title: Bulk RNA-sequencing of hypocotyl explant-derived calli of Arabidopsis WT and rap2.12-2

city: Seoul

first: Soon Hyung

last: Bae

state: South Korea

address: Gwanak-ro 1, Gwanak-gu

country: South Korea

institute: Seoul National University

summary: Plants are aerobic organisms that rely on molecular oxygen for respiratory energy production. Hypoxic conditions, with oxygen levels ranging between 1% and 5%, usually limit aerobic respiration and affect plant growth and development. Here, we demonstrate that hypoxic microenvironment induced by active cell proliferation during the two-step plant regeneration process intrinsically represses the regeneration competence of callus in Arabidopsis thaliana. Hypoxia-repressed plant regeneration was mediated by the RELATED TO APETALA 2.12 (RAP2.12) protein, a member of the Ethylene Response Factor VII (ERF-VII) family. The hypoxia-activated RAP2.12 protein promoted salicylic acid (SA) biosynthesis and defense responses, inhibiting pluripotency acquisition and de novo shoot regeneration in calli. RAP2.12 could bind directly to the SALICYLIC ACID INDUCTION DEFICIENT 2 (SID2) gene promoter and activate SA biosynthesis, repressing plant regeneration via a PLETHORA (PLT)-dependent pathway. The rap2.12 mutant calli exhibited enhanced shoot regeneration, which was impaired by SA treatment. Taken together, our findings demonstrate that cell proliferation-dependent hypoxic microenvironment reduces cellular pluripotency and plant regeneration through the RAP2.12–SID2 module.

overall_design: Hypocotyls of 1-week-old Col-0 (WT) and rap2.12-2 Arabidopsis thaliana seedlings were excised and incubated on callus-inducing medium (CIM). Calli were collected 7 days after incubation on CIM. Three biological replicates for each sample were generated.

first: Dohee

last: Koo

first: Hong Gil

last: Lee

first: Kyounghee

first: Pil Joon

last: Seo


title: Bulk RNA-sequencing of hypocotyl explants and hypocotyl explant-derived calli of Arabidopsis

status: Public on Feb 16 2024

type: Expression profiling by high throughput sequencing

overall_design: Hypocotyls of 1-week-old Col-0 Arabidopsis thaliana seedlings were excised and incubated on callus-inducing medium (CIM). Samples were collected immediately after excision and 7 days after incubation on CIM. Three biological replicates for each time point were generated.


title: Akkermansia muciniphila-induced trained immune phenotype increases bacterial intracellular survival and attenuates inflammation

city: Derio

first: Ana

middle: Maria

last: Aransay

state: BIZKAIA

address: Parque tecnologico de Bizkaia, Building 801-A

department: Genome Analysis Platform

country: Spain

institute: CIC bioGUNE

summary: The initial exposure to pathogens and commensals confers innate immune cells the capacity to respond distinctively upon a second stimulus. This training capacity might play key functions in developing an adequate innate immune response to the continuous exposure to bacteria. However, the mechanisms involved in induction of trained immunity by commensals remain mostly unexplored. A. muciniphila represents an attractive candidate to study the promotion of these long-term responses. Here, we show that priming of macrophages with live A. muciniphila enhances bacterial intracellular survival and decreases the release of pro- and anti-inflammatory signals, lowering the production of TNF and IL-10. Global transcriptional analysis of macrophages after a secondary exposure to the bacteria showed the transcriptional rearrangement underpinning the phenotype observed compared to acutely exposed cells, with the increased expression of genes related to phagocytic capacity and those involved in the metabolic adjustment conducing to innate immune training. Accordingly, key genes related to bacterial killing and pro-inflammatory pathways were downregulated. These data demonstrate the importance of specific bacterial members in the modulation of local long-term innate immune responses, broadening our knowledge of the association between gut microbiome commensals and trained immunity as well as the anti-inflammatory probiotic potential of A. muciniphila.

overall_design: Genome-wide changes in gene expression in mouse bone marrow-derived macrophages exposed to Akkermansia muciniphila at a multiplicity of infection of 10. Groups correspond to unstimulated (U), acutely stimulated (U-A.m) and stimulated and restimulated (A.m-A.m).

first: Juan

last: Anguita

first: Leticia

last: Abecia

first: Ainize

last: Peña

first: Ainhoa

last: Palacios

middle: M


title: Microarray data of lenses with 2ME2

city: Fukui

first: Masaru

last: Takashima

address: 3-9-1 Bunkyo

country: Japan

institute: University of Fukui

summary:
The addition of 2ME2 (HIF-1 inhibitor) to rat model of galactose-induced cataract decreased opacity and therapeutic effect. Genes regulated by 2ME2 addition were identified by microarray analysis.
A total of 7 samples were analyzed: samples with no cataract (Control_Day4, Control_Day6), samples with cataract (Galactose-1, Galactose-2, Galactose-3), samples with decreased lens opacity (2ME2, 2ME2-2)

overall_design: Genes whose expression increased  from Control to Galactose and decreased from Galactose to 2ME2 were extracted.

first: Masaya

last: Nagaya

first: Yoshihiro

last: Takamura

last: Inatani

last: Oki


title: Oxidative phosphorylation is a pivotal therapeutic target of fibrodysplasia ossificans progressiva

city: Kyoto

first: Yonghui

last: Jin

address: 53 Shogoin Kawahara-cho, Sakyo-ku

department: Institute for Life and Medical Sciences

institute: Kyoto University

summary: Heterotopic ossification (HO) is a non-physiological process of bone formation in which progenitor cells in soft tissues differentiate into chondrogenic cells. In the case of fibrodysplasia ossificans progressiva (FOP), a rare genetic disease characterized by progressive and systemic HO, the Activin A/mutated-ACVR1/mTORC1 cascade induces HO in progenitors, one of which is the fibro/adipogenic progenitors (FAPs) in muscle tissues. The relevant biological processes aberrantly regulated by activated mTORC1 remain unclear, however. RNA sequencing analyses revealed the enrichment of genes involved in oxidative phosphorylation (OXPHOS) during Activin A-induced chondrogenesis of mesenchymal stem cells derived from induced pluripotent stem cells (iPSCs) of FOP patients. Functional analyses showed a metabolic transition from glycolysis to OXPHOS during chondrogenesis, in conjunction with an upregulation of mitochondrial biogenesis. mTORC1 inhibition by rapamycin suppressed OXPHOS, and IACS-010759, an inhibitor of OXPHOS, inhibited cartilage matrix formation in vitro, indicating that OXPHOS is principally involved in mTORC1-induced chondrogenesis. Furthermore, IACS-010759 inhibited the muscle injury-induced enrichment of FAP genes and HO in transgenic mice carrying the mutated human ACVR1. These data indicated that OXPHOS is a critical downstream mediator of mTORC1 signaling in chondrogenesis and therefore is a potential FOP therapeutic target.

overall_design: Gene expression at day 0 and day 6 after Activin-A-induced chondrogenesis.

first: Liping

last: Sun

first: Junya

last: Toguchida


overall_design: To investigate the inhibitory effect of OXPHOS blocker-IACS on HO, mouse gastrocnemius was pinched after inducing FOP-ACVR1 expression by doxcycline (DOX) administration. At day 7 after pinch-injury, total RNA was extracted from gastrocnemius. Comparative gene expression profiling analysis of RNA-seq data for 4 groups: G1-DOX(-), PINCH(-); G2-DOX(-), PINCH(+); G3-DOX(+), PINCH(+); G4-DOX(+), PINCH(+), IACS(+).


title: Histone ChIP-seq from BLaER1 (ENCSR580GOD)

status: Public on Feb 27 2024

city: Stanford

first: ENCODE

last: DCC

state: CA

address: 300 Pasteur Dr

country: USA

institute: ENCODE DCC

type: Genome binding/occupancy profiling by high throughput sequencing

summary:
H3K4me1 ChIP-Seq on BLaER1 cell line after 36 h treatment with 100 nM 17β-estradiol, 10 ng/mL Interleukin-3, 10 ng/mL CSF1.

For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODE_Data_Use_Policy_for_External_Users_03-07-14.pdf


title: Analysis of Single Nuclear Chromatin Accessibility Demonstrates Unique Myeloid Populations Differentially Influence Prognosis in Human Pancreatic Ductal Adenocarcinoma [snMultiome]

status: Public on Feb 15 2024

city: Morgantown

first: Gangqing

last: Hu

state: WV

address: 64 Medical Center Drive

institute: West Virginia University

summary:
Background: A better understanding of the pancreatic adenocarcinoma (PDAC) immune microenvironment is critical to improving outcomes. Myeloid cells are of particular importance for PDAC progression. Circulating monocytes are recruited and hijacked by the tumor to become immune suppressive tumor-associated macrophages. Meanwhile, tissue resident macrophages are innately anti-inflammatory and pro-fibrotic. Open chromatin and transcription factor activity provide source and function data for myeloid cells. Thus, we explore single nuclear assay for transposase accessible chromatin (ATAC) sequencing (snATAC-Seq), a method to analyze open gene promoters and transcription factor binding, as an important means for discerning the myeloid composition in human PDAC tumors.
Methods: Frozen pancreatic tissues (benign or PDAC) were digested into single nuclei suspensions. snATAC-Seq and sn-Multiome (ATAC + RNA)  libraries were prepared using 10X Chromium technology and sequenced by an Illumina sequencer. Signac was used for preliminary analysis, clustering, and differentially accessible chromatin region identification. snMultiome data was used to validate cell annotations for the snATAC-Seq data.
Results: Unique populations comprised the benign and tumor samples. Myeloid cell transcription factor activities were higher in tumor samples than benign pancreatic samples, enabling us to further stratify myeloid populations in PDAC tumors. GO enrichment analysis showed myeloid cells can activate immune response in tumors. Subcluster analysis revealed eight distinct myeloid populations. GO enrichment demonstrated unique functions of different myeloid populations, including IL-1b signaling (recruited monocytes) and intracellular protein transport (dendritic cells).
Conclusions: These data suggest snATAC-Seq as a method for retrospective analysis of frozen human pancreatic tissues to identify myeloid populations. Understanding the source specific potential function of myeloid cells is important for patient prognosis in PDAC.

overall_design: To validate findings from snATAC-Seq analysis, nuclei were isolated from frozen pancreatic tissue from one patient undergoing pancreaticoduodenectomy for PDAC. The normal adjacent and PDAC tumor were compared.  Patient received neoadjuvant chemotherapy.

first: Hillary

middle: G

last: Pratt

first: Li

last: Ma


title: Analysis of Single Nuclear Chromatin Accessibility Demonstrates Unique Myeloid Populations Differentially Influence Prognosis in Human Pancreatic Ductal Adenocarcinoma [snATAC]

overall_design: Nuclei were isolated from 50mg of frozen pancreatic tissue from patients undergoing pancreaticoduodenectomy (benign pancreas or PDAC). Four benign pancreata and 4 PDAC tumors were compared. None of the patients received neoadjuvant chemotherapy.


title: Inhibition of BRD4 Attenuates ER Stress-induced Renal Ischemic-Reperfusion Injury

last: Aransay

summary: Renal ischemia-reperfusion injury (IRI) leads to endoplasmic reticulum (ER) stress, thereby initiating the unfolded protein response (UPR). When sustained, this response may trigger the inflammation and tubular cell death that acts to aggravate the damage. Here, we show that knockdown of the BET epigenetic reader BRD4 reduces the expression of ATF4 and XBP1 transcription factors under ER stress activation. BRD4 is recruited to the promoter of these highly acetylated genes, initiating gene transcription. Administration of the BET protein inhibitor, JQ1, one hour after renal damage induced by bilateral IRI, reveals reduced expression of ATF4 and XBP1 genes, low KIM-1 and NGAL levels and recovery of the serum creatinine and blood urea nitrogen levels. To determine the molecular pathways regulated by ATF4 and XBP1, we performed stable knockout of both transcription factors using CRISPR-Cas9 and RNA sequencing. The pathways triggered under ER stress were mainly XBP1-dependent, associated with an adaptive UPR, and partially regulated by JQ1. Meanwhile, treatment with JQ1 downmodulated most of the pathways regulated by ATF4 and related to the pathological processes during exacerbated UPR activation. Thus, BRD4 inhibition could be useful for curbing the maladaptive UPR activation mechanisms, thereby ameliorating the progression of renal disease.

overall_design: Samples were analyzed by duplicate using biological repliques. Untreated  and unstimulated cells were used as control cells. Cells stimulated with Thapsigargin were used to activate ER stress. Analyzed conditions were: untreated and unstimulated cells. cells estimulated with thapsigargin, cells stimulated with thapsigargin and treated with JQ1, cells knocked for XBP1 gene and stimulated with thapsigargin and cells knocked for ATF4 gene and stimulated with thapsigargin

first: Paula

last: Diaz-Bulnes

first: Ramon

last: Rodriguez

first: Elisenda

last: Banon-Maneus

first: Maria

middle: L

last: Saiz

first: Viviana

last: Corte-Iglesias

middle: J

last: Ramirez

first: Marta

last: Lazo-Rodriguez

first: Isaac

last: Tamargo Lopez

first: Raul

last: Rodrigues Diez

first: Carmen

last: Diaz-Corte

last: Ruiz-Ortega

first: Fritz

last: Diekmann

first: Carlos

last: López-Larrea

first: Beatriz

last: Suarez Alvarez


title: Mannose remodels glucose metabolism to antagonize the overwhelming host inflammatory response

city: HongKong

first: Shuofeng

last: Yuan

address: 102 Pok Fu Lam Road

country: Hong Kong

institute: The University of Hong Kong

summary: we performed transcriptomic analysis in A549 cells and demonstrated a reduced proinflammatory response but increased host tolerance upon H1N1 virus infection and mannose therapy, providing further evidence for the beneficial role of mannose in immunometabolism.

overall_design: The lung epithelial A549 cells were pre-treated with 25mM mannose or PBS control overnight. The cells were then infected with H1N1 (2MOI) and incubated in DMEM medium with 25mM mannose or PBS control. At 24hpi, total cellular RNAs of the virus-infected-PBS, virus-infected-mannose, or non-infected groups (n =3) were collected. Total RNA was extracted with the RNeasy Mini Kit(Qiagen, Cat. No. / ID: 74104). Sequencing libraries were constructed and sequenced in the Proteomics and Metabolomics Core (LKS Faculty of Medicine, The University of Hong Kong). After filtering the low-quality reads, clean reads were mapped to reference sequences using HISAT/Bowtie2. The DEGs in treated samples were submitted to DAVID server for pathway enrichment and cluster analysis. Gene Ontology (GO) enrichment analysis of DEGs was implemented using the GOseq R package. KOBAS software was used to examine the statistical enrichment of DEGS in The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

first: Ronghui

last: Liang


title: Non-severe thermal burn injuries induce long lasting gene expression changes in specific cortical neuron and glia cell types

city: Perth

first: Alexander

last: Tang

state: Western Australia

address: 35 Stirling Highway, University of Western Australia

department: School of Biomedical Science

country: Australia

institute: University of Western Australia

summary: Burn injuries are devastating traumas, often leading to life-long consequences that extend beyond the observable burn scar. Burn injury patients commonly develop chronic neurological disorders but the long-lasting impacts of burn injuries on neurons and glia in the brain is unknown. Whole transcriptome RNA-sequencing from cortical excitatory neurons, inhibitory neurons, astrocytes and microglia showed very few changes to the expression of genes with known functions five weeks following a non-severe burn injury in adult mice. However, genes related to GABA-A receptors in excitatory neurons and several cellular functions in microglia was found to be to differentially expressed in burn injured mice. These findings shed light on the long-term effect of burn injuries on the brain and may help identify potential therapeutic targets and windows to prevent neurological dysfunction in burn patients.

overall_design: Differential gene expression analysis of whole transcriptome sequencing data from mice that received a non-severe thermal burn injury compared to a sham injury for four different, isolated cortical cell types (excitatory neurons, inhibitory neurons, astrocytes and microglia).

middle: D

first: Rebecca

middle: C

last: Ong


title: Analysis of Single Nuclear Chromatin Accessibility Demonstrates Unique Myeloid Populations Differentially Influence Prognosis in Human Pancreatic Ductal Adenocarcinoma

first: Gangqing

last: Hu

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

overall_design: Refer to individual Series


title: ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline [RNA-Seq]

status: Public on Feb 14 2024

city: Davis

first: Satoshi

last: Namekawa

address: One Shields Avenue

institute: UC Davis

summary:
H3K9 tri-methylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that, in male meiosis, ATF7IP2 amasses on autosomal and X pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase. Analyses of ATF7IP2-deficient meiosis reveal the protein’s essential roles in the maintenance of MSCI, suppression of retrotransposons, and global activation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.
We performed gene expression analysis using data obtained from RNA-seq of Atf7ip2+/+ and Atf7ip2-/- pachytene spermatocytes.

overall_design: Comparative gene expression analysis of RNA-seq data for Atf7ip2+/+ and Atf7ip2-/- pachytene spermatocytes.

first: Mengwen

middle: H


title: ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline


title: A distinct feed-forward mechanism between GREB1 and steroid receptors dictates hormone-dependent GREB1 action in endometrial function and dysfunction

city: Houston

first: Hari

middle: Krishna

last: Yalamanchili

state: Texas

address: 1250 Moursund St

department: Pediatrics

institute: Children’s Nutrition Research Center

summary: The cellular responses to the steroid hormones, estrogen (E2), and progesterone (P4) are governed by their cognate receptor’s transcriptional output. Although multiple mechanisms including the tissue-specific coactivators dictate these transcriptional signatures, the feed-forward mechanisms, that evolved to coordinate the cell-type specific transcriptional fulcrums for steroid receptors are not yet identified. In this report, we found that a common feed-forward mechanism between GREB1 and steroid receptors governs the differential effect of GREB1 on steroid hormones in a physiological or pathological context. We found that in physiological (receptive) endometrium, GREB1 is essential for progesterone but not estrogenic responses, whereas in endometriosis, an E2-dependent endometrial pathology, it pre-dominantly mediates estrogenic actions. Specifically, the deletion of GREB1 curtails endometrial receptivity and decidualization, owing to impaired P4-dependent stromal cell proliferation. In contrast, GREB1 is not required for E2-mediated epithelial proliferation of the receptive uterus. Of mechanism, in receptive endometrium, progesterone-induced GREB1 physically interacts with the progesterone receptor on chromatin and functions as a cofactor in a positive feedback mechanism to selectively regulate P4-responses. In pathological conditions (endometriosis), ablation of GREB1 led to the development of smaller endometriotic lesions in mice and reduced E2-driven proliferation of human endometriotic cells. Moreover, E2-induced GREB1 modulated the E2-dependent target gene expression in a feed-forward mechanism. Collectively, acting as a downstream effector, GREB1 governs either P4-responses or E2-responses depending on the physiological or pathological setting. Importantly, this differential action of GREB1 is exerted by a common feed-forward mechanism between GREB1 and steroid receptors, which conceptually advances our understanding of the mechanism(s) that underlie the distinct cell- and tissue-specific actions of steroid hormones.

overall_design: Cut n Run for GREB1 binding in human endometrial stromal cells

first: Sangappa

middle: B

last: Chadchan

first: Pooja

last: Popli

first: Zian

last: Liao

first: Eryk

last: Andreas

first: Michelle

last: Dias

first: Tianyuan

last: Wang

first: Stephanie

last: Gunderson

first: Patricia

last: Jimenez

first: Denise

last: Lanza

first: Rainer

last: Lanz

first: Charles

middle: E

last: Foulds

first: Diana

last: Monsiavas

first: Francesco

last: DeMayo

middle: K

first: Emily

middle: S

last: Jungheim

first: Jason

last: Heaney

first: John

middle: P

last: Lydon

first: Kelle

last: Moley

first: Bert

middle: W

last: O’Malley

first: Ramakrishna

last: Kommagani


title: Lactate regulates major zygotic genome activation by H3K18 lactylation in mammals

city: Harbin

first: Hou

middle: wei

last: bo

state: Heilongjiang

address: Xiangfang District

country: China

institute: College of Life Science,Northeast Agricultural University


title: ATF7IP2/MCAF2 directs H3K9 methylation and meiotic gene regulation in the male germline [CUT&RUN, CUT&Tag]

first: Satoshi

last: Namekawa

summary:
H3K9 tri-methylation (H3K9me3) plays emerging roles in gene regulation, beyond its accumulation on pericentric constitutive heterochromatin. It remains a mystery why and how H3K9me3 undergoes dynamic regulation in male meiosis. Here, we identify a novel, critical regulator of H3K9 methylation and spermatogenic heterochromatin organization: the germline-specific protein ATF7IP2 (MCAF2). We show that, in male meiosis, ATF7IP2 amasses on autosomal and X pericentric heterochromatin, spreads through the entirety of the sex chromosomes, and accumulates on thousands of autosomal promoters and retrotransposon loci. On the sex chromosomes, which undergo meiotic sex chromosome inactivation (MSCI), the DNA damage response pathway recruits ATF7IP2 to X pericentric heterochromatin, where it facilitates the recruitment of SETDB1, a histone methyltransferase that catalyzes H3K9me3. In the absence of ATF7IP2, male germ cells are arrested in meiotic prophase. Analyses of ATF7IP2-deficient meiosis reveal the protein’s essential roles in the maintenance of MSCI, suppression of retrotransposons, and global activation of autosomal genes. We propose that ATF7IP2 is a downstream effector of the DDR pathway in meiosis that coordinates the organization of heterochromatin and gene regulation through the spatial regulation of SETDB1-mediated H3K9me3 deposition.
CUT&RUN of H3K9me3 in Atf7ip2+/+ and Atf7ip2-/- pachytene spermatocytes and CUT&Tag of ATF7IP2 in C57/B6 WT pachytene spermatocytes.

overall_design: Chromatin profiling analysis using CUT&Tag in WT pachytene spermatocytes; and quantitative CUT&RUN in Atf7ip2+/+ and Atf7ip2-/- pachytene spermatocytes.

first: Mengwen

last: Hu


title: Gene accessibility analysis of major histocompatibility complex II (MHCII) low and MHCII high, Tspan8-positive and Tspan8-negative medullary thymic epithelial cells.

city: Marburg

first: Kristin

last: Rattay

address: Karl-von-Frisch-Strasse 2

country: Germany

institute: University of Marburg

summary: ATAC sequencing of Tspan8posMHCIIlo, Tspan8posMHCIIhi, Tspan8negMHCIIlo and Tspan8negMHCIIhi mTECs was performed in biological duplicates.

overall_design: Comparative ATAC profiling analysis of ATAC-seq data for MHCII low and MHCII high, Tspan8-positive and Tspan8-negative medullary thymic epithelial cells

first: Sören

last: Lammers

first: Philip

last: Brennecke

first: Lars

last: Steinmetz


title: Mapping the developmental potential of mouse inner ear organoids at single-cell resolution

status: Public on Feb 13 2024

city: Ann Arbor

first: Joerg

last: Waldhaus

state: MI

address: Medical Sciences I Building, 1150 West Medical Center Drive

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

institute: University of Michigan

summary: Inner ear organoids recapitulate development and are intended to generate cell types of the otic lineage for applications such as basic science research and cell replacement strategies. Here, we use single-cell sequencing to study the cellular heterogeneity of late-stage mouse inner ear organoid sensory epithelia, which we validated by comparison with data sets of the mouse cochlea and vestibular epithelia. We resolved supporting cell sub-types, cochlear like hair cells, and vestibular Type I and Type II like hair cells.  While cochlear like hair cells aligned best with an outer hair cell trajectory, vestibular like hair cells followed developmental trajectories similar to in vivo programs branching into Type II and then Type I extrastriolar hair cells. These results highlight the transcriptional accuracy of the organoid developmental program but will also inform future strategies to improve synaptic connectivity and regional specification.

overall_design: Examination of transcriptional profiles of inner ear organoids at 16, 20, and 21 days-in-vitro (DIV). Samples prepared at 21 DIV were divided into GFP high and GFP low groups.

first: Keith

last: Duncan

first: Linghua

last: Jiang

first: Liqian

last: Liu


title: Gene expression analysis of major histocompatibility complex II (MHCII) low and MHCII high, Tspan8-positive and Tspan8-negative medullary thymic epithelial cells.

summary: RNA sequencing of Tspan8posMHCIIlo, Tspan8posMHCIIhi, Tspan8negMHCIIlo and Tspan8negMHCIIhi mTECs was performed in biological duplicates.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for MHCII low and MHCII high, Tspan8-positive and Tspan8-negative medullary thymic epithelial cells


title: Lactate regulates major zygotic genome activation by H3K18 lactylation in mammals [CUT&TAG]

summary: Lactate is present at a high level in the microenvironment of mammalian preimplantation embryos in vivo and in vitro. However, its role in preimplantation development is unclear. Here, we report that lactate is highly enriched in the nuclei of early embryos when major zygotic genome activation (ZGA) occurs in humans and mice. The inhibition of its production and uptake results in developmental arrest at the 2-cell stage, major ZGA failure, and loss of lactate-derived H3K18lac, and the abnormal phenotypes could be recapitulated by overexpression of H3K18R mutation and rescued by addition of Lac-CoA. By profiling the landscape of H3K18lac in human and mouse preimplantation embryos, we show that H3K18lac is enriched on the promoter regions of most major ZGA genes and corelates with their expressions. Taken together, we demonstrate the important role for lactate in major ZGA via H3K18lac, showing a conserved metabolic mechanism underlies preimplantation development of mammalian embryos.

overall_design: CUT&Tag was performed using the Hyperactive In-Situ ChIP Library Prep Kit for Illumina (Vazyme Biotech). Embryos were incubated in 50 µL antibody buffer; 0.5 μg antibody was added and the mixture was incubated at 4℃ overnight. After washing twice with dig-wash buffer, 50 µL dig-wash buffer with 0.2 µg secondary antibody was added and the mixture was incubated at room temperature for 1 h. After washing twice with 100 µL dig-wash buffer, 1 µL pG-Tn5 and 49 µL dig-300 buffer were added, and the samples were incubated at room temperature for 1 h. Next, the samples were washed twice with 100 µL dig-wash buffer. We subsequently added 200 µL tagmentation buffer and incubated the samples at 37°C for 1 h. The reaction was stopped with 7 µL 0.5 M EDTA, 2 µL 10% SDS, and 1.7 µL 20 mg/mL proteinase K; for single-cell CUT&Tag assays, 5 µL stop buffer was used. After extraction with phenol-chloroform and ethanol precipitation, PCR was performed to amplify the libraries under the following conditions: 72°C for 3 min, 98°C for 30 s, 17 cycles of 98°C for 10 s and 60°C for 5 s, and a final extension at 72°C for 1 min with a hold at 4°C. For single-cell CUT&Tag of human embryos, CUT&Tag was performed in well of a microplate. After tagmentation, single human embryos were transferred to 200µl tubes containing 5% Proteinase K. After embryos lysis, PCR buffer and adaptors were directly added to tubes, normal PCR cycle was performed. Post-PCR clean-up was performed by adding a 1.5× volume of DNA Clean Beads (Vazyme Biotech). Libraries were sequenced on the Illumina NovaSeq 6000 platform according to the manufacturer’s instructions.

first: Jingyu

last: Li

first: Weibo

last: Hou

first: Qi

last: Zhao

first: Wei

last: Han

first: Jianzhong

last: Su

first: Shaorong

last: Gao

first: Guoning

last: Huang

first: Qingran

last: Kong


title: Lung lymphatic endothelial cells undergo inflammatory and prothrombotic changes in a model of chronic obstructive pulmonary disease

status: Public on Feb 12 2024

city: New York

first: Tyler

last: Lu

state: New York

address: 1300 York Avenue

institute: Weill Cornell Medicine

summary: The lymphatic vasculature regulates lung homeostasis  through drainage of fluid and trafficking of immune cells and plays a key role in the response to lung injury in several disease states. We have previously shown that lymphatic dysfunction occurs early in the pathogenesis of chronic obstructive pulmonary disease (COPD) caused by cigarette smoke (CS) and that this is associated with increased thrombin and fibrin clots in lung lymph. However, the direct effects of CS and thrombin on lymphatic endothelial cells (LECs) in COPD are not entirely clear. Studies of the blood vasculature have shown that COPD is associated with increased thrombin, which causes endothelial dysfunction after CS exposure and is characterized by changes in the expression of coagulation factors and leukocyte adhesion proteins. Here, we determined whether similar changes occur in LECs. We used an in vitro cell culture system and treated human lung microvascular lymphatic endothelial cells with cigarette smoke extract (CSE) and/or thrombin. We found that CSE treatment led to decreased fibrinolytic activity in LECs, which was associated with increased expression of plasminogen activator inhibitor 1 (PAI-1). LECs treated with both CSE and thrombin together had a decreased expression of TFPI and an increased expression of vascular adhesion molecules. RNA sequencing of lung LECs isolated from mice exposed to CS also showed upregulation of prothrombotic and inflammatory pathways at both acute and chronic exposure time points. Analysis of publicly available single-cell RNA sequencing of LECs as well as immunohistochemical staining of lung tissue from COPD patients supported these data and showed an increased expression of inflammatory markers in LECs from COPD patients compared to those from controls. These studies suggest that in parallel with blood vessels, the lymphatic endothelium undergoes inflammatory changes associated with CS exposure and increased thrombin in COPD. Further research is needed to unravel the mechanisms by which these changes affect lymphatic function and drive tissue injury in COPD.

overall_design:
Murine lung tissues were dissociated by Collagenase Dispase digestion and stained for Lineage cocktail, EPCAM, and CD31. Lymphatic endothelial cells were sorted as live (Dapi negative), Lineage&EPCAM negative, CD31 positive, and Prox-1 positive (eGFP reporter) cells. Sorted lymphatic endothelial cells were processed for RNA isolation using a Qiagen Mini Kit and RNA was sequenced.

*** Raw data will be provided.

first: Anjali

last: Trivedi

first: Raphaël

last: Lis

first: Hasina

middle: O

last: Reed


title: Ankylosing spondylitis patients present a distinct CD8 T-cell subset with osteogenic and cytotoxic potential

city: Bellinzona

first: Veronica

last: Martini

state: Ticino

address: Via Francesco Chiesa 5

country: Switzerland

institute: Institute for Research in Biomedicine

summary: Ankylosing Spondylitis (AS) is a chronic inflammatory rheumatic disease affecting mainly the axial skeleton. Peripheral involvement (arthritis, enthesitis and dactylitis) and extra-musculoskeletal manifestations including uveitis, psoriasis and bowel inflammation occur in a relevant proportion of patients. AS is responsible for chronic and severe back pain caused by local inflammation that can lead to osteoproliferation and ultimately spinal fusion. The association of AS with the Human Leukocyte Antigen (HLA)‑B27 gene, together with elevated levels of chemokines CCL17 and CCL22 in the sera of patients with AS, lead us to study the role of CCR4+ T-cells in the disease pathogenesis.  Here we show that, CD8+ CCR4+ T-cells display a distinct effector phenotype and upregulate the inflammatory chemokine receptors CCR1, CCR5, CX3CR1 and the L‑selectin CD62L, indicating an altered migration ability. Functionally, CD8+CCR4+T‑cells expressing CX3CR1 present an enhanced cytotoxic profile, expressing both perforin and granzymeB. Of note, these cells, isolated from patients in the active state of the disease, upregulate genes promoting the ossification process. Our results shed light to a new molecular mechanism by which T-cells may promote new bone formation and contribute to the pathological ossification process observed in AS.

overall_design: CD8+CCR4+ T-cells were sorted from 5 healthy donors and 6 AS patients and bulk RNA sequencing was perfomred on the isolated RNA using the NextSeq 500 or 600 (Illumina). RNA sequencing reads were aligned with the STAR-aligner. The Ensembl human genome build GRCh38 was used as reference. Gene expression values were computed with the function featureCounts from the R package Rsubread while gene expression analysis was performed using the BigOmics platform.

first: Valentina

last: Cecchinato

first: Ylenia

last: Silvestri

first: Andrea

last: Rinaldi

first: Mariagrazia

last: Uguccioni


title: Neutrophils drive skin autoinflammation by releasing IL-26

city: Lausanne

first: JEREMY

last: DI DOMIZIO

state: Vaud

address: Av Boveresses 155, 1066 Epalinges

institute: CHUV

summary: we profiled the inflammatory transcriptome of skin lesions from pustular psoriasis patients

overall_design: Inflammation-oriented transcriptomics was performed on RNA extracted from skin biopsies using NanoString technologies

first: Jeremy

last: Di Domizio


title: A common cellular response to broad splicing perturbations is characterized by metabolic transcript downregulation driven by the Mdm2-p53 axis.

city: Cambridge

first: Eliezer

last: Calo

state: MA

address: 31 Ames St

institute: Massachusetts Institute of Technology

summary: Disruptions in core cellular processes elicit stress responses that drive cell state changes leading to organismal phenotypes. Perturbations in the splicing machinery cause widespread mis-splicing, resulting in p53-dependent cell-state changes that give rise to cell-type specific phenotypes and disease. However, a unified framework for how cells respond to splicing perturbations, and how this response manifests itself in nuanced disease phenotypes, has yet to be established. Here, we show that a p53-stabilizing Mdm2 alternative splicing event and widespread downregulation of metabolic transcripts are common events that arise under various splicing perturbations in both cellular and organismal models. Together, our results classify a common cellular response to splicing perturbations, put forth a new mechanism behind the cell-type specific phenotypes that arise when splicing is broadly disrupted, and lend insight into the pleiotropic nature of the effects of p53 stabilization in disease.

overall_design: To assay gene expression and splicing changes that occur when the splicing machinery is perturbed, we knocked-down Eftud2 with two independent shRNAs (shRNA1, shRNA2) in R1-mESCs and performed RNA-Seq. Non-targeting shRNA (nt-shRNA) used as control

first: Jade

last: Varineau


title: Agents which upregulate CD55 [decay accelerating factor (DAF)] expression [Array]

city: Cleveland

first: Edward

middle: M Medof

last: MD

state: OH

address: 17 1/2 Peppercreek Dr

department: Pathology

institute: Case western Reserve University

summary: (Submitter supplied) Identification of transcription factors (TFs) which upregulate human CD55 expression. CD55 was originally described as a cellular complement regulator that protects self-cells from autologous complement attack.   It is now known as cellular regulator which controls many functions of cells, as examples T cell commitment to T effector cells vs Foxp3+ T regulatory cells, B2 cell Ab production, and receptor tyrosine kinase (RTK) growth factor receptor function. Overlap of the arrays identified the immunosuppressive TF Kruppel Like Factor 4 (KLF4).  The current study shows that CD55 functions jointly with KLF4.

overall_design:
Total RNA was extracted from either HUVEC or PMA (Phorbol 12-myristate 13-acetate), Simvastatin, Atorvastatin, CRP treated HUVEC and K562 or PMA treated K562 cells by QIAGEN mini RNeasy kit. Gene expression profiling was performed by Case Western Reserve University and the Ireland Cancer Center Gene Expression core facility using Affymetrix technology. All experiments were performed using oligonucleotide-based Human Genome U133 Plus 2.0 chips

*** Submitter states that raw data files were not retained by the Affymetrix Core. ***

first: Feng-Qi

last: An

first: M

last: Medof


title: UBTF tandem duplication acute myeloid leukemias are sensitive to menin inhibition [Cut&Run]

city: Memphis

first: Jeffery

last: Klco

state: TN

address: 262 Danny Thomas Place

institute: St Jude Children's Research Hospital

summary: UBTF tandem duplications (TD) have recently emerged as a subtype-defining alteration in pediatric acute myeloid leukemia (pAML), which is characterized by HOXB gene dysregulation and a poor response to conventional chemotherapy. Here, we use protein-protein interaction studies to show that UBTF-TD maintains interactions with components of the RNA pol I complex, while also engaging with a network of unique protein interactors specific to UBTF-TD, like KMT2A. These data suggest that UBTF- TD both preserves canonical UBTF functions and demonstrates gain of function activities. Furthermore, we show that UBTF-TD and KMT2A co-localize to key genomic targets dysregulated in UBTF-TD myeloid malignancies, like HOXB gene clusters and MEIS1. Using a protein degradation system, we show that stemness, proliferation, and the HOXB molecular signature are dependent on sustained UBTF-TD localization to chromatin. Finally, we show UBTF-TD leukemias are sensitive to menin inhibition—providing a viable therapeutic strategy for children with this high-risk AML subtype.

overall_design: CUT&RUN Genomic Occupancy Analysis of UBTF-TD in hematopoeitic cells and acute myeloid leukemia samples

middle: M

first: Juan

last: Barajas


title: Transcriptional gene expression regulation in medullary thymic epithelial cells by the transcription factors Ehf, Fezf2, Elf3 and Klf4

first: Kristin

last: Rattay

summary: ChIP sequencing of Ehf, Fezf2, Elf3 and Klf4 was performed on medullary thymic epithelial cells to analyze the role of Ehf-, Fezf2-, Elf3- and Klf4-dependent gene regulation in mTECs.

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for Ehf, Fezf2, Elf3, Klf4 and their respective input controls were analyzed for ChIP seq peaks.

first: Victor

last: Burgos

first: Shannan

last: Sui

first: Ulrich

last: von Andrian


title: UBTF tandem duplication acute myeloid leukemias are sensitive to menin inhibition [RNA-seq]

overall_design: Comparative gene expression profiling analysis of RNA-seq data for cbCD34+ cells expressing FKBP12-F36V-tagged UBTF-TD treated with DMSO or dTAG-13, or human bone marrow cells isolated from UBTF-TD PDX model treated with Veh or SNDX-5613


title: Quantitative multiplexed ChIP-Seq (MINUTE-ChIP) from formaldehyde-fixed material

status: Public on Feb 11 2024

city: Solna

first: Simon

last: Elsässer

address: Tomtebodavägen 23A

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

summary: In crosslinked MINUTE-ChIP, formaldehyde-fixed chromatin is fragmented using sonication, blunted and ligated to double-stranded DNA adaptors that include a T7 RNA polymerase promoter and a sample barcode sequence. Finally, samples are combined and subsequent ChIP reactions are performed with the pooled samples. ChIP material is prepared into an Illumina-compatible library using linear amplification by virtue of T7 RNA polymerase, reverse transcription and a low-cycle library PCR amplification. Here, we demonstrate a MINUTE-ChIP for Nanog from formaldehyde-fixed cells using fragmentation by sonication.

overall_design: For fixed anti-Nanog MINUTE-ChIP, Rw4 (129X1/SvJ) mouse embryonic stem cells were cultured in a naive pluripotent state in the presence of LIF (the Nanog-high condition). As a negative control, the cells were cultured in same medium ibut without LIF for 24 hr, then 48h in the presence of 2uM  retonoic acid (Sigma R2625), to yield a Nanog negative condition (Nanog is absent by western blot under this condition). 1 Mio cells in were barcoded in duplicates for each condition and pooled.

first: Banushree

last: Kumar

first: Hannah

last: Schwämmle


title: Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair [AmpliconSeq]

city: San Francisco

first: Kristen

last: Seim

address: 201 Haskins Way

institute: GraphiteBio

summary: Homology Directed Repair (HDR) enables precise genome editing and holds great promise in the gene therapy field. However, the implementation of HDR-based therapies is hindered by limited efficiency in comparison to methods that exploit alternative DNA repair routes, such as Non-Homologous End Joining (NHEJ). In this study, we demonstrate the development of a functional, pooled screening platform utilizing an HDR-based readout to identify protein-based reagents that improve HDR outcomes in human hematopoietic stem and progenitor cells (HSPCs), a clinically relevant cell type for gene therapy. We leveraged this screening platform to explore sequence diversity at the binding interface of the NHEJ inhibitor i53 and its target, 53BP1, and we identified optimized i53 variants that enable new intermolecular bonds and robustly increase HDR. These variants specifically reduce insertion-deletion outcomes and also synergize with a DNAPK inhibitor to increase HDR rates. When applied at manufacturing scale, the incorporation of improved variants results in a significant increase in cells with at least one repaired allele and improved HDR in long-term HSPCs subpopulations, while not increasing off-target editing or gross chromosomal rearrangements. We anticipate the pooled screening platform will enable discovery of future gene editing reagents that improve HDR outcomes, such as the i53 variants reported here.

overall_design: we describe the design and implementation of a pooled screen to identify protein variants with optimized HDR boosting capabilities.

middle: A

last: Perez-Bermejo


title: Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair [i53]

middle: L


title: Nuclear Morphology is Instructed by the Loop Extrusion Program [HiC]

city: SAN DIEGO

first: Indumathi

last: Patta

address: 9500 Gilman Drive, NSB5114A, LaJolla

department: Molecular Biology

institute: UCSD

summary:
More than a century ago Metchnikoff defined neutrophils as small amoeboid-like cells that harbored peculiar polymorphonuclear structures. While since these early studies much has been learned about neutrophil physiology, mechanistic insight into neutrophil polymorphonuclear assembly remains rudimentary. Here we found that depletion of factors, that initiate the loop extrusion program, including NIPBL and MAU2, greatly accelerated the conversion of mononuclear to polymorphonuclear cells and orchestrated the induction of a neutrophil specific transcription signature. Remarkably, mere ablation of either NIPBL or MAU2 expression under conditions that normally prevent neutrophil differentiation, swiftly converted mono-nuclear into polymorphonuclear cells that expressed a neutrophil specific gene program.  Depletion of NIPBL and MAU2 in neutrophil progenitors activated an armamentarium of neutrophil-specific enhancers to establish a neutrophil genome depleted for loop extrusion-induced looping. These observations indicate that extinction of the loop extrusion program converts mononuclear progenitor cells into polymorphonuclear cells and suffices to induce a neutrophil specific program of gene expression.
Expression profiling by high throughput sequencing

overall_design: Hi-C was performed using two independent replicates of NIPBL-FKBP12F36V-EYFP progenitor cells that were cultured in the presence of β-estradiol and in the presence or absence of dTAG-13 for 72hrs

first: Ming

last: Hu

first: Cornelis

last: Murre


title: Nuclear Morphology is Instructed by the Loop Extrusion Program [ChIP-Seq]

summary: More than a century ago Metchnikoff defined neutrophils as small amoeboid-like cells that harbored peculiar polymorphonuclear structures. While since these early studies much has been learned about neutrophil physiology, mechanistic insight into neutrophil polymorphonuclear assembly remains rudimentary. Here we found that depletion of factors, that initiate the loop extrusion program, including NIPBL and MAU2, greatly accelerated the conversion of mononuclear to polymorphonuclear cells and orchestrated the induction of a neutrophil specific transcription signature. Remarkably, mere ablation of either NIPBL or MAU2 expression under conditions that normally prevent neutrophil differentiation, swiftly converted mono-nuclear into polymorphonuclear cells that expressed a neutrophil specific gene program.  Depletion of NIPBL and MAU2 in neutrophil progenitors activated an armamentarium of neutrophil-specific enhancers to establish a neutrophil genome depleted for loop extrusion-induced looping. These observations indicate that extinction of the loop extrusion program converts mononuclear progenitor cells into polymorphonuclear cells and suffices to induce a neutrophil specific program of gene expression.

overall_design: ChIP-Sequencing for RAD21 in progenitors and NIPBL-depleted progenitors


title: Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair

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

overall_design: Refer to individual Series


title: Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair [scRNA-Seq]


title: Nuclear Morphology is Instructed by the Loop Extrusion Program [RNA-seq]

summary:
More than a century ago Metchnikoff defined neutrophils as small amoeboid-like cells that harbored peculiar polymorphonuclear structures. While since these early studies much has been learned about neutrophil physiology, mechanistic insight into neutrophil polymorphonuclear assembly remains rudimentary. Here we found that depletion of factors, that initiate the loop extrusion program, including NIPBL and MAU2, greatly accelerated the conversion of mononuclear to polymorphonuclear cells and orchestrated the induction of a neutrophil specific transcription signature. Remarkably, mere ablation of either NIPBL or MAU2 expression under conditions that normally prevent neutrophil differentiation, swiftly converted mono-nuclear into polymorphonuclear cells that expressed a neutrophil specific gene program.  Depletion of NIPBL and MAU2 in neutrophil progenitors activated an armamentarium of neutrophil-specific enhancers to establish a neutrophil genome depleted for loop extrusion-induced looping. These observations indicate that extinction of the loop extrusion program converts mononuclear progenitor cells into polymorphonuclear cells and suffices to induce a neutrophil specific program of gene expression.
Performed gene expression profiling by RNA-seq in progenitors and differentiated neutrophils.

overall_design: We performed RNA-seq of NIPBL, MAU2, and both NIPBL-MAU2 depleted progenitors as well as differentiated neutrophils for gene expression profiling analysis.

first: Kathleen

last: Fish


title: Functional screening in human HSPCs identifies optimized protein-based enhancers of Homology Directed Repair [Amplicon]


title: STING signalling compensates for low tumour mutation burden to drive anti-tumour immunity

status: Public on Feb 10 2024

city: Duarte

first: Peter

last: Lee

address: 1500 E. Duarte Rd.

department: Immuno-Oncology

institute: Beckman Research Institute at City of Hope

summary: While mutation-derived neoantigens are well recognized in generating anti-tumor T cell responses, increasing evidences highlight the complex association between tumor mutation burden (TMB) and tumor infiltrating lymphocytes (TILs). We examined this relationship across nine major cancer types to identify non-TMB determinants of immune responses within the tumor microenvironment. TMB overall correlated poorly with both TILs and exhausted CD8+ T cells (Tex) as an indicator of tumor-specific T cells. Computational clustering analysis performed on 4,510 tumors from The Cancer Genome Atlas revealed a group of tumors with abundant Tex but low TMB. In those tumors, we observed significantly higher expression of the stimulator of interferon genes (STING) signaling. Dendritic cells, particularly those of BATF3+ lineage, were also found to be essential for accumulation of Tex within tumors. Mechanistically, loss of genomic and cellular integrity, marked by decreased DNA damage repair, defective replication stress response, and increased apoptosis were shown to drive STING activation. These results highlight that TMB alone does not fully predict tumor immune profiles, with STING signaling compensating for low TMB in non-hypermutated tumors to enhance anti-tumor immunity. We further validated these findings using clinical samples via NanoString technology and single cell RNA-seq. Translating these results, STING agonists may benefit patients with non-hypermutated tumors. STING activation may serve as an additional biomarker to predict response to immune checkpoint blockades alongside TMB. Our research also unraveled the interplay between genomic instability and STING activation, informing potential combined chemotherapy targeting the axis of genomic integrity and immunotherapy.

overall_design: We employed single cell sequencing from 11 primary tumors (PTs) of patients with breast cancer (BC), including 6 ER+ and 5 triple negative BC, followed by unbiased Seurat cluster analysis to assess the STING singaling in each cell type. Additional 3 ER+ BC PT scRNA-seq data for this work have been uploaded to GSE248288.

first: Jiayi

last: Tan

first: Weihua

last: Guo

first: Colt

last: Egelston

first: Jinhui

last: Wang

first: Xiwei

last: Wu


title: Nuclear Morphology is Instructed by the Loop Extrusion Program [ATAC-Seq]

overall_design: ATAC-Seq of NIPBL depleted progenitors treated with estradiol and dTAG


title: Characterization of a drug-resilient cancer cell phenotype [RNA-Seq 1]

city: Lund

first: Christopher

middle: Paul

last: Carroll

state: Skåne

address: BMC, B11

department: Experimental Medical Science

institute: Lund University

summary: Resistance to treatment and post-therapy recurrence due to small populations of resistant cells remain core challenges in cancer therapy. The mechanisms underlying therapy resistance are undefined for many patients. Here, we demonstrate that some cancer cells survive therapeutic stress via a transient state of whole genome doubling. At the onset of the polyploidization program, we identified upregulation of key transcriptional regulators, including the early stress-response protein AP-1 and normoxic stabilization of HIF-2α. Moreover, chromatin accessibility is altered and expression of pRB is ablated, and we identified enrichment of AP-1 motif accessibility along with an upregulation of replication-dependent histone variants. Here we demonstrate that AP-1and HIF-2α regulate a therapy resilient and survivor phenotype in cancer cells. Consistent with this, genetic or pharmacologic targeting of AP-1 and HIF-2α reduced the frequency of surviving cells following treatment with chemotherapy. These results suggest a novel avenue for tackling chemotherapy-induced resistance in cancer.

overall_design: To establish a drug resistant populatio we treated cells wit LD50 cisplatin for 72h

first: Kenneth

last: Pienta

first: Arthur

last: Castro

first: Niklas

last: Engstrom

first: Emma

last: Hammarlund

first: Floris

last: Fiojer


title: Characterization of a drug-resilient cancer cell phenotype [ATAC-Seq]


title: HiCrayon reveals distinct layers of multi-state 3D chromatin organization

city: Omaha

first: Michael

middle: Jordan

last: Rowley

state: Nebraska

address: 42nd and Emile street

department: Genetics & Cell Biology

institute: University of Nebraska Medical Center

type:
Other
Third-party reanalysis

summary: The co-visualization of chromatin conformation with 1D 'omics data is key to the multi-omics driven data analysis of 3D genome organization. Chromatin contact maps are often shown as 2D heatmaps and visually compared to 1D genomic data by simple juxtaposition. While common, this strategy is imprecise, placing the onus on the reader to align features with each other. To remedy this, we developed HiCrayon, an interactive tool that facilitates the integration of 3D chromatin organization maps and 1D datasets. This visualization method integrates data from genomic assays directly into the chromatin contact map by coloring interactions according to 1D signal. HiCrayon is implemented using R shiny and python to create a graphical user interface (GUI) application, available in both web or containerized format to promote accessibility. HiCrayon is implemented in R, and includes a graphical user interface (GUI), as well as a slimmed-down web-based version that lets users quickly produce publication-ready images. We demonstrate the utility of HiCrayon in visualizing the effectiveness of compartment calling and the relationship between ChIP-seq and various features of chromatin organization. We also demonstrate the improved visualization of other 3D genomic phenomena, such as differences between loops associated with CTCF/cohesin vs. those associated with H3K27ac. We then demonstrate HiCrayon's visualization of organizational changes that occur during differentiation and use HiCrayon to detect compartment patterns that cannot be assigned to either A or B compartments, revealing a distinct 3rd chromatin compartment. Overall, we demonstrate the utility of co-visualizing 2D chromatin conformation with 1D genomic signals within the same matrix to reveal fundamental aspects of genome organization.

overall_design:
Hi-C samples from 8 cell lines in LCL, with replicates ranging from 3 to 34. The following shows cell line with replicate number; AK1: 9, GM12878: 34 (re-analyzed; raw files not uploaded. Only processed combination), GM12891: 4, GM12892: 3, GM13976: 16, GM13977: 16, GM18526: 20, GM19239: 24
Please note that the GM12878.hic was generated by re-analyzing the 'GSM1551550-GSM1551578' samples (in GSE63525) and the re-analyzed sample details are provided in the 're-analyzed_sample_accns.txt'

first: Ben

last: Nolan

first: Hannah

last: Harris

first: Achyuth

last: Kalluchi

first: Timothy

last: Reznicek

middle: T

last: Cummings

middle: J


title: Transcriptome and epigenome data analysis to reveal the key role of KAKU4 during leaf senescence in Arabidopsis

status: Public on Feb 09 2024

city: Beijing

first: Zhen

last: Su

address: #2 Yuanmingyuan Xilu

department: College of Biological Sciences

institute: China Agricultural University

summary: We conducted the combination analysis of transcriptomic and epigenomic data between kaku4-2 mutant and WT suggested that KAKU4 may inhibit the expression of a series of genes related to hormone signals and H2O2 metabolism by affecting the deposition of H3K27me3, thereby suppressing leaf senescence.

overall_design: Rosette leaves of the 6-week-old kaku4-2 mutant and WT plants were sampled for RNA-seq with three independent biological replicates. The ChIP-seq analyses of H3K27me3 were conducted using the 6-week-old rosette leaves of kaku4-2 mutant and WT.

first: Yaxin

last: Cao

first: Hengyu

last: Yan

first: Minghao

last: Sheng

first: Wenying

last: Xu


title: TOPBP1 Shapes XY Silencing Dynamics during Meiosis to Ensure Male Fertility

city: Ithaca

first: Andrew

last: Grimson

state: NY

address: 526 Campus Rd

department: Molecular and Cell Biology

institute: Cornell University

summary: TOPBP1 and other DNA damage response proteins play an important role in meiotic sex chromosome inactivation. We investigate a mutant mouse with a mutation in the BRCT5 domain of TOPBP1 and use single-cell RNA-seq to investigate the effect of the mutation on XY silencing.

overall_design: Single-cell suspensions were generated from mouse testes and sorted by fluorescence-activated cell sorting to enrich for spermatocytes and analyzed using scRNA-seq (10X Genomics Chromium).

first: Carolline

middle: F

last: Ascencao

first: Alexis

last: Dziubek

first: Elizabeth

last: Fogarthy

first: Paula

last: Cohen

first: Marcus

last: Smolka


title: Coactivator condensation drives cell lineage specification

status: Public on Feb 08 2024

city: Dallas

first: kenian

last: chen

state: TX

address: 5323 Harry Hines Boulevard

department: Population and Data Science

institute: UTsouthwestern

summary: During development, cells make switch-like decisions to activate new gene programs specifying cell lineage. The mechanisms underlying these decisive choices remain unclear. Here we show that the cardiovascular transcriptional coactivator, Myocardin (MYOCD), activates cell identity genes by concentration-dependent and switch-like formation of transcriptional condensates. MYOCD forms such condensates and activates cell identity genes at critical concentration thresholds achieved during smooth muscle cell and cardiomyocyte differentiation. The C-terminal disordered region of MYOCD is necessary and sufficient for condensate formation. Disrupting this region’s ability to form condensates disrupts gene activation. Rescuing condensate formation by replacing this region with disordered regions from functionally unrelated proteins rescues gene activation. Our findings demonstrate that MYOCD condensate formation is required for gene activation during differentiation. We propose that the formation of transcriptional condensates at critical concentrations of cell type-specific regulators provides a molecular switch underlying the activation of key cell identity genes during cell lineage specification.

overall_design: Comparative gene expression profiling analysis of RNA-seq data and MYOCD chromatin occupancy and chromatin accessibility (H3K27ac) by ChIP-seq data for 10T1/2 cells reprogrammed by GFP (control), MYOCD and its different mutants (FUS, deltaTAD, CDT1)

first: Peiheng

last: Gan

first: Mikayla

last: Eppert

first: Nancy

last: De La Cruz

first: Heankel

last: Lyons

first: Reshma

last: Veettil

first: Kenian

last: Chen

first: Akansha

last: Shah

first: Prashant

last: Pradhan

first: Svetlana

last: Bezprozvannaya

first: Lin

first: Ning

last: Liu

first: Eric

middle: N

last: Olson

first: Benjamin

middle: R

last: Sabari


title: Characterization of a drug-resilient cancer cell phenotype [RNA-Seq 2]

first: Christopher

last: Carroll

first: Kenneth

last: Pienta

first: Arthur

last: Castro

first: Niklas

last: Engstrom

first: Emma

last: Hammarlund

first: Floris

last: Fiojer


title: Characterization of a drug-resilient cancer cell phenotype


title: Deciphering cell states and genealogies of human hematopoiesis with single-cell multi-omics [Crispr_Mouse_Batch2]

status: Public on Feb 26 2024

city: Boston

first: Chen

last: Weng

address: 415 Main St, Cambridge

department: Genetics

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

first: Vijay

middle: G

last: Sankaran

first: Jonathan

last: Weissman


title: Nuclear Morphology is Instructed by the Loop Extrusion Program

first: Indumathi

last: Patta


title: Spatial immuno-oncology protein abundance profiles in hepatocellular carcinoma tissues from patients treated with bavituximab and pembrolizumab

first: Yujin

last: Hoshida

address: 5323 Harry Hines Blvd

institute: University of Texas Southwestern Medical Center

summary: Immune checkpoint inhibitors (ICIs) targeting PD-1/L1 have modest efficacy in hepatocellular carcinoma (HCC) as single agents, but combination regimens have demonstrated increased clinical benefit at the expense of treatment toxicities. Exposed phosphatidylserine suppresses the innate immune response in the tumor microenvironment (TME). Targeting phosphatidylserine does not induce tumor responses in the clinical setting when used as monotherapy or in combination with cytotoxic and targeted therapies, but may induce pro-inflammatory and –immune stimulating effects that enhance ICI activity. This hypothesis was tested in a single-arm phase 2 trial evaluating bavituximab, a phosphatidylserine targeting antibody, plus pembrolizumab in patients with unresectable HCC who had not received prior systemic treatments (ClinicalTrials.gov ID: NCT03519997). The primary end point was investigator-assessed objective response rate (ORR) among evaluable patients, and secondary end points included progression-free survival (PFS) and the incidence of adverse events. Among 28 evaluable patients, the ORR was 32.1%, including 2 complete responses, and a disease control rate of 64.3%. Median PFS was 6.3 months (95% CI, 1.3-11.3 months). Treatment related adverse events of any grade occurred in 45.7% of patients with grade 3 or 4 adverse events occurring in 14.3% of patients. Exploratory analyses of baseline tumor specimens showed that a depletion of B cells, and the expression of smooth muscle actin and immune checkpoints in stroma was associated with tumor response. An RNA-based TME assay which distinguishes immune-rich of inflammatory tumors was also shown to be predictive of ORR. These results suggest that targeting phosphatidylserine may lead to synergistic effects with ICIs without increasing toxicity rates, and future studies on this therapeutic strategy may be guided by biomarkers characterizing the TME.

overall_design: Pre-treatment tumor and liver biopsy tissues were obtained from patients with unresectable hepatocellular carcinoma treated with bavituximab and pembrolizumab, and subjected to the Digital Spatial Profiling GeoMx assay.

first: Naoto

last: Kubota

first: Asim

last: Hassan

first: Courtney

last: Katz

first: Soumith

last: Paritala

first: David

last: Hsiehchen


title: fhl2b expression ameliorates muscular dystrophy [5 months EOM and trunk]

city: Umeå

first: Nils

last: Dennhag

address: Linneaus väg 9

institute: Umeå Univeristy

summary: In muscle dystrophies, muscle fibers loose integrity and die, leading to significant suffering and a shorter life. Strikingly, the extraocular muscles (EOMs), controlling eye movements, are spared and function well despite the disease progression. Although EOMs have been shown to have important differences compared to body musculature the mechanisms underlying this inherent resistance to muscle dystrophies remain largely unknown. Here, we demonstrate important differences in gene expression as a response to muscle dystrophies between the EOMs and trunk muscle in zebrafish via transcriptomic profiling. We show that the LIM-protein Fhl2 is upregulated in response to knockout of desmin, plectin and obscurin, intermediate filament proteins causing different muscle dystrophies, and contributes to disease protection of the EOMs. Moreover, we show that ectopic expression of fhl2b can partially rescue the muscle phenotype in the zebrafish Duchenne muscular dystrophy model sapje, significantly improving their survival rate. Therefore, fhl2 is a protective agent and a candidate target gene for therapy of muscle dystrophies.

overall_design: To examine the innate resistance of extraocular muscle to muscular dystrophies we knocked down desmin and sequenced extraocular muscle as well as trunk muscle from zebrafish at two time points. We then performed gene expression analysis between WT and desmin knock-down tissues of the same type at the same time points.

first: Abraha

last: Kahsay

first: Itzel

last: Nissen

first: Maria

last: Cherminina

first: Hanna

last: Nord

first: Jiao

last: Liu

first: Anders

last: Arner

first: Ludvig

last: Backman

first: Silvia

last: Remeseiro

first: Jonas

last: von Hofsten

first: Fatima

last: Pedrosa Domellöf


title: fhl2b expression ameliorates muscular dystrophy [5dpf trunk]

overall_design: To examine the effect of fhl2b overexpression in muscle in the background of the dmd/sapje mutant line at 5 dpf


title: fhl2b expression ameliorates muscular dystrophy [20 months EOM and trunk]


title: A Group of Highly Secretory miRNAs Correlates with Lymph Node Metastasis and Poor Prognosis in Oral Squamous Cell Carcinoma

status: Public on Feb 07 2024

city: Wuhan

first: Gang

last: Chen

state: Hubei

address: Guangba Rd.

institute: Wuhan University

type: Non-coding RNA profiling by high throughput sequencing

summary: MicroRNAs (miRNAs) in oral squamous cell carcinoma (OSCC)-derived small extracellular vesicles (sEVs) play a pivotal role in modulating intercellular communications between tumor cells and other cells in microenvironment, thereby influencing tumor progression and the efficacy of therapeutic interventions. However, a comprehensive inventory of these secretory miRNAs in sEVs and their biological and clinical implications remains elusive. This study aims to profile the miRNA content of OSCC cell lines sEVs and computationally elucidate their biological and clinical relevance. We conducted miRNA sequencing to compare the miRNA profiles of OSCC cells and their corresponding sEVs. Our motif enrichment analysis identified specific sorting motifs that are implicated in either cellular retention or preferential sEVs secretion. Target cell analysis suggested that the sEVs miRNAs potentially interact with various immune cell types, including natural killer cells and dendritic cells. Additionally, we explored the clinical relevance of these miRNAs by correlating their expression levels with TNM stages and patient survival outcomes. Intriguingly, our findings revealed that a distinct sEVs miRNA signature is associated with lymph node metastasis and poorer survival in patients in TCGA-HNSC dataset. Collectively, this research furthers our understanding of the miRNA sorting mechanisms in OSCC and underscores their clinical implications.

overall_design: Extracellular vehicles (EVs) were harvested from cell culture supernatants using a ultracentrifugation protocol. First, we centrifuged the supernatants twice at 3000 g for 20 minutes at 4 °C to remove cell debris. Then, the cleared supernatant underwent ultracentrifugation at 120,000 g for 70 minutes at 4 °C using a Beckman Coulter Optima XE-100, facilitating EV isolation. The EV pellet was resuspended in phosphate-buffered saline (PBS). The quantitative and qualitative analysis of EVs was conducted through nanoparticle tracking analysis (NTA, Particle Metrix, Germany). For transmission electron microscopy (TEM) examination, EV samples were applied onto carbon-coated grids for 2 minutes, followed by dual PBS washes. Post-blotting and air-drying, the specimens were stained with 2% uranyl acetate for enhanced contrast and visualized using a Hitachi transmission electron microscope. miRNAs were extracted from cell and sEVs. miRNA-seq were performed to profiling miRNA transcriptome.

first: Yicun

last: Li

first: Hongyu

last: Yang


title: Effect of S767 treatment on gene expression of OCI-AML5 cells

city: Montreal

first: Guy

last: Sauvageau

state: QC

address: 2950, Chemin de Polytechnique

country: Canada

institute: Institute for Research in Immunology and Cancer

summary: Determine the effect on gene expression of exposure to the small molecule S767. The aim was to identify significantly affected cellular pathways by the molecule.

overall_design: OCI-AML5 cells were treated for 24 hours with 1 uM of S767 and compared to DMSO treated cells

first: Céline

last: Moison

first: Deanne

last: Gracias


title: Transcriptional reprogramming of neural stem cells by a PHF6/EphR signalling pathway

city: Ottawa

first: Arezu

last: Jahani-Asl

state: Ontario

address: 451 Smyth Road

department: Department of Cellular and Molecular Medicine

institute: University of Ottawa


title: Fat-to-blood recirculation of partially dysfunctional PD-1+CD4 Tconv cells is associated with dysglycemia in human obesity

city: Milan

first: Giulia

middle: Maria

last: Scotti

state: Milan

address: Viale Rita Levi Montalcini, 1

department: National Facility for Data Handling and Analysis

country: Italy

institute: Fondazione Human Technopole

summary: Obesity is characterized by accumulation of T cells in insulin-sensitive tissues, including the visceral adipose tissue (VAT), that can interfere with the insulin signaling pathway eventually leading to insulin resistance (IR) and type 2 diabetes. Here, we found that PD-1+CD4 conventional T (Tconv) cells, endowed with a transcriptomic and functional profile of partially dysfunctional cells, are diminished in VAT of obese patients with dysglycemia (OB-Dys). These cells showed enhanced capacity to recirculate into the bloodstream and had a non-restricted TCRβ repertoire divergent from that of normoglycemic obese and lean individuals. PD-1+CD4 Tconv were reduced in the circulation of OB-Dys, exhibited an altered migration potential, and were detected in the liver of patients with non-alcoholic steatohepatitis. The findings suggest that dysglycemia in individuals with obesity is associated with recirculation of a heterogeneous population of partially dysfunctional PD-1+CD4 Tconv cells. These changes may contribute to the inter-organ crosstalk underlying IR

overall_design: In order to investigate the role of PD-1+ CD4 Tconv cells in the development of dysglycemia in obesity we FACS-sorted PD-1+ CD4 Tconv cells from visceral adipose tissue and peripheral blood of obese patients with and without dysglycemia. RNA extraction was performed from bulk PD-1+ CD4 Tconv cells to perform RNAseq and carry out gene expression analysis.  Gene expression profile of PD-1+ CD4 Tconv cells from dysglycemic patients (OB-Dys) was compared with the one from non-diabetic normoglycemic patients (OB-ND).

first: Alessandra

last: Petrelli

last: M Scotti

first: Anna

last: Giovenzana

first: Eugenia

last: Bezzecchi

first: Anita

last: Bichisecchi

first: Sara

last: Cardellini

first: Francesca

last: Ragogna

first: Federica

last: Pedica

last: Invernizzi

first: Luigi

last: Di Filippo

first: Valentina

last: Tomajer

last: Aleotti

first: Carlo

last: Socci

first: Giovanni

last: Cesana

first: Stefano

last: Olmi

first: Marco

last: Morelli

first: Massimo

last: Falconi

first: Andrea

last: Giustina

first: Chiara

last: Bonini

first: Lorenzo

last: Piemonti

first: Eliana

last: Ruggiero


title: Transcriptional reprogramming of neural stem cells by a PHF6/EphR signalling pathway [RNA-seq]

first: Arezu

last: Jahani-Asl

summary: The plant homeodomain zinc finger protein,PHF6, is a transcriptional regulator, with its germline mutations causing the X-linked intellectual disability (XLID), Börjeson-Forssman-Lehmann syndrome (BFLS).The precise mechanisms by whichPHF6regulates transcription and how its mutant forms lead to XLID remain poorly characterized. Here, we show genome-wide binding of PHF6 in the developing cortex, most of which overlap (CA)n-microsatellite repeats near genes involved in central nervous system development and neurogenesis. Mice harbouring BFLS patient mutations exhibit defects in neurogenesis and altered neural stem cell fate. Mechanistically, we find that PHF6 regulatesa panel of Ephrin receptors (EphRs)and that PHF6regulation ofEphRis impaired in BFLS mice and ina conditionalPhf6knock-out mouse model. We report that mice harboring BFLS mutations exhibit an increase in embryonic neural stem cell (eNSC) self-renewal, a significant attenuation of newly born neurons, and thatEphRknockdown phenocopies the PHF6 loss-of-function defects in altering eNSC fate. Our results suggest that alterations in eNSC fate specification via a PHF6/EphRtranscriptional pathway leads to impaired neurogenesis in BFLS

overall_design:
RNA-seq profiling of cortical progenitors treated with siCtl or siPhf6.
Cortical progenitors were established from the cortex and were maintained in culture for 5 days to expand the homogeneous population prior to submitting samples for mRNA-Seq. mRNA-seq raw reads were mapped to mm10 genome using HISAT2 (Kim, Langmead, & Salzberg, 2015), followed by duplicate read removal using samtools. Gene-level read counts were obtained by HTSeq (Anders, Pyl, & Huber, 2015), using gene annotations from GENCODE (release M9). Genes with a minimum of 150 reads in at least one sample were retained. Gene set analysis was performed using ConsensusPathDB (Kamburov et al., 2011).

first: Dilan

last: Rasool

first: Audrey

last: Burban

first: Ahmad

last: Sharanek

first: Ariel

last: Madrigal Aguirre

first: Jingua

last: Hu

first: Keqin

last: Yan

first: Dianbo

last: Qu

first: Anne

middle: K

last: Voss

first: Ruth

last: Slack

first: Tim

last: Thomas

first: Azad

last: Bonni

first: David

last: Picketts

first: Vahab

last: Soleimani

first: Hamed

last: Najafabadi


title: Transcriptional reprogramming of neural stem cells by a PHF6/EphR signalling pathway [ChIP-Seq]

overall_design:
Here, we show genome-wide binding of PHF6 in the developing cortex.
ChIP-seq data were processed as previously described (Hernandez-Corchado & Najafabadi, 2022). Briefly, raw reads were aligned to the mouse genome assembly version mm10 with bowtie2 (version 2.3.4.1) using the “--very-sensitive-local” mode. Duplicate reads were removed using samtools (version 1.9) (Danecek et al., 2021). ChIP-seq peaks were identified using MACS (version 1.4) (Feng, Liu, Qin, Zhang, & Liu, 2012; Zhang et al., 2008) with a permissive p-value threshold of 0.001, using “--nomodel” option. Fragment size was specified using “--shiftsize” argument, with the fragment length obtained by cross-correlation analysis using phantompeakqualtools (Landt et al., 2012). Peak-TSS distances were calculated using bedtools (Quinlan & Hall, 2010) only for peaks that passed p-value threshold of 10–5, with TSS coordinates obtained from GENCODE (Frankish et al., 2019) (release M9).

last: Madrigal  Aguirre


title: microRNAs dysregulated in multiple sclerosis affect the differentiation of CG-4 cells, an oligodendrocyte progenitor cell line

city: Brussels

first: Océane

last: Perdaens

address: avenue Mounier 53

country: Belgium

institute: UCLouvain

summary: Remyelination in multiple sclerosis (MS) is from the start of the disease imperfect, and strongly decreases with its progression, mainly due to the harm on oligodendrocyte progenitor cells (OPCs), causing neurodegeneration and resulting in irreversible neurological deficits. Therapeutic strategies promoting remyelination are still very preliminary and lack within the current treatment panel of MS. In a previous study we have identified 21 microRNAs dysregulated mostly in the CSF of relapsing and/or remitting MS patients. Here, we observed that among these, the majority of 13 transfected microRNA mimics decreased the differentiation of an OPC cell line called CG-4. Herein we support by RNA sequencing and independent RT-qPCR analyses that miR-33-3p, miR-34c-5p, miR-124-5p, and miR-145-5p impede OPC differentiation as evidenced by the downregulation of premyelinating oligodendrocyte (OL) (Tcf7l2, Cnp [except for miR-145-5p]) and mature OL (Plp1, Mbp, Mobp) markers, whereas only miR-214-3p promotes OPC differentiation. We further propose a comprehensive exploration of their change in cell fate through Gene Ontology enrichment analysis. We finally confirm by RT-qPCR analysis the downregulation of several predicted mRNA targets for each microRNA that possibly support their effect on OPC differentiation by very distinctive mechanisms, of which some are still unexplored in OPC/OL physiology. We hereby open new perspectives in the research on OPC differentiation and the pathophysiology of demyelination/remyelination, and possibly even in the research on new remyelinating therapeutic strategies in the scope of MS.

overall_design:
We aimed to investigate the effect of several microRNAs on the differentiation of oligodendrocyte progenitor cells (OPCs). Therefore CG-4 cells (an OPC cell line) were transfected with each microRNA mimic separately and were then cultured in differentiation medium for 48h
We performed RNA-sequencing on the transfected cells (5 different microRNAs) and controls (proliferation vehicle control, differentiation vehicle control, differentiation microRNA negative control)
We compared each microRNA condition to each control regarding differentially expressed genes and Gene Ontology enrichment analysis

first: Pauline

last: Bottemanne

first: Vincent

last: van Pesch


title: Th17 cells secrete TWEAK to trigger epithelial-mesenchymal transition and promote colorectal cancer liver metastasis (WES)

status: Public on Feb 06 2024

city: Guangzhou

first: Lixia

last: Xu

address: 58 Zhongshan Road 2

institute: Sun Yat-sen University First Affiliated Hospital

type: Genome variation profiling by high throughput sequencing

summary: Liver metastasis is the leading cause of mortality in patients with colorectal cancer (CRC). Given the significance of both epithelial-mesenchymal transition (EMT) of tumor cells and the immune microenvironment in CRC liver metastasis (CRLM), the interplay between them could hold the key for developing improved treatment options. We employed multi-omics analysis of 130 samples from 18 synchronous CRLM patients integrated with external datasets to comprehensively evaluate the interaction between immune cells and EMT of tumor cells in liver metastasis. Single-cell RNA sequencing analysis revealed distinct distributions of non-malignant cells between primary tumors from patients with metastatic CRC (mCRC) and non-metastatic CRC, showing that Th17 cells were predominantly enriched in the primary lesion of mCRC. TWEAK, a cytokine secreted by Th17 cells, promoted EMT by binding to receptor Fn14 on tumor cells, and the TWEAK-Fn14 interaction enhanced tumor migration and invasion. In mouse models, targeting Fn14 using CRISPR-induced knockout or lipid-nanoparticle-encapsulated siRNA alleviated metastasis and prolonged survival. Mice lacking Il17a or Tnfsf12 (encoding TWEAK) exhibited fewer metastases compared to wild-type mice, while co-transfer of Th17 with tumor cells promoted liver metastasis. Higher TWEAK expression was associated with a worse prognosis in CRC patients. Additionally, CD163L1+ macrophages interacted with Th17 cells, recruiting Th17 via the CCL4-CCR5 axis. Collectively, this study unveils the role of immune cells in the EMT process and identifies TWEAK secreted by Th17 as a driver of CRLM.

overall_design:
32 tumor samples from 17 patients with colorectal cancer liver metastasis were collected for whole exome sequencing.

***************************************************************
The raw data have been deposited in the Genome Sequence Archive for Human under accession HRA006630
***************************************************************

first: Xin

last: Liu

last: Wang

first: Qingxia

last: Yang

first: Xinming

last: Song

first: Fang


title: Th17 cells secrete TWEAK to trigger epithelial-mesenchymal transition and promote colorectal cancer liver metastasis (RNA-seq)

overall_design:
30 tumor samples from 15 patients with colorectal cancer liver metastasis were collected for bulk RNA-sequencing.

***************************************************************
The raw data have been deposited in the Genome Sequence Archive for Human under accession HRA006630
***************************************************************


title: Adeno-associated virus-mediated trastuzumab delivery to the central nervous system for human epidermal growth factor receptor 2+ brain metastasis

status: Public on Feb 05 2024

city: Philadelphia

first: James

last: Wilson

state: Pennsylvania

address: 125S 31st St

institute: University of Pennsylvania

summary: Trastuzumab improves overall survival for HER2+ breast cancer, but its short half-life in the cerebral spinal fluid (~2-4 days) and delivery limitations restrict the ability to target HER2+ central nervous system (CNS) disease.We developed an adeno-associated virus (AAV) vector expressing a codon-optimized, ubiquitin C (UbC)-promoter-driven trastuzumab sequence (AAV9.UbC.trastuzumab) for intrathecal administration. Transgene expression was evaluated in adult Rag1 knockout mice and rhesus nonhuman primates (NHPs) after a single intracerebroventricular (ICV) or intra-cisterna magna (ICM) AAV9.UbC.trastuzumab injection, respectively, using real-time PCR, ELISA, Western blot, in situ hybridization, single-nucleus RNA sequencing, and liquid chromatography-mass spectrometry; antitumor efficacy was evaluated in brain xenografts using HER2+ breast cancer cell lines (BT-474, MDA-MB-453).Transgene expression was detected in brain homogenates of Rag1 knockout mice following a single ICV injection of AAV9.UbC.trastuzumab (1×1011 vector genome copies [GC]/mouse) and tumor progression was inhibited in xenograft models of breast-to-brain metastasis. In NHPs, ICM delivery of AAV9.UbC.trastuzumab (3×1013 GC/animal) was well tolerated (36–37 days in-life) and resulted in transgene expression in CNS tissues and cerebrospinal fluid at levels sufficient to induce complete tumor remission in MDA-MB-453 brain xenografts. With AAV9’s proven clinical safety record, this gene therapy may represent a viable approach for targeting HER2+ CNS malignancies.

overall_design: Two adult female rhesus macaques were dosed ICM with 3x10^13 GC of AAV9.UbC.trastuzumab and brain tissue was collected at necropsy 36-37 days post-dosing. scRNA-seq was used to determine cell types that were positive for transgene expression in cerebellum and occitital cortex.

first: Marcela

middle: S

last: Werner

first: Shweta

last: Aras

first: Ashleigh

last: Morgan

first: Jillian

last: Roamer

first: Nesteene

last: Param

first: Kanyin

last: Olagbegi

first: R

last: Lamontagne

first: Jenny

last: Greig


title: Clinical and biomarker analyses of hepatic arterial infusion chemotherapy plus lenvatinib and PD-1 inhibitor for patients with advanced intrahepatic cholangiocarcinoma

first: Yexing

last: Huang

address: 651 Dongfeng East Road

institute: Sun Yat-sen University Cancer Center

summary: Intrahepatic cholangiocarcinoma (iCCA) is a highly aggressive cancer with a dismal prognosis and few effective therapeutic approaches. This study aimed to investigate the efficacy, safety, and predictive biomarkers of hepatic arterial infusion chemotherapy (FOLFOX-HAIC) in combination with lenvatinib and PD-1 inhibitor for patients with advanced iCCA. Locally advanced or metastatic iCCA patients receiving the triple combination therapy of lenvatinib, PD-1 inhibitor, and FOLFOX-HAIC were included in this retrospective study. Primary endpoint was the progression-free survival, evaluated using the RECIST criterion. The secondary endpoints included overall survival, objective response rate, and safety. Whole exome and RNA sequencing of tumor biopsy tissues were performed for biomarker exploration. Between May, 2019 and December 2022, a total of 46 patients were included in this study. The primary endpoint showed a median progression-free survival of 9.40 months (95% CI: 5.28-13.52), with a 6-month progression-free survival rate of 76.1%. The median overall survival was 16.77 months (95% CI, 14.20-19.33), with an objective response rate of 47.8% and disease control rate of 91.3% per RECIST. In addition, 4.3% and 8.7% of patients achieved complete response of all lesions and intrahepatic target lesions per mRECIST, respectively. The most common treatment-related adverse events were neutropenia, thrombocytopenia, elevated aspartate aminotransferase and alanine aminotransferase level. Furthermore, integrated analysis of genetic, transcriptomic, and immunohistochemistry data revealed that pre-existing immunity (high expression level of immune-related signatures and intra-tumoral CD8+ T cell density) in baseline tumour tissues was associated with superior clinical benefits. However, the evaluation of tumor mutation burden did not show potential predictive value in this triple combination. FOLFOX-HAIC in combination with lenvatinib and PD-1 inhibitor demonstrated a promising antitumor activity with manageable safety profiles in patients with advanced iCCA. Moreover, our study also revealed new perspectives on potential biomarkers for clinical efficacy.

overall_design: To identify potential biomarkers for this triple combination, we collected the tumor biopsy samples of patients with advanced intrahepatic cholangiocarcinoma and performed the transcriptome profiling.

first: Zefeng

last: Du

first: Anna

last: Kan

first: Minke

last: He


title: HuR promotes castration-resistant prostate cancer progression by altering ERK5 activation via posttranscriptional regulation of BCAT1

city: Chongqing

first: Song

last: Guojing

address: Gaotan Yan Street

institute: The First Affiliated Hospital of the Army Medical University

summary: Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment, and its progression mechanism is not fully uncovered. This study aims to clarify the role and mechanism of Human antigen R (HuR) as a therapeutic target for CRPC progression.

overall_design: HuR was knocked out by Cas9  in CRPC cell lines  PC3 and DU145. Subsequently, we collected RNA from the experimental and control groups.Compare gene expression profile analysis in the RNA-seq data.

first: Gang

first: Hang

last: You

first: Guojing


title: Prior influenza infection mitigates SARS-CoV-2 disease in Syrian hamsters

city: West haven

first: Antariksh

last: Tyagi

state: CT

address: 830 West Campus Dr

institute: Yale University

summary: Seasonal infection rates of individual viruses are influenced by synergistic or inhibitory interactions between coincident viruses.  Endemic patterns of SARS-CoV-2 and influenza infection overlap seasonally in the Northern hemisphere and may be similarly influenced. We explored the immunopathologic basis of SARS-CoV-2 and influenza A (H1N1) interactions in Syrian hamsters. H1N1 given 48 hours prior to SARS-CoV-2 profoundly mitigated weight loss and lung pathology compared to SARS-CoV-2 infection alone.  This was accompanied by normalization of granulocyte dynamics and accelerated antigen presenting populations in bronchoalveolar lavage and blood. Using nasal transcriptomics, we identified rapid upregulation of innate and antiviral pathways induced by H1N1 by the time of SARS-CoV-2 inoculation in 48 hour dual infected animals.  Dual infected animals also experienced significant transient downregulation of mitochondrial and viral replication pathways.  By quantitative RT-PCR, we confirmed reduced SARS-CoV-2 viral load and lower cytokine levels throughout disease course in lung of dual infected animals. Our data confirm that H1N1 infection induces rapid and transient gene expression that is associated with mitigation of SARS-CoV-2 pulmonary disease. These protective responses are likely to begin in the upper respiratory tract shortly after infection.  On a population level, interaction between these two viruses may influence their relative seasonal infection rates.

overall_design: We infected 6-8 week old Syrian hamsters intranasally with Influenza (Influenza virus A/Michigan/45/2015 (H1N1) pdm09), followed by infection with SARS-CoV-2 (SARS-CoV-2 Wuhan/IVDC-HB-01/2019) given intranasally 48 hours later. Four infection groups were used: H1N1 infection only (n=6), SARS-CoV-2 infection only (n=6), H1N1 infection followed by SARS-CoV-2 infection 48 hours later (n=6), and mock infected (n=4). All animals were males. Animals were sacrificed at Day 1 (n=3 per infection type) and Day 2 (n=3 per infection type) after their last infection. Nasal epithelium was collected under sterile conditions for RNA sequencing. . We compared differential gene expression in infected vs mock control animals, as well as differential gene expression between infection types at comparable days post infection.

first: Caroline

last: Zeiss


title: Candida albicans interaction with intestinal epithelial cells

city: Jena

first: Sascha

last: Schäuble

address: Beutenbergstraße 11a

institute: Leibniz Institute for Natural Product Research and Infection Biology

summary: To characterize the interaction of Candida albicans with intestinal epithelial cells (C2BBe1) from early fungal adhesion to invasion (up to 6 h) and the late translocation and damage phase (12 -24 h), we conducted dual RNA-sequencing of C. albicans-infected C2BBe1 cells over a 24 h time course, with sampling at 0 h, 45 min, 3 h, 6 h, 12 h, and 24 h.

sample_organism:
Candida albicans
Homo sapiens

platform_organism:
Candida albicans
Homo sapiens

overall_design: Gene expression profiling analysis of RNA-seq data comparing C2BBe1 cells infected with Candida albicans with C2BBe1 or C. albicans cells cultured alone, with the aim to identify infection-specific gene expression.

first: Jakob

last: Sprague

first: Lydia

last: Kasper

first: Stefanie

last: Allert

first: Peter

last: Großmann

first: Gianni

last: Panagiotou

first: Bernhard

last: Hube


title: The global RNA-RNA interactome of Klebsiella pneumoniae unveils a small RNA regulator of cell division (RIP-Seq)

first: Eric

last: Ruhland

address: Winzerlaer Str. 2

institute: Friedrich Schiller University Jena

summary: The ubiquitous RNA chaperone Hfq is involved in the regulation of key biological processes in many species across the bacterial kingdom. In the opportunistic human pathogen Klebsiella pneumoniae, deletion of the hfq gene affects the global transcriptome, virulence, and stress-resistance, however, the ligands of the major RNA-binding protein in this species have remained elusive. In this study, we have combined transcriptomic, co-immunoprecipitation and global RNA interactome analyses to compile an inventory of conserved and species-specific RNAs bound by Hfq, and to monitor Hfq-mediated RNA-RNA interactions. In addition to dozens of previously unknown RNA-RNA pairs, our study revealed an Hfq-dependent sRNA, DinR, which is processed from the 3’ terminal portion of dinI mRNA. Transcription of dinI is controlled by the master regulator of the SOS response, LexA. As DinR accumulates in K. pneumoniae in response to DNA damage, the sRNA represses translation of the ftsZ transcript by occupation of the ribosome binding site. Ectopic overexpression of DinR causes depletion of ftsZ mRNA and inhibition of cell division, while deletion of dinR antagonizes cell elongation in the presence of DNA damage. Collectively, our work highlights the important role of RNA-based gene regulation in K. pneumoniae and uncovers the central role of DinR in LexA-controlled cell cycle inhibition during the SOS response.

overall_design: Hfq bound RNAs were analyzed. For that, CoIP with Klebsiella pneumoniae MGH78578 wt or hfq-3XFLAG cells was performed and libraries were prepared (RIP-seq)

first: Kathrin

last: Fröhlich


title: The RNA binding protein QKI guides a pro-mesenchymal splicing program driving a subtype switch and limiting outcome in pancreatic cancer [PANC-1 and HPAF-II]

city: Paris

first: Pierre

last: de la Grange

address: 29, rue du Faubourg Saint-Jacques

department: Paris Biotech Santé

country: France

institute: GenoSplice technology

summary: Transcriptomic analyses have identified two molecular PDAC subtypes, one of which, namely basal-like subtype, is associated with chemoresistance and worse clinical outcomes. Splicing dysregulation is known to contribute to PDAC malignancy. Functional studies in PDAC cells lines identified the splicing factor QKI as a key determinant of a basal-like splicing signature associated with increased tumor aggressiveness. Our studies demonstrate that QKI represses splicing events associated with the classical subtype and improved clinical outcome, while promoting basal-like events associated with shorter survival.

overall_design: Briefly, PANC-1 cells were silenced for QKI and RBFOX2 (40nM) for 48 hours.HPAF-II cell silenced with siCtrl. Each samples has 3 biological replicates.

first: Veronica

last: Ruta

first: Chiara

last: Naro

first: Marco

last: Pieraccioli

first: Livia

last: Archibugi

first: Eleonora

last: Cesari

first: Valentina

last: Panzeri

first: Chantal

last: Allgower

first: Paolo

last: Arcidiacono

first: Massimo

last: Falconi

first: Carmine

last: Carbone

first: Giampaolo

last: Tortora

first: Giuseppe

last: Quero

first: Sergio

last: Alfieri

first: Claudio

last: Doglioni

first: Alezander

last: Kleger

first: Gabriele

last: Capurso

last: Sette


title: RNA and miRNA profiles in mouse hepatocytes ApcdeltaHep ROSA or DLK1DeltaWRE

first: Angelique

last: Gougelet

address: 15 rue de l'école de médecine

institute: INSERM U1138

summary: We analyzed the expression of RNAs and microRNAs in hepatocytes isolated from  ApcDeltaHep livers, either ROSA26 or deleted for the b-catenin binding site upstream of the DLK1/DIO3 locus by Crispr/Cas9  (DLK1DeltaWRE). We thus explored if editing of this regulatory site impaired the expression of the entire locus and which signaling pathways were modulated.

overall_design: RNA profiles from hepatocytes isolated from ApcDeltaHep livers either edited for the Rosa26 locus or the beta-catenin binding site identified upstream of the DLK1/DIO3 locus

first: Julie

last: Sanceau

first: Angélique


title: fhl2b expression ameliorates muscular dystrophy

first: Nils

last: Dennhag

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

overall_design: Refer to individual Series