Scientific Papers

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

Efficient reprogramming of human fibroblasts and blood-derived endothelial progenitor cells using non-modified RNA for reprogramming and immune evasion.

Hum Gene Ther. 2015 Oct 1.

Poleganov, M.A., Eminli, S., Beissert, T., Herz, S., Moon, J.I., Goldmann, J.*, Beyer, A., Heck, R., Burkhart, I., Barea Roldan, D., Türeci, Ö., Yi, K., Hamilton, B., and Sahin, U.

mRNA reprogramming results in the generation of genetically stable induced pluripotent stem (iPS) cells while avoiding the risks of genomic integration. Previously published mRNA reprogramming protocols have proven to be inconsistent, time consuming, and mainly restricted to fibroblasts thereby demonstrating the need for a simple, but reproducible protocol applicable to various cell types. So far there have been no published reports using mRNA to reprogram any cell type derived from human blood. Non-modified synthetic mRNAs are immunogenic and activate cellular defense mechanisms, which can lead to cell death and inhibit mRNA translation upon repetitive transfection. Hence to overcome RNA related toxicity we combined non-modified reprogramming mRNAs [OCT4, SOX2, KLF4, cMYC, NANOG and LIN28 (OSKMNL)] with immune evasion mRNAs [E3, K3 and B18R (EKB)] from Vaccinia virus (VACV). Additionally we included mature, double stranded microRNAs (miRNAs) from the 302/367 cluster, which are known to enhance the reprogramming process, to develop a robust reprogramming protocol for the generation of stable iPS cell lines from both human fibroblasts and human blood-outgrowth endothelial progenitor cells (EPCs). Our novel combination of RNAs enables the cell to tolerate repetitive transfections for the generation of stable iPS cell colonies from human fibroblasts within 11 days while requiring only four transfections. Moreover, our method resulted in the first known mRNA-vectored reprogramming of human blood-derived EPCs within 10 days while requiring only eight daily transfections.


CDK7-dependent transcriptional addiction in triple-negative breast cancer.

Cell. 2015 Sep 24;163(1):174-86.

Wang, Y., Zhang, T., Kwiatkowski, N.*, Abraham, B.J.*, Lee, T.I.*, Xie, S., Yuzugullu, H., Von, T., Li, H., Lin, Z., Stover, D.G., Lim, E., Wang, Z.C., Iglehart, J.D,, Young, R.A.*, Gray, N.S., and Zhao, J.J.

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer that exhibits extremely high levels of genetic complexity and yet a relatively uniform transcriptional program. We postulate that TNBC might be highly dependent on uninterrupted transcription of a key set of genes within this gene expression program and might therefore be exceptionally sensitive to inhibitors of transcription. Utilizing kinase inhibitors and CRISPR/Cas9-mediated gene editing, we show here that triple-negative but not hormone receptor-positive breast cancer cells are exceptionally dependent on CDK7, a transcriptional cyclin-dependent kinase. TNBC cells are unique in their dependence on this transcriptional CDK and suffer apoptotic cell death upon CDK7 inhibition. An "Achilles cluster" of TNBC-specific genes is especially sensitive to CDK7 inhibition and frequently associated with super-enhancers. We conclude that CDK7 mediates transcriptional addiction to a vital cluster of genes in TNBC and CDK7 inhibition may be a useful therapy for this challenging cancer.


Tracing dynamic changes of DNA methylation at single-cell resolution.

Cell. 2015 Sep 24;163(1):218-29.

Stelzer, Y.,* Shivalila, C.S.*, Soldner, F.*, Markoulaki, S.*, and Jaenisch, R.*

Mammalian DNA methylation plays an essential role in development. To date, only snapshots of different mouse and human cell types have been generated, providing a static view on DNA methylation. To enable monitoring of methylation status as it changes over time, we establish a reporter of genomic methylation (RGM) that relies on a minimal imprinted gene promoter driving a fluorescent protein. We show that insertion of RGM proximal to promoter-associated CpG islands reports the gain or loss of DNA methylation. We further utilized RGM to report endogenous methylation dynamics of non-coding regulatory elements, such as the pluripotency-specific super enhancers of Sox2 and miR290. Loci-specific DNA methylation changes and their correlation with transcription were visualized during cell-state transition following differentiation of mouse embryonic stem cells and during reprogramming of somatic cells to pluripotency. RGM will allow the investigation of dynamic methylation changes during development and disease at single-cell resolution.


Hallmarks of pluripotency.

Nature. 2015 Sep 24;525(7570):E6-9.

De Los Angeles, A., Ferrari, F., Xi, R., Fujiwara, Y., Benvenisty, N., Deng, H., Hochedlinger, K., Jaenisch, R.*, Lee, S., Leitch, H.G., Lensch, M.W., Lujan, E., Pei, D., Rossant, J., Wernig, M., Park, P.J., and Daley, G.Q.

Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.


Failure to replicate the STAP cell phenomenon.

Nature. 2015 Sep 24;525(7570):E6-9.

De Los Angeles, A., Ferrari, F., Fujiwara, Y., Mathieu, R., Lee, S., Tu, H.C., Ross, S., Chou, S., Nguyen, M., Wu, Z., Theunissen, T.W.*, Powell, B.E.*, Imsoonthornruksa, S.*, Chen, J., Borkent, M., Krupalnik, V., Lujan, E., Wernig, M., Hanna, J.H., Hochedlinger, K., Pei, D., Jaenisch, R.*, Deng, H., Orkin, S.H., Park, P.J., and Daley, G.Q.


The menu of features that define primary microRNAs and enable de novo design of nicroRNA genes.

Mol Cell. 2015 Sep 23. pii: S1097-2765(15)00661-9.

Fang, W.*, and Bartel, D.P.*

MicroRNAs (miRNAs) are small regulatory RNAs processed from stem-loop regions of primary transcripts (pri-miRNAs), with the choice of stem loops for initial processing largely determining what becomes a miRNA. To identify sequence and structural features influencing this choice, we determined cleavage efficiencies of >50,000 variants of three human pri-miRNAs, focusing on the regions intractable to previous high-throughput analyses. Our analyses revealed a mismatched motif in the basal stem region, a preference for maintaining or improving base pairing throughout the remainder of the stem, and a narrow stem-length preference of 35 +/- 1 base pairs. Incorporating these features with previously identified features, including three primary-sequence motifs, yielded a unifying model defining mammalian pri-miRNAs in which motifs help orient processing and increase efficiency, with the presence of more motifs compensating for structural defects. This model enables generation of artificial pri-miRNAs, designed de novo, without reference to any natural sequence yet processed more efficiently than natural pri-miRNAs.


Clinical heterogeneity associated with KCNA1 mutations include cataplexy and nonataxic presentations.

Neurogenetics. 2015 Sep 22.

Brownstein, C.A., Beggs, A.H., Rodan, L., Shi, J.*, Towne, M.C., Pelletier, R., Cao, S., Rosenberg, P.A., Urion, D.K., Picker, J., Tan, W.H., and Agrawal, P.B.

Mutations in the KCNA1 gene are known to cause episodic ataxia/myokymia syndrome type 1 (EA1). Here, we describe two families with unique presentations who were enrolled in an IRB-approved study, extensively phenotyped, and whole exome sequencing (WES) performed. Family 1 had a diagnosis of isolated cataplexy triggered by sudden physical exertion in multiple affected individuals with heterogeneous neurological findings. All enrolled affected members carried a KCNA1 c.941T>C (p.I314T) mutation. Family 2 had an 8-year-old patient with muscle spasms with rigidity for whom WES revealed a previously reported heterozygous missense mutation in KCNA1 c.677C>G (p.T226R), confirming the diagnosis of EA1 without ataxia. WES identified variants in KCNA1 that explain both phenotypes expanding the phenotypic spectrum of diseases associated with mutations of this gene. KCNA1 mutations should be considered in patients of all ages with episodic neurological phenotypes, even when ataxia is not present. This is an example of the power of genomic approaches to identify pathogenic mutations in unsuspected genes responsible for heterogeneous diseases.


A gene regulatory program for meiotic prophase in the fetal ovary.

PLoS Genet. 2015 Sep 17;11(9):e1005531.

Soh, Y.Q.*, Junker, J.P., Gill, M.E.*, Mueller, J.L.*, van Oudenaarden, A., and Page, D.C.*

The chromosomal program of meiotic prophase, comprising events such as laying down of meiotic cohesins, synapsis between homologs, and homologous recombination, must be preceded and enabled by the regulated induction of meiotic prophase genes. This gene regulatory program is poorly understood, particularly in organisms with a segregated germline. We characterized the gene regulatory program of meiotic prophase as it occurs in the mouse fetal ovary. By profiling gene expression in the mouse fetal ovary in mutants with whole tissue and single-cell techniques, we identified 104 genes expressed specifically in pre-meiotic to pachytene germ cells. We characterized the regulation of these genes by 1) retinoic acid (RA), which induces meiosis, 2) Dazl, which is required for germ cell competence to respond to RA, and 3) Stra8, a downstream target of RA required for the chromosomal program of meiotic prophase. Initial induction of practically all identified meiotic prophase genes requires Dazl. In the presence of Dazl, RA induces at least two pathways: one Stra8-independent, and one Stra8-dependent. Genes vary in their induction by Stra8, spanning fully Stra8-independent, partially Stra8-independent, and fully Stra8-dependent. Thus, Stra8 regulates the entirety of the chromosomal program but plays a more nuanced role in governing the gene expression program. We propose that Stra8-independent gene expression enables the stockpiling of selected meiotic structural proteins prior to the commencement of the chromosomal program. Unexpectedly, we discovered that Stra8 is required for prompt down-regulation of itself and Rec8. Germ cells that have expressed and down-regulated Stra8 are refractory to further Stra8 expression. Negative feedback of Stra8, and subsequent resistance to further Stra8 expression, may ensure a single, restricted pulse of Stra8 expression. Collectively, our findings reveal a gene regulatory logic by which germ cells prepare for the chromosomal program of meiotic prophase, and ensure that it is induced only once.


The genome of the vervet (Chlorocebus aethiops sabaeus).

Genome Res. 2015 Sep 16.

Warren, W.C., Jasinska, A.J., Garcia-Perez, R., Svardal, H., Tomlinson, C., Rocchi, M., Archidiacono, N., Capozzi, O., Minx, P., Montague, M.J., Kyung, K., Hillier, L.W., Kremitzki, M., Graves, T., Chiang, C., Hughes, J.*, Tran, N., Wang, Y., Ramensky, V., Choi, O.W., Jung, Y.J., Schmitt, C.A., Juretic, N., Wasserscheid, J., Turner, T.R., Wiseman, R.W., Tuscher, J.J., Karl, J.A., Schmitz, J.E., Zahn, R., O'Connor, DH., Redmond, E., Nisbett, A., Jacquelin, B., Müller-Trutwin, M.C., Brenchley, J.M., Dione, M., Antonio, M., Schroth, G.P., Kaplan, J.R., Jorgensen, M.J., Thomas, G.W., Hahn, M.W., Raney, B., Aken, B., Schmitz, J., Churakov, G., Noll, A., Stanyon, R., Webb, D., Thibaud-Nissen, F., Nordborg, M., Marques-Bonet, T., Dewar, K., Weinstock, G.M., Wilson, R.K., and Freimer, N.B.

We describe a genome reference of the African green monkey or vervet (Chlorocebus aethiops). This member of the Old World monkey (OWM) superfamily is uniquely valuable for genetic investigations of simian immunodeficiency virus (SIV), of which it is the most abundant natural host species, and of a wide range of health-related phenotypes assessed in Caribbean vervets (C. a. sabaeus), whose numbers have expanded dramatically since Europeans introduced small numbers of their ancestors from West Africa during the colonial era. We use the reference to characterize the genomic relationship between vervets and other primates, the intrageneric phylogeny of vervet subspecies, and genome-wide structural variations of a pedigreed C. a. sabaeus population. Through comparative analyses with human and rhesus macaque, we characterize at high resolution the unique chromosomal fission events that differentiate the vervets and their close relatives from most other catarrhine primates, in whom karyotype is highly conserved. We also provide a summary of transposable elements and contrast these with the rhesus macaque and human. Analysis of sequenced genomes representing each of the main vervet subspecies supports previously hypothesized relationships between these populations, which range across most of sub-Saharan Africa, while uncovering high levels of genetic diversity within each. Sequence-based analyses of major histocompatibility complex (MHC) polymorphisms reveal extremely low diversity in Caribbean C. a. sabaeus vervets, as compared to vervets from putatively ancestral West African regions. In the C. a. sabaeus research population we discover the first structural variations that are in some cases predicted to have a deleterious effect; future studies will determine the phenotypic impact of these variations.


Identification of an oncogenic RAB protein.

Science. 2015 Sep 3.

Wheeler, D.B., Zoncu, R., Root, D.E., Sabatini, D.M.*, and Sawyers, C.L.

In an shRNA screen for genes that affect AKT phosphorylation, we identified the RAB35 small GTPase-a protein previously implicated in endomembrane trafficking-as a regulator of the PI3K pathway. Depletion of RAB35 suppresses AKT phosphorylation in response to growth factors, whereas expression of a dominant active GTPase-deficient mutant of RAB35 constitutively activates the PI3K/AKT pathway. RAB35 functions downstream of growth factor receptors and upstream of PDK1 and mTORC2 and co-purifies with PI3K in immunoprecipitation assays. Two somatic RAB35 mutations found in human tumors generate alleles that constitutively activate PI3K/AKT signaling, suppress apoptosis, and transform cells in a PI3K-dependent manner. Furthermore, oncogenic RAB35 is sufficient to drive PDGFRalpha to LAMP2-positive endomembranes in the absence of ligand, suggesting there may be latent oncogenic potential in dysregulated endomembrane trafficking.


*Author affiliated with Whitehead Institute for Biomedical Research

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