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. Oncogene Fog2 function in the mammary gland development NL Manuylov et al 5206 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).

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