UTF1 is required for EC cell differentiation
To study the potential role of mouse UTF1 (mUTF1; hereafter UTF1) in ES and EC cell differentiation, we stably expressed UTF1 and Renilla luciferase (hereafter Renilla) siRNAs in P19CL6 EC cells. UTF1 expression levels were substantially decreased in all clones tested (), whereas expression levels of the pluripotency marker Oct4 were not affected (). Next, DMSO-induced differentiation of wild-type (wt), Renilla, and UTF1 KD cells was analyzed (). wt and Renilla KD cells differentiated normally, which was reflected by a drastic reduction in Oct4 levels around day 4, decreased UTF1 levels between days 4 and 6, and detectable GATA4 (not determined for Renilla) and Troma1 expression by day 8. Actin was used as a protein loading control. In UTF1 KD lines, the differentiation-induced down-regulation of Oct4 was either delayed (#1) or minor (#2), and both GATA4 and Troma1 were not detected. Residual UTF1 protein levels were not further down-regulated, most likely as a consequence of high Oct4 levels, a transcriptional activator of the UTF1 gene.
Figure 1. UTF1 is involved in the differentiation of EC and ES cells. (A) UTF1 expression in P19CL6 EC cells (wt), 14-d DMSO-differentiated EC cells (wt d14), and four independent UTF1 EC KD clones (UTF1 #1–#4). The asterisk indicates a (more ...)
As UTF1 was previously reported to be involved in ES cell proliferation (Nishimoto et al., 2005
), we determined the doubling times of wt, Renilla KD, and UTF1 KD EC cells. UTF1 KD cells showed a 24% and 17% increase in doubling time (8.9 ± 0.3 h) compared with wt EC (7.2 ± 0.1 h) and Renilla KD (7.6 ± 0.3 h) cells, respectively. Next, the differentiation of wt and UTF1 KD EC cells was performed with different cell numbers to rule out potential cell density effects on differentiation (0.5 and 2 times the number of cells: 1.8 × 105
and 7.3 × 105
cells, respectively). Irrespective of the initial number of cells, the differentiation of UTF1 KD cells was always delayed or blocked, whereas wt cells differentiated normally (unpublished data). Summarizing, these data indicate that in EC cells, UTF1 KD results in an abrogated differentiation capacity and persistent Oct4 expression under differentiation-inducing conditions.
UTF1 is involved in ES cell differentiation
To extend these findings to a nontransformed mouse cell line, we tested the effect of UTF1 KD on IB10 ES cell differentiation. Renilla KD clones expressed normal levels of UTF1 and Oct4, whereas in UTF1 KD cell lines, UTF1 levels were reduced, but Oct4 expression was not affected (). In addition, UTF1 and Renilla KD ES cells are positive for AP, confirming their ES cell phenotype (). To determine whether UTF1 down-regulation also affected the differentiation potential of these cells, embryoid bodies (EBs) were generated. Where wt and Renilla KD cells formed normal EBs with high efficiency, UTF1 KD–derived EBs were irregularly shaped, much smaller in size, formed with low efficiency, and compaction was not observed ().
In agreement with observations by Nishimoto et al. (2005)
, UTF1 KD affected (although less dramatically) the doubling time of ES cells: UTF1 KD ES cells have a doubling time of 11.8 ± 0.7 h compared with 9.6 ± 0.7 h and 10.2 ± 0.1 h for wt (23% increase) and Renilla (16% increase) ES cells, respectively. Because UTF1 KD abrogated the ability of EC cells to differentiate, we tested whether EBs from UTF1 KD ES cells also failed to differentiate. AP staining showed that day 8 wt and Renilla EBs are largely AP negative, whereas UTF1 KD EBs still displayed substantial AP activity, suggesting that UTF1 is involved in ES cell differentiation (). To further validate this observation, the expression pattern of several germ layer–specific marker genes during EB development was determined by RT-PCR (). Both wt and Renilla ES cells show a clear up-regulation of various lineage markers. At days 3–5, Brachyury (early mesoderm) was detected, and at day 10, BMP5 (dorsal mesoderm) was detected. Endoderm markers GATA4 and GATA6 were detected at day 10, and ectoderm markers GAP43 and FGF5 were both detected at days 3–10. In contrast, both UTF1 KD cell lines showed either an absence (GATA6), minor (GATA4 and BMP5), or delayed (Brachyury) expression of these markers. However, ectoderm markers FGF5 and GAP43 were detected from day 3. Pluripotency markers like Oct4, REX1, and Nanog were detected at all time points in the various EBs, most likely as a result of the incomplete differentiation of a subset of cells.
The increased doubling time of UTF1 KD ES cells could (partially) be responsible for the observed differentiation defect. However, already after 48 h, when differences in doubling times have not yet resulted in substantial differences in cell numbers, we observed that UTF1 KD cells often failed to form aggregates (unpublished data). This strongly suggests that the observed effects on EB formation and differentiation cannot solely be explained by the increased doubling time of UTF1 KD cells. Collectively, these data show that UTF1 KD in ES cells results in perturbed EB formation and a severely reduced differentiation potential in the endodermal and mesodermal lineages.
UTF1 is a chromatin-associated transcriptional repressor
To understand the mechanistic properties of UTF1 that underlie its involvement in EC and ES cell differentiation, a series of experiments were performed to molecularly characterize the protein. First, we determined the subcellular localization of UTF1 in EC cells (). UTF1 was clearly localized to the nucleus and excluded from the nucleoli. In addition, we found UTF1 to localize to the chromosomes at different stages during cell division. To further characterize this potential interaction between UTF1 and the DNA/chromatin, we performed subnuclear fractionations of EC cells separating free-diffusing proteins (cytosolic and nuclear), weak/strong DNA-associated proteins, and nuclear matrix (associated) proteins. UTF1 is observed exclusively in the ammonium sulfate fraction known to contain strongly DNA-associated proteins, like core histone H2A (). In contrast, Oct4 primarily localized to the free-diffusing fraction and, to some extent, to the nuclear matrix fraction, indicating that Oct4 and UTF1 have distinct chromatin-binding characteristics. To compare the observed behavior of UTF1 to that of chromatin-modifying proteins, we determined the fractions containing histone deacetylase 1 (HDAC1; ). Unlike UTF1, HDAC1 is found in the fractions containing free-diffusing and weak DNA-associated proteins. Collectively, these data suggest that UTF1 is a protein with a high affinity for chromatin, similar to that of core histones and different from chromatin-modifying proteins like HDAC1.
Figure 2. Characterization of localization, fractionation, and reporter activity of UTF1. (A) Immunofluorescence analysis of endogenous UTF1 in EC cells using an antibody directed against UTF1. (B) Subnuclear fractionation of EC cells: F, free-diffusing protein (more ...)
As a chromatin-associated protein, UTF1 is likely to be involved in gene expression regulation. To determine the effect of UTF1 on promoter activity, reporter assays were performed using constructs containing multiple copies of either the Smad-binding element (SBE) or BMP-responsive element (BRE). These reporters were used because we previously identified UTF1 as an SBE-interacting protein in a yeast 1 hybrid screen. However, more detailed analysis showed that UTF1 is not specifically involved in Smad signaling. The reporters are activated by the cotransfection of either Smad3 and 4 (SBE) or Smad1 and 4 (BRE; ). Cotransfection of UTF1 reduced the activity of Smad-stimulated SBE and BRE reporters by approximately twofold and fourfold, respectively. These data indicate that UTF1 is a transcriptional repressor.
Mapping of UTF1 repressor domains
UTF1 contains two conserved domains (CDs): CD1 (aa 55–124), which shares high homology with Myb/SANT DNA-binding domains, and CD2 (aa 271–334), which contains a putative leucine zipper. To identify its repressor domains, the effect of a series of GAL4-UTF1 (deletion) constructs was tested on a thymidine kinase (TK) luciferase reporter containing five copies of the GAL4 target sequence (UAS-TK-Luc; ). As expected, UTF1 repressed UAS-TK-Luc reporter activity (eightfold reduction compared with GAL4). Deletion of the very C-terminal 39 aa resulted in an almost 2.8-fold reduction in repressor activity. Further C-terminal deletions only marginally affected repressor activity, but when the Myb/SANT domain (aa 55–124) was deleted, an additional drop in repressor activity compared with the 1–167 and 1–134 constructs was observed. The finding that both the C terminus and Myb/SANT domain are involved in transcriptional repression was confirmed using a series of progressive N-terminal deletions. Deletion of aa 1–65 resulted in a 3.1-fold reduction of repressor activity (compare 1–339 with 66–339), and further N-terminal deletions did not affect UTF1 repressor activity except for the deletion construct (297–339) that misses part of the CD2 domain, which completely lacked repressor activity. To address the importance of the Myb/SANT domain, we generated a mutant lacking this region, which reduced repressor activity by 1.7-fold, indicating that it is important for transcriptional repression by UTF1. In addition, the Myb/SANT domain alone (33–134) also displayed considerable repressor activity (3.9-fold repression). Collectively, these experiments indicate that both the Myb/SANT domain and the extreme C terminus of UTF1 are important for transcriptional repression by UTF1.
Live cell distribution of UTF1
To study its localization in living cells, UTF1 was fused to enhanced GFP (eGFP), creating eGFP-HA-UTF1 (hereafter GFP-UTF1), and was stably expressed in EC cells. To prevent localization artifacts, we used a clone that underexpressed GFP-UTF1 compared with the endogenous protein (). Subnuclear fractionation showed that GFP-UTF1, like endogenous UTF1, is almost exclusively found in the strongly DNA-associated fraction (). Reporter (UAS-TK-Luc) assays in HepG2 cells showed that GFP-UTF1 acted as a transcriptional repressor as well (see ). These data indicate that fusing GFP to the N terminus of UTF1 does not interfere with the function of the protein.
Figure 3. Cellular localization and subnuclear fractionation of GFP-UTF1 in EC and ES cells. (A) Western blot analysis of wild type (wt) EC cells and a clone stably expressing GFP-UTF1 (#1) using an antibody directed against UTF1. (B) Subnuclear fractionation (more ...)
Figure 5. Analysis of subcellular localization and mobility of wt and mutated GFP-UTF1. (A) A schematic representation and repressor activity of various GFP-UTF1 mutants. The Myb/SANT domain (aa 55–124) and conserved domain 2 (CD2) are indicated by black (more ...)
Confocal microscopy of living cells showed that GFP-UTF1 localized to the nucleus with an inhomogeneous distribution in a similar fashion as the endogenous protein ( and ). GFP-UTF1 is excluded from the nucleoli (, arrows). The punctate localization is more intense around the nucleoli and in the nuclear periphery. Time-lapse imaging of a cell counterstained with Hoechst showed the chromosomal localization of GFP-UTF1 during metaphase, anaphase, and telophase ().
In ES cells, a similar GFP-UTF1 distribution was observed: localized to the nucleus, excluded from the nucleoli (, arrows), and chromosome associated during mitosis (, arrowhead). GFP-UTF1 ES cells were AP positive () and expressed Oct4 (not depicted). Fractionation of GFP-UTF1 ES cells showed that both endogenous UTF1 (αmUTF1) and GFP-UTF1 (αHA) localized to the fraction containing strongly DNA-associated proteins ().
Mobility of UTF1 in living cells
To study the observation that UTF1 is a stably chromatin-associated protein in a more physiological context, we analyzed the dynamic properties of UTF1 in living cells using a FRAP protocol. In EC cells, GFP-UTF1 molecules were bleached in a small strip spanning the nucleus, and subsequent fluorescent recovery in the strip was measured at 20-ms intervals (Hoogstraten et al., 2002
; van den Boom et al., 2004
). The mean fluorescence intensity in the strip of several cells was plotted against time relative to the prebleach level. GFP-expressing cells showed a fast recovery of fluorescence in the strip (, green line), indicating a highly mobile protein. Fluorescence in the strip did not recover to prebleach levels as a result of the permanent bleaching of a fraction of the molecules. In contrast to GFP, GFP-UTF1 (, blue line) showed only little recovery after bleaching, indicating that the vast majority is long-term immobilized, at least for the duration of the FRAP experiments. Because mobility measurements of GFP and GFP-UTF1 in ES cells produced identical results (, green line and blue line, respectively), EC cells were used for all subsequent mobility measurements.
Figure 4. Strip-FRAP analysis of GFP, GFP-UTF1, H2B-GFP, and Oct4-GFP. (A) FRAP analysis of EC cells expressing GFP (green line) or GFP-UTF1 (blue line). The graph shows the relative fluorescent recovery directly after bleaching. The prebleach level is normalized (more ...)
In terms of localization ( and ) and subnuclear fractionation behavior (), UTF1 greatly resembles core histones (Kanda et al., 1998
). To further substantiate this observation, the mobilities of UTF1 and core histone H2B (Kimura and Cook, 2001
) were compared (). FRAP curves for GFP-UTF1 (, blue line) and H2B-GFP (, red line) were virtually identical, indicating the similar molecular kinetics of these proteins. Computer simulations of the FRAP procedure were used to fit the experimental data, yielding diffusion constants, immobile fractions, and residence times of all proteins tested ( and Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200702058/DC1
). Both the population of GFP-UTF1 and H2B-GFP molecules displayed an immobile fraction of ~90% ( and Fig. S1, A and B). The duration of immobilization was much longer than the 20-s time scale of our experiments and, therefore, could only be determined with limited accuracy. For both GFP-UTF1 and H2B-GFP, a residence time in the order of minutes to hours was determined, which is in agreement with the findings of Kimura and Cook (2001)
Diffusion constants, immobile fractions, and residence times of tested constructs derived from FRAP data fitting
Subsequently, we compared the dynamic behavior of GFP-UTF1 and Oct4-GFP (). In contrast to GFP-UTF1 (, blue line), Oct4-GFP (, red line) is largely mobile. Note that Oct4-GFP fluorescence recovery is much slower than that of GFP (, green line). Computer simulations indicated that 10% of the Oct4-GFP molecules are immobile with a residence time in the order of 0.1 s ( and Fig. S1 C). In addition, the diffusion rate of Oct4-GFP (3 μm2
/s) suggested that the protein resides in a high molecular weight complex. The highly dynamic behavior of Oct4-GFP molecules is similar to what is found for several other DNA transacting factors like the transcription/repair factor TFIIH, the homologous recombination protein Rad54, and TFIIB during interphase (McNally et al., 2000
; Phair and Misteli, 2000
; Chen et al., 2002
; Essers et al., 2002
; Hoogstraten et al., 2002
; Phair et al., 2004
; van den Boom et al., 2004
; Houtsmuller, 2005
). These data indicate that the dynamic behavior of UTF1 is similar to that of core histones but not to that of transcription factors like Oct4.
Localization and mobility of GFP-UTF1 mutants
Using GAL4-UTF1 fusions, we identified the putative Myb/SANT domain and the C terminus of UTF1 as repressor domains (). To investigate the requirement of these domains in UTF1 localization and mobility, a series of GFP-UTF1 mutants was generated. First, the repressor activity of wt and mutant GFP-UTF1 proteins was determined in reporter assays. Mutation of aa 63 (W→G) and 67 (E→K; GFP-UTF1 W63G E67K; ), two amino acids highly conserved in Myb/SANT domains, and/or deletion of the C-terminal 39 aa (GFP-UTF1 W63G E67K 1–300; GFP-UTF1 1–300) resulted in a complete loss of UTF1 repressor activity ().
In terms of localization, GFP-UTF1 and GFP-UTF1 W63G E67K display a similar distribution. Deletion of the entire Myb/SANT domain resulted in an almost completely cytoplasmic localized fusion protein (unpublished data). GFP-UTF1 1–300 also interacted with mitotic chromosomes, whereas during interphase, the protein seemed to be more dispersed (). GFP-UTF1 W63G E67K 1–300 showed a completely homogenous nuclear distribution in combination with nucleolar exclusion. Furthermore, association with mitotic chromosomes was never observed (). These data indicate that both the Myb/SANT domain and C terminus of UTF1 are required for proper localization of the protein during interphase as well as mitosis.
To determine the role of the Myb/SANT domain and C terminus in UTF1 mobility, FRAP analyses were performed. GFP-UTF1 W63G E67K–expressing cells showed an increased recovery of fluorescence in the strip (, red line) compared with GFP-UTF1 (, blue line), indicating a reduced binding efficiency. The rate of fluorescence recovery after the initial influx resembled that of GFP-UTF1, implying that the residence time of individual molecules was not affected. Computer simulations showed that the residence time of GFP-UTF1 W63G E67K molecules is similar to GFP-UTF1 molecules (in the order of minutes to hours) but that the mean immobile fraction was smaller (~60%; and Fig. S1 D).
GFP-UTF1 1–300–expressing cells showed a complete recovery after bleaching (, red line), demonstrating that the C terminus is required for the long-term immobilization of GFP-UTF1. However, the initial fluorescence recovery in the strip was substantially slower than that of GFP (), suggesting that UTF1 resides in a high molecular weight complex and/or is still capable of transiently interacting with sites of affinity. Simulations showed that 85% of the GFP-UTF1 1–300 molecules are immobilized with a residence time in the order of 0.25 s ( and Fig. S1 E). These data indicate that the C terminus of UTF1 is required for the long-term stabilization of interactions with sites of affinity, most likely chromatin.
Remarkably, GFP-UTF W63G E67K 1–300 showed a much faster recovery of fluorescence than GFP-UTF1 1–300 and an only slightly slower recovery than GFP (), indicating that this mutant is freely mobile. This was further supported by computer simulations that predicted that 25% of the GFP-UTF1 W63G E67K 1–300 molecules was immobilized with a short residence time of 0.25 s ( and Fig. S1 F). The UTF1 mutants lacking their C-terminal 39 aa (GFP-UTF1 1–300 and GFP-UTF1 W63G E67K 1–300) displayed a marked increase in their diffusion constants compared with GFP-UTF1 and GFP-UTF1 W63G E67K (14 vs. 0.6 μm2/s; ). However, because the model used for fitting the data only included one pair of binding constants (immobile fraction and residence time) and the stable binding of GFP-UTF1 and GFP-UTF1 W63G E67K is dominant in the FRAP curve, the observed low mobility of 0.6 μm2/s is most likely the result of additional transient interactions similar to those of the C-terminal mutants.
To investigate whether the differential mobilities of the mutant proteins are reflected by altered distribution over subnuclear fractions, cell lines stably expressing mutant GFP-UTF1 proteins were analyzed (). As shown before (), GFP-UTF1 localized to the strongly DNA-associated protein fraction. The majority of the GFP-UTF1 W63G E67K proteins was also strongly DNA associated but was detected in the free-diffusing protein fraction as well, indicating the presence of an increased portion of mobile molecules, which is in agreement with the FRAP data. Both GFP-UTF1 1–300 and GFP-UTF1 W63G E67K 1–300 were found in the free-diffusing protein fraction, indicating that both mutants are fully mobile. Note that GFP-UTF1 1–300 was still capable of binding to mitotic chromosomes and had a punctate nuclear localization, suggesting that this protein is capable of transient interactions with sites of affinity. Throughout these experiments, endogenous UTF1 was always detected in the fraction containing strong DNA-associated proteins (unpublished data).