title: Genetic regulation of nascent RNA maturation revealed by direct RNA nanopore sequencing [nanopore]

status: Public on Oct 22 2024

city: Sherbrooke

first: Karine

last: Choquet

state: Québec

address: 3201 rue Jean-Mignault

department: Biochemistry and Functional Genomics

country: Canada

institute: Université de Sherbrooke

summary: Quantitative trait loci analyses have revealed an important role for genetic variants in regulating alternative splicing (AS) and alternative cleavage and polyadenylation (APA) in humans. Yet, these studies are generally performed with mature mRNA, so they report on the outcome rather than the processes of RNA maturation and thus may overlook how variants directly modulate pre-mRNA processing. The order in which the many introns of a human gene are removed can substantially influence AS, while nascent RNA polyadenylation can affect RNA stability and decay. However, how splicing order and poly(A) tail length are regulated by genetic variation has never been explored. Here, we used direct RNA nanopore sequencing to investigate allele-specific pre-mRNA maturation in 12 human lymphoblastoid cell lines. We found frequent splicing order differences between alleles and uncovered significant single nucleotide polymorphism (SNP)-splicing order associations in 17 genes. This included SNPs located in or near splice sites as well as more distal intronic and exonic SNPs. Moreover, several genes showed allele-specific poly(A) tail lengths, many of which also had a skewed allelic abundance ratio. HLA class I transcripts, which encode proteins that play an essential role in antigen presentation, showed the most allele-specific splicing orders, which frequently co-occurred with allele-specific AS, APA or poly(A) tail length differences. Together, our results expose new layers of genetic regulation of pre-mRNA maturation and highlight the power of long-read RNA sequencing for allele-specific analyses.

overall_design: Subcellular direct nanopore RNA sequencing in human lymphoblastoid cell lines

first: Louis-Philippe

last: Chaumont

first: Autum

last: Baxter-Koenigs

first: L

last: Churchman

first: Simon

last: Bache


title: A long context RNA foundation model for predicting transcriptome architecture

city: San Francisco

first: Hani

last: Goodarzi

state: CA

department: Biochemistry and Biophysics

country: USA

institute: UCSF

type: Expression profiling by high throughput sequencing

summary: Linking DNA sequence to genomic function remains one of the grand challenges in genetics and genomics. Here, we combine large-scale single-molecule transcriptome sequencing of diverse cancer cell lines with cutting-edge machine learning to build LoRNASH, an RNA foundation model that learns how the nucleotide sequence of unspliced pre-mRNA dictates transcriptome architecture—the relative abundances and molecular structures of mRNA isoforms. Owing to its use of the StripedHyena architecture, LoRNASH handles extremely long sequence inputs at base-pair resolution (~65 kilobase pairs), allowing for quantitative, zero-shot prediction of all aspects of transcriptome architecture, including isoform abundance, isoform structure, and the impact of DNA sequence variants on transcript structure and abundance. We anticipate that our public data release and the accompanying frontier model will accelerate many aspects of RNA biotechnology. More broadly, we envision the use of LoRNASH as a foundation for fine-tuning of any transcriptome-related downstream prediction task, including cell-type specific gene expression, splicing, and general RNA processing.

overall_design: Cells were cultured until 80% confluence before RNA extraction using TRIzol reagent (ThermoFisher) followed by column purification (Zymo Research). Short-read (Takara Bio SMARTer Stranded Total RNA-seq kit v3) and long-read RNA-sequencing libraries (NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module, PacBio Iso-Seq Express Oligo Kit and SMRTbell express template prep kit 3.0) were then constructed using matched RNA samples.

first: Ali

last: Saberi

first: Benedict

last: Choi

first: Simai

last: Wang

first: Vijay

last: Ramani


title: Chaperone saturation mediates translation and protein folding efficiency

status: Public on Oct 27 2024

city: Baltimore

first: Vincent

middle: J

last: Hilser

state: Maryland

address: 3400 N Charles St

department: Biology

institute: The Johns Hopkins University

summary: Whether the emergence of a nascent protein from the ribosome and the formation of structural elements are synchronized has been a longstanding question. Paradoxically, kinetically efficient translation can induce mis-folding and aggregation despite the presence of molecular chaperones, which in Escherichia coli are induced by unfolded protein via σ32. The molecular mechanisms mediating translation efficiency and protein folding efficiency remain poorly understood. Using ribosome profiling and protein quantitation, we show that synonymous changes to Firefly Luciferase (Luc) mRNA have a direct effect on its translation efficiency. These changes alone cause up to a 70-fold difference in Luc protein levels. However, increased Luc protein is met with at most a ~2-fold increase in chaperone levels, revealing that the σ32 transcriptional response has saturable properties. This response is found to be poised near its midpoint (where it is most sensitive to perturbation) when Luc mRNA has an intermediate translation efficiency. These results suggest not only that chaperone saturation limits the ability of cells to maintain protein folding homeostasis when challenged with highly efficient translation, but that translation efficiency and protein folding efficiency evolved for mutual sensitivity.

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

overall_design:
Cells containing pACYC177 plasmid serve as the control samples expressing no Firefly Luciferase. All experiments were performed in duplicate or triplicate.
Some samples were processed at the same time and sequenced together (5 total series):
C_1, S_1, W_1, H_1, F_1
C_2, S_2, W_2, H_2, F_2
C_3, S_3, W_3, H_3, F_3
SF_1, SF_2, SF_3, FS_1, FS_2, FS_3
SinF_1, SinF_2, FinS_1, FinS_2

mRNA sequencing experiments were performed once.
C_mRNA,S_mRNA,W_mRNA,H_mRNA,F_mRNA

first: Andrew

middle: T

last: Martens


title: Polycomb Repressive Complexes occupancy reveals PRC2- independent PRC1 critical role in the control of limb development [RNA-seq]

status: Public on Oct 21 2024

city: Montréal

first: Fanny

last: GUERARD-MILLET

state: Quebec

address: 110 AVENUE DES PINS OUEST

institute: IRCM

summary: The Polycomb Repressive Complexes (PRC) are key players in the regulation of tissuespecific gene expression through their ability to epigenetically silence developmental genes. They are subdivided into two multicomponent complexes, PRC1 and PRC2, functioning through posttranslational modifications of histone tails. A large body of work revealed functional interactions between PRC1 and PRC2, whereby trimethylation of lysine 27 on histone H3 (H3K27me3) by PRC2 contributes to the recruitment of canonical PRC1 (cPRC1). In parallel, a PRC2-independent binding of PRC1 has been uncovered and referred to as non-canonical PRC1 or variant PRC1 (vPRC1), in which PRC1-dependent ubiquitination of lysine 119 on histone H2A is involved in recruiting PRC2/propagating PRC2-dependent H3K27 trimethylation. While it was initially assumed that cPRC1 and vPRC1 bind distinct targets, subsequent evidence pointed to cPRC1 and vPRC1 sharing a significant subset of targets. How the functional interplay between PRC2, cPRC1 and vPRC1 contributes to gene regulation remains partially understood. Here, we show that, in the developing limb, PRC2 inactivation barely affects PRC1 occupancy, as the majority of PRC2- bound loci are bound by vPRC1 (RYBP-PRC1), both in wild type and PRC2 mutant limbs. Consistent with this, we found that loci bound by CBX2, a PRC1 subunit involved in the recognition of H3K27me3 and thereby recruitment of cPRC1, are, for the vast majority, also bound by vPRC1. Intriguingly, analysis of PRC2 mutant limbs revealed that while a large part of CBX2 occupancy is lost in absence of PRC2 function, as expected from the absence of H3K27me3, there is a significant number of genes retaining CBX2 occupancy as well as a few genes with apparent gain of CBX2 binding. Importantly, among these genes, 56 of them correspond to developmental genes known for playing a key role in limb morphogenesis. Based on the importance of vPRC1 in gene silencing, our findings emphasize the primary role of PRC2-independent PCR1 function in regulating developmental genes and questions the role of PRC2/cPRC1 in controlling developmental programs.

overall_design:
RNAseq analysis in proximal and distal limb tissue at E12.5

UPDATE: [June 06, 2025] The titles of Samples GSM6693221-GSM6693232 were updated.

first: Claudia

last: GENTILE

first: Racheal

last: PAUL

first: Christian

last: POITRAS


title: Polycomb Repressive Complexes occupancy reveals PRC2- independent PRC1 critical role in the control of limb development

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Transcript profiling of wrinkled1 mutant embryos in Arabidopsis thaliana

status: Public on Oct 16 2024

city: Davis

first: John

last: Harada

address: One Shields Avenue

department: Plant Biology

institute: University of California

summary: Understanding the mechanisms of transcriptional regulation for genes involved with FA biosynthesis and TAG accumulation in seeds can represent an important step in the development of crops with increased oil content. Among well studied transcription factors (TFs), WRINKLED1 (WRI1), a member of the APETALA2 (AP2) family of TFs, has been characterized as a central regulator of FA biosynthesis in seeds of many plant species.  Despite its importance for the process, the detailed mechanisms of WRI1 function in regulating FA biosynthesis in seeds is still not well understood. This study aimed to characterize the gene networks controled by WRI1 during soybean embryo development and its function as central regulator of FA biosynthesis and TAG accumulation in seeds.  In this part of the study, we identified genes misregulated in the embryos of the Arabidopsis mutant wrinkled1-1, when compared to wild-type.

overall_design: Embryos from homozygous wrinkled1-1 or wild-type plants were collected, 8 days after pollination.  Three biological replicates per genotype were analyzed.

first: Julie

last: Pelletier

first: Leonardo

last: Jo

first: Robert

middle: B

last: Golberg


title: Key epigenetic and signaling factors in the formation and maintenance of the blood-brain barrier

city: Houston

first: Peeyush Kumar

last: Thankamani Pandit

state: TEXAS

address: 6431 Fannin St,

department: Neurosurgery

institute: University of Texas Health Science Center Houston

summary:
To identify CNS endothelial cell (EC) the gene cohorts that participate in blood brain barrier (BBB) formation versus maintenance. We investigated the mechanism behind the reduced Wnt/β-catenin signaling observed in during later development
We also investigated whether the adult endothelial cells  can re express the EC gene cohorts required for the BBB formation.

overall_design: Ultra low mRNA sequencing of FACS sorted CD31+ve cortical ECs from E-17.5 W.T and HDAC2 ECKO. To conditionally KO HDAC2 E-17.5 endothelial cells we used tamoxifen-inducible Cdh5(PAC)-CreERT2 mice. HDAC2 flox/flox: mother was crossed with HDAC2flox/flox:Cdh5(PAC)-CreERT2 male. Tamoxifen was injected into the mother starting at E-12.5 and on alternate days until E-16.5. E-17.5 brain was harvested and cortex was isolated for Cd31+ve FACS sorting.

first: Peeyush

middle: K


title: TEAL-seq (Targeted Expression Analysis Sequencing)  of S. aureus and S. epidermidis in TSB and skin-relevent conditions

status: Public on Oct 18 2024

city: Farmington

first: Mark

last: Adams

state: CT

address: 10 Discovery Drive

institute: The Jackson Laboratory

summary: Metagenome sequencing enables discovery and genetic characterization of complex microbial communities from diverse ecosystems. However, determining the activity of isolates within a community using transcriptomics presents several challenges including the wide dynamic range of organismal and gene expression abundances, the presence of host RNA, and low microbial biomass at many body sites. To address these limitations, we developed “Targeted Expression Analysis Sequencing” or TEAL-seq. Targeting strategies enabled sensitive species-specific analyses of gene expression using highly multiplexed custom probe pools targeting about 1700 core and accessory genes of Staphylococcus aureus (S.a.) and S. epidermidis (S.e.), two key species of the skin microbiome. Two targeting methods were applied to mixed cultures and nasal swab specimens from human research participants. Both methods showed a high degree of specificity, with >90% reads on target, even in the presence of complex microbial or human background DNA/RNA. Targeting using molecular inversion probes demonstrated excellent correlation in inferred expression levels with bulk RNA-seq. Further, we show that a linear pre-amplification step to increase the amount of input nucleic acids for analysis was quite reproducible . While pre-amplification introduced some noise compared to non-amplified samples, it also enabled profiling of expression from as little as 1 ng of total RNA. TEAL-seq is much less expensive than bulk metatranscriptomic profiling and enables detection across a greater dynamic range. Custom probe pools are readily configurable and this strategy is broadly applicable for determining the transcriptional status of organisms in any microbial community.

sample_organism:
Escherichia coli
Staphylococcus aureus
Staphylococcus epidermidis
Homo sapiens
Mus musculus

overall_design: S. aureus and S. epidermidis were grown in TSB, TSB pH 4.8 or on reconstructed human epidermidis for bulk and targted RNA-seq with and without ribo-depletion and/or SPIA.

first: Georgia

last: Doing

first: Priya

last: Shanbhag

first: Isaac

last: Bell

first: Efthymios

last: Motakis

first: Elizabeth

last: Aiken

first: Julia

last: Oh

middle: D


title: mRNA changes following induction of GmWRI1 activity in Arabidopsis leaf mesophyll protoplasts

summary: Understanding the mechanisms of transcriptional regulation for genes involved with FA biosynthesis and TAG accumulation in seeds can represent an important step in the development of crops with increased oil content. Among well studied transcription factors (TFs), WRINKLED1 (WRI1), a member of the APETALA2 (AP2) family of TFs, has been characterized as a central regulator of FA biosynthesis in seeds of many plant species.  Despite its importance for the process, the detailed mechanisms of WRI1 function in regulating FA biosynthesis in seeds is still not well understood. This study aimed to characterize the gene networks controled by WRI1 during soybean embryo development and its function as central regulator of FA biosynthesis and TAG accumulation in seeds.  In this part of the study, we identified genes regulated by the soybean WRI1s (GmWRI1s) by overexpressing GmWRI1-A (Glyma.08G227700) in Arabidopsis leaf mesophyll protoplasts, and identifying the mRNAs that are induced 16h after transfection.

overall_design: Arabidopsis mesophyll protoplasts were transfected with either 35S:GmWRI1-A or 35S:mCHERRY constructs and harvested after a 16h incubation.  Three biological replicates per treatment were analyzed.


title: Identification of GLYMA.08G227700 binding sites in soybean cotyledon stage embryos

summary: To dissect the gene regulatory networks operating during soybean embryo development, we identified the binding sites genome-wide for transcription factor GLYMA.08G227700  in early maturation embryos using ChIP-seq.

overall_design: Identification of GLYMA.08G227700 binding sites in soybean embryos at the cotyledon stage.  ChIP and total chromatin controls (input) were obtained for two biological replicates.  A total of 4 samples were sequenced using the Illumina NovaSeq platform.

first: Rie

last: Uzawa

first: Min

last: Chen


title: Regulatory T cells and IFN-γ-producing Th1 cells play a critical role in the pathogenesis of Sjögren’s Syndrome

status: Public on Nov 04 2024

city: New York

first: Maxwell

last: McDermott

state: NY

address: 550 1st Ave

department: Pathology

institute: NYU Langone Health

summary: Sjogren’s Disease (SjD) is an autoimmune disorder characterized by progressive inflammation and destruction of both salivary and lacrimal gland leading to dysfunction, as well as extra glandular manifestations. The etiology and pathophysiology of SjD are incompletely understood. Regulatory T (Treg) cells are critical for immunological tolerance. Although Treg abnormalities have been implicated in the pathogenesis of several autoimmune diseases, their role in SjD remains ambiguous. We had previously shown that the function and development of Treg cells depend on store-operated Ca2+ entry (SOCE), which is mediated by ORAI1 Ca2+ channels and stromal interaction molecules (STIM) 1 and 2 following T cell receptor (TCR) stimulation. The ablation of SOCE in Treg cells causes SjD-like disease with increased Th1 and interferon gene expression signatures in the inflamed salivary glands of mice. Here we show enhanced Th1 gene expression signatures and type I and II interferon responses in the peripheral blood mononuclear cells (PBMCs) of human patients with SjD compared to non-SjD controls. We also identified attenuated gene expression in memory Treg cells of patients with SjD. These data from mice and human patients provide new evidence for a critical role of Treg cells and IFNγ producing Th1 cells in the pathogenesis of SjD.

overall_design: PBMCs of patients with SjD and non-SjD controls were acquired from the SICCA consortium. The single-cell transcriptomic profiles of a total of 9 SjD patients and 8 non-SjD controls were assessed using single-cell RNA sequencing (scRNA-seq).

first: Li

last: Wenyi

first: Yin-Hu

last: Wang

first: Stefan

last: Feske


title: The regulatory effect of IRF8 on the gene expression profiles of bone marrow derived dendritic cells

status: Public on Oct 10 2024

city: Hangzhou

first: Zhejun

last: Cai

state: Zhejiang

address: 88 Jiefang Road

department: Department of Cardiology, The Second Affiliated Hospital

country: China

institute: Zhejiang University School of Medicine

summary: To investigate the role of IRF8 in the inflammatory microenvironment of abdominal aortic aneurysm tissue, bone marrow cells were isolated from the tibias and femurs of 8-week-old male mice and cultured with granulocyte-macrophage colony-stimulating factor and Flt3L cytokines to generate CD103-positive dendritic cells. Cells were harvested on days 9 and 15-16 and stimulated with tumor necrosis factor-alpha. Gene ontology analysis revealed upregulation of T cell activation and chemotaxis in bone marrow-derived dendritic cells from IRF8 overexpression mice, indicating IRF8's role in dendritic cell-T cell interactions. Protein-protein interaction analysis highlighted Cd40 and Fcgr1 as key surface markers. IRF8-related genes were enriched in type I interferon signaling pathways, implicating IRF8 in antigen presentation functions of dendritic cells.

overall_design: 8-week-old male mice, both wild-type and genetically modified IRF8 overexpression mice were be used. Bone marrow were extracted from the tibias and femurs, and the cells were cultured in RPMI 1640 medium supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF) and Flt3L cytokines to generate CD103-positive dendritic cells. On days 9 and 15-16, non-adherent dendritic cells were harvested and stimulated with tumor necrosis factor-alpha. Comparisons of  gene expression profiles and enrichment differences were conducted between Irf8-OE mice and control ones

first: Zhen

last: Yuan

last: Shu

first: Yidong


title: CTCF/RAD21 organize the ground state of chromatin-nuclear speckle association

status: Public on Nov 07 2024

city: Philadelphia

first: Ruofan

last: Yu

state: PA

address: 3400 civic center blvd

department: Cell and Developmental Biology

institute: University of Pennsylvania

summary: Recent findings indicate that nuclear speckles, a distinct type of nuclear body, interact with certain chromatin regions in a ground state. Here, we report that the chromatin structural factors CTCF and cohesin are required for full ground-state association between DNA and nuclear speckles. We identified a putative speckle targeting motif (STM) within cohesin subunit RAD21 and demonstrated that the STM is required for chromatin-nuclear speckle association, disruption of which impaired induction of speckle-associated genes.  Depletion of the cohesin-releasing factor WAPL, which stabilizes cohesin on chromatin and reinforces DNA–speckle contacts, results in enhanced inducibility of speckle-associated genes. Additionally, we observed disruption of chromatin–nuclear speckle association in patient-derived cells with Cornelia de Lange syndrome, a congenital neurodevelopmental disorder involving defective cohesin pathways. In summary, our findings reveal a mechanism for establishing the ground state of chromatin–speckle association and promoting gene inducibility, with relevance to human disease.

overall_design: Refer to specific sequencing types.


title: Cytosolic Ribosomal Protein Haploinsufficiency affects Mitochondrial Morphology and Respiration

status: Public on Oct 31 2024

city: Austin

first: ELIF

last: SARINAY CENIK

state: TX

address: 2415 Speedway

institute: University of Texas at Austin

summary: The interplay between ribosomal protein composition and mitochondrial function is essential for sustaining energy homeostasis. Precise stoichiometric production of ribosomal proteins is crucial to maximize protein synthesis efficiency while reducing the energy costs to the cell. However, the impact of this balance on mitochondrial ATP generation, morphology and function remains unclear. Particularly, the loss of a single copy ribosomal protein gene is observed in Mendelian disorders like Diamond Blackfan Anemia and is common in somatic tumors, yet the implications of this imbalance on mitochondrial function and energy dynamics are still unclear. In this study, we investigated the impact of haploinsufficiency for four ribosomal protein genes implicated in ribosomopathy disorders (rps-10, rpl-5, rpl-33, rps-23) in Caenorhabditis elegans and corresponding reductions in human lymphoblast cells. Our findings uncover significant, albeit variably penetrant, mitochondrial morphological differences across these mutants, alongside an upregulation of glutathione transferases, and SKN-1 dependent increase in oxidative stress resistance, indicative of increased ROS production. Specifically, loss of a single copy of rps-10 in C. elegans led to decreased mitochondrial activity, characterized by lower energy levels and reduced oxygen consumption. A similar reduction in mitochondrial activity and energy levels was observed in human leukemia cells with a 50% reduction in RPS10 transcript levels. Importantly, we also observed alterations in the translation efficiency of nuclear and mitochondrial electron transport chain components in response to reductions in ribosomal protein genes’ expression in both C. elegans and human cells. This suggests a conserved mechanism whereby the synthesis of components vital for mitochondrial function are adjusted in the face of compromised ribosomal machinery. Finally, mitochondrial membrane and cytosolic ribosomal components exhibited significant covariation at the RNA and translation efficiency level in lymphoblastoid cells across a diverse group of individuals, emphasizing the interplay between the protein synthesis machinery and mitochondrial energy production. By uncovering the impact of ribosomal protein haploinsufficiency on the translation efficiency of electron transport chain components, mitochondrial physiology, and the adaptive stress responses, we provide evidence for an evolutionarily conserved strategy to safeguard cellular functionality under genetic stress.

overall_design: Ribo-Seq and RNA-Seq libraries of C. elegans heterozygous mutants for four different ribosomal protein genes (rpl-5, rpl-33, rps-23 and rps-10)

first: Elif

last: Sarinay Cenik


title: Fat body-derived cytokine Upd2 regulates the polarity of Drosophila tracheal stem cells [RNA-seq]

status: Public on Oct 28 2024

first: qiang

last: zhao

address: 866 Yuhangtang Rd, Hangzhou

institute: Zhejiang University

summary: Adult stem cells are essential for maintaining tissue homeostasis. We employ Drosophila tracheal progenitors to understand the mechanism controlling the polarity of stem cell migration. Here, we show that polarity of tracheal progenitors depends on the function of fat body. Cytokine, Upd2, produced by fat body, signals to tracheal progenitors and activation JAK/STAT signal transduction. Perturbation of Upd2 production or loss-of-function of JAK/STAT signaling in the trachea cause aberrant bidirectional migration of tracheal progenitors. JAK/STAT signaling promotes the expression of a series of genes involved in planar cell polarity, which generates asymmetric localization of Fat and Pod1. Furthermore, the transport of Upd2 requires Rab5-, Rab7-mediated endocytic sorting and Lbm-dependent vesicle trafficking, where vesicular Upd2 functionally interacts with Rabs, Tetraspanin and Grasp65. These results reveal an inter-organ communication in which cytokines from fat body direct the polarity of ectodermal origin tracheal progenitors and suggest that Upd2-dependent JAK/STAT signaling regulates anterior-posterior polarity of tracheal progenitor movement.

overall_design: We performed RNA sequencing (RNA-seq) analysis of tracheal progenitors from aforementioned fat body defective flies to determine the signal between fat body and trachea.The tracheal progenitors were dissected and collected from 1hr APF pupae.

first: Pengzhen

last: Dong

first: Yue

last: Li

first: Yuying

first: Hai

last: Huang


title: Myometrial specimens

city: RTP

first: Franco

last: Demayo

state: NC

address: 111 TW Alexander Drive

institute: NIEHS


title: Functional assessement of putative myometrial enhancers by the CRISPR activation system

summary: Timely control of parturition is crucial for maternal and fetal health. Failures on this biological process often result in pregnancy complications including preterm birth, labor dystocia, and health disorders on newborn babies. The myometrium is the muscular structure of the uterus maintaining uterine structural integrity and providing contractile force for parturition. The myometrial structure changes in adaptation to the pregnancy via stage-specific transcriptomic profiles. Data from the mouse model indicate that changes of myometrial epigenomic landscape precedes the adoption of stage-specific gene expression pattern at term. The present study documents the transcriptomic profile and epigenomic landscape of term pregnant myometrial tissues and functionally characterize a subset of putative enhancers to further understand the enhancer-gene interaction in human the myometrium.

overall_design: Guide RNAs (gRNA) were uitilized to recruite the dCas9-VPR CRSIPR activators to putative enhancers of interest for promoting gene expression in the immortalized human myometrial cell line hTERT-HM cells. Cells that carried gRNA-expressing and dCas9-VPR-expressing lentiviral vectors were isolated by cell sorters. The single cell RNAseq assay was performed on the sorted cells to detect gRNA expression and profile the single cell transcriptome in the same cell (Perturb-Seq).

first: Elvis

last: Quiroz

first: Xin

last: Xu

first: San-Pin

last: Wu

first: Tianyuan

first: Francesco

last: DeMayo


title: Rbm24 dictates mRNA recruitment for germ plasm assembly

city: qingdao

first: yizhuang

last: zhang

address: binhai road

institute: Shandong university

summary: Ribonucleoprotein (RNP) granules are the most common membrane-less biomolecular condensates. However, the mechanisms underlying their assembly are largely unknown. The aggregation of germ plasm determines the fate of primordial germ cells (PGCs) and serves as a model for RNP granule assembly. Here, we show that maternal RNA binding protein Rbm24a is the key factor governing specific sorting of mRNAs. Mechanistically, Rbm24a complexes with Buc and interacts to dictate the specific grasp of germ plasm mRNAs into phase-separated condensates. Germ plasm particles lacking Rbm24a and mRNAs fail to undergo kinesin-dependent transport towards the cleavage furrows where small granules fuse into large aggregates. Therefore, the loss of maternal Rbm24a causes a complete degradation of germ plasm and the disappearance of PGCs. These findings demonstrate that Rbm24a functions as a nucleating organizer of the germ plasm, highlighting an emerging mechanism for RNA-binding proteins in reading and recruiting RNA components into the phase-separated protein scaffold.

overall_design: To examine the global transcriptome alterations following the loss of maternal Rbm24a, we conducted bulk RNA-seq analysis on Mrbm24a and their sibling embyos at 4-cell, sphere stage and 24 hpf.

first: Yizhuang

last: Zhang

first: Ming

last: Shao


title: Transcriptomic profile of the term pregnant human myometrial specimens

overall_design: Transcriptome of term pregnant not in labor (TNL) human myometrial specimens from three healthy subjects was profiled by the RNAseq assay.

last: Wang


title: Reduction of DNA Topoisomerase Top2 reprograms the epigenetic landscape and extends health and life span across species

city: Chengdu

first: Man

last: Zhu

state: Sichuan

address: No. 4, Section 2, Jianshe North Road, Chenghua District, Chengdu City, Sichuan, China

department: School of Medicine

institute: University of Electronic Science and Technology of China

summary: DNA topoisomerases are essential molecular machines that manage DNA topology in the cell and play important roles in DNA replication and transcription. We find that knocking down the enzyme Topoisomerase IIβ (Top2b) increases the lifespan of S. cerevisiae, C. elegans, and mice. TOP2B reduction also extends the health span of mice and alleviates the characteristics and pathologies of aging in multiple tissues. At the cellular/molecular level, TOP2 reduction attenates the major hallmarks of aging, such as cellular senescence, deregulated nutrient-sensing, epigenetic alterations, and lysosomal biogenesis. We observed that TOP2 reduction significantly alters the epigenetic landscape in various mouse tissues toward those of the young animals, and differentially down-regulates genes with active promoter and high expression. Our observations suggest that TOP2 reduction confers longevity effect across species via a conserved mechanism, and may be used as a novel therapeutic strategy for countering aging.

sample_organism:
Caenorhabditis elegans
Mus musculus

platform_organism:
Caenorhabditis elegans
Mus musculus

overall_design: We performed RNA-seq analyses on Top2 RNAi-treated C. elegans (n=4, each group) and various Top2b knockdown mice tissues including kidney, lung, and skeletal muscle (n=4, each group).

first: Jing

last: Yang

first: Hao

first: Yi

last: Zheng


title: Transcriptome of nonpregnant myometrial specimens with or without the medroxyprogesterone treament

summary: Timely control of parturition is crucial for maternal and fetal health. Failures on this biological process often result in pregnancy complications including preterm birth, labor dystocia, and health disorders on newborn babies. The myometrium is the muscular structure of the uterus maintaining uterine structural integrity and providing contractile force for parturition. The myometrial structure changes in adaptation to the pregnancy via stage-specific transcriptomic profiles. Progesterone signaling plays a crucial role in myometrial remodeling. The present study profiles the transcriptome of human myometrial specimens that are expected to manifest a wide spectrum of progesterone signaling via the RNAseq assay.

overall_design: Nonpregnant myometrial specimens were subject to the RNAseq assay to document gene expression profiles in proliferative, mid-secretary, post-menopausal, and provera (medroxyprogesterone)-treated groups.


title: Nucleolar Pol II interactome reveals TBPL1, PAF1, and Pol I at intergenic rDNA drive rRNA biogenesis

status: Public on Oct 09 2024

city: Miami

first: Stephen

last: Lee

state: FL

department: Biochemistry & Molecular Biology

institute: University of Miami

summary: Nucleolar ribosomal DNA (rDNA) repeats control ribosome manufacturing. rDNA harbors a ribosomal RNA (rRNA) gene and an intergenic spacer (IGS). RNA polymerase (Pol) I transcribes rRNA genes yielding the rRNA components of ribosomes. Pol II at the IGS induces rRNA production by preventing Pol I from excessively synthesizing IGS non-coding RNAs (ncRNAs) that can disrupt nucleoli. At the IGS, Pol II regulatory processes and whether Pol I function can be beneficial remain unknown. Here, we identify IGS Pol II regulators, uncovering nucleolar optimization via IGS Pol I. Compartment-enriched proximity-dependent biotin identification (compBioID) showed enrichment of the TATA-less promoter-binding TBPL1 and transcription regulator PAF1 with IGS Pol II. TBPL1 localizes to TCT motifs, driving Pol II and Pol I and maintaining its baseline ncRNA levels. PAF1 promotes Pol II elongation, preventing unscheduled R-loops that hyper-restrain IGS Pol I and its ncRNAs. PAF1 or TBPL1 deficiency disrupts nucleolar organization and rRNA biogenesis. In PAF1-deficient cells, repressing unscheduled IGS R-loops rescues nucleolar organization and rRNA production. Depleting IGS Pol I-dependent ncRNAs is sufficient to compromise nucleoli. We present the interactome of nucleolar Pol II and show its control by TBPL1 and PAF1 ensures IGS Pol I ncRNAs maintaining nucleolar structure and operation.

overall_design: Iso-seq of nucleolar RNA from unstressed (37degC) and heat shocked (43 degC) MCF-7 cells. Cells were either left unstressed or heat shocked at 43degC for 1 hour, then nucleolus was enriched using sucrose centrifugation method. Total RNA was isolated using TRIZOL and then sequenced using Iso-seq standard protocol.

first: Michael

last: Bokros

first: Negin

last: Khosraviani

first: Karim

last: Mekhail


title: A heterodimer of hemoglobin identifies theranostic targets on brain-metastasizing melanoma cells

status: Public on Oct 03 2024

city: Tel Aviv

first: Metsada

last: Pasmanik-Chor

address: Ramat Aviv

country: Israel

institute: Tel Aviv University

summary: Innate factors such as β-defensins may exhibit anti-cancer functions, serving as a first line of defense against incipient or dormant cancer cells. In previous studies, our lab demonstrated that hemoglobin-derived factors such as the hemoglobin beta chain or an alpha/beta heterodimer of hemoglobin trigger apoptosis and necrosis in neuroblastoma and brain-metastasizing melanoma cells, respectively.  In addition to its cytotoxic function, the alpha/beta hemoglobin dimer downregulated the expression of proteins such as BRD4 and IRS2, which perform essential functions in tumorigenesis and cancer progression. In the present study, we proved the concept, raised for the first time in this study, that in addition to their anti-cancer functions, innate resistance factors could serve as pathfinders to identify theranostic targets on progressing cancer cells.

overall_design: we used small-molecule inhibitors of the above proteins as potential anti-brain-metastasizing melanoma cell agents.         Indeed, a combination of sub-lethal concentrations of BRD4 and IRS2 inhibitors synergistically arrested melanoma cells at the subG1 phase of the cell cycle, killed more than 70% of such cells, and attenuated the virulence of brain-metastasizing melanoma cells.

first: Maharrish

last: Chelladurai

first: Orit

last: Sagi-Assif

first: Shlomit

last: Ben-Menachem

first: Tsipi

last: Meshel

first: Sivan

last: Izraely

first: Dave

middle: S

last: Hoon

first: Isaac

middle: P

last: Witz


title: Drosha loss drives pineoblastoma by upregulating Ccnd2 [RNA-seq of Dicer1 tumors]

city: Dallas

first: Kenneth

last: Chen

address: 5323 Harry Hines Blvd

department: Pediatrics

institute: UT Southwestern

summary: Mutations in the microRNA processing genes DICER1 and DROSHA drive childhood cancers such as pineoblastoma, a brain tumor that resembles undifferentiated precursors of the pineal gland. To understand how microRNAs regulate proliferation of these progenitors, we developed a mouse model of pineoblastoma by ablating Drosha or Dicer1 in the developing pineal gland. The tumors that arise in these models resemble proliferative, undifferentiated embryonic progenitors. They are marked by loss of microRNAs, including the let-7 family, and de-repression of let-7 target genes. One upregulated let-7 target gene is the oncofetal transcription factor Plagl2, which activates expression of key pro-growth genes Igf2 and Ccnd2, and we show that small molecules targeting these two drivers block tumor growth. Thus, our results demonstrate that tumors driven by loss of microRNA processing may be therapeutically targeted by inhibiting downstream drivers of proliferation.

overall_design: We used RNAseq to identify overexpressed genes and enriched pathways in IPDrosha vs IPRb1 tumors.

first: Claudette

middle: R

last: Fraire

first: Kavita

last: Desai

first: Lindsay

last: Mendyka

first: Uma

last: Obalaparum

first: Teja

last: Sebastian

first: Veena

last: Rajaram


title: Investigating cellular transcriptomic alterations caused by VEEV replicase and SARS-CoV-2 mRNA co-transfection

status: Public on Oct 07 2024

city: University Park

first: Maurice

last: Byukusenge

address: 131 Pastureview Rd

department: Veterinary and Biomedical Sciences

institute: Pennsylvania State University

summary: The viral RNA-dependent RNA polymerase (replicase) from Venezuelan equine encephalitis virus constitutes a vital component of the bipartite trans-amplifying mRNA vaccine. In this vaccine strategy aimed at targeting SARS-CoV-2, the replicase mRNA is administered alongside the mRNA encoding the SARS-CoV-2 spike protein. Our investigation sought to determine whether the replicase induces amplification of cellular mRNAs. To this end, cells were transfected with mRNAs encoding the replicase and SARS-CoV-2 spike protein, while control groups received transfections of mRNAs encoding an unrelated protein along with the SARS-CoV-2 spike. We observed no significant upregulation of genes in the treatment group compared to the control group. This suggests that the replicase does not induce off-target amplification of cellular mRNAs.

overall_design: There were two conditions tested in this experiment: In Condition 1, A549 cells underwent treatment involving transfection with mRNAs encoding the VEEV replicase and SARS-CoV-2 spike proteins. In Condition 2, serving as the control, A549 cells were transfected with mRNAs encoding an unrelated mRNA, Xef, along with the SARS-CoV-2 spike protein. Subsequent analysis focused on comparing the cellular transcriptomes between these two conditions at 4 hours and 12hours post-transfection.

first: Abhinay

last: Gontu

first: Suresh

last: Kuchipudi


title: Hypoxia and loss of GCM1 expression prevents differentiation and contact inhibition in human trophoblast stem cells [RNA-Seq I]

status: Public on Oct 02 2024

city: Montreal

first: William

last: Pastor

address: 3655 Promenade Sir-William-Osler rm900

department: Biochemistry

institute: McGill University

summary: The placenta develops alongside the embryo and nurtures fetal development to term. During the first stages of embryonic development, due to low blood circulation, the blood and ambient oxygen supply is normally very low (~1-2% O2) and gradually increases upon placental invasion. While a hypoxic environment is associated with stem cell self-renewal and proliferation, persistent hypoxia may have severe effects on differentiating cells and could be the underlying cause of placental disorders. We find that human trophoblast stem cells (TSC) thrive in low oxygen, whereas differentiation of syncytiotrophoblast (STB) and extravillous trophoblast (EVT) is negatively affected by hypoxic conditions. We find that the pro-differentiation factor GCM1 (human Glial Cell Missing-1) is downregulated in low oxygen, and there is substantial reduction of GCM1-regulated genes in hypoxic conditions. Knock-out of GCM1 in TSC caused impaired EVT and STB formation and function, reduced expression of genes that respond to differentiation, and resulted in maintenance of self-renewal genes. Treatment with a PI3K inhibitor reported to reduce GCM1 protein levels likewise counteracts spontaneous or directed differentiation. Chromatin immunoprecipitation of GCM1 showed enrichment of GCM1-specific binding near key transcription factors upregulated upon differentiation including the contact inhibition factor CDKN1C. Loss of GCM1 resulted in downregulation of CDKN1C and corresponding loss of contact inhibition, implicating GCM1 in regulation of this critical process.

overall_design: To determine how hypoixa is regulating gene expression changes in trophoblast stem cell, we culture 3 trophoblast stem cell lines in 20%, 5%, 2% oxygen conditions. 3 replicates were collect at different passages.

first: Jessica

last: Cinkornpumin


title: Tumor Associated Macrophages And Microglia Drive Tumor Progression And Are Transcriptionally Shaped By Histone Mutations In Pediatric Diffuse Midline Glioma

status: Public on Oct 11 2024

city: NEW YORK

first: Alexander

last: Tsankov

state: New York

address: 1425 Madison Ave

department: Genetics and Genomic Sciences

institute: Icahn School of Medicine at Mount Sinai

summary: Pediatric high-grade gliomas, including diffuse midline gliomas (DMG), harbor mutually exclusive tumor location specific histone mutations. Using immunocompetent genetically engineered mouse models, we demonstrate the predominant non-neoplastic cell population are infiltrating myeloid cells, including peripheral monocytes and brain resident microglia. Single cell RNA sequencing, flow cytometry, and immunohistochemistry demonstrate the presence of unique myeloid cell populations distinctly shaped by the specific histone mutation. Disease associated myeloid cell phenotypes are identified, resembling those found in other neurodegenerative diseases, and demonstrate immune permissive characteristics. H3.3K27M DMGs, the most aggressive DMG subtype, demonstrate enrichment of disease associated myeloid cells as well as proliferating myeloid and tumor cells.

overall_design: Using immunocompetent de novo mouse models of pediatric high grade glioma (pHGG), we generated tumors in both the cortical hemisphere and midline to represent each major subtype of pHGG including H3WT, H3.1K27M, or H3.3K27M pHGGs. Each tumor subtype contains the relevant driver mutations found in their human counterparts. Further experiments utilizing H3.1K27M DMGs in Ccl8/12 total knockout mice were performed, using H3.1K27M samples as wild type controls.Brain tumor cells were isolated at the humane endpoint of these mice, and sc-RNA seq was conducted to compare their functional differences.

first: Dolores

last: Hambardzumyan

first: James

middle: L

last: Ross

middle: M

first: Nishant

last: Soni


title: Hypoxia and loss of GCM1 expression prevents differentiation and contact inhibition in human trophoblast stem cells [ATAC-Seq]

overall_design: ATACseq was perform on 3 replicates of each cell type to uncover changes in open chromatin region when TSC are differentiated into EVT or STB.

first: Soumen

last: Paul


title: Drosha loss drives pineoblastoma by upregulating Ccnd2

first: Kenneth

last: Chen

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Glioblastoma-cortical organoids recapitulate cell state heterogeneity and intercellular transfer

status: Public on Oct 01 2024

city: Rehovot

first: Rony

last: Chanoch

state: Israel

address: Weizmann Institute of Science, 234 Herzl Street

institute: Weizmann Instititue

summary: Glioblastoma is characterized by heterogeneous malignant cells that are functionally integrated within the neuroglial microenvironment. Here, we model this ecosystem by growing glioblastoma into long-term cultured human cortical organoids that contain the major neuroglial cell types found in the cerebral cortex. Single-cell RNA-seq analysis suggests that, compared to matched gliomasphere models, glioblastoma cortical organoids (GCO) more faithfully recapitulate the diversity and expression programs of malignant cell states found in patient tumors. Additionally, we observe widespread transfer of glioblastoma transcripts and GFP proteins to non-malignant cells in the organoids. Mechanistically, this transfer involves extracellular vesicles and is biased towards defined glioblastoma cell states and astroglia cell types. These results extend previous glioblastoma-organoid modeling efforts and suggest widespread intercellular transfer in the glioblastoma neuroglial microenvironment.

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Glioblastoma-cortical organoids were established, dissociated into single cells, and the GFP/tdT positive and negative cells were sorted and profiled separately with scRNA-seq. In select cases, purified EVs were recovered from model systems, and the bulk RNA contained in the EVs was sequenced.

first: Vamsi

last: Mangena

last: Chanoch-Myers

first: Itay

last: Tirosh

first: Mario L.

last: Suva


title: Hypoxia and loss of GCM1 expression prevents differentiation and contact inhibition in human trophoblast stem cells [RNA-Seq 2]

overall_design: To determine how GCM1 LOF is regulating gene expression changes in trophoblast stem cells, we generated GCM1-Knockout via CRISPR gene editing. GCM1KO lines were then differentiated to EVT or STB3D to uncover the impact GCM1 as on the expression profile of these down-stream lineages.


title: RNA-seq samples from male sex organs in Drosophila

status: Public on Oct 15 2024

city: Davis

first: Ben

last: Hopkins

address: One Shields Avenue

department: Evolution & Ecology

institute: UC Davis

summary: Phenotypic evolution can result from gains and losses of genes, mutations in coding sequences, or regulatory mutations affecting gene expression. While the relative importance of these mechanisms is debated, regulatory evolution is recognized as a key driver of phenotypic diversity. In this study, we applied a phylogenetic model to discretized gene expression states (active or inactive) to investigate the evolutionary turnover of organ-specific transcriptomes, which we define as instances where gene expression is activated or deactivated in a particular organ.  We focus on transcriptome turnover in two male reproductive organs in 11 species of the Drosophila melanogaster species group. Using the Bayesian inference method zigzag (Thompson et al. 2020), we estimate that testes express a higher proportion of the genome (65--75% of genes, depending on the species) compared to accessory glands (46--64%), with background expression noise producing less than 1% of transcripts in both organs.  We find that many conserved genes have gained or lost expression in testes and accessory glands.  Our model of joint transcriptome evolution, applied to 8,660 genes conserved as single copy families in all species (singleton genes), revealed similar turnover rates but distinct evolutionary trajectories in the two organs.  We estimate that the genes in our data set transition between active and inactive expression states at rates on the order of 10^-9 yr^-1. The two organs experienced accelerated transcriptome turnover on different branches of the Drosophila phylogeny.  The accessory glands exhibit greater variation in turnover rates among lineages, suggesting a lower baseline rate with bursts of rapid evolution in a subset of branches on the phylogeny.  We do not observe significant differences in turnover rates between X-linked and autosomal genes.  Genes that encode transcription factors transition between active and inactive states slower than non-TF genes in the accessory gland, but slightly faster in the testis.  The results are robust to the choice of probability cut-offs used to discretize gene expression states, and there is good agreement between the estimates of expression states from zigzag and the inferences from the phylogenetic model.  Overall, our study highlights the complex dynamics of transcriptome evolution in male reproductive organs. We discuss the benefits and challenges associated with investigating the evolution of gene expression as a binary trait and suggest potentially fruitful avenues for further methodological development.

sample_organism:
Drosophila ananassae
Drosophila melanogaster
Drosophila simulans
Drosophila yakuba
Drosophila eugracilis
Drosophila takahashii
Drosophila elegans
Drosophila ficusphila
Drosophila kikkawai
Drosophila bipectinata
Drosophila biarmipes

overall_design:
To investigate the evolution of gene expression in drosophila male sex organs, we purified mRNA from the accessory glands and testis of pooled samples of about 25 adult male flies from two biological replicates from each of two different strains in 11 species. Libraries with insufficient read (< about 30 million) were augmented with additional sequencing runs.
Relative expression was quantified and expression state inferred using latent mixture models.
An evolutionay model and phylogenetic tree were fit to the comparitive data to infer evolutionary rates and history.

first: Ammon

last: Thompson

first: Artyom

last: Kopp

first: Nerisa

last: Reidl

first: Olga

last: Barmina


title: PARP-1 as a potential therapeutic target in ERα-positive endocrine therapy-resistant breast cancer

status: Public on Sep 30 2024

city: Chicago

first: Azzurra

last: Zicarelli

state: illinois

address: 929 E 57th St

institute: University of Chicago

summary:
Abstract
Several endocrine therapy (ET) resistance mechanisms for ER-positive (ER+) breast cancer (BC) have been proposed, including acquired ESR1 (ERα gene) mutations. The two most common ESR1 mutations are Y537S and D538G, which give rise to a constitutively active receptor with reduced affinity for agonists and antagonists. The discovery of new effective therapies remains a significant challenge in treating mutated ER+ BC. In this context, Poly (ADP-ribose) polymerase-1 (PARP-1) has captured considerable interest as a target for therapeutic inhibitors in specific types of cancers. Here, we report that crosstalk between PARP-1 and ERα may represent a novel therapeutic approach for ER+ BC. We have demonstrated that the up-regulation of PARP-1 expression, stimulated by 17-β estradiol (E2), was blocked by treatment with fulvestrant (Ful), a potent ERα antagonist, or ESR1 siRNA in ER+ MCF7 and T47D BC cell models that express both wild type ERα and the Y537S mutation, indicating that ERα regulates the expression of PARP-1. In addition, ERα-mediated transcriptional activity depended on PARP-1 activity in these models, as confirmed by ERα target gene expression and ERE reporter gene analyses +/- niraparib (Nira). Notably, PARP-1 modulates the estrogen-dependent genomic binding of ERα and FoxA1, which play a crucial role in the proliferation of ER+ BC. We also showed that PARP-1 inhibition prevented proliferation and cell cycle activities of ERα WT and ERα Y537S cells upon ERα activation. In vivo, Nira and lasofoxifene (Laso), an ERα antagonist, significantly reduced primary tumor growth versus vehicle, both as single agents and in combination. Moreover, RNA-seq analyses demonstrated the downregulation of ERα signaling in the mammary glands of mice treated with Nira versus Vehicle. Our results provide novel insights into the molecular events through which PARP-1 may serve as a more comprehensive therapeutic approach to target ET BC resistance in women with advanced ER+ BC.

overall_design: Identification of transcriptomic profile, by RNA-seq, in the mammary glands of mice injected with ER-positive breast cancer cell lines MCF7, expressing heterozygous ERα Y537S mutations. The mice were treated with either the PARP-1 inhibitor, Niraparib, or  with the selective estrogen-receptor modulator, Lasofoxifene as a single treatment or in combination. The objective is to gain new insights into individualized specific treatments for these specific malignancies.

first: Marianna

last: Talia

first: Muriel

last: Lainè

first: Rosamaria

last: Lappano

first: Marcello

last: Maggiolini

first: Geoffrey

last: Greene


title: Nanopore PCR-free direct cDNA sequencing on a murine model of endotoxin-indcued acute kidney injury

city: Indianapolis

first: Takashi

last: Hato

state: IN

address: 950 W. Walnut St. R2 202

department: Medicine

institute: Indiana University

summary: To investigate the mechanisms of endotoxin-induced acute kidney injury in mice, we performed Nanopore long-read RNA sequencing on bulk kidney tissues using the direct cDNA sequencing kit (SQK-DCS109) and R9.4.1 flow cells.

overall_design: C57BL/6J male mice were subjected to a single-dose, 4 mg/kg LPS (E. coli serotype 0111:B4) tail vein i.v. or vehicle saline in a volume of 300 ul. Kidney tissues were harvested at 0, 1, 4, 16, or 28 hours after LPS injection.


title: H3K27ac and H3K4me1 histone mark profiles in term pregnant human myometrial tissues

first: Franco

last: Demayo

summary: Timely control of parturition is crucial for maternal and fetal health. Failures on this biological process often result in pregnancy complications including preterm birth, labor dystocia, and health disorders on newborn babies. The myometrium is the muscular structure of the uterus maintaining uterine structural integrity and providing contractile force for parturition. The myometrial structure changes in adaptation to the pregnancy via stage-specific transcriptomic profiles. Data from the mouse model indicate that changes of myometrial epigenomic landscape precedes the adoption of stage-specific gene expression pattern at term. The present study documents the transcriptomic profile and putative enhancer landscape of term pregnant myometrial tissues.

overall_design: Term pregnant not in labor (TNL) human myometrial specimens from three healthy individuals were subject to the ChIP-seq assay to profile the genome-wide H3K27ac and H3K4me1 histone marks .

first: Lin

last: Lin

first: Tianyuan

last: Wang

first: Francesco

last: DeMayo

first: San-Pin

last: Wu


title: Genome-wide chromatin conformation capture of the term pregnant human myometrial specimens

summary: Timely control of parturition is crucial for maternal and fetal health. Failures on this biological process often result in pregnancy complications including preterm birth, labor dystocia, and health disorders on newborn babies. The myometrium is the muscular structure of the uterus maintaining uterine structural integrity and providing contractile force for parturition. The myometrial structure changes in adaptation to the pregnancy via stage-specific transcriptomic profiles. Data from the mouse model indicate that changes of myometrial epigenomic landscape precedes the adoption of stage-specific gene expression pattern at term. The present study documents the chromatin interaction profiles in the term pregnant not in labor human myometrial tissues at a genome-wide scale.

overall_design: Term pregnant not in labor (TNL) human myometrial specimens from two healthy individuals were subject to the Hi-C assay to profile the genome-wide chromatin interactions.


title: Role of whiB6 and kdpDE in the successful multidrug-resistant clone Mycobacterium tuberculosis B0/W148 [whiB6_RNA-seq]

status: Public on Sep 25 2024

city: Paris

first: Mickael

last: Orgeur

address: 25-28 rue du Docteur Roux

country: France

institute: Institut Pasteur

summary: Among the multidrug-resistant (MDR) clones of Mycobacterium tuberculosis (Mtb) that were epidemiologically particularly successful, the 100-32 MDR Beijing clone, also called B0/W148 clone, has emerged since the early sixties. These B0/W148 strains belonging to the lineage 2 within the global Mtb phylogeny, are the main contributors to the MDR epidemic in Russia and Eastern Europe, and since the USSR’s fall, have also propagated to Western Europe. Among the various mutations that were identified as being specific for the MDR B0/W148 clone, we focused on two found in the transcriptional regulators KdpDE and WhiB6 and characterized in a H37Rv strain background the transcriptional profile associated with these mutations and their potential impact on the in vitro and in vivo growth characteristics.

overall_design: Gene expression profiling by RNA-seq of the effect induced by the mutation T51P in whiB6 of M. tuberculosis H37Rv cultured in normal growth condition

first: Isabelle

last: Bonnet

first: Azimdine

last: Habib

first: Laure

last: Lemée

first: Alexandre

last: Pawlik

first: Marc

last: Monot

first: Roland

last: Brosch

first: Régis

last: Tournebize


title: A Unified Framework for Systematic Identification of Post-Transcriptional Regulatory Modules [ZNF800_ko]

city: San Francisco

first: Hani

last: Goodarzi

department: Department of Biochemistry and Biophysics

institute: UCSF

summary: RNA-binding proteins (RBPs) are multifunctional regulators of gene expression with complex and context-dependent mechanisms of action, yet the regulatory grammar that underlies RBP-mediated functions remains underexplored. Here, we performed a multi-omic data integration to systematically decipher the context-specific functions of RBPs. First, we report a compendium of in vivo proximity-dependent biotinylation (BioID) datasets of 50 human RBPs, which we used to generate a large-scale map of RBP protein neighborhoods. In parallel, we took advantage of CRISPR-interference with single-cell RNA-seq read-out (Perturb-seq) to capture rich transcriptomic phenotypes downstream of each RBP knockdown. By combining these physical and functional interaction readouts, along with the ENCODE transcriptome-wide atlas of RBP binding sites from eCLIP assays, we generated an integrated map of functional RBP interactions. This map not only captures well-studied post-transcriptional processes, but also reveals numerous previously unknown RBP-mediated regulatory functions. For a number of these cases, we have validated their predicted context-specific RBP functions using biochemical and genetic approaches. By deciphering these complex modes of functional interactions between RBPs, we have taken the first step towards a more comprehensive understanding of post-transcriptional regulatory processes and their underlying molecular grammar.

overall_design: We obtained transcriptome-wide gene expression measurements for 68 RBPs (representing a variety of regulatory processes) knockdowns using single-cell RNA sequencing and used the resulting high-dimensional data to systematically delineate genetic interactions between RBPs.  Perturb-seq experiment was performed as previously described (Datlinger et al. 2017).

first: Matvei

last: Khoroshkin

first: Andrey

last: Buyan

first: Martin

last: Dodel

first: Albertas

last: Navickas

first: Johnny

last: Yu

first: Fathima

last: Trejo

first: Anthony

last: Doty

first: Rithvik

last: Baratam

first: Shaopu

last: Zhou

first: Tanvi

last: Joshi

first: Kristle

last: Garcia

first: Benedict

last: Choi

first: Sohit

last: Miglani

first: Vishvak

last: Subramanyam

first: Hailey

last: Modi

first: Chris

last: Carpenter

first: Daniel

last: Markett

first: Ivan

middle: V

last: Kulakovskiy

first: M

last: Corces

first: Faraz

last: Mardakheh


title: Role of whiB6 and kdpDE in the successful multidrug-resistant clone Mycobacterium tuberculosis B0/W148 [kdpDE_RNA-seq]

first: Mickael

last: Orgeur

overall_design: Gene expression profiling by RNA-seq of the effect induced by the 2-nucleotide deletion in kdpDE of M. tuberculosis H37Rv cultured with and without potassium.

first: Isabelle

last: Bonnet

first: Azimdine

last: Habib

first: Laure

last: Lemée

first: Alexandre

last: Pawlik

first: Marc

last: Monot

first: Roland

last: Brosch

first: Régis

last: Tournebize


title: Role of whiB6 and kdpDE in the successful multidrug-resistant clone Mycobacterium tuberculosis B0/W148


title: QuantSeq from Nutlin-3a-treated MCF-7, U2OS, and RPE-1 cells

status: Public on Sep 18 2024

city: Jena

first: Martin

last: Fischer

state: Thuringia

address: Beutenbergstraße 11

department: Computational Biology

country: Germany

institute: Leibniz Institute on Aging - Fritz Lipmann Institute (FLI)

summary: QuantSeq from MCF-7, U2OS, and RPE-1 cells treated with Nutlin-3a or DMSO control

overall_design: MCF-7 cells were treated with Nutlin-3a 10 µM or DMSO control for 24 h

first: Elina

last: Wiechens

first: Konstantin

last: Riege

first: Steve

last: Hoffmann


title: CAGE-seq from Nutlin-3a-treated MCF-7, U2OS, and RPE-1 cells

summary: CAGE-seq from MCF-7, U2OS, and RPE-1 cells treated with Nutlin-3a or DMSO control


title: Nanopore long-read sequencing from Nutlin-3a-treated MCF-7 cells

summary: Nanopore long-read sequencing from MCF-7 cells treated with Nutlin-3a or DMSO control

last: Bens


title: Cardiac fibroblast BAG3 regulates TGFBR2 signaling and fibrosis in dilated cardiomyopathy

status: Public on Sep 16 2024

city: Boston

first: Seong Won

last: Kim

state: MA

address: 77 Ave Louis Pasteur

department: Genetics

institute: Harvard Medical School

summary: Loss of Bcl2-associated anthanogene 3 (BAG3) is associated with dilated cardiomyopathy (DCM). Recent studies show that BAG3 regulates sarcomere protein turnover in cardiomyocytes (CMs). However, the function of BAG3 in other cell types of the heart is unknown. In this study, we used an isogenic pair of BAG3 knockout and wild-type human induced pluripotent stem cells (hiPSC) to interrogate the function of BAG3 in hiPSC-derived cardiac fibroblasts (CFs). Generating a series of conditional knockout (cKO) engineered heart tissues, we determined the contribution of CF BAG3 to engineered heart tissue function. Despite normal CM genotype, the loss of fibroblast-specific BAG3 (FBKO) caused a reduction in contractile force and an increase in cardiac fibrosis in the engineered heart tissues, recapitulating the phenotype of DCM. Using culture substrates with defined stiffness, we decoupled the mechanical activation of fibroblasts from their ligand response. In BAG3-/- CFs, we observed an increased sensitivity to transforming growth factor β (TGFβ) signaling and activation of a fibrogenic response when cultured at physiological stiffness (8kPa). Mechanistically, loss of BAG3 increased transforming growth factor receptor 2 (TGFBR2) levels. BAG3 binds TGFBR2 and mediates its ubiquitination and proteasomal degradation. To further validate these results, we performed single-nuclei sequencing of cardiac tissue from DCM patients carrying pathogenic BAG3 mutations. Mutations in BAG3 increased fibrotic gene expression in cardiac fibroblasts. Together, these results extend our understanding of the roles of BAG3 in heart disease beyond the cardiomyocyte-centric view. This study also highlights the ability of hiPSC-based models and tissue engineering to answer questions about the cell-type specific aspects of cardiac disease.

overall_design: Control, BAG3 heterozygous knock-out, and BAG3 homozygous knockout hiPSCs cells were differentiated into cardiac fibroblasts. Subsequently, they were grown on 8kPa substrate for 5 days and frozen down. Frozen cell pellets were barcoded using the CellPlex Kit from 10X Genomics. Nuclei from the barcoded cells were extracted, pooled, and sequenced using the 10X Genomics platform. Reads mapping to the barcodes were used for demultiplexing.

first: Bryan

middle: Z

last: Wang

first: Margaretha

last: Morsink

first: Christine

middle: E

last: Seidman

first: Jonathan

first: Gordana

last: Vunjak-Novakovic


title: CSF attenuates methotrexate efficacy

status: Public on Sep 23 2024

city: Minneapolis

first: Peter

last: Gordon

state: MN

address: 420 Delaware S. SE, MMC 366

institute: University of Minnesota

summary: Treatment and prophylaxis of the central nervous system (CNS) is a critical component of acute lymphoblastic leukemia (ALL) therapy. However, CNS-directed therapies are a significant cause of morbidity and CNS relapse remains a cause of treatment failure. CNS-directed ALL therapies must target leukemia cells within cerebrospinal fluid (CSF), a fluid that is compositionally distinct from plasma and has been shown to impact leukemia biology. Herein, we demonstrate that human CSF attenuates the potency and efficacy of anti-folate drugs including methotrexate, the primary CNS-directed chemotherapeutic for over six decades. Importantly, this effect of CSF on leukemia methotrexate sensitivity was reversible. Additional mechanistic studies support that diminished proliferation and activation of the integrated stress response (ISR) in leukemia cells in CSF contribute to this resistance. Our findings suggest potential strategies to enhance methotrexate efficacy in CNS-directed ALL therapy and highlight the need to critically reassess even established standards of care.

overall_design: Human leukemia cells (NALM-6 and REH) were cultured in either stnadard media (RPMI/FCS 10%) or human cerebrospinal fluid (CSF) for 48 hours. Each condition was performed in trilplicate. RNA was then isolated and RNA-seq performed.


title: Hypoxia and loss of GCM1 expression prevents differentiation and contact inhibition in human trophoblast stem cells [ChIP-Seq]

first: William

last: Pastor

state: Quebec

overall_design: ChIP-seq was performed on day3 differentiated EVT to determine GCM1 transcriptional targets. 2 biological replicates were generated for this experiment.

first: Jessica

last: Cinkornpumin

first: Mariyan

last: Jeyarajah

first: Stepahn

last: Renaud


title: Neddylation Status Determines the Therapeutic Sensitivity of BCR-ABL1-targeting Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia

status: Public on Sep 15 2024

city: Hangzhou

first: Peng

last: Xiao

address: No.3 East Qingchun Road

institute: Zhejiang University

summary: BCR-ABL1-targeting tyrosine kinase inhibitors (TKIs) dominate the treatment of chronic myeloid leukemia (CML) over the past decades. In this study, we reported an unexpected role of neddylation inhibitors in desensitizing the therapeutic efficacy of BCR-ABL1-targeting TKIs in CML. Unlike their function in reducing drug resistance in many solid tumors, we revealed that neddylation inhibitors counteracted the cytotoxicity of TKIs against CML cells, both in cellular experiments and in animal model. Conversely, neddylation agonist sensitized the function of TKIs. RNA sequencing data revealed that neddylation inhibitor reversed the transcriptomic changes induced by TKI. Co-immunoprecipitation (co-IP) assay identified ABL1 kinase domain as a novel substrate for neddylation. Furthermore, an artificial intelligence (AI) 3-Dimensional spatial structure binding technology was employed to predict the impact of neddylation on the structure of ABL1 kinase domain. Finally, we provided potential evidence showing that TKI therapy decreased the expression of neddylation enzymes in the bone marrow of CML patients. Hence, our study offers new insights into the post-translational modification (PTM)-mediated drug resistance, and highlights the potential clinical benefits of neddylation agonists in improving the responsiveness of BCR-ABL1 TKIs in CML.

overall_design: Human chronic myelogenous leukemia K562 cells were treated with imatinib (0.5 μM) in the presence or absence of MLN4924 (25 μM) for 12 hours, RNA sequencing was performed.


title: A Multi-omics Study of Epigenetic Changes in Type II Alveolar Cells of A/J Mice Exposed to Environmental Tobacco Smoke

first: Natalia

last: Tretyakova

state: Minnesota

address: 2231 6th St SE Room 2-147

department: Medicinal Chemistry

type: Methylation profiling by high throughput sequencing

summary: Lung cancer remains a major contributor to cancer fatalities, with cigarette smoking known to be responsible for up to 80% of cases. Based on the ability of cigarette smoke to induce inflammation in the lungs and increased lung cancer incidence in smokers with inflammatory conditions such as COPD, we hypothesized that inflammation plays an important role in carcinogenicity of cigarette smoke. In order to test this hypothesis, we performed multi-omic analyses of Type II pneumocytes of A/J mice exposed to cigarette smoke for various time periods. We found that cigarette smoke exposure resulted in significant changes in DNA methylation and hydroxymethylation, gene expression patterns, and protein abundance that were partially reversible and contributed to an inflammatory and potentially oncogenic phenotype.

overall_design: Male and female A/J mice were treated with cigarette smoke or filtered air for 3 weeks, 10 weeks and 10 weeks followed by 4 weeks recovery. There are three mice in each treatment group. The mice were sacrificed after treatment and Alveolar type II epithelial cells (Type II pneumocytes) were isolated from the harvested lung tissues. DNA and RNA were extracted from the isolated Alveolar type II epithelial cells. DNA were used for genome wide mapping of 5mC and 5hmC and RNA were used for RNA-seq.

first: Qiyuan

last: Han

first: Jenna

last: Fernandez

first: Andrew

last: Rajczewski

first: Thomas

last: Kono


title: Image-based spatial transcriptomics identifies molecular niche dysregulation associated with distal lung remodeling in pulmonary fibrosis

status: Public on Sep 12 2024

city: Phoenix

first: Nicholas

last: Banovich

state: AZ

address: 445 North Fifth Street

department: Integrated Cancer Genomics

institute: TGen - Translational Genomics Research Institute

overall_design: Refer to individual Series


title: Engineering covalent small molecule–RNA complexes in living cells

city: Innsbruck

first: Kamila

last: Nykiel

address: Innrain 52

country: Austria

institute: Medical University of Innsbruck

summary: Targeting cellular RNA by small molecules has come to the forefront of biotechnology and holds great promise for therapeutic use. Strategies to identify, validate and optimize these molecules are essential, but are still lacking in some aspects. In particular, the site-specific covalent labeling and modification of RNA in living cells poses many challenges. Here, we describe a general structure-guided approach to engineer non-covalent RNA aptamer–ligand complexes into their covalent counterparts using a molecular tether. The key is to modify the native ligand with an electrophilic handle that allows it to react specifically with a guanine at the RNA ligand binding site. We show that site-specific cross-linking between ligand and RNA is achieved in mammalian cells upon transfection of a genetically encoded version of the preQ1-I riboswitch aptamer. Further, we showcase the versatility of the tether by engineering the first covalent fluorescent light-up aptamer (coFLAP) out of the non-covalent Pepper FLAP. The coPepper system maintains strong fluorescence in live-cell imaging even after repeated washing. Thus, any background signal arising from unspecific fluorophore accumulation in the cell can be eliminated. In addition, we generated a bifunctional Pepper ligand containing a second handle for bioorthogonal chemistry to allow for easily traceable and efficient pulldown of the covalently linked target RNA. Finally, we provide evidence for the suitability of this tethering strategy for specific drug targeting. Taken together, our results show that functionalized ligands generated by rational design can cross-link site-specifically with target RNAs in cells, and hence, open up a wide range of applications in RNA biology that require irreversible small molecule binding.

overall_design: Analysis of differential gene expression between groups "DMSO" and "MsOc3HBC" or "HBC530", respectively.


title: Cyclic stretch and interstial sitffness effects on A549 cells

city: Denton

first: Brian

last: Meckes

email: brian.meckes@unt.edu, bmeckes@gmail.com

address: 3940 N Elm St

department: Biomedical Engineering

institute: University of North Texas

summary: The mechanical properties and forces in the extracellular environment surrounding alveolar epithelial cells modulate their behavior. Particularly, breathing applies 3-dimensional cyclic stretch to the cells, while diseases such as cancer or fibrosis induce stiffening of the interstitium. To model this behavior, a biomimetic device was developed that effectively imitates the active forces in the alveolus, while allowing one to control the interstitium matrix stiffnesses to recreate different mechanical environments to replicate lung diseases. Alveolar epithelial A549 cancer cells were cultured on the platforms and their transcriptome was profiled using RNA sequencing.

overall_design: A549 cells were grown in three conditions, tissue culture plastic and biomimetic devices that have 3D cyclic stretch (15% strain 0.2 Hz) with either stiff (50 kPa) or soft (5 kPa) membranes. The cells were then collected after 7 days of the resepective treatment and RNA was collected and then sequenced.

first: Ala

last: Hessami


title: CRISPR/Cas9-induced  zebrafish mutants for ebf3a

status: Public on Sep 09 2024

city: Worcester

first: Summer

last: Thyme

address: 364 Plantation St

institute: UMass Chan Medical School

summary: Mutations in the transcription factor EBF3 result in a neurodevelopmental disorder, and studies in animal models indicate that it has a critical role in neuronal differentiation. The molecular pathways and neuron types disrupted by its loss, however, have not been thoroughly investigated. Nor have the outcomes of these changes on behavior and brain activity. Here, we generated and characterized a zebrafish ebf3a loss-of-function mutant. We discovered morphological and neural phenotypes, including an overall smaller brain size, particularly in the hypothalamus, cerebellum, and hindbrain. Brain function was also compromised, with activity strongly increased in the cerebellum and abnormal behavior at baseline and in response to visual and acoustic stimuli. RNA-sequencing of developing larvae revealed significant downregulation of genes that mark olfactory sensory neurons, the lateral line, and cerebellar Purkinje neurons. Corroborating the RNA-sequencing, staining revealed fewer lateral line neuromasts and reduced Parvalbumin signal in the cerebellum. This study sets the stage for determining which downstream pathways underlie the emergence of the observed phenotypes and establishes multiple strong phenotypes that could form the basis of a drug screen.

overall_design: Total RNA was extracted from the anterior half of the body from 10-12 5 dpf larvae per sample at using the Rneasy Mini Kit (Qiagen).  RNA for the 2 dpf RNA-seq samples were collected in the same way, but with 15-20 embryos per pool. The 5 dpf RNA-seq was collected also with 10-12 larvae. The posterior portion of the cuts was used for genotyping. The RNA was isolated from the anterior portion as described and submitted to sequencing by GENEWIZ at Azenta Life Sciences.

first: Nan Cher

last: Yeo

first: Nghi

last: Dang

first: Thinh

last: Truong


title: Recovery of Tetrahymena thermophila from degranulation

first: Aaron

last: Turkewitz

state: IL

address: 920 E 58th St

department: MGCB

summary:
The object was to identify genes that are upregulated during the recovery of cells from stimulation of granule exocytosis, when new granules are being synthesized.  The granules are also known as mucocysts.
To distinguish between genes upregulated due to demands of granule synthesis, vs genes upregulated in response to the exocytic stimulus per se, we exploited a mutant cell line, MN173, that does not release its granules on stimulation.

overall_design: Total RNA was isolated from cells prior to stimulation of exocytosis, and then 60 minutes post stimulation.  In parallel, cells from the MN173 mutant line were treated and processed equivalently.

first: Lydia

last: Bright


title: ATAC-seq from Nutlin-3a-treated MCF-7 cells

first: Martin

last: Fischer

summary: ATAC-seq from MCF-7 cells treated with Nutlin-3a or DMSO control

first: Konstantin

last: Riege

first: Steve

last: Hoffmann


title: Effect of slit1 gene overexpression during opc differentiation

status: Public on Aug 31 2024

city: shanghai

first: Ximing

last: Nian

state: Shanghai

address: Chongqing Nanlu 227

institute: Shanghai Jiao Tong University

summary: To investigate the  function of slit1 in the regulation of opcs differentiation, we transfected Vector and Slit1 to Rat OPC by Lonza Transfection.

overall_design: RNA-seq was performed on control or Slit1-overexpressing rat OPCs.

first: Dai

last: Wenxiu

first: Zhang

last: Liang


title: Diet driven differences in host tolerance are linked to shifts in global gene expression in a common avian host-pathogen system

status: Public on Aug 29 2024

city: Fayetteville

first: Carson

middle: Len

last: Stacy

state: AR

address: 850 W. Dickson St. Rm 601

department: Biological Sciences

institute: University of Arkansas

summary: As humans alter the landscape, wildlife have become increasingly dependent on anthropogenic resources, altering interactions between individuals and subsequently disease transmission dynamics. Further, nutritional quantity and quality greatly impact an individual host’s immune capacity and ability to mitigate damage caused by infectious disease. Thus, understanding the impact of dietary nutrition on immune function is critical for predicting disease severity and transmission as human activity both facilitates the dispersal of pathogens and alters dietary options for wildlife. Here, we use transcriptomics to explore the previously unstudied molecular mechanisms underpinning diet-driven differences in pathogen tolerance using a widespread avian bacterial pathogen, Mycoplasma gallisepticum (MG). MG is an ideal model for understanding the dietary drivers of disease as the human supplementation that wild birds commonly rely on, bird feeders, are also an important source for MG transmission. Significant diet-driven differences in the expression of many genes encoding immune response and translational machinery proteins are seen both in the absence of MG and during the recovery period. Prior to infection, protein-fed birds are more transcriptionally primed for infection than lipid-fed birds which translates to greater tolerance in protein-fed birds during the recovery period. Given the significant importance of human supplemented food in wildlife disease systems, the molecular mechanisms by which interactions between diet and infection emerge provide insight into the ecological and immunological consequences of human behavior and wildlife disease.

overall_design: To investigate the effect of high protein and high lipid diet on the recovery of Serinus canaria (common canary) from  Mycoplasma gallisepticum exposure, we performed whole blood gene expression profiling on infected and uninfected control treatment groups, blocked by diet, on days 0, 14, and 21 post infection

first: Erin

last: Sauer

first: Weston

last: Perrine

first: Ashely

middle: C

last: Love

first: Jeffrey

middle: A

last: Lewis

first: Sarah

last: DuRant


title: Xist RNA targets selective autosomal genes to modulate expression in a Repeat B-dependent manner [RNA-seq]

status: Public on Aug 23 2024

first: Shengze

last: Yao

address: 185 Cambridge St

department: Molbio

institute: Massachusetts General Hospital

summary: Xist, a pivotal player in X chromosome inactivation (XCI), has long been perceived as a cis-acting long noncoding RNA that binds exclusively to the inactive X chromosome (Xi). However, Xist’s ability to diffuse under select circumstances has long been documented, leading us to suspect that Xist RNA may have targets and functions beyond the Xi. Here, using female mouse embryonic stem cells (ES) and mouse embryonic fibroblasts (MEF) as a model, we demonstrate that Xist RNA indeed can spread beyond the Xi. However, its binding is limited to ~100 genes in differentiating ES and MEF cells. The target genes are diverse in function but are unified by their active chromatin status. Although active, Xist binding is associated with a downregulation of gene expression. Unlike on the Xi, Xist binding does not lead to full silencing and also does not spread beyond the target gene. Deleting Xist’s Repeat B (RepB) domain reduces Xist’s diffusion away from the Xi and inhibits Xist’s localization to autosomal targets. Nonetheless, Xist transcript lacking RepB fails to down-modulate autosomal gene expression. Altogether, our findings reveal Xist targets beyond the Xi and identify Repeat B as a crucial domain for its in-trans function.

overall_design: Xist localization patterns in various cell lines were examined at nucleotide resolution by CHART-seq. And RNA-seq was used to detect the gene expression level in the cells.

first: Yesu

last: Jeon

first: Barry

last: Kesner

first: Jeannie

last: Lee


title: Epigenetic manipulation of lineage-specific enhancers defines distinct oncogenic and tumor suppressive AR programs

status: Public on Sep 03 2024

city: New York

first: Xuanrong

last: Chen

address: 1300 York Ave

institute: Weill Cornell Medicine

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


title: Nutlin-3a induced changes in the transcriptome of MCF-7 cells

first: Martin

last: Fischer

summary: RNA-seq from MCF-7 cells treated with Nutlin-3a or DMSO control


title: Nutlin-3a induced changes in the transcriptome of HCT116 and RPE-1 cells

summary: RNA-seq from HCT116 and RPE-1 hTERT cells treated with Nutlin-3a or DMSO control

overall_design: HCT116 and RPE-1 cells were treated with Nutlin-3a 10 µM or DMSO control for 24 h

first: Konstantin

last: Riege


title: Adult skull bone marrow is an expanding and resilient hematopoietic reservoir

status: Public on Aug 22 2024

city: Münster

first: Kai

last: Kruse

state: North Rhine-Westphalia

address: Roentgenstr. 20

department: Bioinformatics Core Facility

institute: Max Planck Institute for Molecular Biomedicine

summary: The bone marrow microenvironment is a critical regulator of hematopoietic stem cell self-renewal and fate. While it is appreciated that aging, chronic inflammation and other insults compromise bone marrow function and thereby negatively affect hematopoiesis, it is not known whether different bone compartments exhibit distinct microenvironmental properties and functional resilience. Here, we have employed imaging, pharmacological approaches, and mouse genetics to uncover specialized and highly surprising properties of bone marrow in adult and aging skull. Specifically, we show that the skull bone marrow undergoes lifelong expansion involving vascular growth, which results in an increasing contribution to total hematopoietic output. Furthermore, skull is largely protected against major hallmarks of aging, including upregulation of pro-inflammatory cytokines, adipogenesis and loss of vascular integrity. Striking dynamic and rapid changes to the skull vasculature and bone marrow are induced by physiological alterations, namely pregnancy, but also pathological challenges, such as stroke and experimental chronic myeloid leukemia. These responses are highly distinct from femur, the most extensively studied bone marrow compartment. We propose that skull harbors a protected and dynamically expanding bone marrow microenvironment, which is relevant for experimental studies but, potentially, also clinical treatments in humans.

overall_design:
FACS-sorted hematopoietic stem and progenitor cells (HSPCs) and MACS hematopoietic lineage-depleted stromal cells from skull or femoral bone marrow of young or old mice analyzed using scRNA-seq

UPDATE: [June-12-2025] The titles of GSM8472477 and GSM8472478 were updated ("HSPC" to "Haem").

first: Bong

middle: I

last: Koh

first: Hyun-Woo

last: Jeong

first: Ralf

middle: H

last: Adams


title: Targeted RNA-sequencing of formalin-fixed paraffin-embedded breast cancer tissue samples

status: Public on Sep 13 2024

city: Bangkok

first: Kitiporn

last: Plaimas

address: 254 Phayathai Rd., Wangmai, Patumwan

department: Department of Mathematics and Computer Science

country: Thailand

institute: Chulalongkorn University

summary: The presence of over-expressed receptors in triple-negative breast cancer (TNBC) leads to the absence of targeted therapy. Tumor budding (TB) has emerged as a recent prognostic indicator. It is essential to pinpoint genes unique to high TB cases to prevent tumor advancement and spread. In this study, we utilized TempO-Seq to profile transcriptomic data from breast cancer samples to identify biomarkers for high TB cases.

overall_design: The gene expression profile was subgrouped into 15 breast cancer (BCA) and 13 TNBC cases. BCA samples include 12 high and 3 low TB samples, while TNBC comprises 13 samples, with 11 high and 2 low TB patients.

first: Panisa

last: Janyasupab

first: Kodchanan

last: Singhanat

first: Malee

last: Warnnissorn

first: Peti

last: Thuwajit

first: Apichat

last: Suratanee

first: Chanitra


title: Fibroblast-specific TGF-b signaling mediates dysfunction, fibrosis and hypertrophy in a mouse model of obesity-associated type 2 diabetes [RNA-seq: TSP4 OE]

status: Public on Aug 18 2024

city: NYC

first: Izabela

last: Tuleta

state: NY

address: 1300 Morris Park Avenue

department: Cardiology

institute: Albert Einstein College of Medicine

summary: Our study provides the first direct evidence supporting a fibroblast-mediated mechanism of dysfunction and adverse remodeling in the diabetic heart, highlighting the importance of interstitial cells in the pathogenesis of diabetic cardiomyopathy. Diabetes-associated activation of the TGF-b/Smad3 cascade may promote dysfunction not only by accentuating ECM deposition and crosslinking, but also by modulating cardiomyocyte phenotype and function. Thus, the TGF-b system may be a promising therapeutic target in patients with diabetes-associated heart failure.

overall_design: Comparison of the transcriptomic profile of cardiac fibroblasts with TSP-4 overexpression (4 samples) vs. control (4 samples).

first: Simone

last: Sidoli

first: Claudio

last: Humeres

first: Nikolaos

middle: G

last: Frangogiannis


title: Fibroblast-specific TGF-b signaling mediates dysfunction, fibrosis and hypertrophy in a mouse model of obesity-associated type 2 diabetes [RNA-seq: TSP4 stimulation]

overall_design: Comparison of the transcriptomic profile of cardiac fibroblasts stimulated by TSP-4 (3 samples) vs. control (4 samples).


title: Fibroblast-specific TGF-b signaling mediates dysfunction, fibrosis and hypertrophy in a mouse model of obesity-associated type 2 diabetes [RNA-seq: Smad3 in dbdb]

overall_design: Comparison of the transcriptomic profile of cardiac fibroblasts from Smad3KO vs. Smad3 lean and db/db animals. 3 samples per group (12 samples in total).


title: Induced variation in BRASSINOSTEROID INSENSITIVE 1 (BRI1) confers a compact wheat architecture

status: Public on Aug 14 2024

city: Harpenden

first: Stephen

last: Pearce

state: Hertfordshire

address: West Common

department: Sustainable Soils and Crops

country: United Kingdom

institute: Rothamsted Research

summary: The brassinosteroid (BR) plant hormones regulate numerous developmental processes, including those determining stem height, leaf angle, and grain size that have agronomic relevance in cereals. Indeed, barley (Hordeum vulgare) varieties containing uzu alleles that impair BR perception through mutations in the BR receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) exhibit a semi-dwarf growth habit and more upright leaves suitable for high-density planting. We used forward and reverse genetic approaches to develop novel BRI1 alleles in wheat (Triticum aestivum L.) and investigated their potential for crop improvement. The combination of ethyl methanesulfonate-induced mutations introducing premature stop codons in all three homoeologous TaBRI1 genes resulted in severe dwarfism, malformed leaves and sterility as observed in bri1 mutants in other species. Double mutants had reduced flag leaf angles (FLAs) conferring a more upright canopy but exhibited no differences in height or grain weight. In a forward genetics screen using a double mutant, we identified two BR-insensitive lines with reduced height and FLA that contained amino acid substitutions in conserved regions of BRI-A1. The less severe mutant had a 56% reduction in FLA and was 35% shorter than the wild-type although seed set, seed area and grain weights were also reduced. The most severe mutants contained elevated levels of bioactive BRs and increased expression of BR-biosynthesis genes consistent with reduced feedback suppression of biosynthesis. Our study gives a better understanding of BRI1 function in wheat and provides mutants that could potentially be explored for improving grain yields when sown at high density.

overall_design: RNA-seq was conducted on tabri1-a.1bd, tabri1-a.2bd, tabri1-a.3bd, tabri1-bd mutants and wild-types TaBRI1-NS and Cadenza. The entire above-ground tissue for all genotypes was harvested at 2nd leaf seedling stage and multiple seedlings/genotype were pooled together to make one biological replicate. This experiment was conducted using 4 biological replicates for all genotypes except for 3 reps for tabri1-a.1bd mutant due to tissue limitations. The trimmed paired-end reads were mapped to wheat IWGSC RefSeq v2.1 genome assembly. The raw mapped reads were counted and converted to TPM's (transcripts per million). The raw data (.fastq) and TPM's are included in this project.

first: Manpartik

last: Gill

first: Peter

last: Hedden

last: Thomas


title: Transcriptomic characteristics of quiescent cells in human fallopian tube epithelial organoids detected by a fluorescent membrane dye PKH26

city: Shinjuku

first: Tomohiro

last: Tamura

state: Tokyo

address: Shinanomachi 35

department: Department of Extended Intelligence for Medicine, The Ishii-Ishibashi Laboratory

country: Japan

institute: Keio University School of Medicine

summary: We established fallopian tube epithelial organoids from human fallopian tube tissues and cultured them in the organoid culturing cocktail medium. To investigate the heterogeneity of proliferation in the fallopian tube epithelial organoids, we stained the fallopian tube epithelial cells by a fluorescent membrane dye PKH26. As culturing them, the intensity of PKH26 was reduced in accordance with the proliferative level of each cell. We sorted the organoid cells by FACS into PKH26-retained slow cycling cells and PKH26-reduced proliferative cells, and compared their transcriptomic characteristics by bulk RNA sequencing and gene set enrichment analysis (GSEA). The result of GSEA showed that several pathways related to cell cycle were downregulated in PKH26-retained cells. So this dataset is supposed to be suitable for the analysis of quiescent cells in human fallopian tube epithelium.

overall_design: To investigate the characteristics of quiescent cells in fallopian tube epithelial organoids, we stained the fallopian tube epithelial cells with a fluorescent membrane dye PKH26. After culturing the organoids, we sorted the fallopian tube epithelial cells by FACS into PKH26-retained slow-cycling cells and PKH26-reduced proliferative cells. We compared the transcrioptomic characteristics by bulk RNA  sequencing.

first: Keiyo

last: Imaeda

first: Kenta

last: Masuda


title: Splenic fibroblastic reticular cells regulate dendritic cell maturation and T-DC interactions required for antiviral immunity

status: Public on Aug 16 2024

city: Melbourne

first: Nikita

last: Potemkin

state: Vic

address: 792 Elizabeth St

department: The Peter Doherty Institute for Infection and Immunity

country: Australia

institute: University of Melbourne

summary: Stromal cells construct the architecture of the spleen and provide crucial signals that influence the homeostasis and migration of immune cells in the steady state. Fibroblastic Reticular Cells (FRCs) respond to infection via the regulation of many key structural and functional components. Yet, how spleen FRCs contribute to the induction and maintenance of antiviral T cell responses is still poorly defined. We found that ablation of splenic FRCs impacted the generation of virus-specific CD8 T cell during lymphocytic choriomeningitis virus (LCMV) infection in mice. Imaging revealed that during T cell priming in the presence of FRCs, CD8 T cells clustered in the T cell zone with type 1 conventional dendritic cells (cDC1) before moving to the marginal zone to interact with virus-infected cells. In the absence of FRCs, T cells instead clustered with virus-infected cells and cDC1s in the marginal zone, undergoing suboptimal priming due to reduced TCR signalling and unstable DC-T cell interactions. We found that FRCs regulated the early inflammatory wave required for optimal DC activation in vivo. The absence of FRCs thus resulted in diminished DC activation and impaired capacity to prime T cell responses. We further revealed that the presence of an intact FRC network was crucial for the generation of functional effector T cells. Importantly, these early cellular events orchestrated by FRCs also regulated the generation of protective memory T cells upon resolution of infection. Altogether, our study reveals important roles for FRCs for induction of antiviral T cell responses through support for DC maturation, effector CTL generation and the formation of protective memory T cells.

overall_design: To investigate how FRC regulate early immune responses to LCMV infection, we transferred transgenic tdTomato+ P14 CD8 T cells into control and CCL19-DTR mice, followed by DT treatment to deplete white pulp CCL19+ FRCs and subesquantly infected mice with LCMV Armonsg. Spleens were harvested 1.5 days post-infection, digested and different cell populations were sorted by flow cytometry. We sorted 6 fractions consisting of tdTomato+ P14 cells, CD157+PDPN+ stromal cells, CD157-PDPN- stromal cells, CD11c+ myeloid cells, CD11c- myeloid cells, and total splenocytes. Sorted cells were then mixed in equal ratios and subjected to 10X single cell  sequencing.

first: Yannick

middle: O

last: Alexandre

first: Scott

middle: N

last: Mueller


title: Detection of APOBEC3B's RNA binding sites in T-47D cells using eCLIP-seq

city: London

first: Chi

last: Zhang

address: 15 Cotswold Road

department: Division of Cancer Therapeutics

institute: The Institute of Cancer Research

summary: RNA editing is a crucial post-transcriptional process that diversifies proteomic outcomes and influences gene expression. Particularly, the APOBEC3 family has emerged as a significant player in this mechanism, with APOBEC3A (A3A) showing notable roles in immune response and stress conditions. APOBEC3B (A3B), another family member, has garnered attention for its potential role in breast cancer genomic mutations. In this study, we employ an inducible expression cell model and eCLIP-seq to detect the RNA binding sites for A3B a breast cancer cell model. Our findings indicate that A3B engages in selective RNA editing and targets NEAT1 and MALAT1 long non-coding RNAs. Notably, the binding of these RNAs sequesters A3B’s catalytic activity, and thereby affects A3A’s activity through a feedback loop.

overall_design: eCLIP-seq was conducted on T-47D cell line expressing inducible A3B. The inducible A3B was tagged with flag peptide, allowing affinity purification of protein-RNA complex using anti-Flag (M2) antibody.

first: Yujing

last: Lu

first: Mei

last: Wang

first: Bingjie

last: Chen

first: Feifei

last: Xiong

first: Costas

last: Mitsopoulos

first: Xiuling

last: Li

first: Paul

last: Clarke


title: NEAT1 Modulates the TIRR/53BP1 Complex to Maintain Genome Integrity

status: Public on Sep 04 2024

city: Seville

first: Patrick

last: Toolan-Kerr

address: Calle Américo Vespucio 24 Edificio

country: Spain

institute: University of Seville

summary: Tudor Interacting Repair Regulator (TIRR) is an RNA-binding protein (RBP) that interacts directly with 53BP1, restricting its access to DNA double-strand breaks (DSBs) and its association with p53. We utilized iCLIP to identify RNAs that directly bind to TIRR within cells, identifying NEAT1_1, the short isoform of the long noncoding RNA NEAT1, as the primary RNA partner. The high affinity of TIRR for NEAT1_1 is due to prevalent G-rich motifs in NEAT1_1. This interaction destabilizes the TIRR/53BP1 complex, promoting 53BP1's function. NEAT1_1 is enriched during the G1 phase of the cell cycle, thereby ensuring that TIRR-dependent inhibition of 53BP1’s function is cell cycle-dependent. TDP-43, an RBP that is implicated in neurodegenerative diseases, modulates the TIRR/53BP1 complex by promoting NEAT1_1 production. Together, we infer that NEAT1_1, and factors regulating NEAT1_1, may impact 53BP1-dependent DNA repair processes, with implications for a spectrum of diseases.

overall_design:
iCLIP was used to identify RNA bound by FLAG-tagged TIRR protein. A comparison was made between untreated cells and cells irradiated with 5Gy. Briefly, U2OS cells stably transfected with a Flag version of TIRR, or EV, were UV-crosslinked on ice and lysed in RIPA buffer. For the irradiation experiment, cells were irradiated at 5 Gy with an X-ray source. Two replicates were prepared for each condition. 0.4 Units of RNaseI and 4 Units Turbo DNase were added per 1 mL of cell lysate at 1 mg/mL protein concentration for RNA fragmentation. Antibodies against the Flag coupled to magnetic Protein G beads were used to isolate Protein-RNA complexes, and RNA was ligated to a pre-adenylated infra-red labeled IRL3 adaptor with the following sequence: /5rApp/AG ATC GGA AGA GCG GTT CAG AAA AAA AAA AAA /iAzideN/AA AAA AAA AAA A/3Bio/. For full experimental protocol see: Lee, F. C. Y. et al. An improved iCLIP protocol. bioRxiv, 2021.2008.2027.457890 (2021). https://doi.org/10.1101/2021.08.27.457890

RNA_seq: To identify genes whose expression is impacted by gamma radiation in osteosarcoma cell line U2OS

first: Pascal

last: Drane

first: Chowdhury

last: lab