title: Evolution of gene expression across brain regions in behaviorally divergent deer mice

city: Cambridge

first: Andreas

middle: F

last: Kautt

state: MA

address: 16 Divinity Ave

institute: Harvard University

summary: The evolution of innate behaviors is ultimately due to genetic variation likely acting in the nervous system. Gene regulation may be particularly important because it can evolve in a modular brain-region specific fashion through the concerted action of cis- and trans-regulatory changes. Here, to investigate transcriptional variation and its regulatory basis across the brain, we perform RNA sequencing (RNA-Seq) on ten brain subregions in two sister species of deer mice (Peromyscus maniculatus and P. polionotus) – which differ in a range of innate behaviors, including their social system – and their F1 hybrids. We find that most of the variation in gene expression distinguishes subregions, followed by species. Interspecific differential expression (DE) is pervasive (52–59% of expressed genes), whereas the number of DE genes between sexes is modest overall (~3%). Interestingly, the identity of DE genes varies considerably across brain regions. Much of this modularity is due to cis-regulatory divergence, and while 43% of genes were consistently assigned to the same gene regulatory class across subregions (e.g., conserved, cis-, or trans-regulatory divergence), a similar number were assigned to two or more different gene regulatory classes. Together, these results highlight the modularity of gene expression differences and divergence in the brain, which may be key to explain how the evolution of brain gene expression can contribute to the astonishing diversity of animal behaviors.

sample_organism: Peromyscus maniculatus x Peromyscus polionotus

overall_design: We performed bulk RNA-Seq on ten different brain regions of two species of deer mice (P. maniculatus "BW" strain and P. polionotus "PO" strain) and their F1 hybrids. We sequenced six biological replicates for each subregion, including both sexes ("M" and "F"). In addition, we sequenced whole brain samples of 5-6 replicates of both species, all males.

first: Jenny

first: Hopi

last: Hoekstra


title: Selective gene expression maintains human tRNA anticodon pools during differentiation

status: Public on Oct 22 2023

city: Planegg

first: Danny

last: Nedialkova

state: Bayern

address: Am Klopferspitz 18

country: Germany

institute: Max Planck Institute for Biochemistry

type: Genome binding/occupancy profiling by high throughput sequencing

type: Expression profiling by high throughput sequencing

type: Non-coding RNA profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Aiolos modulates IL-15 responsiveness of intestinal intraepithelial lymphocytes [scRNA-seq]

status: Public on Oct 20 2023

city: St. Louis

first: Marco

last: Colonna

state: Missouri

address: 425 S. Euclid

department: Pathology and Immunology

country: USA

institute: Washington University in St. Louis

summary: Intestinal intraepithelial lymphocytes (IEL) are capable of rapid innate-like responses to microenvironmental cues. While poised to activation, IEL effector functions must be stringently controlled. Aiolos is an Ikaros zinc finger (IKZF) family member encoded by Ikzf3 that promotes histone deacetylation. Here, we found that Aiolos is a crucial regulator of IEL activation. Ikzf3–/– CD8αα+ IEL expressed high levels of innate NK receptors, cytotoxic enzymes, cytokines and chemokines. Single cell RNAseq of IEL revealed that Ikzf3 deficiency mostly amplified the effector machinery of a CD8αα+ IEL subset characterized by high expression CD122, the receptor for IL-15. Furthermore, Ikzf3 deficiency increased IEL responsiveness to IL-15. Aiolos binding sites were close to  those for STAT5 and RUNX transcription factors that promote IL-15 signaling and cytolytic programs, respectively; lack of Ikzf3 increased accessibility and histone acetylation of these chromatin regions. Ikzf3 deficiency increased susceptibility to colitis, demonstrating the functional relevance of Aiolos-mediated regulation of IEL effector functions.

overall_design: single cell transcriptional profiling of Aiolos-deficient and control IELs was performed using the 10x Genomics platform

first: Kentaro

last: Yomogida


title: Aiolos modulates IL-15 responsiveness of intestinal intraepithelial lymphocytes [ATAC-seq]

overall_design: Global chromatin accessibility profiling of Aiolos-deficient IEL and control cells


title: Long 3'UTRs predispose neurons to inflammation by promoting immunostimulatory double-stranded RNA formation

city: New York

first: Hachung

last: Chung

state: New York

address: 701 W 168th St, HHSC Rm 1506

department: Microbiology & Immunology

institute: Columbia University

summary: Loss of RNA homeostasis underlies numerous neurodegenerative and neuroinflammatory diseases. However, the molecular mechanisms that trigger neuroinflammation are poorly understood. Viral double-stranded RNA (dsRNA) triggers innate immune responses when sensed by host pattern recognition receptors (PRRs) present in all cell types. Here, we report that human neurons intrinsically carry exceptionally high levels of immunostimulatory dsRNAs and identify long 3'UTRs as giving rise to neuronal dsRNA structures. We found that the neuron-enriched ELAVL family of genes (ELAVL2, -3, -4) can increase 1) 3'UTR length, 2) dsRNA load, and 3) activation of dsRNA-sensing PRRs such as MDA5, PKR, and TLR3. In wild-type neurons, neuronal dsRNAs signaled through PRRs to induce tonic production of the antiviral type I interferon. Depleting ELAVL2 in WT neurons led to global shortening of 3'UTR length, reduced immunostimulatory dsRNA levels, and rendered WT neurons susceptible to herpes simplex virus and Zika virus infection. Neurons deficient in ADAR1, a dsRNA-editing enzyme mutated in the neuroinflammatory disorder Aicardi-Goutières syndrome, exhibited intolerably high levels of dsRNA that triggered PRR mediated toxic inflammation and neuronal death. Depleting ELAVL2 in ADAR1 knockout neurons led to prolonged neuron survival by reducing immunostimulatory dsRNA levels. In summary, neurons are specialized cells where PRRs constantly sense ‘self’ dsRNAs to pre-emptively induce protective antiviral immunity, but maintaining RNA homeostasis is paramount to prevent pathological neuroinflammation.

overall_design:
For the HuB/C/D ectopic expression experiment (GSE214943; biological triplicates of HEK-293T_Mock, HEK-293T_EV, and HEK-293T_HuB/C/D), RNA was extracted with the Direct-zol RNA MiniPrep (Zymo Research). ERCC RNA Spike-In Mix (Thermo Fisher Scientific) was added to each RNA sample. rRNA was depleted using the RiboMinus™ Eukaryote Kit (Thermo Fisher Scientific). Libraries were prepared with the NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs). The 9 libraries were pooled and sequenced on the Illumina Novaseq platform with a read length of 150 nt in the paired end configuration. For analyzing global 3`UTR extension in neurons (GSE214941), biological triplicates of RNA samples from neurons and HEK 293T were extracted with the Direct-zol RNA MiniPrep (Zymo Research). After adding ERCC RNA Spike-In Mix (Thermo Fisher Scientific), libraries were prepared with the TruSeq stranded Total RNA with Ribozero kit (Illumina). The libraries were sequenced on the Illumina Novaseq platform with a read length of 100 nt in paired end configuration.

Grant ID: 20-SSP-114
Funding agency: Searle Scholars Program


title: The dsRNA-enriched neural transcriptome drives neuroinflammation [neurons]

summary: To confirm 3'UTR lengthening in neurons, we differentiated neurons from HEUS8, a human stem cell line and performed RNA sequenicng. HEK293T cells were used as a control

overall_design: We performed alternative polyadenylation analysis by using DaPars2 v2.1 software with RNA sequencing data

first: Heegwon

last: Shin

first: Tyler

middle: J

last: Dorrity


title: Three-dimensional spherical cellular aggregates of RPTEC as an in vitro tool for evaluation of human renal proximal tubular toxicity and drug disposition

city: Kobe

first: Hijiri

last: Fujioka

address: 6-7-5, Minatojimaminamimachi, Chuouku,

country: Japan

institute: NBI

summary: The proximal tubule plays an important role in the secretion and reabsorption of drugs in the kidney and is a major site of drug interaction and toxicity. Kidney toxicity analysis via in vitro assays is challenging as few provide appropriate proximal tubular cell function. In this study, we aimed to develop a simple and reproducible method to culture human proximal tubular epithelial cells (RPTECs), used in pharmacokinetic and toxicological evaluation by monitoring organic anion transporter (OAT)1 as a selection marker. As a result, by culturing RPTECs in spherical cellular aggregates, OAT1 protein expression, which does not increase in conventional 2D culture, increased over time and reached a similar level to that in human renal cortices from 5 different donors. The expression of proximal tubule markers, AQP1 and CDH6, was maintained, indicating that 3D RPTEC spheroids had proximal tubule characteristics. For SLC transporters, the 3D spheroid culture improved the protein expression of about 7% of the 139 transporter proteins detected and 2.3% of 4,800 proteins detected to about 5-fold that in human renal cortices. Furthermore, the protein expression levels of about 4800 proteins in 3D RPTEC spheroids (cultured for 12 days) were maintained over 20 days. Cisplatin and adefovir exhibited transporter-dependent ATP decreases in 3D RPTEC spheroids. These results indicate that the 3D RPTEC spheroid is a simple and robust in vitro experimental system for pharmacokinetic and toxicological evaluation in drug development.

overall_design: Cortex 5 donor

first: Naoki

last: Ishiguro

first: Etsuhi

last: Takahashi

first: Hiroshi

last: Arakawa

first: Asami

last: Saito

first: Fumihito

last: Kitagawa

first: Masayuki

last: Kondo

first: Gaku

last: Morinaga

first: Masahito

last: Takatani

first: Ryo

first: Takashi

last: Kudo

first: Shin-ich

last: Mae

first: Moeno

last: Kadoguhi

first: Yuya

last: Nakazono

first: Ikumi

last: Tamai

first: Kenji

last: Osafune

first: Yoichi

last: Jimbo


title: Double ratio of microRNA signatures as promising diagnosis biomarkers for mucinous borderline and malignant ovarian tumors

status: Public on Oct 19 2023

city: lille

first: frédéric

last: leprêtre

address: Boulevard du Pr Leclercq

department: CHU of Lille, CBP

country: France

institute: Lille University

summary:
Epithelial ovarian cancer (EOC) are a heterogeneous collection of malignancies, each with their own developmental origins, clinical behavior and molecular profile. With less than 5% of EOC cases, mucinous ovarian carcinoma is a rare form with a poor prognosis with a 5-year survival of 11% for advanced stages (III/IV). Clinically, due to the large size and heterogeneity of mu-cinous tumors, Malignant forms are difficult to distinguish from Borderline (15%) and benign (80%) forms with a better prognosis. Improving their diagnosis is therefore a challenge with regard to the risk of under-treating a malignant form or of unnecessarily undertaking radical surgical excision. The involvement of microRNAs (miRNAs) in cancer and their potential as biomarkers of diagnosis are becoming increasingly recognized. In this study, miRNA expres-sion profiling generates using FFPE patient samples followed by qRT-PCR validation allows to identify 10 down-regulated and 5 up-regulated miRNAs between Malignant and Borderline tumors. To overcome issues linked to data normalization and to improve the accuracy of the results, a ratio analysis combining up-regulated and down-regulated miRNAs was used. Alt-hough 21/50 miRNA expression ratio were significantly different between Malignant and Bor-derline tumor samples, any ratio could perfectly discriminate the two groups. However, a combination of 14 miRNAs expression ratio (double ratio) showed high discriminatory poten-tial, with 100% of accuracy in distinguishing Malignant and Borderline ovarian tumors, and suggests that miRNAs may hold significant clinical potential as a diagnostic tool.
Epithelial ovarian cancer (EOC) are a heterogeneous collection of malignancies, each with their own developmental origins, clinical behavior and molecular profile. With less than 5% of EOC cases, mucinous ovarian carcinoma is a rare form with a poor prognosis with a 5-year survival of 11% for advanced stages (III/IV). Clinically, due to the large size and heterogeneity of mu-cinous tumors, Malignant forms are difficult to distinguish from Borderline (15%) and benign (80%) forms with a better prognosis. Improving their diagnosis is therefore a challenge with regard to the risk of under-treating a malignant form or of unnecessarily undertaking radical surgical excision. The involvement of microRNAs (miRNAs) in cancer and their potential as biomarkers of diagnosis are becoming increasingly recognized. In this study, miRNA expres-sion profiling generates using FFPE patient samples followed by qRT-PCR validation allows to identify 10 down-regulated and 5 up-regulated miRNAs between Malignant and Borderline tumors. To overcome issues linked to data normalization and to improve the accuracy of the results, a ratio analysis combining up-regulated and down-regulated miRNAs was used. Alt-hough 21/50 miRNA expression ratio were significantly different between Malignant and Bor-derline tumor samples, any ratio could perfectly discriminate the two groups. However, a combination of 14 miRNAs expression ratio (double ratio) showed high discriminatory poten-tial, with 100% of accuracy in distinguishing Malignant and Borderline ovarian tumors, and suggests that miRNAs may hold significant clinical potential as a diagnostic tool.

overall_design: microRNA analysis mucinous borderline and malignant ovarian tumors

first: Christophe

last: Denoyelle

first: Matthieu

last: Meryet-Figuiere


title: Cytotoxic CD4+ tissue-resident memory T cells are associated with asthma severity [single-cell RNA-seq]

city: San Diego

first: Pandurangan

last: Vijayanand

state: California

address: 9420 Athena Circle

department: Vaccine Discovery

institute: La Jolla Institute for Immunology

summary: Patients with severe uncontrolled asthma represent a distinct endotype with persistent airway inflammation and remodeling refractory to corticosteroid treatment. CD4+ T cells and their canonical effector molecules, like type 2 cytokines, play a central role in orchestrating asthma pathogenesis, and biologic therapies targeting these cytokine pathways have had promising outcomes. However, not all patients respond well to such treatment, and their effects are not always durable nor reverse airway remodeling. This observation raises the possibility that other CD4+ T cell subsets and their effector molecules may drive airway inflammation and remodeling. To address this issue in a hypothesis-free manner, we performed single-cell transcriptome analysis of >50,000 airway CD4+ T cells isolated from bronchoalveolar lavage (BAL) samples from 30 patients with mild and severe asthma. We observed striking heterogeneity in the nature of CD4+ T cells present in asthmatics' airways with tissue-resident memory (TRM) cells making a dominant contribution. Notably, a subset of CD4+ TRM cells (CD103-expressing) was significantly increased, comprising nearly 65% of all CD4+ T cells in the airways of male patients with severe asthma when compared to mild asthma. This subset was enriched for transcripts linked to T cell receptor (TCR) activation (HLA-DRB1, HLA-DPA1, CD40LG) and cytotoxicity (GZMB, GZMH), and following stimulation expressed high levels of transcripts encoding for pro-inflammatory non-TH2 cytokines (CCL3, CCL4, CCL5, TNF, IL-17A, CSF2, LIGHT) that could fuel persistent airway inflammation and remodeling. Furthermore, we found that CD4+ T cells isolated ex vivo from airways of severe asthmatics displayed signatures linked to corticosteroid resistance (FKBP5, DDIT4), as well as reduced expression of transcripts encoding for molecules that dampen TCR signaling and T cell effector functions (CREM, DUSP1, TNFAIP3).  Overall, our single-cell transcriptomic analysis highlights the pathogenic properties and clinical associations of airway CD4+ T cell subsets in severe asthmatics, features that can potentially trigger their unrestrained activation, and that these findings were more pronounced in males. These pathways may therefore be crucial for driving disease severity in adult male severe asthmatics.

overall_design: Single-cell RNA-seq was performed from total CD4+ T cells in resting and stimulation conditions isolated ex vivo from bronchoalveolar lavage (BAL) samples collected from patients with mild (n=14) and severe (n=16) asthma.

first: Sara

last: Herrera-de la Mata

first: Ciro

last: Ramírez-Suástegui

first: Heena

last: Mistry

first: Mohammad

middle: A

last: Kyyaly

first: Hayley

last: Simon

first: Shu

last: Liang

first: Laurie

last: Lau

first: Clair

last: Barber

first: Monalisa

last: Mondal

first: Syed

middle: H

last: Arshad

first: Ramesh

last: Kurukulaaratchy

first: Grégory

last: Seumois


title: The dsRNA-enriched neural transcriptome drives neuroinflammation [HuBCD]

first: Hachung

last: Chung

summary: To investigate the effects of HuB, HuC, and HuD, which are neuron-specific RNA binding proteins in mRNA lengthening and innate immune responses, we transfected them in HEK293T cells and performed the RNA sequencing experiment.

overall_design: HEK293T cells were transfected with HuB/C/D overexpressing vectors or empty vector. RNA was extracted using Trizol. Three biological replicates were collected.

first: Heegwon

last: Shin

first: Tyler

last: Dorrity


title: Cytotoxic CD4+ tissue-resident memory T cells are associated with asthma severity


title: Cytotoxic CD4+ tissue-resident memory T cells are associated with asthma severity [bulk RNA-seq]

overall_design: Bulk RNA-seq was performed from CD103+ CD69+ (CD103+ TRM), CD103– CD69+ (CD103– TRM), and CD103– CD69– (non-TRM) CD4+ T cells in resting and after stimulating conditions isolated ex vivo from bronchoalveolar lavage (BAL) samples collected from patients with mild (n=14) and severe (n=16) asthma. In total 202 samples containing up to 400 cells/tube were sequenced using the Smart-seq2 RNA-seq assay.


title: Massive horizontal gene transfer and the evolution of nematomorph-driven behavioral manipulation of mantids

city: Fukuoka

first: Tappei

last: Mishina

address: 744 Motooka Nishi-ku

institute: Kyushu University

summary: To complete their life cycle, a wide range of parasites must manipulate the behavior of their hosts. This manipulation is a well-known example of the “extended phenotype,” where genes in one organism have phenotypic effects on another organism. Recent studies have explored the parasite genes responsible for such manipulation of host behavior, including the potential molecular mechanisms. However, little is known about how parasites have acquired the genes involved in manipulating phylogenetically distinct hosts. In a fascinating example of the extended phenotype, nematomorph parasites have evolved the ability to induce their terrestrial insect hosts to enter bodies of water, where the parasite then reproduces. Here, we comprehensively analyzed nematomorphs and their mantid hosts, focusing on the transcriptomic changes associated with host manipulations and sequence similarity between host and parasite genes to test molecular mimicry. The nematomorph’s transcriptome changed during host manipulation, whereas no distinct changes were found in mantids. We then discovered numerous possible host-derived genes in nematomorphs, and these genes were frequently up-regulated during host manipulation. Our findings suggest a possible general role of horizontal gene transfer (HGT) in the molecular mechanisms of host manipulation, as well as in the genome evolution of manipulative parasites. The evidence of HGT between multicellular eukaryotes remains scarce but is increasing and, therefore, elucidating its mechanisms will advance our understanding of the enduring influence of HGT on the evolution of the web of life.

sample_organism:
Tenodera angustipennis
Chordodes fukuii

platform_organism:
Tenodera angustipennis
Chordodes fukuii

overall_design: Chordodes fukuii whole body and Tenodera angustipennis whole brain transcriptomes were analyzed for different manipulation processes. For Chordodes fukuii, samples from before, during and after manipulation were analyzed. For Tenodera angustipennis, before and during manipulation as well as uninfected samples were analyzed.

first: Ming-Chung

last: Chiu

first: Yasuyuki

last: Hashiguchi

first: Sayumi

last: Oishi

first: Atsunari

last: Sasaki

first: Ryuichi

last: Okada

first: Hironobu

last: Uchiyama

first: Takeshi

first: Midori

last: Sakura

first: Hirohiko

last: Takeshima

first: Takuya

last: Sato


title: Stepwise differentiation of follicular helper T cells reveals distinct developmental and functional states [RNA-Seq]

city: Boston

first: Peter

last: Sage

address: 221 Longwood Ave

department: Medicine/Renal

institute: Brigham and Women's Hospital

summary: Assessment of transcriptional programs between TfhProg and TfhFull cells

overall_design: Tfh-Prog versus Tfh-Full RNASeq


title: The ncBAF complex regulates transcription in AML through H3K27ac sensing by BRD9

city: Pittsburgh

first: David

last: Klein

state: PA

address: 4249 Fifth Ave

department: Biological Sciences

institute: University of Pittsburgh


title: Distinct Transcriptional Landscapes Identified by Radiographic Response in Metastatic Triple-Negative Breast Cancer Treated with Carboplatin, Nab-Paclitaxel, and Pembrolizumab in a Single-Arm Phase II Trial

city: Cleveland

first: Vladimir

last: Makarov

state: OH

address: 9500 Euclid Ave

institute: CLEVELAND CLINIC LERNER COM-CWRU

summary: A single arm, Phase II trial of carboplatin, nab-paclitaxel, and pembrolizumab (CNP) in metastatic triple negative breast cancer (mTNBC) was designed to evaluate overall response rate (ORR), progression-free survival (PFS), duration of response (DOR), safety/tolerability, and identify pathologic and transcriptomic correlates of response to therapy.

overall_design: Single arm phase II clinical trial involving treatment of mTNBC patients with carboplatin, nab-paclitaxel, and pembrolizumab (CNP) to assess the overall response rate (ORR), overall and progression-free survival (OS, PFS), and toxicity profile associated with this particular combination of chemo-immunotherapeutic agents.

first: Avia

middle: D

last: Wilkerson

first: Prerana

middle: B

last: Parthasarathy

first: Nickolas

last: Stabellini

first: Carley

last: Mitchell

first: Paul

middle: G

last: Pavicic Jr

first: Pingfu

last: Fu

first: Amit

last: Rupani

first: Hana

last: BA

first: Patricia

last: Rayman

first: Shadi

last: Swaidani

first: Jame

last: Abraham

first: G

middle: T

last: Budd

first: Halle

last: Moore

first: Zahraa

last: Al-Hilli

first: Jennifer

middle: S

last: Ko

first: Joseph

last: Baar

first: Timothy

last: Chan

last: Alban

first: C

middle: M

last: Diaz-Montero

first: Alberto

last: Montero


title: SRCAP Mutations Drive Clonal Hematopoiesis Through Epigenetic and DNA Repair Dysregulation

city: HOUSTON

first: Margaret

last: Goodell

state: TX

address: 1 Baylor Plz, Rm N1030

institute: Baylor College of Medicine


title: SRCAP Mutations Drive Clonal Hematopoiesis Through Epigenetic and DNA Repair Dysregulation [Cut & Run]

summary:
To investigate how Srcap mutations confer mutant hematopoietic stem and progenitor cells (HSPCs) with selective advantage after transplantation
We assessed SRCAP chromatin remodeling and corresponding H2A.Z incorporation using Cut-and-run sequencing

overall_design: We established Srcap heterozygous mutant murine competitive transplantation model. Donor test (CD45.2+) HSPCs in the bone marrow were sored and isolated at 12 weeks post-transplantation for Cut-and-run sequencing

first: Chun-Wei

last: Chen


title: LOX1 and PLP1 dependent transcriptional reprogramming is essential for injury induced conidiophore development in a filamentous fungus

city: Irapuato

first: Alfredo

last: Herrera Estrella

state: Guanajuato

address: Km 9.6 Libramiento Norte Carretera Irapuato-León

department: LANGEBIO

country: Mexico

institute: CINVESTAV

summary: We need to learn more about the components of the fungal damage response that are similar and well-studied in plants and animals, such as the role of oxidized lipids in mechanical stress. Using a genetic approach, mass spectrometry, and phenotypic analysis, we describe the functional role of a patatin-like phospholipase (plp1) and a unique lipoxygenase (lox1) with biosynthetic function in the production of oxidized PUFA derivatives by mechanical injury in the model fungus Trichoderma atroviride. lox1 and plp1 are co-expressed and regulated by damage signaling and sensing components. Phenotypic analysis revealed an essential defect in the emergence of aerial hyphae in the lox1 and plp1 null mutant strains, blocking injury-induced conidiation. In addition, functional loss analysis demonstrated that both genes are essential for producing the wound oxylipin 13-HODE (13-hydroxy octadecadienoic acid) and the transcriptional reprogramming required for conidiation. T. atroviride requires LOX1 and PLP1 to induce at early stages of the response transcription factors involved in asexual development like a BrlA homolog, Hox2, and Azf1, and at a later stage to activate lipid metabolism and structural proteins involved in aerial mycelium emergence. Our study emphasizes how the co-function of lox1 and plp1, during the response to wounding, regulate the molecular and physiological processes that lead to reproductive aerial mycelium development to warrant survival through conidiation.

overall_design: To decipher the biological function of LOX1 and PLP1 in response to wounding, we generated mutant strains of LOX1 and PLP1. We sequenced mRNA obtained from mycelium of WT and mutants strains (dlox1 and dplp1) at three times of response: control (0 h), 90 min and 4 h after mechanical injury. Differential gene expression was performed contrasting control versus injury using data obtained from RNA-seq analysis.

first: Camargo-Escalante

last: Martín Orlando

first: Balcázar-López

last: Edgar

first: Albores-Méndez

middle: E

last: Manuel

first: Winkler

last: Robert

first: Herrera-Estrella

last: Alfredo


title: Effect of sulforaphane on gene expression in Spodoptera exigua and Trichoplusia ni

city: Carlisle

first: Dana

middle: J.

last: Somers

department: Biology

institute: Dickinson College

summary: Cruciferous plants produce sulforaphane (SFN), an inhibitor of nuclear histone deacetylases (HDACs). In humans and other mammals, the consumption of SFN alters enzyme activities, DNA-histone binding, and gene expression within minutes. However, the ability of SFN to act as a HDAC inhibitor in nature, disrupting the epigenetic machinery of insects feeding on these plants, has not been explored. Here, we investigate the effect of SFN, as well as the pharmaceutical HDAC inhibitor Trichostatin A (TSA), consumed in the diet in the generalist grazer Spodoptera exigua and in the co-evolved specialist feeder Trichoplusia ni. To investigate the mechanisms of HDAC inhibition, we profiled transcriptome changes via RNA-seq in S. exigua and T. ni fat body tissues responding to SFN or TSA, in biological triplicate.

sample_organism:
Spodoptera exigua
Trichoplusia ni

platform_organism:
Spodoptera exigua
Trichoplusia ni

overall_design:
To investigate the effect on gene expression of HDAC inhibitors, we supplemented the standard artificial diet of two different species (S. exigua and T. ni) with SFN, TSA, or a control (EtOH).
We obtained RNA-seq data from fat body tissues extracted from 10-day old larvae. To obtain sufficient tissue, fat bodies from 3 – 5 individual larvae were pooled per biological replicate.
Differential gene expression analysis was performed for three biological replicates, comparing the mean expression for all pairwise combinations of treatments.

first: Thomas

last: Arnold

first: David

last: Kushner


title: SRCAP Mutations Drive Clonal Hematopoiesis Through Epigenetic and DNA Repair Dysregulation [RNA-seq]

summary:
To investigate how Srcap mutations confer mutant hematopoietic stem and progenitor cells (HSPCs) with selective advantage after exposure to cellular stressors, transplantation and doxorubicin treatment
We established Srcap heterozygous mutant murine model and utilized competitive bone marrow transplantation to assess the transcriptome of donor cells using RNA-seq

overall_design: Transcriptomic profiling of RNA-seq data for isolated donor test (CD45.2+) cells 20 weeks after competitive bone marrow transplantation


title: SRCAP Mutations Drive Clonal Hematopoiesis Through Epigenetic and DNA Repair Dysregulation [ATAC-Seq]

summary:
To investigate how Srcap mutations confer mutant hematopoietic stem and progenitor cells (HSPCs) with selective advantage after transplantation
We assessed chromatin remodeling and chromatin accessbility before and after transplantation to understand the mechanism of the acquisition of Srcap selective advantage

overall_design: We established Srcap heterozygous mutant murine competitive transplantation model. Donor test (CD45.2+) HSPCs in the bone marrow were sored and isolated at 12 weeks post-transplantation for ATAC-seq


title: TIME-Seq Enables Scalable and Inexpensive Epigenetic Age Predictions.

first: Patrick

last: Griffin

address: 77 Avenue Louis Pasteur

department: Harvard Medical School, Dept. of Genetics

institute: Patrick Griffin

type: Methylation profiling by high throughput sequencing

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series


title: Contribution of PDGFRα Lineage cells in adult mouse hematopoiesis

city: Suita

first: Takashi

last: Shimbo

state: Osaka

address: 2-2 Yamadaoka

institute: Osaka university

summary: Hematopoietic system is composed of various cells with a wide range of functions. Hematopoietic stem cells generate these descendant cells. Recently, it has become clear that mesodermal tissues give rise to HSCs during early development. Here, we examined the properties and functions of mesoderm-derived HSCs in the adult hematopoietic system.

overall_design: We isolated mesoderm-derived HSCs using PDGFRα as a mesodermal marker. Briefly, PDGFRα lineage HPCs were isolated using PDGFRα-Cre;ROSAtdTomato mice.  HSCs were isolated from bone marrow and peripheral blood. We performed single cell RNA-seq analysis on the isolated PDGFRα lineage HPCs to reveal their properties and functions.


title: Sox9 regulates alternative splicing and pancreatic beta cell function [mouse]

status: Public on Oct 18 2023

city: San Francisco

first: Sapna

last: Puri

address: 513 Parnassus Avenue

institute: UCSF

summary: Type 2 Diabetes Mellitus (T2D), a multifactorial disease, can result from perturbations in numerous pancreatic genes. We describe a previously unanticipated role for Sox9, a transcriptional regulator of embryonic pancreas and endocrine cell development, in mature beta cells. Our data demonstrate that Sox9 has continued function in beta cells as they mature, and elimination of Sox9 compromises beta cell activities. Sox9-depleted rodent beta cells fail to appropriately secrete insulin and exhibit glucose intolerance in aging animals, mimicking the progressive degeneration observed in T2D. Human beta cells lacking SOX9 are functionally impaired with stunted first phase insulin secretion. In both rodent and human, Sox9 loss in beta cells disrupts alternative splicing patterns, with significant implications for maintenance of cellular function. Thus, our data uncover a novel, unprecedented role for a developmental transcription factor in mature beta cell function.

overall_design: To investigate the role of SOX9 in pancreatic beta cells, we crossed Ins-Cre mice with Sox 9 flox mice to generate the beta cell-specific Sox9 Knockout mice.

first: Hasna

last: Maachi

first: Gopika

last: Nair

first: Holger

last: Russ

first: Richard

first: Pamela

last: Pulimeno

first: Zachary

last: Cutts

first: Vasilis

last: Ntranos

first: Matthias

last: Hebrok


title: Sox9 regulates alternative splicing and pancreatic beta cell function

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


title: Sox9 regulates alternative splicing and pancreatic beta cell function [human]

overall_design: To investigate SOX9 function in human adult pancreatic beta cells, TALEN technology was used to generate a Mel1-INS-GFP inducible SOX9 knockout reporter line. The line was differentiated to mature beta cells and treated with doxycycline to silence SOX9 expression.

last: Chen


title: Lipid Nanoparticles Allow Efficient and Harmless Ex Vivo Gene Editing of Human Hematopoietic Cells [NHEJ]

city: Milan

first: Stefano

last: Beretta

address: Via Olgettina, 60

country: Italy

institute: San Raffaele Telethon Institute for Gene Therapy

summary: Ex vivo gene editing in T cells and hematopoietic stem/progenitor cells (HSPCs) holds promise for treating diseases. Gene editing encompasses delivery of a programmable editor RNA or ribonucleoprotein, often achieved ex vivo by electroporation and, when aiming to homology-driven correction, of a DNA template often provided by viral vectors together with a nuclease editor. Whereas HSPCs activate robust p53-dependent DNA damage response (DDR) upon nuclease-based editing, the responses triggered in T cells remain poorly characterized. Here, we performed comprehensive multi-omics analyses and found that electroporation is the main culprit of cytotoxicity in T cells, causing death and cell cycle delay, perturbing metabolism and inducing inflammatory response. Nuclease RNA delivery by lipid nanoparticles (LNPs) nearly abolished cell death and ameliorated cell growth, improving tolerance to the procedure and yielding higher number of edited cells compared to electroporation. Transient transcriptomic changes upon LNP treatment were mostly caused by cellular loading with exogenous cholesterol, whose potentially detrimental impact could be overcome by limiting exposure. Notably, LNP-based HSPC editing dampened p53 pathway induction and supported higher clonogenic activity and similar or higher reconstitution by long-term repopulating HSPCs compared to electroporation, reaching comparable editing efficiencies. Overall, LNPs may allow efficient and harmless ex vivo gene editing in hematopoietic cells for treatment of human diseases.

overall_design: Targeted deep sequencing on mPB HSPC donors, treated with different editing conditions, namely, RNP, LNPs, and mRNA in different target regions (B2M exon1 and exon2, AAVS1, and CD40L), or after transplant in different tissues (mPB, BM, and spleen).

first: Valentina

last: Vavassori

first: Samuele

last: Ferrari

first: Ivan

last: Merelli

first: Luigi

last: Naldini


title: CSF1R inhibition promotes neuroinflammation and behavioural deficits during graft-versus-host disease in mice.

city: Brisbane

first: Rachael

last: Adams

state: QLD

address: 300 Herston Road

country: Australia

institute: QIMR Berghofer Medical Research Institute

summary: Chronic graft-versus-host disease (cGVHD) remains a significant complication of allogeneic hematopoietic stem cell transplantation. Central nervous system (CNS) involvement is becoming increasingly recognised, where brain-infiltrating donor MHC class II+ bone marrow-derived macrophages (BMDM) drive pathology. BMDM are also mediators of cutaneous and pulmonary cGVHD, and clinical trials assessing the efficacy of antibody blockade of colony-stimulating factor 1 receptor (CSF1R) to deplete macrophages are promising. We hypothesised that CSF1R antibody blockade may also be a useful strategy to prevent/treat CNS cGVHD. Increased blood-brain barrier permeability during acute GVHD (aGVHD) facilitated CNS antibody access and microglia depletion by anti-CSF1R treatment. However, CSF1R blockade early post-transplant unexpectedly exacerbated aGVHD neuroinflammation. In established cGVHD, vascular changes and anti-CSF1R efficacy were more limited. Anti-CSF1R-treated mice retained donor BMDM, activated microglia, CD8+ and CD4+ T cells, and local cytokine expression in the brain. These findings were recapitulated in GVHD recipients where CSF1R was conditionally depleted in donor CX3CR1+ BMDM. Notably, inhibition of CSF1R signalling post-transplant failed to reverse GVHD-induced behavioural changes. Moreover, we observed aberrant behaviour in non-GVHD control recipients administered anti-CSF1R blocking antibody and naïve mice lacking CSF1R in CX3CR1+ cells, revealing a novel role for homeostatic microglia and indicating that ongoing clinical trials of CSF1R inhibition should assess neurological adverse events in patients. In contrast, transfer of Ifngr-/- grafts could reduce MHC class II+ BMDM infiltration, resulting in improved neurocognitive function. Our findings highlight unexpected neurological immune toxicity during CSF1R blockade and provide alternative targets for the treatment of cGVHD within the CNS.

overall_design: Brains from mice with chronic GVHD following allogenic transplantation with wild-type or IFNG receptor knockout bone marrow, and transplanted non-GVHD controls, were collected at day70 post-transplant. Nuclei were isolated with the 10X Genomics Chromium Nuclei Isolation kit and further purified by sorting for DAPI+ cells.

middle: C

first: Dylan

last: Carter-Cusack

first: Genesis

last: Llanes

first: Christopher

middle: R

last: Hunter

first: Janaki

last: Manoja Vinnakota

first: Marc

last: Ruitenberg

first: Jana

last: Vukovic

last: Bertolino

first: Kirat

middle: K

last: Chand

first: Julie

last: Wixey

first: Sam

middle: P

last: Nayler

first: Geoffrey

last: Hill

first: Scott

middle: N

last: Furlan

first: Robert

last: Zeiser

first: Kelli

last: MacDonald


title: Data for Alternative Splicing of Adhesion-GPCR Latrophilin-3 Controls Synapse Formation

city: Stanford

first: Shuai

last: Wang

state: CA

address: 265 Campus Drive

institute: Stanford University

summary: How synapses are assembled and specified in brain is incompletely understood. Latrophilin-3, a postsynaptic adhesion-GPCR, mediates Schaffer-collateral synapse formation in the hippocampus but the mechanisms involved remained unclear. Here we show that Latrophilin-3 organizes synapses by a convergent dual-pathway mechanism by which Latrophilin-3 simultaneously activates GaS/cAMP-signaling and recruits phase-separated postsynaptic protein scaffolds. We found that cell type-specific alternative splicing of Latrophilin-3 controls its G protein coupling mode, resulting in Latrophilin-3 variants that predominantly signal via Gas and cAMP or via Gα12/13. A CRISPR-mediated genetic switch of Latrophilin-3 alternative splicing from a GaS- to a Gα12/13-coupled mode impaired synaptic connectivity similar to the overall deletion of Latrophilin-3, suggesting that GaS/cAMP-signaling by Latrophilin-3 splice variants mediates synapse formation. Moreover, GaS- but not Gα12/13-coupled splice variants of Latrophilin-3 recruit phase-transitioned postsynaptic protein scaffolds that are clustered by binding of presynaptic Latrophilin-3 ligands. Strikingly, neuronal activity promotes alternative splicing of the synaptogenic variant of Latrophilin-3, thereby enhancing synaptic connectivity. Together, these data suggest that activity-dependent alternative splicing of a key synaptic adhesion molecule controls synapse formation by parallel activation of two convergent pathways, GaS/cAMP signaling and the phase separation of postsynaptic protein scaffolds.

overall_design:
Two experiments included in this submission:
(1) RNA sequencing in primary hippocampal neurons cultured from Cas9-expressing mice (JAX ##024858), to study the transcriptomic alterations after Lphn3 is deleted (Lphn3 KO), or the Exon31 of Lphn3 is disrupted (Lphn3 Exon31 KO). In this experiment, neurons were infected with lentiviruses expressing gRNA which either: has no target as negative control (control group, 3 replicates), or targets Lphn3 constitutive exon7 to induce KO of all Lphn3 (Lphn3 KO group, 3 replicates), or targets the splice acceptor of Exon31 of Lphn3 in order to disrupt the splicing of Exon31 (Lphn3 Exon31 KO group, 3 replicates).
(2) Amplicon sequencing of PDZ-binding motif (PBM) region of Lphn3 in primary hippocampus neurons cultured from Cas9-expressing mice (JAX ##024858), to assess the efficiency of PBM KO in all Lphn3 mRNAs. In this experiment, neurons were infected with lentiviruses expressing gRNA which either: has no target as negative control (control group, 2 replicates), or targets the upstream of PBM of Lphn3 to induce KO of PBM (PBM KO group, 2 replicates).


title: Cytosolic PRRs are required to upregulate interferon gene expression upon EZH2 inhibition with GSK343 in B16-F10 melanoma cells [RNA-seq]

city: London

first: Frederick

middle: Andrew

last: Dick

state: ON

address: 790 Commissioners Road E

department: Pathology

country: Canada

institute: Western University

summary: EZH2 is a histone methyltransferase that deposits the repressive histone H3 lysine 27 trimethylation (H3K27me3). This mark has been reported to silence expression of various repetitive elements in the genome. We sought to identify the effect of EZH2 inhibition on coding and non-coding, repetitive RNA expression. We generated a novel mutant mouse line whose ability to detect induced repeat element expression has been abrogated. We report that the mutant splenic B cells fail to upregulate inflammatory gene expression upon EZH2 inhibition, despite comparable upregulation of various repetitive elements with WT. Unlike WT splenic B cells that do not upregulate interferon gene expression upon EZH2 inhibition, B16-F10 melanoma cells activate their expression under the same inhibitor treatment. Cytosolic pattern recognition receptors, RIG-I and Cgas, are required for such response as their deletion abrogates this pathway.

overall_design: Gene expression profiles of WT and PRR mutant B16-F10 cells upon GSK343 treatment

first: Seung

last: Kim


title: Simultaneous deep transcriptome and proteome profiling in single mouse oocytes

city: Hangzhou

first: Yi-Rong

last: Jiang

state: Zhejiang

address: Room C306, Chemistry Building, Zijingang Campus, No. 866 Yuhangtang Ro

department: Department of chemistry

country: China

institute: Zhejiang University

summary: Nowadays, although single-cell multi-omics technologies are undergoing rapid development, simultaneous transcriptome and proteome analysis of a single-cell individual still faces great challenges. Here, we developed a single-cell simultaneous transcriptome and proteome (scSTAP) analysis platform based on microfluidics, high-throughput sequencing and mass spectrometry technology, to achieve deep and joint quantitative analysis of transcriptome and proteome at the single-cell level, providing an important resource for understanding the relationship between transcription and translation in cells. This platform was applied to analyze single mouse oocytes at different meiotic maturation stages, reaching an average quantification depth of 19948 genes and 2663 protein groups in single mouse oocytes.

overall_design: We analyzed the transciptomes of single mouse oocytes at GV and MII stages using the MATQ-seq.

first: Yirong

first: Le

last: Zhu

first: Lanrui

last: Cao

first: Xudong

last: Fu

first: Yongcheng

first: Qun

last: Fang


title: Competition for H2A.Z underlies the developmental impacts of repetitive element de-repression

status: Public on Oct 23 2023

city: Rochester

first: Fanju

last: Meng

state: NY

address: 601 Elmwood Avenue

department: Biomedical Genetics

institute: University of Rochester Medical Center

summary: The histone variant H2A.Z is central to early embryonic development, determining transcriptional competency through chromatin regulation of gene promoters and enhancers. In addition to genic loci, we find that H2A.Z resides at a subset of evolutionarily young repetitive elements, including DNA transposons, LINEs, and LTRs, during early zebrafish development. Moreover, increases in H2A.Z occur when repetitive elements become transcriptionally active. Acquisition of H2A.Z corresponds with a reduction in the repressive histone modification H3K9me3, and a moderate increase in chromatin accessibility. Notably however, de-repression of repetitive elements also leads to a significant reduction in H2A.Z over non-repetitive genic loci. Genic loss of H2A.Z is accompanied by transcriptional silencing at adjacent coding sequences, but remarkably, these impacts are mitigated by augmentation of total H2A.Z protein, via transgenic over-expression. Our study reveals that levels of H2A.Z protein determine embryonic sensitivity to de-repression of repetitive elements, that repetitive elements can function as a nuclear sink for epigenetic factors, and that competition for H2A.Z greatly influences overall transcriptional output during development. These findings uncover general mechanisms in which counteractive biological processes underlie phenotypic outcomes.

overall_design: H2A.Z genomic profiling and gene expression in DMSO, 5-AzaC, 5-AzadC, and TDCIPP treated zebrafish embryos at 6hpf and 12hpf were generated by next-generation sequencing.


title: SRCAP Mutations Drive Clonal Hematopoiesis Through Epigenetic and DNA Repair Dysregulation [scRNA-Seq]

first: Margaret

last: Goodell

summary: To investigate the transcriptomic regulation of Srcap mutant lymphoid-biased expansion after compeititve transplantation

overall_design: We established Srcap heterozygous mutant murine competitive transplantation model.  Donor test (CD45.2+) HSPCs in the bone marrow were sored and isolated at 20 weeks post-transplantation for scRNA-seq

first: Chun-Wei

last: Chen


title: RNA-seq of CYR61-treated THP1 human monocytes

city: Saint Louis

first: Joseph

last: Lin

state: MO

address: 660 South Euclid Ave, MSC-8096-06-07

department: Ophthalmology & Visual Sciences

institute: Washington University School of Medicine

summary: We assess the effects of CYR61 treatment on the transcriptome of human THP1 monocytes.

overall_design: We used THP1 monocytes (ATCC TIB-202). These cells were maintained in RPMI 1640 media containing 1% L-glutamine, 1% penicillin/streptomycin, and 10% FBS. We treated THP1 cells with recombinant human CYR61 (PeproTech 120-25): 0.00, 0.05, 0.50, and 5.00 μg/ml for 24 hr. After incubation, we washed cells with PBS, isolated total RNA, and prepared samples for RNA-sequencing.

first: Philip

last: Ruzycki

first: Rajendra

last: Apte


title: Splenic CD43- B cells exhibit DNA hypomethylation specifically at repetitive elements compared to CD43+ cells

summary: EZH2 is a histone methyltransferase that deposits H3K27me2/3 in heterochromatin and it is misregulated in tumorigenesis. Inhibition of heterochromatin can induce viral mimicry in cancer cells, where upregulated repetitive elements are detected by cytosolic pattern recognition receptors (PRRs) to activate inflammatory signaling. Here we demonstrate that EZH2 inhibitors stimulate inflammation and immune self-recognition of resting splenic B cells. We generated a PRR loss-of-function mouse model called RIC with mutations in Rigi, Ifih1 (MDA5), and Cgas. In both WT and RIC mutant B cells, EZH2 inhibition caused loss of H3K27me3 at repetitive elements and upregulated their expression. However, expression of inflammatory chemokines in B cells was interrupted by the RIC mutations. Furthermore, recruitment of cytotoxic T cells and neutrophils in response to EZH2 inhibition was blocked in RIC mutants preserving viability of B cells. This study demonstrates a pharmacologically induced mechanism of inflammation that causes self-recognition and B cell death.

overall_design: Bisulphite converted DNA from the 12 samples were hybridised to the Illumina Infinium 285k mouse methylation BeadChip.

middle: J


title: Epigenetic Switch Reshapes Epithelial Stem/Progenitor Cells Signatures and Contributes to Inflammatory Programming in Hidradenitis Suppurativa [ATAC-seq]

city: birmingham

first: lin

last: jin

state: alabama

address: 1824 6th ave s

department: Dermatology

institute: university of alabama at birmingham

summary: By using dual-omics profiling for both chromatin accessibility and gene expression as well as CUT&RUN sequencing for chromatin modification states, we identified that numerous disease-associated inflammatory enhancers were epigenetically activated and co-exist with the transcriptional states of their target genes in basal progenitor cells.

overall_design: The Institutional Review Board of the University of Alabama at Birmingham approved the protocol (IRB-300005214) for obtaining surgically discarded skin tissues from heathy and HS (Hurley II/III stage) subjects. Surgical excisions from 3 patients with HS (axillary and groin lesions) and 3 healthy controls (discarded tissue from breast or abdominoplasty reduction surgery) were collected.

first: Lin

last: Jin

first: Mohammad

last: Athar


overall_design: Chromatin accessibility was examined after TDCIPP and Dacitabine (DAC) treatment in developing zebrafish embryos

first: Claire

last: Makowski

first: Kristin

last: Murphy


title: A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells

first: SHOU-WEN

last: WANG

state: Zhejiang Province

address: Shilongshan ST #18, Xihu District

institute: Westlake University

summary: Cellular lineage histories along with their molecular states encode fundamental principles for tissue development and homeostasis. Current cellular barcoding mouse models have limited barcode diversity and poor single-cell lineage readout, thus precluding their use in tissues composed of millions of cells. Here, we developed DARLIN, an improved Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) to enhance insertion events over 30 CRISPR target sites, stably integrated into 3 distinct genomic loci. DARLIN is inducible, has an estimated ~10^18 lineage barcodes across tissues, and allows detection of reliable barcodes in ~60% of profiled single cells. Using DARLIN, we revealed fate priming within hematopoietic stem cells (HSCs) and evaluated HSC migration across tissues. Additionally, we adapted a method to jointly profile DNA methylation, chromatin accessibility, gene expression, and lineage barcodes in single cells. Applying it to study clonal memory of HSCs over time, we found that cells within a clone have more similar genome-wide DNA methylation than gene expression or chromatin accessibility. In total, our study enables systematically dissecting lineage relationships and their molecular mechanisms across diverse problems in biology.

overall_design: In this study, we generate bulk CARLIN sequence datasets for both the DARLIN and Cas9/CARLIN mouse lines to evaluate the performance of DARLIN. Then, we generated both single-cell CARLIN and transcriptome data to study early fate bias within hematopoietic stem cells (HSCs). Furthermore, we generated bulk CARLIN sequences across different developmental stages to study HSC migration. We also performed single-cell multi-omic lineage tracing (DNA methylation, chromatin accessibility, gene expression, and lineage information) to study the clonal memory of HSCs.

first: Li

last: Li

first: Sarah

last: Bowling

first: Sean

last: McGeary

first: Qi

last: Yu

first: Bianca

last: Lemke

first: Karel

last: Alcedo

first: Yuemeng

last: Jia

first: Xugeng

last: Liu

first: Mark

last: Ferreira

first: Allon

last: Klein

first: Shou-Wen

last: Wang

first: Fernando

last: Camargo


title: Epigenetic Switch Reshapes Epithelial Stem/Progenitor Cells Signatures and Contributes to Inflammatory Programming in Hidradenitis Suppurativa [scRNA-seq]

first: lin

last: jin

summary: In this study, we used single-cell RNA-sequencing to reveal how cellular heterogeneity of Hidradenitis Suppurativa (HS) lesional epidermis was reshaped at the transcriptional level. By comparison with healthy interfollicular epidermal basal cells, in HS we revealed marked gene signatures centered on mitotic chromosome segregation, DNA replication and repair as well as cell-cell adhesion and chromatic remodeling. Combing the pseudotime-ordered single-cell trajectory with the spatial localization analysis of defined basal cells, we further identified and validated a global alteration in cellular diversity within HS epidermis characterized by basal I-III cells hyperproliferation, concomitant decreases in superbasal keratinocytes, and phenotypic shifts toward the S100A cluster highly expressing pro-inflammatory genes S100A7/8/9. Cell-cell communication modeling suggested that HS BIII keratinocytes and S100A population serve as a crucial source to trigger IL-1 and IL-10 inflammatory cascades in the disease progression.

overall_design: The Institutional Review Board of the University of Alabama at Birmingham approved the protocol (IRB-300005214) for obtaining surgically discarded skin tissues from heathy and HS (Hurley II/III stage) subjects. Surgical excisions from 7 patients with HS (axillary and groin lesions) and 5 healthy controls (discarded tissue from breast or abdominoplasty reduction surgery) were collected.

first: Lin

last: Jin


title: RNA-seq of human umbilical vein endothelial cells co-incubated with CYR61-treated mouse bone marrow-derived macrophages

first: Joseph

last: Lin

summary: We assess the transcriptional changes when human umbilical vein endothelial cells are co-incubated with naïve or CYR61-activated mouse bone marrow-derived macrophages.

overall_design: We used primary human umbilical vein endothelial cells (HUVECs) from ATCC (PCS-100-010). We maintained HUVECs according to the manufacturer's recommended instructions. We isolated bone marrow from the tibias and femurs of C57BL/6J mice (2-5 months old). Bone marrow cells were incubated for 5-6 days in macrophage differentiation media: DMEM + 10% FBS + 10% CMG-conditioned media + 1% penicillin/streptomycin + 1% L-glutamine. After differentiation, macrophages were switched to mature macrophage media: DMEM + 10% FBS + 2% CMG-conditioned media + 1% penicillin/streptomycin + 1% L-glutamine. We plated HUVECs in the bottom compartments of 24-well transwell plates (0.4 μm pore size) along with the following in the top compartments: 1) cell-free (n=6 wells); 2) naïve BMDMs (n=6 wells); or 3) CYR61-treated BMDMs (0.5 μg/ml, 24 hr, n=6 wells). After co-incubation for 24 hrs, we washed HUVECs twice with PBS, extracted total RNA from HUVECs, and then prepared samples for RNA-sequencing.

first: Philip

middle: A

last: Ruzycki

first: Rajendra

last: Apte


title: A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells [scRNA-seq datasets from Camellia-seq]

first: SHOU-WEN

last: WANG

summary: Cellular lineage histories along with their molecular states encode fundamental principles of tissue development and homeostasis. Current lineage-recording mouse models have limited barcode diversity and poor single-cell lineage coverage, thus precluding their use in tissues composed of millions of cells. Here, we developed DARLIN, an improved Cas9 barcoding mouse line that utilizes terminal deoxynucleotidyl transferase (TdT) to enhance insertion events over 30 CRISPR target sites, stably integrated into 3 distinct genomic loci. DARLIN is inducible, has an estimated ~10^18 lineage barcodes across tissues, and enables detection of usable barcodes in ~60% of profiled single cells. Using DARLIN, we examined fate priming within developing hematopoietic stem cells (HSCs) and revealed unique features of HSC migration. Additionally, we adapted a method to jointly profile DNA methylation, chromatin accessibility, gene expression, and lineage information in single cells. Using this approach we found that cellular clonal memory is associated with genome-wide DNA methylation rather than gene expression or chromatin accessibility. DARLIN will enable widespread high-resolution study of lineage relationships and their molecular signatures in diverse tissues and physiological contexts.

first: Li

last: Li


title: RNA-seq of CYR61-treated murine bone marrow-derived macrophages

summary: We assess the effect of CYR61 treatment on the transcriptome of mouse bone marrow-derived macrophages.

overall_design: We isolated bone marrow from the tibias and femurs of C57BL/6J mice (2-5 months old). Bone marrow cells were incubated for 5-6 days in macrophage differentiation media: DMEM + 10% FBS + 10% CMG-conditioned media + 1% penicillin/streptomycin + 1% L-glutamine. After differentiation, macrophages were switched to mature macrophage media: DMEM + 10% FBS + 2% CMG-conditioned media + 1% penicillin/streptomycin + 1% L-glutamine. We treated macrophages with 0.00 or 0.50 ug/ml CYR61 (PeproTech 120-25) for 24 hrs. After incubation, we washed cells with PBS, isolated RNA, and prepared samples for RNA-sequencing.


title: A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells [10X scRNA-seq data]

first: Sarah

last: Bowling

first: Sean

last: McGeary


title: Bulk RNA-seq of: HUVECs co-incubated with CYR61-treated mouse bone marrow-derived macrophages; CYR61-treated murine bone marrow-derived macrophages; CYR61-treated HUVECs; and CYR61-treated human THP1 monocytes

overall_design: Refer to individual Series


title: Epigenetic Switch Reshapes Epithelial Stem/Progenitor Cells Signatures and Contributes to Inflammatory Programming in Hidradenitis Suppurativa [RNA-seq]

first: Mohammad

last: Athar


title: RNA-seq of CYR61-treated human umbilical vein endothelial cells

summary: We assess the effect of CYR61 treatment on the transcriptome of human umbilical vein endothelial cells.

overall_design: We used primary human umbilical vein endothelial cells (HUVECs) from ATCC (PCS-100-010). These cells were maintained according to the manufacturer's recommended instructions. We treated HUVECs with recombinant human CYR61 (PeproTech 120-25): 0.00, 0.05, 0.50, and 5.00 μg/ml for 24 hr. After incubation, we washed cells with PBS, isolated total RNA, and prepared samples for RNA-sequencing.


title: In vivo macrophage engineering reshapes the tumor microenvironment leading to eradication of liver metastases [scRNA-seq TCR]

status: Public on Oct 17 2023

first: Stefano

last: Beretta

summary: Liver metastases are associated with poor response to current pharmacological treatments, including immunotherapy. We describe a lentiviral vector (LV) platform to selectively engineer liver macrophages, including Kupffer cells and tumor-associated macrophages (TAMs), to deliver type I interferon (IFNα) to liver metastases. Gene-based IFNα delivery delays the growth of colorectal and pancreatic ductal adenocarcinoma liver metastases in mice. Response to IFNα is associated with TAM immune activation, enhanced MHC-II-restricted antigen presentation and reduced exhaustion of CD8+ T cells. Conversely, increased IL-10 signaling, expansion of Eomes CD4+ T cells, a cell type displaying features of type I regulatory T (Tr1) cells, and CTLA-4 expression are associated with resistance to therapy. Targeting regulatory T cell functions by combinatorial CTLA-4 immune checkpoint blockade and IFNα LV delivery expands tumor-reactive T cells, attaining complete response in most mice. These findings support a promising therapeutic strategy with feasible translation to patients with unmet medical need.

overall_design: Single-cell transcriptomics with corresponding TCRs (VDJ) on metastatic liver in three distinct cohorts: (1) Control: Control LV-treated mice, (2) IFNa: IFNa LV-treated mice and, (3) IFNa + aCTLA4: mice treated with IFNa in combination with a-CTLA-4.

first: Thomas

last: Kerzel

first: Giovanna

last: Giacca

first: Ivan

last: Merelli

first: Luigi

last: Naldini

first: Mario

middle: L

last: Squadrito


title: Cytosolic PRRs are required to upregulate inflammatory gene expression upon EZH2 inhibition with GSK343

first: Frederick

last: Dick

summary: EZH2 is a histone methyltransferase that deposits the repressive histone H3 lysine 27 trimethylation (H3K27me3). This mark has been reported to silence expression of various repetitive elements in the genome. We sought to identify the effect of EZH2 inhibition on coding and non-coding, repetitive RNA expression. We generated a novel mutant mouse line whose ability to detect induced repeat element expression has been abrogated. We report that the mutant splenic B cells fail to upregulate inflammatory gene expression upon EZH2 inhibition, despite comparable upregulation of various repetitive elements with WT.

overall_design: Coding and non-coding repetitive RNA expression profiles of WT and triple mutant splenocytes upon GSK343 treatment

first: Seung

last: Kim


overall_design:
In this study, we generate bulk CARLIN sequence datasets for both the DARLIN and Cas9/CARLIN mouse lines to evaluate the performance of DARLIN. Then, we generated both single-cell CARLIN and transcriptome data to study early fate bias within hematopoietic stem cells (HSCs). Furthermore, we generated bulk CARLIN sequences across different developmental stages to study HSC migration. We also performed single-cell multi-omic lineage tracing (DNA methylation, chromatin accessibility, gene expression, and lineage information) to study the clonal memory of HSCs.

The processed data from 10X single-cell RNA sequencing of hematopoietic cells sourced from various bones is accessible through GSE221501. For the single-cell Camellia-seq datasets spanning Hematopoietic Stem Cells (HSCs) across the AGM, fetal liver, and adult bone marrow stages, the processed scRNA-seq data can be accessed via GSE222246; additionally, the processed data for chromatin accessibility (CpG file) and DNA methylation (GpC) can be found at GSE222328. However, the processed versions of the DARLIN lineage datasets linked with these investigations have not been provided. All original sequencing data, including the DARLIN lineage data, associated with our research have been deposited on the SRA server.


title: EZH2 inhibition with GSK343 reduces H3K27me3 deposition at various repetitive elements in splenic B cells [ChIP-Seq]

summary: EZH2 is a histone methyltransferase that deposits the repressive histone H3 lysine 27 trimethylation (H3K27me3). This mark has been reported to silence expression of various repetitive elements in the genome. We sought to identify the effect of EZH2 inhibition on H3K27me3 peaks and their overlap with repetitive elements in splenic B cells. We generated a novel mutant mouse line whose ability to detect induced repeat element expression has been abrogated. We report that EZH2 inhibition decreases the number of H3K27me3 peaks in both WT and mutants compared to control.

overall_design: Genome-wide examination of H3K27me3 deposition in splenic B cells upon EZH2 inhibition


title: Rad6,an E2 enzymes required for insect-pathogenic lifestyle, UV damage repair, and genomic expression of Beauveria bassiana

first: Xin-Cheng

last: Luo

address: Yuhangtang Road

department: college of life science

summary: This transcriptomic analysis aims at unveiling all possible genes orchestrated by Rad6, which is evidently required for insect pathogenicity , virulence-related cellular events and UV damage repair of Beauveria bassiana

overall_design: Total RNAs were extracted from three 3-day-old hyphal cultures (replicates) of Rad6 disruption and wild-type strains grown under normal culture conditionsand and subjected to deep sequencing on Illumina NovaseqTM 6000 platform. The sequence reads that passed quality filters were mapped to the genome of Beauveria bassiana. All genes differentially expressed in the disruptant versus the wild-type strain were enriched to GO function classes and KEGG pathways.

first: Ming-Guang

last: Feng


title: Epigenomic mapping in B-cell acute lymphoblastic leukemia identifies transcriptional regulators and noncoding variants promoting distinct chromatin architectures [Hi-C]

city: Memphis

first: Daniel

last: Savic

state: Tennessee

address: 262 Danny Thomas Pl

department: Pharmaceutical Sciences

institute: St. Jude Children's Research Hospital

summary: Acute lymphoblastic leukemia (ALL) is the most common malignancy in pediatric patients and represents about a quarter of all pediatric malignancies. Past work has characterized B-cell lineage ALL (B-ALL) into molecular subtypes spanning a range of aberrant chromosomal rearrangements and oncogene chimeric fusions driving malignancy. While transcriptional and DNA methylome profiling of these subtypes has been extensively examined, the accompanying chromatin landscape and corresponding gene regulatory repertoire are not well characterized for many B-ALL subtypes. To better profile the B-ALL epigenome and gene regulatory networks we examined chromatin accessibility of 10 distinct molecular subtypes (BCR-ABL1, DUX4/ERG, ETV6-RUNX1, Hyperdiploid, Low hypodiploid, KMT2A-rearranged, Ph-like, PAX5-altered, TCF3-PBX1, ZNF384-rearranged and B-other) using 156 primary patient samples from across the United States with assay for transposase-accessible chromatin and high-throughput sequencing (ATAC-seq). In complement to ATAC-seq a subset of cell line and patient samples were profiled using promoter capture Hi-C to better define gene regulatory network connections.

overall_design: Promoter capture HiC


title: Intratumoral aluminum hydroxide-anchored IL-12 drives potent anti-tumor activity by remodeling the tumor microenvironment

city: Cambridge

first: Sailaja

last: Battula

state: MA

address: 245 Main St, 2nd Floor

institute: ANKYRA Therapeutics

summary: The goal of this scRNA-sequencing experiment is to study the immune modulatory effects after intratumoral injection of mANK-101 complex in a syngeneic mouse tumor model.

overall_design: C57BL/6 mice bearing MC38 syngeneic tumors (n=4 per group) were administered a single intratumoral injection of vehicle or 5 µg mANK-101 complex (5 µg mIL-12-ABP protein complexed with 50 µg Alhydrogel®). On Day 7, the tumors were harvested for scRNA sequencing analysis.

first: Gregory

last: Papastoitsis

first: Howard

last: Kaufman

first: K

middle: D

last: Wittrup

first: Michael

last: Schmidt


title: Histone retention preserves epigenetic marks during heat stress-induced transcriptional memory

city: Potsdam

first: Isabel

last: Bäurle

address: Karl-Liebknecht-Str. 24-25

department: Institut für Biochemie und Biologie

institute: Universität Potsdam

summary: Plants often experience recurrent stressful events, for example during heat waves. They can adapt to such recurrent heat stress (HS), allowing subsequent survival of more severe HS conditions. At certain genes HS induces sustained expression for several days beyond the actual HS. This transcriptional memory is associated with hyper-methylation of histone H3 lysine 4 (H3K4me3), however, how this is maintained for extended periods of time is unclear. Here, we determined histone turnover by measuring chromatin association of a HS-induced histone H3.3. Genome-wide Histone turnover was not homogenous, in particular, H3.3 was retained longer at HS memory genes compared to HS-induced non-memory genes during the memory phase. While low nucleosome turnover retained H3K4 methylation, its loss did not affect turnover, suggesting that low nucleosome turnover sustains H3K4 methylation (and not vice versa). Together, our results unveil the modulation of histone turnover as a mechanism to retain environmentally-mediated epigenetic modifications.

overall_design: ChIP-seq to estimate histone turnover using pulse-labelled histone H3.3.

first: Loris

last: Pratx

first: Christian

last: Kappel


title: Genome-wide expression analysis of Arabidopsis Col-0 plants under different air humidity

city: Shanghai

first: xiufang

last: xin

address: Xuhui

institute: Chinese Academy of Sciences

summary: The goal of this study is to demonstrate the global expression profile of Arabidopsis Col-0 wild type plant leaves in response to  low, moderate and high humidity.

overall_design: Four-week-old Col-0 plants were treated with  low, moderate and high humidity for 1 h. Leaves were collected at  1 h after different humidity treatment. Total RNA was extracted from the tissues using Trizol Reagent (Invitrogen) based on the manufacture's instruction, and genomic DNA was removed using the DNaseI (Invitrogen). RNA was further purified by using the RNeasy MinElute Cleanup kit (QIAGEN). RNA quality check, library construction, and RNA sequencing were performed by the Majorbio company.

first: Lingya

last: Yao

first: Zeyu

last: Jiang

first: Xiu-Fang

last: Xin


title: BATF represses BIM to sustain tolerant T cells in the periphery (RNA-Seq)

city: Minneapolis

last: Mueller

state: MN

address: 2101 6th Street SE

department: Medicine

institute: University of Minnesota Medical School

summary: T cells that encounter self-antigens after exiting the thymus avert autoimmunity through peripheral tolerance. Pathways for this include an unresponsive state known as anergy, clonal deletion, and T regulatory (Treg) cell induction. The transcription factor cues and kinetics that guide distinct peripheral tolerance outcomes remain unclear. Here, we found that anergic T cells are epigenetically primed for regulation by the non-classical AP-1 family member BATF. Tolerized BATF-deficient CD4+ T cells were resistant to anergy induction and instead underwent clonal deletion due to pro-apoptotic BIM (Bcl2l11) upregulation. During prolonged antigen exposure, BIM de-repression resulted in fewer PD-1+ conventional T cells as well as loss of peripherally-induced FOXP3+ Treg cells. Simultaneous Batf and Bcl2l11 knockdown meanwhile restored anergic T cell survival and Treg cell maintenance. The data identify the AP-1 nuclear factor BATF as a dominant driver of sustained T cell anergy and illustrate a mechanism for divergent peripheral tolerance fates.

overall_design: A combination of ATAC-seq and RNA-seq was used to interrogate freshly isolated spleen and lymph node polyclonal CD4 T cell subsets (Anergic, CD44high FOXP3– CD73high FR4high NRP1+; Teff, CD44high FOXP3– CD73low FR4low; Treg, FOXP3+ CD25+; Naïve, CD44low). Chromatin accessibility in sorted CD4+ T cell subpopulations was determined using the ATAC-seq. Briefly, 5 x 10^4 captured cells from each of the indicated groups (n = 2 or 3) were pelleted, washed with PBS, resuspended in cold sterile-filtered lysis buffer (10mM Tris-Cl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% Igepal CA-630), and immediately centrifuged for 10 minutes (500 x g, 4oC). Pellets were then resuspended in the transposition reaction mix (Nextera TD buffer, Nextera TDE1 transposase, nuclease-free H2O) and incubated for 30 minutes at 37oC. After transposition, DNA was purified with the MinElute PCR Purification kit (QIAGEN) according to the manufacturer’s instructions. Library amplification (Nextera dual-indexed), quality control (Agilent sizing and PicoGreen quantification), and sequencing (Illumina NovaSeq SP flow cell 2x50-bp paired-end run) were carried out at the University of Minnesota Genomics Center. Trimmed ATAC-seq reads (Trimmomatic 0.38.1) were aligned to the mm10 mouse genome assembly using Bowtie2 2.3.3.1 with the ‘--very-sensitive’ preset, then sorted and converted in SAMtools 1.13. Peak-calling with integrated PCR duplicate removal and Tn5 transposase cut site interval adjustment was performed on individual samples with Genrich 0.5 (github.com/jsh58/Genrich) in ATAC-seq mode. Reads mapping to mitochondrial DNA and ENCODE blacklist regions were excluded from analysis. From the individual peak-called samples, a total of 38,509 consensus peak sites were determined by DiffBind 2.10.0 (bioconductor.org/packages/release/bioc/html/DiffBind.html). Peaks were assigned to the gene with the nearest transcription start site using the annotatePeaks program in HOMER v4.11.1  with reference to the mm10 genome assembly and RefSeq annotation coordinates. For the RNA-seq analysis, total RNA was extracted from physically sorted CD4+ T cell populations using a QIAGEN RNeasy Kit according to the manufacturer’s instructions. Purified RNA quality control (Agilent sizing and RiboGreen quantification), library creation (Takara SMARTer Stranded Total RNA-seq Kit v2 Pico Input Mammalian), and sequencing (Illumina NovaSeq SP flow cell 2x50-bp paired-end run) were carried out at the University of Minnesota Genomics Center. RNA-seq reads were processed using an established pipeline (https://github.com/msi-ris/CHURP/wiki/PURR-Manual-Page). This pipeline incorporates the following software components: Trimmomatic, HISAT2, SAMtools, featureCounts, and edgeR. For visualization at select gene loci, ATAC-seq and RNA-seq mapped reads were summed from sample replicates using the bigwigCompare tool for display using Integrated Genomics Viewer.

first: Philip

last: Titcombe

first: Milagros

last: Silva Morales

last: Zhang


title: Epigenomic mapping in B-cell acute lymphoblastic leukemia identifies transcriptional regulators and noncoding variants promoting distinct chromatin architectures [CUT&RUN]

last: Savic

summary: B-cell lineage acute lymphoblastic leukemia (B-ALL) is comprised of diverse molecular subtypes and while transcriptional and DNA methylation profiling of B-ALL subtypes has been extensively examined, the accompanying chromatin landscape is not well characterized for many subtypes. We therefore mapped chromatin accessibility using ATAC-seq for 10 B-ALL molecular subtypes in primary ALL cells from 154 patients. Comparisons with B-cell progenitors identified candidate B-ALL cell-of-origin and AP-1-associated cis-regulatory rewiring in B-ALL. Cis-regulatory rewiring promoted B-ALL-specific gene regulatory networks impacting oncogenic signaling pathways that perturb normal B-cell development. We also identified that over 20% of B-ALL accessible chromatin sites exhibit strong subtype enrichment, with transcription factor (TF) footprint profiling identifying candidate TFs that maintain subtype-specific chromatin architectures. Over 9000 inherited genetic variants were further uncovered that contribute to variability in chromatin accessibility among individual patient samples. Overall, our data suggest that distinct chromatin architectures are driven by diverse TFs and inherited genetic variants which promote unique gene regulatory networks that contribute to transcriptional differences among B-ALL subtypes.

overall_design: AP-1 genomic binding in 697 or SUPB15 cells. N=1 with IgG used for peak calling and E. Coli spike-in for normalization.


title: Cytosolic pattern recognition receptors respond to EZH2-inhibition induced viral mimicry response that eliminates splenic B cells

first: Frederick

last: Dick

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


title: A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells [BiSulfite-seq]

first: SHOU-WEN

last: WANG

overall_design: In this study, we generate bulk CARLIN sequence datasets for both the DARLIN and Cas9/CARLIN mouse lines to evaluate the performance of DARLIN. Then, we generated both single-cell CARLIN and transcriptome data to study early fate bias within hematopoietic stem cells (HSCs). Furthermore, we generated bulk CARLIN sequences across different developmental stages to study HSC migration. We also performed single-cell multi-omic lineage tracing (DNA methylation, chromatin accessibility, gene expression, and lineage information) to study the clonal memory of HSCs.

first: Li

last: Li

first: Sarah

last: Bowling

first: Sean

last: McGeary

first: Qi

last: Yu

first: Bianca

last: Lemke

first: Karel

last: Alcedo

first: Yuemeng

last: Jia

first: Xugeng

last: Liu

first: Mark

last: Ferreira

first: Allon

last: Klein

first: Shou-Wen

last: Wang

first: Fernando

last: Camargo


title: Alternative splicing induced by bacterial pore-forming toxins sharpens CIRBP-mediated cell response to Listeria infection

city: PARIS

first: Stephane

last: Le Crom

address: 46 rue Ulm

department: Genomic Core Facility

institute: IBENS

summary: We aimed at identifying the targets of the protein Cold-Inducible RNA-Binding Protein in a human epithelial colon adenocarcinoma cell line (LoVo ATCC CCL-229) in cells infected by Listeria monocytogenes strain LL195 compared to non-infected cells. This set is linked to a previous one referenced on ENA (https://www.ebi.ac.uk/ena/browser/view/PRJEB26593)

overall_design: Comparative identification of CIRBP targets in LoVo cells infected or not by L. monocytogenes along a 0-10h infection in triplicate.

first: Morgane

last: Corre

first: Alice

last: Lebreton

first: Catherine

last: Senamaud-Beaufort

first: Mehdi

last: Ait Djoudi Oufella


title: Interaction Dynamics Between Innate and Adaptive Immune Cells Responding to SARS-CoV-2 Vaccination in Non-Human Primates

city: Bethesda

first: Chaim

last: Schramm

state: MD

address: 40 Convent Dr

department: Vaccine Research Center

institute: NIAID, NIH

summary: As new variants of SARS-CoV-2 continue to evolve and escape pre-existing immunity, testing updated vaccine antigens in non-human primates remains important for guiding clinical practice in humans. To date, such studies have focused on titers of binding and neutralizing antibodies, and frequencies of antigen-specific B and T cells. Here, we extend our understanding of the macaque immune response to vaccination with mRNA-1273 with an integrated approach that captures key interactions between the adaptive and innate immune responses. We show that a subset of S-specific T cells are characterized by the expression of key cytokines for activating innate immune responses, including CCL3, IL21, and IFNγ. We also observe an increase in CCR5-expressing intermediate monocytes and a shift of natural killer cells to a more mature cytotoxic phenotype. Antigen-specific B cells are polyclonal but demonstrate preferential usage of specific IGHV genes, and we observe two public clones, one of which matches a widely reported human anti-SARS-CoV-2 public B cell clone. The second vaccine dose elicits an increase in circulating germinal center-like B cells, which are more likely to be clonally expanded and target epitopes on the receptor binding domain. Both vaccine doses stimulate the expression of inflammatory response genes which have previously been associated with elevated antibody responses to a range of vaccines. Overall, this study provides a comprehensive picture of bidirectional signaling between innate and adaptive components of the immune system and suggests potential mechanisms for the enhanced response to a secondary exposure.

overall_design: Innate (monocyte, NK, or DC), CD4 memory T cells, or naïve B cells were sorted from 8 monkeys at baseline and/or 2 weeks after each dose of 100ug mRNA-1273. These cells were processed using the 10x Chromium for single cell sequencing. Antigen-specific memory B cells were also sorted from the post-vaccine time points into plates and sequenced using a SmartSeq-based protocol.

first: Damee

last: Moon

first: Lowrey

last: Peyton

first: Noemia

last: Lima

first: Christian

last: Wake

first: Kristin

last: Boswell

first: Amy

last: Henry

first: Farida

last: Labuone

first: David

last: Ambrozak

first: Samuel

middle: W

last: Darko

first: I-Ting

last: Teng

first: Kathryn

last: Foulds

first: Adrea

last: Carfi

first: Darin

last: Edwards

first: Peter

last: Kwong

first: Richard

last: Koup

first: Robert

last: Seder

first: Daniel

last: Douek


title: Single cell RNA-sequencing (scRNA-seq) of ISL1 knockout (KO) or knockdown and NKX2.5 KO compared to WT in cardiac progenitors (CPs); scRNA-seq of ISL1 KO compared to WT in motor neuron progenitors (MNPs)

first: Deepak

last: Srivastava

address: 1650 Owens St

department: GICD

institute: J David Gladstone Institutes

summary: Previous evidence suggested ISL1 plays unique transcriptional roles between vastly different cell types in developing humans. Single cell RNA-sequencing (scRNA-seq) of in vitro progenitor cells detailed ISL1's cell-type-specific roles in human cardiac progenitors (CPs) and motor neuron progenitors (MNPs). In addition, scRNA-seq confirmed ISL1 and NKX2.5 likely regulate common transcriptional pathways.

overall_design: scRNA-seq of WT, ISL1 KO, NKX2.5 KO in D8 CPs (n=3). scRNA-seq of WT vs ISL1 KO in D18 MNPs (n=3). scRNA-seq of scrambled control vs siRNA ISL1 in D8 CPs (n=1).

first: Bonnie

last: Maven

first: Casey

last: Gifford


title: CD8+ T lymphocytes in hypopigmented mycosis fungoides: malignant cells or reactive clone?

city: Pittsburgh

first: Oleg

last: Akilov

state: PA

address: 3601 Fifth Avenue, 5th Floor

department: Department of Dermatology

institute: University of Pittsburgh

summary: We used targeted single cell RNA sequencing using a gene set composed of the BD Rhapsody Human Immune Response Panel, TCR panel, and a custom panel of 61 malignancy-associated genes and T/B cell receptor sequencing to characterize single cell populations in hypopigmented mycosis fungoides (HMF). Using a reference mapping model built on publically deposited classic mycosis fungoides single cell RNA sequencing datasets, we identified a predicted malignant cell population independent from the clonal T cell population in HMF.

overall_design:
Sequencing of skin cells from hypopigmented patches from one patient collected as three punch biopsies.
Raw data not provided due to patient privacy concerns.

first: Simon

last: Cao

last: Kruglov


title: The multi-lineage transcription factor ISL1 controls cardiomyocyte cell fate through interaction with NKX2.5

city: San Francisco

overall_design: Refer to individual Series


title: Hypoblast from human pluripotent stem cells regulates epiblast development [scRNA-seq]

city: Kyoto

first: Yasuhiro

last: Takashima

address: 53 Kawahara-cho, Shogoin, Sakyo-ku

institute: Kyoto University

summary: Recently, several studies using cultures of human embryos together with single-cell RNA-seq (scRNA-seq) analyses have revealed differences between humans and mice, necessitating the study of human embryos. Despite the importance of human embryology, ethical and legal restrictions have limited post-implantation stage studies. Thus, recent efforts have focused on developing in vitro self-organising models using human stem cells. Here, we established a genetic approach to generate authentic hypoblast cells (nHyC) - known to give rise to one of the two extraembryonic tissues essential for embryonic development - from naïve human pluripotent stem cells (hPSCs). Our nHyCs spontaneously assemble with naïve hPSCs to form a three-dimensional bilaminar structure (bilaminoids) with a pro-amniotic-like cavity. In the presence of additional naïve hPSC-derived analogues of the second extraembryonic tissue, the trophectoderm, the efficiency of bilaminoid formation increases from 20% to 40%, and the epiblast within the bilaminoids continues to grow due to IL6 secreted by the trophectoderm. Furthermore, we show that bilaminoids robustly recapitulate the patterning of the anterior-posterior axis and the formation of cells reflecting the pre-gastrula stage, whose emergence can be shaped by genetically manipulating the DKK1/OTX2 hypoblast-like domain. We therefore model and reveal mechanisms by which the two extraembryonic tissues efficiently guide the stage-specific growth and progression of the epiblast as it establishes the post-implantation landmarks of human embryogenesis.

overall_design: Analyzing transcriptional profiles in naïve PSCs, primed PSCs, PDGFRA positive cells induced by GATA6 o/e from naive and primed PSCs; induced by 7F, 4F, and 2F from naïve PSCs and bilaminoids at day 6, day9.

first: Takumi

last: Okubo

first: Mio

last: Kabata

first: Takuya

last: Yamamoto


title: A MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2 targeting [Hi-ChIP]

status: Public on Oct 16 2023

city: Rome

first: Matteo

last: Cassandri

address: Viale di San Paolo 15

department: Department of Oncohematology

institute: Bambino Gesù Children's Hospital

summary: Rhabdomyosarcoma (RMS) is a pediatric mesenchymal-derived malignancy encompassing Fusion Positive (FP)-RMS expressing PAX3/7-FOXO1 and Fusion Negative (FN)-RMS often mutated in the RAS pathway. RMS expresses the master myogenic transcription factor MYOD that, paradoxically, is unable to support differentiation while essential for tumor cell survival. We identify here SKP2, an oncogenic E3-ubiquitin ligase, as a critical driver of tumorigenesis in FN-RMS. SKP2 is overexpressed in RMS at the highest levels among several adult and pediatric cancers and its expression is maintained by MYOD through an intronic enhancer within the gene. In FN-RMS SKP2 promotes cell cycle progression and prevents differentiation directly targeting p27Kip1 and p57Kip2, respectively, unlocking a transcriptional myogenic program, partly MYOD-dependent, resulting in de novo expression of terminal muscle differentiation markers. SKP2 depletion strongly affects stemness and tumorigenic features in vitro and prevents in vivo tumor growth. The in vitro effects are mirrored by the SKP2 inhibitor SMIP004. Moreover, the investigational NEDDylation inhibitor MLN4924 hampers SKP2 functions restraining FN-RMS cell survival and tumor growth. Our results uncover a MYOD-SKP2 axis crucial for the crosstalk between transcriptional and post-translational mechanisms that contribute to FN-RMS tumorigenesis and broaden the understanding of MYOD function. Furthermore, they suggest inhibition of NEDDylation as a potential therapeutic approach in this tumor.

overall_design: HiChIP DNA-sequencing for histone modification H3K27ac in RD FN-RMS and RH4 FP-RMS cells

first: Silvia

last: Pomella

first: Berkley

last: Gryder

first: Rossella

last: Rota


title: Genomic occupancy of TFs ISL1 and NKX2.5 in cardiac and neural populations

summary: ChIP-sequencing demonstrated that ISL1 binds cardiac DNA in a cell-type-specific manner, and that NKX2.5 is both necessary and sufficient for this localization.

overall_design: 3 replicates of ISL1-bound DNA (ChIP-seq) in WT and ISL1–/– hiPSC derived d6 CPs. 3 replicates of NKX2.5 ChIP-seq in WT NKX2.5–/– hiPSC derived d6 CPs. 3 replicates of ISL1 ChIP-seq in NKX2.5–/– hiPSC derived d6 CPs. 3 replicates of ISL1 ChIP-seq in WT and ISL1–/–  hiPSC derived d18 MNPs. One replicate of ISL1 and NKX2.5 ChIP-seq in adCMV::NKX2.5-infected d18 MNPs. One replicate of NKX2.5 ChIP-seq in adCMV::EGFP-infected d18 MNPs.

first: Kaitlen

last: Samse-Knapp


title: Multimodal spatiotemporal transcriptomic resolution of embryonic palate osteogenesis [Spatial Transcriptomics]

first: Ryan

last: Dale

address: Rm 10D39, 10 Center Drive

department: National Institute of Child Health and Human Development

institute: National Institutes of Health

summary: The terminal differentiation of osteoblasts and subsequent formation of bone marks an important phase in palate development that leads to the separation of the oral and nasal cavities. While the morphogenetic events preceding palatal osteogenesis are well explored, major gaps remain in our understanding of the molecular mechanisms driving the formation of this bony union of the fusing palate. Through bulk, single-nucleus, and spatially resolved RNA-sequencing analyses of the developing secondary palate, we identify a shift in transcriptional programming between embryonic days 14.5 and 15.5 pinpointing the onset of osteogenesis. We define spatially restricted expression patterns of key osteogenic marker genes that are differentially expressed between these developmental timepoints. Finally, we identify genes in the palate highly expressed by palate nasal epithelial cells, also enriched within palatal osteogenic mesenchymal cells. This investigation provides a relevant framework to advance palate-specific diagnostic and therapeutic biomarker discovery.

overall_design: Mid-palatal coronal cross sections of whole murine wild-type embryo heads (embryonic day 14.5 and 15.5 were included).

first: Jeremie

middle: O

last: Piña

first: Resmi

last: Raju

first: Daniela

last: Roth

first: Emma

last: Winchester

first: Parna

last: Chattaraj

first: Fahad

last: Kidwai

first: Fabio

last: Faucz

first: James

last: Iben

first: Apratim

last: Mitra

first: Kiersten

last: Campbell

first: Gus

last: Fridell

first: Caroline

last: Esnault

first: Justin

last: Cotney

first: Rena

last: D'Souza


title: Genome-wide expression analysis of Arabidopsis Col-0 plants under BTH treatment and high or low humidity

first: xiufang

last: xin

summary: The goal of this study is to demonstrate the global expression profile of four-week-old Arabidopsis Col-0 wild type plant leaves in response to high or low air humidity, under water or BTH (SA analog) treatment.

overall_design: Col-0 plants were treated with high or low humidity for 24 h and then sprayed with 100 μM BTH. Leaves were collected at 0 and 12 h after BTH treatment. Total RNA was extracted from the tissues using Trizol Reagent (Invitrogen) based on the manufacture's instruction, and genomic DNA was removed using the DNaseI (Invitrogen). RNA was further purified by using the RNeasy MinElute Cleanup kit (QIAGEN). RNA quality check, library construction, and RNA sequencing were performed by the Majorbio company.

first: Lingya

last: Yao

first: Zeyu

last: Jiang

first: Xiu-Fang

last: Xin


first: Stephane

last: Le Crom

summary: We investigated wether host cell gene expression response to a bacterial infection can involve changes at alternative splicing level. To identify these events, we have analyzed the long read sequencing data to identify which transcript isoforms were altered in a human epithelial colon adenocarcinoma cell line (LoVo ATCC CCL-229) along a 0- to 10-h time-course of infection by Listeria monocytogenes strain LL195. This set is linked to a previous short-read set referenced on ENA (https://www.ebi.ac.uk/ena/browser/view/PRJEB26593).

overall_design: Comparative alternative splicing events identification analysis of long read RNA-seq data for LoVo cells infected or not by L. monocytogenes along a 0-10h infection in triplicate.

first: Morgane

last: Corre

first: Alice

last: Lebreton

first: Vinko

last: Besic

first: Ammara

last: Mohammad


title: Nystagmus in the B6(CG)Tyr(c-2J)/J  Albino Mouse: A Functional and RNAseq Analysis

first: Laura

last: Johnson

state: Minnesota

address: 2001 6th ST SE

institute: University of Minnesota

summary: PURPOSE: Infantile nystagmus syndrome (INS) is a gaze-holding disorder characterized by conjugate, uncontrolled eye oscillations that can result in significant visual acuity loss. INS is often associated with albinism, but the mechanism is unclear. Albino mice have nystagmus; however, a pigmented mouse with a tyr mutation making it phenotypically albino, the B6(CG)-Tyr(c-2J)/J (B6 albino), had not been tested. We tested optokinetic nystagmus reflexes (OKN) in B6 albino and control mice. RNA-Seq was performed on extraocular muscles (EOM), tibialis anterior muscle (TA), abducens (CN6), and oculomotor (CN3) neurons to uncover molecular differences that could account for nystagmus.

overall_design: Two genotypes of mice used are B6(CG)Tyr(c-2J)/J and C57BL/6. The B6(CG)Tyr(c-2J)/J is an albino mouse, C57BL/6 is a pigmented mouse. For each genotype there were four replicated of four types of tissues taken, these tissues were the extraocular muscles, the tibialis anterior, the oculormoter neuron, and the abducen motor neuron.

first: Linda

last: McLoon


title: Cereblon influences the timing of muscle differentiation in Ciona tadpoles

city: St Louis

first: Laurence

middle: A.

last: Lemaire

address: 3507 Laclede Ave

department: Biology

institute: Saint Louis University

summary: Thalidomide has a dark history as a teratogen, but in recent years its derivates have been shown to function as a chemotherapeutic agent. These drugs bind cereblon (CRBN), the substrate receptor of an E3 ubiquitin ligase complex and modify its degradation targets. Despite these insights, remarkably little is known about the normal function of cereblon in development. Here we employ Ciona, an invertebrate chordate, to identify endogenous Crbn targets. In Ciona, Crbn is specifically expressed in developing muscles during tail elongation before they acquire contractile activity. Crbn expression is activated by Mrf, the ortholog of MYOD1, a transcription factor important for muscle differentiation. CRISPR/Cas9-mediated mutations of Crbn lead to precocious onset of muscle contractions. By contrast, overexpression of Crbn delays contractions and is associated with decreased expression of contractile protein genes such as troponin. This reduction is possibly due to reduced Mrf protein levels without altering Mrf mRNA levels. Our findings suggest that Myod and Crbn form a negative feedback loop to control the precision of muscle differentiation during tail elongation.

overall_design: Single-cell transcriptomes from mid tailbud embryos expressing either membrane GFP in the tail muscle (control condition) or both membrane GFP and Cereblon (Crbn overexpression condition) were analyzed using Chromium Single Cell Gene Expression (10X Genomics©). 9854 cells for the control condition and 8540 cells for the Crbn overexpression condition, which include 212 and 174 muscle cells, respectively, were sequenced.

first: Juanjuan

last: Long

first: Michael

last: Levine


title: BATF represses BIM to sustain tolerant T cells in the periphery (ATAC-Seq)

first: Daniel

last: Mueller

first: Philip

last: Titcombe

first: Milagros

last: Silva Morales

last: Zhang


title: Genome-wide characterization of TAZ binding sites in mammary epithelial cells in temporary and special manner (RNA-seq)

city: Buffalo

first: Tao

last: Liu

address: Elm and Carlton St

department: Biostatistics and Bioinformatics

institute: Roswell Park Comprehensive Cancer Institute

summary: TAZ, one of the key components of Hippo signaling pathway, is a transcription coactivator and plays an important role in cell proliferation, anti-apoptosis, therapeutic resistance, cancer stemness and tumorigenesis. TAZ mediated transcription program through transcription factors, such as TEAD family members. Genome-wide TAZ occupancy is poorly defined. Here, using a tet-inducible TAZ activation in mammary epithelial MCF10A cells, we characterized genome-wide binding sites in different TAZ activation time points. We found vast majority of TAZ binds to distal enhancer and intergenic regions of genes in the early TAZ activation time point and shift to promoter regions in the later TAZ activation time point. We identified three distinctive TAZ binding patterns to its targets. Furthermore, we found TAZ activation results in chromatin architecture alterations. Our studies provide insight of TAZ regulated transcription program in mammary epithelial cells and indicate potential therapeutic targets for breast cancer.

overall_design: Inducible TAZ-4SA transduced MCF10A cells were treated without Dox or with 0.05µg/ml Dox for 24 h, 48 h and 72 h. Total RNA was extracted using Trizol Reagent (Life Technologies) according to the manufacturer’s protocol. RNAs were further processed for next generation sequencing at Roswell genomic core facility.

first: Jiaojiao

last: Zhou

first: Yanmin

last: Chen

first: Jia

last: Fang

first: Song

first: Costa

last: Frangou

first: Jianmin


title: Genome-wide characterization of TAZ binding sites in mammary epithelial cells in temporary and special manner (ChIP-seq)

overall_design: Chromatin immunoprecipitation (ChIP) assays were performed using the Simple ChIP Enzymatic Chromatin IP Kit. Briefly, inducible TAZ transduced MCF10A cells were treated without or with Dox for 24 h, 48h and 72 h. Cells were cross-linked, lysed, and sonicated to generate DNA fragments. The immunoprecipitation was performed using 5 ug antibody to TAZ (Cell Signaling Technologies) or IgG. ChIPed DNAs were further processed for next generation sequencing at Roswell genomic core facility.


title: RPA and RAD51 ChIP-seq from genetically modified Dmc1-knockout B6xCAST PRDM9-Humanized/CAST mouse testes

city: Oxford

first: Anjali

middle: Gupta

last: Hinch

address: Roosevelt Drive

department: WCHG

country: United Kingdom

institute: Oxford University

summary: Mammalian meiotic recombination proceeds via repair of hundreds of programmed DNA double-strand breaks (DSBs). This process requires choreographed binding of RPA, DMC1 and RAD51 to single-stranded DNA (ssDNA) substrates and in vivo binding maps of these proteins provide insights into the underlying molecular mechanisms. When assayed in F1-hybrid mice, these maps can distinguish the broken chromosome from the homologous chromosome used as template for repair, which reveals further mechanistic detail and enables the structure of the recombination intermediates to be inferred. By applying CRISPR/Cas9 mutagenesis directly on F1-hybrid embryos, we have extended this powerful analysis technique to explore the molecular detail of recombination when a key component is knocked-out. As a proof-of-concept, we have generated biallelic knockouts of Dmc1 and built maps of meiotic binding of RAD51 and RPA in these knockout hybrid mice. Dmc1 mutants undergo meiotic arrest and comparison of these maps with those from wild-type mice is informative about the structure and timing of recombination intermediates in both genotypes. We confirm a complete abrogation of strand exchange in Dmc1 mutants, and observe a redistribution of RAD51 binding across both the distal and proximal ends of the resected DNA. We observe unexpected RPA and DMC1 binding in the wild-type, which suggests multiple rounds of strand invasion with template-switching in mouse. The methodology used involves direct phenotyping of hybrid “founder” mice following CRISPR mutagenesis and provides a high-throughput approach for the analysis of gene function during meiotic recombination, at low animal cost.

overall_design: Comparison of RPA and RAD51 ChIP-seq data from wild-type and genetically modified hybrid mice across tens of thousands of hotspots genome-wide

first: Ben

last: Davies