Expression of p21 has been shown to be up-regulated by the p53 tumor suppressor gene in vitro in response to DNA-damaging agents. However, p21 expression can be regulated independently of p53, and here we show that expression of p21 in various tissues during development and in the adult mouse occurs in the absence of p53 function. However, most tissues tested did require p53 for p21 induction following exposure of the whole animal to ~/irradiation. These results show that normal tissue expression of p21 to high levels is not dependent on p53 and confirm that induction of p21 by DNA-damaging agents does require p53. p21 is expressed upon differentiation of p53-deficient murine erythroleukemia (MEL1 cells, and the kinetics of induction of p21 in this system suggest that it may be involved in the growth arrest that precedes terminal differentiation. The gene is up-regulated in mouse fibroblasts in response to serum restimulation but the kinetics and levels of induction differ between wild-type and mutant cells. Expression of p21 message following serum restimulation is superinducible by cycloheximide in wild-type but not in p53-deficient cells. The increases in p21 mRNA are reflected in changes in p21 protein levels, p21 expression also appears to be regulated at the post-transcriptional level because moderate increases in mRNA expression, during differentiation of MEL cells and upon serum restimulation of fibroblasts, are followed by large increases in protein levels. Regulation of the mouse p21 promoter by p53 depends on two critical p53-binding sites located 1.95 and 2.85 kb upstream from the transcriptional initiation site. The sequences mediating serum responsiveness of the promoter map to a region containing the proximal p53 site. p53 appears to play a critical role in p21 induction following DNA damage. Moreover, p21 can be regulated independently of p53 in several situations including during normal tissue development, following serum stimulation, and during cellular differentiation.

In normal fibroblasts CDKs exist predominantly in p21/PCNA/cyclin/CDK quaternary complexes, whereas in p53-deficient cells, p21 expression is depressed and the kinases are reduced to a cyclin/CDK binary state, p21 is a universal cyclin kinase inhibitor, but we show here that p21-containing complexes exist in both catalytically active and inactive forms. This finding challenges the current view that active cyclin kinases function only in the binary state and reveals the subtlety with which tumor-suppressor proteins modulate the cell cycle.[Key Words: p21-containing cyclin kinase; cell cycle progression; human fibroblasts; quaternary complex; kinase inhibitor]Received May 12, 1994; revised version accepted June 16, 1994.Much of our current understanding of the regulation of the cell division cycle has emerged from studies of a family of protein kinases [cdc2, CDC28, and generically cyclin-dependent kinase (CDK)] and their inhibitors and activators (for review, see Sherr 1993). A critical step in understanding cell cycle control was the discovery that CDKs interact with cyclins, proteins that serve as essential activating subunits and specificity determinants of the kinases (Draetta 1990;Sherr 1993). In human cells, multiple cyclins and CDKs interact in a relatively promiscuous fashion to form a large family of related cyclin kinases, each of which is presumed to play a specific role in cell cycle progression.The view that mammalian cyclin kinases exist predominantly in a binary (cyclin/CDK) state prevailed until these enzymes were examined in normal human fibroblasts, rather than the many oncogenically transformed cell types that had been investigated hitherto (Xiong et al. 1992). In normal fibroblasts the major population of multiple cyclin kinases exists in quatemary complexes consisting of cyclin, CDK, proliferating cell nuclear antigen (PCNA), and a protein of apparent Mr 21,000, p21 (Zhang et al. 1993). However, in fibroblasts that are transformed with a variety of tumor viral oncoproteins the quatemary complexes essentially reduce to a binary state. Deregulation of cell proliferation is a hallmark of neoplastic transformation. Difference in the composition of cell cycle kinases between normal and transformed cells suggests that fundamental changes in cell cycle regulation contribute to neoplastic transformation.Recently, it has become apparent that p21 is a universal inhibitor of cyclin/CDK catalytic activity (Gu et al. 1993;Harper et al. 1993;Xiong et al. 1993b). Each member of the cyclin kinase family is inhibited by p21, but their relative affinities vary with each enzyme (Gu et al. 1993;Harper et al. 1993;Xiong et al. 1993b). Furthermore, several lines of evidence suggest that p21 expression is regulated by the p53 tumor-suppressor protein (E1- Deiry et al. 1993;Xiong et al. 1993b). Thus, cells derived from certain p53-deficient Li-Fraumeni patients lack p21 associated with cyclin kinases (Xiong et al. 1993a). The p21 gene has a p53 transcriptional regulatory motif (E1- Deiry et al. 1993), and cells lac...

Somatic reprogramming induced by defined transcription factors is a low efficiency process that is enhanced by p53 deficiency 1-5. To date, p21 is the only p53 target shown to contribute to p53 repression of iPSC (induced pluripotent stem cell) generation 1, 3, suggesting additional p53 targets may regulate this process. Here, we demonstrated that mir-34 microRNAs (miRNAs), particularly miR-34a, exhibit p53-dependent induction during reprogramming. mir-34a deficiency in mice significantly increased reprogramming efficiency and kinetics, with miR-34a and p21 cooperatively regulating somatic reprogramming downstream of p53. Unlike p53 deficiency, which enhances reprogramming at the expense of iPSC pluripotency, genetic ablation of mir-34a promoted iPSC generation without compromising self-renewal and differentiation. Suppression of reprogramming by miR-34a was due, at least in part, to repression of pluripotency genes, including Nanog, Sox2 and Mycn (N-Myc). This post-transcriptional gene repression by miR-34a also regulated iPSC differentiation kinetics. miR-34b and c similarly repressed reprogramming; and all three mir-34 miRNAs acted cooperatively in this process. Taken together, our findings identified mir-34 miRNAs as novel p53 targets that play an essential role in restraining somatic reprogramming.

Understanding how oncogenic transformation sensitizes cells to apoptosis may provide a strategy to kill tumor cells selectively. We previously developed a cell-free system that recapitulates oncogene dependent apoptosis as ref lected by activation of caspases, the core of the apoptotic machinery. Here, we show that this activation requires a previously identified apoptosis-promoting complex consisting of caspase-9, APAF-1, and cytochrome c. As predicted by the in vitro system, preventing caspase-9 activation blocked druginduced apoptosis in cells sensitized by E1A, an adenoviral oncogene. Oncogenes, such as E1A, appear to facilitate caspase-9 activation by several mechanisms, including the control of cytochrome c release from the mitochondria.

Summary: We report on a major new version of the RMAP software for mapping reads from short-read sequencing technology. General improvements to accuracy and space requirements are included, along with novel functionality. Included in the RMAP software package are tools for mapping paired-end reads, mapping using more sophisticated use of quality scores, collecting ambiguous mapping locations and mapping bisulfite-treated reads.Availability: The applications described in this note are available for download at http://www.cmb.usc.edu/people/andrewds/rmap and are distributed as Open Source software under the GPLv3.0. The software has been tested on Linux and OS X platforms.Contact: andrewds@usc.edu; mzhang@cshl.eduThe RMAP algorithm was introduced by (Smith et al., 2008) as one of the earliest available programs for mapping reads from the Illumina second-generation sequencing technology. One important contribution of RMAP was to incorporate the use of quality scores directly into the mapping process: read positions with too low a quality score were not considered while mapping, and that quality score cutoff could be adjusted by the user. Subsequently, numerous mapping algorithm have appeared (Langmead et al., 2009; Li,H. et al., 2008; Li,R. et al., 2008; Lin et al., 2008; Schatz, 2009; Yanovsky et al., 2008), with improvements in both efficiency and breadth of functionality (e.g. ability to map paired-end reads; integrated SNP calling). Investigators requiring solutions to mapping problems now have many options. As new applications of short-read sequencing emerge, many variations on the analysis task of read mapping emerge. Diversity in performance characteristics of existing mapping tools becomes potentially valuable.We report the first major update to RMAP. The basic algorithmic framework in RMAP is still to preprocess reads and scan the genome, but several modifications have been made and much additional functionality has been included. Importantly, RMAP has a memory footprint that depends on the number of reads being mapped. This feature allows RMAP to be used effectively in cluster environments with commodity nodes, because partitioning the reads allows natural parallelizations with linear reduction in memory requirements per processor core used.Included in this release of the RMAP software package is functionality for mapping paired-end reads, making more sophisticated use of quality scores, collecting mapping locations for ambiguously mapping reads and mapping bisulfite-treated reads.

The bcr-abl chimeric oncoprotein exhibits deregulated protein tyrosine kinase activity and is implicated in the pathogenesis of Philadelphia chromosome (Ph)-positive human leukemias, such as chronic myelogenous leukemia (CML). Recently we have shown that the levels of the protein tyrosine phosphatase PTP1B are enhanced in p210 bcr-ablexpressing cell lines. Furthermore, PTP1B recognizes p210 bcr-abl as a substrate, disrupts the formation of a p210 bcr-abl͞Grb2 complex, and inhibits signaling events initiated by this oncoprotein PTK. In this report, we have examined whether PTP1B effects transformation induced by p210 bcrabl. We demonstrate that expression of either wild-type PTP1B or the substrate-trapping mutant form of the enzyme (PTP1B-D181A) in p210 bcr-abl-transformed Rat-1 fibroblasts diminished the ability of these cells to form colonies in soft agar, to grow in reduced serum, and to form tumors in nude mice. In contrast, TCPTP, the closest relative of PTP1B, did not effect p210 bcr-abl-induced transformation. Furthermore, neither PTP1B nor TCPTP inhibited transformation induced by v-Abl. In addition, overexpression of PTP1B or treatment with CGP57148, a small molecule inhibitor of p210 bcr-abl, induced erythroid differentiation of K562 cells, a CML cell line derived from a patient in blast crisis. These data suggest that PTP1B is a selective, endogenous inhibitor of p210 bcr-abl and is likely to be important in the pathogenesis of CML.

Near the end of the Pleistocene epoch, populations of the woolly mammoth (Mammuthus primigenius) were distributed across parts of three continents, from western Europe and northern Asia through Beringia to the Atlantic seaboard of North America. Nonetheless, questions about the connectivity and temporal continuity of mammoth populations and species remain unanswered. We use a combination of targeted enrichment and high-throughput sequencing to assemble and interpret a data set of 143 mammoth mitochondrial genomes, sampled from fossils recovered from across their Holarctic range. Our dataset includes 54 previously unpublished mitochondrial genomes and significantly increases the coverage of the Eurasian range of the species. The resulting global phylogeny confirms that the Late Pleistocene mammoth population comprised three distinct mitochondrial lineages that began to diverge ~1.0–2.0 million years ago (Ma). We also find that mammoth mitochondrial lineages were strongly geographically partitioned throughout the Pleistocene. In combination, our genetic results and the pattern of morphological variation in time and space suggest that male-mediated gene flow, rather than large-scale dispersals, was important in the Pleistocene evolutionary history of mammoths.

IntroductionOur understanding of the role of micro-RNAs (miRNAs) in processes such as development and differentiation has increased dramatically over the past few years. The most current version of the miRNA reference database miRBase V16.0 describes 672 Mus musculus and 1048 Homo sapiens miRNAs, but predictions of the total number of miRNAs for these species range up to the thousands, 1,2 suggesting that there are more miRNAs to be discovered. Indeed, with the advent of next-generation sequencing technology studies have emerged describing tens to hundreds of new miRNA candidates. [3][4][5][6][7] Overall, these studies suggest that the known miRNAs described to date predominantly represent the most abundant, globally expressed and evolutionary conserved miRNAs. Those miRNAs still to be discovered most probably are those expressed at lower levels, more cell type specific, or species specific. Validation of new miRNAs involves identification of rodent/human equivalents (unless they are species specific), structural predications, and the presence of the miRNA* sequence (which is the lesser abundant single-stranded small RNA sequence generated by Dicer processing of the pre-miRNA hairpin). 5 Studies have shown that miRNAs play important roles in the immune system and in its development. 8 Recently, deep sequencing of small RNAs isolated from 27 immune tissues, 6 provided a partial survey of the miRNAs expressed in various cell types and stages of immune development. Lineage-specific knockout of proteins involved in miRNA generation, such as Dicer, halt immune cell development. [9][10][11] Further studies in which specific miRNAs have been eliminated further support this view. [12][13][14][15][16][17][18] B cells represent perhaps the best-characterized developmental lineage in the immune system, 19,20 and the role of miRNAs in their development has been established. Deletion of Dicer early in B-cell development leads to a block at the pro to pre-B-cell transition. 10 miR-15a and miR-16-1 regulate several mRNAs involved in apoptosis or cell cycle progression (eg, Mcl1, Bcl2, Ets1, and Jun). 21 In human chronic lymphocytic leukemia chromosomal region 13q14 containing miR-15a and miR-16-1 is often deleted, 21 and deletion of this region in mice in vivo causes a B-cell autonomous lymphoproliferative disorder. 22 Overexpression of miR-155 from the VH promoter-Ig heavy chain E enhancer leads to pre-B-cell proliferation and lymphoma in mice, 23 and it has been reported that this miRNA is important for regulation of the germinal center response. 14 The oncomir-1/miR-17-92 cluster controls Bim expression 15,18 and miR-150 represses c-Myb during B-cell development, 17 both proteins playing pivotal roles in early B-cell development. Finally, miR-34a inhibits the transition of pro-B cells into pre-B cells by inhibiting Foxp1, a known B-cell oncogene. 24 Thus, miRNAs are strongly implicated in the B-cell lineage.To understand the contribution of the expression of individual miRNAs (or miRNA sets) to normal B-cell development an...