Research Paper
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Acta Biochim Biophys Sin
2005,37: 819-825 |
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doi:10.1111/j.1745-7270.2005.00117.x |
Loss of Posterior Silk Gland Transcription Specificity of Fibroin Light Chain Promoter due to Absence of 41 bp Sequence Containing Possible Inhibitor Binding Sites
Ting-Qing GUO1,2#, Jian-Yang WANG1,2,3#, Sheng-Peng WANG1,2,3, Xiu-Yang GUO1,2,
Ke-Wei HUANG3, Jun-Ting HUANG3, and Chang-De LU1*
1
Institute
of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai 200031, China;
2 Graduate School of
the Chinese Academy of Sciences, Shanghai 200031, China;
3 Sericultural
Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang 212018,
China
Received: September
1, 2005
Accepted: October
17, 2005
This work was supported
by the grants from the National Natural Science Foundation of China (No.
30370326, No. 30470350)
# These authors
contributed equally to this work
*Corresponding
author: Tel, 86-21-54921234; Fax, 86-21-54921011; E-mail, [email protected]
Abstract������� The gene encoding fibroin light chain protein (FibL) is
specifically expressed in the posterior silk gland of silkworm and repressed in
other tissues. The binding sites of several transcription factors involved in
the silk gland transcription specificity of fibl promoter have been
recognized, including SGFB, PSGF and BMFA. Here we report the leak expression
of the enhanced green fluorescent protein (EGFP) reporter gene in
tissues other than the posterior silk gland in vivo when under the
control of a shortened fibl promoter with deletion of the 5'
terminal 41 bp sequence, which is located at -650 nt to -610 nt upstream
of the fibl transcription starting site. Assay of silk gland specificity
of the promoters was performed by �observation of green fluorescence in tissues
of silkworm larvae following inter-haemocoelic injection of recombinant Autographa
californica multiple nuclear polyhedrosis virus carrying the EGFP
reporter gene controlled by different lengths of fibl promoters. Our
results indicated that availability of the binding sites of several known
factors, including SGFB, PSGF and BMFA, is not sufficient for intact silk gland
transcription specificity of fibl promoter, and there are possible
inhibitor binding sites in the 41 bp sequence (-650 nt to -610 nt) upstream
of the transcription starting site which may be required to repress the
activity of fibl promoter in other tissues.
Key words������� promoter specificity; fibroin light chain; silkworm; Bombyx
mori; recombinant AcMNPV
The silk fiber spun by the silkworm Bombyx mori is a mixture
of sericins and fibroin, the latter composed of three kinds of major silk
proteins. During the insect�s fifth larval instar, fibroin proteins are
synthesized in the cells of a pair of posterior silk glands (PSG), secreted
into the lumen of PSG and transported to the lumen of the middle silk gland
(MSG). Here the fibroin is coated with sericin secreted by cells of MSG, then
transported toward the anterior silk gland to form and spin the silk fiber. All
three kinds of fibroin proteins, fibroin heavy chain protein (FibH), fibroin
light chain protein (FibL) and fibrohexamerin/p25 protein (FHX/P25), are
expressed in PSG of Bombyx mori with strict territorial and
developmental specificities [1,2]. The developmental regulation of silk genes
is mainly �hormone-mediated transcription regulation and related to chromatin
topology [3-5]. Many genes have been �identified to be involved in the
regulation of silk genes [1,6-8]. By �nuclease protection and mobility shift assays, three
PSG-expressed genes were shown to share similarity in �upstream and intron
sequences. The cis-elements and trans-�activators of silk genes
have been identified [9-14].
Understanding the mechanisms underlying the tissue-specific
expression of fibroin genes would shed light on the transcription regulation
and development of the silk gland. So far, many efforts have been made to
identify the cis-regulatory elements and trans-acting factors �responsible
for the silk gland-specific activity of these fibroin promoters, especially for
the expression of the fhx/p25 gene. Of the elements identified,
the binding sites of BMFA, SGFB and PSGF are considered to be responsible for
silk gland specificity. BMFA is a ubiquitous protein proposed to be involved in
the repression of fibroin genes at molting [3]. SGFB is a silk gland-specific
regulatory protein �expressed in both PSG and MSG, but has access to its target
sequence only in PSG cells [15]. PSGF is a factor deduced from DNase I
protection assay, supposed to be expressed only in PSG and to facilitate the
recruitment of SGFB [15]. The control mechanism by which the �expression of the
fhx/p25 gene is restricted in PSG but not in MSG was illustrated in
vivo using organ �transplantation and transgenic methods [15-17]. It was
shown that the proximal 254 bp sequence of fhx/p25 �promoter contains
all sequences required for its specific expression in PSG. In order to
understand the PSG-specific� �expression of the fhx/p25 gene, the
binding sites of SGFB and PSGF were combined with a synthetic TATA box of the
A3 gene and this synthetic promoter was sufficient to drive PSG-�specific
expression of the reporter gene. It was also hypothesized that inactivation of
fibroin promoters in tissues other than PSG may be due to the combination of
the absence of PSGF and the attachment of BMFA to the �promoters [15].
Among silk genes, the fibroin light chain (fibl) gene is
relatively less studied on the regulation of gene expression. The nucleotide
sequence of the fibl gene was determined [18] and the possible elements
were analyzed [19]. Imamura et al. [17] has reported a 691 bp (-650 nt to +41
nt) �functional fibl promoter with PSG transcriptional specificity, in
which the binding sites of SGFB, BMFA, and PSGF could be recognized. However,
it has not yet been determined whether the availability of these binding sites
is sufficient for PSG specificity of fibl promoter.
Autographa californica
nucleopolyhedrovirus (AcNPV), whose permissive host is Trichoplusia ni,
is commonly applied for large-scale expression of eukaryotic proteins in
permissive cell lines or insects [20]. In our previous studies, recombinant
AcNPV has been used as a highly efficient transient in vivo gene
delivery vector to some strains of silkworm larvae by haemocoel injection
[21,22]. Recombinant AcNPV vector has also been demonstrated to facilitate the
study of silk gland-specific protein �expression and secretion, using the
enhanced green �fluorescent protein (EGFP) reporter gene fused to
the sericin signal peptide coding sequence and under the �control of the �promoter
of the sericin 1 gene [23].
In this study, we delivered recombinant AcNPVs �harboring the EGFP
cassettes controlled by different lengths of fibl promoter into Sf9
cells and silkworm larvae, and found that a shortened fibl promoter with
the deletion� of a 41 bp sequence (-650 nt to -610 nt) at its 5'
terminal could be activated in Sf9 cells, hemocytes, MSG and fat body in
addition to PSG, as revealed by expression of EGFP reporter. The binding
sites of SGFB, BMFA, and PSGF remained intact. Our results suggested the �existence
of binding sites of other unrecognized factors necessary for repressing the
activity of fibl promoter in other tissues� but not in PSG within the 41
bp (-650 nt to -610 nt) sequence.
Materials and Methods
Silkworm strains and cell lines
Silkworm B. mori strain 54A, which is AcNPV permissive, was
provided by the Sericultural Research Institute, Chinese Academy of
Agricultural Sciences (Zhenjiang, China). The Sf9 cells were maintained in
Grace�s medium (Invitrogen, Carlsbad, USA) �supplemented with 10% fetal bovine
serum (FBS; Invitrogen) at 27 �C. The Escherichia coli DH10BacDEGT cell line
was established in our laboratory [21].
Construction of recombinant baculoviruses
Fibroin light chain promoters FL1 (-650 nt to +41 nt) and FL2 (-609 nt to +41
nt) were amplified by polymerase chain reaction (PCR) using genomic DNA
extracted from the silk gland of fifth instar silkworm larvae as the template.
Primers for amplification of FL1 were: U1, 5'-gagctcTGCATATTGGACATCC-3'
(SacI site in lowercase) and L1, 5'-gaattcTTTAGTGGTCTGTTA-3' (EcoRI
site in lowercase). Primers for amplification of FL2 were: U2, 5'-gagctcgtaattctcggtacggttcg-3' (SacI
site in lowercase) and L1. The PCR products were cloned into pGEM-T vector
(Promega, Madison, USA) to generate plasmids pTV-FL1 and pTV-FL2. They were
confirmed by sequencing. Then the promoters were digested out with SacI/EcoRI
and ligated into the same sites in plasmid pEGFP-N3 (Clontech, Palo Alto, USA)
to produce �plasmids pFL1-EGFP and pFL2-EGFP, which were then� �digested by SacI/PstI
and inserted into plasmid pFFa2 [21] to �produce donor plasmid pFFa2FL1-EGFP
and pFFa2FL2-EGFP, respectively. The restriction maps showed the clones were
correct (data not shown). pFFa2FL1-EGFP and pFFa2FL2-EGFP were transformed into
E. coli DH10BacDEGT cells to make recombinant bacmid AcFL1egfpDEGT and
AcFL2egfpDEGT.
Recombinant baculovirus preparation
Generation and large-scale harvest of recombinant baculoviruses
followed the instruction manual "Bac to Bac baculovirus expression systems"
(Invitrogen) using the Sf9 cell line. Stocks of virus were concentrated by �centrifugation
at 35,000 g for 60 min, and pelleted virus was resuspended in phosphate
buffered saline (PBS, �pH 7.5) supplemented with 1% (V/V) FBS
before being stored at -70 �C or subjecting to insect injection. Virus titer was determined
by the Tissue Culture Infectious Dose 50 method, which is based on end-point
dilution.
Insect inoculation and dissection
Larvae of silkworm B. mori strain 54A were routinely reared
on mulberry leaves. Recombinant baculoviruses were injected into the haemocoel
of newly-ecdysed fifth instar larvae. For analysis of EGFP reporter
expression in various kinds of tissues, larvae were dissected at appropriate
time points. Tissues of hemocytes, fat body, MSG and PSG were collected after
they were rinsed in PBS (pH 7.5).
Observation of EGFP fluorescence
Fluorescence of EGFP in cultured cells and silkworm tissues was
observed with fluorescence microscopes BX50 (Olympus, New York, USA) or MZ FL
III (Leica, Wetzlar, Germany).
Results
Recombinant AcNPVs harboring EGFP reporter controlled by
different fibl promoters
Fibroin light chain promoters FL1 and FL2 were cloned and sequenced
before they were used to drive EGFP expression. Then FL1- and FL2-driven
EGFP cassettes were transferred to the bacmid AcDEGT to generate the
recombinant bacmids AcFL1egfpDEGT and AcFL2egfpDEGT (Fig. 1). Recombinant bacmids were confirmed by PCR
analysis using wild-type bacmid-specific M13 reverse oligo and FL1 or FL2
specific oligo (U1 or U2) as primers. The PCR products were approximately 2 kb
in length (Fig. 2, lanes 3 and 4). Another PCR assay using M13 reverse
oligo and EGFP-specific primer EGFP-����1 (5'-aagcttgtcgacagatctgcatgcatggtgagc-3')
produced a band of approximately 1.3 kb (Fig. 2, lanes 1 and 2). The
purity of bacmid was assayed by PCR using wild-type bacmid-specific M13 reverse
oligo and M13 forward oligo primers, with AcFFa2DEGT as the template, and a
2.2 kb band was observed (Fig. 2, lane 5). With AcFL1egfpDEGT or AcFL2egfpDEGT as the
template, no 2.2 kb band was observed. Purified bacmids AcFL1egfpDEGT and
AcFL2egfpDEGT were used to transfect cultured Sf9 cells with Cellfectin
(Invitrogen) to produce recombinant viruses.
Leak expression of EGFP in Sf9 cells under control of the shortened fibl
promoter
Baculoviruses of AcFL1egfpDEGT and AcFL2egfpDEGT were incubated with Sf9
cells at a multiplicity of infection of 10, and productive infection of
baculovirus was achieved. In Sf9 cells infected by vAcFL1egfpDEGT, no green
fluorescence derived from expression of EGFP was observed even 3 d after
incubation, when infectious �symptoms were obvious [Fig. 3(B)]. This was
in �accordance with the fact that FL1 promoter could only be activated in PSG,
not in Sf9 cells. However, green �fluorescence could be observed in Sf9 cells
infected with vAcFL2egfpDEGT [Fig. 3(A)], where EGFP was �controlled by FL2
promoter, which is 41 bp shorter at the 5' terminal compared with FL1
promoter. The activation of FL2 promoter in Sf9 cells strongly suggested that
the missing sequence of 41 bp contains information �necessary to silence fibl
promoter in Sf9 cells.
Leak expression of EGFP in tissues of silkworm larvae under control
of the shortened fibl promoter
Leak expression of FL2 promoter-controlled EGFP in Sf9 cells
intrigued us to ask whether deletion of the 41 bp sequence would also cause the
loss of PSG transcriptional specificity of fibl promoter in vivo.
Budded virus of vAcFL1egfpDEGT or vAcFL2egfpDEGT was injected into the haemocoel of newly-ecdysed fifth instar
larvae of silkworm 54A at the amount of 2�105 pfu per
larva. Between day 3 and 7 after injection, green fluorescence derived from
expression of EGFP reporter was gradually observed in PSG cells, but not
other tissues, when the reporter was controlled by FL1 promoter [Fig. 4(A)].
However, when EGFP expression was controlled by FL2 promoter, green
fluorescence could be observed in fat body [Fig. 4(C)] and hemocytes
(data not shown) in addition to PSG; green fluorescence could also be observed
in MSG [Fig. 4(B)]. Expansion of the reporter expression pattern caused
by the deletion of the 41 bp sequence from FL1 promoter indicated that the 41
bp sequence contains functional �elements necessary for repression of fibl
promoter in fat body.
Discussion
In this study, we addressed the necessary length of fibl
promoter for its PSG transcriptional specificity in a �loss of function�
manner. Expression of EGFP reporter was restricted in PSG cells under
the control of FL1 promoter, indicating FL1 promoter does contain cis-elements
�sufficient for PSG transcriptional specificity, which is �consistent with a
previous report [17]. However, when driven by FL2 promoter, which is 41 bp (-650 nt to -610 nt) shorter
than FL1 promoter, leak expression of EGFP �reporter was observed in
MSG, fat body, hemocytes and even Sf9 cells, in addition to PSG. Our results
strongly suggested the 41 bp sequence contains possible elements required for
binding of unrecognized inhibitory factors necessary for repressing the activity
of fibl promoter in other tissues but not in PSG. Previous research
performed by Horard et al. [15] revealed a synthetic promoter �containing
binding sites of SGFB and PSGF, located �upstream of the TATA box from Bombyx
cytoplasmic �actin 3 promoter, has strict PSG transcriptional
specificity. They further suggested that inactivation of fibroin promoters in
tissues other than PSG might be due to the combination of the absence of PSGF
and the attachment of BMFA to the promoters [15]. However, this is not the case
in fibl promoter. In the sequence of FL2 promoter, which could be
activated in MSG, fat body, hemocytes and Sf9 cells in addition to PSG, binding
sites of SGFB, PSGF and BMFA were recognized (Table 1). As shown in Table
1, binding sites of SGFB, PSGF or BMFA are located from -366 nt to -15 nt, so
deletion of the 41 bp sequence (-650 nt to ��-610 nt) did not
remove or destroy any binding sites of the three known factors, but rather of
some unrecognized factors. Previous studies have focused on the intact cis-regulatory
elements responsible for the PSG-specific �activity of the three silk
promoters, but no inhibitory �elements or factors for repressing expression of
silk genes in other tissues have yet been reported. Our results �implied that
there are two kinds of mechanisms involved in �controlling the expression of
silk genes: one activates �expression of silk genes in PSG, and another
represses the �expression of silk genes in other tissues. What is the
inhibitory element? It needs to be further investigated.
In the 41 bp (-650 nt to -610 nt) sequence, we noticed 5'-TATAAA-3' (-619 nt to -614 nt), which
is a TATA box-like sequence. Srinivasan et al. reported a TATA box �binding
factor (TBF), which they designated P43 TATA box binding factor, that could
inhibit in vitro transcription of the tRNAGly multi-gene family and RNA polymerase II
�transcription from actin5C promoter when binding to motifs including
TATATAA and TATAAA [24]. P43 TBF was �purified from PSG nuclear extracts of B.
mori, and �proposed to negatively modulate the transcription of tRNAGly genes. No evidence shows that P43 TBF
is �involved in transcription regulation of the fibl gene, but the
regulation mechanism that binding of some TBF to TATA box-like elements helps
to inhibit transcription may also apply to fibl promoter.
For many years, in vitro transcription assay and �chromatin
footprinting have proved to be valuable tools in helping to determine factors
and their binding sites involved in the activation of silk gland-specific
genes. Our �knowledge of SGFB, BMFA, PSGF and many other �factors associated
with silk gland transcription regulation is either partially or totally derived
from these in vitro assays [9,13]. However, it also means that many of
these conclusions have not yet been tested in vivo, due to the absence
of appropriate in vivo gene delivery and reporter assay �systems for
silkworm. In recent years, PSG specificity of fibroin promoters such as fhx/p25
and fibl promoters has been readdressed, utilizing newly-established
transgenic techniques [17] or transient in vivo gene delivery systems,
like the ballistic method [15,25]. Organ transplantation needs skilled
workers,� whereas obtaining a transgenic
silkworm strain takes a long time, therefore results from in vivo
experiments were limited. In this report, we �delivered in vivo the
reporter cassettes using recombinant AcNPV bacmid as the vector. High
efficiency of gene transfer, demonstrated by the expression of EGFP
reporter in vivo, indicates recombinant AcNPV bacmid could serve as a
vector for transcription regulation research in silkworm� [21-23].
Acknowledgement
We are grateful to Prof. Guo-Zheng ZHANG (Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, China) for kindly providing silkworm eggs and adults.
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