(Received for publication, August 16, 1994; and in revised form, December 28, 1994)
From the
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.
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) ()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.
Restriction enzymes and DNA modification enzymes were
purchased from Life Technologies, Inc. and New England Biolabs.
Radioactive nucleotides, [-
P]dCTP,
[
S]dATP, and
[
C]chloramphenicol were obtained from Amersham
Corp. Thermus aquaticus (Taq) DNA polymerase and Vent
DNA polymerase were obtained from Life Technologies, Inc. and New
England Biolabs, respectively.
-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.
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.
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.
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. ()
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. 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.