title: X-linked deletion of Crossfirre, Firre, and Dxz4 in vivo uncovers diverse phenotypes and combinatorial effects on autosomes

city: Munich

first: Tim

last: Hasenbein

address: Biedersteiner Str. 29

department: Institute of Pharmacology and Toxicology

institute: Technical University Munich

summary: The lncRNA Crossfirre was identified as an imprinted X-linked gene, and is transcribed antisense to the trans-acting lncRNA Firre. The Firre locus forms an inactive-X-specific interaction with Dxz4, both loci providing the platform for the largest conserved chromatin structures. Here, we characterized the epigenetic profile of these loci, revealing them as the most female-specific accessible regions genome-wide. To address their in vivo role, we performed one of the largest X-linked knockout studies by deleting Crossfirre, Firre, and Dxz4 individually and in combination (TKO). Despite their distinct epigenetic features observed on the X chromosome, our allele-specific analysis uncovers these loci as dispensable for imprinted and random X chromosome inactivation. However, we provide evidence that Crossfirre affects autosomal gene regulation but only in combination with Firre. To shed light on the functional role of these sex-specific loci, we performed an extensive standardized phenotyping pipeline and uncovered diverse knockout and sex-specific phenotypes. Collectively, our study provides the foundation for exploring the intricate interplay of conserved X-linked loci in vivo.

overall_design: RNA-seq from Crossfirre, Crossfirre-Firre and Crossfirre-Firre-Dxz4 deletion mouse strains (extraembryonic and adult organs). scRNA-seq from heterozygous Crossfirre-Firre-Dxz4 deletion mouse strains (adult spleen). RNA-seq from heterozygous Crossfirre-Firre-Dxz4 deletion mouse strains (adult spleen).

first: Daniel

last: Andergassen


title: Analysis of expression profiles in miR-195 transduced Ebf1–/– Lin– cells

status: Public on Oct 28 2024

city: Saga

first: Ryo

last: Yanagiya

state: Saga

address: 5-1-1 Nabeshima

department: Department of Internal Medicine

country: Japan

institute: Saga University

summary:
To further investigate whether miR-195 transduction lead  Ebf1–/– Lin– cells to develop into later stages than pro B, we have employed microarray expression profiling.
For the purpose, we compared B cell and other lineages (T cell, NK cell and myeloid) related gene expressions among these samples.

overall_design: miR-195 transduced or not Ebf1–/– Lin– cells were cultured with SCF, IL-7 and Flt-ligand containg medium.

first: Yuji

last: Miyatake

first: Ai

last: Kotani


title: Characterization of tumor microenvironment using single cell RNA sequencing in Lewis Lung Carcinoma cell transplantation mouse model

city: Seoul

first: Minsu

country: South Korea

institute: Catholic University of Korea

type: Expression profiling by high throughput sequencing

summary: The tumor microenvironment holds significant importance in cancer development and progression. Research conducted using mouse models of tumor cell transplantation can enhance our comprehension of the tissue microenvironment that allows tumor growth at an early stage. Among these models, those featuring the Lewis Lung Carcinoma (LLC) cell line are noteworthy due to their low immunogenic features. Since numerous cancer patients do not respond effectively to current immunotherapy, it is imperative to investigate models with low immunogenic features. Consequently, this study employed single-cell RNA sequencing on an orthotopic lung cancer mouse model utilizing the LLC cell line to profile the characteristics of diverse cell types that emerge in the lung during tumor engraftment. The lung microenvironment exhibited discernible transformations, including fibrogenic gene expression, increased cell-to-cell and cell-to-extracellular matrix adhesion, and reduced infiltration of T cells compared to lungs without tumor engraftment. In conclusion, these findings reveal cellular and molecular alterations that can be targeted for tumor prevention and treatment for low-immunogenic tumors.

overall_design: Eight to nine-week-old week old female C57BL/6 mice were intracardially injected with 1 x 10⁴-1 x 10⁵  LL/2-Luc2 cells. The mice were sacrificed on days 19 to 24 after tumor injection. Tissue specimens were analyzed by H&E staining, scRNAseq(10x genemics).

first: Hae-Ock

last: Lee


title: Transcriptomic analysis of colon tissues of stressed and resting wild-type mice.

city: Shanghai

first: Xiaofei

last: Yu

address: 2005 Songhu Rd

country: China

institute: Fudan University

summary: To study the transcriptional change on colon tissue during stress, we performed RNA-seq with colon tissues from stressed and resting wild-type mice.

overall_design: After the 3rd episode of 30-min restraint stress, the colon tissues were dissected immediately. RNA were purified and sequenced.

first: Zheyu

last: Jin


title: Yin Yang 1 and guanine quadruplexes protect dopaminergic neurons from cellular stress via transmissive dormancy [BG4 CUT&RUN]

city: Dallas

first: Peter

middle: M

last: Douglas

state: Tx

address: 6000 Harry Hines Blvd

department: Molecular Biology

country: USA

institute: University of Texas Southwestern Medical Center

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Neurons deploy diverse adaptive strategies to ensure survival and neurotransmission amid cellular stress. When these adaptive pathways are overwhelmed, functional impairment or neurodegeneration follows. Here, we report a scenario where stressed neurons actively induce a state of transmissive dormancy as a protective measure. Examination of dopaminergic neurons surviving severe cellular challenges revealed decreased spontaneous activity accompanied by dynamic control of dopamine metabolism through the transcriptional regulator Yin Yang 1 (YY1). Under these conditions, YY1 increases expression of the vesicular monoamine transporter 2, promoting sequestration of dopamine into synaptic vesicles. Following oxidative stress and subsequent DNA damage, YY1 inhibits dopamine synthesis through stabilization of a guanine quadruplex in intron 10 of tyrosine hydroxylase. This cytoprotective response has the potential to drive circuit inactivation and safeguard neurons by minimizing the toxic accumulation of cytosolic dopamine and inducing a state of neuronal dormancy. In essence, neurons appear to actively prioritize viability over functionality.

overall_design: Differentiated LUHMES cells were treated with 10 nM TG or DMSO and collected 24 hours later. 3 biological replicates were collected and combined for CUT&RUN. CUT&RUN targets included Flag control or BG4+Flag anitbodies.

first: Kielen

last: Zuurbier


title: Clonal Haematopoiesis-related Mutant ASXL1 Promotes Atherosclerosis in Mice via Dysregulated Innate Immunity [scRNA-seq]

status: Public on Oct 25 2024

city: Tokyo

first: Naru

last: Sato

address: 7-3-1 Hongo, Bunkyo-ku

department: Graduate School of Pharmaceutical Sciences

institute: University of Tokyo

summary: Certain somatic mutations confer a fitness advantage in hematopoietic stem cells, resulting in the clonal expansion of mutant blood cells, known as clonal haematopoiesis (CH). Among the top 3 CH mutations, ASXL1 mutations present the highest risk for developing cardiovascular diseases (CVDs). However, how ASXL1 mutations induce CVDs remains totally elusive. Here we show that haematopoietic cells harbouring C-terminally truncated form of ASXL1 mutant (ASXL1-MT) accelerated development of atherosclerosis in Ldlr–/– mice. Transcriptome analyses of plaque-cells showed inflammatory signatures of monocytes and macrophages expressing ASXL1-MT. Mechanistically, wild-type ASXL1 inhibited innate immune signalling through the inhibition of IRAK1-TAK1 interaction in the cytoplasm, indicating an unexpected non-epigenetic role of ASXL1. In contrast, ASXL1-MT lost this regulatory function, leading to NF-κB activation. Intriguingly, IRAK1/4 inhibition decreased inflammatory monocytes and atherosclerosis driven by ASXL1-MT. The present work connects ASXL1 mutations with inflammation and CVDs, giving a clue to prevent CVDs in ASXL1-CH.

overall_design: scRNA-seq (wild-type vs VAV1-Cre+/- ASXL1-MTfl/fl)

first: Susumu

last: Goyama

first: Yu-Hsuan

last: Chang

first: Masashi

last: Miyawaki

first: Takeshi

last: Fujino

first: Shuhei

last: Koide

first: Tamami

last: Denda

first: Xiaoxiao

last: Liu

first: Koji

last: Ueda

first: Keita

last: Yamamoto

last: Asada

first: Reina

last: Takeda

first: Taishi

last: Yonezawa

first: Yosuke

last: Tanaka

first: Hiroaki

last: Honda

first: Yasunori

last: Ota

first: Takuma

last: Shibata

first: Motohiro

last: Sekiya

first: Tomoya

last: Isobe

first: Chrystelle

last: Lamagna

first: Esteban

last: Masuda

first: Atsushi

last: Iwama

first: Hitoshi

last: Shimano

first: Jun-ichiro

last: Inoue

first: Kensuke

last: Miyake

first: Toshio

last: Kitamura


title: Analysis of gene expression levels in ovaries of wild-type and ABCG5/G8 knockout mice

first: Yoshihide

last: Yamanashi

department: Pharmacy

institute: The University of Tokyo Hospital

summary: A heterodimer of ATP-binding cassette transporters G5 and G8 (ABCG5/G8) functions as a lipid exporter in the intestine. Homozygous ABCG5/G8 knockout female mice exhibit infertility under normal chow diet conditions due to impaired folliculogenesis and poor egg quality. Interestingly, this infertility was restored by changing the diet from a chow diet (CD) to a vegetable oil-free diet (VOFD). To elucidate mechanisms underlying the diet-induced infertiility in ABCG5/G8 KO mice, RNA-seq analyses were conducted and found that multiple genes potentially involved in the reproductive system and embryogenesis, in addition to those involved in sterol biosynthesis, were down regulated in the ovaryes from CD-fed infertile KO mice compared to those from fertile CD-fed wild-type mice and VOFD-fed KO mice.

overall_design: RNA-seq analyses were conducted to analyze differences in gene expression in the ovaries of CD-fed fertile wild-type mice, CD-fed infertile KO mice, and VOFD-fed fertile KO mice.


title: Clonal Haematopoiesis-related Mutant ASXL1 Promotes Atherosclerosis in Mice via Dysregulated Innate Immunity

first: Naru

last: Sato

overall_design: To investigate how ASXL1-MT KI macrophages exerted their effects on atherosclerotic lesions compared to normal cells , we carried out  RNA-sequencing of the CD11b+ F4/80+ cells dissociated from the aortae of Ldlr-/- mice reconstituted with ASXL1-MT KI or control BMCs. Three biological replicates were analyzed for each of 2 groups (two genotypes).

first: Susumu

last: Goyama

first: Yu-Hsuan

last: Chang

first: Masashi

last: Miyawaki

first: Takeshi

last: Fujino

first: Shuhei

last: Koide

first: Tamami

last: Denda

first: Xiaoxiao

last: Liu

first: Koji

last: Ueda

first: Keita

last: Yamamoto

last: Asada


title: Spatial transcriptomical analysis of livers from patients with COVID19

first: Zhong

last: Fang

address: Dongan Road 131#

summary: In this study, we stained liver tissues from COVID-19 cases with abnormal liver functions, and found viral proteins were detectable from dead or severe cases, but not from moderate patients. We then conducted spatial transcriptomics to analyze effects of SARS-COV-2 on livers.

overall_design: 10x Genomics Visium CytAssist Spatial Gene Assay was designed to analyze viral RNA and mRNA isolated from FFPE liver tissue sections from patients with COVID19Spatial Barcodes are used to associate the reads back to the tissue section images for spatial mapping of gene expression. Figure images were generated via Loupe Browser 6.5.0 downloaded from the website of 10x Genomics Inc.

first: Shiqi

last: Chen

first: Quan

last: Yuan


title: Hepatic stellate cell single cell atlas reveals a highly similar activation process across liver disease aetiologies

city: Jette

first: Leo

middle: A.

last: van Grunsven

address: Laarbeeklaan 103

country: Belgium

institute: Vrije Universiteit Brussel

summary: It is still unclear whether hepatic stellate cells (HSC) activate in a similar manner in different chronic liver disease aetiologies.  Here, we investigate how human stellate cells activate in vitro at the transcriptome level.

overall_design: Ultrapure primary human HSCs were isolated by FACS (excitation: 355 nm, emission: 450 nm) (n = 4)  and compared with plated primary human HSCs after 1 (n = 3)  and 3 passages (n = 3).

first: Vincent

last: Merens

first: Ayla

last: Smout

first: Stefaan

last: Verhulst


title: H3K36 methylation regulates cell plasticity and regeneration in the intestinal epithelium [regen_rnaseq_mouse]

city: Boulder

first: Alison

middle: Rae Swearingen

last: Pashos

state: CO

address: 1945 Colorado Avenue

department: MCDB

institute: University of Colorado Boulder

summary: Cell plasticity is needed during development and homeostasis to generate diverse cell types from stem and progenitor cells. Following differentiation, plasticity must be restricted in specialized cells to maintain tissue integrity and function. For this reason, specialized cell identity is highly stable under homeostatic conditions; however, cells in some tissues regain plasticity during injury-induced regeneration. While precise gene expression is needed to control these processes, the regulatory mechanisms that restrict or promote cell plasticity are poorly understood. Here, we use the mouse small intestine as a model system to study cell plasticity. We find that H3K36 methylation reinforces expression of cell type-associated genes to maintain specialized cell identity in intestinal epithelial cells. Depleting H3K36 methylation leads to defects in lineage commitment and activates a plastic, regenerative gene expression signature. Correspondingly, we observe rapid and reversible remodeling of H3K36 methylation following injury-induced regeneration. Together, these data suggest a fundamental role for H3K36 methylation in regulating cell plasticity and regeneration.

overall_design: RNA-sequencing for WT mice at 0, 3 and 5 days after irradiation, 2 biological replicates each.

middle: R

first: Justin

last: Brumbaugh


title: H3K36 methylation regulates cell plasticity and regeneration in the intestinal epithelium [cutandtag_enzyme_profiling]

overall_design: CUT&Tag for EZH2, NSD2, and SETD2 in WT H3 and H3K36M organoids, 2 biological replicates each.


title: Multiparameter imaging reveals clinically relevant cancer cell – stroma interaction dynamics in head and neck cancer

city: Münster

first: Sara

middle: A

last: Wickström

address: Röntgenstraße 20

department: Cell & Tissue Dynamics

country: Germany

institute: Max Planck Institute for Molecular Biomedicine

summary: Epithelial tumors are characterized by abundant inter- and intra-tumor heterogeneity, which complicates diagnostics and treatment. The contribution of cancer-stroma interactions to this heterogeneity is poorly understood. Here we report a paradigm to quantify phenotypic diversity in head and neck squamous cell carcinoma (HNSCC) with single cell resolution. By combining cell state markers with morphological features, we identify phenotypic signatures that correlate with clinical features, including metastasis and recurrence. Integration of tumor and stromal signatures reveals that partial epithelial-mesenchymal transition (pEMT) renders disease outcome highly sensitive to stromal composition, generating a strong prognostic and predictive signature. Spatial transcriptomics and subsequent analyses of cancer spheroid dynamics identifies cancer-associated fibroblast–pEMT axis as a nexus for intercompartment signaling that reprograms pEMT cells into an invasive phenotype. Taken together, we establish a paradigm to identify clinically relevant tumor phenotypes, and discover a cell state-dependent interplay between stromal and epithelial compartments that drives cancer aggression.

overall_design: Fresh samples from eight tongue carcinoma patients were collected from patients undergoing surgery at Helsinki University Hospital in 2022. Samples were fresh frozen in OCT and 10 µm sections were cut and mounted onto the ST arrays (10X Genomics Visium).

first: Karolina

last: Punovuori


title: H3K36 methylation regulates cell plasticity and regeneration in the intestinal epithelium [regen_cutandtag_mouse]

overall_design: CUT&Tag for H3K27me3, H3K36me3, and H3K36me2 in WT mice at 0, 3 and 5 days after irradiation, 2 biological replicates each.


title: Bulk RNA-sequencing of wild-type (Ler) and dme-2 calli harvested 7 days after incubation on callus-inducing medium and of de novo regenerated shoots harvested 14 days after incubation on shoot-inducing medium

first: Seunga

last: Lee

address: 1, Gwanak-ro, Gwanak-gu

institute: Seoul National University

summary: Plants possess high potential for somatic cell reprogramming. Despite extensive studies on the molecular network and key genetic factors during regeneration, the underlying epigenetic landscape remains incompletely understood. Here, we explored methylome and transcriptome dynamics during two-step plant regeneration. During leaf-to-callus transition, genic CG methylation shifts, while pericentromeric regions undergo substantial CG and extensive CHH hypomethylation. Upon shoot regeneration, methylation increases across all cytosine contexts, particularly in pericentromeric regions, coinciding with RNA-directed DNA methylation (RdDM) pathway activation. However, the DEMETER (DME)-deficient dme-2 mutant exhibited significant genic CG redistribution and global non-CG hypomethylation, partly due to RdDM downregulation. The dme-2 mutant affects gene expression in pluripotency and shoot meristem development, promoting regeneration through a reprogrammed state by pericentromeric hypomethylation. Our study demonstrates the intimate linkage between DME demethylase and RdDM underlying pericentromeric non-CG methylation maintenance, providing insight into the epigenetic mechanisms balancing pluripotency states and genome stability during plant regeneration.

overall_design: Leaves of 2-week-old Arabidopsis thaliana seedlings were excised and incubated on callus-inducing medium. Three biological replicates were generated for each sample.

first: Soon

middle: H

last: Bae

first: Yunji

last: Jeon

first: Pil

middle: J

last: Seo

first: Yeonhee

last: Choi


title: Genome-wide loss-of-function CRISPR/Cas9 screen of Triple Negative Breast Cancer GSI-resistant MB157R cells treated with GSI or vehicle.

city: Lausanne

first: Nadine

last: Fournier

state: Vaud

address: Agora Cancer Research Center, Bugon 25A

country: Switzerland

institute: Agora Cancer Research Center & Swiss Institute of Bioinformatics

summary: Cell plasticity of Triple negative breast cancer (TNBC) contributes to tumor heterogeneity and is one of the major reasons for the limited success of chemo- or combination therapies in the clinics. The molecular mechanisms of therapy-induced tumor cell plasticity and associated resistance to chemo or targeted therapies are largely unknown.  Using a genome wide CRISPR-Cas9 screen we investigated the escape mechanisms of Notch driven TNBC, when treated with targeted therapy. We describe molecularly a reciprocal inhibitory feedback mechanism between Notch signaling and the pluripotency associated transcription factor SOX2, which shapes divergent cell states, EMT, cancer stem cell features and associates with therapeutic response and escape to targeted therapy. Moreover, we performed and assessed monotherapy and drug combination treatments in Notch-inhibitor sensitive and resistant TNBC xenotransplant and identified combination and second line treatment options which were able to induce tumor control and reduce metastatic burden.

overall_design: Comparative analysis of MB157R cells, stably expressing Cas9, infected with the human GeCKO v2 CRISPR libraries and treated with either vehicle or GSI for 14 days.

first: Morgane

first: Joaquim

last: Javary

first: Freddy

last: Radtke


title: A novel small molecule Enpp1 inhibitor improves tumor control following radiation therapy by targeting stromal Enpp1 expression

city: Portland

first: venkatesh

last: rajamanickam

state: OR

address: 4805 NE Glisan St, Suite 2N35

institute: Earle A. Chiles Research Institute

summary: Through variation in the cancer cell and their immune infiltrates each tumor represents a unique problem, but therapeutic targets can be found in the shared features. Radiation therapy can change the interaction between the cancer cells and the stroma through release of innate adjuvants including the STING agonist cGAMP. Enpp1 is a phosphodiesterase that can be expressed by cancer cells and can degrade cGAMP. Enpp1 can therefore limit innate adjuvant availability following radiation therapy. We observed that many cancer cells lack Enpp1 expression, but that Enpp1 expression is retained in cells of the tumor stroma and this expression in the tumor stroma limits tumor control by radiation therapy. We demonstrate the efficacy of a novel Enpp1 inhibitor and show that this inhibitor improves tumor control by radiation even where the cancer cells lack Enpp1. We show that the mechanism requires STING and type I IFN receptor expression by non-cancer cells, and is dependent on CD8 T cells as a final effector mechanism of tumor control. This suggests that Enpp1 inhibition may be an effective partner for radiation therapy regardless of whether cancer cells express Enpp1, and identifies a novel therapeutic Enpp1 inhibitor suitable for combination therapies in immuno-oncology.

overall_design: Tumors were implanted subcutaneously into the right flank as follows: C57BL/6 2x105 MC38; BALB/c 2x105 CT26. When tumors were approximately 5mm in average diameter at approximately 10-14 days following implantation, mice were randomized to receive treatment with CT-guided radiation using the Small Animal Radiation Research Platform (SARRP) from XStrahl. Dosimetry was performed using Murislice software from XStrahl. The SARRP delivered a single dose of 12Gy to an isocenter within the tumor using a 10mm x 10mm collimator and a 45° beam angle to minimize dose delivery to normal tissues. The Enpp1 inhibitor VIR3 was delivered through oral gavage starting 1 day prior to RT daily. 4d following RT, the tumors were harvested, debulked and immediately flash frozen by rapid immersion in liquid nitrogen. Frozen tumors were crushed, treated with 500µl of RNAlater, then frozen at -80oC. To extract RNA, the samples were thawed on ice, and 500µl of buffer RLT (QIAGEN Cat. No. 79219) was added and samples were homogenized. Following centrifugation, RNA was isolated using a QIAGEN RNeasy Plus Mini Kit (Cat No. 74134), according to the manufacturer’s instructions. Quality was checked on nanodrop (ND-1000) and the quantity of RNA was determined on Qubit 4 flourometer. To prepare for sequencing, samples were processed using an Illumina TruSeq® Stranded mRNA Library Prep kit with with Illumina TruSeq RNA Single Indexes Set A and Set B barcoding kits. An Illumina NovaSeq 6000 S1 Reagent Kit v1.5 was used to make RNA libraries along with a NovaSeq XP 2-Lane Kit v1.5. RNA libraries were sequenced on NovaSeq 6000.

first: Jason

last: Baird

first: Venkatesh

last: Rajamanickam

first: Michael

last: Gough


title: PI3K/AKT signalling regulates mouse Inner Cell Mass (ICM) progenitor specification [FLASH-seq]

city: Paris

first: Michel

address: 25 rue du Dr Roux

country: France

institute: Institut Pasteur

summary: The inner cell mass (ICM) of early mouse embryos is specified into Epiblast (Epi) and primitive endoderm (PrE) lineages during blastocyst formation. The antagonistic transcription factors (TFs) NANOG and GATA6 in combination with FGF/ERK signaling are central actors in ICM fate choice. However, what initiates the specification of the bipotent ICM progenitor and whether other factors are involved in this process is not fully understood yet. Here, we report the key role of PI3K in mouse ICM progenitors specification. Surprisingly, while the PI3K/AKT signaling pathway is known for almost two decades to participate to the maintenance of pluripotency in stem cells, no role for this pathway in ICM cell fate decisions has been reported so far. This is likely due to the dynamic and asynchronous nature of ICM specification combined with the pleiotropic and rapidly evolving functions mediated by PI3K/AKT. By limiting the perturbations of the pathway to a short time-window corresponding to the early phase of ICM specification, we unravelled a dual role of PI3K in ICM progenitor, being required on the one hand for the maintenance of pluripotency TFs and, on the other hand, for the competence to engage into PrE differentiation in response to FGF signaling. Thus, our work identifies PI3K as a novel critical regulator of ICM progenitor specification in the mouse embryo.

overall_design: Our dataset includes 26 single-ICM RNA-seq from E2.75 embryos cultured ex vivo for 24 hours with vehicle (DMSO), the PI3K inhibitor LY294002 (LY) , FGF4 and heparin (FGF4) or both LY and FGF4/heparin (LY+FGF4). Embryos from five litters were mixed and distributed between the four conditions. Some embryos were fixed and analysed by immunofluorescence to verify the effectivness of the treatments.

first: Adèle

last: Micouin

first: Almira

last: Chervova

first: Pablo

last: Navarro

last: Cohen-Tannoudji


title: Propafenone facilitates mitochondrial-associated ferroptosis and synergizes with immunotherapy in melanoma

status: Public on Oct 26 2024

city: changsha

first: yating

last: dian

address: xiangya hospital

institute: Xiangya Hospital Central South University

summary: Despite the successful application of immunotherapy, both innate and acquired resistance are typical in melanoma. Ferroptosis induction appears to be a potential strategy to enhance the effectiveness of immunotherapy. However, the relationship between the status of ferroptosis and the effectiveness of immunotherapy, as well as viable strategies to augment ferroptosis, remain unclear. In this study, through large-scale drug screening of cardiovascular drugs, we identified propafenone, an anti-arrhythmia medication, as capable of synergizing with ferroptosis inducers in melanoma. Furthermore, we observed that propafenone, in combination with RSL3, collaboratively induces mitochondrial-associated ferroptosis. Mechanistically, propafenone transcriptionally upregulates mitochondrial HMOX1 via activation of the JNK/JUN signaling pathway under RSL3 treatment, leading to overloaded ferrous iron and ROS within the mitochondria. In xenograft models, the combination of propafenone with ferroptosis induction led to nearly complete tumor regression. Consistently, propafenone enhances immunotherapy-induced antitumor immunity and tumoral ferroptosis in tumor-bearing mice. Significantly, patients exhibiting high levels of ferroptosis/JUN/HMOX1 exhibited improved efficacy of immunotherapy and prolonged progression-free survival. These findings suggest that propafenone holds promise as a candidate drug for enhancing the efficacy of immunotherapy and other ferroptosis-targeted therapies in the treatment of melanoma.

overall_design: Immunocompetent C57BL/6 mice implanted with B16F10 melanoma cells received intra-peritoneal injections of anti-PD-1 treatment or IgG control with or without propafenone on the indicated day after the tumor reached approximately 100 mm3 and tumor were excised for RNA sequencing on day 16.

first: Qian

last: Zhou


title: Reciprocal inhibition of NOTCH and SOX2 shapes tumor cell plasticity and therapeutic escape in triple negative breast cancer.

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

overall_design: Refer to individual Series


title: The histone acetylation module and histone chaperone in the Arabidopsis NuB4 complex have synergistic effect on chromatin accessibility (RNA-Seq)

city: Beijing

first: xinjian

last: He

address: No. 7, zhongguancun life science park road, changping district

institute: National Institute of Biological Sciences

summary: Although the conserved type B histone acetyltransferase is known to regulate both cytoplasmic histone acetylation and nuclear nucleosome assembly, the coordination between the dual functions remains poorly understood. Our study revealed that the Arabidopsis thaliana type B histone acetyltransferase HAG2 interactions with the histone chaperones MSI2, MSI3, and NASP, as well as histones H3 and H4, resulting in the formation of a conserved NuB4 complex. Within this complex, HAG2, MSI2, and MSI3 constitute a histone acetylation module that acetylates newly synthesized histone H4 at lysine 5 and 12 sites in the cytoplasm. This module collaborates with NASP to maintain the abundance of H3 and H4 proteins in the cytoplasm, but it is not necessary for nucleosome assembly in the nucleus, which differs from the role of NuB4 complexes in other eukaryotes. Furthermore, we found that, within the nucleus, the collaborative actions of the histone acetylation module and NASP enhance chromatin accessibility near the transcription start sites of protein-coding genes, while showing a negative impact on chromatin accessibility near the transcription termination sites. This suggests that the Arabidopsis NuB4 complex is responsible for modulating chromatin accessibility across the entire genome, highlighting a plant-specific function of the NuB4 complex.

overall_design:
Examination of the mRNA expression of 12-day-old wild type and four mutants in Arabidopsis
Examination of the mRNA expression of 20-day-old wild type and four mutants in Arabidopsis

first: Xin-Jian


title: The histone acetylation module and histone chaperone in the Arabidopsis NuB4 complex have synergistic effect on chromatin accessibility (ChIP-Seq)

overall_design: Examination of H4K12Ac levels in wild type and two mutants


title: H3K36 methylation regulates cell plasticity and regeneration in the intestinal epitheliumm (RNA-seq IV)

first: Alison

last: Pashos

overall_design: RNA-sequencing for sorted intestinal cells: Stem, Paneth, Goblet, Tuft, Enteroendocrine, and enterocyte cells, 2 biological replicates each.

middle: S

first: Justin

last: Brumbaugh


title: Subchronic elevation in ambient temperature drives alterations to the sperm epigenome and accelerates early embryonic development in mice

city: Philadelphia

first: Natalie

middle: Alyce

last: Trigg

state: Pennsylvania

address: 421 Curie Blvd

institute: Children's Hospital of Philadelphia

type: Non-coding RNA profiling by high throughput sequencing

summary: Forecasted increases in the prevalence and severity of extreme weather events accompanying changes in climatic behavior pose potential risk to the reproductive capacity of animals of ecological and agricultural significance. While several studies have revealed that heat stress induced by challenges such as testicular insulation can elicit a marked effect on the male reproductive system and in particular the production of spermatozoa, comparatively less is known about the immediate impact on male reproductive function following sub-chronic whole-body exposure to elevated ambient temperature. To address this knowledge gap, here we exposed unrestrained male mice to heat stress conditions that emulate a heatwave (daily cycle of 8 h at 35°C followed by 16 h at 25°C) for a period of seven days. Neither the testes or epididymides of heat exposed mice exhibited evidence of gross histological change and similarly, the spermatozoa of exposed males retained their functionality and ability to support embryonic development. Notably, however, the embryos sired by heat exposed spermatozoa experienced pronounced changes in gene expression linked to acceleration of early embryo development, aberrant blastocyst hatching and increased fetal weight. Such changes were causally associated with altered sperm small non-coding RNA (sncRNA) profiles, such that these developmental phenotypes were recapitulated by microinjection of wild-type embryos sired by control spermatozoa with RNAs extracted from heat exposed spermatozoa. Such data highlight that even a relatively modest excursion in ambient temperature can affect male reproductive function and identify the sperm sncRNA profile as a particular point of vulnerability to this imposed environmental stress.

overall_design: To investigate the impact of paternal heat exposure on the sperm small RNA payload and subsequent transcriptome of the embryo we sequenced the small RNA fraction of sperm from control and heat-exposed mice (n=3 biological replicates per treatment) and performed RNA-seq on single 4-cell and morula stage embryos fertilized by control or heat-exposed sperm

first: John

middle: E

last: Schjenken

first: Jacinta

last: Martin

first: David

last: Skerrett-Byrne

first: Shannon

middle: P

last: Smyth

first: Ilana

last: Bernsteirn

first: Amanda

middle: L

last: Anderson

first: Simone

last: Stanger

first: Ewan

middle: N

last: N. Simpson

first: Archana

last: Tomar

first: Raffaele

last: Teperino

first: Colin

middle: C

last: Conine

first: Geoffry

last: De Iuliis

first: Shaun

middle: D

last: Roman

first: Matthew

last: Dun

first: Andrew

last: Eamens

first: Brett

last: Nixon


title: Chromatine immunoprecipitation (ChIP-seq) for RBPJ and SOX2 in Triple negative breast cancer MB157 cells and GSI-resistant MB157R cells upon induction of Notch1 or Sox2 signaling.

first: Nadine

last: Fournier

overall_design: Comparative analysis of RBPJ and SOX2 chromatine occupancy in MB157 and MB157R cells upon induction of Notch1 or Sox2 signaling.

first: Morgane

first: Joaquim

last: Javary

first: Freddy

last: Radtke


title: Transcriptome Analysis of Human Cancer Cells with Depleted or Overexpressed STELLA Proteins [RNA-seq]

city: Guangzhou

first: Wenbin

last: Gu

address: 190 Kaiyuan Avenue, Huangpu District, Guangzhou

department: Chinese Academy of Sciences

institute: Guangzhou Institutes of Biomedicine and Health

summary: UHRF1 maintains DNA methylation by recruiting DNA methyltransferases (DNMT’s) to chromatin. These dynamics are well defined for mouse STELLA (mSTELLA) but poorly characterized for human STELLA (hSTELLA). Herein, we demonstrate that hSTELLA is defective, while mSTELLA is fully proficient in associating with UHRF1 and inhibiting the abnormal DNA methylation and oncogenic functions of UHRF1 in human cancer cells. We illustrate, in structural studies a region of low sequence homology between the above STELLA orthologs, allows mSTELLA but not hSTELLA to bind tightly to the essential histone binding domains of UHRF1, thus mediating their above functional differences. The ortholog-specific binding modes of STELLA proteins with UHRF1 prompt us to explore a rationale for designing UHRF1 inhibitors for cancer therapy. For this, we use a lipid nanoparticle (LNP)-mediated mRNA delivery approach demonstrating the short mSTELLA, but not hSTELLA regions are required to reverse cancer-specific DNA methylation abnormalities and impair CRC tumor growth.

overall_design:
To investigate the role of hSTELLA versus mSTELLA in regulating abnormal DNA hypermethylation associated tumor suppressor genes (TSGs) silencing, we established hSTELLA-knockout isogenic cell clones in BeWo and NCCIT cells. The transcriptome changes induced by hSTELLA-knockout were studied on the cell clones transduced with control sgRNAs (sgSCR) and hSTELLA-targeting sgRNAs. Next, we stably over-expressed both hSTELLA and mSTELLA in both HCT116 and RKO cells, and compared the transcriptome differences (relative to EV) between cells with hSTELLA overexpression and cells with mSTELLA overexpression
To define the key regions mediating the functional differences between hSTELLA and mSTELLA, we designed two chimeric mutants for C-terminal mSTELLA which replace the corresponding sequences from hSTELLA. The two mutants were designated as mSTELLA-Swap1 and mSTELLA-Swap2, respectively. We compared the transcriptome changes induced by these mutants with those induced by mSTELLA and hSTELLA, upon their stable overexpression in UHRF1-deficient HCT116 cells (HCT116_UHRF1_def or HCT116-UHRF1+/-).
Based on the above findings, we utilized a lipid nanoparticle (LNP) approach for delivering mRNAs encoding mSTELLA and mSTELLA-Swap1 into HCT116 cells. Cells treated with LNP-mSTELLA and LNP-mSTELLA-Swap1 were collected at Days 2, 6 and 10, and the transcriptome differences for each group were compared at each time point.

first: W

last: Bai

first: Y

last: Cui

first: H

last: Guo

first: C

last: Xia

first: S

middle: B

last: Baylin

first: X

last: Kong


title: Single-cell transcriptomics unveils molecular signatures of neuronal vulnerability in a mouse model of prion disease that overlap with Alzheimer’s disease

city: Winnipeg

first: Jessy

middle: Andrew

last: Slota

state: Manitoba

address: 1015 Arlington St.

department: One Health Division

country: Canada

institute: National Microbiology Laboratory; Public Health Agency of Canada

summary: Understanding why certain neurons are more sensitive to dysfunction and death caused by misfolded proteins could provide therapeutically relevant insights into neurodegenerative disorders. Here, we harnessed single-cell transcriptomics to examine live neurons isolated from prion-infected female mice, aiming to identify and characterize prion-vulnerable neuronal subsets. Our analysis revealed distinct transcriptional responses across neuronal subsets, with a consistent pathway-level depletion of synaptic gene expression in damage-vulnerable neurons. By scoring neuronal damage based on the magnitude of depleted synaptic gene expression, we identified a diverse spectrum of prion-vulnerable glutamatergic, GABAergic, and medium spiny neurons. Comparison between prion-vulnerable and resistant neurons highlighted baseline gene expression differences that could influence neuronal vulnerability. For instance, the neuroprotective cold-shock protein Rbm3 exhibited higher baseline gene expression in prion-resistant neurons and was robustly upregulated across diverse neuronal classes upon prion infection. We also identified vulnerability-correlated transcripts that overlapped between prion and Alzheimer’s disease. Our findings not only demonstrate the potential of single-cell transcriptomics to identify damage-vulnerable neurons, but also provide molecular insights into neuronal vulnerability and highlight commonalties across neurodegenerative disorders.

overall_design: Live single cell suspensions were isolated from prion (RML) and Mock infected mice, enriched for neurons, and supplied for 10x genomics 3' v3 single cell RNA sequencing

first: Lise

last: Lamoureux

first: Kathy

last: Frost

first: Babu

middle: V

last: Sajesh

first: Stephanie

last: Booth


title: DNA Methylome Analysis of Human Cancer Cells with Depleted or Overexpressed STELLA Proteins [Array]

type: Methylation profiling by genome tiling array

summary: Genome-wide DNA methylation profiling of the human STELLA (hSTELLA) knockout cell clones derived from TGCT cell lines (BeWo and NCCIT). The DNA methylome profiles are also studied on the CRC cells lines (HCT116 and RKO) stably expressing hSTELLA and mouse STELLA (mSTELLA), as well as on the HCT116 cells treated by LNP-delivered mRNA encoding mSTELLA. The Infinium MethylationEPIC 850k array was used to obtain DNA methylation profiles across more than 850,000 CpGs in 26 samples. Samples included 6 cell clones derived from BeWo cells with hSTELLA knockout or sgRNA control, 5 cell clones derived from NCCIT cells with hSTELLA knockout or sgRNA control, 6 stable CRC cells expressing STELLA orthologs, and 9 LNP-mRNA treated HCT116 cells.

overall_design: Bisulphite converted DNA from the 26 samples were hybridized to the Infinium MethylationEPIC 850k Beadchip


title: H3K36 methylation regulates cell plasticity and regeneration in the intestinal epithelium (CUT&Tag III)

first: Alison

last: Pashos

overall_design: CUT&Tag for H3K27me3 and H3K36me3 in sorted intestinal cells: Stem, Paneth, Goblet, Tuft, Enteroendocrine, and enterocyte cells, 2 biological replicates each.

first: Justin

last: Brumbaugh


title: H3K36 methylation regulates cell plasticity and regeneration in the intestinal epithelium (RNA-Seq II)

overall_design: RNA-sequencing for WT H3 and H3K36M mouse intestine samples, 2 biological replicates each.


title: H3K36 methylation regulates cell plasticity and regeneration in the intestinal epithelium (RNA-seq III)

overall_design: RNA-sequencing for WT H3 and H3K36M organoids at time 0, 1, 2, 4, and 8 weeks after doxycycline induction. 2 biological replicates each.


title: H3K36 methylation regulates cell plasticity and regeneration in the intestinal epithelium (CUT&Tag II)

overall_design: CUT&Tag for H3K27me3, H3K36me3, and H3K36me2 in WT H3 and H3K36M mouse intestine samples, 2 biological replicates each.


title: The histone acetylation module and histone chaperone in the Arabidopsis NuB4 complex have synergistic effect on chromatin accessibility (ATAC-Seq)

first: xinjian

last: He

overall_design: Examination of chromatin accessibility in wild type and four mutants

first: Xin-Jian


title: H3K36 methylation regulates cell plasticity and regeneration in the intestinal epithelium (scRNA-Seq)

overall_design: scRNA-seq of Paneth cells isolated from the proximal small intestine of Defa4-cre Rosa26 tdTomato mice by fluorescence activated cell sorting.


title: Spinster homolog 1 (spns1)-dependent endocardial autophagy-lysosomal pathway drives valve morphogenesis through the control of Notch1 signaling.

city: Bern

first: Nadia

last: Mercader

address: Baltzerstrasse 2

institute: University of Bern

summary: Autophagy-lysosomal degradation is an evolutionarily conserved process key to cellular homeostasis, differentiation, and stress survival, which is particularly important to the pathophysiology of the cardiovascular system. What is more, both experimental and clinical observations indicate that autophagy-lysosomal degradation affects correct cardiac morphogenesis, and in particular valve development. However, it is still unclear which cells upregulate autophagy-lysosomal degradation and for which specific cellular processes it is required.  Here, we introduce novel zebrafish transgenic models to visualize autophagosomes and lysosomes in vivo and to follow their temporal and cellular localization in the larval heart. This allowed us to determine the kinetics of autophagosome and lysosome vesicle formation and to observe  significant accumulation of lysosomal vesicles during the development of the atrioventricular and bulboventricular valves. We then addressed the functional role of lysosomal degradation in cardiovascular development using a spns1 mutant as a zebrafish model of lysosomal impairment. We found that spns1 mutants displayed morphologically and functionally abnormal heart development, including abnormal endocardial organization, impaired cardiac valve formation and high incidence of retrograde blood flow. Single-nuclear transcriptome analysis revealed endocardial-specific differences in the expression of lysosome-related genes and alterations of notch1 signaling in the mutant larval heart. Further, endocardial-specific overexpression of spns1 and notch1 rescued features of valve formation and function as well as overall cardiac morphogenesis. Altogether, our study provides an improved description of the autophagy and lysosomal events that take place during zebrafish heart development and reveals a cell-autonomous role of lysosomal processing during cardiac valve formation upstream of notch1 signaling.

overall_design: Sibling and spns mutant zebrafish larval hearts were obtained at 3 dpf by manual dissection. Two replicates, each consisting of four pools(50 hearts), were obtained for each experimental group (sibling, mutant). Single nuclei suspensions containing 3800-4000 nuclei/ µL were prepared using the Chromium Nuclei Isolation Kit. The Transposition, GEM generation & barcoding, reverse transcription, and preparation of the gene expression and ATAC libraries was performed according to the 4Chromium Next GEM Single Cell Multiome ATAC + Gene Expression. The cDNA libraries were pooled and sequenced with a loading concentration of 300 pM, asymmetric paired-end and dual indexed, using an Illumina NovaSeq 6000 S1 Reagent Kit v1.5 100 cycles (Illumina, 20028319) on an Illumina NovaSeq 6000.

first: Myra

last: Chavez

first: Alexander

last: Ernst

first: Prateek

last: Arora

first: Marco

last: Meer

first: Rodrigo

last: Morales


title: PI3K/AKT signalling regulates mouse Inner Cell Mass (ICM) progenitor specification [scRNA-seq]

first: Michel

overall_design: Our dataset includes 184 single-cells RNA-seq from inner cells recovered from mouse embryos: freshly collected E3.25 (20), E3.75 (24), and  E4.0 (18) as well as  E2.75 cultured ex vivo for 16 hours (64) or 24 hours (58) with the PI3K inhibitor LY294002 (LY) or with vehicle (DMSO) . Single cells from each stage and condition were obtained from two to three independent experiments, each including multiple litters. For each experiment, some embryos were fixed and analysed by immunofluorescence to verify the development stage of the embryos and the effectivness of LY treatment.

first: Anna

last: Geiselmann

first: Vincent

last: Laville

first: Pablo

last: Navarro

last: Cohen-Tannoudji


title: Divergent clinical and immunologic outcomes based on STK11 co-mutation status in resectable KRAS-mutant lung cancers following neoadjuvant immune checkpoint blockade

status: Public on Oct 24 2024

city: Baltimore

first: Kellie

last: Smith

address: 1550 Orleans St

institute: Johns Hopkins University

summary: Co-mutations of the KRAS and STK11 genes in advanced non-small cell lung cancer (NSCLC) are associated with immune checkpoint blockade (ICB) resistance. While neoadjuvant chemoimmunotherapy is now a standard of care treatment for early-stage NSCLC, the clinical and immunologic impact of KRAS and STK11 co-mutations in this setting are unknown. We evaluated the clinical outcomes and intratumoral T cell profiles of resected KRAS-mutant tumors treated with neoadjuvant ICB. Relative to KRASmut/STK11wt tumors, KRASmut/STK11mut exhibited significantly higher recurrence risk. Single-cell transcriptomics showed enhanced oxidative phosphorylation with evidence of decreased PGE-2 signaling and increased IL-2 signaling in CD8+ tumor-infiltrating lymphocytes (TIL) from KRASmut/STK11mut tumors, a finding that was mirrored in KRASwt tumors that relapsed. TIL from KRASmut/STK11mut tumors expressed high levels of molecules associated with tumor residence, including CD39 and ZNF683 (HOBIT). These divergent T cell transcriptional fates suggest T cell maintenance and residence may be detrimental to anti-tumor immunity in the context of neoadjuvant ICB for resectable NSCLC, regardless of KRAS mutation status. Our work provides a basis for future investigations into the mechanisms underpinning PGE-2 and IL-2 signaling as they relate to T cell immunity to cancer and to divergent clinical outcomes in KRASmut/STK11mut NSCLC treated with neoadjuvant ICB.

overall_design: To better understand the immunological underpinnings of the increased risk of recurrence in patients with KRASmut/STK11mut tumors, we performed single cell TCRseq/RNAseq on biospecimens from 6 patients with KRASmut/STK11mut disease and 7 patients with KRASmut/STK11wt disease treated with neoadjuvant ICB. All KRASmut/STK11mut tumors were confirmed to have functionally and/or clinically relevant STK11 mutations.

first: Sydney

last: Connor


title: Inhibition of discoidin domain receptor (DDR)-1 with nilotinib alters CSF miRNAs and is associated with reduced inflammation and vascular fibrosis in Alzheimer's Disease

city: Washington, DC

first: Max

last: Stevenson

state: DC

address: 3970 Reservoir Rd. NW

institute: Georgetown University

summary: Discoidin Domain Receptor (DDR)-1 is activated by collagen. Nilotinib is a tyrosine kinase inhibitor that is FDAapproved for leukemia and potently inhibits DDR-1. Individuals diagnosed with mild–moderate Alzheimer’s disease (AD) treated with nilotinib (versus placebo) for 12 months showed reduction of amyloid plaque and cerebrospinal fluid (CSF) amyloid, and attenuation of hippocampal volume loss. However, the mechanisms are unclear. Here, we explored unbiased next generation whole genome miRNA sequencing from AD patients CSF

overall_design: miRNAs were matched with their corresponding mRNAs using gene ontology. Changes in CSF miRNAs were confirmed via measurement of CSF DDR1 activity and plasma levels of AD biomarkers. Approximately 1050 miRNAs are detected in the CSF but only 17 miRNAs are specifically altered between baseline and 12-month treatment with nilotinib versus placebo.

first: Rency

last: Varghese

first: Charbel

last: Moussa


title: H3K36 methylation regulates cell plasticity and regeneration in the intestinal epithelium

first: Alison

last: Pashos

overall_design: Refer to individual Series


title: DNA methylome analysis of parental and p53-knockout MCF-7 and T47D breast cancer cells treated with sodium arsenite

city: PETALING JAYA

first: Felicia

last: Chung

state: Selangor

address: Sunway University, 5, Jalan Universiti, Bandar Sunway, 47500 Petaling Jaya, Selangor

country: Malaysia

institute: Sunway University

summary: The study aimed to test the hypothesis that mutations in p53, a gene commonly altered in human malignancies, may influence cellular responses to an environmental non-mutagenic agent through potential epigenetic mechanisms. Utilizing the CRISPR-Cas9 system, p53 knockouts were generated in MCF7 and T47D breast cancer cell lines. DNA methylation patterns in these cell lines were characterized using targeted pyrosequencing and methylome-wide Infinium MethylationEPIC BeadChip arrays following exposure to sodium arsenite, a well-established human carcinogen known to impact the epigenome. The results demonstrated that the knockout of p53 alone was associated with extensive alterations in DNA methylation, characterized by predominant CpG hypermethylation alongside global demethylation.

overall_design: MCF7 and T47D breast cancer cell lines harboring p53 knockout mutations were generated using CRISPR-Cas9 gene editing. DNA methylation profiling was conducted using the Illumina Infinium MethylationEPIC BeadChip arrays to investigate the effects of the p53 knockout mutations on the epigenome, and to determine if these mutations affect cellular susceptibility to sodium arsenite-induced epigenetic alterations. Reconstitution of p53 in MCF7 knockout cell lines was conducted to evaluate its effects on restoring epigenetic stability.

first: Rita

last: Khoueiry

first: Aurélie

last: Sallé

first: Cyrille

last: Cuenin

first: Maria

last: Boskovic

first: Zdenko

last: Herceg


title: Expression of a murine model of Rheumatoid Arthritis with or without Intermittent fasting

city: Chihuahua

first: Mercedes

last: Bermúdez

state: Chihuahua

address: Chihuahua

department: Dentistry

country: Mexico

institute: Universidad autónoma de Chihuahua

summary: Intermittent fasting (IF) reduces the inflammatory symptoms of Rheumatoid Arthritis (RA); however, its anti-inflammatory underlying molecular mechanisms are unknown. Aim: To investigate the effect of IF on joint inflammatory molecular mechanisms in collagen-induced arthritis (CIA) through DNA microarray and clinical and histopathological evaluations. Methods: CIA was induced in sixteen male DBA/1 mice divided into two groups, one received every other day IF for four weeks. The clinical, histological, and transcriptomic analyses, through DNA microarray, were done to evaluate the effect of IF on joint inflammation and remodeling. Results: CIA mice with IF show lower arthritis incidence and severity. IF also decreased the histological inflammation and joint damage. DNA microarrays dataset revealed 364 overexpressed and 543 subexpressed genes. The highest over- and subexpressed genes were the Ereg and MUPs, respectively. Retinol biosynthesis and the macrophage alternative activation pathways were associated with a positive z-score. Pathways with negative z-scores were related to the inflammatory process, including systemic lupus erythematosus in B cells and osteoarthritis, in which the RA-related genes Cd72, Cd79a, Ifna, Il33, and Bglap2 were found to be decreased. The bioinformatic algorithm identified four central possible regulators: CEBPA, FOXO1, HIF1A, PPARG, and PPARA. In the analysis of diseases and biofunctions, molecular transport, small molecule biochemistry, and lipid and carbohydrate metabolism tended to increase; conversely, organismal injury and abnormalities, inflammatory response, metabolic diseases, cancer, and endocrine, reproductive, gastrointestinal, skeletal, and muscular system disorders, were decreased by IF

overall_design:
The Collagen induced arthritis was induced, all animals were fed with a standard chow diet and water ad libitum until day 21, when the second collagen injection was done. On this day,mice were randomly divided into two groups of eight mice each; one received every-other-day Intermittent Fasting (IF) for four weeks (experimental group), and the other did not (control group).
The effect of IF on transcriptome was evaluated in the Affymetrix RNA microarray by comparing the IF group (experimental group) and the control group. One hind paw of each mouse was used for this analysis.

first: Rubén

last: Cuevas-Martínez

first: César

last: Pacheco-Tena

first: Susana

last: González-Chávez


title: Targeting HDAC1 enzymatic activity in T cells protects against experimental autoimmune encephalomyelitis

city: Vienna

first: Wilfried

last: Ellmeier

address: Lazarettgase 19

department: Institute of Immunology

country: Austria

institute: Medical University of Vienna

summary: Histone deacetylases are key epigenetic regulators that control T cell-mediated immunity. A T cell-specific deletion of Hdac1 (HDAC1cKO) protects mice against experimental autoimmune encephalomyelitis (EAE). However, it remains elusive whether inhibition of HDAC1 enzymatic activity and thus mimicking HDAC1 inhibitor treatment is sufficient to block EAE induction. In order to address this question, we generated a novel mouse strain that expresses catalytically inactive HDAC1 (HDAC1Off) from the Rosa26 locus in HDAC1cKO CD4+ T cells to mimic selective inhibition of HDAC1 enzymatic activity in vivo. Mice expressing wildtype HDAC1 in HDAC1cKO CD4+ T cells (HDAC1On) were generated as corresponding controls. In contrast to HDAC1On mice, HDAC1Off mice did not develop EAE, and this correlated with diminished leukocyte CNS infiltration. HDAC1Off CD4+ T cells in the CNS displayed a severe reduction of IFNγ, IL-17A and TNFα proinflammatory cytokine expression. In vivo activated HDAC1Off CD4+ T cells downregulated gene sets associated with T cell activation, inflammatory response and cell migration, indicating impaired effector functions of HDAC1Off CD4+ T cells. Thus, our study demonstrates that the inhibition of the catalytic activity of HDAC1 is sufficient to achieve a clinical benefit in EAE disease development. This raises the exciting translational perspective that targeting HDAC1 enzymatic activity is a promising therapeutic strategy in treating human T cell-mediated autoimmune diseases.

overall_design: Cells from draining (popliteal) lymph nodes were isolated and RNA was extracted from single cell suspension. Two mice were pooled for one biological replicate and three biological replicates were generated for each genotype (group). One WT group and one catalalytically inactive group were included.

first: Ci

last: Zhu

first: Valentina

last: Stolz

first: Christian

last: Seiser


title: Global transcriptome of Arabidopsis plants (Col-0, Rld2 and Shahdara genotypes) inoculated by the fungal pathogen Sclerotinia sclerotiorum following temperate, mediterranean and continental acclimation

city: Castanet tolosan

first: Sylvain

last: Raffaele

address: 28 chemin de borde rouge

institute: INRAE

summary: The short-term, reversible process that allows plants to cope with immediate environmental fluctuations is often referred to as acclimation. Plant acclimation help maintain the balance of natural systems, supporting biodiversity and the services that ecosystems provide, such as carbon sequestration and water regulation. With climate change modifying the distribution area of plants and causing more frequent and severe weather events, knowledge of how plants acclimate can inform strategies to manage ecosystems and agriculture. Analyses of plant immune responses under abiotic constraints generally focus on pathogen inoculation under prolonged and stable abiotic conditions. Daily fluctuations of the environment may alter plant metabolism, growth and flowering as well as gene regulation and invasive growth of fungal pathogens. Yet, how plant immunity acclimates to daily temperature fluctuations remains largely unexplored. To study the molecular bases of quantitative disease resistance acclimation, we performed a global transcriptome analysis of Arabidopsis thaliana accessions Col-0, Rld-2 and Sha grown in temperate, continental and Mediterranean climates, followed or not by Sclerotinia sclerotiorum inoculation.

overall_design: Periphery of S. sclerotiorum disease lesions collected 24 hours post-inoculation on detached leaves of A. thaliana Col-0, Rld2 and Shahdara. Plants were previously acclimated under three different day/night temperature regimes. Mock inoculated plants acclimated at the same time serve as controls. Samples were collected in three independent replicates of the experiment.

first: Justine

last: Sucher

first: Marie

last: Didelon


title: Identification of miR-141 as a new regulator of epidermal homeostasis, in cooperation with miR-203

city: Valbonne - Sophia-Antipolis

first: Kevin

last: Lebrigand

address: 660 route des lucioles

institute: IPMC/CNRS

summary: To identify putative novel specific targets of miR-141-3p, we overexpressed this miRNAs in primary keratinocytes using a synthetic mimic (pre-miR-141-3p) or a synthetic “negative” control mimic (pre-miR-ctrl). RNA samples were harvested 30 hours post-transfection and 3 independent experiments were carried out.

overall_design: 3 independent experiments performed in a one color design, corresponding to 2 conditions (pre-miR-ctrl and pre-miR-141-3p) for a total of 6 samples.

first: C

last: Marionnet

first: R

last: Rezzonico

first: B

last: Mari

first: K

first: P

last: Barbry


title: Gene expression profiling of Triple Negative Breast Cancer MB157 cells and GSI-resistant MB157R cells upon induction of Notch1 or Sox2 signaling.

first: Nadine

last: Fournier

summary: Cell plasticity of Triple negative breast cancer (TNBC) contributes to tumor heterogeneity and is one of the major reasons for the limited success of chemo- or combination therapies in the clinics. The molecular mechanisms of therapy-induced tumor cell plasticity and associated resistance to chemo or targeted therapies are largely unknown.  Using a genome wide CRISPR-Cas9 screen we investigated the escape mechanisms of Notch driven TNBC, when treated with targeted therapy. We describe molecularly a reciprocal inhibitory feedback mechanism between Notch signaling and the pluripotency associated transcription factor SOX2, which shapes divergent cell states, EMT, cancer stem cell features and associates with therapeutic response and escape to targeted therapy. Moreover, we performed and assessed monotherapy and drug combination treatments in Notch-inhibitor sensitive and resistant TNBC xenotransplant and identified combination and second line treatment options which were able to induce tumor control and reduce metastatic burden.

overall_design: Comparative gene expression profiling of MB157 cells between different conditions: MB157R vs MB157; MB157R-NICD vs MB157R-EV; MB157-SOX2 vs MB157-EV.

first: Morgane

first: Joaquim

last: Javary

first: Freddy

last: Radtke


title: Pantothenate kinase 4 controls skeletal muscle substrate metabolism

city: Nuthetal

first: Maximilian

last: Kleinert

address: Arthur-Scheunert-Allee 114-116, , Arthur-Scheunert-Allee 114-116

department: Molecular Physiology of Exercise and Nutrition

institute: German Institute of Human Nutrition

summary: Metabolic flexibility in skeletal muscle is essential for maintaining healthy glucose and lipid metabolism, and its dysfunction is closely linked to metabolic diseases. Exercise enhances metabolic flexibility, making it an important tool for discovering mechanisms that promote metabolic health. Here we show that pantothenate kinase 4 (PanK4) is a new conserved exercise target with high abundance in muscle. Muscle-specific deletion of PanK4 impairs fatty acid oxidation which is related to higher intramuscular acetyl-CoA and malonyl levels. These elevated acetyl-CoA levels persist regardless of feeding state and are associated with whole-body glucose intolerance, reduced insulin-stimulated glucose uptake in glycolytic muscle, and impaired glucose uptake during exercise. Conversely, increasing PanK4 levels in glycolytic muscle lowers acetyl-CoA and enhances glucose uptake. Our findings highlight PanK4 as an important regulator of acetyl-CoA levels, playing a key role in both muscle lipid and glucose metabolism.

overall_design: Male PanK4 WT and PanK4 mKO mice (age 28 weeks) were fasted for 4 hours and then injected with glucose (2 g/ kg body weight) and 40 min later mice were humanely euthanized and gastrocnemius muscles were collected and analyzed.

first: Markus

last: Jähnert


title: Investigating the transcriptional heterogeneity of the beta cells experiencing Ca2+ excitotoxicity

city: Dresden

first: Mohammad

middle: Nadeem

last: Akhtar

state: Saxony

address: Fetscherstrasse 105

department: CRTD

institute: TU Dresden

summary: Beta cells are remarkably heterogeneous. However, beta cells response to chronic stress at a single cell level remains largely unknown. Here, we used a chemo-genetic model of beta cell excitotoxcity coupled with single cell RNA sequencing to understand beta cells response to chronic stress. Remarkably, we found that a subset of stressed beta cells progressed towards death while the other group evaded the destruction by dedifferentiation. Vulnerable beta cells that progressed towards death showed upregulation of genes implicated in apoptosis, inflammation and immune system activation. The resilient beta cells that evaded the stress showed increased expression aldh1a3, a canonical marker of mammalian beta cell dedifferentiation.

overall_design:
We used three different treatment groups for this study. Tg(ins:Kaede) larvae (n=20) treated with capsaicin (csn) for 48 hours i.e. from 3 to 5 days post fertilization (dpf) were used as control. Tg(ins:TRPV);Tg(ins:Kaede) larvae (n=20) treated with csn for 24 hours i.e. from 4 to 5 dpf were used to identify genes involved in immediate stress response and Tg(ins:TRPV);Tg(ins:Kaede) larvae (n=20) treated with csn for 48 hours i.e. from 3 to 5 dpf were used to identify the genes involved in chronic stress response. Post csn treatment, pancreatic islets from the larvae were manually dissected and then subsequently dissociated via enzymatic digestion. Post dissociation the beta cells were stained with calcein (viability marker dye) and FACS sorted. Individual beta cells were collected in a 384 well plate and their transcriptomes were profiled using smart seq2.
RNA_01_H19, RNA_02_G11, RNA_03_P18 do not correspond to the other samples (eg, Ctrl, CNS24, CNS48-2).


title: Mutability and hypermutation antagonize immunoglobulin codon optimality; Analysis of human MLN B cells.

city: New York

first: Patrick

last: Wilson

state: NY

address: 413 E 69th St

department: Drukier Institute

institute: Weill Cornell Medicine

summary: The main objective of this study was to analyze codon optimality in relation to somatic hypermutation in natural V(D)J repertoires.

overall_design: Human mediastinal lymph node (10L/10R/12L/12R//2L/4R/7) B cells were sorted and analyzed by 10x Genomics 5' scRNA-seq. 5' GEX and V(D)J libraries are included.

first: Joshua

last: McGrath

first: Chloe

last: Troxell

first: Jordan

last: Chervin


title: Interleukin-1α release during necrotic-like cell death generates myeloid-driven immunosuppression that restricts anti-tumor immunity

city: Tampa

first: Xiaoxian

last: Liu

state: FL

address: 13131 USF Magnolia Drive

institute: Moffitt Cancer Center

summary: Necroptosis can promote antigen-specific immune responses, suggesting induced necroptosis as a therapeutic approach for cancer. Here we sought to determine the mechanism of immune activation but found the necroptosis mediators RIPK3 and MLKL dispensable for tumor growth in genetic and implantable models of breast or lung cancer. Surprisingly, inducing necroptosis within established breast tumors generated a myeloid suppressive microenvironment that inhibited T cell function, promoted tumor growth, and reduced survival. This was dependent upon the release of the nuclear alarmin interleukin 1a(IL1a) by dying cells. Critically, IL1arelease occurred during chemotherapy and targeting this molecule reduced the immunosuppressive capacity of tumor myeloid cells and promoted CD8+ T cell recruitment and effector function. Neutralizing IL1a enhanced the efficacy of single agent paclitaxel or combination therapy with PD-1 blockade in preclinical models. LowIL1Alevels correlated with positive patient outcome in several solid malignancies, particularly in patients treated with chemotherapy.

overall_design: PyMT C57 mouse tumor cells expressing non-targeting control (NTC) or Il1a-targeting guide RNA together with Cas9 were implanted into mammary fat pad of female C57BL/6 mice at 8 weeks of age. Tumors were harvested 14 days post implantation in the absence of any treatment and tumor volume did not show any difference between groups. CD45 positive cells were harvested from these tumors by fluorescence-activated cell sorting on a FACS ARIA II and used for single cell sequencing analysis.

first: Kay

last: Hänggi

first: Brian

last: Ruffell

first: Xiaoqing

last: Yu


title: Mutability and hypermutation antagonize immunoglobulin codon optimality; Analysis of mouse splenic B cells.

overall_design: Spleens from 5 mice previously infected (34wpi) with influenza A NL09 were pooled. Pooled splenocyte B cells were sorted and analyzed by 10x Genomics 5' scRNA-seq. V(D)J libraries are provided.


title: H3K36 methylation regulates cell plasticity and regeneration in the intestinal epithelium (RNA-Seq I)

city: Boulder

first: Alison

middle: Rae Swearingen

last: Pashos

address: 1945 Colorado Avenue

institute: University of Colorado Boulder

summary: Cell plasticity is needed during development and homeostasis to generate diverse cell types from stem and progenitor cells. Following differentiation, plasticity must be restricted in specialized cells to maintain tissue integrity and function. For this reason, specialized cell identity is highly stable under homeostatic conditions; however, cells in some tissues regain plasticity during injury-induced regeneration. While precise gene expression is needed to control these processes, the regulatory mechanisms that restrict or promote cell plasticity are poorly understood. Here, we use the mouse small intestine as a model system to study cell plasticity. We find that H3K36 methylation reinforces expression of cell type-associated genes to maintain specialized cell identity in intestinal epithelial cells. Depleting H3K36 methylation leads to defects in lineage commitment and activates a plastic, regenerative gene expression signature. Correspondingly, we observe rapid and reversible remodeling of H3K36 methylation following injury-induced regeneration. Together, these data suggest a fundamental role for H3K36 methylation in regulating cell plasticity and regeneration.

overall_design: RNA-sequencing for WT H3 organoids at time 0, 24, 48, and 96 hours after irradiation and H3K36M organoids after 4 weeks of induction, 2 biological replicates each.

first: Justin

last: Brumbaugh


title: Response of Vibrio cholerae to exogenous peptidoglycan

city: Basel

first: Knut

last: Drescher

state: BS

address: Spitalstrasse 41

department: Biozentrum

institute: University of Basel

summary: Using transcriptomics, we studied the transcriptional response of Vibrio cholerae to 10 min of exogenously supplied peptidoglycan at 300 µg/mL.

overall_design: Vibrio cholerae cells unexposed or exposed to peptidoglycan for 10 min in microfluidic flow chambers were collected and analyzed using RNA-seq with 3 replicates for each condition.

first: Sanika

last: Vaidya

first: Kazuki

last: Nosho


title: The DNA demethylase TET1 modifies the impact of maternal folic acid status on embryonic brain development

city: Leuven

first: Lehua

last: Chen

address: Herestraat 49

institute: KU Leuven

summary: Folic acid (FA) prevents neural tube defects (NTDs), but it remains unclear why many cases of human NTDs still occur despite FA supplementation. Here, we investigate how the DNA demethylase TET1 interacts with maternal dietary FA status to regulate embryonic brain development. We determined that cranial NTDs in Tet1-/- embryos display higher penetrance in non-inbred than in inbred genetic backgrounds and are resistant to FA supplementation across strains. Maternal diets that are either too rich or deficient in FA are linked to an increased incidence of mild cranial deformities in wild type and Tet1+/- offspring and altered DNA hypermethylation in Tet1-/- embryos primarily at neurodevelopmental loci. Excess FA in Tet1-/- embryos results in phospholipid metabolite loss and reduced expression of multiple membrane solute carriers, including a FA transporter gene which exhibits increased promoter DNA methylation, thus mimicking a pseudo-FA deficiency. FA deficiency reveals an impact of Tet1 haploinsufficiency that contributes to DNA hypermethylation and susceptibility to NTDs. Overall, our study reveals that epigenetic dysregulation may underlie the resistance of NTDs to FA supplementation.

overall_design: 129S6.cg Tet1 HET dams were adapted to different FA diets: 30 ppm, 3 ppm, 0.1 ppm minimally for 4 weeks. After a successful timed-mating, dams were euthanized at E11.5 for brain tissue collection. WT, Tet1 HET, Tet1 KO brains from each diet group were selected for RNA-seq.

first: Bernard

middle: K

last: van der Veer


title: Mutability and hypermutation antagonize immunoglobulin codon optimality; Analysis of human tonsil B cells.

overall_design: Human tonsil B cells were sorted from one donor, and analyzed by 10x Genomics 5' scRNA-seq. 5' GEX and V(D)J libraries are included.


type: Methylation profiling by high throughput sequencing

summary: Folic acid (FA) prevents neural tube defects (NTDs), but it remains unclear why many cases of human NTDs still occur despite FA supplementation. Here, we investigate how the DNA demethylase TET1 interacts with maternal dietary FA status to regulate embryonic brain development. We determined that cranial NTDs in Tet1-/- embryos display higher penetrance in non-inbred than in inbred genetic backgrounds and are resistant to FA supplementation across strains. Maternal diets that are either too rich or deficient in FA are linked to an increased incidence of mild cranial deformities in wild type and Tet1+/- offspring and altered DNA hypermethylation in Tet1-/- embryos primarily at neurodevelopmental loci. Excess FA in Tet1-/- embryos results in phospholipid metabolite loss and reduced expression of multiple membrane solute carriers, including a FA transporter gene which exhibits increased promoter DNA methylation, thus mimicking a pseudo-FA deficiency. FA deficiency reveals an impact of Tet1 haploinsufficiency that contributes to DNA hypermethylation and susceptibility to NTDs. Overall, our study reveals that epigenetic dysregulation may underlie the resistance of  NTDs to FA supplementation.

overall_design: 129S6.cg Tet1 HET dams were adapted to different FA diets: 30 ppm, 3 ppm, 0.1 ppm minimally for 4 weeks. After a successful timed-mating, dams were euthanized at E11.5 for brain tissue collection. WT, Tet1 HET, Tet1 KO brains from each diet group were selected for RRBS.


title: FOXA2 and ASCL1 Cooperate in Driving Neuronal Lineage Commitment in Prostate Cancer [RNA-seq]

status: Public on Oct 23 2024

city: Vancouver

first: Amina

last: Zoubeidi

state: BC

address: 2660 Oak Street

institute: Vancouver Prostate Centre

summary: In castration-resistant prostate cancer, lineage plasticity mediates resistance to androgen receptor pathway inhibitors (ARPIs) and progression from adenocarcinoma to neuroendocrine prostate cancer (NEPC), a highly aggressive and poorly understood subtype. ASCL1 has emerged as a central regulator of the NEPC phenotype, driving neuroendocrine differentiation. However, ASCL1’s influence on neuronal lineage switching and maturation, as well as its partners in NEPC, remain largely unknown. Here, we provided insights into ASCL1’s cistrome reprogramming in ARPI-induced NEPC versus terminal NEPC and showed that ASCL1 binding pattern tailors the subsequent expression of transcription factor combinations that underlie discrete terminal NEPC identity. We identified FOXA2 as a major co-factor of ASCL1 in terminal NEPC that it is highly expressed  in ASCL1-driven NEPC. FOXA2 and ASCL1  interact and work in concert to orchestrate terminal neuronal differentiation in prostate cancer, and regulate key neuroendocrine-associated genes including PROX1. Our findings provide insights into the molecular conduit underlying the interplay between different lineage determinant transcription factors to support the neuroendocrine identity in prostate cancer.

overall_design: To investigate the role of FOXA2 in Neuro endocrine prostate cancer, FOXA2 gene was knocked out using shRNA in NCI-H660 cell line and gene expression analysis was performed using RNAseq data from two biological replicates.

first: Shaghayegh

last: Nouruzi

first: Takeshi

last: Namekawa

first: Nakisa

last: Tabrizian

first: Maxim

last: Kobelev

first: Olena

last: Sivak

first: Joshua M

last: Scrull

first: Cassandra Jingjing

last: Cui

first: Dwaipayan

last: Ganguli


title: Transcriptome profiling of maize transcription factor mutants to probe gene regulatory network predictions

city: St Paul

first: Nathan

last: Springer

state: MN

address: 1479 Gortner Ave

department: Plant and Microbial Biology

institute: University of Minnesota

summary: Loss-of-function mutant alleles were characterized for 22 maize transcription factors to test the functional relevance of prior yeast-one-hybrid and co-expression gene regulatory network predictions. The transcriptome and phenolic metabolome of these mutants were profiled to document the functional roles of these transcription factors. A subset of the prior gene regulatory network predictions were supported but had limited overall power in predicting changes in gene expression or phenolic compounds. The limitations of testing transcription factor-target predictions with single TF knockouts are discussed.

overall_design: Loss-of-function mutant alleles were isolated for 22 maize transcription factors. Transcriptome profiling by RNA-seq was performed for each TF mutant allele to characterize genome-wide perturbations of expression. Enrichment tests were performed to determine if predicted target genes were enriched for differential expression.

first: Erika

last: Ellison

first: Peng

last: Zhou

first: Yi-Hsuan

last: Chu

first: Peter

last: Hermanson

first: Erich

last: Grotewold

middle: M

first: Lina

last: Gomez-Cano

first: Zachary

middle: A

last: Myers

first: Ankita

last: Abnave

first: John

last: Gray

first: Candice

last: Hirsch


title: H3K36 methylation regulates cell plasticity and regeneration in the intestinal epithelium (CUT&Tag I)

first: Alison

last: Pashos

overall_design: CUT&Tag for H3K27me3, H3K36me3, and H3K36me2 in WT H3 organoids at time 0, 24, 48, and 96 hours after irradiation, and H3K36M organoids after 4 weeks of induction, 2 biological replicates each.

first: Justin

last: Brumbaugh


title: Gain of function of Antp in developing flight muscle prevents determination of the fibriallar muscle fate

city: Marseille

first: Andrew

middle: J

last: Saurin

address: Campus de Luminy

institute: IBDM / CNRS

summary: Muscle development and diversity require a large number of spatially and temporally regulated events controlled by transcription factors (TFs). Drosophila has long stood as a model organism to study myogenesis due to the highly conserved key TFs involved at all stages of muscle development, from specification to differentiation. While many studies focused on the diversification of Drosophila larval musculature, how distinct adult muscle types are generated is much less characterised. Here, we identify a novel essential regulator of Drosophila thoracic flight muscle development, the Hox TF Antennapedia (Antp). Correcting a long standing misconception that flight muscle development occurs without the input of Hox TFs, we show that Antp intervenes at several stages of adult flight muscle development, from the establishment of the progenitor pool in the embryo to myoblast differentiation in the early pupa. Furthermore, the precisely regulated clearance of Hox at the pupal stage in the developing indirect flight muscle fibers is required to allow for stretch-activated fibrillar muscle fate diversification that sets these flight muscles apart from all other adult tubular muscle types.

overall_design: For the role of Antp in developing IFM once normal Antp expression is lost, Act88F-Gal4 flies were crossed to either UAS-GFP  or UAS-Antp. IFM were then dissected at 72h APF, RNA prepared and sequenced. Samples represent triplicate independent replicates of each condition (WT and Antp GOF)

first: Gabreila

last: Poliacikova

first: Aïcha

last: Aouane

first: Nathalie

last: Caruso

first: Nicolas

last: Brouilly

first: Corinne

last: Maurel-Zaffran

first: Yacine

last: Graba


title: The Hox protein Antennapedia orchestrates Drosophila adult flight muscle development

summary:
Muscle development and diversity require a large number of spatially and temporally regulated events controlled by transcription factors (TFs). Drosophila has long stood as a model organism to study myogenesis due to the highly conserved key TFs involved at all stages of muscle development, from specification to differentiation. While many studies focused on the diversification of Drosophila larval musculature, how distinct adult muscle types are generated is much less characterised. Here, we identify a novel essential regulator of Drosophila thoracic flight muscle development, the Hox TF Antennapedia (Antp). Correcting a long standing misconception that flight muscle development occurs without the input of Hox TFs, we show that Antp intervenes at several stages of adult flight muscle development, from the establishment of the progenitor pool in the embryo to myoblast differentiation in the early pupa. Furthermore, the precisely regulated clearance of Hox at the pupal stage in the developing indirect flight muscle fibers is required to allow for stretch-activated fibrillar muscle fate diversification that sets these flight muscles apart from all other adult tubular muscle types.

This SuperSeries is composed of the SubSeries listed below.

overall_design: Refer to individual Series


title: Loss of Antp function in Drosophila flight muscle precursors : RNAi in larval wing disc-associated myoblasts

overall_design: For the role of Antp in 0h APF pupal myoblasts, that will fuse and differentiate into adult flight muscle, Mef2-Gal4;UAS-GFPnls flies were crossed to either UAS-Y RNAi or UAS-Antp RNAi. Myoblasts were dissociated from 0h APF dissected wing discs, FACS sorted, RNAi prepared and sequenced. All samples represent each genotype repeated with three  independent biological replicates (triplicates)


title: Skin autonomous antibody production regulates host-microbiota interactions

city: Bethesda

first: Verena

last: Link

address: 4 Memorial Drive

department: NIAID

institute: NIH

summary: Host symbiosis with its microbiota can represent a liability for the host. Indeed, while these communities of microbes broadly control host physiology, the microbiota can also promote inflammation and represent a source of local and systemic infection. The unique strategies employed by each tissue to maintain and control coexistence with commensal microbes remain largely unclear. Here we uncover that, within the skin, host protection from its microbiota depends on the remarkable ability of the skin to act as an autonomous lymphoid organ. Notably, an encounter with a new skin microbe is associated with two parallel responses both, under the control of Langerhans cells. On one hand, skin commensal promotes the development of classical germinal centers within secondary lymphoid organs leading to IgG1 and IgG3 responses. On the other hand, microbial colonization also leads to the development of tertiary lymphoid organs that can locally sustain and mature IgG2b and IgG2c responses. The ability of the skin to develop as a lymphoid compartment is at least in part mediated by the ability of the local Treg pool to convert into T follicular helper cells accumulating within organized tertiary lymphoid organs surrounding the hair follicles. Remarkably, skin autonomous production of antibodies is sufficient to control both microbial colonization within the skin as well as subsequent infection with the same commensal. Collectively these results reveal a profound compartmentalization of humoral responses between the lymph nodes and the skin. Further, this work uncovers a previously unappreciated function for the skin as a compartment able to develop, in the absence of inflammation, powerful and long-lived antibody responses independently of secondary lymphoid organs.

overall_design: T cells were extracted for single cell RNA-seq from skin of mice treated with Stapholococcus epidermidies


title: Automal Random Allelic Expression (aRMAE) analysis in clonally expanded T cells from healthy donors

first: Dusan

last: Bogunovic

address: 650 W 168th Street

department: Pediatrics

institute: Columbia University Medical Center

summary: Inborn errors of immunity (IEIs) are genetic disorders that underlie susceptibility to infection, autoimmunity, autoinflammation, allergy, and/or malignancy. Incomplete penetrance is common among IEIs despite their monogenic basis. Herein we investigate the contribution of autosomal random monoallelic expression (aRMAE), a somatic commitment to expression of one allele, to phenotypic variability observed in families with IEIs. Using a clonal primary T cell system to assess aRMAE status of genes in healthy individuals, we find 4.30% of IEI genes and 5.20% of all genes undergo aRMAE. Perturbing H3K27me3 and DNA methylation alters allele expression commitment, supporting two proposed mechanisms for regulation of aRMAE. We tested PBMCs from individuals sharing genetic lesions but with discordant clinical phenotypes in seven families with different IEIs for aRMAE. Among relatives heterozygous for a mutation in PLCG2, an antibody deficiency phenotype corresponds with selective mutant allele expression in B cells. In contrast, among relatives heterozygous for a mutation in JAK1, the unaffected carrier T cells predominantly expressed the wild-type JAK1 allele, while the affected carrier T cells exhibited biallelic expression. Selective expression of a single allele was documented in phenotypically discordant family members carrying an AD Loss-of-function (LoF) mutation in STAT1, CARD11, as well as gain-of-function (GoF) mutations in STAT1. This study highlights the importance of not only considering genotype but also the “transcriptotype” in the analyses of the penetrance and expressivity of monogenic disorders.

overall_design: To asses aRMAE in T cell clones we expanded 50 clones across 10 healthy donors. Whole exome sequencing identified heterozygous SNPs in donors and RNA-seq determined expression of each allele.

first: O'Jay

last: Stewart

first: Conor

last: Gruber

first: Haley

last: Randolph

first: Roosheel

last: Patel

first: Meredith

last: Ramba


title: Blood gene expression profiling of interstitial lung disease samples

city: VANCOUVER

first: Daniel

last: He

address: 1081 Burrard St Room 166

institute: University Of British Columbia

summary: Expression of 180 genes in IPF, HP, SSc-ILD, IPAF patients (n=59)

overall_design: Blood samples from ILD patients were collected in PAXgene tubes and profiled using a custom NanoString nCounter assay originally designed to differentiate between asthma subtypes

first: Scott

last: Tebbutt

first: Christopher

last: Ryerson


title: Glaucoma Inducing Retinal Ganglion Cell Degeneration Alters Diurnal Rhythm of Key Molecular Components of the Central Clock and Locomotor Activity in Mice

city: Lübeck

first: Leonardo

last: de Assis

address: Marie Curie

institute: University of Lübeck

summary: Glaucoma is a chronic optic neuropathy characterized by the progressive de- generation of retinal ganglion cells (RGC). These cells play a crucial role in transmitting visual and non-visual information to brain regions, including the su- prachiasmatic nucleus (SCN), responsible for synchronizing biological rhythms. To understand how glaucoma affects circadian rhythm synchronization, we in- vestigated potential changes in the molecular clock machinery in the SCN. We found that the progressive increase in intraocular pressure (IOP) negatively correlated with spontaneous locomotor activity (SLA). Transcriptome analy- sis revealed significant alterations in the SCN of glaucomatous mice, including downregulation of genes associated with circadian rhythms. In fact, we showed a loss of diurnal oscillation in the expression of vasoactive intestinal peptide (Vip), its receptor (Vipr2), and period 1 (Per1) in the SCN of glaucomatous mice. These findings were supported by the 7-h phase shift in the peak expression of arginine vasopressin (Avp) in the SCN of mice with glaucoma. Despite maintaining a 24-h period under both light/dark (LD) and constant dark (DD) conditions, glaucoma- tous mice exhibited altered SLA rhythms, characterized by decreased amplitude. Taken altogether, our findings provide evidence of how glaucoma affects the regulation of the central circadian clock and its consequence on the regulation of circadian rhythms.

overall_design: We used the DBA2J strain as a model of glaucoma development. These mice carry a spontaneous mutation in the GPNMB (transmembrane glycoprotein) gene resulting in high IOP and retinal damage (Anderson et al., 2001). As control mice we used DBA/2J-Gpnmb+SjJ that do not show GPNMB mutation (Jackson Laboratory (Stock No: 007048 and 000671). Experimental procedures were conducted in accordance with national and international ethical guidelines and regulations and approved by the Ethical Committee for Animal Use (ICB/USP, number 8143290819).  Mice were group housed (2 to 5 mice per cage) at 25°C ± 1 in a 12h light: 12h dark (12:12 LD) cycle. The light source consisted of LED lights providing illumination at 400 lux (wavelength range from 420 to 750 nm).  Food and water were available ad libitum throughout the study. The lights were turned on at 7 AM (zeitgeber time - ZT0) and turned off at 7 PM (ZT12). Control and advanced glaucoma (n=3-4) mice were euthanized for SCN microdissection, which was used for RNA isolation and transcriptome analysis. Sections of 600 μm were obtained in a cryostat from the hypothalamic region, where the SCN is located (Bregma -0.22 mm to -0.82 mm), according to the coordinates of Paxinos & Watson atlas (The Mouse Brain in Stereotaxic Coordinates, 2008).

last: Monteiro de Assis

first: Pietra

middle: S

last: Barsanele

first: Maria

last: Moraes


title: Overexpression of Malat1 drives metastasis through inflammatory reprogramming of the tumor microenvironment

city: New Haven

first: Nadya

last: Dimitrova

state: CT

address: 260 Whitney Ave

department: Molecular, Cellular and Developmental Biology

institute: Yale University

summary: Although the expression of some long noncoding RNAs (lncRNAs), including MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), is predictive of metastasis, their impact and mechanism of action remain elusive. Here we use CRISPR activation (CRISPRa) to model MALAT1/Malat1 overexpression in patient-derived lung adenocarcinoma (LUAD) cell lines and in the autochthonous K-ras/p53 LUAD mouse model. The results indicate that Malat1 overexpression alone is sufficient to enable the progression of LUAD to metastatic disease. We show that overexpressed MALAT1/Malat1 enhances cell mobility and promotes the recruitment of pro-tumor macrophages to the tumor microenvironment through paracrine secretion of the CCL2/Ccl2 cytokine. We determine that Ccl2 upregulation results from an increase in global chromatin accessibility upon Malat1 overexpression. Importantly, macrophage depletion and Ccl2 blockade counteracted the effects of Malat1 overexpression. These data demonstrate that a single lncRNA can drive LUAD metastasis through reprogramming of the tumor microenvironment.

overall_design: To examine the impact of Malat1 overexpression on chromatin accessibility in lung adenocarcinoma cells, we performed CRISPRa using luciferase (con) or Malat1 (mA2) targeting dRNAs in a cell line derived from KP mouse lung tumor. We then performed ATAC-seq in two biological replicate cell samples per treatment group.

first: Elena

last: Martinez-Terroba

first: Leah

last: Plasek Hegde

first: Ioannis

last: Chiotakakos

first: Vincent

last: Li

first: Fernando

last: de Miguel

first: Camila

last: Robles-Oteiza

first: Antariksh

last: Tyagi

first: Katerina

last: Politi

first: Jesse

middle: R

last: Zamudio


title: Single Cell RNA seq data for fibroblast paper

city: Pittsburgh

first: Kathryn

last: Torok

state: PA

address: 4401 Penn Ave

department: Pediatric Rheuamatology

institute: University of Pittsburgh

summary:
In this study we compared 14 healthy and 14 LS samples evaluated by 10X Genomics single cell sequencing. Samples were processed and clustered for DEG analysis.
LS = Localized Scleroderma, also called Morphea

overall_design: Investigation on fibroblast expression in certain cell types across all samples

first: Giffin

last: Werner

first: Anwesha

last: Sanyal

first: Emily

last: Mirizio

first: Theresa

last: Hutchins

first: Tracy

last: Tabib

first: Robert

last: Lafyatis

first: Heidi

last: Jacobe


title: Identification of miR-141 as a new regulator of epidermal homeostatis, in cooperation with miR-203

city: Aulnay-sous-Boi

first: Claire

last: Marionnet

address: 1 avenue Eugène Schueller

institute: L'Oréal

summary: MicroRNAs, small endogenous non-coding RNAs, are involved in the regulation of epidermal homeostasis. Among them, miR-203 was the most described and expressed in human epidermis, promoting keratinocyte differentiation by repressing genes involved in proliferation. To identify other miRNAs involved in this process, the miRNomes of normal human keratinocytes cultured in monolayer (2D) or in 3D reconstructed skin were compared. Besides miR-203, mR-141 was one of the most expressed miRNAs in 3D culture and was overexpressed in 3D vs 2D condition, i.e. during keratinocyte differentiation. Functional experiments revealed that, mostly expressed in the basal layer, miR-141 decreased keratinocyte proliferation and clonogenicity while promoting their differentiation. Target prediction algorithm coupled with transcriptomic data of keratinocytes overexpressing miR-141, as well as 3’UTR luciferase assays enabled to evidence CCND2 mRNA as a direct target of miR-141, leading to its down regulation by miR-141 overexpression. Finally, CCND2 silencing decreased keratinocyte proliferation and induced their differentiation, revealing that miR-141 action was mediated by CCND2. MiR-141 features were also compared with miR-203 in parallel experiments. Although miR-141 displayed similar functions to the ones of miR-203, it exhibited different localization and targets, suggesting a joint participation of miR-141 and miR-203 to engage and maintain keratinocyte towards differentiation, respectively.

overall_design: To discover microRNAs which could be involved in the proliferation / differentiation balance of epidermal keratinocytes, we determined differentially expressed miRNA between normal primary human keratinocytes (NHK) grown in 2D culture (monolayer, n=3 biological replicates) and NHK grown in 3D culture (reconstructed epidermis, 3 biological replicates).


title: UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation

city: Grand Rapids

first: Scott

last: Rothbart

state: MI

address: 333 Bostwick Avenue NE

institute: Van Andel Research Institute

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


title: UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation [BiSulfite-seq]

summary: The E3 ubiquitin ligase UHRF1 is an established cofactor for replication-coupled DNA methylation inheritance. The current model posits a stepwise pathway in which UHRF1 engages nucleosomes through histone and DNA binding. This multivalent nucleosome readout directs UHRF1 ubiquitin ligase activity toward several lysines on N-terminal tails of histone H3, binding sites for DNMT1 through tandem ubiquitin interacting motifs (UIM1 and UIM2). While this elegant chromatin regulatory mechanism is now taking shape, the extent to which ubiquitin signaling through UHRF1 contributes to DNMT1-dependent DNA methylation maintenance genome-wide is not known. Here, we present comparative, genome-scale analysis of DNA methylation inheritance in human colon cancer cells with compromised UHRF1 ubiquitin ligase and DNMT1 ubiquitin reading activities. We reveal that DNA methylation maintenance in contexts of low CpG density is particularly vulnerable to disruption of UHRF1 ubiquitin ligase activity. This low CpG density hypomethylation signature, a common feature of partially methylated domains (PMDs) in human cancers, is also seen when DNMT1 ubiquitin reading activity through UIM1 is disrupted. Notably, disrupting UIM2 function affects DNA methylation inheritance to a similar extent as a catalytically dead form of the enzyme. Collectively, these studies demonstrate that DNMT1-dependent DNA methylation inheritance is a ubiquitin-regulated process and suggest that a disrupted UHRF1-DNMT1 ubiquitin signaling axis contributes to the development of PMDs in human cancers.

overall_design: Genomic DNA was isolated from HCT116 cells lines modified with stable integration of a doxycycline-inducible shRNA targeting the 3’UTR of either UHRF1 or DNMT1. Bulk (shDNMT1 = 2) and clonally expanded (shUHRF1 = 3 clones) cell populations were used. In all cases, Baseline (NoDox) samples were grown in culture for 2 weeks in the absence of doxycycline, and serve as the baseline methylome control for Knockdown and Recovery samples. Knockdown samples were treated with doxycycline (10 ng/mL) for two weeks to induce expression of the shRNA. Recovery samples were treated with doxycycline for two weeks (to induce knockdown of the target protein), and then doxycycline was removed for an additional two weeks to measure recovery methylation dynamics with re-expression of the either UHRF1 or DNMT1.

first: Rochelle

last: Tiedeman

first: Joel

last: Hrit

first: Qian

last: Du

first: Ashley

last: Wiseman

first: Hope

middle: E

last: Eden

first: Bradley

last: Dickson

first: Xiangqian

last: Kong

first: Alison

last: Chomiak

last: Vaughan

first: Jakob

last: Hebert

first: Yael

last: David

first: Wanding

last: Zhou

first: Stephen

last: Baylin

first: Peter

last: Jones

first: Susan

last: Clark


title: UHRF1 ubiquitin ligase activity supports the maintenance of low-density CpG methylation [ChIP-seq]

overall_design: Chromatin immunoprecipitation for H3K9me3 and H3K27me3 was perfromed on HCT116 shUHRF1 Clone 3 and Clone 6 for the Baseline distribution of these marks.

first: Robert


title: RNA-seq analysis of target genes  in Noonan syndrome model mice  with cardiac hypertrophy

city: Sendai

first: Taiki

last: Abe

address: Seiryo-machi 1-1, Aobaku

institute: Tohoku University

summary: Leucine-zipper-like transcriptional regulator 1 (LZTR1) is a member of the BTB-Kelch superfamily, which regulates the RAS proteostasis. Autosomal dominant (AD) mutations in LZTR1 have been identified in patients with Noonan syndrome (NS), a congenital anomaly syndrome. However, it remains unclear whether LZTR1 regulates the proteostasis of the RAS subfamily molecules and activate MAPK signaling pathway in heart.  To investigate the impact of the LZTR1 mutant on gene expression, we performed RNA-seq analyses using LVs.

overall_design: To investigate the impact of the LZTR1 on gene expression, we performed RNA-seq analyses using LVs from wild-type and mutant mice (n = 3).

first: Yoko

last: Aoki