Fog2 Excision in Mice Leads to Premature Mammary Gland Involution and Reduced Esr1 Gene Expression

Oncogene (2007) 26, 5204–5213 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Fog2 excision in mice leads to premature mammary gland involution and reduced Esr1 gene expression

NL Manuylov1,3, FO Smagulova1,3 and SG Tevosian1,2

1Department of Genetics, Dartmouth Medical School, Hanover, NH, USA and 2Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, NH, USA

The critical role for GATA family proteins in maintaining differentiation of multiple lineages (reviewed in Cantor the normal (non-transformed) cell state is corroborated by and Orkin, 2005). FOG proteins (FOG1 and FOG2) are the recent findings of mutations or methylation in GATA cofactors for the GATA family of transcriptional genes both in primary cancers and tumor lines including regulators; FOG proteins are intimately linked with breast. Previously, microarray profiling studies deter- GATA function (Tsang et al., 1997; Crispino et al., mined that the highest expression of both GATA3 and 1999; Lu et al., 1999; Svensson et al., 1999; Tevosian ESR1 (estrogen receptor a) is seen in tumors associated et al., 1999, 2002). with the most favorable survival outcomes, whereas the Currently there is no proof of FOG2 involvement in lowest expression of GATA3 is detected in tumor subtypes mammary gland development or breast cancer. The showing the worst outcomes. At this time, genes and essential role for GATA family proteins in maintaining pathways that are regulated by GATA3 in the mammary the normal non-transformed state of the cells is gland are not well defined. We have previously established corroborated by recent findings of mutations (Lacroix a requirement for FOG (Friend Of GATA) cofactors and Leclercq, 2004; Usary et al., 2004) or methylation during mouse development. Here we report that in the (Akiyama et al., 2003) of GATA genes both in primary murine mammary gland Fog2 gene expression is upregu- cancers and tumor lines. Mutations of GATA1 in Down lated upon pregnancy and lactation with prominent syndrome myeloid leukemogenesis provide support for expression in the epithelial cells of the gland during the hypothesis that mutations of GATA factors occur post-lactational regression. Mammary-specific deletion of early in cancer (Wechsler et al., 2002; Mundschau et al., Fog2 identified a role for this gene during gland 2003; Crispino, 2005). Finally, several studies have involution; excision of the Fog2 gene leads to the already implicated GATA proteins as potential tumor accelerated involution of the gland despite diminished suppressors in various tissues, including the mammary levels of the remodeling enzymes. Importantly, the levels gland (Bertucci et al., 2000, 2004; Lacroix and Leclercq, of several genes linked to the control of cancerous 2004; Usary et al., 2004; Shen et al., 2005). In particular, transformation in the breast (Esr1, Prg and Foxa1) are microarray profiling studies have shown that GATA3 is significantly reduced upon Fog2 excision. This implicates highly expressed in a subset of human breast tumors FOG2 in the maintenance of epithelial cell differentiation (Perou et al., 2000; Gruvberger et al., 2001; West et al., in the mammary gland and in performing a protective role 2001; van’t Veer et al., 2002) and that GATA3 in breast cancer. expression in breast tumors highly correlates with the Oncogene (2007) 26, 5204–5213; doi:10.1038/sj.onc.1210333; expression of the estrogen receptor a-gene/protein published online 19 February 2007 (ESR1) (Hoch et al., 1999; van de Rijn et al., 2002). Analysis of 115 breast tumor samples confirmed that Keywords: GATA; FOG2; estrogen receptor; transcription expression of GATA3 correlated with the expression of factor; mammary; involution a subset of genes considered important in breast luminal epithelial function (Sorlie et al., 2003). Importantly, the highest expression of GATA3 and ESR1 is seen in tumors of the ‘luminal A’ subtype, which is associated Introduction with the most favorable survival outcomes (Parikh et al., 2005). The lower expression of GATA3 and ESR1 is Multi-zinc finger FOG (Friend Of GATA) proteins play characteristic of the ‘luminal B’ ER þ subtype, which is critical roles in mammalian development and the associated with poor survival; the lowest expression of GATA3 is seen in ‘HER2 þ ’ and ‘basal-like’ tumor subtypes, which show the worst outcomes (Sorlie et al., Correspondence: Dr S Tevosian, Department of Genetics, Dartmouth 2003; Mehra et al., 2005; Parikh et al., 2005). Somatic Medical School, Hanover, NH 03755, USA. mutations of GATA3 have been identified in five breast E-mail: [email protected] 3These two authors have contributed equally to this work. tumors (and not in 192 control individuals (388 Received 8 December 2006; revised 4 January 2007; accepted 10 January chromosomes)) (Usary et al., 2004). Despite these 2007; published online 19 February 2007 important advances we do not yet understand which Fog2 function in the mammary gland development NL Manuylov et al 5205 genes and pathways are regulated by GATA3, or FOG2, or both in the mammary gland. In our inspection of the mice harboring a knock-in of the b-galactosidase marker gene in the Fog2 locus (Manuylov et al., submitted), we noted a prominent Fog2 expression in the pregnant and involuting mammary glands. To identify the processes and cells that require FOG2 protein for their function, we deleted the Fog2 gene specifically in the luminal mammary epithelium. Here, we characterize the morphology and gene expression in the mammary gland following conditional deletion of the Fog2 gene. We report here a premature mammary gland involution in female mice homozygous for a loss-of-function mutation in the Fog2 gene that correlates with a reduction in the expression of Bcl2 proto-oncogene. Although gene expression pattern in the involuted glands in the Fog2 mutant mice remains largely intact, the Esr1 and Foxa1 gene expression levels are significantly downregulated; the level of ESR1 protein is also reduced. These data implicate FOG2, a cofactor for GATA family proteins, in the transcriptional control of gene expression in mammary gland.

Figure 1 Whole-mount LacZ staining of the mammary gland in Results the virgin (a), pregnant (b), lactating (c) and involuting (d) Fog2LacZ-ires-eGFP females. The lymph node (arrowhead) staining is a nonspecific staining also present in the lacZ-negative Fog2 expression in the mammary gland animals. To examine Fog2 expression we generated a FOG2- LacZ-eGFP line of mice. In these animals the lacZ gene is incorporated (‘knocked-in’) into the Fog2 locus to In the involuting gland, Fog2 expression appears in allow b-galactosidase expression as a fusion protein in- the luminal epithelial cells by the second day and by day frame with the first 235 amino acids of the FOG2 four of involution Fog2 is strongly expressed in the protein. This creates a null allele of Fog2 (Manuylov remnants of the collapsed lobular/alveolar structures et al., submitted). Unexpectedly, in our analysis of Fog2 (epithelial cords/clusters) that are surrounded by adipo- expression in neonatal animals we observed a pro- cytes (Figure 3a–c). It also continues to be expressed in nounced X-gal staining in epidermal appendages (hair the myoepithelium surrounding these clusters. Upon follicles and teeth primordia) that was confirmed by completion of the remodeling and clearance of the anti-FOG2 antibody staining (data not shown). Since a alveolar remnants, Fog2 expression nearly disappears, number of genes that are known to play a role in hair although it can still be easily detected on day 10 of and teeth development are also involved in mammary involution (Figure 3d). gland morphogenesis we examined Fog2 expression in the mammary gland. Fog2 expression during mouse mammary gland Targeted excision of Fog2 in the mammary gland development appears to be highly dynamic. In the We determined that Fog2 is highly expressed in the virgin gland Fog2 level is very low (Figure 1a); however, mammary epithelium of the gland during the involution during pregnancy, lactation and involution a pro- stage. As strong and specific promoters are available to nounced X-gal staining of the whole-mount gland express Cre recombinases in the lactating mammary preparation could be observed (Figure 1b–d). Histolo- epithelium, it is possible to examine the consequences of gical evaluation of the X-gal-stained tissue confirmed Fog2 gene loss in this cell population. We used a whey that in the virgin gland stromal fibroblasts of the gland acidic protein (WAP)-Cre line of animals available from as well as the vasculature were Fog2 positive, whereas the NIH-NCI Frederick Mouse Repository. WapCre during pregnancy Fog2 expression appears in the recombinase is activated upon lactation specifically in myoepithelial cells (Figure 2a and b). Upon lactation, luminal mammary epithelium (Wagner et al., 1997, X-gal staining could be clearly observed in whole-mount 2001). We reasoned that the WapCre-mediated excision preparations of the gland in myoepithelial cells of the of the Fog2 gene should permit studying the conse- alveoli (Figure 2c–d). To ascertain that X-gal staining quences of Fog2 ablation in luminal breast epithelium. faithfully mimics the expression of the FOG2 protein in To examine the consequences of tissue-specific the myoepithelium, the lactating gland preparations Fog2 gene loss, a conditionally targeted line of mice, were reacted with antibody that recognizes the N- Fog2flox/flox, has been generated. Fog2flox/flox animals are fully terminal region of the FOG2 protein (Figure 2e). viable and fertile and exhibit no obvious abnormalities.

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Figure 2 The X-gal-stained mammary gland from the virgin (a), pregnant (b) and lactating dame (L10, c–d) were sectioned and photographed. The arrows point to the myoepithelial cells (d). The X-gal staining as a ‘read-out’ for the Fog2 expression is conﬁrmed by the aFOG2 antibody staining of the lactating gland (e). Magniﬁcation: (a–c) and (e), Â 100; (d), Â 200.

WapCre animals were bred to Fog2 þ /À females to generate WapCreFog2 þ /À males. These males were mated to Fog2flox/flox females to generate female mice that carry Cre recombinase and are hemizygous flox, WapCreFog2flox/À. To confirm the excision of Fog2 we performed a Southern-blot analysis with DNA extracted from the mammary gland of WapCreFog2flox/À females after completion of lactation (Figure 4). As expected from the previously published data, we detected a considerable amount of the deleted allele in the mammary gland thus confirming that the gene is excised (the non-deleted allele is likely contributed by the non- luminal cells). We also examined a loss of FOG2 at the protein level to confirm that while FOG2 is retained in the myoepithelial cells (that do not express WapCre), the luminal cells are no longer positive for FOG2 expression Figure 3 The X-gal-stained I4 (a–c) and I10 (d) mammary glands (Figure 5a–f). from the Fog2LacZ-ires-eGFP mouse were sectioned, stained with WapCreFog2flox/À mice became pregnant and were hematoxylin and photographed. Note LacZ expression in the fertile and able to support the normal size litters during collapsing alveoli (a–d). High power picture clearly shows that the X-gal staining marks luminal epithelial cells at I4 (c). Expression is lactation (data not shown), so pregnancy and lactation still readily detectable at I10 (d). Magnification: (a) and (d), Â 100; are not obviously affected by the absence of Fog2 in (b), Â 200; (d), Â 400. mammary epithelial cells. We also performed histological evaluation of the mammary glands in the pregnant

Oncogene Fog2 function in the mammary gland development NL Manuylov et al 5207 and lactating WapCre-positive animals and did not (before the bulk of the cells is cleared from the control observe any visible abnormalities (data not shown). gland). We also observed a slight increase in the amount of apoptotic cells (scored both by the histological appearance of apoptotic bodies on the HandE sections Fog2 is required for normal mammary gland involution (data not shown) and by the TUNEL assay) in the Fog2- We compared the mammary gland samples of the deleted gland that, however, was not statistically flox/ control mice (Fog2 À) with the glands of experimental significant (Figure 7c–e). Cell proliferation (defined as flox/À (WapCreFog2 ) mice, where Fog2 is excised on days the % of the PCNA (proliferating cell nuclear antigen)- 3, 4 and 6 of involution (I). We observed that in the positive cells) in Fog2-deleted I3 glands was found to be experimental sample involution occurs faster on both similar to the control sample (data not shown). After 6 days I3 and I4 compared with the control samples. days of involution no differences could be observed Whereas at I3 the control gland still consists mainly of between the experimental and the control samples distended alveoli, the fat tissue is already reappearing (Figure 6c–f). (lipid is accumulating) in the excised gland (Figure 6a– d). Similarly, while the control gland on I4 contained Analysis of the gene expression changes upon some distended alveoli, in the Fog2-excised sample the Fog2 ablation alveoli have completely collapsed into clusters of In an effort to understand the downstream events epithelial cells (Figure 6b–e) and the area containing involved in the regulation of cell survival by Fog2,we adipocytes has greatly expanded (Figure 7a–b). To analysed several potential signaling pathways and target examine the apoptosis we compared the samples of the genes (Supplementary Table 1). Quantitative real-time Fog2-deleted and control gland at day 3 of involution polymerase chain reaction (qRT-PCR) analysis confirmed that Fog2 expression in the mammary gland is at its peak on days 3 and 4 of the involution (data not shown). Hence, in our analysis of the WapCre-induced Fog2 deletion we compared the Fog2-excised glands with controls at this stage. WapCre excision leads to a significant reduction in Fog2 expression in the glands on both days (Figure 8a); the remaining Fog2 expression is likely a product of myoepithelial cells that do not Figure 4 Southern-blot genotyping analysis for deletion of Fog2 express WapCre (see Figures 4 and 5). in mammary gland. Genomic DNA extracted from mammary We examined the expression of the genes that are glands of Fog2flox/À, WapCreFog2flox/À and WapCreFog2wt/À (top to involved in the regulation and execution of apoptosis bottom) was digested with HindIII and hybridized with a probe to and survival; we also queried genes that have been show bands for wild-type (wt), nonrecombined (flox) and recombined Fog2 alleles (D). Fog2À (null) allele is not detectable implicated in both the first and the second stage of the by this analysis and was independently confirmed by second probe involution (e.g. IGF and TGF-b pathways; Clu (cluster- as previously described (Tevosian et al., 2000) (data not shown). in); Mmp3 (matrix metalloproteinase)). qRT-PCR

Figure 5 Immunoﬂuorescent analysis of the control (a–c) and the WapCre-deleted gland (d–f). The glands were obtained at I4, sectioned and stained with anti-FOG2 antibody (a and c; d and f) or anti-FOG2 antibody þ DAPI (to detect nuclear staining, b and e). The FOG2 expression is detected in the luminal cells in the control (c, arrows), but not Fog2-excised gland (f) although expression in the myoepithelial cells appears unchanged.

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Figure 6 Accelerated involution in the I3 and I4 Fog2-excised glands. Mammary glands from the control (Fog2flox/À, a–c) and flox/À experimental (WapCreFog2 , d–f)I3(a and d), I4 (b and e) and Figure 7 Adipogenesis and apoptosis in the I3 Fog2-excised I6 (c and f) mammary glands have been sectioned and stained with glands. Frozen sections from the I3 control (a and c) and hematoxylin and eosin. Magnification: (a–f), Â 100 experimental WapCre-deleted (b and d) mammary glands were stained with Oil Red (to visualize adipocytes) and hematoxylin nuclear stain (a–b) or processed for a TUNEL assay (c–d). TUNEL-positive cells have been counted; notice that an increase flox/ revealed that in the WapCreFog2 À glands expression in the number of the apoptotic cells/bodies in the experimental of most genes was not altered significantly compared sample is non-significant (e). with the control samples; however, statistically significant changes have been observed for several others (e.g. Bcl2 and Bax, caspase 3 (Casp3), metalloproteinase Discussion Mmp2, the plasminogen activator urokinase and its receptor (Plau(uPA) and Plaur)) (Supplementary Table 1 To examine the mechanism of Fog2 function in and Figure 8b). involution we compared gene expression in the control Previous work has established a link between the and Fog2-excised mammary glands. We demonstrate FOG2 protein partner GATA3 and ESR1 as well as two here that ablation of GATA cofactor FOG2 in other transcription factors, FOXA1 (HNF3A) and mammary epithelium results in the significantly reduced XBP1 (e.g. Lacroix and Leclercq, 2004; Laganiere level of Esr1 and Pgr RNA expression as well as et al., 2005; Eeckhoute et al., 2006). Hence, we also decreased ESR1 protein expression in these cells. We examined the effect of Fog2 deletion on these genes’ also observe a dramatic downregulation of expression expression. We observed a statistically significant for another transcription factor, Foxa1, upon Fog2 reduction in both estrogen and progesterone receptor excision. We hence propose that FOG2 is required to RNA expression in the Fog2-deleted gland (Figure 8c); control (upregulate) Esr1 and Foxa1 expression in the the level of Foxa1 expression was dramatically (B4 epithelial cells of the breast. Since our previous work has times at I4) reduced, whereas Xbp1 was downregulated established an essential requirement for a FOG/GATA only slightly (Figure 8d). To confirm that the difference partnership, we also hypothesize that it is a GATA/ in RNA expression translates into a reduced protein FOG complex that maintains the differentiated state of level we examined the ESR1 expression in the frozen a breast epithelium and that inactivation/downregula- sections in the control and Fog2-deleted mammary tion of either gene could lead to downregulation of Esr1 gland on day 4 of involution. We observed a significant and Foxa1 expression. Gata3 expression in the mam- decrease in ESR1 expression in the Fog2-deleted gland mary epithelium during involution was previously (Figure 9). We conclude that FOG2 is essential for the described (Master et al., 2002); however, other GATA regulation of ESR1 expression in mammary epithelial family members (e.g. GATA4 and GATA6; Huggins cells during gland involution. et al., 2006) are expressed in the gland as well. It is

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Figure 8 Analysis of gene expression in the control and Fog2-excised glands. (a) Fog2 RNA level is signiﬁcantly reduced upon WapCre excision in the experimental samples (grey bars) on days 3 and 4 of the involution compared with the controls (black bars, 100%). (b) Gene expression analysis on day 3 of involution. (c) Expression of the Esr1 and Prg receptor genes in the control (black bars) and Fog2-excised (grey bars) glands on days 3 and 4 of the involution. (d) Expression of the Foxa1 and Xbp1 genes in the control (black bars) and Fog2-excised (grey bars) glands on days 3 and 4 of the involution. *Po0.05; **Po0.01; ***Po0.001. important to emphasize that FOG2 is likely to serve as a In contrast to the long-term protective effect of common cofactor for all/any GATA proteins; hence pregnancy, pregnancy is associated with a short-term eliminating Fog2 will interfere with Fog-dependent increase in breast cancer risk in young women. functions of GATA proteins even in tissues where Furthermore, breast cancer that is diagnosed shortly multiple members of the GATA family play redundant after a woman gives birth has a worse clinical prognosis roles. than tumors not associated with a recent pregnancy. It Recent data underscored the importance of epithelial has been suggested that remodeling of the cellular (and not stromal) ESR1 for proliferation and morpho- microenvironment during pregnancy and involution genesis in the mammary gland (Booth and Smith, 2006; may contribute to the enhanced invasive and metastatic Mallepell et al., 2006). Although the positive correlation potential of breast carcinomas and worsen the clinical between the Gata3 expression and Esr1 expression in the outcome (see Polyak, 2006 for review). We hypothesize breast has been well documented, the exact nature of the that involution could be a particularly vulnerable stage relationship between Gata3 and Esr1 is not known. Our during the pregnancy cycle, and the inability of data suggest that GATA/FOG complex is upstream and GATA3/FOG2 complex (e.g. due to mutation in either is able to regulate Esr1 expression during the involution gene) to maintain the differentiated state of the luminal stage. While the function of estrogen in mammary epithelium in the face of extensive tissue remodeling epithelium viability is overall well established (e.g. see during involution could be the initial step in cancerous Jordan, 2004 for review), estrogen pathway contribution transformation. to cell survival speciﬁcally during the involution stage is The differences in the RNA level that we observe are not well understood. Based on the essential role estrogen likely due to changes in the gene expression program in plays in breast function it is likely, however, that the absence of Fog2; they are not simply the con- reduction in the Esr1/Pgr levels is a contributing factor sequences of the decrease in the cell number in the in the decreased viability of Fog2-null epithelial cells. excised gland. Indeed, the expression of several genes

Oncogene Fog2 function in the mammary gland development NL Manuylov et al 5210 metalloproteinase gene upregulation (Han et al., 2003). Alternatively, it is possible that the mammary glands from the Fog2-excised animals are downregulating pathways involved in tissue remodeling in an attempt to compensate for the increased number of epithelial cells undergoing apoptosis. Our studies have potential implications for human disease. We hypothesize that Fog2 is essential in maintaining the differentiated state of the mammary luminal epithelium. Although this remains to be proven experimentally, the loss of Fog2 is likely to increase the pool of Esr1-negative cells that could be targeted by an oncogenic event leading to a more severe clinical outcome. The relatively low level of Fog2 RNA in the mammary gland (which is substantially lower than that of Gata3 (our unpublished observations)) may have precluded the detection of Fog2 differential expression by microarray profiling screens; however, our results Figure 9 Immunohistochemical analysis of the ESR1 expression in the suggest that the status of the Fog2 gene in tumor control (Fog2flox/À)(a and c)andFog2-excised (WapCreFog2flox/À) (b and d) I4 glands. Slides were reacted with anti-ESR1 antibody samples should be evaluated for possible mutations and/ followed by a horseradish peroxidase (HRP)-coupled secondary or expression abnormalities. Further studies will address antibody. No expression was detected upon staining with the the mechanisms of GATA/FOG2 action during involu- secondary antibody alone (data not shown). Magnification: tion and should afford new insights into how the (a–b), Â 200; (c–d), Â 400. GATA/FOG-regulated pathways are corrupted in cancer. that are strictly specific to mammary epithelium (e.g. While this work was under review, Werb and co- Igfbp5 and C/ebpd; Allar and Wood, 2004; Thangaraju workers (Kouros-Mehr et al., 2006) described the et al., 2005) (Figure 8b) remains constant or only subtly animals with the conditional knockout of Gata3 in down in the WapCreFog2flox/À glands indicating that the mammary epithelium. Although the consequences of expression differences we observe for other genes (e.g. Gata3 gene ablation are more severe than that of Fog2, Esr1 and Foxa1) are not solely due to the decrease in Kouros-Mehr et al. (2006) also placed Esr1 and Foxa1 epithelial cell count upon Fog2 excision. downstream of GATA3 and demonstrated that FOXA1 The acceleration of involution coincides with the promoter binds GATA3 in vivo. Hence it is likely that decrease in the expression of Bcl2 proto-oncogene upon these genes are regulated by the GATA3/FOG2 com- Fog2 deletion. Although Bcl2 was shown to promote plex, whereas other prospective GATA3 target genes epithelial cell survival in the gain-of-function experi- (e.g. Xbp1) do not require Fog2. Additionally, the ments in the gland (Murphy et al., 1999; Schorr et al., recently published study adds to a framework for 1999) its role in the control of apoptosis during the early understanding the role of FOXA1 in the mammary phase of normal involution process may be limited gland as it highlights its role as a growth inhibitor and a (Metcalfe et al., 1999; Schorr et al., 1999). Nevertheless, favorable prognostic factor in human breast cancer we cannot exclude that a decrease in Bcl2 gene (Wolf et al., 2006). Thus, Fog2 is required for normal expression in the WapCreFog2flox/À mice is a contribut- expression of two genes (Esr1 and Foxa1) that are ing factor that leads to an accelerated involution in these correlated with a favorable outcome in breast cancer. animals. Recent work suggests that cardiac expression of Bcl2 is directly regulated by a GATA family member (GATA4) in vivo, although the role of FOG2 has not Materials and methods been explored (Kobayashi et al., 2006). Other Bcl2 Animals family genes we have examined (see Supplementary To generate animals with a conditionally targeted Fog2 locus Table 1), both proapoptotic and death suppressors, are we performed a homologous recombination in the ES cells. not affected by Fog2 excision. The first LoxP site was introduced into the XhoI site in the Epithelial Fog2 expression is limited to the involution seventh intron and the second LoxP site into the XbaI site of stage of mammary development and WapCre-mediated the 30 downstream sequence in the Fog2 locus (Zfpm2, Ensembl ablation of Fog2 accelerates mammary gland involution. database); the details of the construct are available upon Fog2 excision likely affects both the cell survival and request. WapCre mice were obtained from the NIH Reposi- remodeling in the involuting mammary gland. Despite tory and were maintained on their original C57BL/6NCr accelerated involution in the Fog2-excised glands, the background. Fog2LacZiresEGFP mice (Manuylov et al., submitted) and Fog2 þ /À heterozygous mice (Tevosian et al., 2000) expression of the remodeling proteolytic enzymes is were maintained on a mixed 129/C57BL/6J background. For downregulated in the absence of Fog2. This could expression studies Fog2LacZiresEGFP males were crossed with be explained by a separate role GATA/FOG2 transcrip- the C57BL6/6J females. As variations in mouse genetic back- tion complex plays in the regulation of these genes as ground are known to affect the involution (e.g. Thangaraju GATA family members have been implicated in et al., 2004), we followed a standard mating scheme to generate

Oncogene Fog2 function in the mammary gland development NL Manuylov et al 5211 primiparous experimental animals during the conditional Total RNA extraction excision. Specifically, we crossed Fog2 þ /À mice with WapCre RNA was extracted using TRIZOL (Invitrogen, Carlsbad, animals to generate WapCreFog2 þ /À males. These males were CA, USA); lymph nodes were removed and mammary tissue mated to homozygous floxed (Fog2flox/flox) females to generate was homogenized in liquid nitrogen. Isolated RNA was further both experimental (WapCre Fog2À/floxed) and control (Fog2À/flox) cleaned with RNAeasy columns (Qiagen, Valencia, CA, USA). female siblings. For involution experiments these females were A first strand cDNA synthesis kit (Invitrogen) was used for bred to the wild-type males. To equalize the lactation pressure, cDNA preparation from 2 mg of total RNA. the number of pups for each dam was adjusted to six and maintained through the lactation period. For analysis of the Quantitative Real Time RT-PCR involuting gland the dams were allowed to nurse (lactate) for a The RT-PCR was performed using a PCR SYBR Green I Kit total of 10 days at which time the pups were removed; 2, 3, 4, 6 (Applied Biosystems, Foster City, CA, USA) according to the and 10 days later the mice were euthanized and the glands manufacturer’s instructions in a 7500 fast RT-PCR system removed for analysis. For the analysis of the lactating glands machine and a standard amplification protocol was established mice were euthanized and glands removed on day 10 of for each gene. A standard curve for the Fog2 gene was lactation. Mammary tissue was snap frozen in liquid nitrogen generated from serial dilutions of a plasmid (Tevosian et al., for DNA/RNA extraction, frozen in the OCT compound 1999) containing a full-length Fog2 cDNA; calibration points (VWR, West Chester, PA, USA) for both antibody staining for serial dilutions of the plasmids were from 106 to 102 copies. and TUNEL assay or fixed in formalin/glutaraldehyde mix for For the rest of the tested genes calibration curves were the whole-mount X-gal staining. generated using a gel-purified RT-PCR product; dilution for calibration points was as described above. The value for each Mouse genotyping gene was normalized to the Gapdh gene value and these rations Mice were genotyped both by PCR and Southern-blot analysis were compared between the control and the Fog2 mutant using genomic DNA prepared from tail snips. Fog2À (null) mammary sample. Gene copy number was calculated with ABI allele was detected by a Southern-blot analysis as described SDS Software Version 1.3.1. Values from at least three previously (Tevosian et al., 2000). Fog2LacZiresEGFP animals independent experiments were compared; standard deviation (Manuylov et al., submitted) were genotyped using primers and a Student’s t-test were calculated using Excel (Microsoft, complementary to the LacZ fragment (LacZf 50-taaccgtcac Redmond, WA, USA). gagcatcatc-30 and LacZrev 50-gcgtacatcgggcaaataat-30); Fog2flox allele was distinguished from the wild-type locus using a PCR Immunohistochemistry and immunofluorescence reaction followed by a HindIII enzyme digestion of a PCR Sections of the frozen glands were cut on the cryostat (Leica, product with F2Gen1: 50-ggtcttcgacatccatgtttcacagc-30 F2Gen2 Bannockburn, IL, USA). For FOG2 protein detection slides 50-tgcgagaacaggtgttggtggaagtt-30 primers. To detect a WapCre were reacted with goat anti-FOG2 antibody (Santa Cruz allele, Cre-specific primers were used: Cre1 50-caaaacaggtagt Biotechnology, Santa Cruz, CA, USA) and stained with anti- tattcgg-30 and Cre2 50-cgtatagccgaaattgccag-30. Littermates goat Alexa Flour 555 (Molecular Probes, Carlsbad, CA, with genotypes Fog2flox/À (controls) and WapCreFog2flox/À USA). Slides were mounted in Vectashield with DAPI (40,6- (mutants) were used for further analysis. Diamidino-2-phenylindole, Vector) and photographed. For PCNA staining, an anti-PCNA antibody (AbCam, Cam- Whole-mount X-gal staining bridge, MA, USA) was used. PCNA-positive epithelial nuclei Whole-mount X-gal staining was performed as previously were counted in 10 random microscopic fields. Three mice of described (Adameyko et al., 2005) overnight at 371C. 10 mM each genotype (Fog2flox/À and WapCre Fog2flox/À) were ana- thick tissue sections were cut on the microtome (Olympus, lysed. For the immunodetection of the ESR1 protein slides Center Valley, PA, USA) and floated onto SuperFrost þ slides were reacted with the rabbit anti-ESR1 antibody (AbCam) and stained with haematoxylin and eosin (Richard Allan followed by anti-rabbit HRP-conjugated antibody (Bio-Rad, Scientific, Kalamazoo, MI, USA). Hercules, CA, USA). Slides were stained using a DAB kit (Vector). Detection of apoptotic cells Mammary glands were dissected at I3 and apoptotic cells were Acknowledgements detected by terminal deoxynucleotide UTP nick-end labeling (TUNEL), using an In Situ Cell Death Detection kit (Roche, We thank Christian Lytle for his advice on qRT-PCR, James Indianapolis, IN, USA). Nuclei were counterstained with DiRenzo for his advice on the whole-mount gland prepara- Vectashield with DAPI (Vector, Burlingame, CA, USA). tions and William Pu for the advice on mouse genotyping. This TUNEL-positive epithelial nuclei were counted in 10 random work was supported by a grant from the Department of microscopic fields. Three mice of each genotype (Fog2flox/À and Defense Congressionally Mandated Medical Research Pro- WapCre Fog2flox/À) were analysed. gram on Breast Cancer (W81XWH-06-1-0394) to SGT.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

Oncogene