title: Nascent RNA antagonises the interaction of a set of regulatory proteins with chromatin

city: London

first: Richard

last: Jenner

address: 72 Huntley Street

department: Cancer Biology

institute: UCL Cancer Institute

summary: A number of regulatory factors are recruited to chromatin by specialised RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonised the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors including BAF, NuRD, EHMT1 and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonising the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.

overall_design: iCLIP for P-TEFb, LARP7 and input in mESC treated with either DMSO or pla-B. Also RNA-seq from chromatin or whole cell extract from mESC treated with triptolide or flavopiridol for 0, 1, 3 or 9 hours and also from permeabilised mock or RNaseA-treated mESC. Total RNA and polyA+ RNA was sequenced from the chromatin fraction to measure mature polyA+ and nascent RNA associated with chromatin and polyA+ RNA was sequenced from the whole cell extract samples to measure mature polyA+ RNA.

first: Lenka

last: Skalska

first: Victoria

last: Begley

first: Manuel

last: Beltran

first: Saulius

last: Lukauskas

first: Garima

last: Khandelwal

first: Peter

last: Faull

first: Amandeep

last: Bhamra

last: Tavares

first: Rachel

last: Wellman

first: Andrey

last: Tvardovskiy

first: Benjamin

last: Foster

first: Igor

last: Ruiz de los Mozos

first: Javier

last: Herrero

first: Silvia

middle: P

last: Surinova

first: Ambrosius

last: Snijders

first: Till

last: Bartke

middle: G


title: HMGB1 as a rheostat of chromatin topology and RNA homeostasis on the path to senescence [sCLIP]

status: Public on Jun 23 2021

city: Cologne

first: Yulia

last: Kargapolova

address: Robert-Koch-Straße 21

department: CMMC

country: Germany

institute: Cologne University

type: Expression profiling by high throughput sequencing

summary: Spatial organization and gene expression of mammalian chromosomes are maintained and regulated in conjunction with cell cycle progression. This link is perturbed once cells enter senescence. The highly abundant HMGB1 protein, known to associate with bent and looped DNA, is actively depleted from senescent cell nuclei to act as an extracellular proinflammatory stimulus. Despite its physiological importance, we still lack understanding of the positioning and functional roles of HMGB1 on chromatin in vivo. To address this, we mapped HMGB1 binding genome-wide in different primary cells using a tailored protocol. We then integrated ChIP-seq and Hi-C data with a knot theory approach to uncover HMGB1 demarcation at the boundaries of particular topologically-associating domains (TADs). These TADs harbor genes involved in the key proinflammatory leg of the senescent transcriptional program. Moreover, we used sCLIP and siRNA-mediated knockdown to show that HMGB1 is a bona fide RNA-binding protein also affecting splicing choices. Together, our findings highlight a broader than hitherto assumed role for HMGB1 in chromatin homeostasis connected to cell cycle potency, and allow us to postulate a rheostat model for HMGB function with implications in cancer biology.

overall_design: sCLIP was performed to assess the RNA-binding properties of HMGB1 and HMGB2 proteins in human fibroblast cell line IMR90-79 .

first: Argyris

last: Papantonis


title: ActD timecourse in RAW264.7 cells

city: Mannheim

first: Johanna

middle: Daniela

last: Schott

address: Franz-Volhard-Straße 6

department: Mannheim Institute for Innate Immunoscience

institute: Heidelberg University

summary: mRNAs are generally assumed to be loaded instantly with ribosomes upon entry into the cytoplasm. To measure ribosome density on nascent mRNA, we developed nascent Ribo-Seq (nRibo-Seq) by combining Ribo-Seq with progressive 4-thiouridine labelling. In mouse macrophages, we experimentally determined, for the first time, the lag between the appearance of nascent RNA and its association with ribosomes, which was calculated to be 20 - 22 min for bulk mRNA, and approximated the time it takes for mRNAs to be fully loaded with ribosomes to be 41 - 44 min. Notably, ribosomal loading time is adapted to gene function as rapid loading was observed with highly regulated genes. The lag and ribosomal loading time correlate positively with ORF size and half-life, and negatively with tRNA adaptation indices. Similar results were obtained in mESCs, where the lag between IN and FP was even more pronounced with 35 - 38 min. We validated our measurements after stimulation of macrophages with lipopolysaccharide, where the lag between cytoplasmic and translated mRNA leads to a corresponding uncoupling between input and ribosome-protected fragments. Uncoupling is stronger for mRNAs with long ORFs or half-lives, a finding we also confirmed at the level of protein production by nascent chain proteomics. As a consequence of the lag in ribosome loading, ribosome density measurements are distorted when performed under conditions where mRNA levels are far from steady state expression, and transcriptional changes affect ribosome density in a passive way. This study uncovers an unexpected and considerable lag in ribosome loading, and provides guidelines for the interpretation of Ribo-Seq data taking passive effects on ribosome density into account.

overall_design: Transcriptional shut-off with ActD followed by RNA-Seq to determine mRNA half-lives on a transcriptome-wide scale in RAW264.7 cells

first: Sonja

last: Reitter

first: Doris

last: Lindner

first: Jan

last: Grosser

first: Marius

last: Bruer

first: Anjana

last: Shenoy

first: Tamar

last: Geiger

first: Arthur

last: Mathes

first: Gergana

last: Dobreva

first: Georg

last: Stoecklin


title: Cardiomyocyte-specific Srsf3 deletion reveals a mitochondrial regulatory role

city: Sherbrooke

first: Michelle

middle: S

last: Scott

state: Quebec

address: 3201, rue Jean-Mignault

country: Canada

institute: University of Sherbrooke

summary: Serine-rich splicing factor 3 (SRSF3) was recently reported as being necessary to preserve RNA stability via an mTOR mechanism in a cardiac mouse model in adulthood. Here, we demonstrate the link between Srsf3 and mitochondrial integrity in an embryonic cardiomyocyte-specific Srsf3 conditional knockout (cKO) mouse model. Fifteen-day-old Srsf3 cKO mice showed dramatically reduced (below 50%) survival and reduced the left ventricular systolic performance, and histological analysis of these hearts revealed a significant increase in cardiomyocyte size, confirming the severe remodeling induced by Srsf3 deletion. RNA-seq analysis of the hearts of 5-day-old Srsf3 cKO mice revealed early changes in expression levels and alternative splicing of several transcripts related to mitochondrial integrity and oxidative phosphorylation. Likewise, the levels of several protein complexes of the electron transport chain decreased, and mitochondrial complex I-driven respiration of permeabilized cardiac muscle fibers from the left ventricle was impaired. Furthermore, transmission electron microscopy analysis showed disordered mitochondrial length and cristae structure. Together with its indispensable role in the physiological maintenance of mouse hearts, these results highlight the previously unrecognized function of Srsf3 in regulating the mitochondrial integrity.

overall_design: Mouse heart tissues expressing Srsf3 are compared with Srsf3 cardiomyocyte conditional knockout, each with 3 biological replicates

first: Audrey-Ann

last: Dumont

first: Lauralyne

first: Delong

last: Zhou

first: Hugo

last: Giguère

first: Chantal

last: Pileggi

first: Mary-Ellen

last: Harper

first: Denis

last: Blondin

first: Mannix

last: Auger-Messier


title: RNA-seq analysis of human pancreas cell line SU8686 treated with DOT1L inhibitor (SGC0946) or DMSO

city: Shanghai

first: Zhiwei

last: Liu

address: Zuchongzhi Road 555

country: China

institute: Chinese Academy of Sciences

summary: To understand the pathway alteration associated with DOT1L inhibition, we performed RNA-seq analysis of DOT1L inhibitor treated human pancreas cell line SU8686. We reported the changed pathways.

overall_design: The SU8686 cells were treated with 20 μM SGC0946 or DMSO for 72 hr (triplicate)

first: Yingluo

first: Lili

last: Qian

first: Shangwen

last: Jiang

first: Xiameng

last: Gai

first: Shu

last: Ye

first: Yuehong

last: Chen

first: Xiaomin

last: Wang

first: Linhui

last: Zhai

first: Jun

last: Xu

first: Congying

last: Pu

first: Jing

last: Li

first: Fuchu

last: He

first: Min

last: Huang

first: Minjia

last: Tan


title: Deep Representation Learning Improves Prediction of LacI-mediated Transcriptional Repression

status: Public on Jun 22 2021

city: Boston

first: Alexander

last: Garruss

state: MA

address: 77 Avenue Louis Pasteur, Rm 238

department: Genetics

country: USA

institute: Harvard Medical School

summary: We analyze LacI mutations with the goal to understand and better predict basal transcriptional repression function after mutation. This study utilized lac repressor variants from the Taylor, et al. 2015 Nature Methods study including ten tiles of synthesized single mutations (NCBI GEO Series GSE75009), and an additional six tiles corresponding to higher-order mutations (this study).

overall_design: Identification of protein variants depleted after negative selection by next-generation sequencing.

first: Noah

middle: D

last: Taylor

first: Srivatsan

last: Raman

first: George

middle: M

last: Church


title: Transcriptomic analysis of mdx mouse muscles reveals a signature of early human Duchenne muscular dystrophy

city: Bethesda

first: Stephen

middle: R

last: Brooks

state: MD

address: 50 SOUTH DR, RM 1140

department: Biodata Mining and Discovery Section

institute: NIAMS/NIH

summary: The mdx mouse (C57BL/10ScSn-DMDmdx/J), like Duchenne muscular dystrophy (DMD) patients, lacks the protein dystrophin. However, the mdx mouse has a normal lifespan and mild pathology while DMD remains a severe, fatal disease. New mouse models have been developed that are more severely affected but they have not replaced the mdx mouse in DMD research. A few years ago RNA-sequencing (RNA-seq) results of biopsies from DMD and normal human muscle were published but we could not find equivalent data for the mouse. We now report RNA-sequencing of three wild-type and mdx mouse muscles: the flexor digitorum brevis (FDB), the extensor digitorum longus (EDL), and the soleus (SOL). The FDB, a plantar foot muscle is often used for in vivo and ex vivo experiments but may be less affected by the lack of dystrophy than the hindlimb muscles. We compared muscles from 2- and 5-month old mice to investigate the time-course of the mdx pathology. The results show a muscle- and age-dependent parallel between mdx and DMD muscles. Although the FDB is less affected than EDL and SOL at 2 months, the three muscles show, at both ages, activation of close to 100 genes from 7 pathways that are affected in presymptomatic DMD patients and have been called "DMD disease signature".

overall_design: We examined the transcriptome of three hindlimb muscles, comparing 2- and 5-month old WT and mdx mice to investigate degree and time-course of mdx pathology and comparing against published human DMD transcriptome.

first: Evelyn

last: Ralston

first: Gustavo

last: Gutierrez-Cruz

first: Aster

last: Kenea


title: Next Generation Sequencing Facilitates Quantitative Analysis of healthy controls and RSA patients Transcriptomes

city: Wuhan

first: Long

last: Yu

state: Hubei

address: 13 Hangkong Road, Hankou

institute: Tongji medical college

summary: To explore the changes of local microenvironment in the feto-maternal interface during early pregnancy in RSA, we next examined the transcriptional profiles of the decidua tissue by RNA sequencing using samples from 3 RSA patients and 3 healthy controls

overall_design: decidua samples from 3 healthy controls and 3 RSA patients were bulk sequenced for gene expression

first: Ying

last: Zha

first: Qi

last: Kang

first: Pan

last: Zhi


title: Dynamic association of IκBα to chromatin during intestinal differentiation is regulated by Acetylation and N-terminal cleavage of histone H4

status: Public on Jun 21 2021

city: Barcelona

first: Lluis

last: Espinosa

state: Barcelona

address: Dr. Aiguader 88

department: Institut Municipal d'Investigacions Mèdiques, (IMIM)

country: Spain

institute: Hospital del Mar

type: Genome binding/occupancy profiling by high throughput sequencing

summary: IκBs exert a principal function as cytoplasmic inhibitors of the NF-kB transcription factors. Additional functions for specific IκB homologues have also been described including their association to chromatin to directly regulate gene transcription. Phosphorylated and SUMOylated IκBα (pS-IκBα) specifically binds histones H2A and H4 in the stem and progenitor compartment of skin and intestine, but the mechanisms that control the recruitment of nuclear pS-IκBα to the chromatin are largely unstudied. We here show thatserine 32-36 phosphorylation of IκBα favors its binding with nucleosomes and demonstrated that p-IκBαassociation to H4 is favored by acetylation at specific H4 lysine (K) residues. Acetylated N-terminal tail of H4 is lost during intestinal cell differentiation due to histone cleavage at amino acids 17-19 by the action of trypsin or chymotrypsin, which interferes p-IκBα association to chromatin. Paradoxically, pharmacologic or genetic inhibition of trypsin and chymotrypsin activity in HT29 cells increased p-IκBα chromatin binding, associated to impaired goblet cell differentiation, which was comparable to IκBα deletion. Together our results indicate that dynamic binding of IκBα to chromatin is a requirement for intestinal cell differentiation and provide a molecular base for the restricted nuclear distribution of p-IκBα at specific stem cell compartments.

overall_design: HCT116 ChIP-seq with various antibodies.  5 analyzed samples.

first: Laura

last: Marruecos

first: Joan

last: Bertran

first: Anna

last: Bigas


title: Identification of the potential gene regulatory networks and therapeutics in aged mice with postoperative neurocognitive disorder (circRNA)

city: Guangzhou

first: Wensi

last: Wu

address: No. 135, West Xingang Street

institute: SUN YAT-SEN UNIVERSITY

type: Non-coding RNA profiling by high throughput sequencing

summary: Postoperative neurocognitive disorder (PND) is one of the most common postoperative neurological complications in aged patients.  In order to detect the differential expression profiles of genes caused by PND, a total of 26 18-month-old male C57BL/6 mice were randomly assigned to control group and PND group.  Behavioral tests showed that mice in the PND group had impaired cognitive function compared with the control group.  Three mice in each group were randomly selected to harvest the brain for analysis the expressions of circRNAs, miRNAs and mRNAs in the prefrontal cortex by next-generation sequencing (NGS) technology.  Differentially expressed genes, including 1192 circRNAs, 27 miRNAs and 266 mRNAs were identified, and its accuracy was further confirmed by qRT-PCR.

overall_design: circular RNA expression profiles in the prefrontal cortex of 18-months-old control and PND mice

first: Yongpai

last: Peng

first: Jiaxin

last: Zhou

first: Xiaojun

last: Zhang

first: Lin

last: Cao

first: Wei-jye

last: Lin

first: Yanan

last: Lu

first: Jing

last: Wen

first: Zhi

last: Wang


title: Low-input ATAC&mRNA-Seq: a simple and robust method for simultaneous dual-omics profiling with low cell number

city: RTP

first: ruifang

last: li

state: NC

address: 111 T.W. Alexander Drive

institute: NIEHS

summary: Deciphering epigenetic regulation of gene expression requires measuring the epigenome and transcriptome jointly. However, multi-omics profiling remains challenging for low-input samples. Therefore, we developed low-input ATAC&mRNA-Seq, a simple and robust method for studying the role of chromatin structure in gene regulation in a single experiment with thousands of cells, to maximize insights from limited input material by obtaining ATAC-seq and mRNA-seq data simultaneously from the same cells with data quality comparable to conventional mono-omics assays. Remarkably, integrative data analysis revealed similar strong association between promoter accessibility and gene expression using the data of low-input ATAC&mRNA-Seq as using single-assayed data, underscoring the accuracy and reliability of our dual-omics assay to generate both data types simultaneously with just thousands of cells. We envision our method to be widely applied in many biological disciplines with limited materials.

overall_design: Low-input ATAC&mRNA-Seq was performed on E14 mESCs with 5K, 10K, and 20K cells. Two independent experiments were carried out on different days to assess data reproducibility. To benchmark our method, Omni-ATAC-seq was also conducted with 5K, 10K, and 50K of E14 mESCs.

first: Ruifang

last: Li

first: Sara

middle: A

last: Grimm


title: RNA-seq of Arc/Arg3.1-positive versus Arc/Arg3.1-negative skin migratory dendritic cells from healthy mice

city: Hamburg

first: Manuel

last: Friese

address: Falkenried 94

department: Institut für Neuroimmunologie und Multiple Sklerose

institute: Universitätsklinikum Hamburg-Eppendorf

summary: The key function of migratory dendritic cells (migDCs) is to take up antigens in peripheral tissues and migrate to draining lymph nodes (dLN) to initiate immune responses. Recently, we discovered in mice that in the immune system activity-regulated cytoskeleton associated protein/activity-regulated gene 3.1 (Arc/Arg3.1) is exclusively expressed by migDCs and is a central driver of fast inflammatory migration. However, the frequency of Arc/Arg3.1 expressing cells in different migDC subsets including Langerhans cells (LC), their phylogenetic origin, transcription factor dependency and functional role remains unclear. Here we found that Arc/Arg3.1+ migDCs derived from common DC precursors (CDPs) and radio-resistant LCs. They were present in all migDC subsets and showed a consistent superiority in inflammatory migration but were independent of the transcription factors Irf4 or Batf3. In intradermal Staphylococcus aureus infection, a model that relies on effective inflammatory antigen transport, Arc/Arg3.1 deletion strongly reduced T cell responses. By contrast, Arc/Arg3.1 deficiency did not hamper the immune response to systemic Listeria monocytogenes infection, which does not require antigen transport. Thus, our results show that Arc/Arg3.1 is a molecular marker for defining a population of DCs with superior migratory capacity that spans across all migDC subsets irrespective of ontogeny and phenotype.

overall_design: Arc/Arg3.1-eGFP-positive and Arc/Arg3.1-eGFP-negative migratory dendritic cells (MHC-II-high, CD11c-intermidiate) were isolated by fluorescent cell sorting from skin draining lymph nodes of Arc/Arg3.1-eGFP mice.

first: Joseph

last: Tintelnot

first: Friderike

last: Ufer

first: Jan

middle: B

last: Engler


title: Identification of the potential gene regulatory networks and therapeutics in aged mice with postoperative neurocognitive disorder

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

overall_design: Refer to individual Series


title: C3G regulates STAT3, ERK, and adhesion signaling, and is essential for differentiation of mouse embryonic stem cells

city: Hyderabad

first: Divya Tej

last: Sowpati

state: Telangana

address: Habsiguda

country: India

institute: CSIR Centre for Cellular and Molecular biology

summary:
Purpose: Here we report the differential gene expression profile between WT and C3G knock out E14Tg2a cells. The cells were grown in equel seeding density in GMEM  Serum +LIF media. RNA was isolated after 48 hr and sequenced
Methods:  C3G knock out E14Tg2a mouse ES cells were made by CrisprCAS9 mediated knock out strategy. RNA was collected using Trizol method from WT and knock out clone 48 hr after seeding in equel density in a 6 well plate, precoated with gelatin.
Results: Using an optimized data analysis workflow, we mapped about 75 million sequence reads per sample to the mouse genome (build mm10) and identified 5797 genes were differentially expressed with adjusted p-value < 0.05(significant) between the WT and C3G-/-.  Approximately 17% of the genes showed differential expression between the WT and C3g−/− ESCs, with a fold change ≥1.5 and p value <0.05. Biological pathways and gene ontology enrichment analysis of differentially expressed genes reveals that C3G is  required  to maintain adhesion of ES cells and for differentiation of mouse ES cells.
Conclusions: The study revealed the first transcriptome profile of C3G gene knock out in E14Tg2a mouse embryoinic tem cells. In our study we found that C3G acts as a mediaator of differentiation by activatingERK and integrin signaling and supressing STAT3 and other pluripotency factors like ESRRB and KLF4.

overall_design: Examination of differential gene expression between WT and C3G knock out E14Tg2a mouse embryonic stem cells.

first: Vijay

middle: V

last: Vishnu

first: Bh

last: Muralikrishna

first: Archana

last: Verma

first: Sanjeev

middle: C

last: Nayak

first: Divya

middle: T

first: Vegesna

last: Radha

first: P

last: Shekar


title: The transcriptome of StRIK-silenced periderms suggests a role of this potato gene in RNA processing and stress

city: Girona

first: Olga

last: Serra

state: Girona

address: C/ Maria Aurèlia Capmany, 40

department: Biologia

institute: Universitat de Girona

summary: The periderm is a protective barrier crucial for land plants survival, but genetic factors involved in its regulation are fairly unknown. In a previous transcriptomic approach in the cork oak periderm (Quercus suber) we identified a RS2-INTERACTING KH PROTEIN (RIK) homolog, of unknown function and with a K homology (KH)-domain RNA-binding protein, as a regulatory candidate gene in the periderm. To gain insight into the function of RIK in periderm, the potato (Solanum tuberosum) tuber periderm was used as a model. The StRIK transcript profile showed ubiquitous accumulation in all vegetative tissues analyzed, including periderm and other suberized tissues such as root and wound-healing tissues. Downregulation of StRIK in potato by RNA interference (StRIK-RNAi) did not show evident effects in tuber periderm anatomy but unexpectedly unlike Wild type, the transgenic plants flowered. Nevertheless, the periderm of StRIK-RNAi lines did show altered expression of genes associated with RNA metabolism, stress and signaling, mirroring the biological processes found enriched within the in silico co-expression network of the Arabidopsis ortholog. Altogether those findings suggest that the potato StRIK might play roles in RNA maturation and stress response throughout diverse plant developmental processes such as flowering and periderm formation.

overall_design: Sample: Tuber skin (periderm) of freshly-harvested potatoes of plants growing in soil under long-day conditions (12 h light/12 h dark)  Replicates: 3 Wild-type potato (Solanum Tuberosum) plants; 3 StRIK-RNAi transgenic line 9 potato plants, 3 StRIK-RNAi transgenic line 12 potato plants, 3 StRIK-RNAi transgenic line 47 potato plants.

first: Pau

last: Boher

first: Marçal

last: Soler

first: Sandra

last: Fernández-Piñán

first: Xènia

last: Torrent

first: Sebastian

last: Mueller

first: Krys

last: Kelly

first: Mercè

last: Figueras


title: Epitranscriptomic addition of m6A regulates HIV-1 RNA stability and alternative splicing

city: Taipei

first: Kevin

last: Tsai

address: 128 Sec. 2, Academia Rd. Nankang

department: Institute of Biomedical Sciences

country: Taiwan

institute: Academia Sinica

summary: Several prior reports have demonstrated that the epitranscriptomic addition of m6A to viral transcripts promotes the replication and pathogenicity of a wide range of viruses yet the underlying mechanism(s) causing this positive effect has remained unclear. It is known that m6A function is largely mediated by cellular m6A binding proteins or readers, however, how these m6A reader proteins contribute to the regulation of HIV-1 gene expression has remained controversial. Here, we confirm that m6A indeed enhances HIV-1 gene expression. We demonstrate that this effect is collectively mediated by the cytoplasmic reader YTHDF2, which increases HIV-1 transcript stability, and the nuclear m6A reader YTHDC1, which binds HIV-1 RNA at 7 distinct m6A methylated sites, regulating viral transcript alternative splicing.

sample_organism:
Homo sapiens
Human immunodeficiency virus 1

overall_design: GFP (control) or YTHDC1 PAR-CLIP on HIV-1-infected CD4+ 293T cells.

first: Bryan

last: Cullen


title: Mechanism of CYB561A3 in iron metabolism of Burkitt Lymphoma cells

first: Benjamin

last: Gewurz

address: 181 Longwood Avenue

institute: Brigham and Women's Hospital

summary: To confirm the role of CYB561A3 in Burkitt cell lines, we knocked out the CYB561A3 gene in P3HR1. Total RNA was extracted and RNA sequencing library was constructed for next high-throughput sequencing analysis. Sequencing results revealed that knocking out CYB561A3 would induce an iron starvation response in P3HR1, resulting in changes in gene expression related to iron metabolism such as TFRC, indicating that CYB561A3 plays a key role in iron metabolism in Burkitt cells.

overall_design: The non-targeted control sgRNA group and the CYB561A3 targeted sgRNA group each had three biological replicates.

first: Zhonghao

last: Wang

middle: E


title: Integrated -Omics Analyses Demonstrate that Patient-Derived Colon Organoids Faithfully Recapitulate the Tumor Tissue of Origin, Highlighting Altered Pathways in Colorectal Cancer Progression

city: Udine

first: Emiliano

last: Dalla

state: UD

address: Piazzale Kolbe 4

department: Department of Medicine

country: Italy

institute: Università degli Studi di Udine

summary:
Purpose: methodically characterize colorectal cancer patient-derived organoids, at the molecular level, under different culturing conditions, by evaluating genomic, transcriptomic and phosphoproteomic profiles in order to define their ability to stably recapitulate patients-derived tissues features.
Outcome: integrated molecular and bioinformatics analyses demonstrated that PDCOs faithfully recapitulate the tumor tissue of origin. Our results indicate that the in vitro culture of biopsy-derived organoids does not introduce substantial variation in the genomic landscape of CRC but still promotes the expansion of the more aggressive subset of cancer cells. In conclusion, our findings strongly support the use of CRC organoids as a novel and very promising and stable tool for personalized medicine that could represent a powerful alternative to animal experimentation in vivo.

overall_design: Patient-derived colon organoids (PDCOs) were obtained from surgical resected tissue of tumor and normal intestinal mucosa of patients affected by colon cancer (P12, P14 and P16). The crypts, derived from normal tissue, and the isolated cells derived from tumor were considered as primary tissues. From primary tissues, we generated normal (P12N, P14N, P16N) and tumor (P12T, P14T, P16T) PDCOs, respectively. Paired-end Whole Exome Sequencing allowed detecting the temporal acquisition of somatic variants, in a patient-specific manner, having deleterious effects on driving genes associated with colorectal cancer. Moreover, a targeted NGS approach confirmed that organoids faithfully recapitulated patients’ tumor tissue. Finally, using paired-end RNA-seq experiments, we identified differentially expressed genes in tumor versus normal organoids at different time points, underlining several pathways altered in a time-dependent fashion.

first: M

last: Codrich

first: E

first: C

last: Mio

first: G

last: Antoniali

last: Malfatti

first: S

last: Marzinotto

last: Pierobon

last: Baldelli

last: Di Loreto

last: Damante

last: Terrosu

last: Pucillo

last: Tell


title: Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma

city: Vienna

first: Olaf

last: Merkel

address: Spitalgasse 23

department: Department of Pathology

country: Austria

institute: Medical University of Vienna


title: Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma [ChIP-Seq]

summary: Systemic anaplastic large cell lymphoma (ALCL) is an aggressive T-cell lymphoma comprising ALK-positive (ALK+) and ALK-negative (ALK−) as well as breast implant-associated (BIA)-ALCL. The prognosis for ALCL, ALK− in particular is poor, and already in second line there is an unmet medical need for effective treatment options. To identify genes defining ALCL cell state and dependencies, we here started by an unbiased characterization of super-enhancer regions by genome-wide H3K27ac chromatin immunoprecipitation sequencing (ChIP-seq). We identified, in addition to known key regulators, the AP-1 transcription factor member BATF3 and IL-2 receptor (IL2R) components among the genes with most extensively H3K27-acetylated regulatory regions. Consistently, specific and high-level expression of the IL-2R subunits, IL-2Rα, IL-2Rβ and IL-2Rγ in ALCL correlate with BATF3 expression. Confirming a regulatory link, expression of IL-2R subunits decreases following BATF3 knockout, and BATF3 is recruited to AP-1 sites in IL2R regulatory regions. Functionally, IL-2, IL-15 and Neo-2/15, a hyper-stable IL-2/IL-15 mimic, accelerate growth of ALCL cells and activate STAT1, STAT5 and ERK, but not STAT3. In line, strong IL-2Rα expression in ALCL patients is linked to more aggressive clinical presentation. Finally, we demonstrate highly efficient killing of ALCL cells by an IL-2Rα-targeting antibody-drug conjugate in vitro and in vivo. Our results highlight the importance of the BATF3/IL-2R module for ALCL biology and identify IL-2Rα as the target of a promising treatment strategy for ALCL.

overall_design: Genome binding/occupancy profiling by high throughput sequencing in anaplastic large cell lymphoma cell lines

first: Huan-Chang

last: Liang

first: Nicole

last: Prutsch

first: Mark

last: Zimmerman

first: Brian

middle: J

last: Abraham

first: Thomas

last: Look


title: Super-enhancer-based identification of a BATF3/IL-2R-module reveals vulnerabilities in anaplastic large cell lymphoma [RNA-Seq]

overall_design: Expression profiling by high throughput sequencing in CRISPR/Cas9-mediated BATF3-knockout anaplastic large cell lymphoma cells

first: Cosimo

last: Lobello

first: Jan

last: Oppelt

first: Šárka

last: Pospíšilová


title: JUN promotes hypertrophic skin scarring via CD36

city: Tucson

first: Kellen

last: Chen

state: AZ

address: 1501 N Campbell Ave

department: Surgery

institute: University of Arizona

summary: Hypertrophic skin scarring following dermal injury causes extreme pain and psychological trauma for patients. Unfortuately, we do not have effective treatments to prevent or reverse skin scarring. Using RNA and ATAC sequencing of mouse and human fibroblasts, we show that JUN expressing fibroblasts are responsible for skin scarring by regulating CD36 expression. In summary,  we show that CD36 antagonism by represent a therapeutic target to overcome JUN hypertrophic skin scarring.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: RNA was obtained from FACS-isolated mouse fibroblast subpopulations (reticular and lipofibroblasts) from JUN+ and JUN- mice at post opeartive day 7 (POD), and human cultured JUN KO and JUN non-KO fibroblasts obtained from normal skin, scar, hypertrophic scar and keloid tissue. For mice samples, biological replicates were included; and for human cell samples technical replicates were included. To examine chromatin accessibility changes in response to JUN state in fibroblasts, an average of 50,000 cells were isolated per replicate by FACS in same fibroblasts types as above for both mouse and human samples. Biological replicates were included for mouse samples and technical replicates for human cell samples. To examine chromatin accessibility changes in response to JUN state in fibroblasts, an average of 50,000 cells were isolated per replicate by FACS in same fibroblasts types as above for both mouse and human samples. Biological replicates were included for mouse samples and technical replicates for human cell samples.

first: Julia

last: Garcia

first: Mimi

last: Borrelli

first: Michelle

middle: F

last: Griffin

first: Michael

last: Januzyk


title: YAP and TAZ Dictate Binary Pan-Cancer Superclasses [ChIP-seq]

city: Toronto

first: Rod

last: Bremner

state: ON

address: 600 University Ave

department: Lunenfeld-Tanenbaum Research Institute

institute: Mount Sinai Hospital

summary: We defined pan-cancer binary classes based on distinct expression of YAP (and its paralog TAZ/ WWTR1) and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we showed that opposite pro- or anti-cancer YAP activity functionally defines binary YAPon or YAPoff cancer classes that express or silence YAP, respectively. Essentially all leukemia and lymphoma fall into the YAPoff class, as do multiple neural and neuroendocrine YAPoff solid cancers. YAPoff solid cancers are frequently RB1-/-, such as retinoblastoma, small cell lung cancer and neuroendocrine prostate cancer. YAP-silencing was intrinsic to the cell-of-origin, or acquired with lineage-switching and drug-resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior, and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, whereas YAP induces cell cycle genes in YAPon cancers, extensive RNAseq data showed that forced YAP expression in YAPoff cancers instead activated adhesion genes that are normally co-silenced with YAP. YAP regulates both of these anti-cancer adhesive or pro-cancer cell cycle programs through the TEAD DNA binding family (TEAD1-4). YAP/TEAD targets AP1-bound enhancers in YAPon cancers, but Chipseq studies revealed that in YAPoff cancers, YAP/TEAD instead targeted elements co-bound with neural and neuroendocrine lineage-defining basic helix-loop-helix (bHLH) and Homeobox transcription factors (e.g. NEUROD, ASCL1, NKX2, OTX2).  A CRISPR screen revealed that, among the adhesion regulators, ITGAV/ITGB5 pair are required for YAP induced cytostasis in YAPoff cancers. YAP is thus pivotal across all cancer, but in opposite pro- or anti-cancer ways, which define contrasting genetic and drug sensitivities.

overall_design: TEAD4 ChIP-Seq in 2 retinoblastoma cell lines and 2 small cell lung cancer (SCLC) cell lines expressing an Empty vector (control sample) or YAP (Y79 - wild type; other lines overactive 5SA mutant) in duplicate.

first: Joel

last: Pearson

first: Katherine

last: Huang


title: YAP and TAZ Dictate Binary Pan-Cancer Superclasses

summary:
This SuperSeries is composed of the SubSeries listed below.
We defined pan-cancer binary classes based on distinct expression of YAP (and its paralog TAZ/ WWTR1) and YAP-responsive adhesion regulators. Combining informatics with in vivo and in vitro gain- and loss-of-function studies across multiple murine and human tumor types, we showed that opposite pro- or anti-cancer YAP activity functionally defines binary YAPon or YAPoff cancer classes that express or silence YAP, respectively. Essentially all leukemia and lymphoma fall into the YAPoff class, as do multiple neural and neuroendocrine YAPoff solid cancers. YAPoff solid cancers are frequently RB1-/-, such as retinoblastoma, small cell lung cancer and neuroendocrine prostate cancer. YAP-silencing was intrinsic to the cell-of-origin, or acquired with lineage-switching and drug-resistance. The binary cancer groups exhibit distinct YAP-dependent adhesive behavior, and pharmaceutical vulnerabilities, underscoring clinical relevance. Mechanistically, whereas YAP induces cell cycle genes in YAPon cancers, extensive RNAseq data showed that forced YAP expression in YAPoff cancers instead activated adhesion genes that are normally co-silenced with YAP. YAP regulates both of these anti-cancer adhesive or pro-cancer cell cycle programs through the TEAD DNA binding family (TEAD1-4). YAP/TEAD targets AP1-bound enhancers in YAPon cancers, but Chipseq studies revealed that in YAPoff cancers, YAP/TEAD instead targeted elements co-bound with neural and neuroendocrine lineage-defining basic helix-loop-helix (bHLH) and Homeobox transcription factors (e.g. NEUROD, ASCL1, NKX2, OTX2).  A CRISPR screen revealed that, among the adhesion regulators, ITGAV/ITGB5 pair are required for YAP induced cytostasis in YAPoff cancers. YAP is thus pivotal across all cancer, but in opposite pro- or anti-cancer ways, which define contrasting genetic and drug sensitivities.


title: Whole genome expression profiling of Arabidopsis thaliana OsRR6ox transgenic vis-à-vis wild-type

city: New Delhi

first: Jitendra

middle: Paul

last: Khurana

address: Benito Juarez Road

department: Department of Plant Molecular Biology

institute: University of Delhi South Campus

summary: We found that Arabidopsis plants constitutively expressing OsRR6 exhibit reduced cytokinin sensitivity, adventitious root formation and enhanced anthocyanin accumulation. In addition they are hypersensitive to red, far-red and blue light in hypocotyl growth assay and also flower little early as compared to wild type.Therefore, to identify the downstream pathways affected in the OsRR6 overexpression plants, we performed transcriptome profiling of overexpression line vs wild type using Affymetrix microarray platform.

overall_design: Ten days old Arabidopsis seedlings for each genotype were grown on 1/2MS medium and 100 mg tissue was harvested, snap frozen in liquid nitrogen. Total RNA was isolated and hybridized on Arabidopsis GeneChips with two replicates for each genotype. Microarray using Affymetrix platform was performed for total of four samples, two for wild type and two for overexpression line.

first: Avantika

last: Bhaskar

first: Laju

middle: K

last: Paul

first: Eshan

last: Sharma

first: Mukesh

last: Jain


title: The CD200-CD200R axis promotes squamous cell carcinoma metastasis via regulation of Cathepsin K

status: Public on Jun 19 2021

city: New York

first: Anqi

last: Shao

state: NY

address: 1150 St. Nicholas Ave.

institute: Columbia University

summary: The CD200-CD200R immunoregulatory signaling axis plays an etiological role in the survival and spread of numerous cancers primarily through suppression of anti-tumor immune surveillance. Our previous work outlined a pro-metastatic role for the CD200-CD200R axis in cutaneous squamous cell carcinoma (cSCC) that is independent of direct T cell suppression but modulates the function of infiltrating myeloid cells. To identify effectors of the CD200-CD200R axis important for cSCC metastasis, we conducted RNA-Seq profiling of infiltrating CD11b+Cd200r+ cells isolated from Cd200+ versus Cd200 null cSCCs and identified the cysteine protease Cathepsin K (Ctsk) to be highly upregulated in Cd200+ cSCCs. CD11b+Cd200r+ cells, which expressed phenotypic markers associated with myeloid-derived suppressor cell-like cells and tumor-associated macrophages, were the primary source of Ctsk expression in cSCC. Using a Cd200r+ myeloid cell-cSCC co-culture system, we observed that induction of Ctsk in Cd200r+ tumor-infiltrating cells was dependent on engagement of the CD200-CD200R axis, indicating that Ctsk is a target gene of this pathway in the cSCC tumor microenvironment. Inhibition of Ctsk, but not matrix metalloproteinases (MMPs), significantly blocked cSCC cell migration through collagen gels in vitro. Finally, both targeted Cd200 disruption in tumor cells and Ctsk pharmacological inhibition dramatically reduced cSCC metastasis in vivo. Collectively, our findings identify a novel direct role for Cd200r+ infiltrating myeloid lineages in tumor metastasis and support the conclusion that CD200 stimulates cSCC invasion and metastasis via induction of Ctsk in CD200r+ infiltrating cells.

overall_design: Profiling Cd200-dependent genes in Cd200r+ cSCC-infiltrating myeloid cells in Cd200r+ myeloid cells purified from orthotopic Wt (CD200+) or EpiKO(CD200-) cSCCs.

first: David

last: Owens

first: Jiyoon

last: Cho


title: mRNA-seq analysis of Caenorhabditis elegans daf-18(ok480) and WT starved L1 larvae shortly after hatching

city: Eugene

first: Amy

last: Webster

state: Oregon

address: Institute of Ecology and Evolution

institute: University of Oregon

summary: Whole L1 larvae were collected from GC1459 [naSi2 [pGC550 (mex-5p::mCherry::H2B::nos-2 3'UTR +unc-119(+))] II; unc-119(ed3) III ?; daf-18(ok480) IV] and GC1171 [naSi2 [pGC550 (mex-5p::mCherry::H2B::nos-2 3'UTR +unc-119(+))] II; unc-119(ed3) III] strains up to two hours after hatching without food.

overall_design: 2 conditions (GC1459 and GC1171) and 4 biological replicates per condition.

first: Amanda

middle: L

last: Fry

first: Rojin

last: Chitrakar

first: L

last: Baugh

first: E

last: Hubbard


title: Evaluating the Lettuce Metatranscriptome with MinION Sequencing for Future Spaceflight Food Production Application.

status: Public on Jun 20 2021

city: Merritt Island

first: Jamie

last: Foster

state: FL

address: 505 Odyssey Way

institute: University of Florida

summary: Healthy plants are vital for successful, long-duration missions in space, as they provide the crew with life support, food production, and psychological benefits. The microorganisms that associate with plant tissues play a critical role in improving plant growth, health, and production. To that end, it is necessary to develop methodologies that investigate the metabolic activities of the plant’s microbiome in orbit to enable rapid responses regarding the care of plants in space. In this study, we developed a protocol to characterize the endophytic and epiphytic microbial metatranscriptome of red romaine lettuce, a key salad crop that was grown under International Space Station (ISS)-like conditions. Microbial transcripts enriched from host-microbe total RNA were sequenced using the Oxford Nanopore MinION sequencing platform. Results showed that this enrichment approach was highly reproducible and effective for rapid on-site detection of microbial transcriptional activity. Taxonomic analysis based on 16S and 18S rRNA transcripts identified that the top five most abundant phyla in the lettuce microbiome were Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Ascomycota. The metatranscriptomic analysis identified the expression of genes involved in many metabolic pathways, including carbohydrate metabolism, energy metabolism, and signal transduction. Network analyses of the expression data show that, within the signal transduction pathway of the fungal community, the Mitogen-Activated Protein Kinase signaling pathway was tightly regulated across all samples and could be a potential driver for fungal proliferation. Our results demonstrated the feasibility of using MinION-based metatranscriptomics of enriched microbial RNA as a method for rapid, on-site monitoring of the transcriptional activity of crop microbiomes, thereby helping to facilitate and maintain plant health for on-orbit space food production.

overall_design: Red romaine lettuce, Lactuca sativa cv ‘Outredgeous’ were grown in environmentally controlled growth chambers at Kennedy Space Center (KSC), FL, under conditions simulating those aboard the ISS (RH-50%, CO2-3000 ppm, and temperature 23 ºC). The growth cycle was established at 16h/8h light dark cycles with 200-300 µmol fluorescent lights and samples were harvested at day 28 for leafy greens.  Leaf tissue from each of the three pots (L93, L96, and L101) were placed immediately into RNAlater™  and stored at -80 ºC until ready for RNA extraction.Total RNA was extracted from each of the three lettuce leave replicates with the RNeasy Plant Mini Kit and was DNase-treated. A modified enrichment method was developed to remove plant mRNA and rRNA for compatibility with Nanopore library preparation protocols. Modified Invitrogen Dynabeads polyA enrichment protocol was performed to deplete the plant mRNA. Invitrogen RiboMinusTM plant kit was performed to removal of plant ribosomal RNA. Host depleted RNA form each sample was then A-tailed using Poly(A) polymerase.  Library preparation for cDNA-PCR sequencing was performed using the SQK-PCS109 sequencing kit following the manufacturer’s instructions (Oxford Nanopore Technologies, Oxford, UK) with the following cycling conditions, an initial denaturing step at 95°C for 5 min, then 14 cycles of 15 s at 95°C, 15 s at 62°C, and 1 min at 65°C, followed by a final extension step at 65°C for 6 mins and a hold at 4°C.  cDNA library using 100 fmol of amplified cDNA with adapters was loaded onto a FLO-MIN106 flowcell and the RNA library was sequenced on a MinION device for 48 h. Basecalling was perform in real-time using the MinIT device. L93, L96 and L101 replicates were sequenced on individual flowcells to provide information about the process reproducibility and variability.

first: Natasha

last: Sng

first: Christina

last: Khodadad

first: Anirudha

last: Dixit

first: Artemis

last: Louyakis

first: Gioia

last: Massa

first: Kasthuri

last: Venkateswaran


title: Epicardial Differentiation Drives Fibro-fatty Remodeling in Arrhythmogenic Cardiomyopathy [scRNA-seq]

city: Utrecht

first: Eva

last: van Rooij

state: Utrecht

address: Uppsalalaan 8

country: Netherlands

institute: Hubrecht Institute

summary: We performed single cell RNA sequencing (scRNAseq) on epicardial cells generated from human induced pluripotent stem cells (hiPSCs) of an arrhythmogenic cardiomyopathy patient and their PKP2 reverted isogenic control. The goal of this experiment was to analyze the process of epicardial to fibro-fatty celluar differentiation in diseased cells and compare the cellular transcirptomes of diseased and healthy cells.

overall_design: scRNAseq of PKP2 mutant and reverted hiPSC-derived epicardial cells at day 70 of differentiation

first: Arwa

last: Kohela

first: Sebastiaan

last: van Kampen

first: Tara

last: Moens

first: Martijn

last: Wehrens

first: Bas

last: Molenaar

first: Cornelis

last: Boogerd

first: Jantine

last: Monshouwer-Kloots

first: Ilaria

last: Perini

first: Marie-José

last: Goumans

first: Anke

last: Smits

first: J

middle: P

last: van Tintelen


title: Epicardial Differentiation Drives Fibro-fatty Remodeling in Arrhythmogenic Cardiomyopathy [bulk RNA-seq]

summary: We performed bulk cell RNA sequencing (RNAseq) on epicardial cells generated from human induced pluripotent stem cells (hiPSCs) of an arrhythmogenic cardiomyopathy patient and their PKP2 reverted isogenic control. The goal of this experiment was to compare the transcriptomes of diseased and healthy cells.

overall_design: Bulk RNAseq of PKP2 mutant and reverted hiPSC-derived epicardial cells at day 30 of differentiation


title: microRNA-132 regulates adult neural stem cells and their progeny to restore adult hippocampal neurogenesis and memory deficits in Alzheimer’s disease

city: Leuven

first: Bart

last: de Strooper

address: Campus Gasthuisberg, Herestraat 49, bus 602

department: VIB-KU Leuven Center for Brain & Disease Research

country: Belgium

institute: KULeuven

summary: Neural stem cells residing in the hippocampal neurogenic niche sustain life-long neurogenesis in the adult brain. Adult hippocampal neurogenesis (AHN) is functionally linked to mnemonic and cognitive plasticity in humans and rodents. In Alzheimer’s disease (AD), the process of generating new neurons at the hippocampal neurogenic niche is impeded, yet the mechanisms involved are unknown. Here we identify miR-132, one of the most consistently downregulated microRNAs in AD, as a potent regulator of AHN, exerting cell-autonomous pro-neurogenic effects in the adult neural stem cells and their progeny. Using distinct AD mouse models, cultured human primary and established neural stem cells, and human patient material, we demonstrate that AHN is directly impacted by AD pathology. miR-132 replacement in adult mouse AD hippocampus restores AHN and relevant memory deficits. Our findings corroborate the significance of AHN in AD and reveal the possible therapeutic significance of targeting miR-132 in neurodegeneration.

overall_design: Nestin:GFP+-niche cells were FACS-sorted from the AD dentate gyrus of Nestin:GFP mice at 2 months of age. Four miR-132-injected and four control-injected Nestin:GFP male mice were used and 94 cells were initially sorted per mouse.

first: Hannah

last: Walgrave

first: Sriram

last: Balusu

first: Sarah

last: Snoeck

first: Elke

last: Vanden Eynden

first: Katleen

last: Craessaerts

first: Nicola

last: Thrupp

first: Leen

last: Wolfs

first: Katrien

last: Horré

first: Yannick

last: Fourne

first: Alicja

last: Ronisz

first: Edina

last: Silajdžić

first: Amber

last: Penning

first: Giorgia

last: Tosoni

first: Zsuzsanna

last: Callaerts-Vegh

first: Rudi

last: D’Hooge

first: Dietmar

last: Thal

first: Henrik

last: Zetterberg

first: Sandrine

last: Thuret

first: Mark

last: Fiers

first: Carlo

last: Sala Frigerio

last: De Strooper

first: Evgenia

last: Salta


title: Comparing mRNA and sncRNA profiles during the maternal-to-embryonic transition in bovine  IVF and scNT embryos [mRNA]

status: Public on Jun 18 2021

city: Logan

first: Abby

last: Benninghoff

state: UT

address: 4815 Old Main Hill

department: Animal, Dairy and Veterinary Sciences

institute: Utah State University

summary: Production of embryos with high developmental competence by somatic cell nuclear transfer (scNT) is far less efficient than for in vitro fertilized (IVF) embryos, likely due to an accumulation of errors in genome reprogramming that results in aberrant expression of RNA transcripts, including messenger RNAs (mRNA) and, possibly, microRNAs (miRNA).  Thus, our objectives were to use RNAseq to determine the dynamics of mRNA expression in early developing scNT and IVF embryos in the context of the maternal-to-embryonic transition (MET) and to correlate apparent transcriptional dysregulation in cloned embryos with miRNA expression profiles.  Comparisons between scNT and IVF embryos indicated large scale transcriptome differences, which were most evident at the 8-cell and morula stages for genes associated with biological functions critical for the MET.  For two miRNAs previously identified as differentially expressed in scNT morulae, miR-34a and miR-345, negative correlations with some predicted mRNA targets were apparent, though not widespread among the majority of predicted targets.  Moreover, although large-scale aberrations in expression of mRNAs were evident during the MET in cattle scNT embryos, these changes were not consistently correlated with aberrations in miRNA expression at the same developmental stage, suggesting that other mechanisms controlling gene expression may be involved.

overall_design: mRNA profiles of bovine oocytes; fibroblasts; and embryos produced via in vitro fertilization or somatic cell nuclear transfer at 2-cell, 8-cell, morula, and blastocyst developmental stages and blastocyst-derived cells.  Four pools of 20 2-cell, 8-cell, morula, and blastocyst stage embryos were collected at 2, 3, 6, and 8 days post-IVF or scNT, washed in PBS, snap frozen in cryotubes containing RNA/DNA Shield, and stored at -80 C until RNA isolation

first: Jocelyn

last: Cuthbert

first: Stewart

last: Russell

first: Irina

last: Polejaeva

first: Qinggang

last: Meng

first: Kenneth

last: White


title: Dynamics of small non-coding RNAs in bovine scNT embryos through the maternal-to-embryonic transition [non-coding RNA]

summary: The efficiency of somatic cell nuclear transfer (scNT) for production of viable offspring is relatively low as compared to in vitro fertilization (IVF), presumably due to deficiencies in epigenetic reprogramming of the donor cell genome. Such defects may also involve the population of small non-coding RNAs (sncRNAs), which are important during early embryonic developmeNT.The objective of this study was to examine dynamic changes in relative abundance of sncRNAs during the maternal-to embryonic transition (MET) in bovine embryos produced by scNT as compared to IVF by using RNA sequencing. When comparing populations of miRNA in scNT versus IVF embryos, only miR-2340, miR-345, and miR34a were differentially expressed in morulae, though many more miRNAs were differentially expressed when comparing across developmental stages. Also of interest, distinct populations of piwi-interacting like RNAs (pilRNAs) were identified in bovine embryos prior to and during embryonic genome activation (EGA) as compared bovine embryos post EGA and differentiated cells. Overall, sncRNA sequencing analysis of preimplantation embryos revealed largely similar profiles of sncRNAs for IVF and scNT embryos at the 2-cell, 8-cell, morula and blastocyst stages of developmeNT.However, these sncRNA profiles, including miRNA, piRNA and tRNA fragments, were notably distinct prior to and after completion of the MET.

overall_design: Small non-coding RNA (sncRNA) profiles (focused in microRNAs, piwi-like RNAs, and transfer RNA fragments) of bovine oocytes; fibroblasts; and embryos produced via in vitro fertilization or somatic cell nuclear transfer at 2-cell, 8-cell, morula, and blastocyst developmental stages and blastocyst-derived cells.  Initially, four pools of 20 2-cell, 8-cell, morula, and blastocyst stage embryos were collected at 2, 3, 6, and 8 days post-IVF or scNT.


title: Circadian clock, carcinogenesis, chronochemotherapy connections

city: Chapel Hill

first: Yanyan

last: Yang

address: 120 Mason Farm Road

department: Biochemistry & Biophysics

institute: University of North Carolina  at Chapel Hill

summary: The circadian clock controls the expression of nearly 50% of protein coding genes in mice, and most likely in humans as well. Therefore, disruption of the circadian clock is presumed to have serious pathological effects including cancer. However, epidemiological studies on individuals with circadian disruption because of night shift or rotating shift work have produced contradictory data not conducive to scientific consensus as to whether circadian disruption increases the incidence of breast, ovarian, prostate or colorectal cancers. Similarly, genetically engineered mice with clock disruption do not exhibit spontaneous or radiation-induced cancers at higher incidence than wild-type controls. Because many cellular functions including the cell cycle and cell division are, at least in part, controlled by the molecular clock components (CLOCK, BMAL1, CRYs, PERs), it has also been expected that appropriate timing of chemotherapy may increase the efficacy of chemotherapeutic drugs and ameliorate their side effect. However, empirical attempts at chronochemotherapy have not produced beneficial outcomes. Using mice without and with human tumor xenografts, sites of DNA damage and repair following treatment with the anticancer drug cisplatin have been mapped genome-wide at single nucleotide resolution and as a function of circadian time. The data indicate that mechanism-based studies such as these may provide information necessary for devising rational chronochemotherapy regimens.

overall_design: Patient’s tumor samples were collected under Duke Institutional Review Board (IRB) approved protocol (Pr000089222) and written informed consent were obtained from all patients who participated in the study. The generation of PDX was performed as described in our previous studies (Somarelli et al, MCT 2020). Briefly, the resected tumor was washed with phosphate buffered saline (PBS) and minced to small fragments (<2mm). To produce single cell suspension, tumor fragments were further dissociated with tissue dissociation kit (gentleMACS Dissociator). Subsequently, 150 µL of homogenized tumor tissue suspension (150 mg/ml concentration) was injected into the flank of 8-10-week-old JAX NOD.CB17-PrkdcSCID-J mice (obtained from the Duke University Rodent Genetic and Breeding Core). Mice were observed daily and after first sign of tumor appearance, tumor size was checked every other day by digital caliper Vernier. Once the tumor size reached a size of 1cm X 1cm in diameters (1st generation), it was harvested, and passaged to make 2nd generation PDX. PDX were deemed stable after the 3rd generation. All animal procedures were according to Duke University Institutional Animal Care and Use Committee.  We use the Excision Repair-sequencing (XR-seq) method developed in our lab to study cisplatin repair in both human Patient Derived Xenografts (PDXs) cultivated in mice and in mouse liver tumors. XR-seq maps repair sites genome-wide at nucleotide resolution.   Two hours after injecting cisplatin, the mice were sacrificed by carbon dioxide exposure, the liver and kidneys were removed and washed extensively with cold PBS, and then homogenized in 5 mL ice-cold PBS using 15 strokes of a Teflon homogenizer, at which point the plunger moved freely. The homogenized tissues were transferred into 50-mL tubes and pelleted by centrifugation in a centrifuge (Model CL2, cat. no. 004260F; Thermo Fisher) at 2,500 rpm for 4 min, the supernatant was discarded, and the pellets were washed three times to remove fatty material. The pellets were suspended in 5 mL ice-cold Buffer A [25 mM Hepes (pH 7.9)], 100 mM KCl, 12 mM MgCl2, 0.5 mM EDTA, 2 mM DTT, 12.5% glycerol, 0.5% Nonidet P-40)/per liver or two kidneys and incubated for 10 min on ice. Resuspended cells were transferred to an ice-cold Dounce homogenizer and lysed on ice with 60 strokes using a tight plunger. The chromatin fraction was then pelleted by centrifugation for 30 min at 14,000 rpm at 4 °C in a centrifuge (Model 5418, cat. no. 022620304; Eppendorf). The supernatants were harvested for the first immunoprecipitation [anti-TFIIH, p89 antibody (G-10) and p62 antibody (H-10); Santa Cruz Biotechnology]. The DNAs were subjected to a second round of immunoprecipitation with anti-cisplatin antibody. Excision products were treated with NaCN to remove Pt before PCR. Remaining steps, including oligonucleotides and adaptors were according to the previously described XR-seq procedure.

first: Laura

last: Lindsey-Boltz

first: Courtney

last: Vaughn

first: Christopher

last: Selby

first: Xuemei

last: Cao

first: Zhenxing

last: Liu

first: David

last: Hsu

first: Aziz

last: Sancar


title: YAP and TAZ Dictate Binary Pan-Cancer Superclasses [RNA-seq]

first: Rod

last: Bremner

overall_design: Identification of YAP regulated genes in 2 retinoblastoma cell lines and 3 small cell lung cancer (SCLC) cell lines: Y79 (retinoblastoma) and SHP77 (SCLC) cell lines expressing an Empty vector (control sample), wild type YAP or S94A mutant YAP in duplicate, and WERI-RB1 (retinoblastoma), NCI-H209 (SCLC) and NCI-H2171 (SCLC) cell lines expressing an Empty vector (control sample) or 5SA mutant YAP in triplicate.

first: Joel

last: Pearson

first: Katherine

last: Huang


title: Downregulation of exhausted cytotoxic T cells in gene expression networks of multisystem inflammatory syndrome in children

first: Alexander

last: Charney

state: New York

address: 1424 Madison Ave, 14-52

institute: Icahn Medical School at Mount Sinai

summary: An unbiased, transcriptome-wide survey implicates downregulation of NK cells and cytotoxic T cell exhaustion in the pathogenesis of MIS-C.

overall_design: RNA-seq of a total 19 samples were analyzed. 8 MISC samples, 7 pediatric Covid samples, and 4 controls

middle: W

first: Noam

last: Beckman

first: Phillip

middle: H

last: Comella

first: Esther

last: Cheng


title: Epicardial Differentiation Drives Fibro-fatty Remodeling in Arrhythmogenic Cardiomyopathy

first: Eva

last: van Rooij

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

overall_design: Refer to individual Series


title: CRISPRi/a functional screen with PARP7 inhibitor in NCI-H1373 cells

city: Cambridge

first: Mario

last: Niepel

address: 35 Cambridgepark Dr Suite 300

institute: Ribon Therapeutics, Inc.

summary: PARP7 is a monoPARP that catalyzes the transfer of single units of ADP-ribose onto substrates to change their function.  PARP7 expression is increased by aromatic hydrocarbons and cellular stress, and the PARP7 gene is amplified in cancers, especially in those of the upper aerodigestive tract.  PARP7 is a negative regulator of nucleic acid sensing in tumor cells.  Inhibition of PARP7 restores Type I IFN signaling responses to nucleic acids in tumor models.  Restored signaling can directly inhibit cell proliferation and activate the immune system, both of which contribute to tumor regression.  RBN-2397 is a potent and selective inhibitor of PARP7.  To further explore how RBN-2397 inhibits NCI-H1373 cell proliferation, we performed unbiased genetic screens using whole-genome CRISPRi and CRISPRa libraries (le Sage et al., 2017; Jost and Weissman, 2018).  Comparison of CRISPRi and CRISPRa phenotype scores highlights genes with opposing functionality upon RBN-2397 treatment.

overall_design: CRISPR-Cas9 dual screening with RBN-2397 (2.5 nM) in NCI-H1373 cells was performed at Horizon Discovery (Cambridge, UK).  Briefly, cells were transduced with either whole-genome CRISPR activation (CRISPRa) or interference (CRISPRi) libraries, then selected and maintained in culture to allow CRISPR-driven gene perturbations to occur (le Sage et al., 2017).  Genomic DNA was extracted using the QIAamp DNA Blood Maxi kit (Qiagen #51194).  Samples were prepared and purified for amplicon sequencing using an Illumina NextSeq next generation sequencing platform.  Analysis of NGS data sets e.g. sgRNA abundance was achieved using Horizon’s data processing scripts, based on published analysis tool MAGeCK v0.5.04,5.  Dual screen analysis was achieved by normalizing average log-fold changes in guide abundance by the difference in population doubling for each screen track: Phenotype score = LFC (DMSO vs RBN)/ΔPD (DMSO vs RBN).  Vehicle (DMSO) treated cells were used as control.

first: Joseph

last: Gozgit


title: Human corneal endothelial cells, iPS cells, CECSi cells, and neural crest cells

city: Tokyo

first: Shin

last: Hatou

address: #305, 1-13-7 Nihombashi-Muromachi, Chuo

country: Japan

institute: Cellusion Inc

summary: Transcriptional profiling of  human iPS cells, CECSi cells and iPS-derived neural crest cells (NCC) comparing human corneal endothelial cells (HCE)

overall_design: HCEC vs iPS, CECSi and NCC, Biological replicates: 3 HCE replicates, 3 iPS replicates, 3 CECSi replicates, 2 CECSi replicates.


title: Investigation of the PCNA binding site in the DNA of cells in S-phase

city: Malmö

first: Maria

middle: Alvarado

last: Kristensson

address: 59 Jan Waldenströms street

country: Sweden

institute: Lund University

summary: In a cell, g-tubulin establishes a cellular network of threads named g-string meshwork. However, the functions of this meshwork remain to be determined. We investigated the traits of the meshwork and show that g-strings have the ability to connect the cytoplasm and the nuclear DNA together. We also show that g-tubulin has a role in determining the positioning of PCNA during DNA-replication.

overall_design:
The experiment was performed in an MCF10A cell line and a total number of 8 ChIP samples were included. There are two samples per timepoint (one and two hours) and antibody (anti-PCNA) per cell line. A whole cell lysate (WCE) is included as a control for each sample; these WCE samples are published under GEO Series GSE107975.
Please note that processed data was generated from two samples (replicates *.1 and *.2) and is linked to the first replicate (i.e. *1.1, *2.1) of each pair.

last: Alvarado-Kristensson

first: Lisa

last: Lindström


title: Identification of immune correlates of fatal outcomes in critically ill COVID-19 patients

city: Oxford

first: Nicholas

last: Lim

state: Oxfordshire

address: 26 Bullingdon Road

department: Paediatrics

country: United Kingdom

institute: University of Oxford

summary: Single-cell RNA-sequencing reveals a shift from focused IFN alpha-driven signals in COVID-19 ICU patients who survive to broad pro-inflammatory responses in fatal COVID-19 – a feature not observed in severe influenza. We conclude that fatal COVID-19 infection is driven by uncoordinated inflammatory responses that drive a hierarchy of T cell activation, elements of which can serve as prognostic indicators and potential targets for immune intervention.

overall_design: scRNA-seq of PBMCs from severe COVID patients using nanowell based method Seq-Well.

first: Jonathan

last: Youngs

middle: M

last: Provine

first: Philip

last: Goulder

first: Paul

last: Klenerman

first: Tihana

last: Bicanic


title: Neonatal Thyroxine Activation Modifies Epigenetic Programming Of The Liver [RNA-Seq]

city: Chicago

first: Tatiana

middle: L.

last: Fonseca

state: IL

address: 5841 S. Maryland Ave. MC1027, room N246B

department: Medicine

institute: University of Chicago

summary: In the neonatal liver, a peak of type 2 deiodinase (D2) activity accelerates local T3 production and the expression of thyroid hormone (TH)-responsive genes. Here we show that this acute increase in T3 signaling permanently modifies hepatic gene expression. Liver-specific Dio2 inactivation (Alb-D2KO) transiently increased H3K9me3 levels during post-natal days 1-5 (P1-P5) in discrete chromatin areas, and methylation of 1,508 DNA sites (H-sites) that remained in the adult mouse liver. These sites were associated with 1,551 areas of reduced chromatin accessibility (RCA; Atac-seq) within core promoters and 2,426 within intergenic regions, with reduction in the expression of 1,525 genes (RNA-seq). There was strong correlation between H-sites and RCA sites (r=0.85; p<0.0002), suggesting a cause-effect relationship. The analysis of chromosome conformation capture (Hi-C) data revealed a set of 57 repressed genes that have a promoter RCA in close contact with an intergenic RCA ~300 Kbp apart, including Foxa2 that plays an important role during development. Thus, the post-natal surge in hepatic D2 activity and TH-signaling prevents discrete DNA methylation and modifies the transcriptome of the adult mouse. This explains how the systemic T3 hormone acts locally during development to define future chromatin accessibility and expression of critically relevant hepatic genes.

overall_design: Examination of gene expression in liver of animals with Liver-specific Dio2 inactivation (Alb-D2KO).

first: Antonio

last: Bianco

first: Tzintzuni

last: Garcia

first: Gustavo

last: Fernandes

first: T

last: Nair


title: The g-tubulin meshwork assists to the transport of cytosolic PCNA to chromatin in mammalian cells

summary: Changes in the location of g-tubulin ensure cell survival and preserve genome integrity. We investigated whether the nuclear accumulation of g-tubulin facilitates the transport of proliferating cell nuclear antigen (PCNA) between the cytosolic and the nuclear compartment in mammalian cells. We found that the g-tubulin meshwork assists to the recruitment of PCNA to chromatin. Also, decreased levels of g-tubulin affected the nuclear pool of PCNA. In addition, the g-tubulin C terminus encodes a PCNA-interacting peptide (PIP) motif, and a g-tubulin–PIP-mutant affects the nuclear accumulation of PCNA. In a cell-free system, PCNA and g-tubulin formed a complex. In tumors, there is a significant positive correlation between TUBG1 and PCNA expression. Thus, we report a novel mechanism that constitutes the basis for tumor growth by which the g-tubulin meshwork maintains indefinite proliferation by acting as an opportune scaffold for the transport of PCNA from the cytosol to the chromatin.

overall_design: The experiment was performed in two cell lines and a total number of 64 samples were included.There are two samples for each antibody (gamma tubulin, PCNA, FoxM1  and Mcm5 in U2OS cells, and gamma tubulin, and PCNA for shTUBG U2OS cells) and timepoint (three, seven and nine hours, and Non-synchronous) per cell line and a whole cell lysate (WCE) is included as a control for each sample.

last: Alvarado Kristensson


title: CTCF and transcription orchestrate chromatin structure re-configuration after mitosis

status: Public on Jun 17 2021

city: Philadelphia

first: Yemin

last: Lan

state: PA

address: 3400 Civic Center Blvd

institute: University of Pennsylvania


title: Human PUS7 in tRNA pseudouridylation

city: San Francisco

first: Kailin

last: Yin

state: California

address: 1650 Owens street

institute: Gladstone Institutes

summary: Pseudouridine is the first discovered and the most frequent modification in RNA. However, its biological functions in physiology and human diseases are largely unknown. Here, we show that pseudouridine synthase PUS7 is differentially expressed in glioblastoma patient tissues verse non-tumor brain tissues, and highly expressed in patient brain-derived cancer stem cells, compared to normal brain-derived neural stem cells. Upregulated expression of PUS7 predicts worse survival in glioblastoma patients in multiple databases. Indeed, we show that PUS7 plays an important role in regulating the self-renewal and tumorigenesis of glioblastoma stem cells. Overexpression of the wild type but not the catalytically inactive PUS7 increases the growth and self-renewal of GSCs. In contrast, knockdown of PUS7 dramatically suppresses GSC growth, self-renewal and tumorigenesis. Mechanistically, knockdown of PUS7 activates interferon pathway through translational control of TYK2 via PUS7-regulated tRNAs. Moreover, we have identified chemical inhibitors for PUS7 in this study. These chemical compounds target pseudouridine modification and suppress GSC growth and tumorigenesis, providing a potential therapeutic tool for GBM treatment.

overall_design: mRNA, rRNA, and small RNA Ψ-seq to detect PUS7 dependent pesudouridne sites in PBT003 cells, NSC006 and G13 treatment cells

first: Xiaoting

last: Zhang

first: Chengqi

last: Yi


title: Neonatal Thyroxine Activation Modifies Epigenetic Programming Of The Liver [ATAC-Seq]

overall_design: Examination of chromatin accessibility in liver of animals with Liver-specific Dio2 inactivation (Alb-D2KO).


title: Chitinase-3 Like 1 Is a Profibrogenic Factor Overexpressed in  Aging Livers and in Patients with  Cirrhosis in the Aging Liver and in Patients with Liver Cirrhosis

city: Bethesda

first: Ronald

last: Engle

state: MD

address: 50 South Drive 6345

department: NIAID

institute: NIH

summary: Older age at the time of infection with hepatitis viruses is associated with an increased risk of liver fibrosis progression, but the mechanisms remain unclear. We hypothesized that the pace of liver fibrosis progression may reflect changes in gene expression within the aging liver. To test this hypothesis, we compared gene expression in liver specimens from 54 adult liver donors, including 36 over 40 years of age, that we defined older  and 18 between 18 and 40 years of age that we defined yonger.  Microarray was performed using Affymetrix Human U133 Plus 2 arrays.  Comparison of the genes between older and younger donors identified 13 genes that were differentially expressed with a p-value <0.001.

first: Patrizia

last: Farci

first: Norihisa

last: Nishimura


title: Effect of pivalate-induced secondary carnitine deficiency on hepatic transcriptome and plasma metabolome in growing pigs

city: Gießen

first: Robert

last: Ringseis

address: Heinrich-Buff-Ring 26-32

department: Institute of Animal Nutrition and Nutrition Physiology

institute: JLU Gießen

summary: Administration of pivalate has been demonstrated to be suitable for induction of secondary car-nitine deficiency (CD) in pigs, as model objects for humans. In order to comprehensively charac-terize the metabolic effects of secondary CD in the liver of pigs, the present study aimed to carry out comparative analysis of hepatic transcriptome and plasma metabolome of a total of 12, male 5-weeks-old pigs administered either pivalate (group PIV, n = 6) or vehicle (group CON, n = 6) for 28 days. Pigs of group PIV had approximately 40-60% lower concentrations of free carnitine and acetylcarnitine in plasma, liver and different skeletal muscles than pigs of group CON (p < 0.05). Transcript profiling of the liver revealed 140 differentially expressed genes (DEGs) between group PIV and group CON (fold change > 1.2 or < −1.2, p-value < 0.05). Biological process terms dealing with the innate immune response were found to be enriched with the DEGs (p < 0.05). Using a targeted metabolomics approach for the simultaneous quantification of 630 metabolites, 13 me-tabolites were identified to be lower and 5 metabolites to be higher in group PIV than in group CON (p < 0.05). Despite pivalate-induced CD caused only weak alterations of the hepatic tran-scriptome and the plasma metabolome, the changes observed indicate that secondary CD modu-lates the innate immune response of pigs.

overall_design: For the experiment a total of 12, 5-weeks-old, male cross-bred piglets (Pietrain x (German Landrace x German Edelschwein)) were used, which were kept in pairs in flat-deck pens under controlled conditions (23 ± 2°C room tem-perature, 50-60 % relative humidity, light from 06.00 a.m. to 07.00 p.m.). The piglets were randomly allocated to two groups (group CON, group PIV) of n = 6 piglets each, and fed identical nutrient-adequate diets [48] according to a phase feeding system (phase I: until a body weight of 15 kg; phase II: until the end of the experiment) throughout the experimental period of 28 days. The diet in phase I consisted of (g/kg): wheat, 381.7; barley, 315; soybean meal (44% crude protein), 250; soybean oil, 15; min-eral and vitamin premix, 33.5; L-lysin, 2.6; DL-methionine, 1.0 and L-threonine, 1.2. The diet of phase II consisted of (g/kg): wheat, 401.9; barley, 302; soybean meal (44% crude protein), 240; soybean oil, 15; mineral and vitamin premix, 33.4; L-lysin, 1.5; DL-methionine, 0.5 and L-threonine, 0.7 and a mixture of organic acids (containing 45% formic acid, 7.7% lactic acid, 5% sodium acetate), 5. The carnitine concentration in the diets was below 10 mg/kg diet as analyzed by LC-MS/MS according to [49]. In ad-dition, piglets were given orally either (per kg body weight (BW)): NaHCO3 (Sig-ma-Aldrich, Steinheim, Germany) solution (60 mg/100 mL) (group CON) or 30 mg so-dium pivalate (95% purity; Sigma-Aldrich, Steinheim, Germany) dissolved in NaHCO3 solution (group PIV) each day throughout the experimental period of 28 days. Water was constantly available ad libitum from nipple drinkers during both experiments. At the end of the experiment, the pigs were killed in the post-prandial period (2 h after their last meal) by electronarcosis followed by exsanguination. Blood was col-lected into heparinized polyethylene tubes (Sarstedt, Nümbrecht, Germany) and plasma obtained by centrifugation at 1100 x g for 10 min at 4°C and subsequently stored at -80°C. Aliquots from liver and selected muscles (longissimus dorsi, semitendi-nosus, superficial biceps femoris) were collected, snap-frozen in liquid nitrogen and stored at -80°C pending analysis.

first: Sarah

last: Grundmann

first: Klaus

last: Eder


title: Perivenous stellate cells are the main source of myofibroblasts and cancer-associated-fibroblasts formed after chronic liver injuries

city: Shanghai

first: Bo O.

last: Zhou

address: 320 Yueyang Road

department: Shanghai Institute of Biochemistry and Cell Biology

institute: Chinese Academy of Sciences

summary: We employed scRNA-seq to elucidate the transcriptome of all non-parenchymal cell (NPC) types from the liver. We have identified 9 different cell types based on the expression patterns of known cell type-specific marker genes. From the top differentially expressed genes, we identified Tcf21 as a novel HSC-specific gene. And Tcf21 is the only one that distinguishes quiescent HSCs from other liver cell types of the normal livers, as well as from activated HSCs in the fibrotic liver.

overall_design: Single-cell transcriptomic data (10x) are presented from healthy mice liver.

first: Bo

middle: O

first: Shan

middle: S

last: Wang

first: Xinyu

last: Tang

first: Minghui

last: Lin


title: Profiling of indolent and aggressive mammary lesions

city: Houston

first: Yuan

last: Qi

state: TX

address: 1400 Pressler St

department: Bioinformatics & Computational Biology

institute: University of Texas M.D. Anderson Cancer Center


title: Network Inference of Transcriptional Regulation in Germinating Low Phytic Acid Soybean Seeds

city: Blacksburg

first: Song

last: Li

state: VA

institute: Virginia Tech

summary: The low phytic acid trait in soybeans can be conferred by loss-of-function mutations in genes encoding myo-inositol phosphate synthase and two epistatically interacting genes encoding multidrug-resistance protein ABC transporters. However, perturbations in phytic acid biosynthesis are associated with poor seed vigor. Since the benefits of the low phytic acid trait, in terms of end-use quality and sustainability, far outweigh the negatives associated with poor seed performance, a fuller understanding of the molecular basis behind the negatives will assist crop breeders and engineers in producing variates with low phytic acid and better germination rate. The gene regulatory network for developing low and normal phytic acid soybean seeds was previously constructed, with genes modulating a variety of processes pertinent to phytic acid metabolism and seed viability being identified. In this study, a comparative time series analysis of low and normal phytic acid soybeans was carried out to investigate the transcriptional regulatory elements governing the transitional dynamics from dry seed to germinated seed. Gene regulatory networks were reverse engineered from time series transcriptomic data of three distinct genotypic subsets composed of low phytic acid soybean lines and their normal phytic acid sibling lines. Using a robust unsupervised network inference scheme, putative regulatory interactions were inferred for each subset of genotypes. These interactions were further validated by published regulatory interactions found in Arabidopsis thaliana and motif sequence analysis. Results indicate that low phytic acid seeds have increased sensitivity to stress, which could be due to changes in phytic acid levels, disrupted inositol phosphate signaling, disrupted phosphate ion homeostasis, and altered myo-inositol metabolism. Putative regulatory interactions were identified for the latter two processes. Changes in abscisic acid signaling candidate transcription factors putatively regulating genes in this process were identified as well. Analysis of the gene regulatory networks reveal altered regulation in processes that may be affecting the germination of low phytic acid soybean seeds. Therefore, this work contributes to the ongoing effort to elucidate molecular mechanisms underlying altered seed viability, germination and field emergence of low phytic acid crops, understanding of which is necessary in order to mitigate these problems.

overall_design:
The four genotypic lines used in this study are designated as “MRP,” contains four near isogenic lines, the lpa line “2mlpa” (mrp-l and mrp-n mutations) and three normal phytic acid lines, “2MWT” (no mutation), “2MWT-L” (mrp-n mutation only), and “2MWT-N” (mrp-l mutation only). These lines were developed from a cross between the lpa lines “CX-1834” (mrp-1 and mrp-n mutations) and the normal phytic acid line V99-5089. The mutations conferring the lpa trait in 2mlpa and CX-1834 are the result of point mutations in the epistatically interacting loci, MRP-L and MRP-N, on chromosomes 19 and 3, respectively (Wilcox et al., 2000; Walker et al., 2006; Maroof et al., 2009). In the associated manuscript, eight experimental lines were used – 1mlpa, 1MWT, 2mlpa, 2MWT, 2MWT-L, 2MWT-N, 3mlpa, and 3MWT.  The eight experimental lines represent three distinct subsets of genotypes and data from four lines are included in this dataset. Other four lines have been released from our previous publication (data in GSE101692).
Line "mips1/mrp-l/mrp-n" is also called "3mlpa" which contains mips1, mrp-1, and mrp-n mutations and has low phytic acid content. The normal phytic acid line "MIPS1/MRP-L/MRP-N" (no mutation) is also called 3MWT. These lines were developed from a cross between CX-1834 and V99-5089. These files also include the lpa line "1mlpa" (mips1 mutation) and the normal phytic acid line "1MWT" (no mutation). These lines are isogenic and were developed from a cross between the normal phytic acid line "Essex"" (no MIPS1 mutation) and the lpa line "V99-5089" (mips1 mutation).

first: Lindsay

last: DeMers

first: M

middle: A

last: Saghai-Maroof


title: CTCF and transcription orchestrate chromatin structure re-configuration after mitosis [ChIP-seq]

first: Yemin

last: Lan

summary: During mitosis, transcription is globally attenuated and chromatin architecture is dramatically reconfigured. Here we exploited the M-phase to G1-phase progression to interrogate the contributions of the architectural factor CTCF and the process of transcription to re-sculpting the genome in newborn nuclei. While CTCF appears to be dispensable for large scale post-mitotic compartmentalization, depletion of CTCF specifically during the M-phase to G1-phase transition alters the re-establishment of local short-range compartmentalization after mitosis. Without CTCF, structural loops fail to reform, leading to illegitimate contacts between cis-regulatory elements (CREs) and altered gene expression in G1-phase. Transient CRE contacts that are normally resolved after telophase persist deeply into G1-phase in CTCF depleted cells. Boundary reformation is largely disrupted upon CTCF loss. Yet, a subset (~27%) of boundaries emerges normally in the absence of CTCF and is characterized by transitions in chromatin states. Reformation of gene domains can occur prior to the full onset of transcription and can be linked to tri-methylation at lysine 36 of histone 3 (H3K36me3), a mark stable throughout mitosis. The focus on the de novo formation of nuclear architecture during G1 entry yielded novel insights into how CTCF and the process of transcription contribute to the dynamic re-configuration of chromatin architecture during the mitosis to G1 phase progression.

overall_design: Uninduced G1E-ER4 (-GATA1) cells were synchronized to pro-metaphase with nocodazole treatment and treated with or without auxin to remove CTCF. Cells from both CTCF depleted and replated groups were released from nocodazole for a variety of durations. For PolII ChIP-seq: Cells were harvested at 0min (prometa), 60min (early-G1), 120min (mid-G1) or 240min (late-G1) after nocodazole release to assess post-mitotic PolII binding with or without CTCF. 2-3 biological replicates per time point were collected

first: Haoyue

first: Gerd

last: Blobel


title: Neonatal Thyroxine Activation Modifies Epigenetic Programming Of The Liver [ChIP-Seq]

first: Tatiana

last: Fonseca

overall_design: Examination of H3K9me3 histone modification in liver of animals with Liver-specific Dio2 inactivation (Alb-D2KO).

first: Antonio

middle: C

last: Bianco

first: Tzintzuni

last: Garcia

first: Gustavo

last: Fernandes

first: T

last: Nair


title: Role of Gpnmb in response to virally expressed mutant SNCA

city: South San Francisco

first: Brad

last: Friedman

state: CA

address: 1 DNA Way

department: Bioinformatics

institute: Genentech, Inc.

summary:
Effect of Gpnmb knockout on development of differential expression in AAV-aSyn model was studied.
The "samid" sample characteristic is a sample identifier internal to Genentech.
The ID of this project in Genentech's ExpressionPlot database is PRJ0036339

overall_design: RNA from contralateral or ipsilateral whole striatum or midbrain of male mice 10 weeks after injection with AAV expressing either human aSyn-A53T or null virus.

first: Bob

last: Brendza


title: Transcriptional profiling of indolent and aggressive mammary lesions

summary: We performed transcriptional profiling of tumor cells from indolent and aggressive lesions isolated from a mouse model of breast cancer. To initiate formation of mammary lesions, we delivered lentiviral particles carrying constitutively-activated Erbb2 tagged with HA and GFP (caErbB2) through the lactiferous glands of adult virgin female. Mammary glands bearing lesions were visualized under a fluorescence stereoscope, and regions harboring aggressive (>2mm, comprised of invasive lesions) and indolent (<2mm, enriched for in situ lesions) lesions were dissected away from each other. Lesions from multiple animals were pooled to obtain sufficient material for downstream analysis. Following tissue digestion, GFP-expressing epithelial cells were isolated by fluorescence-activated cell sorting (FACS), and then subjected to bulk RNA-seq.

overall_design: Transcriptional profiles of tumor cells from indolent and aggressive mouse mammary lesions were generated from multiple pools of animals

first: Helen

last: Piwnica-Worms

first: Vidya

last: Sinha


title: Single-cell transcriptional profiling of indolent and aggressive mammary lesions

summary: We performed transcriptional profiling of tumor cells from indolent and aggressive lesions isolated from a mouse model of breast cancer. To initiate formation of mammary lesions, we delivered lentiviral particles carrying constitutively-activated Erbb2 tagged with HA and GFP (caErbB2) through the lactiferous glands of adult virgin female. Mammary glands bearing lesions were visualized under a fluorescence stereoscope, and regions harboring aggressive (>2mm, comprised of invasive lesions) and indolent (<2mm, enriched for in situ lesions) lesions were dissected away from each other. Lesions from multiple animals were pooled to obtain sufficient material for downstream analysis. Following tissue digestion, GFP-expressing epithelial cells and CD45+ immune cells were isolated by fluorescence-activated cell sorting (FACS), and then subjected to single-cell RNA-sequencing.

overall_design: Single-cell transcriptional profiles of tumor and immune cells from indolent and aggressive mouse mammary lesions were generated from a pool of animals


title: Transcriptomic, proteomic and phosphoproteomic underpinnings of daily exercise performance and Zeitgeber activity of endurance training in mouse skeletal muscle

status: Public on Jun 16 2021

city: Basel

first: Christoph

last: Handschin

address: Spitalstrasse 41

country: Switzerland

institute: Biozentrum, University of Basel

summary:
Running at different times of the day resulted in a vastly different activation of signaling pathways, e.g., related to stress response, vesicular trafficking, repair, and regeneration.
Second, voluntary wheel running at the opposite phase of the dark, feeding period surprisingly revealed minimal Zeitgeber (i.e., synchronizing) activity of training.
This integrated study provides important insights into the circadian regulation of endurance performance and the control of the circadian clock by exercise.

overall_design: Investigating the effect of acute treadmill exercise and voluntary chronic wheel running exercise on the transcriptomic, proteomic and phosphoproteomic level at different times of the day in mouse skeletal muscle

first: Geraldine

last: Maier

first: Julien

last: Delezie

first: Pål

last: Westermark

first: Gesa

last: Santos

first: Danilo

last: Ritz


title: Genome-wide effect of STING-mediated acivation in endothelial cells in response to TNF

city: Boston

first: Alexander

last: Poltorak

state: Massachusetts

address: 150 Harrison avenue

institute: Tufts University

summary: We report the role of STING in mediating responses to TNF in the mouse endothelial cells

overall_design: primary endothelisal cells from mouse heart were plated on gelatin support, let adhere, and activated for 4 hours with TNF

first: Marina

last: Anastasiou


title: RNA seq analysis of U251 and A549 cells transduced with tet-pLKO1-SHC016 control shRNA

city: Kraków

first: Joanna

last: Bereta

address: Gronostajowa 7

department: Cell Biochemistry

country: Poland

institute: Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University

summary: The goal of this study was to find a target of SHC016 common to both cell lines

overall_design: The cells were transduced with tetracycline (doxycycline)- inducible SHC016 shRNA, non targeting control for MISSION shRNA library. The expression of SHC016 was induced with doxycycline at a concentration of 100 ng/ml for 24 h and than RNA was isolated.

first: Maria

last: Czarnek


title: CTCF and transcription orchestrate chromatin structure re-configuration after mitosis [Hi-C]

first: Yemin

last: Lan

overall_design: Uninduced G1E-ER4 (-GATA1) cells were synchronized to pro-metaphase with nocodazole treatment and treated with or without auxin to remove CTCF. Cells from both CTCF depleted and replated groups were released from nocodazole for a variety of durations. Cells at different cell cycle stages were purified with FACS. Hi-C experiments were performed on purified populations to examine chromatin structure with or without CTCF at designated cell cycle stage. 2-3 biological replicates were performed for each time point

first: Haoyue

last: Zhang

first: Gerd

last: Blobel


title: Gene expression atlas of energy balance brain regions

city: New York

first: Claudia

last: Doege

address: 1150 Saint Nicholas Ave

department: Pathology

institute: Columbia University

summary: This project involved bulk-RNAseq analysis of mouse brain regions involved in energy balance. Two mouse embryonic stem cell controls were included

overall_design: 70 samples across 22 brain regions and two stem cell lines

first: Hannah

last: Glover

last: De Rosa


title: Investigation of Measurable Residual Disease in Acute Myeloid Leukemia by DNA Methylation Patterns

city: Aachen

first: Chao-Chung

last: Kuo

state: NRW

address: Pauwelsstrasse 30

department: IZKF Genomics Facility

institute: RWTH Aachen University Hospital

type: Methylation profiling by high throughput sequencing

summary: Assessment of measurable residual disease (MRD) upon treatment of acute myeloid leukemia (AML) remains a challenge. It is usually addressed by highly sensitive PCR-based screening of disease-specific mutations or multiparametric flow cytometry. However, not all patients have suitable mutations to track the disease and heterogeneity of applied surface markers hampers standardization in clinical routine. In this study, we propose an alternative approach to estimate MRD based on AML-associated DNA methylation (DNAm) patterns. We identified four CG dinucleotides (CpGs) that commonly reveal aberrant DNAm in AML and their combination could reliably discern healthy and AML samples. Interestingly, many of these aberrations are shown to affect both alleles. Using bisulfite amplicon sequencing (BA-seq), we captured local DNAm patterns flanking these AML associated CpGs, and trained shallow-learning and deep-learning algorithms to identify anomalous, non healthy DNAm patterns within the amplicons. The method was then tested on follow-up samples with and without MRD status. Notably, many MRD negative samples revealed higher anomaly ratios than healthy controls. Our results demonstrate that targeted DNAm analysis facilitates reliable discrimination of malignant and healthy samples. However, even after successful treatment aberrant DNAm patterns persist in some samples that were classified as MRD negative.

overall_design:
Assays for targeted bisulfite amplicon sequencing (BA-seq) were designed for four regions with the AML associated CpGs (cg15289427, cg22797031, cg27630153, and cg19586199). The methylation statuses on each read were identified and analyzed for the pattern for stratification of disease status.
Definitions:
PB: Peripheral Blood
BM: Bone Marrow
NA: Not Available
Mixed DNA: Mixture of different sources of DNA.

Chromosome coordinates of CpGs used in this study:
cg15289427(FAM65B), chr6:24910743-24911059 (hg19)
cg22797031, chr1:170630003-170630204 (hg19)
cg27630153(FAM38A), chr16:88844790-88845210 (hg19)
cg19586199(PRKACA), chr19:14224943-14225293 (hg19)

first: Tanja

last: Božić

first: Jan

last: Hapala

first: Julia

last: Franzen

first: Monika

last: Eipel

first: Uwe

last: Platzbecker

first: Martin

last: Kirschner

first: Fabian

last: Beier

first: Edgar

last: Jost

first: Christian

last: Thiede

first: Wolfgang

last: Wagner


title: The LysR-type transcriptional regulator BsrA (PA2121) is engaged in the control of vital metabolic pathways in Pseudomonas aeruginosa (ChIP-seq)

city: Warszawa

first: Adam

last: Kawałek

address: Pawinskiego 5A

department: Department of Microbial Biochemistry

institute: Institute of Biochemistry and Biophysics, PAS

summary: Pseudomonas aeruginosa, a facultative human pathogen causing nosocomial infections, has complex regulatory systems involving many transcriptional regulators. The LTTR family (LysR-Type Transcriptional Regulators) consists of proteins involved in regulation of various processes including stress response, motility, virulence or amino acid metabolism. The aim of this study was characterization of the LysR-type regulator BsrA (PA2121), identified previously as a negative regulator of biofilm formation in P. aeruginosa. To identify the BsrA binding sites in P. aeruginosa the ChIP-seq analysis was performed. It revealed 765 BsrA binding sites in P. aeruginosa PAO1161 genome, among them 367 was localized in the intergenic regions. Parallel transcriptomic analysis identified altered expression of 157 genes in response to BsrA excess, among them 35 had a BsrA binding site in the corresponding promoter regions, indicating direct influence of BsrA on expression of these genes. BsrA-repressed loci encompass genes encoding proteins engaged in key metabolic pathways including the tricarboxylic acid cycle. A group of directly activated genes by BsrA, consists of several loci encoding proteins involved in pili/fimbriae assembly as well as secretion and transport systems. Results also confirmed that BsrA acts as an autorepressor. Presented data uncover the regulon of BsrA protein with its role as transcriptional regulator of genes engaged in vital cellular processes in P. aeruginosa.

overall_design: Pseudomonas aeruginosa PAO1161 (leu-, r-, RifR) derivative of PAO1, was used in the experiment (Kawalek et al., 2020; BMC Genomics, 21:14). Chromatin immunoprecipitation and sequencing (ChIP-seq) analysis was performed on P. aeruginosa PAO1161 ΔbsrA / pMEB99 (lacIQ-tacp-bsrA-flag) (hereafter referred to as BsrA-F) as well as PAO1161 /pABB28.1 (lacIQ-tacp-flag), empty vector control (hereafter called EV-F) strains. Cells were grown under selection in L broth with 0.05 mM IPTG untillOD600 = 0.5). For each biological replicate, two independent cultures of each strain were pooled together. The immunoprecipitation was performed with commercially available polyclonal anti-FLAG antibodies (DYKDDDDK Tag polyclonal  antibodies; PA1-985B; Invitrogen).

first: Magdalena

last: Modrzejewska

last: Kawalek

first: Aneta

last: Bartosik


title: Neonatal Thyroxine Activation Modifies Epigenetic Programming Of The Liver

first: Tatiana

last: Fonseca

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

overall_design: Refer to individual Series


title: Characterization of a RAD51C-Silenced High Grade Serous Ovarian Cancer Model During PARP Inhibitor Resistance Development

city: Rochester

first: Hu

last: Li

state: MN

address: 200 First Street, Gonda Building G19-408

department: Molecular Pharmacology & Experimental Therapeutics

institute: Mayo Clinic

type: Methylation profiling by genome tiling array