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(Journal of Leukocyte Biology. 2001;69:21-32.)
© 2001 by Society for Leukocyte Biology

The role of architectural transcription factors in cytokine gene transcription

M. F. Shannon*, L. S. Coles{dagger}, J. Attema* and P. Diamond{dagger}

* Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra
{dagger} Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, South Australia

Correspondence: Dr. M. Frances Shannon, Division of Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, GPO Box 334, Canberra, ACT 2601, Australia. E-mail: frances.shannon{at}anu.edu.au


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ABSTRACT
 
The strict control of cytokine gene transcription is required for the correct regulation of an immune response. Cytokine gene transcription is generally inducible and can also be cell-type specific. Promoter and enhancer regions that control the expression of these genes assemble complex arrays of transcription factors known as enhanceosomes. One important aspect of the organization of these multi-protein complexes is the presence of proteins known as architectural transcription factors. Architectural proteins influence structural aspects of enhanceosomes through protein:DNA as well as protein:protein interactions. The high mobility group I(Y) and the cold shock domain families of architectural proteins have been shown to play roles in cytokine gene transcription and will be discussed here. These families of proteins interact with specific structural features of DNA, modulate transcription factor binding to DNA, and interact directly with other transcription factors. The mechanisms by which they affect inducible cytokine gene transcription will be discussed.

Key Words: DNA architecture • inducible transcription • immune system • enhanceosome


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INTRODUCTION
 
A range of cell types in the immune and hemopoietic systems play important roles in the response of the body to infection and parasitism, and in allergy. The appropriate cells respond to such attacks by activating a cascade of intracellular signaling events that result in altered gene expression. The genes that are required to mount an appropriate response are activated by these signaling events, whereas other genes that maintain cells in an inactive or undifferentiated state are repressed. Such genes encode cytokines, chemokines and their receptors, and other cell surface molecules. Cytokines are an important group of proteins that serve as communicators between different cell types in immune or inflammatory responses. The controlled expression of cytokines is therefore a critical event in the regulation of immune and inflammatory responses.

There is enormous variety in the expression patterns of cytokine genes, i.e., they can be either constitutively expressed or can be activated or repressed when a cell is exposed to a particular signal. They can also have cell-restricted expression or be widely expressed in many cell types. Often both cell-specific and activation-responsive mechanisms need to be integrated for the same gene. Most of these responses appear to be controlled at the level of transcription, with large increases in the rate of transcription after activation. For inducible genes the response after cell activation is usually rapid (within 1–4 h) and transient, with transcription being switched off again after the appropriate time of activation.

The promoter and enhancer regions of many cytokine genes have been extensively mapped and the functional domains determined [reviewed for some genes in refs. 1 2 3 ]. A wealth of information has accumulated describing the inducible and constitutive transcription factors that bind to these functional regions. Most of the binding studies have been carried out using electrophoretic mobility shift assays (EMSA) or footprinting assays in vitro but recently chromatin immunoprecipitation assays (ChIPs) are being used to determine binding sites in vivo [4 ]. Deletion or mutation of transcription factors in mice is now serving to determine the physiological role of individual factors and to confirm their involvement in the transcription of individual genes [5 ].

The assembly of higher-order protein complexes on promoter/enhancer regions, to generate functional transcriptional units, is an important consideration in the activation and control of cytokine gene expression. The promoter or enhancer regions of many cytokine genes comprise arrays of closely aligned transcription factor binding sites [1 2 3 , 6 ]. In many cases, individual binding sites have only weak affinity for the cognate factor but cooperative binding of adjacent proteins leads to the generation of a high affinity functional complex. Even in the absence of cooperative binding multiple proteins binding to adjacent sites can generate cooperative functional responses. There is evidence that these higher-order protein complexes are essential for the recruitment of coactivators of transcription and for the activity of the RNA polymerase enzyme [6 ]. It is thought that the presence of the appropriate array of transcription factors bound in the correct configuration on the DNA forms a surface that can recruit coactivators or other basal transcription factors.

The architecture of the DNA plays an important role in the assembly of these functional transcription units, which have become known as enhanceosomes. DNA architecture may be affected by the presence of nucleosomes and higher-order chromatin structure. In addition, there are a number of protein families that can bind to and modify DNA structure. Proteins that play a structural role on DNA and thereby affect transcription are collectively referred to as architectural transcription factors. Many such proteins have now been characterized and depending on their own structure, sequence, or function, are classified into family groups. Here we will discuss two families of architectural proteins that have been shown to function in inducible cytokine gene transcription.

One of these families is referred to as the high mobility group I(Y) [HMGI(Y)] proteins and are a subgroup of the larger family of HMG proteins [7 , 8 ]. The HMGI(Y) proteins have been implicated in the control of cytokine gene transcription and probably play a major architectural role in the assembly of enhanceosome complexes on cytokine promoters and enhancers. The other family of architectural factors that will be discussed here is known as the cold shock domain (CSD) proteins [9 10 11 12 ]. These proteins are a group of evolutionarily conserved proteins that function in both transcription and translation and have the distinct feature of binding to single-stranded nucleic acids.


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THE HMGI(Y) FAMILY OF ARCHITECTURAL TRANSCRIPTION FACTORS
 
The HMGI(Y) family of proteins consists of three members, HMGI, HMGY, and HMGI-C [7 , 13 ]. HMGI and HMGY arise from two differentially spliced mRNAs from the same gene and are usually referred to together as HMGI(Y) [14 ] (Fig. 1 ). HMGI-C is derived from a separate gene but is highly related to the other family members [7 ]. The proteins are approximately 10 kDa in size and have in common three conserved DNA binding motifs (Fig. 1) . They bind to the minor groove of A/T-rich DNA and appear to recognize structural rather than sequence features of the DNA. The presence of three DNA binding domains allows the proteins to interact at several distinct A/T-rich regions along a linear stretch of DNA [15 ]. Recently, it has been shown in vitro that HMGI(Y) can bind sites at some distance from each other, causing DNA looping [16 ]. The solution structure of a fragment of HMGI(Y), containing binding domains II and III (BDsII and III), in a complex with DNA has recently been solved [17 ] and has significantly contributed to our understanding of HMGI(Y) function. Both domains utilize the same Arg-Gly-Arg motif in the conserved DNA binding domains (Fig. 1) to make contact with the DNA deep in the minor groove. Surrounding residues at either end of the core mediate hydrostatic interactions with the DNA backbone. In addition, for BDII an extended six amino acids past the carboxy-terminal end of the core interacts with the sugar phosphate backbone leading to the higher affinity observed for BDII interactions with DNA. The structural analysis also showed that the so-called A/T-hook motifs of the protein bound to A/T DNA stretches in a directional manner. It has also been shown that HMGI(Y) binds directionally to four-way junction DNA and to DNA assembled into nucleosomes [18 19 20 ]. This manner of binding may be important in generating recognition surfaces for the recruitment of other proteins.



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  		Figure 1. Structure of the HMGI family of architectural transcription factors. HMGI, HMGY, and HMGI-C are shown and their linear amino acid sequence is represented by a line with the number of the carboxy-terminal amino acid shown. Location of the three DNA binding domains (BDI-III) are indicated by stippled boxes with the first and last amino acid of each box shown. The consensus sequence for the DNA binding domains is given where the subscripted amino acid occurs less frequently at indicated positions.

The HMGI(Y) proteins have many features and functions by which they can affect gene transcription (Table 1 ). HMGI(Y) binding to DNA has been shown to significantly alter DNA structure, including the introduction of bends [21 , 22 ]. The intact HMGI(Y) protein (which contains three independent DNA binding regions) has the ability to introduce significant bends in DNA but the extent of such bending critically depends both on the number and organization of the binding sites available in the substrate [Reeves, unpublished observations]. In some cases, HMGI(Y) binding appears to cause only subtle alterations in DNA structure which, nonetheless, are important in assembly of transcription factor complexes [22 ]. HMGI(Y) has also been shown to alter DNA superhelicity on closed circular DNA templates [23 ], a feature that may also affect transcription factor:DNA interactions.


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  		Table 1. HMG-I(Y) Mechanisms of Action

HMGI(Y) proteins have been shown in many cases to alter transcription factor binding to sites overlapping or adjacent to their A/T-rich binding sites [7 , 13 ]. Both enhancement and inhibition of binding has been observed. The transcription factors that have so far been found to be affected by HMGI(Y) are described in Table 2 . In most cases, transcription factor binding is enhanced, e.g., NF-{kappa}B, ATF, AP-1 proteins but in other cases such as the NFAT proteins both enhancement and inhibition has been reported (see Table 2 for references). Direct protein:protein contact has also been shown to be important for the ability of HMGI(Y) to promote transcription factor binding, and studies have shown that HMGI(Y) can interact directly with many transcription factors in the absence of DNA. The proteins with which HMGI(Y) can interact are listed in Table 2 . Both protein:protein and protein:DNA interactions appear to be important for the role of HMGI(Y) in the assembly of an active enhanceosome complex on cytokine gene promoters [24 , 25 ].


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  		Table 2. HMG-I(Y) Modulates Transcription Factor Binding to Cytokine Gene Promoters

HMGI(Y) may be the target of signaling cascades in the cell and its function affected by the signaling-directed modifications. HMGI(Y) proteins can be modified by phosphorylation and acetylation, both of which can affect protein:DNA and protein:protein interactions. A number of kinases including cdc2 (also known as histone H1 kinase), casein kinase 2 (CK-2), mitogen-activated kinase (MAPK), and protein kinase C (PKC) have been shown to phosphorylate HMGI(Y) either in vitro or in vivo [19 , 26 27 28 29 30 ]. In the cases of cdc2, CK-2, and PKC, phosphorylation alters the affinity of the protein for DNA. Two histone acetyltransferases, CBP and P/CAF have been shown to acetylate HMGI(Y) but on distinct residues [31 ]. CBP acetylation, like phosphorylation, inhibits HMGI(Y):DNA interactions [31 ]. The outcome of these modifications has not yet been studied in detail for many genes but such studies should add to our understanding of HMGI(Y) function.

HMGI(Y) may also play a role in chromatin structure or modification of that structure. HMGI(Y) proteins can bind to DNA packaged into nucleosomes and has been shown to alter the localized rotational setting of nucleosome core particles on DNA [20 , 32 , 33 ]. HMGI(Y) appears to bind to the entry and exit points of nucleosomes and thus may play a role in positioning nucleosomes or moving them along the DNA [32 ]. HMGI(Y) has also been shown to be associated with H1-depleted chromatin and to antagonize H1-mediated transcriptional repression [34 ]. Although all of the above functions have been attributed to HMGI(Y) proteins, the relative importance of each is not clear in a physiological setting. There are examples, however, where each of the functions of HMGI(Y) described above are implicated in the control of cytokine gene transcription and these will be discussed below.


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HMGI(Y) PLAYS A MAJOR ROLE IN CYTOKINE GENE TRANSCRIPTION
 
There are now numerous reports describing the activity of HMGI(Y) proteins in gene transcription and the genes that have been so far studied are listed in Table 3 . Most of the genes are inducible but this does not exclude the possibility that HMGI(Y) is important in the regulation of constitutively expressed or developmentally regulated genes. For example the TCR-{alpha} chain gene is controlled by a distal enhancer that appears to require HMGI(Y) for activity [5 ]. Many of the inducible cytokine genes contain a number of closely spaced HMGI(Y) binding sites overlapping or adjacent to other transcription factor binding sites in their promoter or enhancer regions. The multiple abilities of HMGI(Y) to alter DNA structure, modulate transcription factor binding, and also bind to nucleosomes may contribute to their influence on these gene promoters/enhancers. A general model of the many roles of HMGI(Y) in inducible cytokine gene transcription is derived from the examples discussed below and is illustrated in Figure 2 . There are several well-studied examples of HMGI(Y) involvement in the assembly of transcription factor complexes on cytokine gene promoter/enhancer regions and the subsequent functional outcome. Three of these will be discussed here and will serve to illustrate the various ways in which HMGI(Y) can influence cytokine gene transcription.


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  		Table 3. HMG-I(Y)-Mediated Positive and Negative Regulation of Gene Transcription



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  		Figure 2. A model of HMGI(Y) function in the activation of a cytokine gene promoter. The possible roles of HMGI(Y) are devided into four steps, highlighting the individual roles of HMGI(Y). Binding sites for HMGI(Y) are shown as while circles on the DNA in a nucleosome (step 1) and as striped bars on the linear diagram. The HMGI(Y) protein is a black circle in the enhanceosome structure (step 4). Filled shapes indicate other transcription factors, coactivators, and the basal machinery.

Interferon-ß (IFN-ß)
The IFN-ß promoter is the prototype cytokine promoter where HMGI(Y) was first implicated in transcription. IFN-ß is specifically induced by virus infection in mammalian cells, and the transcription factor complexes involved have been studied in detail. HMGI(Y) appears to have an important role in the activation of IFN-ß transcription by facilitating the assembly of the enhanceosome at several organizational levels (see Fig. 2 ).

HMGI(Y) binds in vitro to four sites across the IFN-ß promoter and promotes the binding of NF-{kappa}B and ATF/c-Jun complexes to the so-called PRDII and PRDIV sites, respectively [6 , 35 36 37 38 ] (Fig. 3A ). The relative arrangement of the transcription factor and HMGI(Y) binding sites appears to be critical for the enhancement of factor binding by HMGI(Y). The specific directional binding as well as intra- and inter-molecular cooperativity of the two HMGI(Y) molecules bound to the promoter are critical for enhanceosome assembly [15 ]. Recruitment of transcription factors appears to be mediated by allosteric changes induced in the DNA by HMGI(Y) binding [24 ]. HMGI(Y) binding has been shown to alter the DNA structure of the IFN-ß promoter by unbending an intrinsic bend in the promoter (Fig. 2 , step 3) [22 ]. Direct contact between HMGI(Y) and the transcription factors is also required for the completion of enhanceosome assembly [15 , 24 ] (Fig. 2 , step 4).



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  		Figure 3. The promoter regions of IFN-ß (A), IL-2 (B), IL-2R{alpha} (C), and IL-4 (D) illustrating the HMG-I(Y) binding sites.The IFN-ß promoter region from the start of transcription (+1) to position -110 is shown. The PRDII, PRDIII-I, and PRDIV control elements are indicated with solid bars. Nucleotide sequences of PRDII and PRDIV are shown and HMG-I(Y) binding site at A/T sequences represented by dashed lines. The IL-2 minimal promoter from -300 bp to the transcription start site (+1) is illustrated. Black bars represent functional transcription factor binding sites and the designated names of elements are shown. Dashed lines highlight the HMG-I(Y) binding sites on both the linear representation and the nucleotide sequence of individual elements. For the IL-2R{alpha} gene, the two control regions that respond to mitogenic signals (PRRI and PRRII) as well as the IL-2 responsive region (PRRIII) are shown with black bars. Three sites for HMG-I(Y) binding are indicated with dashed lines on the PRRII sequence below. The IL-4 promoter from -260 to +1 is shown. Control elements containing transcription factor binding sites are shown as black solid boxes and names are indicated. The sequences of Pu-bb and BoxII are shown highlighting A/T sequences to which HMG-I(Y) binds.

The assembled enhanceosome on the IFN-ß promoter appears to act as a recruiting complex for coactivators as well as components of the basal transcriptional machinery (Fig. 2 step 4). It has been shown that the correctly structured enhanceosome forms a specific interacting surface with contributions from several of the protein components that allow recruitment of coactivators such as the histone acetyltransferase CBP/p300 [31 ]. It has recently been shown that localized hyperacetylation of histones H3 and H4 occurs across the IFN-ß promoter in response to virus infection [39 ]. This supports the in vitro finding that the assembled enhanceosome recruits histone acetyltransferases. The enhanceosome has also been shown to recruit preinitiation complex components such as TFIIB and the polymerase II holoenzyme [40 ]. Thus the assembly of a precisely organized complex of transcription factors on the IFN-ß promoter appears necessary for the generation of a functional transcription unit. Although the model of enhanceosome structures recruiting chromatin-modifying complexes such as CBP is an attractive one, it begs the question of what comes first. Does chromatin need to be remodeled before the enhanceosome can assemble on the DNA or does the recruitment of CBP lead to remodeling of adjacent nucleosomes? The use of chromatin immunoprecipitation assays to monitor the complexes that bind to DNA in cells in an activation- and time-dependent manner should help to answer this question.

Recently, it has been shown that HMGI can be acetylated at specific and distinct lysine residues by the coactivators CBP and P/CAF [41 ]. Acetylation by CBP but not P/CAF leads to destabilization of HMGI(Y):DNA interactions. It is interesting that the lysine residue at position 65 is most highly acetylated by CBP but not by P/CAF. This lysine residue is located in the region of extended contact for the BDII region of HMGI(Y) with DNA described above and may explain why its acetylation may affect DNA binding. Acetylated HMGI cannot promote NF-{kappa}B binding to the IFN-ß promoter nor can it participate in enhanceosome assembly in vitro [41 ]. These results have lead to a proposal where HMGI(Y) is involved not only in assembly but also disassembly of the enhanceosome complex and thus in the switching off of the gene after transient activation. If this model applies then the acetylation of HMGI(Y) would need to be temporally controlled but this issue has not yet been addressed. In any case, HMGI(Y) appears to be an important component of the activation and deactivation of IFN-ß transcription, functions that may extend to many inducible genes.

It has to be pointed out, however, that all of the experiments on the role of HMGI(Y) in IFN-ß gene regulation have been carried out through the use of in vitro binding assays or transfection of reporter constructs into cells. It has not been shown whether HMGI(Y) affects expression from the endogenous IFN-ß gene or whether HMGI(Y) is part of an enhanceosome complex in vivo.

Interleukin-2 (IL-2) and the IL-2 receptor autocrine loop
The proliferation of naive T cells is dependent on the cytokine IL-2, which is produced by the activated T cells themselves and acts as an autocrine growth factor. The IL-2 gene is expressed in a cell-type-restricted as well as an inducible manner and these characteristics as well as its importance in an immune response have made its regulation the subject of intense study. The regions of the IL-2 gene that are responsible for inducible expression have been well defined, as have the transcription factors that bind to and control the function of these regions (Fig. 3B) [2 , 3 ]. The proximal promoter/enhancer controls most of the inducible transcription and lies between +1 and -300 bp but it is likely that regions far upstream are involved in cell-type-specific expression [42 , 43 ].

The first 300 bp of the IL-2 promoter is highly A/T-rich (~65%) and it was therefore not surprising that in DNaseI footprinting experiments HMGI(Y) bound to many sites across the promoter [25 , 44 ] (Fig. 3B) . Functional studies using antisense expression for HMGI(Y) RNA showed that HMGI(Y) was a positive activator of the IL-2 promoter [25 , 44 ]. Indeed, of several T cell promoters tested [IL-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-3, and HIV], IL-2 was the most sensitive to loss of HMGI(Y) proteins [unpublished observations]. Most of the HMGI(Y) binding sites are overlapping or close to known functional transcription factor sites. Further experiments showed that HMGI(Y) could affect the in vitro binding of many of the major transcription factor families that drive IL-2 transcription, i.e., NFAT, NF-{kappa}B, and AP-1 [25 , 44 , 45 ].

There were two interesting findings from an investigation of the binding of HMGI(Y) to the promoter region of IL-2 and the related cytokine, GM-CSF. First, it appears that HMGI(Y) may have differential impacts on transcription factors of the same family. This is illustrated by the fact that HMGI(Y) has a major influence on the in vitro binding of c-Rel but not RelA to the CD28RR of the IL-2 and GM-CSF genes [44 ]. A similar situation has been described for members of the ATF family where two ATF-2 isoforms are differentially affected by HMGI(Y). HMGI(Y) can interact directly with one [ATF-2(195)] but not with the other [ATF-2(192)] and as a result HMGI(Y) promotes the binding of ATF-2(195) but inhibits the binding of ATF-2(192) [46 ]. This example illustrates the importance of protein:protein interactions in the modulation of transcription factor binding to DNA as discussed below.

The other important finding was that DNA binding by HMGI(Y) was not essential for the promotion of transcription factor binding. This was shown by generating a mutant HMGI(Y) protein unable to bind to DNA and testing its effect in the promotion or inhibition of transcription factor binding. It was surprising to find that this HMGI(Y) mutant was able to enhance transcription factor binding to several sites tested [25 ]. This mutant, however, failed to inhibit the binding of certain transcription factors such as NFATp at high concentrations, a phenomenon observed for the wild-type protein [25 ]. These results have led to a model in which HMGI(Y):transcription factor interactions are important for the enhancement of transcription factor binding but when HMGI(Y) is in excess and able to bind to DNA alone it can act as an inhibitor of transcription factor binding. It has been shown that HMGI(Y) can interact with many transcription factors in the absence of DNA (Table 2) , thus supporting the idea that protein:protein interactions may enhance transcription factor binding to DNA.

When the non-DNA binding mutant of HMGI(Y) was tested for function in transfection assays it acted as a dominant-negative protein inhibiting the activity of the IL-2 promoter [25 ]. This implies that the DNA binding capacity of HMGI(Y) is important for transcriptional activity and that the non-DNA binding mutant, while promoting transcription factor binding, may form non-productive complexes. Experiments on the IFN-ß promoter have also shown that both DNA binding and direct HMGI(Y):transcription factor interactions are important for the formation of a functional IFN-ß enhanceosome [24 ].

IL-2 interacts with the IL-2 receptor to generate a proliferative signal for T cells [47 ]. The IL-2 receptor is composed of three polypeptide chains, which together form a high-affinity receptor [47 ]. The IL-2 receptor alpha chain (IL-2R{alpha}) is synthesized in activated T cells to generate the high-affinity receptor. Like the IL-2 gene, the IL-2R{alpha} gene promoter is also A/T rich (60%) and HMGI(Y) appears to contribute to its activity. HMGI(Y) has been shown to functionally cooperate, in transfection experiments using a reporter construct, with the ETS family member, Elf1, on a region of the promoter known as PRRII [48 ] (Fig. 3C) . It has recently been shown that this region of the IL-2R{alpha} promoter assembles into a highly positioned nucleosome in vitro and there is in vivo evidence for a similarly positioned nucleosome in unstimulated T cells [20 ]. It is interesting that HMGI(Y) can bind to the IL-2R{alpha} PRRII sequence even when it is assembled into a nucleosome in vitro (Fig. 2 , step 1) [20 ]. It is interesting that the two HMGI(Y) molecules that have been shown to bind to the nucleosome-assembled promoter do so in a directional manner without, however, any overt disruption of the nucleosome structure. It can be speculated that such a directional binding may form a stereospecific recognition signal that could recruit proteins involved in nucleosome disruption or remodeling (Fig. 2 , step 2). We have also recently shown that the IL-2 promoter assembles a highly positioned nucleosome in vitro and while many factors such as AP-1, c-Rel, and NFATp are now excluded from binding to their recognition sites, HMGI(Y) can still bind to its specific recognition sequences [Attema and Shannon, unpublished data]. It is possible that HMGI(Y) bound to these nucleosomes acts as a recruiting agent for complexes needed to disrupt or remodel the nucleosomes, thus allowing other transcription factors to bind and activate transcription. It will be necessary to design experiments in cells that allow us to distinguish between the role of HMGI(Y) in chromatin remodeling and enhanceosome assembly.

All of the functional experiments described above have been carried out in transformed cell lines in culture and by transfection of promoter/reporter constructs. What is the evidence that HMGI(Y) actually plays a role in gene expression from endogenous cytokine genes? Recently, we have performed a study examining the effect of modulating HMGI(Y) levels on the expression of the endogenous IL-2 gene both in Jurkat T cells and in peripheral blood lymphocytes (PBLs). Both expression of antisense HMGI(Y) or of the non-DNA-binding mutant dramatically reduced the level of IL-2 protein produced by either the Jurkat T cells or the PBLs [25 ]. This correlated with a reduced proliferative ability of the IL-2-dependent PBLs but not the Jurkat cells. Conversely, increased expression of HMGI(Y) lead to increased IL-2 production and proliferation of PBLs [25 ].

In this context it is interesting to note that HMGI(Y) may also be important for the expression of the endogenous IL-2R{alpha} gene in primary T cells [25 ]. Thus, the HMGI(Y) proteins may play a role in controlling T cell function by influencing two crucial genes that generate an autocrine loop for T cell proliferation.

Interleukin-4 (IL-4)
Studies on the role of HMGI(Y) in IL-4 gene expression and IL-4 signal transduction have shown the importance of phosphorylation in HMGI(Y) activity. IL-4 is a cytokine produced primarily by T cells during an immune response. It can interact with its own receptor on T cells, promote the expression of more IL-4, and therefore generate a Th2-like phenotype in the T cell population [49 , 50 ]. There are several HMGI(Y) binding sites in the IL-4 promoter [51 ] (Fig. 3D) . These sites are associated with functional binding sites for NFAT or NFAT/AP-1 complexes and Oct proteins [51 ]. It has been shown that HMGI(Y) inhibits the binding of NFAT factors to one specific region known as the Pu-bB region of the IL-4 promoter [52 ]. Not only can HMGI(Y) inhibit the binding of NFATp but if NFATp is in excess the opposite effect occurs, implying mutually exclusive binding of these two factors. Furthermore, there is functional evidence of antagonism between HMGI(Y) and NFATp in transfection experiments [52 ]. When the HMGI(Y) binding site was mutated, leaving the NFATp binding site intact, an increase in IL-4 promoter activity was observed. The relative levels of NFATp and HMGI(Y) may be important in determining the functional outcome of the interaction between NFATp and HMGI(Y). It has also been observed that HMGI(Y) can promote NFATp binding to certain sites in the IL-2 and ICAM promoters at low concentrations but at higher concentration an inhibition of NFAT binding was observed [25 , 45 ]. This phenomenon may be explained by the fact that both HMGI(Y) and NFATp make contacts with the minor groove of the DNA and thus may inhibit each other’s binding. On the other hand, protein:protein interactions may promote NFATp binding by a distinct mechanism.

HMGI(Y) has also been shown to inhibit transcription from the germline immunoglobulin epsilon (Ig{varepsilon}) gene [53 ]. It is interesting that this gene is induced by IL-4 and thus may share some similarity with the IL-4 promoter itself. In an attempt to investigate the mechanism of HMGI(Y) inhibition of the Ig{varepsilon} promoter it was found that IL-4 treatment of cells could lead to the serine phosphorylation of HMGI(Y) [29 ]. Phosphorylated HMGI(Y) has a reduced affinity for DNA [29 ], which could, in turn, lead to an increase in transcription from those genes that are repressed by HMGI(Y) such as IL-4 or Ig{varepsilon}. On the other hand, promoters of genes such as IL-2 or GM-CSF, that are activated by HMGI(Y), may have decreased activity after HMGI(Y) phosphorylation.

The phosphorylation sites on HMGI(Y) that are modified in response to IL-4 signaling have been mapped in detail. These sites correspond to CK-2 consensus sites at the carboxy-terminal end of the protein and lead to reduced binding to DNA. IL-4-driven phosphorylation of HMGI(Y) was rapamycin but not genestein sensitive, suggesting a role for pp70 S6 kinase but not tyrosine kinases such as the IL-4R-associated JAKs in the pathway [28 , 29 ]. Further experiments have identified a precise motif in the IL-4 receptor that is required for HMGI(Y) phosphorylation [28 ]. This motif contains a tyrosine residue that is a docking site for IRS-1, an adaptor protein involved in insulin receptor signaling. This in turn leads to enhancement of p70 S6 kinase activity through a PI 3-kinase pathway. These experiments serve to illustrate that HMGI(Y) can be the target of signal transduction pathways within the cell and thus its function in the control of cytokine gene may be controlled by the many diverse signals that activate cells of the immune system.

Although it is intriguing that HMGI(Y) may act as repressor or activator on different genes, it is also possible that on one gene, as described for INF-ß it can both establish transcriptionally active complexes or disassemble these complexes in a time-dependent manner depending on its acetylation or phosphorylation status.


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CSD PROTEINS: STRUCTURE AND SUBTYPES
 
CSD proteins, also known as Y-box proteins, have a central 100-amino-acid domain that is highly conserved throughout evolution. This region, called the cold shock domain, was named due to its conservation with bacterial cold shock proteins [reviewed in refs. 11 12 ]. The CSD proteins of higher organisms have been reported to bind double- and single-strand DNA and RNA, and can interact with a diverse range of proteins. By virtue of their diverse nucleic acid and protein interaction abilities CSD proteins have been shown to be involved in multiple aspects of gene regulation, including transcriptional repression, activation, and coactivation and in mRNA packaging, transport, localization, stability, and translation [reviewed refs. 11 12 54 55 ]. A primary function for CSD proteins appears to be in the transcriptional regulation of growth factor, stress response and cell proliferation-associated genes (Table 4 ).


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  		Table 4. Regulation of Gene Transcription by CSD Proteins

CSD proteins have three functional domains, an amino-terminal, the central CSD and a carboxy-terminal domain (Fig. 4A ). The highly conserved central CSD domain has a five-stranded ß-barrel structure and contains a motif called RNP1, which is required for sequence-specific single-strand RNA and DNA binding [56 57 58 ]. The RNP1 motif is conserved in a number of single-strand nucleic acid binding proteins [59 , 60 ]. The amino-terminal domain has not been well characterized but it has been shown to contribute to single-strand DNA binding and hence potentially repression mechanisms [58 , 61 ]. The carboxy-terminal domains of CSD proteins are generally composed of alternating basic and acidic regions and the primary function of these regions is in interaction with heterologous proteins [62 63 64 ]. The main function for the carboxy-terminal domain appears to be in transcriptional activation [65 and our unpublished data]. Both the abilities of CSD proteins to bind single-strand DNA and to interact with heterologous proteins may be involved in regulation of gene transcription.



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  		Figure 4. (A) Outline structure of a typical CSD protein showing the functional domains. (B) Conservation of single-strand CSD site arrangement across cytokine gene promoters. The sequence of a region of the proximal promoters of GM-CSF, G-CSF, and IL-3 are shown. The regularly spaced CSD binding sites are highlighted and boxed. Functional activating regions of the promoter are indicated by bars and named. The consensus CSD binding sites that emerge from these promoters are shown. Py, pyrimidine.

There are two major CSD subtypes expressed in avian and mammalian species, YB-1 and dbpA [66 , 67 ]. YB-1 is also called dbpB, EF1A, p50, TSEP-1, and MSY-1 depending on the species from which the protein was isolated. Homologues of dbpA are called EF-II, M1Y, YB-2 and most recently ZONAB. YB-1 and dbpA are encoded by separate genes and are ubiquitously expressed [66 67 68 ]. Truncated proteins resulting from alternative mRNA splicing have also been reported. The major splice variant reported arises from the dbpA gene [66 , 69 70 71 ]. This splicing event results in a 69-amino-acid deletion in the carboxy-terminal region, which does not affect single-strand DNA binding but could have an effect on the ability of the truncated protein to interact with other regulatory proteins [61 ]. In addition to these ubiquitously expressed proteins, germ cell-specific CSD proteins (contrin, MSY2, FRGY2; [56 , 72 , 73 ]) and CSD-related proteins such as UNR, which contains multiple CSD domains [74 ], have been identified.


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ROLE OF CSD PROTEINS IN CYTOKINE GENE TRANSCRIPTION
 
As described above, the CSD proteins can be activators or repressors of gene transcription and this seems also to be the case for cytokine genes. We have shown that overexpression of CSD proteins in embryonic fibroblasts leads to repression of GM-CSF promoter activity linked to a reporter construct [75 ]. However, this has not yet been extended to an analysis of the endogenous GM-CSF gene. The binding of CSD proteins to the promoter regions of several cytokine genes has now been examined in detail [1 , 75 76 77 78 ]. For example, we have identified four single-strand DNA binding sites for nuclear and recombinant (YB-1, dbpA) CSD proteins across the proximal human GM-CSF promoter (Fig. 4B) . A general consensus for CSD binding sites in the genes of higher organisms has not yet been determined but it does appear that CSD proteins have a preference for CT-rich sequences [reviewed in refs. 1 10 11 ] and the GM-CSF binding sites conform to this general framework. One of the most intriguing aspects of the GM-CSF promoter study is that the spacing between the four CSD sites on the GM-CSF promoter is conserved (13bp), bringing about an ordered regularly spaced arrangement of CSD sites across the proximal GM-CSF promoter. They are arranged with two sites on one strand and two sites on the opposite DNA strand (Fig. 4B) . We have subsequently found that the IL-3 and G-CSF genes have the same arrangement of CSD binding sites across their proximal promoters (Fig. 4B) . As for the GM-CSF gene, these sites are adjacent to or overlap activator elements, some of which are in common with the GM-CSF gene (SP1, CBF, NF-{kappa}B, and CD28-responsive complex sites). This conserved spacing on several genes suggests that a particular protein:DNA structure may play a role in repression for all of these genes. The simplest interpretation is that these proteins generate a single-stranded structure capable of blocking the binding of transcriptional activators that are dependent on double-strand DNA for binding and activity (Fig. 4) . This, however, has not been shown for the GM-CSF or any other gene repressed by CSD proteins. The finding that all four CSD sites are required for maximal promoter repression [11 ] supports the model of an altered DNA conformation. Consistent with this model, CSD proteins have been shown in vitro to induce or stabilize single-strand regions within double-strand DNA containing CSD sites [58 , 78 , 79 ]. Also consistent with the above model we have observed that repression of the GM-CSF promoter absolutely requires the presence of the central CSD single-strand DNA binding domain [unpublished data]. Although it is generally believed that most promoter regions exists as double-stranded DNA, a role for CSD proteins in the regulation of gene transcription has clearly been established, and whether single-stranded binding is important remains to be determined.

There is ample evidence that the GM-CSF promoter elements that bind the CSD proteins act as repressor elements in many cell types including fibroblasts, myeloid cells, endothelial cells, and T cells [1 , 75 , 76 , 80 81 82 ]. Signaling pathways activated in different cell types may determine the ability to overcome the repressive effects of CSD proteins to allow gene activation. Consistently, the domain 2 region in the G-CSF gene has repressor activity in CHU-2 cells [83 ] and we have confirmed that overexpression of CSD proteins in fibroblasts represses G-CSF promoter activity in a reporter construct [unpublished data]. It has also been demonstrated that the regions of the IL-3 gene containing CSD sites have repressor activity in T cells [84 ]. It appears therefore that a common promoter structure may be formed across the three cytokine genes to bring about repression. It is also possible that contact with specific transcriptional activators may be involved in repression as well. For example, it has been reported that contact of CSD proteins with YY-1 and NF-Y activators may in part be involved in transcriptional repression of the grp78 and MHC class II I-Aß genes [63 , 85 ].

The mechanisms by which CSD-repressed promoters are derepressed to allow gene activation are unclear. Transcriptional activators such as NF-{kappa}B p65 and SP-1 have been shown to be able to inhibit CSD protein binding to single-strand DNA in vitro [86 , 87 ] and we have observed that NF-{kappa}B p65 can also remove recombinant YB-1 CSD protein from single-strand GM-CSF promoter DNA [unpublished data]. It is therefore possible that upon cell stimulation, that up-regulated transcription factors could remove CSD proteins from their single-strand DNA binding sites, allowing the promoter to return to double-strand form and permitting transcription factors to bind. Both NF-{kappa}B and SP-1 are key regulators of GM-CSF expression in T cells [1 ].

There is as yet no evidence for a single-strand structure across the endogenous GM-CSF gene but a structural analysis of the CSD proteins interacting with the GM-CSF promoter would greatly increase our understanding of the function of these proteins.


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CONCLUSIONS
 
The HMGI(Y) and CSD families of proteins clearly have the capacity to influence the architecture of promoter/enhancer regions of many cytokine genes. The HMGI(Y) proteins are critical for the assembly of enhanceosome complexes on the control regions of these genes. Although the importance of HMGI(Y) has been shown for endogenous gene transcription in activated primary T cells, its role in an in vivo immune response remains to be determined. This will require the conditional deletion of the gene in specific cells of the immune system. One important aspect of the CSD proteins is their preference for binding single-strand DNA. It would appear that this binding is important in their ability to repress cytokine gene transcription. It will be important to determine the structure of a cytokine promoter (e.g., GM-CSF) bound to CSD proteins to further elucidate their mechanism of action.

Received August 31, 2000; revised September 27, 2000; accepted October 3, 2000.


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