Scientific Papers

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

Growth of human breast tissues from patient cells in 3D hydrogel scaffolds.

Breast Cancer Res. 2016 Mar 1;18(1):19.

Sokol, E.S.*, Miller, D.H.*, Breggia, A., Spencer, K.C., Arendt, L.M., and Gupta, P.B.*

BACKGROUND: Three-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells. METHODS: Primary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue growth was monitored over time using bright-field and fluorescence microscopy, and maturation was assessed using morphological metrics and by immunostaining for markers of stem cells and differentiated cell types. The hydrogel tissues were also studied by fabricating physical models from confocal images using a 3D printer. RESULTS: When seeded into these 3D hydrogels, primary human breast epithelial cells rapidly self-organized in the absence of stromal cells and within 2 weeks expanded to form mature mammary tissues. The mature tissues contained luminal, basal, and stem cells in the correct topological orientation and also exhibited the complex ductal and lobular morphologies observed in the human breast. The expanded tissues became hollow when treated with estrogen and progesterone, and with the further addition of prolactin produced lipid droplets, indicating that they were responding to hormones. Ductal branching was initiated by clusters of cells expressing putative mammary stem cell markers, which subsequently localized to the leading edges of the tissue outgrowths. Ductal elongation was preceded by leader cells that protruded from the tips of ducts and engaged with the extracellular matrix. CONCLUSIONS: These 3D hydrogel scaffolds support the growth of complex mammary tissues from primary patient-derived cells. We anticipate that this culture system will empower future studies of human mammary gland development and biology.

  

Improved ribosome-footprint and mRNA measurements provide insights into dynamics and regulation of yeast translation.

Cell Rep. 2016 Feb 23;14(7):1787-99.

Weinberg, D.E., Shah, P., Eichhorn, S.W.*, Hussmann, J.A., Plotkin, J.B., and Bartel, D.P.*

Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a ten-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5' UTRs. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast.

 

Visualizing antibody affinity maturation in germinal centers.

Science. 2016 Feb 18.

Tas, J.M.*, Mesin, L.*, Pasqual, G.*, Targ, S.*, Jacobsen, J.T.*, Mano, Y.M.*, Chen, C.S.*, Weill, J.C., Reynaud, C.A., Browne, E.P., Meyer-Hermann, M., and Victora, G.D.*

Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza.

 

Condensin and Hmo1 mediate a starvation-induced transcriptional position effect within the ribosomal DNA array.

Cell Rep. 2016 Feb 9;14(5):1010-7

Wang, D., Mansisidor, A., Prabhakar, G.*, and Hochwagen, A.

Repetitive DNA arrays are important structural features of eukaryotic genomes that are often heterochromatinized to suppress repeat instability. It is unclear, however, whether all repeats within an array are equally subject to heterochromatin formation and gene silencing. Here, we show that in starving Saccharomyces cerevisiae, silencing of reporter genes within the ribosomal DNA (rDNA) array is less pronounced in outer repeats compared with inner repeats. This position effect is linked to the starvation-induced contraction of the nucleolus. We show that the chromatin regulators condensin and Hmo1 redistribute within the rDNA upon starvation; that Hmo1, like condensin, is required for nucleolar contraction; and that the position effect partially depends on both proteins. Starvation-induced nucleolar contraction and differential desilencing of the outer rDNA repeats may provide a mechanism to activate rDNA-encoded RNAPII transcription units without causing general rDNA instability.

 

Models of human core transcriptional regulatory circuitries.

Genome Res. 2016 Feb 3.

Saint-Andre, V.*, Federation, A.J., Lin, C.Y., Abraham, B.J.*, Reddy, J.*, Lee, T.I.*, Bradner, J.E., and Young, R.A.*

A small set of core transcription factors (TFs) dominates control of the gene expression program in embryonic stem cells and other well-studied cellular models. These core TFs collectively regulate their own gene expression, thus forming an interconnected auto-regulatory loop that can be considered the core transcriptional regulatory circuitry (CRC) for that cell type. There is limited knowledge of core TFs, and thus models of core regulatory circuitry, for most cell types. We recently discovered that genes encoding known core TFs forming CRCs are driven by super-enhancers, which provides an opportunity to systematically predict CRCs in poorly studied cell types through super-enhancer mapping. Here, we use super-enhancer maps to generate CRC models for 75 human cell and tissue types. These core circuitry models should prove valuable for further investigating cell-type-specific transcriptional regulation in healthy and diseased cells.

 

Structurally defined alphaMHC-II nanobody-drug conjugates: A therapeutic and imaging system for B-cell lymphoma.

Medicine (Baltimore). 2016 Feb;95(5):e2588.

Fang, T.*, Duarte, J.N.*, Ling, J.*, Li, Z.*, Guzman, J.S.*, and Ploegh, H.L.*

Antibody-drug conjugates (ADCs) of defined structure hold great promise for cancer therapies, but further advances are constrained by the complex structures of full-sized antibodies. Camelid-derived single-domain antibody fragments (VHHs or nanobodies) offer a possible solution to this challenge by providing expedited target screening and validation through switching between imaging and therapeutic activities. We used a nanobody (VHH7) specific for murine MHC-II and rendered "sortase-ready" for the introduction of oligoglycine-modified cytotoxic payloads or NIR fluorophores. The VHH7 conjugates outcompeted commercial monoclonal antibodies (mAbs) for internalization and exhibited high specificity and cytotoxicity against A20 murine B-cell lymphoma. Non-invasive NIR imaging with a VHH7-fluorophore conjugate showed rapid tumor targeting on both localized and metastatic lymphoma models. Subsequent treatment with the nanobody-drug conjugate efficiently controlled tumor growth and metastasis without obvious systemic toxicity.

 

Chemical genomics-based antifungal drug discovery: Targeting glycosylphosphatidylinositol (GPI) precursor biosynthesis.

ACS Infect Dis. 2015 Jan 9;1(1):59-72.

Mann, P.A., McLellan, C.A.*, Koseoglu, S., Si, Q., Kuzmin, E., Flattery, A., Harris, G., Sher, X., Murgolo, N., Wang, H., Devito, K., de Pedro, N., Genilloud, O., Kahn, J.N., Jiang, B., Costanzo, M., Boone, C., Garlisi, C.G., Lindquist, S.*, and Roemer, T.

ACS infectious diseases 1, 59-72.Steadily increasing antifungal drug resistance and persistent high rates of fungal-associated mortality highlight the dire need for the development of novel antifungals. Characterization of inhibitors of one enzyme in the GPI anchor pathway, Gwt1, has generated interest in the exploration of targets in this pathway for further study. Utilizing a chemical genomics-based screening platform referred to as the Candida albicans fitness test (CaFT), we have identified novel inhibitors of Gwt1 and a second enzyme in the glycosylphosphatidylinositol (GPI) cell wall anchor pathway, Mcd4. We further validate these targets using the model fungal organism Saccharomyces cerevisiae and demonstrate the utility of using the facile toolbox that has been compiled in this species to further explore target specific biology. Using these compounds as probes, we demonstrate that inhibition of Mcd4 as well as Gwt1 blocks the growth of a broad spectrum of fungal pathogens and exposes key elicitors of pathogen recognition. Interestingly, a strong chemical synergy is also observed by combining Gwt1 and Mcd4 inhibitors, mirroring the demonstrated synthetic lethality of combining conditional mutants of GWT1 and MCD4. We further demonstrate that the Mcd4 inhibitor M720 is efficacious in a murine infection model of systemic candidiasis. Our results establish Mcd4 as a promising antifungal target and confirm the GPI cell wall anchor synthesis pathway as a promising antifungal target area by demonstrating that effects of inhibiting it are more general than previously recognized.

 

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

Neurogenetics. 2016 Jan;17(1):11-6.

Brownstein, C.A., Beggs, A.H., Rodan, L., Shi, J.H.*, Towne, M.C., Pelletier, R., Cao, S.Q., 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.

 

Mediobasal hypothalamic overexpression of DEPTOR protects against high-fat diet-induced obesity.

Mol Metab. 2015 Dec 8;5(2):102-12.

Caron, A., Labbe, S.M., Lanfray, D., Blanchard, P.G., Villot, R., Roy, C., Sabatini, D.M.*, Richard, D., and Laplante, M.

BACKGROUND/OBJECTIVE: The mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that functions into distinct protein complexes (mTORC1 and mTORC2) that regulate energy homeostasis. DEP-domain containing mTOR-interacting protein (DEPTOR) is part of these complexes and is known to dampen mTORC1 function, consequently reducing mTORC1 negative feedbacks and promoting insulin signaling and Akt/PKB activation in several models. Recently, we observed that DEPTOR is expressed in several structures of the brain including the mediobasal hypothalamus (MBH), a region that regulates energy balance. Whether DEPTOR in the MBH plays a functional role in regulating energy balance and hypothalamic insulin signaling has never been tested. METHODS: We have generated a novel conditional transgenic mouse model based on the Cre-LoxP system allowing targeted overexpression of DEPTOR. Mice overexpressing DEPTOR in the MBH were subjected to a metabolic phenotyping and MBH insulin signaling was evaluated. RESULTS: We first report that systemic (brain and periphery) overexpression of DEPTOR prevents high-fat diet-induced obesity, improves glucose metabolism and protects against hepatic steatosis. These phenotypes were associated with a reduction in food intake and feed efficiency and an elevation in oxygen consumption. Strikingly, specific overexpression of DEPTOR in the MBH completely recapitulated these phenotypes. DEPTOR overexpression was associated with an increase in hypothalamic insulin signaling, as illustrated by elevated Akt/PKB activation. CONCLUSION: Altogether, these results support a role for MBH DEPTOR in the regulation of energy balance and metabolism.

 

Tet1 and Tet2 protect DNA methylation canyons against hypermethylation.

Mol Cell Biol. 2015 Nov 23;36(3):452-61.

Wiehle, L., Raddatz, G., Musch, T., Dawlaty, M.M.*, Jaenisch, R.*, Lyko, F., and Breiling, A.

DNA methylation is a dynamic epigenetic modification with an important role in cell fate specification and reprogramming. The Ten eleven translocation (Tet) family of enzymes converts 5-methylcytosine to 5-hydroxymethylcytosine, which promotes passive DNA demethylation and functions as an intermediate in an active DNA demethylation process. Tet1/Tet2 double-knockout mice are characterized by developmental defects and epigenetic instability, suggesting a requirement for Tet-mediated DNA demethylation for the proper regulation of gene expression during differentiation. Here, we used whole-genome bisulfite and transcriptome sequencing to characterize the underlying mechanisms. Our results uncover the hypermethylation of DNA methylation canyons as the genomic key feature of Tet1/Tet2 double-knockout mouse embryonic fibroblasts. Canyon hypermethylation coincided with disturbed regulation of associated genes, suggesting a mechanistic explanation for the observed Tet-dependent differentiation defects. Based on these results, we propose an important regulatory role of Tet-dependent DNA demethylation for the maintenance of DNA methylation canyons, which prevents invasive DNA methylation and allows functional regulation of canyon-associated genes.

 

*Author affiliated with Whitehead Institute for Biomedical Research

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