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J. Biol. Chem., Vol. 281, Issue 21, 14882-14892, May 26, 2006
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1
From the
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520-8024, the Department of Orthopedic Surgery and Cell Biology, New York University School of Medicine, New York, New York 10003, and the ¶Department of Internal Medicine, Cardiology, Yale University School of Medicine, New Haven, Connecticut 06520-8024
Received for publication, October 31, 2005 , and in revised form, February 15, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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Among the two best characterized members of the murine p200 family proteins is p202a (which was designated earlier as p202) (16-26). p202a modulates transcription, cell proliferation, and apoptosis. It functions primarily by binding various sequence-specific transcription factors and inhibiting their activity generally, but not exclusively, by binding them and blocking their sequence-specific binding to DNA (24). p202a overexpression was implicated in the susceptibility of mice to the autoimmune disease lupus (27).
The other much studied p200 family protein p204 is encoded by the Ifi204 gene (5). p204 is structurally related to p202a and is inducible by interferons, as is p202a. Depending on the cell type and the state of differentiation, p204 can be located in the nucleolus, the nucleoplasm, or the cytoplasm (28-30). Its overexpression in cultured mammalian cells is growth-inhibitory (28, 31). It can delay the progression of cells from the G0/G1 to the S phase of the cell cycle (31). p204 can inhibit the transcription of ribosomal RNA by binding to the ribosomal DNA-specific UBF transcription factor and by inhibiting its binding to DNA (30). p204, similarly to p202a, contains the pRb-, p107-, and p130-binding motif LXCXE and can bind these pocket proteins (19, 30, 32).
Among 10 adult mouse tissues tested, the level of p204 is highest in the heart and second highest in skeletal muscle (29). During the differentiation of cultured C2C12 mouse skeletal muscle myoblasts to myotubes, the p204 (and p202a) levels increased severalfold as a consequence of the transactivation of the Ifi204 gene by the muscle-specific MyoD, myogenin, and E12/E47 transcription factors (29). This increase did not depend on interferon action (33). p204 is required for the differentiation of C2C12 myoblasts to myotubes. It enables the differentiation, at least in part, by overcoming the inhibition of the activities of MyoD, E12/E47, and other myogenic basic region-helix-loop-helix transcription factors by the inhibitor of differentiation (Id)2 proteins Id1, Id2, and Id3 (33).
p204 is also involved in osteogenesis. It is expressed in osteoblasts of various tissues in embryonic and neonatal mice. It is induced in the course of the BMP-2-triggered differentiation of C2C12 cells to osteoblasts. p204 binds and acts as a cofactor of Cbfa-1 (Core binding factor 
-1), which is an essential transcription factor in osteoblasts differentiation and bone formation (26).
The aim of this study has been to explore whether p204 is involved in and is required for cardiac myocyte differentiation and the basis of the high level of expression of p204 in mouse heart.
As a model system for cardiac myocyte differentiation, we used cultured murine P19 embryonal carcinoma cells (34-37). The P19 line of pluripotent embryonal stem cells was derived from a teratocarcinoma induced in C3H/HC mice (38). The cells can be maintained in culture in an undifferentiated state. When injected into early mouse embryos, P19 cells are capable of contributing to a variety of apparently normal tissues suggesting that the mechanisms of their differentiation are similar to those of normal embryonic cells (39). P19 cells can also be induced to differentiate in vitro into multiple cell types, including beating cardiac myocytes, skeletal myocytes, and myotubes, as well as neurons (34, 36).
The differentiation of P19 cells to cardiac and skeletal myocytes in vitro is initiated by plating P19 cells grown in tissue culture dishes and dissociated with trypsin into bacteriological culture dishes to which the cells do not attach and in which they form aggregates. The cultures are supplemented with DMSO. The aggregates are transferred to tissue culture dishes and further incubated in the presence of DMSO. Proliferating cells migrate out from the aggregates, including many that are differentiated to cardiac myocyte-type beating cells (and later also skeletal myocyte-type cells). In the absence of DMSO (whose mode of action remains to be clarified), there occurs little or no differentiation of P19 cells in vitro (34, 36). DMSO causes an immediate, transient increase in Ca2+ ions in the cells, and it was proposed that permeabilization and membrane penetration by DMSO may alter molecular interactions in the cells (40, 41).
We report here that in P19 cells (i) DMSO induced p204 formation, (ii) ectopic 204AS RNA blocked the DMSO-induced differentiation (as well as p204 formation), and (iii) ectopic p204 could partially substitute for DMSO in inducing the differentiation of P19 cells to beating myocytes. p204 contains an NES, and in the course of differentiation to myocytes a portion of p204 was translocated from the nucleus to the cytoplasm of P19 cells (29). p204 lacking an NES or carrying a mutated NES was not translocated in P19 cultures treated with DMSO and could not substitute for p204 in triggering differentiation.
The 5'-flanking region of the gene encoding p204 (i.e. Ifi204) (11) contains several binding sites for the Gata4, Nkx2.5, and Tbx5 transcription factors. All these factors are important transcriptional regulators of cardiac gene expression and are members of protein families conserved in evolution from Drosophila to vertebrates (42-47). Nkx2.5 is an NK-2 class homeodomain protein (48). Mouse Nkx2.5 is expressed throughout the development of the heart and is required for this process. In DNA it binds to the 5'-AAGTG-3' sequence (NKE element), among others. Gata4 is a zinc finger protein (49). It is expressed in the developing mouse heart almost simultaneously and spatially overlapping with Nkx2.5 (46, 50-52). Gata4 binds in DNA to a sequence element containing a core 5'-GATA-3' motif. This motif occurs in the promoter region of many genes encoding cardiac proteins, including the atrial natriuretic factor gene (53). Tbx5 is a member of the family of transcription factors containing T box-type DNA binding domains (42). It is also expressed throughout heart development. Its consensus DNA-binding sequence is 5'-(A/G/T)GGTG(T/C)(C/T/G)(A/G) (43). This also occurs in the promoter regions of various cardiac-specific genes, including that encoding atrial natriuretic factor (43). Nkx2.5, Gata4, and Tbx5 can pairwise bind one another and can cooperatively or synergistically activate gene expression (52, 54-56).
We report results showing that ectopic Gata4, Nkx2.5, and Tbx5 synergistically transactivated the expression of reporter constructs, including segments from the 5'-flanking region of the Ifi204 gene in murine 10T1/2 fibroblasts and in cardiac myocytes from rats, and also cooperatively transactivated the expression of endogenous p204 in 10T1/2 cells. Ectopic p204 (also DMSO) triggered P19 cells (to differentiate and) to increase their expression of the Gata4 and Nkx2.5 transcription factors. An examination of P19 cells differentiated to myocytes by chromatin coimmunoprecipitation assays with Gata4 or Nkx2.5 antibodies and PCR revealed the binding of endogenous Gata4 and Nkx2.5 to a segment of the 5'-flanking region of the Ifi204 gene, which contains a cluster of Gata4- and Nkx2.5-specific sequences. These findings indicate the following: (i) p204 is required for cardiac type myocyte differentiation from P19 embryonal stem cells, and (ii) the level of p204 increases during myocyte differentiation in consequence of the transactivation of the Ifi204 gene by cardiac transcription factors. The various mechanisms by which p204 enabled the differentiation of P19 cells to cardiac-type myocytes are reported in the accompanying article (76).
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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NES (29); pCMV204NES, pCMV204MNES; pCMVFLAG-p204, pCMVFLAG-p204NES, and FLAG-p204MNES; pCGNGata4 and pCGNNkx2.5 (52); and pCMVTbx5, pCMVHATbx5, pACCMVTbx5 (57). The various Ifi204-based reporters (based on the pGL3 expression vector (Promega)) included wild type Ifi204 gene sequences or sequences with deletions or mutations. For further details see supplement M. Cell Culture—P19 cells (a gift from M. W. McBurney) were derived from mouse teratocarcinoma induced in C3H/HC mice. The cells were cultured and induced to differentiate to cardiac-type myocytes as described previously (34) and in supplement M. Neonatal rat cardiac myocytes were isolated from hearts of 3-day-old rats as described previously (58, 59). 10T1/2 cells, cloned murine embryonic fibroblasts (ATCC 226 CCL) (60), were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For further details see supplement M.
Transfection—P19 cells were transfected as specified with expression plasmids encoding p204, p204ASRNA, p204MNES, p204NES, Id3, p204 plus Id3, or p204NES plus FLAGId3 using Lipofectamine 2000 in minimum essential medium- (Invitrogen), supplemented with 2.5% fetal bovine serum (Invitrogen) and 7.5% donor calf serum (TerraCell). Stable cell lines were established by selection with G418. Clones were assayed to ascertain high level expression of the ectopic RNA or protein of interest by Northern or Western blotting, respectively. For further details see supplement M.
Assay of p204, Gata4, Nkx2.5, and MHC by Immunofluorescence Microscopy—P19 cells growing on 35-mm culture dishes were treated as described previously (34) and incubated as specified with mouse MF20, the Developmental Studies Hybridoma Bank, and M2 monoclonal FLAG (Sigma), rabbit p204 and rabbit Id3 (Santa Cruz Biotechnology), and goat Gata4 or Nkx2.5 antibodies (Santa Cruz Biotechnology). As secondary antibodies, fluorescein isothiocyanate or Texas Red-linked goat anti-rabbit and anti-mouse IgG and donkey anti-goat IgG (Santa Cruz Biotechnology) were used. Nuclei were stained with DAPI.
RT-PCR—P19 cells in 35-mm tissue culture dishes were lysed in 1 ml of RNA STAT-60TM (Tel-Test Inc.), and total RNA was isolated following the instructions of the manufacturer and subjected to RT-PCR using the Omniscript RT kit (Qiagen). Sequences of primers are shown in supplement M.
Western Blotting—P19 cells were lysed as described previously (61), and 100 µg of total protein was subjected to 4-20% gradient PAGE and transferred to a polyvinylidene difluoride membrane and blotted with rabbit anti-p204 antibody (30). The blots were visualized using ECL (Pierce, SuperSignal Western Blotting kit). Rat anti-HA antibodies (Santa Cruz Biotechnology) and mouse anti-
-tubulin antibody (the Developmental Studies Hybridoma Bank) were used to detect HA-tagged Tbx5 and -tubulin, respectively. Gata4 and Nkx2.5 antibodies were used for detection of Gata4 and Nkx2.5, respectively.
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-galactosidase were assayed using the luciferase reporter gene assay (Roche Applied Science) and the -galactosidase assay system (Promega), respectively. The experiments were repeated 3-4 times. Sequence Analysis—To search for Gata4, Nkx2.5, and Tbx5 recognition sequences in the Ifi204 gene 5'-flanking region (11) (GenBankTM accession number AC006944 [GenBank] ), the MatInspector, version 2.1, data base was used, and the parameter selected for both core similarity and matrix was 0.8.
Chromatin Immunoprecipitation—The chromatin immunoprecipitation assay kit (Upstate) was used following the instructions of the manufacturer. Published procedures (63) were followed in the treatment of the cells with formaldehyde to cross-link the DNA protein interactions, the preparation of the nuclear extracts, the shearing of the chromatin by sonication, and the immunoprecipitation of DNA-protein complexes using immobilized antibodies (i.e. Gata4 and Nkx2.5). The nucleotide sequence of the 5'-flanking region of the Ifi204 gene was reported (GenBankTM accession number AC006944
[GenBank]
). The sequences of the primers used in the PCR analysis of the DNA segments immunoprecipitated are available upon request.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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As shown in the Western blots in Fig. 1A in P19 cells proliferating in tissue culture dishes (in the absence or presence of DMSO), no p204 was detected (day 0). Cells aggregated in Petri dishes without DMSO contained very low levels, if any, of p204, which increased slightly after their shift to tissue culture dishes (between day 6 and day 8). Cells aggregated in the presence of DMSO contained very low levels of p204 (on days 2-4) but much higher levels after their shift to tissue culture dishes (between day 6 and day 8). Thus, DMSO induced p204 in differentiating P19 cultures.
The immunofluorescence microscopic pictures (day 8; Fig. 1B) illustrate the following: (i) the need for DMSO in the triggering of the formation of beating cardiac myocyte-like cells expressing MHC proteins (Fig. 1B, compare panels a and e); (ii) the ability of ectopic p204 to trigger the formation of beating myocyte-like cells expressing MHC (in the absence of DMSO (Fig. 1B, panel b)) (see supplemental Videos 1 and 2); and (iii) the inhibition of the induction of beating and MHC-expressing cells by DMSO in P19 cells that expressed 204ASRNA (Fig. 1B, panel f). (As will be demonstrated in Fig. 2E, the ectopic 204ASRNA inhibited the induction of p204 by DMSO.) The curves in Fig. 1C represent the time course of the increase in the percent of cell aggregates with beating myocytes in P19 cultures exposed to various conditions.
The cultures that were transfected with expression plasmids were selected in the presence of G418 to eliminate untransfected cells. The conditions (Fig. 1C, curves 1-11) are specified in the figure. Curve 2, by day 12 of the differentiation process, 38% of the P19 cell aggregates exposed to 0.8% DMSO included beating myocytes. Transfection of pCMV204 did not increase significantly the yield of beating aggregates (39%) in a culture exposed to 0.8% DMSO (curve 1). Curve 6, no beating cultures arose in the absence of DMSO or (curve 8) in the presence of 0.8% DMSO if pCMV204AS (a plasmid encoding 204AS RNA) was transfected. Curve 5, transfection of pCMV204 resulted in the formation of aggregates, including beating myocytes (even) in the absence of DMSO. However, the percent of such aggregates (10%) was lower than in the corresponding culture (29%) (curve 3), which was transfected with pCMV, the control vector, and was incubated in the presence of DMSO. Curve 4, DMSO at a concentration as low as 0.1% increased the yield of aggregates with beating cells in a culture expressing ectopic p204 (16%), whereas as already noted (curve 5) ectopic p204 alone resulted only in a lower yield (10%). Curve 11, 0.1% DMSO treatment of cells expressing pCMV vector produced no beating culture.
The data in Fig. 1, A-C, are in line with the following conclusions: (i) DMSO induced p204 expression in aggregated P19 cells; (ii) p204 was required for the induction of differentiation by DMSO; and (iii) p204 could partially substitute for DMSO in inducing the differentiation.
The RT-PCR assays in Fig. 2A reveal that DMSO did not trigger the transcription of the Ifi204 gene to 204 RNA in proliferating P19 cells (day 0). DMSO did, however, trigger the transcription to 204 RNA in differentiating P19 cells, and the 204 RNA was present from day 6 at least until day 12, the last day when it was tested. Transfection of pCMV204 (but not of pCMV) into P19 cells (without or after elimination of the untransfected cells) resulted in 204 RNA expression even in proliferating cells (Fig. 2B). This expression persisted during the differentiation of the cells at least until day 12 (data not shown). The ectopic p204 level was determined by Western blotting to confirm that 204 RNA in P19 cells was translated into p204 (Fig. 2C). Transfection of a pCMV204AS plasmid resulted in the expression of a high level of 204AS RNA in the cells (Fig. 2D). This ectopic 204AS RNA (but not ectopic pCMV vector) decreased the amount of p204 induced by DMSO to a barely detectable level (Fig. 2E). These results suggest that the inhibition of the DMSO-induced differentiation of P19 cells to beating myocytes by 204AS RNA was a consequence of the inhibition by 204AS RNA of the accumulation of p204 as induced by DMSO.
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NES). The generation of p204NES involved the deletion of a 12-amino acid segment from p204. It is possible that this might have altered the structure of p204 resulting in the loss of some of its activities. Therefore, as a control we also generated p204 with a mutated NES (p204MNES), in which only three amino acid residues were replaced (Fig. 3A-(3)). This shows the altered amino acid sequence of p204MNES in which three Leu residues from the NES were replaced by Ala residues. The mutated nucleotide sequence encoding the altered amino acid sequence is also shown. The Western blots in Fig. 3, A-(4), A-(5), and A-(6), established that the pCMV plasmids encoding p204, p204NES, and p204MNES transfected into P19 cells resulted in the expression of similar amounts of protein.
The fluorescence microscopic analysis in Fig. 3B involved the use of two types of fusion proteins as follows: (i) green fluorescent protein-204 (GFP-p204) and (ii) GFP linked to a p204 derivative from which the NES oligopeptide segment was lacking (GFP-p204NES). GFP serving as a control was shown to be distributed between the nuclei and the cytoplasm in both proliferating (Fig. 3B, PROLIF.) and differentiated (DIFF.) P19 cells (Fig. 3B, bottom panels). GFP-p204 was nuclear in proliferating cells, but a portion of GFP-p204 was translocated from the nucleus to the cytoplasm in differentiated cells (Fig. 3B, top panels). GFP-p204NES, however, was nuclear in proliferating P19 cells and remained nuclear also in differentiated P19 cells (Fig. 3B, middle panels).
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NES, and FLAG-p204MNES. Confirming the observations in Fig. 3B, the immunofluorescence microscopic analysis (in Fig. 3C, bottom panel) revealed that in proliferating P19 cells FLAG-p204 was nuclear (and so were FLAG-p204NES and FLAG-p204MNES (not shown)). In P19 cells whose differentiation to cardiac myocytes cells was triggered by DMSO, much of the FLAG-p204 was translocated to the cytoplasm; however, FLAG-p204NES and FLAG-p204MNES, which lacked an intact NES, remained in the nuclei (Fig. 3C, upper panels). The level of endogenous p204 in proliferating P19 cells is too low for immunofluorescent detection. However, we have reported earlier that in proliferating C2C12 myoblasts, in which the endogenous p204 level is higher and thus detectable, p204 is nuclear (29). In differentiating P19 cells the endogenous 204 was distributed between the nuclei and the cytoplasm (Fig. 3C, middle panel).
The findings in Fig. 1C indicate that (in addition to controlling the subcellular location of p204, as shown in Fig. 3, B and C) the NES was also required for the differentiation of P19 cells to beating cardiac-type myocytes. Thus, although the transfection of pCMV204 triggered the differentiation of P19 cells (even in the absence of DMSO) (Fig. 1C (curve 5)), the transfection of pCMV204NES or pCMV204MNES did not trigger the formation of beating myocytes from P19 cells in the absence of DMSO (Fig. 1C (curves 9 and 10)). Furthermore, ectopic p204NES or p204MNES did not substitute for ectopic p204 in triggering MHC protein formation in aggregated P19 cells in the absence of DMSO (data not shown).
p204, but Not p204 Lacking the NES, Could Substitute for DMSO in the Triggering of the Expression of the Gata4 and Nkx2.5 Transcription Factors in Aggregated P19 Cultures—As reported earlier (49, 65), two of the transcription factors expressed early in the course of differentiation of aggregated P19 cultures to cardiac-type myocytes in the presence of DMSO (and also in the course of murine heart development) are Gata4 and Nkx2.5. Moreover, in the absence of DMSO, the P19 aggregates (even if transfected with an empty vector) contained primarily undifferentiated carcinoma cells, and neither Gata4 nor Nkx2.5 expression could be detected (65, 66).
As shown in the Western blots (Fig. 4A), aggregated P19 cells, incubated with DMSO or incubated without DMSO but transfected with pCMV204 and thus expressing p204 (p204), expressed both Gata4 and Nkx2.5 (left panel). Aggregated cells incubated without DMSO and transfected with empty vector (Fig. 4A, Con), however, did not express either Gata4 or Nkx2.5 (left panel). Moreover, the expression of ectopic 204ASRNA strongly inhibited the expression of Nkx2.5 and Gata4 as induced by DMSO (Fig. 4A, middle and right panels). P19 cells transfected with pCMV204NES and incubated in the absence of DMSO expressed little if any Gata4 and Nkx2.5 (Fig. 4A, left panel). This was the case although the level of p204NES expressed was similar to the level of p204 in cells transfected with pCMV204 (see Fig. 3, A-(4) and A-(5)). These results are in line with the observations that p204NES or p204MNES could not substitute for wild type p204 in inducing MHC either (data not shown). All these findings are in accord with the results in Fig. 1C in demonstrating that p204 lacking an active NES was unable to trigger the differentiation of P19 cells to beating cardiac myocytes.
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NES) (see Fig. 4B), some of the p204 has spread into the cytoplasm. This (as noted earlier, see Fig. 3C) is in consequence of the differentiation of the P19 cells induced by p204 (but not by p204NES).
The RT-PCR patterns in Fig. 4C revealed that the induction of MHC protein by DMSO or ectopic p204 in differentiating P19 cells (Fig. 1B) was (i) the consequence of the induction of - and MHCRNA, and (ii) the ectopic 204ASRNA inhibited the induction of the transcription of the MHC genes by DMSO.
The Expression of the Ifi204 Gene Could Be Synergistically Transactivated in 10T1/2 Cells, Rat Cardiac Myocytes, and P19 Cells Differentiating to Myocytes by the Gata4, Nkx2.5, and Tbx5 Cardiac Transcription Factors—Searching for cardiac transcription factor recognition sequences in the 5'-flanking region of the murine Ifi204 gene (11), we noted the occurrence of numerous recognition sequences for Gata4, Nkx2.5, and Tbx5 (Fig. 5A).
These transcription factors that function in cardiac differentiation can bind each other and are capable of synergistically transactivating gene expression (51, 52, 54-56, 67). We assayed the activity of the three factors to transactivate Ifi204 gene expression in transfected 10T1/2 murine embryonic fibroblasts (Figs. 5B and 6), cultured myocytes from newborn rats (Fig. 7), and proliferating and differentiating P19 cultures (Fig. 8). In the bulk of the assays we used reporter constructs, including various segments from the 5'-flanking region of the Ifi204 gene driving luciferase expression.
One of the short Ifi204 segments (of 160 nucleotides) used in a reporter construct (Fig. 5A) included parts of exon 1 and intron 1 with three recognition sites for Gata4 (designated as GATA), one for Nkx2.5 (designated as NKE), and two for Tbx5 (designated as TBX). It should be noted that a cluster of several short (about 6-8 bp) transcription factor recognition sites, such as in this region, is rare in the genome, although single recognition sites are common. Multiple sequence-specific factors in such a cluster typically function synergistically (68).
The expression of the reporter construct, including the Ifi204 segment in Fig. 5A in transfected 10T1/2 cells, is shown in Fig. 5B (wild type panel). The low level of expression in control cells (lane 1) (taken as 1 to serve as the standard for comparison) was only slightly increased by ectopic Gata4, Nkx2.5, or Tbx5 (lanes 2-4). Any two of these transcription factors activated the expression synergistically (lanes 5-7), and all three factors activated it even further (25-fold) (lane 8). Mutation in the reporter of any one of the two TBX sequences and especially of both strongly decreased the synergistic boost of the expression by TBX5 but affected the boosts by Gata4 and Nkx2.5 less, if at all (Fig. 5B, panels Mutated TBX (1), Mutated TBX (2), and Doubly mutated TBX). These results are in accord with the finding that synergistic transactivation by Tbx5 (with Nkx2.5 or Gata4) requires the binding of Tbx5 to DNA (54-56).
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The effects of ectopic Nkx2.5, Gata4, and Tbx5 on the expression of endogenous p204 in 10T1/2 cells, together with the levels of expression of the three ectopic transfection factors, are shown in the Western blots in Fig. 6. The numbers below the p204 panel in Fig. 6 reveal the cooperativity or in some cases the slight synergy between any two of the three ectopic transcription factors. (The lack of a further increase in the level of endogenous p204 upon addition of the third transcription factor may only be apparent because it may be due, at least in part, to the fact that in this case in order to keep the total DNA concentration constant less DNA was transfected for each factor (2 µg) than in all other cases (3 µg).) The lesser increase in the level of p204 protein (Fig. 6) than of p204 RNA in 10T1/2 cells (Fig. 5B) by ectopic Gata4, Nkx2.5, and Tbx5 is likely to be the consequence of, at least in part, a post-transcriptional control of p204 expression. Such control was reported in the case of the expression of p202b, another protein of the p200 family (17, 23).
To establish whether the increase in the expression of p204 by ectopic Nkx2.5, Gata4, and Tbx5 in 10T1/2 cells may be relevant for cardiac myocyte differentiation, we also tested the effect of these transcription factors on p204 expression in cultures of primary cardiac myocytes from newborn rats (Fig. 7). Both the endogenous level of Nkx2.5 protein and the endogenous Gata4 activity were reported to be very low in cultured newborn rat myocytes (70, 71). We transfected newborn rat myocytes in culture with a reporter construct, including a sequence from the Ifi204 gene 5'-flanking segment (shown in Fig. 5A). The Gata4, Nkx2.5, and Tbx5 transcription factor binding sites in the construct are shown in Fig. 7. Transfection of an expression plasmid encoding any one of the above listed transcription factors transactivated p204 expression weakly (less than 2-fold). Transfection of any two of them had a synergistic effect (an over 8-fold increase). Transfection of all three had an even stronger synergistic effect (an over 14-fold increase). These results establish that ectopic Gata4, Nkx2.5, and Tbx5 can synergistically transactivate p204 expression not only in 10T1/2 cells but also in cardiac myocytes.
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Coimmunoprecipitation of Chromatin Segments from the 5'-Flanking Region of the Ifi204 Gene Containing Gata4- and Nkx2.5-specific Sequences by Antibodies to Gata4 and Nkx2.5—As indicated earlier, undifferentiated proliferating P19 cells do not contain detectable levels of Gata4 and Nkx2.5 transcription factors (65, 66, 72) and express the Ifi204 gene very weakly, if at all (Fig. 2A). P19 cells differentiating to myocytes, however, contain pronounced levels of Gata4 and Nkx2.5 (73) and strongly express the Ifi204 gene (Figs. 1A and 2B). These findings prompted us to test if antibodies to Gata4 and Nkx2.5 (but not control IgG) can coimmunoprecipitate chromatin segments (generated by sonication (Fig. 9A, panel b)) containing Gata4- and Nkx2.5-specific sequences from the 5'-flanking region of the Ifi204 gene in nuclear extracts from differentiating P19 cells but not from proliferating P19 cells. The data in Fig. 9A, panel a, indicate that this was the case.
As determined by PCR, of the five chromatin segments tested (i.e. four from the 5'-flanking region and one from the coding region, including the DNA with the last exon of p204 RNA), two segments, segment 1 (extending from nucleotide -5124 to -4705) and segment 4 (extending from nucleotide -184 to +93), were coimmunoprecipitated by antibodies to Gata4 and to Nkx2.5 (but not by IgG), but only in a nuclear extract from differentiating P19 cells and not in that from proliferating P19 cells (Fig. 9A, panel a). (As noted earlier, the first nucleotide of intron 1 of the gene Ifi204 was taken as +1.) Segments 2 and 3, located between segments 1 and 4, were not coimmunoprecipitated despite the occurrence in them of potential Gata4- and Nkx2.5-specific sequences. This indicates that the presence of such a sequence does not ensure the binding of the appropriate transcription factor.
The coimmunoprecipitable segment 4 included a sequence (Fig. 5A) that was used (i) in a reporter construct whose expression in transfected cells was synergistically transactivated by Gata4 and Nkx2.5) (Fig. 5B), and (ii) to which Gata4 and Nkx2.5 bound in an electrophoretic mobility shift assay in vitro (not shown).
The reporter construct (in Fig. 9B) contained an 
5.5-kb sequence from the Ifi204 5'-flanking region. This included all the chromatin segments tested in the chromatin immunoprecipitation in Fig. 9A. The diagram in Fig. 9B reveals that the expression of this construct was cooperatively transactivated by Gata4 and Nkx2.5.
The results of the chromatin coimmunoprecipitation assays indicated that Gata4 and Nkx2.5 were bound in vivo to the chromatin from the 5'-flanking region of the Ifi204 gene in P19 cells differentiating to myocytes but not in proliferating P19 cells. These findings are in accord with other findings in this study by suggesting that the expression of p204 in P19 cells differentiating to myocytes is the consequence, at least in part, of the transactivation of the gene by Gata4 and Nkx2.5. Chromatin segment 1, which was also coimmunoprecipitated by antibodies to Gata4 and to Nkx2.5, also contained Gata4- and Nkx2.5-specific sequences. This segment was part of the 3'-terminal transcribed but not translated region of the Ifi203a gene, the 5'-terminal neighbor of the Ifi204 gene (11). According to a recent report (74), about a third of the transcription factor binding sites (as examined in human chromosomes 21 and 22) are located within or immediately 3' to well characterized genes and are significantly correlated with noncoding RNAs. Furthermore, overlapping pairs of protein coding and noncoding RNAs are often coregulated. Consequently, it will be interesting to see whether this segment 1 is a site in which a noncoding RNA transcript is initiated, and if so whether the transcript affects the expression of the Ifi203a and/or Ifi204 genes.
Various Inducers and Transcription Factors Can Promote the Tissue-specific Expression of p204—p204 was discovered as an interferon-inducible protein (5). Studies on the differentiation of skeletal muscle revealed that in the course of this process p204 expression is activated by the skeletal muscle-specific MyoD and myogenin transcription factors (29). The results presented in this study established that in cardiac myocyte differentiation, p204 expression is synergistically activated by the cardiac Gata4, Nkx2.5, and Tbx5 transcription factors. In all the above cases, distinct transcription factors and cis-acting sequences in the Ifi204 gene mediated p204 expression. Furthermore, the expression of p204 in differentiating T cells was reported to be activated by Notch 1 (75); however, the mediators of the activation remain to be identified. Thus the expression of p204 in different tissues can be activated by a variety of tissue-specific mechanisms, i.e. by distinct inducers, transcription factors, and cis-acting sequences.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplement M, Fig. S1, and Videos 1 and 2.
1 To whom correspondence should be addressed: Dept. of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar St., New Haven, CT 06520-8024. Tel.: 203-737-2061; Fax: 203-785-7979; E-mail: peter.lengyel{at}yale.edu.
2 The abbreviations used are: Id, inhibitor of differentiation; NES, nuclear export signal; 204AS, 204 antisense; protein (e.g. Gata4), antiserum to the protein in question (e.g. to Gata4); DMSO, dimethyl sulfoxide; MHC, myosin heavy chain; p204NES, p204 lacking the NES; p204MNES, p204 with a mutated NES; DAPI, 4,6-diamidino-2-phenylindole; HA, hemagglutinin; RT, reverse transcription.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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