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Originally published In Press as doi:10.1074/jbc.M401258200 on February 14, 2004

J. Biol. Chem., Vol. 279, Issue 17, 17842-17849, April 23, 2004
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Factors Binding a Non-classical Cis-element Prevent Heterochromatin Effects on Locus Control Region Activity*

Faith Harrow{ddagger}, Jeanne U. Amuta{ddagger}§, Shauna R. Hutchinson||, Frank Akwaa**, and Benjamin D. Ortiz{ddagger}{ddagger}

From the Department of Biological Sciences, City University of New York, Hunter College, New York, New York 10021

Received for publication, February 4, 2004 , and in revised form, February 13, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A locus control region (LCR) is a cis-acting gene-regulatory element capable of transferring the expression characteristics of its gene locus of origin to a linked transgene. Furthermore, it can do this independently of the site of integration in the genome of transgenic mice. Although most LCRs contain subelements with classical transcriptional enhancer function, key aspects of LCR activity are supported by cis-acting sequences devoid of the ability to act as direct transcriptional enhancers. Very few of these "non-enhancer" LCR components have been characterized. Consequently, the sequence requirements and molecular bases for their functions, as well as their roles in LCR activity, are poorly understood. We have investigated these questions using the LCR from the mouse T cell receptor (TCR) {alpha}/Dad1 gene locus. Here we focus on DNase hypersensitive site (HS) 6 of the TCR{alpha} LCR. HS6 does not support classical enhancer activity, yet has gene regulatory activity in an in vivo chromatin context. We have identified three in vivo occupied factor-binding sites within HS6, two of which interact with Runx1 and Elf-1 factors. Deletion of these sites from the LCR impairs its activity in vivo. This mutation renders the transgene locus abnormally inaccessible in chromatin, preventing the normal function of other LCR subelements and reducing transgene mRNA levels. These data show these factor-binding sites are required for preventing heterochromatin formation and indicate that they function to maintain an active TCR{alpha} LCR assembly in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A locus control region (LCR)1 confers integration site-independent expression upon a linked transgene in vivo. Thus, LCR-driven transgene expression is present in all transgene positive mice and is generally copy number-dependent with predictable and consistent tissue distribution (1, 2). LCRs are usually composed of a group of linked DNase I hypersensitive sites (HS) that synergize in a manner that is very poorly understood (3). Most LCRs contain at least one subelement that supports a "classical" transcriptional enhancer activity. Although some enhancers have recently been shown to support a variety of activities that affect transcription (4, 5), enhancer elements remain united by the ability to directly affect the activity of RNA polymerase, leading to modulation of transcription levels (6, 7). This ability allows these elements to be assayed in the kind of transient reporter gene transcription assays that are still commonplace in studies of gene regulation. However, it has become clear that the activity of such classical enhancer elements does not completely explain the complexities of LCR activity (8).

A small number of important functional LCR subelements that are devoid of classical enhancer activity have been described (9-12). The sequence requirements and molecular bases of the function of such "non-classical" cis-elements, as well as their roles in LCR activity, remain largely uncharacterized. This is partially because of the low number of complete bona fide LCRs identified and the even lower number of such LCRs that have been studied in mechanistic detail. This latter subset includes the LCRs of the human {beta}-globin (13), human CD2 (14), human growth hormone (15), and the mouse T cell receptor (TCR) {alpha} encoding gene loci (11, 12).

We studied the LCR derived from the mouse TCR{alpha} genomic locus (see Fig. 1A) (16). This gene locus contains three differentially expressed genes, TCR{alpha}, TCR{delta}, and Dad1 (17). The TCR{alpha} and TCR{delta} genes encode subunits of the antigen receptors on {alpha}{beta} and {gamma}{delta} T cells, respectively. The expression of these genes is normally restricted to T cells. The ubiquitously expressed Dad1 gene encodes a subunit of an oligosaccharyl transferase enzyme involved in N-linked glycosylation (18). The LCR is flanked by the TCR{alpha} and Dad1 genes and is composed of nine HS dispersed over 12 kb (see Fig. 1A). HS1 contains the TCR{alpha} classical enhancer element (19, 20). HS1' is just 3' of HS1 and plays a major role in the tissue specificity of LCR activity (11). HS2-HS6 lie 3' to HS1'. None of the HS 3' of HS1 contain any classical enhancer activity (19). HS7 and HS8 lie 5' to HS1 and do not appear to significantly contribute to LCR activity (11). HS1 and HS1' comprise the main "tissue specificity determinants" of LCR activity (see Fig. 1B) (11). When these sites are removed from the LCR, the remaining HS (HS2-6) drive widely active integration site-independent transgene expression (11, 21). HS6 was subsequently found to contain the bulk of this widespread activity (12).



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  		FIG. 1.
The DNase hypersensitive sites of the TCR{alpha} LCR. A, scale diagram of the 3'-end of the TCR{alpha}/Dad1 genomic locus with the nine LCR hypersensitive sites (HS) indicated as vertical arrows. Horizontal arrows indicate the transcriptional orientations of the TCR{alpha} and Dad1 genes. Dark boxes indicate exon sequences. C{alpha}, TCR{alpha} constant region exons; E{alpha}, TCR{alpha} classical transcriptional enhancer region. B, transgene constructs used in this study. Numbers indicate the HS of the LCR included in the construct and linked 3' of the human {beta}-globin reporter gene fragment. The filled box indicates the BclI-PstI region within HS6 that is deleted in the mutant transgene. This region contains the TF1, TF2, and TF3 factor-binding sites identified previously by in vivo footprinting (46). The sequences of these footprint regions are shown.

 
When a 1.8-kb fragment containing HS6 is linked alone to a reporter transgene, its position-effect-suppressing activity is evident in all tissues of transgenic mice examined (12). However, in the context of the full LCR, HS6 appears to be active only in lymphoid organs (11, 21). We have hypothesized that HS6 activity is modified by TCR{alpha} proximal upstream elements (mainly HS1') to establish the tissue-specific activity of the TCR{alpha} LCR. This hypothesis was supported by in vivo footprint data. These experiments revealed three factor-binding sites (called thymic footprints (TF)) in HS6 that were named TF1, TF2, and TF3 (collectively referred to as the TF1-2-3 region). TF1 binds an as yet unidentified tissue-unrestricted factor, whereas TF2 and -3 bind complexes that contained the tissue-restricted proteins AML-1/Runx1 and Elf-1, respectively. TF2 and -3 are occupied only when the tissue specificity elements of the LCR are linked in cis, suggesting that factors binding to these sites are important for the ability of HS6 to contribute to LCR activity (12). However, the roles of the TF1-2-3 sequences in the functions of HS6 and the TCR{alpha} LCR are unknown.

Here we present data demonstrating the important functional contribution of the TF1-2-3 region DNA to the activity of the TCR{alpha} LCR in vivo. Although the TF1-2-3 region does not appear to contribute to the widely active function of HS6 as modeled in stable transfected fibroblasts, internal deletion of these sites from an LCR-driven transgene results in a marked reduction in transgene expression levels/copy in all independent transgenic mouse lines examined. DNase hypersensitivity analyses of these mutant transgenes indicate that this phenotype is associated with the formation of an abnormally inaccessible chromatin configuration at the mutant transgene locus. These data reveal a role for TF1-2-3 binding factors in regulating long range chromatin structure. This activity is required for maintaining the function of the TCR{alpha} LCR in T cells in vivo.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reporter Transgene and Plasmid Constructions—Construction of the 12.3-kb {beta}:1-6 transgene has been described previously (11). To construct the {beta}:1-6{Delta}TF123 transgene, a deletion mutant of the 238-bp region within HS6 that includes the sites TF1, -2, and -3 was created as follows. A 622-bp BclI-MluI fragment was excised from the previously described pSP72:{beta}:1-6 vector and replaced with a 384-bp PstI-MluI fragment that represents the 3'-portion of the initial deletion. The effective deletion is a 238-bp BclI-PstI fragment. XhoI and ClaI were used to liberate the transgene from the vector prior to microinjection.

The pTHCYFP reporter vectors used to generate stable transfectants of NIH3T3 mouse fibroblasts were constructed as follows. A cytomegalovirus promoter-driven yellow fluorescent protein (YFP) reporter gene (Clontech) was subcloned into the NotI and EcoRV sites of the pBlue-script KS+ vector (Stratagene). A hygromycin resistance gene driven by a herpes simplex virus thymidine kinase promoter was inserted into the ApaI and XhoI sites of this vector. A 1.8-kb MfeI-SacI HS6-containing fragment of the TCR{alpha} LCR was inserted 3' of the YFP reporter gene to generate the pTHCYFP-HS6 construct. The pTHCYFP-HS6{Delta}1.3 derivative contains a 1.3-kb BlpI deletion within the HS6-containing fragment. The pTHCYFP-HS6{Delta}TF123 construct contains the same 238-bp BclI-PstI deletion described above, whereas the pTHCYFP-HS6{Delta}316 construct contains a 316-bp PstI-BglII deletion just 3' of the TF1-2-3 region.

Transgenic Mice—DNA fragments for microinjection were doubly purified by gel electrophoresis on low melting point agarose (Seaplaque) followed by digestion with {beta}-agarase (New England Biolabs, Beverly, MA). DNA fragments were microinjected into the male pronucleus of (C57BL x CBA) F1 fertilized mouse eggs and transferred into pseudo-pregnant female mice. Transgenic founders were identified using polymerase chain reaction and/or Southern blot analyses of the DNA derived from ear-punch or tail tip biopsy tissue. Transgenic founders were outcrossed to C57BL mice (Taconic, Germantown, NY), and transgene heterozygous offspring were analyzed. The relative transgene copy number was determined by PhosphorImager (Molecular Dynamics) analyses of Southern blots. For all new lines described here, the relative copy number was determined from tail DNA samples from at least two individuals/line. These samples were analyzed simultaneously using the same restriction enzymes and hybridized to the same probe preparations on the same Southern blot. A representative sample of the previously analyzed {beta}:1-6 lines was always included in these Southern blots to enable us to relate the copy number estimates for the previously analyzed {beta}:1-6 lines to those of the new {beta}:1-6{Delta}TF123 transgenic lines. The probes and restriction enzyme digestions were designed to allow simultaneous detection of the transgene locus as well as a non-overlapping fragment of the endogenous TCR{alpha} locus. Transgene signals were then normalized to the endogenous TCR{alpha} locus signal to control for sample loading variation.

RNA Analyses—RNA was prepared according to the single step RNA isolation protocol (22) from mouse tissues that were dissected of fat, washed with phosphate-buffered saline to minimize contamination with blood, and minced. Thymus and spleen tissues were disrupted between frosted glass slides. Five µg of total RNA/sample were run on a 0.8% agarose gel and transferred to nylon membrane for Northern blot analyses. A 428-bp BamHI-NcoI genomic fragment of human {beta}-globin gene coding region was used as a probe for the mRNA product of the transgene. To normalize for variations in sample loading, blots were stripped and reprobed with either a 500-bp Sau3AI fragment of the TCR{alpha} constant region cDNA or a probe to 18 S rRNA (Ambion, Austin, TX). All probes were labeled with [{alpha}-32P]ATP using the random primers method (Invitrogen). Transgene mRNA signals were normalized to the loading control signal and quantified by PhosphorImager analysis (Molecular Dynamics).

DNase I Hypersensitivity Assays—Thymocyte nuclei from {beta}:1-6{Delta}TF123 transgenic mice were prepared as described previously (21) and resuspended in DNase digestion buffer at 6 x 107 nuclei/ml. Samples were treated on ice for 10 min with increasing amounts (0-1.25 µg/2 x 107 nuclei) of DNase I (Worthington), and reactions were stopped with 1/10 volume of 5% SDS/100 mM EDTA. Genomic DNA was purified after protease K treatment (200 µg/ml overnight at 55 °C) and phenol/chloroform extraction. For analysis of hypersensitivity at the transgene locus, DNase I-treated samples were digested with SwaI and SacI to generate a 9.2-kb parent fragment of the transgene. Separate aliquots of the same DNase I-treated samples were digested with NheI to generate an 11-kb parent TCR{alpha} locus fragment for analysis of hypersensitivity at the endogenous TCR{alpha} gene. Samples were run on 0.8% agarose and transferred to nylon membrane for Southern blot analysis. The transgene locus fragment was probed from the 5'-end with a 547-bp SwaI-PstI human {beta}-globin fragment. The endogenous TCR{alpha} locus fragment was also probed from the 5'-end with a 1.2-kb NheI-XhoI TCR{alpha} locus fragment not present in the transgene.

Fibroblast Transfections—NIH3T3 mouse fibroblasts were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1% penicillin/streptomycin, 1% Glutamax (Invitrogen). Using LipofectAMINE Plus (Invitrogen) and following the manufacturer's instructions, the linearized pTHCYFP-HS6 construct, and deletion mutants thereof, were transfected into NIH3T3 cells. Cells were transferred to selective medium containing 0.25 mg/ml hygromycin 24-48 h following transfection. Individual hygromycin-resistant colonies emerged after 14-21 days of selection. The YFP expression phenotype of each individual colony in the live cultures was assessed on an inverted phase contrast/fluorescence microscope.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Assessing LCR Activity Using Randomly Integrated Reporter Transgenes in Mice—For many years we (11, 12, 21) and others (1, 2, 23, 24) have used a 4.9-kb fragment of the human {beta}-globin locus as a reporter gene for testing LCR activity in transgenic mouse studies. This fragment, which contains the {beta}-globin promoter, exons, introns, and a 3'-enhancer, is highly susceptible to integration site-dependent position effects in the absence of additional control elements (25, 26). Each transgenic line carrying a given construct represents an assay of the activity of that construct at a random position of integration in the genome. A position effect occurs when sequences at a particular integration site either silence or otherwise alter the pattern and level of gene expression from the construct. This is a common confounding variable in transgenic studies (27). The activity of a complete intact LCR eliminates these position effects, thus yielding predictable expression patterns with mRNA levels in proportion with the number of integrated transgene copies.

Fig. 1B shows a diagram of the previously analyzed {beta}:1-6 transgene that contains the human {beta}-globin fragment linked 5' of DNase hypersensitive sites 1-6 of the TCR{alpha} LCR. These seven HS (see Fig. 1A) support complete LCR activity (11). The {beta}:1-6 transgene is expressed in a copy number-dependent manner consistent with T-lineage specificity independent of the site of integration. Using this transgene as a basis, we engineered a 238-bp internal deletion in the LCR of this transgene. The deleted region contains the in vivo factor-occupied sites TF1, -2, and -3 identified within the HS6 region of the LCR (12). This mutant LCR-driven transgene was named {beta}:1-6{Delta}TF123 (Fig. 1B). Five independent lines of transgenic mice were generated with this construct. By comparing the activity of the mutant LCR to that of the wild type intact LCR, we aimed to discern the functional contribution of the TF1-2-3 region to LCR function in vivo.

The {Delta}TF123 Mutation Reduces Transgene Expression Levels/Copy—We examined the mRNA expression levels generated by the {Delta}TF123 mutant LCR in comparison to that generated by the wild type LCR. Northern blot experiments (Fig. 2A) were performed including all five independent lines of {beta}:1-6{Delta}TF123 mice and two representative lines of the previously analyzed {beta}:1-6 transgenic mice (11). Reporter transcript levels were examined in the thymus and spleen. PhosphorImager analyses (Fig. 2B) of the experiments shown in Fig. 2A were then conducted. The human {beta}-globin mRNA signal was normalized to that obtained with a probe to TCR{alpha} mRNA. This value was then divided by the relative copy number estimates.



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  		FIG. 2.
Deletion of the TF1-2-3 region reduces the level of reporter transcript/transgene copy. A, Northern blot analyses of thymus RNA from the indicated transgenic lines. The {beta}-globin probe detects the transgene product. The TCR{alpha} probe serves as a loading control. The five mutant transgene lines (line numbers left to right, 3, 5, 6, 30, 33) were compared with two wild type transgenic lines representative of those reported previously (45). B, Northern blot analyses of spleen RNA from the same lines as in A. C, PhosphorImager analyses of the Northern blots shown. The {beta}-globin signal is normalized to the TCR{alpha} signal for each lane. This figure was then divided by the copy number and graphed. Each filled circle represents an independent transgenic line carrying the indicated construct. With the exception of the spleen in a single line (line 3), all mutant transgene lines express lower levels of transcript/copy than the wild type transgene lines.

 
The data show that in the thymus, removal of the TF1-2-3 region resulted in a marked reduction of transgene activity. Transgene expression levels/copy in the thymus of mutant LCR lines are 2.0-5.1-fold lower than those seen in the wild type. The magnitude of this effect in the thymus is similar to that observed when the TCR{alpha} classical enhancer (HS1) is deleted from the LCR (11). Additionally, in four of five {beta}:1-6{Delta}TF123 lines, spleen mRNA levels/copy are severely reduced (3.4-17-fold) in comparison to the wild type. The exceptional line is {beta}:1-6{Delta}TF123 line 3. However, unlike the other four lines that show a tissue distribution typical of wild type LCR-driven transgenes (Fig. 3), line 3 displays a severely abnormal tissue distribution. Normally, transgenes under the control of the wild type TCR{alpha} LCR are most highly expressed in the thymus of transgenic animals. Expression in the spleen is typically between 10 and 30% of the level of thymic expression. In the non-lymphoid organs, transgene activity is very low (less than 10% of thymic expression levels) (11, 21). In contrast, in line 3 (Fig. 3A, stippled bars), the spleen was the highest expressing organ, and thymic expression was only 13% of spleen, a level comparable with those seen in non-lymphoid organs. Therefore, we surmise that the apparent "wild type" levels of transgene expression observed in the spleen of line 3 are results secondary to a severe positive position effect in the spleen of this line. From these observations we can conclude that the absence of TF1-2-3 region sequences reduces the ability of the TCR{alpha} LCR to consistently drive high level expression in the lymphoid organs.



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  		FIG. 3.
Varying tissue distribution of {beta}:1-6{Delta}TF123 transgene mRNA among independent lines. A, Northern blot analysis of the indicated tissues of three {beta}:1-6{Delta}TF123 lines. The {beta}-globin probe detects the transgene product. The 18 S probe is used as a loading control. B, graph of PhosphorImager analyses of all five independent {beta}:1-6{Delta}TF123 lines. {beta}-Globin signal was normalized to that detected by the probe to 18 S rRNA. To facilitate presentation on the same graph, for each line, the organ expressing the highest level of transgene mRNA was designated as 100% of maximum expression. Note that expression of transgene in the spleen of line 3 (stippled bars) is much higher, relative to thymic expression, than one would expect from wild type LCR-driven reporter gene. In contrast, the other lines display tissue distributions that resemble more the wild type LCR-driven transgene.

 
The {beta}:1-6{Delta}TF123 Transgene Locus Exists in an Abnormally Inaccessible Chromatin Configuration—The DNase hypersensitive assay is used as an indication of chromatin structure at a given locus (28). We performed this assay on thymocyte nuclei derived from {beta}:1-6{Delta}TF123 transgenic lines. We compared the hypersensitivity pattern at the transgene locus with that observed at the endogenous TCR{alpha} LCR in the same cells. We have shown previously that wild type LCR-driven transgenes display a normal DNase hypersensitivity pattern that is similar to that seen at the endogenous TCR{alpha} LCR (11, 21). Fig. 4A shows the DNase hypersensitivity at the endogenous locus of {beta}:1-6{Delta}TF123 line 5. Here, it is clear that the DNase titration went to completion in this experiment. The parent fragment is no longer visible at higher DNase I concentrations. Moreover, we observed bands that represent all of the hypersensitive sites that normally form at this locus. To examine the chromatin structure of the transgene locus in these same cells, separate aliquots of DNA from the same DNase-treated nuclei were digested with restriction enzymes to generate an appropriate fragment of the transgene. The blot was then hybridized with a probe specific for the transgene. In contrast to the normal conformation at the endogenous locus, DNase hypersensitivity at the transgene appeared to be compromised (Fig. 4B). The parent fragment persists even at high DNase I titration points. The absence of a band representative of HS6 was not unexpected as the TF1-2-3 deletion is within this hypersensitive site. Interestingly, HS4 also is not observed, and the formation of HS1 and HS1' is significantly impaired. This indicates that the vast majority of transgene copies at the integration site are in an abnormal inaccessible chromatin configuration. Similar observations were made in a second transgenic line (data not shown). Thus in contrast to the classical enhancer in HS1, which has no apparent role in controlling chromatin structure (11), removal of the TF1-2-3 region prevents the LCR from establishing the normal accessible chromatin state at its locus of integration. This in turn seems to prevent the function of the other LCR subelements. These observations may explain why expression levels/copy driven by the mutant LCR are lower than wild type.



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  		FIG. 4.
The {beta}:1-6{Delta}TF123 transgene locus is relatively inaccessible to DNase I. A, DNase hypersensitivity assay of the endogenous TCR{alpha} LCR locus. The genomic DNA on this blot was cut with NheI and probed with endogenous TCR{alpha} locus sequences not present on the transgene. Here, the restriction fragment is being probed from the 5'-end. B, DNase hypersensitivity assay of the {beta}:1-6{Delta}TF123 transgene locus in line 5. The DNA for this blot was cut with SwaI and SacI and was probed with a human {beta}-globin fragment recognizing the 5'-end of the restriction fragment examined. Samples analyzed in both panels are aliquots of the same DNase-treated samples. The blot shown in A demonstrates that the DNase titration went to completion in this experiment with efficient formation of all expected HS of the LCR (arrows). In contrast the broken arrows in B indicate missing hypersensitive sites (HS) in the mutant LCR-driven transgene in the same cell population. Furthermore, formation of HS1 (the TCR{alpha}-enhancer site) is impaired. Similar results were obtained in a second transgenic line (line 6).

 
The Widespread Activity of HS6 DNA Can Be Modeled in Cell Culture—The above data indicate that sequences of HS6 do indeed participate in the activity of the tissue-specific TCR{alpha} LCR in vivo. However, HS6 DNA, when linked by itself to a reporter gene, also displays a position-effect-suppressing activity that is not tissue-restricted (12). We next investigated the role of TF1-2-3 DNA in the widespread functions of HS6. To accomplish this in a more rapid manner than is possible with transgenic mouse models, we developed a reporter vector for assessing the activity of HS6 in stable transfected cells. We reasoned that the widely active function of HS6 would manifest itself in any cell line. Therefore, we decided to begin these experiments with NIH3T3 mouse fibroblasts.

The basic vector we constructed (named pTHCYFP) contains a YFP reporter gene driven by a cytomegalovirus promoter (Fig. 5A). It also contains a gene that confers resistance to hygromycin under the control of a herpes simplex virus thymidine kinase promoter. In the pTHCYFP-HS6 construct, a 1.8-kb HS6 fragment is linked 3' of the YFP reporter. This is the same HS6 fragment that was shown previously to be functional in vivo (12). In contrast, the pTHCYFP-HS6{Delta}1.3 contains a severely truncated HS6 fragment missing a 1.3-kb region in between two BlpI sites (Fig. 5A). On the hypothesis that the widely active sequences would be found within this large region, we used this latter construct as a negative control for HS6 activity. The constructs were linearized as shown in Fig. 5A such that the HS6 variant is proximal to the YFP gene and distal from the hygromycin resistance gene. Because LCR activity appears to favor the gene nearest to it (29-32), we reasoned that HS6 would be more likely to affect YFP activity than hygromycin resistance. This linearizing scheme also minimizes the likelihood of transcriptional interference by ensuring that the two transcription units would be separated by several kilobases of vector DNA and be in divergent transcriptional orientations (33). These constructs were transfected into NIH3T3 fibroblasts to generate individual stable transfected colonies. Indeed, no significant difference is seen in the number of hygromycin-resistant colonies generated by the test constructs indicating that HS6 does not measurably affect the incidence of hygromycin resistance in this assay. These results allow the number of hygromycin-resistant colonies to be used as a normalizing control in assessing the effect of HS6 DNA on the efficiency of YFP expression.



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  		FIG. 5.
HS6 DNA suppresses position effects in transfected fibroblasts. A, diagram of the yellow fluorescence protein (YFP) reporter vectors used to establish a cell culture system for the widespread activity of HS6. The constructs are shown as linearized prior to transfection. The HS6{Delta}1.3 fragment represents a 1.3-kb deletion within HS6 DNA. The arrows indicate the transcriptional orientations of the YFP and hygromycin resistance (HygroR) genes. TKp, thymidine kinase promoter; CMVp, cytomegalovirus promoter; pBS indicates the position of 3 kb of Bluescript vector sequences separating the two transcription units. B, microscopy of NIH3T3 mouse fibroblast colonies that are representative of the three YFP expression phenotypes observed in stable transfection experiments. Colonies were grown in selective medium for 14 days prior to fluorescence microscopy. C, graph of data from three representative experiments showing the percent incidence of the YFP expression phenotypes among the stable transfected colonies. The negative or variegating plot represents the sum of both phenotypes in each data set. A total of 468 HS6 colonies and 449 HS6{Delta}1.3 colonies were counted in these three experiments. Error bars represent the variation in the percent incidence of the indicated YFP expression phenotype between the three experiments.

 
In contrast to its apparent lack of effect on hygromycin resistance, HS6 does affect the expression of the more proximal YFP gene. Three YFP expression phenotypes are seen among the colonies: positive, negative, and variegating (Fig. 5B). In positive colonies, nearly all of the cells express YFP. In negative colonies, none of the member cells express YFP. The variegating phenotype occurs when only a subset of scattered cells within a colony is YFP-positive. Both the negative and variegating phenotypes would indicate the inability of the YFP transcription unit to consistently maintain expression at its site of integration. The presence of intact HS6 sequences on the construct (pTHCYFP-HS6) lowers the incidence of YFP-negative and variegating colonies from that which results from transfection of the mutant HS6 containing construct (pTHCYFP-HS6{Delta}1.3). Fig. 5C shows a graph of results from three separate experiments. The incidence of negative colonies is reduced 6.8-fold on average, and the incidence of variegation is reduced 3.3-fold. Overall, the incidence of variegating and/or negative colonies is reduced over 4.3-fold. These data come from scoring 468 HS6 colonies and 449 HS6{Delta}1.3 colonies in total. In short, the deleted sequences within HS6 decrease the probability of reporter gene silencing by position effects at a random site of integration. This activity is consistent with that which HS6 displays in vivo (12).

Sequences Suppressing Position Effects in Fibroblasts Reside Outside the TF1-2-3 Region—We next wanted to determine the sequences within HS6 responsible for its widespread activity, as modeled in this fibroblast assay. We created two more deletion mutants in the HS6 fragment (Fig. 6A). The first of these, the HS6{Delta}TF123 mutant, contains the identical 238-bp BclI-PstI deletion of the TF1-2-3 region that was studied in transgenic mice. The second deletion, the HS6{Delta}316 mutant, removes a 316-bp PstI-BglII fragment just 3' of the TF1-2-3 region. These HS6 variants were inserted into the pTHCYFP vector. These vectors were linearized as shown in Fig. 6A and used to generate stable transfectants of NIH3T3 fibroblasts. We used the pTHCYFP-HS6 and pTHCYFP-HS6{Delta}1.3 constructs as positive and negative controls, respectively.



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  		FIG. 6.
A 316-bp region of HS6 (but not the TF1-2-3 region) is required for the position-effect-suppressive activity of HS6 DNA in cultured fibroblast transfectants. A, diagram of the HS6 internal deletion mutants tested in the pTHCYFP vector. B, graph of data from three representative transfection experiments using the HS6 fragment or the indicated deletion mutant. Stable transfected colonies were scored using fluorescence microscopy. The percentages reported indicate the total of hygromycin-resistant colonies that displayed either a YFP negative or variegating phenotype. The data represent scoring of the following total numbers of colonies: HS6 (202), HS6{Delta}TF123 (217), HS6{Delta}316 (160), HS6{Delta}1.2 (186). The experiments in this graph are totally separate from the experiments represented in the graph in Fig. 5.

 
The results of three separate experiments are shown in Fig. 6B. In this set of experiments, the negative control HS6{Delta}1.3 fragment produced a 5.5-fold higher total of negative and variegating colonies than the positive control HS6 fragment. These experiments are completely separate from those shown in Fig. 5C. Removal of the TF1-2-3 region from HS6 had no apparent effect on the incidence of negative and variegating colonies. The activity of the {Delta}TF123 mutant fragment is indistinguishable from the intact HS6 positive control. This would indicate that TF1-2-3 region sequences do not function in fibroblasts. In contrast, a deletion of 316-bp just 3' of the TF1-2-3 region resulted in a 3.5-fold increase in the incidence of negative and variegating colonies over that obtained with wild type HS6. These data indicate that the TF1-2-3 region does not participate in the widespread function of HS6 and that this activity is supported by different sequences outside the HS6 core.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Despite over a decade and a half of investigation, the LCR mechanism of action remains largely a mystery. This is partially because of the low number of LCRs that have been studied in-depth, and the very time consuming and expensive technology typically employed to dissect LCR activity. There have been two major experimental approaches to structure/function studies of LCRs: targeted alteration of LCR sequences in endogenous loci and LCR-driven transgenic reporter genes. The former approach assesses the non-redundant role of LCR sequences in the context of its locus of origin. The latter technology asks what the LCR is capable of at an ectopic site of integration in the genome. Answers to both questions are of great significance to the field of gene regulation.

Although results from some gene targeting studies have questioned the role of an LCR in regulating chromatin in its native locus (34, 35), analyses of LCR-driven transgenes at ectopic integration sites have strongly supported the hypothesis that these elements somehow overcome heterochromatin-induced position-effect variegation that would silence a transgene at a subset of integration sites (14). They are not seen to alter the rate of transcription initiation per se, as would a classical enhancer element such as a promoter or enhancer. As such, LCRs contain "non-classical" functional components that are key to their activity. The investigation of enhancer-like (15) and even promoter-like elements (36, 37) associated with LCRs has given some insight into the LCR mechanism of action. Similarly, investigation of non-classical LCR subelements is of great interest. In addition to their apparent importance to LCR activity, these LCR subelements may represent novel cis-acting elements in their own right with important functions in the genome outside of LCRs. Here we have further characterized HS6, an important non-enhancer component of the TCR{alpha} LCR. Our data have linked the activity of identified factor-binding sites within HS6 to the prevention of heterochromatin formation and TCR{alpha} LCR function in vivo.

The TF1-2-3 Region Is Required for LCR Activity in Vivo—Removal of the TF1-2-3 region of HS6 from the LCR subjects the transgene locus to the formation of abnormally inaccessible chromatin and impairs its ability to consistently drive high level expression of a linked transgene in lymphoid organs. Given that accessible chromatin structures in the TCR{alpha} (38) and other gene loci (39) precede active transcription, this indicates a role for the TF1-2-3 region in the attraction of regulatory mechanisms combating the formation of chromatin structures that are non-permissive for gene expression. AML-1/Runx1 and Elf-1 transcription factors have been found to interact with TF2 and TF3 sequences, respectively. Elf-1 is an enhancer-binding protein important for the regulation of many T cell expressed genes and is expressed at all stages of T cell development (40). Runx1 has been implicated in lymphocyte gene enhancer function (41) as well as lineage-specific gene silencing (42). These factors have not previously been implicated in LCR activity. How might these factors participate in the regulation of chromatin structure? Elf-1 is a member of the Ets winged helix family of proteins. The winged helix domain is shared by proteins involved in nucleosome positioning such as linker histones and the HNF3{alpha} transcription factor (43). Runx family proteins have been shown to interact with histone-modifying complexes (41, 44). Mechanisms involving the alteration of histone modification patterns have been associated with LCR activity in some (15, 36, 45) but not all (35, 46) cases. It is possible that normal histone modification and/or nucleosome phasing patterns might be perturbed by TF1-2-3 deletion with consequent effects on chromatin structure and gene expression. Using individual binding site mutants, we will be investigating these possibilities in light of the recently described histone code hypothesis (47). DNA methylation has been molecularly linked to histone deacetylation and chromatin closure (48, 49). We have identified previously a discrete region of the LCR (near HS4) that is selectively demethylated in LCR-driven transgenes and the endogenous locus (50). However, we did not detect any effect of the TF1-2-3 deletion on this selective DNA demethylation (data not shown).

Tissue-unrestricted Position-Effect-suppressing Sequences of HS6—We have developed a cell culture model for the widely active position-effect-suppressing function of HS6 using a YFP reporter gene and fibroblasts. In this assay we score for YFP expression phenotypes in a background of hygromycin-resistant colonies. Intact HS6 DNA reproducibly lowers the incidence of YFP-negative and -variegating phenotypes among stable transfected colonies. The system we devised to study position-effect-suppressing elements such as HS6 adds to the somewhat limited array of cell culture approaches that have been utilized to study the activity of LCRs. These include the use of recombination-mediated cassette exchange (51, 52), targeted deletion of endogenous sequences in cell lines (53), stable integrated antibiotic resistance markers (54), and cell fusion (55). Continued development of such assays may provide alternatives to time-consuming and expensive transgenic animal models.

We have utilized our assay to determine that the TF1-2-3 region of HS6 does not participate in the widely active function of HS6 in fibroblasts. The Runx1 and Elf-1 factors are not present in fibroblasts. Our results would indicate that other TF1-2-3 binding factors that are present in fibroblasts (12) are not functional in this assay. Furthermore, the TF2/Runx1 and TF3/Elf-1 sites are not occupied unless HS6 is linked in cis to HS1' of the LCR (12). Therefore, context dependence and tissue specificity are both likely to be key aspects of the function of this element.

In contrast, a 316-bp region was found to be required for the position-effect-suppressing function of HS6 in fibroblasts. This finding indicated two very important things about our cell culture reporter system. First, it demonstrated that this assay is robust enough to screen for functional sequences within a position-effect-suppressing element. Second, it showed that the position-effect-suppressive activity of HS6 we observe in this assay is extremely unlikely to be because of a nonspecific "spacer DNA"-type effect. The {Delta}TF123 and {Delta}316 mutant fragments of HS6 only differ from each other by 78 bp (less than 5%). Yet, the former fragment is functional, whereas the latter is significantly impaired. Because the HS6{Delta}1.3 construct still contains roughly 0.5-kb of the HS6 region sequence, other functional regions may yet be found within the 1.8-kb HS6 fragment.

The 316-bp region contains DNA outside of the HS6 core that had been sequenced previously (21). Therefore we present the sequence of this newly identified functional region in Fig. 7. We examined the DNA for sites of factor binding that might be involved in its function. We reasoned that binding sites that appear in the 316-bp region that do not appear in the {Delta}TF1-2-3 region would be the most plausible candidates for involvement in the activity we observe. Using MacVector software, we detected two binding sites for the SP-1 transcription factor. Binding sites for this factor have also been implicated in the activity of the non-enhancer HS3 component of the {beta}-globin LCR (56). We also detected binding sites for two inducible transcription factors, AP-1 and NF-IL6. These factors are usually associated with activation-induced expression and have not been implicated in the action of elements that suppress position effects. However, an example of a transcriptional activator element with some position-effect-suppressing capacity has been described (57). Thus, a role for these factors in the activity of the non-enhancer HS6 element is plausible. Naturally, these speculations will need to be tested in future deletion mutagenesis experiments. Moreover, future in vivo footprinting experiments will perhaps be more important in pointing out which sequences within the 316-bp region of HS6 are most likely to be responsible for its activity. As ubiquitous factors have been implicated in the activity of the human CD2 (10) and human {beta}-globin (56) LCRs, it is also possible that the 316-bp region that functions in fibroblasts has a role to play in TCR{alpha} LCR activity in vivo. This possibility will also be tested in future experiments.



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  		FIG. 7.
DNA sequence of the newly identified 316-bp functional region of HS6. MacVector 7.0 software was used to detect consensus recognition sequences for transcription factors in the {Delta}316 region of HS6 that do not appear in the neighboring {Delta}TF123 region. The MacVector 7.0 subsequence file used was TFDSites.SUBSEQ (GenBankTM accession number AY54598).

 

   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY54598.

* This work was supported by National Science Foundation Career Award MCB-0236964, the Professional Staff Congress of City University of New York Research Awards Program, the Hunter College Foundation, and National Institutes of Health Grant AI-053050 (to B. D. O.). This work was also supported by the Support for Continued Research Excellence (SCORE) program funded by the National Institutes of Health Grant GM-60654 to Hunter College. Support of the infrastructure and instrumentation in the Department of Biological Sciences at Hunter College was provided by the Research Centers in Minority Institutions (RCMI) Award RR-03037 from the National Center for Research Resources (NCRR) of the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Supported by National Institutes of Health Grant GM-060665 for the Research Initiative for Scientific Enhancement (RISE) program. Back

§ Present address: Albert Einstein College of Medicine, Bronx, NY 10461. Back

Supported by National Institutes of Health Grant GM-007823 for the Minority Access to Research Careers program. Back

|| Present address: Tufts University School of Medicine, Boston, MA 02111. Back

** Present address: University of Rochester School of Medicine, Rochester, NY 14642. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Biological Sciences, City University of New York, Hunter College, 695 Park Ave., Rm. 927-N, New York, NY 10021. Tel.: 212-772-5670; Fax: 212-772-5227; E-mail: ortiz{at}genectr.hunter.cuny.edu.

1 The abbreviations used are: LCR, locus control region(s); TCR, T cell receptor; HS, hypersensitive site; TF, thymic footprint, YFP, yellow fluorescence protein. Back


   ACKNOWLEDGMENTS
 
We thank D. Brazill, S. Knirr, and D. Marotta for helpful comments on this manuscript and the Rockefeller University Transgenic Service Laboratory for the generation of transgenic mice.



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 EXPERIMENTAL PROCEDURES
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