title: Cell type directed design of synthetic enhancers

status: Public on Nov 19 2023

city: Leuven

first: Gert

last: Hulselmans

address: Herestraat 49 PO Box 602

department: Center for Brain and Disease Research

country: Belgium

institute: VIB

summary: Transcriptional enhancers act as docking stations for combinations of transcription factors and thereby regulate spatiotemporal activation of their target genes. A single enhancer, of a few hundred base pairs in length, can autonomously and independently of its location and orientation drive cell-type specific expression of a gene or transgene. It has been a long-standing goal in the field to decode the regulatory logic of an enhancer and to understand the details of how spatiotemporal gene expression is encoded in an enhancer sequence. Recently, deep learning models have yielded unprecedented insight into the enhancer code, and well-trained models are reaching a level of understanding that may be close to complete. As a consequence, we hypothesized that deep learning models can be used to guide the directed design of synthetic, cell type specific enhancers, and that this process would allow for a detailed tracing of all enhancer features at nucleotide-level resolution. Here we implemented and compared three different design strategies, each built on a deep learning model: (1) directed sequence evolution; (2) directed iterative motif implanting; and (3) generative design. We evaluated the function of fully synthetic enhancers to specifically target Kenyon cells or glial cells in the fruit fly brain using transgenic animals. We then exploited this concept further by creating “dual-code” enhancers that target two cell types, and minimal enhancers smaller than 50 base pairs that are fully functional. By examining the trajectories followed during state space searches towards functional enhancers, we could accurately define the enhancer code as the optimal strength, combination, and relative distance of TF activator motifs, and the absence of TF repressor motifs. Finally, we applied the same three strategies to successfully design human enhancers, finding highly similar design principles as in Drosophila. In conclusion, enhancer design guided by deep learning leads to better understanding of how enhancers work and shows that their code can be exploited to manipulate cell states.

sample_organism:
Drosophila melanogaster
Homo sapiens

platform_organism:
Drosophila melanogaster
Homo sapiens

overall_design: Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for Zeb2 in MM001. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) in MM001 and MM099 transduced with a lentiviral pool of 10 synthetic enhancers. ATAC-seq in MM001 transduced with a lentiviral pool of repressed synthetic enhancers as well as with random sequences or evolved synthetic enhancers. ATAC-seq in Drosophila melanogaster lines containing synthetic enhancer constructs.

first: Ibrahim

middle: I

last: Taskiran

first: Katina

last: Spanier

first: Hannah

last: Dickmänken

first: Niklas

last: Kempynck

first: Alexandra

last: Pančíková

first: Eren

last: Can Ekşi

first: Joy

middle: N

last: Ismail

first: Koen

last: Theunis

first: Roel

last: Vandepoel

first: Valerie

last: Christiaens

first: David

last: Mauduit

first: Stein

last: Aerts


title: Mixed-Lineage Leukemia 1 Inhibition Enhances the Differentiation Potential of Bovine Embryonic Stem Cells by Increasing H3K4 Mono-Methylation at Active Promoters. (RNA-seq)

city: Hohhot

first: jing

last: wang

state: Nei Mongol

address: Zhaojun Road

country: China

institute: Inner Mongolia University

type: Expression profiling by high throughput sequencing

summary: Mixed-lineage leukemia 1 (MLL1) introduces 1-, 2- and 3-methylation into histone H3K4 through the evolutionarily conserved set domain. In this study, bovine embryonic stem cells (bESCs, known as bESCs-F7) were established from in vitro-fertilized (IVF) embryos via Wnt signaling inhibition; however, their contribution to the endoderm in vivo is limited. To improve the quality of bESCs, MM-102, an inhibitor of MLL1, was applied to the culture. The results showed that MLL1 inhibition along with GSK3 and MAP2K inhibition (3i) at the embryonic stage did not affect bESCs’ establishment and pluripotency. MLL1 inhibition improved the pluripotency and differentiation potential of bESCs via the up-regulation of stem cell signaling pathways such as PI3K-Akt and WNT. MLL1 inhibition decreased H3K4me1 modification at the promoters and altered the distribution of DNA methylation in bESCs. In summary, MLL1 inhibition gives bESCs better pluripotency, and its application may provide high-quality pluripotent stem cells for domestic animals.

overall_design: In this study, a CTFR-bESC culture system was applied to establish bovine- ESCs from MM-102 (50 μM)-, 2i (0.5 μM PD0325901 and 3 μM CHIR99021)- and 3i (2i plus 30 μM MM-102)-treated bovine IVF embryos. When embryos formed blastocysts at 7–8 days, the blastocysts, removed from the zona pellucida, were plated to establish bESCs using mouse fetal fibroblast (MEF) feeder cells. The obtained bESC cell lines were named bESCs-102, bESCs-2i and bESCs-3i. Given that MM-102 treatment of embryos altered the pluripotency of the established bESCsone involved the addition of 5 μM of MM-102 to CTFR in long-term cultures, and in the other, bESCs were cultured for 7 days with the addition of 50 μM MM-102, and then switched back to CTFR. The derived cell lines were named bESCs-102-5 and bESCs-102-50

first: Chen

last: Li

first: Xuejie

last: Han

first: Jing

last: Wang

first: Xueling


title: Mixed-Lineage Leukemia 1 Inhibition Enhances the Differentiation Potential of Bovine Embryonic Stem Cells by Increasing H3K4 Mono-Methylation at Active Promoters. (ChIP-seq)

type: Genome binding/occupancy profiling by high throughput sequencing


title: Mixed-Lineage Leukemia 1 Inhibition Enhances the Differentiation Potential of Bovine Embryonic Stem Cells by Increasing H3K4 Mono-Methylation at Active Promoters. (BS-seq)

type: Methylation profiling by high throughput sequencing


title: Distinct functional constraints driving conservation of the cofilin N-terminal regulatory tail

status: Public on Nov 18 2023

city: New Haven

first: Ben

last: Turk

state: CT

address: 333 Cedar Street, SHM B386

department: Pharmacology Department

country: USA

institute: Yale University

summary: Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specifics aspects driving this conservation are unclear. Here, we screened a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants revealed distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explained sequence constraints on phosphoregulation, which were instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We found remarkably loose sequence requirements for actin binding and phosphoinhibition, but collectively they restricted the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation.

overall_design: The S. cerevisiae TeTO7-COF1 strain31 (Horizon Discovery TH_5610) was transformed sequentially with pRS415-GAL1-FLAG-LIMK1 and the cofilin library plasmid pool and frozen in aliquots to conduct replicate screens. For each screen, transformed yeast were pooled and grown at 30 °C with shaking in liquid culture with selective media (SC-His- Leu) containing 2% glucose at a starting OD600 of 0.1. After 4 - 5 population doublings, a portion was diluted to an OD600 of 0.1 with SC-His-Leu media containing 2% raffinose and 10 mg/L DOX. After the culture reached an OD600 of 1 - 1.5, a portion was reserved for plasmid recovery (T0 sample), and the remaining culture was split and diluted into SC-His-Leu liquid media containing 10 mg/L DOX and either 2% glucose or 2% raffinose/1% galactose to OD600 = 0.1. Cultures were subjected to three growth and dilution cycles in which they were grown to an OD600 of 1 - 2 and diluted back to an OD600 of 0.1, saving a portion for plasmid recovery at each step (T1 – T3 samples). After the screen was complete, plasmids were recovered from cell pellets using a QIAGEN Spin Miniprep kit. The N-terminal variable region of the plasmid pool was PCR amplified to attach sequencing adaptors and add barcodes and sequenced on an Illumina NovaSeq instrument. The relative representation of each cofilin variant at each timepoint was calculated by dividing the number of reads that variant by the total read count. The enrichment or depletion of a cofilin-1 library sequence in the absence of LIMK1 induction was calculated as the ratio of its relative representation at T0 and T2 in glucose. Enrichment or depletion following LIMK1 induction was calculated from timepoints T1 and T3 in glucose or galactose. Sequences were ranked by the average log fold changes in representation across three replicate screens. Data were processed using Microsoft Excel.

first: Joel

middle: A

last: Sexton

first: Benjamin

middle: E


title: Gene regulatory network analysis predicts cooperating transcription factor regulons required for FLT3-ITD+ AML growth [ChIP-seq]

status: Public on Nov 20 2023

city: Birmingham

first: Peter

last: Keane

address: Vincent Drive

department: Institute for Cancer and Genomic Sciences

country: United Kingdom

institute: University of Birmingham

summary: AML is a heterogenous disease caused by different mutations. We have previously shown that each mutational sub-type develops its specific gene regulatory network (GRN) with transcription factors interacting with multiple gene modules, many of which are transcription factor genes themselves. Here we hypothesized that highly connected nodes within such networks comprise crucial regulators of AML maintenance. We tested this hypothesis using FLT3-ITD mutated AML as model and conducted an shRNA drop-out screen informed by this analysis. Our screen showed that AML-specific GRNs are predictive for identifying crucial regulatory modules required for AML but not normal cellular growth. Furthermore, our work shows that all modules are highly connected and regulate each other. The careful multi-omic analysis of the role of one (RUNX1) module by shRNA and chemical inhibition shows that this transcription factor and its target genes stabilize the GRN of FLT3-ITD AML and that its removal leads to GRN collapse and cell death

overall_design: RUNX1 ChIP-Seq in FLT3-ITD primarly patient cells with CBFBi and control

first: Daniel

last: Coleman

last: Cockerill

first: Constanze

last: Bonifer


title: Deciphering cell states and genealogies of human hematopoiesis with single-cell multi-omics [Young 1]

city: Boston

last: Weng

state: OH

address: 415 Main St, Cambridge

department: Genetics

institute: Broad Institute and Whitehead Institute

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

overall_design: We developed single-cell Regulatory multi-omics with Deep Mitochondrial mutation profiling (ReDeeM), which is a modified massive parallel single cell protocol to simultaneously profile multi-omics with deep mtDNA sequencing based on the 10X Genomics platform. With ReDeeM, three separate libraries are generated, including enriched mtDNA library for deep sequencing and mutation profiling, RNA library for gene expression, as well as ATAC library for chromatin accessibility profiling, all of which are linked via matchable single cell barcodes. Following the principle of our previous work, we firstly modified the droplet-based 10X genomics multi-omics protocol (Catalog #100283) by processing the whole cell instead of nuclei with fixation and mild permeabilization to maximally retain mitochondrial DNA. Next, we designed mtDNA specific probe sets to enrich the mitochondrial fragments using DNA hybridization. The RNA and ATAC library preps follow the standard 10X protocol with some modifications.  More method details are described in the method section and an open ReDeeM Protocol is available. ReDeeM is further computationally supported by consensus variant calling pipeline redeemV , and an inhouse R package redeemR for the downstream mutation quality control, as well as single cell phylogenetic and integrative analysis.

last: van Galen

first: Vijay

middle: G

last: Sankaran

first: Jonathan

middle: S

last: Weissman


title: Deciphering cell states and genealogies of human hematopoiesis with single-cell multi-omics [Aged 2]


title: Deciphering cell states and genealogies of human hematopoiesis with single-cell multi-omics [Aged 1]

last: van  Galen


title: Chemically defined and scalable culture system for intestinal stem cells derived from human intestinal organoids (scRNA-Seq)

city: Daejeon

first: Mi-Young

last: Son

address: 111 Gwahangno, Yuseong-gu

country: South Korea

institute: Korea Research Institute of Bioscience and Biotechnology (KRIBB)

summary: we established a human intestinal stem cell (ISC) culture method expanded under feeder-free and fully defined conditions through selective enrichment of ISC populations (ISC3D-hIO) within hIO derived from human pluripotent stem cells.

overall_design: Single cell RNA-sequencing of human intestinal stem cells (ISCs) derived from 3D human intestinal organoids (3D HIOs)

first: Ohman

last: Kwon


title: Gene regulatory network analysis predicts cooperating transcription factor regulons required for FLT3-ITD+ AML growth [ATAC-seq]

overall_design: ATAC-Seq of FLT3-ITD AML patient cells treated with CBFBi, FLT3i, shRNA and controls


title: Characterisation of the sensory phenotype of the oesophageal mucosa in gastroesophageal reflux disease

city: London

first: Ahsen

last: Ustaoglu

state: England

address: Blizard Institute

institute: Queen Mary University of London

summary: Identification of Novel Immune Cell Signature in Gastroesophageal Reflux Disease: Altered Mucosal Mast Cells and Dendritic Cell Profile. The mechanisms underlying the most troublesome symptom of gastroesophageal reflux disease (GERD), heartburn, remain incompletely understood. The pathogenesis of heartburn in GERD is likely to involve not only central mechanisms of sensitization including hypervigilance, but also multiple mucosal factors including maintenance of epithelial barrier integrity via tight junction proteins, expression of acid-sensing ion channels on nerve endings, and mucosal inflammation . We hypothesized  that immune cell components play a regulatory role in mucosal mechanisms of sensitization in GERD, and aimed to identify differences in the immune cell signature and sensory mucosal markers between reflux phenotypes and healthy asymptomatic subjects.

overall_design: A total of 37 patients with heartburn symptoms were phenotyped endoscopically and with objective reflux studies into  erosive reflux disease (ERD) (N=10), nonerosive reflux disease (NERD) (N=9), functional heartburn (FH) (N=9), and Barrett’s esophagus (BE) (N=9). RNA extracted from mucosal biopsies taken at endoscopy from 37 GERD patients and 8 healthy controls bulk mRNA sequencing, and sequencing data was analysed using Partek Flow. Immune enrichment analysis was performed using xCell.

first: Philip

last: Woodland

first: Madusha

last: Peiris

last: Sifrim

first: David

last: Kelsell

first: Diana

last: Blaydon

first: James

last: Boot


title: Active growth signalling promotes senescence and cancer cell sensitivity to CDK7 inhibition

status: Public on Nov 17 2023

city: Toronto

first: Micheal

middle: D

last: Tyers

state: Ontario

address: 555 University Avenue

country: Canada

institute: The Hospital for Sick Children

summary: Tumour growth is driven by continued cellular growth and proliferation. Cyclin Dependent Kinase 7’s (CDK7) role in activating mitotic CDKs and globalgene expression makes it therefore an attractive target for cancer therapies. However, what makes cancer cells particularly sensitive to CDK7 inhibition (CDK7i) remains unclear. Here we address this question. We show that CDK7i, by samuraciclib, induces a permanent cell cycle exit, known as senescence, without promoting DNA damage signalling or cell death. A chemogenetic genome-wide CRISPR knock-out screen identified that active mTOR (mammalian target of rapamycin) signalling promotes samuraciclib-induced senescence. mTOR inhibition decreases samuraciclib sensitivity, and increased mTOR-dependent growth signalling correlates with sensitivity in cancer cell lines. Reverting a growth promoting mutation in PIK3CA to wild-type decreases sensitivity to CDK7i. Our work establishes that enhanced growth alone promotes CDK7i sensitivity, providing an explanation for why some cancers are more sensitive to CDK inhibition than normally growing cells.

overall_design: Genome-wide CRISPR/Cas9 screen to test for gene deletions that affect fitness in human cells treated with CDK7 inhibitor samuraciclib.

first: Gemma

last: Wilson

first: Karla

last: Vuina

first: Georgina

last: Sava

first: Caroline

last: Huard

first: Leticia

last: Meneguello

first: Jasmin

last: Coulombe-Huntington

first: Thierry

last: Bertomeu

first: Rory

middle: J

last: Maizels

first: Josh

last: Lauring

first: Janos

last: Kriston-Vizi

first: Michael

first: Simak

last: Ali

first: Cosetta

last: Bertoli

first: Robertus

last: de Bruin


title: Deciphering cell states and genealogies of human hematopoiesis with single-cell multi-omics [Young2]

first: Chen

last: Weng

first: Peter

last: van Galen

first: Vijay

last: Sankaran

first: Jonathan

last: Weissman


title: Chemically defined and scalable culture system for intestinal stem cells derived from human intestinal organoids

first: Mi-Young

last: Son

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

overall_design: Refer to individual Series


title: Transcriptome analysis of mammospheres from MCF-7 and T47D cells

city: Yogyakarta

first: Adam

last: Hermawan

state: DI Yogyakarta

address: Sekip Utara II

department: Pharmacy

country: Indonesia

institute: Universitas Gadjah Mada

summary: Breast cancer cells were cultured in spheroids and analyzed mRNA expression through mRNA sequencing.

overall_design: We performed mRNA sequencing to understand differentially expressed genes in mammospheres from two different breast cancer cells, MCF-7 and T47D.

first: Herwandhani

last: Putri

first: Nurul

last: Fatimah

first: Heri

middle: H

last: Prasetio


title: Deciphering cell states and genealogies of human hematopoiesis with single-cell multi-omics [Young1_T2]


title: Vasopressin V2 Receptor, Tolvaptan, and ERK1/2 Phosphorylation in the Renal Collecting Duct II

city: Bethesda

first: Lihe

last: Chen

state: MD

address: 10 center drive MSC 1603, Building 10, Room 6N228

institute: NHLBI

summary: Activation of ERK1 and ERK2 is essential in regulation of a wide variety of cellular and physiological processes. In native inner medullary collecting ducts, vasopressin (AVP) working through the V2 subtype vasopressin receptor (V2R)-mediated activation of Gαs, inhibits ERK1 and ERK2 activity. However, it has been reported that V2R can signal independently of Gαs through the activation of β-arrestin, which activates ERK1 and ERK2. Vaptans, V2R antagonists that function as so-called “inverse agonists”, have the potential of promoting cell proliferation via β-arrestin-dependent ERK activation. Here we use the mpkCCD cell line which natively expresses V2R to investigate the effects of AVP, the V2-selective analog dDAVP, and tolvaptan on ERK1 and ERK2 phosphorylation and activation. We demonstrated that ERK1 and ERK2 phosphorylation in mpkCCD cells was significantly reduced by either AVP or dDAVP, in contrast to the increases seen in non-collecting duct cells overexpressing V2R. We also found that tolvaptan has a strong effect to increase ERK1 and ERK2 phosphorylation in the presence of dDAVP and that the tolvaptan effect to increase ERK1 and ERK2 phosphorylation is absent in PKA-null mpkCCD cells. Thus, it appears that the tolvaptan effect to increase ERK activation is PKA-dependent and, therefore, not mediated by the β-arrestin pathway. Overall, the studies show that AVP decreases and that tolvaptan increases ERK1 and ERK2 activation in cells expressing V2R at endogenous levels, and provide no evidence for a role for β-arrestin in the regulation of ERK1 and ERK2 activity.

overall_design: Here we use the mpkCCD cell line to address the following questions regarding the endogenously expressed V2 vasopressin receptor: 1) What are the effects of AVP and dDAVP on ERK1 and ERK2 phosphorylation/activation? 2) What is the effect of tolvaptan on ERK1 and ERK2 phosphorylation/activation in the presence and absence of dDAVP? 3) What is the role of PKA in vasopressin-mediated regulation of ERK1 and ERK2 phosphorylation/activation and on the effect of tolvaptan?


title: VEGFA mRNA-LNP promote biliary epithelial cell-to-hepatocyte conversion in acute and chronic liver diseases and reverses steatosis and fibrosis

first: Pushpinder

last: Bawa

state: Massachusetts

address: 670 Albany St

department: Boston University Medical Campus

institute: Center for Regenerative Medicine

summary: The goal of this experiment was to analyse transcriptomic changes between Biliary epithelial cells or BECs from the liver of mice treated with Acetaminophen (a drug that causes acute liver injury) in combination with AAV8-Tbg-p21 viral vector to induce hepatocyte senecence. The aim was to understand the impact of injury on transcriptional status of biliary epithelial cells. Briefly, we analyzed mouse liver cells (hepatocytes and non-parenchymal cells fractions) obtained by in-situ liver perfusion 48 h after injury. We further extracted cells corresponding to the hepatocyte and biliary epithelial cell gene signature for detailed analysis. Two separate clusters of BECs (BEC1 and BEC2) were further identified and analyzed to study their transcriptional changes and corresponding pathways.

overall_design: Two male mice are treated with AAV8-Tbg-p21 viral vectors one week prior to injection with acute liver injury causing drug, acetaminophen. 48 h after injury, liver is perfused in-situ and digested to obtain liver cells as two fractions. In the first step, hepatocyte fraction is recovered which is composed of predominantly hepatocytes and also some non-parenchymal cells. This was followed by subsequent liver digestion to procure non-parenchymal cells (NPCs) fraction consisting of blood cells, immune cells, cholangiocytes, stellate cells as well as smaller hepatocytes. Both hepatocytes and NPCs fractions from each mouse were harvested and submitted separately for library preparation.

first: Fatima

last: Rizvi

first: Valerie

last: Gouon-Evans


title: Tachycardia-induced metabolic rewiring as a driver of contractile dysfunction

city: Palo Alto

first: Yu

last: Liu

state: CA

address: 1215 Welch Road

department: CVI

institute: Stanford University

summary: Bulk RNA-Sequencing of EHTs subjected to tachypacing to probe the mechanism of tachycardia-induced cardiac dysfunction.

overall_design:
EHTs were made from iPSC derived cardiomyocytes and matured. EHTs were paced at 3Hz for varying durations to probe the effect of tachycardia on cardiac transcriptome.
*** Submitter states that missing raw files are due to patient privacy concerns. ***

first: Chengyi

last: Tu


title: Integrative single-cell transcriptome analysis of human pancreatic cancer reveals an intermediate cancer cell population associated with poor prognosis [RNA]

first: Jong-Eun

last: Park

address: 291, Daehak-ro, Yuseong-gu

institute: Korea Advanced Institute of Science and Technology

summary: We identified five distinct functional subclusters of pancreatic cancer cells and six distinct cancer-associated fibroblast subclusters. We deeply profiled their characteristics, and we found that these subclusters successfully deconvoluted most of the features suggested in bulk transcriptome analysis of pancreatic cancer. Among those subclusters, we identified a novel cancer cell subcluster, Ep_VGLL1, showing intermediate characteristics between the extremities of basal-like and classical dichotomy, despite its prognostic value. Molecular features of Ep_VGLL1 suggest its transitional properties between basal-like and classical subtypes, which is strongly supported by spatial transcriptome data.

overall_design: We performed single cell RNA sequencing using enriched non-immune cell populations from 17 pancreatic cancer tumor tissues, and also generated paired spaital transcriptome data from 7 of the patient samples. We also generated immune cell scRNA-seq dataset from 3 pancreatic cancer tumor tissues.

first: Galam

last: Leem

first: Seongryong

last: Kim


title: Integrative single-cell transcriptome analysis of human pancreatic cancer reveals an intermediate cancer cell population associated with poor prognosis


title: Assay for Transposase-Accessible Chromatin using sequencing of PD-1+ Tfh cells and CD62L+ Tfh cells

city: Beijing

first: Chen

last: Dong

address: Haidian District

department: Institute for Immunology and School of Medicine

institute: Tsinghua University

summary: T follicular helper (Tfh) cells, essential for germinal center reactions, are not identical, with different phenotypes reported. Whether, when and how they generate memory cells are still poorly understood. Here, through single-cell RNA-sequencing analysis of CXCR5+Bcl6+ Tfh cells generated under different conditions, we discovered, in addition to PD-1hi effector Tfh cells, a CD62L+PD1low subpopulation. CD62L-expressing Tfh cells developed independently from PD-1+ cells, and not in direct contact with B cells. More importantly, CD62L+ Tfh cells expressed memory- and stemness-associated genes; with more superior long-term survival, they readily generated PD-1hi cells in the recall response. Finally, KLF2 and IL7R, also highly expressed by CD62L+ Tfh cells, were required to regulate their development. Our work thus demonstrates a novel Tfh memory-like cell subpopulation, which may benefit our understanding on immune responses and diseases.

overall_design: PD1+ Tfh cells, CD62L+ Tfh cells, non-Tfh effector cells were sorted from KLH immunized Bcl6RFPFoxp3GFP dual reporter mice at day 8 post immunization. The genome DNA were extracted and performed ATAC-sequencing

first: Han

last: Feng

first: Xiaohong

last: Zhao


title: Global gene expression profile of PD1+ Tfh cells, CD62L+ Tfh cells, non-Tfh effector cells

overall_design: PD1+ Tfh cells, CD62L+ Tfh cells, non-Tfh effector cells were sorted from KLH immunized Bcl6RFPFoxp3GFP dual reporter mice at day 8 post immunization. The total RNA were extracted and sequenced


title: Depletion of SAM leading to loss of heterochromatin drives muscle stem cell ageing

city: Los Angeles

first: Jengmin

last: Kang

address: 615 Charles E Young Dr S

institute: University of California, Los Angeles

summary: The global loss of heterochromatin during aging has been observed in eukaryotes from yeast to humans, and this has been proposed to be one of the causes of aging. However, the cause of this age-associated loss of heterochromatin has remained enigmatic. Here we show that heterochromatin markers, including histone H3K9 di-/tri-methylation and HP1, decrease with age in murine muscle stem cells (MuSCs) as a consequence of the depletion of the methyl donor SAM. We find that restoration of intracellular SAM in aged MuSCs restores heterochromatin content to youthful levels and rejuvenates age-associated features including DNA damage accumulation, increased cell death, and defective muscle regeneration. SAM is not only a methyl group donor for transmethylation but it is also an aminopropyl donor for polyamine synthesis.  Excessive consumption of SAM in polyamine synthesis may reduce its availability for transmethylation. Consistent with this premise, we observe that perturbation of increased polyamine synthesis by inhibiting spermidine synthase (Srm) restores the intracellular SAM content as well as heterochromatin formation, leading to improvements in aged MuSC function and regenerative capacity. Together, our studies demonstrate a direct causal link between polyamine metabolism and epigenetic dysregulation during MuSC aging.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for FACS-isolated muscle stem cells from young (3-month-old) and old (20-month-old) C57BL/6 mice.


overall_design: Chromatin accessibility analysis of ATAC-seq data for FACS-isolated muscle stem cells from young (3-month-old) mice, old (20-month-old) mice treated with vehicle, MCHA, or SAM.


title: BACH1 deficiency impedes differentiation of vascular smooth muscle cell from hESCs via reducing the CARM1-catalyzed H3R17me2

city: Shanghai

first: Jieyu

last: Guo

state: Shanghai

address: 138 Yixueyuan Rd,

institute: fudan university

summary: Vascular smooth muscle cells (VSMCs) derived from human embryonic stem cells (hESCs) have been considered as a potential therapeutic application in vascular diseases. The transcription factor BTB and CNC homology 1 (BACH1) participates in stem cell development and has an increased expression during the VSMCs differentiation process. Knockout of BACH1 inhibits the differentiation of hESCs into VSMCs, whereas overexpression of BACH1 promotes VSMCs differentiation after the mesoderm stage. Mechanistically, BACH1 interacts with Coactivator-associated arginine methyltransferase 1 (CARM1) in a bZIP domain-dependent manner. BACH1 recruits CARM1 and its specific histone mark H3R17me2 to VSMC marker genes promoters, thus up-regulating target gene expression. BACH1-induced promotion of VSMC marker genes was partially abolished by the knockdown of CARM1 or H3R17me2. Thus, our results demonstrate the regulatory role of BACH1 and CARM1 in VSMCs differentiation.

overall_design: RNA-Seq + CUT&Tag

first: Dan

last: Meng

first: Yunquan

last: He


title: T3 and Glucose Increase Expression of Phosphoenolpyruvate Carboxykinase (Pck1) Leading to Increased ß-Cell Proliferation

city: New York

first: Liora

middle: s

last: Katz

state: NY

address: One Gustave L. Levy Place, Box 1152, Atran building 5-17

institute: Icahn School of Medicine at Mount Sinai

summary: Thyroid hormone (T3) and high glucose concentrations are critical components of β-cell maturation and function. In the present study, we asked whether T3 and glucose signaling pathways coordinately regulate transcription of genes important for β-cell function and proliferation

overall_design: RNA-seq analysis of cadaveric human islets from five different donors in response to low and high glucose concentrations (6 and 20 mM, respectively) and in the presence or absence of T3 (10 nM). All donors were between the age of 24 to 61 and with body mass index (BMI) of 18-26

first: Donald

middle: K

last: Scott


title: Impact of the dopamine transporter knockdown in human colorectal cancer cells

city: Ottawa

first: Yannick

middle: D.

last: Benoit

address: 451 Smyth road

institute: University of Ottawa

summary: Here, we performed transcriptome-profiling experiments on control scramble shRNA and shSLC6A3 HCT116 cells to study the impact DAT silencing on gene networks associated with neoplastic stemness. We also treated control HCT116 cells with the DAT agonist vanoxerine (VXN) to identify commonly regulated networks between DAT knockdown and pharmacological antagonism.

overall_design: RNA-seq transcriptome profiling, HCT116 human cell line. 3 replicates each of: shCTRL DMSO-treated, shSLC6A3 DMSO-treated,  shCTRL VXN-treated

first: Christopher

last: Bergin


title: Analysis of the role of SMARCB1 in bladder cancer cell line T24 [ATAC-seq]

city: Houston

first: H. Courtney

last: Hodges

state: TX

address: 1 Baylor Plaza, BCM 229

department: Department of Molecular & Cellular Biology

institute: Baylor College of Medicine

summary: To identify DNA accessibility targets regulated by the SWI/SNF subunit SMARCB1 in bladder cancer, we compared the ATAC-seq signals in T24 cells engineered for SMARCB1 knockout, non-targeting control, or SMARCB1 re-expression following knockout. Analysis of altered DNA accessibility profiles revealed new roles for SMARCB1 in the regulation of gene expression in bladder cancer, and suggested new therapeutic opportunities.

overall_design: ATAC-seq analysis of control T24 cells, SMARCB1 knockout T24 cells, and SMARCB1 rescue T24 cells.

first: Chandra

last: Amara

first: Yuen

last: San Chan

first: Pavlos

last: Msaouel

first: H

middle: C

first: Nagireddy

last: Putluri


title: Dysregulated lncRNAs regulate human umbilical cord mesenchymal stem cell differentiation into insulin-producing cells by forming a regulatory network with mRNAs

status: Public on Nov 16 2023

city: Nanchang

first: Jianping

last: Liu

address: Building 1, Central University

institute: The Second Affiliated Hospital of Nanchang University

type: Non-coding RNA profiling by high throughput sequencing

summary: In recent years, cell therapy has emerged as a new research direction in the treatment of diabetes. However, the underlying molecular mechanisms of mesenchymal stem cell (MSC) differentiation necessary to form such treatment have not been clarified.

overall_design: In this study, human umbilical cord mesenchymal stem cells (HUC-MSCs) isolated from newborns were progressively induced into insulin-producing cells (IPCs) using small molecules. HUC-MSC (S0) and four induced stage (S1-S4) samples were prepared. We then performed transcriptome sequencing experiments to obtain the dynamic expression profiles of both mRNAs and long noncoding RNAs (lncRNAs).

first: Tianqin

last: Xie

first: Qiming

last: Huang

first: Qiulan


title: Integrative single-cell transcriptome analysis of human pancreatic cancer reveals an intermediate cancer cell population associated with poor prognosis [Spatial]

first: Jong-Eun

last: Park

overall_design: We performed single cell RNA sequencing using enriched non-immune cell populations from 17 pancreatic cancer tumor tissues, and also generated paired spatial transcriptome data from 7 of the patient samples.

first: Galam

last: Leem

first: Junjeong

last: Choi

first: Seongryong

last: Kim


title: miR-238, miR-239a, and miR-239b microRNAs in Caenorhabditis elegans Aging

city: Cambridge

first: Laura

last: Chipman

state: MA

address: 1 Portland St

institute: Pfizer

summary: MicroRNAs (miRNAs) regulate gene expression by base-pairing to target sequences in messenger RNAs (mRNAs) and recruiting factors that induce translational repression and mRNA decay. In animals, nucleotides 2-7 at the 5-prime end of the miRNA, called the seed region, are often necessary and sometimes sufficient for functional target interactions. MiRNAs that contain identical seed sequences are grouped into families where individual members have the potential to share targets and act redundantly. A rare exception seemed to be the miR-238/239ab family in C. elegans, as previous work indicated that loss of miR-238 reduced lifespan while deletion of the miR-239ab locus resulted in enhanced longevity and thermal stress resistance. Here, we re-examined these potentially opposing roles using new strains that individually disrupted each miRNA sister. We confirmed that loss of miR-238 is associated with a shortened lifespan but could detect no longevity or stress phenotypes in animals lacking miR-239a or miR-239b, individually or in combination. Additionally, dozens of genes were mis-regulated in miR-238 mutants but almost no gene expression changes were detected in miR-239a or miR-239b mutants compared to wild type animals. We present evidence that the lack of redundancy between miR-238 and miR-239ab is independent of their sequence differences; miR-239a or miR-239b could substitute for the longevity role of miR-238 when expressed from the miR-238 locus. Altogether, these studies disqualify miR-239ab as negative regulators of aging and demonstrate that expression, not sequence, dictates the specific role of miR-238 in promoting longevity.

overall_design: Small RNA sequencing was performed on five independent replicates of synchronized wildtype (N2), alg-1(gk214), and alg-2(ok304) strains collected on day 5 of adulthood. RNA sequencing from wildtype (N2), miR-238(n4112), miR-239a(ap439), and miR-239b(ap432) day 5 adults.

middle: B

first: San

last: Luc

first: Ian

last: Nicastro

first: Jesse

last: Hulahan

first: Delaney

last: Pagliuso

first: Devavrat

middle: M

last: Bodas

first: Amy

last: Pasquinelli


title: Distinct mouse models of Stargardt disease display differences in pharmacological targeting of ceramides and inflammatory responses

city: Warsaw

first: Marcin

last: Tabaka

address: Kasprzaka 44/52

country: Poland

institute: International Centre for Translational Eye Research

summary: We wanted to transcriptomically compare single cells taken from the retinas of C57BL6/J mice, Abca4-/-Rdh8-/- (dKO) mutant mice, and Abca4PV/PVRdh8-/- (PVR) mutant mice before and at an early (24-hour post-bleach) timepoint post-bleaching, to see if the two Abca4 mutants displayed different transcriptomic shifts in response to intense light, which initiates retinal degeneration. Additionally, we wanted to determine if the two mutants had different transcriptional responses to the immunomodulatory drug maraviroc. The wild-type C57BL6/J mice were included as controls for each treatment condition. We profiled the three cohorts of mice under three separate treatment conditions: without light-induced degeneration, 24-hours post-bleaching with intense light and vehicle treatment, and 24-hours post-bleaching with intense light and maraviroc treatment. Single cell isolates were collected, fixed, barcoded, and sequenced using kits from Parse Biosciences.

overall_design: In each of the six experimental groups, pairs of retinae from three mice were independly processed; the resulting single-cell suspensions were fixed and frozen using the Parse Biosciences WT V2 chemistry fixation kit instructions. For each of the 27 independent biological samples, the fixed cells were simultaneously thawed and suspensions were loaded into independent wells on a Parse Biosciences WT V2 barcoding plate for library preparation (~2,360 cells/ biological sample), according to the manufacturer's instructons.

first: Zachary

last: Engfer

first: Damian

last: Panas


title: Antitumor efficacy of sequence specific DNA-targeted gamma PNA based cMyc inhibitor

city: Storrs

first: Shipra

last: Malik

address: 69 North Eagleville Road, Unit 3092

department: Pharmaceutical Sciences

institute: University of Connecticut

summary: Targeting oncogenes at the genomic DNA level can open new avenues for precision medicine. Significant efforts are ongoing to target oncogenes using diverse RNA-targeted and protein-targeted platforms, but no progress has been made to target genomic DNA for cancer therapy. Here, we introduce a gamma peptide nucleic acid (PNA)-based genomic DNA-targeted platform to silence oncogenes in vivo. gPNAs can efficiently invade the mixed sequences of genomic DNA with high affinity and specificity. As a proof-of-concept, we established that gPNA could inhibit c-Myc transcription in multiple cell lines. In vivo efficacy and safety of the DNA-targeted approach were demonstrated in three pre-clinical models. We also established that anti-transcription gPNA in combination with histone deacetylase inhibitors (HDACi) and chemotherapeutic drugs results in high and robust antitumor activity in cell-line and patient-derived xenografts. Overall, this strategy offers a unique therapeutic platform to target genomic DNA to inhibit oncogenes for cancer therapy.

overall_design: human diffused large B cell lymphoma (U2932) cells were used in this study. Nucleic acid mediated knockdown of c-myc was achieved. mRNA profiles in U2932 cells after knockdown was compared with PBS treated cells after 48 and 72 hours.

first: Sai Pallavi

last: Pradeep

first: Vikas

last: Kumar

first: Yong

last: Xiao

first: Yanxiang

last: Deng

first: Rong

last: Fan

first: Juan

last: Vasquez

first: Vijender

last: Singh

first: Raman

last: Bahal


title: Smad4 sequestered in SFPQ condensates prevents TGF-β tumor-suppressive signaling

city: Hangzhou

first: Mu

state: Zhejiang

address: 866 Yuhangtang Road

department: Life Sciences Institute

institute: Zhejiang University

summary: Loss of TGF-beta growth-inhibitory responses is a hallmark of human cancer. However, the molecular mechanisms underlying the TGF-beta resistance of cancer cells remain to be fully elucidated. Splicing factor proline- and glutamine-rich protein (SFPQ) is a prion-like RNA-binding protein that is frequently upregulated in human cancers, such as Hepatocellular carcinoma (HCC). In this study, we identified SFPQ as a potent suppressor of TGF-beta signaling. The ability of SFPQ to suppress TGF-beta responses depended on its prion-like domain (PrLD) that drives phase separation (LLPS). Mechanistically, SFPQ physically restrained Smad4 in its condensates, which excluded Smad4 from the Smad complex and chromatin occupancy, and thus functionally dampened Smad-dependent transcriptional responses. Accordingly, SFPQ deficiency or loss of LLPS rendered cells hypersensitive to TGF-beta responses. Together, our data reveal a unique function of SFPQ through LLPS that suppresses Smad transcriptional activation and TGF-beta tumor-suppressive activity.

overall_design: mRNA profiles of six HCC cells siControl and siSFPQ cells with or without TGF-beta were generated by deep sequencing, using Illumina HiSeq X Ten. There are 24 samples for transcriptome sequencing (RNA-Seq).

first: Fei

last: Wang

first: Nuo

last: Chen

first: Hanchenxi

last: Zhang

first: Li

last: Shen

first: Xia

last: Lin

first: Xin-hua

last: Feng


title: Single cell RNA-sequencing of Tfh cells and Tfr cells

first: Chen

last: Dong

overall_design: Tfh and Tfr cells from indicated model were isolated and perform single cell RNA-sequencing

first: Han

first: Xiaohong

last: Zhao

first: Weiwei

last: Fu

first: Boxi

last: Kang


title: Pharmacological modulation of H3K9me2 deposition in human colorectal cancer cells and transformed ES cells

first: Yannick

last: Benoit

summary: Transformed variant of H9 human ES cells (t-hESC) represent a surrogate model for cancer stem cells in adherent cultures, which faithfully recapitulate the main functional, transcriptional, and epigenetic characteristics of CSCs in culture and in vivo. The use of G9a inhibitor BIX-01294 reduced the levels of H3K9me2 and global DNA methylation in t-hESCs. Thus, we performed transcriptome-profiling experiments on control and BIX-treated t-hESC to study the impact of G9a inhibition on gene networks associated with neoplastic stemness. Moreover, using a phenotypic chemical screening assay, we identified vanoxerine (VXN) as a new small molecule decreasing H3K9me2 deposition in t-hESCs. In addition to t-hESCs, transcriptome profiling was performed on colorectal cancer cell line HT29 upon treatment with VXN. In addition to annotaded genes, the analysis included the quantification of human retrotransposable elements.

overall_design: RNA-seq transcriptome profiling, HT29 and t-hESC human cell line. 2 biological replicates of each condition.

first: Christopher

last: Bergin


title: CBir1 T cells acquire an effector phenotype during inflammation (ATAC-seq)

status: Public on Nov 15 2023

city: Stockholm

first: Eduardo

last: Villablanca

address: CMM

department: Department of Medicine

country: Sweden

institute: Karolinksa Institute

summary: CD4+ T cells specific for commensal bacterial antigens are expanded in inflammatory bowel disease (IBD) patients compared to healthy individuals. How and where commensal-specific CD4+ T cells get activated is yet to be fully understood. We used TCR-transgenic CD4+ T cells specific to a commensal bacterial antigen (CBir1 T cells) and the dextran sulfate sodium (DSS) model of IBD to characterize how and where the activation of commensal-specific CD4+ T cells occurs. We found that CBir1 T cells proliferate following intestinal damage and cognate antigen presentation is mediated by CD11c+ cells in the colon-draining mesenteric lymph nodes (cMLNs). Using assay for transposase-accessible chromatin sequencing (ATAC-seq) and flow cytometry, we showed that activated CBir1 T cells preferentially acquire an effector rather than regulatory phenotype, which is plastic over time.

overall_design: To understand the functional polarization of CBir1 cells during intestinal inflammation, we analyzed the chromatin landscape of CBir1 T cells following DSS-mediated activation. Naïve CD4+ CBir1 T cells were enriched from the spleen of CBir1 transgenic mice, using Miltenyi kit 130-104-453, stained with CellTraceTM Violet and injected into four WT C57BL/6J recipient mice. Starting on the following day, recipient mice were treated with 2% dextran sulfate sodium (DSS) in drinking water for 7 days, followed by normal drinking water, to induce intestinal barrier disruption. Recipient mice received 1 mg/kg of body weight FTY720 on days 7, 10 and 13 after the beginning of DSS administration, to retain cells in the lymph nodes. On day 14 after the beginning of DSS treatment, we isolated cells from the colon-draining mesenteric lymph nodes (cMLNs) of recipient mice, and sorted them into 3 fractions: 1) undivided (CTVhi) CBir1 T cells, 2) divided (CTVdim) CBir1 T cells, and 3) total non-CBir1 endogenous CD4+ T cells (polyclonal Th cells). Analogous fractions from two mice were pooled, to guarantee a sufficient number of cells for library construction, in duplicate. Libraries were prepared using the method described in Buenrostro et al. (Nature Methods 2013).

first: Chiara

last: Sorini

first: Rebeca

middle: F

last: Cardoso

first: Kumar

middle: P

last: Tripathi


title: Carnosine Regulation of Intracellular pH Homeostasis Promotes Lysosome-dependent Tumor Immunoevasion

city: Hefei

first: Ronghui

last: Yan

state: Anhui

address: Huangshan road 443

institute: University of Science and Technology of China

summary: To explore which genes are suppressed by NFX1 under hypoxic conditions, we performed RNA-seq analysis in PLC cells overexpressing EV or NFX1 under hypoxia.

first: Pinggen

first: Ping

last: Gao

first: Huafeng


title: Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA [ATAC-seq]

city: Boston

first: Richard

middle: Stefan

last: Isaac

address: 77 Avenue Louis Pasteur, NRB 356

institute: Harvard Medical School

summary: Cellular metabolism relies on the regulation and maintenance of mitochondrial DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist in each cell, yet because mitochondria lack histones or other machinery important for nuclear genome compaction, it remains unresolved how mtDNA is packaged into individual nucleoids. In this study, we used long-read single-molecule accessibility mapping to measure the compaction of individual full-length mtDNA molecules at near single-nucleotide resolution. We found that, unlike the nuclear genome, human mtDNA largely undergoes all-or-none global compaction, with most nucleoids existing in an inaccessible, inactive state. Highly accessible mitochondrial nucleoids are co-occupied by transcription and replication components and selectively form a triple-stranded displacement loop structure. In addition, we showed that the primary nucleoid-associated protein TFAM directly modulates the fraction of inaccessible nucleoids both in vivo and in vitro, acting consistently with a nucleation-and-spreading mechanism to coat and compact mitochondrial nucleoids. Together, these findings reveal the primary architecture of mtDNA packaging and regulation in human cells.

overall_design: ATAC-seq performed on mitochondria isolated from human HeLa S3 cells to assess bulk averaged DNA accessibility across the human mitochondrial genome.

first: Thomas

middle: W

last: Tullius

first: Katja

last: Hansen

first: Danilo

last: Dubocanin

first: Mary

last: Couvillion

first: Andrew

last: Stergachis

first: Stirling

middle: L

last: Churchman


title: Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA

overall_design: PacBio sequencing of exogenously methylated mitochondrial DNA from: HeLa cells; HeLa cells with perturbed TFAM levels, inhibited mitochondrial transcription, and disrupted complex III function; human skeletal myoblasts across three differentiation time points; U2OS cells; and in vitro generated mtDNA with varying levels of recombinant TFAM.

first: L


title: Effect of RBM5 knockout or overexpression on transcription in MOLM13 AML cells

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:
Background: The oncogenic protein HOXA9 plays a critical role in leukemia transformation and maintenance, and its aberrant expression is a hallmark of most aggressive acute leukemia. Although inhibiting the upstream regulators of HOXA9 has been proven as a significant therapeutic intervention, the comprehensive regulation network controlling HOXA9 expression in leukemia has not been systematically investigated.

Results: Here, we perform genome-wide CRISPR/Cas9 screening in the HOXA9-driven reporter acute leukemia cells. We identify a poorly characterized RNA-binding protein, RBM5, as the top candidate gene required to maintain leukemia cell fitness. RBM5 is highly overexpressed in acute myeloid leukemia (AML) patients compared to healthy individuals. RBM5 loss triggered by CRISPR knockout and shRNA knockdown significantly impairs leukemia maintenance in vitro and in vivo. Through domain CRISPR screening, we reveal that RBM5 functions through a noncanonical transcriptional regulation circuitry rather than RNA splicing, such an effect depending on DNA-binding domains. By integrative analysis and functional assays, we identify HOXA9 as the downstream target of RBM5. Ectopic expression of HOXA9 rescues impaired leukemia cell proliferation upon RBM5 loss. Importantly, acute protein degradation of RBM5 through auxin-inducible degron system immediately reduces HOXA9 transcription.

Conclusions: We identify RBM5 as a new upstream regulator of HOXA9 and reveal its essential role in controlling the survival of AML. These functional and molecular mechanisms further support RBM5 as a promising therapeutic target for myeloid leukemia treatment.

Keywords: Acute myeloid leukemia; CRISPR screen; Genome editing; HOXA9; RBM5.

overall_design: To investigate the RNA binding protein, RBM5, in the regulation of MLL-r leukemia maintenance, we established CRISPR KO MOLM13 cell lines in which RBM5 has been targeted by Cas9/sgRNA against coding exons. Lentiviral cDNA was delivered in to MOLM13 for overexpression. We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 biological replicates in sgNT, sgRBM5, OE RBM5 groups. Differential gene expression was compared between sgNT vs sgRBM5,  NT. Vs RBM5 OE.

first: Mengli

last: Zhang

first: Chunliang

last: Li

first: Peng


title: Prenatal alcohol exposure alters expression of genes involved in cell adhesion, immune response, and toxin metabolism in adolescent rat hippocampus

status: Public on Nov 14 2023

city: FORT WAYNE

first: Amal

last: Khalifa

state: Indiana

address: 2101 E. Coliseum Blvd, ET-125

department: Computer Science

institute: Purdue University  Fort Wayne

summary: Alcohol exposure during fetal development is associated with a wide range of behavioral and physical symptoms that are observed from childhood throughout adolescence and beyond. It is believed that this exposure may alter gene expression patterns permanently by changing genomic architecture, but the actual changes themselves are still unclear. In this study we examined gene expression patterns in rats exposed to ethanol during gestation. These were compared to control rats where dams were fed a similar liquid diet without ethanol as well as rats fed a normal diet. We identified the top differentially expressed genes and performed gene ontology analysis to identify both genes and pathways important in the response to ethanol during fetal development. We focused on adolescent rats since PAE has been shown at this stage to influence behavior. We indeed found a number of significant changes in gene expression, suggesting that PAE has ongoing consequences throughout and likely beyond adolescence into adulthood. Pathways such as cell adhesion, immune response, and toxin response were all highlighted. Future work will focus on making connections between these gene expression changes and behavioral changes observed at this same life stage.

overall_design: RNA was isolated  from the hippocampus of adolescent rats exposed to ethanol during prenatal development and compared gene expression to controls. Dams were  either given free access to standard chow ad libitum (AD), pair-fed a liquid diet (PF) or were given a liquid diet with ethanol (6.7% ethanol, ET) throughout gestation (gestational day (GD) 0-20). All dams were given control diet ad libitum beginning on GD 20 and throughout parturition and lactation. A total of 17 RNA samples were sequenced representing the three treatment groups: 5 ET samples (2 females and 3 males), 6 PF samples (3 Females and 3 Males), 6 AD samples (3 Females and 3 Males) with three technical replicates for each sample. Differential gene expression analysis was performed using the DESeq method as implemented within the Bioinformatics Toolbox in Matlab (R2022a).

first: Rebecca

last: Palu

first: Amy

middle: E

last: Perkins

first: Avery

last: Volz


title: Single-nucleoid architecture reveals heterogeneous packaging of mitochondrial DNA [RNA-seq]

summary: Cellular metabolism relies on the regulation and maintenance of mitochondrial DNA (mtDNA). Hundreds to thousands of copies of mtDNA exist in each cell, yet how mtDNA is packaged into individual mitochondrial nucleoids remains poorly resolved, as mitochondria lack histones or other chromatin machinery important for nuclear genome compaction. Here, we used long-read single-molecule accessibility mapping to resolve the compaction of individual full-length mitochondrial nucleoids at single-nucleotide resolution. We find that, unlike the nuclear genome, human mtDNA largely undergoes an all-or-none global compaction, with the majority of nucleoids existing in an inaccessible, inactive state. Highly accessible mitochondrial nucleoids are occupied by transcription and replication machinery, and selectively form a D-loop structure. In addition, we demonstrate that the primary nucleoid-associated protein TFAM directly modulates the fraction of inaccessible nucleoids both in vivo and in vitro, and acts via a ‘nucleation and spreading’ mechanism to coat and compact mitochondrial nucleoids. Overall, these findings expose the primary architecture of mtDNA packaging and regulation in humans.

overall_design: RNA-seq of human skeletal muscle myoblasts at three time points: 0d, 3d, and 6d in differentiation media.

middle: G


title: Multi-omic Analysis of Primary Human Kidney Tissues Identifies Medulla-Specific Gene Expression Patterns

city: Seattle

first: Shreeram

last: Akilesh

state: WA

address: 1959 NE Pacific St, Box 356100

department: Laboratory Medicine and Pathology

institute: University of Washington

summary: The renal medulla is a specialized region of the kidney with important homeostatic functions. It has also been implicated in genetic and developmental disorders and ischemic and drug-induced injuries. Despite its role in kidney function and disease, the medulla’s baseline gene expression and epigenomic signatures have not been well described in the adult human kidney. Here we generate and analyze gene expression (RNA-seq), chromatin accessibility (ATAC-seq), chromatin conformation (Hi-C) and digital spatial profiling data from adult human kidney cortex and medulla. Using data from our carefully annotated specimens, we assign samples in the larger public GTEx database to cortex and medulla, thereby identifying several misassignments and extracting meaningful medullary gene expression signatures. Using integrated analysis of gene expression, chromatin accessibility and conformation profiles, we reveal insights into medulla development and function. Our datasets will also provide a valuable resource for researchers in the GWAS community for functional annotation of genetic variants.

overall_design: Human nephrectomy tissues were fixed in formalin and paraffin embedded (FFPE). Single tissue sections of kidney cortex and medulla from 3 different donors (RAM012, 013, 015) were applied to 3 SuperFrost charged slides. In preparation for digital spatial profiling, the slides were baked and then subjected to heat-induced antigen retrieval and proteinase K digestion (1µg/ml). Human whole transcriptome atlas probes were applied to each slide and incubated at 37°C overnight. The following day, after stringent washes, slides were blocked and then counterstained with SYTO13 (labels DNA) and the following 2 antibodies: Alexafluor-532 conjugated anti-pan-cytokeratin (labels some epithelial cells in kidney) and Alexafluor-647 conjugated anti-CD10 (labels glomerular podocytes and proximal tubule cells in kidney). The slides were then loaded into the Nanostring GeoMx DSP instrument and scanned at 20x magnification (exposure times: SYTO13, 100ms; pan-CK, 400ms; CD10, 400ms). Regions of interest (ROI) were selected and bound probes were captured by the instrument. The captured probes were converted into an Illumina NGS compatible library using the SeqCode A barcode kit from Nanostring.  The library was QC'ed using an Agilent TapeStation and quantified using a Qubit fluorometer. The library was seqeunced using a NextSeq 2000 SP flowcell.


title: The nuclear factor ID3 endows macrophages with a potent anti-tumor activity

first: Zihou

last: Deng

address: 165 E 118th St, Apt 3N

department: Immunology program

institute: MSKCC

summary: Macrophage activation is controlled by a balance between activating and inhibitory receptors which protect normal tissues from excessive damage during infection but promote tumor growth and metastasis in cancer. We report here that the Kupffer cell (KC)-lineage determining factor ID3 controls this balance and selectively endows KC with the ability to phagocytose live tumor cells and orchestrate the recruitment, proliferation, and activation of NK and CD8 T lymphoid effector cells in the liver to restrict the growth of a variety of tumors. ID3 shifts the macrophage inhibitory/activating receptor balance to unleash the phagocytic and lymphoid response, at least in part by buffering binding of the transcription factors ELK1 and E2A at the Sirpα locus. Furthermore, loss- and gain-of-function experiments demonstrate that ID3 is sufficient to confer this potent anti-tumor activity to mouse bone marrow derived macrophages and human induced pluripotent stem cells derived macrophages. Expression of ID3 is therefore necessary and sufficient to endow macrophages with the ability to form an efficient anti-tumor niche, which could be harnessed for cell therapy in cancer.

overall_design: Kupffer cells were sorted from mice intraportal injected with KPC tumor for 2 weeks or from non tumor bearing mice. RNA were extracted for RNA sequencing.

first: Li

first: Bhavneet

last: Bhinder

first: Olivier

last: Elemento

first: Frederic

last: Geissmann


title: Gene based immune profiling of vermiform appendix GALT of adult patients with pancreatic cancer or colon diseases.

city: Rotterdam

first: Eveline

middle: Emma

last: Vietsch

state: South Holland

address: Dr. Molewaterplein 40

country: Netherlands

institute: Erasmus MC

summary: Differential immune gene expression analysis in appendix GALT between patients with pancreatic cancer versus benign or malignant colon disease using the nanostring ncounter platform.

overall_design: In this study the GALT of appendix was analyzed by RNA expression analysis of ~700 immune related genes using the Pan Cancer Immune Panel from nanostring. Non inflamed normal looking FFPE appendix tissue from sixteen pancreatic cancer patients, nine colon cancer patients, and nine benign colon disease patients was used to compare GALT immune profiles among the three groups.

first: Casper

last: van Eijck


title: BRAF and MEK inhibitor combinations induce potent molecular and immunological effects in NRAS-mutant melanoma cells – insights into mode of action and resistance mechanisms

city: Dresden

first: Dana

last: Westphal

address: Fetscherstrasse 74

department: Department of Dermatology

country: Germany

institute: University Hospital Dresden

summary: About 25% of melanoma harbor activating NRAS mutations, which are associated with aggressive disease therefore requiring a rapid anti-tumor intervention. However, no efficient targeted therapy options are currently available for patients with NRAS-mutant melanoma. MEK inhibitors (MEKi) appear to display a moderate anti-tumor activity and also immunological effects in NRAS-mutant melanoma, providing an ideal backbone for combination treatments. In this study, the MEKi binimetinib, cobimetinib, and trametinib combined with the BRAF inhibitors (BRAFi) encorafenib, vemurafenib, and dabrafenib were investigated for their ability to inhibit proliferation, induce apoptosis and alter the expression of immune modulatory molecules in sensitive NRAS-mutant melanoma cells using 2D and 3D cell culture models and transcriptome analyses. Furthermore, NRAS-mutant melanoma cells resistant to the three BRAFi/MEKi combinations were established to characterize the mechanisms contributing to their resistance. All BRAFi induced a stress response in the sensitive NRAS-mutant melanoma cells thereby significantly enhancing the anti-proliferative and pro-apoptotic activity of the MEKi analyzed. Furthermore, BRAFi/MEKi combinations upregulated immune relevant molecules, such as ICOS-L, components of antigen-presenting machinery and the “don’t eat me signal” molecule CD47 in the melanoma cells. The BRAFi/MEKi-resistant, NRAS-mutant melanoma cells counteracted the molecular and immunological effects of BRAFi/MEKi by upregulating downstream mitogen-activated protein kinase pathway molecules, inhibiting apoptosis and promoting immune escape mechanisms. Together, this study reveals potent molecular and immunological effects of BRAFi/MEKi in sensitive NRAS-mutant melanoma cells that may be exploited in new combinational treatment strategies for patients with NRAS-mutant melanoma.

overall_design: Bulk RNA sequencing was carried out  from the sensitive WM1366 melanoma cell line and 3 BRAF and MEK inhibitor-resistant daugther cell lines. The resistant cell lines were cultured in presence of the BRAF and MEK inhibitors, while the sensitive cell line was cultured in DMSO. Samples from 3 independent experiments were sequenced.

first: Alexander

last: Schulz

middle: A

last: Wurm

first: Mathias

last: Lesche

first: Andreas

last: Dahl


title: Microarray expression analysis depicting MHCII-dependent features of FOXP3-expressing CD4 T regulatory cells

city: Philadlephia

first: Daniel

last: Beiting

state: Pennsylvania

address: 380 S. University Avenue

institute: University of Pennsylvania

summary: Analysis comparing K14 and MCHII+/- Tregs. The hypothesis was that the lack of MHCII+/- in the spleen and intestine would result in a large shift in K14 Treg gene expression away from an activated and effector phenotype.  Results demonstrate how a lack of MHCII-antigen presentation  in the small intestine,  where Tregs exhibit an effector penotype, does not greatly reduce effector features of T regulatory cells in K14 mice.

overall_design: RNA was isolated from splenic and intestinal FOXP3 Tregs and conventional T cells from control MHCII+/-, FOXP3GFP mice and experimental K14Aβb, FOXP3GFP mice which lack expression of MHCII outside of the thymus. Mice were pooled for each condition.

first: Lisa

last: Korn

first: Terri

last: Laufer


title: Direct Reprogramming of Non-limb Fibroblasts to Cells with Properties of Limb Progenitors [bulkRNA-seq]

city: BOSTON

first: CHANGHEE

last: LEE

address: 77 AVENUE LOUIS PASTEUR

summary: The early limb bud consists of mesenchymal progenitors (limb progenitors) derived from the lateral plate mesoderm (LPM) that produce most of the tissues of the mature limb bud. The LPM also gives rise to the mesodermal components of the trunk, flank and neck. However, the mesenchymal cells generated at these other axial levels cannot produce the variety of cell types found in the limb bud, nor can they be directed to form a patterned appendage-like structure, even when placed in the context of the signals responsible for organizing the limb bud. Here, by taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28) normally expressed in the early limb bud, that are capable of imparting limb progenitor-like properties to non-limb fibroblasts. Cells reprogrammed by these factors show similar gene expression profiles, and can differentiate into similar cell types, as endogenous limb progenitors. The further addition of Lin41 potentiates proliferation of the reprogrammed cells while suppressing differentiation. These results suggest that these same four key factors may play pivotal roles in the specification of endogenous limb progenitors.

overall_design: RNA-seq of mesenchymal cells between different source tissues (Forelimb, Hindlimb, Neck, Flank)

first: Yuji

last: Atsuta

first: ChangHee

last: Lee

first: Alan

middle: R

last: Rodrigues

first: Charlotte

last: Colle

first: Reiko

last: Tomizawa

first: Ernesto

last: Lujan

first: Patrick

last: Tschopp

first: Danos

last: Christodoulou

first: Joshua

last: Gorham

first: Jean-Pierre

last: Vannier

first: Christine

last: Seidman

first: Jonathan

last: Pourquié

first: Clifford

middle: J

last: Tabin


title: Sensory Neurons Promote Immune Homeostasis in the Lung

city: New York

first: BiNGS

last: Core

address: 1470 Madison Ave

institute: Icahn School of Medicine at Mount Sinai

summary: Cytokines employ downstream Janus kinases (JAKs) to promote many chronic inflammatory disorders. JAK1-dependent type 2 cytokines drive allergic inflammation and patients with JAK1 gain-of-function (GOF) variants develop atopic dermatitis (AD) and asthma. To explore the tissue-specific role of JAK1, we inserted a human JAK1 GOF variant into mice and observed the spontaneous development of AD-like skin inflammation but unexpected resistance to allergic lung inflammation when JAK1 GOF expression was restricted to the stroma. Further, we identified the chemical denervation of both vagal and spinal visceral afferents exacerbated allergic lung inflammation, though selective denervation of spinal visceral afferent did not change the disease severity compared to control groups. Collectively, we suggested that neuron-intrinsic JAK1 in the vagal afferents protect against allergic lung inflammation.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for mouse lung of control and experimental samples. (Bone marrow (BM) chimeric mice [wild-type (WT)_to_WT mice vs. WT_to_human JAK1 GOF mice], selective denervation of spinal visceral afferents [intrathecal vehicle injection vs. intrathecal resiniferatoxin injection], systemic denervation of both vagal and spinal visceral afferents [subcutaneous vehicle injection vs. subcutaneous resiniferatoxin injection])

first: Brian

middle: S

last: Kim

first: Masato

last: Tamari

first: Dan

last: Hasson

first: Deniz

last: Demircioglu

first: Gargi

last: Damle


title: Direct Reprogramming of Non-limb Fibroblasts to Cells with Properties of Limb Progenitors [scRNA-seq]

first: CHANGHEE

last: LEE

sample_organism:
Gallus gallus
Mus musculus

platform_organism:
Gallus gallus
Mus musculus

overall_design: scRNA-seq for non limb fibroblasts undergoing reprogramming into limb progenitors, with controls

first: Yuji

last: Atsuta

first: ChangHee

last: Lee

first: Alan

last: Rodrigues

first: Laura

last: Galan

first: Meng

last: Zhu

first: Marian

last: Ros

first: Olivier


title: Human neural networks with sparse TDP-43 pathology reveal NPTX2 misregulation in ALS/FTLD

city: Zurich

first: Izaskun

last: Mallona

address: Winterthurerstrasse 190

country: Switzerland

institute: University of Zurich

summary: Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating these age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies, which involve human-specific mechanisms that cannot be directly studied in animal models. To explore the emergence and consequences of TDP-43 pathologies, we generated iPSC-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors.

overall_design: Bulk RNA-seq and Chromium v3 single-cell RNA-seq.

first: Magdalini

last: Polymenidou


title: Autocrine Sfrp1 inhibits lung fibroblast invasion during transition to injury induced myofibroblasts

city: Neuherberg

first: Johannes

last: Beckers

address: Ingolstaedter Landstr. 1

department: Institute of Experimental Genetics

institute: Helmholtz Zentrum Muenchen

summary: Fibroblast to myofibroblast conversion is a major driver of tissue remodeling in organ fibrosis. Several distinct lineages of fibroblasts support homeostatic tissue niche functions, yet, specific activation states and phenotypic trajectories of fibroblasts during injury repair have remained unclear. Here, we combined spatial transcriptomics, longitudinal single-cell RNA-seq and genetic lineage tracing to study fibroblast fates during mouse lung regeneration. We discovered a transitional fibroblast state characterized by high Sfrp1 expression, derived from both Tcf21-Cre lineage positive and negative cells. Sfrp1+ cells appeared early after injury in peribronchiolar, adventitial and alveolar locations and preceded the emergence of myofibroblasts. We identified lineage specific paracrine signals and inferred converging transcriptional trajectories towards Sfrp1+ transitional fibroblasts and Cthrc1+ myofibroblasts. Tgfβ1 downregulated Sfrp1 in non-invasive transitional cells and induced their switch to an invasive Cthrc1+ myofibroblast identity. Finally, using loss of function experiments we show that autocrine Sfrp1 directly inhibits fibroblast invasion by regulating the RhoA pathway. In summary, our study reveals the convergence of spatially and transcriptionally distinct fibroblast lineages into transcriptionally uniform myofibroblasts and identifies Sfrp1 as an autocrine inhibitor of fibroblast invasion during early stages of fibrogenesis.

overall_design: We performed gene expression microarray analysis in primary human pulmonary fibroblasts treated with Sfrp1-siRNA and controls

first: Christoph

middle: H

last: Mayr

first: Arunima

last: Sengupta

first: Meshal

last: Ansari

first: Jeanine

last: Pestoni

first: Paulina

last: Ogar

first: Ilias

last: Angelidis

first: Andreas

last: Liontos

first: Alberto

last: Rodriguez-Castillo

first: Niklas

last: Lang

first: Maximilian

last: Strunz

first: Sara

last: Asgharpour

first: Diana

last: Porras-Gonzalez

first: Bettina

last: Oehrle

first: Valeria

last: Viteri-Alvarez

first: Isis

last: Fernandez

first: Martin

last: Irmler

first: Oliver

last: Eickelberg

first: Mircea

last: Stoleriu

first: Nikolaus

last: Kneidinger

first: Ali

middle: O

last: Yildirim

first: Katrin

last: Ahlbrecht

first: Rory

last: Morty

first: Christos

last: Samakovlis

first: Fabian

last: Theis

first: Gerald

last: Burgstaller

first: Herbert

last: Schiller


title: Identification of fibrotinflammatory and fibrotic transcriptomic subsets of human sclerotic cutaneous chronic graft-versus-host disease

city: Bethesda

first: Stephen

last: Brooks

address: 50 SOUTH DR, RM 1140

department: Biodata Mining and Discovery Section

institute: NIAMS/NIH

summary: Cutaneous sclerotic chronic graft-versus-host disease (cGVHD) is a common and highly morbid complication of allogeneic hematopoietic stem cell transplantation (HSCT). We identified genes that are significantly upregulated in the skin of sclerotic cGVHD patients (n = 17) compared to HSCT patients without sclerotic cGVHD (n = 9) by bulk RNA sequencing. This revealed two transcriptomic groups of affected patients: those with fibrotic and inflammatory/Th1 gene expression, the fibroinflammatory group, and those with predominantly fibrotic/TGF-associated expression, the fibrotic group. This transcriptomic heterogeneity was also associated with histopathologic heterogeneity, with the fibroinflammatory sub-cluster showing more histopathologic features of epidermal cGVHD and inflammation. Further study will help elucidate if these gene expression and histopathologic findings can be used to tailor treatment decisions. Multiple highly induced genes in the skin (SFRP4, SERPINE2, COMP) were also found to be significantly induced in sclerotic cGVHD patient plasma (n = 16) compared to control HSCT patients without sclerotic cGVHD (n = 17), suggesting these TGF and Wnt pathway mediators as candidate blood biomarkers of disease.

overall_design: Comparative gene expression profiling analysis of bulk RNA-seq data from human skin of hematopoietic stem cell transplant patients (HSCT) with chronic sclerotic graft-versus-host disease compared to HSCT patients without chronic sclerotic graft-versus-host disease.

first: Rachel

middle: K

last: Rosenstein

first: Jeremy

last: Rose

first: Wanxia

last: Tsai

first: Steven

middle: Z

last: Pavletic

first: Keisuke

last: Nagao

first: Edward

last: Cowen


title: Direct Reprogramming of Non-limb Fibroblasts to Cells with Properties of Limb Progenitors

first: CHANGHEE

last: LEE

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

overall_design: Refer to individual Series


title: Effect of HDAC1/2 Inhibitor compound 60 (Cmpd60) on multiple organ systems in aged Mice

status: Public on Nov 13 2023

city: Amsterdam

first: Man

middle: Iman

last: Hu

address: Meibergdreef 9

department: Laboratory Genetic Metabolic Diseases

institute: Amsterdam UMC Location University of Amsterdam

summary: In the current work, we performed multiple in silico drug screens using transcriptional profiles of 2837 small molecules testing their ability to mimic known genetic longevity interventions. We identified one compound that was most commonly found to mimic the transcriptional profile of the genetic longevity interventions. This benzamide-based small molecule, termed compound 60 (Cmpd60; aka Merck60 or BRD 692), is a selective histone deacetylase 1 and 2 (HDAC1/2) inhibitor.  Here, we used a combination of molecular, phenotypic, and bioinformatic analyses in multiple disease cell models and mouse models for age-related disease to establish if Cmpd60 acts as a geroprotector.

overall_design:
Aged mice: Natural aged, approx. 20 months old, male BL6 mice were acquired from Taconic and single housed under a 12:12-hour light-dark cycle in a room set to 23°C (+/- 0.2°C). All animals were fed a regular chow diet. Mice received daily i.p. injections with either Cmpd60 (n=6) or vehicle (n=7) for 14 days total. Body weight was monitored every 5 days to adjust i.p. volumes to body weight. Cmpd60 treated animals received a dose of 22.5 mg/kg with an i.p. volume of 7.5 ml/kg. Cmpd60 was dissolved in 2% DMSO, 49% PEG400, and 49% saline solution (= vehicle) resulting in a 3 mg/ml concentration.
APPSWE-1349 mice: The APPSWE-1349 mice62 (BL6 background) were acquired from Taconic. The transgenic mice possess a transgene coding for the 695-amino acid isoform of human Alzheimer β-amyloid (Aβ) precursor protein carrying the Swedish mutation. Animals (~6-7 months old) were single housed under a 12:12-hour light-dark cycle in a room set to 23°C (+/- 0.2°C). All animals were fed a regular chow diet. Mice received daily i.p. injections with either Cmpd60 (n=12) or vehicle (n=12) for 14 days total. Body weight was monitored every 5 days to adjust i.p. volumes to body weight. Cmpd60 treated animals received a dose of 22.5 mg/kg with an i.p. volume of 7.5 ml/kg. Cmpd60 was dissolved in 2% DMSO, 49% PEG400, and 49% saline solution (= vehicle) resulting in a 3 mg/ml concentration.

first: Alessandra

last: Tammaro

first: Eileen

last: Daniels

first: Iman

middle: M

first: Kelly

middle: C

last: Hart

first: Kim

last: Reid

first: Rio

last: Juni

first: Loes

last: Butter

first: Goutham

last: Vasam

first: Rashmi

last: Kamble

first: Aldo

last: Jongejan

first: Richard

middle: I

last: Aviv

first: Joris

last: Roelofs

first: Eleonora

last: Aronica

first: Reinier

last: Boon

first: Keir

last: Menzies

first: Riekelt

last: Houtkooper

first: Georges

last: Janssens


title: Comparative single cell epigenomic analysis of gene regulatory programs in the rodent and primate motor cortex [marmoset]

city: La Jolla

first: Joseph

last: Ecker

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

summary: Sequence divergence of cis-regulatory elements drives species-specific traits, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains to be elucidated. We investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset, and mouse with single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome, and chromosomal conformation profiles from a total of over 180,000 cells. For each modality, we determined species-specific, divergent, and conserved gene expression and epigenetic features at multiple levels. We find that cell type-specific gene expression evolves more rapidly than broadly expressed genes and that epigenetic status at distal candidate cis-regulatory elements (cCREs) evolves faster than promoters. Strikingly, transposable elements (TEs) contribute to nearly 80% of the human-specific cCREs in cortical cells. Through machine learning, we develop sequence-based predictors of cCREs in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Lastly, we show that epigenetic conservation combined with sequence similarity helps uncover functional cis-regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

overall_design: The motor cortex of  Macaque were disected and nuclei were prepared for library preparation. In-situ 3C treatment was done during the nuclei preparation that allows capturing the chromatin conformation modality as described previously. These steps were carried out using the Arima-3C BETA Kit (Arima Genomics). The nuclei were isolated and sorted into 384-well plates using previous methods. Briefly, single-nuclei were stained with AlexaFluor488-conjugated anti-NeuN antibody (MAB377X, Millipore) and Hoechst 33342 (62249, ThermoFisher) followed by FANS using a BD Influx sorter with single-cell (1 drop single) mode.  The snm3C-seq samples followed the library preparation protocol detailed previously. This protocol has been automated using the Beckman Biomek i7 instrument to facilitate large-scale applications. The snm3C-seq libraries were sequenced on an Illumina NovaSeq 6000 instrument, utilizing one S4 flow cell per 16 384-well plates and employing a 150 bp paired-end mode.

first: Wenliang

last: Wang

first: Anna

last: Bartlett

first: Hanqing

last: Liu

first: Jordan

last: Altshul

first: Rosa

last: Gomez Castanon

last: Nery

first: Huaming

last: Chen


title: 3D Culture of human lung fibroblasts decreases proliferative and increases extracellular matrix remodeling genes

city: Groningen

first: Janette

last: Burgess

state: Groningen

address: Hanzeplein 1

institute: Umcg

summary: Fibroblasts are the main producers of extracellular matrix (ECM) responsible for ECM maintenance and repair, a process often disrupted in chronic lung diseases. The accompanying mechanical changes adversely affect resident cells and overall lung function. Numerous models have been used to elucidate fibroblast behavior which are now evolving towards complex 3D models incorporating ECM, aiming to replicate the cells’ native environment. Little is known about the cellular changes that occur when moving from 2D to 3D cell culture. This study compared gene expression profiles of primary human lung fibroblasts from 7 subjects with normal lung function, cultured for 24 hours on 2D collagen I-coated tissue culture plastic and in 3D collagen I hydrogels, which are commonly used to mimic ECM in various models, from contraction assays to organ-on-a-chip models. Comparing 3D to 2D cell culture, 6,771 differentially expressed genes (2,896 up, 3,875 down) were found; enriched gene sets within the downregulated genes, identified through Gene Set Enrichment Analysis and Ingenuity pathway analysis, were involved in the initiation of DNA replication which implied downregulation of fibroblast proliferation in 3D. Observation of cells for 72 hours in 2D and 3D environments confirmed the reduced progression through the cell cycle in 3D. A focused analysis examining the Hippo pathway and ECM-associated genes, showed differential patterns of gene expression in the 3D versus 2D culture. Altogether, the transcriptional response of fibroblasts cultured in 3D indicated inhibition of proliferation, and alterations in Hippo and ECM pathways indicating a complete switch from proliferation to ECM remodeling.

overall_design: Control human primairy lung fibroblasts from 7 donors were cultured either on top of collagen I coated tissue culture plastic (2D) or inside of collagen 1 hydrogels (3D) for 24 hours. This generated 7 2D samples and 7 3D samples. The RNA was sequenced for each sample and were compared via gene set enrichment analysis and Ingenuity Patway analysis (IPA). IPA analysis was done unbiased as well as a biased analysis on Extracellular matrix and Hippo signaling gene sets.

first: Nataliya

last: Migulina

first: Roderick

last: de Hilster

first: Sabine

last: Bartel

first: Rolf

last: Vedder

first: Maarten

last: van den Berge

first: Anika

middle: P

last: Nagelkerke

first: Wim

last: Timens

first: Martin

last: Harmsen

first: Machteld

middle: N

last: Hylkema


title: Effect of overexpression and depletion of MCEMP1 following stimulation of THP1 cells with TGF-beta

city: Tampa

first: Theodoros

last: Karampitsakos

address: 560 Channelside D

institute: University of South Florida

overall_design: THP1 cells were transduced with lentivirus to stably overexpress or knockdown MCEMP1 expression, and subsequently treated with TGF-beta for 24 hours to see how changes in MCEMP1 expression affects THP1 response to TGF-beta.

first: Jose

middle: D

last: Herazo-Maya

first: Carole

middle: Y

last: Perrot


title: Epigenetic priming targets tumor heterogeneity to shift transcriptomic phenotype of pancreatic ductal adenocarcinoma towards a Vitamin D susceptible state

city: palo alto

first: Jiangbin

last: ye

state: California

address: 269 Campus Drive CCSR-South 1245

institute: Stanford university

summary: As a highly heterogeneous tumor, pancreatic ductal adenocarcinoma (PDAC) exhibits non-uniform responses to therapies across subtypes. Overcoming therapeutic resistance due to tumor heterogeneity in PDAC remains a challenge. Here, we report that Vitamin D-resistant PDAC cells hijacked Vitamin D signaling to promote tumor progression, whereas epigenetic priming with glyceryl triacetate (GTA) and 5-Aza-2′-deoxycytidine (5-Aza) overcame Vitamin D resistance and shifted the transcriptomic phenotype of PDAC toward a Vitamin D-susceptible state. Increasing overall H3K27 acetylation with GTA and reducing overall DNA methylation with 5-Aza not only elevated the Vitamin D receptor (VDR) expression but also reprogrammed the Vitamin D-responsive genes. Consequently, Vitamin D inhibited cell viability and migration in the epigenetically primed PDAC cells by activating genes involved in apoptosis as well as genes involved in negative regulation of cell proliferation and migration, while the opposite effect of Vitamin D was observed in unprimed cells. Studies in genetically engineered mouse PDAC cells further validated the effects of epigenetic priming for enhancing the anti-tumor activity of Vitamin D. Using gain- and loss-of-function experiments, we further demonstrated that VDR was necessary for augmenting original Vitamin D responses, but was insufficient for shifting transcriptomic phenotype of PDAC toward a therapeutic response to Vitamin D, highlighting that both the VDR and Vitamin D-responsive genes were prerequisites for Vitamin D response. These data reveal a previously undefined mechanism in which epigenetic state orchestrates the expression of both VDR and Vitamin D-responsive genes, and determines the response to Vitamin D in PDAC.

overall_design: To understand the underlying mechanism by which the epigenetic priming with GTA and 5-Aza shift the transcriptomic phenotype of PDAC towards a Vitamin D susceptible state, we treated the MiaPaca2 cells for 24 hours with DMSO(Control),  or CPT (Vitamin D analogue),  or GTA+5-Aza, or CPT+GTA+5-Aza. Total RNA was extracted using TRIzol Reagent (Invitrogen) protocol. RNA sequencing was performed by Novogene.

first: Bo

last: He

first: Lauren

last: Stoffel

first: Clifford

first: Kumsun

last: Cho

first: Albert

last: Li

first: Brittany

last: Flowers

first: Sofia

last: Ferreira

first: Kha

middle: T

last: Nguyen

first: Kelly

middle: W

last: Wang

first: Audrey

last: Zhao

first: Meng-Ning

last: Zhou

first: Laura

last: Attardi

last: Ye


title: Effect of 9-HODE and 13-HODE on gene expression of young male C57BL6 mouse livers

city: Tianjin

first: JingJie

last: Duan

state: Tianjin

address: No22.Qixiangtai Rd.,Heping Dist,Tianjin P.R. China

institute: tianjin medical university

summary: RNA-sequencing of 9-HODE and 13-HODE-treated and control mouse livers.

overall_design: 8-week-old male mice were treated with 9/13-HODE(a mixture of 9-HODE and 13-HODE in equal amounts) or vechicle once a day for 9 days. After that, mouse livers were collected to perform RNA-seq and gene expression analysis.

first: Jinjie

first: Wenhui

last: Dong

first: Guangyan

first: Wenjing

last: Xiu

first: Guangyin

last: Pu

first: Jingwen

last: Xu

first: Chenji

first: Xu

last: Zhang

first: Yi

last: Zhu

first: Chunjiong


title: Gestational diabetes in mice induces hematopoietic memory that impacts the long-term health of the offspring.

city: Cincinnati

first: Soona

last: Shin

state: Cincinnati

address: 3333 Burnet Ave, OH

institute: Cincinnati Children's Hospital Medical Center

summary: Gestational diabetes is a common medical complication of pregnancy that is associated with adverse perinatal outcomes and an increased risk of metabolic diseases and atherosclerosis in adult offspring. The mechanisms responsible for this delayed pathological transmission remain unknown. In mouse models, we found that the development of atherosclerosis in adult offspring born to diabetic pregnancy can be in part linked to hematopoietic alterations. Although they do not show any gross metabolic disruptions, the adult offspring maintain hematopoietic features associated with diabetes, indicating the acquisition of a lasting diabetic hematopoietic memory. We show that the induction of this hematopoietic memory during gestation relies on the activity of the pattern recognition receptor AGER and the NLRP3 inflammasome, which leads to increased placental inflammation. In adult offspring, we find that this memory is associated with DNMT1 upregulation and epigenetic changes in hematopoietic progenitors. Altogether, our results demonstrate that the hematopoietic system can acquire a lasting memory of gestational diabetes, and that this memory constitutes a new pathway connecting gestational health to adult pathologies.

overall_design: ATAseq analysis on WT LSK cells isolated from the BM of adult offspring born to nomal and diabetic  pregnancy

first: Vinothini

last: Govindarajah

first: Masahide

last: Sakabe

first: Samantha

last: Good

first: Michael

last: Solomon

first: Ashok

last: Arasu

first: Nong

last: Chen

first: Xuan

first: H

middle: L

last: Grimes

first: Ady

last: Kendler

first: Mei

last: Xi

first: Damien

last: Reynaud


title: Incorporation of human iPS cell-derived stroma creates a pancreatic cancer organoid with heterogeneous cancer-associated fibroblasts [AmpliSeq]

city: Minato Ku

first: kenta

last: takeuchi

state: Tokyo To

address: 4-6-1, Shirokanedai

country: Japan

institute: The Institute of Medical Science, The University of Tokyo

summary: The aggressiveness of pancreatic ductal adenocarcinoma (PDAC) is affected by a tumor microenvironment (TME). In this study, to recapitulate PDAC TME ex vivo, we cocultured patient-derived PDAC cells with mesenchymal and vascular endothelial cells derived from human induced-pluripotent stem cells (hiPSCs) to create a fused pancreatic cancer organoid (FPCO) in air–liquid interface. FPCOs were further induced to resemble two distinct parts of a PDAC tissue. Owing to various types of cancer associated fibroblasts (CAFs) derived from hiPSCs, the TME consisted of abundant extracellular matrix proteins, which likely conferred strong drug resistance to PDAC cells in one type of FPCOs. Because of re-proliferation capacity of PDAC cells after anticancer drug treatment, the other FPCO is the first culture system for investigating PDAC recurrence. Introducing hiPSC technology, we have created, for the first time, the PDAC organoids representing the heterogeneity of PDAC tissue, a potential platform for screening anticancer drugs.

overall_design: Two types of fused pancreatic cancer organoid (FPCO) generated by patiend-derived pancreatic cancer organoids(PCOs), hiPSC-derived mesenchymal cells (hiPSC-MC) and vascular endothelial cells (hiPSC-EC), and their materials. In addintion, bone marrow mesenchymal stem cell (BMMSC) and patient-derived cancer associated fibroblast (CAF) as comparison target with FPCO-CAF.

first: Kenta

last: Takeuchi

first: Shunsuke

last: Tabe

last: Takahashi

first: Megumi

last: Matsuo

first: Yasuharu

last: Ueno

first: Yoichi

last: Furukawa

first: Kiyoshi

last: Yamaguchi

first: Masayuki

last: Ohtsuka

first: Soichiro

last: Morinaga

first: Yohei

last: Miyagi

first: Tomoyuki

first: Naok

last: i Tanimizu

first: Hideki

last: Taniguchi


title: A novel acetyltransferase regulates the RNA binding capacity of the RNA chaperone Hfq in Escherichia coli

city: Lebanon

first: Christine

last: Alewine

state: NH

address: One Medical Center Drive, Williamson 651,

institute: Dartmouth Cancer Center

summary: Bacterial small regulatory RNAs (sRNAs) regulate gene expression by base-pairing to their target mRNAs. In Escherichia coli and many other bacteria, this process is dependent on the RNA chaperone Hfq, which binds sRNAs and mRNAs on different faces. YhbS (renamed here as HqbA), a putative Gcn5-related N-acetyltransferase (GNAT), was identified as a novel silencer of sRNA signaling in a genomic library screen. Here, we studied how HqbA regulates sRNA signaling and determined its physiological roles in modulating Hfq activity. Using fluorescent reporter assays, we found that HqbA overproduction suppresses all tested Hfq-dependent sRNA signaling. Chromosomal HqbA suppresses the signaling of the ChiX sRNA when the C-terminus of Hfq was deleted. Direct interaction between HqbA and Hfq was demonstrated both in vivo and in vitro, and mutants that blocked interaction also interfered with HqbA suppression of Hfq. However, an acetylation-deficient HqbA mutant still disrupted sRNA signaling, suggesting that HqbA is bifunctional, with separate roles for regulating via Hfq interaction and via acetylation of undefined substrates. Gel shift assays indicated that HqbA strongly reduced the interaction between the Hfq distal face and low-affinity RNAs, but not high-affinity RNAs. Hfq-IP RNA-Seq in WT and chromosomal hqbA mutants led to the identification of two tRNA precursors, metZWV and proM, that were enriched in Hfq binding in the absence of HqbA interaction. Our results suggest that HqbA provides a level of quality control for Hfq by competing with low-affinity RNA binders.

sample_organism: Escherichia coli str. K-12 substr. MG1655

overall_design: There are 3 strains of E.coli K12 MG1655: wild type (WT), deletion of HbqA (dely) and D98AE102A mutant of HbqA (d98); two growth conditions: aerobic(ae) and anaerobic (an), and two extraction methods: total RNA or anti-Hfq precipitated, and each has two replicates. Paired-end sequencing were performed.

first: Xing

last: Liuo

first: Aixia

last: Zhang

first: Chin-Hsien

last: Tai

first: Jiandong

last: Chen

first: Nadim

last: Majdalani

first: Gisela

last: Storz

first: Susan

last: Gottesman


title: The Transcriptomic Profiling of Blood CD4 and CD8 T-cells in Narcolepsy Type I

city: Toulouse

first: Leila

last: Khajavi

address: TOULOUSE

country: France

institute: INSERM

summary: Background Narcolepsy Type I (NT1) is a rare, life-long sleep disorder arising as a consequence of the extensive destruction of orexin-producing hypothalamic neurons. The mechanisms involved in the destruction of orexin neurons are not yet elucidated but the association of narcolepsy with environmental triggers and genetic susceptibility (strong association with the HLA, TCRs and other immunologically-relevant loci) implicates an immuno-pathological process. Several studies in animal models and on human samples have suggested that T-cells are the main pathogenic culprits.Methods RNA sequencing was performed on four CD4 and CD8 T-cell subsets (naive, effector, effector memory and central memory) sorted by flow cytometry from peripheral blood mononuclear cells (PBMCs) of NT1 patients and HLA-matched healthy donors as well as (ageand sex-) matched individuals suffering from other sleep disorders (OSD). The RNAseq analysis was conducted by comparing the transcriptome of NT1 patients to that of healthy donors and other sleep disorder patients (collectively referred to as the non-narcolepsy controls) in order to identify narcolepsy- specific genes and pathways.We determined narcolepsy-specific differentially expressed genes, several of which are involved in tubulin arrangement found in CD4 (TBCB, CCT5, EML4, TPGS1, TPGS2) and CD8 (TTLL7) T cell subsets, which play a role in the immune synapse formation and TCR signaling. Furthermore, we identified genes (GZMB, LTB in CD4 and NLRP3, TRADD, IL6, CXCR1, FOXO3, FOXP3 in CD8) and pathways involved in various aspects of inflammation and inflammatory response. More specifically, the inflammatory profile was identified in the "naive" subset of CD4 and CD8 T-cell.We identified NT1-specific differentially expressed genes, providing a cell-type and subset specific catalog describing their functions in T-cells as well as their potential involvement  in NT1. Several genes and pathways identified are involved in the formation of the immune synapse and TCR activation as well as inflammation and the inflammatory response.An inflammatory transcriptomic profile was detected in both "naive" CD4 and CD8 T-cell subsets suggesting their possible involvement in the development or progression of the narcoleptic process.

overall_design: RNA sequencing was performed on four CD4 and CD8 T-cell subsets (naive, effector, effector memory and central memory) sorted by flow cytometry from peripheral blood mononuclear cells (PBMCs) of NT1 patients and HLA-Matched healthy donors as well as (age- and sex-) matched individuals suffering from other sleep disorders (OSD). The RNAseq analysis was conducted by comparing the transcriptome of NT1 patients to that of healthy donors And other sleep disorder patients (collectively referred to as the non-narcolepsy controls) in order to identify Narcolepsy-specific genes and pathways.

first: KHAJAVI

middle: F

last: Leila

first: NGUYEN

last: Xuan-Hung

first: QUERIAULT

last: Clemence

first: CHABOD

last: Marianne

first: BARATEAU

last: Lucie

first: DAUVILLIERS

last: Yves

first: ZYTNICKI

last: Matthias

first: LIBLAU

last: Roland