Acidic nuclear phosphoprotein 32kDa (ANP32)B-deficient mouse reveals a hierarchy of ANP32 importance in mammalian development Patrick T. Reillya, Samia Afzalb,c, Chiara Gorrinib, Koren Luib, Yury V. Bukhmanb,1, Andrew Wakehamb, Jillian Haightb, Teo Wei Linga, Carol C. Cheungb, Andrew J. Eliab, Patricia V. Turnerd, and Tak Wah Maka,b,2 aDivision of Cellular and Molecular Research, National Cancer Centre Singapore, Singapore 169610; bCampbell Family Cancer Research Institute, University Health Network, Toronto, ON, Canada M5G 2C1; cGraduate Program in Immunology, University of Toronto, Toronto, ON, Canada M5S 1A8; and dDepartment of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1 Contributed by Tak Wah Mak, April 20, 2011 (sent for review February 18, 2011) The highly conserved ANP32 proteins are proposed to function in these studies has specifically excluded a particular ANP32 protein a broad array of physiological activities through molecular mech- as contributing to the activities examined. anisms as diverse as phosphatase inhibition, chromatin regulation, More recent work has demonstrated functions that are ex- caspase activation, and intracellular transport. On the basis of clusive to the ANP32B protein, at least in humans. First, previous analyses of mice bearing targeted mutations of Anp32a ANP32B, but not ANP32A, controls the expression of the den- or Anp32e, there has been speculation that all ANP32 proteins play dritic cell maturation factor CD83 by regulating the transport of redundant roles and are dispensable for normal development. its mRNA to the cytoplasm (22). Second, ANP32B modulates However, more recent work has suggested that ANP32B may in the activity of the transcription factor Kruppel-like factor 5 fact have functions that are not shared by other ANP32 family (KLF5), whereas ANP32A cannot (32). Finally, ANP32B is a members. Here we report that ANP32B expression is associated caspase substrate, whereas ANP32A is not (33). with a poor prognosis in human breast cancer, consistent with Reported loss-of-function mutants for ANP32 family members Anp32b include two independently targeted ANP32A-deficient mice (34, the increased levels of mRNA present in proliferating fi wild-type (WT) murine embryonic fibroblasts and stimulated WT 35), an ANP32E-de cient mouse (35), and a presumptive null mutant of mapmodulin in Drosophila (36). All of these mutants B and T lymphocytes. Moreover, we show that, contrary to pre- Anp32b were viable and fertile with no obvious abnormalities. Here, we vious assumptions, is very important for murine embryo- fi −/− fi report on the phenotype of Anp32b-de cient (Anp32b ) mice genesis. In a mixed genetic background, ANP32B-de cient mice and, using compound mutants, show that ANP32 family members displayed a partially penetrant perinatal lethality that became are not completely redundant in mammals. We demonstrate that fully penetrant in a pure C57BL/6 background. Surviving ANP32B- fi ANP32B is the most important ANP32 family member for mam- de cient mice showed reduced viability due to variable defects malian development and likely plays a complex role in sustaining in various organ systems. Study of compound mutants lacking viability that has yet to be completely defined. In addition, we ANP32A, ANP32B, and/or ANP32E revealed previously hidden roles provide evidence that the level of ANP32B mRNA expression in for ANP32A in mouse development that became apparent only in human breast cancers may serve as a prognostic marker. the complete absence of ANP32B. Our data demonstrate a hierarchy of importance for the mammalian Anp32 genes, with Anp32b be- Results ing the most critical for normal development. ANP32B as a Potential Prognostic Marker in Human Cancer. Reports that ANP32B expression was related to cell proliferation (15, 17) cell growth | mouse embryogenesis | premature aging prompted us to investigate whether ANP32B expression was al- tered in human tumor samples. We examined the relationship NP32B (a.k.a. APRIL, PAL31, PHAP1b) is a mamma- between ANP32B mRNA expression and breast cancer patient Alian member of the highly conserved acidic nuclear phos- prognosis using information from three available studies, namely phoprotein 32kDa (ANP32) family of gene products (1). These the NEJM (New England Medical Journal) (37), the BMC (BMC metazoan-specific factors are characterized by the presence of Genomics) (38), and the PNAS datasets (39). When we com- an amino-terminal leucine-rich repeat domain and a carboxyl- pared the survival of these three groups of patients among all terminal region that is highly enriched in acidic amino acid three datasets, we found that patients whose tumors showed the fi highest ANP32B mRNA levels had signi cantly shorter survival GENETICS residues (2). These features are found in ANP32 proteins from − times using the Cox proportional hazard model (P < 10 4). To mapmodulin, the single representative in Drosophila, and in the fi three vertebrate family members identified ANP32A, ANP32B, display this effect, we strati ed the patients in these datasets into three major groups: those with tumors expressing high levels of and ANP32E (1, 3). ANP32B mRNA (highest 33.3% or tertile), those with tumors The ANP32 proteins have been implicated in a broad array of with medium ANP32B mRNA levels (middle tertile), and those physiological processes, including cell differentiation (4–7), apo- with tumors with low ANP32B mRNA levels (lowest tertile). ptotic cell death (8–14), and cell proliferation (15–17). Diverse mechanisms have been postulated for how these proteins perform their function(s). Some studies indicate that ANP32 proteins may Author contributions: P.T.R. and T.W.M. designed research; P.T.R., S.A., C.G., K.L., A.W., directly control enzymatic activities, such as via inhibition of J.H., T.W.L., C.C.C., and A.J.E. performed research; P.T.R., S.A., C.G., Y.V.B., P.V.T., and protein phosphatase 2A (PP2A) (18–20) or activation of caspases T.W.M. analyzed data; and P.T.R. and T.W.M. wrote the paper. (10, 11, 13, 14, 21). Other studies suggest that they may regulate The authors declare no conflict of interest. – intracellular transport at nuclear pores or microtubules (22 25). Freely available online through the PNAS open access option. Several studies present evidence that nuclear ANP32 proteins 1Present address: Great Lakes Bioenergy Research Center, University of Wisconsin, Mad- may influence transcription either through the inhibitor of acetyl ison, WI 53706. transferases complex (26–29) or by direct effects on transcription 2To whom correspondence should be addressed. E-mail: [email protected]. factors (30, 31). Most of the above reports have focused on This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ANP32A, the founding member of the ANP32 family, but none of 1073/pnas.1106211108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1106211108 PNAS | June 21, 2011 | vol. 108 | no. 25 | 10243–10248 Downloaded by guest on October 2, 2021 Fig. 2. Elevated Anp32b mRNA expression correlates with increased cell pro- liferation. (A) Correlation with highly proliferative tissues. A commercial mouse tissue blot was examined by Northern blotting to detect Anp32b mRNA. Gapdh: Fig. 1. ANP32B mRNA expression is a marker for aggressive breast cancer. relative expression control. Anp32b mRNA is decreased relative to Gapdh in brain Survival analyses of patients with tumors expressing the highest levels of and muscle. (B) Correlation with nutrient-induced proliferation. WT MEFs were ANP32B mRNA (highest tertile: dashed line), and patients with tumors deprived of serum for 72 h and then resupplied with medium containing 10% expressing the lowest levels of ANP32B mRNA (lowest tertile: solid line). The FBS for 24 h. Anp32b mRNA levels were determined by qPCR relative to β-actin. middle tertile is not shown. Values were acquired from three separate Data shown are the mean ± SD of two independent WT MEF cultures. **P < 0.01. datasets: NEMJ (37), PNAS (39), and BMC (38). (C) Correlation with lymphocyte stimulation. (Left)Purified B cells from WT mice were treated with 1 μg/mL LPS for 24 h. (Right)PurifiedTcellsfromWTmicewere stimulated with plate-bound anti-CD3 (2 μg/mL) plus anti-CD28 (0.2 μg/mL) for Here, the survival effect of the highest and lowest ANP32B- 36 h. In both cases, Anp32b mRNA levels were determined by qPCR relative to expressing tumors is clearly demonstrated (Fig. 1; middle tertile 18S rRNA. Results shown are the mean ± SD of triplicates and are expressed as not shown). These results imply that elevated ANP32B expres- fold increase over levels in untreated controls. Results are representative of sion in a tumor may be a marker of poor patient prognosis. two trials. **P < 0.01. Anp32b mRNA Levels Are Linked to Cell Proliferation in Mice. The less favorable prognosis of breast cancer patients with high mRNA levels (Fig. 2C). These results indicate that Anp32b is up- ANP32B-expressing tumors suggested that the elevated ANP32B regulated under conditions that favor cell proliferation. inthesesamplesmightbelinkedtomoreaggressivetumor Anp32b cell proliferation. To investigate this hypothesis, we performed Gene Targeting of in Mice. To investigate the physiological Northern blot analysis of a wide range of tissues from adult wild- functions of ANP32B in vivo, we generated mice in which the type (WT) mice. We found that Anp32b expression was low Anp32b gene was disrupted by replacing the genomic region (relative to Gapdh) in tissues where less cell proliferation usually containing exons 2, 3, and 4 with a pgk-neo expression cassette occurs, i.e., in brain tissue (Fig. 2A). In contrast, tissues that (Fig. 3A). This mutation results in an mRNA in which the last 17 generally have high cell proliferation rates, i.e., spleen and thy- codons of exon 1 are linked to an out-of-frame exon 5. Southern mus, showed higher relative levels of Anp32b mRNA expression.