Isolation, cloning and characterization of the FSH beta gene promoter of the chinook salmon (oncorhynchus tshawytscha)

Comparison of the amino acid sequence of the tilapia FSHβ and LHβ with β subunits from other fish species showed that the FSHβ subunit protein sequence is less conserved than that of the

females) In some species, it may also be responsible for regulating the synthesis and subsequent release of progesterone by the corpus luteum (Norris, 1997)

In the teleost fish, it was initially thought that only a primordial gonadotropin (GtH)

existed However in 1979, Ng and Idler reported the presence of two types of gonadotropin in plaice and flounder pituitaries Subsequently, two different forms of GtH, GtH-I and GtH-II, were

isolated from salmon and shown to have different steroidogenic activities (Suzuki et al., 1988) The studies of the amino acid (Itoh et al., 1988) and the cDNA sequences (Sekine et al., 1989)

revealed close similarity of GtH-I and GtH-II to the mammalian FSH and LH, respectively, and thus established the duality of gonadotropins in the teleost fish With additional information on the GtH structures from numerous other fish species, the mammalian nomenclature has been adopted

The roles of the pituitary gonadotropins in controlling gonadal function and subsequent fertility in the teleosts are less clear than in mammals Nevertheless, studies have shown that in the salmonid species, the two gonadotropins have distinct roles in reproduction It has been suggested that FSH regulates the early stages of gonadal development, vitellogenesis and spermatogenesis, while LH is responsible for stimulating the events leading to final oocyte maturation and ovulation

in females, and spermiation in males (Suzuki et al., 1988; Swanson et al., 1991; Gomez et al.,

1999) This is in agreement with the observation that FSH levels in the pituitary and plasma

increase with gonadal development and are also correlated to the levels of 11-ketotestosterone and

estradiol in the male and female coho salmon (Swanson et al., 1991) FSH levels decrease while the LH levels begin to increase and reach a maximum during spawning (Swanson et al., 1991; Slater et al., 1994; Figure 1) This marked elevation of LH is most probably responsible for the

single and synchronized spawning behavior of the salmonids This is further supported by electron microscopy studies which demonstrated the differential biosynthesis of FSH and LH during the

reproductive cycle of the rainbow trout (Naito et al., 1991)

Plasma levels of FSH and LH

Figure 1: Plasma levels of FSH and LH in coho salmon during reproductive

maturation (modified from Dickhoff and Swanson, 1990)

The expression and pulsatile secretion of these hormones are temporally regulated

throughout the reproductive cycle and are subject to the complex control of hypothalamic releasing factors, paracrine factors from within the pituitary, and through the feedback actions of gonadal hormones The secretion of the hypothalamic factors in turn is also regulated by feedback actions

of the gonadal hormones (summarized in Figure 2)

GnRH

LH FSH

- ve +ve -ve

Steriods

(T, E, P)

+ve inhibin, activin, follistatin

LH and FSH which stimulate the production of steroid hormones in their gonads Testosterone (T),

estrogen (E) and progesterone (P) negatively or positively regulate the synthesis of the gonadotropins

directly at the pituitary, or indirectly by modulating GnRH secretion from the hypothalamus The

gonadal peptides, inhibin, activin and follistatin, also regulate of gonadotropin gene expression by

exerting positive or negative feedback (modified from Brown and McNeilly, 1999)

Unlike the mammalian FSH and LH, which are largely co-expressed in the same cells of the mature pituitary, teleost FSH and LH are produced in distinct cells, in different locations within the proximal pars distalis (PPD) FSHβ is produced in cells in close association with the

somatotrophs, surrounding the nerve ramifications, while the LHβ subunit is produced in the peripheral regions of the PPD The common α subunit, however, is produced in both types of cells

(Nozaki et al., 1990 and Naito et al., 1991)

1.2 Genomic organization of the gonadotropins

The gonadotropins are encoded by separate genes localized in different chromosomes

(Naylor et al., 1983; Julier et al., 1984) The genomic sequences encoding the LHβ and FSHβ

genes of several mammalian and a few teleost species have been elucidated, and analysis has revealed that like the α subunit genes, the genomic organization of β subunit genes is generally well conserved Unlike the α subunit gene however, which is composed of four exons and three

introns (Fiddes and Goodman, 1979; Godine et al., 1982; Nilson et al., 1983; Gordon et al., 1988),

all the β subunit genes comprise three exons and two introns Furthermore, the β genes are smaller due to the considerably smaller introns, with a total length of about 1.5 kb for LHβ and 4 kb for FSHβ (Figure 3) The positions of the β subunit introns are also conserved The first intron

interrupts the region of either the signal peptide or the 5’ untranslated sequences whereas the second intron is located three amino acids downstream from the fifth cysteine residue in the coding

region (Jameson et al., 1984; Talmadge et al., 1984; Virgin et al., 1985; Gharib et al., 1989; Ezashi

et al., 1990; Xiong et al., 1994a; Rosenfeld et al., 1997; Sohn et al., 1998) or the sixth cysteine residue in the goldfish (Yoshiura et al., 1997) However, compared to the LHβ subunit gene, the

introns and 3’ untranslated region (3’ UTR; Figure 3) Compared to the 0.3 – 0.35 kb size of intron

1 of the LHβ genes, the size of the corresponding intron in the FSHβ genes is more variable,

ranging from 0.28 kb to 1.35 kb (Figure 3) Furthermore, the mammalian FSHβ is different from other β subunit genes in that it possesses an extremely long 3’UTR that is over 1 kb, as seen in the rat (1 kb), cow (1.2 kb) and human (1.5 kb) Within this 3’UTR, there are five highly conserved

segments (Gharib et al., 1989) whose significance is presently not known but it is possible that

they may be involved in determining RNA stability, as shown for other genes (Shaw and Kamen, 1986)

Limited information is available for the teleost FSHβ genomic sequence as only two FSHβ

genes (goldfish and tilapia) have been isolated and sequenced to date (Rosenfeld et al., 1997; Sohn

et al., 1998) The genomic organization in both fish conforms to the three exons and two introns,

as reported in the mammalian FSHβ genes, although the exon 3 of the goldfish and tilapia FSHβ genes contains a much shorter 3’UTR than those of mammals (Figure 3) The location of the first and second introns in both fish shows a well-conserved pattern similar to that of mammalian FSHβ genes For the tilapia, the gene is found to be coded by a single copy gene In contrast, two distinct genes (GTHIβ-1 GTHIβ-2) encoding the FSHβ in the goldfish were isolated and sequence analysis revealed high sequence identity between the coding regions Initial studies revealed that both goldfish FSHβ genes are expressed, although one of these is expressed in higher proportion in sexually immature goldfish, while both are expressed in equal amounts in the mature fish This suggests differential regulation of the goldfish GTHIβ genes at various reproductive stages

Comparison of the amino acid sequence of the tilapia FSHβ and LHβ with β subunits from other fish species showed that the FSHβ subunit protein sequence is less conserved than that of the LHβ gene The tilapia LHβ amino acid sequence shares the highest homology with the grouper at 96%, while the highest homology of the tilapia FSHβ amino acid sequence is with that of the

bonito and is only 60% (Rosenfeld et al., 1997)

Figure 3: Comparison of the LH β and FSHβ subunit genes

The open reading frames are represented by yellow boxes and the untranslated region of exons (E) are

represented by open boxes Blue lines between the boxes indicate introns (In) Numbers below the introns show the approximate length in kb References for LH β subunit genes: porcine (Ezashi et al., 1990);

human (Talmadge et al., 1984); bovine (Virgin et al., 1985); rat (Jameson et al., 1984); equine (Sherman, 1992); tilalia (Rosenfeld et al., 1997) References for FSH β subunit genes: goldfish (Sohn et al., 1998);

mouse (Kumar et al., 1995); rat (Gharib et al., 1989); ovine (Guzman et al., 1991); bovine (Kim et al.,

1988); porcine (Hirai et al., 1990); human (Jameson et al., 1988); tilapia (Rosenfeld et al , 1997)

(Modified from Bousfield et al., 1994)

FSH β subunit gene organization

1.3 Transcriptional regulation of the gonadotropin subunit genes

The regulation of gonadotropin genes comprises basal gene expression which targets these genes specifically to the gonadotropes, and hormonal stimulation in which GnRH and gonadal steroids up-regulate expresson at puberty To date, much less is known about the regulation of these genes in teleost fish than in mammals and information regarding the regulation of the FSHβ subunit gene expression lags far behind what is known about the LHβ subunit

(Kumakura et al., 2003) and salmonid species (Weil and Marcuzzi, 1990; Kitahashi, et al., 1998; Dickey and Swanson, 1998; Melamed et al., 2002)

The degree of stimulatory effects of GnRH on the gonadotropin genes is dependent largely

on the reproductive stage of the fish and the species, and to a lesser extent, the gender In the study

of gonadotropin response to GnRH during sexual ontogeny of the common carp, gonadotropin levels in juvenile fish were unresponsive to GnRH administration In contrast, FSH- and LHβ mRNA of the maturing females increased up to three fold over controls, while there was only a slight increase of the LHβ mRNA in the male counterparts In the post-vitellogenic females, LHβ

increased FSHβ and LHβ mRNA levels were not observed (Kandel-Kfir et al., 2002) In immature

striped bass, GnRH in combination with testosterone, stimulated the β subunit mRNA levels to

various extents but had no effect in the maturing fish (Hassin et al., 2000) A study on female

rainbow trout revealed that GnRH did not significantly stimulate FSHβ secretion at any stage of gametogenesis, even when the FSHβ levels increased after ovulation, whereas LHβ secretion

increased following mid-vitellogenesis (Breton et al., 1998) Likewise, in the sham- and

testosterone-implanted sea bass, GnRH had no effect on FSHβ mRNA levels, but increased LHβ

mRNA levels (Mateos et al., 2002) In contrast, GnRH treatment induced the expression of all three gonadotropin subunit genes in the immature sea bream (Kumakura et al., 2003) In the

goldfish, however, GnRH treatment increased both gonadotropin subunit mRNAs in the sexually

matured fish but decreased the level of FSHβ mRNA in the sexually regressed fish (Khakoo et al., 1994; Sohn et al., 2001) Taken together, these studies indicate that the gonadotropin genes are

regulated through various GnRH-induced signaling pathways and are also influenced by the

reproductive stage of the fish

1.3.1.1 GnRH signaling pathway

The stimulation of gonadotropin subunit genes by GnRH is activated through intracellular signaling transduction pathways following the binding of GnRH to G protein-coupled, seven-transmembrane receptors (GnRHR) on the pituitary gonadotrope (Marshall and Kelch, 1986;

Gharib et al., 1990) In mammals it has been shown that the receptor activates L-type calcium channels, allowing extracellular calcium into the cell (Naor, 1990) Phospholipase C (PLC) is also activated, leading to cleavage of phosphatidylinositol-diphosphate (PIP2) located in the cell

membrane, into inositol 1,4,5-triphosphate (IP3), which mediates calcium release from

intracellular stores, and diacylglycerol (DAG) Increased concentrations of intracellular calcium together with DAG production lead to activation of protein kinase C (PKC), which in turn leads to activation of other protein kinases, such as the ubiquitous mitogen-activated protein kinases

(MAPK) known to be involved in various cellular functions including cell growth, differentiation,

transformation, cell cycle and apoptosis (Roberson et al, 1995; Haisenleder et al, 1998) The

activated MAPKs translocate to the nucleus and stimulate sequence-specific transcription factors via phosphorylation Once the transcription factors are activated, they bind to promoter DNA

sequences and trigger the expression of the gonadotropin subunit genes (Naor et al., 2000 and Shacham et al., 2001)

In the mammalian gonadotrope, GnRH is able to differentially regulate LH and FSH

biosynthesis through preferential sensitivity of the gonadotropin subunit gene promoters to either

calcium influx or PKC/MAPK cascade (Roberson et al., 1993; Schoderbek et al., 1993; Weck et al., 1998; Saunders et al., 1998) In the report by Saunders et al (1998), PKC-dependent pathways

seemed to play a larger role in the GnRH-mediated transcriptional control of the LHβ- and FSHβ genes while the α-subunit appeared to be more sensitive to calcium influx However, in a recent study involving exposure of tilapia pituitary cells to GnRH, the PKC-extracellular signal-regulated kinase (ERK) cascade elevated α- and LHβ mRNAs, whereas FSHβ transcript induction was ERK-

independent and was regulated by the cAMP-PKA pathway (Gur et al., 2002) The frequency and

amplitude of the GnRH pulses delivered to the anterior pituitary, which vary during different phases of the estrous cycle, is also known to direct differential regulation of the gonadotropin gene

expression (Levine and Ramirez, 1982; Crowley et al., 1985) In general, higher GnRH amplitude stimulates LH release while FSH production is increased at lower GnRH doses (Papavasiliou et

al., 1986) The effect of the variation in GnRH pulse pattern is also associated with differential LH

and FSH release, with higher frequency resulting in greater LH secretion while lower frequency

stimulates FSH release (Dalkin et al., 1989)

1.3.1.2 GnRH-responsive elements on the common α subunit promoter

Gonadotropin α-subunit promoter elements required to mediate GnRH responsiveness have

been mapped in human (Kay and Jameson, 1992), cow (Hamernik et al., 1992), and mouse

(Schoderbeck et al., 1993), but not in fish In the mouse, two DNA elements, the pituitary

glycoprotein hormone basal element (PGBE) and GnRH response element (GnRH-RE;

TGCCTGTT) were found to be important for GnRH stimulation (Schoderbeck et al., 1993) It was

postulated that the GnRH-RE identified at the proximal promoter (-406 to -399 bp) might respond

to GnRH by binding an Ets-like transcription factor that is activated through the MAPK pathway

(Roberson et al., 1995), while the PGBE, at a more distal region, could be targeted by a homeodomain protein (Roberson et al., 1994) In the human α-subunit promoter, two or more

LIM-GnRH responsive regions have been mapped to between -420 to -244 bp, and these are responsible

for transcriptional activation by calcium influx (Holdstock et al., 1996) There are two cAMP

response elements (CRE) within this region whose mutation greatly reduces the basal activity, and

are thus likely to be involved in the GnRH responsiveness of this promoter (Andersen et al., 1990)

cAMP response element binding protein (CREB) is known to bind the CRE and a recent report showed that GnRH stimulates the phosphorylation of CREB in α-T3 cells, which was necessary

for GnRH stimulation of the human α-subunit gene (Duan et al., 1999)

1.3.1.3 GnRH-responsive elements on the LHβ subunit promoter

Sequence analysis of the teleost LHβ gene promoters reveals a number of cis-acting

elements, some of which have also been shown to be essential in the basal and GnRH-stimulated transcription in the mammalian homologs Since the first teleost genomic sequence for the LHβ gene was isolated from the Chinook salmon (csLHβ; Xiong and Hew, 1991), most functional studies on the putative elements have so far been restricted to this fish The csLHβ gene promoter contains a strong proximal tripartite element targeted by steriodogenic factor-1 (SF-1), pituitary homeobox-1 (Ptx-1) and estrogen receptor (ER) whose synergistic interaction, in estradiol-

dependent manner, drives the expression of the gene (Le Dréan et al., 1996, 1997; Melamed et al.,

2002) Moreover, this proximally bound ER appears to interact with another ER bound at a distal

ERE (dERE), and both EREs were suggested to be involved in mediating the GnRH effect (Liu et al., 1995) It was further suggested that this interaction is facilitated by the bending of the target

DNA, induced by Ptx-1 dimers binding four Ptx-1 binding sites located between these two EREs

(Melamed et al., 2002)

GnRH responsive regions have been identified in the LHβ subunit promoter of several

mammalian species including sheep (Brown et al., 1993), cow (Keri et al., 1994) and rat (Fallest et al., 1995) Unlike in teleosts however, mammalian LHβ gene promoters contain two early growth

response factor-1 (Egr-1) REs which cooperate synergistically with SF-1 and Ptx-1 to mediate

GnRH stimulation (Tremblay and Drouin, 1999; Halvorson et al., 1999) In addition, the rat LHβ

promoter contains two binding sites for Sp-1 which were demonstrated to play an important role in

conferring GnRH responsiveness (Kaiser et al., 1998) Further investigation showed that the 3’

Sp-1 site is also critical for basal expression and that the Sp-Sp-1 binding sites act synergistically with the

downstream SF-1 and Egr-1 binding sites to incite maximal response to pulsatile GnRH (Weck et al., 2000)

1.3.1.4 GnRH-responsive elements on the FSHβ subunit promoter

Inspection of the 1.7 kb 5’-flanking region of the tilapia FSHβ gene has revealed several putative regulatory sequences which have been shown to be essential for inducible and tissue-

specific transcriptional regulation of other gonadotropin genes (Rosenfeld et al., 2001) These

regulatory sequences include the gonadotrope-specific element (GSE), the binding site for

steroidogenic factor-1 (SF-1); half sites of estrogen response elements (½ ERE) and a cAMP response element (CRE), the recognition site for the Fos and Jun transcription factors or activating protein-1 (AP-1) Transient transfection studies of the tilapia FSHβ promoter fused to a luciferase reporter vector showed that the promoter responds to GnRH treatment in a dose-dependent manner although the region that mediates this stimulation was not fully defined 5’ deletion analysis of the gene construct showed that this 1.7 kb DNA sequence contains both positive and negative

regulatory elements (Rosenfeld et al., 2001; Yaron et al., 2001) Examination of the 5’-flanking

region of the two goldfish FSHβ genes also revealed the presence of several putative cis-acting elements including a GSE, GnRH REs and several half androgen responsive elements (½ ARE) and EREs located contiguously between –187 and –124 bp upstream from a TATAA sequence, which may be involved in the transcriptional control of the goldfish FSHβ gene (Sohn et al.,

1998) However, functional studies of these putative GnRH REs in the goldfish FSHβ subunit gene promoter have not been performed

The mammalian FSHβ gene has been more extensively analyzed In recent studies, AP-1 recognition sites (TGAC/GTCA) located at -120 and –83 bp of the ovine FSHβ (oFSHβ) promoter

were shown to mediate the GnRH response (Strahl et al., 1997, 1998; Huang et al., 2001b) Using

HeLa and COS-7 cells, both AP-1 sites were required by GnRH to induce a threefold increase in oFSHβLuc transcription, and mutation of these AP-1 sites completely abolished the GnRH effect

(Strahl et al., 1997, 1998) It was also shown that these AP-1 sites could support GnRH-mediated

induction of oFSHβLuc in pituitary cultures derived from transgenic mice harboring the

oFSHβLuc DNA constructs (Huang et al., 2001b)

A 1 binding site on the rat FSHβ (rFSHβ) promoter that is similar to the consensus

Ptx-1 recognition sequence (TAAT/GCC; Zakaria et al., 2002), was also found to be important for

both basal and GnRH-stimulated expression of the gene Conversely, the GnRH-stimulated

promoter activity was significantly reduced when the Ptx-1 site was mutated suggesting that Ptx-1

could mediate GnRH induction of the FSHβ gene (Zakaria et al., 2002) Ptx-1, a member of the Bicoid protein family of TFs (Lamonerie et al., 1996), is expressed in almost all cell-types in the

pituitary including the gonadotropes Ptx-1 has been found to activate the promoters of most

pituitary-specific genes (Szeto et al., 1996; Tremblay et al., 1998)

1.3.2 Regulation by gonadal steroids

The action of gonadal steroids, testosterone and estrogen, on gonadotropin production in fish can be negative or positive, depending largely on the stage in the reproductive cycle and possibly also the species Studies in several species showed that treatment of immature fish (e.g salmon, European eel, goldfish and catfish) with gonadal steroids resulted in positive feedback

effects on the LHβ mRNA levels (Trinh et al., 1986; Dickey and Swanson, 1998; Quérat et al., 1991; Huggard et al., 1996, 2002; Rebers et al., 1997) Similar results were observed in cultured pituitary cells of immature rainbow trout, European eel, tilapia and goldfish (Xiong et al., 1994b; Huang et al., 1997; Melamed et al., 1997; Huggard et al., 1996, 2002) Contrary to the results

observed in immature fish, treatment of pituitary cells from the mature or spawning fish with

steroids did not affect LHβ transcript levels (Xiong et al., 1994b; Huggard et al., 1996; Sohn et al.,

1998) Furthermore, circulating LH levels were reduced in the mature salmon injected with

estrogen (Billard et al., 1977) There seems to be a shift in the sensitivity of the pituitary gland to steroid treatment between early and late recrudescent Atlantic Croaker and goldfish (Khan et al., 1999; Huggard et al., 2002) although this is not true for the Atlantic salmon (Borg et al., 1998) In

the early recrudescence phase of the gonadal cycle of Atlantic Croaker, estrogen and testosterone exerted positive effects on LH secretion In the early recrudescent goldfish, however, high doses of estrogen were needed to achieve maximal stimulation of both α- and LHβ subunit mRNAs, but a lower concentration of estrogen was required for similar response in late recrudescent fish

(Huggard et al., 2002)

Fewer studies have been carried out on the effects of steroids on FSHβ gene expression However, for the tilapia FSHβ subunit gene, response to testosterone depends on the reproductive stage of the fish In cultured pituitary cells from immature or maturing tilapia, FSHβ mRNA levels

increased after exposure to low doses of testosterone (Melamed et al., 1997) This effect was not

observed in regressed fish while those at the end of the spawning season showed reduced mRNA

levels after treatment with testosterone or estrogen (Melamed et al., 1998) In salmonids, treatment

with testosterone had no significant effects on the FSHβ levels in immature rainbow trout and coho

salmon (Xiong et al., 1994b; Dickey and Swanson, 1998), and likewise, steroid treatment did not

affect immature coho salmon FSHβ mRNA levels, although the level decreased in early

recrudescent fish (Swanson et al., 1991)

Steroids may regulate gonadotropin gene expression by acting on the hypothalamus or the pituitary At the hypothalamic level, steroids negatively regulate the transcription of mammalian gonadotropin genes by altering the GnRH pulse pattern A direct effect of steroids has been shown

on the rat FSHβ gene promoter, which contains progesterone-like responsive elements (PREs) sequences Gel shift assays showed that these PRE-like sequences bind the progesterone receptor with high affinity and specificity Moreover, transient transfection studies on the rat FSHβ

promoter showed that progesterone positively regulates the rat FSHβ gene (O’Conner et al., 1997, 1999) Dihydrotestosterone-bound androgen receptor also acts at the pituitary to negatively

regulate basal and GnRH-stimulated α-subunit gene expression (Clay et al., 1993) This

transcriptional repression occurs through direct interaction of the ligand bound receptor with Sp1 and possibly Egr-1, so disrupting the interactions of these transcription factors with their binding

sites on the promoter (Curtin et al., 2001) Jorgensen et al (2001a ; 2001b) reported that the

suppression of the bovine LHβ promoter activity by the ligand bound androgen receptor is

mediated through interactions with the SF-1 transcription factor; while for the human α-subunit, it

is mediated via interactions with the c-jun and activation transcription factor 2 (ATF2) In other studies, estrogen potentiates GnRH analog-induced LHβ secretion in goldfish and black porgy fish

(Trudeau et al., 1991, 1993; Yen et al., 2002) The mechanism was suggested to be through acting

on the GnRH receptor number (Hamernik et al., 1995), post-translational processes (Gardner et al.,

1.3.2.1 Molecular mechanisms of steroid actions

The transcriptional regulation of gene expression by steroid hormones is based on a step model that involves high affinity binding of the hormone to specific steroid hormone

two-receptors, usually within the cytoplasm of the target cells, followed by activation of the receptor complexes which translocate into the nucleus The activated receptors bind to the

hormone-hormone responsive elements of target promoters as monomers (Johnston et al., 1997),

homodimers (Vanacker et al., 1999) or heterodimers (Beato and Klug, 2000) to activate

transcription

While both positive and negative steroid effects on gonadotropin production have been observed in various fish species, the molecular mechanisms of these steroidal actions are still relatively unknown and most studies have been restricted to the csLHβ subunit gene promoter In this promoter, one full ERE and several ½ EREs have been mapped, of which the proximal ERE (pERE) is suggested to de-repress the proximal silencer resulting in increased transcription of the

LHβ subunit gene (Xiong et al., 1994b) There is also evidence that the other ½ EREs may be

involved in estrogen responsiveness through the synergistic cooperation with the proximal ERE

(Liu et al., 1995) The ER also interacts synergistically with SF-1 to stimulate the csLHβ subunit gene promoter in an estradiol-dose dependent manner (Le Dréan et al., 1996) In the study, SF-1

alone could stimulate promoter but the stimulation was dramatically enhanced when combined with ER This synergistic SF-1/ER effect was dependent on binding to the ERE and two putative

SF-1 binding sites located in the proximal region of the promoter (Le Dréan et al., 1996)

Recently, transient transfection studies in α-T3 cells revealed that ER can also be activated

by the PKA or the PKC/MAPK signaling pathways leading to the transactivation of an

ER-responsive promoter in a ligand-independent manner (Demay et al., 2001; Schreihofer et al.,

2001) This ligand-independent ER activity likely depends on its phosphorylation in AF-1,

allowing interaction with specific co-activators (Hall et al., 2001)

increase in FSHβ mRNA levels and FSH secretion that was not observed in LHβ and common α

subunit levels (Carroll, et al., 1989) Moreover, GnRH stimulation of FSHβ mRNA may be

activin-dependent, because the increase in FSHβ mRNA level induced by GnRH was attenuated

when the activin-binding follistatin was added to the perifusion system (Besecke, et al., 1996)

Conversely, female rats treated with a GnRH antagonist secrete FSH when stimulated by activin

(River, et al., 1991) Although activin signaling in a variety of cells has been studied to some

extent, the specific signal pathways through which activin enhances FSHβ gene expression are at present unclear and have not been studied in the fish

1.3.3.1 Mechanisms of activin-stimulated gene expression

Activin regulates FSH transcription through interaction with receptors that are members of the TGFβ family of transmembrane receptor kinases, resulting in both the elevation of FSHβ

mRNA levels and FSH secretion (Matthews and Vale, 1991; Attisano et al., 1992) Activin signal

transduction is mediated by a heterotetrameric receptor complex consisting of two ligand-binding type II receptors (ActRII) and two signal transducing type I receptors (ActRI; Massagué and Wotton, 2000) Upon ligand binding, the ActRII phosphorylates the ActRI on multiple serine and threonine residues located in a conserved GS-rich motif located just N-terminal to the kinase domain The phosphorylated receptor is then activated to signal to its downstream targets, the Smad family of proteins which transmit TGFβ signals from the cell surface into the nucleus

(Attisano and Wrana, 2000; Zimmerman and Padgett, 2000)

The Smad family of proteins is classified into three major classes The first class is the receptor-regulated Smads (R-Smads) that includes the activin receptor-targeted Smad 2 and 3 These Smad proteins are phosphorylated by the type I receptor on the last two serines at the

extreme carboxy terminus (Abdollah et al., 1997) The second class of Smad proteins is the

common mediator Smad (Co-Smad) comprising of the Smad4 found in mammals and the highly

related Smad4β/Smad10 in Xenopus These Co-Smads associate with phosphorylated R-Smads

and the resulting R-Smad/Co-Smad complex then translocates to the nucleus to induce specific

gene responses (Lagna et al., 1996; Howell, et al., 1999; Masuyama, et al., 1999) Activin

signaling is prevented by the third class of Smad proteins known as the inhibitory Smads

(I-Smads) These proteins antagonize signaling by interacting directly with the receptor to prevent access to and phosphorylation of R-Smads, or by interfering with R-Smad/Smad4 complex, or by

recruiting an E3 ligase that induces degradation of the receptor complex (Welt et al., 2002)

Once in the nucleus, the R-Smad/Co-Smad complex may bind specific gene promoters through the consensus motif GCTC or AGAC (Massagué and Wotton, 2000) However, this

binding is of low affinity and specificity, and might not be required on all promoters For example,

Pardali et al (2000) showed that Smad3 and Smad4 functionallycooperate with Sp-1 to activate the human p21 promoter in hepatomaHepG2 cells This activation requires the ubiquitous Sp-1 to bind to the proximal promoter Using Smad3and Smad4 DNA-binding site mutants, the Smad proteins could still transactivate the p21 promoter as efficientlyas wild type Smads indicating that the Smad3 and Smad4 binding sites are not crucial The same Smad proteins synergize with c-jun/c-fos at the AP-1 binding site of the collagenase I promoter to induce transcriptional activation

in response to TGF-β Mutational analyses of the c-jun protein and the AP-1 binding site in the promoter revealed that the interaction of c-jun with DNA is necessary for the transcriptional

activation, whereas similar analysis of Smad3 revealed that its binding to the DNA, is required

although there appears to be little importance for the actual DNA binding sequence (Qing et al.,

2000)

These studies, together with the short consensus Smad recognition sequence, suggest that Smad-dependent gene expression only involves weak binding of Smad to its recognition sequence,

and requires interactions with other transcription factors (Welt et al., 2002) The activin-mediated

regulation of gene expression therefore likely involves a series of interconnecting signal

transduction pathways, culminating with the concerted actions of the DNA-binding Smad proteins and other transcription factors and associated co-factors

It was reported that the Smad3 and Smad4 transcription factors are responsible for

mediating the stimulation of the rat FSHβ promoter by activin in LβT2 cells Functional analysis identified the target site of the promoter through which the transcription factors act Moreover, the pituitary-specific Ptx-2 transcription factor and its corresponding binding site on the promoter were also shown to be involved in regulating the FSHβ subunit gene in a tissue-specific manner This was the first paper to identify Smad transcription factors acting in concert with a pituitary-specific nuclear transcription factor to mediate the activin stimulation of the FSHβ subunit gene

(Suszko, et al., 2003) In another recent study in primary ovine pituitary and LβT2 cells, activin

activated several signaling pathways with different time course but only the Smad2 pathway

appeared to be directly implicated in the expression and release of FSH in LβT2 cells (Dupont et al., 2003) Taken together, these two studies suggest that Smad3/Smad4 complex is at least partly

responsible for the activin-induced expression of the FSHβ gene via the Smad2 pathway

1.3.3.2 Hormonal modulators of activin action on the FSHβ subunit gene

The actions of activin are regulated by the extracellular proteins inhibin and follistatin Inhibin interferes with the activin transduction pathway by disrupting the formation of functional activin-ActRII-ActRI complexes This inhibitory regulation occurs through at least two inhibin-activated intracellular pathways The first involves betaglycan, a type III TGFβ receptor that functions as an inhibin co-receptor that binds to the ActRII with high affinity, thus preventing the

activin from doing so (Lewis et al., 2000) The second is the inhibin-binding protein,InhBP

(p120), that associates strongly with the ActRI(type IB) in a activin-responsive manner It

antagonizes the activin signal transduction through modulatingthe activin heteromeric receptor complex assembly (Chapman and Woodruff, 2001) Follistatin binds to activin with high affinity

and prevents it from binding to its own receptor on the cell surface (Nakamura et al., 1990; de Winter et al., 1996) Huang and co-workers (2001a) showed that both inhibin and follistatin

decreased the activin-stimulated basal expression of the oFSHβ in the LβT2 cell cultures Similar results were also obtained with the mouse FSHβ promoter using activin and follistatin in the LβT2

cells (Jacobs et al., 2003) In this respect, it is reasonable to assume a regulatory function of these

extracellular proteins for the fish FSHβ subunit gene transcription, although the detailed

mechanism remains to be elucidated

1.4 Hypothesis

The promoter of the Chinook salmon FSHß gene contains regulatory sequences that are responsible for the cell-specific and basal expression of this subunit, and also those that mediate the stimulatory effects of GnRH and activin during puberty and sexual maturation

1.5 Aim

The aim of this project is to initiate research on the mechanism of transcriptional regulation

of the FSHβ subunit gene promoter of the Chinook salmon The Chinook salmon FSHβ subunit gene and 5’ flanking sequence will be isolated, sequenced and the transcriptional start site mapped using primer extension The promoter will then be cloned to a reporter construct to test for

functionality in the pituitary-derived LβT2 as well as a heterogeneous cell line Subsequent

transfection studies will be carried out to determine responsiveness of the promoter and the

sequence will be analyzed to identify putative regulatory elements that may mediate these effects

2 MATERIALS AND METHODS

2.1 Extraction and isolation of genomic DNA

Female Chinook salmon brain (200 mg) in 4 mL of DNAzol reagent (Life Technologies) was homogenized using a tissue grinder according to the manufacturer’s instructions The

homogenate was centrifuged for 10 min at 10, 000 x g at 4 0C Following centrifugation, the

resulting supernatant was transferred to a fresh 1.5 mL microcentrifuge tube DNA was

precipitated from the lysate/homogenate by adding 2 mL of 100 % ethanol The sample was mixed

by inversion and stored at room temperature for 2 min The DNA precipitate was spooled with a pipette tip and transferred to a clean 1.5 mL microcentrifuge tube Following this, the DNA

precipitate was washed twice with 1 mL 75 % ethanol At each wash, the DNA was suspended in ethanol by inverting the tube three times The DNA was allowed to settle to the bottom of the tube before removing the ethanol by pipetting After the DNA was air dried for 15 s, it was dissolved in

400 µL of 8 mM NaOH

The integrity of the genomic DNA (gDNA) was first determined One µg of gDNA and control human gDNA were loaded onto a 1.0 % agarose/EtBr gel to check for the size of the

gDNA The quality of the genomic DNA was also analyzed by Dra I digestion The

electrophoresis was carried out at 100 V for 1 h A 1 kb DNA ladder (GeneRulerTM, MBI

Fermentas) was used as a molecular weight marker for size estimation of the DNA fragments The EtBr-stained linear DNA fragments were observed under UV illumination and the gel was

photographed using a digital camera

3.0 2.0 1.5 1.0 0.5

M 1 2 3 4

Figure 4: Overall view of the Universal GenomeWalker TM Kit method

2.2.1 Construction of GenomeWalker TM libraries

GenomeWalkerTM libraries were constructed by digesting the extracted gDNA with BsrBI, DraI, EcoRV, HpaI and MlyI followed by phenol:chloroform purification method as outlined in Sambrook et al (1989) The digested blunt DNA fragments were then ligated with

GenomeWalkerTM adaptors (Table 1) The ligation reaction was carried out at 16 0C for 18 h

2.2.2 PCR-based DNA walking in GenomeWalker TM libraries

After the libraries had been constructed, two PCR amplifications were carried out per library The primary PCR used the adaptor primer (AP1: 5’-GTAATACGACTCACTATAGGGC-3’) provided by the kit and gene specific primer 1 (GSP1: 5’-

TACCTGCAGTCTGTCCCCGCCCTCACTGGT-3’) The components and cycling parameters of the PCR are outlined in Table 2 and 3, respectively

Table 2: The components in primary PCR cycle

Step Cycle Temperature ( o C) Time

The primary PCR mixtures were then diluted 50 times and used as templates for secondary PCR (Table 4)

The adaptor primer 2 (AP2: 5΄-ACGCGTGGTCGACGGCCCGG-3΄) and GSP2

(5’-GTCACCCACAGCGTTGCCATGACGACCAGT-3’) were used The GSP2 that was a sequence

at the 5’ end of the unpublished csFSHβ cDNA (Suzuki and Hew) was designed to extend into the unknown 5’ flanking gDNA sequence

Step Cycle Temperature ( o C) Time

2.3 Cloning of the 2.2 kb PCR fragment

The PCR fragment (40 µL) was run on a 1 % agarose gel and purified from the gel by the GeneCleanIII kit (BIO 101) according to instructions provided The purified PCR fragment was then cloned into the pT7Blue-2 vector (Novegen) as described in the manufacturer’s protocol The insert DNA was first converted to a blunt, phosphorylated form in a single, brief reaction (10 min; Table 5)

Following heat inactivation, the DNA insert was ligated to dephosphorylated pT7Blue vector using 1:2 molar ratio of vector: insert (Table 6) The ligation reaction was performed at 16°C for 18 h

2.3.1 Preparation of competent E.coli cells

A tube of glycerol stock of DH5-α Escherichia coli (E.coli) cells was thawed and 50 µL was used to innoculate 2 mL of Lauria Bertiani (LB) medium This was shaken in a 37 °C

incubator set at 230 rpm for 18 h Of this culture, 100 µL was transferred to 250 mL of LB

medium and left to shake at 230 rpm at 37 °C till the optical density at 600 nm (OD600) reached a value of 0.3 to 0.6 The bacterial culture was centrifuged at 4000 x g for 15 min at 4 °C The pellet was resuspended on ice in 1/20 volume TSS [LB broth (pH 6.1), 10 % polyethylene glycerol, 5 % DMSO (dimethylsulfoxide, Sigma), 10 mM MgCl2, 10 mM MgSO4; final pH 6.5] The cells were kept on ice for 10 min, then aliquoted in 100 µL and snap-frozen in liquid nitrogen before storage

at -80 °C

2.3.2 Transformation of E.coli competent cells

The ligation mixture (12 µL) was added to 100 µL KCM (100 mM KCl, 30 mM CaCl2, 50

mM MgCl2) Frozen DH5-α competent cells (100 µl) were added to the DNA/KCM mixture They were gently mixed and kept on ice for 60 min They were then plated onto a LB agar plate

pretreated with 40 µL of 10% (w/v) 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and 4 µL of 100 mM isopropyl-β-D-thiogalactopyranoside (IPTG) The plate was incubated

overnight at 37 °C

2.3.3 Small scale plasmid DNA isolation and purification

Selected colonies were grown in 5 mL LB medium supplemented with 100 µg/µL

by centrifuging 2 mL of the cultures at 10,000 x g for 10 min The plasmid DNA was extracted using the Wizard® Plus SV Minipreps DNA Purification System (Promega)

2.3.4 Analysis of DNA purity and concentration

To analyze the amount and purity of DNA, spectrophotometric readings at OD260 nm and

OD280 nm were taken after DNA purification The ratios of OD260 nm /OD280 nm were taken as the basis of DNA purity

2.3.5 Verification of 2.2 kb-pT7Blue vector construct

The plasmid DNAs and control vector were digested with SacI restriction enzyme (New

England Biolabs), according to the manufacturer’s instructions Each reaction mixture was

incubated at 37 °C for 2 h The reaction mixtures were stopped by heating at 75°C for 15 min 5

µL of each reaction mixture was analyzed by gel electrophoresis to verify for the correct size

2.3.6 Large-scale plasmid DNA isolation and purification

After verifying the result from the digestion reaction, the selected colony was grown in 5

mL LB-ampicillin medium After about 8 h, the culture was poured into a 500 mL conical flask containing 250 mL Terrific Broth (3g bacto-tryptone, bacto-yeast extract, 1 mL glycerol, 225 mL water and 25 mL of 0.17 M KH2PO4 and 0.72 M K2HPO4) containing ampicillin (100 µg/mL) The culture was shaken in a 37 °C incubator set at 250 rpm for about 16 h The Quantum Prep plasmid maxiprep kit (BIO-RAD) was used to isolate plasmid DNA The large-scale plasmid preparation was air-dried and the DNA was resuspended in 300 µL deionized water

2.3.7 DNA sequencing of the 2.2 kb PCR fragment

The 2.2 kb PCR fragment was then sequenced using ABI PRISMTM BigDye® Terminator v 3.1 Cycle Sequencing kit (PE Applied Biosystems, Perkin Elmer) from both sense and antisense orientations The primers used are shown in Table 22 The DNAMAN sequence analysis software (Lynnon BioSoft, version 4.15) was then used to assemble and analyze the DNA sequences

obtained The components and cycling parameters of the sequencing reaction are shown in Table 7 and 8 respectively

The precipitation of the sequencing PCR products was carried out according to the manufacturer’s protocol Automated sequencing was then performed using the ABI Prism® 3100 DNA Automated Sequencer (Perkin Elmer)

Components Volume ( µL)

Double-stranded DNA template (0.4 µg) 1

Terminator Ready Reaction Mix 4

Table 7: The components in DNA sequencing PCR

Step Cycle Temperature ( o C) Time

2.4 Subcloning of the 1 kb csFSHβ intronic enhancer into expression vector for functional studies

The 1 kb intronic enhancer was subcloned into an expression vector containing the

chloroamphenicol acetyl-transferase (CAT6) reporter gene and into the promoterless pEGFP-1 expression vector (Clontech) containing the enhanced green fluorescent protein (EGFP) for

promoter functionality studies

2.4.1 PCR amplification of 1 kb intronic enhancer

The 1 kb intronic enhancer was PCR amplified by two primers

(CCCCAAGCTTGGGCGACGGCCCGGGCTGGTAT-3’ and

5’-CGCGGATCCGCGGCGTCTGCATAGCCAGGCAC-3’) from the 2.2 kb - pT7Blue vector

(Table 9 and 10) The primers used in the PCR were specially designed to incorporate HindIII and BamHI restriction sites to the flanks of the 1 kb fragment for subsequent cloning into the

2.4.2 Cloning of the 1kb intronic enhancer into the vectors

Both the amplified 1 kb fragment and the CAT6 and EGFP plasmid vectors were digested

with HindIII and BamHI restriction enzymes (New England Biolabs) according to the

manufacturer’s instructions The cut DNA fragments were analyzed by gel electrophoresis and gel purified A 1:3 molar ratio of vector : insert was used for the cloning of the 1 kb intronic enhancer into both vectors (Table 11) The ligation was performed at 16 0C for 18 h

The overnight ligation mixture was then transformed into the DH5-α competent cells following which the plasmid DNA was extracted using the Wizard® Plus SV Minipreps DNA Purification

System (Section 2.3.3) The strategy for cloning the intronic enhancer into the CAT6 vector is summarized in Figure 5

Step Cycle Temperature ( o C) Time

Hind III and BamHI

Double digest + Geneclean + Ligation

1kb csFSH β intronic enhancer

PCR + Geneclean

Figure 5: Construction of 1kb csFSH β - CAT6 gene construct

The 1 kb csFSHβ intronic enhancer was PCR amplified from the 2.2 kb PCR insert-pT7Blue2 vector using

primers that were designed to incorporate the HindIII and BamHI restriction sites to the flanks of the 1 kb

fragment Both amplified fragment and pCAT6 vector were then double digested with HindIII and BamHI

restriction enzyme, gel purified and ligated to become the 1 kb csFSHβ-CAT6 vector construct

pBLCAT6

1kb csFSH β intronic enhancer

BamHI

2.4.3 Verification and sequence analysis of the 1 kb csFSH β gene constructs

The correct 1 kb csFSHβ-CAT6 vector and 1 kb csFSHβ-EGFP vector constructs were

verified by NdeI and NheI restriction enzymes (New England Biolabs) double digestion according

to the manufacturer’s instructions Each reaction mixture (5 µl) was analyzed by gel

electrophoresis to check for the correct size and then confirmed by sequencing

Consensus promoter sequences and putative transcription factor binding sites on the 1 kb csFSHβ intronic enhancer were found using the DNAMAN software and through the Transcription Factor Database, TRANSFAC (http://transfac.gbf.de/TRANSFAC/)

2.5 Construction of truncated and site-deleted gene constructs

Truncated and site-directed gene constructs were made from the 1 kb csFSHβ-CAT6 gene construct in collaboration with other students in the lab The 1 kb csFSHβ and CAT6 vector

sequence were analyzed using the Webcutter 2.0 (http://www.firstmarket.com/cutter/cut2.html) for identification of suitable restriction enzymes to generate truncated constructs The truncated

constructs (64 bp, 378 bp and 587 bp from the putative transcription start site) were then

constructed using four restriction enzymes (Table 12)

Truncated constructs Restriction enzymes

The 1 kb csFSHβ-CAT6 gene construct was double digested with the restriction enzymes and then gel-purified The overhang 5’ ends of the linearized DNA were then blunted using Klenow

fragment (Promega; Table 13)

The reaction was carried out at room temperature (25 0C) for 10 min before heat inactivation at 16

0C for a further 10 min The constructs were then re-ligated (Table 14) at 16 0C for 18 h

Deletion of the putative half estrogen response element (ERE) sites (675 to 667 bp and 559 to 542

bp upstream of the first base of the unpublished cDNA sequence [Suzuki and Hew]) was

performed using the QuikChange site-directed mutagenesis kit (Stratagene) according to the

manufacturer’s instructions (Table 15 and 16)

The primers (containing the desired mutation) used in the site-directed mutagenesis reactions are shown in Table 17

Dpn I (10 U) restriction enzyme was then added to each reaction mixture The reaction mixture

was gently mixed, then spin down in a microcentrifuge before incubating at 370C for 1 h Of the

Dpn I treated-DNA, 1 µL was used for transformation into DH5-α compentent cells

Transformation of the truncated or site-deleted DNA into DH5-α cells (Section 2.3.2) was carried out This was followed by small- and large scale DNA preparation (Section 2.3.3 and 2.3.6) and verification by sequencing (Section 2.3.7)