The Zinc Finger Ikaros Transcription Factor Regulates Pituitary Growth ...

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Molecular Endocrinology 19(4):1004–1011 Copyright © 2005 by The Endocrine Society doi: 10.1210/me.2004-0432

The Zinc Finger Ikaros Transcription Factor Regulates Pituitary Growth Hormone and Prolactin Gene Expression through Distinct Effects on Chromatin Accessibility Shereen Ezzat, Shunjiang Yu, and Sylvia L. Asa Department of Medicine (S.E., S.Y.), Mount Sinai Hospital and University of Toronto, Department of Pathology (S.L.A.), University Health Network and University of Toronto, The Freeman Centre for Endocrine Oncology (S.E., S.Y., S.L.A.), and The Ontario Cancer Institute, Toronto, Ontario, Canada M5G 2M9 The Ikaros transcription factors perform critical functions in the control of lymphohematopoiesis and immune regulation. Family members contain multiple zinc fingers that mediate DNA binding but have also been implicated as part of a complex chromatin-remodeling network. We show here that Ikaros is expressed in pituitary mammosomatotrophs where it regulates the GH and prolactin (PRL) genes. Ikaros was detected by Northern and Western blotting in GH4 pituitary mammosomatotroph cells. Wild-type Ikaros (Ik1) inhibits GH mRNA and protein expression but stimulates PRL mRNA and protein levels. Ikaros does not bind

directly to the proximal GH promoter but abrogates the effect of the histone deacetylation inhibitor trichostatin A on this region. Ikaros selectively deacetylates histone 3 residues on the proximal transfected or endogenous GH promoter and limits access of the Pit1 activator. In contrast, Ikaros acetylates histone 3 on the proximal PRL promoter and facilitates Pit1 binding to this region in the same cells. These data provide evidence for Ikaros-mediated histone acetylation and chromatin remodeling in the selective regulation of pituitary GH and PRL hormone gene expression. (Molecular Endocrinology 19: 1004–1011, 2005)

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binding domains. The intact dimerization domains of these isoforms render them capable of forming inactive heterodimers. The various isoforms can act as either activators or repressors in a functionally diverse chromatin remodeling network (3). We recently identified expression of Ikaros in the pituitary where it is thought to play a role in the regulation of fibroblast growth factor receptor 4 (FGFR4) (5). Altered expression of Ikaros isoforms was also implicated in pituitary tumorigenesis through its actions on FGFR4 promoter acetylation (6, 7). In this report we sought further evidence for functional properties of Ikaros in pituitary hormone gene expression. In particular, Ikaros was abundantly expressed in mammosomatotroph cells that express GH and prolactin (PRL). Introduction of wild-type and dominantnegative (dn) forms of Ikaros revealed reciprocal effects on GH and PRL regulation attributable to selective chromatin remodeling and histone acetylation of the respective promoters.

HE PROCESS OF pituitary hormone regulation is dependent on a number of cis- and trans-active elements that are necessary for tissue-specific gene expression. Several putative transcription-regulating proteins identified in the pituitary have been implicated as key elements in determining cell-specific phenotypes and the regulation of hormone genes uniquely expressed by these cells (1, 2). Which of the lineageregulating factors predominates in determining the lineage choice can be decided not only by the expression and gradient of the transcription factor but also possibly by accessibility to their cognate sites. Ikaros was initially described as a transcription factor that recognizes regulatory sequences of genes expressed in lymphoid cells (3, 4). The N terminus encodes zinc finger motifs that recognize cognate DNA-binding sites. In contrast, the C terminus shared by all Ikaros isoforms contains a dimerization domain. Alternative splicing results in isoforms that lack DNA-

First Published Online December 23, 2004 Abbreviations: AcH3, Acetyl histone 3; ChIP, chromatin immunoprecipitation; dn, dominant negative; FGFR4, fibroblast growth factor receptor 4; GAPDH, glyceraldehyde-3phosphate dehydrogenase; HDAC, histone deacetylase; PRL, prolactin; SCID, severe combined immunodeficient. Molecular Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.

RESULTS Ikaros Is Abundantly Expressed in Pituitary Mammosomatotrophs Using Northern blotting to investigate the distribution of Ikaros, we detected an mRNA doublet of approxi1004

Ezzat et al. • Ikaros Regulates GH and PRL

mately 2.7 kb in mammosomatotroph GH4 cells. This product corresponds to the expected Ik1 and Ik2 mRNA isoforms and was of the same size as that expressed by pro-B lymphocytes (Fig. 1A). The Ikaros mRNA transcripts were translated in GH4 cells as demonstrated by Western immunoblotting (Fig. 1B). In contrast, this mRNA species was not sufficiently detectable by Northern blotting in lactotroph PRL235 cells. Nevertheless, Ikaros protein was detected by Western blotting in these cells (5). Moreover, we could not detect mRNA transcripts for the Ikaros-related family member Eos in GH4 or PRL 235 cells (data not shown). Ikaros Regulates GH and PRL Gene Expression Having identified Ikaros expression in pituitary mammosomatotrophs, we examined whether the GH and PRL genes were subject to regulatory control by this zinc finger protein (Fig. 2). GH4 cells stably transfected with Ikaros (Ik1) or the dn, non-DNA-binding isoform (Ik6) were examined by Northern blotting. GH4 cells stably transfected with Ik1 demonstrated consistent reduction of endogenous GH mRNA expression (Fig. 2A). In contrast, PRL mRNA expression in the same cells was induced by overexpression of Ikaros (Fig. 2C). Western blotting of corresponding cell lysates confirmed that these changes were also reflected in endogenous GH (Fig. 2B) and PRL protein levels (Fig. 2D). To further investigate the effects of Ikaros on GH and PRL gene regulation, stably transfected GH4 cells were inoculated into severe combined immunodeficient (SCID) mice and examined by immunohistochemistry after 2 wk of growth in vivo (Fig. 2E). GH4 cells overexpressing Ik1 showed appreciable up-regulation of PRL, but this was not evident in cells overexpressing Ik6 (Fig. 2E). In contrast, GH expression

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was attenuated selectively in cells overexpressing Ik1 (Fig. 2E). To determine the mechanism responsible for Ikaros differential regulation of the GH and PRL genes, we tested the response of the GH and PRL promoters to cotransfection with Ikaros or the Ikaros dn (non-DNAbinding) Ik6 isoform. Figure 3 demonstrates the effect of Ik1 on the proximal GH and PRL promoters in transfected GH4 cells. Ik1 inhibited the activity of a 320-bp GH proximal promoter by a modest 40%, and transfection of the dn Ik6 resulted in near doubling of GH promoter activity. In contrast to the effects on the GH promoter, Ik1 transfection resulted in consistent activation of the 422-bp proximal PRL promoter by 50%, an effect not shared with Ik6. Ikaros Does Not Bind the GH or the PRL Promoter A search of the proximal GH and PRL promoter failed to identify any potential Ikaros-binding sites. This prediction was further supported by DNA footprinting using nuclear extracts from GH4 cells and EMSA assays using overlapping fragments of the GH and PRL proximal promoters as previously described (5)(data not shown). Ikaros Alters Acetylation of the GH and PRL Promoters Given the recognized ability of Ikaros to associate with components of the nucleosome remodeling and histone deacetylation (NuRD) transcriptional repression complex complex that includes the chromatinremodeling histone deacetylase 1 (HDAC1) and HDAC2 (8), we examined the status of histone acetylation of the GH and PRL promoters using a chromatin immunoprecipitation (ChIP) assay. Transfec-

Fig. 1. Identification of Ikaros Expression in Pituitary Mammosomatotroph GH- and PRL-Producing Cells Northern blotting of poly-A RNA from GH/PRL-expressing pituitary mammosomatotrophic GH4 and PRL-producing PRL 235 cells using Ikaros cDNA (top) identifies a doublet transcript of 2.7 and 2.5 kb consistent with Ik1 and Ik2 mRNA expression in GH4 cells. This doublet comigrates with that from pro-B lymphocytes but is not detectable in prolactin-producing PRL 235 cells. The GAPDH loading control is shown immediately below. B, Western blotting using specific antibodies to Ikaros identifies a 57- and 50-kDa doublet consistent with Ik1/Ik2 in untransfected GH4 cells and in GH4 cells transfected with empty vector (GH4-pcDNA); this band comigrates with that from control pro-B-lymphocytes. Transfection of Ik1 enhances expression of this band. Transfection of the dn Ik6 isoform (Ik6) yields the expected truncated 35-kDa transcript.

1006 Mol Endocrinol, April 2005, 19(4):1004–1011


Fig. 2. Ikaros Suppresses GH but Induces PRL Gene Expression in Pituitary Mammosomatotroph Cells GH4 cells were stably transfected with Ikaros (Ik1) or its dn (Ik6) isoform and examined by Northern blotting of independent clones for GH (panel A) and PRL (panel C) mRNA expression. Note the contrasting effects where GH expression is diminished but PRL mRNA levels are induced by Ik1 overexpression. The GAPDH loading control is shown immediately below. Ikaros-stably transfected GH4 cell lysates from independent clones were also examined by Western immunoblotting. Note the contrasting effects of Ik1 on GH attenuation (panel B) but increased PRL expression (panel D) compared with Ik6 or empty vector-transfected cells. The actin loading controls are shown immediately below. E, The effect of Ikaros on GH and PRL expression was examined in Ik1-, Ik6-, or empty vector-transfected GH4 cells inoculated into SCID mice. Two weeks after inoculation, resected tissue reveals diminished GH immunoreactivity (upper panels) in cells stably expressing Ik1 (middle), and enhanced PRL immunoreactivity (lower panel) in the same cells compared with cells expressing Ik6 or empty vector (pcDNA)-transfected cells.

tion of Ik1 resulted in deacetylation of histone 3 complexes on the GH proximal promoter (Fig. 4A). Introduction of Ik6 resulted in reversal of this effect consistent with acetylation of the GH promoter. Moreover, transfection of Ikaros reversed the activating effect of the pharmacological deacetylation inhibitor, trichostatin A, on the GH promoter (Fig. 4B). Trichostatin A treatment alone resulted in activation of the GH (⬃4-fold increase) and PRL (⬃2fold increase; Fig. 4B) promoters consistent with the significance of histone acetylation in control of these genes. Ik1 resulted in appreciable acetylation of histone 3 on the PRL proximal promoter as determined by

ChIP assay (Fig. 4A). In contrast to the effect on the proximal GH promoter, Ik1 transfection did not abrogate the effect of trichostatin A on the PRL promoter (Fig. 4B). That the action of Ikaros on GH and PRL gene expression is mediated through differential mechanisms on histone acetylation was further tested by examining the effect of Ikaros on the endogenous genes. Examination of the endogenous GH and PRL promoters by ChIP assay demonstrated changes similar to those identified on the transfected promoters. Cells overexpressing Ik1 demonstrated a selective absence of GH-derived PCR product, consistent with histone deacetylation of this promoter (Fig. 4C). In contrast, Ik6 resulted in



or with empty vector (Fig. 5B; left panel). The selectivity of this Pit1 restriction was also evident when the ability of Pit1 to activate the PRL promoter was examined (Fig. 5B; right panel). In contrast to the effect on the GH promoter, Pit1 activation of the PRL promoter was not impeded but somewhat enhanced in the face of Ikaros overexpression.

DISCUSSION Fig. 3. Ikaros Confers Distinct Effects on GH and PRL Promoter Activity GH4 cells were transiently cotransfected with the GH (left) or PRL (right) promoter along with Ikaros (Ik1) or its dn isoform (Ik6) and subjected to a luciferase reporter assay. Note the suppressive effect on GH promoter activity by Ik1. In contrast, transfection of Ik6 results in activation of the GH reporter. Note the activation of the PRL promoter by Ik1, an effect not shared by Ik6. All transfections included corresponding empty control vectors along with 20 ng of pCMV␤gal to normalize for transfection efficiency. Data are presented as the mean luciferase activity adjusted for ␤-gal activity (⫾SD) and compared with control wells of three separate experiments, each performed in triplicate. The asterisk denotes a P value ⬍ 0.005 compared with empty vector (pcDNA)-transfected cells.

selective deacetylation of the endogenous PRL promoter in the same cells (Fig. 4C). Ikaros Alters Pit1 Accessibility Selectively to the GH and PRL Promoters To further determine the effect of Ikaros-mediated histone acetylation on the GH and PRL promoters, we examined the ability of this zinc finger protein to modulate the binding of the well-recognized activator Pit1. Using a ChIP-based approach, we compared the ability of Ikaros to alter Pit1 binding to the GH promoter vs. the PRL promoter (Fig. 5A). Ikaros significantly restricted the access of Pit1 to the GH promoter (Fig. 5A). In marked contrast and consistent with the effects on endogenous PRL gene regulation, Ikaros facilitated Pit1 binding to the PRL promoter. These effects on the GH and PRL promoters were not shared by Ik6, which tended to show the opposite effect of that mediated by Ik1, consistent with the heterodimerizing properties of Ikaros isoforms. To determine the functional impact of altered Pit1 accessibility by Ikaros to the GH and PRL promoters, we reasoned that Pit1 action on GH might be more restricted compared with PRL when Ikaros is overexpressed. We thus examined the ability of Pit1 to activate the GH and PRL promoters in cells overexpressing Ikaros, Ik6, or their empty vector control. Consistent with the ChIP-derived data, the ability of Pit1 to activate the GH promoter was significantly restricted in the background of Ik1 overexpression, an effect not evident in the same cells transfected with Ik6

Ikaros is expressed in the anterior pituitary gland (5) where, in addition to its wild-type form, the gene undergoes alternative splicing in tumors to generate the dn Ik6 isoform in human pituitary adenomas (6). Forced expression of Ikaros results in nuclear protein compared with the cytoplasmic localization of overexpressed Ik6. The different subcellular localizations of Ikaros isoforms made it possible to demonstrate the nearly exclusive cytoplasmic expression of Ik6 in more than half of primary human pituitary tumors (6). These two Ikaros isoforms result in differential effects on histone acetylation on the FGFR4 promoter. The net effect of these Ikaros isoform interactions results in an environment that silences the wild-type proximal promoter and favors utilization of a cryptic downstream site leading to the genesis of a unique tumor-derived FGFR4 isoform (7). In this study, we investigated the effect of Ikaros on pituitary hormone gene expression. We focused on the GH and PRL genes as we found significant levels of Ikaros expression in mammosomatotroph cells that coexpress both genes. We provide here the first evidence that Ikaros regulates both genes in a reciprocal fashion in the same cell type. Despite the absence of specific Ikaros-binding sites in either the GH or PRL proximal promoters, we show that Ikaros suppresses GH but activates PRL gene expression in cells of the mammosomatotroph lineage. The mechanism for this differential hormone effect appears to rely on the state of chromatin accessibility mediated by Ikaros. The formation of Ikaros homo- and heterodimers among the DNA-binding Ikaros family members increases their affinity for DNA, whereas heterodimers between the DNA-binding isoforms and non-DNAbinding isoforms are unable to bind DNA. Ikaros proteins with fewer than three N-terminal zinc fingers can negatively interfere with the activity of Ikaros isoforms that bind DNA (9, 10). Histones have been shown to be underacetylated in the vicinity of Ikaros recruitment sites whereas the HDAC inhibitor trichostatin abrogates transcriptional repression mediated by Ikaros (11). An abundance of the non-DNA-binding, alternatively-spliced Ikaros isoforms results in deregulated expression of target genes that are essential for normal development. Ikaros isoforms with DNA-binding domains when bound in cis to Ik-binding sites have been shown to activate gene transcription (9, 10, 12). In contrast, Ikaros represses transcription when re-


Fig. 4. Ikaros Confers Distinct Effects on Chromatin Histone Acetylation on the GH and PRL Promoters A, Distinct effects of Ikaros on histone 3 (H3) acetylation of the GH and PRL promoters was examined by ChIP assay. GH4 cells stably expressing Ikaros (Ik1), its dn Ik6 isoform, or their empty vector control were transiently transfected with the GH (upper) or PRL (lower) promoter and subsequently examined by ChIP. Cross-linked chromatin was immunoprecipitated with antibody to AcH3 followed by PCR analysis. Input DNA represents PCR products without prior immunoprecipitation (IP). DNA from plasmid was amplified as positive controls (⫹); negative controls omitted the DNA template (⫺). Note the effect of Ikaros on histone deacetylation on the GH promoter resulting in absence of PCR product. In contrast, Ikaros acetylates histone 3 on the PRL promoter (lower panel). B, Impact of Ikaros on trichostatin A-mediated activation of the GH and PRL promoters. GH4 cells stably expressing Ikaros or Ik6, or their empty vector, were transiently transfected with the GH-luciferase or PRL-luciferase minimal promoters followed by incubation with trichostatin A (dark bars). Note the ability of trichostatin A to activate the GH and PRL promoter. Results represent the mean ⫹ SE derived from three independent experiments, each performed in triplicate wells. Cells transfected with empty vector were set as con-


cruited to DNA through a heterologous DNA-binding domain (11). This transcriptional repression has been regarded to be mediated through at least two main repression domains at the N and C termini, which interact with HDAC 1 and 2 containing mSin3 (11) and Mi-2 (13) proteins. The extent to which this mechanism of gene repression is global or gene specific, however, remained to be determined. We show here that Ikaros in the pituitary opposes the effects of trichostatinmediated inhibition of histone deacetylation on the GH promoter but not on the PRL promoter. These data are more in support of a gene-specific, as opposed to a cell-specific, effect for Ikaros-mediated gene regulation. Ikaros isoforms that contain the DNA-binding domain have also been shown to function as activators. Indeed, Ikaros-mediated gene activation with localization in heterochromatin complex has been described (14). These data provided the framework for an alternative model for Ikaros action. In this capacity, Ikaros has been suspected to be associated with target genes in a predominantly restrictive chromatin environment that houses tightly regulated genes (8). We propose that GH and PRL reflect examples of tightly regulated genes that are typically repressed in most adult tissues. Under unique conditions in which specific activators such as Pit1 and Ikaros are coexpressed, chromatin remodeling can be repackaged accordingly. In this model, Ikaros functions as a potentiator by indirectly remodeling the densely packaged chromatin favoring pituitary transcription factor access. Here, we provide evidence that access of the well-described Pit1 activator is modulated in a genespecific manner by Ikaros. Consistent with this model, Pit1-mediated activation of the GH promoter was abrogated in the presence of Ikaros overexpression, an effect not shared with the PRL promoter. Pit1 is a well-recognized member of the homeobox family of developmental regulatory proteins (12, 15). The presence of an additional domain, conserved in Pit1 and the proteins OCT-1, OCT-2, and UNC-86, gave rise to the term “POU-domain” that characterizes this family of homeodomain proteins (16, 17). As the name suggests, Pit1 exhibits pituitary-restricted expression where it activates the structurally related GH and PRL genes in rat and human (17). The role of Pit1 in cytodifferentiation is also well recognized when it was found that pit-1 expression in the developing rodent pituitary is associated with the onset of GH and PRL production (10, 18). Moreover, isoforms of Pit1

trols at 100% activity. *, P ⬍ 0.05 compared with empty vector-transfected control. C, The distinct effects of Ikaros on histone 3 (H3) acetylation of the endogenous GH and PRL promoters were examined by ChIP assays as in panel A. Input DNA represents PCR products without prior immunoprecipitation. DNA from plasmid was amplified as positive controls (⫹); negative controls included omission of the DNA template (⫺).



Fig. 5. Ikaros Confers Distinct Effects on Pit1 Binding and Action on the GH and PRL Promoters A, Effects of Ikaros on Pit1 binding to the GH and PRL promoters were examined by a ChIP assay as indicated in Fig. 4 except Immunoprecipitation (IP) of the DNA-protein complex was performed using an anti-Pit1 antibody. Positive PCR control (⫹) included plasmid DNA; negative control (⫺) omitted the DNA template. PCR products from input DNA not immunoprecipitated (input DNA) indicate products from all reactions. B, GH4 cells stably expressing Ik1, Ik6, or their empty vector were transiently transfected with Pit1 and the GH-luciferase or PRL-luciferase reporter. Note the inability of Pit1 to activate the GH promoter, consistent with restricted access to this promoter in the presence of Ik1 overexpression (Fig. 5B; left panel). The selectivity of this Pit1 restriction was also evident when the ability of Pit1 to activate the PRL promoter was examined (Fig. 5B; right panel). Results represent the mean ⫾ SE derived from three independent experiments, each performed in triplicate. Cells transfected with empty vector were set as controls at 100% activity. *, P ⬍ 0.05 compared with empty vector-transfected control.

that result from alternative mRNA splicing, Pit1␤ (8, 19, 20) and Pit1T (21, 22), have different selective effects on pituitary gene transcription. In particular, Pit1 isoformspecific repression of Ras signaling to the PRL promoter was demonstrated to be histone acetylation sensitive (23). It is also known that the differential effects of Pit1 on GH in somatotrophs vs. lactotrophs are related to the ability of Pit1, in combination with other DNA-binding factors, to recruit a corepressor complex that includes the nuclear receptor corepressor (24). Our findings of distinct Ikaros-mediated deacetylation recruitment to the proximal GH and PRL promoters in the same cell type provide another line of evidence in favor of chromatin remodeling as a critical contributor to selective transcription factor-mediated gene regulation in the pituitary. Our current data on the opposing effects of Ikaros on the GH and PRL genes in the same cell type are consistent with a gene-specific effect. Whereas the Ikaros family members, Aiolos, Helios, and Eos, can associate with Ikaros corepressors, the potential for their involvement in mediating Ikaros-like effects is less likely, given their negligible expression in pituitary mammosomatotrophs and lactotrophs. Instead, the possibilities of functional networks between Ikaros and other pituitary transcription factors including Pit1 appear more likely. Given the immune modulatory properties of the GH/PRL family of cytokines (25) and the critical contribution of Ikaros to immune cell development (8), the current findings predict an interfac-

ing role for Ikaros in governing development of the growth axis.

MATERIALS AND METHODS Cell Culture The rat clonal pituitary lactotroph PRL235, and mammosomatotroph GH4 cell lines were purchased from the ATCC (Manassas, VA) and cultured in Ham F10 media supplemented with 15% horse serum, 2.5% fetal bovine serum (Sigma, Oakville, Ontario, Canada) and 2 mM glutamine, 100 IU/ml penicillin, and 100 ␮g/ml of streptomycin. The pro-Blymphocyte BaF3 cells were propagated in DMEM (Life Technologies, Inc., Gaithersburg, MD) with high glucose supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 IU/ml penicillin, and 100 ␮g/ml of streptomycin. Stable clones expressing Ikaros (Ik1) or its isoform (Ik6) were generated using a conventional antibiotic selection procedure (Geneticin; Invitrogen, San Diego, CA). Plasmids The expression vectors encoding full-length Ikaros (CDM8Ik1) and dn Ikaros 6 (CDM8-Ik6) were generously provided by Dr. K. Georgopolous (Boston, MA) and used for cloning into the pcDNA 3.1 (Invitrogen) expression vector. The full-length mouse Pit1 expression vector (kindly provided by Dr. H. Elsholtz, Toronto, Ontario, Canada) was also cloned into pcDNA3.1. The orientation and sequence of all constructs were confirmed by restriction digestion and nucleotide sequencing.


Promoter analyses were performed with assistance from the transcription factor Database TRANSFAC 4.0 (http:// transfac.gbf.de/cgi-bin/matSearch/matsearch.pl). The convention for sequence coordinates with ⫹1 as the first base of the coding sequence in exon 1 was adopted. Transient Transfection and Luciferase Assays Plasmid reporters and expression vectors were prepared by column chromatography (QIAGEN, Missisauga, Ontario, Canada) for sequencing and transfections. Cells were transfected by the lipofectamine method (Invitrogen) according to the manufacturer’s protocol. Cells were plated into six-well cluster dishes (7 ⫻ 105 cells per well), transfected the following day with 3 ␮l or 5 ␮l/well of lipofectamine and 1 or 2 ␮g of DNA per well. The total amount of transfected DNA was equalized by adding empty vector. PRL promoter activity was analyzed with reporter constructs pSV2A-rPRL-luc containing a ⫺422-bp fragment of the rPRL promoter or pSV2ArGH-luc containing the ⫺320-bp of the rat (r) GH promoter (both kindly provided by Dr. H. Elsholtz, Toronto, Ontario, Canada). To normalize for transfection efficiency variation within and between experiments, 20 ng/well of pSV-␤-galactoside control vector (Promega Corp., Madison, WI) was included with each transfection. Forty-eight hours after transfection, cells were lysed in buffer containing 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1% Triton X, and 1 mM dithiothreitol. Luciferase activity was measured for 20 sec in a luminometer. ␤-Galactosidase activity was measured to normalize for variations in transfection efficiency. In chromatin acetylation studies, cells were transfected and subsequently treated with trichostatin A (200 ng/ml) for at least 16 h before luciferase assay. Promoter activity of each construct was expressed as firefly luciferase/␤-gal activity. Each experiment was independently performed on three separate occasions with triplicate wells in each experiment. RNA Isolation and Northern Blotting Analysis RNA was prepared from exponentially growing cells (2–5 ⫻ 107) using Isolation FastTrack 2.0 Kit (Invitrogen, San Diego, CA) following the manufacturer’s instructions. PolyA-enriched RNA (2.5 ␮g) was electrophoresed on a 1% formaldehyde-agarose gel and transferred to a nylon membrane. Hybridization was performed with probes labeled randomly (labeling kit, Roche Diagnostics, GmbH, Mannheim, Germany) with [32P]dCTP from the cDNAs of Ikaros, Eos, GH, PRL, and glyceraldehyde-3phosphate dehydrogenase (GAPDH). The Ikaros probe consisted of a 1200-bp fragment encoding the C terminus BamHI/ EcoRV fragment of the Ikaros cDNA. The Eos probe was a 587-bp BamHI/HindIII fragment of the mouse Eos cDNA (kindly provided by Dr. M. Crossley, Sydney, New South Wales, Australia) (26). The rGH and rPRL cDNA probes for Northern blotting were used as previously described (27). The GAPDH probe was generated by RT-PCR using specific primers to yield a 687-bp fragment. Western Blotting Whole lysates (40 ␮g) were separated on 10% sodium dodecyl sulfate (SDS) denaturing polyacrylamide gels, after which the proteins were transferred onto a nylon membrane (Millipore Corp., Bedford, MA) at 100 V for 1 h at room temperature. Blots were blocked with 5% nonfat milk and incubated with a mouse monoclonal antibody that recognizes the C-terminal fragments of Ikaros proteins (4E9; kindly provided by K. Georgopolous, Boston, MA) (10) or polyclonal antisera to rPRL or rGH [donated by the National Hormone and Pituitary Program (NHPP), NIDDK, NICHHD, Bethesda, MD] applied at dilutions of 1:8,000 and 1:50,000, respectively, or actin (Sigma; at 1:500) in PBS-5% nonfat milk with


0.1% Tween 20 at 4 C overnight, followed by washing four times with PBS-Tween 20 for 10 min at room temperature and incubated with secondary antibody of peroxidase-conjugated goat antimouse or antirabbit IgG (1:2000) for 1 h at room temperature with agitation. Proteins were detected using a chemiluminescence method (ECL, Amersham Pharmacia Biotech, Arlington Heights, IL). ChIP Assay Cells were cotransfected with the proximal 5⬘-rGH ⫺320 or the rPRL ⫺422-bp promoters and either the Ik1 or Ik6 expression vector or their empty control vector as indicated. The ChIP assay was performed in accordance with the manufacturer’s recommendations (Upstate Biotechnology, Inc., Lake Placid, NY) and as previously described (6). In some experiments, as indicated, the effect of Ikaros was examined on the endogenous rGH or rPRL promoters. Briefly, histone was cross-linked to DNA by the direct addition of 37% formaldehyde in cells, and cells were washed with cold PBS containing protease inhibitors before cells were lysed; the lysates were sonicated to shear DNA lengths between 200 and 1000 bp. After centrifugation, cell suspensions were further diluted, and 20 ␮l of lysate from each sample were kept and used to quantitate the amount of DNA present (input DNA) for PCR detection. The rest of the lysate was cleared with salmon sperm DNA/protein G-agarose beads. Half of the cleared lysate was incubated with acetyl histone 3 (AcH3) or Pit-1 antibody (as indicated) and protein G-agarose beads overnight at 4 C with agitation, and the other non-antibodyimmunoprecipitated protein was used as a negative control, both of which were either examined by immunoblotting with anti-AcH3 or Pit-1 antibody (BabCO, Berkeley, CA) or by PCR. For PCR analysis, the histone-DNA cross-links of the eluates were reversed at 65 C, and the immunocomplexes were digested with proteinase-K for 1 h at 50 C, and DNA was finally purified by phenol extraction and used for PCR amplification. The PCR conditions for both rGH and rPRL amplification were: 95 C for 4 min followed by 35 cycles of 95 C for 40 sec, 55 C for 40 sec, and 72 C for 1 min, and finally 72 C for 7 min using the following primers: rGH, forward (5⬘-GTGACCATTGCCCATAAACC-3⬘) and reverse (5⬘-TGCATGCCCTTTTTATACCC-3⬘) corresponding to nucleotides 1522– 1541 and 1738–1719 of the rGH promoter sequence, respectively (GenBank accession no. X12967) yielding a 216-bp PCR product from the ⫺320 bp GH promoter. Similarly, the following primers: rPRL, forward (5⬘-GCAATGGCACACATTGCAGA-3⬘) and reverse (5⬘-AGTCCTAAGAGAACCACTGC-3⬘) were used to generate a 364-bp fragment from the ⫺422-bp PRL promoter. Immunohistochemistry GH4 cells stably transfected with Ikaros, Ik6, or vector alone were injected sc into SCID mice (5 ⫻ 106 cells per injection). Animals were handled in accordance with Ontario Cancer Institute institutional guidelines and protocol approval. Tumors were examined histologically and immunohistochemically 2 wk after injection to confirm GH4 origin and to examine impact on GH and PRL expression as described previously (28). Statistical Analysis Data are presented as mean ⫾ SE. Differences were assessed by Student’s paired t test. Significance level was assigned at P ⬍ 0.05.

Acknowledgments We thank Drs. Katia Georgopoulos and Stephen Smale for Ikaros antibodies, and Dr. Merlin Crossley for Eos reagents.


We also thank Dr. Harry Elsholtz for insightful discussions. The technical assistance of Mr. K. So is gratefully acknowledged.

Received October 22, 2004. Accepted December 16, 2004. Address all correspondence and requests for reprints to: Dr. S. Asa, Department of Pathology, University Health Network, 610 University Avenue, Suite 4-302, Toronto, Ontario, Canada M5G 2M9. E-mail: [email protected]. This work was supported by the Canadian Institutes of Health Research (Grant MT-14404) and the Toronto Medical Laboratories.

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