©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Role of Intron I in Expression of the Human Factor IX Gene (*)

(Received for publication, August 16, 1994; and in revised form, December 28, 1994)

Sumiko Kurachi Yoshinori Hitomi Midori Furukawa (§) Kotoku Kurachi (¶)

From the Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan 48109-0618

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The first intron (intron I) of the human factor IX gene, which has been previously suggested of having an expression-augmenting activity, was systematically studied for its potential enhancer activity. When tested with the chloramphenicol acetyltransferase expression vector with a minimal factor IX promoter, subregions of intron I showed only marginal enhancing activities (1.7-1.9-fold enhancement at the highest). Smaller subregions encompassing nucleotides 5660-6350 of the intron sequence even showed some weak negative regulatory activities (50% suppression at the highest), while a cytomegalovirus enhancer sequence, which was used as the positive control, had a 7-fold enhancement. A set of three factor IX minigene expression vectors with the same factor IX promoter were then constructed: p-416FIXc which contained the factor IX cDNA, p-416FIXm1 which contained the factor IX cDNA with a largely truncated intron I, and p-416FIXm2 which contained the factor IX cDNA with the intron I sequence further truncated. The p-416FIXm1 and p-416FIXm2 constructs showed 7-9-fold higher expression activities than p-416FIXc. The elevated factor IX antigen levels agreed well with the grossly elevated factor IX clotting activity and mRNA levels. These results indicate that the expression enhancing activity of intron I is not due to specific enhancer elements present in the intron subsequences, but is due to functional splicing sequences present in the precursor mRNAs produced from the minigene constructs containing intron I. By being efficiently assembled into spliceosome complexes, transcripts with splicing sequences may be better protected in the nucleus from random degradations than those without such sequences.


INTRODUCTION

Factor IX plays a critical role in the middle phase of blood coagulation(1) , and its deficiency results in hemophilia B (Christmas disease), an abnormal bleeding disorder. Because of several fascinating characteristics of the factor IX gene regulation, such as abnormal regulations which are represented by the Leyden phenotype mutant genes (1, 2) , factor IX gene has been extensively studied in recent years (3, 4, 5, 6, 7, 8) . Like most other mammalian genes, expression of the factor IX gene is regulated by a complex mechanism requiring multiple cis-acting elements which are present not only in the 5`-flanking region at proximal as well as distal locations, but also within various structural regions of the gene including 5`- and 3`-untranslated regions (UTRs) (^1)in addition to introns(1) . The maximal promoter activity of the human factor IX gene is contained within the minimal 5`-flanking region approximately up to nt -400(3, 4, 9) , with a 5` upstream major transcription start site at or near nt -176 in human liver(9) . These elements, which interact with various trans-acting factors in specific contexts, are responsible directly or indirectly for the regulation of this gene(1) . Hemophilia B Leyden is a unique phenotype which shows a unique late-onset amelioration of abnormal bleeding. The Leyden-specific region (LS region) containing all the known mutations of the Leyden phenotype of hemophilia B is located in a region approximately spanning nt +20 to -40 within the 5`-UTR(1, 9) .

Jallat et al.(10) recently reported a systematic analysis of various factor IX minigenes, constructed with and without various introns, on recombinant human factor IX expression levels in transgenic mice. In this study, the transgene construct containing only the factor IX cDNA expressed factor IX at a non-detectable level, while factor IX minigenes containing either all eight introns with various truncations of their middle portions or only intron I expressed factor IX at a level equivalent to or substantially higher than that of the intact factor IX gene. The liver specificity was maintained in these minigene expressions. The study suggests that such enhancer-like activity of the intron I sequence may be due 1) to enhancer elements present in intron I, particularly in the intron I sequence with its middle 4.8-kb portion deleted out of the intact 6.2-kb sequence, 2) to the presence of legitimate splicing sequences (donor, acceptor, and branch sites), or 3) to a possible combination of these two or other unknown mechanisms. The study, however, was not extended to determine the responsible mechanism underlying the observed enhancer-like activity.

In this paper, we report our systematic analyses of the human factor IX intron I using a cultured cell assay system, which support the conclusion that higher levels of expression of factor IX from the minigene constructs are due to the presence of splicing sequences in intron I, and not due to the presence of various enhancer-like sequence elements within the intron.


MATERIALS AND METHODS

Restriction enzymes and DNA modification enzymes were purchased from Life Technologies, Inc. and New England Biolabs. Radioactive nucleotides, [alpha-P]dCTP, [S]dATP, and [^14C]chloramphenicol were obtained from Amersham Corp. Thermus aquaticus (Taq) DNA polymerase and Vent(R) DNA polymerase were obtained from Life Technologies, Inc. and New England Biolabs, respectively. beta-Galactosidase expression plasmid vector (pCH110) and acetyl-CoA were obtained from Pharmacia P-L Biochemical. pRc/CMV plasmid and FastTrack mRNA kit was purchased from Invitrogen Inc. The DNA sequencing kit was purchased from United States Biochemical Corp. The supplies for ELISA were from Bio-Rad. Mouse monoclonal anti-human factor IX (AHIX-5041) was purchased from Hematology Technologies Inc. Rabbit polyclonal anti-human factor IX used for ELISA was described previously(11) . Synthetic oligonucleotides were made at our Biomedical Research Core facility. All other reagents were of the highest quality commercially available.

Construction of Chloramphenicol Acetyltransferase (CAT) Expression Vectors

Unless otherwise mentioned, the nucleotide numbering system used here follows that of the complete human factor IX gene sequence we have previously reported(12) .

CAT expression vector pUMS-416/29CAT, which was previously constructed and successfully used to analyze the 5`-end-flanking sequence of the factor IX gene for its promoter activity(3, 4) , was used in this study for testing the potential enhancer-like effects present in the intron I sequence with its middle portion deleted (Fig. 1). DNA fragments containing various subregions of the intron sequence were generated by polymerase chain reactions (PCR) with the human genomic DNA template using primers containing the KpnI linker sequence (Table 1). After digestion with KpnI, these fragments were subcloned into pUMS-416/29CAT at its unique KpnI site (158 bp 5` to the factor IX promoter) in the UMS (mouse upper sequence containing multiple polyadenylation signal sequences)(3) . PCRs were carried out as described previously (9) , with minor modifications. DNA fragments prepared by PCR were purified by using Ultrafree-MC Filters (Millipore) before they were inserted into pUMS-416/29CAT. The positive control expression vector for enhancer activity, pUMS(CMV)-416/29CAT, was constructed by inserting a 382-bp CMV enhancer sequence into pUMS-416/29CAT at the KpnI site in UMS. This enhancer corresponding to a 5`-end region spanning nt -584 to nt -217 of the CMV promoter (13) was prepared by PCR amplification with pRc/CMV.


Figure 1: Structure of the CAT expression vector pUMS-416/29CAT. Subregion sequences of the factor IX intron I or the CMV enhancer element were inserted at the unique KpnI site in the UMS, 158 bp 5` to the factor IX promoter. FIX-416/29 indicates the factor IX promoter sequence. Arrow indicates the transcriptional start site.





Construction of Factor IX Minigene Expression Vectors

Three new human factor IX minigene expression vectors, p-416FIXc, p-416FIXm1, and p-416FIXm2 were constructed with pUC19 (Fig. 2). These vectors contained the minimal transcriptional control region of the human factor IX gene identical to that used in the above CAT vectors, linked to either one of the three factor IX minigenes, -416FIXc, -416FIXm1, or -416FIXm2. p-416FIXc, which contains the human factor IX cDNA, was prepared by ligating two DNA fragments. The first, a 485-bp fragment encompassing a region of nt -416 to nt 69 (the unique StuI site at nt 69 in exon I) was the minimal factor IX promoter sequence prepared by StuI digestion of a PstI/KpnI fragment encompassing nt -416 to nt 650 (which was generated from the genomic DNA template by PCR using a 5`-primer with PstI linker and a 3`-primer with KpnI linker). The second fragment was a 1466-bp StuI/BamHI fragment (containing the same StuI site at nt 69 at the 5`-end and a BamHI site at the 3`-end) which was prepared by StuI and BamHI digestion of the factor IX cDNA sequence in pFIX/pD2 (14) . pFIX/pD2, a factor IX expression vector, contained the human factor IX cDNA sequence as a BamHI fragment which was modified from the original factor IX cDNA clone(15) , and the adenovirus 2 major late promoter(14) . The fragment resulting from the ligation of the above two fragments, which contained the factor IX promoter sequence (up to nt -416 at the 5`-side) linked to a factor IX cDNA sequence (FIXc) encompassing the entire coding region and 29-bp 3`-UTR, was then ligated at its 3`-BamHI end with the 5`-BamHI end of a 380-bp BamHI/PstI fragment encompassing nt 32691-33071. This BamHI/PstI fragment was prepared by PCR amplification with the genomic DNA template using a 5`-primer containing a BamHI linker and a 3`-primer containing a PstI linker. The genomic sequence region amplified contained the polyadenylation signal (AATAAA) sequence as well as the polyadenylation site at nt 32757, in addition to its contiguous 314-bp 3`-flanking sequence. The resulting 2289-bp fragment -416FIXc was subcloned into pUC19 vector at the PstI site in an orientation with its 5`- and 3`-ends in close proximity to the SphI and KpnI sites in the pUC19 vector sequence, respectively. PCR-amplified regions and fragment ligation sites were sequenced before further use.


Figure 2: Structure of human factor IX minigene constructs. Relevant portions of pUC19-based factor IX minigene expression vectors p-416FIXc, p-416FIXm1, and p-416FIXm2 are shown. Open boxes, hatched boxes, and solid boxes indicate 5`- or 3`-flanking genomic sequences, 5`- or 3`-UTRs, and coding regions of the factor IX cDNA, respectively. Solid lines and broken lines indicate the retained and deleted regions of the intron I sequence, respectively. Thin lines at the both 5`- and 3`-ends represent portions of pUC19 vector sequence. Numbers indicate nucleotide positions for relevant sites cited in constructing these vectors. Relevant restriction sites and their nucleotide numbering positions are shown with thin vertical lines. Asterisks () indicate the polyadenylation site. Figures are not exactly proportional to the actual sizes. The nucleotide numbering is based on the complete nucleotide sequence previously reported for this gene(12) .



The second expression vector p-416FIXm1, which contained a factor IX minigene with the intron I sequence with its middle 4.8-kb region deleted, was constructed by sequential ligations of five DNA fragments. All the DNA manipulations were done by utilizing pUC19. The first three fragments included (i) PstI/XmnI fragment (809 bp) encompassing nt -416 to nt 393 (the first XmnI site in intron I), which was generated by XmnI digestion of a PCR-amplified fragment (PstI linker at the 5`-end) encompassing nt -416 to nt 650; (ii) XmnI/PvuII fragment encompassing nt 394 (XmnI site in intron I) to nt 1098 (the first PvuII site in intron I) prepared by digestion of a PCR-amplified fragment (nt 98-1312) with XmnI and PvuII; (iii) PvuII/HaeIII fragment spanning from nt 5882 (the last PvuII site in intron I) to nt 6365 (HaeIII site in exon II) prepared by PvuII and HaeIII digestion of a PCR-amplified fragment (spanning from nt 5660 to 6385). Ligation of these three fragments into pUC19 in the sequential order listed above resulted in a 1997-bp factor IX sequence linked to a PstI linker at the 5`-end, and a HaeIII restriction sequence corresponding to the endogenous HaeIII site in exon II. This fragment encompassed a region of nt -416 to nt 6365 with its middle portion (nt 1099-5881, 4783 bp in length) of the intron I sequence deleted. This fragment was isolated from the pUC19 vector by digestion with SphI (which cuts pUC19 sequence just outside of the 5`-PstI site of the factor IX sequence) and HaeIII, and then ligated to a HaeIII/BamHI fragment generated by digestion of p-416FIXc with HaeIII and BamHI, containing the 3`-half of exon II beginning with the unique HaeIII site and the rest of the cDNA sequence with the BamHI site at the 3`-end. The resulting SphI/BamHI fragment was then inserted into p-416FIXc at SphI/BamHI sites, replacing the -416FIXc insert freed by SphI and BamHI digestion, finally generating a factor IX minigene expression vector p-416FIXm1 with a 3714-kb factor IX minigene (FIXm1) containing 981 and 443 bp sequences of the 5`- and 3`-end regions of intron I, respectively.

The third factor IX minigene expression vector p-416FIXm2 was prepared by further deleting the middle portion of the largely truncated intron I sequence of p-416FIXm1. p-416FIXm1 was subjected to a partial digestion with ScaI. A construct digested only at two unique ScaI sites (nt 258 and 6168) within the intron I sequence maintained in FIXm1, but not digested at a ScaI site present in the pUC19 vector sequence, was screened by restriction mappings. This construct which contained only 141- and 157-bp sequences of the 5`- and 3`-end region of intron I, respectively, was then self-ligated generating the third factor IX minigene expression vector, p-416FIXm2.

Cell Transfection

All expression vector DNAs including CAT vectors, factor IX minigene expression vectors, and pCH110 (internal control for transfection efficiency) used for transient assays were purified by passing through Qiagen-tip anion-exchange resin columns (16) , and carried out as described previously(9) . Transient assays of expression vectors with HepG2 cells were carried out as described before (3, 4) using a high efficiency CaPO(4)-DNA precipitation method(17, 18) . SV40 promoter of pCH110 used as the internal control for transfection was previously shown to have little effect on the factor IX promoter activity, serving as an excellent internal control for the present study(3) . After transfection, cells were incubated at 37 °C under 5% CO(2) in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum for CAT expression vectors, or Dulbecco's modified Eagle's medium supplemented with 10% barium sulfate-treated fetal calf serum (19) and 1 µg of vitamin K (12) for factor IX expression vectors.

CAT Assay

CAT and beta-galactosidase assays were carried out as described previously(3, 4) , with minor modifications. Seventy-two h after transfection, cells were harvested for CAT and beta-galactosidase activity assays. Aliquots (leq100 µl) of the cell lysates, which varied depending on the beta-galactosidase activity, were taken for CAT assays, and the Betascope 603 blot analyzer (Betagen) was used to quantitate acetylated forms of chloramphenicol. All the transfection experiments were repeated three to five times, and averages of the results are presented.

Factor IX Clotting Assay

Factor IX activity was determined by one-stage clotting assay (aPTT) as described before(20) , with minor modifications. The culture medium was collected 92 h after cell transfection for factor IX assay, and cells were collected for beta-galactosidase assay. All assays were performed three times for each sample with two different dilutions in duplicates. The standard curve for the activity quantitation was generated by using pooled normal human plasma (George King Bio-Medical, Inc.).

ELISA

Human factor IX protein was assayed by ELISA as described before(11) . Mouse monoclonal antibody to human factor IX (AHIX5041) and affinity-purified rabbit polyclonal anti-human FIX antibody were used as the first and second antibody, respectively. ELISA was duplicated for each diluted sample, and the reaction products were quantitated at room temperature by reading absorbency at 450 nm with a microtiter plate reader (Bio-Tek Instruments, model EL312c). This ELISA system reproducibly detected human FIX antigen as low as 0.2 ng/ml. Pooled normal human plasma was used to prepare a standard curve for quantitation.

Northern Blot Analysis

Northern blot analysis of HepG2 cells transiently transfected with factor IX expression vectors was carried out as described previously(9) . Aliquots (5 µg) of poly(A) RNAs, prepared with an oligo(dT) column (FastTrack kit) from about 3 times 10^7 HepG2 cells transfected with factor IX expression vectors, were used. All other conditions were the same as those mentioned in the cell transfection section, except pUMS-416/29CAT vector was used in the mock control transfection. Factor IX cDNA (14) labeled with P was used as the hybridization probe. Quantitation of RNA bands hybridized with the probe was carried out with the Betascope 603 blot analyzer.


RESULTS AND DISCUSSION

PCR-amplified subregions of intron I which were tested in this study are summarized in Fig. 3A. In order to include the 5`-splice donor and 3`-acceptor sequences in the analysis, subregion sequences a1, a4, b1, and b2 (which were amplified by PCR) contained parts of the adjacent exon I and II sequences. These subregion fragments were analyzed for their potential enhancer activities by inserting them into a CAT expression vector pUMS-416/29CAT, at the unique KpnI site in the UMS located immediately upstream of the factor IX promoter (Fig. 1). The control CMV enhancer inserted at the KpnI site gave 7-fold enhancement in expression over that of pUMS-416/29CAT which contained no enhancer element at the site, confirming the effectiveness of this vector system for assessing enhancer activity. When the intron sequence containing the entire subregions under testing (fragments a1 + b1) was inserted at the KpnI site, only a weak enhancer activity (1.8-fold increase in CAT activity at the highest) was observed indicating that these sequences have only marginal enhancer activities. Smaller subregion sequences generated from the intron also showed independently only weak enhancer activities or even weak suppressor activities, in good agreement with the overall marginal enhancer activity observed for the a1 + b1 fragment. These results indicate that in spite of the significant numbers of known enhancer-like sequence elements present within the partial intron I sequence under investigation (such as three AP-1s, two AP-3s, one octamer, one NF-A, and possibly others), these structural elements only marginally contribute in enhancing the factor IX expression. The weak enhancer activity associated with the intron I sequence cannot fully explain the increased expression activity observed in the transgenic mice(10) , strongly suggesting a different mechanism to be responsible.


Figure 3: A, subregions of intron I tested for potential enhancer activities. Solid horizontal lines indicate the intron I sequence, while portions of exons I and II are shown by open boxes. Subregion DNA fragments prepared by PCR amplification are shown by labeling with the 5`- and 3`-end nucleotide positions. Dotted lines indicate the corresponding regions. B, effects of the intron I subregions on the factor IX promoter. CAT activities (averages of three to five independent assays) are shown by hatched or solid bars for the reverse or forward orientation (respectively, relative to the factor IX promoter) of the subregion sequences at the KpnI site. Standard deviations are shown by thin horizontal lines with short vertical lines. The CAT activity of pUMS-416/29CAT, which contained no enhancer element at the KpnI site, was defined as 100%. A CMV enhancer element in pUMS(CMV)-416/29CAT served as the positive control.



In order to further study the underlying mechanism of the enhancing activity of intron I on the factor IX promoter, we then constructed three factor IX expression vectors, p-416FIXc, p-416FIXm1, and p-416FIXm2 (Fig. 2). p-416FIXc contained factor IX cDNA under the transcriptional control of the factor IX minimal promoter. p-416FIXm1 and p-416FIXm2 contained factor IX minigenes FIXm1 or FIXm2 with the middle portion-truncated intron I sequences inserted into FIXc at the identical position as in the natural gene. FIXm1 contained intron I with a 4.8-kb deletion of its middle region spanning nt 1099-5881, while FIXm2 had a greater deletion of intron I sequence, spanning nt 259-6167. Both FIXm1 and FIXm2 contained legitimate splicing sequences (donor, acceptor and branch sites). The factor IX minimal promoter used in these expression vectors included the 5` immediate-flanking sequence up to nt -416. This factor IX promoter was identical to that used in the CAT expression vectors in testing the intron I subregions for potential activities. p-416FIXm1 and p-416FIXm2 were designed to have exactly the same structure as p-416FIXc, except FIXc in p-416FIXc was replaced with FIXm1 or with FIXm2, respectively. Transient expression of p-416FIXm1 in HepG2 cells produced 88.8 ng of recombinant factor IX into 10 ml of culture medium after 92 h of transfection. This was equivalent to an 8.9-fold higher expression activity over that of p-416FIXc which produced 10.2 ng factor IX in simultaneous experiments (Table 2). The increase in human factor IX antigen secreted into the culture medium with the p-416FIXm1 construct correlated well with the substantial increase (7.4-fold) in the factor IX clotting activity. This increase in activity and factor IX protein levels also agreed with the 7.8-fold increase in the factor IX mRNA level within the experimental variations.



The mature mRNAs produced by p-416FIXm1 were of the fully spliced form with an expected size of 1.8 kb for the minigene (Fig. 4). The actual size may vary slightly depending on the length of poly(A) tail added. The molecular size of this major mRNA band is consistent with the 5`-upstream transcription start site at or in the vicinity of nt -176, but not with the site at nt +1(9) . Some minor bands with lower molecular mass, including one of 1 kb in size, are likely generated by minor alternative processings of precursor RNA, but due neither to possible nonspecific cross-hybridization, because they are absent in the control lane, nor to nonspecific degradations, because the bands are highly discrete.


Figure 4: Northern blot analysis of HepG2 cells transiently transfected with factor IX expression vectors. Lane a, mock control; lane b, transfected with p-416FIXm1; lane c, transfected with p-416FIXc. Mature factor IX mRNAs which are 1.8 kb in size are shown by the arrow. Size marker positions are shown on the right.



As described above, intron I lacks any substantial enhancer activity (Fig. 3, A and B), and all the factor IX minigene constructs used have the identical promoter sequence, ensuring a very similar, if not identical, promoter activity for all these constructs. Consequently, the increase in the mRNA level for p-416FIXm1 construct is highly likely due to the much increased protection of precursor mRNA molecules with an intron sequence from degradation in comparison to those without any intron sequence produced from FIXc. It is speculated that with the presence of legitimate splicing signal sequences, the minigene precursor mRNAs in the nucleus are efficiently recognized and assembled into spliceosome complexes(21, 22, 23, 24) , resulting in an increased protection of the precursor mRNAs from random degradations before being transported out of the nucleus, and consequently in an elevated level of the mature mRNAs.

This conclusion was further supported by the results obtained with the third minigene construct, p-416FIXm2. The factor IX minigene in this vector had a much greater deletion of its first intron sequence than that of p-416FIXm1 and contained no known enhancer sequence elements which were still present in FIXm1. p-416FIXm2 showed an equally elevated expression as p-416FIXm1 in comparison to p-416FIXc. These results indicate that any other enhancer elements, if present in the remaining regions, may also have only insignificant activities. They also indicate that the shortening of the intron I sequence did not change the efficiency of intron splicing events.

It has been known for other genes that splicing sequences augment their expression with various mechanisms(25, 26, 27, 28, 29) . The present study demonstrates that intron I of the factor IX gene also has a strong overall expression enhancing activity. However, the augmented expression is not due to specific enhancer elements present in intron I, but rather to the increased precursor mRNA stability mediated by its splicing sequences. It is interesting to note that in transgenic mice experiments, the presence of multiple introns in the factor IX expression vector provided neither synergistic, nor additive enhancing activity above that observed for intron I(10) . This may suggest that the presence of an intron, which happens to be intron I for the factor IX gene constructs used in the present study, is sufficient for an efficient assembly of precursor mRNAs into the spliceosome complex, resulting in increased protection from random degradations. More recently, we also observed that the factor IX minigene construct (FIXm1) in retroviral expression vectors under the control of heterologous promoters also showed a 10-12-fold increase in expression level over that of the factor IX cDNA (FIXc) construct, further supporting the present results. (^2)

The transgenic mice study reported by Jallat et al.(10) also suggested that intron I as well as all other introns (introns II-VII) may not play any significant role in determination of the liver-specificity of the factor IX gene expression. In their study, a 5-kb 5`-flanking sequence of the factor IX gene was used as the promoter. Our recent transgenic mice study with -416FIXc as well as -416FIXm1 minigene also showed a high (although not exclusive) liver-specific expression.^2 These observations together with the high expression activities observed in HepG2 cells for p-416FIXm1 and p-416FIXm2 in the present study support the conclusion that none of the intron sequences in the factor IX gene plays a significant role in the liver specificity of factor IX expression.


FOOTNOTES

*
This work was in part supported by National Institutes of Health Grants HL38644 (to K. K.) and M01-RR00042 to the University of Michigan General Clinical Research Center.

§
Awardee of an American Heart Association of Michigan postdoctoral fellowship.

To whom all correspondence should be addressed.

(^1)
The abbreviations used are: UTR, untranslated region; CAT, chloramphenicol acetyltransferase; UMS, upper mouse sequence; PCR, polymerase chain reactions; FIX, factor IX; CMV, cytomegalovirus; ELISA, enzyme-linked immunosorbent assay; nt, nucleotide(s); kb, kilobase(s); bp, base pair(s).

(^2)
S. Kurachi, Y. Hitomi, M. Furukawa, and K. Kurachi, unpublished data.


ACKNOWLEDGEMENTS

We thank Dewesh Agrawal for his critical reading of the manuscript.


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