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We describe the successful application of a modified gene-trap approach, the secretory trap, to systematically analyze the functions in vivo of large numbers of genes encoding secreted and membrane proteins. Secretory-trap insertions in embryonic stem cells can be transmitted to the germ line of mice with high efficiency and effectively mutate the target gene. Of 60 insertions analyzed in mice, one-third cause recessive lethal phenotypes affecting various stages of embryonic and postnatal development. Thus, secretory-trap mutagenesis can be used for a genome-wide functional analysis of cell signaling pathways that are critical for normal mammalian development and physiology.

ATP-dependent chromatin remodeling complexes are a notable group of epigenetic modifiers that use the energy of ATP hydrolysis to change the structure of chromatin, thereby altering its accessibility to nuclear factors. BAF250a (ARID1a) is a unique and defining subunit of the BAF chromatin remodeling complex with the potential to facilitate chromosome alterations critical during development. Our studies show that ablation of BAF250a in early mouse embryos results in developmental arrest (about embryonic day 6.5) and absence of the mesodermal layer, indicating its critical role in early germ-layer formation. Moreover, BAF250a deficiency compromises ES cell pluripotency, severely inhibits self-renewal, and promotes differentiation into primitive endoderm-like cells under normal feeder-free culture conditions. Interestingly, this phenotype can be partially rescued by the presence of embryonic fibroblast cells. DNA microarray, immunostaining, and RNA analyses revealed that BAF250a-mediated chromatin remodeling contributes to the proper expression of numerous genes involved in ES cell self-renewal, including Sox2, Utf1, and Oct4. Furthermore, the pluripotency defects in BAF250a mutant ES cells appear to be cell lineage-specific. For example, embryoid body-based analyses demonstrated that BAF250a-ablated stem cells are defective in differentiating into fully functional mesoderm-derived cardiomyocytes and adipocytes but are capable of differentiating into ectodermderived neurons. Our results suggest that BAF250a is a key component of the gene regulatory machinery in ES cells controlling self-renewal, differentiation, and cell lineage decisions.BAF250a (ARID1a) ͉ lineage commitment ͉ mesoderm R egulatory factors that control chromatin architecture (directly or indirectly) are potential key proteins for maintaining the pluripotent state or directing differentiation of early embryonic cells into distinct cell types (1). Such factors include ATP-dependent chromatin remodeling complexes that hydrolyze ATP to noncovalently restructure, mobilize, or eject nucleosomes to modulate transcription factor access to chromosomal DNA (2). Among the various members of the ATPdependent chromatin remodeling superfamily is the SWI/SNF subfamily, consisting of two closely related SWI/SNF remodeling complexes BAF and PBAF in mammalian cells (3, 4).BAF250a, a defining subunit of the BAF chromatin remodeling complex (5, 6), is a trithorax group (TrxG) protein (7). TrxG proteins were initially identified by their ability to antagonize the Polycomb group (PcG) proteins to maintain proper expression of many differentiation regulators during development (8). Interestingly, many PcG and TrxG proteins are chromatin modifying factors. Recent studies mapping the targets of PcG action in mouse and human ES cells suggest these proteins also play a role in sustaining a heritable epigenetic state uniquely associated with pluripotency (9, 10).Given the influence of epigenetic factors in determining developmental potential (embryonic development, cellular ...

MeCP2 is a chromosomal protein that is concentrated in the centromeric heterochromatin of mouse cells. In vitro, the protein binds preferentially to DNA containing a single symmetrically methylated CpG. To find out whether the heterochromatic localization of MeCP2 depended on DNA methylation, we transiently expressed MeCP2-LacZ fusion proteins in cultured cells. Intact protein was targeted to heterochromatin in wild-type cells but was inefficiently localized in mutant cells with low levels of genomic DNA methylation. Deletions within MeCP2 showed that localization to heterochromatin required the 85-amino-acid methyl-CpG binding domain but not the remainder of the protein. Thus MeCP2 is a methyl-CpG-binding protein in vivo and is likely to be a major mediator of downstream consequences of DNA methylation.Widespread methylation of genomic DNA is a characteristic of the vertebrates. The target of methylation is the dinucleotide CpG, which acquires a methyl group at the 5 position on the cytosine ring. Functions of methylation have long been the subject of experiment and speculation, but recent experiments provide evidence for an essential role in development. Li et al. (16) disrupted the gene that encodes the DNA methyltransferase (MTase) enzyme in mice and found that homozygous mutant embryos had greatly reduced levels of methylation, were developmentally retarded, and died at midgestation. Undifferentiated embryonic stem (ES) cells, on the other hand, behaved normally in culture, despite very low levels of methylation, indicating that methylation may be less important at this totipotent stage. Why methylation is essential for development is not yet completely clear. One possibility is that methylation-mediated repression of genes is somehow built into the developmental program (discussed in reference 1).To understand the biology of DNA methylation, it is necessary to identify all components of the system. Several proteins that bind specifically to methylated DNA are known (reviewed in reference 31). Two of these, MeCP1 and MeCP2, bind to symmetrical methyl-CpG pairs in any sequence context and may therefore be of general significance (14,17,18). Studies of MeCP1 have implicated it in methylation-mediated gene inactivation (2, 3). The second protein, MeCP2, consists of a single chain of 492 amino acids in the rat, including an 85-amino-acid domain near the N terminus that encodes all of the specificity required for binding to methylated DNA (22). The mouse protein (484 amino acids) is very similar to its rat homolog, showing an overall identity of 95% at the amino acid level and absolute conservation of the methylated DNA binding domain (29). This methyl-CpG binding domain (MBD) can bind as a monomer to a single symmetrically methylated CpG pair. Immunofluorescence analysis of mouse chromosomes shows that MeCP2 is preferentially localized in pericentromeric heterochromatin (14), which is also the region of highest 5-methylcytosine concentration (19). The predominant DNA sequence in mouse heterochromatin is the m...

To address the long-known relationship between supernumerary centrosomes and cancer, we have generated a transgenic mouse that permits inducible expression of the master regulator of centriole duplication, Polo-like-kinase-4 (Plk4). Over-expression of Plk4 from this transgene advances the onset of tumour formation that occurs in the absence of the tumour suppressor p53. Plk4 over-expression also leads to hyperproliferation of cells in the pancreas and skin that is enhanced in a p53 null background. Pancreatic islets become enlarged following Plk4 over-expression as a result of equal expansion of α- and β-cells, which exhibit centrosome amplification. Mice overexpressing Plk4 develop grey hair due to a loss of differentiated melanocytes and bald patches of skin associated with a thickening of the epidermis. This reflects an increase in proliferating cells expressing keratin 5 in the basal epidermal layer and the expansion of these cells into suprabasal layers. Such cells also express keratin 6, a marker for hyperplasia. This is paralleled by a decreased expression of later differentiation markers, involucrin, filaggrin and loricrin. Proliferating cells showed an increase in centrosome number and a loss of primary cilia, events that were mirrored in primary cultures of keratinocytes established from these animals. We discuss how repeated duplication of centrioles appears to prevent the formation of basal bodies leading to loss of primary cilia, disruption of signalling and thereby aberrant differentiation of cells within the epidermis. The absence of p53 permits cells with increased centrosomes to continue dividing, thus setting up a neoplastic state of error prone mitoses, a prerequisite for cancer development.