The genome sequence of Molossusnigricans (Chiroptera, Molossidae; Miller, 1902).

We present a genome assembly from an individual male Molossus nigricans (Chordata; Mammalia; Chiroptera; Molossidae). The genome sequence is 2.41 gigabases in span. The majority of the assembly is scaffolded into 24 chromosomal pseudomolecules, with the X sex chromosome assembled.

Bat pluripotent stem cells reveal unusual entanglement between host and viruses.

Bats are distinctive among mammals due to their ability to fly, use laryngeal echolocation, and tolerate viruses. However, there are currently no reliable cellular models for studying bat biology or their response to viral infections. Here, we created induced pluripotent stem cells (iPSCs) from two species of bats: the wild greater horseshoe bat (Rhinolophus ferrumequinum) and the greater mouse-eared bat (Myotis myotis). The iPSCs from both bat species showed similar characteristics and had a gene expression profile resembling that of cells attacked by viruses. They also had a high number of endogenous viral sequences, particularly retroviruses. These results suggest that bats have evolved mechanisms to tolerate a large load of viral sequences and may have a more intertwined relationship with viruses than previously thought. Further study of bat iPSCs and their differentiated progeny will provide insights into bat biology, virus host relationships, and the molecular basis of bats' special traits.

Multisite Evaluation of Next-Generation Methods for Small RNA Quantification.

Small RNAs (smRNAs) are important regulators of many biologic processes and are now most frequently characterized using Illumina sequencing. However, although standard RNA sequencing library preparation has become routine in most sequencing facilities, smRNA sequencing library preparation has historically been challenging because of high input requirements, laborious protocols involving gel purifications, inability to automate, and a lack of benchmarking standards. Additionally, studies have suggested that many of these methods are nonlinear and do not accurately reflect the amounts of smRNAs in vivo. Recently, a number of new kits have become available that permit lower input amounts and less laborious, gel-free protocol options. Several of these new kits claim to reduce RNA ligase-dependent sequence bias through novel adapter modifications and to lessen adapter-dimer contamination in the resulting libraries. With the increasing number of smRNA kits available, understanding the relative strengths of each method is crucial for appropriate experimental design. In this study, we systematically compared 9 commercially available smRNA library preparation kits as well as NanoString probe hybridization across multiple study sites. Although several of the new methodologies do reduce the amount of artificially over- and underrepresented microRNAs (miRNAs), we observed that none of the methods was able to remove all of the bias in the library preparation. Identical samples prepared with different methods show highly varied levels of different miRNAs. Even so, many methods excelled in ease of use, lower input requirement, fraction of usable reads, and reproducibility across sites. These differences may help users select the most appropriate methods for their specific question of interest.

Genome-wide analyses of Nkx2-1 binding to transcriptional target genes uncover novel regulatory patterns conserved in lung development and tumors.

The homeodomain transcription factor Nkx2-1 is essential for normal lung development and homeostasis. In lung tumors, it is considered a lineage survival oncogene and prognostic factor depending on its expression levels. The target genes directly bound by Nkx2-1, that could be the primary effectors of its functions in the different cellular contexts where it is expressed, are mostly unknown. In embryonic day 11.5 (E11.5) mouse lung, epithelial cells expressing Nkx2-1 are predominantly expanding, and in E19.5 prenatal lungs, Nkx2-1-expressing cells are predominantly differentiating in preparation for birth. To evaluate Nkx2-1 regulated networks in these two cell contexts, we analyzed genome-wide binding of Nkx2-1 to DNA regulatory regions by chromatin immunoprecipitation followed by tiling array analysis, and intersected these data to expression data sets. We further determined expression patterns of Nkx2-1 developmental target genes in human lung tumors and correlated their expression levels to that of endogenous NKX2-1. In these studies we uncovered differential Nkx2-1 regulated networks in early and late lung development, and a direct function of Nkx2-1 in regulation of the cell cycle by controlling the expression of proliferation-related genes. New targets, validated in Nkx2-1 shRNA transduced cell lines, include E2f3, Cyclin B1, Cyclin B2, and c-Met. Expression levels of Nkx2-1 direct target genes identified in mouse development significantly correlate or anti-correlate to the levels of endogenous NKX2-1 in a dosage-dependent manner in multiple human lung tumor expression data sets, supporting alternative roles for Nkx2-1 as a transcriptional activator or repressor, and direct regulator of cell cycle progression in development and tumors.

Connecting microRNA genes to the core transcriptional regulatory circuitry of embryonic stem cells.

MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity.

Aberrant chromatin at genes encoding stem cell regulators in human mixed-lineage leukemia.

Mixed-lineage leukemia (MLL) fusion proteins are potent inducers of leukemia, but how these proteins generate aberrant gene expression programs is poorly understood. Here we show that the MLL-AF4 fusion protein occupies developmental regulatory genes important for hematopoietic stem cell identity and self-renewal in human leukemia cells. These MLL-AF4-bound regions have grossly altered chromatin structure, with histone modifications catalyzed by trithorax group proteins and DOT1 extending across large domains. Our results define direct targets of the MLL fusion protein, reveal the global role of epigenetic misregulation in leukemia, and identify new targets for therapeutic intervention in cancer.

Control of developmental regulators by Polycomb in human embryonic stem cells.

Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.

Program-specific distribution of a transcription factor dependent on partner transcription factor and MAPK signaling.

Specialized gene expression programs are induced by signaling pathways that act on transcription factors. Whether these transcription factors can function in multiple developmental programs through a global switch in promoter selection is not known. We have used genome-wide location analysis to show that the yeast Ste12 transcription factor, which regulates mating and filamentous growth, is bound to distinct program-specific target genes dependent on the developmental condition. This condition-dependent distribution of Ste12 requires concurrent binding of the transcription factor Tec1 during filamentation and is differentially regulated by the MAP kinases Fus3 and Kss1. Program-specific distribution across the genome may be a general mechanism by which transcription factors regulate distinct gene expression programs in response to signaling.

Transcriptional regulatory networks in Saccharomyces cerevisiae.

We have determined how most of the transcriptional regulators encoded in the eukaryote Saccharomyces cerevisiae associate with genes across the genome in living cells. Just as maps of metabolic networks describe the potential pathways that may be used by a cell to accomplish metabolic processes, this network of regulator-gene interactions describes potential pathways yeast cells can use to regulate global gene expression programs. We use this information to identify network motifs, the simplest units of network architecture, and demonstrate that an automated process can use motifs to assemble a transcriptional regulatory network structure. Our results reveal that eukaryotic cellular functions are highly connected through networks of transcriptional regulators that regulate other transcriptional regulators.

Two transcripts, differing at their 3' ends, are produced from the Candida albicans SEC14 gene.

A search for Candida albicans mutants defective in filamentous growth led to the isolation of a mutant strain with an insertion mutation in the SEC14 gene. SEC14 encodes the phosphatidylinositol/phosphatidylcholine transfer protein, an essential protein in the yeast Saccharomyces cerevisiae. In the dimorphic yeast Yarrowia lipolytica, SEC14 is needed for growth only in the hyphal form and is not required for growth in the yeast form. However, unlike Y. lipolytica SEC14, C. albicans SEC14 is probably essential for growth. Northern blot analysis and PCR amplification of transcripts produced from the SEC14 gene demonstrated that two transcripts differing at their 3' ends were produced. The two transcripts may regulate the activity of SEC14 so that Sec14p can perform two functions in C. albicans. One function may be an essential function analogous to the function of Sec14p in S. cerevisiae and the second function may be important during filamentous growth, analogous to the function of Sec14p in Y. lipolytica.