We have previously cloned and characterized a new gene: zebra fish foxi one
. It encoded a nuclear protein with 419 amino acids and was expressed specifically in a small number of tissues in the developing zebra fish embryo. FoxI1 has been shown to possess complex signaling connections with other genes, such as fibroblast growth factors, pax8
, band3 anion exchangers (AE1
), and possibly, the ATP6B
subunit of the vacuolar H+
). The mechanism of these multi-interactions is not clear. We speculate that FoxI1 may play a central role in a complex signaling network necessary for ear and jaw development.
We created two zebra fish cell lines where we could regulate FoxI1 expression under the control of a doxycycline responsive system. Unlike most transcription factors that are actively excluded from the condensed chromatin during mitosis (35
), we demonstrated that the zebra fish forkhead transcription factor FoxI1 remains bound to condensed chromatin throughout the cell cycle. Only two examples of transcription factors have been shown to remain bound to the mitotic chromosomes: the general transcription factor TATA binding protein (11
) and the ubiquitous Drosophila
proteins GAGA factor and Prod (49
). Thus, FoxI1 is the first example of a transcription factor that is expressed tissue specifically yet is able to stably bind to condensed chromatin. The developmental defects demonstrated in both zebra fish and mouse when FoxI1 is mutated suggest that this stable chromatin binding is an essential aspect of the early developmental program. The punctate nuclear staining of FoxI1 is reminiscent of the subcellular localizations of GAGA and Prod in Drosophila simulans
and Drosophila manuritiana
brain interphase nuclei (49
). Platero interpreted this granular pattern as the protein predominately binding to euchromatic sites, with the intense spots representing interphase binding of the proteins to satellite DNA targets. We believe that the forkhead transcription factors could be part of a class of factors including GAGA and prod that bind chromatin stably and modulate developmental responses of the cells by altering the genomic transcriptional template in a stable manner, in contrast to other types of transcription factors and nuclear proteins which have rapid DNA association dynamics (47
). While the cell lines we generated create a carefully controlled environment for studying FoxI1, there is the caveat that this artificial system, because of overexpression or inappropriate cellular context, may not represent the function of FoxI1 in vivo. What we can conclude is that FoxI1 demonstrates unusual characteristics for a transcription factor in that it is able to maintain its DNA interactions in condensed chromatin, and we feel strongly that this ability to bind cannot be explained by an overexpression of the protein. The logical conclusion is that this binding to condensed chromatin is related to the actual function of FoxI1 in vivo.
Remarkably, the induction of FoxI1 expression has a very modest effect on gene expression in these cells. Only 12 genes of the approximately 20,000 (0.06%) sampled by microarray changed in levels by any significant amount. Given that FoxI1 can be shown to widely bind to the chromatin (Fig. ), it can be concluded that FoxI1 is not likely to be a potent activator or repressor on its own and more likely requires a combinatorial effect with other transcription factors to cause significant changes in expression. This is consistent with a role in global chromatin remodeling. Similarly, linker histone H1 does not have a major effect on global transcription but can act as either a positive or negative gene-specific regulator of transcription in vivo (55
). There is also the possibility that by making a fusion of FoxI1 to GFP or the V5 epitope, the transcriptional regulation activity of FoxI1 is disrupted or altered. Because this is an artificial system and gene regulation is a complex interaction from multiple transcription factors, it is not clear whether any of the genes changed in the array analysis are bona fide targets of FoxI1 regulation. We do not argue that the direction of the gene changes are relevant to the normal developmental context, as many modifying factors may be present in the developing organism that are not present in the tissue culture system, but we do believe that many of these genes will prove to be relevant targets of regulation by FoxI1. The genes that are affected do appear to be enriched for genes known to be involved in ear and jaw development or kidney function (the precise locations of FoxI1 expression). Sox9a is involved in both ear and jaw development (72
). MMP-9 is a key enzyme in cartilage formation (43
), and MCT4 is expressed specifically in the mouse kidney (41
). The establishment of these genes as genuine targets of regulation in zebra fish embryos is ongoing, although a clear genetic relationship between FoxI1 and Sox9a has already been established (34
The nature of association of FoxI1 with the heterochromatic satellite DNA is not clear. It may be a simple coincidence that these repetitive DNAs contain multiple copies of the consensus binding site of Fox proteins but are not functionally relevant sites in vivo. Alternatively, it may be a consequence of the coevolution of forkhead genes with satellite DNAs. This concept follows from the theory of mitotic borrowing proposed by Csink and Henikoff (16
). The authors proposed that expansion of a new satellite DNA repeat would borrow an appropriate DNA-binding protein, which ensures packaging of this satellite DNA array during mitosis. Only those repeat motifs that are able to borrow appropriate mitotic proteins can be expanded into large blocks of satellite DNA arrays. In interphase, these DNA-binding proteins would have other functions unrelated to satellite DNAs (16
). In that case, forkhead gene expansion may be responding or correlating with satellite DNA evolution. Fitting with that hypothesis, the forkhead class of transcription factor has an overall structure similar to those of the linker histones H1 and H5. This similarity of structure may allow the forkhead transcription factors to bind a DNA context (i.e., condensed chromatin) from which most transcription factors are excluded. Additionally, Cirillo and Zaret showed that the forkhead protein HNF3 bound to DNA more stably in the context of nucleosomes than when bound to naked DNA (13
), again suggesting that the similarity of three-dimensional structure implies a preferred chromatin context for the forkhead transcription factors and providing for a possible “crossover” role as described by Csink and Heinkoff. This would not necessarily imply that FoxI1 is directly competing with H1 for DNA binding, merely that the general three-dimensional structure of the forkhead proteins conveys an ability to bind condensed chromatin, in sharp contrast to most transcription factors.
To examine the influence of FoxI1 expression on chromatin structure, we performed DNase I hypersensitivity assays using the sequences we isolated from ChIP enrichment. We found that most of the confirmed genomic targets bound by FoxI1 maintained or decreased DNase I sensitivity in the presence of FoxI1. A smaller number of sites bound by FoxI1 displayed increases in DNase I sensitivity that were maintained even in mitotically condensed chromatin. In fact, these differences in DNA structure were magnified in condensed chromatin compared to unsynchronized cells. In some cases, this sensitivity to DNase I could change more than 10-fold, with some locations becoming more sensitive and others becoming less sensitive. Importantly, some of the changes in DNase I sensitivity were not correlated with FoxI1-binding sites, suggesting that FoxI1 was also having longer-range effects on chromatin structure. The DNase I studies suggest that FoxI1 has two effects on chromatin: direct effects on specifically bound targets and indirect effects on the global organization of chromatin. In support of this argument, Carroll et al. demonstrated that the estrogen receptor bound to many sites on chromosomes 21 and 22, many of which were far from regulated genes (10
). They were then able to demonstrate that the binding of the estrogen receptor required the presence of FoxA1 binding in close proximity. This is consistent with the permissive role we hypothesized for FoxI1.
Additional chromatin structural analyses demonstrated that FoxI1 was strongly enriched in insoluble chromatin, with a much smaller amount of FoxI1 associated with the active chromatin S1 fraction. No FoxI1 was detected in the S2 fraction, which is enriched for the linker histones H1 and H5 (74
). The association of FoxI1 with both the transcriptionally active chromatin (S1) and competent chromatin (P) again demonstrates the two modes of FoxI1 function, with the S1 fraction having effects on specific transcripts and the P fraction of FoxI1 involved in overall chromatin architecture. Studies have shown that repetitive elements could influence chromatin structure in two ways: (i) dispersed repeated copies containing binding sites for chromatin-organizing proteins can form a basis for local chromatin structure (54
) and (ii) tandemly repetitive sequences can nucleate the highly compacted structure called “heterochromatin” and negatively affect the expression of genetic loci at distances of many kilobase pairs. Both types of position effect variegation are well documented in fruit flies (68
). Consistent with a FoxI1 role in the association with satellite DNA, the Domina (Dom) protein, another member of the FKH/WH transcription factor gene family in Drosophila
, was shown to be accumulated in the chromocenter and function as a suppressor of position-effect variegation (PEV) (63
). In addition, the GAGA protein which has a similar staining pattern to FoxI1 does not have structural similarity to the FKH/WH family but, like Dom, was also originally identified as being involved in PEV in Drosophila
. It may not, therefore, be a coincidence that both the zebra fish and mouse mutations in FoxI1 displayed somewhat variable phenotypes (27
), which could suggest PEV effects in a vertebrate context. It is widely believed that living cells can use repetitive DNA sequences in various ways to affect the expression of coding sequences. Our results provide evidence that FoxI1-expressing cells can modulate chromatin structures, both local effects of FoxI1 binding and global or long distance changes in nucleosome organization. The model for regulation by forkhead transcription factors becomes one where the protein is bound stably to the chromatin with both isolated and global effects on chromatin structure, essentially establishing a genomic “template.” This template will allow cells expressing the forkhead protein to rapidly and appropriately respond to the external induction factors in the context of the developing embryo. We demonstrated previously that FoxI1 was necessary for an appropriate response to fibroblast growth factor signaling in the zebra fish embryo, and the present study gives a framework for how that response could take place.