quiescence exhibit differences in gene regulatory mechanisms compared to cells that are continuously cycling. Studies of cycling cells revealed that certain E2F-regulated genes are expressed without significant fluctuations following cell cycle reentry from mitosis (26). In addition, E2F4 and E2F5 are dispensable in quiescent cells, but necessary in cycling cells for pocket protein-mediated G1 arrest (17). Embryonic fibroblasts isolated from E2F4- and E2F5-null mice proliferate normally and reenter the cell cycle from G0 with kinetics similar to those of wild-type cells. However, they fail to arrest in G1 in response to p16INK4 (17). Moreover, it has been suggested that an E2F6 complex consisting of HP1, Polycomb group proteins, and a novel histone methyltransferase preferentially occupies target promoters in G0 rather than G1 cells (34), suggesting that chromatin modifiers contribute to silencing of E2F-responsive genes in quiescent but not G1 cells. More recent studies, however, suggest that E2F6 functions to repress certain genes in S phase, thereby limiting their expression to the G1/S transition (19).
It is becoming clear that elucidating the regulatory mechanisms that differentiate quiescent, mitogenically stimulated, and cycling cells will be essential, as it will allow the identification of specific targets that are regulated under growth-suppressive conditions and whose improper regulation could lead to cellular transformation. Thus, it has been shown that the disruption of normal regulatory networks that maintain adult stem cells in a stable, quiescent state may convert them to the malignant progenitors of cancer (2).
Several new experimental approaches, including combined use of chromatin immunoprecipitation (ChIP) and DNA microarrays (termed ChIP-on-chip or location analysis), have extended our understanding regarding cell cycle control by E2F and pocket protein complexes. This technique allows identification of novel targets of a given transcription factor on a genomic scale. These studies have revealed a diverse array of processes controlled by E2Fs, including DNA replication and repair, chromatin assembly and modification, mitosis, and cell cycle checkpoints (5, 39, 49). Although these data have immensely expanded our understanding of how E2F and pRB regulate transcription, these studies focused on transcriptional regulation of E2F-dependent genes under conditions of growth arrest or mitogenic stimulation.
Here, we have investigated how E2F-dependent genes are regulated in continuously cycling cells. Location analysis reveals differences between pocket proteins and shows that E2F4, p107, and p130 regulate common and distinct sets of genes: some genes appear to be regulated exclusively by one protein or diverse combinations of two or more of these proteins. Moreover, we have shown that these groups have distinct histone acetylation and mammalian Sin3B corepressor recruitment signatures. Interestingly, we have identified new target genes in cycling cells unique to E2F4 and p107 that are involved in stress and immune responses. Thus, we suggest that location analysis provides a unique advantage over genetic methods to discriminate between targets bound by highly related and functionally overlapping family members. We conclude that E2F4, p107, and p130 regulate distinct classes of targets, enabling them to play distinct roles in the regulation of cell cycle progression.
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MATERIALS AND METHODS |
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Western blot analysis. Western blot analysis was performed with antibodies against pRB (G3-245; BD Biosciences), p107 (sc-318), p130 (sc-317) and cyclin B1 (sc-245) from Santa Cruz Biotechnologies, and Ser 10-phosphorylated histone H3 (clone RR002) purchased from Upstate Biotechnologies.
Construction of Hu13K promoter DNA microarrays.
The construction and use of the custom DNA microarray (Hu13K) containing 13,000 human gene promoter regions spanning 
700 bp upstream and 200 bp downstream of the transcription start sites have been described previously (5, 33). The complete ChIP-on-chip data set and supplemental information can be found at http://www.med.nyu.edu/Research/dynlab01.html.
Chromatin immunoprecipitation. Chromatin immunoprecipitation (ChIP) was performed as described previously (39, 43), with some modifications. Briefly, cells were cross-linked with 1% formaldehyde and sonicated to chromatin fragments of 500 to 1,000 bp in length. Crude chromatin was incubated with 2 µg of antibodies against p107 (sc-18), E2F4 (sc-1082), or p130 (sc-317) (all from Santa Cruz). Recovered immunoprecipitates were treated as described previously. The DNA was purified from resulting samples and assayed by semiquantitative PCR using corresponding primers for the indicated E2F-regulated promoter (sequences available upon request). The resultant PCR products were separated on either a polyacrylamide gel (if a radioactive nucleotide was used in PCRs) and visualized by exposure to X-ray film, or on 2% agarose gels (visualized by ethidium bromide staining). In some experiments, as indicated in figure legends, the intensities of the enrichment in ChIP assays were quantitated using Quantiti One and normalized to the input. The data are presented in graphs where the y axis shows input-normalized intensity (Int x mm2) of each ChIP band: (ChIP volume – mock volume)/(input volume – mock volume).
Location analysis. Preparation of the hybridization probe was carried out essentially as described previously (38) with some modifications. Briefly, immunoprecipitated DNA was amplified by ligation-mediated PCR and 0.5 to 1 µg of amplification product was used for labeling. The absorbance of Cy3- and Cy5-labeled DNA was monitored at 550 nm for Cy3 and 650 nm for Cy5. Only the probes that contained at least 80 to 100 pmol of the dye were used for the hybridization on microarray slides. The hybridization was carried out in a hybridization buffer containing 3x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate, 20 µg human Cot-1 DNA, and 40 µg yeast tRNA at 60°C for 16 to 18 h. Following washing, promoter arrays were scanned with a Genomic Solutions LS IV or Axon 4000B scanner, and scanned images were analyzed with ImaGene and GenePix Pro software, respectively. A previously described error model (39) was used to assess the relative numbers of enriched targets. Data from three independent experiments were used to calculate average P value and enrichment ratios. The binding of a factor to DNA was deemed significant if the average P value is <0.005.
RNAi. Small interfering RNA (siRNA) oligonucleotides targeting p130 and E2F4 were designed by and purchased from Dharmacon Inc. The sequences were as follows: p130 (5'-AAGAGCAGAGCTTAATCGAAT-3' and 5'-AAGGACTTAGTTTATGGAAAT-3'), and E2F4 (5'-AAGGAGATTGCTGACAAACTG-3'). T98G cells were plated at the density of 6 x 105 per 100-mm tissue culture dish. At 24 h, cells were switched to serum-free Dulbecco's modified Eagle's medium. Following 36 h incubation in serum-free medium, cells were transfected with 400 pmol of siRNA oligonucleotides per 100-mm dish using sImporter transfection reagent (Upstate) per the manufacturer's instructions. At 16 h posttransfection, cells were switched to DMEM plus 10% FBS containing 2 mM final concentration of hydroxyurea. Following a 21-h hydroxyurea treatment, cells were released into DMEM plus 10% FBS and harvested at 3, 9, and 14 h for reverse transcription (RT)-PCR, Western blotting, and fluorescence-activated cell sorting (FACS) analysis.
RT-PCR. Total RNA was extracted using TRIzol reagent (Invitrogen). First-strand cDNA synthesis was performed using SuperScript First-Strand synthesis system for RT-PCR (Invitrogen) as described by the manufacturer. The resulting cDNA was amplified using gene-specific primers. Linear amplification was assured for each gene, and three independent reactions were performed in all cases. Primer sequences are available upon request.
Affymetrix DNA microarrays.
Hybridization to human U133Av2.0 microarrays was carried out according to the manufacturer's instructions. Briefly, total RNA was isolated with TRIzol from synchronized T98G cells; 10 µg of total RNA was used for reverse transcription and subsequent in vitro cRNA synthesis. We designated a gene as derepressed if its relative signal in RNA interference (RNAi)- treated cells was 
1.5-fold higher compared to nonspecific oligonucleotide-treated cells. Data extraction and analysis were performed with dChip (27). Each experiment was performed two times. The complete Affymetrix data set is available at http://www.med.nyu.edu/Research/dynlab01.html.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Promoter occupancy by E2F4, p107, and p130 throughout the cell cycle. E2F4 represses transcription in quiescent cells through the recruitment of p130 to target promoters (5, 43). Interestingly, p107 is bound to a number of promoters in asynchronously growing cells, but not in quiescent or serum-restimulated human cells (5, 43). Figure 1B shows that p130, but not p107 or pRB, is expressed in G0 cells. However, all three pocket proteins are expressed in continuously cycling cells (Fig. 1B). This suggests that the occupancy of E2F-regulated promoters by transcriptional repressors in quiescent cells and cycling cells may not be equivalent. Therefore, we investigated the occupancy of E2F-responsive promoters in the S, G2, M, early G1, and G1/S phases of cycling cells.
We used chromatin immunoprecipitation to show that p130 and E2F4, but not p107, are recruited to E2F-regulated promoters in quiescent cells (Fig. 2A), in agreement with previously published results (43). Interestingly, p107, p130, and E2F4 were bound to several E2F-regulated promoters in the early G1 phase of cycling cells (14 h after release from hydroxyurea; Fig. 2A). These data suggest that transcriptional repression in cycling cells is mediated by both p107 and p130. pRB was not detected on several E2F-regulated promoters in cycling cells, consistent with biochemical and genetic studies in human and mouse cells showing that pRB does not directly regulate these genes (data not shown).
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We used ChIP to investigate the cell cycle-dependent patterns of histone acetylation on a subset of E2F-responsive promoters. As shown in Fig. 2B, histones H3 and H4 are extensively acetylated at E2F-responsive promoters, in the S, G2 and M phases of the cell cycle. In contrast, both histones are deacetylated in early G1, corresponding to the period during which p107 and p130 are recruited to these promoters (Fig. 2A), although the extent of deacetylation varied among the promoters (Fig. 2B). In stark contrast with quiescent cells, either histone H3 or H4, but generally not both, was deacetylated on certain promoters. Interestingly, we could distinguish three different patterns of deacetylation: promoters that are deacetylated on histone H3 alone (p107 and CDC6), histone H4 alone (cyclin A), or a combination of both (CDC2 and CDC25A). These findings suggest that promoters may have specific histone acetylation signatures that must be reset with each cell cycle and that deacetylation of either histone may suffice in some cases to resilence each during early G1 of cycling cells. This could occur through cycles of recruitment of one or more HDAC with distinct specificities for a given histone.
ChIP-on-chip analysis reveals binding of E2F4 and pocket proteins to common and distinct sets of targets.
To further investigate the role of E2F4, p107, and p130 in cell cycle-dependent gene regulation, we performed genome-wide location analysis to identify promoters bound by each factor during early G1 in cycling cells. For this analysis, we made use of a custom DNA microarray (Hu13K) containing 13,000 human gene promoter regions spanning 700 bp upstream and 200 bp downstream of the transcription start sites (5, 33). Figure 3A shows representative scatter plots for the G1-phase location analyses of E2F4, p130, and p107 on the Hu13K promoter microarray. Using a P value cutoff of 
0.005, we found that E2F4 occupied 301 promoters, while p130 and p107 were bound to 226 and 244 genes, respectively (supplemental data S1, located at http://www.med.nyu.edu/Research/dynlab01.html).A comparison of targets revealed that the majority (171) of targets were bound by all three factors (Fig. 3B). Remarkably, a substantial number of targets (74) identified in the E2F4 ChIP-on-chip experiment did not overlap with either p107 or p130 targets and were thus unique for E2F4 (Fig. 3B). Moreover, we identified another subset of genes (40) that were uniquely bound by p107. We estimated the false positive and false negative rates for all three antibodies in our genomewide location analysis experiments to be 12% and 18%, respectively, when a P value cutoff of 0.005 was used.
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Functional categories of targets. E2F/pocket protein complexes are known to regulate the G1-S phase transition and DNA replication. Recent studies have shown that the role of E2F/pRB family complexes extends beyond these well-established functions to mitosis, chromatin assembly, chromatin modification, and apoptosis (5, 6, 22, 36, 39, 50). We used Gene Ontology to categorize the novel early G1 phase targets of E2F4, p130, and p107 into functional categories and found that E2F/p107/p130 complexes regulate genes within a diverse range of functional categories, including apoptosis, mitochondrial biogenesis, transcriptional regulation, protein ubiquitination and turnover, and organelle assembly and function (Fig. 4A and supplemental data S3). We compared the functional categories of targets found in cycling cells to those identified in growth-inhibited conditions (5). Interestingly, we found that genes in three functional categories-stress response, signal transduction, and immune response—were uniquely bound by E2F4 and/or p107 in the early G1 phase of cycling cells (Fig. 4A) (5).
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Functional analysis of targets. (i) Subsets of targets are differentially regulated in pocket protein-null cells. In contrast with observations that E2F4 and p130 function together to repress transcription in growth-arrested cells (5), we identified a number of novel genes that were bound by E2F4, but not by p107 or p130, in early G1 cells (Fig. 3B and 3C). These findings suggest that certain genes can be regulated by E2F4 in a p107/p130-independent manner in early G1. To confirm this finding, we analyzed expression of E2F target genes in primary mouse embryo fibroblasts that are wild type, deficient for p107 and p130 (double knockout), or all three pRB family members (triple knockout). Cells were synchronized as before by hydroxyurea treatment and released for 14 h to obtain early G1 cells prior to performing semiquantitative RT-PCR.
As expected, loss of p107 and p130 resulted in derepression of genes bound by both p107 and p130 (Fig. 5A; p107/p130/E2F4 targets). Interestingly, a subset of genes, including POLA, ATM, CLSPN, MTHFD1, and FBXO5, that are bound exclusively by E2F4 were derepressed in triple-knockout cells, but not in double-knockout cells, suggesting that these genes may be direct or indirect targets of pRB (Fig. 5A; E2F4 targets, class I). We have performed ChIP experiments to address whether pRB binds these promoters directly. However, we have failed to detect pRB on several class I promoters that we tested (data not shown). We note that we have only examined proximal promoters using this assay, and we cannot rule out the possibility that RB may regulate these genes by binding to another region beyond the boundaries of our assay or that it may indirectly regulate this class of promoters.
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We investigated whether the ablation of E2F4 by specific siRNAs would deregulate E2F4-only targets. We observed no dramatic changes in the expression of these targets, most likely as a result of functional compensation by either E2F5 or E2F6 (17, 19, 38). When we examined E2F6 binding to a subset of these E2F4-only promoters (CTDSP2, NVL1, GBF1, and RPL26) in wild-type and triple-knockout MEFs, we found that E2F6 is recruited to most of the targets that we tested, consistent with this possibility (data not shown).
We also examined the expression levels of genes identified as p107-specific by comparing wild-type, double-knockout, and triple-knockout cells. As expected and as shown in Fig. 5B (p107 targets), expression of certain p107 target genes (e.g., SUV39H1 and others) increases in cells lacking p107 and p130 and in cells devoid of all three pocket proteins. Interestingly, we found that the observed derepression of p107-specific genes in double-knockout and triple-knockout MEFs is specific to cycling cells, and derepression of the same genes is not seen in quiescent cells (Fig. 5B, compare early G1 and G0). These data further support the existence of a cadre of genes specifically regulated by p107 in cycling cells.
Next, we investigated the regulation of unique p107 targets in single-knockout MEFs that are deficient for p107 or p130. Strain-dependent defects have been observed in mice lacking either the p107 or p130 gene (23, 24). Interestingly, as shown in Fig. 5B, several genes bound exclusively by p107 (ATF2, SUV39H1, MRPL4, and EAF1) are up-regulated in p107–/– cells compared to wild-type and p130–/– cells. These data provide the first evidence for novel regulation of p107-selective targets in both human and mouse cells. Of particular interest is SUV39H1, a histone methyltransferase whose activity is required for heterochromatin formation and whose overexpression leads to growth retardation (11). Taken together with our observation that heterochromatin protein HP1 is also a target of pocket protein-E2F4 repression (5), our data indicate that expression of the SUV39H1-HP1 module may be entrained to the cell cycle via pocket protein-mediated regulation.
In an effort to uncover p130-specific targets, we performed genome-wide expression profiling of cells treated with p130 siRNAs using Affymetrix microarrays. Two genes that were bound exclusively by p130 in our ChIP-on-chip experiments were significantly up-regulated (4.1- and 1.7-fold) in cells treated with two different p130 siRNAs compared with cells treated with control siRNA (data not shown). These genes encode phospholipid scramblase 1 (PLSCR1) and Treacher-Collins syndrome factor 1 (TCOF1). We confirmed the effect of p130 knockdown by both p130 siRNAs using RT-PCR, and the expression of these genes did not change in cells treated with control or E2F4-specific siRNAs, in agreement with our previous ChIP-on-chip results (Fig. 5D and data not shown). Finally, we tested the expression levels of these genes in double-knockout and triple-knockout MEFs. Importantly, the RNA levels of both genes were increased in double-knockout and triple-knockout cells compared to wild-type cells (Fig. 5D).
(ii) E2F4 binding to promoters in triple-knockout MEFs. Our previous findings indicated that p130 and p107 play a role in the nuclear localization of E2F4 in G0, and that binding of E2F4 to certain promoters is undetectable in quiescent cells lacking p107 and p130 (37). This suggests that regulation of a subset of genes bound exclusively by E2F4 in cycling cells may be driven by a novel mechanism. Thus, we investigated whether the binding of E2F4 to these promoters is affected in MEFs lacking all three pocket proteins. Interestingly, ChIP analysis revealed that E2F4 binding persists in cells lacking pocket proteins on the promoters that were bound exclusively by E2F4 in our ChIP-on-chip experiments (Fig. 6A). These data offer further support that E2F4 is indeed capable of binding to certain promoters independently of pocket proteins.
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Sin3 can be recruited to promoters as a Sin3/HDAC complex by several transcription factors, altering their histone acetylation state and repressing gene expression (1, 37, 41). Our previous studies showed that the decrease in histone acetylation levels coincides with transcriptional repression of E2F-responsive genes (43). Thus, we compared the histone acetylation state of class I and class II E2F4-specific targets, as well as E2F4 targets that also recruit p107/p130. Interestingly, in cycling early G1 cells, we observed persistent acetylation at the promoters of class II E2F4-bound genes that do not recruit Sin3B (Fig. 6B and C), but not at class I targets, or E2F4 targets that are also bound by p107/p130 as well as Sin3B (Fig. 2B and Fig. 6B and C).
Finally, in order to further characterize the set of genes we identified as E2F4-specific, we examined previously published cDNA microarray experiments that monitored the expression of 29,600 human genes during cell cycle progression to determine whether E2F4-specific genes show cell cycle periodicity (51). Interestingly, we found that the vast majority of genes (85%) identified as E2F4-specific in our ChIP-on-chip analysis are not cell cycle regulated (supplemental data S1). To confirm this experimentally, we examined the cell cycle periodicity of expression of class I and class II E2F4-specific target genes. Strikingly, we found that the expression of class I E2F4-only targets fluctuates throughout the cell cycle, while class II E2F4 targets do not (Fig. 6D).
That class I targets are deregulated in triple-knockout but not double-knockout MEFs (suggesting a role for pRB) is particularly noteworthy in light of these observations (Fig. 5). Furthermore, the data in Fig. 6D augment the other differences we see in these two classes of genes: class I targets are bound by mSin3B and exhibit diminished histone acetylation at their promoters during early G1, while class II targets do not appear to recruit mSin3B and the levels of promoter histone acetylation remain nearly constant through early G1 (Fig. 6). This suggests that E2F-dependent cell cycle oscillations in gene expression may be driven by the periodic recruitment of an HDAC/mSin3B complex in early G1 phase in cycling cells.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Numerous molecular events contribute to transcriptional repression. One such event is chromatin condensation, which limits the accessibility of the DNA template to transcription factors and RNA polymerases. However, recent studies have shown that certain basal transcription factors remain associated with cell cycle-regulated promoters in asynchronous and mitotic human cell populations, arguing that such factor-promoter complexes withstand condensation of chromatin into transcriptionally silent chromosomes (8, 20, 30). Unlike these basal transcription factors, E2F4, p130, and p107 are not detected on E2F-regulated promoters in the S, G2, or M phase of cycling cells, but only in the G0 and early G1 phases. These data suggest that the binding of these pocket proteins and E2F4 to the promoter is restricted to cell cycle stages in which transcription is repressed.
E2F/pocket protein complexes repress transcription through the recruitment of histone-modifying enzymes, in particular HDACs, to target promoters. Recruitment of histone deacetylases results in histone H3 and H4 deacetylation and transcriptional silencing. We have found that, although histones associated with E2F-regulated promoters are generally acetylated throughout the cell cycle in growing cells, acetylation is significantly decreased in early G1 phase. Interestingly, our data show that histone acetylation profiles differ from promoter to promoter. These data are in agreement with previously published studies in Saccharomyces cerevisiae (13) showing that specific activators confer distinct patterns of histone acetylation on target promoters, and transcriptional activation is not necessarily associated with increased acetylation.
Our data show that promoters are acetylated in the S, G2 and M phases of the cell cycle. However, we detect activator E2F (E2F1 and E2F2) binding to promoters only in S phase, not in G2 or M phase (data not shown), in agreement with recently published data (19). This suggests that transcription might be silenced in the G2 and M phases of cell cycle, despite the acetylation of H3 and H4 that persists during these phases. Furthermore, promoter-selective deacetylation of histones H3 and H4 in early G1 suggests that distinct histone deacetylases may preferentially bind specific E2F targets and could imply the existence of an additional layer of specificity at E2F-responsive promoters. Alternatively, these promoters could recruit a single HDAC that acquires new specificities for histone H3 or H4 in a promoter-dependent context.
ChIP-on-chip and functional analysis of targets. Here we investigated the differences between highly related pocket protein family members in cycling human cells. Previous studies revealed that p130 and E2F4 share highly overlapping functions in growth-arrested cells. In contrast, our findings suggest that E2F4, p130 and p107 regulate common and unique sets of targets in proliferating cells (Fig. 7). In addition, we have found evidence that E2F4 and p107 may regulate gene transcription independently of pRB family members and E2F4, respectively. These findings indicate that there may be at least five distinct groups of targets regulated by combinations of E2F4, p107, and p130. Additional classes may exist, given evidence suggesting promoter-specific patterns of histone acetylation.
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Furthermore, our data provide new insights into the transcriptional regulatory mechanisms of subgroups of E2F-regulated genes and suggest distinctive patterns of mSin3B corepressor recruitment to their promoters, resulting in differential acetylation: Differential recruitment of Sin3B (and HDACs) in quiescent or early G1 cells (or both) could establish a mark that mechanistically distinguishes certain E2F4 target genes and ensures that genes will be deacetylated and silenced under both conditions or exclusively during cell cycle arrest.
Our genomewide location analysis identified new classes of genes that are differentially regulated in continuously cycling cells. In particular, our examination of E2F4-specific genes raises the possibility that the recruitment of E2F4 to promoters does not necessarily correlate with gene regulation in normal conditions. Thus, E2F4 may be bound to specific promoters in an inactive state, awaiting activation of its repressive function through recruitment of additional transcriptional regulators in response to currently unknown cues. Such a poised state could allow rapid responses to changing extracellular cues (4, 5).
We have also found that certain genes (TCOF1 and PLSCR1) are derepressed in cells in which p130 expression has been suppressed. These genes were also identified as p130-specific targets. PLSCR1 has been shown to play a role in cell differentiation, apoptosis, and proliferation (16, 52). The TCOF1 gene product is involved in ribosomal RNA gene transcription by interacting with an upstream binding factor (46). Mutations in the Treacher-Collins syndrome gene, TCOF1, result in craniofacial defects, and Tcof+/– mouse embryos exhibit craniofacial defects and growth retardation (46). It will be interesting in future studies to test a role for regulation of PLSCR1 and/or TCOF1 transcription in cell cycle control and differentiation. This may offer further insights into the biological functions of p130 and the contribution of these functions to the phenotypes of knockout animals. We surmise that in the future, similar genome-scale analyses will uncover distinctions among targets of other fully or partially redundant regulatory proteins and reveal novel mechanisms through which they function.
Finally, our observations regarding gene expression of the two subsets of E2F4-only target genes (class I and class II, Fig. 5 and 6) demonstrate that these are truly distinct classes of E2F4 target genes, regulated by separate but not yet fully understood mechanisms. This observation highlights how careful studies of gene regulation during the early G1 phase of cycling cells reveal important phenomena that may not be disclosed by studies with quiescent and serum-stimulated cell populations.
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ACKNOWLEDGMENTS |
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N.H.L. was initially supported by an NRSA postdoctoral fellowship (1F32-AG025617-01) and is now supported by a Susan G. Komen postdoctoral fellowship (PDF0504345). This work was initiated and completed using funds from the American Cancer Society (#RSG-0034204CCG) and NIH (2R01 CA77245-07), respectively, to B.D.D.
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FOOTNOTES |
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