Transposons are mobile DNA segments that can disrupt gene function by inserting in or near genes. Here we show that insertional mutagenesis by the PiggyBac transposon can be used for cancer gene discovery in mice. PiggyBac transposition in genetically engineered transposon/ transposase mice induced cancers whose type (hematopoietic versus solid) and latency were dependent on the regulatory elements introduced into transposons. Analysis of 63 hematopoietic tumors revealed the unique qualities of PiggyBac for genome-wide mutagenesis and discovered many cancer genes not identified in previous retroviral or Sleeping Beauty transposon screens, including Spic, which encodes a PU.1-related transcription factor, and Hdac7, a histone deacetylase gene. PiggyBac and Sleeping Beauty have different integration preferences. To maximize the utility of the tool, we engineered 20 mouse lines to be compatible with both transposases in constitutive, tissue-or temporal-specific mutagenesis. Mice with different transposon types, copy numbers and chromosomal locations support wide applicability.Genetic screening in higher organisms has been hampered for decades by the lack of efficient insertional mutagenesis tools. Retroviruses have been used for cancer gene discovery in mice, but their application has been limited to the study of hematopoietic and mammary tumors due to viral tropism for these tissues (1). DNA transposons, which are the key insertional mutagens in lower organisms, were inactivated in vertebrate genomes millions of years ago. Only recently have new transposons been engineered to be active in mammalian cells, a development that provides opportunities for their use as genetic tools in higher organisms (2). Sleeping Beauty (SB), a TC1/mariner transposon, was reconstructed from dormant elements in fish genomes and optimized to transpose in multiple cell types (3), including mouse embryonic stem cells (4). Further improvements of SB led to its successful application for somatic mutagenesis in mice (5, 6). Another transposon, PiggyBac (PB) from the cabbage looper moth, was recently engineered to be highly active in mammalian cells (7) and has been shown to have biological properties distinct from those of SB (2, 7-9). PB can move larger DNA fragments (allowing complex transgene designs to be incorporated into the transposon) and it has a weaker tendency for local hopping in vitro (which makes it an attractive candidate for genome-wide mutagenesis). Furthermore, in contrast to SB, PB does not leave undesired footprint mutations after transposition. Finally, PB and SB have different integration preferences. (10)). All transposons possess PB and SB inverted terminal repeats (ITRs), allowing mobilization with both transposases. Promoter/enhancer elements, a splice donor, bidirectional SV40 polyAs and two splice acceptors were introduced in between the ITRs to allow gain or loss of function mutations, depending on the transposon orientation and its spatial relation to genes. Transgenic mice were generated with three va...

Emerging high-throughput screening technologies are rapidly providing opportunities to identify new diagnostic and prognostic markers and new therapeutic targets in human cancer. Currently, cDNA arrays allow the quantitative measurement of thousands of mRNA expression levels simultaneously. Validation of this tool in hospital settings can be done on large series of archival paraffin-embedded tumor samples using the new technique of tissue microarray. On a series of 55 clinically and pathologically homogeneous breast tumors, we compared for 15 molecules with a proven or suspected role in breast cancer, the mRNA expression levels measured by cDNA array analysis with protein expression levels obtained using tumor tissue microarrays. The validity of cDNA array and tissue microarray data were first verified by comparison with quantitative reverse transcriptase-polymerase chain reaction measurements and immunohistochemistry on full tissue sections, respectively. We found a good correlation between cDNA and tissue array analyses in one-third of the 15 molecules, and no correlation in the remaining twothirds. Furthermore, protein but not RNA levels may have prognostic value; this was the case for MUC1 protein, which was studied further using a tissue microarray containing ϳ600 tumor samples. For THBS1 the opposite was observed because only RNA levels had prognostic value. Thus, differences extended to clinical prognostic information obtained by the two methods underlining their complementarity and the need for a global molecular analysis of tumors at both the RNA and protein levels.

Aneuploidy is a hallmark of human solid cancers that arises from errors in mitosis and results in gain and loss of oncogenes and tumor suppressors. Aneuploidy poses a growth disadvantage for cells grown in vitro, suggesting that cancer cells adapt to this burden. To understand better the consequences of aneuploidy in a rapidly proliferating adult tissue, we engineered a mouse in which chromosome instability was selectively induced in T cells. A flanked by Lox mutation was introduced into the monopolar spindle 1 (Mps1) spindle-assembly checkpoint gene so that Cre-mediated recombination would create a truncated protein (Mps1 DK ) that retained the kinase domain but lacked the kinetochore-binding domain and thereby weakened the checkpoint. In a sensitized p53 +/− background we observed that Mps1 DK/DK mice suffered from rapid-onset acute lymphoblastic lymphoma. The tumors were highly aneuploid and exhibited a metabolic burden similar to that previously characterized in aneuploid yeast and cultured cells. The tumors nonetheless grew rapidly and were lethal within 3-4 mo after birth.

Key Points• We demonstrate a critical role for histone deacetylase 1 and 2 (HDAC1/2) in T-cell development and the maintenance of genomic stability. IntroductionHistone deacetylase (HDAC) 1 and 2 are sister proteins (ϳ 80% identical), functionally redundant in many cell types, 1-3 which are recruited together into 3 main transcriptional complexes: Sin3A, 4 NuRD 5,6 and CoREST. 7,8 Classically, HDAC1 and 2 (HDAC1/2) function has been viewed in the context of transcriptional repression, because deacetylation of histone tails results in the tightening of nucleosomal arrays. 9,10 However, genome-wide mapping of HDAC1 (or Rpd3) binding sites in human cells 11 and yeast, 12 reveals a positive correlation with gene activity, suggesting a role for HDACs in the cyclical acetylation of histones within the vicinity of active promoters. 13 HDAC1/2 may thus have roles in both gene activation and repression.In the clinic, the HDAC inhibitor SAHA is used to treat patients with cutaneous T-cell lymphoma. 14 Therefore a greater understanding of the key pathways regulated by HDAC1/2 activity in T-lymphocyte development will help inform their use as drug targets. Moreover, as a dispensable cell type in a standard pathogen free environment, it is an excellent model to study essential genes, such as HDAC1/2. T-cell development, from immature double negative (DN), to double positive (DP) intermediates and mature (but naive) CD4 single positive (CD4SP, helper) and CD8 single positive (CD8SP, cytotoxic) populations is regulated by a number of transcription factors and chromatin modifying complexes. 15,16 Runx1 and Runx3 have nonredundant roles in the regulation of CD4 expression. [17][18][19] The zinc-finger transcriptional regulator, Gata3, is crucial for the development of the earliest T-cell progenitors. 20 ThPOK expression is necessary to direct CD4 expression in MHC II restricted cells and is also sufficient to redirect class-I MHC T cells into the CD4 T-helper lineage. 21 Individual HDACs have also been implicated in T-lymphocyte function. HDAC7 regulates cell survival and TCR signaling in DP thymocytes, 22 HDAC6 and HDAC9 regulate the activity of T-regulatory cells, 23,24 while HDAC11 directly controls IL-10 expression levels in antigen presenting cells. 25 Deletion of either Sin3A 26 or Mi2␤, 27 central components of the HDAC1/2 containing Sin3A and NuRD complexes, respectively, perturbs thymopoiesis. Loss of Sin3A causes a deficiency in the DN to DP transition and a significant reduction in the number of CD8SP cells. Mi2␤ also plays a role in DN to DP transition, and in addition is required for transcriptional activation of the Cd4 gene. These 2 studies clearly implicate HDAC1/2 in T-cell development. However, deletion of Hdac1 alone at the double positive stage of development produced a relatively mild phenotype 28 ; suggesting that, as in many other tissue systems, deletion of both Hdac1 and For personal use only. on May 9, 2018. by guest www.bloodjournal.org From Hdac2 is required to observe a more substantial phenotypic...