title: The kinase Rio1 and a ribosome collision-dependent decay pathway survey the integrity of 18S rRNA cleavage

city: Juptier

first: Katrin

last: Karbstein

state: FL

address: 120 Scripps Way

institute: The Herbert Wertheim UF Scripps Institute for Biomedical Innovation and Technology

summary: The 18S rRNA sequence is highly conserved, particularly at its 3’-end, which is formed by the endonuclease Nob1. How Nob1 identifies its target sequence is not known, and in vitro experiments have shown Nob1 to be error-prone. Moreover, the sequence around the 3’-end is degenerate with similar sites nearby. Here we used yeast genetics, biochemistry, and next generation sequencing to investigate a role for the ATPase Rio1 in monitoring the accuracy of the 18S rRNA 3’-end. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control step, but not in healthy cells with intact quality control mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weaker than its binding to accurately processed 18S rRNA. Accordingly, excess Rio1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA can translate, albeit more slowly, thereby inviting collisions with trailing ribosomes. These collisions result in degradation of the defective ribosomes utilizing parts of the machinery for mRNA quality control. Altogether, the data support a model in which Rio1 inspects the 3’-end of the nascent 18S rRNA to prevent miscleaved 18S rRNA-containing ribosomes from erroneously engaging in translation, where they induce ribosome collisions. The data also demonstrate how ribosome collisions purify cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.

overall_design: To examine the accuracy of 18S rRNA 3’-end formation in vivo, we performed 3’-RACE (rapid amplification of cDNA ends) sequencing on 18S rRNA and 25S rRNA to survey the 3'-ends of 18S rRNA and 25S rRNA. Saccharomyces cerevisaea strains were engineered and grown in conditions to deplete endogenous Pno1 and Dim1 or Rps14 and Dim1 protein expression, while expressing plasmid-derived Pno1 or Pno1-KKKF mutant proteins and Dim1 or Dim1-E85A mutant proteins, or plasmid-derived Rps14 and Dim1 proteins. 18S rRNA 3’-end formation was investigated using 8 biological replicates of yeast cells expressing plasmid-derived Pno1 and Dim1-E85A (wild type control), 8 biological replicates of yeast cells expressing plasmid-derived Pno1-KKKF and Dim1-E85A (ribosome assembly quality control bypass condition), and 2 biological replicates of yeast cells expressing plasmid-derived Rps14 and Dim1 (control for the effects of the Dim1-E85A mutant on 18S rRNA 3’-end miscleavage frequency). 25S rRNA 3’-end formation was investigated using 2 biological replicates of yeast cells expressing plasmid-derived Pno1 and Dim1-E85A and 2 biological replicates of yeast cells expressing plasmid-derived Pno1-KKKF and Dim1-E85A as controls to test whether bypass conditions specifically altered 18S rRNA miscleavage frequency. Yeast strains were grown to early stationary phase (between OD600 1.2-1.8) at 25˚C or 30˚C, 40S and 60S ribosomal subunits were purified, and 18S and 25S rRNAs were extracted for cDNA library preparation and 3’-RACE sequencing.

first: Melissa

last: Parker

first: Adam

last: Getzler


title: RNA sequencing in the ventral hippocampus

status: Public on Mar 03 2024

city: Shenzhen

first: songqiang

last: huang

address: Xili Academy Avenue

country: China

institute: Southern University of Science and Technology

type: Expression profiling by high throughput sequencing

overall_design: We performed RNA sequencing (RNA-seq) analyses on tissues from freshly isolated vHIP among CRS-WT,CRS-NKAα1+/-  and CRS-WT + DR5-12D mice


title: Gene expression profile at single cell level of stromal cells(SCs) from the periosteum and bone marrow

city: Shanghai

first: Yiming

last: Liu

state: Shanghai

address: yueyang road

institute: Sibcb

summary: Stromal cells in bone are considered as a heterogenous population that have different locations and functions, though all of them can self-renew and differentiate into osteoblasts. We used single cell RNA sequencing(scRNA-seq) to dissect the heterogeneity of Stromal cell in the bone.

overall_design: Stromal cell in bone marrow and bone periosteum were digested and isolated by Fluorescence-activated cell sorting (FACS). Then cells were analyzed using scRNA-seq.

first: Bo

middle: O

last: Zhou

middle: L

first: Xinyu

middle: T

last: Tang


title: KDM4 inhibition disrupts the core regulatory transcriptional circuitry in MYCN-driven neuroblastoma [ATAC-seq]

status: Public on Mar 04 2024

city: Memphis

first: Hongjian

last: Jin

state: TN

address: 262 Danny Thomas Place

department: Center for Applied Bioinformatics

country: USA

institute: St Jude Children's Research Hospital

type: Genome binding/occupancy profiling by high throughput sequencing

summary: A small set of interconnected lineage-specific transcription factors (TFs) form a core regulatory circuitry (CRC) that establishes and maintains cell identity and grants selective dependencies of distinct cancer types. However, effective therapies to targeting CRC TFs remain lacking. Here, we show that the best-in-class KDM4 inhibitor QC6352 has a potent anticancer activity in MYCN-driven high-risk neuroblastoma and significantly represses the CRC TFs including MYCN, HAND2, ASCL1, PHOX2B by disrupting the super-enhancers that dominate the expression of CRC TFs. Furthermore, we have developed a combination therapy by integrating QC6352 into cytotoxic chemotherapy, which leads to a complete response of MYCN amplified tumors and a better animal survival. This study reveals that targeting histone lysine demethylase 4 family may transform into a therapeutic strategy in clinic for cancers driven by CRC TFs.

overall_design: Examination of chromatin accessibility, histone modification post QC6352 treatment in BE2C.

first: Fang

last: Jie

first: Jin

last: Hongjian

first: Yang

last: Jun


title: KDM4 inhibition disrupts the core regulatory transcriptional circuitry in MYCN-driven neuroblastoma [ChIP-seq]


title: Chromatin accessibility and transcriptomic responses to HDAC inhibitors in TP53 mutated patient-derived colon cancer organoids [RNA-seq]

city: Basel

first: Emanuela

last: Kerschbamer

state: Basel Stadt

address: Mattenstrasse 28

department: Department of Biomedicine

country: Switzerland

institute: University of Basel

summary: Here we present the generation and characterization of patient-derived organoids (PDOs) from colorectal cancer patients. Two PDOs derived from patients with TP53 mutations were tested with two different HDACIs (SAHA and NKL54). Induction of cell death, transcriptome, and chromatin accessibility changes were analyzed. HDACIs promote the upregulation of low expressed genes and suppress the expression of highly expressed genes. A similar differential effect is also observed at the level of chromatin accessibility. Only SAHA is a potent inducer of cell death. The induction of apoptosis is characterized by the upregulation of BH3-only genes (especially BIK and BMF). Up-regulation of BIK is associated with increased accessibility in an intronic region. SAHA, but not NKL54, also causes downregulation of BCL2L1 and decreases chromatin accessibility in three distinct regions of the BCL2L1 locus. Both inhibitors upregulate the expression of innate immunity genes and members of the MHC family. In conclusion, our studies explain the mechanisms of action of SAHA and cast doubt on the use of NKL54 as a single agent for efficient apoptosis induction.

overall_design:
To investigate the effect of HDAC inhibitors on the survival and proliferation of PDOs derived from CRC patients, we performed RNA-seq experiments
We perfromed gene expression profiling analysis using data obtained from RNA-seq of 2 different CRC PDOs treated with DMSO (control), with SAHA (5µM) or with NKL54 (10µM) for 6 hours.  3 biological replicates for each condition were generated and analyzed.

first: Teresa

last: Gagliano

first: Umberto

last: Baccarani

first: Martina

last: Minisini

first: Eros

last: Di Giorgio

first: Emiliano

last: Dalla

first: Christian

middle: X

last: Weichenberger

first: Vittorio

last: Cherichi

first: Giovanni

last: Terrosu

first: Claudio

last: Brancolini


title: Phosphorylation of AGO2 by TBK1 Promotes the Formation of Oncogenic miRISC in NSCLC

first: Jianxiu

last: Yu

address: Room 202, Building 7, 280 South Chongqing Road,

department: Department of Biochemistry and Molecular Cell Biology

institute: Shanghai Jiao Tong University School of Medicine

summary:
Non-small-cell lung cancer (NSCLC) is a highly lethal tumor that often
develops resistance to targeted therapy. It is shown that Tank-binding kinase 1
(TBK1) phosphorylates AGO2 at S417 (pS417-AGO2), which promotes NSCLC
progression by increasing the formation of microRNA-induced silencing
complex (miRISC). High levels of pS417-AGO2 in clinical NSCLC specimens
are positively associated with poor prognosis. Interestingly, the treatment
with EGFR inhibitor Gefitinib can significantly induce pS417-AGO2, thereby
increasing the formation and activity of oncogenic miRISC, which may
contribute to NSCLC resistance to Gefitinib. Based on these, two therapeutic
strategies is developed. One is jointly to antagonize multiple oncogenic
miRNAs highly expressed in NSCLC and use TBK1 inhibitor Amlexanox
reducing the formation of oncogenic miRISC. Another approach is to combine
Gefitinib with Amlexanox to inhibit the progression of Gefitinib-resistant
NSCLC. This findings reveal a novel mechanism of oncogenic miRISC
regulation by TBK1-mediated pS417-AGO2 and suggest potential therapeutic
approaches for NSCLC.

overall_design: We generated stably expressing Flag-AGO2-WT or Flag-AGO2-S417A in H1299 cells, in which endogenous AGO2 was silenced by using the lentiviral shRNA system. AGO2-RNA immunoprecipitation sequencing (AGO2-RIP-seq) was performed to detect AGO2-bound miRNAs, and miRNA-seq and RNA-seq were used to detect the miRNA expression and mRNA expression profile in above stable cell lines.

first: Xian

last: Zhao


title: The TET-BMP regulatory axis in pathogenesis of CFM [RNA-Seq]

city: Kunming

first: Liangliang

last: Wang

state: Yunnan

address: South Section, East Outer Ring Road, Chenggong District

institute: Yunnan University

summary: Craniofacial microsomia (CFM) is a congenital defect that usually results from aberrant development of embryonic pharyngeal arches. However, the molecular basis of CFM pathogenesis is largely unknown. Here we employ zebrafish model to investigate the mechanism of CFM pathogenesis. In early embryos, tet2 and tet3 are highly expressed and are essential for pharyngeal cartilage development. Single-cell RNA sequencing and genetic analyses reveal that loss of Tet2/3 impaired chondrocyte differentiation largely due to insufficient BMP signaling. Mechanistically, Tet2/3-mediated 5-hydroxymethylcytosine modification allows the 5-hydroxymethylcytosine “reader”, Sall4, to specifically bind the bmp4 promoter, thereby promoting bmp4 expression and enabling efficient BMP signaling. These findings indicate the TET-BMP regulatory axis via 5-hydroxymethylcytosine to be critical for pharyngeal cartilage development. Whole-exome sequencing of CFM patient samples show that single nucleotide polymorphisms in TET and BMP pathway genes increase the risk of CFM. Collectively, our study provides novel insights into understanding craniofacial development and CFM pathogenesis.

overall_design: Total RNAs were purified using TRIzol reagent (Life Technologies, 343702). Libraries were constructed using a VAHTS Universal V8 RNA-seq Library Prep Kit for MGI (Vazyme) according to the manufacturer’s instructions and then sequenced on the MGISEQ-2000 platform.

first: Dahua

last: Chen

first: Wenxin

last: Zhang


title: The TET-BMP regulatory axis in pathogenesis of CFM [scRNA]

overall_design: Single-cell suspensions were processed for library construction using a Chromium Next GEM Single Cell 30 Reagent Kit v3 following the manufacturer’s instructions. Libraries were then sequenced on the Illumina Novaseq 6000 platform.


title: Mismatch repair protein MLH1 suppresses replicative stress in BRCA2 deficient breast tumors

city: Frederick

first: Yongmei

state: MD

address: Post Office Box B

department: Biomedical Informatics and Data Science (BIDS) Directorate

institute: Frederick National Labroatory for Cancer Research

summary:
To investigate the cooperative function KDM4D/NFIB/MLL1 complex in the regulation of Mirin Pretreatment, we established 10T1/2 cell lines in which each target gene has been knocked down by shRNA.
We then performed gene expression profiling analysis using data obtained from RNA-seq of 4 different cells at two time points.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for mESC cells and its KD derivatives (shKDM4D, shNFIB, and shMLL1).

first: Shyam

middle: K

last: Sharan

first: Satheesh

last: Sengodan


title: The TET-BMP regulatory axis in pathogenesis of CFM [hMeDIPseq]

type: Methylation profiling by high throughput sequencing

overall_design: DNA was purified from tissues according to the manufacturer’s instructions. 5hmC DNA immunoprecipitation (hMeDIP) was performed as previously described. Briefly, genomic DNA was digested with proteinase K at 55°C overnight and then with RNase A at 37°C for 1 h. Genomic DNA was purified by phenol/chloroform extraction and recovered by ethanol precipitation. Purified genomic DNA was sonicated to an average size of 200–400 bp and heat-denatured (95°C, 10 min). Then, 5 μg of fragmented genomic DNA was diluted in 500 μL DNA-IP buffer [10 mM sodium phosphate (pH 7.0), 140 mM NaCl, 0.05% Triton X-100] and pre-cleared with 50 μL protein A/G beads at 4°C for 3 h. Five percent of the pre-cleared genomic DNA was saved as an input sample. The DNA-IP sample was incubated with 5 μg 5hmC antibody (Active Motif, 39769) at 4°C overnight and then with 50 μL protein A/G beads for 4 h at 4°C. Three washes for a total of 30 min were then performed to remove non-specifically bound antibodies. The beads were then treated with 200 μL TE buffer (pH 8.0) plus proteinase K for at least 3 h at 1000 rpm, 55°C in a thermomixer. The DNA was then purified by phenol/chloroform extraction followed by ethanol precipitation. Purified DNA was dissolved in ultrapure H2O, and libraries were constructed using a VAHTS Universal DNA Library Prep Kit for MGI (Vazyme) according to the manufacturer’s instructions. Libraries were then sequenced on the MGISEQ-2000 platform.


title: Effect of depletion of METTL3 on gene expression of A549 cells

city: Guangzhou

first: Xia

last: Yang

address: No. 74, Zhongshan 2nd Road

institute: Zhongshan School of Medicine, Sun Yat-sen University

summary: To investigate the function of METTL3 in lung cancer, we established shMETTL3 A549 cell line.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for METTL3 knockdown and control.

first: Qiaorui

last: Bai


title: Impaired innate and adaptive immune responses to BNT162b2 SARSCoV-2 vaccination in systemic lupus erythematosus [CTD]

status: Public on Mar 02 2024

city: Stanford

first: Allan

last: Feng

state: CA

address: 269 Campus Dr

department: Imm/Rheum

institute: Stanford School of Medicine

summary: We measued IgG autoantibodies associated with Connective Tissue Diseases (CTDs) and Anti-Cytokine Antibodies (ACA) in SLE and healthy controls who received the BNT162b2 vaccine.

platform_organism:
Escherichia coli
Triticum aestivum
Spodoptera frugiperda
Homo sapiens
Sus scrofa
Bos taurus
Capra hircus

overall_design: We constructed protein arrays by  first coupling recombinant protein antigens to uniquely fluorescent carboxylated magnetic beads. We then combined the beads into two seperate arrays that were used to probe patient SLE serum samples, healthy control samples, and autoimmune prototype samples. A Luminex FLEXMAP 3D® system was used to quantitatively measure autoantibody reactivity in each sample in units of mean flourescent intensity (MFI).

first: Kavita

middle: Y

last: Sarin

first: Hong

last: Zheng

first: Yashaar

last: Chaichian

first: Prabhu

middle: S

last: Arunachalam

first: Gayathri

last: Swaminathan

first: Alec

last: Eschholz

first: Fei

last: Gao

first: Oliver

last: Wirz

first: Brandon

last: Lam

first: Emily

first: Lori

last: Lee

first: Matthew

middle: A

last: Lewis

first: Janice

last: Lin

first: Holden

last: Maecker

first: Scott

middle: D

last: Boyd

first: Mark

middle: M

last: Davis

first: Kari

middle: C

last: Nadeau

first: Bali

last: Pulendran

first: Purvesh

last: Khatri

first: Paul

middle: J

last: Utz

first: Lisa

last: Zaba


title: Impaired innate and adaptive immune responses to BNT162b2 SARSCoV-2 vaccination in systemic lupus erythematosus [ACA]

first: Allan

last: Feng

platform_organism:
Escherichia coli
Homo sapiens

overall_design: We constructed protein arrays by  first coupling recombinant protein antigens to uniquely fluorescent carboxylated magnetic beads. We then combined the beads into two separate arrays that were used to probe patient SLE serum samples, healthy control samples, and autoimmune prototype samples. A Luminex FLEXMAP 3D® system was used to quantitatively measure autoantibody reactivity in each sample in units of mean flourescent intensity (MFI).

first: Kavita

last: Sarin

first: Hong

last: Zheng

first: Yashaar

last: Chaichian

first: Prabhu

last: Arunachalam

first: Gayathri

last: Yang


title: 3D reconstruction of the mouse cochlea from scRNA-seq data suggests morphogen-based principles in apex-to-base specification

city: Ann Arbor

first: Joerg

last: Waldhaus

state: MI

address: Medical Sciences I Building, Rm. 5315A, 1150 West Medical Center Drive

department: Department of Otolaryngology–Head and Neck Surgery, Kresge Hearing Research Institute

institute: University of Michigan

summary: In the mammalian auditory system, frequency discrimination depends on numerous morphological and physiological properties of the organ of Corti that gradually change along the longitudinal (tonotopic) axis of the organ. For example, the basilar membrane stiffness changes tonotopically, thus affecting the tuning properties of individual hair cells. At the molecular level, those frequency-specific characteristics are mirrored by gene expression gradients; however, the molecular mechanisms controlling tonotopic gene expression in the mouse cochlea remain elusive. Through analyzing scRNA-seq data from two developmental time points, we predicted that morphogens, rather than a timing-associated mechanism, confer spatial identity in the cochlea. Subsequently, we reconstructed the developing cochlea in 3D space from scRNA-seq data to investigate the molecular pathways mediating tonotopic information. The retinoic acid and sonic hedgehog pathways were found to form opposing tonotopic gradients, and functional interrogation using mouse cochlear explants suggested that both pathways jointly specify the tonotopic axis during development.

overall_design: We investigated the transcriptional heterogeneity from the developing cochlear duct at embryonic day (E)12.5 and E14.5 via single-cell RNA sequencing (scRNA-seq). Inner ears samples were dissected from the Sox2-GFP mouse line and cochlea was separated from vestibular compartments. Cochlea samples were digested to single-cell level and subjected to fluorescence activated cell sorting of Sox2-GFP+ cells. For each time point, two independent libraries were generated using the 10x Genomics platform.

first: Shuze

last: Wang

first: Saikat

last: Chakraborty

first: Mary

middle: P

last: Lee


title: Hippo-YAP/TAZ signaling coordinates adipose plasticity and energy balance by uncoupling leptin expression from fat mass

city: Daejon city

first: Ju Gyeong

last: Kang

address: Daehak-Ro 291

department: Biological sciences

country: South Korea

institute: KAIST

summary: Adipose tissue contributes to systemic energy homeostasis by serving as an energy reservoir and endocrine organ. Coordinated regulation of these functions is essential for metabolic health, but the mechanisms that underlie cross-talk between them have been unknown. Here we show that the transcriptional coregulators YAP and TAZ of the Hippo signaling pathway in adipose tissue control systemic energy balance. Mechanistically, we identify a YAP/TAZ-TEAD axis that upregulates leptin expression by directly binding to an upstream enhancer site of the leptin gene.

overall_design: TAZ (WWTR1) ChIP-seq data of adipocytes differentiated from C3H10T1/2 cells. RNA-seq data obtained from mouse adipose tissue (iWAT)

first: Ju-Gyeong

first: Taejun

last: Seol

first: Sungwoo

last: Choi

first: Jae Myoung

last: Suh

first: Dae-Sik

last: Lim


title: Deleterious, protein-altering variants in the transcriptional coregulator ZMYM3 in 27 individuals with a neurodevelopmental delay phenotype [RNA-seq]

city: Huntsville

first: Brian

middle: Scott

last: Roberts

state: AL

address: 601 Genome Way

institute: HudsonAlpha Institute for Biotechnology

summary: Neurodevelopmental disorders (NDDs) result from highly penetrant variation in hundreds of different genes, some of which have not yet been identified. Using the MatchMaker Exchange, we assembled a cohort of 27 individuals with rare, protein-altering variation in the transcriptional coregulator gene ZMYM3, located on the X chromosome. Most (n=24) individuals were males, 17 of which have a maternally inherited variant and four of which have de novo variants. Overlapping features included developmental delay, intellectual disability, behavioral abnormalities, and a specific facial gestalt in a subset of males. Variants in almost all individuals (n=26) are missense, including six with variants that have arisen independently at the same codon at least two times. Four unrelated probands were identified with inherited variation at Arg441 (Arg441Trp or Arg441Gln), a site at which variation has been previously seen in NDD-affected siblings, and two individuals have de novo variation at Arg1294 (Arg1294Cys). All variants affect evolutionarily conserved sites, and most are predicted to damage protein structure or function. ZMYM3 is relatively intolerant to variation in the general population, is widely expressed across human tissues, and encodes a component of the KDM1A-RCOR1 chromatin-modifying complex. ChIP-seq experiments on one mutant, Arg1274Trp, indicate dramatically reduced genomic occupancy, supporting a hypomorphic effect. Similarly, RNA-seq comparing mutant cells to reference revealed numerous expression changes. Such changes were not seen in edited cells harboring a likely benign variant, Arg688His. While we are unable to perform statistical evaluations to support a conclusive causative role for variation in ZMYM3 in disease, the totality of the evidence, including 27 affected individuals, recurrent variation affecting at least two codons, overlapping phenotypic features, protein-modeling data, evolutionary constraint, and experimentally-confirmed functional effects strongly support ZMYM3 as a novel NDD gene.

overall_design: HepG2 cells were edited by CRISPR/Cas9 to add a FLAG epitope tag to ZMYM3 and to introduce putatively deleterious variants or reference controls. Cells were cultured and 3’ RNA-seq was performed in triplicates.

first: Susan

last: Hiatt

first: Gregory

last: Cooper

first: E

last: Partridge

first: Ivan

last: Rodriguez-Nunez


title: Single-cell sequencing analysis of spinal cord motor neuron organoids (scMN-Organs)

status: Public on Mar 01 2024

city: Beijing

first: sun

last: zheng

address: NO.1 West Beichen Road,Chaoyang District

institute: Institute of Genetics and Developmental Biology, Chinese Academy of Sciences

summary:
Neonatal spinal cord tissues exhibit remarkable regenerative capabilities as compared to adult spinal cord tissues
after injury, but the role of extracellular matrix (ECM) in this process has remained elusive. Here, we found
that early developmental spinal cord had higher levels of ECMproteins associated with neural development and
axon growth, but fewer inhibitory proteoglycans, compared to those of adult spinal cord. Decellularized spinal
cord ECM from neonatal (DNSCM) and adult (DASCM) rabbits preserved these differences. DNSCM promoted
proliferation, migration, and neuronal differentiation of neural progenitor cells (NPCs) and facilitated axonal
outgrowth and regeneration of spinal cord organoids more effectively than DASCM. Pleiotrophin (PTN) and Tenascin
(TNC) inDNSCMwere identified as contributors tothese abilities. Furthermore,DNSCMdemonstrated superior
performance as a delivery vehicle forNPCs and organoids in spinal cord injury (SCI)models. This suggests
that ECMcues from early development stages might significantly contribute to the prominent regeneration ability
in spinal cord.

overall_design: To analyze the cell subtypes and molecular expression profiles contained in spinal motor neuron organoids constructed in DNSCM and DASCM, respectively, we performed single-cell sequencing of scMN-Organs cultured to day 28.

first: Zheng

last: Sun

first: Man

last: Yin

first: Jianwu

last: Dai

first: Yannan

last: Zhao


title: KDM4 inhibition disrupts the core regulatory transcriptional circuitry in MYCN-driven neuroblastoma [RNA-seq]

first: Hongjian

last: Jin

overall_design: Examination of mRNA profiles post QC6352 treatment in BE2C, KELLY, SIMA, SKNAS, SKNSH and SKNFI.

first: Abuzaid

last: Ahmed

first: Fang

last: Jie

first: Jin

last: Hongjian

first: Yang

last: Jun


title: Identifying new markers of angiogenic sprouting using transcriptomics: New role for RND3

city: Bryan

first: Colette

middle: Ann

last: Abbey

state: TX

address: 8447 Riverside Pkwy

department: Medical Physiology

institute: Texas A&M Health

summary: To determine, using HUVEC in a 3D collagen model, gene expression in invading endothelial compared to non-invading at the single-cell level.

overall_design: 2 individual HUVEC donors were collected for each experimental condition.  Total, non-invading and invading HUVEC were collected from a 3D collagen invasion assay for single-cell sequencing.

first: Kayla

last: Bayless

first: Ashley

last: Coffell


title: Elucidation of genes enhancing natural product biosynthesis through co-evolution analysis

first: Xinran

last: Wang

state: Guangdong

address: No.1068 Xueyuan Avenue

department: Center for Synthetic Biochemistry

institute: Shenzhen Institute of Advanced Technology, Chinese Acadamy of Science

summary: Streptomyces has the largest repertoire of natural product biosynthetic gene clusters (BGCs), yet developing a universal engineering strategy for each Streptomyces species is challenging. Given that some Streptomyces species have larger BGC repertoires than others, we hypothesized that a set of genes co-evolved with BGCs to support biosynthetic proficiency must exist in those strains, and that their identification may provide universal strategies to improve the productivity of other strains. We show here that genes co-evolved with natural product BGCs in Streptomyces can be identified by phylogenomics analysis. Among the 597 genes that co-evolved with polyketide BGCs, 11 genes in the “coenzyme” category have been examined, including a gene cluster encoding for the co-factor pyrroloquinoline quinone (PQQ). When the pqq gene cluster was engineered into 11 Streptomyces strains, it enhanced production of 16,385 metabolites, including 36 known natural products with up to 40-fold improvement and several activated silent gene clusters. This study provides a new engineering strategy for improving polyketide production and discovering new biosynthetic gene clusters.

sample_organism:
Streptomyces griseus
Streptomyces rapamycinicus

overall_design: To test the effect of pyrroloquinoline quinone gene operon on the expression of biosynthetic gene clusters, comparative RNA-seq of Streptomyces griseus and Streptomyces rapamycinicus with and without PQQ operon were performed.

first: Xiaozhuo

last: Luo

first: Jay

last: Keasling


title: KDM4 inhibition disrupts the core regulatory transcriptional circuitry in MYCN-driven neuroblastoma [Cut&Tag]

overall_design: Examination of chromatin accessibility, histone modification post QC6352 treatment in BE2C.


title: Harnessing developmental dynamics of spinal cord extracellular matrix improves regenerative potential of spinal cord organoids

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design: To investigate the different effects of DNSCM and DASCM on the cell fate differentiation of spinal cord neural precursor cells (scNPC),  we cultured scNPC in DNSCM and DASCM. RNA-seq was performed after 7 and 14 days of 3D culture for differential analysis of gene expression. In addition, RNA-seq and differential analysis were performed on spinal motor neuron organoids(scMN-Organs) cultured in DNS CM, DASCM and Matrigel for 21 days.

first: jianwu

first: yannan


title: Root growth in Orchid Dendrobium cv. Sonia requires shade avoidance response of PHYB along with regulation of auxin pathway genes

city: Khurda

first: Kishore

middle: Cs

last: Panigrahi

state: Odisha

address: NISER, P.O Jatni

department: School of Biological Sciences

country: India

institute: National Institute of Science Education and Research

summary: Orchid Dendrobium Sonia (Sonia) has wide demand in floral market throughout the world due to its vibrant coloured flowers, flowering recurrence and dense inflorescence. Effects of different monochromatic light qualities (red, far-red, blue, white) on different developmental and growth responses such as seed gemination, shoot and root growth, chlorophyll and carotenoid accumulation were studied in Sonia. Asymbiotic seed germination was the highest under blue or white light (80%, p<0.001) and least under far-red (55%, p<0.001) suggesting that it could either be controlled by an exceptional novel function of Cryptochrome 1 or the blue wavelengths perceived by PHYA or PHYB in Sonia. All analyses were done in 6-months-old plants till 1 year of age. Shoot length increased significantly in white or red light (3.7-fold, 3.5-fold respectively) while was least under far-red (decreased by 1.6-fold) indicating the major regulatory role of PHYB for shoot growth. Root growth in red in Sonia was more similar to hypocotyl adventitious root (HAR) formation genetically considering PHYB transcript expression. We suggest that the root growth (HAR) under red in Sonia is partly attributed to PHYB, negatively regulated with HY5 transcription factor and positively associated with auxin biosynthesis and accumulation gene BABY BOOM (BBM2) and efflux carriers such as LIKE AUXIN RESISTANT 2 and 3 (LAX2, LAX3). Highest chlorophyll content under far-red and blue might be a hyper-response of SAR under FR light in Sonia. Taxonomic tree analysis finds Sonia closer to Phalenopsis and Dendrobium catenatum Lindl. The three phytochromes and one cryptochrome sequences which were similar to PHYA, B, C and CRY1 respectively. CRY1 was retrieved with one more isoform CRY1_X2, with all these sequences more similar to those of Oryza.sativa. Further study can clarify the indicating reason of a probable gene loss which is evident from the absence of any sequence similar to CRY2 in root RNA isolates of Dendrobium Sonia.

overall_design: De novo transcirptome assembly of Dendrobium Sonia plants grown for 6 months under white light. Root sample from plants in pre-flowering stage were used for Total RNA isolation.

first: Durga

last: Biswal

first: Brajabandhu

last: Pradhan

first: Sulagna

last: Jena

first: Sony

last: Kumari

first: Soumya

last: Rao

first: Nihar

middle: R

last: Nayak

first: Madhusmita

last: Panigrahy


title: Dux promotes metabolism-lactylation-MET cascaded network during early iPSC reprogramming with Brg1 as the histone lactylation reader [RNA-Seq]

city: Harbin

first: Xinglin

last: Hu

state: Heilongjiang

address: Harbin Medical University, 194 Xuefu Rd, Nangang District, Harbin, Heilongjiang Province, P.R. China 150081

department: Histology and Embryology

institute: Harbin Medical University

overall_design: Comparative gene expression profiling analysis of  RNA-seq data for MEF, D4, D4-OE-DUX, and iPSC .

first: Xingwei

last: Huang

first: Lei

last: Lei


title: METTL16 deficiency attenuates apoptosis through translational control of extrinsic death receptor during nutrient deprivation

city: nanjing

first: Zhang

last: Jun

state: jiangsu

address: longmiandadao

institute: China Pharmaceutical University

summary: METTL16 is a well-characterized m6A methyltransferase that has been reported to contribute to tumorigenesis in various types of cancer. However, the effect of METTL16 on tumor progression under restricted nutrient conditions, which commonly occur in tumor microenvironment, has yet to be elucidated. Herein, our study initially reported the inhibitory effect of METTL16 depletion on apoptosis under amino acid starvation conditions.

overall_design: To investigate the potential role of METTL16 under nutrient deprivation, stable METTL16-knockdown (M16-KD) cell lines were initially constructed using classic A549 NSCLC cells, through a shRNA-based gene interference approach.

first: Qiujie

last: Li

first: Jun

last: Zhang


title: Role of the histone variant H2A.Z.1 in memory, transcription, and alternative splicing is mediated by lysine modification

city: Mississauga

first: Iva

last: Zovkic

state: Ontario

address: 3359 Mississauga Rd

department: Department of Psychology

country: Canada

institute: University of Toronto

summary: Creating long-lasting memories relies on learning-induced changes in gene expression, which are regulated by epigenetic modifications of DNA and associated histone proteins. Post-translational modifications (PTMs) of histone proteins are key regulators of transcription, with different PTMs producing unique effects on gene activity and behavior. Although recent studies began to investigate histone variants as key regulators of memory formation, PTMs are rarely considered in relation to their function.  Here, we analyze the role of acetylation of histone variant H2A.Z.1 in memory and gene expression.  To this end, we developed AAV constructs to overexpress mutated H2A.Z.1 isoforms that either mimic acetylation (acetyl-mimetic) by replacing lysines 4, 7 and 11 with glutamine (KQ), or that have impaired acetylation (acetyl-defective) by replacing the same lysine with alanine (KA).  We found that overexpression of the acetyl-mimetic (H2A.Z.1KQ) or the acetyl-defective (H2A.Z.1KA) isoforms of H2A.Z.1 in the hippocampus produces different effects on memory depending on strength of the task and sex, with H2A.Z.1KQ generally having a beneficial effect on memory, while H2A.Z.1KA results in impaired memory. We further analyzed gene expression data and found that H2A.Z.1KQ and H2A.Z.1KA uniquely impact the expression of different classes of genes in females and males. However, different genes are regulated by different isoforms in the 2 sexes. Finally, we describe, for the first time, that H2A.Z is involved in the alternative splicing of neuronal genes. Notably, H2A.Z depletion and its replacement with different isoforms influence transcription and splicing of different genes, suggesting that H2A.Z.1 can regulate genes through splicing and expression levels.  This is the first study demonstrating that direct manipulation of the levels of AcH2A.Z regulates memory, gene expression and splicing. Overall, this study adds another layer of complexity to the contribution of histone variants to higher brain functions, consolidating H2A.Z as a key regulator of memory.

overall_design: To investigate the effect of  knocking down, and of acetylation on the histone variant H2A.Z, we injected AAVs expressing Cre into the hippocampus of H2A.Z.1/H2A.Z.2 floxed mice to konck down endogenous H2A.Z.1/H2A.Z.2 and expressing our H2A.Z.1 mutants, acetyl mimic, acetyl block or a wildtype overexpression, or overexpressing Myc as the control.


title: Modeling T cell temporal response to cancer immunotherapy rationalize development of combinatorial treatments protocols

city: Rehovot

first: Ido

last: Amit

address: 234 Herzl st.

department: Immunology

country: Israel

institute: Weizmann Institute of Science

summary: Successful immunotherapy relies on triggering complex responses involving T-cells dynamics in tumors and the periphery. Characterizing these responses remains challenging using static human single-cell atlases or mouse models. To address this, we developed a framework for in vivo tracking of tumor-specific CD8+ T cells over time and at single-cell resolution. Our tools facilitate the modeling of gene program dynamics in the tumor microenvironment (TME) and tumor-draining lymph node (tdLN). Using this approach, we characterize two modes of anti-PD1 (aPD1) activity, decoupling induced differentiation of tumor-specific activated precursor cells from cDC1-dependent proliferation and recruitment to the TME. We demonstrate that combining aPD1 with anti-4-1BB agonist enhances the recruitment and proliferation of activated precursors resulting in tumor control. These data suggest that effective response to aPD1 therapy is dependent on sufficient influx of activated precursor CD8+ cells to the TME, and highlight the importance of understanding system-level dynamics in optimizing immunotherapies.

overall_design: Dissecting the temporal dynamics of CD8+ T cell in the tumor microenvironment using single-cell RNA sequencing. Mice were inoculated with different tumor cell lines (B16-OVA,MC38 and LLC) cells and treated with Anti-PD1, Anti-41BB, or both.OT1-CD8+,Bystanders CD8+, and endogenous CD8+ T Cells were harvested from tumors, tdLN, and spleen at different time points and underwent scRNAseq. We also utilized XCR1-DTR mice in various settings. Ex vivo incubation of CD8+ with tumor cell line cells was performed.

first: Oren

last: Barboy

first: Akhiad

last: Bercovich

first: Hanjie

first: Yaniv

last: Eyal-Lubling

first: Adam

last: Yalin

first: Yuval

last: Shapir-Itai

first: Kathleen

last: Abadie

first: Mor

last: Zada

first: Eyal

last: David

first: Shir

last: Shlomi-Loubaton

first: Yonatan

last: Katzenelenbogen

first: Diego

last: Jaitin

first: Chamutal

last: Gur

last: Yofe

first: Tali

last: Feferman

first: Merav

last: Cohen

first: Rony

last: Dahan

first: Evan

middle: W

last: Newell

first: Aviezer

last: Lifshitz

first: Amos

last: Tanay


title: SPARC boost cholesterol-dependent aggressive phenotype and sorafenib resistance in hepatocellular carcinoma

city: Suzhou

first: Shan

last: Wan

address: Renai Road 199

department: Pathology

institute: Soochow University

summary: Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is characterized as a highly aggressive tumor entity and has become a health challenge worldwide. Intracellular secreted protein acidic and rich in cysteine (SPARC) has been described as secreted protein that serves as paracrine mediator between cells extracellular matrix (ECM), however, its intracellular role remains unclear. Genetically modified HCC cell lines, cancer patient-derived organoids, and mouse models were used to analyze SPARC on metabolic processes, as well as the invasive behavior and sorafenib resistance of HCC cells. Transcriptome, interactome, and biochemical analyses were performed to study how SPARC regulate cholesterol homeostasis. High expression of intracellular SPARC was significantly associated with elevated cholesterol levels and an enhanced invasive phenotype in HCC. Our findings unveil a previously unrecognized interplay between SPARC and cholesterol homeostasis. Targeting SPARC-triggered cholesterol-dependent oncogenic signaling serves as a promising strategy for treating advanced HCC.

overall_design: To investigate the impact of SPARC overexpression on cholesterol metabolism and PI3K-AKT signaling in HCC cell lines and tumor tissues. HepG2 cells stably expressing SPARC were generated by lentiviral vector system. HepG2 cells transfected with empty vector served as a negative control. Three biological replications were performed to assess the transcriptome in SPARC-overexpression cells relative to the control HepG2 cells.

first: Hua

last: Wu


title: Telomeric Chromosomal Instability Fosters Paracrine Mediated Cell Competition to Initiate Intestinal Cancer

city: Houston

first: Jiexi

last: Li

address: 1881 East Road

institute: The University of Texas MD Anderson Cancer Center

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

overall_design: Refer to individual Series


title: Telomeric Chromosomal Instability Fosters Paracrine Mediated Cell Competition to Initiate Intestinal Cancer [bulk RNA-seq]

summary: Telomere dysfunction drives chromosomal instability (CIN) during the transition from benign adenoma to malignant adenocarcinoma. While CIN provides a mutator mechanism for cancer- relevant genomic events, its role in shaping tumor biology during carcinogenesis is not well understood. Here, we explored the molecular and biological impact of telomere dysfunction and associated CIN in vivo in a faithful model of CRC. In vivo lineage tracing revealed that CIN increased the rate of neoplastic cell clonal expansion through accelerated differentiation of neighboring stem cells, resulting in increased number of adenomas and decreased survival in CIN-high Apcmin mice. Mechanistically, CIN represses EZH2 leading to upregulation of secreted Wnt antagonists, which resulted in a growth advantage to CIN-high neoplastic cells. Correspondingly, pharmacological activation of intrinsic WNT signaling enhanced intestinal stem cells fitness, leading to reduced neoplastic cell clonal expansion and adenoma burden. Thus, the CIN-EZH2-WNT axis enhances intestinal cancer initiation in the nascent tumor microenvironment, providing a preventive strategy for patients harboring germline APC mutations.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for CIN-low and CIN-high Apcmin colonoids.

first: Kyle

last: LaBella


title: Comparison of mRNA expression between naive CD4+ T (TN) cells treated or not treated with IL-1b

city: Ichikawa

first: Takashi

last: Sekiya

state: Chiba

address: 1-7-1 Kohnodai

country: Japan

institute: National Center for Global Health and Medicine

summary: To analyze effects of IL-1b on TN cells, RNA-seq analysis was performed on freshly prepared TN cells (0 h) and TN cells cultured with or without recombinamt IL-1b.

overall_design: Comparative gene exprssion profiling analysis of RNA-seq data for freshly isolated TN cells and TN cells cultured in the presence or absence of recombinant IL-1b (10 ng/mL) in vitro for 20 h.

first: Sekiya

last: Takashi


title: Comparison of mRNA expression between spleen and mesenteric lymph node (mLN) naive CD4+ T (TN) cells

summary: To analyze difference between spleen and mLN TN cells, RNA-seq analysis was performed on TN cells freshly prepared from each organ.

overall_design: Comparative gene exprssion profiling analysis of RNA-seq data for total TN cells freshly  isolated from speen or mLN.


title: Elucidation of the effects of tonic TCR stimulation and tissue environments of the  chromatin status of naive CD4 T (TN) cells

summary: Naive CD4 T (TN) cells show some degree of heterogeneity. However, mechanisms that alter TN cells are still largely unknown, and  physiological importance in the phenotypic alteration of TN cells is unclear. Thus, to reveal the mechanisms that alter TN cell characteristics, we performed transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) on TN cells from different secondary lymphoid organs with different strength of tonic TCR stimulation.

overall_design: Transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) on TN cells with Nr4a1-dsGFP low and high from three different secondary lymphoid organs (spleen, mesenteric lymph node, axillary lymph node).


title: Telomeric Chromosomal Instability Fosters Paracrine Mediated Cell Competition to Initiate Intestinal Cancer [CUT&RUN]

overall_design: Profiling of EZH2 binding and H3K27me3 modifications in CIN-low and CIN-high Apcmin colonoids.


title: Transcriptome profiling of the hepatic acute-phase response in LPS-treated dHepaRG cells.

city: Jena

first: Charlotte

middle: Sophie

last: Ehle

address: Hans-Knoell Str. 2

department: Institute of Biochemistry and Biophysics

country: Germany

institute: Friedrich-Schiller-University

summary: HepaRG cells are a human hepatic cell line that upon differentiation (dHepaRG) provide many features of primary hepatocytes, including high metabolic activity, detoxification, bile acid and urea synthesis. They are also able to undergo the acute-phase response upon inflammatory stimulation - the massive upregulation of serum proteins which support the systemic immune response. The aim of this study was to investigate the transcriptomic reprogramming of dHepaRG cells upon Lipopolysaccharide (LPS) treatment. LPS potently activates immune responses in dHepaRG cells by inducing autocrine IL6-signalling and the expression of acute-phase genes. However, it is known that homeostatic liver functions are downregulated consecutively. The RNA-Seq data should deliver an overview about affected pathways and factors which are involved in this reprogramming process.

overall_design: Comparative gene expression profiling of dHepaRG cells treated with LPS for 0 h, 6 h or 24 h.

first: Aishwarya

last: Iyer-Bierhoff

first: Yunchen

first: Shaojun

last: Xing

first: Maren

last: Godmann

first: Thorsten

last: Heinzel


title: RNA-seq analsis of siRNA knockdown of cadherin-11 (CDH11) gene in human intestinal myofibroblasts (HIMFs)

city: Cleveland

first: Pranab

last: Mukherjee

state: OH

address: 9500 Euclid Avenue

department: Infection and Immunity

institute: Cleveland Clinic

summary: The goal of this experiment was to determine the effect of cadherin-11 (CDH11) knockdown on transcriptional profile of HIMFs

overall_design: RNA was isolated, cDNA libraries (paired end) were made at the University of Chicago Genomics Facility, and sequenced on an Illumina HiSeq4000, in four replicates, according to standard protocols

first: Jiannan

first: Florian

last: Rieder


title: Impact of NAA50 depletion on the transcriptome of rosette leaves from the reference plant Arabidopsis thaliana (Col-0).

city: Mannheim

first: Carolina

last: Delatorre

address: Theodor-Kutzer-Ufer 1-3

institute: University Heidelberg

summary: In humans and plants, 40 % of the proteome is co-translationally acetylated at the N-terminus by a single Nα-acetyltransferase (Nat) termed NatA. The core NatA complex consists of the catalytic subunit NAA10 and the ribosome-anchoring subunit NAA15. The regulatory subunit HYPK and the acetyltransferase NAA50 join this complex in humans. Even though both are conserved in Arabidopsis thaliana, only AtHYPK is known to interact with AtNatA. Here we aimed to determine the global transcriptome change in the reference plant Arabidopsis thaliana leaves upon depletion of the acetyltransferase NAA50 (AT5G11340). The wild type and the amiNAA50 #13 mutant were grown on soil for seven weeks on soil under short-day conditions (8.5 h of light, 70-100 μE light photon flux density, 24 °C/18 °C day/night temperature, and 50-60 % humidity) and RNA was extracted with the Universal RNA Kit (Roboclon) according to the manufacturer´s instructions.

overall_design: Comparison of two genotypes with 4 replicates

first: Markus

last: Wirtz

first: Laura

last: Armbruster


title: Transcriptomic Profiling of Plasma Extracellular Vesicles Enables Reliable Annotation of the Cancer-specific Transcriptome and Molecular Subtype (plasma_healthy)

status: Public on Feb 29 2024

city: New York

first: Anirban

last: Maitra

state: NY

address: Smilow 910, 522 1st Ave

department: Perlmutter Cancer Center

institute: New York University Grossman School of Medicine

summary: Longitudinal monitoring of patients with advanced cancers is crucial to evaluate both disease burden and treatment response. Current liquid biopsy approaches mostly rely on the detection of DNA-based biomarkers. However, plasma RNA analysis can unleash tremendous opportunities for tumor state interrogation and molecular subtyping. Through the application of deep learning algorithms to the deconvolved transcriptomes of RNA within plasma extracellular vesicles (evRNA), we successfully predict consensus molecular subtypes in metastatic colorectal cancer patients. We further demonstrate the ability to monitor changes in transcriptomic subtype under treatment selection pressure and identify molecular pathways in evRNA associated with recurrence. Our approach also identified expressed gene fusions and neoepitopes from evRNA. These results demonstrate the feasibility of transcriptomic-based liquid biopsy platforms for precision oncology approaches, spanning from the longitudinal monitoring of tumor subtype changes to identification of expressed fusions and neoantigens as cancer-specific therapeutic targets, sans the need for tissue-based sampling.

overall_design: To investigate the potential of utilizing RNA isolated from plasma extracellular vesicles for the longitudinal monitoring of patients with metastatuc colorectal cancers

first: Kimal

middle: I

last: Rajapakshe

first: Paola

last: Guerrero

first: Vahid

last: Bahrambeigi

first: Jaewon

last: Lee


title: RNA-seq data of melon fruits produced in  different locations

city: College station

first: Hisashi

last: Koiwa

state: Texas

address: 495 Horticulture Rd.

department: Department of Horticultural Sciences

institute: Texas A&M University

summary: cDNA libraries of Melon fruits produced in different locations in the USA

overall_design: RNA sequencing using fruits harvested at 45 days after flowering (cultivar: F39 & TTDV in 2018 and F39, TTDV, PRIMO, OC164, CRUISER, HD-150, HD-252, infinite Gold, TH-5 and TH-6 in 2019).

first: Yukihiro

last: Nagashima

first: Seyednami

last: Niyakan

first: Xiaoning

last: Qian


title: Effects of IL-1b inhibition on the phenotypes of spleen naive CD4+ T (TN) cells.

first: Takashi

last: Sekiya

summary: To analyze the influence of IL-1b on TN cells, RNA-seq analysis was performed on spleen TN cells from  mice treated or not treated with IL-1b inhibitors.

overall_design: Comparative gene exprssion profiling analysis of RNA-seq data for total TN cells isolated from speen of mice treated or not treated with anti-IL-1b neutralizing antibodies or MCC950 for 10 consecutive days.

first: Sekiya

last: Takashi


title: A human iPSC-derived multilineage liver organoid model containing Kupffer cells to study human sepsis-associated liver dysfunction [KuLOs]

city: Minato

first: Yang

last: Li

state: Tokyo

address: Shirokanedai 4-6-1

institute: The University of Tokyo

summary:
To recapitulate the pathology of human septic liver, we established hiPSC-derived liver organoids containing Kupffer cells (KuLOs).
We performed gene expression profiling analysis using data obtained from RNA-seq of KuLOs with/without LPS/IFNr stimulation and the KuLOs during recovery after endotoxin elimination.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for KuLOs.

first: Yunzhong

last: Nie

first: Hideki

last: Taniguchi


title: Single-cell transcriptomic analysis reveals increased regeneration in diseased human inner ears

city: Nashville

first: Taha

last: Jan

state: TN

address: 2220 Pierce Ave, PRB 752

department: Otolaryngology

institute: VUMC

summary: Surgically procured utricles from organ donors and vestibular schwannoma patients underwent scRNAseq revealing unique transcriptional signatures among highly diverse sensory and non-sensory cells.

overall_design: Utricles from vestibular schwannoma patients or organ donors were acquired and enzymatically digested followed by single cell dissociation. The cells then underwent single cell dissociation followed by 10x capture and sequencing.

first: Jan

last: Taha

first: Cheng

middle: G

last: Alan


title: Transcriptomic Profiling of Plasma Extracellular Vesicles Enables Reliable Annotation of the Cancer-specific Transcriptome and Molecular Subtype (tumor tissues)

first: Anirban

last: Maitra

overall_design: To evaluate the consensus molecular subtypes (CMS) and immune cell subtypes of tumor tissues


title: Telomeric Chromosomal Instability Fosters Paracrine Mediated Cell Competition to Initiate Intestinal Cancer [scRNA-seq]

first: Jiexi

overall_design: scRNAseq was performed on single cell supsensions generated from G0+T and G4+T distal adenomas of Apcmin mice.

first: Kyle

last: LaBella


title: Comparative cofactor screens reveal the influence of transactivation domains and core promoters on the mechanisms of transcription [CRISPR TAD]

city: Melbourne

first: Charles

last: Bell

state: VICTORIA

address: VCCC, Gate 1, 14 Flemington Road

department: Cancer Research

country: Australia

institute: Peter MacCallum Cancer Centre

summary: Eukaryotic transcription factors (TFs) activate gene expression by recruiting cofactors to promoters. However, the relationships between TFs, promoters and their associated cofactors remain poorly understood. Here, we combine GAL4-transactivation assays with comparative CRISPR-Cas9 screens to identify the cofactors required by nine different TFs in human cells. Using this dataset, we associate key TFs with their cofactors, classify cofactors as ubiquitous or specific, discover novel transcriptional co-dependencies and demonstrate that certain TFs use the tail 2 and kinase submodules of Mediator to potentiate transcriptional elongation. By employing a reductionistic and comparative approach, we demonstrate that TFs do not display discrete mechanisms of activation. Instead, each TF is dependent on a unique combination of cofactors, which influences distinct steps in the transcriptional process. We also extend our screens to nine different core promoters to explore how core promoter elements influence cofactor dependence. Our data suggest that different classes of promoter are constrained by either initiation or pause release, which influences their dynamic range of gene expression and compatibility with specific cofactors. Overall, our comparative cofactor screens uncover the interplay between TFs, cofactors and core promoters and reveal general principles by which they influence transcription.

overall_design: GFP negative cells were isolated on D5, D6 and D7 after guide infection to identify the genes required for the activity of nine different transcription factors.. Sufficient cells were used to maintain 1000-fold representation at all stages of the screening process. The cells were transduced with an appropriate volume of viral supernatant to ensure only a single guide was present in most cells (MOI=0.3). At day 5, 6 and 7 day after guide infection, guide positive, GFP negative cells (< 25% of the MFI of the entire population) were sorted. Guide positive cells were also sorted as a library control at each time point to provide a library control reference to calculate enrichment. Four of the Gal4-TAD cell lines from the comparative CRISPR screens were also maintained until day 14 after guide infection to test for genes required for cell growth. These four TAD lines were used as independent replicates for the dropout analysis. Genomic DNA was extracted using Monarch Genomic DNA purification kit (New England Biolabs), according to the manufacturer’s instructions. PCR was conducted to maintain guide representation, using Q5 High Fidelity DNA Polymerase (New England Biolabs). PCR products were pooled and sequenced on the NextSeq500 using 75bp paired-end chemistry.

first: Mark

last: Dawson

first: Laure

last: Talarmain

first: Andrea

last: Gillespie


title: A human iPSC-derived multilineage liver organoid model containing Kupffer cells to study human sepsis-associated liver dysfunction [hiPSC-EMP-KC-pKC]

summary:
To incorporate Kupffer cells into hiPSC-LOs, we differentiated erythro-myeloid progenitors (EMPs) from hiPSCs.
We compares the gene expression profiles of hiPSC-EMPs with cord-blood hematopoietic stem and progenitor cells (CB-HSPCs); and EMP-generated Kupffer cells (EMP-KC) with human primary Kupffer cells.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for hiPSC, hiPSC-EMP, CB-HSPC, EMP-derived kupffer cells, and primary Kupffer cells.


title: A human iPSC-derived multilineage liver organoid model containing Kupffer cells to study human sepsis-associated liver dysfunction

overall_design: Refer to individual Series


title: Transcriptomic Profiling of Plasma Extracellular Vesicles Enables Reliable Annotation of the Cancer-specific Transcriptome and Molecular Subtype (plasma_cancer)

first: Kimal

last: Rajapakshe

first: Paola

last: Guerrero

first: Vahid

last: Bahrambeigi

first: Jaewon

last: Lee


title: Transcriptomic Profiling of Plasma Extracellular Vesicles Enables Reliable Annotation of the Cancer-specific Transcriptome and Molecular Subtype (cell mixture)

overall_design: To test the computational deconvolution pipeline on in vitro cellRNA mixtures containing known proportions of cancer and normal RNAs to infer cancer-derived RNA proportions and test whether the deconvoluted cancer-specific transcriptome can be used for the downstream analysis


title: Comparative cofactor screens reveal the influence of transactivation domains and core promoters on the mechanisms of transcription.


title: MHC class II antigen presentation by tumoral lymphatics is required for tumor specific signature and suppressive functions of Tregs

city: Geneva

first: Sylvain

last: LEMEILLE

address: 1 rue Michel-Servet

department: Department of Pathology and Immunology

institute: University of Geneva Medical School

summary: A number of solid malignancies triggers lymphangiogenesis, facilitating metastasis. Recent studies further indicate that tumor-associated lymphatic vessels significantly contribute to the generation of an immunosuppressive tumor microenvironment. Here, we have investigated the ability of tumor-associated lymphatic endothelial cells (LECs) to function as MHC class II restricted antigen-presenting cells in the regulation of anti-tumor immunity. Using murine models of lymphangiogenic tumors engrafted under the skin, we show that tumor LECs upregulate MHCII and the MHCII antigen processing machinery, and promote Treg expansion ex vivo. Using mice with a LEC-restricted lack of MHCII expression, we demonstrate that tumor growth is severely impaired, whereas tumor-infiltrating T effector cells are increased. Reduction of tumor growth and reinvigoration of tumor-specific T cell responses both result from alterations of the tumor-infiltrating regulatory T cell (Treg) transcriptome and phenotype. Treg suppressive functions are consequently profoundly altered in tumors lacking MHCII in LECs. No difference in effector T cell responses or Treg phenotype and functions were observed in tumor-draining lymph nodes, indicating that MHCII restricted antigen presentation by LECs is required locally in the tumor microenvironment (TME) to confer potent suppressive functions to Tregs. Altogether, our study advocates a role for MHCII-restricted antigen-presenting tumor LECs that function as a local brake, dampening tumor T cell immunity and promoting intratumoral Treg suppressive functions.

overall_design: mRNA profiles of regulatory T cells derived from tumors in which lymphatic endothelial cells express MHCII or not after injection with lymphangiogenic mouse melanoma cell line

first: Juan

last: Dubrot

first: Anastasia-Olga

last: Gkountidi

first: Dale

last: Brighouse

last: Lemeille

first: Stéphanie

last: Hugues


title: Transcriptomic Profiling of Plasma Extracellular Vesicles Enables Reliable Annotation of the Cancer-specific Transcriptome and Molecular Subtype (cell lines)

overall_design: To investigate if extracellular vesicles transcriptomic profiles recapitulate the transcriptomic profiles of the cells of origin in vitro


title: Transcriptomic Profiling of Plasma Extracellular Vesicles Enables Reliable Annotation of the Cancer-specific Transcriptome and Molecular Subtype


title: RSC and Bud27 influences chromatin organization and alternative polyadenylation

city: Sevilla

first: Gabriel

last: Gutierrez

address: PO BOX 1095

department: Genetics

country: Spain

institute: University of Seville

summary: The association of the RSC remodeler complex with the chromatin depends on the prefoldin-like Bud27 and determines the nucleosome positioning and polyadenylation sites usage in Saccharomyces cerevisiae

overall_design: Mnase-Seq

first: A

last: Cuevas-Bermúdez

first: V

last: Martínez-Fernández

last: Garrido-Godino

last: Jordán-Pla

first: X

last: Peñate

first: M

last: Martín-Expósito

first: G

last: Gutiérrez

first: J

middle: E

last: Perez-Ortin

first: C

middle: K

last: Govind

first: S

last: Chávez

last: Pelechano

first: F

last: Navarro


title: Pre-Hypertrophic Chondrogenic Enhancer Landscape of Limb and Axial Skeleton Development [RNA-seq]

first: Guillaume

last: Andrey

address: Rue Michel-Servet 1

department: Department of Genetic Medicine and Development

institute: University of Geneva

summary: The precisely orchestrated differentiation of chondrocytes during skeleton development is a critical determinant of human height and body shape. Disruptions of this process can cause severe skeletal abnormalities. The ultimate size and shape of each of over 200 bones depends on the intricate spatiotemporal regulation of chondrogenic and chondrocyte differentiation genes, but the genomic architecture coordinating these events remains poorly defined. Here we provide a comprehensive map of transcriptional enhancers specifically active in chondrocytes and show that they provide a mechanistic framework through which noncoding genetic variants can influence human stature. We isolated limb and trunk fetal chondrocytes from mice with a Col2a1 fluorescent regulatory sensor and used RNA-seq to identify 780 genes that are specifically expressed during chondrogenesis. To create cell type-specific enhancer maps, we performed ATAC-seq to map open chromatin regions and ChIP-seq for H3K27ac, an enhancer-associated histone modification, and identified 2'704 putative chondrogenic enhancer regions. Most of these enhancers (74%) showed pan-chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. We found that chondrogenic enhancers are enriched for the binding of several chondrogenic transcription factors including SOX9. Moreover, we find that genetic variation overlapping chondrogenic enhancers explains a higher fraction of the heritability of human adult height than the one overlapping non-chondrogenic enhancers. Finally, targeted deletions of identified enhancers at the Fgfr3, Col2a1, Hhip and, Nkx3-2 loci each exhibited a significant reduction of cognate gene expression, therefore demonstrating their functional importance. This data provides a comprehensive mapping of the chondrogenic enhancer repertoire, paving the way to interpreting the role of non-coding sequence polymorphisms in phenotypic variation and bone diseases.

overall_design: We devised a Col2a1 fluroescent reporter approach to sort chondrocytes from embryonic trunk and limbs. We validate the specficity of the reproter using scRNAseq. We then analyse Col2a1-EGFP positive and negative cells to profile their transcriptome, open chromatin and H3K27ac coverage to define enhancers and asssooicated genes. We also engineered four deletions at Col2a1, Fgfr3, Hhip and Nkx3-2 removing chondrogenic enhancers and measuring changes in genes expressions by bulk RNA-seq.

first: Fabrice

last: Darbellay


title: Regulation of transcription patterns, poly(ADP-ribose), and RNA-DNA hybrids by the ATM protein kinase

city: Austin

first: Tanya

middle: T.

last: Paull

address: 2500 Speedway MBB 2.448

department: Molecular Biosciences

institute: Univ. of Texas at Austin

summary: The ATM protein kinase is a master regulator of the DNA damage response and also an important sensor of oxidative stress in mammals. Loss of ATM or its ability to be activated by reactive oxygen species (ROS) generates high levels of ROS and an excess of single-strand DNA breaks in non-dividing human cells. Here we map locations of poly-ADP-ribose and RNA-DNA hybrid accumulation genome-wide in post-mitotic human neurons with ATM inhibition and find that these locations are coincident with promoter regions, active transcription histone marks, and sequences with high GC content. Antioxidant treatment reverses the accumulation of R-loops in transcribed regions, consistent with the central role of ROS in promoting these lesions. In A-T patient cerebellum tissue, RNAseq analyses show large-scale transcriptional changes that correlate with both expression level and GC content of transcribed genes. Based on these results we postulate that transcription-associated lesions accumulate in ATM-deficient neurons and that the single-strand breaks and PARylation at these sites ultimately generate changes in transcription that compromise cerebellum function and lead to neurodegeneration of this tissue over time in A-T patients.

overall_design: chromatin immunoprecipitation (poly-ADP-ribose ChIPseq and R-ChIP), RNAseq

first: Phillip

last: Woolley

first: Xuemei

last: Wen

first: Olivia

last: Conway

first: Nicolette

last: Ender

first: Ji-Hoon

last: Lee


title: Biallelic variants in SNUPN cause a limb girdle muscular dystrophy with myofibrillar-like features

city: San Sebastian

first: Lorea

last: Blazquez

address: Dr Begiristain

institute: Biogipuzkoa Health Research Institute

summary: Alterations in RNA-splicing are a molecular hallmark of several neurological diseases, including muscular dystrophies where mutations in genes involved in RNA metabolism or characterised by alterations in RNA splicing have been described. Here, we present five patients from two unrelated families with a limb-girdle muscular dystrophy (LGMD) phenotype carrying a biallelic variant in SNUPN gene. Snurportin-1, the protein encoded by SNUPN, plays an important role in the nuclear transport of small nuclear ribonucleoproteins (snRNPs), essential components of the spliceosome. We combine deep phenotyping, including clinical features, histopathology and muscle magnetic resonance image (MRI), with functional studies in patient-derived cells and muscle biopsies to demonstrate that variants in SNUPN are the cause of a new type of LGMD according to current definition. Moreover, an in vivo model in Drosophila melanogaster further supports the relevance of Snurportin-1 in muscle. SNUPN patients show a similar phenotype characterised by proximal weakness starting in childhood, restrictive respiratory dysfunction and prominent contractures, although interindividual variability in terms of severity even in individuals from the same family was found. Muscle biopsy showed myofibrillar-like features consisting of myotilin deposits and Z-disc disorganisation. MRI showed predominant impairment of paravertebral, vasti, sartorius, gracilis, peroneal and medial gastrocnemius muscles. Conservation and structural analyses of Snurportin-1 p.Ile309Ser variant suggest an effect in nuclear-cytosol snRNP trafficking. In patient-derived fibroblasts and muscle, cytoplasmic accumulation of snRNP components is observed, while total expression of Snurportin-1 and snRNPs remains unchanged, which demonstrates a functional impact of SNUPN variant in snRNP metabolism. Furthermore, RNA-splicing analysis in patients’ muscle showed widespread splicing deregulation, in particular in genes relevant for muscle development and splicing factors that participate in the early steps of spliceosome assembly. In conclusion, we report that SNUPN variants are a new cause of limb girdle muscular dystrophy with specific clinical, histopathological and imaging features, supporting SNUPN as a new gene to be included in genetic testing of myopathies. These results further support the relevance of splicing-related proteins in muscle disorders.

overall_design: Comparative transcriptomic profiling analysis of total RNA-seq data from muscle biopsies derived from patients with biallelic SNUPN c.926T>G variant and control individuals.


title: RNA-seq analysis of spleen and mesenteric lymph node (mLN) hematopoietic cells

first: Takashi

last: Sekiya

summary: To search for factors that affect naive CD4+ T (TN) cell phenotypes in spleen, we performed RNA-seq analysis to compare mRNA expression between  freshly-prepared spleen and mLN hematopoietic cells.

overall_design: Comparative gene exprssion profiling analysis of RNA-seq data for total CD45+ cells freshly sorted from spleen (Spl) and mLN.

first: Sekiya

last: Takashi


title: Transcriptome analysis during the growth and development of asparagus stems

city: chengdu

first: lin

middle: mao

last: he

state: sichuan

address: wenjiang

institute: sichuan agricultural university

summary: Asparagus is a nutritionally dense stem vegetable whose growth and development are correlated with its quality and yield. For height of 25 cm asparagus , 17-20 cm is still viable, in which the cells can continue to elongate. When 40 cm, the growth in the 17-20 cm range basically stops. Therefore, we picked periods with significant growth differences for transcriptome comparison to find the dynamic changes and underlying mechanisms during the elongation and growth process of asparagus stems. The results showed that the differentially expressed genes were enriched in many aspects, including the metabolism of plant hormones and carbohydrates.

overall_design: To investigate the dynamic changes and underlying mechanisms during the elongation and growth process of asparagus stems, we sampled asparagus stems at different ages. We selected only  asparagus stems of different plant heights with a stem diameter of 13-15 mm, including 10, 25, 40, and 60 cm, with three replicates for each treatment. For different growth and development stages of asparagus, 3-6 cm stem segments from the surface were collected from 10 cm, while 17-20 cm of stem segments from the surface were collected from 25, 40, and 60 cm as the test materials.

first: Maolin

last: He

first: Peiran

last: Chen

first: Yangxia

last: Zheng

first: Mengyao

last: Li


title: β-catenin and Hoxa9 preserve DNA replication dynamics, genomic integrity and stem cell functionality in a Prmt1-dependent manner [CUT&RUN]

city: London

first: Eric

last: So

address: School of Cancer and Pharmaceutical Sciences

country: United Kingdom

institute: KCL

summary: Replication stress is a main driver of functional decline of hematopoietic stem cells (HSCs), which carry the highest burden of maintaining genomic integrity and functionality within the hematopoietic system. Here, we identify a novel signaling axis in which β-catenin and Hoxa9 function in a compensatory manner to preserve HSC functionality by protecting DNA replication dynamics and genomic integrity, in part via regulation of Prmt1 activity. Co-inactivation of β-catenin and Hoxa9 induces severe hematopoietic defects accompanied by accumulating replication stress and DNA damage resulting in functional HSC decline. Furthermore, we illustrate how β-catenin and Hoxa9 pathways converge on the pivotal downstream target, Prmt1, which functions to maintain an adequate supply of DNA replication and repair factors. Prmt1 aids in alleviating replication stress and DNA damage accumulation thereby preserving HSC integrity and functionality.

overall_design: CUT&RUN was performed using 0.5 million HSPCs from WT BL6 mice or Ctnnb1-Biotin-3xFLAG knockin mice (JAX:029511) according to Epicypher CUT&RUN Protocol V2.0.

first: Jennifer

last: Lynch

first: Estelle

last: Troadec

first: Tsz

last: Fung

first: Kornelia

last: Gladysz

first: Clemence

last: Virely

first: Priscilla

last: Lau

first: Ngai

last: Cheung

first: Bernd

last: Zeisig

first: Jason

last: Wong

first: Massimo

last: Lopes

first: Suming

last: Huang

first: Chi


title: β-catenin and Hoxa9 preserve DNA replication dynamics, genomic integrity and stem cell functionality in a Prmt1-dependent manner [RNA-seq dKO_HSPCs]

overall_design: RNA-sequencing analysis was conducted following ectopic PRMT1 expression in dKO HSPCs harvested from primary transplanted mice.


title: DOS-3 mediates cell-non-autonomous DAF-16/FOXO activity in antagonizing age-related loss of C. elegans germline stem

city: Shanghai

first: Zhifei

last: Zhang

address: NO.1239 Siping Road

institute: Tongji university

summary: Age-related depletion of stem cells causes tissue degeneration and failure to tissue regeneration, driving aging at the organismal level. Previously we reported a cell-non-autonomous DAF-16/FOXO activity in antagonizing the age-related loss of germline stem/progenitor cells (GSPCs) in C. elegans, indicating that regulation of stem cell aging occurs at the organ system level. Here we discover the molecular effector that links the cell-non-autonomous DAF-16/FOXO activity to GSPC maintenance over time by performing a tissue-specific DAF-16/FOXO transcriptome analysis. Our data show that dos-3, which encodes a non-canonical Notch ligand, is a direct transcriptional target of DAF-16/FOXO and mediates the effect of the cell-non-autonomous DAF-16/FOXO activity on GSPC maintenance through activating Notch signaling in the germ line. Importantly, expression of a human homologous protein can functionally substitute for DOS-3 in this scenario. As Notch signaling controls the specification of many tissue stem cells, similar mechanisms may exist in other aging stem cell systems.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for the proximal region of the somatic gonad (PSG) cells of daf-2(rf) and daf-16(0); daf-2(rf) hermaphrodite nematodes on day1 (D1) and day 7 (D7) of adulthood.

first: Haiyan

last: Yang

first: Lei

last: Fang

first: Guangrong

last: Zhao

first: Jun

last: Xiang

first: Jialin

last: C Zheng

first: Zhao

last: Qin


title: β-catenin and Hoxa9 preserve DNA replication dynamics, genomic integrity and stem cell functionality in a Prmt1-dependent manner [RNA-seq LSK]

first: Eric

last: So

overall_design: RNA-sequencing profiles of LSKs isolated from WT, single KO and dKO primary transplanted mice six weeks post tamoxifen treatment.

first: Jennifer

last: Lynch

first: Estelle

last: Troadec

first: Tsz


title: β-catenin and Hoxa9 preserve DNA replication dynamics, genomic integrity and stem cell functionality in a Prmt1-dependent manner [ATAC-Seq]

overall_design: ATAC-sequencing were performed on LSKs isolated from Prmt1KO primary transplanted mice.

last: Fung

first: Kornelia

last: Gladysz

first: Clemence

last: Virely

first: Priscilla

first: Ngai


title: β-catenin and Hoxa9 preserve DNA replication dynamics, genomic integrity and stem cell functionality in a Prmt1-dependent manner

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


title: Modelling host-Trypanosoma brucei gambiense interactions in vitro using human induced pluripotent stem cell-derived cortical brain organoids

city: Manchester

first: Juan

middle: Fernando

last: Quintana

address: 2.18c CTF, Grafton Street

department: Lydia Becker Institute of Immunology and Inflammation

institute: University of Manchester

summary: Background: Sleeping sickness is caused by the extracellular parasite Trypanosoma brucei and is associated with neuroinflammation and neuropsychiatric disorders, including disruption of sleep/wake patterns, and is now recognised as a circadian disorder. Sleeping sickness is traditionally studied using murine models of infection due to the lack of alternative in vitro systems that fully recapitulate the cellular diversity and functionality of the human brain. The aim of this study is to develop a much-needed in vitro system that reduces and replaces live animals for the study of infections in the central nervous system, using sleeping sickness as a model infection. Methods: We developed a co-culture system using induced pluripotent stem cell (iPSC)-derived cortical human brain organoids and the human pathogen T. b. gambiense to model host-pathogen interactions in vitro. Upon co-culture, we analysed the transcriptional responses of the brain organoids to T. b. gambiense over two time points. Results: We detected broad transcriptional changes in brain organoids exposed to T. b. gambiense, mainly associated with innate immune responses, chemotaxis, and blood vessel differentiation compared to untreated organoids. Conclusions: Our co-culture system provides novel, more ethical avenues to study host-pathogen interactions in the brain as alternative models to experimental infections in mice. Although our data support the use of brain organoids to model host-pathogen interactions during T. brucei infection as an alternative to in vivo models, future work is required to increase the complexity of the organoids ( e.g., addition of microglia and vasculature). We envision that the adoption of organoid systems is beneficial to researchers studying mechanisms of brain infection by protozoan parasites. Furthermore, organoid systems have the potential to be used to study other parasites that affect the brain significantly reducing the number of animals undergoing moderate and/or severe protocols associated with the study of neuroinflammation and brain infections.

overall_design: Single cell transcriptomics of human brain rganoids c-cultured with the human pathogen Trypanosoma brucei gambiense for a period of 24 and 72 hours

first: Praveena

last: Chandrasegaran

first: Agatha

last: Nabilla Lestari

first: Matthew

last: Sinton

first: Jay

last: Gopalakrishnan

middle: F


title: Mechanisms of Innate Immune Injury in Arrhythmogenic Cardiomyopathy

status: Public on Feb 28 2024

city: Saint Louis

first: Kory

last: Lavine

state: Missouri

address: 660 S. Euclid Ave

department: Internal Medicine - Cardiology

institute: Washington University School of Medicine

summary:
Introduction: We have reported that iPSC-cardiac myocytes from patients with arrhythmogenic cardiomyopathy (ACM) mount an intense innate immune response under basal conditions in vitro. In addition, blocking innate immune signaling via NFκB prevents disease in a mouse model of ACM (Dsg2mut/mut mice). Here, we defined the relative pathogenic roles of immune signaling in cardiac myocytes vs. infiltrating inflammatory cells in Dsg2mut/mut mice.
Methods: To define the role of immune signaling in cardiac myocytes, we bred Dsg2mut/mut mice with mice with cardiac myocyte-specific expression of a dominant-negative form of IκBα (DN-IκBα), which prevents nuclear translocation and, thereby, activation of NFκB signaling only in cardiac myocytes. To define the role of inflammatory cells in ACM, we focused on cells expressing C-C motif chemokine receptor-2 (CCR2+ cells), known to mediate adverse cardiac remodeling and fibrosis. We bred Dsg2mut/mut mice with germline deletion of Ccr2 mice (Ccr2-/- mice). We compared phenotypes in Dsg2mut/mut mice with double-mutant Dsg2mut/mut X DN-IκB and Dsg2mut/mut X Ccr2-/- lines.
Results: Dsg2mut/mut mice develop marked myocardial fibrosis, reduced LV ejection fraction (EF) and many PVCs. These disease features were all normalized in Dsg2mut/mut X DN-IκB mice. Hearts of Dsg2mut/mut and Dsg2mut/mut X DN-IκB mice contained equal numbers of CD68+ macrophages, but Dsg2mut/mut hearts had many more CCR2+ cells. Cardiac myocyte expression of Tlr4 which activates NFκB, was increased in Dsg2mut/mut mice but not in Dsg2mut/mut X Ccr2-/- mice. Dsg2mut/mut X Ccr2-/- mice showed greatly reduced myocardial fibrosis and PVCs but LV EF was markedly depressed. This suggested that contractile dysfunction in ACM is due not only to loss of muscle but to immune signaling in viable cardiac myocytes as well. To test this hypothesis, we treated Dsg2mut/mut and Dsg2mut/mut X Ccr2-/- mice with the NFκB inhibitor Bay 11-7082. Before treatment, LV EFs were significantly reduced in both groups. Untreated Dsg2mut/mut mice had ongoing deterioration of LV function, whereas treated Dsg2mut/mut mice showed no further disease progression and actually had some improvement in contractile function. However, LV function in treated Dsg2mut/mut X Ccr2-/- mice was virtually normalized.
Conclusions: NFκB signaling in cardiac myocytes drives myocardial injury, LV dysfunction and arrhythmias in Dsg2mut/mut mice. It also mobilizes CCR2+ cells to the heart, where they promote myocardial injury and arrhythmias. CCR2+ cells alter gene expression in cardiac myocytes. LV dysfunction in Dsg2mut/mut mice is caused not only by muscle loss but also by negative inotropic effects of inflammation mediated by NFκB in viable cardiac myocytes. These results have obvious implications for ACM patients and suggest that anti-inflammatory therapy may be beneficial even in patients with established disease.

overall_design: snRNA-seq and CITE-seq in WT, Dsg2mut, and Dsg2mutxCcr2-/- mice

first: Stephen

last: Chelko

first: Vinay

last: Penna

first: Morgan

last: Engel

first: Maicon

last: Landim-Vieira

first: Elisa

middle: N

last: Cannon

first: Jeffrey

last: Saffitz


title: Chromatin binding by HORMAD proteins regulates meiotic recombination initiation

first: Carolyn

last: Milano

email: crmilano@gmail.com, cm5497@nyu.edu

address: 15 Washington Place, Apartment 3M

institute: New York University

summary: The meiotic chromosome axis coordinates chromosome organization and interhomolog recombination in meiotic prophase, and is essential for fertility. In S. cerevisiae, the HORMAD protein Hop1 mediates an enrichment of axis proteins at nucleosome-rich genomic islands through a central chromatin-binding region (CBR). Here, we use cryoelectron microscopy to show that the Hop1 CBR directly recognizes bent nucleosomal DNA through a composite interface in its PHD and winged helix-turn-helix domains. Targeted disruption of the Hop1 CBR-nucleosome interface causes a loss of axis-protein enrichment at nucleosome-rich genomic islands, reduces meiotic DNA double-strand breaks (DSBs), and causes defects in chromosome synapsis. Synthetic effects with the disassemblase Pch2 suggest that nucleosome binding delays a conformational switch in Hop1 from a DSB-promoting, Pch2-inaccessible state to a DSB-inactive, Pch2-accessible state to regulate the extent of meiotic DSB formation . Phylogenetic analyses of meiotic HORMADs reveal an ancient origin of this domain,  suggesting that these mechanisms are broadly conserved.

overall_design: ChIP-seq analysis of binding dynamics of meiotic factors in S. cerevisiae during meiosis, and CC-seq analysis of DSB occurences in the S. cerevisiae genome during meiosis. Each txt file is the location of detected DSB locations, represented in Hits per Million results for the CC-seq samples. Each bedgraph file is the results from paired Input and ChIP samples.

first: Sarah

last: Ur

first: Yajie

last: Gu

first: Elco

last: Tromer

first: Luis

middle: A

last: Vale-Silva

first: Jessie

last: Zhang

first: Andreas

last: Hochwagen

first: Kevin

middle: D

last: Corbett


title: Linker Histone H1.0 Couples Cellular Stiffness to Chromatin Structure [RNA-seq]

city: Los Angeles

first: Thomas

middle: Martin

last: Vondriska

state: CA

address: 650 Charles Young Dr

department: Anesthesiology

institute: UCLA

summary: Eukaryotic nuclei encase the genome and differentially package it for the various needs of distinct cell types. Tuning of genome structure and function is accomplished by chromatin binding proteins, which are responsive to cellular stress, determining the transcriptome and phenotype of the cell. We sought to investigate the connection between extracellular stress and chromatin structure to regulate cellular stiffness. We demonstrate that the linker histone H1.0, which compacts nucleosomes into higher order chromatin fibers, controls genome structure and cellular response to stress. Histone H1.0 has privileged expression in tension-responsive fibroblasts across tissue types in mouse and humans, and is necessary and sufficient to mount a myofibroblast phenotype in these cells. Loss of histone H1.0 prevents transforming growth factor beta (TGF-b)-induced fibroblast contraction, proliferation and migration in an isoform-specific manner via inhibition of a transcriptome targeting extracellular matrix molecules. Histone H1.0 is associated with local regulation of gene expression by chromatin fiber compaction and histone acetylation, rendering the nucleus and cell stiffer in response to cytokine stimulation. Knockdown of H1.0 decreased levels of HDAC1 and the chromatin reader BRD4, thereby preventing transcription of a fibrotic gene program. Transient depletion of histone H1.0 in vivo decompacts chromatin and prevents fibrosis in cardiac muscle, lung, and kidney, thereby linking chromatin structure with fibroblast phenotype in response to extracellular stress. Our work identifies an unexpected role of linker histones to sense and respond to cellular stress, directly coupling cellular tension, nuclear organization and gene transcription.

overall_design: RNA-seq of isolated adult mouse cardiac fibroblasts treated with siRNA against H1.0 or siScramble control, in the presence and absence of TGFb. There are 3 biological replicates per condition.

middle: M

first: Douglas

middle: J

last: Chapski


title: Differential DNA methylation occurs in RUNX1 heterozygous mutations harboring hematopoietic progenitor cells [YX-FLI1_EM]

status: Public on Feb 26 2024

city: Yokohama

first: Yuki

last: Tanaka

state: Kanagawa

address: 1-7-22 Suehiro-cho, Tsurumi-ku

department: Center for Integrative Medical Sciences

institute: RIKEN

summary:
Background: Familial platelet disorder (FPD) is an autosomal dominant disease caused by a heterozygous germline mutation inRUNX1. FPD patients show not only thrombocytopenia with platelet dysfunction, but also a high level of developing hematological malignancies, strongly suggesting that FPD is in a precancerous state. However, the DNA methylation status of FPD has not yet been elucidated due to no animal models for FPD and the difficulty in obtaining FPD patient-derived samples.
Results: We found that differentiation efficiencies into HPCs and megakaryocytes was reduced in the FPD-mimicking cells, which were established by genome editing for human iPS cells as a FPD-model, compared with those of wild-type cells. The FPD-mimicking HPCs were subjected to DNA methylation analysis, and the HPCs showed the distinct DNA methylation patterns compared to wild-type HPCs. Furthermore, we demonstrated a putative causative transcription factor of the differential DNA hypermethylation, which are involved in both promoting the binding site-directed DNA demethylation and regulating megakaryopoiesis.

overall_design: To characterize the DNA methylation changes between FLI1-overexpressing hematopoietic progenitor cells harboring FPD-mimicking RUNX1WT/Y287X mutation and mock-control cells, single-base resolution methylome analyses were performed.

first: Ken-ichi

last: Nakada

first: Rino

last: Maruyama

first: Harukazu

last: Suzuki

first: Takahiro


title: Linker Histone H1.0 Couples Cellular Stiffness to Chromatin Structure [ChIP-seq]

overall_design: ChIP-seq on isolated cardiac fibroblasts, using an antibody against H1.0, FLAG, or H3K27ac.


title: Differential DNA methylation occurs in RUNX1 heterozygous mutations harboring hematopoietic progenitor cells [WES]

type: Genome variation profiling by high throughput sequencing

overall_design:
To establish the human iPS cell lines with FPD-mimicking RUNX1 heterozygous mutations, we performed CRISPR-Cas9-mediated genome editing of RUNX1, followed by single-cell cloning of the resulting genome-edited cells.
We performed whole exome sequencing (WES) analysis to confirm potential off-target editing on protein cording regions in the established cell lines.


title: Linker Histone H1.0 Couples Cellular Stiffness to Chromatin Structure

overall_design: Refer to individual Series


title: Deciphering cell states and genealogies of human hematopoiesis with single-cell multi-omics [Crispr_Mouse_Batch1]

city: Boston

first: Chen

last: Weng

address: 415 Main St, Cambridge

institute: Broad Institute and Whitehead Institute

summary: The human blood system is maintained through the differentiation and massive amplification of a limited number of long-lived hematopoietic stem cells (HSCs). Perturbations to this process underlie a diverse set of diseases, but the clonal contributions to human hematopoiesis and how this changes with age remain incompletely understood. While recent insights have emerged from barcoding studies in model systems, simultaneous detection of cell states and phylogenies from natural barcodes in humans has been challenging, which has limited the ability to explore functional differences between HSC clones. Here, we introduce an improved single-cell lineage tracing system based on deep detection of naturally-occurring mitochondrial DNA (mtDNA) mutations with simultaneous readout of transcriptional states and chromatin accessibility. We use this system to define the clonal architecture of HSCs, and map the physiological state and output of these clones. We uncover functional heterogeneity in HSC clones, which is stable over months and manifests as differences in total HSC output as well as biases toward the production of different mature blood and immune lineages. We also find that the diversity of HSC clones decreases dramatically with age leading to an oligoclonal structure with multiple distinct clonal expansions. Our study thus provides the first clonally-resolved and cell-state aware atlas of human hematopoiesis at single-cell resolution revealing an unappreciated functional diversity of human HSC clones both in young and aged individuals and more broadly paves the way for refined studies of clonal dynamics across a range of tissues in human health and disease.

overall_design: We developed single-cell Regulatory multi-omics with Deep Mitochondrial mutation profiling (ReDeeM), which is a modified massive parallel single cell protocol to simultaneously profile multi-omics with deep mtDNA sequencing based on the 10X Genomics platform. With ReDeeM, three separate libraries are generated, including enriched mtDNA library for deep sequencing and mutation profiling, RNA library for gene expression, as well as ATAC library for chromatin accessibility profiling, all of which are linked via matchable single cell barcodes. Following the principle of our previous work, we firstly modified the droplet-based 10X genomics multi-omics protocol (Catalog #100283) by processing the whole cell instead of nuclei with fixation and mild permeabilization to maximally retain mitochondrial DNA. Next, we designed mtDNA specific probe sets to enrich the mitochondrial fragments using DNA hybridization. The RNA and ATAC library preps follow the standard 10X protocol with some modifications.  More method details are described in the method section and an open ReDeeM Protocol is available. ReDeeM is further computationally supported by consensus variant calling pipeline redeemV , and an inhouse R package redeemR for the downstream mutation quality control, as well as single cell phylogenetic and integrative analysis. This SubSeries contains data of CRISPR mouse lineage tracing with ReDeeM.  All mouse and human sample data were generated using the same protocol. In Extended Data Fig. 5 of the manuscript, the data were processed using an earlier version of our mapping approach (using the bam files from Cellranger without mtDNA mask for redeemV variant calling). In the latest version, we have processed raw reads using our in-house masked bowtie2 alignment. The other steps remain exactly the same. The agreement between ReDeeM and CRISPR is consistently observed in both analyses. We have provided the latest version of the processed RawGenotype data with this accession.

first: Vijay

last: Sankaran

first: Jonathan

middle: S

last: Weissman


title: Differential DNA methylation occurs in RUNX1 heterozygous mutations harboring hematopoietic progenitor cells [FLI1_EM]

overall_design: We performed the methylome analysis of iPS cells overexpressed FLI1, a putative causative transcription factor for differential DNA hypermethylation in FPD-mimicking HPCs, by doxycycline (DOX)-inducible manner to validate whether FLI1 induces the DNA demethylation at its binding sites in vitro.


title: The impact of HPV8 on hair follicle stem cell populations

city: Cardiff

first: Girish

last: Patel

address: Maindy Road

institute: Cardiff University


title: Differential DNA methylation occurs in RUNX1 heterozygous mutations harboring hematopoietic progenitor cells [EM]

overall_design: To characterize the global DNA methylation status of hematopoietic progenitor cells harboring FPD-mimicking RUNX1 heterozygous mutations, the single-base resolution methylome analyses were performed.


title: Biological nitrogen fixation maintains C/N balance and photosynthesis at elevated CO2

city: Urbana

first: Matthew

middle: D.

last: Brooks

state: Illinois

address: 1201 W Gregory Drive

department: GCPRU

institute: USDA

summary: To investigate the effect that  biological nitrogen fixation will have on plant responses to nitrogen dose at elevated CO2, alfalfa (Medicago sativa) lines were grown at three nitrogen doses and ambient or elevated CO2. Four lines were used in the study, two lines that can form nodules capable of fixing nitrogen (effective lines) and two lines that can not form nodules capable of nitrogen fixation (ineffective lines). The ineffective lines are the result of a complementary mutation in the same gene.

overall_design: RNA-seq


title: Muti-Omics Integrated Analysis Reveals Altered metabolism in Sarcopenia.

status: Public on Feb 22 2024

city: Chengdu

first: Tao

last: Li

address: No. 88, Keyuan South Road, High-tech Zone, Chengdu

department: Department of Anesthesiology

institute: Sichuan Universiy West China Hospital

summary:
To investigate the metabolic dysfunction in the process of sarcopenia, we collected the skeletal muscles from the participants of healthy aged, pre-sarcopenia and sarcopenia.
We then performed gene expression profiling analysis using data obtained from RNA-seq of skeletal muscle tissue from  healthy aged, pre-sarccopenia and sarcopenia.


title: Tcaim delays mitochondria ER contact formation and thereby activation-induced upregulation of anabolism and CD8+ T cell effectr differentiation

status: Public on Feb 25 2024

city: Berlin

first: Karsten

last: Jürchott

address: Luisenstraße

department: Rahel Hirsch Center

institute: BIH at Charité

summary: T cells need to adapt their cellular metabolism for full effector cell differentiation upon activation and it has been reported that this relies on alterations in mitochondrial physiology including cristae resolution, fission and an increase in membrane potential, mROS production as well as metabolite supply for anabolic pathways. However, which signals and molecules regulate those activation‑induced alterations of mitochondria and to which extend inter-organelle communications such as interactions between mitochondria and the ER are required for the change in cellular metabolism still remains unclear. We recently reported that the mitochondrial protein TCAIM is downregulated upon T cell activation and inhibits effector cell differentiation of conventional CD4+ T cells. Our previous investigations also indicated that it interferes with activation-induced changes in mitochondrial morphology and function. Thus, TCAIM seems to function as an important link between activation-induced mitochondrial alterations and changes in cellular metabolism driving full effector cell differentiation. Therefore, we here utilized conditional Tcaim KI and KO mice as a model to investigate which molecular processes at the mitochondria control effector T cell differentiation. First, we could show here that Tcaim KI causes also for CD8+ T cells comparable effects of impaired proliferation, effector/ effector‑memory differentiation and cytokine production as we previously reported for CD4+ T cells. Furthermore, our data on Tcaim KO CD8+ T cells point towards an advantage for effector/ effector memory differentiation in vivo. This further supports the regulatory role of TCAIM for activation and differentiation of T cells in general. Metabolomics and transcriptional data indicated that Tcaim KI inhibits activation‑induced enforcement of TCA cycle activity and glutaminolysis. More importantly, up-regulation of anabolic pathways, especially of the mevalonate pathway and cholesterol biosynthesis, was blocked upon Tcaim KI. Indeed, addition of cholesterol could rescue T cell activation and proliferation of Tcaim KI CD8+ T cells. At the molecular level, we could show that TCAIM delays activation-induced interaction between mitochondria and the ER, which might be mediated by binding of TCAIM to proteins known to promote MERC formation such as RMD3 and VDAC. Collectively, we provide evidence that TCAIM negatively regulates activation‑induced increase in MERC formation and thereby upregulation of anabolic processes, especially towards increased mevalonate pathway and cholesterol biosynthesis, resulting in impaired CD8+ T cell activation and proliferation.

overall_design: mRNA expression profiles of naïve and 24 to 48 h polyclonally activated CD8+ T cells from conditional Tcaim KI (n=3), KO (n=2) or wt (n=3) mice by bulk RNA sequencing

first: Christina

last: Iwert

first: Birgit

last: Sawitzki

first: Tatiana

last: Borodina

first: Madlen

last: Sohn