title: CRISPR adenine and cytosine base editors with reduced RNA off-target activities [CBE]

city: New York

first: Caleb

last: Lareau

state: New York

address: 417 E 68th St, Zuckerman - ZRC 1132

institute: Memorial Sloan Kettering

summary:
CRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively.
We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.

overall_design: Overexpression of cytosine base editors in HEK293T, GFP-based cell sorting and RNA-seq for evaluation of off-target RNA editing. Here, AID, A3A, and eA3A were screened and analyzed in triplicate with a GFP control.

first: Julian

last: Grunewald


title: CRISPR adenine and cytosine base editors with reduced RNA off-target activities

status: Public on Jul 11 2019

country: USA

type: Expression profiling by high throughput sequencing

summary: This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: CRISPR adenine and cytosine base editors with reduced RNA off-target activities [ABE]

overall_design: Overexpression of adenine base editors in HEK293T, GFP-based cell sorting and RNA-seq for evaluating the degree of off-target editing from ABEs.


title: Genome-wide CRISPR-Cas9 screen identifies SLC1A3 as a key contributor to L-asparaginase Resistance in Solid tumors

city: Amsterdam

first: Pierre-Rene

last: Körner

address: Plesmanlaan 121

department: Oncogenomics

country: Netherlands

institute: Netherlands Cancer Institute (NKI)

summary: L-asparaginase (ASNase) has been incorporated in the treatment of acute lymphoblastic leukemia for decades. However, intolerable toxicity due to increasing ASNase dosage has been observed in clinical trials with solid tumors. Here, we performed a genome-wide CRISPR-Cas9 functional screen in ASNase-resistant PC3 prostate cancer cells and identified SLC1A3, an aspartate transporter highly expressed in brain tissues but also in several tumor types. Interestingly, combined ASNase treatment and SLC1A3 inhibition caused cell cycle arrest and metabolic alterations in the urea cycle, nucleotide synthesis, energy production by both TCA cycle and glycolysis, and impaired lipid metabolism. Additionally, the introduction of SLC1A3 into SLC1A3-negative mouse and human breast cancer cells increased tumor and metastasis burden, and promoted ASNase tolerance in vitro and in vivo. These findings uncover an important role of SLC1A3 in ASNase resistance and indicated limited aspartate uptake for enhancement of ASNase efficacy with solid tumors.

overall_design: PC3, DU145 and DU145-SLC1A3oe cells were cultured under 4 conditions for 3 days. Total RNA were extracted and libraries were prepared following TruSeq Stranded mRNA sample preparation kit (Illumina).

first: Alejandro

last: Piñeiro-Ugalde

first: Abhijeet

last: Pataskar

first: Jianhui

last: Sun


title: Eμ-TCL1xMyc Mice as a Therapeutic Model of Accelerated B-cell Malignancy

city: Columbus

first: Pearlly

last: Yan

state: OH

department: Division of Hematology

institute: The Ohio State University

summary: Richter’s syndrome (RS) is an aggressive B-cell lymphoma arising from chronic lymphocytic leukemia (CLL). RS patients are generally unresponsive both to conventional and B-cell receptor-targeted agents such as ibrutinib. Mouse models that mimic the biology and therapeutic response of RS are lacking, which hampers the development of alternative treatment strategies urgently needed to improve the dismal outcome of RS patients. Aberrant Myc expression is a major factor of RS pathogenesis. To investigate Myc overexpression in the context of CLL, we generated Eµ-TCL1 mice with B-cell restricted Myc expression. Eµ-TCL1xMyc mice uniformly developed highly aggressive lymphoid disease with histologically and immunophenotypically distinct concomitant CLL and B-cell lymphoma, leading to a significantly reduced lifespan. Injection of cells from diseased Eμ-TCL1xMyc into WT mice established a disease similar to that in double-transgenic mice. Both Eμ-TCL1xMyc mice and mice with disease after adoptive transfer failed to respond to ibrutinib. Effective and durable disease control was however observed by selective inhibition of nuclear export protein exportin-1 (XPO1) using a compound currently in clinical development for relapsed/refractory malignancies, including CLL and lymphoma. Altogether, the Eµ-TCL1xMyc mouse model represents a new preclinical tool for experimental drug testing of aggressive lymphoma in the context of CLL, mimicking clinical behavior and therapeutic responses seen in RS patients.

overall_design: Eµ-TCL1 transgenic mice developing CLL and Eµ-Myc transgenic mice developing B-cell lymphoma have previously been described (Bichi et al, 2002, Adams et al, 1985). c-Myc and wild type (WT) mice were purchased from Jackson laboratories (Bar Harbor, ME). C57BL/6J females were crossed with Eµ-Myc hemizygous males to produce Eµ-Myc hemizygous or non-transgenic (ntg) pups. Eµ-TCL1-B6 homozygous females were crossed with Eµ-Myc hemizygous males to produce Eµ-TCL1B6 hemizygous/Eµ-Myc hemizygous (abbreviated to Eµ-TCL1xMyc or double transgenic (dTG)) or Eµ-TCL1B6 hemizygous/ntg pups. Survival of transgenic mice was assessed in all transgenic colony mice born within a 24 month period. Histopathological and immunophenotype assessments were conducted in spleen, lymph node, bone, and blood to characterize malignancy.

first: Fabienne

last: Lucas

first: Kerry

middle: A

last: Rogers

first: Bonnie

middle: K

last: Harrington

first: Alexander

last: Pan

first: Lianbo

last: Yu

first: Justin

last: Breitbach

first: Virginia

middle: M

last: Goettl

first: Zachary

middle: H

last: Hing

first: Parviz

last: Kanga

first: Rose

last: Mantel

first: Deepa

last: Sampath

first: Lisa

middle: L

last: Smith

first: Ronni

last: Wasmuth

first: Danielle

last: White

first: Ralf

last: Bundschuh

first: John

middle: C

last: Byrd

first: Rosa

last: Lapalombella

first: Jennifer

last: Woyach


title: The RNA exosome nuclease complex regulates human embryonic stem cell differentiation [HITS-CLIP]

city: Frederick

first: Sandra

last: Wolin

state: Maryland

address: 1050 Boyles Street, BLDG 560 Room 11-39

department: NCI

institute: NIH

summary: This work shows that the exosome modulates the levels of LINE-1 retrotransposons, miRNAs, lncRNAs and specific mRNAs that encode developmental regulators or affect their expression. The exosome restrains differentiation in part by degrading transcripts encoding FOXH1, a transcription factor crucial for mesendoderm formation.

overall_design: RNA exosome targets were identified in human embryonic stem cells by performing HITS-CLIP using antibodies against two exosome core subunits, EXOSC3 and EXOSC4

first: Cedric

last: Belair

first: Soyeong

last: Sim

first: Kun-Yong

last: Kim

first: Yoshiaki

last: Tanaka

first: In-Hyun

last: Park


title: Profiling chromatin state by single-cell itChIP-seq [E7.75_E8.25_heart]

city: Beijing

first: Chen

last: Li

state: Beijing

address: Yihe Yuan Road No.5

department: Institute of Molecular Medicine

country: China

institute: Peking University

type: Genome binding/occupancy profiling by high throughput sequencing

summary: We present a low-cost, generalizable ChIP-seq (itChIP), compatible to both low-input and single cells for profiling chromatin states. This method combines chromatin opening, simultaneous cellular indexing and chromatin tagmentation in a single tube. Single-cell itChIP data yield ~ 5000 unique reads per cell, sufficiently defining cell identifies and subpopulations of a given cell type. Our results demonstrate that itChIP is a generalizable technology for single-cell chromatin profiling of samples limited to ultra-low number of cells.

overall_design: Based on Tn5 transposition of chromatin, the generalizable itChIP combines chromatin opening, simultaneous cellular indexing and chromatin tagmentation in a single tube.

first: Shanshan

last: Ai

first: Haiqing

last: Xiong

first: Yingjie

last: Luo

first: Qiang

last: Shi

first: Aibin

last: He


title: Profiling chromatin state by single-cell itChIP-seq [itChIP-seq]


title: Mta2, a component of the NuRD complex, regulates B cell development and cooperates with OCA-B in controlling the pre-B to immature B cell transition (ChIP-Seq)

first: Chi-Shuen

last: Chu

state: NY

address: 1230 York Ave

institute: The Rockefeller University

summary: ChIP-sequencing of human 697 cells.

overall_design: ChIP-sequencing of human 697 cells.

first: Xiangdong

last: Lu


title: Rapid accumulation of motility-activating mutations in resting liquid culture of Escherichia coli

city: Cambridge

first: Darren

middle: John

last: Parker

state: MA

address: 31 Ames Street

department: Biology

institute: MIT

summary: Expression and use of motility genes is a potentially beneficial but costly process in bacteria. Interestingly, many isolate strains of Escherichia coli possess motility genes but have lost the ability to activate them in conditions in which motile cells are advantageous, raising the question of how they respond to these situations. Through transcriptome profiling of strains in the E. coli single-gene knockout Keio collection, we noticed drastic upregulation of motility genes in many of the deletion strains as compared to its typically non-motile parent strain (BW25113). This switch to a motile phenotype is not a direct consequence of the genes deleted, but is instead due to a variety of secondary mutations that increase the synthesis of the major motility regulator, FlhDC. We found that a phenotypic switch to motility at a population level can be induced in non-motile E. coli strains by incubation in non-shaking liquid medium overnight but not in shaking media. Individual isolates after the overnight incubation acquired distinct mutations upstream of the flhDC operon, including different insertion sequence (IS) elements and, to a lesser extent, point mutations. The rapid sweep in the non-shaking population shows that non-motile strains without existing regulatory mechanisms can quickly adapt to a motile lifestyle by quickly acquired genetic changes.

overall_design: 71 RNA-seq experiments on knockout strains from the Keio E. coli knockout collection grown in MOPS Minimal liquid media

middle: J

first: Pinar

last: Demetci

first: Gene-Wei


title: Profiling chromatin state by single-cell itChIP-seq [ATAC-seq]

overall_design: ATAC-seq was processed with 50K mouse embryonic cells as previously described. Tn5-MEA/B was used to tagment the native chromatin.


title: Rnase L reprograms translation by widespread mRNA turnover escaped by antiviral mRNAs

status: Public on Jul 10 2019

city: Boulder

first: Roy

middle: Robert

state: CO

address: 3415 Colorado Ave

department: Chemistry and Biochemistry

institute: University of Colorado at Boulder

summary: In response to foreign and endogenous double-stranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2  phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by only allowing for the translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such as IFN- . Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNaseL reprograms translation in response to dsRNA.

overall_design: Total RNA from parental (WT) and RNase L knockout (RLKO) A549 cells before and after 6-hours post-transfection of poly(I:C) (500-ng/ml) was isolated for library preparation. Three independent samples from each cell line and condition were analyzed. ERRC spike-in controls were added prior to rRNA depletion and library preparation.

first: James

last: Burke

first: Stephanie

last: Moon

first: Evan

middle: T

last: Lester

first: Tyler

last: Matheny


title: Genome-wide identification of binding sites and gene targets of Alx1, a pivotal regulator of echinoderm skeletogenesis

city: Pittsburgh

first: Jian Ming

last: Khor

state: PA

address: 4400 Fifth Ave

department: Biological Sciences

institute: Carnegie Mellon University

summary: ChIP-seq provides a genome-wide view of the binding sites of Alx1, a pivotal transcription factor in a gene regulatory network that controls skeletogenesis in sea urchins and other echinoderms.

overall_design: Three independent cultures of S. purpuratus were collected and processed at the mesenchyme blastula stage (24 hpf). For each culture, ChIP was performed using α-Sp-Alx1 and normal rabbit IgG (Sigma-Aldrich, Cat. No. 12370) was used for mock IP.

first: Charles

last: Ettensohn


title: Selective ribosome profiling to study interactions of translating ribosomes in yeast

city: Heidelberg

first: Kristina

middle: Rebekka

last: Doering

state: Baden-Württemberg

address: Im Neuenheimer Feld 282

department: University of Heidelberg

country: Germany

institute: ZMBH

summary: A number of enzymes, targeting factors and chaperones engage ribosomes to support fundamental steps of nascent protein maturation, including enzymatic processing, membrane targeting and co-translational folding. The selective ribosome profiling (SeRP) method is a new tool for studying the co-translational activity of maturation factors that provides proteome-wide information on a factor’s nascent interactome, the onset and duration of binding and the mechanisms controlling factor engagement. SeRP is based on the combination of two ribosome-profiling (RP) experiments, sequencing the ribosome-protected mRNA fragments from all ribosomes (total translatome) and the ribosome subpopulation engaged by the factor of interest (factor-bound translatome). We provide a detailed SeRP protocol, exemplified for the yeast Hsp70 chaperone Ssb (stress 70 B), for studying factor interactions with nascent proteins that is readily adaptable to identifying nascent interactomes of other co-translationally acting eukaryotic factors. The protocol provides general guidance for experimental design and optimization, as well as detailed instructions for cell growth and harvest, the isolation of (factor-engaged) monosomes, the generation of a cDNA library and data analysis. Experience in biochemistry and RNA handling, as well as basic programing knowledge, is necessary to perform SeRP. Execution of a SeRP experiment takes 8–10 working days, and initial data analysis can be completed within 1–2 d. This protocol is an extension of the originally developed protocol describing SeRP in bacteria.

overall_design: Method description. Control mixing experiment to show that no post-lysis binding occurs.

first: Carla

middle: V

last: Galmozzi

first: Dorina

last: Merker

first: Ulrike

last: Friedrich

last: Döring

first: Günter

last: Kramer


title: Analysis of TEAD4 protein-depleted mouse embryos after Trim-away

city: Muenster

first: Michele

last: Boiani

address: Roentgenstrasse 20

institute: Max Planck Institute Muenster

summary:
In order to study early developmental events in the mammalian embryo it is often desirable to be able to impair expression of specific genes. While DNA and RNA methods are routine, protein methods are still at the very beginning. When a specific antibody is supplied to mouse oocytes expressing the ubiquitin-protein ligase TRIM21, a ternary complex forms with the target protein, leading to its rapid and acute degradation - hence the name 'Trim-away' (PMID 29153837). However, there are many unknowns in this new endeavour. First and foremost, the extent to which endogenous proteins can be depleted depends on their amount, in relation to the amount of exogenous antibody, which is limited by the microinjection procedure. Secondly, the depletion of the protein must be sustained over days. Using the iBAQ algorithm we show that proteins found in preimplantation mouse embryos range from 3,5E-10 to 2,6E-02 picomoles. These amounts are tractable with our microinjection method, which supplies up to 100 picoliters and up to 6,7E-4 picomoles antibody before incurring in toxic effects on mouse development. Building on these data, we demonstrate the feasibility of protein knock-down for a gene which is essential in the preimplantation mouse embryo, namely TEAD4 (TEA domain family member 4). Protein knock-down persists for sufficient time to result in a phenotype which is entirely consistent with that of the null mutation (Tead4 -/-) and of the RNA interference, namely: significantly reduced mRNA expression of TEAD4 target genes Cdx2 and Gata3, and embryo’s inability to implant. We conclude that for a time window of 3-4 days of preimplantation development the protein depletion method can be a valid alternative to DNA and RNA methods.
After in vivo fertilization and short culture in KSOM(aa) medium, pronuclear-stage oocytes (B6C3F1 x CD1) were microinjected with Trim21 mRNA and dextran beads as tracer (named 'group 4'), or Trim21 mRNA, dextran beads and anti-GFP antibody (named 'group 5'), or Trim21 mRNA, dextran beads and anti-TEAD4 antibody (named 'group 6'), two replicates each ('a' and 'b'). On day 4 after microinjection, the most advanced embryos were examined for phenotype or lysed for transcriptome analysis. TEAD4-depleted embryos formed only 30% blastocysts and these were not able to implant in the outgrowth assay, in contrast to the almost full rates of the embryos injected with Trim21 mRNA or Trim21 mRNA + anti-GFP antibody. Transcriptome analysis revealed that the TEAD4 target genes Cdx2 and Gata3 are significantly reduced in the embryos that received the anti-TEAD4 antibody compared to the embryos that received Trim21 mRNA only, while the embryos that received anti-GFP antibody were much similar to those that received Trim21 mRNA only.

overall_design: All three groups of embryos (Trim21 mRNA; Trim21 mRNA + anti-GFP antibody; Trim21 mRNA + anti-TEAD4 antibody) were fertilized and injected on the same day, and cultured in parallel for 4 days, until collection and lysis for microarray analysis (10 embryos per group).


title: Adaptive molecular responses of mouse half-embryos grown in different culture media

summary:
Mouse zygotes are suspected to mount adaptive responses (e.g. physiology, metabolism, gene expression) to the environment, which can be the natural environment of the female genital tract or the artificial environment of a culture medium. The jury is still out when it comes to the possible long-term effects of culture media on embryo development. In 2016 the ESHRE took a stance about this subject, saying that the environment the early embryo is exposed to can cause reprogramming of embryonic, fetal and postnatal development, with negative consequences for the conceptus (Sunde et al., Hum Reprod. 31(10):2174-82, 2016). Of course, in order to possibly measure these long-term effects, the embryo has to develop to term. On the one hand, the impact of prior adaptive responses on postimplantation development is contingent on the presence of sufficient cells in the epiblast compartment of the blastocyst-stage embryo. In mice the viable epiblast number is considered to have a minimum at 4 (Morris et al., Cell Rep. 2(4):756-65, 2012) and a maximum at 8 (Soriano and Jaenisch, Cell 46(1):19-29, 1986). On the other hand, regular embryos have reserve capacities and lie well above the minimum level, whereby detrimental effects of culture conditions are probably underestimated. In plain words, there are embryos that might have suffered from in vitro culture, but they make it because they already secured the minimum epiblast number. We reasoned that these effects may be exposed if the embryos had half the number of total cells, thereby bringing them closer to the critical minimum epiblast threshold. These half embryos live on the knife’s edge: if they make it above the threshold, then they can survive, otherwise they fail. Therefore, if any adaptation during embryo culture were to occur, it could either raise the half embryos above the critical threshold or deliver the final blow. This would give us the opportunity to work with a clear dichotomy and to measure clear responses in terms of development vs. no development. We used the model system of mouse half-embryo system to assess the adaptive responses to culture media in terms of gene expression.
After in vivo zygote production and short culture in KSOM(aa) medium to complete the first cell cycle, 2-cell embryos were split, and the individual blastomeres were cultured in KSOM(aa), GM501 or SAGE 1step medium. Resultant blastocysts were compared and contrasted with each other. Transcriptome analysis revealed differences of gene expression due to culture media.

overall_design: Starting pair associations (e.g. blastomere 'a' and 'b' from the same 2-cell embryo) were recorded.

first: Verena

last: Nordhoff


title: Increased expression of cytokine receptor Ccr2 is a hallmark of the aged mouse intestine epithelium

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

country: United Kingdom

institute: Altos Labs

summary: We characterize changes in gene expression during late ageing in intestinal epithelium-derived cells through RNA-Seq approaches and show that gene expression is highly stable during ageing.

overall_design: RNA-Seq  profile from sorted mouse small intestinal differentiated enterocytes and stem cells performed in triplicates of adult and aged animals. A subset of differentially expressed genes was validated by RT-qPCR on additional samples.

first: Juri

last: Kazakevych

first: Patrick

last: Varga-Weisz


title: Kinome profiling of Non-Hodgkin’s lymphoma reveals Tyro3 is important in primary effusion lymphoma survival

city: St. Louis

first: Jason

middle: Paul

last: Wong

state: MO

address: Neuroscience Research Building

department: Pediatrics

institute: Washington University in St. Louis

summary: Non-Hodgkin’s lymphomas (NHL) make up the majority of lymphoma diagnoses and represent a very diverse set of malignancies. We sought to identify kinases uniquely upregulated in different NHL subtypes. Using Multiplexed Inhibitor Bead-mass spectrometry (MIB/MS), we found Tyro3 was uniquely upregulated and important for cell survival in primary effusion lymphoma (PEL). We developed an inhibitor against Tyro3 named UNC3810A, which inhibited cell growth in PEL but not in other NHL subtypes. UNC3810A also significantly inhibited tumor progression in a PEL xenograft mouse model compared to the vehicle treated animals. Our data suggests Tyro3 may be a therapeutic target for PEL.

overall_design: RNASeq and analysis of 24 NHL cell lines. RNA was harvested from cell lines using the RNeasy Plus Mini Kit according to manufacturer’s instructions. mRNA-Seq libraries were generated using the Stranded mRNA-Seq kit and multiplexed with Illumina TruSeq adapters. Samples were run on two 75-cycle single-end sequencing runs with an Illumina NextSeq-500.

middle: P

first: Timothy

last: Stuhlmiller

first: Louise

last: Giffin

first: Carolina

last: Lin

first: Steven

middle: I

last: Park

first: Gary

last: Johnson

first: Blossom

last: Damania


title: Single cell analysis of sister blastomeres of parthenogenetic 2-cell mouse embryos respecting pair associations

summary: Following fertilization in mammals, it is generally accepted that totipotent cells are exclusive to the zygote and to each of the two blastomeres originating from the first mitotic division. We counter that this classic view needs to be revised, because we have presented compelling evidence that the sister blastomeres are both totipotent in only a subset of 2-cell stage mouse embryos (PMID 28811525). Building on our previous findings, we here ask the question if the interblastomere differences depend - at least in part - on the contribution of sperm, since the area of sperm entry is inherited preferentially by one blastomere. To this end, we created sister 2-cell stage blastomeres without sperm entry point, by parthenogenesis. We compare the transcriptomes of the sister blastomeres, to see if the interblastomere mRNA differences observed after fertilization (GSE94050) are still there when the sperm entry point is missing.

overall_design: After parthenogenetic activation and short culture in KSOM(aa) medium, 2-cell embryos were split, and the individual blastomeres were lysed immediately, keeping track of the original pair associations (e.g. blastomere '1a' and blastomere '1b' both derived from same 2-cell embryo number '1'). Transcriptome analysis followed by intra-pair mRNA ratio analysis revealed differences between the embryos of the same pair. These differences were fewer, but not substantially fewer, than the differences observed in the sister blastomeres of fertilized 2-cell embryos (GSE94050). Starting pair association (e.g. blastomere 'a' and 'b' from the same 2-cell embryo) was rigorously observed. Samples were subjected to Affymetrix microarray analysis, performed by Anika Witten c/o Institut fuer Humangenetik, Universitaetsklinikum Muenster.


title: Expression data from CSC and Non-CSC of three primary HCC samples

first: Yanying

last: Wang

address: 15 Datun Road, Chaoyang District,Beijing 100101, China

institute: Institute of Biophysics, Chinese Academy of Sciences

type:
Expression profiling by array
Non-coding RNA profiling by array

summary:
Hepatocellular carcinoma (HCC) represents the major subtype of liver cancer, characterized with a high rate of recurrence and heterogeneity. Liver cancer stem cells (CSCs) may account for a hierarchical organization of heterogeneous cancer cells. However, how liver CSCs sustain their self-renewal remains largely unknown.
We used microarrays to discover the long non-coding RNAs (lncRNAs) expression underlying cell stem cell (CSC) and non cell stem cell (non-CSC) and identified distinct lncRNAs during this process.

overall_design: We sorted CD13+CD133+ and CD13-CD133- cells from three primary HCC patients as liver CSCs and non-CSCs, then hybridized on Aligent microarrays. We sought to identify distinct lncRNAs in liver CSCs.


title: Profiling chromatin state by single-cell itChIP-seq [ESC_EpiLC]

first: Chen

last: Li

first: Shanshan

last: Ai

first: Haiqing

last: Xiong

first: Yingjie

last: Luo

first: Qiang

last: Shi

first: Aibin

last: He


title: Profiling chromatin state by single-cell itChIP-seq [sonication ChIP]

overall_design: Sonication-based ChIP was the traditional method to profile modification of chromatin. Chromatin was sonicated by Q800R to 200-800 bp fragments. After being pulled down by antibody and Dynabeads protein A beads, fragments were ligated with adaptors by ChIPmentation method. After being amplified, libraries were sequenced on Illumina HiSeq 4000 for paired end 150 bp. Sonication-ChIP data played as the positive control of our itChIP-seq.


title: Profiling chromatin state by single-cell itChIP-seq

overall_design: Refer to individual Series


title: Gene expression profiles of young and aged mammary epithelial and stromal cell populations from Wild Type and Slug/Snai2-null mice

status: Public on Jul 09 2019

city: Boston

first: Charlotte

last: Kuperwasser

address: 150 Harrison Ave

department: School of Medicine

institute: Tufts Univeristy

summary: This experiment shows RNA-Seq gene expression profiles of Wild Type and Slug/Snai2-null luminal, basal, and stromal mammary cell populations from young (3 mo) and aged (12 mo) mice.

overall_design: Luminal, basal, and stromal populations were sorted from Wild Type and Slug/Snai2-null mice at young and aged timepoints based on expression of EpCAM and CD49f, followed by RNA sequencing using Illumina HiSeq 2500

first: Kayla

last: Gross


title: Sleep Deprivation Alters the Pituitary Stress Transcriptome in male and female Mice

city: Bethesda

first: Mario

middle: G

last: Oyola

state: MD

address: 4301 Jones Bridge Rd

institute: Uniformed Services University

summary:
We performed a comparative, whole-transcriptome, analysis to identify stress-induced genes and relevant pathways that may be affected by sleep deprivation.
Methods: One day following 12 hours of Paradoxical Sleep Deprivation (PSD), mice were restrained for 20 minutes. Gene expression changes in the pituitary were assessed via RNA-Seq and Gene Ontology in PSD and/or restrained groups compared to controls.
Results: We show that restraint triggers transcriptional responses involved in hormone secretion, the glucocorticoid response, and apoptosis in both sexes, with 285 differentially expressed genes in females and 93 in males. When PSD preceded restraint stress, the numbers of differentially expressed genes increased to 613 in females and 580 in males. The pituitary transcriptome of restraint+PSD animals was enriched for microglia and macrophage proliferation, cellular response to corticosteroids, and apoptosis, among others. Finally, we show sex-specific differences in restraint-induced genes following PSD.
Conclusion: The results indicate striking differences in the male and female stress-induced transcriptome, as well as in the PSD-induced changes. When PSD preceded the restraint stress challenge, the effects on the pituitary transcriptome were striking. While the male and female PSD + restraint-induced transcriptome was similar, we detected remarkable differences, perhaps indicating different strategies used by each sex to cope with challenges to homeostasis. We hope that these data illuminate future research elucidating how sleep deprivation impacts the vital response to stress and motivate the analysis of male and female subjects when designing experiments.

overall_design: Gene expression changes in the pituitary were assessed via RNA-Seq and Gene Ontology in Paradoxical Sleep Deprivation and/or restrained groups compared to controls.

first: Elizabeth

last: Shupe

first: Anthony

middle: R

last: Soltis

first: Gauthaman

last: Sukumar

first: Marcelo

last: Paez-Pereda

first: Darwin

middle: O

last: Larco

first: Matthew

last: Wilkerson

first: Stephen

last: Rothwell

first: Clifton

last: Dalgard

first: T

last: Wu


title: Sen1 is required for robust RNA Polymerase III transcription termination

city: Sherbrooke

first: François

last: Bachand

state: Québec

address: 3201 Jean Mignault

department: Biochimie

country: Canada

institute: Université de Sherbrooke

summary: R-loop disassembly by the human helicase Senataxin contributes to genome stability and to proper transcription termination at a subset of RNA polymerase II genes. Whether Senataxin-mediated R-loop disassembly also contributes to transcription termination at other classes of genes has remained unclear. Here we show that Sen1, one of two fission yeast homologues of Senataxin, promotes efficient termination of RNA Polymerase III (RNAP3) transcription in vivo. In the absence of Sen1, RNAP3 accumulates downstream of the primary terminator at RNAP3-transcribed genes and produces long exosome-sensitive 3’-extended transcripts. Importantly, neither of these defects was affected by the removal of R-loops. The finding that Sen1 acts as an ancillary factor for RNAP3 transcription termination in vivo challenges the pre-existing view that RNAP3 terminates transcription autonomously. We propose that Sen1 is a cofactor for transcription termination that has been co-opted by different RNA polymerases in the course of evolution.

overall_design: ChIP-seq of Rpc1 and Rpc2 in WT or sen1 mutant and ChIP-seq of Sen1 in WT.

first: Julieta

last: Rivosecchi

first: Marc

last: Larochelle

first: Camille

last: Teste

first: Frédéric

last: Grenier

first: Amélie

last: Malapert

first: Emiliano

last: Ricci

first: Pascal

last: Bernard

first: Vincent

last: Vanoosthuyse


title: H3K27me3 ChIP-seq profiling studies of HEK293 cells

status: Public on Jul 08 2019

city: Durham

first: Greg

last: Wang

state: NC

address: 3 Genome Court

department: Duke Department of Pharmacology and Cancer Biology  and Duke Cancer Institute

institute: Duke University School of Medicine, and Department of Biochemistry and Biophysics, UNC at Chapel Hill (adjunct)

summary: Tri-methylation of histone H3 lysine 27 is catalyzed by Polycomb Repressive Complex 2 (PRC2). By genome-wide mapping of H3K27me3 sites in HEK293 cells, we are able to identify potential sites for PRC1-mediated repression and use these data to inform gene expression experiments in the presence and absence of pharmacological inhibition or KD/KO of various Polycomb subunits.

overall_design: Genome wide mapping for H3K27me3 was generated by ChIP-seq using specific antibody in HEK293 cells.

first: Huitao

last: Fan


title: Response of triple negative breast cancer to BAZ2A/B inhibition and BET bromodomain inhibition alone and in combination

first: Bradley

middle: E

last: Bernstein

state: Massachusetts

address: 450 Brookline Ave

department: Cancer Biology

institute: Dana Farber Cancer Institute

summary: This SuperSeries is composed of the SubSeries listed below.


title: Supraphysiological Androgens Repress Prostate Cancer Growth and Induce DNA Damage Augmented by PARP Inhibition

city: Seattle

first: Ilsa

last: Coleman

state: WA

address: 1100 Fairview Ave N, E2-112

department: Human Biology

institute: Fred Hutchinson Cancer Center

summary: Prostate cancer (PC) is initially dependent on androgen receptor (AR) signaling for survival and growth. Therapeutics designed to repress AR activity, such as those reducing circulating androgen levels, serve as the primary intervention for advanced disease. However, supraphysiological androgen (SPA) concentrations, can produce a paradoxical response leading to growth inhibition. We sought to determine the mechanisms by which SPA represses PC growth and determine if molecular context associates with anti-tumor effects.

overall_design: RNA sequencing of LNCaP human prostate tumor cell lines using Illumina TruSeq Library prep and sequenced on Illumina HiSeq 2500.

first: Payel

last: Chatterjee

first: Jared

middle: M

last: Lucas

first: Michael

middle: D

last: Nyquist

first: Robin

last: Tharakan

first: Elahe

last: Mostaghel

first: Ming

last: Lam

first: Eva

last: Corey

last: Schweizer

first: Peter

middle: S

last: Nelson


title: Response of triple negative breast cancer to BAZ2A/B inhibition and BET bromodomain inhibition alone and in combination (ChIPseq)

summary: Three triple negative breast cancer cell lines (MDAMB231, SUM159, and HCC1806) were treated with small molecule inhibitors (JQ1, BET bromodomain inhibitor; GSK2801, BAZ2A/B bromodomain inhibitor) or BAZ siRNA alone and in combination  with JQ1 for 48 hours

overall_design: 34 experimental samples analyzed, 2 chromatin input control samples

first: Gary

middle: L

last: Johnson

first: Samantha

last: Bevill


title: Response of triple negative breast cancer to BAZ2A/B inhibition and BET bromodomain inhibition alone and in combination (RNAseq)

summary: Three triple negative breast cancer cell lines (MDAMB231, SUM159, and HCC1806) were treated with small molecule inhibitors (JQ1, BET bromodomain inhibitor; GSK2801, BAZ2A/B bromodomain inhibitor) alone and in combination for 72 hours

overall_design: 12 experimental samples


title: The Molecular Dissection of the Oncogenic Role of ETS1 in the Mesenchymal Subtypes of Head and Neck Squamous Cell Carcinoma [RNA-seq Cell lines]

city: Buffalo

first: Satrajit

last: Sinha

address: 701 Ellicott Street

department: Department of Biochemistry

institute: State University of New York at Buffalo

summary: RNA-Sequencing Analysis was performed on 7 Head and Neck Squamous Cell Carcinoma Cells Lines in order to examine their overall gene expression profiles.

overall_design: In order to understand the transcriptomic heterogeneity that exists between different Head and Neck Squamous Carcinoma (HNSCC) Cell Line Models, RNA-Seq analysis was performed on a cohort of 7 different cell lines.  These profiles were assessed for their overall variance in gene expression profile via unsupervised clustering analysis and were shown to match established intrinsic subtypes of HNSCC such as the Atypical, Basal and Mesenchymal classes of tumors.

first: Christian

last: Gluck

first: Isha

last: Sethi


title: Polynucleotide phosphorylase promotes the stability and function of Hfq-binding sRNAs by degrading target mRNA-derived fragments

city: Houston

first: Nicholas

last: De Lay

state: Texas

address: 6431 Fannin St

department: Microbiology and Molecular Genetics

institute: University of Texas Health Science Center

summary: In many gram-negative and some gram-positive bacteria small regulatory RNAs (sRNAs) that bind the RNA chaperone Hfq have a pivotal role in modulating virulence, stress responses, metabolism, and biofilm formation. These sRNAs recognize transcripts through base-pairing, and sRNA-mRNA annealing consequently alters the translation and/or stability of transcripts leading to changes in gene expression. We have previously found that the highly conserved 3'-to-5' exoribonuclease polynucleotide phosphorylase (PNPase) has an indispensable role in paradoxically stabilizing Hfq-bound sRNAs and promoting their function in gene regulation in Escherichia coli. Here, we report that PNPase uniquely contributes to the degradation of specific mRNA cleavage products, the majority of which bind Hfq and are derived from targets of sRNAs. Specifically, we found that these mRNA-derived fragments accumulate in the absence of PNPase or its exoribonuclease activity and interact with PNPase. Additionally, we show that mutations in hfq or in the seed pairing region of a sRNA eliminated the requirement of PNPase for sRNA stability. Altogether, our results are consistent with a model that PNPase degrades mRNA-derived fragments that could otherwise deplete cells of Hfq-binding sRNAs through pairing mediated decay.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: Examination of mRNA expression in four samples of strains carrying wild type or mutant genes of rph and/or pnp. Additional examination of four samples from pnp wild type and mutant strains of RNA from Hfq co-immunoprecipitation input and output fractions. All samples were sequenced with two biological replicates.


title: The Molecular Dissection of the Oncogenic Role of ETS1 in the Mesenchymal Subtypes of Head and Neck Squamous Cell Carcinoma [RNA-seq knock-down]

summary: RNA-Sequencing Analysis of gene expression changes due to ablation of Ets1 in SCC25 Cells.

overall_design: Global transcriptomic characterization of the effects of Ets1 knockdown in SCC25 Head and Neck Squamous Cell Carcinoma cells was performed via RNA-Seq Analysis.  The knockdown of ETS1 was performed using transient siRNA based approach as well as stable cell lines were generated with targeted shRNA. As controls, we used SCC25 cells transduced with siRNAs and shRNAs with non-silencing control hairpin sequences that do not match any known mammalian genes and hence are not expected to not target any gene product.


title: The Molecular Dissection of the Oncogenic Role of ETS1 in the Mesenchymal Subtypes of Head and Neck Squamous Cell Carcinoma


title: The Airn RNA does not require any DNA elements within its locus to silence distant imprinted genes

status: Public on Jul 07 2019

city: Munich

first: Daniel

last: Andergassen

address: Biedersteiner Straße 29

institute: Technical University Munich

summary: Long non-coding (lnc) RNAs are numerous and found throughout the mammalian genome, and many are thought to be involved in the regulation of gene expression. However, the majority remain relatively uncharacterised and of uncertain function making the use to model systems to uncover their mode of action valuable. Imprinted lncRNAs target and recruit epigenetic silencing to a cluster of imprinted genes on the same chromosome, making them one of the best characterized lncRNAs to silence distant genes in cis. In this study we examined silencing of the distant imprinted gene Slc22a3 by the lncRNA Airn in the Igf2r imprinted cluster in mouse. Previously we proposed that imprinted lncRNAs may silence distant imprinted genes by disrupting promoter-enhancer interactions by being transcribed through the enhancer, which we called the enhancer interference hypothesis. Here we tested this hypothesis by first using allele-specific chromosome conformation capture (3C) to detect interactions between the Slc22a3 promoter and the locus of the Airn lncRNA that silences it on the paternal chromosome. In agreement with the model we found interactions enriched on the maternal allele, but across the entire Airn gene consistent with multiple enhancer-promoter interactions. Therefore, to further test the enhancer interference hypothesis we devised an approach to delete the entire Airn gene. However, the deletion showed that there are no essential enhancers for Slc22a3 within the Airn gene, strongly indicating that the Airn RNA rather than its transcription is responsible for silencing distant imprinted genes. Therefore we propose an alternative hypothesis whereby the chromosome interactions may initially guide the lncRNA to target imprinted promoters and recruit repressive chromatin, and that these interactions are lost once silencing is established.

overall_design: RNA-seq and ChIP-seq from F1 extraembryonic tissues extracted from reciprocal crosses between RSDEL mouse and CAST/EiJ

first: Florian

last: Pauler

first: Quanah

last: Hudson


title: Identification of transcriptomic drivers of squamous cell carcinoma development through a preneoplastic intermediate [mouse RNA-Seq]

first: Kenneth

last: Tsai

state: TX

address: 2130 West Holcombe Blvd , LSP9.4218

department: Dermatology & Translational Molecular Pathology

institute: University of Texas MD Anderson Cancer Center

summary: Cutaneous squamous cell carcinoma (cuSCC) comprises 15-20% of all skin cancers, accounting for over 700,000 cases in the U.S. annually. Most cuSCC arise in association with a distinct precancerous lesion, the actinic keratosis (AK). In order to identify potential targets for molecularly targeted chemoprevention, we performed integrated cross-species genomic analysis of cuSCC development through the preneoplastic AK stage using matched human samples and a solar UV-driven Hairless mouse model. We identified the major transcriptional drivers of this sequence showing that the key genomic changes in cuSCC development occur in the normal skin to AK transition. Our data validate the use of this UV-driven mouse cuSCC model for cross-species analysis and demonstrate that cuSCC bears deep molecular similarities to multiple carcinogen-driven SCCs from diverse sites, suggesting that cuSCC may serve as an effective, accessible model for multiple SCC types and that common treatment and prevention strategies may be feasible.

overall_design: We sought to identify important genetic events that drive squamous cell carcinoma development through combined analysis of next generation sequencing of matched patient samples with a UV-driven mouse model to identify key pathways.

middle: Y

first: Kimal

last: Rajapakshe

first: Hussein

middle: A

last: Abbas

first: Cristian

last: Coarfa

first: Weimin

last: Xiao

first: Preethi

last: Gunaratne

first: Li

last: Shen

first: Hao

last: Yui

first: Xiaoping

last: Su

first: Erika

last: Thompson

first: Yoko

last: Takata

first: Jianjun


title: A homeostatic Arid1a-dependent permissive chromatin state licenses hepatocyte responsiveness to liver injury-associated YAP signaling (ChIP-seq)

city: Shanghai

first: Lijian

last: Hui

address: yueyang road 320

department: SIBCB

institute: CEMCS, CAS

summary: Following injury, differentiated epithelial cells can serve as a stem cell-independent source for tissue regeneration by undergoing reprogramming into other cell types. The intrinsic molecular basis underlying plasticity of differentiated cells remains largely unaddressed. Here we show that Arid1a, a key component of the SWI/SNF chromatin remodeling complex, controls liver regeneration and gene expression associated with emergence of injury-induced progenitor-like cells (LPLCs). Hepatocyte-specific Arid1a ablation reduces LPLC gene expression in several models of periportal liver injury and impairs liver regeneration, leading to organ dysfunction. Arid1a establishes a permissive chromatin state at LPLC-enriched genes during homeostasis, suggesting it endows hepatocytes with competence to respond to injury-induced signals.  Consistently, Arid1a facilitates binding of YAP, a critical regeneration signaling pathway, to LPLC-enriched genes, and Arid1a deletion prevents their YAP-associated induction following injury. Together, these findings provide a framework for studying the contributions of injury-induced LPLCs to periportal liver regeneration.

overall_design:
(i) Hepatocytes were perfused and isolated from DDC-injured mice livers, analysis of genomic occupancy of Yap in hepatocytes from DDC-injured Arid1a WT and Arid1a liver-specific KO mice by ChIP-seq
(ii) Hepatocytes were perfused and isolated from normal diet feeding and DDC diet feeding mice livers, analysis of genomic occupancy of H3K27ac and H3k4me1 in hepatocytes from Arid1a WT and Arid1a liver-specific KO mice by ChIP-seq

first: Weiping

last: Li


title: Sox7 regulates lineage decisions in cardiovascular progenitor cells

status: Public on Jul 06 2019

city: Minneapolis

first: Lauren

last: Mills

state: MN

address: 420 Delaware St SE

department: Department of Pediatrics

institute: University of Minnesota

summary: Specification of the mesodermal lineages requires a complex set of morphogenetic events orchestrated by interconnected signaling pathways and gene regulatory networks. The transcription factor Sox7 has critical functions in differentiation of multiple mesodermal lineages, including cardiac, endothelial, and hematopoietic. Using a doxycycline-inducible mouse embryonic stem cell (mESC) line, we have previously shown that expression of Sox7 in cardiovascular progenitor cells promotes expansion of endothelial progenitor cells. Here, we show that the ability of Sox7 to promote endothelial cell fate occurs at the expense of the cardiac lineage. Using ChIP-Seq coupled with ATAC-Seq we identify downstream target genes of Sox7 in cardiovascular progenitor cells and, by integrating these data with transcriptomic analyses, we define Sox7-dependent gene programs specific to cardiac and endothelial progenitor cells. Further, we demonstrate a protein-protein interaction between SOX7 and GATA4 and provide evidence that Sox7 interferes with the transcriptional activity of Gata4 on cardiac genes. In addition, we show Sox7 modulates WNT and BMP signaling during cardiovascular differentiation. Our data represent the first genome-wide analysis of Sox7 function and reveal a critical role for Sox7 in regulating signaling pathways that affect cardiovascular progenitor cell differentiation.

overall_design: Sox7 function during mesoderm specification was investigated using doxycycline-inducible embryonic stem (ES) cells. ATAC-seq was used to identify changes in chromatin accessibility induced by Sox7 upon 1-day induction in cardiac (CPC) and endothelial (EPC) progenitors. ChIP-seq was used to determine Sox7 genomic occupancy in mouse ES cells undergoing specification (1-day and non-induced). Gene expression changes induced by Sox7 in mouse differentiating ES cells (12-hrs and non-induced) were assessed by RNA-seq. All data was collected from day 4 EBs.

first: Michelle

middle: J

last: Doyle

first: Alessandro

last: Magli

first: Cindy

last: Martin


title: A homeostatic Arid1a-dependent permissive chromatin state licenses hepatocyte responsiveness to liver injury-associated YAP signaling (ATAC-seq)

overall_design:
Normal hepatocytes and DDC-injured hepatocytes were isolated from Arid1a wild type and Arid1a liver specific KO mouse livers by a two-step liver collagenase perfusion and further purified by a series of low speed gravity centrifugation (3x2minx50g). DDC-injured hepatocytes were isolated from Sox9CreERT2:RFP reporter mice by gravity purification, and Sox9+ reprogrammed hepatocytes (LPLCs) were further isolated by sorting RFP+ hepatocytes based on the RFP expression via FACS. Biliary epithelia cells (BECs) were isolated from CK19CreERT2:RFP reporter mice by sorting RFP+ non parenchymal cells based on the RFP expression via FACS. The chromatin accessibility of normal hepatocytes, DDC-injured hepatocytes, LPLCs and BECs were identified via ATAC-seq.
Please note that each processed data .bw file was generated from both replicate samples and is linked to the corresponding Rep1 sample records.


title: The Molecular Dissection of the Oncogenic Role of ETS1 in the Mesenchymal Subtypes of Head and Neck Squamous Cell Carcinoma [ChIP-seq]

first: Satrajit

last: Sinha

summary: Mapping the genome-wide binding profile of ETS1 in a Mesenchymal HNSCC Cell Line using ChIP-Seq techniques

overall_design: The global binding loci of ETS1 in SCC25 cells, were mapped using ChIP-Sequencing analysis.  We performed three replicate experiments of high quality, using two different antibodies, C20 (Santa Cruz Biotechnology) and 501A (Bethyl Laboratories), and consensus binding events were determined using IDR analysis.  The ETS1 binding profiles in SCC25 were intersected with the global ETS1 Knockdown RNA-Seq analysis in order to identify the direct transcriptional targets of ETS1 in a Mesenchymal model of HNSCC.

first: Christian

last: Gluck

first: Isha

last: Sethi


title: PIP degron-stabilized Dacapo/p21Cip1 and mutations in ago act in an anti- versus pro-proliferative manner, yet both trigger an increase in Cyclin E levels

city: St Lucia

first: Stefan

last: Thor

state: QLD

address: Otto Bldg (81) room 620

department: School of Biomedical Sciences

country: Australia

institute: University of Queensland

summary: During cell cycle progression, the activity of the CycE-Cdk2 complex gates S-phase entry. CycE-Cdk2 is inhibited by CDK inhibitors (CKIs) of the Cip/Kip family, which include the human p21Cip1 and Drosophila Dacapo proteins. Both the CycE and Cip/Kip proteins are under elaborate control via protein degradation, mediated by the Cullin-Ring-Ligase (CRL) family of ubiquitin ligase complexes. The CRL complex SCFFbxw7/Ago targets phosphorylated CycE while p21Cip1 and Dap are targeted by the CRL4Cdt2 complex, binding to the PIP degron. The role of CRL-mediated degradation of CycE and Cip/Kip proteins during CNS development is not well understood. Here, we analyse the role of ago (Fbxw7) mediated CycE degradation and of Dap/p21Cip1 degradation during Drosophila CNS development. We find that ago mutants display over-proliferation, accompanied by elevated CycE expression levels. In contrast, expression of PIP degron mutant Dap/p21Cip1 transgenes inhibit proliferation. However, surprisingly, this is also accompanied by elevated CycE levels. Hence, ago mutation and PIP degron Cip/Kip transgenic expression trigger opposite effects on proliferation, but similar effects on CycE levels.

overall_design: Transcriptome analysis of Drosophila melanogaster embryos, at developmental stage 15-16, overexpressing the cell cycle inhibitor dacapo, both the wild type gene and degradation-stabilized versions.


title: A homeostatic Arid1a-dependent permissive chromatin state licenses hepatocyte responsiveness to liver injury-associated YAP signaling (RNA-seq)

overall_design: Liver transcriptional profiling in 2 weeks DDC-injured  Arid1a-WT and Arid1a-KO livers were generated by RNA-seq analysis.


title: ChIP-seq data for 2 NFKB1 subunits (p50, p65) from two donors

status: Public on Jul 05 2019

first: Diego

last: Calderon

address: 3720 15th Ave NE

institute: University of Washington

summary: To validate p50 binding at predicted regions of interest, we performed ChIP-seq on activated CD4+ T cells sorted from two donors.

overall_design: ChIP was measured from 2 NFKB1 subunits (p50, p65) from two donors.

last: Nguyen

first: Anja

last: Mezger


title: Transcriptomic profiling of the liver after Roux-en-Y gastric bypass in a humanized murine model of the metabolic syndrome

city: New York City

first: Dimitris

last: Nasias

state: New York

address: 550 1st Avenue

department: Langone Medical School

institute: New York University

summary: The metabolic syndrome (MetS) is attributed to a number of risk factors related to obesity and its comorbidities such as hypertension, increased blood glucose, hypertriglyceridemia and cardiovascular disease (CAD). Several mouse models have been used trying to understand the metabolic abnormalities occur in obesity and MetS. However, The ApoE*3Leiden.human Cholesteryl Ester Transfer Protein (ApoE3L.CETP) mouse model better describes the physiology and pathophysiology of the MetS upon high fat diet (HFD) feeding. Towards MetS treatment, Roux-en-Y gastric bypass (RYGB) is a surgical bariatric approach that allows to achieve sustained and long-term weight loss and to improve comorbidities of the MetS. The aim of this study was to elucidate whether improvements in lipid and glucose metabolism after RYGB surgery are body weight-dependent or not and to identify transcriptomic signatures related to these phenotypic observations.

overall_design: For this purpose, male ApoE*3Leiden.CETP (ApoE3L.CETP) mice fed Western type diet for 6 weeks underwent RYGB or Sham surgery. Sham groups were either fed ad libitum group (Sham AL) or were body weight-matched (BWm) to the RYGB mice, in order to identify surgical effects from body weight loss associated effects. The metabolic profile of mice was thoroughly assessed before and after surgery. Mice were sacrificed 20 days after surgery and the livers were collected to assess metabolic, histological and global gene expression changes after surgery. RNA from the three different cohorts of mice (Rygb group, 6 replicates/ Sham AL group, 5 replicates/ BWm group, 4 replicates) was labeled and hybridized to GeneChip™ Mouse Gene 2.0 ST Affymetrix expression arrays. The total of 15 arrays was analyzed to the Affymetrix Expression Console Software Version 1.4.1.46 by applying the robust multi-array average (RMA) algorithm. To determine the differentially expressed genes in the Affymetrix® Transcriptome Analysis Console (TAC) Software (Version 3.1.0.5), we applied the comparisons of the Rygb versus the Sham AL group and the Rygb versus BWm group under specific p-value and fold change thresholds (One Way Analysis of Variance: p-value <0.05 and absolute fold change ≥1.5).

last: Kardassis


title: RNA sequencing based comparative analysis of human hepatic progenitor cells and their niche from alcoholic steatohepatitis livers

city: Brussels

first: Stefaan

last: Verhulst

address: Laarbeeklaan 103, D022

department: BMWE

country: Belgium

institute: Vrije Universiteit Brussel

summary: Hepatic progenitor cells (HPCs) are small cells with a relative large oval nucleus and a scanty cytoplasm situated in the canals of Hering, that express markers of (immature) hepatocytes and cholangiocytes. HPCs are present in large numbers in alcoholic steatohepatitis (ASH), one of the leading causes of chronic liver disease. To date, the mechanisms responsible for proliferation and differentiation of human HPCs are still poorly understood and the role of HPCs in ASH development is unknown. In this study, we aimed to characterise human HPCs and their interactions with other cells through comparison, on both protein and RNA level, of HPC-enriched cell populations from adult human liver tissue using different isolation methods.   Fresh human liver tissue was collected from ASH explant livers and HPC-enriched cell populations were obtained via three different isolation methods: side population (SP), EpCAM and TROP-2 membrane marker isolation. Gene expression profiles of fluorescent activated cell sorted HPCs, whole liver extracts and laser microdissected HPC niches were determined by RNA sequencing.   Immunohistochemical evaluation of the isolated populations indicated the enrichment of HPCs in the SP, EpCAM+ and TROP-2+ cell populations. Pathway analysis of the transcription profiles of human HPCs showed an enrichment and activation of known HPC pathways like Wnt/β-catenin, TWEAK and HGF. Integration of the HPC niche profile suggests autocrine signalling by HPCs (TNF, PDGFB and VEGFA) as well as paracrine signalling from the surrounding niche cells including MIF and IGF-1. In addition, we identified IL-17A signalling as a potentially novel pathway in HPC biology.   In conclusion, we provide the first RNA-seq based, comparative transcriptome analysis of isolated human HPCs from ASH patients and revealed active signalling between HPCs and their surrounding niche cells in ASH livers and suggest that HPCs can actively contribute to liver inflammation.

overall_design: Via FACS isolation SP, EpCAM+ and TROP-2+ populations were isolated from 4 alcoholic hepatitis patients. From these patients, whole liver samples were taken containging all types of liver cell types. 3 of these patients were also used to isolate the HPCs with their surrounding niche via LMD.

first: An

last: Ceulemans

first: Matthias

last: Van Haele

first: Olivier

last: Govaere

first: Juan-Jose

last: Ventura

first: Leo

last: van Grunsven

first: Tania

last: Roskams


title: Gene expression of human T cells stimulated in the presence of TGF-β

city: Montreal

first: Jean-Sebastien

last: Delisle

state: Quebec

address: 5415, boulevard de l’Assomption

institute: Centre de recherche de l’Hôpital Maisonneuve-Rosemont

summary: Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine that dictates T-cell fates in various settings. Despite a wealth of data implicating TGF-β in the differentiation and homeostasis of both suppressive and inflammatory T cells, little is known about the role of TGF-β signaling in the development of memory T cells. This experiment was to correlate gene expression with the finding that TGF-β exposure during human T-cell stimulation ex vivo leads to the accumulation of T cells with central memory (tcm) markers.

overall_design: Gene expression quantification of TGF-β-treated (n=4) or untreated (n=3) CD4 and CD8 T cells was generated by deep sequencing, using Ion AmpliSeq™ Transcriptome Human Gene Expression Kit.

first: Amina

last: Dahmani

first: Valérie

last: Janelle

first: Jean-Sébastien


title: PHF7 Links H3K4 Methylation to H2A Ubiquitination During Spermiongenesis [RNA-seq]

first: Qing

last: Li

address: 320 Yueyang Road

department: institute of biochemistry and cell biology

institute: Chinese Academy of Science

summary: Histone ubiquitination has been suggested to serve as a “tag” for nucleosome removal during histone-to-protamine exchange that is essential for chromatin packaging in round spermatids. Here, we screen for putative E3 ligase and identify that the PHF7, containing both RING finger and PHD domains, is critical for H2A ubiquitination and histone removal. Mechanistically, its PHD domain as a histone code reader can specifically bind H3K4me3/me2 and its RING domain as a histone writer can ubiquinate H2A. PHF7 deficiency results in male infertility in mice by generating Phf7 knockout mice using CRISPR-Cas9 technology. Futhermore, we find impaired histone-to-protamine exchange leads to reduced chromatin compaction in Phf7 knockout sperm. Immunostaining and western blot further confirme abnormal retention of  core histones and reduced levels of protamine PRM1 and PRM2 in Phf7 knockout sperm. In order to exclude the possibility that Phf7 regulate the expession of protamine, we sorte the round spermatids from WT and Phf7 knockout females for RNA-seq. The transcriptomes in round spermatids are very similar between WT and Phf7 knockout mice. There are few differentially expressed genes in the two groups and the expression of protamine is also unchanged even if PHF7 deficiency. Therefore we find PHF7 has no effects on gene regulation in histone-to-protamine exchange.

overall_design: We isolated round spermatids from testes of WT and Phf7 knockout mice according to reported protocol (Bastos et al., 2005) as different sample for downstream analysis. Round spermatids mRNA profiles of 8-week old WT and Phf7 knockout mice were generated by deep sequencing, in triplicate, using Illumina HiSeq X10.

first: Xiukun

last: Wang

first: Jinsong


title: CRISPR-Cas9 mutagenesis of U937 cells for variants resistant to Yersinia pestis intoxication

status: Public on Jul 04 2019

city: Chicago

first: Yan

state: IL

address: 900 East 57th St.

department: Center for Research Informatics

institute: The University of Chicago

summary: The plague agent, Yersinia pestis, employs a type III secretion system (T3SS) to selectively destroy human immune cells, thereby enabling its replication in the bloodstream and transmission to new hosts via fleabite. The host factors responsible for the selective destruction of immune cells by plague bacteria were not known. Here we show that LcrV, the needle cap protein of the Y. pestis T3SS, binds N-formylpeptide receptor (FPR1) on human immune cells to promote the translocation of bacterial effectors.

overall_design: Y. pestis AM18, a DyfeAB variant of the vaccine strain EV76, is defective for iron and manganese scavenging. In broth cultures, Y. pestis AM18 secretes the T3SS effector named YopE via the lcrV-dependent T3SS pathway. Compared with the DlcrV variant (AM46), which cannot kill immune cells above control levels, Y. pestis AM18 infection resulted in modest killing of U937 human histiocytic leukemia cells differentiated into macrophages (U937 cells). Y. pestis POO1 is a variant of AM18 expressing yopE-dtx, a translational hybrid between the YopE effector and the mature domain of diphtheria toxin. Dtx is an ADP-ribosyltransferase that modifies elongation factor 2 (EF2) at its diphthamide residue to promote cell death. Y. pestis POO1 secretes YopE-Dtx and causes death of U937 macrophages in an lcrV-dependent manner. U937 cells were transduced with lentiviral libraries expressing RNA guided endonuclease Cas9 and short guide RNAs (sgRNA – 3 per gene) targeting 19,052 genes. After 12 hours of infection with Y. pestis POO1 or POO2 (yopE-dtx ΔlcrV), resistant cells were cultured with antibiotics to approximately 70% confluence and re-infected. The sgRNA sequences from surviving U937 cells were identified via next-generation sequencing (NGS) and candidate genes ranked based on the number of unique sgRNAs versus NGS reads. FPR1 stood out in three independent screens with the most abundant sgRNAs. FPR1 is a member of the GPCR family that activates immune cell chemotaxis and cytokine release in response to N-formylpeptides contained in bacteria or mitochondria.

first: Patrick

last: Osei-Owusu

first: Dominique

last: Missiakas


title: PHF7 Links H3K4 Methylation to H2A Ubiquitination During Spermiongenesis

summary: Histone ubiquitination has been suggested to serve as a “tag” for nucleosome removal during histone-to-protamine exchange that is essential for chromatin packaging in round spermatids. Here, we screen for putative E3 ligase and identify that the PHF7, containing both RING finger and PHD domains, is critical for H2A ubiquitination and histone removal. Mechanistically, its PHD domain as a histone code reader can specifically bind H3K4me3/me2 and its RING domain as a histone writer can ubiquinate H2A. So we want to use ChIP-seq to see whether all PHF7-binding peaks overlapped with those of H3K4me3. We report the application of ChIP-seq for high-throughput profiling of histone modifications in spermatids. By obtaining over 240M of sequences from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps and PHF7 binding position of late spermatids. We find binding peaks of PHF7, H3K4me3 and ub-H2A were mainly enriched at gene-regulatory elements (-5kb to +5kb from transcriptional start site), including promoter and enhancers, indicative of important roles in gene regulations. More than 96% (n=15419) of all PHF7-binding peaks overlapped with those of H3K4me3, supporting a specific binding of PHF7 to H3k4me3. Moreover, the common binding sites between PHF7 and H3K4me3 were associated with ub-H2A. Taken together, these results demonstrate that PHF7 ubiquitinate H2A through binding with H3K4me3.

overall_design: Examination of different histone modifications (H3K4me3 and ub-H2A) and PHF7 binding in late spermatids. We isolated late spermatids from testes of Phf7-KI-flag mice according to reported protocol (Bastos et al., 2005) as different sample for downsrream analysis.


title: Genome-wide alteration of 5-hydroxymethylcytosine in hypoxic-ischemic neonatal rat model of cerebral palsy

first: Yunpeng

last: Zhang

address: Longhang Road 1508 Jinshan distinct

department: jinshan hospital

institute: Fudan university

type: Methylation profiling by high throughput sequencing

summary: Our results revealed that hypoxic-ischemic brain injury decreased the overall 5hmC abundance in rat temporal cortex, and these results suggest that 5hmC modifications are involved in the cerebral palsy pathogenesis.

overall_design: Examination of DNA hydroxymethylation modifications in temporal lobe cortex of control rats and hypoxic-ischemic rats.

first: Bing


title: Expressing data from endothelial cells overexpressing the purinergic receptor P2Y2

city: Vienna

first: Wolfgang

last: Holnthoner

address: Donaueschingenstrasse 13

department: Ludwig Boltzmann Institute for Experimental and Clinical Traumatology

country: Austria

institute: Austrian Cluster for Tissue Regeneration

summary: Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation and vascular tone. A role for ATP release and P2Y receptors in angiogenesis has been implicated, however, the involvement of purinergic signalling in endothelial sprouting and vascular growth remains poorly understood. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Inhibition of P2Y2 using a selective antagonist or modulation of P2Y2 receptor expression significantly influenced sprouting and vascular tube formation. Mechanistically, overexpression of P2Y2 in endothelial cells resulted in increased expression of the pro-angiogenic molecules CXCR4, CD34 and angiopoietin-2, while expression of Tie2 and VEGFR-2 decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed, indicating that enhanced receptor expression, and not activation, is the primary trigger for angiogenesis. Our data suggest that P2Y2 receptors have an essential function in angiogenesis through cross-talk of P2Y2 and VEGFR-2 and that P2Y2 may represent a therapeutic target to regulate blood vessel growth.

overall_design: 4 HUVEC samples total, two samples per group, control and treatment are represented with two biological replicates (= two different batches of cells)

first: Severin

last: Mühleder

first: José

last: Basílio

first: Johannes

last: Pröll

first: Christian

last: Siehs


title: RNA-seq analysis for characterization of the TCR repertoire

status: Public on Jul 02 2019

first: Ying

address: 117 Pleasant St S.E.

department: Minnesota Supercomputing Institute

summary: Immune tolerance to allografts has been pursued for decades as an important goal in transplantation. Administration of apoptotic donor splenocytes effectively induces antigen-specific tolerance to allografts in murine studies. Here we show that two peritransplant infusions of apoptotic donor leukocytes under short-term immunotherapy with antagonistic anti-CD40 antibody 2C10R4, rapamycin, soluble tumor necrosis factor receptor and anti-interleukin 6 receptor antibody induce long-term (≥1 year) tolerance to islet allografts in 5 of 5 nonsensitized, MHC class I-disparate, and 1 MHC class II DRB allele-matched rhesus macaques. Tolerance in our preclinical model is associated with a regulatory network, involving antigen-specific Tr1 cells exhibiting a distinct transcriptome and indirect specificity for matched MHC class II and mismatched class I peptides. Apoptotic donor leukocyte infusions warrant continued investigation as a cellular, nonchimeric and translatable method for inducing antigen- specific tolerance in transplantation.

overall_design:
Animals (Amazo, Rocket and Nebula) were infused with Apoptotic Donor Leucocytes on day -7 and day +1. PBMCS were isolated from serial blood samples collected post-ADL infusion and used for TCR sequencing.
Total RNA was extracted from frozen PBLs using Rneasy Plus Universal (Qiagen) and first strand cDNA was created from the poly A tailed fraction of total RNA, SMART™ Approach was used for Full-Length cDNA Library Construction.
Enriched VDJ amplicons from each sample were uniquely dual-indexed by PCR for multiplexing compatibility during sequencing.
Indexed amplicon libraries were pooled equimolarly and cleaned with SPRI beads (Ampure XP, Beckman Coulter). The final pool was sequenced on a MiSeq (Illumina) 300 bp paired end run (v3 kit).
Raw, QC-ed short reads were cleaned via trimmomatic81 using following  parameters (ILLUMINACLIP:all_illumina_adapters.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:70).
The preprocessed reads were directly input into MiXCR82 for TCR profiling with default setting (https://mixcr.readthedocs.io/en/latest/rnaseq.html).
KnownTRB clonetypes in rhesus monkeys was used as reference.
The resulted TRB clonetypes were further filtered using customized threshold with a clone fraction of ≥ 0.5%.
Data from Rocket is presented in the manuscript.

first: Sabarinathan

last: Ramachandran

first: Amar

last: Singh

first: Nagendra

last: palani

first: Bernhard

last: Hering


title: Transcriptome analysis of extruded germline from wild-type C. elegans at different temperatures and under inhbition of germ stem cell proliferation

status: Public on Jul 03 2019

city: Cologne

first: David

last: Vilchez

state: NRW

address: Joseph Stelzmann Strasse, 26

institute: CECAD - Cluster of Excellence

summary: To assess the mechanism by which adult germ cells induce cbs-1 expression in the intestine at cold temperature, we performed transcriptome analysis of extruded germ lines from wild-type worms upon iff-1 knockdown or temperature increase

overall_design: We extruded germ line of iff-1 RNAi-treated worms at 15°C and empty vector (EV) RNAi-treated worms at 20°C and compared to the germ line of EV RNAi-treated worms at 15°C.

first: Hyun Ju

last: Lee

first: Alireza

last: Noormohammadi

first: Seda

last: Koyuncu

first: Milos

last: Simic

first: Giuseppe

last: Calculli

first: Marija

last: Herholz

first: Aleksandra

last: trifunovic


title: Microbiota-driven transcription signatures in lung stroma cells

city: London

first: Andreas

last: Wack

address: 1 Midland Road

institute: The Francis Crick Institute

summary: Commensal bacteria have been shown to influence the reactivity of immune cells in the gut and in other organs. This study aims to assess the impact of microbiota on transcription signatures in lung stroma cells. Samples were generated from mice that were either left on plain water, on water with antibiotics, or on water with antibiotics followed by faecal transplant to reverse the antibiotics effect. CD45 negative cells were MACS-purified from the lung, and RNA seq was performed on samples.

overall_design: Biological triplicates or quadruplicates for each of the three treatment groups were prepared and processed.

first: Stefania

last: Crotta


title: Transcriptome Analysis of IL-33-Deficient Early Developing B Cells

city: Nashville

first: Matthew

middle: Tyler

last: Stier

state: TN

address: 1161 21st Avenue South

department: Internal Medicine

institute: Vanderbilt University Medical Center

summary: We identified that IL-33 is expressed in pro-B (B220+ IgM- CD19+ CD43+) and large pre-B cells (B220+ IgM- CD19+ CD43- FSC-Ahi SSC-Ahi) in mice.  We assessed for IL-33-dependent gene expression differences in these B cell stages by performing bulk RNA-sequencing of FACS-purified pro-B and large pre-B cells from WT and IL-33-deficient 8 week old adult, naive female mice.

overall_design: Examination of poly-A enriched transcriptome of WT and IL-33-deficient pro-B and large pre-B cells.

first: R

last: Stokes Peebles

first: Christine

last: Eischen


title: Single-cell protein-expression map of bone marrow reveals stromal cell subset selection with stress

city: Boston

middle: T

last: Scadden

state: MA

address: 185 cambridge street

department: CRM

institute: MGH

summary: We identified CD73+ bone marrow stromal cells as hematopoietic niche candidates. To further validate our finding, we tested the functional role of CD73 during bone marrow transplantation using CD73-/- mice. We evaluated the transcriptome of transplanted WT hematopoietic stem and progenitor cells from WT or CD73-/- recipient mice and found changes in metabolic, cell cycle and signaling genes.

overall_design: We isolated LKS (Lin-Kit+Sca1+) cells from bone marrow of WT and CD73-/- recipient mice by flow cytometry, collected RNA, and ran RNA-seq.


title: Microarray based gene expression comparison between wild type and yap-1(ys38) C. elegans embyos

city: Seoul

first: Junho

address: Gwanak-ro 1

department: Department of Biological Sciences

country: South Korea

institute: Seoul National University

summary: Although multiple determinants for establishing polarity in membranes of epithelial cells have been identified, the mechanism for maintaining the apicobasal polarity is not fully understood. Here, we show that the conserved Hippo kinase pathway plays a role in the maintenance of apicobasal polarity in the developing intestine of Caenorhabditis elegans. We screened suppressors of the mutation in wts-1, the gene encoding the LATS kinase homolog, whose deficiency leads to the disturbance of the apicobasal polarity of the intestinal cells and the eventual death of organisms. Multiple alleles of yap-1 and egl-44, each encoding the worm homologs of YAP/Yki and TEAD/Sd, respectively, were identified as suppressors. WTS-1 directly bound to YAP-1 and inhibited its nuclear accumulation in intestinal cells. We also found that NFM-1, the worm homolog of NF2/Merlin, functioned in the same genetic pathway as WTS-1 to regulate YAP-1 for maintaining cellular polarity. The transcriptome analysis using microarray identified several target candidates of the YAP-1-EGL-44 complex including TAT-2, encoding a putative P-type ATPase. In summary, we have delineated the conserved Hippo pathway in worms consisting of NFM-1-WTS-1-YAP-1-EGL-44 and prove that the proper regulation of YAP-1 by upstream NFM-1 and WTS-1 is esssential for the maintenance of apicobasal membrane identities of the growing intestine

overall_design: To identify target genes of the Hippo pathway for cellular polarity maintenance, we compared the expression levels of all the genes of yap-1(ys38) mutants to those of wild type worms using a microarray experiment. To collect embryos of N2 and yap-1(ys38), gravid adults from 10 plates of 10cm NGM were bleached. Total RNA was extracted from 3 biological replicates by the freezing-thawing method. The isolated RNA was qualified and used as templates for cDNA synthesis in the Genome Research Facility of the Seoul National University using an Affymetrix chip (Seolin bio, Seoul, Korea) containing 22,625 gene probes targeting 22,150 genes from C. elegans. The chip data were analyzed using the Microarray Suite version 5.0 (MAS 5.0) with global scaling performed as the normalization method using Genplex ver.3.0 (istech.inc). The results of one sample of N2 were significantly different from those of the other two replicates, therefore were excluded from the analysis. Significant genes were obtained by calculating fold-change and Welch’s T-test (P-value ≤ 0.05) using Volcano Plot and filtering A (absent) by detection call.

first: Hanee

first: Junsu

last: Kang

first: Soungyub

last: Ahn


title: Reorganization of 3D Genome Structure May Contribute to Gene Regulatory Evolution in Primates

city: South San Francisco

first: Ittai

middle: Ethan

last: Eres

state: CA

address: 750 Gateway Boulevard

department: CRADI

institute: Amgen

summary: A growing body of evidence supports the notion that variation in gene regulation plays a crucial role in speciation and adaptation. However, a comprehensive functional understanding of the mechanisms underlying regulatory evolution remains elusive. In primates, one of the crucial missing pieces of information towards a better understanding of regulatory evolution is a comparative annotation of interactions between distal regulatory elements and promoters. We used Hi-C to characterize 3D regulatory interactions in induced pluripotent stem cells (iPSCs) from humans and chimpanzees (Pan troglodytes). We overlaid these data with previously-collected RNA-seq data from the same cell lines. Generally, we observe that fine-scale distal 3D genomic interactions are conserved in humans and chimpanzees, but higher order genomic organization of contacts, such as TADs, are not as conserved. Inter-species differences in fine-scale 3D locus-locus interactions are often associated with gene expression differences between the species. To provide additional functional context to our observations, we also considered previously published chromatin data from human iPSCs. We found strong enrichment for both active and repressive marks in distal 3D genomic interactions that are both divergent between species and that are associated with inter-species differences in gene expression. Overall, our data demonstrates that, as expected, an understanding of 3D genome reorganization is key to explaining regulatory evolution.

sample_organism:
Pan troglodytes
Homo sapiens

platform_organism:
Pan troglodytes
Homo sapiens

overall_design: Examination of Hi-C contacts in 4 individual iPSC lines from humans and 4 individual iPSC lines from chimpanzees (both sex balanced).

first: Kaixuan

last: Luo

first: Chiaowen

last: Hsiao

first: Lauren

last: Blake

first: Yoav

last: Gilad


title: An Lkb1-Sik axis suppresses tumor growth and controls differentiation [Tumor Barcode Sequencing]

city: Stanford

first: Christopher

last: Murray

state: California

address: 1291 Welch Rd. B261, Beckman Center

department: Genetics

institute: Stanford University

summary:
Background: LKB1 is among the most frequently altered tumor suppressors in lung adenocarcinoma. Despite being implicated in the regulation of a variety of cellular processes, the mechanisms by which LKB1 constrains lung tumor growth and progression remains an area of intense investigation.
Purpose: To assess the impact of CRISPR/Cas9-mediated targeting of the canonical substrates of Lkb1 on tumor size within the context of a genetically engineered mouse model of oncogenic Kras-driven lung adenocarcinoma.
Approach: Comparisons of tumor size across genetic perturbations were conducting by measuring tumor sizes via tumor barcode sequencing (Rogers, et al. 2017 Nature Methods). Briefly, tumors were initiated in KrasLSL-G12D/+;R26LSL-Tomato, KrasLSL-G12D/+;Lkb1flox/flox;R26LSL-Tomato;H11LSL-Cas9, and KrasLSL-G12D/+;R26LSL-Tomato;H11LSL-Cas9 mice using a pool of Lenti-sgRNA/Cre vectors that encode a cassette comprised of an sgRNA-specific ID along with clonal identifier. Following, tumor development/progression, the two-component lentiviral cassettes integrated within transduced populations were amplified from genomic DNA extracted from whole lungs homogenates and subjected to next-generation sequencing. From the resulting reads, barcode pileups were generated and filtered prior to translation to absolute numbers of cancer cells via normalization to spiked-in samples of known quantities of cancer cells. The resultant tumor size distribution were then analyzed by multiple statistical measures as described by Rogers, et al.
Results: Lkb1 targeting dramatically increased tumor size in the Kras-only setting relative to functionally inert sgRNAs. Unlike other families of Lkb1 substrates, the targeting of the salt inducible kinase (Sik) family increased tumor growth comparable to Lkb1 targeting. In contrast to targeting the paralogs Sik1 and Sik3 individually, dual targeting of Sik1 and Sik3 enhanced tumor growth. Sik targeting in the Lkb1-deficient setting also modestly increased tumor size whereas Lkb1 targeting had no effect.
Conclusions: Siks are potent tumor suppressors belonging the family of canonical Lkb1 substrates. Siks retain some tumor-suppressive activity in the absence of Lkb1.

overall_design: 23 mice total. Eight replicates for KrasLSL-G12D/+;Lkb1flox/flox;R26LSL-Tomato;H11LSL-Cas9, 5 replicates for KrasLSL-G12D/+;R26LSL-Tomato, and ten KrasLSL-G12D/+;R26LSL-Tomato;H11LSL-Cas9.

middle: W

first: Monte

last: Winslow


title: The Toll signaling pathway targets the insulin-like peptide Dilp6 to inhibit growth in Drosophila

city: Charlottesville

first: Miyuki

last: Suzawa

state: VA

address: 1340 Jefferson Park Ave 5015 Pinn Hall

department: Pharmacology

institute: University of Virginia

summary: To identify genes that mediate altered communication between fat body and peripheral tissues, we report the gene expression changes in Drosophila third instar larval fat bodies with or without constitutively-active Toll (Toll10b) to activate innate immune signaling, myristoylated Akt (myrAkt) to activate insulin signaling, or both transgenes to bypass the block from Toll signaling to the upstream part of the insulin signaling pathway

overall_design: Comparison of RFP/GFP (Control), Toll10b/GFP (Toll10b), RFP/myrAkt (myrAkt), and Toll10b/myrAkt (Toll10b + myrAkt)

first: Nigel

last: Muhammad

first: Michelle

last: Bland


title: A distinct motif for numerous CTCF binding sites in the murine IgH locus

first: Yuka

last: Takeuchi

address: 185 Cambridge Street

department: Molecular biology

type: Genome binding/occupancy profiling by genome tiling array

summary: Our studies have revealed that a large class of CTCF binding sites – namely the upstream sites – conform neither to a conformational nor a local recombinase activating role. Their conserved spatial distances upstream of V gene segments suggests a possible role in insulating V gene segments from neighboring V gene segments. In any case, understanding the sequence determinants of CTCF binding to the murine IgH locus should facilitate future studies evaluating how IgH locus accessibility regulates CTCF binding as well as the functions that CTCF plays in regulating the recombinational accessibility of VH gene segments during B cell development.

overall_design: The sequence determinants of CTCF binding to the murine IgH locus in murine primary Rag-deficient and wild type proB cultured in the presence of IL-7.

last: Namiki

first: David

middle: N

last: Ciccone

first: Changfeng

last: Chen

first: Katrina

middle: B

last: Morchead

first: Andrew

last: Wood

first: Colette

last: Johnston

first: John

last: Morris

first: Yanqun

last: Wang

first: Ruslan

last: Sadreyev

first: Anne

last: Corcoran

first: Adam

last: Matthews

first: Marjorie

last: Oettinger


title: RNA sequencing of mTC and mTN cells

city: Stockholm

first: Martin

last: Bergö

address: Hälsovägen 7C (Neo building)

department: Department of Biosciences and Nutrition

country: Sweden

institute: Karolinska Institute

summary: The goal of the study is to define the impact of long-term administration of antioxidants on lung cancer progression. mTC and mTN cell lines were established from mice with KrasG12D/+ lung tumors 58 weeks post intranasal Cre-delivery. mTC cells comes from control mice, mTN cells come from NAC-treated mice. Mice were maintained on a mixed C57BL/6-129/Sv genetic background

overall_design: 3 and 4 biological replicates respectively (3 mTC and 4 mTN cell lines) were analyzed

last: Bergo

first: Volkan

middle: I

last: Sayin

first: Clotilde

last: Wiel


title: Tissue resident macrophages promote renal cystic disease

city: Oklahoma City

first: Kurt

last: Zimmerman

state: Oklahoma

address: 940 Stanton L Young Blvd

institute: University of Oklahoma Health Sciences Center

summary: We show that resident macrophages accumulate in cilia mutant mice prior to cyst formation and that inhibition of resident macrophage accumulation reduces cystic kidney disease.

overall_design: We analyzed gene expression of R2a (CD11c+) and R2b (CD11c-) resident macrophages isolated from adult, wild type mice. In these experiments, we isolated RNA from 6-8 mice and pooled the RNA together prior to sequencing.

first: Cheng

last: Song

first: Zhang

last: Li

first: Jeremie

last: Lever

middle: K

last: Crossman

first: Addison

last: Rains

first: Ernald

last: Aloria

first: Nancy

last: Gonzalez

middle: R

last: Bassler

first: Juling

last: Zhou

first: Michael

last: Crowley

first: Dustin

middle: Z

last: Revell

first: Zhaoqi

last: Yan

first: Dan

last: Shan

first: Etty

last: Benveniste

first: James

middle: F

last: George

first: Michal

last: Mrug

first: Bradley

last: Yoder


title: Targeted RNA-sequencing Facilitates Quantitative Analysis of RB and E2F3 in mouse Sertoli cells

city: Turku

first: Emmi

last: Rotgers

address: Kiinamyllynkatu 10

department: Institute of Biomedicine

country: Finland

institute: University of Turku

summary: A Sertoli cell specific knockout of retinoblastoma protein (SC-RbKO) results in a complete disruption of spermatogenesis in mice. In this project the SC-RbKO mice were cross-bred with E2F3 floxed mice to see if a double knockout would rescue the phenotype associated with RB loss.

overall_design: Gene expression analysis by Targeted RNA-sequencing between SC-E2F3KO and Wild type at 2 and 5 months old testes and among SC-Rb-/-E2F3+/+, SC-Rb-/-E2F3+/-, SC-Rb-/-E2F3-/- and Wild type at 2,5 months old testes were performed.


title: FICC-Seq: A method for enzyme-specified profiling of methyl-5-uridine in cellular RNA

city: Bath

first: Shobbir

last: Hussain

address: Claverton Down

department: Department of Biology and Biochemistry

institute: University of Bath

summary: Methyl-5-uridine (m5U) is one the most abundant non-canonical bases present in cellular RNAs, and is formed via catalytic conversion of uridines. In yeast, m5U is known to be present at position 54 of tRNAs where modification is catalysed by the methyltransferase Trm2. Although the mammalian enzymes that catalyse m5U formation are yet to be identified via experimental evidence, based on sequence homology to Trm2, two candidates currently exist, TRMT2A and TRMT2B. Here we developed a genome-wide single-nucleotide resolution mapping method, Fluorouracil-Induced-Catalytic-Crosslinking-Sequencing (FICC-Seq), in order to map the relevant enzymatic targets in cellular RNA. We demonstrate that TRMT2A is responsible for the majority of m5U present in human RNA, and that it ubiquitously targets U54 of cytosolic tRNAs. By comparison to current methods, we show that FICC-Seq is a particularly robust method for accurate and reliable detection of relevant enzymatic target sites. Our associated demonstration of irreversible human m5U enzyme-RNA crosslinking in vivo following 5-flurouracil exposure is further intriguing, as it suggests a tangible mechanism for a previously suspected RNA-dependent route of 5-fluorouracil-mediated cytotoxicity.

overall_design: Examination of novel FICC-Seq method in comparison with iCLIP and miCLIP.

first: Jean-Michel

last: Carter

first: Warren

last: Emmett

first: Igor

last: Ruiz de los Mozos

first: Annika

last: Kotter

first: Mark

last: Helm

first: Jernej

last: Ule


title: Identification of novel microRNAs and characterization of microRNA expression profiles in zebrafish ovarian follicular cells

status: Public on Jul 01 2019

city: Toronto

first: Chun

last: Peng

state: ONTARIO

address: 4200 keele street

department: Biology

institute: YorkU

type: Non-coding RNA profiling by high throughput sequencing

summary: MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression primarily at the post-transcriptional levels and thereby play important roles in regulating many physiological and developmental processes. Oocyte maturation in fish is induced by hormones produced from the hypothalamus, pituitary, and ovary. Gonadotropin-releasing hormone (GnRH) stimulates the secretion of luteinizing hormone (LH), which in turn, induces the secretion of maturation-inducing hormone (MIH) from the ovary.  It is documented that small early vitellogenic (or stage IIIa) follicles are unable to undergo oocyte maturation whereas oocytes in mid- to late vitellogenic (stage IIIb) follicles can be induced by LH and MIH to become mature.  To determine whether miRNAs may be involved in the growth and acquisition of maturational competency of ovarian follicles, we determined the miRNA expression profiles in follicular cells collected from stage IIIa and IIIb follicles using next-generation sequencing.  It was found that miRNAs are abundantly expressed in the follicular cells from both stages IIIa and IIIb follicles. Furthermore, bioinformatics analysis revealed the presence of 214 known, 31 conserved novel and 44 novel miRNAs in zebrafish vitellogenic ovarian follicular cells. Most mature miRNAs in follicular cells were found to be in the length of 22 nucleotides. Differential expression analysis revealed that 11 miRNAs were significantly up-regulated, and 13 miRNAs were significantly down-regulated in the stage IIIb follicular cells as compared with stage IIIa follicular cells. The expression of four of the significantly regulated miRNAs, dre-miR-22a-3p, dre-miR-16a, dre-miR-181a-3p, and dre-miR-29a, was validated by real-time PCR. Finally, gene enrichment and pathway analyses of the predicted targets of the significantly regulated miRNAs supported the involvement of several key signaling pathways in regulating ovarian function, including oocyte maturation. Taken together, this study identifies novel zebrafish miRNAs and characterizes miRNA expression profiles in somatic cells within the zebrafish ovarian follicles.  The differential expression of miRNAs between stage IIIa and IIIb follicular cells suggests that these miRNAs are important regulators of zebrafish ovarian follicle development and/or oocyte maturation.

overall_design: miRNA seq analysis in stage IIIa and IIIb vitellogenic zebrafish follicular cells.

first: Yara

last: Zayed

first: Xin

last: Qi


title: Nut/Hos1 and Sas2/Rpd3 control H3 acetylation of two different sets of osmotic stress-induced genes

city: Burjassot

first: Paula

last: Alepuz

state: Valencia

address: Doctor Moliner, 50

department: Biochemistry and molecular biology

country: Spain

institute: University of Valencia

summary:
Epigenetic information is able to interact with the cellular environment and therefore it could be especially useful for reprogramming gene expression in response to a physiological perturbation. In fact, genes induced or repressed by osmotic stress undergo significant changes in the levels of various histone modifications, especially the acetylation levels of histone H3. Exposure of yeast to high osmolarity results in the activation of p38-related SAPK, Hog1, which plays a central role in the control of gene expression. Therefore, two types of mechanisms converge in gene regulation by stress: that of the epigenetic signals and that of the trans-acting protein factor. We have evaluated the connection between the presence of Hog1 and the changes on acetylation of histone H3 in stress-regulated genes. Furthermore, we asked which enzymatic activities are involved in the changes produced in the acetylation of H3 linked to gene activation by osmotic stress.
We have demonstrated that increase of acetylation of lysines 9 and 14 of H3 produced on induced genes during stress positively correlates between them and are largely dependent on Hog1 at genome-wide level. Conversely, decrease in acetylation on repressed genes was not dependent on Hog1. However, the absence of Hog1 produces different and even opposite effects on the induction and acetylation of H3 of each gene, which suggests that Hog1 influences both processes through different mechanisms. Additionally, we found that in genes up-regulated during osmotic stress in a Msn2/Msn4-independent manner, the state of acetylation of lysine 9 of H3 was altered in strains deficient in Nut1 HAT and Hos1 HDAC; meanwhile for Msn2/Msn4-dependent induced genes H3k9 acetylation varies in strains deficient in Sas2 HAT and Rpd3 HDAC. During induction by osmotic stress at least two different mechanism act on H3k9 acetylation of two differentiated set of induced genes, one of them would act through Nut1/Hos1, while the other through Sas2/Rpd3. The results presented here show new and unexpected participants in the process of the regulation of gene in response to environmental perturbations.

overall_design:
Histone H3 acetylation at lysines K9 and K14 respect signal for total H3 protein, both measured by ChIP, in wt (BY4742) and hog1delta yeast cells under non-stress and 10 min of 0.4 M NaCl treatment. One replica experiment.
The hog1Δ data obtained in this study was compared to the wild type data obtained by Magraner-Pardo et al., 2014 (GSE41587).

first: María

middle: E

last: Pérez-Martínez

first: Marta

last: Benet

first: Vicente

last: Tordera


title: Gene expression profiling by RNA-seq in hTert-HME1 cell line treated with control or BRCA2 siRNAs and grown with or without EGF (epithelial growth factor)

city: Palo Alto

first: Michael

last: Snyder

address: 3165 Porter Dr

summary: Mammary epithelial cells were treated with control or BRCA2 siRNAs, then allowed to recover for 4 passages, then grown in mammary epithelial growth media supplemented with or without EGF (epithelial growth factor) for 2 passages (6 days).

overall_design: Control siRNA transfection grown in media +EGF (3 replicates), control siRNA transfection grown in media -EGF (2 replicates), BRCA2 siRNA transfection grown in media +EGF (3 replicates), BRCA2 siRNA transfection grown in media -EGF (3 replicates)

first: Joshua

last: Gruber

middle: P


title: ChIP sequencing of BACH1 in mTC and mTN cells

first: Martin

last: Bergö

overall_design: Examination of BCH1 binding to DNA in mTC, and mTN cells (3+3 biological replicates) and 1 mTN KO for BACH1

middle: O

last: Bergo

first: Volkan

last: Sayin

first: Clotilde

last: Wiel


title: RNA sequencing for transcriptional profiling of cardiac and cavity macrophages

status: Public on Jul 16 2019

city: Calgary

first: Justin

last: Deniset

address: University of Calgary 2500 University Dr. NW

institute: University of Calgary

summary: We report that cavity macrophages from the pericardial, pleural and peritoneal cavities have distinct expression profile from cardiac macrophages

overall_design: Comparison of sorted macrophages from the pericardial cavity, pleural cavity, peritoneal cavity, and heart under baseline conditions. 4 replicates per cavity or tissue were included.

first: Paul

last: Kubes

first: Stephen

last: Brooks