Recent Scientific Papers

For more papers, visit a faculty member's page from the listing on Whitehead Faculty and access the PubMed link.


e-Cell analysis reveals that noncoding RNAs dontribute to dlonal heterogeneity by modulating transcription factor recruitment.
Mol Cell. 2012 Feb 24;45(4):470-82.
Bumgarner, S.L.*, Neuert, G., Voight, B.F., Symbor-Nagrabska, A.*, Grisafi, P.*, van Oudenaarden, A., and Fink, G.R.*
Mechanisms through which long intergenic noncoding RNAs (ncRNAs) exert regulatory effects on eukaryotic biological processes remain largely elusive. Most studies of these phenomena rely on methods that measure average behaviors in cell populations, lacking resolution to observe the effects of ncRNA transcription on gene expression in a single cell. Here, we combine quantitative single-molecule RNA FISH experiments with yeast genetics and computational modeling to gain mechanistic insights into the regulation of the Saccharomyces cerevisiae protein-coding gene FLO11 by two intergenic ncRNAs, ICR1 and PWR1. Direct detection of FLO11 mRNA and these ncRNAs in thousands of individual cells revealed alternative expression states and provides evidence that ICR1 and PWR1 contribute to FLO11's variegated transcription, resulting in Flo11-dependent phenotypic heterogeneity in clonal cell populations by modulating recruitment of key transcription factors to the FLO11 promoter.

Strict evolutionary conservation followed rapid gene loss on human and rhesus Y chromosomes.
Nature. 2012 Feb 22;483(7387):82-6.
Hughes, J.F.*, Skaletsky, H.*, Brown, L.G.*, Pyntikova, T.*, Graves, T., Fulton, R.S., Dugan, S., Ding, Y., Buhay, C.J., Kremitzki, C., Cho, T.J.*, Koutseva, N.*, Rozen, S., Muzny, D.M., Warren, W.C., Gibbs, R.A., Wilson, R.K., and Page, D.C.*
The human X and Y chromosomes evolved from an ordinary pair of autosomes during the past 200-300 million years. The human MSY (male-specific region of Y chromosome) retains only three percent of the ancestral autosomes' genes owing to genetic decay. This evolutionary decay was driven by a series of five 'stratification' events. Each event suppressed X-Y crossing over within a chromosome segment or 'stratum', incorporated that segment into the MSY and subjected its genes to the erosive forces that attend the absence of crossing over. The last of these events occurred 30 million years ago, 5 million years before the human and Old World monkey lineages diverged. Although speculation abounds regarding ongoing decay and looming extinction of the human Y chromosome, remarkably little is known about how many MSY genes were lost in the human lineage in the 25 million years that have followed its separation from the Old World monkey lineage. To investigate this question, we sequenced the MSY of the rhesus macaque, an Old World monkey, and compared it to the human MSY. We discovered that during the last 25 million years MSY gene loss in the human lineage was limited to the youngest stratum (stratum 5), which comprises three percent of the human MSY. In the older strata, which collectively comprise the bulk of the human MSY, gene loss evidently ceased more than 25 million years ago. Likewise, the rhesus MSY has not lost any older genes (from strata 1-4) during the past 25 million years, despite its major structural differences to the human MSY. The rhesus MSY is simpler, with few amplified gene families or palindromes that might enable intrachromosomal recombination and repair. We present an empirical reconstruction of human MSY evolution in which each stratum transitioned from rapid, exponential loss of ancestral genes to strict conservation through purifying selection.

Protein ligation in living cells using sortase.
Traffic. 2012 Feb 20.
Strijbis, K.*, Spooner, E.*, and Ploegh, H.L.*
Sortagging is a versatile method for site-specific modification of proteins as applied to a variety of in vitro reactions. Here we explore possibilities of adapting the sortase method for use in living cells. For intracellular sortagging we employ the Ca(2+) -independent SrtA from Streptococcus pyogenes. Substrate proteins were equipped with the C-terminal sortase-recognition motif (LPXTG); as nucleophiles we used proteins with a N-terminal (oligo)glycine. We show that sortase-dependent protein ligation can be achieved in Saccharomyces cerevisiae and in mammalian HEK293T cells, both in the cytosol and in the lumen of the endoplasmic reticulum (ER). ER-luminal sortagging enables secretion of the reaction products, among which circular polypeptides. Protein ligation of substrate and nucleophile occurs within 30 minutes after translation. The versatility of the method is shown by protein ligation of multiple substrates with GFP-based nucleophiles in different intracellular compartments.

Prions are a common mechanism for phenotypic inheritance in wild yeasts.
Nature. 2012 Feb 15;482(7385):363-8.
Halfmann, R.*, Jarosz, D.F.*, Jones, S.K.*, Chang, A.*, Lancaster, A.K.*, and Lindquist, S.*
The self-templating conformations of yeast prion proteins act as epigenetic elements of inheritance. Yeast prions might provide a mechanism for generating heritable phenotypic diversity that promotes survival in fluctuating environments and the evolution of new traits. However, this hypothesis is highly controversial. Prions that create new traits have not been found in wild strains, leading to the perception that they are rare 'diseases' of laboratory cultivation. Here we biochemically test approximately 700 wild strains of Saccharomyces for [PSI(+)] or [MOT3(+)], and find these prions in many. They conferred diverse phenotypes that were frequently beneficial under selective conditions. Simple meiotic re-assortment of the variation harboured within a strain readily fixed one such trait, making it robust and prion-independent. Finally, we genetically screened for unknown prion elements. Fully one-third of wild strains harboured them. These, too, created diverse, often beneficial phenotypes. Thus, prions broadly govern heritable traits in nature, in a manner that could profoundly expand adaptive opportunities.

Chromosome- and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation.
Nat Cell Biol. 2012 Feb 12;14(3):311-7.
Kiyomitsu, T.*, and Cheeseman, I.M.*
Mitotic spindle positioning by cortical pulling forces defines the cell division axis and location, which is critical for proper cell division and development. Although recent work has identified developmental and extrinsic cues that regulate spindle orientation, the contribution of intrinsic signals to spindle positioning and orientation remains unclear. Here, we demonstrate that cortical force generation in human cells is controlled by distinct spindle-pole- and chromosome-derived signals that regulate cytoplasmic dynein localization. First, dynein exhibits a dynamic asymmetric cortical localization that is negatively regulated by spindle-pole proximity, resulting in spindle oscillations to centre the spindle within the cell. We find that this signal comprises the spindle-pole-localized polo-like kinase (Plk1), which regulates dynein localization by controlling the interaction between dynein-dynactin and its upstream cortical targeting factors NuMA and LGN. Second, a chromosome-derived RanGTP gradient restricts the localization of NuMA-LGN to the lateral cell cortex to define and maintain the spindle orientation axis. RanGTP acts in part through the nuclear localization sequence of NuMA to locally alter the ability of NuMA-LGN to associate with the cell cortex in the vicinity of chromosomes. We propose that these chromosome- and spindle-pole-derived gradients generate an intrinsic code to control spindle position and orientation.

Accessory molecules for Toll-like receptors and their function.
Nat Rev Immunol. 2012 Feb 3;12(3):168-79.
Lee, C.C.*, Avalos, A.M.*, and Ploegh, H.L.*
Toll-like receptors (TLRs) are essential components of the innate immune system. Accessory proteins are required for the biosynthesis and activation of TLRs. Here, we summarize recent findings on TLR accessory proteins that are required for cell-surface and endosomal TLR function, and we classify these proteins based on their function as ligand-recognition and delivery cofactors, chaperones and trafficking proteins. Because of their essential roles in TLR function, targeting of such accessory proteins may benefit strategies aimed at manipulating TLR activation for therapeutic applications.

Enhancer decommissioning by LSD1 during embryonic stem cell differentiation.
Nature. 2012 Feb 1;482(7384):221-5.
Whyte, W.A.*, Bilodeau, S.*, Orlando, D.A.*, Hoke, H.A.*, Frampton, G.M.*, Foster, C.T., Cowley, S.M., and Young, R.A.*
Transcription factors and chromatin modifiers are important in the programming and reprogramming of cellular states during development. Transcription factors bind to enhancer elements and recruit coactivators and chromatin-modifying enzymes to facilitate transcription initiation. During differentiation a subset of these enhancers must be silenced, but the mechanisms underlying enhancer silencing are poorly understood. Here we show that the histone demethylase lysine-specific demethylase 1 (LSD1; ref. 5), which demethylates histone H3 on Lys 4 or Lys 9 (H3K4/K9), is essential in decommissioning enhancers during the differentiation of mouse embryonic stem cells (ESCs). LSD1 occupies enhancers of active genes that are critical for control of the state of ESCs. However, LSD1 is not essential for the maintenance of ESC identity. Instead, ESCs lacking LSD1 activity fail to differentiate fully, and ESC-specific enhancers fail to undergo the histone demethylation events associated with differentiation. At active enhancers, LSD1 is a component of the NuRD (nucleosome remodelling and histone deacetylase) complex, which contains additional subunits that are necessary for ESC differentiation. We propose that the LSD1-NuRD complex decommissions enhancers of the pluripotency program during differentiation, which is essential for the complete shutdown of the ESC gene expression program and the transition to new cell states.

Maternal and paternal genomes contribute equally to the transcriptome of early plant embryos.
Nature. 2012 Jan 22;482(7383):94-7.
Nodine, M.D.*, and Bartel, D.P.*
In animals, maternal gene products deposited into eggs regulate embryonic development before activation of the zygotic genome. In plants, an analogous period of prolonged maternal control over embryogenesis is thought to occur based on some gene-expression studies. However, other gene-expression studies and genetic analyses show that some transcripts must derive from the early zygotic genome, implying that the prevailing model does not fully explain the nature of zygotic genome activation in plants. To determine the maternal, paternal and zygotic contributions to the early embryonic transcriptome, we sequenced the transcripts of hybrid embryos from crosses between two polymorphic inbred lines of Arabidopsis thaliana and used single-nucleotide polymorphisms diagnostic of each parental line to quantify parental contributions. Although some transcripts seemed to be either inherited from primarily one parent or transcribed from imprinted loci, the vast majority of transcripts were produced in near-equal amounts from both maternal and paternal alleles, even during the initial stages of embryogenesis. Results of reporter experiments and analyses of transcripts from genes that are not expressed in sperm and egg indicate early and widespread zygotic transcription. Thus, in contrast to early animal embryogenesis, early plant embryogenesis is mostly under zygotic control.  

Functional Association of Gdown1 with RNA Polymerase II Poised on Human Genes.
Mol Cell. 2012 Jan 13;45(1):38-50.
Cheng, B., Li, T.D., Rahl, P.B., Adamson, T.E., Loudas, N.B., Guo, J.N., Varzavand, K., Cooper, J.J., Hu, X.P., Gnatt, A., Young, R.A.*, and Price, D.H.
Most human genes are loaded with promoter-proximally paused RNA polymerase II (Pol II) molecules that are poised for release into productive elongation by P-TEFb. We present evidence that Gdown1, the product of the POLR2M gene that renders Pot II responsive to Mediator, is involved in Pol II elongation control. During in vitro transcription, Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. Supporting these mechanistic insights, ChIP-Seq demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Our results establish that Gdown1 stabilizes poised polymerases while maintaining their responsiveness to P-TEFb and suggest that Mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function.

Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells.
Proc Natl Acad Sci U S A. 2012 Jan 10;109(2):535-40.
Giraud, M., Yoshida, H., Abramson, J., Rahl, P.B.*, Young, R.A.*, Mathis, D., and Benoist, C.
Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50-100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II-rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases.

*Whitehead Institute for Biomedical Research

Last updated March 5, 2012.