FASEB J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by MORGAN, H.
Right arrow Articles by CARTER, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by MORGAN, H.
Right arrow Articles by CARTER, D.
(The FASEB Journal. 2000;14:1109-1116.)
© 2000 FASEB

The transactivation-competent carboxyl-terminal domain of AF-9 is expressed within a sexually dimorphic transcript in rat pituitary

HELEN MORGAN, MARTIN SMITH, ZOE BURKE and DAVID CARTER1

School of Biosciences, Cardiff University, Cardiff CF1 3US. U.K.

1Correspondence: School of Biosciences, Cardiff University, Biomedical Buildings, Museum Avenue, Cardiff CF1 3US, U.K. E-mail: smbdac{at}cardiff.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have investigated the biological role of the cellular counterpart of the leukemogenic AF-9 gene by cloning the rat AF-9 (rAF-9) cDNA and defining the regulation of an anterior pituitary-specific rAF-9 transcript that is expressed in a sexually dimorphic manner. Expression of this transcript is down-regulated after puberty in females and can be subsequently up-regulated in adults by ovariectomy. Hormone replacement studies have provided direct evidence that rAF-9 mRNA expression is suppressed by estrogen. Mapping the 1.9 kb anterior pituitary transcript has shown that it corresponds in size to the rAF-9 cDNA clone, which contains an open reading frame (ORF) that is truncated compared with the human AF-9 ORF, but encodes a previously defined transcriptional activation domain. Thus, the cellular AF-9 gene is alternatively expressed in a manner that reflects the presence of translocated, functionally active (oncogenic) AF-9 sequences in leukemias. Using a novel antisera raised against a rAF-9 peptide, we have also demonstrated tissue- and sex-specific expression of a nuclear 41 kDa anterior pituitary protein and have localized this protein to a major population of growth hormone synthesizing cells. By localizing the expression and defining the physiological regulation of rAF-9, our studies have provided novel insights into the AF-9 gene that will facilitate an understanding of both oncogenic and endocrine roles.—Morgan, H., Smith, M., Burke, Z., Carter, D. The transactivation-competent carboxyl-terminal domain of AF-9 is expressed within a sexually dimorphic transcript in rat pituitary.


Key Words: leukemia • estrogen • oncogene


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ABNORMALITIES ASSOCIATED WITH band q23 of human chromosome 11 are involved in over 20% of acute leukemias. A gene termed ALL-1(MLL or HRX) located at the 11q23 breakpoint region (1) is involved in multiple different translocations that fuse the amino-terminal of ALL-1 to a variety of different partner proteins. A common translocation in acute myeloid leukemia is t(9, 11)(p22;q23) in which the AF-9 gene (ALL-1 fused gene from chromosome 9) encodes the oncogenic partner protein (2 , 3) . The biological activity of AF-9 has not been completely defined, although studies have demonstrated transcriptional regulatory activity within a carboxyl-terminal domain (4 ; also see refs 5 , 6 ), which is suggested to be oncogenic in the context of the ALL-1 fusion. Current approaches to a greater understanding of fusion oncogene function include the development of mouse models (7) and analysis of the normal physiological role of partner proteins. In the present study, we have investigated the developmental and physiological regulation of a novel transcript derived from the rat AF-9 (rAF-9) gene, which was identified during a screen for novel regulatory genes that control endocrine function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatments
Animal studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Sprague-Dawley rats were maintained in standard laboratory conditions on a 14:10 light:dark cycle. Estrous cycle stage was determined by microscopic analysis of vaginal washings. For the analysis of gonadal steroid dependency, female rats were either ovariectomized using standard surgical procedures and injected subcutaneously (s.c.) with sesame oil daily on days 8–10 after surgery, ovariectomized, and injected with 17ß-estradiol (s.c. 20 µg/100 g body weight, Sigma, St. Louis, Mo.) or sham-ovariectomized. Male rats were castrated using standard surgical procedures. After surgery, rats were maintained for 10 days prior to killing. Rats were killed by cervical dislocation and sampled tissues were either frozen on dry ice (RNA analysis) or Tissue-Tek embedding medium (in situ hybridization and immunohistochemical analysis) or prepared for protein extraction. Tissue, RNA, and protein samples were stored at -70°C.

cDNA library screening
A rat anterior pituitary cDNA library in Uni-ZAP XR (Stratagene, San Diego, Calif.) was screened using a 32P-dCTP random primer-labeled 267 bp NcoI/TaqI 5' fragment of the rat glucocorticoid receptor cDNA (M14053). Positive clones were excised using the Exassist/SOLR system (Stratagene) and sequenced on both strands using the Thermo Sequenase cycle sequencing kit (Amersham, Little Chalfont, U.K.). Insert sequences were analyzed using a Li-Cor automated fluorescent sequencer 4000L and compared with database sequences using the FASTA program (8) .

Northern analysis and reverse transcription-polymerase chain reaction (RT-PCR)
Total cellular RNA was extracted, fractionated on formaldehyde-agarose gels, and visualized by Northern analysis as described (9) . Both rAF-9 (see Results) and GAPDH (380 bp rat RT-PCR product; ref 10 ) probes were labeled by random priming using 32P-dCTP (Amersham). Densitometric analysis of mRNA levels between treatment groups was performed using Imagequant software (Pharmacia, Uppsala, Sweden).

RT-PCR was performed essentially as described (11) using oligonucleotide primers designed according to the rAF-9 cDNA sequence (P1: 5'-CCTTCCAGGGATCACAAC-3', bases 398–415; P2: 5'-AGCTGACCCTGCGACTTC-3', complementary to bases 1062–1079; P3: 5'-CCTGGAGACATCTGGAACATCCTGAGG-3', bases 1423–1449) and a universal oligo d(T) primer 5'-GTCGGATCCCTCGAG(T)15-3' that contains BamHI and XhoI cloning sites. Amplified cDNA fragments were analyzed by agarose gel electrophoresis and compared with a molecular weight marker (1 kb ladder, Life Technologies, Paisley, U.K.). RT-PCR products were blunted and ligated into linearized pUC18 using standard procedures (11) and sequenced as described above.

Primer extension
Primer extension was performed as described (11) using a 32P-ATP T4 polynucleotide kinase-labeled oligonucleotide (5'-GGTCCCCTCCTGCCTTCAGCAACTTTCTTC-3' corresponding to bases 132–164 of the rAF-9 cDNA sequence) and 5 µg of total cellular RNA. The sizes of extension products were compared with 32P-dCTP-labeled markers (1 kb ladder, Life Technologies).

In vitro transcription/translation and immunoblotting
The rAF-9 cDNA in pBS KS(-) was transcribed and translated in vitro using the Promega TNT and Transcend systems according to the manufacturer’s instructions (Promega, Southampton, U.K.). The Transcend system incorporates biotinylated lysine residues into the nascent proteins, permitting detection by binding with streptavidin-horseradish peroxidase (HRP), followed by chemiluminescent detection of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved protein products.

A polyclonal antiserum was raised in rabbits using a KLH[cys]-linked rAF-9 peptide ([C]EVKSPIKQSKSDKQ; residues 282–295 of the largest rAF-9 ORF) as immunogen (Genemed Synthesis Inc., San Francisco, Calif.). The peptide sequence, which is within both major rAF-9 open reading frames (ORFs), was chosen on the basis of minimal homology to ENL and has been used previously (12) . After affinity purification and enzyme-linked immunoassay analysis (Genemed), the antiserum was used to probe Western blots of whole cell and nuclear protein fractions as described previously (13) . Protein concentrations were determined by the method of Bradford (14) . Two different molecular weight markers were used: Rainbow Markers (Amersham) and Broad Range Protein Markers (New England Biolabs, Beverly, Mass.). The primary antisera were used at a final concentration of 0.1 µg/ml and secondary antisera (donkey anti-rabbit-HRP-linked, Amersham) at 1:5000. Proteins were detected using chemiluminescence (HPRL kit, National Diagnostics, Somerville, N.J.). Protein bands were compared using densitometric analysis (Imagequant, Pharmacia). Specificity of the rAF-9 antiserum was determined by peptide neutralization using the rAF-9 peptide sequence listed above and an unrelated 24 mer peptide. Aliquots of the antisera were incubated overnight at 4°C in a 10-fold excess of peptide in phosphate-buffered saline (PBS) or in PBS alone, then used to probe Western blots as described above.

In situ hybridization and immunocytochemistry
In situ hybridization histochemical analysis of rAF-9 transcript expression in the anterior pituitary was performed essentially as described previously (15) using antisense and sense RNA probes transcribed from the rAF-9 cDNA in pBSKS by using an RNA transcription kit (Stratagene) and 35S-UTP (Amersham). Probe hybridization (5x106 cpm/ml) was performed overnight at 42°C, prior to washing and exposure to X-ray film (Genetic Research Instruments-AX, Braintree, U.K.) and subsequent coating in photographic emulsion (LM-1, Amersham).

Immunocytochemistry was performed on 8 µm sections of anterior pituitary, postfixed in 4% paraformaldehyde in PBS (5 min) and permeabilized in methanol (-20°C, 2 min). Proteins were detected using a Vectastain Elite ABC kit (Vector Laboratories, Burlingame, Calif.) according to the manufacturer’s protocol and localized with DAB. Tissue sections were incubated with either preimmune rabbit serum, 1:500; rAF-9 antiserum, 1:500; rat GH antiserum (NIDDK-anti-rGH-IC-1; AFP411S; [1:500]), or rat alpha subunit antiserum (NIDDK-anti-rAlpha subunit-IC-1;AFP66P9986; [1:500]). After development of the chromogen, slides were counterstained with Meyer’s hematoxylin.

Dual in situ hybridization/immunocytochemistry was performed using a previously described protocol (16) . Briefly, immunocytochemical detection of GH cells was first performed according to the basic protocol described above, but all solutions were prepared in diethylpyrocarbonate-treated water and also contained RNasin ribonuclease inhibitor (Promega; 0.1 U/µl). After the DAB reaction, slides were washed, dried, and stored at -70°C prior to in situ hybridization analysis. After development of the photographic emulsion, sections were counterstained with hematoxylin.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The rAF-9 clone was obtained during the screening of a rat anterior pituitary cDNA library for tissue-specific expressed sequences homologous to the glucocorticoid receptor (GR). The 1591 bp rAF-9 clone (accession number AJ006295) did not exhibit clear sequence homology with GR cDNA, but was selected for further study because it fulfilled the expression criteria and was highly homologous at the nucleotide level (91.3%) to the human putative transcription factor AF-9 (MLLT3, LTG9). The sequence homology is highest within the central open reading frame-containing region of the cDNA (see below) and minimal at the 5' and 3' terminal regions of the clone. The rAF-9 clone contains an ORF (Fig. 1 ) with two potential initiator methionines (cDNA positions 335 and 488), terminating at position 1448, that code for putative proteins of 35.4 and 41.4 kDa, respectively. The initiation codon at position 488 is associated with Kozak’s consensus sequence (17) , but in vitro transcription/translation (TNT and Transcend systems) of the clone indicated that the 5' initiation codon may be preferentially used. Thus, the major in vitro protein product was found to be 49 kDa (data not shown), which corresponds to the larger ORF given a predicted incorporation of biotinylated lysines at 35% (Transcend system, Promega), which would add 7.6 kDa to the mass of the protein. Translation of the 41.4 kDa protein is also indicated by immunoblotting of cellular proteins (see below). The predicted 41.4 kDa rat protein is 97.8% identical to the predicted human AF-9 amino acid sequence (Fig. 1) ; like the human sequence, it contains consensus nuclear localization signals (Fig. 1 ; PSORT program, 18 ).



View larger version (48K):
[in this window]
[in a new window]
  		Figure 1. A) Comparison of the 5' sequence of the rAF-9 clone with the human AF-9 cDNA sequence (accession no. L13744), showing the region leading up to the first ATG (in boldface). B) Predicted amino acid sequence of the rAF-9 open reading frame that starts at position 335 in the rAF-9 cDNA clone. Alignment with the equivalent human sequence (hAF-9) is shown. Colons indicate conservative sequence differences. Nuclear localization sequences and a second potential initiation methionine at position 52 are shown in boldface. Predicted helices (see ref 6 ) are underlined. The transregulatory domain described by Prasad et al (ref 4 ) spanning amino acids 478–568 of the human sequence, which is fully conserved in the rat sequence, is indicated by a dashed line replacing the vertical bars.

Tissue-specific expression of an rAF-9-related transcript in rats was demonstrated with Northern blots using the full-length rAF-9 clone as a probe (Fig. 2 ). Thus, in the anterior pituitary (AP) a 1.9 kb transcript was abundantly expressed, but was not observed in other tissues. In contrast, low abundance transcripts of ~2.6, 3.9, and 4.2 kb (Fig. 2) were apparent in numerous tissues and were clearly observed after prolonged autoradiograph exposure. The presence of these lower abundance transcripts is reflective of previous observations of AF-9 expression in a study that included various cell lines but did not examine pituitary AF-9 expression (see ref 2 ). The possibility that rAF-9 transcript detection in Northern blots was due to hybridization to the extensive CAG repeat sequence in the clone was eliminated by probing similar blots with a CAG repeat-free 1253 bp VspI 3'-end fragment of the clone (e.g., Fig. 3D ) Expression of the rAF-9 sequence in rat AP was also confirmed by cloning and sequencing a rAF-9-specific RT-PCR product amplified from AP (primers P1 and P2; see Materials and Methods; 11 ); using the same approach, a similar product was amplified from mouse (C57Bl6/CBA/J F1) AP (data not shown). We have also noted that several mouse expressed sequences overlap the rAF-9 sequence (accession numbers: AA209036; W42123; W53460).



View larger version (65K):
[in this window]
[in a new window]
  		Figure 2. Tissue-specific expression of rAF-9 transcripts in the rat. Northern blot of total cellular RNA extracted from male Sprague-Dawley rats (12 µg/lane) hybridized first with a full-length rAF-9 cDNA probe and second with a GAPDH cDNA probe. Lane 1, anterior pituitary; lane 2, brain cortex; lane 3, adrenal gland; lane 4, eye; lane 5, liver; lane 6, kidney. Horizontal bars indicate the position of the 28S and 18S ribosomal RNA bands. The arrow indicates the prominent 1.9 kb (anterior pituitary) transcript; arrowheads indicate the position of the 2.6, 3.9, and 4.2 kb transcripts, which are clearly visible only on longer exposure times. Exposure times were 72 (rAF-9) and 16 h (GAPDH).



View larger version (25K):
[in this window]
[in a new window]
  		Figure 3. Sexually dimorphic expression of rAF-9. A) Regulation of the anterior pituitary (AP) 1.9 kb rAF-9 transcript by gonadal steroids. Northern blot of total cellular RNA (10 µg/lane) extracted from the AP of paired rats hybridized first with a 32P-labeled, full-length rAF-9 cDNA probe and second with a 32P-labeled, GAPDH cDNA probe. Lane 1, sham-operated female rats; lane 2, ovariectomized rats treated with vehicle; lane 3, ovariectomized rats treated with 17ß-estradiol; lane 4, male rats. Exposure times were 72 h (rAF-9) and 72 h (GAPDH). B) Histogram of relative density (rd) measurements of the 1.9 kb AF-9 transcript after correction against the level of GAPDH mRNA. C) Effect of castration on rAF-9 expression in male rats. Northern blot of total cellular RNA (15 µg/lane) extracted from the AP of paired rats hybridized first with a 32P-labeled, full-length rAF-9 cDNA probe and then with a 32P-labeled, GAPDH cDNA probe. Lane 1, untreated male rats; lane 2, castrated rats; lane 3, sham-operated male rats. Exposure times were 24 h (rAF-9) and 48 h (GAPDH). D) Ontogeny of 1.9 kb rAF-9 transcript expression in the anterior pituitary (AP) of female rats. Northern blot of total cellular RNA extracted from the AP of paired rats (4 µg/lane) hybridized first with a 32P-labeled, VspI-3'end fragment rAF-9 cDNA probe and second with a 32P-labeled, GAPDH cDNA probe. Lane 1, 5 day; lane 2, 10 day; lane 3, 15 day; lane 4, 20 day; lane 5, 30 day; lane 6, adult. Exposure times were 72 h (rAF-9) and 48 h (GAPDH).

Expression of the 1.9 kb AP transcript was sexually dimorphic, being markedly higher in males (Fig. 3A ), whereas other rAF-9-related transcripts were of approximately equal abundance in males and females (data not shown). Densitometric analysis of the 1.9 kb transcript in multiple animals revealed a 12-fold higher expression in males than in females (12.1±3.5-fold after correction against GAPDH mRNA levels, n=3; P<0.05, paired t test). The basis of the sex difference in expression of the 1.9 kb transcript was investigated in two additional experiments. It was first shown in a developmental study that expression of the 1.9 kb transcript in females is reduced after puberty (Fig. 3D ), indicating regulation by gonadal steroids. This mode of physiological regulation was then directly demonstrated in another experiment in which ovariectomy and subsequent estrogen replacement in adult females was shown to exert reciprocal effects on expression of the 1.9 kb transcript (Fig. 3A, B ). In contrast, castration of male rats was not associated with a significant change in expression of the 1.9 kb transcript (Fig. 3C ). Similar results were obtained in duplicate experiments. No marked variation in expression of the 1.9 kb transcript was observed in APs sampled from female rats on each morning of the estrous cycle (data not shown).

The AP-specific rAF-9 1.9 kb transcript was characterized by using a combination of primer extension and RACE (rapid amplification of cDNA ends) approaches. By comparison with the human AF-9 cDNA sequence (3) , which contains a 568 amino acid ORF that extends beyond the 5' end of the cloned rat sequence, it is apparent that the rAF-9 clone may represent a 5' truncated clone that partially represents a larger transcript corresponding in size to the human homologue. Alternatively, the rAF-9 clone may be largely, if not fully, representative of one of the smaller rAF-9-hybridizing transcripts such as the 1.9 kb transcript. Although the former appears unlikely because of the nonhomologous 5' terminal sequence in the rAF-9 clone, experiments were undertaken to investigate these possibilities. To map the 5' end of the anterior pituitary 1.9 kb transcript, we were able to take advantage of the differential expression of the transcript between both male and female animals and tissues. Thus, primer extension from an oligonucleotide that maps to bases 132–164 of the rAF-9 clone produced a single major (~160 bp) product that was 11-fold more abundant in male vs. female mRNA extension reactions (Fig. 4A ) The relative levels of the 160 bp product in males and females mirror the sex difference in relative abundance of the 1.9 kb transcript on Northern blots (see above). Because other rAF-9-hybridizing transcripts are approximately equally abundant in male and female APs (data not shown), this result indicates that the product represents extension from the 1.9 kb transcript.



View larger version (51K):
[in this window]
[in a new window]
  		Figure 4. A) Primer extension analysis of anterior pituitary rAF-9 transcripts. The analysis was performed as described (see Materials and Methods) using a 32P-labeled oligonucleotide primer (5'-GGTCCCCTCCTGCCTTCAGCAACTTTCTTC-3', corresponding to bases 132–164 of the rAF-9 cDNA sequence), and 5 µg of total cellular RNA. A) Lane 1, male anterior pituitary; lane 2, female anterior pituitary; lane 3, probe alone. DNA size markers (32P-dCTP-labeled 1 kb ladder, Life Technologies) are indicated by horizontal bars. The major, ~160 bp product is indicated by an arrow. Exposure time was 16 h. B–D) Tissue- and sex-specific expression of rAF-9 protein in the rat. Immunoblots of cellular extracts (20 µg/lane) were probed using an antibody raised against a rAF-9 peptide. Horizontal bars indicate the sizes of protein molecular weight markers. B) Lane 1, male anterior pituitary: whole cell; lane 2, male anterior pituitary: nuclear; lane 3 male liver: nuclear. C) Lane 1, male anterior pituitary: nuclear; lane 2, female anterior pituitary: nuclear. D) All three lanes contain male anterior pituitary nuclear extracts. Lane 1, probed with rAF-9 antisera preincubated with PBS; lane 2, probed with rAF-9 antisera preincubated with rAF-9 peptide; lane 3, probed with rAF-9 antisera preincubated with unrelated peptide sequence.

Using the same primer, extension products were also compared between the AP and the adrenal of male rats because the latter tissue contains a relative abundance of the 2.6 kb transcript compared with the AP (not observed on the exposure shown in Fig. 2 ) and does not express the 1.9 kb transcript (Fig. 2) . This experiment also revealed a single major (160 bp) product in the AP only, indicating extension from the 1.9 kb rather than the 2.6 kb transcript (data not shown). The size of the primer extension product furthermore indicates that the 5' end of the rAF-9 cDNA clone is approximately, if not fully, representative of the 1.9 kb transcript.

To map the 3' end of the AP transcript, a simplified RACE procedure (19) was employed using a 3'-end rAF-9-specific primer (P3; see Materials and Methods) and a universal oligo d(T) primer adaptor (see Materials and Methods). This procedure consistently resulted in the amplification of a 162 bp fragment from AP mRNA (data not shown) that, on sequencing, was shown to correspond to the 3' terminal of the rAF-9 sequence between the 3'end primer sequence and a stretch of 20 (A) residues between bases 1554 and 1573. Rather than amplifying from the poly (A) tail, the oligo d(T) primer clearly is selectively amplifying from this extensive internal stretch of A’s. Nevertheless, this procedure served to confirm the 3'-end region sequence of the rAF-9 clone, and by Northern blotting we also demonstrated that the 162 bp PCR fragment hybridized to the 1.9 kb rAF-9 transcript (data not shown). Although the 3' end of the transcript has not been fully mapped, the highly AT-rich sequence beyond the stop codon at position 1445 is characteristic of 3' terminal sequence and precedes a poly (A) signal in the hAF-9 (MLLT3) cDNA cloned by Iida et al. (2) . Assuming a poly (A) tail of 200–250 bases, the rAF-9 clone of 1591 bases would therefore appear to represent an almost full-length cDNA of the 1.9 kb AP transcript. The genomic origin of the 1.9 kb transcript is currently unknown; examination of human genomic sequence from chromosome band 9p22 (accession no. AC002053) has revealed homologous sequence starting at position 307 of the rat cDNA that is preceded by a consensus splice acceptor site, indicating that this position of the cDNA may represent an exon start. Unfortunately, the current extent of chromosome 9 sequencing is not sufficient to reveal more 5' sequence homologous to the rat cDNA. In additional experiments, we have used RT-PCR to amplify sequences from rat AP RNA that correspond to 5' regions of the hAF-9 cDNA that are not represented in the 1.9 kb transcript (data not shown). Therefore, the larger rat transcripts that hybridize to the rAF-9 cDNA in Northern blots (Fig. 2) may enclose different and larger ORFs; the sequence of other rAF-9 transcripts is currently being investigated.

Using an antiserum raised against a rAF-9 peptide sequence, we next demonstrated tissue- and sex-specific expression of a rAF-9-related protein in immunoblots (Fig. 4B ). A protein of ~41 kDa was detected in AP, but not in liver (Fig. 4B ) or brain cortex (not shown) extracts, and was found to be concentrated in nuclear fractions as compared with whole cell extracts (Fig. 4B ). Both the size and cellular localization of this protein are consistent with the predicted rAF-9 protein sequence. A fainter protein band of ~67 kDa was also observed in whole cell AP and nuclear liver extracts (Fig. 4B ); it was less abundant in nuclear AP extracts, but was detectable in longer autoradiograph exposures (data not shown). The 41 kDa protein was more abundant in adult male as compared with adult female nuclear AP extracts (Fig. 4C ); expression was found to be fourfold higher in males (Fig. 4C ). The specificity of the rAF-9 antisera was confirmed through peptide-specific neutralization (Fig. 4D ).

In situ hybridization and immunocytochemical approaches were then used to localize rAF-9 expression to a major subpopulation of AP cells (Fig. 5 ). An antisense RNA probe was shown to hybridize to widely distributed cells within the AP (Fig. 5d ) whereas an equivalent sense probe did not exhibit significant levels of hybridization (Fig. 5c ), confirming that the orientation of the cDNA clone has been correctly designated. Immunocytochemical analysis also confirmed a wide distribution of expression (Fig. 5b ); the primarily nuclear site of expression was observed in a high proportion (>70%) of cells throughout the AP. Nuclear expression is clearly observed in the higher magnification image (Fig. 5g ) in which intensely brown rAF-9-positive nuclei contrast with a relatively limited number of negative hematoxylin-stained nuclei. It should be noted that some cytoplasmic staining was also obtained with the rAF-9 antiserum, which is not clearly observed above background on the exposures shown here; this may reflect detection of the lower abundance, larger molecular weight rAF-9 protein observed by immunoblotting (Fig. 4B ). The pattern of rAF-9 expression was noted to be quantitatively similar to the (cytoplasmic) expression of growth hormone (GH) on similar AP sections (Fig. 5e ), but was in contrast to the sparser distribution of other AP hormone proteins such as the glycoprotein {alpha}-subunit (Fig. 5f ). More studies were therefore conducted to examine the possibility that rAF-9 is expressed in somatotrophs. Using a dual in situ hybridization/immunocytochemical approach, we observed that rAF-9 and GH were indeed extensively colocalized throughout the AP (Fig. 5h ). There was also a low abundance of rAF-9-negative, GH-positive cells and, conversely, evidence of rAF-9 hybridization over GH-negative cells. Further studies are required to confirm the indicated possibility that rAF-9 is expressed in additional cell types.



View larger version (124K):
[in this window]
[in a new window]
  		Figure 5. Localization of rAF-9 protein and mRNA within the anterior pituitary of male rats. a, b, e–g) Immunocytochemical analyses. Tissue sections were incubated with either preimmune serum (a), rAF-9 antiserum (b, g), rat GH antiserum (e), or rat alpha subunit antiserum (f). Immunoreactivity is observed as a brown reaction product (DAB), which contrasts with the blue/purple counterstain (hematoxylin). c, d) In situ hybridization analyses. Tissue sections were hybridized with either sense (c) or antisense (d) RNA probes, and emulsion autoradiographs were developed after 12 days exposure. Under bright-field illumination, the antisense hybridization signal (silver grains, black) can be observed over a wide population of anterior pituitary cells that are counterstained with hematoxylin. h) Dual immunocytochemical/in situ hybridization analysis. Tissue sections were first incubated with rat GH antiserum, prior to hybridization with the antisense rAF-9 RNA probe, and emulsion autoradiographs were developed after 12 days exposure. The sections were also counterstained with hematoxylin. x200 (a–f); x1000 (g, h).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The human AF-9 gene (3 ; alternatively termed MLLT3 or LTG-9, see ref 2 ) was originally identified as one of multiple chromosomal regions involved in reciprocal translocations with the ALL-1 gene that are observed in acute myeloid leukemias. The resultant fusion proteins are thought to induce leukemia through aberrant transcriptional regulation. In the case of the NH2-ALL-1/AF-9-COOH fusion, it has been suggested that a DNA binding motif in ALL-1 functions in combination with a transcriptional regulatory domain in AF-9 (3 , 4) . Support for this hypothesis has come from a recent study (6) , which has shown that 1) multiple DNA binding structures in ALL-1 are required for functional activity, and 2) ENL, a fusion partner protein similar to AF-9, contains transcriptional activation domains that are both necessary and sufficient for function when fused to ALL-1. Creation of an AF-9 fusion gene in mice by homologous recombination has also been shown to be associated with acute leukemia (7) . The physiological function of the ALL-1 partner genes in their normal chromosomal context is unknown. The human AF-9 gene has been cloned and shown to encode a serine-rich protein that encloses a nuclear targeting sequence (3) . Expression studies have been limited, but transcripts of ~3.9 and 4.2 kb have been detected in human lymphocytes, spleen, and thymus (2) .

Using a cloned rat AF-9 cDNA, we have now demonstrated a novel expression pattern for AF-9 that indicates a physiological role within the endocrine system. Our rAF-9 cDNA encodes a protein that is highly homologous to the hAF-9 sequence, but is truncated with respect to the 568 amino acid protein predicted from the human sequence (3) . However, our analysis has shown that the rAF-9 cDNA size is consistent with a 1.9 kb transcript that is specifically expressed in the AP. Multiple larger rat transcripts that appear to correspond in size to human transcripts (2) may possibly encode a protein of similar size to the predicted human sequence. With respect to the potential for regulatory function, the rat AP cDNA encodes the region homologous to the carboxyl-terminal domain of AF-9, which has been shown to transactivate transcription (4) . Indeed, a carboxyl-terminal 90 amino acid region of hAF-9, which is fully conserved in rAF-9, is also highly conserved in ENL (5) and corresponds to the region shown to necessary and sufficient for transcriptional activation in the study by Slany et al. (6) .

The particular biological activity of AF-9 remains to be defined. Two conserved, possibly amphipathic, helical structures have been suggested to underlie the transactivation function of AF-9 and ENL (Fig. 1 ; 6 ). However, the absence of a defined DNA binding domain in AF-9 and ENL has led to the suggestion that these proteins may function as coactivators, interacting with unidentified DNA-bound factors (6) . The conserved regions of AF-9 and ENL proteins have also been shown to be highly similar to TFG3 (20 , 21) , a component of the yeast SW1/SNF chromatin remodeling complex. This 2 MDa protein complex, which has a mammalian equivalent (22) , acts to facilitate the transcription of certain genes by antagonizing chromatin-mediated transcriptional repression. It has been suggested that deregulated activity of AF-9 in the context of an ALL-1 fusion protein may therefore initiate leukemias by enabling the persistent antagonism of this form of repression (20) . Further studies are now required to investigate the role of AF-9 proteins in SWI/SNF function (see ref 23 ). The association of AF-9 with a chromatin remodeling function is intriguing with regard to our identification of rAF-9 in the pituitary, because mammalian homologues of SWI/SNF components have been shown to act cooperatively with both glucocorticoid and estrogen receptors that mediate endocrine feedback actions within the pituitary (24 , 25) . Furthermore, selective expression of rAF-9 in the AP is consistent in this respect with both tissue-specific SWI/SNF components in mammalian systems (26) and evidence of pituitary cell-specific chromatin structures that determine hormone gene expression (27) . The transcriptional regulatory activity of carboxyl-terminal hAF-9 in heterologous systems was shown by Prasad et al. (4) to be both cell- and promoter-specific.

The marked dimorphism in rAF-9 expression between males and females is clearly indicative of a role in sexually dimorphic aspects of GH regulation. The dimorphism in rAF-9 protein expression detected by immunoblotting reflects sex differences in GH levels in the AP, both qualitatively and quantitatively (e.g., ref 28 ). Differences in growth between males and females are, in fact, believed to be largely mediated through hypothalamic mechanisms that result in divergent patterns of pulsatile hormone secretion (29 , 30) . However, pituitary growth hormone-releasing factor (GRF) receptor mRNA levels in female rats are only 15% of male levels (31) , and female pituitaries exhibit a significantly reduced secretory response to GRF (32) . In the latter study, pulse analysis demonstrated that sex differences in GRF-induced GH pulses were evident only in postpubertal animals, which correlates with the ontogeny of sexually dimorphic rAF-9 expression. It can therefore be speculated that sex differences in GH regulation induced at the pituitary level (32) may be related to rAF-9 activity. Our finding that expression of the novel rAF-9 transcript remains under estrogen-mediated suppression in the adult female pituitary suggests the possibility of additional latent activities in reproductive cycles, pregnancy, lactation, and senescence. It may also be speculated that changes in rAF-9 activity may accompany pathological GH secretion, for example, in adenomas associated with acromegaly. In summary, our finding that a conserved homologue of the human AF-9 gene exhibits physiologically regulated expression in the rat pituitary gland has provided a new and important insight into the role of this cellular proto-oncogene. Through the localization of cellular expression, it may now be possible to identify target genes that are transactivated via AF-9, leading to a greater understanding of oncogenic and potential endocrine roles.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Zhen-Ping Chen and Professor Stafford Lightman (University of Bristol, U.K.) for the gift of the cDNA library and to Dr. A. F. Parlow and the National Hormone and Pituitary Program for the gifts of hormone antisera. This work was supported by an MRC (U.K.) project grant to DAC.


   FOOTNOTES
 
Received for publication August 3, 1999. Revised for publication December 22, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Zieman-Van Der Poel, S., McCabe, N. R., Gill, H. J., Espinosa, R., Patel, Y., Harden, A., Rubinelli, P., Smith, S. D., Lebeau, M. M., Rowley, J. D., Diaz, M. O. (1991) Identification of a gene MLL that spans the breakpoint in 11q23 translocations associated with human leukemias. Proc. Natl. Acad. Sci. USA 88,10735-10739[Abstract/Free Full Text]
  2. Iida, S., Seto, M., Yamamoto, K., Komatsu, H., Tojo, A., Asano, S., Kamada, N., Ariyoshi, Y., Takahashi, T., Ueda, R. (1993) MLLT3 gene on 9p22 involved in t(9;11) leukemia encodes a serine/proline rich protein homologous to MLLT1 on 19p13. Oncogene 8,3085-3092[Medline]
  3. Nakamura, T., Alder, H., Gu, Y., Prasad, R., Canaani, O., Kamada, N., Gale, R. P., Lange, B., Crist, W. M., Nowell, P. C., Croce, C. M., Canaani, E. (1993) Genes on chromosomes 4, 9 and 19 involved in 11q23 abnormalities in acute leukemia share sequence homology and/or common motifs. Proc. Natl. Acad. Sci. USA 90,4631-4635[Abstract/Free Full Text]
  4. Prasad, R., Yano, T., Sorio, C., Nakamura, T., Rallapalli, R., Gu, Y., Leshkowitz, D., Croce, C. M., Canaani, E. (1995) Domains with transcriptional regulatory activity within the ALL1 and AF4 proteins involved in acute leukemia. Proc. Natl. Acad. Sci. USA 92,12160-12164[Abstract/Free Full Text]
  5. Rubnitz, J. E., Morrissey, J., Savage, P. A., Cleary, M. L. (1994) ENL, the gene fused with HRX in t(11;19) leukaemias encodes a nuclear protein with transcriptional activation potential in lymphoid and myeloid cells. Blood 84,1747-1752[Abstract/Free Full Text]
  6. Slany, R. K., Lavau, C., Cleary, M. L. (1998) The oncogenic capacity of HRX-ENL requires the transcriptional transactivation activity of ENL and the DNA binding motifs of HRX. Mol. Cell. Biol. 18,122-129[Abstract/Free Full Text]
  7. Corral, J., Lavenir, I., Impey, H., Warren, A. J., Forster, A., Larson, T. A., Bell, S., McKenzie, A. N. J., King, G., Rabbitts, T. H. (1996) An Mll-AF-9 fusion gene made by homologous recombination causes acute leukaemia in chimeric mice: a method to create fusion oncogenes. Cell 85,853-861[Medline]
  8. Pearson, W. R., Lipman, D. J. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. USA. 85,2444-2448[Abstract/Free Full Text]
  9. Carter, D. A., Murphy, D. (1989) Independent regulation of neuropeptide mRNA level and poly (A) tail length. J. Biol. Chem. 264,6601-6603[Abstract/Free Full Text]
  10. Dukas, K., Sarfati, P., Vayesse, N., Pradayrol, L. (1993) Quantitation of changes in the expression of multiple genes by simultaneous polymerase chain reaction. Anat. Biochem. 215,66-72
  11. Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecular Cloning: A laboratory Manual 2nd Ed Cold Spring Harbor Laboratory Press Cold Spring Harbor, N. Y..
  12. Joh, T., Kagami, Y., Yamamoto, K., Segawa, T., Takizawa, J., Takahashi, T., Ueda, R., Seto, M. (1996) Identification of MLL and chimeric MLL gene products involved in 11q23 translocation and possible mechanisms of leukemogenesis by MLL truncation. Oncogene 13,1845-1953
  13. Carter, D. A. (1994) A daily rhythm of activator protein-1 activity in the rat pineal is dependent upon trans-synaptic induction of JunB. Neuroscience 62,1267-1278[Medline]
  14. Bradford, M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Biochemistry 72,248-252
  15. Zeng, Q., Carter, D. A., Murphy, D. (1994) Cell-specific expression of a vasopressin transgene in rats. J. Neuroendocr. 6,469-477[Medline]
  16. Allen, D. L., Mitchner, N. A., Uveges, T. E., Nephew, K. P., Khan, S., Ben-Jonathan, N. (1997) Cell-specific induction of c-fos expression in the pituitary gland by estrogen. Endocrinology 138,2128-2135[Abstract/Free Full Text]
  17. Kozak, M. (1996) Interpreting cDNA sequences: some insights from studies on translation. Mammalian Genome 7,563-574
  18. Nakai, K., Kanehisa, M. (1992) A knowledge base for predicting protein localization sites in eukaryotic cells. Genomics 14,897-911[Medline]
  19. Berchtold, M. W. (1989) A simple method for direct cloning and sequencing cDNA b the use of a single specific oligonucleotide and oligo (dT) in a polymerase chain reaction (PCR). Nucleic Acids Res. 17,453[Free Full Text]
  20. Cairns, B. R., Henry, N. L., Kornberg, R. D. (1996) TFG3/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukaemogenic proteins ENL and AF-9. Mol. Cell. Biol. 16,3308-3316[Abstract]
  21. Welch, M. D., Drubin, D. G. (1994) A nuclear protein with sequence similarity to proteins implicated in human acute leukaemias is important for cellular morphogenesis and actin cytoskeletal functions in Saccharomyces cerevisiae. Mol. Biol. Cell 5,617-632[Abstract]
  22. Armstrong, J. A., Emerson, B. M. (1998) Transcription of chromatin: these are complex times. Curr. Opin. Genet. Dev. 8,165-172[Medline]
  23. Jacobson, S., Pillus, L. (1999) Modifying chromatin and concepts of cancer. Curr. Opin. Genet. Dev. 9,175-184[Medline]
  24. Muchardt, C., Yaniv, M. (1993) A human homologue of saccharomyces cerevisiae SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the glucocorticoid receptor. EMBO J 12,4279-4290[Medline]
  25. Chiba, H., Muramatsu, M., Nomoto, A., Kato, H. (1994) Two human homologues of Saccharomyces cerevisiae SW12/SNf2 and Drosophila brahma are transcriptional coactivators cooperating with the estrogen receptor and the retinoic acid receptor. Nucleic Acids Res. 22,1815-1820[Abstract/Free Full Text]
  26. Wang, W., Xue, Y., Zhou, S., Kuo, A., Cairns, B. R., Crabtree, G. R. (1996) Diversity and specialization of mammalian SWI/SNF complexes. Genes Dev 10,2117-2130[Abstract/Free Full Text]
  27. Aizawa, A., Kazahari, K., Yoneyama, T. (1996) Significance of the chromatin structure in expression of the rat prolactin gene. J. Biochem. 119 ,296-301
  28. Charlton, M., Clark, R. G., Robinson, I. C. A. F., Porter-Goff, A. E., Ciox, B. S., Bugnon, C., Bloch, B. A. (1988) Growth hormone-deficient dwarfism in the rat: a new mutation. J. Endocrinol. 119,51-58[Abstract]
  29. Clark, R. G., Carlsson, L. M. S., Robinson, I. C. A. F. (1987) Growth hormone secretory profiles in conscious female rats. J. Endocrinol. 114,399-407[Abstract]
  30. Wehrenberg, W. B. (1986) The role of growth hormone-releasing factor and somatostatin on somatic growth in rats. Endocrinology 118,489-494[Abstract]
  31. Ono, M., Miki, N., Murata, Y., Osaki, E., Tamitsu, K., Ri, T., Yamada, M., Demura, H. (1995) Sexually dimorphic expression of pituitary growth hormone-releasing factor in the rat. Biochem. Biophys. Res. Commun. 216,1060-1066[Medline]
  32. Fishman, A., Hertz, P., Hochberg, Z. (1993) Ontogenesis of the sexual dimorphism of growth hormone secretion by perifused rat pituitaries. Neuorendocrinology 57,782-788




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by MORGAN, H.
Right arrow Articles by CARTER, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by MORGAN, H.
Right arrow Articles by CARTER, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS