title: Blocking the Angiopoietin-2-dependent Integrin beta-1 signaling axis abrogates small cell lung cancer invasion and metastasis

status: Public on Mar 28 2024

city: Erlangen

first: Lydia

last: Meder

state: Bavaria

address: Glückstr. 6

department: Experimentelle Medizin 1

summary: Small cell lung cancer (SCLC) is the most aggressive lung cancer entity with an extremely limited therapeutic outcome. Most patients are diagnosed at an extensive stage. However, the molecular mechanisms driving SCLC invasion and metastasis remain largely elusive. We used an autochthonous SCLC mouse model and matched primary and metastatic SCLC patient samples to investigate the molecular characteristics of tumor metastasis. We demonstrate that tumor cell invasion and liver metastasis in SCLC are triggered by an Angiopoietin-2/Integrin beta-1-dependent pathway in tumor cells, mediated by focal adhesion kinase-Src kinase signaling. Strikingly, CRISPR-Cas9 knock out of Integrin beta-1 or blocking Integrin beta-1 signaling by an anti-Angiopoietin-2 treatment abrogates liver metastasis formation in vivo. Interestingly, analysis of a unique collection of matched primary and metastatic SCLC patient samples confirmed a strong increase of Integrin beta-1 in liver metastasis in comparison to the primary tumor. We further show that Angiopoietin-2 blockade combined with anti-VEGFR and PD-1-targeted treatment displays synergistic treatment effects in SCLC. Together, our data demonstrate a fundamental role of Angiopoietin-2/Integrin beta-1 signaling in SCLC cells for tumor cell invasion and liver metastasis and provide a new effective treatment strategy for patients with SCLC.

overall_design: To investigate the role of ITGB1 in SCLC metastasis formation we created a ITGB1KO clones and compared them to the correspondin wild type.

first: Charlotte

middle: I

last: Orschel

first: Christoph

middle: J

last: Otto

first: Mirjam

last: Koker

first: Johannes

last: Brägelmann

first: Meryem

last: Ercanoglu

first: Sabrina

last: Dähling

first: Anik

last: Compes

first: Carolin

last: Selenz

first: Marieke

last: Nill

first: Felix

last: Dietlein

first: Alexandra

last: Florin

first: Marie-Lisa

last: Eich

first: Sven

last: Borchmann

first: Margarete

last: Odenthal

first: Raquel

last: Blazquez

first: Frank

last: Hilberg

first: Florian

last: Klein

last: Hallek

first: Reinhard

last: Büttner

first: Christian

last: Reinhardt

first: Roland

last: Ullrich


title: Transcriptional control of the Cryptosporidium lifecycle

status: Public on Apr 01 2024

city: Philadelphia

first: Katelyn

middle: Ann

last: Walzer

state: PA

address: 380 South University Avenue

department: Pathobiology

country: USA

institute: University of Pennsylvania

type: Expression profiling by high throughput sequencing

summary: The parasite Cryptosporidium is a leading agent of diarrheal disease in young children, and a cause and consequence of chronic malnutrition. There are no vaccines and only limited treatment options. The parasite infects enterocytes, where it engages in asexual and sexual replication, both of which are essential to continued infection and transmission. However, their molecular mechanisms remain largely unknown. Here we use single-cell RNA sequencing to reveal the gene expression program of the entire C. parvum life cycle in culture and infected animals. Diverging from the prevailing model, we find support for only three intracellular stages: asexual type-I meronts, male gamonts, and female gametes. We uncover a highly organized program for the assembly of components at each stage. Dissecting the underlying regulatory network, we identify the transcription factor Myb-M as the earliest determinant of male fate, in an organism that lacks genetic sex determination. Conditional expression of this factor overrides the developmental program and induces widespread maleness, while conditional deletion ablates male development. Both have a profound impact on the infection. A large set of stage-specific genes now provides the opportunity to understand, engineer, and disrupt parasite sex and life cycle progression to advance the development of vaccines and treatments.

overall_design: Bulk and single-cell RNA sequencing (scRNA-seq) data were generated in this study. Samples for scRNA-seq and stage-specific bulk RNA sequencing were flow sorted to enrich for infected cells. For bulk analysis of stage-specific reporters, asexual and female datasets were already available (GEO:GSE129267) while the male in vitro dataset, consisting of four biological replicates, was produced in this study. For bulk analysis of Myb-M gain-of-function mutants, three biological replicates were used for each strain and condition collected at 30 hours after 6 hours of vehicle or Shield-1 treatment. For scRNA-seq analysis, infected cells were captured from culture or a mouse. In vitro samples were collected at 24, 36, 42, 46, and 54 hours while the in vivo sample was collected from an acutely infected mouse on day 6.

middle: A

first: Jayesh

last: Tandel

first: Jessica

middle: H

last: Byerly

first: Abigail

middle: M

last: Daniels

first: Jodi

last: Gullicksrud

first: Eoin

middle: C

last: Whelan

first: Stephen

middle: D

last: Carro

first: Elise

last: Krespan

first: Daniel

middle: P

last: Beiting

first: Boris

last: Striepen


title: E2F7-GFP binding sites in primary airway mouse epithelial cells (mTECs) cultured at air-liquid interface for three days.

status: Public on Mar 30 2024

city: San Francisco

first: Semil

last: Choksi

email: semilc@gmail.com, semil.choksi@ucsf.edu

state: CA

address: 555 Mission Bay Blvd. South

institute: UCSF

type: Genome binding/occupancy profiling by high throughput sequencing

summary: The canonical mitotic cell cycle coordinates cell growth, DNA replication, centriole duplication and cytokinesis to generate two cells from one. In certain contexts, such as in mammalian trophoblast giant cells or hepatocytes, cells employ cell cycle variants like the endocycle or endomitosis to increase DNA content without undergoing cytokinesis. Multiciliated cells, found in the mammalian airway, brain ventricles and reproductive tracts, generate hundreds of centrioles during differentiation, each of which extend a motile cilium. We found that multiciliated cells utilize a variant of the cell cycle, which we refer to as the multiciliation cycle. The multiciliation cycle redeploys much of the mitotic cell cycle regulatory framework, including cell division kinases (CDKs) and cyclins, to generate hundreds of centrioles without undergoing DNA synthesis or cytokinesis. Unlike the mitotic cycle, a transcriptional repressor, E2F7, is upregulated during the multiciliation cycle. E2F7 directly represses genes encoding DNA synthesis machinery during the multiciliation cycle. Loss of E2F7 results in aberrant DNA synthesis and disruption of centriole biogenesis in differentiating multiciliated cells. Thus, multiciliation is coordinated by a variant cell cycle that uses E2F7 to uncouple centriole synthesis from DNA replication.

overall_design: mTECs were transduced with E2F7-GFP or NLS-GFP lentivirus, differentiated, and grown at air/liquid interface for three days. Cells were dissociated and analyzed by CUT&RUN with a GFP antibody to detect genomic binding sites of E2F7.

first: Lauren

last: Byrnes

first: Mia

last: Konjikusic

first: Rachel

last: Deleon

first: Quanlong

last: Lu

first: Christopher

last: Westlake

first: Jeremy

last: Reiter


title: Gene expression profile at single cell level of in vitro monoculture and co-culture cells of ovarian clear cell carcinoma (OCCC) [OCCC_scRNAseq_invitro]

city: Tokyo

first: Yutaro

last: Mori

address: 2-21-1 Kaga, Itabashi-ku

department: Advanced Comprehensive Research Organization

country: Japan

institute: Teikyo University

summary: We performed single cell RNA sequencing (scRNA-seq) to examine potential cross-talk between Cancer cells and Cancer associated fibroblasts (CAFs) in OCCC.

overall_design:
Spheroid cancer cells and CAFs were mixed for co-culture or unmixed for monoculture and analyzed by scRNA-seq using the Single Cell 3' Reagent Kit v3 and 3’ CellPlex Kit Set A (10X Genomics) following the manufacturer’s protocol.

The number of cells for each sample:
Coculture_cells: 3,232
Mono_CAF: 3,347
Mono_Cancer: 2,725

first: Koji

last: Okamoto


title: Airway mouse epithelial cells (mTECs) cultured at air-liquid interface

status: Public on Mar 29 2024

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

The canonical mitotic cell cycle coordinates DNA replication, centriole duplication and cytokinesis to generate two cells from one1. Some cells, such as mammalian trophoblast giant cells, use cell cycle variants like the endocycle to bypass mitosis2. Differentiating multiciliated cells, found in the mammalian airway, brain ventricles and reproductive tract, are post-mitotic but generate hundreds of centrioles, each of which matures into a basal body and nucleates a motile cilium3,4. Several cell cycle regulators have previously been implicated in specific steps of multiciliated cell differentiation5,6. Here we show that differentiating multiciliated cells integrate cell cycle regulators into a new alternative cell cycle, which we refer to as the multiciliation cycle. The multiciliation cycle redeploys many canonical cell cycle regulators, including cyclin-dependent kinases (CDKs) and their cognate cyclins. For example, cyclin D1, CDK4 and CDK6, which are regulators of mitotic G1-to-S progression, are required to initiate multiciliated cell differentiation. The multiciliation cycle amplifies some aspects of the canonical cell cycle, such as centriole synthesis, and blocks others, such as DNA replication. E2F7, a transcriptional regulator of canonical S-to-G2 progression, is expressed at high levels during the multiciliation cycle. In the multiciliation cycle, E2F7 directly dampens the expression of genes encoding DNA replication machinery and terminates the S phase-like gene expression program. Loss of E2F7 causes aberrant acquisition of DNA synthesis in multiciliated cells and dysregulation of multiciliation cycle progression, which disrupts centriole maturation and ciliogenesis. We conclude that multiciliated cells use an alternative cell cycle that orchestrates differentiation instead of controlling proliferation.

overall_design: Refer to individual Series


title: RNA-Seq analysis of SUN1 knockout and control CT26 cells and tumors

city: Calgary

first: Edwin

last: Wang

state: Alberta

address: 2500 University Drive N.W.

country: Canada

institute: University of Calgary

summary: T cell infiltration is essential for immune checkpoint inhibitors to be effective in treating solid cancers. Through a bioinformatic pipeline, we identified a target gene SUN1 that might relate to modulating immune cell infiltration and immune response. Thus, we generated one Sun1_knockout CT26 cell line (Sun1_KO) and two control CT26 cell lines (Sun1_Control) using CRISPR-Cas9. By performing RNA-seq on cultured cells, tumors grown in syngeneic model, and purified tumor cells from tumors grown in syngeneic model, we set out to understand how mouse Sun1 can affect immune-related pathways and immune cell infiltration and anti-PD1 efficacy in BALB/c mice.

overall_design: 1)Sun1_KO and Sun1_Control cells cultured in complete RPMI medium were used for RNA extraction and RNA-seq; 2)Sun1_KO and Sun1_Control cells were subcutaneously injected in the right flank. Tumors were collected at day 14 post-inoculation, and RNA was extracted for RNA-seq; 3)Sun1_KO and Sun1_Control cells were subcutaneously injected in the right flank. Tumors were collected at day 14 post-inoculation, prepared in single cell, and then tumor cells were purified using a mouse tumor cells isolation kit. RNA was extracted from the purified cell lines (Sun1_KO and Sun1_Control) and used for RNA-seq.

first: Sahar

last: Nasr

first: Lin

last: Li


title: TDP43 proteinopathy exhibits disease, tissue, and context-specific cryptic splicing signatures

city: South San Francisco

first: Brad

last: Friedman

address: 1 DNA Way

department: Neuroinformatics

institute: Genentech, Inc.

summary: Mislocalization of the nuclear protein TDP43 is a hallmark of ALS and FTD and leads to de-repression and inclusion of cryptic exons, which represent promising biomarkers of TDP43 pathology. However, most cryptic exons to date have been identified from in vitro models, limiting our understanding of any tissue and/or cell-specific splices. We meta-analyzed published bulk RNA-Seq datasets representing 1,778 RNAseq profiles of ALS and FTD post mortem tissue, and in vitro models with experimentally depleted TDP43. We identified novel cryptic splices and mapped out their tissue-specificity, demonstrating subsets with distinct cortical and spinal cord enrichment. Novel events were validated by RNA-Seq and RT-qPCR in a new spinal cord cohort, and analysis of single-nucleus datasets localized cortical splices to layer-specific neuronal populations. This catalog of cryptic splices is the first step towards the development of biomarkers for cell type-specific TDP43 pathology.

overall_design:
Post mortem lumbar spinal cord samples were obtained (n=10 healthy controls, n=10 ALS), and bulk RNA was isolated. We performed differential splicing anlayses with SGSeq/DEXseq to characterize TDP43-associated cryptic splicing.

**Raw data are not provided due to privacy concerns**

first: Dwight

middle: F

last: Newton

first: Rena

last: Yang

first: Johnny

last: Gutierrez

first: Jeffrey

middle: W

last: Hofmann

first: Felix

last: Yeh

first: Anne

last: Biever


title: Proximity-dependent biotinylation map of the RNAPII CTD reveals that the primary role of Serine 2 phosphorylation is in the suppression of pervasive antisense transcription and not in RNA 3’ end processing [ChIP-seq]

city: Namur

first: Carlo

last: Yague-Sanz

address: 61 rue de Bruxelles

department: Medecine

country: Belgium

institute: Namur University

summary: The RNA polymerase II carboxy-terminal domain (CTD) consists of conserved repeats of the consensus sequence Y1-S2-P3-T4-S5-P6-S7, which can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although affinity purification coupled with mass spectrometry has defined CTD-associated proteins, phospho-dependent CTD interactions have remained largely elusive. Proximity-dependent biotinylation (PDB) provides an alternative approach to identify protein-protein associations in the native cellular environment. Here we present a PDB-based map of the fission yeast RNAPII CTD in live cells. The proteomic screen identified known CTD-associated proteins, but also captured new and unexpected CTD proximal proteins. We also used PDB to identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. Surprisingly, CTD-mediated biotinylation of most 3’ end processing factors was not affected in the S2A mutant, consistent with RNA-seq and ChIP-seq analysis indicating that CTD Ser2 phosphorylation is not required for 3’ end processing and transcription termination. Conversely, we found that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation. We show that loss of CTD Ser2 phosphorylation disables the Set2-Clr6(II) axis, resulting in a global increase in cryptic antisense transcription that correlates with elevated levels of histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation.

overall_design: Gene expression,  chromatin modification (H3K36me3 and H3K9ac),  RNAPII  occupancy, and RNAPII-interacting proteins (Set2 and Pcf11) occupancy, in wild-type and Rpb1 CTD mutants.

first: Damien

last: Hermand

first: François

last: Bachand

first: Cedric

last: Boulanger


title: Colon IEL gene expression

city: Aurora

first: Kristine

last: Kuhn

state: Colorado

address: 1775 Aurora Ct. Mail Stop B115

department: Medicine

institute: University of Colorado School of Medicine

summary: Comparison of gene expression by colon IELs versus small intestine IELs and colon lamina propria T cells

overall_design:
T cells from the colon epithelium, lamina propria, and small intestine epithelium were magenetically enriched by negative selection from C57Bl/6 mice ages 8-10 weeks. 10 total mice were pooled (5 male and 5 female). One group of mice were treated with broad-spectrum antibiotics (vancomycin, ampicillin, neomycin, and metronidazole) for one week then recolonized by co-housing with unmanipulated littermates for one week prior to harvesting the colon IELs.
Four samples were analyzed for gene expression by microarray:
Colon lamina propria T cells (LP), colon intraepithelial lymphyocytes from unmanipulated mice (WT_IEL), colon IELs from mice treated with antibiotics followed by bacterial recolonization (Recolonized_IEL), and IELs from the small intestine of unmanipuatled mice (SI_IEL). Methods for the acquisition of these cell types are as follows:
A total of 10 C57Bl/6 mice (5 males and 5 females) at 8-10 weeks of age were pooled for each sample. Mice were treated with 1% grape flavored Koolaid (Kraft), 0.01% ampicillin, 0.01% neomycin, 0.05% metronidazole (Research products International), and 0.05% vancomycin (Alfa Aesar) delivered ad libitum in the drinking water for 7 days. Re-colonization of antibiotic-treated SPF housed animals was done using mixed dirty bedding from untreated littermates for 7 days. Mice were then euthanized and colon and small intestine (SI) harvested. Tissue was minced and shaken at maximum speed on a benchtop vortex for ten minutes in 10ml PBS (Gibco) supplemented with  1 mM EDTA and 7.5 mM HEPES to isolate the epithelial fraction. Cells were passed through a 70um cell strainer to collect the epithelial fraction containing intraepithelial lymphocytes (IELs). Lamina propria cells were isolated after epithelial-isolation. Tissue was finely minced and incubated for 30 minutes at 37C in 0.5 mg/ml type IV collagenase (Worthington) in RPMI 1640 (Gibco) supplemented with 10% FBS (Thermo), 1mM EDTA, and 7.5mM HEPES. Digested tissue was passed through a 70um cell strainer (Corning). T cells from the SI epithelial fraction, colon epithelial fraction, and colon lamina propria fraction through magnetic enrichment using the EasySep Negative T cell isolation kit (Stemcell) following the manufacturer   s recommended protocol with the addition of 0.5 ng/mL biotin-labeled anti-mouse EpCAM (clone G8.8, Biolegend).


title: Single-cell RNA-sequencing of wildtype murine tracheal epithelial cells cultured at air-liquid interace for one, three, nine, and thirty-six days.

first: Semil

last: Choksi

overall_design: Mouse tracheal epithelial cells grown at air-liquid interface for one, three, nine, or thirty-six days from were analyzed using scRNAseq.

first: Lauren

last: Byrnes

first: Mia

last: Konjikusic

first: Rachel

last: Deleon

first: Quanlong

last: Lu

first: Christopher

last: Westlake

first: Jeremy

last: Reiter


title: Metabolism of L-arabinose converges with virulence regulation to promote enteric pathogen fitness

city: Newcastle upon Tyne

first: Curtis

last: Cottam

address: The Medical School, Newcastle University, Framlington Place

department: Biosciences Institute

country: United Kingdom

institute: Newcastle University

summary: Role of L-arabinose on gene expression in E. coli O157:H7 TUV93-0 where CTRL samples were grown in the absence of L-arabinose and ARA samples were grown in the presence of L-arabinose.

overall_design: 3 independent biological replicates were grown in the presence or absence of L-arabinose (total of 6 samples )

first: James

last: Connolly


title: Proximity-dependent biotinylation map of the RNAPII CTD reveals that the primary role of Serine 2 phosphorylation is in the suppression of pervasive antisense transcription and not in RNA 3’ end processing [RNA-seq]

summary: he RNA polymerase II carboxy-terminal domain (CTD) consists of conserved repeats of the consensus sequence Y1-S2-P3-T4-S5-P6-S7, which can be phosphorylated to influence distinct stages of the transcription cycle, including RNA processing. Although affinity purification coupled with mass spectrometry has defined CTD-associated proteins, phospho-dependent CTD interactions have remained largely elusive. Proximity-dependent biotinylation (PDB) provides an alternative approach to identify protein-protein associations in the native cellular environment. Here we present a PDB-based map of the fission yeast RNAPII CTD in live cells. The proteomic screen identified known CTD-associated proteins, but also captured new and unexpected CTD proximal proteins. We also used PDB to identify phospho-dependent CTD interactions by using a mutant in which Ser2 was replaced by alanine in every repeat of the fission yeast CTD. Surprisingly, CTD-mediated biotinylation of most 3’ end processing factors was not affected in the S2A mutant, consistent with RNA-seq and ChIP-seq analysis indicating that CTD Ser2 phosphorylation is not required for 3’ end processing and transcription termination. Conversely, we found that CTD Ser2 phosphorylation is critical for the association between RNAPII and the histone methyltransferase Set2 during transcription elongation. We show that loss of CTD Ser2 phosphorylation disables the Set2-Clr6(II) axis, resulting in a global increase in cryptic antisense transcription that correlates with elevated levels of histone acetylation in gene bodies. Our findings reveal that the fundamental role of CTD Ser2 phosphorylation is to establish a chromatin-based repressive state that prevents cryptic intragenic transcription initiation.


title: Biochemical-free enrichment or depletion of RNA classes in real-time during direct RNA sequencing with RISER (cell lines)

city: Canberra

first: Alexandra

last: Sneddon

state: ACT

address: 131 Garran Rd Acton

country: Australia

institute: The Australian National University

summary: The heterogeneous composition of cellular transcriptomes poses a major challenge for detecting weakly expressed RNA classes, as they can be obscured by abundant RNAs. Although biochemical protocols can enrich or deplete specified RNAs, they are time-consuming, expensive and can compromise RNA integrity. Here we introduce RISER, a biochemical-free technology for the real-time enrichment or depletion of RNA classes. RISER performs selective rejection of molecules during direct RNA sequencing by identifying RNA classes directly from nanopore signals with deep learning and communicating with the sequencing hardware in real time. By targeting the dominant messenger and mitochondrial RNA classes for depletion, RISER reduced their respective read counts by more than 85%, resulting in an increase in sequencing depth of up to 93% for long non-coding RNAs. We also applied RISER for the depletion of globin mRNA in whole blood, achieving a decrease in globin reads by more than 90% as well as a significant increase in non-globin reads. Furthermore, using a GPU or a CPU, RISER is faster than GPU-accelerated basecalling and mapping. RISER’s modular and retrainable software and intuitive command-line interface allow easy adaptation to other RNA classes. RISER is available at https://github.com/comprna/riser.

overall_design: Nanopore direct RNA sequencing was performed using 2 blood samples from human donors for training RISER, and 1 blood sample from another human donor for testing RISER during live MinION sequencing.

first: Agin

last: Ravindran

first: Somasundhari

last: Shanmuganandam

first: Madhu

last: Kanchi

first: Nadine

last: Hein

first: Simon

last: Jiang

first: Nikolay

last: Shirokikh

first: Eduardo

last: Eyras


title: BRCA2 suppresses “head-on” transcription-replication conflicts incurred by dormant origin firing at transcription termination sites [RNA-seq]

city: New York

first: Tony

middle: T

last: Huang

state: NY

address: 450 East 29th St, Rm 807

department: Biochemistry and Molecular Pharmacology

institute: NYU School of Medicine

summary: BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from genotoxicity and replication stress, but the mechanism(s) by which this is achieved remains elusive. Here, we provide evidence that BRCA2 acts as a critical suppressor of “head-on” transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified new origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNaseH2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results identify a new source of genomic instability caused by HO-TRCs in BRCA2-deficient ovarian cancer precursor cells, providing a new strategy in modulating HO-TRCs to enhance genomic instability

overall_design: RNA sequencing was performed on fallopian tube epithelial cells (FTEs) with a doxycyline (DOX)-inducible shRNA against BRCA2 after 0 days (untreated) and 3 days of BRCA2-knockdown. Four replicates of RNA were performed per sample.

first: Liana

last: Goehring

first: Sarah

last: Keegan

first: David

last: Fenyo


title: NF-κB transcription factors RelA and c-Rel selectively control CD4+ T-cell function in multiple sclerosis and cancer

city: Lyon

first: Yenkel

last: Grinberg-Bleyer

address: 28 rue Laennec

department: Immunity, viruses and inflammation

country: France

institute: Cancer Research Center of Lyon

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: NF-κB transcription factors RelA and c-Rel selectively control CD4+ T-cell function in multiple sclerosis and cancer [bulk_mouse]

summary: The outcome of cancer and autoimmunity is dictated by the recruitment and effector functions of CD4+ conventional T cells (Tconv cells). The NF-kappaB (NF-κB) family of transcription factors is implicated in different aspects of Tconv cell biology, but the cell-autonomous roles of NF-κB subunits are elusive. Here, we explored the contributions of canonical RelA and c-Rel subunits to Tconv function in health and disease. We found that RelA, rather than c-Rel, shaped the transcriptome of mouse and human Tconv cells at steady-state and was required for Tconv polarization toward the TH17 lineage in vitro. RelA-deficient mice were fully protected against neuro-inflammation in a mouse model of multiple sclerosis (MS), because of defective transition to a pathogenic TH17 gene expression program. In contrast, Tconv-restricted ablation of c-Rel, but not RelA, impaired their function in the microenvironment of transplanted tumors, resulting in enhanced cancer burden. c-Rel in Tconv cells was required for the response to PD-1-blockade therapy, and a c-Rel-dependent gene signature was correlated with better response and prognosis in anti-PD-1-treated patients. Our data demonstrate a division of labor between different subunits of the NF-κB pathway, paving the way for subunit-targeted immunotherapies.

overall_design: CD4+CD25- Tconv cells were isolated from spleen and LNs of control and cKO mice, and stimulated with anti-CD3/CD28 and IL-2 for 4 hours. Cells were then subjected to bulk RNAseq

first: Grinberg-Bleyer

last: Yenkel

first: Lalle

last: Guilhem

first: Lautraite

last: Raphaëlle

first: Tonon

last: Laurie


title: NF-κB transcription factors RelA and c-Rel selectively control CD4+ T-cell function in multiple sclerosis and cancer [bulk_human]

overall_design: CD4+CD25- Tconv cells were isolated from heathy donors' PBMCs, stimulated with anti-CD3/CD28 and IL-2 for 3 days and electroporated with Cas9 RNPs. After another 3 days of culture, ATTO550+ cells were sorted and stimulated as above for 4 hours. Cells were then subjected to bulk RNAseq

first: Guilhem

last: Lalle

first: Raphaëlle

last: Lautraite

first: Laurie

last: Tonon


title: Biochemical-free enrichment or depletion of RNA classes in real-time during direct RNA sequencing with RISER

first: Alexandra

last: Sneddon


title: The long non-coding RNA Meg3 mediates imprinted gene expression  during stem cell differentiation

city: Gif-sur-Yvette

first: Benoit

last: Moindrot

address: 1, avenue de la terrasse

institute: Institute for Integrative Biology of the Cell

summary: The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain’s imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian-/- mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression.  lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5’-side of Meg3.  Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome.  The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for CTCF in F1-hybrid mouse embryonic stem cells (BJ1 cells = C57BL/6J x JF1; JB1 cells = JF1 x C57BL/6J). Cells are described in Kota et al. (2014). Input is also provided

first: Daan

last: Noordermeer


title: Subtype Heterogeneity and Epigenetic Convergence in Neuroendocrine Prostate Cancer [scRNA-seq]

status: Public on Mar 27 2024

city: Boston

first: Yingtian

last: Xie

state: MA

address: 450 BROOKLINE AVE

institute: DANA FARBER CANCER INSTITUTE INC

summary: We analyzed chromatin accessibility, scATAC-seq and RNA-seq across neuroendocrine carcinomas from distinct anatomic origins including Merkel cell carcinomas, neuroendocrine prostate cancer, small cell lung cancer and gastrointestinal NECs.

overall_design: scRNA-seq


title: Single-stranded Pre-methylated 5mC Adapters Uncover the Methylation Profile of Plasma Ultrashort Single-stranded Cell-free DNA

city: Los Angeles

first: Jordan

last: Cheng

institute: UCLA

type: Methylation profiling by high throughput sequencing

summary: Whole-genome bisulfite sequencing (BS-Seq) measures cytosine methylation changes at single-base resolution and can be used to profile cell-free DNA (cfDNA). In plasma, ultrashort single-stranded cfDNA (uscfDNA, ~50 nt) has been identified together with 167 bp double-stranded mononucleosomal cell-free DNA (mncfDNA). However, the methylation profile of uscfDNA has not been described. Conventional BS-Seq workflows may not be helpful because bisulfite conversion degrades larger DNA into smaller fragments, leading to erroneous categorization as uscfDNA. We describe the “5mCAdpBS-Seq” workflow in which pre-methylated 5mC (5-methylcytosine) single-stranded adapters are ligated to heat-denatured cfDNA before bisulfite conversion. This method retains only DNA fragments that are unaltered by bisulfite treatment, resulting in less biased uscfDNA methylation analysis. Using 5mCAdpBS-Seq, uscfDNA had lower levels of DNA methylation (~15%) and was enriched in promoters and CpG islands. Hypomethylated uscfDNA fragments were enriched in upstream transcription start sites (TSSs), and the intensity of enrichment positively affected gene expression of hemopoietic cells. Using tissue-of-origin deconvolution, we inferred that uscfDNA is derived primarily from eosinophils, neutrophils, and monocytes. As proof-of-principle, we show that characteristics of the methylation profile of uscfDNA can distinguish non-small cell lung carcinoma from non-cancer samples. The 5mCAdpBS-Seq workflow is recommended for any cfDNA methylation-based investigations.

overall_design: 5 non-cancer matched samples were processed with BRcfDNA-Seq, BS-Seq, and 5mCAdpBS-Seq and 4 NSCLC samples were processed with 5mCAdpBS-Seq

first: Neeti

last: Swarup

first: Marco

last: Morselli

first: Matteo

last: Pellegrini

last: Wong


title: CRISPRi mediated treatment of dominant rhodopsin-associated retinitis pigmentosa.

city: Iowa City

first: Nathaniel

middle: Kevin

last: Mullin

state: IA

address: 375 Newton Road

institute: University of Iowa

summary: Rhodopsin (RHO) mutations such as Pro23His, are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive S. aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying GFP cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ~74%-84% reduction in RHO promoter activity in RHOpCRISPRi treated vs plasmid only controls. Following subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in ER-stress and apoptosis markers and preservation of photoreceptor cell layer thickness.

overall_design: Human donor retinal explants were transduced with AAV5-RHOp-CRISPR-dSaCas9. RNA was isolated from transduced and control explants and used to prepare RNA sequencing libraries to examine gene expression changes between donors and following treatment.

middle: K

first: Erin

middle: R

last: Burnight

first: Budd

last: Tucker


title: BRCA2 suppresses “head-on” transcription-replication conflicts incurred by dormant origin firing at transcription termination sites [Ok-seq]

first: Tony

last: Huang

overall_design: Okazaki-fragment (Ok-seq) sequencing was performed on fallopian tube epithelial cells (FTEs) with a doxycyline (DOX)-inducible shRNA against BRCA2 after 0 days (untreated), 3 days, and 6 days of BRCA2-knockdown. Two replicates of Ok-seq were performed per sample.

first: Liana

last: Goehring

first: Sarah

last: Keegan

first: David

last: Fenyo


title: DNA methylation profiles of 4T1 invasively-distinct subpopulations

city: Atlanta

first: Adam

last: Marcus

state: Georgia

address: 1365 Clifton Rd NE, Building C, Room 4092

institute: Emory University

summary: Solid tumors exhibit significant phenotypic heterogeneity. This phenotypic heterogeneity yields the formation of phenotypically distinct subpopulations of cells within a single tumor. Within the 4T1 murine mammary adenocarcinoma cell line, we observe cells that invade either in collective chains or as single cells. Through this study, we sought to dissect the epigenetic differences between these invasively-distinct subpopulations.

overall_design: Five total groups were assessed, each in triplicate (15 samples total). No controls or reference groups were included, and the data for each group was compared to each other group.

first: Sung

middle: B

last: Yoon


title: DBF4, not DRF1, is the crucial regulator of CDC7 kinase at replication forks.

city: PARIS

first: Chrystelle

last: Maric

address: 15 rue Hélène Brion

department: UMR7592 Institut Jacques-Monod

institute: CNRS

summary: In eukaryotes, CDC7 kinase is crucial for DNA replication initiation and has been involved in fork processing and replication stress response. Human CDC7 requires the binding of either one of two regulatory subunits, DBF4 and DRF1, for its activity. However, it is unclear whether the two regulatory subunits target CDC7 to a specific set of substrates, thus having different biological functions, or if they act redundantly. Using genome editing technology, we generated an isogenic set of cell lines deficient in either one of the two CDC7-activating subunits: these cells are viable but present signs of genomic instability, indicating that both DBF4 and DRF1 can independently support CDC7 for bulk DNA replication. Nonetheless, DBF4-deficient cells show altered replication efficiency, including partial deficiency in MCM helicase phosphorylation and alterations in the replication timing of discrete genomic regions. Notably, we find that CDC7 function at replication forks is entirely dependent on DBF4 and not DRF1. Thus, DBF4 is the primary regulator of CDC7 activity, likely mediating most of its functions in unperturbed DNA replication and during replication fork processing upon replication interference.

overall_design: nascent-DNA from early S-phase versus nascent-DNA from late S-phase

first: Anja

last: Göder

first: Jean-Charles

last: Cadoret

first: Corrado

last: Santocanale


title: ATAC-seq of DP thymocytes from Suv39h1 and Suv39h2 double knockout chimeric mice

city: Parkville

first: Gordon

last: Smyth

state: Vic

address: 1G Royal Pde

department: Bioinformatics

institute: Walter and Eliza Hall Institute of Medical Research

summary: H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes for healthy cellular function. Within all eukaryotic nuclei, heterochromatin and euchromatin are spatially segregated, with euchromatin typically located in the nuclear interior and heterochromatin at the nuclear periphery. Here we investigate the changes in DNA accessibilty in the absence of H3K9me3-dependent heterochromatin by performing ATAC-seq in primary immune cells deficient in both Suv39h1 and Suv39h2 (Suv39DKO), the major mammalian histone methyltransferase enzymes which catalyse heterochromatic H3K9me3 deposition.

overall_design: CD4+CD8+ double positive mouse thymocyte cells were flow-sorted (Ly5.2+CD19-CD4+CD8+) and analysed. ATAC-seq libraries from two replicates from Suv39h1+/y Suv39h2+/- (control) mice and two replicates from Suv39h1-/y Suv39h2-/- double knock-out (Suv39DKO) mice were independently prepared and sequenced . For the bioinformatics analysis, fastq files were aligned, filtered and then biological replicates were merged.

first: Christine

last: Keenan

first: Hannah

last: Coughlan

first: Rhys

middle: S

last: Allan


title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer V

city: Bethesda

first: Kent

last: Hunter

state: MD

address: 37/4048 37 Convent Drive

department: Center for Cancer Research

institute: National Cancer Institute

summary: Patients with ER-negative breast cancer have the worst prognosis of all breast cancer subtypes, often experiencing rapid recurrence or progression to metastatic disease shortly after diagnosis. Given that metastasis is the primary cause of mortality in most solid tumors, understanding metastatic biology is crucial for effective intervention. Using a mouse systems genetics approach, we previously identified 12 genes associated with metastatic susceptibility. Here, we extend those studies to identify Resf1, a poorly characterized gene, as a novel metastasis susceptibility gene in ER- breast cancer. Resf1 is a large, unstructured protein with an evolutionarily conserved intron-exon structure, but with poor amino acid conservation. CRISPR or gene trap mouse models crossed to the PyMT GEMM demonstrated that reduction of Resf1 resulted in a significant increase in tumor growth, a shortened overall survival time, and increased incidence and number of lung metastases, consistent with patient data. Furthermore, an analysis of matched tail and primary tissues revealed loss of the wildtype copy in tumor tissue, consistent with Resf1 being a tumor suppressor. Mechanistic analysis revealed a potential role of Resf1 in transcriptional control through association with compound G4 quadruplexes in expressed sequences, particularly those associated with ribosomal biogenesis. These results suggest that loss of Resf1 enhances tumor progression in ER- breast cancer through multiple alterations in both transcriptional and translational control.

overall_design: To investigate the potential affect of RESF1 depletion on transcription in human ER- breast cancer cells, MBA-MD-231 cells were infected with either scramble or RESF1 shRNAs and total RNA-sequencing performed.

first: Megan

last: Majocha

first: Devin

middle: E

last: Jackson

first: Ngoc-Han

last: Ha

first: Ruhul

last: Amin

first: Marie

last: Pangracova

first: Christina

last: Ross

first: Howard

last: Yang

first: Maxwell

last: Lee


title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer III


title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer

overall_design: Refer to individual Series


title: Next Generation Sequencing Facilitates Quantitative Analysis of chondrocyte from the inject prrx1-iWATMSC inducible and age- and gender-matched  native prrx1 expression .

city: shanghai

first: hongshang

last: chu

address: 800 Dongchuan RD. Minhang District, Shanghai, China

country: China

institute: Shanghai Jiao Tong University

summary: We had one goal to perform this study:  Analysis ofchondrocyte from the inject prrx1-iWATMSC inducible and age- and gender-matched  native prrx1 expression .

overall_design: Comparison of  chondrocyte from the inject prrx1-iWATMSC inducible and age- and gender-matched  native prrx1 expression .

first: Hongshang


title: NF-κB transcription factors RelA and c-Rel selectively control CD4+ T-cell function in multiple sclerosis and cancer [sc_rna]

first: Yenkel

last: Grinberg-Bleyer

overall_design: CD4+ T cells were FACS-sorted from tumor-bearing or EAE-induced draining lymph nodes (DLN), tumor tissues and central nervous sytem (CNS, pooled brains and spinal cords), respectively.

first: Grinberg-Bleyer

last: Yenkel

first: Lalle

last: Guilhem

first: Lautraite

last: Raphaëlle

first: Tonon

last: Laurie


title: ENVIRONMENT MODULATES PROTEIN HETEROGENEITY THROUGH TRANSCRIPTIONAL AND TRANSLATIONAL STOP CODON RECODING

city: Dresden

first: Agnes

last: Toth-Petroczy

address: Pfotenhauerstrasse 108

country: Germany

institute: Max Planck Institute of Molecular Cell Biology and Genetics

summary: In order to systematically assess the frequency and origin of stop codon recoding events, we designed a library of reporters. We introduced premature stop codons into mScarlet that enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon recoding may occur with a rate as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon recoding events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon recoding. The RNA polymerase is more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon recoding by mass spectrometry revealed that temperature regulates the expression of cryptic sequences generated by stop codon recoding in E. coli. Overall, our findings suggest that the environment influences the accuracy of protein production which increases protein heterogeneity when the organisms need to adapt to new conditions.

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

overall_design: We designed several reporters for stop codon recoding events. The strategy dwells on introducing a premature stop codon into an mScarlet allele. Thus, only upon stop codon recoding, full-length and, therefore, functional mScarlet will be synthesized. Here, we studied the mRNA sequence of 12 reporters for stop codon recoding events and the mScarlet wild-type, expressed in wild-type E. coli grown at 18℃ in LB media. The reporters for stop codon recoding were ordered from Twist Biosciences.

first: Maria

middle: L

last: Romero-Romero

first: Anastasiia

last: Kirilenko

first: Jonas

last: Poehls

first: Doris

last: Richter

first: Tobias

last: Jumel

first: Anna

last: Shevchenko


title: Estrogen receptor 1 chromatin profiling in human breast tumors reveals high inter-patient heterogeneity with enrichment of risk SNPs and enhancer activity at most-conserved regions

city: Amsterdam

first: Wilbert

last: Zwart

state: Noord-Holland

address: Plesmanlaan 121

department: Oncogenomics

country: Netherlands

institute: Netherlands Cancer Institute

first: Stacey

last: Joosten

first: Sebastian

last: Gregoricchio


title: Genome-wide identification of target genes for the transcription factors Nkx2.2 and Zfp488 in differentiating CG4 cells as a model for differentiating oligodendrocytes

city: Erlangen

first: Michael

last: Wegner

address: Fahrstr. 17

department: Institut of Biochemistry

summary: In an attempt to validate the CG4 cell line as a model for oligodendrocytes, we determined the expression profiles in both cell types and the alterations during differentiation and found a striking overlap. Using CG4 cells as a model system for oligodendroglial cells,  we generated several Nkx2.2 and Zfp488 overexpressing CG4 lines and compared their expression profile under differentiating conditions to regular CG4 cells to identify target genes of the two transcription factors in a genome-wide and unbiased approach. Several target genes were shared between Nkx2.2 and Zfp488, among them Gpr37.

overall_design: We generated mRNA profiles of control (pCAG-IRES-EGFP transduced), Zfp488 (pCAG-myc-Zfp488-IRES-EGFP transduced) and Nkx2.2 (pCAGT7-Nkx2.2-IRES-EGFP transduced) overexpressing CG4 cells after 3 days of differentiation using the Illumina NovaSeq 6000, PE 2x150 platform. Transcriptomic profiles of primary rat oligodendroglia under proliferative and differentiating conditions (3 days) were genereted on the Illumina NovaSeq 6000, PE 2x150 platform as well. Transcriptomic profiles of wild-type CG4 cells under proliferative conditions resulted from sequencing on an Illumina NovaSeq 6000, PE 2x100 platform.

first: Aberle

last: Tim


title: Selective refueling of CAR T cells using ADA1 and CD26 boosts antitumor immunity

city: HOUSTON

first: Xiaotong

last: Song

state: Texas

address: 2121 W. Holcombe Blvd

department: CIID

institute: Texas A&M University

summary: CAR T cell therapy has revolutionized the treatment of hematologic malignancies, but its efficacy in solid tumors remains limited due to the immunosuppressive nature of the tumor microenvironment and the inability of T cells to persist and traffic to the tumor site. While current strategies focus on enhancing CAR T cell activity through costimulatory molecules and cytokines, a critical yet often overlooked factor is the competition for nutrients between tumor cells and T cells in the nutrient-deprived tumor microenvironment. To address this challenge, we employed a selective metabolic refueling (MR) strategy by providing T cells with inosine as an alternative fuel source for growth and functionality. In this study, we engineered CAR T cells to co-express a membrane-bound CD26 and a cytoplasmic adenosine deaminase 1 (ADA1) fused to an anti-CD3 scFv. ADA1 irreversibly converts both intracellular and extracellular adenosine to inosine, overcoming adenosine-mediated immunosuppression and providing T cells with inosine for growth. The inclusion of an anti-CD3 scFv fusion partner and overexpressing CD26 boosts ADA1 capture in a membrane proximal manner, providing inosine for T cells and minimizing feeding the tumor cells. We demonstrate that ADA1 is conditionally secreted in an autocrine manner in response to CD3/CD26 stimulation and that it activates CAR T cells through trans-signaling in a tumor-specific manner. In addition, we show that, compared to unmodified CAR T cells, CD26-overexpressing CAR T cells have better migration capacity and are less susceptible to TGF-β1 suppression. Finally, we demonstrate that, in mice models of human hepatocellular carcinoma (GPC3-MR-CAR) and human non-small cell lung cancer (HER2-MR-CAR), metabolically refueled CAR T cells (MR-CAR) are more efficient in reducing tumor growth than unmodified CAR T cells. Thus, selective refueling CAR T cells using ADA1 and CD26 holds promise for improving the efficacy of CAR T cell therapy of solid tumors.

overall_design: Comprehensive analyses of RNA-seq profiles compared between nontransduced and different CAR-T cells

first: Abhijit

last: Sarkar


title: Subtype Heterogeneity and Epigenetic Convergence in Neuroendocrine Prostate Cancer [scATAC-seq]

first: Yingtian

last: Xie

overall_design: scATAC-seq


title: IFI35 regulates non-canonical NF-κB signaling to maintain glioblastoma stem cells and recruit tumor-associated macrophages [RNA-seq]

city: Chapel Hill

first: Daqi

last: Li

address: 450 West Dr

institute: Univerity of North Carolina at Chapel Hill

summary: Glioblastoma (GBM) is the most aggressive malignant primary brain tumor characterized by a highly heterogeneous and immunosuppressive tumor microenvironment (TME). The symbiotic interactions between glioblastoma stem cells (GSCs) and tumor-associated macrophages (TAM) in the TME are critical for tumor progression. Here, we identified that IFI35, a transcriptional regulatory factor, plays both cell-intrinsic and cell-extrinsic roles in maintaining GSCs and the immunosuppressive TME. IFI35 induced non-canonical NF-kB signaling through proteasomal processing of p105 to the DNA-binding transcription factor p50, which heterodimerizes with RELB (RELB/p50), and activated cell chemotaxis in a cell autonomous manner. Further, IFI35 induced recruitment and maintenance of M2-like TAMs in TME in a paracrine manner. Targeting IFI35 effectively suppressed in vivo tumor growth and prolonged survival of orthotopic xenograft-bearing mice. Collectively, these findings reveal the tumor-promoting functions of IFI35 and suggest that targeting IFI35 or its downstream effectors may provide effective approaches to improve GBM treatment.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for 839 cell and its KO derivatives (sgIFI35/sgRELB).

first: Xiefeng

last: Wang

first: Kexin

last: Chen

first: Danyang

last: Shan

first: Wei

last: Yuan

first: Qiankun

last: Lin

first: Ryan

last: Gimple

first: Deobrat

last: Dixit

first: Chenfei

last: Lu

first: Danling

last: Gu

first: You

last: Hao

first: Jiancheng

last: Gao

first: Yangqing

first: Tao

last: Kang

first: Junlei

last: Yang

first: Hang

last: Yu

first: Kefan

first: Zhumei

last: Shi

first: Yun

first: Junxia

last: Zhang

first: Xiao

last: Fan

first: Qiulian

last: Wu

first: Linjie

last: Zhao

first: Zhe

last: Zhu

first: Jianghong

last: Man

first: Qianghu

first: Fan

first: Weiwei

last: Tao

first: Stephen

last: Mack

first: Chaojun

first: Nu

first: Yongping

last: You

first: Xu

last: Qian

first: Kailin

first: Jeremy

middle: N

last: Rich

first: Qian

first: Xiuxing


title: BRCA2 suppresses “head-on” transcription-replication conflicts incurred by dormant origin firing at transcription termination sites

first: Tony

last: Huang


title: ChIP-seq of histone marks of DP thymocytes from Suv39h1 and Suv39h2 double knockout chimeric mice

first: Gordon

last: Smyth

summary: H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes for healthy cellular function. Within all eukaryotic nuclei, heterochromatin and euchromatin are spatially segregated, with euchromatin typically located in the nuclear interior and heterochromatin at the nuclear periphery. Here we investigate the histone modifications (H3K27ac, H3K27me3, H3K4me3 and H3K9me3) in the absence of H3K9me3-dependent heterochromatin by performing ChIP-seq in primary immune cells deficient in both Suv39h1 and Suv39h2 (Suv39DKO), the major mammalian histone methyltransferase enzymes which catalyse heterochromatic H3K9me3 deposition.

overall_design: CD4+CD8+ double positive mouse thymocyte cells were flow-sorted (Ly5.2+CD19-CD4+CD8+) and analysed. H3K27ac, H3K27me3, H3K4me3 and H3K9me3 ChIP-seq libraries from Suv39h1+/y Suv39h2+/- (control) mice were prepared and sequenced along with an IgG and whole-cell extract (input) library. H3K27ac, H3K27me3 and H3K4me3 ChIP-seq libraries from Suv39h1-/y Suv39h2-/- double knock-out (Suv39DKO) mice were prepared and sequenced along with an IgG and whole-cell extract (input) library. For the bioinformatics analysis, the reads were aligned to mm10 and duplicates were removed.

first: Christine

last: Keenan

first: Hannah

last: Coughlan

first: Rhys

last: Allan


title: Interleukin-6 supplementation improves bovine conceptus elongation and transcriptomic indicators of developmental competence

city: Blacksburg

first: Alan

middle: D.

last: Ealy

state: VA

address: 175 W Campus Dr

institute: Virginia Tech

summary: A high incidence of pregnancy failures occurs in cattle during the second week of pregnancy as blastocysts transition into an elongated conceptus. This work explored whether interleukin-6 (IL6) supplementation during in vitro embryo production would improve subsequent conceptus development. Bovine embryos were treated with 0 or 100 ng/mL recombinant bovine IL6 beginning on day 5 post-fertilization. At day 7.5 post-fertilization, blastocysts were transferred into estrus synchronized beef cows (n=5 recipients/treatment, 10 embryos/recipient). Seven days after transfer (day 14.5), cows were euthanized to harvest reproductive tracts and collect conceptuses. Individual conceptus lengths and stages were recorded before processing for RNA-sequencing. Increases in conceptus recovery, length, and the proportion of tubular and filamentous conceptuses were detected in conceptuses derived from IL6-treated embryos. The IL6 treatment generated 591 differentially expressed genes (DEG) in conceptuses (n=9-10/treatment). Gene ontology enrichment analyses revealed changes in transcriptional regulation, DNA-binding, and antiviral actions. Only a few DEG were associated with extraembryonic development, but several DEG were associated with embryonic regulation of transcription, mesoderm and ectoderm development, organogenesis, limb formation, and somatogenesis. To conclude, this work provides evidence that IL6 treatment before embryo transfer promotes pre-implantation conceptus development and gene expression in ways that resemble the generation of a robust conceptus containing favorable abilities to survive this critical period of pregnancy.

overall_design: Savannah L. Speckhart

first: Mary

middle: A

last: Oliver

first: Jessica

last: Keane

first: Nicholas

last: Dias

first: Vitor

last: Mercadante

first: Fernando

last: Biase


title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer IV

first: Kent

last: Hunter

first: Megan

last: Majocha

first: Devin

last: Jackson

first: Ngoc-Han

last: Ha

first: Ruhul

last: Amin

first: Marie

last: Pangracova

first: Christina

last: Ross

first: Howard

middle: H

last: Yang

first: Maxwell

last: Lee


title: Barcode lineage tracking of ferret cortical progeny using TrackerSeq (Mayer lab)

city: Sant Joan d’Alacant

first: Lucia

last: Del-Valle-Anton

address: Av. Santiago Ramón y Cajal s/n

country: Spain

institute: Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández

summary: Diversity of cortical radial glia cells (RGCs) and their complex relationships to generate neurons in species with expanded germinal zones and a folded cortex, remains unclear. We used TrackerSeq, a technique that integrates DNA barcodes into the genome of electroporated RGCs, to identify the distinct cell lineages that shape ferret cortex.

overall_design: E34 ferret embryos were electroporated with a piggyBac transposon-based library and eGFP+ cells were isolated at E37 by FACS and procesed for their parallel transcriptomic and barcode-lineage tracing characterization.

first: Salma

first: Daniela

last: Cimino

first: Florian

last: Neuhaus

first: Elena

last: Dvoretskova

first: Virginia

last: Fernández

first: Yigit

last: Babal

first: Cristina

last: Garcia-Frigola

first: Anna

last: Prieto-Colomina

first: Raquel

last: Murcia-Ramón

first: Yuki

last: Nomura

first: Adrián

last: Cárdenas

first: Chao

last: Feng

first: Juan Antonio

last: Moreno-Bravo

first: Magdalena

last: Götz

first: Christian

last: Mayer

first: Víctor

last: Borrell


title: Ethyl Acetate Extract of Antenoron Filiforme Inhibits the Proliferation of Triple Negative Breast Cancer Cell via Suppressing SKP2/P21 Signaling Axis

city: Chengdu

first: Yumei

last: Wang

address: 1166 LiuTai

institute: Chengdu University of TCM

summary:
Purpose: Antenoron Filiforme is a traditionally used herb medicine in China. Our findings indicate that the ethyl acetate extract of Antenoron Filiforme (AF-EAE) has the ability to inhibit the proliferation of triple negative breast cancer cells (TNBC). However, little is known about the underlying mechanism of AF-EAE's action on TNBC.
Methods: Here, we performed RNA sequencing on MDA-MB-231 treated by AF-EAE with two concentrations for 24 hours. The sequence reads that passed quality filters were analyzed at the transcript isoform level with hisat2 followed by HTSeq.
Results: Using an optimised data analysis workflow, RNA-seq data showed 1,392 differentially expressed genes (DEGs) after low concentration drug treatment and 2,847 DEGs in the high concentration drug treatment group, with a fold change of ≥1.5 and p-value <0.05. Expression changes in six genes were confirmed by qRT-PCR, demonstrating the high sensitivity of the RNA-seq method. Based on the KEGG pathway enrichment analysis results, cell growth relevant pathways such as "p53 signaling pathway" and "cell cycle" were greatly enriched. In addition, GSEA analysis indicated that DEGs in AF-EAE treated MDA-MB-231 cells were also well enriched in "cell cycle", "DNA replication" and "G2/M cell cycle".
Conclusion: Our study indicated that AF-EAE could inhibit the proliferation of  TNBC by inducing cell cycle arrest. Meanwhile, we provide a new potential drug treating breast cancer.

overall_design: mRNA profiles of AF-EAE-treated MDA-MB-231 cell line with 2 different concentrations, in duplicate, using Illumina NovaSeq 6000.

first: Yile

last: Liao

first: Dong

first: Yu

last: Gui


title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer II

first: Kent

last: Hunter

overall_design: To investigate the potential affect of RESF1 depletion on transcription in human ER- breast cancer cells, 6DT1murine mammary tumor cells were infected with either scramble or RESF1 shRNAs and total RNA-sequencing performed.

first: Megan

last: Majocha

first: Devin

last: Jackson

first: Ngoc-Han

last: Ha

first: Ruhul

last: Amin

first: Marie

last: Pangracova

first: Christina

last: Ross

first: Howard

last: Yang

first: Maxwell

last: Lee


title: Effect of PELI2 deficiency on the gene expression of bone marrow B220+ cells

city: Jinan City

first: Yan

last: Xu

address: No. 44 Wenhua West Road

institute: Shandong University

summary: To investigate the effect of PELI2 in the regulation of bone marrow B220+ cells in PELI2 knockout mice. We then performed gene expression profiling analysis using data obtained from RNA-seq of 2 different cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for bone marrow B220+ cells from WT and PELI2 knockout mice.

first: Baobing

last: Zhao


title: Resf1 is a compound G4 quadruplex-associated tumor suppressor for triple negative breast cancer I


title: Identification of HTRA4 as a trasncritpional target of P63 in trophoblast

city: La Jolla

first: Robert

last: Morey

address: 2880 Torrey Pines Scenic Dr

department: Pathology

institute: UCSD

summary:
The placenta plays a crucial role in pregnancy success. deltaNp63alpha (p63), a transcription factor from the TP53 family, is highly expressed in villous cytotrophoblasts (CTBs), the epithelial stem cells of the human placenta, and has been shown to be involved in CTB maintenance and differentiation. In this study, we set out to identify mechanism(s) of action of p63, by identifying its downstream targets. We evaluate gene expression changes following overexpression and knockdown of p63 in the JEG3 choriocarcinoma cell line, using microarray-based RNA profiling. We identify High temperature requirement A4 (HTRA4), a placenta-specific serine protease involved in trophoblast differentiation and altered in preeclampsia, as a gene reciprocally regulated by p63, and characterize its expression in primary human placental tissues by RNAseq (PMID 34540826; GSE173372) and in situ hybridization.
Forced overexpression of deltaNp63alpha in JEG-3 was obtained by pCMV lentiviral vector infection (empty vector used as control). ) The pLKO.1-based non-targeting scramble and p63-specific short hairpin (shRNA) lentiviral plasmids were used for gene knock-down. Vectors were previously described in Li et al Am J Pathol. 2014 PMID: 25307348.

overall_design: Mary E. Donohoe

first: Yingchun

first: Don

last: Pizzo

first: Sampada

last: Kallol

first: Hee

middle: Y

last: Cho

first: Francesca

last: Soncin

first: Mana

middle: M

last: Parast


title: Study of the translation initiation by Selective TCP-seq in the context of eIF1-eIF4G1 inhibitor

city: Rehovot

first: Benjamin

last: Weiss

address: 234 Herzl Street

country: Israel

institute: Weizmann Institute of Science

summary: Start codon recognition by the 48S complex is a critical step in translation. However, understanding the in vivo initiation and its regulation at a global scale is limited. To better understand the mechanism in vivo, we have screened for small molecules that specifically inhibit the function of eIF1-eIF4G1 interaction. We performed Selective-48S footprinting against eIF1 (HA-tagged), eIF2a (HA-tagged), eIF4G1 and eIF3c to answer questions regarding the function of that interaction in the context of the scanning and initiating 48S ribosome.

overall_design: Selective 48S footprinting (TCP-seq) of eIF4G1, HAeIF1,HA eIF2a, eIF3 from DMSO or i14G1-12 treated cells in HEK 293T cells.

first: Rivka

last: Dikstein


title: Study of the eIFG1-eIF1 interaction by TCP-seq on translation initiation in HEK 293T cells

summary: Start codon recognition by the 48S complex is a critical step in translation. However, understanding the in vivo initiation and its regulation at a global scale is limited. Here, we analyzed translation complex profiling (TCP-seq) data to determine the impact of eIF4G1-eIF1 inhibition and the 48S organization. Our analysis provides the first global view of leaky scanning and reveal the central roles of mRNA features and eIF4G1-eIF1 in its regulation. Specifically, non-leaky genes are enriched with a Kozak bearing  a C at -1 position while those with short 5’ UTR with TISU. Additionally, the stability of the 48S complex and its integrity during scanning are impaired upon eIF4G1-eIF1 inhibition. Detailed analysis of initiation site footprints revealed three main classes conserved from yeast to human. Our analysis provides a general overview of AUG selection and evidence for conformational rearrangements in vivo.

overall_design: Comparative initiation profiling analysis of TCP-seq and RNA-seq data for HEK 293T cells treated with DMSO or with i14G1-12 (3 replicates per conditions)


title: Transcriptomic characterization of Paneth-derived and Intestinal stem cell-derived murine intestinal tumors

city: Rotterdam

first: Mathijs

last: Verhagen

address: Doctor Molewaterplein 40

institute: Erasmus Medical Center

summary: To investigate the effect of the cell-of-origin on the transcriptional phenotype of the tumor, we performed bulk RNAseq on macroscopically dissected intestinal tumors from mice carrying lineage-specific Apc/Kras mutations in Paneth or ISC cells. Where indicated, tumorigenisis was triggered in an inflammatory context by administration of Dextran Sodium Sulfate (DSS).

overall_design: Comparative gene expression profiling analysis of RNA-seq data from Paneth-derived versus ISC-derived tumors

first: M

middle: P

first: R

last: Joosten

last: Fodde


title: Effect of western-style diet on gene expression of mouse intestinal Paneth cells

summary: To investigate the effect of wetern-style diet on Paneth cells, we performed bulk RNAseq on sorted Paneth cells from mice fed for 3 months with western-style diet (NWD1) and control diet (AIN76A)

overall_design: Comparative gene expression profiling analysis of RNA-seq data from Paneth cells exposed to NWD1 versus AIN76A diets

last: Schmitt


title: scRNA-seq of ferret cortical germinal zones from splenial gyrus and lateral sulcus (Borrell lab)

first: Lucia

last: Del-Valle-Anton

summary: Diversity of cortical radial glia cells (RGCs) and their complex relationships to generate neurons in species with expanded germinal zones and a folded cortex, remains unclear. We used single-cell RNA sequencing (scRNA-seq) of microdissected cortical germinal layers (ventricular zone (VZ) and outer subventricular zone (OSVZ)), from two cortical regions (splenial gyrus (SG) and neighboring lateral sulcus (LS)) at two critical time points for ferret cortex development (embryonic day (E) 34 and postnatal day (P) 1) to distinguish the molecular diversity of progenitors and newborn neurons, and study their transcriptomic trajectories.

overall_design: Individually microdissected germinal zones from living brain slices were isolated from the six following conditions (E34_VZ_SG, E34_VZ_LS, P1_VZ_SG, P1_VZ_LS, P1_OSVZ_SG, P1_VZ_LS) and processed for scRNA-seq. We obtained each condition from 3 independent biological replicas, each replica profiled individually and containing cells from 2-4 sibling E34 embryos or 1 P1 kit, from a total of 9 litters.

first: Salma

last: Amin

first: Daniela

last: Cimino

first: Florian

last: Neuhaus

first: Elena

last: Dvoretskova

first: Virginia

last: Fernández

first: Yigit

last: Babal

first: Cristina

last: Garcia-Frigola

first: Anna

last: Prieto-Colomina

first: Raquel

last: Murcia-Ramón

first: Yuki

last: Nomura

first: Adrián

last: Cárdenas

first: Chao

last: Feng

first: Juan Antonio

last: Moreno-Bravo

first: Magdalena

last: Götz

first: Christian

last: Mayer

first: Víctor

last: Borrell


title: Estrogen receptor 1 chromatin profiling in human breast tumors reveals high inter-patient heterogeneity with enrichment of risk SNPs and enhancer activity at most-conserved regions [ChIP-seq]

first: Wilbert

last: Zwart

summary: Estrogen Receptor 1 (ESR1; also known as ERα, encoded by ESR1 gene) is the main driver and prime drug target in luminal breast cancer. ESR1 chromatin binding is extensively studied in cell lines and a limited number of human tumors, using consensi of peaks shared among samples. However, little is known about inter-tumor heterogeneity of ESR1 chromatin action, along with its biological implications. Here, we use a large set of ESR1 ChIP-seq data from 70 ESR1+ breast cancers to explore inter-patient heterogeneity in ESR1 DNA binding to reveal a striking inter-tumor heterogeneity of ESR1 action. Of note, commonly shared ESR1 sites show the highest estrogen-driven enhancer activity and are most engaged in long-range chromatin interactions. In addition, the most commonly shared ESR1-occupied enhancers are enriched for breast cancer risk SNP loci. We experimentally confirm SNVs to impact chromatin binding potential for ESR1 and its pioneer factor FOXA1. Finally, in the TCGA breast cancer cohort, we can confirm these variations to associate with differences in expression for the target gene. Cumulatively, we reveal a natural hierarchy of ESR1–chromatin interactions in breast cancers within a highly heterogeneous inter-tumor ESR1 landscape, with the most common shared regions being most active and affected by germline functional risk SNPs for breast cancer development.

overall_design:
Estrogen receptor alpha ChIP-seq (ERa, SX-543, Santa Cruz) has been performed on 5 female primary breast tumor tissues.  Raw data are available with restricted access at the EGA under accession number EGAS50000000008.

***************************************************************
Submitter states that missing raw files are due to patient privacy concerns.
***************************************************************

first: Stacey

last: Joosten

first: Sebastian

last: Gregoricchio

first: Suzan

last: Stelloo

first: Elif

last: Yapıcı

first: Chia-Chi

middle: F

last: Huang

first: Maria

last: Donaldson Collier

first: Tunc

last: Morova

first: Umut Berkay

last: Altintas

first: Yongsoo

last: Kim

first: Sander

last: Canisius

first: Gozde

last: Korkmaz

first: Nathan

last: Lack

first: Michiel

last: Vermeulen

first: Sabine

middle: C

last: Linn


title: IFI35 regulates non-canonical NF-κB signaling to maintain glioblastoma stem cells and recruit tumor-associated macrophages

first: Daqi

last: Li

overall_design: Comparative gene expression profiling analysis of ATAC-seq data for 839 cell and its KO derivatives (sgIFI35).

first: Xiefeng

last: Wang

first: Kexin

last: Chen

first: Danyang

last: Shan

first: Wei

last: Yuan

first: Qiankun

last: Lin

first: Ryan

last: Gimple

first: Deobrat

last: Dixit

first: Chenfei

last: Lu

first: Danling

last: Gu

first: You

last: Hao

first: Jiancheng

last: Gao

first: Yangqing

first: Tao

last: Kang

first: Junlei

last: Yang

first: Hang

last: Yu

first: Kefan

last: Song

first: Zhumei

last: Shi

first: Yun

first: Junxia

last: Zhang

first: Xiao

last: Fan

first: Qiulian

last: Wu

first: Linjie

last: Zhao

first: Zhe

last: Zhu

first: Jianghong

last: Man

first: Qianghu

first: Fan

first: Weiwei

last: Tao

first: Stephen

last: Mack

first: Chaojun

first: Nu

first: Yongping

last: You

first: Xu

last: Qian

first: Kailin

first: Jeremy

last: Rich

first: Qian

first: Xiuxing


title: RNA-seq profiles of DP thymocytes from control and Suv39h1/Suv39h2 double knockout mice

first: Gordon

last: Smyth

summary: H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes for healthy cellular function. Here we investigate the transcriptomic effects of heterochromatin loss in primary immune cells deficient in both Suv39h1 and Suv39h2 (Suv39DKO), the major mammalian histone methyltransferase enzymes which catalyse heterochromatic H3K9me3 deposition.

overall_design: Six samples of CD4+CD8+ double positive mouse thymocyte cells were analysed. There were three replicates from Suv39h1+/y Suv39h2+/- (control) mice and three replicates from Suv39h1-/y Suv39h2-/- double knock-out (Suv39DKO) mice. All samples were taken from individual mice.

first: Alexandra

last: Garnham

first: Christine

last: Keenan

first: Wing

last: Chan

first: Timothy

last: Johanson

first: Hannah

middle: D

last: Coughlan

first: Rhys

last: Allan


title: RNA-seq, LaminB1 ChIP-seq and in situ Hi-C of double positive thymocytes from control and Suv39h1/Suv39h2 double knockout mice

summary: H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes for healthy cellular function. Within all eukaryotic nuclei, heterochromatin and euchromatin are spatially segregated, with euchromatin typically located in the nuclear interior and heterochromatin at the nuclear periphery. We investigated the effects of loss of H3K9me3-dependent heterochromatin in primary immune cells deficient in both Suv39h1 and Suv39h2 (Suv39DKO), the major mammalian histone methyltransferase enzymes which catalyse heterochromatic H3K9me3 deposition.

overall_design: CD4+CD8+ double positive thymocyte cells from control and Suv39h1/Suv39h2 double knockout mice were analysed by RNA-seq, LaminB1 ChIP-seq and in situ Hi-C. Three biological replicates were undertaken for RNA-seq and two biological replicates for LaminB1 ChIP-seq and Hi-C.


title: Proximity-dependent biotinylation map of the RNAPII CTD reveals that the primary role of Serine 2 phosphorylation is in the suppression of pervasive antisense transcription and not in RNA 3’ end processing

city: Namur

first: Carlo

last: Yague-Sanz

address: 61 rue de Bruxelles

institute: Namur University

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

overall_design: Refer to individual Series


title: in situ Hi-C profiles of DP thymocyltes from control and Suv39h1/Suv39h2 double knockout mice

summary: H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes for healthy cellular function. Here we investigate the disruption of genome organization in the absence of H3K9me3-dependent heterochromatin by performing in situ Hi-C in primary immune cells deficient in both Suv39h1 and Suv39h2 (Suv39DKO), the major mammalian histone methyltransferase enzymes which catalyse heterochromatic H3K9me3 deposition.

overall_design: CD4+CD8+ double positive mouse thymocyte cells were extracted from two Suv39h1+/y Suv39h2+/- (control) mice and two Suv39h1-/y Suv39h2-/- double knock-out (Suv39DKO) mice and profiled by in situ Hi-C. All samples correspond to individual mice.


title: Multiple parallel cell lineages in the developing mammalian cerebral cortex

first: Lucia

last: Del-Valle-Anton

summary:
Cortical neurogenesis follows a simple lineage: apical Radial Glia Cells (RGC) generate basal progenitors, and these produce neurons. How this occurs in species with expanded germinal zones and a folded cortex, like human, remains unclear. We used single-cell RNA sequencing from individual cortical germinal zones in ferret, and barcoded lineage tracking, to determine the molecular diversity of progenitor cells and their lineages. We identified multiple RGC classes that initiate parallel lineages, converging onto a common class of newborn neuron. Parallel RGC classes and transcriptomic trajectories were repeated across germinal zones and conserved in ferret and human, but not mouse. Neurons followed parallel differentiation trajectories in gyrus and sulcus, with different expression of human cortical malformation genes. Progenitor cell lineage multiplicity is conserved in the folded mammalian cerebral cortex.

This SuperSeries is composed of the SubSeries listed below.

sample_organism:
Mustela putorius
Mustela putorius furo

platform_organism:
Mustela putorius
Mustela putorius furo

overall_design:
Refer to individual Series

Single-cell RNA sequencing (Borrell lab):
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE219254
Barcoded lineage tracking (Mayer lab):
https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE233587


title: Metallothionein 1 (Mt1) Regulates Growth and Survival of DNMT3A;NPM1-Mutant Acute Myeloid Leukemia

status: Public on Mar 26 2024

city: Bar Harbor

first: Jennifer

last: Trowbridge

state: ME

address: 600 Main Street

institute: The Jackson Laboratory

summary: Adult de novo acute myeloid leukemia (AML) is a hematologic malignancy with poor prognosis, commonly driven by mutations in genes including the DNA methyltransferase DNMT3A and nucleophosmin NPM1. We previously generated sequentially inducible mouse models of these mutations and observed transformation from clonal hematopoiesis (CH) to myeloproliferative disorder to AML. In transformed AML, leukemia-propagating cells have a myeloid-restricted progenitor cell phenotype (c-Kit+). Here, to identify mechanisms that sustain tumorigenesis, we performed single-cell RNA-seq of c-Kit+ cells from four primary Dnmt3a;Npm1-mutant AML samples. The most primitive subset of c-Kit+ cells, a multipotent progenitor population Multi-Lin-2, had increased expression of the antioxidant and heavy metal chelator metallothionein 1 (Mt1) in all AML samples. Cas9-mediated Mt1 knockout in primary Dnmt3a;Npm1-mutant AML resulted in reduced cell cycling and proliferation and increased pyroptosis in vitro and increased overall survival following transplant into congenic recipient mice.

overall_design: To investigate the transcriptional differences observed in CRISPR-Cas9 mediated knock-down of Metallothionein 1 (Mt1) in Dnmt3aR878H/+;Npm1cA/+ associated AML in C57BL/6 mice.

first: Patricia

last: Colom

first: Nathan

last: Boyer

first: Tim

last: Sterns


title: LaminB1 ChIP-seq of DP thymocytes from wildtype and Suv39h1 and Suv39h2 double knockout mice

city: Parkville

address: 1G Royal Pde

summary: H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes for healthy cellular function. Within all eukaryotic nuclei, heterochromatin and euchromatin are spatially segregated, with euchromatin typically located in the nuclear interior and heterochromatin at the nuclear periphery. Here we investigate the disruption of lamina-association in the absence of H3K9me3-dependent heterochromatin by performing LaminB1 ChIP-seq in primary immune cells deficient in both Suv39h1 and Suv39h2 (Suv39DKO), the major mammalian histone methyltransferase enzymes which catalyse heterochromatic H3K9me3 deposition.

overall_design: CD4+CD8+ double positive mouse thymocyte cells were analysed. ChIP and whole-cell extract (WCE) libraries from two replicates from Suv39h1+/y Suv39h2+/- (control) mice and two replicates from Suv39h1-/y Suv39h2-/- double knock-out (Suv39DKO) mice were independently prepared and sequenced to confirm successful immunoprecipitation. For the bioinformatics analysis, the replicate FASTQ files were merged. LaminB1 peaks were then called separately for the knockout and control mice using WCE as the input control in each case.


title: Timecourse of transcriptional changes upon eIF4E1 depletion in Toxoplasma gondii

city: Indianapolis

first: Michael

middle: James

last: Holmes

state: IN

address: 635 Barnhill Drive

department: Pharmacology & Toxicology

institute: Indiana University School of Medicine

summary: The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and latent cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5’-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogenous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence.

overall_design: Expression changes in Toxoplasma upon IAA-induced knockdown of eIF4E1mAID for 24h and 48h in RH and ME49 strain backgrounds

middle: J

first: William

last: Sullivan Jr


title: RNA-seq analysis of Arabidopsis root in response to temperature

first: Ling

last: Zhang

address: 10010 N Torrey Pines Rd

institute: Salk Institute for Biological Studies

summary: We reported the temperature responses in roots affect the gene expression. To identify the transcriptome profiles response to temperature, we performed an mRNA-seq experiments in Col-0, hy5-215 and chl1.5 with 2 different temperature conditions (21 and 28C). The differential expressed genes in response to temperature were analyzed. It has 36 samples in total, with 3 biogical replicates for each condition.

overall_design: 4 days old seedling of Col-0, hy5-215, and chl1.5 were grown on 1/2 ms plate and transfer to the either 21 or 28C for additional 5 days, respectively.

first: Wolfgang

last: Busch

first: Sanghwa

last: Lee


title: Semaglutide ameliorates cardiac remodeling in male mice by optimizing energy substrate utilization through the Creb5/NR4a1 axis

city: Wuhan

first: Yu-lan

last: Ma

state: None Selected

address: No. 9 Zhangzhidong Road, Wuchang District, Wuhan City, Hubei Province, China号

institute: The First Clinical College of Wuhan University

summary: Semaglutide, a glucagon-like peptide-1 receptor agonist, is clinically used as a glucose-lowering and weight loss medication due to its effects on energy metabolism. In heart failure, energy production is impaired due to altered mitochondrial function and increased glycolysis. However, the impact of semaglutide on cardiomyocyte metabolism under pressure overload remains unclear. Here we demonstrate that semaglutide improves cardiac function and reduces hypertrophy and fibrosis in a mouse model of pressure overload-induced heart failure. Semaglutide preserves mitochondrial structure and function under chronic stress. Metabolomics reveals that semaglutide reduces mitochondrial damage, lipid accumulation, and ATP deficiency by promoting pyruvate entry into the tricarboxylic acid cycle and increasing fatty acid oxidation. Transcriptional analysis shows that semaglutide regulates myocardial energy metabolism through the Creb5/NR4a1 axis in the PI3K/AKT pathway, reducing NR4a1 expression and its translocation to mitochondria. NR4a1 knockdown ameliorates mitochondrial dysfunction and abnormal glucose and lipid metabolism in the heart. These findings suggest that semaglutide may be a therapeutic agent for improving cardiac remodeling by modulating energy metabolism.

overall_design: All male mice injected with Sema and its vehicle are treated as controls to receive TAC surgery or sham surgery. Mice are intraperitoneally injected with a dose of 60.0 mg/kg/day or control drug solution vehicle for 8 or 4 weeks after ultrasound, and left ventricular heart tissue is taken for RNA-seq

first: Yu-Lan

first: Chun-Yan

last: Kong

first: Zhen

last: Guo

first: Qi-Zhu

last: Tang


title: miR-619-5p and cardiogenic shock in patients with ST-Segment-Elevation Myocardial Infarction

city: Barcelona

first: Lina

last: Badimon

state: BARCELONA

address: Carrer de Sant Antoni Maria Claret, 167

department: ICCC-Cardiovascular Program

institute: Research Institut- HSCSP

summary:
Affymetrix microarray (GeneChip microRNA 4.0) profilling of circulating miRNAs.
We used miRNA arrays to profile miRNAs isolated from plasma of ST-segment elevation acute myocardial infarction (STEMI)-patients with cardiogenic shock (CS) or without cariogenic shock (Non-CS)

overall_design: We isolated circulating miRNAs from plasma samples of STEMI-patients and profiled them to determine miRNA signature.

first: Rafael

last: Escate

first: Sandra

last: Camino

first: Teresa

last: Padró


title: Gene expression profile at single cell level of Paneth cells from the small intestine upon genetic targeting and in inflammatory conditions

first: Mathijs

last: Verhagen

summary: We combined lineage tracing with single cell RNA sequencing (scRNA-seq) to study the mechanisms underlying the conversion of Paneth cells into bona fide tumor cells.

overall_design: Crypts were purified from the small intestine, and enzymatically dissociated with TryplE to obtain a single cell suspension. Cells were stained with fluorescent antibodies (Cd24, cKit) and with Hashing antibodies. Paneth cells were enriched by Fluorescence-activated cell sorting (FACS) according to the SSChiCD24hicKithi gate or by the presence of Yfp/td-Tomato signal. Sorted cells from several mice (controls + genetic targeting) were mixed and processed as Paneth Pool (PP) with 10X Gene expression profiling technology with feature barcoding).

first: M

first: R

last: Joosten

first: A

last: Sacchetti

last: Fodde


title: DNAJC9 prevents CENP-A mislocalization and chromosomal instability by maintaining the fidelity of H3-H4 supply chains

city: Bethesda

first: Vinutha

last: Balachandra

address: 10 Center Drive

department: National Cancer Institute, Genetics Branch

institute: National Institute for Health

summary: The centromeric histone H3 variant CENP-A is overexpressed in many cancers. Mislocalization of CENP-A to non-centromeric regions contributes to chromosomal instability (CIN), a hallmark of cancer. Despite these observations, pathways that promote and prevent CENP-A mislocalization remain poorly defined. Here, we performed a genome-wide RNAi screen to identify regulators of CENP-A localization. We identified DNAJC9, a J-domain protein as a lead candidate from the screen and showed that cells depleted for DNAJC9 exhibit mislocalization of CENP-A, enrichment of CENP-A in chromatin, and CIN phenotypes. Global interactome analysis showed an enhanced interaction of CENP-A with the replication-associated H3-H4 chaperone MCM2 in DNAJC9-depleted cells and co-depletion of MCM2 and DNAJC9 suppressed CENP-A mislocalization. Furthermore, we showed that cells ablated for the ability of DNAJC9 to promote the proper folding of H3–H4, exhibit CENP-A mislocalization. CUT&RUN Sequencing analysis of genome-wide CENP-A occupancy in DNAJC9-depleted cells identified 16,603 sites of non-centromeric localization, that broadly overlapped with open chromatin regions. Our comprehensive analysis has identified factors that prevent mislocalization of CENP-A and has defined DNAJC9 as an important safeguard that prevents CENP-A mislocalization and CIN.

overall_design: CUT&RUN Sequencing of CENP-A in siNegative and siDNAJC9 transfected hTERT-RPE1-Tet-GFP-CENP-A cells treated with 1 ug/ml DOX for 48 hrs. Two biological repeats per condition were analysed with Mouse anti-CENP-A antibody and Mouse anti-IgG was used as control.

first: Roshan

last: Shrestha

first: Colin

last: Hammond

first: Shinjen

last: Lin

first: Samantha

last: Sevilla

first: Munira

last: Basrai


title: Activation of hepatocyte growth factor (HGF)/MET signaling as a mechanism of acquired resistance to a novel YAP1/TEAD small molecule inhibitor.

city: Boston

first: Eric

last: Muise

address: 33 Ave. Louis Pasteur

department: GPGX

institute: Merck

summary: Many tumor types harbor alterations in the Hippo pathway, including mesothelioma, where a high percentage of cases are considered YAP1/TEAD dependent. Identification of autopalmitoylation sites in the hydrophobic palmitate pocket of TEADs, which may be necessary for YAP1 protein interactions, has enabled modern drug discovery platforms to generate compounds that allosterically inhibit YAP1/TEAD complex formation and transcriptional activity. We report the discovery and characterization of a novel YAP1/TEAD inhibitor MRK-A from an aryl ether chemical series demonstrating potent and specific inhibition of YAP1/TEAD activity. In vivo, MRK-A showed a favorable tolerability profile in mice and demonstrated pharmacokinetics suitable for twice daily (BID) oral dosing in preclinical efficacy studies. Importantly, monotherapeutic targeting of YAP1/TEAD in preclinical models generated regressions in a mesothelioma CDX model; however, rapid resistance to therapy was observed. RNA-sequencing of resistant tumors revealed mRNA expression changes correlated with the resistance state and a marked increase of hepatocyte growth factor (HGF) expression. In vitro, exogenous HGF was able to fully rescue cytostasis induced by MRK-A in mesothelioma cell lines. Additionally, co-administration of small molecule inhibitors of the MET receptor tyrosine kinase suppressed the resistance generating effect of HGF on MRK-A induced growth inhibition. In this work, we report the structure and characterization of MRK-A demonstrating potent and specific inhibition of YAP1/TAZ-TEAD mediated transcriptional responses, with potential implications for treating malignancies driven by altered Hippo signaling, including factors resulting in acquired drug resistance.

overall_design: RNA from two cell lines grown in vitro and treated with either vehicle or MRK-A for either 6 hr or 48 hr were sequenced. In addition, RNA from tumors grown in vivo in NSG mice following treatment with either Vehicle or MRK-A for either 5 days, or until the tumors reached 500 mm3. For the 500mm3 samples, MRK-A was dosed either for only 5 days, or continuously until 500mm3.

first: Casey

last: Moure

first: Evan

last: Barry


title: Vitamin A-treated NK cells reduce IFN-γ production and support regulatory T cell differentiation

city: Mannheim

first: Carolina

last: Delatorre

address: Theodor-Kutzer-Ufer 1-3

institute: University Heidelberg

summary: Vitamin A induces changes in NK cell transcriptome, phenotype and function

overall_design: NK cells were sorted from spleen, and treated for 7 days with all-trans retinoic acid or equvalent volume of DMSO (solvent control), in presence of IL-2. Three independent experiments were performed.

first: Mingeum

last: Jeong

first: Ana

last: Stojanovic


title: Circulating miRNA profile and coronary calcification in asymptomatic patients with familial hypercholesterolemia.

first: Lina

last: Badimon

summary:
Affymetrix microarray (GeneChip microRNA 4.0) profilling of circulating miRNAs.
We used miRNA arrays to profile miRNAs isolated from plasma from familial hypercholesterolemia patients with (FH-W_CCS) or without (FH-WO_CCS) coronary calcium score by computed tomographic angiography

overall_design: We isolated circulating miRNAs from plasma samples of familial hypercholesterolemia patients and profiled them to determine miRNA signature.

first: Rafael

last: Escate

first: Sandra

last: Camino

first: Teresa

last: Padró


title: Proteus mirabilis UreR Coordinates Cellular Functions required for Urease Activity

status: Public on Mar 25 2024

city: Ann Arbor

first: Madison

last: Fitzgerald

state: MI

address: 1150 W. Medical Center Drive

department: Microbiology & Immunology

institute: University of Michigan

summary: Catheter-associated urinary tract infections (CAUTIs) account for over 30% of acute nosocomial infections in the U.S. and generate $340 million in healthcare costs annually. A major causative agent of CAUTIs is Proteus mirabilis, an understudied Gram-negative pathogen noted for its ability to form urinary stones via the activity of urease. Urease mutants cannot induce stones and are attenuated in a murine UTI model, indicating this enzyme is essential to P. mirabilis pathogenesis. The ability to induce urinary stone formation requires an active urease, a nickel metalloenzyme that hydrolyzes urea. This reaction produces ammonia as a byproduct, which can serve as a nitrogen source and weak base that raises the local pH. The resulting alkalinity induces the precipitation of polyvalent cations and anions to form stones. Expression of urease genes is activated by transcriptional regulator UreR in a urea-dependent manner. Thus, urease genes are highly expressed in the urinary tract where urea is abundant (~400 mM in human urine). Production of mature urease also requires the import of nickel into the cytoplasm and its incorporation into the urease apoenzyme. Urease accessory proteins primarily acquire nickel from the Ynt transporter and facilitate the incorporation of nickel to form mature urease. P. mirabilis encodes a second, low-affinity transport system (Nik) that can provide nickel when this metal is abundant. In this study, we identified UreR as the first defined regulator of nickel transport in P. mirabilis. We also offer evidence for direct regulation of the ynt promoter by UreR. Using bioinformatics, we identified UreR-regulated urease loci in 15 Morganellaceae family species across three genera. Additionally, we located two mobilized UreR-regulated urease loci that also encode the ynt transporter, implying that UreR regulation of ynt is a conserved regulatory relationship. Our study demonstrates that UreR regulates all genes required to produce mature urease, an essential virulence factor for P. mirabilis uropathogenesis.

overall_design: These RNA-seq data measure gene expression in P. mirabilis wildtype HI4320, ureC::bla, and ureR::aphA during growth in either buffered LB medium or buffered LB medium supplemented with 50 mM urea 15 minutes prior to sample collection. There are three biological replicates per construct for each condition

first: Melanie

last: Pearson

first: Harry

middle: T

last: Mobley


title: Improved RNA stability estimation through Bayesian modeling reveals most bacterial transcripts have sub-minute half-lives [RIF-seq]

city: Würzburg

first: Laura

last: Jenniches

address: Josef-Schneider-Straße 2/D15

institute: Helmholtz Institute for RNA-based Infection Research

summary: RNA decay is a crucial mechanism for regulating gene expression in response to environmental stresses. In bacteria, RNA-binding proteins (RBPs) are known to be involved in post-transcriptional regulation, but their global impact on RNA half-lives has not been extensively studied. To shed light on the role of the major RBPs ProQ and CspC/E in maintaining RNA stability, we performed RNA sequencing of Salmonella enterica over a time course following treatment with the transcription initiation inhibitor rifampicin (RIF-seq) in the presence and absence of these RBPs. We developed a hierarchical Bayesian model that corrects for confounding factors in rifampicin RNA stability assays and enables us to identify differentially decaying transcripts transcriptome-wide. Our analysis revealed that the median RNA half-life in Salmonella in early stationary phase is less than 1 minute, a third of previous estimates. We found that over half of the 500 most long-lived transcripts are bound by at least one major RBP, suggesting a general role for RBPs in shaping the transcriptome. Integrating differential stability estimates with CLIP-seq revealed that approximately 30% of transcripts with ProQ binding sites and more than 40% with CspC/E binding sites in coding or 3' untranslated regions decay differentially in the absence of the respective RBP. Analysis of differentially destabilized transcripts identified a role for both proteins in the control of respiration, and for ProQ in the oxidative stress response. Our findings provide new insights into post-transcriptional regulation by ProQ and CspC/E, and the importance of RBPs in regulating gene expression.

sample_organism: Salmonella enterica subsp. enterica serovar Typhimurium

overall_design:
Sample 1-12: CLIP-seq, Sample 13-132 RIF-seq
RNA sequencing of Salmonella (SL1344) in early stationary phase over a time course following treatment with the transcription initiation inhibitor rifampicin (RIF-seq)

first: Charlotte

last: Michaux

first: Linda

last: Popella

first: Sarah

last: Reichardt

first: Jörg

last: Vogel

first: Alexander

last: Westermann

first: Lars

last: Barquist


title: Gene expression profiling of MDA-MB-231 cells incubated with therapy induced senescence secretome

city: Singapore

first: Matius

last: Robert

state: Singapore

address: 28 Medical Drive

department: Physiology

country: Singapore

institute: National University of Singapore

summary: TNBC, associated with poor prognosis and high tumour recurrence, are often treated with anti-mitotic drugs. However, cells may bypass treatment-induced cell death via mitotic slippage, resulting in multinucleated polyploid cells and senescence activation. Senescent cancer cells represent a population of residual disease and are highly secretory. The SASP elicited is enriched in soluble cytokines linked to tumor recurrence and distant metastasis. In contrast, sEVs derived from senescent cancer cells represent an underappreciated aspect of SASP and its mechanistic role in mediating paracrine effects remains poorly-understood. Here, we show senescent sEVs as a distinct population of SASP that could elicit anti-tumor activity.

overall_design: To investigate the effect of TIS secretome, MDA-MB-231 breast cancer cells were incubated with either No CM (media only), or with CM collected from non-senescent (Cont CM) or from senescent MDA-MB-231 cells induced by 72h treatment of 100 ng/ml Nocodozole. Cells are incubated in CM for 48h prior to RNA extraction.

first: Karen

last: Crasta

first: Rekha

last: Jakhar