heterogeneous nuclear ribonucleoprotein a/b and g inhibits ...heterogeneous nuclear...

www.elsevier.com/locate/ymcne

Mol. Cell. Neurosci. 37 (2008) 69–84

Heterogeneous nuclear ribonucleoprotein A/B and G inhibits thetranscription of gonadotropin-releasing-hormone 1

Sheng Zhao, Wayne J. Korzan, Chun-Chun Chen, and Russell D. Fernald⁎

Department of Biological Sciences & Program in Neuroscience, Stanford University, Stanford, California 94305-5020, USA

Received 18 December 2006; revised 14 August 2007; accepted 21 August 2007Available online 29 August 2007

Gonadotropin-releasing hormone 1 (GnRH1) causes the release ofgonadotropins from the pituitary to control reproduction. Here wereport that two heterogeneous nuclear ribonucleoproteins (hnRNP-A/Band hnRNP-G) bind to the GnRH-I upstream promoter region in acichlid fish Astatotilapia burtoni. We identified these binding proteinsusing a newly developed homology based method of mass spectrometricpeptide mapping. We show that both hnRNP-A/B and hnRNP-G co-localize with GnRH1 in the pre-optic area of the hypothalamus in thebrain. We also demonstrated that these ribonucleoproteins exhibitsimilar binding capacity in vivo, using immortalized mouse GT1–7 cellswhere overexpression of either hnRNP-A/B or hnRNP-G significantlydown-regulates GnRH1 mRNA levels in GT1–7 cells, suggesting thatboth act as repressors in GnRH1 transcriptional regulation.© 2007 Elsevier Inc. All rights reserved.

Keywords: Gonadotropin-releasing hormone 1 (GnRH1); Heterogeneousnuclear ribonucleoprotein A/B (hnRNP-A/B); Heterogeneous nuclearribonucleoprotein G (hnRNP-G); Transcriptional regulation; Mass spectro-metric peptide mapping; Hypothalamic–pituitary–gonadal (HPG) axis

Introduction

In all vertebrates, reproduction is controlled by the hypotha-lamic–pituitary–gonadal (HPG) axis (Cheng et al., 1998; Faulkesand Abbott, 1991; Gahr, 2004; McComb, 1987) via gonadotropin-releasing hormone (GnRH1) released from the brain into thepituitary to regulate reproductive capacity. In many species, socialinteractions provide important control of the HPG axis, as in adultmales of an African cichlid fish, Astatotilapia burtoni. In thisspecies, gaining or losing social dominance induces specific changesin the structure and function of GnRH-containing neurons in thehypothalamus allowing analysis of the molecular basis of changes in

⁎ Corresponding author. Department of Biological Sciences, Gilbert Hall,Room 316, 371 Serra Mall, Stanford University, Stanford CA 94305-5020,USA. Fax: +1 650 723 6132.

E-mail address: [email protected] (R.D. Fernald).Available online on ScienceDirect (www.sciencedirect.com).

1044-7431/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.mcn.2007.08.015

reproductive capacity (Davis and Fernald, 1990; Fernald, 2002;Greenwood and Fernald, 2004; White and Fernald, 1993).

At the cellular level, GnRH1 is known to be controlled bymultiple signals including growth factors, secondary messengers,steroid hormones, neurotransmitters, and neuropeptides (Belshamand Lovejoy, 2005; Hapgood et al., 2005; Nelson et al., 1998).Using immortalized GnRH1 releasing cell lines (GT1–7) (Lipositset al., 1991; Mellon et al., 1990; Weiner et al., 1992; Wetsel, 1995),a proximal promoter (Eraly and Mellon, 1995; Kepa et al., 1992)and distal enhancer regions (Kepa et al., 1996; Lawson et al., 1996;Whyte et al., 1995) critical for mammalian GnRH1 gene regulationhave been identified. The proximal promoter is important for basalGnRH1 gene expression and the distal promoter for GnRH1neuron-specific expression because of an enhancer region. Bothregions could bind to numerous dsDNA-binding proteins.

Transcription factors, including the GATA-factor families (Law-son et al., 1998, 1996), octamer-binding transcription factor-1 (Oct-1) (Clark and Mellon, 1995; Eraly et al., 1998), Otx2 (Kelley et al.,2000), and three-amino acid loop extension (TALE) homeodomainproteins (Rave-Harel et al., 2004) have been shown to be importantin the transactivation of the rat GnRH1 gene. In contrast, COUP-TFIor CCAAT/enhancer binding protein-β (C/EBP-β) may be involvedin the melatonin mediated repression of GnRH1 gene expression(Gillespie et al., 2004). Interactions between the proximal promoterand enhancer are required for optimal expression of the GnRH1 genein GT1 cells (Nelson et al., 2000).

The regulatory mechanisms controlling GnRH1 expression incold-blooded vertebrates remain unknown, in part because there isno well-characterized GnRH releasing cell line derived from thosespecies. Nevertheless, bioinformatic analysis suggests that there aremultiple upstream putative consensus protein binding sites inGnRH1 for adequate regulation (Kitahashi et al., 2005).

To understand how GnRH1 transcription is regulated, weanalyzed GnRH1 upstream binding proteins in the teleost A.burtoni. Here we describe two new GnRH1 upstream bindingproteins that may repress GnRH1 transcription: hnRNP-A/B andhnRNP-G. Both proteins belong to a previously reported ssRNAbinding protein super family of heterogeneous nuclear ribonucleo-proteins. Our results suggest that this newly described function ofhnRNP-A/B and hnRNP-G may be conserved across vertebrates.

mailto:[email protected]://dx.doi.org/10.1016/j.mcn.2007.08.015

Fig. 1. Scheme for identifying novel GnRH1 upstream binding protein. Thiswhole procedure consisted of four steps: in vitro DNA/protein binding assayand purification of the sequence-specific DNA-binding proteins; massspectrometric peptide mapping to identify the novel binding protein basedon homology database searching; verification of the mass mapping results bymolecular cloning; functional tests including co-localization of the identifiedprotein in GnRH releasing neurons, in vivo binding ability of the identifiedprotein to GnRH1 upstream and functional tests of transcriptional regulation.

Fig. 2. Mapping DNA/protein binding sites in the GnRH1 upstream by immobilizinbiotinylated double stranded fragments generated by PCR using 5′ biotin labeled secontrol fragment from the cDNA coding sequence of PCNA; MK, 1 kb DNA laddebinding proteins in SDS–PAGE gel. Compared with the control, 37 kDa (gray arrowSDS–PAGE gel. SB, SeeBlue pre-stained protein ladder; SB+, SeeBlue-plus pre-staupstream fragments (gray lines) and the deduced binding sites of the 37 kDa, 42 k

70 S. Zhao et al. / Mol. Cell. Neurosci. 37 (2008) 69–84

Results

To identify the DNA-binding proteins that interact with theupstream region of the A. burtoni GnRH1 gene, we combined massmapping with classic homology based molecular cloning followedby functional tests (Fig. 1).

GnRH1 gene upstream sequence

Using inverse PCR, we cloned a total of 7235-bp of A. burtoniGnRH1 gene sequence. Double stranded biotinylated DNAfragments (~1150 bp) of the upstream region were generated byPCR (Fig. 2A). These fragments covered 3489 bp upstream regionwith 500–600 bp overlapping with each other (Fig. 2C). Sincemultiple transcription initiation sites for GnRH1 have been foundin other species (Dong et al., 1996, 1993; Kepa et al., 1992;Radovick et al., 1990; Von Schalburg and Sherwood, 1999), wepredicted that there might be more transcription initiation sites thanpreviously predicted (White and Fernald, 1998). To avoid missingany upstream sequence, we included the sequence correspondingto the GnRH1 mRNA 5′ untranslated region to the translation startcodon (+165).

Mapping the DNA/protein binding sites upstream of the GnRH1 gene

Using mass spectrometry compatible silver staining, GnRH1upstream binding proteins were visualized in an SDS–PAGE gel. Atleast seven novel bands could be unambiguously identified. In thisstudy, three bands with molecular weight (MW) around 37 kDa,42 kDa, and 48 kDa (Fig. 2B) were analyzed further. Based on their

g upstream fragments using magnetic beads. (A) DNA electrophoresis of thense primers. G1 to G5, upstream fragments of GnRH1 gene sequence; Ctr, ar (Invitrogen); (B) mass spectrometry compatible silver stain of the captureds), 42 kDa (black arrows), and 48 kDa (white arrows) bands were observed inined protein ladder (Invitrogen). (C) Schematic representation of the GnRH1Da, and 48 kDa binding proteins.

71S. Zhao et al. / Mol. Cell. Neurosci. 37 (2008) 69–84

molecular weights and high band intensity, we labeled these bindingproteins as G1–37, G1–42, and G5–48. The DNA-binding sites inGnRH1 upstream sequence could then be deduced by comparing thecovered regions of the fragments (Table 1 and Fig. 2C). For example,there may not be a 42 kDa protein binding site in −2692 to −383region due to the absence of the 42 kDa band in lane G2, G3, andG4.Similarly, G1–37 binding sites may be located in −3325 to −2693,around −1536, and −382 to +164. For binding protein G5–48, theremight be no binding site in −2692 to −1537 region due to theabsence of the 48 kDa band. The fact that lane G1 (for 37 kDa and42 kDa) and lane G5 (for 48 kDa) had much stronger signals thanother lanes indicate that either there are multiple binding sites or thebinding sites there have higher binding affinity to these proteins.

Mass spectrometric peptide mapping of the binding proteins

To characterize the putative binding proteins, we performedmass spectrometric (MS) peptide mapping for 37 kDa, 42 kDa, and48 kDa bands. Both MS and MS/MS data were analyzed (Mascotsever and NCBInt database). Band G1–42 matched an unnamedprotein (gi|47221330) from puffer fish (Tetraodon nigroviridis)with the highest protein score, 80, higher than the threshold 78(pb0.05). There are 4 MS matches for G1–42 band, in which oneMS/MS peptide sequence (IFVGG-LNPEA-TEETI-R) was alsofound. Total coverage of the whole protein sequence is about 12%.BLAST showed this unnamed puffer fish protein to be homologousto heterogeneous nuclear ribonucleoproteins A/B (hnRNP-A/B).Proteins in this hnRNP subfamily have been reported astranscriptional regulators in other species (Campillos et al., 2003;Chen et al., 2003; Gao et al., 2005, 2004a,b). The 37 kDa bandmatched best to hnRNP-A0 (MS data not shown) with a lowerprotein score than the threshold score (68 vs. 78), indicating apossible false positive match due to sequence variance duringevolution. In rodents, two isoforms of hnRNP-A/B, p40 and p37,have been reported previously (Yabuki et al., 2001) and we suspectthat the 37 kDa we observed here might be the low molecularweight isoform of A. burtoni hnRNP-A/B.

Band G5–48 matched an unnamed protein (gi|47225325) frompuffer fish (T. nigroviridis) with the highest protein score equal tothe threshold: 78 ( p=0.05). There are 10 MS numbers matched(circles in Fig. 3), in which two MS/MS peptide sequences(RGPPP-MHSR and GPAPP-VERGY-PPR) were also found(arrows in Fig. 3). Total coverage of the whole protein sequence

Table 1Summary of the GnRH1 upstream binding proteins deduced by the in vitro DNA/

Protein ID G1 G2

G1–37 Strongest No bandG1–42 Strongest No bandG5–48 Less strong No bandPosition −3325 to −2133 −2692 to −1537Fragment size (bp) 1193 1156

Protein ID Matched protein Deduced minimum site

G1–37 hnRNP-A0 3

G1–42 hnRNP-A/B 2G5–48 hnRNP-G 3

Identified binding proteins G1–37, G1–42, and G5–48 matched to hnRNP-A0, hnG1–37, 2 sites for G1–42, and 4 sites for hnRNP-G protein in A. burtoni GnRH1

is about 26%. BLAST showed this unnamed puffer fish protein tobe homologous to hnRNP-G, also named RNA binding motifprotein, X chromosome (RBMX). This family of proteins, such asRNA binding motif protein, Y chromosome (RBMY), hnRNP-GT,and hnRNP-G, is known to play important roles in braindevelopment and in the reproductive system, especially forspermatogenesis in males.

Internal sequencing of hnRNP-A/B to confirm the mass mappingresult

To verify the reliability of the results produced by massmapping and database searching based on homology amongspecies, we performed internal de novo sequence for the 42 kDaband with large scale purification of pooled G1–42 bands stainedby Coomasie Blue. Two peptides were sequenced (Peptide1:DLKDY-FSK and Peptide2: IFVGG-LNPEA-TEETI-R). We thencombined both peptide sequences and looked for short, nearlyexact matches using BLAST program and GenBank proteindatabase. BLAST results found both peptides in hnRNP-A/B frompuffer fish, chick, mice, rat, and human with no or only onemismatched amino acid (data not shown), which confirmed theresult from mass mapping. Furthermore, Peptide2 was also foundin the original MS and MS/MS data.

Molecular cloning of hnRNP-A/B and hnRNP-G

The full-length 951-bp coding sequence (CDS) of hnRNP-A/B(GenBank Accession No. DQ630738) predicted a protein of 316amino acids (GenBank Accession No. ABG02277) which wascloned from A. burtoni. The full-length 1176-bp coding sequence(CDS) of A. burtoni hnRNP-G predicted a protein of 391 aminoacids and had a calculated molecular weight of ~43.085 kDa. Thisis a smaller molecular weight than the band observed on SDS–PAGE (48 kDa) which could be explained by glycosylation ofhnRNP-G (Soulard et al., 1993). Multiple alignment (Figs. 3A andC) and a phylogenetic test (Figs. 3B and D) were performed forboth hnRNP-A/B and hnRNP-G (or RBMX) with their homologsfrom other species. Identical branching patterns in the phylogenetictrees were generated regardless of method used to produce the tree(data not shown). Analysis across species proved that theseproteins are conserved from fish to humans (Fig. 3): Identi-ties=147/182 (80%), Positives=164/182 (90%) for hnRNP-A/B

protein binding assay

G3 G4 G5

Very weak No band Very weakNo band No band Very weakLess strong Less strong Strongest−2132 to −997 −1536 to −383 −996 to +1641136 1154 1160

s Deduced binding regions

−3325 to −2693, around −1536, and−382 to +164−3325 to −2693 and −382 to +164−3325 to −2691, −1536 to +164

RNP-A/B, and hnRNP-G respectively. There are at least 3 binding sites forupstream.


and Identities=210/401 (52%), Positives=231/401 (57%) forhnRNP-G. hnRNP-A/B is highly conserved in its RNA recognitiondomain (RRD, at 57 to 129 and 141 to 213 amino acid) and morevariable at the C-terminals. hnRNP-G is highly conserved in itsRNA recognition domain (RRD, between 9 and 82 amino acid) andmore variable at the C-terminals. Phylogenetic analysis reveals thatthe cichlid sequences are closer to T. nigroviridis than Danio rerio.

Fig. 3. Molecular cloning of hnRNP-A/B and hnRNP-G from A. burtoni. Vector-Nprotein sequences for A. burtoni hnRNP-A/B (A) or hnRNP-G (RBMX) (C) and seqRRM domains were indicated by an open box and peptides matched in massphylogenetic tree for hnRNP-A/B (B) or hnRNP-G (RBMX) (D) was generated bysequence co-segregates with sequences from the other fish species.

Confirmation of the mass mapping results by analysis of clonedA. burtoni sequences of hnRNP-A/B and hnRNP-G

Following submission of the cloned sequences of hnRNP-A/B andhnRNP-G to GenBank, we conducted a new search (Mascot) usingthe original mass mapping data to probe the updated NCBInt data-base. As expected, the new search found the A. burtoni hnRNP-A/B

T software (Invitrogen) was used to generate multiple alignment of predicteduences from other fish species, mouse, and human. Two conserved RRD andmapping (black) and internal sequencing (red) data were underlined. TheMega 3.1 using neighbor-joining and bootstrap test. Note that the A. burtoni

Fig. 3 (continued ).


sequence with a much higher protein score of 102 and better sequencecoverage of 25% (Table 2). In addition, three more observed MSnumbers were found in A. burtoni hnRNP-A/B sequence (Table 2).Furthermore, the peptide sequences obtained by internal sequencing(DLKDY-FSK and IFVGG-LNPEA-TEETI-R) were found inA. burtoni hnRNP-A/B (Table 2 and Fig. 3A). Similarly, the newsearch for G5–48 band found A. burtoni hnRNP-G sequence with avery high protein score of 381 and sequence coverage of 55%. Inaddition, 23 observedMS numbers and sixMS/MS peptide sequenceswere found in A. burtoni hnRNP-G sequence (Table 3). Peptide

sequence GPAPP-VERGY-PPR found in the original search with asmall ion score (only 5) was not found in the new search (Table 3),indicating a false positive match. Nevertheless, five new MS/MSpeptide sequences were discovered in the subsequent search(DGYGG-GREPR, DRDPY-GPPPP-R, SYMDR-PSGGS-YR,SAPSG-PMSRP-PMSR, and SLDGK-PIKVE-QATKP-QFESA-GR,Table 3 and Fig. 3C). These results strongly confirmed that bands G1–42 and G5–48 are indeed A. burtoni hnRNP-A/B and hnRNP-Grespectively and clearly demonstrated the feasibility of this newhomology based strategy designed to identify novel binding proteins.

Table 2Confirmation of mass mapping result after cloning A. burtoni hnRNP-A/B

Gi|108744009: heterogeneous nuclear ribonucleoprotein A/B [Astatotilapia burtoni]

Mass: 35160 Protein score: 102 Sequence coverage: 25%

Observed Mr(expt) Mr(calc) Delta Start End Miss Ion score Peptide

1144.5867 1143.5794 1143.644 −0.0646 181 189 1 – RGFIFITYK1219.5385 1218.5312 1218.5727 −0.0415 46 57 1 – IDASKGEEDAGK1318.5928 1317.5855 1317.6776 −0.0921 106 117 1 – DSASVDKVLEQK1343.5743 1342.567 1342.6227 −0.0557 58 69 0 – MFVGGLSWDTSK

Oxidation (M)1455.6241 1454.6168 1454.7227 −0.1059 58 70 1 – MFVGGLSWDTSKK1745.8159 1744.8086 1744.8995 −0.0909 142 157 0 65 IFVGGLNPEATEETIR1875.7379 1874.7306 1874.8138 −0.0832 79 95 1 – FGEVSDCTIKMDSNTGR

Oxidation (M)

Peptides in bold were confirmed by MS/MS peptide sequencing and internal sequencing data.


Location of hnRNP-A/B and hnRNP-G mRNA

To discover where hnRNP-A/B and hnRNP-G are located inA. burtoni, we used reverse transcription PCR in a variety of tissues(Fig. 4A). These data showed that hnRNP-A/B is highly expressedin most tissues we sampled, including brain, retina, pituitary,muscle, gill, gut, liver, kidney, ovary, testicle, and heart; withreduced expression in spinal cord and no expression in stomach.hnRNP-G is highly expressed in retina, pituitary, muscle, liver,testicle, and heart, with reduced expression in brain, gill, spleen, gut,kidney, and ovary and no expression in spinal cord and stomach.

To locate expression in the brain, we performed in situ hybrid-ization for hnRNP-A/B and hnRNP-G. We found the mRNA of both

Table 3Confirmation of mass mapping result after cloned A. burtoni hnRNP-G

gi|108744011: heterogeneous nuclear ribonucleoprotein G [Astatotilapia burtoni]

Mass: 43085 Protein score: 381

Observed Mr(expt) Mr(calc) Delta Start E

1005.4341 1004.4268 1004.4675 −0.0407 198 21034.4856 1033.4783 1033.5239 −0.0456 95 11050.4774 1049.4701 1049.5188 −0.0487 95 11063.4479 1062.4406 1062.4842 −0.0436 259 21133.4094 1132.4021 1132.4421 −0.0400 281 21149.5458 1148.5385 1148.521 0.0175 223 21193.5573 1192.55 1192.5948 −0.0448 347 31260.5419 1259.5346 1259.5894 −0.0548 333 31266.569 1265.5617 1265.6153 −0.0536 180 11266.569 1265.5617 1265.6153 −0.0536 180 11292.543 1291.5357 1291.5792 −0.0435 297 31375.5479 1374.5406 1374.5986 −0.0580 269 21391.5385 1390.5312 1390.5935 −0.0623 269 21422.6649 1421.6576 1421.7401 −0.0825 101457.6398 1456.6325 1456.6915 −0.0590 166 11473.6305 1472.6232 1472.628 −0.0048 320 31486.6534 1485.6461 1485.7139 −0.0678 501547.7191 1546.7118 1546.7739 −0.0621 811553.6458 1552.6385 1552.6721 −0.0336 128 11627.7599 1626.7526 1626.691 0.0616 233 21899.8485 1898.8412 1898.9162 −0.0750 502047.839 2046.8317 2046.9322 −0.1005 123 12386.1687 2385.1614 2385.2651 −0.1037 73

Peptides in bold were confirmed by MS/MS peptide sequencing data.

binding proteins in many brain regions (data not shown) including thepre-optic area (Fig. 4B) where GnRH1 neurons are located. Double insitu hybridization showed that hnRNP-A/B or hnRNP-G mRNA andGnRH1 mRNA were co-localized and that some nearby cells alsoexpress hnRNP-A/B and hnRNP-G (Fig. 4C). The successful cloningofmouse hnRNP-A/B (mhnRNP-A/B) and hnRNP-G (mhnRNP-A/B)from GT1–7 cells suggests that they are also present in this culturedmouse GnRH1 releasing neuron (data not shown).

In vivo binding of mouse hnRNP-A/B and hnRNP-G tomouseGnRH1

Although we successfully identified hnRNP-A/B and hnRNP-Gfrom the pool of the in vitroDNA/protein complex, it is possible that

Sequence coverage: 55%

nd Miss Ion score Peptide

05 1 – RDDYPSPR03 1 25 RGPPPMHSR03 1 – RGPPPMHSR68 1 31 DGYGGGREPR90 0 – DSYDGYGNSR32 1 – DSRDYGPPSR57 1 – QDRGPAPPAER43 1 – SDPYPPSRGER90 1 35 DRDPYGPPPPR90 1 – DRDPYGPPPPR Oxidation (M)10 0 – GPPPSYGGSSGSSR80 0 7 SYMDRPSGGSYR80 0 – SYMDRPSGGSYR22 0 – LFIGGLNTETTEK79 0 11 SAPSGPMSRPPMSR32 1 – DGYGSRDSYPNSR63 0 – GFAFVTFESPADAK94 0 – VEQATKPQFESAGR40 0 – DYYDSGSVEPFFK45 1 – DYSYREYSNSTSR67 1 – GFAFVTFESPADAKDAAR40 1 – GPPSRDYYDSGSVEPFFK94 1 96 SLDGKPIKVEQATKPQFESAGR

Fig. 4. Localization of hnRNP-A/B and hnRNP-G mRNA in A. burtoni. (A) mRNA of hnRNP-A/B and hnRNP-G in various A. burtoni tissues. SC, spinal cord;Br, brain; Re, retina; Pit, Pituitary gland; Mu, muscle; Gill, gill; Sp, spleen; St, stomach; Gut, gut; Li, liver; Ki, kidney; Ov, ovary; Te, testicle, He, heart; andCtr, control (water). Beta-Actin was used as an internal control. (B (20× magnification) and C (40× magnification)) In situ hybridization of hnRNP-A/B orhnRNP-G mRNA and GnRH1 mRNA in the pre-optic area of the A. burtoni brain. GnRH1 releasing neurons were stained by DAB (brown in upper panel, brightfield) and hnRNP-A/B or hnRNP-G mRNAwere visualized by silver grains developed in emulsion (black dots in upper panel, bright field, or white dots in lowerpanel, dark field). Cresyl violet staining (blue in upper panel) was used to visualize cell bodies.


this is a false positive due to the buffering conditions in vitro. Toconfirm the in vivo binding capacity of hnRNP-A/B and hnRNP-Gto the upstream region of mouse GnRH1 gene, we performedchromatin immunoprecipitation. Since there is no available antibodyagainstA. burtoni hnRNP-A/B or hnRNP-G, we utilized an antibodyagainst mouse hnRNP-A2/B1 or mammalian hnRNP-G (C-17) to

immunoprecipitate the in vivo DNA/protein complex from GT1–7cells. Only expected PCR products were observed from the samplestreated with hnRNP-A2/B1 or C-17 antibody. All negative controls(bacteria genomic DNA, sample without antibody or with anantibody against hnRNP-M1-4) did not identify any immunopreci-pitant relevant sequence. Thus, the ChIP result did confirm that


mhnRNP-A/B and mhnRNP-G proteins are able to bind to theupstream region of mouse GnRH1 gene (mGnRH1) (Fig. 5).

Overexpression of hnRNP-A/B and hnRNP-G down-regulatesmouse GnRH1 in GT1–7 cells

We conducted a functional test of hnRNP-A/B and hnRNP-G onthe transcriptional regulation of mGnRH1 in the GT1–7 cell line.Overexpression of A. burtoni hnRNP-A/B (aA/B), ahnRNP-G (aG),mouse hnRNP-A/B (mA/B), or mhnRNP-G (mG) reduced thetranscripts of mGnRH1 to 72.60%, 78.33%, 66.72%, and 77.11% ofthe control cells transfected with empty plasmid (Fig. 6A). Over-expression of hnRNP-A/B and hnRNP-G were further confirmed bywestern blot analysis (Fig. 6B). In each case, despite the over-expression of the hnRNP-A/B or hnRNP-G, the inhibition of thehigh level of mGnRH1 expression in GT1–7 cells is moderate,indicating that the overexpression of hnRNP-A/B or hnRNP-Galone is insufficient to totally repress transcription. This phenom-

Fig. 5. In vivo binding of hnRNP-A/B and hnRNP-G to GnRH1 gene upstream in musing specific primers against mouse GnRH1 upstream sequence. mGnRH1 upstre(651 bp) were amplified from the immunocomplex pulled down by antibody againelectrophoresis (arrows). Bg, bacteria genomic DNA; NA, no antibody; A2/B1, antplus DNA Marker (Invitrogen).

enon has also observed in other hnRNP-A/B targeted genes such asthe apo very low density lipoprotein II (apoVLDL II) gene (Smidtet al., 1995).

Putative transcription factor binding sites and hormone responseelements in GnRH1 gene

Finally, theA. burtoniGnRH1 (GenBankAccessionNo.AF076961)and mouse GnRH1 (GenBank Accession No.NT_039606) genesequences were analyzed by a transcription factor predictionprogram, MatInspector. A stringent searching condition was usedto reduce the false positive predictions by choosing 0.85 as thematrix similarity threshold resulting in 721 putative protein bindingsites as opposed to the default optimized matrix similarity threshold(b0.85, found 1142 binding sites). Binding sites of well-knowntranscriptional factors and hormone response elements were plotted(Fig. 7). In summary, there are 9 sites for AP1, 5 for SP1, 6 CAAT-boxes, 20 for CREB sites, 3 for estrogen receptor, 2 for androgen and

ouse GnRH1 releasing neuron, GT1–7 cells. After ChIP, PCR was performedam −2612 to −2085 (532 bp), −909 to −387 (523 bp), and −378 to +273st hnRNP-A2/B1 (A) or mhnRNP-G (B). PCR products were visualized byi-hnRNP-A2/B1; C-17, anti-hnRNP-G, M, anti-hnRNP-M1-4; and MK, 1 kb

Fig. 6. Transcriptional repression of mouse GnRH1 gene by overexpressinghnRNP-A/B and hnRNP-G in mouse GnRH releasing neuron, GT1–7 cells.Control empty Flag plasmid (Ctr), Flag tagged A. burtoni hnRNP-A/B (aA/B),mouse hnRNP-A/B (mA/B), A. burtoni hnRNP-G (aG), and mouse hnRNP-G(mG) plasmids were transfected into GT1–7 cells for 48 h. (A) mGnRH1mRNA levelwas analyzed by real-timePCRand normalized usingmouse beta-Actin mRNA (nN5). Two-tail homoscedastic t-test (two samples assumingequal variance) was used for statistic analysis. ⁎⁎ Indicates that significantdifference was found between the experimental group and control group( pb0.01). (B) Western blots were performed to verify the overexpression ofFlag tagged hnRNP-A/B and hnRNP-G (αFlag, antibody against Flag tag).Mouse beta-Actin (αActin, antibody against beta-Actin) was used as an in-ternal control.


glucocorticoid receptor, 28 for Oct-1/B/P, 8 for nuclear hormonereceptor, and 19 TATA-boxes. For hormone response elements, themouse GnRH1 gene contains 2 for nuclear hormone receptor, 3 forER, 8 for AR, and 3 for GR (Fig. 7A). Based on the known bindingsites of hnRNP-A/B (Table 4), we found the 7 consensus sequences(DRTWWKGWVA) upstream of A. burtoni GnRH1 gene and 6 inmouse GnRH1 gene (Fig. 7B). The positions of some of thesepredicted binding sites are consistent with the results of the bindingassay (Fig. 2, Table 1, Fig. 3A, and Fig. 5A). Moreover, some of thehormone response elements and putative hnRNP-A/B binding sitesare located close to each other in GnRH1 upstream (especiallymGnRH1) (Fig. 7B), implying an interaction between hnRNP-A/Band the hormone receptors which have been shown in other hnRNPsin regulating other genes (Chen et al., 2005; Eggert et al., 2001,1997; Smidt et al., 1995).

Discussion

GnRH1 transcriptional regulation

Our results (Fig. 7) showed that upstream of A. burtoni GnRH1genes contain predicted potential transcription factor binding motifsfor various DNA-binding proteins, including nuclear receptors,consistent with reports from other cichlid species (Kitahashi et al.,2005). Although searching for transcription factor binding sitesbased on short consensus sequence may produces false positives,novel binding sites may remain undiscovered. In cold-blooded

vertebrates, none of the identified putative binding sites in GnRH1gene has been investigated for possible direct binding activity.However, some evidence of transcriptional regulation of GnRH1gene (e.g. overexpression or down-regulation) has been reported forputative binding proteins such as the sex hormone receptors, ER(Parhar et al., 2000), PR (Parhar et al., 1998), TR (Dubois et al.,2001; Iela et al., 1994), and GR (Fox et al., 1997;White et al., 2002).In this study, we sought direct evidence for regulation of GnRH1 atthe level of the transcription.

Discovery of hnRNP-A/B and hnRNP-G as transcriptional regulatorof GnRH1 in A. burtoni

To understand the transcriptional regulation of GnRH1, weassessed very small quantities of proteins bound to the GnRH1gene, analyzing the sequence and then testing its role experimen-tally in cultured cells (Fig. 1). We identified several novel GnRH1upstream binding proteins including hnRNP-A/B and hnRNP-G, astranscriptional repressors.

The hnRNP family is a large nucleoprotein family including morethan 20 known proteins (34 kDa to 120 kDa) that were identified assplicing factors modulating RNA processing (Krecic and Swanson,1999). The hnRNP-A/B subfamily is typified by its characteristicprimary structure containing two RNA recognition domains (RRDs)followed by a glycine-rich C-terminus (Mayeda et al., 1994). TheDNA-binding sequence domain of hnRNP-A/B is different from thetranscriptional regulation domain (Gao et al., 2004a). In rodent,the larger hnRNP-A/B isoform, p40, exhibits an inhibitory activity onthe target gene (Saitoh et al., 2002). In A. burtoni, we also observedtwo hnRNP proteins (42 kDa and 37 kDa bands) that bind to theGnRH1 upstream region with similar molecular weight as rodent’shnRNP-A/B isoforms (Table 1 and Fig. 2). The predicted binding sitesfor hnRNP-A/B based on the known core sequencewere also found inthe fragments where both proteins bind (Fig. 7).

In the transcriptional regulation of osteopontin (OPN), hnRNP-A/Band hnRNP-U proteins function antagonistically (Gao et al., 2005):hnRNP-A/B dissociates from promoter binding site with subse-quent derepression of OPN promoter activity (Gao et al., 2004a,b).hnRNP-U then binds to the same site to further activate OPNpromoter activation (Gao et al., 2005), indicating that aninteraction among hnRNP proteins might be important for theirin vivo functions (Kim et al., 2000). Here we also found that twohnRNP proteins bind to the upstream of the same gene at similarregions (Fig. 7), implying that a possible interaction between themmight exist. Chicken single-strand D-box binding factor (ssDBF),a homolog of mammalian hnRNP-A/B, acts as transcriptionalsilencer of apoVLDL II and heterologous viral promoter viabinding to the D-box and CArG-box respectively. Rat hnRNP-A/Bhomolog, termed AlF-C1, is involved in repressing rat aldolase B(AldB) gene expression and in DNA replication initiation (Saitohet al., 2002; Yabuki et al., 2001). In addition to its repressivefunction, hnRNP-A/Bs have also been shown to be necessary forthe transcriptional activation of the rat serine protease inhibitor 2(spi-2) gene (Leverrier et al., 2000).

hnRNP-G belongs to another hnRNP subfamily with two moremembers, RBMY and hnRNP-GT, with similar structures: an N-terminal RNA recognition motif (RRM) followed by amino acidsrich in serine, arginine, and glycine (Elliott, 2004). All three arenuclear proteins with a previously reported role in regulating pre-mRNA splicing (Elliott, 2004). Interestingly, members of thissubfamily have been reported as potential factors modulating the

Fig. 7. Schematic representation of A. burtoni GnRH1 gene with putative protein binding sites. A. burtoni and mouse GnRH1 gene were analyzed byMatInspector with a stringent threshold (0.85). The putative transcriptional regulatory motif in GnRH1 gene upstream region was plotted in A. Well-knownhormone response elements and putative hnRNP-A/B binding sites (DRTWWKGWVA) were plotted in B. Symbols for binding sites on the upper side of thegene indicate that they are on sense strand. Lower side indicates the antisense strand. Double stranded DNA fragments used for the in vitro DNA/protein bindingassay were indicated by the pink lines and the possible binding sites for hnRNP-A0, hnRNP-A/B, and hnRNP-G were deduced from the in vitro DNA/proteinbinding assay (Table 1 and Fig. 2).


reproductive system of males (Delbridge et al., 1999; Ma et al.,1993; Venables et al., 2000; Westerveld et al., 2004). HumanhnRNP-G (also named RBMX) gene is coded on the X chromosomeand is expressed in many tissues (Lingenfelter et al., 2001) with adifferential expression pattern with a characteristic tissue specificity(Nasim et al., 2003) supported in our PCR data in various fish tissues(Fig. 4A).

The wide distribution of hnRNP-G protein suggests that it mightplay an important role in somatic cells other than the testis. Thehuman hnRNP-G gene is located on the X chromosome at Xq26where several X-linked mental retardation (XLMR) syndromes have

Table 4Consensus binding sequence of hnRNP-A/B

Consensus sequence DRTWWKG DVA

apoVLVD II D-box TTCG GAT T T G G TAARSV CArG-box TCCT A ATT A GG TAAOPN promoter GGGT A GTT A T G AC ATCGTTCATC5′ end of Feline Parvovirus TC T A TA A GG T GAACT

Based on known binding sequences found in other genes, a consensus binding se

also been mapped to (Delbridge et al., 1999), suggesting a possibleinvolvement of hnRNP-G in these mental disorders. In zebrafish,knockdown experiment of hnRNP-G by antisense morpholinoindicated that hnRNP-G is required for normal embryonic develop-ment (Tsend-Ayush et al., 2005). Although current studies on thehnRNP-G protein subfamily have been focused on their function ofpre-mRNA binding and splicing, other hnRNPs have already beenidentified as ssDNA/dsDNA-binding proteins.

Transcriptional activation or repression of RNA polymerase IIdependent genes such as GnRH1 is often mediated by sequence-specific DNA-binding proteins or transcription factors which re-

Format Function References

ssDNA Transcriptional repressor Smidt et al. (1995)ssDNA Transcriptional repressor Smidt et al. (1995)dsDNA Transcriptional repressor Gao et al. (2004a)ssDNA Modify virus replication Wang and Parrish (1999)

quence is deduced (DRTWWKGDVA).


spond to extracellular signals by interacting with key cis-actingregulatory elements. Transcription factors generally bind to double-stranded DNA recognition sites distal to the TATA-box andinfluence promoter activity (Johnson, 1995; Tjian and Maniatis,1994). Although traditional transcription factors are double-strandedDNA (dsDNA) binding proteins, in recent years, many sequence-specific, single-stranded DNA (ssDNA)/RNA (ssRNA)-bindingproteins have been discovered that either activate (Duncan et al.,1996; Haas et al., 1995b; Tomonaga and Levens, 1996) or repress(Altiok and Groner, 1993; Gupta et al., 2003; Haas et al., 1995a;Kelm et al., 1999; Lindzey et al., 1994; Pan et al., 1990; Tanumaet al., 1995; Wilkison et al., 1990) transcriptional activity.

Many such single-strand DNA-binding proteins are importantfor gene transcriptional regulation. For example, the far upstreamelement binding proteins (FBPs) and heterogeneous nuclearribonucleoproteins K have been shown to utilize cis elements,forming non-B-DNA structures, as docking sites to effect geneexpression (Bazar et al., 1995; Davis-Smyth et al., 1996; Michelottiet al., 1996). A yeast 45 kDa protein binds specifically to the lowerstrand of an estrogen responsive element (ERE) to facilitate thebinding of an estrogen receptor (Mukherjee and Chambon, 1990).ER has also been shown to bind single-strand ERE (Lannigan andNotides, 1989; Nardulli et al., 1993). Chicken single-strand D-boxbinding factor (ssDBF), a homolog of mammalian hnRNP-A/B, actsas transcriptional silencer of apoVLDL II via ssDNA-binding.However, there is also some evidence that the DNA recognition siteis in a normal double-strand conformation, in which other generaldouble strand binding proteins might involved (Smidt et al., 1995;Wijnholds et al., 1991). Besides binding to ssDNA, hnRNP-A/B hasalso shown dsDNA-binding activity to the OPN promoter (Gaoet al., 2005, 2004a,b) and hnRNP-A1 was recently reported to bindto a 36-bp double stranded sequence in vitro (Donev et al., 2002). Inour experiment, since PCR generated dsDNA fragments were usedfor the in vitro binding assay, binding between hnRNP-A/B orhnRNP-G and GnRH1 gene may be accomplished in a dsDNA/protein complex format.

Relationship between the hnRNP family and hormones

Many members of the hnRNP family have been reported astranscriptional regulators, acting via their ssDNA or dsDNA-bindingactivity (Campillos et al., 2003; Chen et al., 2003; Eggert et al., 2001;Eggert et al., 1997; Gao et al., 2005 2004a,b; Tay et al., 1992; Tolnayet al., 1999; Tomonaga and Levens, 1995). Some act on the stresssignaling and reproductive cascade. For example, hnRNP-A/B wasfound to play an essential role during oogenesis (Matunis et al.,1994; Steinhauer and Kalderon, 2005). In vitro experiments showeda repressive function of ssDBF (hnRNP-A/B) on estrogen dependentactivity of the apoVLDL II promoter (Smidt et al., 1995). hnRNP-A/B is also involved in the growth hormone dependent transcrip-tional activation of spi-2 genes (Leverrier et al., 2000). OtherhnRNPs, e.g. hnRNP-U, have been identified as repressingglucocorticoid receptor (GR)-dependent transcription by interactingwith GR (Eggert et al., 2001, 1997). hnRNP-C like protein, alsocalled estrogen-response element binding protein (ERE-BP), wasidentified as a competitor of ER for ERE and can cause estrogenunresponsiveness (Chen et al., 2005). A previous work also showedthat there is an association between hnRNP-G and hnRNP-C asshown by co-immunoprecipitation experiments (Choi et al., 1986;Soulard et al., 1991). Both testosterone (Sheflin and Spaulding,2000) and estrogen (Arao et al., 2002) can regulate the expression of

hnRNP-D. We found that some of the hormone response elementsand putative hnRNP-A/B consensus binding sites are located closeto each other in GnRH1 upstream (Fig. 7B), implying a possibleinteraction between hnRNP-A/B and the hormone receptors.

Experimental methods

Animals and materials

We used tissue from an African cichlid fish, A. (Haplochromis) burtoni,bred from wild-caught stock (Fernald, 1977; Fernald and Hirata, 1977) andraised in laboratory aquaria. Animals were maintained under conditions thatmimicked those of the natural habitat (27 °C; 12:12 light/dark cycle withfull spectrum lights; pH 7.6–8.0) and were fed daily (Wardleys, Secaucus,NJ) (Fernald, 1977). All fish used in this study were sexually mature maleswith body sizes ca. 7–9 cm and body weights ca. 12–18 g. Fish were killedby rapid cervical transection and tissue was immediately collected foranalysis. All procedures were in accordance with the National Institutes ofHealth protocol for animal experimentation and approved by the AnimalCare and Use Committee of Stanford University.

Nucleotide primers were obtained from Invitrogen (Carlsbad, CA)(Supplementary Table 1) and all other reagents were purchased fromSigma-Aldrich (St. Louis, MO) if not otherwise specified.

Genomic DNA extraction and inverse PCR

Genomic DNAwas extracted from fish (DNeasy, Qiagen, Valencia, CA)and inverse PCR, based on the known GnRH1 gene sequence (GenBankAccession No.AF0769961), was repeatedly performed until a 7235-bpsequence of A. burtoni GnRH1 gene had been cloned. Briefly, 1 μggenomic DNA was digested with 5–10 units restriction enzyme (NewEngland Biolabs, Ipswich, MA) in 50 μl reaction for 2 h. Several enzymeswere utilized in each digestion to guarantee production of a suitablefragment with the appropriate length for forming a loop. The enzyme wasinactivated at 65 °C for 20 min. Genomic DNA fragments were diluted to500 μl and then circularized by 10 units T4-DNA ligase (Invitrogen,Carlsbad, CA) at 16 °C overnight followed by ethanol precipitation at−20 °C overnight. Touchdown PCR was used for cloning experiments inthis study: 3 min 95 °C initial denature process followed by 16 touchdowncycles from 68 °C to 60 °C (annealing temperature, decrease 0.5 °C everycycle) and continued for another 25 cycles with 60 °C annealingtemperature. All primers were designed with 60 °C melting temperaturebased on the known sequence of A. burtoni.

DNA/Protein in vitro binding assay

The protocol for identifying the GnRH1 upstream binding proteins wasbased on Nordhoff’s method (Nordhoff et al., 1999). Thirteen fish brainswere homogenized in 12 ml buffer A (10 mM Tris pH 7.4, 1 mM EDTA,0.5 mM EGTA, 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mMNa4P2O7, 1 mM Na3VO4, with 1× proteinase inhibitors: 5 μg/ml aprotinin,5 μg/ml leupeptin, and 1 mM PMSF). The crude lysate was mixed with3 ml 5× large-volume low-salt (LVLS) binding buffer (100 mM HEPES,pH 7.9, 250 mM KCl, 5 mM MgCl2, 50% glycerol, 5 mM DTT, and0.5 mg/ml poly-d(IC)). The brain lysate was then pre-cleared with 1 mgDynabeads (Invitrogen, Carlsbad, CA) without immobilized DNA probefor 2 h at 4 °C with slow rotation. Insoluble pellets were spun down at14,000 rpm for 10 min at 4 °C and the supernatant was collected.Biotinylated double stranded GnRH1 upstream DNA fragments and acontrol DNA fragment from the cDNA coding sequence of proliferatingcellular nuclear antigen (PCNA) were generated by PCR in which the senseprimers were 5′ biotin labeled. These double stranded DNA fragments werethen immobilized by Dynal M-280 streptavidin-coated magnetic beads(Invitrogen) following the manufacturer’s instructions. The pre-clearedsupernatant was then aliquoted and incubated with the immobilized dsDNA


fragments (approximately 200 μg protein per 1 mg beads) at 4 °C overnighton rotator. After magnetic separation, the beads were washed six times with500 μl washing buffer (LVLS buffer with 2 mM PMSF, 0.5 mM DTT, and0.1 mg/ml poly-d(IC)). The captured proteins were analyzed by SDS–PAGE gel followed by SilverSNAP Stain for Mass spectrometry (Pierce,Rockford, IL) or Coomasie Blue staining when large-scale purification wasneeded.

Mass spectrometric peptide mapping based on homology

Novel bands were extracted from the silver stained gel and submittedfor mass mapping (Pan Facility, Stanford, CA). Mass spectrometricpeptide mapping data were collected and analyzed on Applied Biosystems4700 Proteomics Analyzer. Mass spectrometry (MS) and MS/MS datawere analyzed by Mascot server using NCBInt database (www.matrixscience.com). In Mascot results, the “ion score” for an MS/MSmatch is −10Log(P) where P is the absolute probability that the observedmatch between the experimental data and the database sequence is arandom event. The protein score is derived from the ion scores. Proteinscores greater than the threshold (=78) are significant ( pb0.05) (Perkinset al., 1999). The highest protein score was considered as the best match tothe binding protein. A BLAST search of the best match against GenBankwas then performed to find more homologs to use in designing degenerateprimers. After cloning the identified DNA-binding proteins from A.burtoni, the predicted protein sequences from the cDNA sequence weresubmitted to GenBank and the original MS and MS/MS data wereanalyzed again by Mascot server with the updated NCBInt database toverify whether the cloned protein could also be found in the resultedmatches with a significant protein score.

Internal sequencing

The pooled Coomasie Blue stained protein bands with the samemolecular weight found in silver stained gel were used for internalsequencing. After partially digesting of the protein, peptides were purifiedby HPLC (Michrom Bioresources, Magic 2002) using columns (ReliasilC18, 1×150 mm, 5 micron particle size, 300 Å pore size) with a gradientfrom 6% acetonitrile/0.1% trifluoroacetic acid to 42% acetonitrile/0.09%trifluoroacetic acid over 40 min. Separated peptides were sequenced byEdman sequencing (Applied Biosystems sequenator, Procise HT).

Molecular cloning of A. burtoni hnRNP-A/B and hnRNP-G

Fish brains were homogenized in 1 ml Trizol (Invitrogen, Carlsbad, CA)followed by 250 μl chloroform to isolate RNA. Rapid amplification ofcDNA ends (RACE) from brain total RNAwas performed (SMART RACEcDNA Amplification Kit, Clontech, Palo Alto, CA). Based on publishedsequences (D. rerio—GenBank Accession No.BC066454; T. nigroviridis—GenBank Accession No.CNS0EWP5 and CNS0GPM1), degenerate primersfor hnRNP-A/B were designed. Degenerate primers for hnRNP-G weredesigned based on published sequences (D. rerio—GenBank Accession No.AJ717349; Macropus eugenii—GenBank Accession No.AF034741; T.nigroviridis—GenBank Accession No.CNS0G9EN). Based on the partialsequence from the degenerate PCR, RACE PCR primers used for both 3′ and5′ ends of hnRNP-A/B and hnRNP-G cDNAwere then designed. All primerswere designed with 60 °Cmelting temperature. Touchdown PCR was used forall cloning experiments. The resulting sequences for A. burtoni hnRNP-A/B(GenBank Accession No.DQ630738) and hnRNP-G (GenBank AccessionNo.DQ630739) cDNA containing the complete coding sequence wereverified by sequencing in both directions (Sequetech, Mountain View,CA). Multiple sequence alignment analysis of A. burtoni compared withother species’ hnRNP-A/B or hnRNP-G was performed (Vector-NTsoftware, Invitrogen). The phylogenetic tree for hnRNP-A/B acrossvarious species was generated (Mega 3.1) using neighbor-joining andbootstrap tests (Kumar et al., 2004).

RNA localizations in organs

PCR was performed on selected tissues from adult A. burtoni (spinalcord, brain, retina pituitary gland, muscle, gill, spleen, stomach, gut, liver,kidney, ovary, testicle, and heart). Tissue was collected and homogenizedbefore extraction of their total RNA (RNeasy Micro Kit, Qiagen Inc.,Valencia, CA). 3′-RACE cDNA for each tissue was synthesized (SMARTcDNA synthesis kit, Clontech Laboratories Inc., Palo Alto, CA). Touch-down PCR was then conducted using specific primers of A. burtonihnRNP-A/B and hnRNP-G.

In situ hybridization

To co-localize simultaneously hnRNP-A/B, hnRNP-G with GnRH1expression, double in situ hybridization was used. Methods developed in ourlaboratory (Chen and Fernald, 2006) were used with minor modifications.Animals (1 year old reproductive male fish, ~8 cm in length and ~18 g inweight) were killed by rapid cervical transection, brains were immediatelyembedded in OCT Compound (Tissue-Tek, Torrence, CA) and flash frozen.Coronal tissue sections for were cut using a cryostat (Microm, Zeiss,Thornwood, NY) at 14 μm and thawmounted onto slides (Superfrost, Fisher,Santa Clara, CA). Templates for radioactively labeled RNA probes specificfor hnRNP-A/B and hnRNP-G were generated by PCR. In the PCR reaction,one of the primers was designed to contain an additional T7 promotersequence on its 5′ end so that the PCR product could be used as a reversetranscription template for making RNAprobe from the endwith T7 promoter.PCR products were obtained by gel purification kit (Qiagen). RNA probeswere then synthesized by T7 transcriptionase (Ambion, Austin, TX) in thepresence of 35S labeled UTP (AmershamBiosciences, Piscataway, NJ). Brainslices were hybridized with the 35S labeled sense or antisense probes, dippedin nuclear emulsion (NBT-2; Kodak, Rochester, NY), and exposed forapproximately 1 month at −20 °C. To identify GnRH1, the probe was labeledwith digoxigenin (DIG) labeled nucleotide triphosphates (NTPs) (RocheApplied Science, Indianapolis, IN) and visualized by 3,3′-Diaminobenzidine(DAB) staining using anti-DIG-peroxidase primary antibody (Roche) andTyramide Signal Amplification kit (NENLife Sciences, Boston,MA). Cresylviolet staining was used to visualize cell bodies. Digital photomicrographswere acquired under both bright field and dark field illumination (Spotcamera, Diagnostic Instruments, Sterling Heights, MI, Axioscope, Zeiss).

Cell culture

All cell culture reagents were supplied by Invitrogen. GT1–7 cells werecultured in 100-mm master plate in Dulbecco’s Modified Eagle’s Medium(DMEM, CA#11995-065) and supplemented with 10% fetal calf serum(FCS), 100 U/ml of penicillin, and 100 μg/ml of streptomycin in ahumidified atmosphere of 5% CO2 at 37 °C. Media were replaced every 2–3 days until confluence was reached. For experiments, cells were seededinto 24-well plates and cultured under the same condition. Cells utilizedwere of similar passage (passage 6–15) and confluence (~90%).

Chromatin immunoprecipitation

To isolate chromatin, an immunoprecipitation (ChIP) protocol modifiedfrom Braunstein and co-workers was used (Alberts et al., 1998; Braunstein etal., 1993; Meluh and Koshland, 1997). Chromatin/Protein complexes inculturedGT1–7 cells were cross-linked by addition of formaldehyde to a finalconcentration of 1% in phosphate buffered saline (PBS). Cross-linkingreaction was stopped after 10 min by adding glycine to a final concentrationof 0.125 M. Cells were then spun down at 5000 rpm for 5 min andresuspended in buffer A-NP40 (85 mM KCl, 5 mM Pipes (pH 8.5), 50 mMNaF, 10 mM Na4P2O7·10H2O, 1 mM Na3VO4, 0.5% NP-40, 1× proteinaseinhibitors: 5 μg/ml aprotinin, 5 μg/ml leupeptin, and 1mMPMSF) and placedon ice for 10 min followed by centrifugation at 7000 rpm for 10 min. Lysisbuffer (10 mM EDTA, 50 mM Tris–HCl (pH 8.0), 1% SDS, 50 mM NaF,

http://www.matrixscience.comhttp://www.matrixscience.com


10 mM Na4P2O7·10H2O, 1 mM Na3VO4, 1× proteinase inhibitors: 5 μg/mlaprotinin, 5 μg/ml leupeptin, and 1 mM PMSF) was then added to pellet(Nuclear) and sat on ice for 10 min. The cells were then sonicated five timesfor 10 s (Power level 3, Branson Sonifier 250, VWR International, WestChester, PA) and spun down at 14000 rpm for 10 min. The supernatant wasthen diluted tenfold in IP buffer (1.2mMEDTA, 16.7mMTris–HCl (pH 8.0),0.01% SDS, 1.1% Triton X-100, 167 mM NaCl, 50 mM NaF, 10 mMNa4P2O7·10 H2O, and 1 mM Na3VO4, 1× proteinase inhibitors: 5 μg/mlaprotinin, 5 μg/ml leupeptin, and 1 mM PMSF) with 20 mg/ml sonicatedbacterial genomic DNA and 1 mg/ml bovine serum albumin (BSA). Thechromatin solution was then pre-cleared for 1 hat 4 °C on a rotating wheel byprotein A-Magnetic beads (Invitrogen, preadsorbed in IP buffer withbacterial genomic DNA and BSA for 1 hour). Next the supernatant wasincubated with/without antibody for 1 h at 4 °C. Antibody against hnRNP-A2/B1 was purchased from Abcam (Cambridge, MA). Antibody againsthnRNP-G (C-17) or hnRNP-M1–4 was purchased from Santa Cruz Biotech(Santa Cruz, CA). The preadsorbed protein A-magnetic beads were added toprecipitate the immune complexes overnight at 4 °C. The beads were washedtwice byW1 buffer (2 mMEDTA, 20mMTris–HCl (pH 8.0), 0.1% SDS, 1%Triton X-100, and 150 mMNaCl), once byW2 buffer (1 mMEDTA, 10 mMTris–HCl (pH 8.0), 1% NP-40, 0.25 M LiCl, and 1% deoxycholate), andtwice by TE buffer (10mMTris–Cl (pH 7.5) and 1mMEDTA) for 5min eachtime. The immune complexeswere eluted by adding Elute buffer (0.25ml 1%SDS, 0.1 M NaHCO3, and 0.3 M NaCl) and rotating for 15 min at roomtemperature. The supernatant was then incubated in a 67 °C for 4 to 5 h toreverse the formaldehyde cross-link followed by phenol/chloroformextraction and then ethanol precipitated at −20 °C overnight. PCR wasperformed to detect the presence of the upstream promoter region of mouseGnRH1 followed by electrophoresis analysis.

Overexpression and transcriptional regulation in GT1–7 cells

Cloned full-length A. burtoni hnRNP-A/B (aA/B), hnRNP-G (aG),mouse hnRNP-A/B (mA/B), and hnRNP-G (mG, from GT1–7 cells) weresubcloned into p3XFLAG-CMV-7.1 Expression Vector (Sigma) betweenHindIII and EcoRI. Mouse GT1–7 cells were transfected by 1 μg emptyplasmid or plasmid containing aA/B, mA/B, aG, or mG with 3 μl TransFastreagent (Promega, Madison, WI) in 24-well plate according to manufacturer’sprotocol. Total RNA from these cells was purified (RNeasy Mini-plus Kit,Qiagen) after 48 h of transfection. Real-time PCRwas then performed and theraw fluorescent data were analyzed using the real-time PCR Miner program(Zhao and Fernald, 2005). Transfected cells were also analyzed using westernblot to confirm the overexpression of the transfected genes. Cells were treatedwith 0.5 ml of ice-cold total lysis buffer (10 mM Tris pH 7.4, 1 mM EDTA,0.5 mM EGTA, 150 mM NaCl, 1% Triton X-100, 50 mM NaF, 10 mMNa4P2O7, 1 mM Na3VO4, 5 μg/ml aprotinin, 5 μg/ml leupeptin, and 1 mMPMSF). Lysate was sheared with a 1 ml syringe three times beforecentrifugation under 14000 rpm for 10 min. The supernatant was thenanalyzed using a 4–12% Tris–Glycine SDS–PAGE gel with MES runningbuffer (Invitrogen) followed by western blotting. The nitrocellulosemembrane with the transferred proteins was blocked in Tris-buffered salineTween-20 (TBS-T, 25mMTris–HCl, pH 8.0; 125mmNaCl; 0.1%Tween-20)buffer with 5% nonfat powderedmilk for overnight at 4 °C and then incubatedwith 200 ng/ml primary antibody (Anti-Flag-M2 from Sigma) in TBS-T for2 h. This was followed by 3 rinses and 4 washes for 15min each time by TBS-T. It was then incubated with 10 ng/ml horseradish peroxidase secondaryantibody (Pierce) in TBS-T for 1.5 h followed by 9 10-min washes. Themembrane was then visualized (SuperSignal West Femto kit, Pierce). Mousebeta-Actin was used as an internal control for both real-time PCR and westernblot.

Prediction of transcription factor binding sites

Both A. burtoni GnRH1 gene (GenBank Accession No. AF076961) andmouse GnRH1 gene were analyzed by MatInspector (www.genomatix.de)(Cartharius et al., 2005; Quandt et al., 1995) to predict the putative proteinbinding sites.

Acknowledgments

We thank D. Winant for technical advice and help forinterpretation of mass spectrometry, Drs. P.L. Mellon and Dr. R.I.Weiner for donating GT1–7 cell line, B.P. Grone and Dr. L. Harbottfor valuable comments on drafts of the manuscript. This researchwas supported by NIH NS 34950 Javits Award to R.D.F.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.mcn.2007.08.015.

References

Alberts, A.S., Geneste, O., Treisman, R., 1998. Activation of SRF-regulatedchromosomal templates by Rho-family GTPases requires a signal thatalso induces H4 hyperacetylation. Cell 92, 475–487.

Altiok, S., Groner, B., 1993. Interaction of two sequence-specific single-stranded DNA-binding proteins with an essential region of the beta-casein gene promoter is regulated by lactogenic hormones. Mol. Cell.Biol. 13, 7303–7310.

Arao, Y., Kikuchi, A., Ikeda, K., Nomoto, S., Horiguchi, H., Kayama, F.,2002. A+U-rich-element RNA-binding factor 1/heterogeneous nuclearribonucleoprotein D gene expression is regulated by oestrogen in the ratuterus. Biochem. J. 361, 125–132.

Bazar, L., Meighen, D., Harris, V., Duncan, R., Levens, D., Avigan, M.,1995. Targeted melting and binding of a DNA regulatory element by atransactivator of c-myc. J. Biol. Chem. 270, 8241–8248.

Belsham, D.D., Lovejoy, D.A., 2005. Gonadotropin-releasing hormone:gene evolution, expression, and regulation. Vitam. Horm. 71, 59–94.

Braunstein, M., Rose, A.B., Holmes, S.G., Allis, C.D., Broach, J.R., 1993.Transcriptional silencing in yeast is associated with reduced nucleosomeacetylation. Genes Dev. 7, 592–604.

Campillos, M., Lamas, J.R., Garcia, M.A., Bullido, M.J., Valdivieso, F.,Vazquez, J., 2003. Specific interaction of heterogeneous nuclearribonucleoprotein A1 with the −219T allelic form modulates APOEpromoter activity. Nucleic Acids Res. 31, 3063–3070.

Cartharius, K., Frech, K., Grote, K., Klocke, B., Haltmeier, M., Klingenhoff,A., Frisch, M., Bayerlein, M., Werner, T., 2005. MatInspector andbeyond: promoter analysis based on transcription factor binding sites.Bioinformatics 21, 2933–2942.

Chen, C.C., Fernald, R.D., 2006. Distributions of two gonadotropin-releasing hormone receptor types in a cichlid fish suggest functionalspecialization. J. Comp. Neurol. 495, 314–323.

Chen, H., Hewison, M., Hu, B., Adams, J.S., 2003. Heterogeneous nuclearribonucleoprotein (hnRNP) binding to hormone response elements: acause of vitamin D resistance. Proc. Natl. Acad. Sci. U. S. A. 100,6109–6114.

Chen, H., Stuart, W., Hu, B., Nguyen, L., Huang, G., Clemens, T.L., Adams,J.S., 2005. Creation of estrogen resistance in vivo by transgenicoverexpression of the heterogeneous nuclear ribonucleoprotein-relatedestrogen response element binding protein. Endocrinology 146,4266–4273.

Cheng, M.F., Peng, J.P., Johnson, P., 1998. Hypothalamic neuronspreferentially respond to female nest coo stimulation: demonstration ofdirect acoustic stimulation of luteinizing hormone release. J. Neurosci.18, 5477–5489.

Choi, Y.D., Grabowski, P.J., Sharp, P.A., Dreyfuss, G., 1986. Heterogeneousnuclear ribonucleoproteins: role in RNA splicing. Science 231,1534–1539.

Clark, M.E., Mellon, P.L., 1995. The POU homeodomain transcriptionfactor Oct-1 is essential for activity of the gonadotropin-releasinghormone neuron-specific enhancer. Mol. Cell. Biol. 15, 6169–6177.

http://www.genomatix.dehttp://dx.doi.org/doi:10.1016/j.mcn.2007.08.015


Davis, M.R., Fernald, R.D., 1990. Social control of neuronal soma size.J. Neurobiol. 21, 1180–1188.

Davis-Smyth, T., Duncan, R.C., Zheng, T., Michelotti, G., Levens, D., 1996.The far upstream element-binding proteins comprise an ancient family ofsingle-strand DNA-binding transactivators. J. Biol. Chem. 271,31679–31687.

Delbridge, M.L., Lingenfelter, P.A., Disteche, C.M., Graves, J.A., 1999. Thecandidate spermatogenesis gene RBMY has a homologue on the humanX chromosome. Nat. Genet. 22, 223–224.

Donev, R.M., Doneva, T.A., Bowen, W.R., Sheer, D., 2002. HnRNP-A1binds directly to double-stranded DNA in vitro within a 36 bp sequence.Mol. Cell. Biochem. 233, 181–185.

Dong, K.W., Yu, K.L., Roberts, J.L., 1993. Identification of a major up-streamtranscription start site for the human progonadotropin-releasing hormonegene used in reproductive tissues and cell lines. Mol. Endocrinol. 7,1654–1666.

Dong, K.W., Duval, P., Zeng, Z., Gordon, K., Williams, R.F., Hodgen, G.D.,Jones, G., Kerdelhue, B., Roberts, J.L., 1996. Multiple transcription startsites for the GnRH gene in rhesus and cynomolgus monkeys: a non-human primate model for studying GnRH gene regulation. Mol. Cell.Endocrinol. 117, 121–130.

Dubois, E.A., Slob, S., Zandbergen, M.A., Peute, J., Goos, H.J., 2001.Gonadal steroids and the maturation of the species-specific gonadotro-pin-releasing hormone system in brain and pituitary of the male Africancatfish (Clarias gariepinus). Comp. Biochem. Physiol. Part B Biochem.Mol. Biol. 129, 381–387.

Duncan, R., Collins, I., Tomonaga, T., Zhang, T., Levens, D., 1996. Aunique transactivation sequence motif is found in the carboxyl-terminaldomain of the single-strand-binding protein FBP. Mol. Cell. Biol. 16,2274–2282.

Eggert, M., Michel, J., Schneider, S., Bornfleth, H., Baniahmad, A.,Fackelmayer, F.O., Schmidt, S., Renkawitz, R., 1997. The glucocorti-coid receptor is associated with the RNA-binding nuclear matrix proteinhnRNP U. J. Biol. Chem. 272, 28471–28478.

Eggert, H., Schulz, M., Fackelmayer, F.O., Renkawitz, R., Eggert, M., 2001.Effects of the heterogeneous nuclear ribonucleoprotein U (hnRNP U/SAF-A) on glucocorticoid-dependent transcription in vivo. J. SteroidBiochem. Mol. Biol. 78, 59–65.

Elliott, D.J., 2004. The role of potential splicing factors including RBMY,RBMX, hnRNPG-T and STAR proteins in spermatogenesis. Int. J.Androl. 27, 328–334.

Eraly, S.A., Mellon, P.L., 1995. Regulation of gonadotropin-releasinghormone transcription by protein kinase C is mediated by evolutionarilyconserved promoter-proximal elements. Mol. Endocrinol. 9, 848–859.

Eraly, S.A., Nelson, S.B., Huang, K.M., Mellon, P.L., 1998. Oct-1 bindspromoter elements required for transcription of the GnRH gene. Mol.Endocrinol. 12, 469–481.

Faulkes, C.G., Abbott, D.H., 1991. Social control of reproduction inbreeding and non-breeding male naked mole-rats (Heterocephalusglaber). J. Reprod. Fertil. 93, 427–435.

Fernald, R.D., 1977. Quantitative behavioural observations of Haplochro-mis burtoni under semi-natural conditions. Anim. Behav. 25, 643–653.

Fernald, R.D., 2002. Social regulation of the brain: sex, size and status.Novartis Found. Symp. 244, 169–184 (discussion 184–166, 203–166,253–167).

Fernald, R.D., Hirata, N.R., 1977. Field study of Haplochromis burtoni:habitats and co-habitants. Environ. Biol. Fisches 2, 299–308.

Fox, H.E., White, S.A., Kao, M.H., Fernald, R.D., 1997. Stress anddominance in a social fish. J. Neurosci. 17, 6463–6469.

Gahr, M., 2004. Hormone-dependent neural plasticity in the juvenile andadult song system: what makes a successful male? Ann. N. Y. Acad. Sci.1016, 684–703.

Gao, C., Guo, H., Wei, J., Mi, Z., Wai, P., Kuo, P.C., 2004a. S-nitrosylationof heterogeneous nuclear ribonucleoprotein A/B regulates osteopontintranscription in endotoxin-stimulated murine macrophages. J. Biol.Chem. 279, 11236–11243.

Gao, C.,Mi, Z., Guo, H.,Wei, J., Wai, P.Y., Kuo, P.C., 2004b. A transcriptional

repressor of osteopontin expression in the 4T1 murine breast cancer cellline. Biochem. Biophys. Res. Commun. 321, 1010–1016.

Gao, C., Guo, H., Mi, Z., Wai, P.Y., Kuo, P.C., 2005. Transcriptionalregulatory functions of heterogeneous nuclear ribonucleoprotein-U and-A/B in endotoxin-mediated macrophage expression of osteopontin.J. Immunol. 175, 523–530.

Gillespie, J.M., Roy, D., Cui, H., Belsham, D.D., 2004. Repression ofgonadotropin-releasing hormone (GnRH) gene expression by melatoninmay involve transcription factors COUP-TFI and C/EBP beta binding atthe GnRH enhancer. Neuroendocrinology 79, 63–72.

Greenwood, A.K., Fernald, R.D., 2004. Social regulation of the electricalproperties of gonadotropin-releasing hormone neurons in a cichlidfish (Astatotilapia burtoni). Biol. Reprod. 71, 909–918.

Gupta, M., Sueblinvong, V., Raman, J., Jeevanandam, V., Gupta, M.P.,2003. Single-stranded DNA-binding proteins PURalpha and PURbetabind to a purine-rich negative regulatory element of the alpha-myosinheavy chain gene and control transcriptional and translational regulationof the gene expression. Implications in the repression of alpha-myosinheavy chain during heart failure. J. Biol. Chem. 278, 44935–44948.

Haas, S., Steplewski, A., Siracusa, L.D., Amini, S., Khalili, K., 1995a.Identification of a sequence-specific single-stranded DNA bindingprotein that suppresses transcription of the mouse myelin basic proteingene. J. Biol. Chem. 270, 12503–12510.

Haas, S., Thatikunta, P., Steplewski, A., Johnson, E.M., Khalili, K., Amini,S., 1995b. A 39-kD DNA-binding protein from mouse brain stimulatestranscription of myelin basic protein gene in oligodendrocytic cells.J. Cell Biol. 130, 1171–1179.

Hapgood, J.P., Sadie, H., van Biljon, W., Ronacher, K., 2005. Regulation ofexpression of mammalian gonadotrophin-releasing hormone receptorgenes. J. Neuroendocrinol. 17, 619–638.

Iela, L., D'Aniello, B., Di Meglio, M., Rastogi, R.K., 1994. Influence ofgonadectomy and steroid hormone replacement therapy on the gonado-tropin-releasing hormone neuronal system in the anterior preoptic area ofthe frog (Rana esculenta) brain. Gen. Comp. Endocrinol. 95, 422–431.

Johnson, A.D., 1995. The price of repression. Cell 81, 655–658.Kelley, C.G., Lavorgna, G., Clark, M.E., Boncinelli, E., Mellon, P.L., 2000.

The Otx2 homeoprotein regulates expression from the gonadotropin-releasing hormone proximal promoter. Mol. Endocrinol. 14, 1246–1256.

Kelm Jr., R.J., Cogan, J.G., Elder, P.K., Strauch, A.R., Getz, M.J., 1999.Molecular interactions between single-stranded DNA-binding proteinsassociated with an essential MCAT element in the mouse smooth musclealpha-actin promoter. J. Biol. Chem. 274, 14238–14245.

Kepa, J.K., Wang, C., Neeley, C.I., Raynolds, M.V., Gordon, D.F., Wood,W.M., Wierman, M.E., 1992. Structure of the rat gonadotropin releasinghormone (rGnRH) gene promoter and functional analysis in hypotha-lamic cells. Nucleic Acids Res. 20, 1393–1399.

Kepa, J.K., Spaulding, A.J., Jacobsen, B.M., Fang, Z., Xiong, X., Radovick,S., Wierman, M.E., 1996. Structure of the distal human gonadotropinreleasing hormone (hGnrh) gene promoter and functional analysis inGt1–7 neuronal cells. Nucleic Acids Res. 24, 3614–3620.

Kim, J.H., Hahm, B., Kim, Y.K., Choi, M., Jang, S.K., 2000. Protein–protein interaction among hnRNPs shuttling between nucleus andcytoplasm. J. Mol. Biol. 298, 395–405.

Kitahashi, T., Sato, H., Sakuma, Y., Parhar, I.S., 2005. Cloning andfunctional analysis of promoters of three GnRH genes in a cichlid.Biochem. Biophys. Res. Commun. 336, 536–543.

Krecic, A.M., Swanson, M.S., 1999. hnRNP complexes: composition,structure, and function. Curr. Opin. Cell Biol. 11, 363–371.

Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software forMolecular Evolutionary Genetics Analysis and sequence alignment.Brief. Bioinform. 5, 150–163.

Lannigan, D.A., Notides, A.C., 1989. Estrogen receptor selectively binds the“coding strand” of an estrogen responsive element. Proc. Natl. Acad. Sci.U. S. A. 86, 863–867.

Lawson, M.A., Whyte, D.B., Mellon, P.L., 1996. GATA factors are essentialfor activity of the neuron-specific enhancer of the gonadotropin-releasing hormone gene. Mol. Cell. Biol. 16, 3596–3605.


Lawson, M.A., Buhain, A.R., Jovenal, J.C., Mellon, P.L., 1998. Multiplefactors interacting at the GATA sites of the gonadotropin-releasinghormone neuron-specific enhancer regulate gene expression. Mol.Endocrinol. 12, 364–377.

Leverrier, S., Cinato, E., Paul, C., Derancourt, J., Bemark, M., Leanderson,T., Legraverend, C., 2000. Purification and cloning of type A/B hnRNPproteins involved in transcriptional activation from the Rat spi 2 geneGAGA box. Biol. Chem. 381, 1031–1040.

Lindzey, J., Kumar, M.V., Grossman, M., Young, C., Tindall, D.J., 1994.Molecular mechanisms of androgen action. Vitam. Horm. 49, 383–432.

Lingenfelter, P.A., Delbridge, M.L., Thomas, S., Hoekstra, H.E., Mitchell,M.J., Graves, J.A., Disteche, C.M., 2001. Expression and conservationof processed copies of the RBMX gene. Mamm. Genome 12, 538–545.

Liposits, Z., Merchenthaler, I., Wetsel, W.C., Reid, J.J., Mellon, P.L.,Weiner, R.I., Negro-Vilar, A., 1991. Morphological characterization ofimmortalized hypothalamic neurons synthesizing luteinizing hormone-releasing hormone. Endocrinology 129, 1575–1583.

Ma, K., Inglis, J.D., Sharkey, A., Bickmore, W.A., Hill, R.E., Prosser, E.J.,Speed, R.M., Thomson, E.J., Jobling, M., Taylor, K., et al., 1993. A Ychromosome gene family with RNA-binding protein homology: candi-dates for the azoospermia factor AZF controlling human spermatogenesis.Cell 75, 1287–1295.

Matunis, E.L., Kelley, R., Dreyfuss, G., 1994. Essential role for aheterogeneous nuclear ribonucleoprotein (hnRNP) in oogenesis: hrp40is absent from the germ line in the dorsoventral mutant squid. Proc. Natl.Acad. Sci. U. S. A. 91, 2781–2784.

Mayeda, A., Munroe, S.H., Caceres, J.F., Krainer, A.R., 1994. Function ofconserved domains of hnRNPA1 and other hnRNPA/B proteins. EMBOJ. 13, 5483–5495.

McComb, K., 1987. Roaring by red deer stags advances the date of oestrusin hinds. Nature 330, 648–649.

Mellon, P.L., Windle, J.J., Goldsmith, P.C., Padula, C.A., Roberts, J.L.,Weiner, R.I., 1990. Immortalization of hypothalamic GnRH neurons bygenetically targeted tumorigenesis. Neuron 5, 1–10.

Meluh, P.B., Koshland, D., 1997. Budding yeast centromere compositionand assembly as revealed by in vivo cross-linking. Genes Dev. 11,3401–3412.

Michelotti, G.A., Michelotti, E.F., Pullner, A., Duncan, R.C., Eick, D.,Levens, D., 1996. Multiple single-stranded cis elements are associatedwith activated chromatin of the human c-myc gene in vivo. Mol. Cell.Biol. 16, 2656–2669.

Mukherjee, R., Chambon, P., 1990. A single-stranded DNA-binding proteinpromotes the binding of the purified oestrogen receptor to its responsiveelement. Nucleic Acids Res. 18, 5713–5716.

Nardulli, A.M., Greene, G.L., Shapiro, D.J., 1993. Human estrogen receptorbound to an estrogen response element bends DNA. Mol. Endocrinol. 7,331–340.

Nasim, M.T., Chernova, T.K., Chowdhury, H.M., Yue, B.G., Eperon, I.C.,2003. HnRNP G and Tra2beta: opposite effects on splicing matched byantagonism in RNA binding. Hum. Mol. Genet. 12, 1337–1348.

Nelson, S.B., Eraly, S.A., Mellon, P.L., 1998. The GnRH promoter: target oftranscription factors, hormones, and signaling pathways. Mol. Cell.Endocrinol. 140, 151–155.

Nelson, S.B., Lawson, M.A., Kelley, C.G., Mellon, P.L., 2000. Neuron-specific expression of the rat gonadotropin-releasing hormone gene isconferred by interactions of a defined promoter element with theenhancer in GT1–7 cells. Mol. Endocrinol. 14, 1509–1522.

Nordhoff, E., Krogsdam, A.M., Jorgensen, H.F., Kallipolitis, B.H., Clark, B.F.,Roepstorff, P., Kristiansen, K., 1999. Rapid identification of DNA-bindingproteins by mass spectrometry. Nat. Biotechnol. 17, 884–888.

Pan, W.T., Liu, Q.R., Bancroft, C., 1990. Identification of a growth hormonegene promoter repressor element and its cognate double- and single-stranded DNA-binding proteins. J. Biol. Chem. 265, 7022–7028.

Parhar, I.S., Soga, T., Sakuma, Y., 1998. Quantitative in situ hybridization ofthree gonadotropin-releasing hormone-encoding mRNAs in castratedand progesterone-treated male tilapia. Gen. Comp. Endocrinol. 112,406–414.

Parhar, I.S., Soga, T., Sakuma, Y., 2000. Thyroid hormone and estrogenregulate brain region-specific messenger ribonucleic acids encodingthree gonadotropin-releasing hormone genes in sexually immature malefish, Oreochromis niloticus. Endocrinology 141, 1618–1626.

Perkins, D.N., Pappin, D.J., Creasy, D.M., Cottrell, J.S., 1999. Probability-based protein identification by searching sequence databases using massspectrometry data. Electrophoresis 20, 3551–3567.

Quandt, K., Frech, K., Karas, H., Wingender, E., Werner, T., 1995. MatIndand MatInspector: new fast and versatile tools for detection of con-sensus matches in nucleotide sequence data. Nucleic Acids Res. 23,4878–4884.

Radovick, S., Wondisford, F.E., Nakayama, Y., Yamada, M., Cutler Jr., G.B.,Weintraub, B.D., 1990. Isolation and characterization of the humangonadotropin-releasing hormone gene in the hypothalamus and placenta.Mol. Endocrinol. 4, 476–480.

Rave-Harel, N., Givens, M.L., Nelson, S.B., Duong, H.A., Coss, D., Clark,M.E., Hall, S.B., Kamps, M.P., Mellon, P.L., 2004. TALE homeodomainproteins regulate gonadotropin-releasing hormone gene expressionindependently and via interactions with Oct-1. J. Biol. Chem. 279,30287–30297.

Saitoh, Y., Miyagi, S., Ariga, H., Tsutsumi, K., 2002. Functional domainsinvolved in the interaction between Orc1 and transcriptional repressorAlF-C that bind to an origin/promoter of the rat aldolase B gene. NucleicAcids Res. 30, 5205–5212.

Sheflin, L.G., Spaulding, S.W., 2000. Testosterone and dihydrotestosteroneregulate AUF1 isoforms in a tissue-specific fashion in the mouse. Am. J.Physiol: Endocrinol. Metab. 278, E50–E57.

Smidt, M.P., Russchen, B., Snippe, L., Wijnholds, J., Ab, G., 1995. Cloningand characterisation of a nuclear, site specific ssDNA binding protein.Nucleic Acids Res. 23, 2389–2395.

Soulard, M., Barque, J.P., Della Valle, V., Hernandez-Verdun, D., Masson,C., Danon, F., Larsen, C.J., 1991. A novel 43-kDa glycoprotein isdetected in the nucleus of mammalian cells by autoantibodies from dogswith autoimmune disorders. Exp. Cell Res. 193, 59–71.

Soulard, M., Della Valle, V., Siomi, M.C., Pinol-Roma, S., Codogno, P.,Bauvy, C., Bellini, M., Lacroix, J.C., Monod, G., Dreyfuss, G., et al.,1993. hnRNP G: sequence and characterization of a glycosylated RNA-binding protein. Nucleic Acids Res. 21, 4210–4217.

Steinhauer, J., Kalderon, D., 2005. The RNA-binding protein Squid isrequired for the establishment of anteroposterior polarity in the Droso-phila oocyte. Development 132, 5515–5525.

Tanuma, Y., Nakabayashi, H., Esumi, M., Endo, H., 1995. A silencerelement for the lipoprotein lipase gene promoter and cognate double-and single-stranded DNA-binding proteins. Mol. Cell. Biol. 15,517–523.

Tay, N., Chan, S.H., Ren, E.C., 1992. Identification and cloning of a novelheterogeneous nuclear ribonucleoprotein C-like protein that functions asa transcriptional activator of the hepatitis B virus enhancer II. J. Virol.66, 6841–6848.

Tjian, R., Maniatis, T., 1994. Transcriptional activation: a complex puzzlewith few easy pieces. Cell 77, 5–8.

Tolnay, M., Vereshchagina, L.A., Tsokos, G.C., 1999. Heterogeneousnuclear ribonucleoprotein D0B is a sequence-specific DNA-bindingprotein. Biochem. J. 338 (Pt 2), 417–425.

Tomonaga, T., Levens, D., 1995. Heterogeneous nuclear ribonucleoproteinK is a DNA-binding transactivator. J. Biol. Chem. 270, 4875–4881.

Tomonaga, T., Levens, D., 1996. Activating transcription from singlestranded DNA. Proc. Natl. Acad. Sci. U. S. A. 93, 5830–5835.

Tsend-Ayush, E., O'Sullivan, L.A., Grutzner, F.S., Onnebo, S.M., Lewis,R.S., Delbridge, M.L., Marshall Graves, J.A., Ward, A.C., 2005.RBMX gene is essential for brain development in zebrafish. Dev. Dyn.234, 682–688.

Venables, J.P., Elliott, D.J., Makarova, O.V., Makarov, E.M., Cooke, H.J.,Eperon, I.C., 2000. RBMY, a probable human spermatogenesis factor,and other hnRNP G proteins interact with Tra2beta and affect splicing.Hum. Mol. Genet. 9, 685–694.

Von Schalburg, K.R., Sherwood, N.M., 1999. Regulation and expression of


gonadotropin-releasing hormone gene differs in brain and gonads inrainbow trout. Endocrinology 140, 3012–3024.

Wang, D., Parrish, C.R., 1999. A heterogeneous nuclear ribonucleoproteinA/B-related protein binds to single-stranded DNA near the 5′ end orwithin the genome of feline parvovirus and can modify virus replication.J. Virol. 73, 7761–7768.

Weiner, R.I., Wetsel, W., Goldsmith, P., Martinez de la Escalera, G., Windle, J.,Padula, C., Choi, A., Negro-Vilar, A., Mellon, P., 1992. Gonadotropin-releasing hormone neuronal cell lines. Front. Neuroendocrinol. 13, 95–119.

Westerveld, G.H., Gianotten, J., Leschot, N.J., van der Veen, F., Repping, S.,Lombardi, M.P., 2004. Heterogeneous nuclear ribonucleoprotein G-T(HNRNP G-T) mutations in men with impaired spermatogenesis. Mol.Hum. Reprod. 10, 265–269.

Wetsel,W.C., 1995. Immortalized hypothalamic luteinizing hormone-releasinghormone (LHRH) neurons: a new tool for dissecting the molecular andcellular basis of LHRH physiology. Cell. Mol. Neurobiol. 15, 43–78.

White, S.A., Fernald, R.D., 1993. Gonadotropin-releasing hormone-containing neurons change size with reproductive state in female Ha-plochromis burtoni. J. Neurosci. 13, 434–441.

White, R.B., Fernald, R.D., 1998. Genomic structure and expression sites of

three gonadotropin-releasing hormone genes in one species. Gen. Comp.Endocrinol. 112, 17–25.

White, S.A., Nguyen, T., Fernald, R.D., 2002. Social regulation ofgonadotropin-releasing hormone. J. Exp. Biol. 205, 2567–2581.

Whyte, D.B., Lawson, M.A., Belsham, D.D., Eraly, S.A., Bond, C.T.,Adelman, J.P., Mellon, P.L., 1995. A neuron-specific enhancer targetsexpression of the gonadotropin-releasing hormone gene to hypothalamicneurosecretory neurons. Mol. Endocrinol. 9, 467–477.

Wijnholds, J., Muller, E., Ab, G., 1991. Oestrogen facilitates the binding ofubiquitous and liver-enriched nuclear proteins to the apoVLDL IIpromoter in vivo. Nucleic Acids Res. 19, 33–41.

Wilkison, W.O., Min, H.Y., Claffey, K.P., Satterberg, B.L., Spiegelman, B.M.,1990. Control of the adipsin gene in adipocyte differentiation. Identifica-tion of distinct nuclear factors binding to single- and double-strandedDNA.J. Biol. Chem. 265, 477–482.

Yabuki, T., Miyagi, S., Ueda, H., Saitoh, Y., Tsutsumi, K., 2001. A novelgrowth-related nuclear protein binds and inhibits rat aldolase B genepromoter. Gene 264, 123–129.

Zhao, S., Fernald, R.D., 2005. Comprehensive algorithm for quantitativereal-time polymerase chain reaction. J. Comput. Biol. 12, 1047–1064.

Heterogeneous nuclear ribonucleoprotein A/B and G inhibits the transcription of gonadotropin-re.....IntroductionResultsGnRH1 gene upstream sequenceMapping the DNA/protein binding sites upstream of the GnRH1 geneMass spectrometric peptide mapping of the binding proteinsInternal sequencing of hnRNP-A/B to confirm the mass mapping resultMolecular cloning of hnRNP-A/B and hnRNP-GConfirmation of the mass mapping results by analysis of cloned A.burtoni sequences of hnRNP-A/.....Location of hnRNP-A/B and hnRNP-G mRNAIn vivo binding of mouse hnRNP-A/B and hnRNP-G to mouse GnRH1Overexpression of hnRNP-A/B and hnRNP-G down-regulates mouse GnRH1 in GT1–7 cellsPutative transcription factor binding sites and hormone response elements in GnRH1 gene

DiscussionGnRH1 transcriptional regulationDiscovery of hnRNP-A/B and hnRNP-G as transcriptional regulator of GnRH1 in A. burtoniRelationship between the hnRNP family and hormones

Experimental methodsAnimals and materialsGenomic DNA extraction and inverse PCRDNA/Protein in vitro binding assayMass spectrometric peptide mapping based on homologyInternal sequencingMolecular cloning of A. burtoni hnRNP-A/B and hnRNP-GRNA localizations in organsIn situ hybridizationCell cultureChromatin immunoprecipitationOverexpression and transcriptional regulation in GT1–7 cellsPrediction of transcription factor binding sites

AcknowledgmentsSupplementary dataReferences

heterogeneous nuclear ribonucleoprotein a/b and g inhibits ...heterogeneous nuclear...

Documents