title: Single Cell Transcriptomics-Informed Induced Pluripotent Stem Cells Differentiation to Tenogenic Lineag

first: Dmitriy

last: Sheyn

address: 127 S. San Vicente Blvd

institute: Cedars-Sinai Medical Center

summary: During vertebrate embryogenesis, axial tendons develop from the paraxial mesoderm and differentiate through specific developmental stages to reach the syndetome stage. While the main roles of signaling pathways in the earlier stages of the differentiation have been well established, pathway nuances in syndetome specification from the sclerotome stage have yet to be explored. Here, we show stepwise differentiation of human iPSCs to the syndetome stage using chemically defined media and small molecules that were modified based on single cell RNA-sequencing and pathway analysis. We identified a significant population of branching off-target cells differentiating towards a neural phenotype overexpressing Wnt. Further transcriptomics post-addition of a WNT inhibitor at the somite stage and onwards revealed not only total removal of the neural off-target cells, but also increased syndetome induction efficiency. Fine-tuning tendon differentiation in vitro is essential to address the current challenges in developing a successful cell-based tendon therapy.

overall_design: In vitro differentiation

first: Papalamprou

last: Angela

first: Yu

last: Victoria

first: Jiang

last: Wensen

first: Sheyn

last: Julia

first: Stefanovic

last: Tina

first: Chen

last: Angel

first: Castaneda

last: Chloe

first: Chavez

last: Melissa

last: Dmitriy


title: Comprehensive curation and validation of genomic datasets for chestnut [2]

status: Public on Dec 24 2024

city: Beijing

first: Yi

last: Zheng

address: No. 7, Beinong Road, Huilongguan Town, Changping District

country: China

institute: Beijing University of Agriculture

type: Expression profiling by high throughput sequencing

summary: The Chinese chestnut (Castanea mollissima) stands out as a plant with significant ecological and economic value, excellent nutritional quality and natural resistance to pests and diseases. Recent strides in high-throughput techniques have enabled the continuous accumulation of genomic data on chestnuts, presenting a promising future for genetic research and advancing traits in this species. To facilitate the accessibility and utility of this data, we have curated and validated a collection of genomic datasets for eight Castanea species, 213 RNA-Seq samples, and 348 resequencing samples. These datasets are publicly available on figshare, providing a robust resource for researchers studying Castanea genetics, functional genomics, and evolutionary biology. Additionally, the Castanea Genome Database (CGD, http://castaneadb.net) serves as a complementary platform, offering advanced data mining and analysis tools, including BLAST, Batch Query, GO/KEGG Enrichment Analysis, and Synteny Viewer, to enhance the usability of the curated datasets.

overall_design:
This study analyzes gene expression differences in embryos, somatic embryos, callus, roots, and mixed samples using RNA sequencing, in addition to the samples provided in the table, including 181 samples obtained from third-party sources. RNA-Seq reads were processed with FastQC (v0.11.9), trimmed with Trimmomatic, aligned using STAR (v2.7.10b), and normalized to FPKM values.
Somatic embryo and callus dataset.
In addition, third-party reanalysis of RNA-Seq samples from SRA database (see TABLE below).


title: Comprehensive curation and validation of genomic datasets for chestnut [3]

overall_design:
This study analyzes gene expression differences in embryos, somatic embryos, callus, roots, and mixed samples using RNA sequencing, in addition to the samples provided in the table, including 181 samples obtained from third-party sources. RNA-Seq reads were processed with FastQC (v0.11.9), trimmed with Trimmomatic, aligned using STAR (v2.7.10b), and normalized to FPKM values.
Root dataset.
In addition, third-party reanalysis of RNA-Seq samples from SRA database (see TABLE below).


title: Sex-specific transcriptome dynamics of Anopheles gambiae during embryonic development [CUT&Tag]

status: Public on Dec 20 2024

city: Mainz

first: Frank

last: Rühle

address: Ackermannweg 4

department: Bioinformatics Core Facility

country: Germany

institute: Institute of Molecular Biology (IMB)

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Malaria-transmitting mosquitoes are extremely sexually dimorphic in their anatomy and behaviour. Sex-specific gene expression in Anopheles gambiae is well-studied in adult stages, but its onset during embryogenesis, apart from sex-determination factors like Yob, remains largely unknown. Here, we report a comprehensive single-embryo transcriptome atlas of A. gambiae males and females to understand the earliest stages of establishing the sex-specific expression networks. Our dataset reveals embryonic RNA isoform diversity including a global shift towards distal alternative polyadenylation (APA) events sites during the maternal-to-zygotic genome transition. Sex-biased gene expression and alternative splicing are limited during embryogenesis, with most sex-specific patterns emerging post-embryonically. X chromosome dosage compensation is established shortly after zygotic genome activation concomitant with direct binding of the master regulator protein SOA to X-linked promoters. Unlike the dosage compensation regulators in Drosophila or mammals, SOA operates in a one-step fashion, directly binding CA-rich promoter motifs without prioritizing certain gene groups over others. We propose that the Anopheles dosage compensation system represents an evolutionary endpoint of a gene-by-gene regulatory mechanism that evolved to a chromosome-wide scale.

overall_design: CUT&Tag libraries generated from WT and SOA-KI pooled embryos are analyzed. Each group has two biological replicates. The IgG antibody is a negative control for unspecific binding of the antibodies.

first: Agata

middle: I

last: Kalita

first: Eric

last: Marois

first: Claudia

last: Keller Valsecchi


title: Comprehensive curation and validation of genomic datasets for chestnut [1]

overall_design:
This study analyzes gene expression differences in embryos, somatic embryos, callus, roots, and mixed samples using RNA sequencing, in addition to the samples provided in the table, including 181 samples obtained from third-party sources. RNA-Seq reads were processed with FastQC (v0.11.9), trimmed with Trimmomatic, aligned using STAR (v2.7.10b), and normalized to FPKM values.
Embryo dataset.
In addition, third-party reanalysis of RNA-Seq samples from SRA database (see TABLE below).


title: Sex-specific transcriptome dynamics of Anopheles gambiae during embryonic development [RNA-seq]

overall_design: RNA sequencing of single embryos of predetermined sex (males and females) and genotype (wild-type and SOA-KI loss-of-function allele) collected at 18 hours of embryonic development


title: Let-7 miRNAs and isoforms of miR-203a involve in regulation of colon cancer metastasis by receptor CD44

status: Public on Dec 19 2024

city: Moscow

first: Anton

last: Zhiyanov

address: 20 Myasnitskaya ulitsa

department: Faculty of Biology and Biotechnology

country: Russia

institute: HSE University

type: Non-coding RNA profiling by high throughput sequencing

summary: Metastasis formation poses a significant challenge to oncologists, as it severely limits the survival of colorectal cancer (CRC) patients. Recently, we demonstrated that CD44 promotes spontaneous distant metastasis in a CRC xenograft model. The depletion of CD44 was associated with reduction in hypoxia, EMT, as well as improved mitochondrial metabolism in primary tumor.Collectively, these effects decreased the metastatic potential of the CRC xenograft tumors under investigation. In this study we explore the molecular mechanisms by which CD44 knockdown (kd) leads to such substantial changes of tumor properties.Methods: Using miRNA-Seq data combined with bioinformatic analysis, we investigated the role of miRNA expression changes in the metastasis prevention observed with CD44 kd.Results: Among the differentially expressed miRNAs, three members of Let-7 family (let-7a-5p, let-7b-5p, and let-7c-5p), two isoforms of miR-203a (canonical miR-203a-3p and its +1 5'-isoform), miR-101-3p, miR-200b-3p|+1 5'-isoform, miR-125a-5p, and miR-185-5p were identified as potentially involved in regulating CD44-mediated metastasis. Gene set analysis of differentially expressed mRNA targets of these miRNAs, along with an examination of key regulators driving the observed changes in both mRNA and miRNA expression profiles, suggests that the CD44-STAT3- Let-7 miRNA axis as one of the most relevant in regulation of colon cancer metastasis via the CD44 receptor.Discussion: Our findings suggest a regulatory relationship between CD44, Let-7 miRNAs, and STAT3 in HT-29 tumors. Additionally, we propose the potential involvement of both isoforms of miR-203a (canonical and its +1 5'-isoform) in this regulatory network, and suggest a role for miR-101-3p and miR-125a-5p in metastasis regulation through CD44 kd.

overall_design: Two types of cells were injected into SCID mice: HT-29 cell lines with shRNA-mediated knockdown of CD44 (shCD44) and control HT-29 cell lines transduced with shRNA against firefly luciferase (shLUC). miRNA was isolated from xenograft tumor tissues and analyzed using RNA-seq.

first: Diana

last: Maltseva

first: Tobias

last: Lange

first: Alexander

last: Tonevitsky


title: RNA-sequencing of mammary epithelial cells from pubertal mice following enrichment of mammary ductal tissue or terminal end buds (TEBs).

city: Parkville

first: Yunshun

last: Chen

state: VIC

address: 1G Royal Parade

department: ACRF Cancer Biology and Stem Cells

country: Australia

institute: Walter and Eliza Hall Institute of Medical Research

summary: Transcriptomic profilifing of the mammary epithelial cells in pubertal ducts and TEBs. Cells were enriched from Elf5-GFP and PR-cre/YFP mouse models to obtain basal, luminal GFP/YFP+ and luminal GFP/FYP-.

overall_design: Mammary luminal epithelial cell subpopulations (Lin- EpCAM+ Cd29hi basal, Lin- EpCAM+ Cd 29lo Elf5-GFP+/PR-cre/YFP+ luminal GFP/YFP+, Lin- EpCAM+ Cd29lo Elf5-GFP-/PR-cre/YFP- luminal GFP/YFP- cells) were isolated from pubertal mammary tissue microdissected for ductal tissue and TEBs and sorted by FACS then subjected to bulk RNA-sequencing (n=2).

first: Michael

last: Milevskiy

first: Jane

last: Visvader


title: Hormone-responsive progenitors have a unique identity and migrate during mammary gland development

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

overall_design: Refer to individual Series


title: RNA-sequencing of luminal epithelial cells from adult murine mammary glands.

summary: Transcriptomic profilifing of the mammary luminal compartment using a refined purification strategy. This strategy uses constitutive expression of Pgr and the progenitor marker CD14.

overall_design: Mammary luminal epithelial cell subpopulations (Lin- CD14+ PR-cre/YFP- HR-LP, Lin- CD14+ PR-cre/YFP+ HR+ LP, Lin- CD14- PR-cre/YFP- ML cells) were isolated from adult mammary tissue by FACS and subjected to bulk RNA-sequencing (n=3).


title: Sex-specific transcriptome dynamics of Anopheles gambiae during embryonic development


title: RNA-seq of IMR90 cells during OIS under TPR depletion

city: Edinburgh

first: Yatendra

last: Kumar

email: yaten2020@gmail.com, ykumar@ed.ac.uk

address: Crewe Road south

department: MRC HGU

country: United Kingdom

institute: MRC Human Genetics Unit, The University Of Edinburgh

summary: During oncogene-induced senescence there are striking changes in the structure of the nucleus and the organisation of heterochromatin. This is accompanied by activation of a pro-inflammatory gene expression programme – the senescence associated secretory phenotype (SASP) – driven by transcription factors such as NF-κB. Here we show that TPR, a protein of the nuclear pore complex basket, is required for the very early activation of NF- κB signalling during the stress-response phase of oncogene-induced senescence. This is prior to activation of the SASP and occurs without affecting NF-κB nuclear import. We show that TPR is required for the activation of TBK1 signalling at these early stages of senescence and we link this to the formation of heterochromatin-enriched cytoplasmic chromatin fragments thought to bleb off from the nuclear periphery. These cytoplasmic chromatin fragments appear to lack nuclear pore components. Our data suggest that TPR at the nuclear pore is involved in the loss of structural integrity of the nuclear periphery during senescence. We propose that this acts as a trigger for activation of cytoplasmic nucleic acid sensing, NF-κB signalling, and activation of the SASP, during senescence.

overall_design: RNA-seq profiling of IMR90 Cells at day 3 and day 8 of RAS mediated senescence.

first: Bethany

last: Bartlett


title: Identification of novel prognostic biomarkers for thyroid cancer by integrated transcriptome analysis of metastasis-associated genes

city: Riyadh

first: yufei

last: shi

address: PO Box 3354

country: Saudi Arabia

institute: King Faisal Specialist Hospital and Research Centre

summary: Distant metastasis (DM) is the most important prognostic factor affecting overall survival (OS) of thyroid cancer. The current study aimed to discover prognostic biomarkers to predict thyroid cancer survival, particularly PTC, the most common subtype of thyroid cancer. Four RNA-seq datasets of experimental lung metastasis from 4 transgenic mouse models of PTC, follicular thyroid cancer (FTC), poorly-differentiated thyroid cancer (PDTC), and anaplastic thyroid cancer (ATC) were integrated to screen for candidate genes involved in DM. TCGA-THCA dataset were used to validate the candidate genes. A total of 105 up-regulated and 25 down-regulated differentially expressed genes (DEGs) were identified to be present in all 4 datasets. Regulation of cytokine production, inflammation, immune checkpoint regulation, MAPK/ERK cascade were major enriched pathways in metastatic tumor cells. We identified 7 genes whose overexpression was present in 63 of 498 PTC samples (13%) and was associated with poor OS (p<0.01). Clinically, the 7–gene expression signature was associated with older age at the diagnosis, late stage of tumor, tall-cell variant, and higher aneuploidy and hypoxia score. Mutation load was increased in samples with 7–gene expression signature: 26 samples had more than one driver mutations (47%, 26/55). Deep deletions in other chromosomal locus were frequently found in samples with BRAFV600E point mutations.  In contrast, only 7% samples without the 7-gene expression signature had more than one driver mutations (24/243). Increased copy number variants (CNVs) were also observed in metastatic as compared to primary tumor cells such as large deletions and duplications. We conclude that the 7–gene expression signature is associated with poor prognosis and chromosomal instability. It may be a useful biomarker for risk stratification for DM and help decision-making in initial surgical recommendations.

overall_design: Comparative gene expression profiling analysis of RNA-seq datasets between primary and metastatic mouse thyroid cancer cell lines.

first: Bushra

last: Alnwisser

first: Yufei

last: Shi


title: Affymetrix OncoScan SNP arrays for profiling somatic copy-number alterations in primary renal diffuse large B cell lymphoma

status: Public on Dec 16 2024

city: Lübeck

first: Axel

last: Künstner

address: Ratzeburger Allee 160

department: LIED

institute: University of Lübeck

summary: Molecular profiling of primary renal diffuse large B cell lymphoma unravels a proclivity for immune-privileged organ-tropism. Here, we used Affymetrix OncoScan CNV arrays to characterise somatic copy number variations in 29 prDLBCL cases.

overall_design: Total DNA was extracted and prepared for OncoSan CNV arrays from 29 FFPE prDLBCL cases following the manufacturer protocol.

first: Künstner

last: Axel

first: Gebauer

last: Niklas

first: Busch

last: Hauke


title: Single-cell transcriptome analysis reveals the molecular mechanisms underlying age-related changes in human testis

status: Public on Dec 13 2024

first: Jingtao

last: Guo

address: Beichen West Road

institute: Chinese Academy of Sciences

summary: The decline in reproductive health observed in aging males is a well-documented phenomenon. However, the molecular and cellular mechanisms underlying testis aging remain poorly understood. In an effort to gain deeper insights into this process, we conducted a comprehensive analysis of 99,130 single-cell transcriptomes derived from testicular cells spanning an age range of 21 to 69 years. Our machine learning analysis revealed a more pronounced response to aging in somatic cells compared to germ cells. Our analysis identified two distinct waves of aging-related changes within testicular cell clusters. Notably, age-related alterations in somatic cells exhibited consistent and specific responses to the aging process. These age-related changes were found to involve immune response pathways, while cell-specific pathways, such as the response to insulin in Sertoli cells at 50s, and extracellular matrix and NOTCH signaling in TPC cells at 30s, were also observed. Of particular significance was the substantial decrease in the frequency of haploid germ cells in individuals with higher BMI across different age groups, suggesting a synergistic interplay between BMI and age

overall_design: We profiled the single-cell transcriptomes of the testes of 25 adults and combined them with our previous single-cell transcriptomes of the testes of 12 adults, resulting in a dataset covering samples from different age groups ranging from 17 to 67 years old. We studied the transcriptional changes over time in each major testicular cell type and discovered a set of molecular mechanisms underlying human testicular aging.

first: Xichen

last: Nie

first: Bradley

last: Cairns


title: ERBB2 R599C variant is associated with left ventricular outflow tract obstruction defects in human

status: Public on Dec 14 2024

city: Porvoo

first: Sabina

middle: Henrika

last: Selenius

address: Mika Waltarin katu 49

department: Stem Cells and Metabolism Research Program (STEMM), New Children's Hospital Pediatric Research Center

country: Finland

institute: Univeristy of Helsinki, Helsinki University Hospital

summary: Non-syndromic congenital heart defects (CHD) are occasionally familial and left ventricular out flow tract obstruction (LVOTO) defects are among the subtypes with the highest hereditability. The aim of this study was to evaluate the pathogenicity of a heterozygous ERBB2 variant R599C identified in three families with LVOTO defects.

overall_design: To examine the difference in expressed genes between ErBB2-mutation cells and wild types we created four hIPS-cell lines, which were differentiated into endothelial cells.


title: The Heterogenicity of Virtual Memory CD8 T Cells in Relation to Their Cytokine Dependent Development

status: Public on Dec 12 2024

city: Seoul

first: Jaeil

last: Lee

address: 103 Daehak-ro, Jongno-gu

country: South Korea

institute: Seoul National University

summary: The development of virtual memory CD8 T cells is dependent on IL-4, type I interferon, and IL-15. However, it remains unclear whether these cytokines individually contribute to the generation of specific subsets of virtual memory CD8 T cells. In this study, virtual memory CD8 T cells were categorized into four subsets based on Ly6C and Sca-1 expression, and their development was examined using knock-out mice lacking IFNAR1, IL-4, or IL-15Rα. Notably, both Ly6C+ Sca-1+ and Ly6C- Sca-1+ subsets were significantly reduced in the spleen of IFNAR1 knock-out mice, while the proportion of Ly6C+ Sca-1- VM CD8 T cells was reduced in IL-4-deficient mice. In IL-15Rα knock-out mice, both the Ly6C+ Sca-1- and Ly6C- Sca-1- subsets were significantly reduced. Bulk RNA sequencing analysis revealed distinct gene expression patterns in naïve cells, true memory cells, and four virtual memory cells subsets. Specifically, Ly6C+ subsets were enriched with IL-15 signal-related genes, whereas Ly6C- subsets and true memory cells were enriched for cell cycle-related genes. Functionally, the Ly6C+ subsets exhibited higher production of IFN-γ, TNF-α, and perforin compared to the Ly6C- subsets. Overall, this study demonstrates the heterogeneity of virtual memory CD8 T cells and highlights the cytokine-dependent nature for their development.

overall_design: Comparison of gene expression patterns of naïve T cells, true memory T cells, and four virtual memory T cell subsets (Ly6c+ Sca-1-, Ly6c+ Sca-1+, Ly6c- Sca-1+, and Ly6c- Sca-1-) by bulk RNA sequencing analysis

first: Hi Jung

last: Park

first: Sung Min

last: Choi

first: Eun A

first: Hyun Ji

last: Boo

first: Jae Il

first: Kyeong Cheon

last: Jung


title: Impaired T cell and neoantigen retention in time serial analysis of lung cancer patients treated with TIL cell therapy

city: Tampa

first: Dongliang

last: Du

address: 111 Magnolia

country: USA

institute: Moffitt

summary: We mapped TIL clones into individual transcriptomes and found that neoantigen-reactive clonotypes expressed a dysfunctional program and lacked stem-like features among patients who lacked clinical benefit.

overall_design: Isolated and cultured tumor infiltrating T cells from NSCLC were collected and analyzed using single cell transcriptome sequencing paired with single cell TCR sequencing

first: Ben

last: Creelan

first: Chao

last: Wang

first: Xiaoqing

last: Yu

first: Jamie

middle: T

last: Teer

first: Cheryl

last: Cox

first: Ana

middle: M

last: Landin

first: Sean

middle: J

last: Yoder

first: Shari

middle: A

last: Pilon-Thomas

first: Jose

middle: R

last: Conejo-Garcia

first: Scott

last: Antonia

first: Eric

middle: B

last: Haura


title: Discovery of NANOG enhancers and their essential roles in self-renewal and differentiation in human embryonic stem cells

city: New York

first: Hyein

middle: Sophia

last: Cho

state: NY

address: 430 E 67th St

department: Developmental Biology

institute: Memorial Sloan Kettering Cancer Center

summary: Human embryonic stem cells (hESCs) are an important system to study development and regeneration. NANOG is a core regulator in hESCs, but it is transcriptionally regulated is not fully understood. In this study, we report the discovery of two NANOG enhancers from a CRISPR interference enhancer screen in hESCs. These enhancers are essential to hESCs, and deletion of a single copy of either enhancers significantly reduced NANOG expression. In addition, hESCs with heterozygous deletion of either enhancers had compromised self-renewal and increased tendency to differentiate. Interestingly, these two NANOG enhancers are involved in a tandem duplication event, but the duplicated counterparts do not regulate NANOG. This work expands our knowledge of functional enhancers in hESCs, and highlights the sensitivity of the hESC state to the dosage of a core regulator.

overall_design:
Bulk RNA-seq of WT and NANOG enhancer knockout on embryonic stem cell and neuroectoderm

***************************************************************
The table below lists GEO accessions reused/reanalyzed for this study.
***************************************************************

first: Jielin

last: Yan

middle: S

first: Danwei

last: Huangfu


title: BCL11A is required for normal erythropoiesis [CUT&Run]

status: Public on Dec 09 2024

city: Memphis

first: Lance

last: Palmer

state: TN - Tennessee

address: 262 Danny Thomas Pl

department: Hematology

institute: St. Jude Children's Research Hospital

summary: Genetic depletion of the transcriptional repressor BCL11A in red blood cell precursors alleviates b-hemoglobinopathies by inducing the fetal g-globin genes. However, several additional erythroid genes are regulated by BCL11A and the effects of its deficiency on erythropoiesis are insufficiently described. We discovered that Cas9 disruption of the BCL11A intron 2 erythroid enhancer in CD34+ hematopoietic stem and progenitor cells (HSPCs) according to a clinically approved strategy caused impaired expansion and increased apoptosis of erythroid precursors in vitro and in vivo. Mutant colony-forming unit erythroid (CFU-e), proerythroblasts, and basophilic erythroblasts exhibited dysregulation of 94 genes (> 2-fold change, FDR < 0.05). 25 of which are likely direct targets of BCL11A. Differentially expressed genes were associated with numerous biological pathways that impact cell expansion and survival. Our findings show that BCL11A regulates additional aspects of erythropoiesis beyond g-globin gene repression.

overall_design: CUT&RUN assay was performed to identify chromatin occupancy of BCL11A.  Three samples with BCL11A antibody and 3 samples with control IgG antibody were analyzed.

first: Yoonjeong

last: Jang

first: Ruopeng

last: Feng

middle: E

first: Yong

last: Cheng

first: Jonathan

last: Yen

first: Mitchell

last: Weiss


title: BCL11A is required for normal erythropoiesis [RNA-seq_Sorted]

overall_design: Comparative gene expression profiling analysis of RNA-seq data forCD34+ derived erytroid cells at different stage (CFU-E, ProE and Baso-E) after edited by CD33 or BCL11A enhancer gRNA.


title: Comparison of IL10-eGFP+ cells with IL10-eGFP- CD4+ T cells induced by in vivo immunisation

city: Birmingham

first: David

last: Bending

address: IBR

department: Institute of Immunology and Immunotherapy

institute: University of Birmingham

summary: Here we perform QuantSeq 3' mRNA sequencing of RNA extracted from flow sorted splenic CD4+ Il10-eGFP+ or CD4+Il10eGFP- T cells from Tg4 Nr4a3-Tocky Il10-eGFP mice 24 hours after immunisation with 4 mg/kg of [4Y]-MBP. Our aim was to confirm the Tr1 cell phenotype within the Il10-eGFP+ T cell subset

overall_design: Tg4 Nr4a3-Tocky Il10-GFP mice were immunized through subcutaneous injection of 4 mg/kg [4Y] MBP peptide PBS into the flank. 24 hrs later mice were then euthanised and CD4+ Nr4a3-Timer+Il10-eGFP+ and CD4+Nr4a3-Timer+Il10-eGFP- T cells isolated and RNA extracted for 3' mRNA sequencing. 2 biological replicate mice were obtained for the 2 experimental groups. Each sample was run on 4 lanes, resulting in 48 FASTQ files which were then merged into single FASTQ files.

first: Bending

last: David


title: BCL11A is required for normal erythropoiesis [ATAC-seq]

overall_design: ATAC-seq experiments were performed inCD34+ derived erythroidblast cells treated with CD33 sgRNA and BCL11A enhancer sgRNA


title: mTOR Signaling Regulates Multiple Metabolic Pathways in Human Lung Fibroblasts After TGF-β and in Pulmonary Fibrosis

city: Chicago

first: Kun Woo

last: Shin

state: IL

address: 5841 S Maryland Ave

department: Medicine

institute: The University of Chicago

summary: We investigated the effects of mTOR and ATF4 on gene expression in human lung fibroblasts using RNA-sequencing. We found both ATF4 and mTOR regulate expression of genes that are involved in controlling amino acid synthesis and transportation. mTOR additionally regulates genes that are involved in glycolysis and oxidative phosphorylation.

overall_design: Comparative gene expression profiling analysis of RNA-seq data from Normal Human Lung Fibroblasts (HLFs) under TGF-beta treatment. ATF4 was inhibited by siRNA knock down, and mTOR was inhibited with Torin1 treatment.

first: Mutlu

last: Gokhan

first: Hamanaka

last: Robert

first: Shin

last: Kun Woo

first: Cetin-Atalay

last: Rengul

first: O’Leary

last: Erin


title: Mutual suppression between mutations in the Dictyostelium greenbeard pathway restores wild-type development

status: Public on Dec 06 2024

city: Houston

first: Gad

last: Shaulsky

state: TX

address: One Baylor Plaza

department: Molecular and Human Genetics

institute: Baylor College of Medicine

summary: The greenbeard allorecognition pathway of Dictyostelium discoideum is mediated by the polymorphic transmembrane proteins TgrB1 and TgrC1. TgrC1 is a ligand and TgrB1 is its receptor, and together they mediate allorecognition with accompanying altruism during the transition from unicellular to multicellular development. A genetic screen for suppressors of developmental defects in the non-compatible allotype TgrB1-C1 pathway revealed activating mutations in tgrB1 and inactivating mutations in rapgapB which encodes a regulator of the small GTPase protein RapA. Inactivation of either tgrB1, tgrC1, or rapgapB leads to developmental defects. However, surprisingly, the respective double-mutant strains rapgapB-tgrB1- and rapgapB-tgrC1- develop well and produce wild-type-like fruiting bodies. This mutual suppression could result from the induction of an alternative developmental pathway or restoration of the wild-type pathway, but morphological analyses alone could not resolve this question. Here we show that the mutual suppressors restore wild-type development. We used RNA-sequencing analyses to compare the transcriptomes of the wild type to those of six mutant strains and found that the single-gene mutations resulted in attenuated transcriptome progression over developmental time, whereas the double-gene mutation strains exhibited near wild-type transcriptomes. We also analyzed a strain that carries an activated tgrB1 allele in the wild type and found evidence for interactions between the activated and the resident alleles. Our findings suggest that tgrB1, tgrC1, and rapgapB are involved in a regulatory feedback pathway in which rapgapB negatively regulates tgrB1 and tgrC1 whereas tgrB1 and tgrC1 positively regulate rapgapB. These findings suggest that the D. discoideum greenbeard pathway interfaces with the central RapGAPB-RapA regulatory pathway, providing molecular insight into a mutual suppression mechanism in which combining two deleterious mutations leads to the restoration of wild-type behavior.

overall_design: Total 108 samples were used for this analysis. 6 developmental timepoints of wild-type AX4 and 5 mutant strains: tgrB1-, tgrC1-, rapgapB-, rapgapB-tgrB1-, and AX4 tgrB1L846F, were analyzed with 3 biological replication.

first: Mariko

last: Katoh-Kurasawa

first: Lena

last: Trnovec

first: Blaz

last: Zupan


title: BCL11A is required for normal erythropoiesis [RNA-seq_NBSGW]

first: Lance

last: Palmer

overall_design: RNA-seq experiments were performed in human CD34+ derived CD235a+ erythroid cells treated with BCL11A enhancer sgRNA from Xenotransplantation

first: Yoonjeong

last: Jang


title: Sample multiplexed scRNA-seq using Nanocoding method for sample barcode labeling

city: Urbana

first: Yujun

last: Feng

address: 1304 W Green St.

department: Materials Science and Engineering

institute: University of Illinois, Urbana-Champaign

summary: Sample multiplexed scRNA-seq is a promising strategy to overcome current barriers in high cost and potential technical variations by multiple scRNA-seq tests. In this study, we developed a highly efficienct novel sample barcode labeling method using DNA-encoded Lipid Nanoparticles ('Nanocoding') that could label cells with minimal dependence on their type or sample conditions. This method provids a roubust and general protocol for sample barcoding and multiplexing in scRNA-seq. We demonstrated the performance of Nanocoding through three scRNA-seq studies, which include:  1. mouse spleen cells mix (one dataset including 6 mouse spleen tissues samples); 2. HeLa-mouse Stromal Vascular Fraction(SVF) cells mix (one dataset containing mixed HeLa cell and SVF cell); 3. Aged-Young SVF cells mix (one dataset containing two SVF samples) tests. These studies showcased the biomodal distribution of barcode counts in different models with high signal-to-background ratio, as well as pan-cell labeling activity for efficient and accurate sample-multiplexing. By using Nanocoding, we profiled obsity and age related change in lipid metabolism associated genes or inflammatory related features, in various cell types from spleen or adipose tissues.

overall_design: Male C57BL/6J Diet-Induced-Obese (DIO, N=3) and age-matched C57BL/6J control lean mice (8 month old, N=3) were used for speen collection and following multiplexed scRNA-seq. Each sample is labeled with a unique sample barcode using Nanocoding before mixing. Male C57BL/6J DIO mice that are 8 month old are used for visceral adipose tissue (VAT) collections and SVF cell collection in HeLa-SVF cell mixing test. Male C57BL/6J DIO mice that are 11 month old (N=3) or 4 month old (N=3) are used for visceral adipose tissue (VAT) collections and SVF cell collation in Young-Aged SVF cell mix test.

first: Fangxiu

last: Xu

first: Chris

last: Wright

first: Andrew

last: Smith


title: BCL11A is required for normal erythropoiesis

status: Public on Dec 08 2024

overall_design: Refer to individual Series


title: Piezo1 mediated mechanotransduction modulates macrophages proliferation during bone remodeling via PI3K-AKT-Ccnd1 axis

city: Nanjing

first: Bin

last: Yan

address: 140Hanzhong Road

institute: Nanjing Medical University

summary: Macrophages play a pivotal role in mechanical force-induced inflammatory bone remodeling. Yet, how macrophages perceive mechanical stimuli and thereby modulate biological behaviors remain elusive. The orthodontic tooth movement (OTM) model was established to access the role of Piezo1 in modulating macrophage response upon mechanical stimuli. The potential functions and molecule mechanisms of Piezo1 were explored by bone marrow-derived macrophages (BMDMs) using mechanical stretch system, western blotting, immunofluorescence, flow cytometry and RNA sequencing. We first found macrophage proliferative phenotype enhanced at the later stage of force application. The biological variation mediated by mechanical force could be suppressed by Piezo1 inhibition. Furthermore, Piezo1 activated PI3K-AKT signaling was closely associated with macrophage proliferation upon mechanical stimuli.  Additionally, Ccnd1 was authenticated as a critical downstream factor of PI3K-AKT signaling and conditional ablation of Ccnd1 in macrophages inhibited macrophage proliferation in mechanical force-induced bone remodeling procedure.

overall_design: To probe the mechanisms by which Piezo1 promotes macrophage proliferation upon mechanical stimuli, RNA-seq experiments was conducted on BMDMs transfected with si-NC and si-Piezo1 and exposed to mechanical stretch

first: Hao


title: Low-density Lipoprotein Regulates Intestinal Stem Cell Homeostasis via PPAR Pathway

status: Public on Dec 05 2024

first: Ruicheng

last: Shi

address: 2001 South Lincoln Avenue, room 36122

department: Comparative Biosciences

institute: University of Illinois at Urbana-Champaign

summary: Epidemiological studies have highlighted a strong association between hyperlipidemia and an increased risk of cancer in the gut. Intestinal stem cells (ISCs) have been demonstrated as the cells of origin for tumorigenesis in the gut. However, the impact of hyperlipidemia on ISC homeostasis remains unclear. Here, we show that hyperlipidemia induced by low-density lipoprotein receptor (Ldlr) deficiency enhances ISC proliferation in vivo. Additionally, LDL treatment impairs organoid survival but increases ISC stemness ex vivo, as evidenced by the formation of poorly differentiated spheroid and higher ISC self-renewal capacity. Mechanistically, LDL treatment activates PPAR pathways, and pharmacological inhibition of PPAR and its downstream targets, including CPT1A and PDK4, mitigates the effect of LDL on ISCs. Furthermore, although Ldlr-/- intestines exhibit increased susceptibility to irradiation-induced crypt damage, they regenerate completely within a similar timeframe as controls. These findings demonstrate that hyperlipidemia modulates ISC homeostasis, providing new insights into the mechanism linking hyperlipidemia with tumorigenesis in the gut.

overall_design: To evaluate the effect of LDL in intestineal stem cell ex vivo,  duodenum and jejunum crypts from 3 mice were isolated and cultured as organoids for 5 days. Organoids were either treated with low density lipoprotein (LDL) or PBS control. Three biological replicates were analyzed for each of 2 treatment conditions

first: Bo

last: Wang


title: Transcriptional changes of tumoral dendritic cells with tumor progession

status: Public on Dec 02 2024

city: Hangzhou

first: Ting

last: Zhou

state: Zhejiang

address: No. 600 Dunyu Road, Xihu District

department: Immunology and Microbiology

institute: Westlake University

summary: Dendritic cells (DCs) play important role of orchestrating anti-tumor immune responses both in tumor context. Following tumor progession, DCs were dysfunctioned by chaotic tumor microenvironment. To understand the change in the molecular level, we purfied conventional DCs from tumor at early stage (day 7 and around 200mm3) or late stage (day14 and larger than 600mm3) and sent for sequencing. Additonally, we would like to understand how pde5 deletion affects DCs function.

overall_design:
Tumoral DCs were sorted from MC38-bearing Zbtb46-Tdtomato mice at early- (day7) or late-stage (day14) for seqencing.
BMDCs derived from WT or Pde5-/- mice were stimulated with or without 200 ng/ml LPS for 12h and then collected for sequencing.


title: Generation of somatic chimeras using genetically-reprogrammed rabbits induced pluripotent stem cells [KEPi]

status: Public on Dec 03 2024

city: Bron

first: Yannicke

last: Pijoff

state: Rh^ne-Alpes

address: 18 Avenue du Doyen Lépine

country: France

institute: INSERM

summary: Little is known about the molecular underpinnings of pluripotent stem cells (PSCs)’ ability to colonize preimplantation embryo epiblast to generate chimeras. Using rabbit PSCs as a model system, we conducted unbiased screening of a cDNA library encoding a panel of 36 pluripotency factors. From this, we identified KLF2, ERAS and PRMT6 whose overexpression endows the ability to self-renew in a KOSR/FGF2-free culture medium supplemented with LIF, activin A, PKC and WNT inhibitors. The reprogrammed cells acquired transcriptomic and epigenetic features of naïve pluripotency, including the reactivation of the 2nd X-chromosome. Leveraging on these PSC lines, we determined the transcriptomic signature of embryonic colonization-competence, demonstrating transcriptional repression of genes involved in MAPK, WNT, HIPPO, and EPH signaling pathways, and activation of genes involved in amino-acid metabolism, NF-kB signaling, and p53 pathway. Remarkably, these reprogrammed cells acquired the ability to produce chimeric embryos and fetuses with high contribution from PSCs in all major organs.

overall_design: Transcriptomic profiling of chimeric competent KEP rabbit iPS cells.

first: Hong-Thu

last: Pham

first: Pierre

last: Savatier

first: Nathalie

last: Beaujean


title: Generation of somatic chimeras using genetically-reprogrammed rabbits induced pluripotent stem cells [KEPc]


title: RNA-seq analysis of human multiple myeloma cell lines treated with pyridostatin and panobinostat

status: Public on Dec 01 2024

city: Montpellier

first: Jerome

last: MOREAUX

address: 141, rue de la Cardonille 34396 Montpellier Cedex 5 FRANCE

institute: IGH

summary: Multiple Myeloma is the second most frequent blood cancer, characterized by clonal proliferation of terminally differentiated plasma cells (PCs) in the bone marrow (BM)1. Despite considerable progress in the treatment of MM, particularly with the emergence of immune-based therapies, it remains unclear how to overcome drug resistance, especially in high-risk patients with TP53 abnormalities including del17p and/or TP53 mutations2–4. Deregulated oncogene expression can alter replication timing and transcriptional programs, resulting in increased replication stress (RS), generally referred to as oncogene-induced RS5. Accordingly, MM cells (MMCs) but not normal PCs exhibit constitutive phosphorylation of the RS marker H2A.X (termed H2A.X)6. Oncogene overexpression also results in hyperactivation of the transcriptional machinery required to sustain increased proliferation and cell growth7,8. Hypertranscription thus exacerbates RS by impeding replication fork progression, often resulting in transcription-replication conflicts (TRCs). MMCs must cope with both oncogene-induced RS and a high level of transcription linked to immunoglobulin (Ig) synthesis, thus constituting a positive loop that sustains genomic instability. In line with this, increased transcription in MM has been associated with increased mutation rate9. This genomic instability is therefore an Achilles heel for MMCs, which are expected to be more dependent on the DNA damage response (DDR) for survival than normal PCs, offering exploitable targets for the development of new therapeutic strategies.

overall_design: To understand how pyridostatin (PDS) increases the potency of lenalidomide (Lena), we performed a transcriptomic analysis using three different multiple myeloma cell lines. In this experiment, we evaluate the response after 48h of treatment with the combination of compound PDS at 2.5µM, and of compound lenalidomide (XG7, JJN3: 25µM), XG20:50µM) in three multiple myeloma cell lines (XG20, XG7, JJN3)

first: Laure

last: Dutrieux

first: Sara

last: Ovejero

first: Miss Leriem

last: Zellagui

first: Antoine

last: Guillemin

first: Elke

last: De Bruyne

first: Catharina

last: Muylaert

first: Lien

last: Van Hemelrijck

first: Yea-Lih

last: Lin

first: Elle

last: Loughran

first: Armelle

last: Choquet

first: Talha

last: Magat

first: Soumya

last: Bouchouika

first: Caroline

last: Bret

first: Guilhem

last: Requirand

first: Nicolas

last: Robert

last: Vincent

first: Guillaume

last: Cartron

first: Charles

last: Herbaux

first: Raphaël

last: Rodriguez

first: Michel

last: Cogné

first: Eric

last: Rivals

first: Jean-Christophe

last: Andrau

first: Alexandre

last: David

first: Philippe

last: Pasero

first: Jérôme

last: Moreaux


title: Generation of somatic chimeras using genetically-reprogrammed rabbits induced pluripotent stem cells

first: Yannicke

last: Pijoff

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


first: Jerome

last: MOREAUX

overall_design: To understand how pyridostatin (PDS) increases the potency of panobinostat (Pano), we performed a transcriptomic analysis using three different multiple myeloma cell lines. In this experiment, we evaluate the response after 48h of treatment with the combination of compound PDS at 1.25µM for XG7 and JJN3 and 0.63µM for XG2, and 5nM of panobinostat in three multiple myeloma cell lines (XG2, XG7, JJN3)

first: Laure

last: Dutrieux

first: Sara

last: Ovejero

first: Antoine

last: Guillemin

first: Elke

last: De Bruyne

first: Catharina

last: Muylaert

first: Lien

last: Van Hemelrijck

first: Yea-Lih

last: Lin

first: Elle

last: Loughran

last: Choquet


title: Characterizing the tumor suppressor activity of FLCN in Birt-Hogg-Dube syndrome through transcriptiomic and proteomic analysis

status: Public on Nov 28 2024

city: Cardiff

first: Iain

last: Perry

state: South Glamorgan

address: Longwood Drive

department: Wales Gene Park

institute: Cardiff University

summary: Birt-Hogg-Dube (BHD) syndrome patients are uniquely susceptible to all renal tumour subtypes. The underlying mechanism of carcinogenesis is unclear. To study cancer development in BHD, we used human proximal kidney (HK2) cells with short-term and long-term folliculin (FLCN) knockdown. HK2 cells lacking FLCN had an altered transcriptome profile with cell cycle control gene enrichment. G1/S cell cycle checkpoint signaling was compromised with heightened protein levels of cyclin D1 (CCND1) and hyperphosphorylation of retinoblastoma 1 (RB1). Taken together with our proteomic work, our findings indicate that long-term FLCN  loss and associated cell cycle defects in BHD patients could contribute to their increased risk of cancer.

overall_design: RNAseq profiling of human kidney proximal tubule cells (HK2) with short-term (< 30 passages) and long-term (>120 passages) knockdown of FLCN, compared to their wild-type controls.

first: Andrew

last: Tee

first: Rachel-Ann

last: Jones

first: Elaine

last: Dunlop

first: Jesse

last: Champion

first: Peter

last: Doubleday

first: Tijs

last: Claessens

first: Zahra

last: Jalali

last: Seifan

last: Giles

first: Oliver

last: Harrison

first: Barry

last: Coull

first: Arjan

last: Houweling

first: Arnim

last: Pause

first: Bryan

last: Ballif


title: EFFICIENT RIBOSOMAL RNA DEPLETION FROM DROSOPHILA TOTAL RNA FOR NEXT-GENERATION SEQUENCING APPLICATIONS

city: Mandi

first: Baskar

last: Bakthavachalu

state: Himachal Pradesh

address: Kamand

department: School of Biosciences and Bioengineering

country: India

institute: Indian Institute of Technology

summary: We developed a cost-effective enzyme-based rRNA-depletion method tailored for Drosophila melanogaster, addressing the limitations of existing commercial kits and the lack of peer-reviewed alternatives. Our method employs single-stranded DNA probes complementary to Drosophila rRNA, forming DNA-RNA hybrids. These hybrids are then degraded using RNAse H enzyme, effectively removing rRNA and enriching all non-ribosomal RNAs, including mRNA, lncRNA and small RNA. When compared to a commercial rRNA Fly Kit, our approach demonstrated superior rRNA removal efficiency and mapping percentage, confirming its effectiveness. Additionally, our method successfully enriched the non-coding transcriptome, making it a valuable tool for studying ncRNA in Drosophila. The probe sequences and rRNA-depletion protocol is made freely available, offering researchers a reliable alternative for rRNA-depletion experiments.

overall_design: RNA-seq profiling of Drosophila adult brains processed by PolyA enrichment and rRNA depletion

first: Omkar

last: Koppaka

first: Shweta

last: Tandon

first: Ankita

last: Chodankar

first: Awadhesh

last: Pandit


title: The histone chaperone Spt6 controls chromatin structure through its conserved N-terminal domain [ChIP-seq]

status: Public on Nov 25 2024

city: Boston

first: James

middle: L

last: Warner

state: MA

address: 77 Avenue Louis Pasteur, Room 358

department: Genetics

institute: Harvard Medical School

summary: The disassembly and reassembly of nucleosomes by histone chaperones is an essential activity during eukaryotic transcription elongation. This highly conserved process maintains chromatin integrity by transiently removing nucleosomes as barriers and then restoring them in the wake of transcription. While transcription elongation requires multiple histone chaperones, there is little understanding of how most of them function and why so many are required. Here, we show that the histone chaperone Spt6 acts through its acidic, intrinsically disordered N-terminal domain (NTD) to bind histones and control chromatin structure. The Spt6 NTD is essential for viability and its histone binding activity is conserved between yeast and humans. The essential nature of the Spt6 NTD can be bypassed by changes in another histone chaperone, FACT, revealing a close functional connection between the two. Our results have led to a mechanistic model for dynamic cooperation between multiple histone chaperones during transcription elongation.

overall_design: Chromatin immunoprecipitation sequencing (ChIP-seq) analysis to determine whether the Spt6 NTD contributes to chromatin binding. Since the Spt62-238 protein does not support viability in Saccharomyces cerevisiae, we used a strain in which the genome-encoded Spt6 could be rapidly depleted using an auxin-inducible degron while expressing either wild-type (Spt6) or mutant (Spt6Δ2-238) versions of Spt6 from a plasmid. We could distinguish the two versions of Spt6 as genome-encoded allele was tagged with a V5 epitope tag and the plasmid-encoded allele was tagged with a Flag epitope tag. We then performed ChIP to determine levels of V5, FLAG, and Rpb1 association with chromatin in non-depleted (DMSO) and depleted (IAA) conditions. Experiments were performed in biological triplicate (here, rep2, rep3, and rep4). Replicate 1 was not analyzed due to poor sonication of chromatin.

first: Vanda

last: Lux

first: Vaclav

last: Veverka

first: Fred

last: Winston


title: The histone chaperone Spt6 controls chromatin structure through its conserved N-terminal domain [MNase-Seq]

overall_design: Micrococcal nuclease sequencing (MNase-seq) analysis to determine whether the Spt6 NTD is required for normal chromatin structure. Since the Spt6Δ2-238 protein does not support viability in Saccharomyces cerevisiae, we used a strain in which the genome-encoded Spt6 could be rapidly depleted using an auxin-inducible degron while expressing either no (empty vector), wild-type (Spt6), or mutant (Spt6Δ2-238) versions of Spt6 from a plasmid. We then performed MNase digestion on cells grown in non-depleted (DMSO) and depleted (IAA) conditions. Experiments were performed in biological triplicate (rep1, rep2, and rep3).


title: Multimodal Spatial Profiling Reveals Immune Suppression and Microenvironment Remodeling in Fallopian Tube Precursors to High-Grade Serous Ovarian Carcinoma

status: Public on Nov 22 2024

first: Clemens

last: Hug

address: 200 Longwood Avenue

department: Harvard Program in Therapeutic Science

summary: High-Grade Serous Ovarian Cancer (HGSOC) originates from fallopian tube (FT) precursors. However, the molecular changes that occur as precancerous lesions progress to HGSOC are not well understood. To address this, we integrated high-plex imaging and spatial transcriptomics to analyze human tissue samples at different stages of HGSOC development, including p53 signatures, serous tubal intraepithelial carcinomas (STIC), and invasive HGSOC. Our findings reveal immune modulating mechanisms within precursor epithelium, characterized by chromosomal instability, persistent interferon (IFN) signaling, and dysregulated innate and adaptive immunity. FT precursors display elevated expression of MHC-class I, including HLA-E, and IFN-stimulated genes, typically linked to later-stage tumorigenesis. These molecular alterations coincide with progressive shifts in the tumor microenvironment, transitioning from immune surveillance in early STICs to immune suppression in advanced STICs and cancer. These insights identify potential biomarkers and therapeutic targets for HGSOC interception and clarify the molecular transitions from precancer to cancer.

overall_design: We have 44 specimens from 43 pre-cancer patients with and without cancer. Out of 44, we performed microregional spatial transcriptomics (GeoMx) on 35 specimens. Mostly these precancer leisons are incidental p53 signatures, incidental STIC, and STIC associated with cancer. Of the 44 specimens, all but nine had matched FT and/or fimbriae (Fim) within the same tissue section.

first: Tanjina

last: Kader

first: Jia-Ren

last: Lin

first: Sandro

last: Santagata

first: Ronny

last: Drapkin


title: Microbial metabolite ammonia disrupts CEACAM1-TGF-β signaling to promote colon cancer

status: Public on Nov 16 2024

city: Manhasset

first: Lopa

last: Mishra

address: 350 Community Drive

institute: The Feinstein Institutes for Medical Research & Cold Spring Harbor Laboratory

summary: Colorectal cancer (CRC) incidence is rising globally and anticipated to become the leading cause of cancer death in younger individuals. Potential risk factors are diet induced obesity and altered microbiomes that lead to accumulation of toxic metabolite accumulation. However, how ammonia and other microbial metabolites impact key signaling pathways, such as TGF-β signaling, to promote CRC remains unclear. Our study investigates a critical link between gut microbiome alterations, ammonia, and their toxic effects on the TGF-β signaling pathway, to drive CRC progression. We found that in an obesity induced mouse model of cancer, altered microbial populations and ammonia promote Caspase-3-mediated cleavage of SMAD3 adaptor βII-spectrin (SPTBN1). Cleaved SPTBN1 fragments form adducts with ammonia to induce pro-inflammatory cytokine expression and alter TGF-β signaling driving CRC. Extending on Alphafold docking simulations, we identified that ammonia interacts with six polar residues at SPTBN1 (S553, Y556, S663, Y666, N986, and T1178) of cleaved SPTBN1 fragments to form hydrogen bonds that disrupt downstream SMAD3 signaling, altering TGF-β signaling to a protumorigenic phenotype. Blocking SPTBN1, through an SPTBN1 specific siRNA blocks ammonia toxicity and restore TGF-β signaling by reducing the abundance of SPTBN1 cleaved fragments. Importantly, SPTBN1 siRNA blocks ammonia toxicity and restore normal TGF-β signaling in CRC cells. Moreover, our research establishes crosstalk between TGF-β signaling and a microbial sensor, carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), which is significantly overexpressed in CRC patients. We identified CEACAM1-SPTBN1 interactions at specific residues (E517 and Y520) within the immunoreceptor tyrosine-based inhibitory motif (ITIM) of CEACAM1 cytoplasmic domain, with both molecules playing pivotal roles in CRC progression. Our study identifies mechanistic insights into how microbial metabolites target TGF-β a major signaling pathways to promote CRC.

overall_design: Using RNA-seq to characterize transcriptional alterations in colon cancer cell line (HCT116) under different conditions: Control, TGF-β, NH4Cl, and NH4Cl plus TGF-β.

first: Krishanu

last: Bhowmick

first: Xiyan

last: Xiang


title: Effects of the deletion of tomato m6A demethylase SlALKBH2 on transcriptome-wide gene expression profiles during the initiation of fruit ripeing.

city: Beijing

first: Leilei

last: Zhou

address: Xianshan street

institute: Institute of Botany, Chinese Academy of Sciences

summary: RNA-seq analysis was conducted to elucidate the transcriptome-wide gene expression differences between the wild-type and the slalkbh2 mutants (slalkbh2-25 and slalkbh2-28). In comparison to the wild-type, we identified 602 downregulated genes and 833 upregulated genes in the slalkbh2-25 mutant, as well as 556 downregulated genes and 470 upregulated genes in the slalkbh2-28 mutant (with a cutoff criterion of FPKM fold change ≥ 1.5 and P value < 0.05). Consequently, 281 downregulated genes and 126 upregulated genes were consistently identified in both slalkbh2 mutants, which were considered differentially expressed genes.

overall_design: Tomato fruits were harvested from the wild-type, slalkbh2-25, and slalkbh2-28 mutants at the MG (39 dpa) stage and then subjected to RNA-seq analysis, in triplicate, using the Illumina Hiseq X system.

first: Guozheng

last: Qin


title: Effects of the deletion of tomato m6A demethylase SlALKBH2 on m6A RNA methylomes during the initiation of fruit ripeing.

summary: We conducted m6A-seq on the fruits of both wild-type and slalkbh2 mutants (slalkbh2-25 and slalkbh2-28) at the mature green (MG) stage, which represents the onset of fruit ripening, to identify putative SlALKBH2 targets. Approximately 10,000 high-confidence m6A peaks were identified in the fruit samples of wild-type, slalkbh2-25, and slalkbh2-28, predominantly situated near the stop codons and in the 3’ UTR regions of gene transcripts. Analysis of differential m6A methylomes revealed 117 and 79 hypermethylated m6A peaks in slalkbh2-25 and slalkbh2-28, respectively, along with only 8 and 15 hypomethylated m6A peaks compared to the wild-type. This suggests a notable increase in total m6A methylation under SlALKBH2 deficiency during ripening initiation. These hypermethylated m6A peaks located in numerous gene transcripts that potentially serve as targets of SlALKBH2.

overall_design: Tomato fruits were harvested from the wild-type, slalkbh2-25, and slalkbh2-28 mutants at the MG (39 dpa) stage and then subjected to m6A-seq analysis, in triplicate, using the Illumina Hiseq X system.


title: Multimodal Genome-wide Survey of Progressing and Non-progressing Breast Ductal Carcinoma In-Situ

status: Public on Nov 15 2024

city: Baltimore

first: Michael

last: Considine

state: Maryland

address: 550 North Broadway

institute: Johns Hopkins University

type: Methylation profiling by genome tiling array

summary: Background: Ductal carcinoma in-situ (DCIS) is a pre-invasive form of invasive breast cancer (IBC). Due to improved breast cancer screening, it now accounts for ~25% of all breast cancers. While the treatment success rates are over 90%, this comes at the cost of considerable morbidity, considering that the majority of DCIS never become invasive and our understanding of the molecular changes occurring in DCIS that predispose to invasive disease is limited. The aim of this study is to characterize molecular changes that occur in DCIS, with the goal of improving DCIS risk stratification.   Methods: We identified and obtained a total of 197 breast tissue samples from 5 institutions (93 DCIS progressors, 93 DCIS non-progressors, and 11 adjacent normal breast tissues) that had at least 10-year follow-up. We isolated DNA and RNA from archival tissue blocks and characterized genome-wide mRNA expression, DNA methylation, DNA copy number variation, and RNA splicing variation.    Results: We obtained all four genomic data sets in 122 of the 197 samples. Our intrinsic expression subtype-stratified analyses identified multiple molecular differences both between DCIS subtypes and between DCIS and IBC. While there was heterogeneity in molecular signatures and outcomes within intrinsic subtypes, several gene sets that differed significantly between progressing and non-progressing DCIS were identified by Gene Set Enrichment Analysis.   Conclusions: DCIS is a molecularly highly heterogenous disease with variable outcomes, and the molecular events determining DCIS disease progression remain poorly defined. Our genome-wide multi-omic survey documents DCIS-associated alterations and reveals molecular heterogeneity within the intrinsic DCIS subtypes. Further studies investigating intrinsic subtype-stratified characteristics and molecular signatures are needed to determine if these may be exploitable for risk assessment and mitigation of DCIS progression. The highly significant associations of specific gene sets with IBC progression revealed by our Gene Set Enrichment Analysis may lend themselves to the development of a prognostic molecular score, to be validated on independent DCIS cohorts.

overall_design: 197 breast tissue samples from 5 institutions (93 DCIS progressors, 93 DCIS non-progressors, and 11 adjacent normal breast tissues) that had at least 10-year follow-up. No replicates.

first: Marija

last: Debeljak

first: Soonweng

last: Cho

first: Bradley

last: Downs

first: Brittany

last: Avin-McKelvey

first: Yongchun

last: Wang

first: Phillip

middle: N

last: Perez

first: William

last: Grizzle

first: Katherine

last: Hoadley

first: Charles

middle: F

last: Lynch

first: Brenda

middle: Y

last: Hernandez

first: Paul

last: van Diest

first: Wendy

last: Cozen

first: Ann

last: Hamilton

first: Debra

last: Hawes

first: Edward

last: Gabrielson

first: Ashley

last: Cimino-Mathews

first: Liliana

middle: D

last: Florea

first: Leslie

last: Cope

first: Christopher

last: Umbricht


title: Oxidative Stress and Inflammation-Related mRNAs Are Elevated in Serum of a Finnish Wet Age-Related Macular Degeneration Cohort

city: Kuopio

first: Kai

last: Kaarniranta

address: Yliopistonrinne 3

department: Department of Ophthalmology

institute: University of Eastern Finland

summary: Purpose: Localized diseases are affected by and affect the systemic environment via blood circulation. In our studies, we explored the differences in different types of circulating serum RNAs between wet age-related macular degeneration (wAMD) patients and controls. Methods: Blood samples were obtained from 60 Finnish wAMD patients and 64 controls. After serum preparation and RNA sequencing, the count data was examined for differentially expressed genes (DEGs) and further checked for enriched molecular pathways and ontology terms as well as links to clinical data. Results: In our mRNA study, we found many DEGs and some enriched pathways, including the inflammation and cell survival-associated pathway tumor necrosis factor alpha (TNFA) signaling via nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB). The related DEGs were oxidized low-density lipoprotein receptor 1 (OLR1), salt inducible kinase 1 (SIK1), and coagulation factor III (F3). DEGs from degradative macular and retinal processes were also examined, many of which were also related to cardiovascular disease and maintenance. Additionally, DEG counts were inspected in relation to clinical and anti-vascular endothelial growth factor (anti-VEGF) treatment parameters, and glutamine amidotransferase-like class 1 domain-containing 3A (GATD3A) levels were found to be significantly lower in wAMD patients who had been receiving anti-VEGF treatments compared to those who had not yet received any. Conclusions: Since regulatory factors of mitochondrial function, oxidative stress, and inflammation have a significant role in the pathology of AMD, our mRNA study results may support the presence of interplay between systemic factors and wAMD development and progression.

overall_design: Blood serum samples from wet age-related macular degeneration and control patients (cataract surgery patients without signs of retinal degeneration) were sequenced (one sample per patient) and total RNA counts were extracted. DESeq2 analysis was used to find differentially expressed genes / RNAs from filtered datasets, such as mRNAs, between the two patient groups as per the package documentation. In the mRNA study, an additional model with the additions of sampling age, BMI, gender, and smoking habits was also created.

first: Mikko

last: Liukkonen

first: Hanna

last: Heloterä

first: Leea

last: Siintamo

first: Bishwa

last: Ghimire

first: Pirkko

last: Mattila

first: Niko

last: Kivinen

first: Joanna

last: Kostanek

first: Cezary

last: Watala

first: Maria

last: Hytti

first: Juha

last: Hyttinen

first: Ali

last: Koskela

first: Janusz

last: Blasiak


title: Multimodal Genome-wide Survey of Progressing and Non-progressing Breast Ductal Carcinoma In-Situ [RNA-seq]

first: Michael

last: Considine

summary: Background: Ductal carcinoma in-situ (DCIS) is a pre-invasive form of invasive breast cancer (IBC). Due to improved breast cancer screening, it now accounts for ~25% of all breast cancers. While the treatment success rates are over 90%, this comes at the cost of considerable morbidity, considering that the majority of DCIS never become invasive and our understanding of the molecular changes occurring in DCIS that predispose to invasive disease is limited. The aim of this study is to characterize molecular changes that occur in DCIS, with the goal of improving DCIS risk stratification. Methods: We identified and obtained a total of 197 breast tissue samples from 5 institutions (93 DCIS progressors, 93 DCIS non-progressors, and 11 adjacent normal breast tissues) that had at least 10-year follow-up. We isolated DNA and RNA from archival tissue blocks and characterized genome-wide mRNA expression, DNA methylation, DNA copy number variation, and RNA splicing variation.  Results: We obtained all four genomic data sets in 122 of the 197 samples. Our intrinsic expression subtype-stratified analyses identified multiple molecular differences both between DCIS subtypes and between DCIS and IBC. While there was heterogeneity in molecular signatures and outcomes within intrinsic subtypes, several gene sets that differed significantly between progressing and non-progressing DCIS were identified by Gene Set Enrichment Analysis. Conclusions: DCIS is a molecularly highly heterogenous disease with variable outcomes, and the molecular events determining DCIS disease progression remain poorly defined. Our genome-wide multi-omic survey documents DCIS-associated alterations and reveals molecular heterogeneity within the intrinsic DCIS subtypes. Further studies investigating intrinsic subtype-stratified characteristics and molecular signatures are needed to determine if these may be exploitable for risk assessment and mitigation of DCIS progression. The highly significant associations of specific gene sets with IBC progression revealed by our Gene Set Enrichment Analysis may lend themselves to the development of a prognostic molecular score, to be validated on independent DCIS cohorts.

overall_design:
197 breast tissue samples from 5 institutions (93 DCIS progressors, 93 DCIS non-progressors, and 11 adjacent normal breast tissues) that had at least 10-year follow-up. No replicates.

***************************************************************
Raw files for human/patient samples were not submitted to GEO due to concerns about submitting personally identifiable sequence data for open access.
***************************************************************

first: Marija

last: Debeljak

first: Soonweng

last: Cho

first: Bradley

last: Downs

first: Brittany

last: Avin-McKelvey

first: Yongchun

last: Wang

first: Phillip

last: Perez

first: William

last: Grizzle

first: Katherine

last: Hoadley

first: Charles

last: Lynch

first: Brenda

last: Hernandez

first: Paul

last: van Diest

first: Wendy

last: Cozen

first: Ann

last: Hamilton

first: Debra

last: Hawes

first: Edward

last: Gabrielson

first: Ashley

last: Cimino-Mathews


title: CRISPR with Transcriptional Readout reveals influenza transcription is modulated by NELF and can precipitate an interferon response

status: Public on Nov 14 2024

city: San Diego

first: Alistair

middle: Brian

last: Russell

state: CA

address: 9500 Gilman Dr,

department: Biological Sciences

institute: University of California, San Diego

summary: Transcription of interferons upon viral infection is critical for cell-intrinsic innate immunity. This process is influenced by many host and viral factors. To identify host factors that modulate interferon induction within cells infected by influenza A virus, we developed CRISPR with Transcriptional Readout (CRITR-seq). CRITR-seq is a method linking CRISPR guide sequence to activity at a promoter of interest. Employing this method, we find that depletion of the Negative Elongation Factor complex increases both flu transcription and interferon expression. We find that the process of flu transcription, both in the presence and absence of viral replication, is a key contributor to interferon induction. Taken together, our findings highlight innate immune ligand concentration as a limiting factor in triggering an interferon response, identify NELF as an important interface with the flu life cycle, and validate CRITR-seq as a tool for genome-wide screens for phenotypes of gene expression.

sample_organism:
Homo sapiens
Influenza A virus

platform_organism:
Homo sapiens
Influenza A virus

overall_design: To perform a genome-wide CRISPR screen of factors influencing type III interferon induction by influenza virus infection we developed a new method, called CRITR-seq. These files are the sequencing output of that method. A549-Cas9 cells bearing our vector were allowed to undergo editing for ten days, after which they were infected by influenza A virus lacking NS1 at an MOI of 2 and gDNA and RNA harvested for sequencing and analysis 8 hours post infection.

first: Alistair B.

first: Alison C.

last: Vicary


title: Deciphering the role of RNA in regulating CTCF’s DNA binding affinity in leukemia cells [CTCF-ChIP]

status: Public on Nov 11 2024

city: Memphis

first: Beisi

last: Xu

state: Tennessee

address: 262 Danny Thomas Pl

department: Center for Applied Bioinformatics

institute: St Jude Children's Research Hosipital

summary: CTCF, a highly studied transcription factor, is essential for chromatin interaction maintenance. Several independent studies have reported that CTCF interacts with RNAs in vitro and in cells. Yet continuous debates about the authenticity of the RNA-binding affinity of CTCF and its biological role remain in large part due to limited research techniques available, such as CLIP-seq. Here, we investigated the role RNA plays on CTCF’s transcription factor function through its chromatin occupancy. To systematically investigate whether RNAs affect CTCF’s ability to bind DNA, we perturbed CTCF-RNA interactions by three independent approaches and examined CTCF genome occupancy by ChIP-seq. Although RnaseA A and Triptolide treatment each affected a certain number of CTCF-binding peaks, few peaks overlapped between treatment groups indicating the effect of RNA in regulating CTCF’s DNA binding affinity was modest and variable between loci. In addition, limited transcriptional or chromatin accessibility changes were observed between cells expressing wild-type CTCF or CTCF lacking the RNA binding region. Our data provide a complementary approach and in silico evidence to reconsider the significance of RNA affecting CTCF’s DNA-binding affinity in a global manner.

overall_design: For CTCF-ChIP samples, CTCFAID2/WT cells were treated with 1 mM Triptolide or DMSO for 8 h concurrent with 5-Ph-IAA and doxycycline treatment. For RnaseA A ChIP, RnaseA A treatment conditions were adapted from those previously reported (Saldana-Meyer et al. 2019, Beltran et al. 2016). Cells were permeabilized before fixation by 0.05% TWEEN-20 in PBS for 10 minutes on ice, washed once with PBS and resuspended in PBS plus 1mg/mL RnaseA A or mock treated and rotated for 45 minutes at room temperature. Each treatment group (+RnaseA A, -RnaseA A) was performed in triplicate. For both Triptolide and RnaseA A ChIP groups, chromatin was prepared as described above. Spike-in antibody and chromatin (Active Motif, 61686 and 53083) along with CTCF antibody (10 mgs, Diagenode, C15410210-50) were added to the chromatin and incubated at 4 °C overnight with gentle rotation. The next day, pre-washed Protein G beads (Dynabeads, Invitrogen, 10004D) were added and incubated 4 °C for 4 h with gentle rotation. On a magnetic stand, the beads were washed twice with wash buffer 1 (50 mM Tris HCL pH 7.4, 1 M NaCl, 1 mM EDTA, 1 % NP-40, 0.1 % SDS, 0.5 % Na Deoxycholate plus protease inhibitors) and once with wash buffer 2 (20 mM Tris HCL pH 7.4, 10 mM MgCl2, 0.2 % Tween-20 plus protease inhibitors). The beads in wash buffer 2 were transferred to a new 1.5 mL Eppendorf tube and placed on a magnetic stand to remove the wash buffer. Decrosslinking was carried out in 1X TE plus 1 % SDS, proteinase K, and 400 mM NaCl at 65 °C for 4 h, followed by phenol, chloroform, and isopropyl alcohol precipitation of the DNA. NEBNext Ultra II NEB Library Prep Kit and NEBNext Multiplex oligos for Illumina were used to construct libraries for sequencing.

first: Judith

last: Hyle

first: Wenjie

last: Qi

first: Mohamed

last: Djekidel

first: Wojciech

last: Rosikiewicz

first: Chunliang

last: Li


title: Regulatory T cell therapy is associated with distinct immune regulatory lymphocytic infiltrates in kidney transplants

city: Oxford

first: Oliver

last: McCallion

address: John Radcliffe Hospital, Headley Way, Headington

institute: University of Oxford

summary: BACKGROUND: Adoptive transfer of autologous regulatory T cells (Tregs) is a promising therapeutic strategy aimed at enabling immunosuppression minimization following kidney transplantation. In our phase I clinical trial of Treg cell therapy in living donor renal transplantation, the ONE Study (NCT02129881), we observed focal lymphocytic infiltrates in protocol kidney transplant biopsies that are not regularly seen in biopsies of patients receiving standard immunosuppression. METHODS: We present seven years of follow-up data on patients treated with adoptive Treg cell therapy early post-transplantation, who exhibited focal lymphocytic infiltrates on a nine-month protocol biopsy. We phenotyped their adoptively transferred and peripherally circulating Treg compartments using CITEseq and investigated the focal lymphocytic infiltrates with spatial proteomic and transcriptomic technologies. FINDINGS: Graft survival rates were not significantly different between Treg-treated patients and the control reference group. None of the Treg-treated patients experienced clinical rejection episodes or developed de novo donor-specific antibodies, and three of ten successfully reduced their immunosuppression to tacrolimus monotherapy. All Treg-treated patients who underwent a protocol biopsy nine months post-transplantation exhibited focal lymphocytic infiltrates. Spatial profiling analysis revealed a prominent CD20+ B cell signature within cell therapy-associated immune infiltrates, distinct from the pro-inflammatory myeloid signature typically associated with the rejection biopsies. A regulatory gene expression program (IKZF2, IL10, PD-L1, TIGIT) characterized the cell therapy-associated immune infiltrates at a transcriptional level. CONCLUSIONS: We demonstrate for the first time that immune cell infiltrates in transplanted kidneys can occur following adoptive Treg cell therapy in humans, potentially facilitating within-graft T:B cell interactions that promote local immune regulation. FUNDING: 7th EU Framework Programme, Grant/Award Number: 260687; National Institute for Health Research (NIHR).

overall_design: Treg were FACS sorted from PBMC samples (as CD4+CD127-CD25hi) of four patients from two time points (week 24 and week 60) and combined with adoptively transferred Treg from the cell therapy product. CITEseq was performed using the BD Rhapsody platform

first: McCallion

last: Oliver

first: Trzupek

last: Dominik

first: Ferreira

last: Ricardo

first: Hester

last: Joanna

first: Issa

last: Fadi


title: Nanopore PCR-free direct cDNA sequencing of human kidney tissue

status: Public on Nov 10 2024

city: Indianapolis

first: Takashi

last: Hato

state: IN

address: 950 W. Walnut St. R2 202

institute: Indiana University

summary: Anonynous donor kidneys were sequenced using the Nanopore long-read direct cDNA sequencing kit (SQK-DCS109) and R9.4.1 flow cells.

overall_design: Total RNA was extracted from frozen kidney tissues (anonymous human donors) using the Zymo Direct-zol RNA miniprep kit, including on-column DNase I digetsion. PolyA mRNA enrichment was performed using the Dynabeads mRNA DIRECT Micro kit. Reverse transcription and strand-switching were carried out using Maxima H Minus Reverse Transcriptase following the Nanopore protocol (SQK-DCS109). After end-prep, adapter ligation, and AMPure XP bead binding, sequencing was conducted on R9.4.1 flow cells using the GridIon platform.


title: An in vivo repertoire of zebrafish cardiomyocyte-specific cis-regulatory elements [ChIP-Seq]

city: Clayton

first: Hieu

middle: Tri

last: Nim

address: Monash University Clayton Campus, Wellington Rd

department: Australian Regenerative Medicine Institute

institute: Monash University

summary: cis-Regulatory elements (cREs) are essential for the spatio-temporal control of gene expression during development and disease. However, cRE activity is highly dependent on cell and tissue type. The developing heart is composed of several cell-types, predominantly cardiomyocytes. Therefore, cardiomyocytes-specific modelling is required to understand the cis-regulation of the developing heart. Zebrafish are an ideal model to study heart development, as they share a number of physiological features with the human heart during development. Therefore, we present a comprehensive cardiomyocyte-specific repertoire of cREs isolated from zebrafish larvae. This data combines live transcriptomics and epigenetic profiling, providing insights into cREs and their associated genes involved in heart development. We further perform a transgenic reporter assay for the identified cREs of bmp10 and popdc2 genes, validating these genomic regions as cardiac regulatory elements. We share this comprehensive, reproducible cardiomyocyte-specific cRE resource as an interrogable web tool for understanding the epigenetic and transcriptomic mechanisms underlying heart development and emergence of congenital heart defects.

overall_design: Cardiomyocytes were isolated from the Tg(myl7::GFP) zebrafish transgenic line. Larvae were collected 72 hpf (hours post fertilization). GFP-positive (30,000 cells, n=3 biological replicates) and GFP-negative cells (30,000 cells, n=3 biological replicates) were collected, and ChIP-sequencing was performed on these samples. The raw ChIP-seq FASTQ files are provided in SRA. The supplementary files are: the BED file containing differential peaks only found in GFP-negative samples (GSM7995224_GFP-_only.bed.gz), the BED file containing differential peaks only found in GFP-positive samples (GSM7995227_GFP+_only.bed.gz).

first: Marcus

last: Tondl

first: Gulrez

last: Chahal

first: Michael

last: Eichenlaub

first: Mirana

last: Ramialison


title: Cholangiocytes contribute to hepatocyte regeneration after partial liver injury in larval zebrafish

status: Public on Nov 13 2024

city: Greater Noida

first: Sumeet

middle: Pal

last: Singh

state: Uttar Pradesh

address: NH91, Tehsil Dadri

department: Life Sciences

institute: Shiv Nadar Institution of Eminence

summary: Liver possesses robust regenerative ability, characterized by flexibility in the cellular source of regeneration based on the extent of the injury. After partial hepatectomy or minor injuries, hepatocytes, the primary liver cells, undergo self-duplication to replenish the liver mass. In contrast, when the damage is extensive, or hepatocyte proliferation is impaired, cholangiocytes contribute to hepatocyte recovery. This current paradigm of regenerative flexibility in the liver has been established for animals with little or no growth. However, the regenerative mechanisms during periods of growth in young animals remain unexplored. Here, we establish two new partial liver injury protocols in the zebrafish model of rapid growth during late larval stage and observe emergence of de novo hepatocytes in the presence of spared hepatocytes. Using single-cell RNA sequencing and lineage tracing, we identify cholangiocytes as the source of de novo hepatocytes. Our study offers a new perspective on the current paradigm of liver regenerating by proposing cholangiocyte-to-hepatocyte transdifferentiation as the default mechanism of hepatocyte recovery in late larval stage zebrafish.

overall_design: We conducted single-cell RNA Sequencing using the 10x platform of larval zebrafish liver. This was performed in the basal state (Control1, Control2), after partial ablation (MTZ_0dppa, MTZ_1dppa and MTZ_9dppa) and after partial hepatectomy (PHx_4dpi and PHx_11dpi). The two control samples were not treated with 4-OHT, and were also not injured. The control samples are from 13 dpf zebrafish raised under normal rearing condition. For all experiments Tg(fabp10a:CellCousin) line was used.

first: Elif

last: Sema


title: CTCF/cohesin organize the ground state of chromatin-nuclear speckle association [hela rad21-aid RNA-seq]

status: Public on Nov 08 2024

city: Philadelphia

first: Ruofan

last: Yu

state: PA

address: 3400 civic center blvd

department: Cell and Developmental Biology

institute: University of Pennsylvania

summary: The interchromatin space in the cell nucleus contains various membrane-less nuclear bodies. Recent findings indicate that nuclear speckles, comprising a distinct nuclear body, exhibit interactions with certain chromatin regions in a ground state. Key questions are how this ground state of chromatin-nuclear speckle association is established and what are the gene regulatory roles of this layer of nuclear organization.  We report here that chromatin structural factors CTCF and cohesin are required for full ground state association between DNA and nuclear speckles. Disruption of ground state DNA-speckle contacts via either CTCF depletion or cohesin depletion had minor effects on basal level expression of speckle-associated genes, however we show strong negative effects on stimulus-dependent induction of speckle-associated genes. We identified a putative speckle targeting motif (STM) within cohesin subunit RAD21 and demonstrated that the STM is required for chromatin-nuclear speckle association.  In contrast to reduction of CTCF or RAD21, depletion of the cohesin releasing factor WAPL stabilized cohesin on chromatin and DNA-speckle contacts, resulting in enhanced inducibility of speckle-associated genes. In addition, we observed disruption of chromatin-nuclear speckle association in patient derived cells with Cornelia de Lange syndrome (CdLS), a congenital neurodevelopmental diagnosis involving defective cohesin pathways, thus revealing nuclear speckles as an avenue for therapeutic inquiry. In summary, our findings reveal a mechanism to establish the ground organizational state of chromatin-speckle association, to promote gene inducibility, and with relevance to human disease.

overall_design: RNAseq on hela rad21-aid cells with plasmid transfection

first: Shelley

last: Berger


title: CTCF/cohesin organize the ground state of chromatin-nuclear speckle association [CUT&RUN, CUT&Tag and RNA-seq]

overall_design: Cut &Run against speckle protein SON and corresponding IGG control, under designated treatment.


title: CTCF/cohesin organize the ground state of chromatin-nuclear speckle association [CUT&RUN]


title: Genomic and transcriptomic data analyses highlight KPNB1 and MYL4 as novel risk genes for congenital heart disease

city: Helsinki

first: Martin

last: Broberg

address: Viikinkaari 5D

department: Biosciences

institute: University of Helsinki

summary: Congenital heart defects (CHD) are structural defects of the heart affecting approximately 1% of newborns. CHDs exhibit a complex inheritance pattern. While genetic factors are known to play an important role in the development of CHD, relatively few variants have been discovered so far and very few genome-wide association studies (GWAS) have been conducted. We performed a GWAS of general CHD and five CHD subgroups in FinnGen followed by functional fine-mapping through eQTL analysis in the GTEx database, and target validation in human induced pluripotent stem cell -derived cardiomyocytes (hiPS-CM) from CHD patients. We discovered that the MYL4-KPNB1 locus (rs11570508, beta = 0.24, P = 1.2x10-11) was associated with the general CHD group. An additional four variants were significantly associated with the different CHD subgroups. Two of these, rs1342740627 associated with left ventricular outflow tract obstruction defects and rs1293973611 associated with septal defects, were Finnish population enriched. The variant rs11570508 associated with the expression of MYL4 (normalized expression score (NES) = 0.1, P = 0.0017, in the atrial appendage of the heart) and KPNB1 (NES = -0.037, P = 0.039, in the left ventricle of the heart). Furthermore, lower expression levels of both genes were observed in human induced pluripotent stem cell -derived cardiomyocytes (hiPSC-CM) from CHD patients compared to healthy controls. Together, the results demonstrate KPNB1 and MYL4 as in a potential genetic risk loci associated with the development of CHD.

overall_design: hIPSC lines from 4 patients and 2 pooled controls were sequenced and compared for gene expression.

first: Minna

last: Ampuja

first: Samuel

last: Jones

first: Tiina

last: Ojala

first: Otto

last: Rahkonen

first: Riikka

last: Kivelä

first: James

last: Priest

first: Hanna

last: Ollila

first: Emmi

last: Helle


title: Comparative transcriptomics reveal contrasting fungal strategies in a plant pathogen versus an endophyte during initial host colonization

status: Public on Nov 05 2024

city: East Lansing

first: Frances

last: Trail

state: Michigan

address: 612 Wilson Road

department: Plant Biology

institute: Michigan State University

summary: Conidial germination marks the beginning of the fungal life cycle, and understanding the genes associated with conidial germination provides insights into fungal pathogenicity and host interactions. Here, we use comparative transcriptomics to demonstrate the transcriptional similarities and differences during conidial germination and initial colony establishment in a plant pathogenic and an endophytic fungus, Fusarium graminearum and M. anisopliae, respectively. We compared the transcriptomes of F. graminearum and M. anisopliae across four stages of conidial germination: fresh conidia, polar growth, hyphal extension, and either first hyphal branching (on medium) or appressorium formation (on barley). F. graminearum exhibited a higher upregulation of CAZyme, specialized metabolite and effector genes compared to M. anisopliae during interaction with the host, particularly in the appressorium stage, reflecting its pathogenic nature. The appressorium structures formed when M. anisopliae conidia germinated on the host. The transcriptome analysis revealed that the fungus produced reduced transcript levels of CAZyme and specialized metabolite genes reflecting a less aggressive host penetration approach. The candidate genes associated with IAA synthesis were upregulated in M. anisopliae during the appressorium stage, supporting its endophytic lifestyle and suggests that the fungus uses a phytohormone based strategy to interact with plant hosts. Collectively, our findings expand the transcriptome resources and provide valuable insights into the gene networks involved in conidial germination and initiation of infection in pathogenic versus endophytic fungus.

overall_design: The gene expression profile of conidial germination stages of Metarhizium anisopliae (ARSEF 549) on a standard medium (Potato Dextrose Agar) and host (Barley) was generated. Four designated germination stages, including fresh conidia (stage 1), polar growth (stage 2), doubling of the long axis (stage 3), and first hyphal branching/appressoria formation (stage 4) was examined to generate the gene expression profile.

first: Soumya

last: Moonjely


title: Comparative transcriptomics reveal contrasting fungal strategies in a plant pathogen (Fusarium graminearum) versus an endophyte (Metarhizium anisopliae) during initial host colonization

overall_design: The gene expression profile of conidial germination stages of Fusarium graminearum PH-1 on a standard medium (Potato Dextrose Agar) and host (Barley) was generated. Four designated germination stages, including fresh conidia (stage 1), polar growth (stage 2), doubling of the long axis (stage 3), and first hyphal branching/appressoria formation (stage 4) was examined to generate the gene expression profile.


title: CTCF/cohesin organize the ground state of chromatin-nuclear speckle association [RNA-Seq]

overall_design: Comparative gene expression profiling analysis of full-length RNA-seq data for HeLa, IMR90, and patient derived LCL cells with deficiency in CTCF, RAD21 or WAPL, with or without designated external stimuli.


title: Transcriptomic Profile of Neuroinflammation in Irradiated Brain Following C5aR1 Inhibition, With and Without Brain Tumor

city: Irvine

first: Munjal

last: Acharya

address: 811, Health Sciences Dr, ROOM B-149, Medical Sciences B

department: Anatomy & Neurobiology

institute: University of California Irvine

summary: Expression of 757 genes related to neuroinflammation was analyzed across 12 treatment groups of male mice to investigate the neuroprotective effects of C5a Receptor 1 (C5aR1) inhibition against cranial irradiation for brain cancer therapy, utilizing both a pharmacologic (PMX205) and genetic (C5aR1-KO) approach.

overall_design: Samples from 58 male mice, ages 23-24 weeks at time of perfusion, were analyzed using the commercially available nCounter® Mouse Neuroinflammation Panel from NanoString™, with 3-4 samples for each of the 15 treatment groups. For the pharmacologic model, C57BL/6J mice were randomly assigned to treatments groups with the following parameters: with or without brain tumor (astrocytoma, CT2A cell line, or glioblastoma, GL261 cell line); with or without single-dose cranial irradiation (9 Gy vs 0 Gy);  and with or without C5aR1 inhibitor drug treatment (PMX205, 1mg/kg body weight injected subcutaneously for 7 days and 100 µg daily in drinking water for 30 days). For the transgenic model, C5aR1 KO mice were randomly assigned to the following treatment groups: with or without brain tumor (astrocytoma, CT2A cell line); and with or without single-dose cranial irradiation (9 Gy vs 0 Gy).   Note: in the treatment group C5aR1 KO + 0 Gy, samples “PMX 25a” and “PMX25b”, as well as “PMX26a” and “PMX26b”, are each pairs of technical replicates, for a total of 2 biological replicates for this group; all other treatment groups contain 4 biological replicates.   Sample ID’s “PMX60” thru “PMX71” originate from a glioblastoma model and were run separately from Sample ID’s “PMX13” thru “PMX59.” Both sample sets underwent the same protocols described below.

first: Robert

middle: P

last: Krattli

first: Do

last: An


title: Integrated Profiling and Immunohistochemistry Analyses Link circ-BRIP1, circ-EPHB4 and Their Host Genes with Imatinib Resistance in Gastrointestinal Stromal Tumors

status: Public on Nov 01 2024

city: Jinshan Distric

first: Jin

last: Wang

state: Shanghai

address: 2901 Caolang Road, Scientific Research Center, RM 319

department: Shanghai Public Health Clinical Center

institute: Fudan University

type:
Expression profiling by array
Non-coding RNA profiling by array

summary: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the digestive tract. The majority of GIST patients eventually develop resistance to imatinib therapy. To identify the responsible mechanisms, we investigated the differentially expressed mRNAs and circRNAs in imatinib-resistant GISTs using SBC ceRNA microarrays. We found that 107 mRNAs and 521 circRNAs were significantly differentially expressed between imatinib-naïve and imatinib-resistant GIST tissue samples. qRT-PCR analyses validated that circ-BRIP1, circ-EPHB4 and circ-RECQL4 and their host genes were upregulated in imatinib-resistant GISTs, and circ-BRIP1, circ-EPHB4, and RECQL4 were associated with imatinib resistance, tumor relapse and progression, and metastasis in GIST patients. The expression levels of circ-BRIP1, circ-EPHB4 and their host genes were also evaluated using TMAs with 150 human GISTs. The expression level of EPHB4 was significantly increased in high-grade GISTs in comparison to low-grade GISTs and correlated with imatinib resistance. Specifically, we first developed a method for high-throughput analysis of the expression of differentially expressed circRNAs by ISH-IHC in a set of FFPE-tissue microarrays in GIST. Our results also suggested a possible role for circ-BRIP1, circ-EPHB4, and their host genes in the progression of GISTs.

overall_design: To identify the responsible mechanisms, we investigated the differentially expressed mRNAs and circRNAs in imatinib-resistant GISTs using ceRNA microarrays. The expression levels of circ-BRIP1, circ-EPHB4 and their host genes were evaluated using quantitative real-time PCR and in situ hybridization-immunohistochemistry (ISH-IHC) / IHC analyses in GIST tissue microarrays (TMAs).

first: Hanxing

last: Tong


title: DNA Methylation Biomarkers for Cervical Cancer Risk Prediction in HIV-Positive Nigerian Women

city: Chicago

first: Yinan

last: Zheng

state: Illinois

address: 680 N Lake Shore Drive, Suite 1400

department: Department of Preventive Medicine

institute: Northwestern University

summary: Cervical cancer (CC) remains a significant public health issue in low- and middle-income countries (LMICs), especially in Western sub-Saharan Africa and Nigeria. While global CC incidence and mortality have declined, these regions continue to face high rates due to inadequate screening and the high prevalence of HIV, which increases CC risk by promoting persistent HPV infections. This study aimed to identify DNA methylation (DNAm) biomarkers for cervical intraepithelial neoplasia (CIN) and CC in HIV-positive Nigerian women and to assess their potential for clinical risk prediction. From 2018 to 2020, 538 participants were recruited from Nigerian tertiary hospitals. Cervical tissue samples were analyzed for DNAm using the Infinium MethylationEPIC BeadChip array, and HPV genotyping was conducted via next-generation sequencing. An epigenome-wide association study revealed 24 significant DNAm biomarkers associated with CIN and CC. These biomarkers showed hypermethylation in tumor suppressor genes (e.g., PRMD8), hypomethylation in oncogenes (e.g., MIR520H), and aberrant methylation in genes related to HIV/HPV infection and oncogenesis (e.g., GNB5, LMO4, FOXK2, NMT1). A machine learning-based DNAm classifier achieved 92.9% sensitivity and 88.6% specificity in predicting CC risk, with higher risk observed in adjacent normal cervical samples from CIN/CC patients and HIV/HPV co-infected women. DNAm biomarkers offer a promising approach to enhancing CC screening and early detection, particularly for HIV-positive women in LMICs. The DNAm-based model developed in this study shows potential for more accurate CC risk stratification, highlighting the need for further optimization, validation, and implementation in low-resource settings.

overall_design: In this study, we focus on identifying epigenetic markers for CIN/CC among Nigerian women with HIV, and then build and evaluate a machine-learning-based model to predict the risk of CIN/CC among a Nigerian population. The study encompassed four groups with reliable HIV and cancer definitions: HIV-positive CC, HIV-positive CIN, HIV-positive CC-free, and HIV-negative with CC.

first: Lifang

last: Hou

last: Murphy

first: Folasade

middle: T

last: Ogunsola

first: Atiene

last: Sagay


title: Enhanced metabolic resilience and exercise adaptation of skeletal muscle in males by Esr1-induced remodeling of mitochondrial cristae-nucleoid architecture

status: Public on Oct 30 2024

city: Los Angeles

first: Zhengyi

last: Zhang

address: Westwood

institute: UCLA

summary: Reduced estrogen action is associated with obesity and insulin resistance. However, the cell and tissue-specific actions of estradiol in maintaining metabolic health remain inadequately understood especially in men. We observed that skeletal muscle ESR1/Esr1 (encodes estrogen receptor a), including expression of specific ESR1 variants is positively correlated with insulin sensitivity and metabolic health in humans and mice. Because skeletal muscle is a primary tissue involved in oxidative metabolism and insulin sensitivity, we generated muscle-selective Esr1 loss- and gain of-expression mouse models. We determined that Esr1 links mitochondrial DNA replication and cristae-nucleoid architecture with metabolic function and insulin action in skeletal muscle of male mice. Overexpression of human ERα in muscle protected male mice from diet-induced disruption of metabolic health and enhanced mitochondrial adaptation to exercise training intervention. Our findings indicate that muscle expression of Esr1 is critical for the maintenance of mitochondrial function and metabolic health in males, and that tissue-selective activation of ERα can be leveraged to combat metabolic-related diseases in both sexes.

overall_design: Isolated nuclei from HFD-fed Control f/f and mERaOE mice were used for ATAC-seq

first: Zhenqi

last: Zhou

first: Andrea

last: L. Hevener


title: Identification of IL-10 producing T cells in CSF and blood during treatment of subarachnoid neurocysticercosis

city: Bethesda

first: Elise

middle: M

last: O'Connell

state: MD

address: 4 Center Drive

department: Laboratory of Parasitic Diseases

institute: National Institute of Allergy and Infectious Diseases

summary: Neurocysticercosis (NCC) is a parasitic infection that affects the central nervous system and is increasingly found in patients in non-endemic regions. Subarachnoid neurocysticercosis (SANCC) is the most severe form of this disease and is largely responsible for mortality associated with NCC. Inflammation is often an important factor in the treatment of SANCC as overactivity can lead to serious complications, but suppression induced by anti-inflammatory molecules – namely interleukin 10 (IL-10) – can lead to an increased time to cure. To better understand the local inflammatory milieu associated with SANCC, we performed single cell RNA sequencing to achieve an unbiased profile of immune cell populations in the cerebrospinal fluid (CSF), and we used flow cytometry to identify important producers of IL-10 in the CSF and blood. These analyses revealed a diverse combination of immune cell populations within the CSF including multiple myeloid and lymphoid populations. We identified three primary sources of IL-10 in CSF – natural regulatory T cells (nTregs), induced regulatory T cells (iTregs), and Th17 cells – with iTregs being the single largest contributor in this compartment. Our data also show that these populations – and the abundance of IL-10 producing CD4+ T cells as a whole – differed between CSF and blood. Interestingly, these IL-10 producing nTregs, iTregs, and Th17 cells persist in the CSF over the course of SANCC treatment, indicating that immunosuppression within the central nervous system is unaffected despite decreases in parasite antigen over time. Overall, this study deepens our understanding of the suppressive phenotype that deters SANCC treatment success and provides a comprehensive blueprint of the local and systemic immunology associated with this important disease process.

overall_design: These are cerebrospinal fluid samples collected from patients treated at the NIH for subarachnoid neurocysticercosis. All patients provided two samples from different timepoints during anthelmintic treatment.

first: Nina

last: Tang

first: Paul

last: Schaughency

first: Thomas

last: Nutman