title: MBNL1 regulates programmed postnatal switching between regenerative and differentiated cardiac states [RIP-seq]

city: Seattle

first: Jennifer

last: Davis

state: WA

address: 815 Mercer Street D342

department: Bioengineering & Pathology & Laboratory Medicine

institute: University of Washignton

summary:
Background: Discovering determinants of cardiomyocyte maturity will be critical to understanding the maintenance of differentiated states and potentially reawakening endogenous regenerative programs in adult mammalian hearts as a therapeutic strategy. Recent evidence has suggested that forced dedifferentiation paired with oncogene expression is sufficient to drive cardiac regeneration. However, elucidation of endogenous developmental determinants of the switch between regenerative and mature cardiomyocyte cell states is necessary for optimal design of regenerative approaches for heart disease. Muscleblind-like 1 (MBNL1) regulates both fibroblast and erythroid differentiation and proliferation. Hence, we examined whether MBNL1 promotes and maintains mature cardiomyocyte states while antagonizing cardiomyocyte proliferation.
Methods: MBNL1 gain- and loss-of-function mouse models were studied at several developmental timepoints and in surgical models of heart regeneration. Multi-omics approaches were combined with biochemical, histological, and in vitro genetic work to determine the mechanisms through which MBNL1 exerts its effects.
Results: MBNL1 is co-expressed with a maturation-association genetic program in the heart and is regulated by the MEIS1/Calcineurin signaling axis. Targeted MBNL1 overexpression early in development prematurely transitioned cardiomyocytes to hypertrophic growth, hypoplasia, and dysfunction, while loss of MBNL1 function increased cardiomyocyte cell cycle entry and proliferation through altered cell cycle inhibitor transcript stability. Moreover, MBNL1-dependent stabilization of estrogen-related receptor signaling was essential for maintaining cardiomyocyte maturity in adult myocytes. In accordance with these data, modulating MBNL1 dose tuned the temporal window of neonatal cardiac regeneration, where increased MBNL1 expression arrested myocyte proliferation and regeneration, and MBNL1 deletion promoted regenerative states with prolonged myocyte proliferation. However, MBNL1 deletion was not sufficient to promote regeneration in the adult heart due to cell-cycle checkpoint activation.
Conclusions: Here, MBNL1 was identified as an essential regulator of cardiomyocyte differentiated states, their developmental switch from hyperplastic to hypertrophic growth, and their regenerative potential through controlling an entire maturation program by stabilizing adult myocyte mRNAs during postnatal development and throughout adulthood. Additionally, MBNL1-dependent perturbations of a myocyte’s preferred growth mechanism at a given developmental state had detrimental consequences to cardiac function, and targeting loss of maturity and removing cell cycle inhibitors was not insufficient to promote adult regeneration.

overall_design: In order to determine the transcripts that MBNL1 binds in cardiomyocytes, FLAG-tagged MBNL1 was transduced into neonatal rat ventricular cardiomyocytes. MBNL1 and its bound RNA was immunoprecipitated with a FLAG antibody and RNA sequencing was performed on input/whole cell lysate (Input) and MBNL1 RNA immunoprecipitaiton (RIP) to identify enriched transcripts in the MBNL1 pulldown.

first: Logan

last: Bailey


title: MBNL1 regulates programmed postnatal switching between regenerative and differentiated cardiac states [RNA-seq]

status: Public on Mar 07 2024

country: USA

type: Expression profiling by high throughput sequencing

overall_design: In order to examine normal patterns of postnatal gene expression and the effects of altering MBNL1 dose on postnatal cardiac maturity, mouse ventricles from control (Ctl), MBNL1 gain-of-function (Tg), and MBNL1 loss-of-function (KO) models were subjected to RNAseq at several developmental timepoints.


title: MBNL1 regulates programmed postnatal switching between regenerative and differentiated cardiac states [scRNA-seq]

overall_design: In order to determine the role of MBNL1 in controlling cardiomyocyte maturity and cell cycle entry following myocardial infarction (MI), ventricles from MBNL1 loss-of-function (KO) and control (Ctl) animals were subjected to single nucleus RNA sequencing 2 weeks following myocardial infarciton.


title: MBNL1 regulates programmed postnatal switching between regenerative and differentiated cardiac states [ActD RNA-seq]

overall_design: In order to determine transcripts with MBNL1-dependent stability, isolated adult cardiomyocytes from MBNL1 loss-of-function (KO) and control (WT) mice were treated with the transcription inhibitor actinomysin D (ActD). RNA was isolated at baseline and 4- and 8-hours after ActD treatment to measure RNA abundance and identify transcripts with different stability.


title: Effect of NIBR-LTSi treatment on gene expression in mouse intestinal organoids

city: Basel

first: Clara

last: Baader

address: Maulbeerstrasse 66

country: Switzerland

institute: FMI

summary: The YAP/Hippo pathway is a key regulator of organ growth and size regulation and is involved in safeguarding multiple tissue stem cell compartments. LATS kinases phosphorylate and thereby inactivate YAP, thus representing a potential direct drug target for promoting tissue regeneration. We report on the gene expression changes associated with YAP activation by the  selective small molecule LATS kinase inhibitor NIBR-LTSi in mouse intestinal organoids. We show that, LATS-LTSi activates YAP signalling, promotes proliferation, induces a regenerative phenotype and blocks differentiation.

overall_design: Mouse intestinal organoids were grown from single cells until crypt-villus structures emerged (96h). Subsequently, organoids where treated with 5uM NIBR-LTSi or DMSO respectively and harvested after 24 h for transcriptome analaysis by RNAseq.

first: Prisca

last: Liberali

first: Jan

middle: S

last: Tchorz


title: Progeria-based vascular model identifies networks associated with cardiovascular aging and disease

city: Ottawa

first: William

middle: L.

last: Stanford

state: Ontario

address: 501 Smyth

department: The Sprott Centre for Stem Cell Research,

country: Canada

institute: The Ottawa Hospital Research Institute

summary:
To model Hutchinson-Gilford Progeria syndrome (HGPS), we differentiated patient-derived induced pluripotent stem cells (iPSCs) to vascular smooth muscle cells (VSMCs).
We then performed gene expression profiling analysis using data obtained from RNA-seq of the serially passaged cells at passage7 and passage 14.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for control and HGPS-patient derived VSMCs.

first: Mzwanele

last: Ngubo

first: Amit

last: Shrestha

first: Zhaoyi

last: Chen

first: Julien

last: Yockell-Lelièvre

first: Christopher

last: Porter

first: Darin

middle: L

last: McDonald

first: Rana

last: Karimpour

first: Aurelie

last: Laurent

first: Ojong Tabi

last: Ojong Besong

first: Eve

middle: C

last: Tsai

first: Jeffrey

last: Dilworth

first: Michael

middle: J

last: Hendzel


title: A MTA2-SATB2 chromatin complex restrains colonic plasticity toward small intestine by retaining HNF4A at colonic chromatin

city: Shanghai

first: Wei

last: Gu

state: Shanghai

address: 51 Rijing Road

department: Beigene Institute

country: China

institute: Beigene

type: Genome binding/occupancy profiling by high throughput sequencing

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

overall_design: Refer to individual Series


title: Bulk RNA-seq (>20M reads) of colonic epithelium from Vil-CreER;MTA2F/F (MTA2KO) and Wild Type mice

summary: RNA-sequening colonic epithelium with deeper reads to evaluate consequence of mta2 gene loss in the colonic epithelium.

overall_design: Two batches of MTA2 intestinal conditional KO experiments to validate the mta2 gene's function in colonic epithelium

first: Xiaofeng

last: Huang

first: Qiao

last: Zhou


title: Bulk RNA-seq of mouse colonic organoids: CRISPR functional Screening of Satb2-interact proteins (NuRD and SWI/SNF complex)

summary: CRISPR knock out the NuRD and SWI/SNF complex gene from mouse colonic organoids to validate the potential function with SATB2.

overall_design: Genomic modified mouse colonic organoids (Ctrl, Satb2, Mta2, Chd4, Gatad2a, Smarca5, Smarca4, Smarcd2 and Ctbp2 sgRNAs) were differentiated and collected for Bulk RNA-Seq


title: Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations (RNA-Seq)

status: Public on Mar 06 2024

city: Palo Alto

first: Chris

last: Hsiung

state: CA

address: 3181 Porter Dr

institute: Arc Institute

summary: Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein:DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.

overall_design: Nanopore sequencing analysis of high molecular weight genomic DNA surrounding CRISPR RNA (crRNA) target sites, using Cas9-based enrichment of targeted regions.

first: Caroline

middle: M

last: Wilson

first: Nicholas

middle: A

last: Sambold

first: Raymond

last: Dai

first: Qingzhou

first: Sara

last: Misiukiewicz

first: Abofazl

last: Arab

first: Noam

last: Teyssier

first: Tom

last: O'Loughlin

first: Joshua

last: Cofsky

first: Luke

last: Gilbert


title: Phosphorus deficiency alleviates cyanobacterial iron-limitation through a direct PhoB_x0002_ mediated gene regulation

city: Wuhan

first: wencan

last: zheng

state: Hubei

address: NO.152 Luoyu Road

department: School of Life Sciences

institute: Central China Normal University

summary: Iron (Fe) and phosphorus (P) are essential nutrients with close geochemical association. They exist at low concentrations in surface waters and may be co-limiting resources for phytoplankton growth. However, the adaptive strategies of photosynthetic organisms to Fe/P co-limitation remain largely unknown. Here, we show that phosphorus deficiency increases the growth of Fe-limited cyanobacteria through a PhoB-mediated regulatory network. In addition to its well-recognized role in controlling phosphate homeostasis, PhoB regulates key metabolic processes crucial for Fe-limited cyanobacteria, including ROS detoxification and Fe uptake. Transcript abundances of PhoB-targeted genes are enriched in samples from the P-deplete ocean, and a conserved PhoB binding site is widely present in the promoters of the targets, suggesting that the strategy we discovered may be highly conserved. Our findings provide important molecular insights into the response of cyanobacteria to simultaneous Fe/P nutrient limitation and help in understanding how nutrient availability affects primary productivity in aquatic environments.

overall_design: The phoB-FLAG3 strains were grown in phosphate limited medium to an OD730 of  0.5 and then cross-linked by 1% formaldehyde treatment for 15 min at room temperature,followed by quenching with 125 mM glycine. Sample preparation for ChIP-Seq was performed  as previously described  and libraries were prepared from two biological replicates. The  library sequencing was performed with Illumina NovaSeq 6000 by IGENEBOOK  Biotechnology Co., Ltd. The MACS2 program (version 2.1.1.20160309) was used for peak  calling, and the data were visualized by IGV (version 2.16.0)

first: Guo-Wei

last: Qiu

first: Wen-Can

last: Zheng

first: Hao-Ming

last: Yang

first: Yu-Ying

last: Wang


title: Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations (CRISPR screen)

sample_organism:
Homo sapiens
synthetic construct

platform_organism:
Homo sapiens
synthetic construct

overall_design: Cas12a CRISPRi pooled screens using single-plex crRNA and 6-plex crRNA libraries in K562 cells, using cell fitness as readout.

last: Chen


title: Satellite glial GPR37L1 and its ligand maresin 1 regulate potassium channel signaling and pain homeostasis.

city: durham

first: sangsu

last: Bang

state: North Carolina

address: 3 genome st, 6200 MSRB3

institute: duke

summary: G protein-coupled receptor 37-like 1 (GPR37L1) is an orphan GPCR, and its function remains largely unknown. Here we report that Gpr37l1 and GPR37L1 are among the most highly expressed GPCR transcripts in mouse and human dorsal root ganglia (DRGs) and are selectively expressed in satellite glial cells (SGCs). Peripheral neuropathy following PTX-induced pain resulted in a downregulation of GPR37L1 plasma membrane expression in DRGs. Transgenic mice with Gpr37l1 deficiency exhibited impaired resolution of neuropathic pain symptoms following PTX-induced pain, whereas overexpression of Gpr37l1 in mouse DRGs reversed pain. GPR37L1 regulates the surface expression and function of these potassium channels. Thus, GPR37L1 in SGCs offers a new target for neuropathy protection and pain control.

overall_design: To investigate the function of GPR37L1 in the DRG during pain, we established PTX injection mice and collected DRG at 4 weeks in WT and Gpr37L1 KO mice.

first: Sangsu

first: Ru-Rong

last: Ji


title: Exploring MicroRNA Patterns as Biomarkers of FOLFOX Chemotherapy-Induced Peripheral Neuropathy in Patients with Colorectal Cancer

city: Gimcheon

first: Yeongdon

last: Ju

state: Gyeongsangbuk-do

address: 214, Daehak-ro, Gimcheon-si

department: Department of Biomedical Laboratory Science

country: South Korea

institute: Gimcheon University

summary: This study aimed to identify potential miRNA-based biomarkers that can predict FOLFOX-CIPN symptoms. High throughput microRNA sequencing was performed on the RNA circulating in the plasma of eight patients with CRC who underwent FOLFOX chemotherapy. miRNA expression profiles were evaluated according to two groups: those who underwent ≤three cycles and those who underwent ≥six cycles of FOLFOX chemotherapy.

overall_design: Comparative gene expression profiling analysis of RNA-seq data for plasma samples of patients with colorectal cancer who underwent three or fewer cycles and those who underwent six or more cycles

first: Dong-Hoon

last: Baek

first: Go-Eun

last: Choi

first: Aelee

last: Jang


title: Effect of Small Mouse Cage Intervention on skeletal muscle gene expression, male and female

city: Salt Lake City

first: Katsuhiko

last: Funai

state: UT

department: Molecular Medicine

institute: University of Utah

summary: Adult male and female C56BL6J mice were subjected to 8 weeks of small mouse cage (SMC) intervention to model physical inactivity. Widely-used physical inactivity models such as hindlimb unloading and immobilization do not phenocopy metabolic adaptations that occur with human sedentary behavior, whereas many aspects of metabolic adaptations with SMC appear to reproduce this effect. The RNAseq analyses were performed in muscles from these mice to capture changes in skeletal muscle gene expression landscape.

overall_design: To test the effects of 8 wk SMC intervention on skeletal muscle gene expression in adult male and female mice.

first: Piyarat

last: Siripoksup


title: Effects of Oct4 depletion on BM-related gene expression during the initial stages of post-implantation amniotic sac embryoid (PASE) development.

city: Vienna

first: Markus

last: Hengstschläger

address: Währinger Straße 10

department: Institute of Medical Genetics

country: Austria

institute: Medical University of Vienna

summary: Basement membranes (BMs) are sheet-like structures of specialized extracellular matrix (ECM), which provide structural support for many tissues and play a central role in signalling. They are key regulators of cell behavior and tissue functions and defects in their assembly or composition are involved in numerous human diseases. Due to the differences between human and animal embryogenesis, ethical concerns, legal constraints, the scarcity of human tissue material and the inaccessibility of the in vivo condition, BM regulation during human embryo development remained elusive. Making use of a recently established embryoid allowed us to draw a picture of BM assembly during human embryogenesis and to examine BM disintegration during primitive streak (PS) cell dissemination. Further, we show that the transcription factor Oct4 regulates the expression of BM structural components and receptors and controls BM development during embryogenesis by regulating Akt signalling and the small GTPase Rac1. These results represent a relevant step towards a more comprehensive understanding of early human development. The deposited RNA-seq data aimed at evaluating the effects of siRNA-mediated depletion of the master transcription factor Oct4 on BM-related gene expression during initial stages of embryoid formation.

overall_design:
To investigate the role of the transcription factor Oct4 on BM-related gene expression during early human embryogenesis, we performed tansient, siRNA-mediated knockdown of Oct4 in WA19 human embryonic stem cells (hESC) and analysed its effect in early stage, WA19-derived post-implantation amniotic sac embryoids (PASE) at two different time points in three independent biological replicates.
We then performed gene expression profiling analysis of control and Oct4-depleted samples using the data obtained from RNA-seq
Comparative gene expression profiling analysis of RNA-seq data for control (WA19 hESC, siControl) and Oct4-depleted (WA19 hESC, siOct4) samples at time point 1 (0h PASE) and time point 2 (24h PASE) in three biological replicates (rep1,2,3).

last: HENGSTSCHLÄGER

first: Margit

last: ROSNER


title: The germline regulates longevity and somatic repair in a sex-specific manner (RNA-Seq)

city: Jerusalem

first: Itamar

last: Harel

address: Givat Ram

department: Genetics

country: Israel

institute: The Hebrew University of Jerusalem

summary: Classical evolutionary theories propose tradeoffs between reproduction, damage repair, and lifespan. However, the specific role of the germline in shaping vertebrate aging remains largely unknown. Here, we use the turquoise killifish (N. furzeri) to genetically arrest germline differentiation at discrete stages, and examine how different ‘flavors’ of infertility impact life-history. We first constructed a comprehensive single-cell gonadal atlas, providing cell-type-specific markers for downstream phenotypic analysis. Next, investigating our genetic models revealed that only germline ablation enhanced female damage repair, while arresting germline differentiation did not. Conversely, germline-ablated males were significantly long-lived, indicating that the mere presence of the germline can negatively affect lifespan. Transcriptomic analysis highlighted enrichment of pro-longevity pathways and genes, with functional conservation in germline-ablated C. elegans. Finally, germline depletion extended male healthspan through rejuvenated metabolic functions. Our results suggest that different germline manipulation paradigms can yield pronounced sexually dimorphic phenotypes, implying alternative mechanisms to classical evolutionary tradeoffs.

overall_design: Livers from killifish were used, and each group varied by sex (males or females), age (young or old), and genotype (wild-type or dnd1 mutant).

first: Moses

last: Eitan

first: Tehila

last: Atlan

first: Xue

last: Sun

first: Georgi

last: Marinov

first: William

last: Greenleaf

first: Oren

last: Ram


title: Mitotic chromosomes are self-entangled and disentangle through a Topoisomerase II-dependent two stage exit from mitosis

city: Worcester

first: Job

last: Dekker

state: MA

address: 368 Plantation Street

department: Program in Systems Biology

institute: University of Massachusetts Medical School/HHMI

summary: The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations and multi-contact 3C, and propose that, in contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a Topoisomerase II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias Topoisomerase II activity towards decatenation. At the second stage, the loops are released, and formation of new entanglements is prevented by lower Topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed, in experiment and models, a normal interphase state cannot be acquired.

overall_design: Hi-C and MC-3C experiments in HeLa S3 and Hi-C experiments in HCT116 + Rad21-mAC cell lines. Hi-C experiments have 1-3 replicates each, MC-3C experiments have 3 replicates each.

first: Erica

last: Hildebrand

first: Kirill

last: Polovnikov

first: Bastiaan

first: Yu

last: Liu

first: Denis

last: Lafontaine

first: A. Nicole

last: Fox

first: Ying

last: Li

first: Sergey

middle: V

last: Venev

first: Leonid

last: Mirny


title: Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations (Nanopore)

first: Chris

last: Hsiung

first: Caroline

last: Wilson

first: Nicholas

last: Sambold

first: Raymond

last: Dai

first: Qingzhou

last: Chen

first: Sara

last: Misiukiewicz

first: Abofazl

last: Arab

first: Noam

last: Teyssier

first: Tom

last: O'Loughlin

first: Joshua

last: Cofsky

first: Luke

last: Gilbert


title: H3K27Ac ChIP-seq of colonic intestine epithelium from Vil-CreER;MTA2F/F (MTA2KO) and Wild Type mice

first: Wei

last: Gu

summary: Understanding the enhancer activity in MTA2 regulates colonic intestine epithelial fate and tissue plasticity.

overall_design: H3K27ac histone modification analysis on intestine colonic epithelium. Genotypes include Vil-CreER;MTA2F/F (MTA2KO) and MTA2f/f (WT).

first: Pratik

middle: N

last: Singh

first: Ramesh

last: Shivdasani

first: Qiao

last: Zhou


title: Cardiometabolic characteristics of people with metabolically healthy and unhealthy obesity [ThighMuscle]

city: St. Louis

first: Jinsheng

last: Yu

state: MO

address: 660 S. Euclid Ave.

institute: Washington University School of Medicine

summary: Some people with obesity are resistant to the adverse metabolic effects of excess adiposity and are considered to have “metabolically healthy obesity” (MHO). However, the mechanisms responsible for MHO are not clear. We performed comprehensive cardiometabolic profiling in 20 adults with MHO (defined by features of normal insulin sensitivity), 20 age-, sex-, and BMI-matched adults with metabolically unhealthy obesity (MUO), and 15 adults who were metabolically healthy and lean (MHL). More than 120 variables were evaluated, including body composition, beta-cell function, 24-hour plasma metabolic profile, rates of de novo lipogenesis and cholesterol synthesis, plasma adipokine concentrations, oxidative stress, plasma lipoprotein profiles, cardiovascular structure/function, skeletal muscle bioactive lipids, and both adipose tissue and skeletal muscle transcriptomics. Using dimensionality reduction approaches, measures of insulinemia and skeletal muscle ceramides were the strongest discriminators of MHO and MUO status. These data clarify the characteristics associated with preserved metabolic health in response to obesity.

overall_design: Comprehensive cardiometabolic profiling was conducted in 20 adults with metabolically healthy obesity (MHO, defined as normal fasting glucose, glucose tolerance, HbA1c, intrahepatic triglyceride, serum triglycerides, and whole-body insulin sensitivity), 20 age-, sex-, and BMI-matched adults with metabolically unhealthy obesity (MUO, defined as prediabetes and hepatic steatosis), and 15 adults who were metabolically healthy and lean (MHL). Vastus lateralis muscle biopsy samples were subjected to bulk RNA-sequencing.

first: Max

last: Petersen

first: Gordon

middle: I

last: Smith

first: Hector

middle: H

last: Palacios

first: Mihoko

last: Yoshino

first: Jun

first: Kevin

last: Cho

first: Victor

middle: G

last: Davila-Roman

first: Mahalakshmi

last: Shankaran

first: Ruteja

last: Barve

first: Bruce

middle: W

last: Patterson

first: Marc

middle: K

last: Hellerstein

first: Gerald

last: Shulman

first: Gary

last: Patti

first: Samuel

last: Klein


title: Yin Yang 1 regulates cohesin complex protein SMC3 in mouse hematopoietic stem cells

city: Madison

first: Xuan

last: Pan

state: WI

address: 4470 Veterinary Medicine Building

institute: University of Wisconsin

summary: Yin Yang 1 (YY1) and Structural Maintenance of Chromosomes 3 (SMC3) are two critical chromatin structural factors that mediate long-distance enhancer-promoter interactions and promote developmentally regulated changes in chromatin architecture in hematopoietic stem/progenitor cells (HSPCs). While YY1 plays critical functions in maintaining hematopoietic stem cell (HSC) quiescence, SMC3 is required for proper lineage differentiation. However, many questions remain unanswered regarding how YY1 and SMC3 interact with each other and impact hematopoiesis. We found that YY1 physically interacts with SMC3 and co-occupies with SMC3 at a large cohort of promoters genome-wide, and YY1 deficiency deregulates the genetic network governing cell metabolism. YY1 occupies the Smc3 promoter and represses SMC3 expression in HSPCs. While deletion of one Smc3 allele partially restores HSC numbers and quiescence in YY1 knockout mice, HSC self-renewal remains defective. YY1 regulates HSC metabolic pathways and maintains proper intracellular reactive oxygen species (ROS) levels in HSCs, and this regulation is independent of YY1- SMC3 axis. Our results establish a distinct YY1-SMC3 axis and its impact on HSC quiescence, self-renewal and metabolism

overall_design: A total of 12 samples were chosen for RNA sequencing (RNA-Seq), 4 from Yy1+/+, 4 from Yy1-/- and 4 from Yy1-/-Smc3+/- mice.  Total RNA was purified from sorted bone marrow LSK (Lin-Sca1+c-Kit+ )cells.   Yy1+/+ was used as a wild type control.

first: Zhanping

last: Lu

first: Peng

last: Liu

first: Sean

last: McIlwain


title: Chromosome conformation capture (Hi-C) of the turquoise killifish

first: Itamar

last: Harel

summary: This approach was used to increase the continuity of the killifish genome, and assess the relative distance of specific genes from the sex locus.

overall_design: Hi-C procedure was used to map 3D genomic interactions in killifish

first: Eitan

last: Moses

first: Tehila

last: Atlan

first: Roman

last: Franek

first: Atif

last: Siddiqui

first: Georgi

last: Marinov

first: Sagiv

last: Shifman

first: David

last: Zucker

first: Adi

last: Oron-Gottesman

first: William

last: Greenleaf

first: Ehud

last: Cohen

first: Oren

last: Ram


title: Medial prefrontal cortex transcriptome of mice susceptible or resilient to chronic stress

city: Seoul

first: Sihwan

last: Seol

address: 84, Heukseok-ro

institute: Chung-Ang University

summary: Stressful circumstances are significant contributors to mental illnesses, such as major depressive disorder. Anhedonia, which is the loss of the ability to enjoy pleasure, including rewarding activities or social contexts, is considered a key symptom of depression. Although stress-induced depression is associated with anhedonia in humans and animals, the underlying molecular mechanisms of anhedonic responses remain poorly understood. In this study, we conducted RNA sequencing to profile the medial prefrontal cortex which was substantially associated with the CUS-induced anhedonic behavioral phenotypes. Employing chronic unpredictable stress (CUS), we determined two subpopulations based on sucrose preference, which was highly correlated with social reward: susceptible (SUS, anhedonic) vs. resilient (RES, non-anhedonic) groups. We identified Syt4 as a hub gene in a gene network unique to anhedonia by conducting a weighted gene co-expression network analysis of the RNA sequencing data from the mPFC of SUS and RES mice. We also confirmed that Syt4 overexpression in the mPFC was pro-susceptible, while the Syt4 knockdown was pro-resilient; the pro-susceptible effects of SYT4 were mediated through the reduction of brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase B (TrkB) signaling in the mPFC. These findings suggested that SYT4-BDNF interactions in the mPFC could be a crucial regulatory mechanism of anhedonic susceptibility to chronic stress.

overall_design: RNA sequencing of medial prefrontal cortex samples from 9 mice after chronic unpredictable stress (n = 3 per group).

first: Jeongseop

last: Kim

first: Tae-Eun

first: Joonhee

last: Lee

first: Ja

last: Koo

first: Hyo

last: Kang


title: Periportal macrophages protect against commensal-driven liver inflammation

city: Suita

first: Daisuke

last: Motooka

state: Osaka

address: 3-1, Yamadaoka

country: Japan

institute: NGS core facility, Research Institute for Microbial Diseases, Osaka University

summary: The liver is the main gateway from the gut and the unidirectional sinusoidal flow from portal to central veins constitutes heterogenous zonation, such as peri-portal vein (PV) and peri-central vein (CV) zones; however, the functional and molecular differences among liver macrophages in these zones remain poorly understood. Here, intravital multiphoton imaging revealed significantly suppressed in PV zones. Zonation-specific single-cell transcriptome analyses detected an immuno-suppressive macrophage subset highly expressing IL-10 and Marco, a scavenger receptor, enriched in PV zones. Inhibited IL-10 signaling and Marco-deficient conditions impaired the suppressive function of these macrophages. The reduced number of Marco-positive suppressive macrophages in germ-free or antibiotic-treated conditions suggested that gut commensal bacteria were responsible for inducing this specific population. Dextran sulfate sodium-induced colitis led to inflammation in liver PV zones, which was more prominent under Marco-deficient conditions. Marco-positive inflammatory macrophages in the human liver are diminished in primary sclerosing cholangitis (PSC), an intractable disease characterized by chronic inflammation around the portal veins and bile ducts. Collectively, commensal bacteria and their pathogenic substances induce Marco-positive immunosuppressive macrophages, consequently limiting excessive inflammation in PV zones. Failure of this self-limiting system may cause hepatic inflammatory disorders, such as PSC.

overall_design: Single-cell RNA-sequencing on CD45+ cells from portal vein and central vein zones of the healthy mouse liver.

first: Yu

last: Miyamoto

last: Okuzaki

first: Masaru

last: Ishii


title: Signals from the reproductive system regulate somatic regeneration and vertebrate lifespan

first: Itamar

last: Harel

summary: In many vertebrate species, age at maturity is proportional to organismal lifespan. A number of theories have attempted to provide a conceptual framework for this fascinating apparent co-evolution. The ‘disposable soma’ theory predicts that costly resources that are invested in germline preservation come at the expense of repairing age-related somatic damage. On the other hand, classical experiments in C. elegans have reported that germline ablation promotes longevity via endocrine signals mediated by the somatic gonad. However, how these seemingly opposing mechanisms may affect vertebrate aging is largely unknown. Here, we use the naturally short-lived turquoise killifish (N. furzeri) to demonstrate that germline ablation can enhance damage repair and promote vertebrate longevity. This was achieved by producing a detailed single-cell atlas of the killifish ovary and testis, and generating a panel of genetic perturbations to manipulate the killifish reproductive state. Specifically, we either block costly stages of germline differentiation by mutating gonadotropins and their receptors, or alternatively, ablate the germline altogether by mutating the dnd1 gene (dead end). Interestingly, only germline ablation enhances somatic repair, particularly in females, by improving tail regeneration following genotoxic stress. To investigate the observed sexual dimorphism, we directly compare several somatic cell types from WT and dnd1 fish. We identify a surprising male-specific upregulation of pro-longevity pathways, including protein homeostasis and stress resistance. Accordingly, only germline-depleted males experience a significant extension in maximal lifespan. Together, dissecting distinct paradigms of germline manipulations, reveals a functional link between the reproductive state, damage repair, and longevity. The sex-specific effect further supports the concept that phenotypes are independent of resource allocation, and instead, could be the result of a possible endocrine effect. Our findings may therefore have exciting implications for uncoupling the reproductive state from vertebrate aging.

overall_design: Sexually mature one-month old fish were used, and each group varied by either sex (males or females) or genotype (wild-type or dnd1 knock-out). For wild-type fish, 3 gonads were combined from either males or female, while for dnd1 mutants, multiple fish were used per group (as the gonads are very small).

first: Moses

last: Eitan

first: Tehila

last: Atlan

first: Xue

last: Sun


title: Humanized APOE genotypes influence lifespan independently of tau aggregation in the P301S mouse model of tauopathy

city: Atlanta

first: Karen

last: McFarland

state: GA

address: 100 Woodruff Circle

department: Pharmacology

institute: Emory University

summary: To gain insight into the role of human APOE on tau-associated neurodegeneration, we examined P301S mutant tau transgenic mice carrying humanized APOE alleles

overall_design: We performed gene expression profiling of the forebrains of P301S mutant tau transgenic mice carrying humanized APOE alleles alongside mice with humanized APOE alleles. Brains from mice that reached end stage phenotype (hindlimb paralysis) were collected.

first: Paramita

last: Chakrabarty


title: Basal cell extrusion primes pluripotent cells for differentiation

city: Berlin

first: Helene

last: Kretzmer

address: Ihnestraße 63

department: Genome Regulation

country: Germany

institute: Max Planck Institute for Molecular Genetics

type: Methylation profiling by high throughput sequencing

summary: The blueprint of the mammalian body plan is laid out during gastrulation, when a trilaminar embryo is formed. This process entails a burst of embryonic cell proliferation, the ingression of pluripotent epithelial cells at the primitive streak, and their priming towards primitive streak fates. How these different events are coordinated remains unknown. Here, we developed a 3D culture of self-renewing pluripotent and epithelial cells that recapitulates the transcriptional and architectural features of the gastrulating mouse embryo. Using this system in combination with microfabrication and in vivo experiments, we found that proliferation triggers basal extrusion in cells that express high levels of the apical polarity protein aPKC. Upon extrusion, cells are more sensitive to Bmp, Wnt, and Nodal signaling, and upregulate the expression of primitive streak markers. This mechanistic coupling between cell position and gene expression ensures that the right cell types become specified at the right place during embryonic development.

overall_design: scRNA-Seq, RNA-Seq, BS-Seq

first: Nanami

last: Satoh

first: Viviane

last: Rosa

first: Aly

last: Makhlouf

first: Abhishek

last: Kumar

first: Stefanie

last: Grosswendt

first: Alexandra

last: Mattei

first: Olivia

last: Courbot

first: Steffen

last: Wolf

first: Katie

last: McDole

first: Frederic

last: Langevin

first: Michal

last: Wiacek

first: Daniel

last: Karpinski

first: Alberto

last: Elosegui-Artola

first: Alexander

last: Meissner

first: Magdalena

last: Zernicka-Goetz

first: Marta

last: Shahbazi


title: Characterization of the host defense against influenza virus infection and a secondary bacterial pneumonia infection

city: Tel Aviv

first: ofir

last: cohn

address: Tel Aviv University

institute: Tel Aviv University

summary: Secondary bacterial infections (‘superinfection’) are a major reason for excessive mortality and hospitalizations during influenza virus infections. Here we present a longitudinal study of gene-expression changes in murine lungs during superinfection, with an initial influenza A virus (IAV) infection and a subsequent S. pneumonia (SP) infection. In addition to the well characterized impairment of the innate immune response, we identified superinfection-specific alterations in endothelial functions, including rapid downregulation in angiogenic activity and vascular regulators. Superinfection-specific alterations were also evident in analysis of cellular states related to the host’s immune resistance against pathogens. We found that only a few hours after secondary bacterial challenge, superinfected mice manifested an excessive induction of immune resistance, and in addition, there was a substantial rewiring of the resistance program: interferon-regulated genes are switched from positive to negative correlations with resistance, whereas genes of fatty-acid metabolism are switched from negative to positive correlations with resistance. Thus, the transcriptional resistance state is reprogrammed toward repressed interferon signaling and induced fatty acid metabolism. Our findings suggest new insights into the remodeling of the host defense upon superinfection, providing promising targets for future therapeutic interventions.

overall_design: For the secondary pneumococcal infection model: anesthetized mice were administered with IAV and 5 days later were infected with S. pneumoniae.

first: Ofir

last: Cohn

first: Gal

last: Yankovitz

first: Naama

last: Pashes-Yaloz

last: Mandelboim

first: Eran

last: Bacharach

first: Irit

last: Gat-Viks


title: MBNL1 regulates programmed postnatal switching between regenerative and differentiated cardiac states

city: Seattle

first: Jennifer

last: Davis

address: 815 Mercer Street D342

institute: University of Washignton

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

sample_organism:
Mus musculus
Rattus norvegicus

platform_organism:
Mus musculus
Rattus norvegicus

overall_design: Refer to individual Series


title: Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations (amplicon)

status: Public on Mar 05 2024

first: Chris

last: Hsiung

summary: Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding genetic elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective for perturbing non-coding regulatory elements in pooled assays. However, current CRISPRi pooled screening approaches are limited to targeting 1-3 genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional interference AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation that stabilizes the ribonucleoprotein:DNA complex via DNA nicking. The multiAsCas12a-KRAB fusion improves CRISPRi activity over DNase-dead AsCas12a-KRAB fusions, often rescuing the activities of lentivirally delivered CRISPR RNAs (crRNA) that are inactive when used with the latter. multiAsCas12a-KRAB supports CRISPRi using 6-plex crRNA arrays in high-throughput pooled screens. Using multiAsCas12a-KRAB, we discover enhancer elements and dissect the combinatorial function of cis-regulatory elements in human cells. These results instantiate a group testing framework for efficiently surveying numerous combinations of chromatin perturbations for biological discovery and engineering.

overall_design: Analysis of indel frequencies by short-read Illumina sequencing of PCR amplicons surrounding genomic sites targeted by Cas12a variant fusion proteins using CRISPR RNAs (crRNA).

first: Caroline

last: Wilson

first: Nicholas

last: Sambold

first: Raymond

last: Dai

first: Qingzhou

last: Chen

first: Sara

last: Misiukiewicz

first: Abofazl

last: Arab

first: Noam

last: Teyssier

first: Tom

last: O'Loughlin

first: Joshua

last: Cofsky

first: Luke

last: Gilbert


title: Role of the histone variant H2A.Z.1 in memory, transcription, and alternative splicing is mediated by lysine modification II

city: Mississauga

first: Iva

last: Zovkic

address: 3359 Mississauga Rd

department: Department of Psychology

institute: University of Toronto

summary: Creating long-lasting memories relies on learning-induced changes in gene expression, which are regulated by epigenetic modifications of DNA and associated histone proteins. Post-translational modifications (PTMs) of histone proteins are key regulators of transcription, with different PTMs producing unique effects on gene activity and behavior. Although recent studies began to investigate histone variants as key regulators of memory formation, PTMs are rarely considered in relation to their function.  Here, we analyze the role of acetylation of histone variant H2A.Z.1 in memory and gene expression.  To this end, we developed AAV constructs to overexpress mutated H2A.Z.1 isoforms that either mimic acetylation (acetyl-mimetic) by replacing lysines 4, 7 and 11 with glutamine (KQ), or that have impaired acetylation (acetyl-defective) by replacing the same lysine with alanine (KA).  We found that overexpression of the acetyl-mimetic (H2A.Z.1KQ) or the acetyl-defective (H2A.Z.1KA) isoforms of H2A.Z.1 in the hippocampus produces different effects on memory depending on strength of the task and sex, with H2A.Z.1KQ generally having a beneficial effect on memory, while H2A.Z.1KA results in impaired memory. We further analyzed gene expression data and found that H2A.Z.1KQ and H2A.Z.1KA uniquely impact the expression of different classes of genes in females and males. However, different genes are regulated by different isoforms in the 2 sexes. Finally, we describe, for the first time, that H2A.Z is involved in the alternative splicing of neuronal genes. Notably, H2A.Z depletion and its replacement with different isoforms influence transcription and splicing of different genes, suggesting that H2A.Z.1 can regulate genes through splicing and expression levels.  This is the first study demonstrating that direct manipulation of the levels of AcH2A.Z regulates memory, gene expression and splicing. Overall, this study adds another layer of complexity to the contribution of histone variants to higher brain functions, consolidating H2A.Z as a key regulator of memory.

overall_design: Chromatin Immunoprecipitation DNA-sequencing for acetylated histone variant AcH2A.Z in primary hippocampal neurons. Investigate the binding of AcH2A.Z


title: Spon1+ inflammatory monocytes promote collagen remodeling and lung cancer metastasis through lipoprotein receptor 8 signaling.

city: Chapel Hill

first: Alessandro

last: Porrello

state: NC

address: 450 West Drive

department: Lineberger Comprehensive Cancer Center

institute: University of North Carolina at Chapel Hill

summary: Lung cancer is the leading cause of cancer-related deaths in the world, and non-small cell lung cancer (NSCLC) is the most common subset. Although immune checkpoint inhibitors have improved outcomes in a subset of NSCLC patients, these effects are usually not durable. Thus, a more complete understanding of how immune niches within the tumor microenvironment (TME) promote NSCLC progression will allow us to build upon these advances. We previously found that infiltration of tumor inflammatory monocytes (TIMs) into lung squamous carcinoma (LUSC) tumors is associated with increased metastases and poor survival. To further understand how TIMs promote metastases, we compared RNA-seq profiles of TIMs from several LUSC metastatic models with IMs of non-tumor bearing controls. We identified Spon1, a secreted extracellular matrix glycoprotein, as upregulated in TIMs and that Spon1 expression in LUSC tumors corresponds with poor survival. Bioinformatic analyses of the Cancer Genome Atlas (TCGA) revealed that LUSC tumors with high SPON1 expression significantly enriched for collagen extracellular matrix (ECM) signatures. Unexpectedly, we observed SPON1+ TIMs mediate their effects directly through LRP8 (ApoER2) on NSCLC cells, which results in TGFβ1 activation and robust production of fibrillar collagens. Using several orthogonal approaches, we demonstrate that SPON1+ TIMs are sufficient to promote NSCLC metastases. Additionally, we find that Spon1 loss in the host, or Lrp8 loss in cancer cells, results in a significant decrease of both high-density collagen matrices and metastases. Finally, we confirm the relevance of the SPON1/LRP8/TGFβ1 axis with collagen production and survival in NSCLC patients. Taken together, our study describes how SPON1+ TIMs promotes collagen remodeling and NSCLC metastases through an LRP8-TGFβ1 signaling axis in lung cancer cells.

overall_design:
Characterize the transcriptome of IMs in healthy and tumor-bearing mice using two immune competent models of lung squamous cancer.
IMs from healthy FVB and DBA2 mouse strains include 3 replicates each and each tumor-bearing group contains 5 replicates.

first: Chad

last: Pecot


title: Clock-dependent chromatin accessibility rhythms regulate circadian transcription

city: Ann Arbor

first: Swathi

last: Yadlapalli

address: 109 Zina Pitcher Pl

institute: University of Michigan

summary: Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.

overall_design: Transposase-accessible chromatin sequencing (ATAC-seq) of Drosophila melanogaster clock neurons. Cells were isolated by fluorescence-activated cell sorting (FACS) using Clk-GAL4>UAS-GFP-NLS line developed in the lab. GFP-positive and DAPI-negative cells were sorted to be clock neurons and each sample consist 1~2 thousand cells. GFP-negative and DAPI-negative cells were sorted to be non-clock cell controls.

first: Ye

last: Yuan

first: Margarita

last: Brovkina

first: E

last: Clowney


title: Cardiometabolic characteristics of people with metabolically healthy and unhealthy obesity

first: Jinsheng

last: Yu


title: Cardiometabolic characteristics of people with metabolically healthy and unhealthy obesity [AbdomFat]

overall_design: Comprehensive cardiometabolic profiling was conducted in 20 adults with metabolically healthy obesity (MHO, defined as normal fasting glucose, glucose tolerance, HbA1c, intrahepatic triglyceride, serum triglycerides, and whole-body insulin sensitivity), 20 age-, sex-, and BMI-matched adults with metabolically unhealthy obesity (MUO, defined as prediabetes and hepatic steatosis), and 15 adults who were metabolically healthy and lean (MHL). Subcutaneous abdominal adipose tissue biopsy samples were subjected to bulk RNA-sequencing.

first: Max

last: Petersen

first: Gordon

last: Smith

first: Hector

last: Palacios

first: Mihoko

last: Yoshino

first: Jun

first: Kevin

last: Cho

first: Victor

last: Davila-Roman

first: Mahalakshmi

last: Shankaran

first: Ruteja

last: Barve

first: Bruce

last: Patterson

first: Marc

last: Hellerstein

first: Gerald

last: Shulman

first: Gary

last: Patti

first: Samuel

last: Klein


title: Single cell approaches define two mammalian oligodendrocyte precursor cell populations and their evolution over developmental time [scATAC-seq]

city: Vancouver

first: Freda

last: Miller

state: BC

address: 2185 East Mall

department: Division of Neurology

institute: The University of British Columbia

summary: Here, we have used single-cell RNA sequencing (scRNA-seq), single-cell ATAC sequencing (scATAC-seq) and spatial transcriptomics to characterize murine cortical OPCs throughout postnatal life. During development, we identify two differentially-localized PDGFRα-positive OPC populations that are transcriptionally and epigenetically distinct. One population (active or actOPCs) is metabolically active and is enriched in white matter. The second (homeostatic or hOPCs) is less active, enriched in grey matter, and predicted to derive from actOPCs. In adulthood, these two groups are transcriptionally but not epigenetically distinct, and relative to developing OPCs are less active metabolically with much less open chromatin. When adult oligodendrogenesis is enhanced following experimental demyelination, adult OPCs do not reacquire a developmental open chromatin state, and the oligodendrogenesis trajectory is distinct from that seen neonatally. These data support a model where two OPC populations subserve distinct postnatal functions, and where neonatal and adult OPC-mediated oligodendrogenesis are fundamentally different.

overall_design: High-throughput single cell transcriptomic profiles of P7, P60 and P90 (control, cuprizone-rapamycin treated mice) from the mouse brain (SVZ and cortex) generated by single cell ATAC sequencing using the 10X Genomics platform. Replicates are indicated (biol 1, biol 2).

first: Daniel

last: Dennis

first: Beatrix

last: Wang

first: Konstantina

last: Karamboulas

first: David

middle: R

last: Kaplan

middle: D


title: Alternative splicing of a chromatin modifier alters the transcriptional regulatory programs of embryonic stem cell maintenance and neuronal differentiation.

city: LOS ANGELES

first: Chia-ho

last: Lin

address: 6577 MRL. 675 CHARLES E YOUNG DR. SOUTH

institute: UCLA


title: RNAseq transcriptomic comparison of mouse tracheal pseudostratified vs hillock basal stem cells

city: Boston

first: Brian

address: 136 Harrison Avenue

department: DMCB

institute: Tufts University

summary: Airway hillocks are stratified epithelial structures of unknown function. We show that the luminal cells of hillocks are squamous barrier cells that shield a unique population of underlying hillock basal stem cells. Hillocks occur in characteristic locations and persist for months in uninjured mice. Following injury, hillock basal stem cells undergo massive clonal expansion, resurface denuded airway, and demonstrate extreme plasticity, generating classic airway epithelium with its six component cell types. Remarkably, hillocks resist a very broad spectrum of injuries, including toxins, infection, acid, and physical injury. Furthermore, hillock basal stem cells are characterized by enhanced expression associated with barrier function, cell adhesion, and retinoic acid catabolism. Indeed, hillock basal cells preferentially expand, stratify, and keratinize in the setting of retinoic acid signaling inhibition, a known clinical cause of squamous metaplasia. Finally, we identify human hillocks whose basal cells demonstrate preferential sensitivity to retinoic acid inhibition and conserved barrier properties. Given their capacity for clonal expansion, their plasticity, and their retinoic acid sensitivity, the existence of hillocks and hillock basal stem cells have broad implications for injury repair and for defining the origin of squamous metaplasia, long thought to be a precursor of lung cancer.

overall_design: Cells from K13-CreER;LSL-tdTomato;p63gfp/+ tracheas were dissociated and sorted. tdTomato+ GFP+ hillock basal cells and tdTomato- GFP+ pseudostratified basal cells were expanded in vitro for 10 days prior to sample collection for bulk RNA/ATAC sequencing. Total RNA samples were provided to Novogene for processing according to standard protocols. Reads were mapped by Novogene to genome build grcm38_p6_gca_000001635_8.

first: Jiawei

last: Sun

first: Viral

last: Shah

first: Chaim

last: Chernoff

first: Jayaraj

last: Rajagopal


title: ATACseq comparison of mouse tracheal pseudostratified vs hillock basal stem cells

overall_design: Cells from K13-CreER;LSL-tdTomato;p63gfp/+ tracheas were dissociated and sorted. tdTomato+ GFP+ hillock basal cells and tdTomato- GFP+ pseudostratified basal cells were expanded in vitro for 10 days prior to sample collection for bulk RNA/ATAC sequencing. Frozen cell pellets were provided to Novogene for processing according to standard protocols. Reads were mapped by Novogene to genome build grcm38_p6_gca_000001635_8.


title: Mouse tracheal pseudostratified vs hillock basal stem cells


title: Reconstructing regulative progenitor behaviors in synthetic embryos

first: Helene

last: Kretzmer

summary: Advanced stem cell-based in vitro models of embryogenesis offer great promise to decipher developmental programs with high-throughput and resolution. However, the rapid changes in morphology and cell state that underly these models mask multiple higher order parameters, including the regulative balance between differentiation and self-renewal that govern transient progenitor cell populations. Here, we apply a continuous single-cell Cas9-based molecular recorder to mouse embryonic stem cells, which enables the recovery of can reproducibly recovere and high-content information regarding the historical relationship between individual cells. We apply our system to Trunk-Like Structures (TLS), a complex in vitro model of embryogenesis that captures differentiation and morphological dynamics from pluripotency exit to the specification of the somitic and neural lineages. By recording cell lineages alongside the transcriptional identities of extant cells, we are able to build high-resolution phylogenetic trees, identify clonal subpopulations, and reveal recover the step-wisestepwise series of events that govern germ layer specification. Moreover, we  examine are able to functionally characterize the specification and maintenance of multipotent neuro-mesodermal progenitor (NMP) populations as they coordinate self-renewal and commitment into more specialized cell types, including how thehow  highly disparateplastic individual  nature of individual branches trajectories ultimately aggregateultimately aggregate to ensure to generate the generation of highly consistent embryonic structures. Finally, we demonstrate the potential of this technology for high-throughput perturbation screening by combining small molecule treatment with individual structure labeling, allowing us to we demonstrate that these parameters can be independently tuned to modulate TLS morphology, including in reversible ways that extend the duration through which NMPs can contribute to both lineages of the embryonic trunkconnect perturbation of early differentiation pathways to progenitor cell behavior and structure morphology. Our work represents the first continuous molecular recording approach applied to dynamic in vitro models, a toolkit that can be combined with inhibitor libraries or other screening modalities to uncover the genetic and epigenetic mechanisms that enable highly orchestrated and robust changes in cell state composition during critical windows of morphogenesis.

sample_organism:
Mus musculus
synthetic construct

platform_organism:
Mus musculus
synthetic construct

overall_design: Single-cell RNAseq data were collected from Tracer TLS at 120h after aggregation (2 individual structures and one Multiseq experiment containing 24 structures, 12 Tracer TLS and 12 Tracer TLSCL). Libraries were prepared and sequenced with 10X Genomics single cell RNA sequencing system (RNAseq fraction). Individul Amplicon seq libraries for evolving, lenti and multiseq barcodes have been prepared separately from the 10x cDNA from each single-cell reaction. Bulk Amplicon seq data were collected from Tracer mESCs at various timepoints after Cas9 activation (Cas9-GFP+ sorted Tracer mESCs at day 0, 1, 2, 3, 4, 5 after aggregation) and from 8 individual Tracer TLS 120h after aggregation. Libraries were prepared from both gDNA and cDNA from the same cell suspensions.

first: Adriano

last: Bolondi

first: Benjamin

last: Law

first: Sehr

last: Gassalog

first: Christina

last: Riemenschneider

first: Dian

last: Yang

first: Rene

last: Buschow

first: Maria

last: Walther

first: Christopher

last: McGinnis

first: Zev

last: Gartner

first: Jesse

last: Veenvliet

first: Alexander

last: Meissner

first: Zachary

last: Smith

first: Michelle

last: Chan


title: Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations

city: Palo Alto

first: Chris

last: Hsiung

address: 3181 Porter Dr

institute: Arc Institute


title: Chronic opioid treatment arrests neurodevelopment and alters synaptic activity in human midbrain organoids

city: New York

first: Kam

last: Leong

state: NY

address: 3600 Broadway, RM450C

department: Biomedical Engineering

institute: Columbia University

summary: Understanding the impact of long-term opioid exposure on the embryonic brain is critical due to the surging number of pregnant mothers with opioid dependency, but has been limited by human brain inaccessibility and cross-species differences in animal models. Here we establish a human midbrain model that uses hiPSC-derived midbrain organoids and apply it to assess cell-type-specific responses to acute and chronic fentanyl treatment and fentanyl withdrawal. Single-cell mRNA sequencing of 25,510 cells from organoids in different treatment groups indicated that chronic fentanyl treatment arrests neuronal subtype specification during early midbrain development and alters synaptic activity and neuron projection. In contrast, acute fentanyl treatment increases dopamine release but does not significantly alter gene expression related to cell lineage development. These results provide the first examination of the effects of opioid exposure on human midbrain development at the single cell level.

overall_design: Midbrain organoids derived from human FA11 iPSC cells were cultured and later treated with or without fentanyl. The acute treatment with Fentanyl was administered at a concentration of 74nM for a duration of 4 hours , on day 53 (KH002_D53_AC_FTY). The chronic treatment with Fentanyl was administered at a concentration of 74nM for a duration of 77 days, from Day 1 to Day 77 (KH004_D77_FTY). Withdrawal sample was treated with Fentanyl (74nM for 77 days, Day 1 to Day 77) and then replaced with fresh media for 2 days (KH005_D79_WD).

first: Kim

last: Hye Sung

first: Xiao

first: Xu

last: Bin

first: Leong

last: Kam


title: Telomeric Chromosomal Instability Fosters Paracrine Mediated Cell Competition to Initiate Intestinal Cancer (WGS)

city: Houston

first: Ronald

last: DePinho

state: TX

address: 515 Holcombe Blvd

department: Cancer Biology

institute: MD Anderson

summary: Telomere dysfunction drives chromosomal instability (CIN) during the transition from benign adenoma to malignant adenocarcinoma. While CIN provides a mutator mechanism for cancer- relevant genomic events, its role in shaping tumor biology during carcinogenesis is not well understood. Here, we explored the molecular and biological impact of telomere dysfunction and associated CIN in vivo in a faithful model of CRC. In vivo lineage tracing revealed that CIN increased the rate of neoplastic cell clonal expansion through accelerated differentiation of neighboring stem cells, resulting in increased number of adenomas and decreased survival in CIN-high Apcmin mice. Mechanistically, CIN represses EZH2 leading to upregulation of secreted Wnt antagonists, which resulted in a growth advantage to CIN-high neoplastic cells. Correspondingly, pharmacological activation of intrinsic WNT signaling enhanced intestinal stem cells fitness, leading to reduced neoplastic cell clonal expansion and adenoma burden. Thus, the CIN-EZH2-WNT axis enhances intestinal cancer initiation in the nascent tumor microenvironment, providing a preventive strategy for patients harboring germline APC mutations.

overall_design: For whole genome sequencing, genomic DNA was isolated from distal colonic adenomas of telomere intact (G0) and telomere dysfunctional (G2) Apcmin mice to evaluate the level of copy number alterations.

first: Kyle

last: LaBella

first: Chia-Chin

last: Wu


title: The Alk receptor tyrosine kinase regulates Sparkly, a novel activity regulating neuropeptide precursor in the Drosophila CNS

city: Göteborg

first: Vimala

last: Anthonydhason

address: Medicinaregatan 9 a-b

department: Institute of Biomedicine

country: Sweden

institute: University of Gothenburg

summary: Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the CNS, however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analysed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk/scRNA-seq and protein datasets from larval brains in which Alk signaling was manipulated, identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (Clock) neurons, and Spar mutants exhibit defects in sleep and circadian rhythm control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.

overall_design: Targeted DamID (TaDa) transcriptional profiling to identify transcriptional targets of Alk signaling in the larval CNS.

first: Ruth

last: Palmer

first: Sanjay

last: Sukumar


title: BRG1 contributes to the global chromatin accessibility revealed by its acute depletion

city: Bethesda

first: Keji

last: Zhao

state: MD

address: Building 10 Room 7B06A

department: National Heart, Lung, and Blood Institute

institute: national Institute of Health

summary: DNase I hypersensitivity (DHS) of chromatin indicates the presence of important regulatory elements of transcription such as enhancers and promoters and plays critical roles for their activities. However, the mechanisms that control the global DHS have not been fully understood. In this report, we show that acute depletion of BRG1 by the auxin-dependent degron system, the essential ATPase subunit of the mammalian chromatin remodeling SWI/SNF-like BAF complexes, severely compromised genome-wide DHSs of chromatin, while the traditional conditional deletion of the Brg1 gene using Cre/Flox system led to only very modest changes of DHSs. The global decrease of DHSs is associated with a genome-wide decrease of transcription. We demonstrate that the DHS change is related with changes in nucleosome positioning at promoters and enhancers. Our data suggest that acute depletion of proteins reveal target genes of BRG1 that are not detected by the traditional models.

overall_design: To address the direct effect of BRG1 depletion in gene regulation and chromatin landscape, we employ auxin-dependent degron (AID) system to acute deplete BRG1 in mouse T cells and hepatocytes. We further validated these directly effects by comparison with the traditional conditional deletion of the Brg1 gene using Cre/Flox system. Integration analysis of multi-omics datasets, we uncover the novel mechanism for BRG1 dependent regulation of gene expression and chromatin landscape, which are not detected by the traditional models.

first: Gang

last: Ren

first: Wai

last: Ku

first: Jackson

last: Hoffman

first: Qingsong

last: Tang

first: Kairong

last: Cui

first: Chengyu

last: Liu

first: Trevor

last: Archer


title: KCNJ2 inhibition mitigates mechanical injury in human brain organoids [scRNA-seq]

status: Public on Mar 04 2024

city: Los Angeles

first: Joshua

last: Berlind

address: 1425 San Pablo St

institute: University of Southern California

summary: Traumatic brain injury (TBI) strongly correlates with neurodegenerative disease. However, it remains unclear which neurodegenerative mechanisms are intrinsic to the brain itself and which strategies most potently mitigate these processes, particularly in individuals genetically predisposed to neurodegeneration. We developed a high-intensity ultrasound platform to inflict mechanical injury to iPSC-derived cortical organoids. Mechanically injured organoids elicit several classic hallmarks of TBI including neuron death, tau phosphorylation, and TDP-43 phosphorylation and nuclear egress. We found that deep layer excitatory neurons were particularly vulnerable to injury and that TDP-43 proteinopathy promotes loss-of-function and cell death. Injured organoids derived from C9ORF72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) patients displayed exacerbated TDP-43 dysfunction. Using genome-wide CRISPR interference screening, we identified a mechanosensory channel, KCNJ2, whose inhibition potently mitigated neurodegenerative processes in vitro and in vivo, including in C9ORF72 ALS/FTD organoids. Thus, targeting KCNJ2 may reduce acute neuron death after brain injury, and we present a scalable, genetically-flexible cerebral organoid model that may enable identification of additional modifiers downstream of mechanical stress.

overall_design: Sham and HIFU-injured organoids were collected after 7 days for scRNA seq. Conditions were multiplexed to save on cost.

first: Justin

last: Ichida


title: Ribo– RNA-seq of psoriasis mouse model delivered (i.v.) EPIC by LNP at D1.5

city: Shanghai

first: Li

last: Yang

address: 131 Dong-An Road

department: Institutes of Biological Sciences

institute: Fudan University

summary: Efforts to advance RNA aptamers as a novel therapeutic modality have been limited by their susceptibilty to degradation and immunogenicity. In a previous study, we demonstrated synthesized double-stranded circular RNAs (ds-cRNAs) with minimal immunogenicity targeted to dsRNA-activated Protein Kinase R (PKR). Here, we test the therapeutic potential of ds-cRNAs in a mouse model of imiquimod-induced psoriasis. We find that genetic supplementation of ds-cRNAs leads to inhibition of PKR, resulting in alleviation of downstream interferon alpha (IFNα)/dsRNA signals and attenuation of psoriasis phenotypes. Delivery of ds-cRNAs by lipid nanoparticles to the spleen attenuates PKR activity in examined splenocytes, resulting in reduced epidermal thickness. These findings suggest that ds-cRNAs represent a promising approach to mitigate excessive PKR activation for therapeutic purposes.

overall_design: To investigate whether delivery of in vitro synthesized RNA circle EPIC can affect endogenous circRNAs expression, we established psoriasis mouse model via IMQ (imiquimod) treatment at D0 and D1, and delivered (i.v.) EPIC by LNP at D1.5. We then performed total RNA sequencing of mouse (C57BL/6) spleens at D2.5 after intravenous injection. Each sequencing sample contained RNA isolated from spleen of one mouse.

first: Si-Kun

last: Guo

first: Chu-Xiao

first: Yi-Feng

last: Xu

first: Fang

first: Ling-Ling

last: Chen


title: Alternative splicing of a chromatin modifier alters the transcriptional regulatory programs of embryonic stem cell maintenance and neuronal differentiation [ChIP-seq]

first: Chia-ho

last: Lin

summary: Maintenance of embryonic stem cells (ESCs) and their differentiation to neuronal lineages requires complex genetic regulation of transcription, splicing, and translation, but the interconnections of these processes is not well understood. We found that the splicing regulator polypyrimidine tract binding protein 1 (PTBP1) alters the neuronal transcriptional program. Transcripts for DPF2, a subunit of the BRG1/BRM-associated factor (BAF) chromatin remodeling complex, contain an additional exon (exon 7) in brain. This exon encodes a 14 amino acid insert in the C2H2-type zinc finger domain generating a longer DPF2-L isoform, compared to the canonical short DPF2-S isoform. We find that inclusion of exon 7 in Dpf2 mRNA is strongly inhibited by PTBP1, and loss PTBP1 switches expression from DPF2-S to DPF2-L early in neuronal differentiation. We created CRISPR/Cas9-mediated genome-edited cells that force expression of either DPF2-S or DPF2-L to examine the consequences of this switch in ESCs, in ESC-derived neural progenitor cells (NPCs), and in glutamatergic neurons (GNs). We found substantial changes in gene expression induced by DPF2-L in ESCs and by loss of this isoform in neurons. Chromatin immunoprecipitation in ESCs revealed that DPF2-S but not DPF2-L preferentially bound to genomic regions associated with the stem cell pluripotency transcription factors OCT4, SOX2, and NANOG. In NPC’s, DPF2-S preferentially bound to genomic regions co-occupied by the transcription factor NF1, while DPF2-L preferred sites bound by the CTC binding factor, CTCF. The correlation of DPF2 isoform binding with chromatin states identified by ChromHMM revealed that DPF2-S binds to sites enriched in enhancers, whereas DPF2-L-bound sites are enriched for promoters. Altogether, our study identifies a new neuronal BAF isoform that alters its functional targets, and demonstrates how splicing regulatory events during development elicit wide ranging impacts on chromatin organization and transcription.

overall_design: To investigate chromatin targeting of alternative DPF2 isoforms we engineered genome-edited mouse ESCs that force express either DPF2-S or DPF2-L. We then performed Chromatin immunoprecipitation of DPF2 folllowed by DNA sequencing (ChIP-seq) in ESCs and ESC-derived NPCs.

first: Mohammad

last: Nazim

first: An-Chieh

last: Feng

first: Chia-Ho

first: Douglas

last: Black


title: KCNJ2 inhibition mitigates mechanical injury in human brain organoids

overall_design: HIFU and sham treated organoids were collected 24 hours after injury and processed for bulk RNA sequencing


title: Therapeutic application of circular RNA aptamers in a mouse model of psoriasis

overall_design: Refer to individual Series


title: Single cell approaches define two mammalian oligodendrocyte precursor cell populations and their evolution over developmental time [Xenium In Situ Gene Expression]

first: Freda

last: Miller

overall_design: Multiplexed in situ hybridization-based spatial transcriptomics using the Xenium platform was used to analyze coronal P7 forebrain sections containing the V-SVZ, corpus callosum and cortex with a probeset that included marker genes for relevant cell types, including actOPCs and hOPCs. We analyzed a region of interest that included the V-SVZ, the corpus callosum and the cortical grey matter from two different brains (2 sections from one brain, 1 section from the other). Replicates are indicated (biol 1, biol 2, biol 3).

first: Daniel

last: Dennis

first: Beatrix

last: Wang

first: Konstantina

last: Karamboulas

first: David

last: Kaplan


title: Single cell approaches define two mammalian oligodendrocyte precursor cell populations and their evolution over developmental time [scMultiome-seq]

overall_design: High-throughput single cell transcriptomic profiles of P6 from the mouse brain (SVZ) generated by single cell multiome sequencing using the 10X Genomics platform.


title: Single cell atlas of the aging mouse trachea

first: Gergana

last: Shipkovenska

state: Massachusetts

address: 185 Cambridge Street

department: Center for Regenerative Medicine

institute: Massachusetts General Hospital

summary: The airways conduct gases to and from the gas-exchanging alveoli of the lung and are the site of age-related diseases, such as chronic obstructive pulmonary disease, asthma, and bronchogenic carcinoma. Here we set out to study the effects of aging on the cellular composition and gene expression of murine airway. We present a comprehensive single-cell RNA-sequencing atlas of tracheas from young (2 month) and aged (24 month) mice, comprising diverse populations of cells, including epithelial, endothelial, immune, fibroblast, and cartilage cells.

overall_design: To study the effects of aging on cellular composition and gene expression of mouse airway, tracheas from two young (2 month) and two aged (24 month) animals were subjected to single cell dissociation and 3’-droplet based sequencing, without targeting or enrichment of specific cell types.

first: Yanxin

first: Andrew

last: Leduc

first: Jiajie

first: Jayaraj

last: Rajagopal

first: Zhixun

last: Dou

first: Nikolai

last: Slavov


title: Chromatin accessibility and transcriptomic responses to HDAC inhibitors in TP53 mutated patient-derived colon cancer organoids

city: Basel

first: Emanuela

last: Kerschbamer

state: Basel Stadt

address: Mattenstrasse 28

department: Department of Biomedicine

institute: University of Basel


title: Chromatin accessibility and transcriptomic responses to HDAC inhibitors in TP53 mutated patient-derived colon cancer organoids [ATAC-seq]

summary: Here we present the generation and characterization of patient-derived organoids (PDOs) from colorectal cancer patients. Two PDOs derived from patients with TP53 mutations were tested with two different HDACIs (SAHA and NKL54). Induction of cell death, transcriptome, and chromatin accessibility changes were analyzed. HDACIs promote the upregulation of low expressed genes and suppress the expression of highly expressed genes. A similar differential effect is also observed at the level of chromatin accessibility. Only SAHA is a potent inducer of cell death. The induction of apoptosis is characterized by the upregulation of BH3-only genes (especially BIK and BMF). Up-regulation of BIK is associated with increased accessibility in an intronic region. SAHA, but not NKL54, also causes downregulation of BCL2L1 and decreases chromatin accessibility in three distinct regions of the BCL2L1 locus. Both inhibitors upregulate the expression of innate immunity genes and members of the MHC family. In conclusion, our studies explain the mechanisms of action of SAHA and cast doubt on the use of NKL54 as a single agent for efficient apoptosis induction.

overall_design:
To investigate the effect of HDAC inhibitors on the survival and proliferation of PDOs derived from CRC patients, we performed ATAC-seq experiments
We analyzed chromatin accessibility using data obtained from ATAC-seq of 2 different CRC PDOs treated with DMSO (control), with SAHA (5µM) or with NKL54 (10µM) for 6 hours.  2 biological replicates for each condition were generated and analyzed.

first: Teresa

last: Gagliano

first: Umberto

last: Baccarani

first: Martina

last: Minisini

first: Eros

last: Di Giorgio

first: Emiliano

last: Dalla

first: Christian

middle: X

last: Weichenberger

first: Vittorio

last: Cherichi

first: Giovanni

last: Terrosu

first: Claudio

last: Brancolini


title: Differential Effects of JAK1 vs. JAK2 Inhibition in Mouse Models of Hemophagocytic Lymphohistiocytosis

city: Memphis

first: Kim

last: Nichols

state: TN

address: 262 Danny Thomas Pl

department: Oncology

institute: St. Jude Children's Research Hospital

summary: Hemophagocytic lymphohistiocytosis (HLH) comprises a severe hyperinflammatory phenotype driven by the overproduction of cytokines, many of which signal via the JAK/STAT pathway. Indeed, the JAK1/2 inhibitor ruxolitinib has demonstrated efficacy in pre-clinical studies and early-phase clinical trials in HLH. Nevertheless, concerns remain for ruxolitinib-induced cytopenias, which are postulated to result from the blockade of JAK2-dependent hematopoietic growth factors. To explore the therapeutic effects of selective JAK inhibition in mouse models of HLH, we carried out studies incorporating the JAK1 inhibitor itacitinib, the JAK2 inhibitor fedratinib and the JAK1/2 inhibitor ruxolitinib. All three drugs were well-tolerated and at the doses tested, they suppressed interferon-gamma (IFNg)-induced STAT1 phosphorylation in vitro and in vivo. Itacitinib, but not fedratinib, significantly improved survival and clinical scores in CpG-induced secondary HLH. Conversely, in primary HLH, where perforin-deficient (Prf1-/-) mice are infected with lymphocytic choriomeningitis virus (LCMV), itacitinib and fedratinib performed suboptimally. Ruxolitinib demonstrated excellent clinical efficacy in both HLH models. RNA-sequencing of splenocytes from LCMV-infected Prf1-/- mice revealed that itacitinib targeted inflammatory and metabolic pathway genes in CD8 T cells, while fedratinib targeted genes regulating cell proliferation and metabolism. In monocytes, neither drug conferred major transcriptional impacts. Consistent with its superior clinical effects, ruxolitinib exerted the greatest transcriptional changes in CD8 T cells and monocytes, targeting more genes across several biologic pathways, most notably JAK-dependent pro-inflammatory signaling. We conclude that JAK1 inhibition is sufficient to curtail CpG-induced disease, but combined inhibition of JAK1 and JAK2 is needed to best control LCMV-induced immunopathology.

overall_design: RNA sequencing analysis of CD8 T cells and CD11b+  Ly6C+Ly6G- monocytes sorted on day 9 p.i.  from Prf1-/- naïve mice, LCMV-infected untreated, or treated in vivo with itacitinib, fedratinib, or ruxolitinib (day 4-8 p.i.)

first: Camille

last: Keenan

first: Ninad

last: Oak

middle: E


title: Effect of MOF mediated UHRF1 acetylation on global DNA methyaltion maintenance in mouse embryonic stem cells.

city: Hong Kong

first: Zhongjun

last: Zhou

state: Hong Kong

address: Room 3-53, Laboratory Block,, 21 Sassoon Road,

country: Hong Kong

institute: The University of Hong Kong

summary: The multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 for DNA methylation maintenance during DNA replication. Here, we show that MOF (Males absent On the First) is an acetyltransferase of UHRF1 to acetylate UHRF1 at Lys670 in the pre-RING linker region whereas HDAC1 is a deacetylase of UHRF1 at the same site. The MOF/HDAC1-mediated acetylation in UHRF1 is cell-cycle regulated and peaks at G1/S phase, in line with the function of UHRF1 in recruiting DNMT1 to maintain DNA methylation. In addition, UHRF1 acetylation significantly enhances its E3 ligase activity and elimination of UHRF1 acetylation at these sites attenuates UHRF1-mediated H3 ubiquitination, which in turn impairs the DNMT1 recruitment and DNA methylation. Taken together, these findings not only identify MOF as a new acetyltransferase for UHRF1 but also reveal a novel mechanism underlying the regulation of DNA methylation maintenance through MOF-mediated UHRF1 acetylation.

overall_design: Mouse WT ESCs, Uhrf1-/- ESCs were cultured on 0.1% gelatin (Sigma) coated dishes in DMEM (high glucose, Gibco, 11965), supplemented with 15% ES-fetal bovine serum (Gibco, 16141-079), 100 units/mL Leukemia Inhibitory Factor (Millipore, ESG1107), GlutaMAX Supplement (Life Technologies, 35050), Sodium Pyruvate (Life Technologies, 11360), and 2-Mercaptoethanol (Life Technologies, 31350), maintained in a 37°C incubator with 5% CO2, and passaged every two days. Plasmids containing flag-myc-tagged human wild-type UHRF1, various domain deleted UHRF1, or different point mutated UHRF1 were transfected to Uhrf1 knockout ES cells using Lipofectamine 3000 (Invitrogen, L3000015) according to the manufacturer’s protocol. Cells were selected by Hygromycin and single colonies were picked to obtain cells with homogeneous gene expression levels. Total genomic DNA from different mESCs was extracted using TIANamp Genomic DNA Kit (TIANGEN, DP304). The double-enzyme RRBS experiments were performed according to a previously published protocol (PMC3570491).

first: Linsheng

last: Wang

first: Shengshuo

last: Huang

first: Yiming

last: Qin


title: Histone lysine demethylase 4 family proteins maintain the transcriptional program and adrenergic cellular state of MYCN-amplified neuroblastoma [ATAC-seq]

first: Hongjian

last: Jin

address: 262 Danny Thomas Place

department: Center for Applied Bioinformatics

institute: St Jude Children's Research Hospital

summary: Neuroblastoma with MYCN amplification (MNA) is a high-risk disease that requires long-term intensive multimodal therapies. Despite this, high-risk patients have a poor survival rate, which has prompted extensive studies aimed at identifying more effective therapies against neuroblastomas with MNA. Neuroblastoma displays cellular heterogeneity, including more differentiated (adrenergic) and more primitive (mesenchymal) cellular states. Here, we demonstrate that MYCN oncoprotein can promote a cellular state switch in mesenchymal cells to an adrenergic state. This cellular state transition is accompanied by induction of histone lysine demethylase 4 family members (KDM4A-C), which act in concert to control the expression of MYCN and adrenergic core regulatory transcription factors (CRC TF). Pharmacologic inhibition of KDM4 blocks expression of MYCN and the adrenergic CRC transcriptome with genome-wide induction of transcriptionally repressive H3K9me3, resulting in potent anticancer activity against neuroblastomas with MNA by inducing neuroblastic differentiation, apoptosis, and a type I interferon response. Further, KDM4 inhibition in combination with conventional, cytotoxic chemotherapy results in complete tumor responses of xenografts with MNA, without overt toxicity in animals. Thus, KDM4 blockade may be a novel and transformative strategy to target the adrenergic CRC dependencies in MNA neuroblastomas.

overall_design: To compare genome-wide chromatin accessibility changes between BE2C parental and BE2C-QC6352 resistant cells, ATAC-seq was performed with BE2C parental in DMSO (0.025%) and BE2C-QC6352-resistant (maintained in 2.5 µM of QC6352) cells.

first: Fang

last: Jie

first: Ahmed

last: Abu-Zaid

first: Jin

last: Hongjian

first: Yang

last: Jun


title: Effect of KDM5 histone demethylase in 3T3-L1 differentiation

first: Karen

last: Reue

state: CA

address: 695 Charles E. Young Drive South

institute: University of California, Los Angeles

summary: The KDM5 histone demethylases regulate histone methylation to modulate chromatin structure and gene expression. Using a KDM5 enzyme inhibitor (KDM5-C70) and transcriptome profiling by RNA-sequencing in 3T3-L1 cells at different states of differentiation, we determined that KDM5 activity influences the induction of genes associated with cell cycle regulation and mitochondrial function.

overall_design: 3T3-L1 preadipocytes were treated with 10 µM KDM5-C70 (KDM5 inhibitor) or DMSO control at differentiation Day –2 (100% confluency). Cells were collected for RNA at differentiation Day 0, 1, 2, and 7.

first: Laurent

last: Vergnes

first: Carrie

middle: B

last: Wiese


title: Nutri-cereal tissue-specific transcriptome atlas during development: Functional integration of gene expression to identify mineral uptake pathways in little millet (Panicum sumatrense)

city: Saskatoon

first: Sateesh

last: Kagale

state: SK

address: 110 Gymnasium Place

department: Aquatic and Crop Resource Development

institute: National Research Council Canada

summary: We present a developmental transcriptome atlas of little millet. It has superior nutritional properties including high micronutrients (Fe, Zn, Ca, Mn), dietary fiber content, and low glycemic index with potential health prospective. This crop is cultivated by tribal people in the marginal areas, and it is adapted to a wide range of growing environments.

overall_design: Seeds of Little millet, Panicum sumatrense, genotype JK-8, were obtained from the University of Agricultural Sciences, Bengaluru, India. Ten different plant tissues were sampled at Agriculture and Agri-Food Canada, Saskatoon, Canada; Germinating seeds (2 days after sowing (DAS)), plumule and radicle (3 DAS), young leaf and young root (13 DAS), crown meristem (26 DAS), vegetative stem (48 DAS), panicle early (61 DAS), panicle mid (73 DAS), panicle late (85 DAS).  Total RNA was extracted from 100 mg of harvested tissues using RNeasy Plant Mini Kit (Qiagen) or Triazol (Sigma-Aldrich) and  purified using the RNeasy MinElute cleanup kit (Qiagen) according to manufacturer's protocol. Purified RNA was quantified using a NanoDrop ND‐100 spectrophotometer (Thermo Scientific), and its integrity evaluated using Agilent 2100 Bioanalyzer (Agilent Technologies). cDNA libraries were prepared using a TruSeq1 RNA sample preparation Kit (Illumina). De novo transcriptome assembly was performed with Trinity.  TPM (transcripts per million) were estimated using Kallisto.

first: Raju

last: Soolanayakanahally


title: Neuronal BST2: A Pruritic Mediator alongside Protease-Activated Receptor 2 in the IL-27–Driven Itch Pathway

city: Kaifeng, Henan Province

first: Yanqing

last: Li

address: Jinming Avenue

institute: Henan University

summary: In the  present study, RNA-seq were performed To explore biological significance of IL-27 and BST2 in the neuro-cutaneous signaling network of chronic itch. mRNA profiles were generated by deep sequencing, in triplicate, using MGISEQ-2000. We found that both human keratinocytes and murine sensory neurons responded to IL-27 and BST2, resulting in enrichment of genes promoting itch and inflammation. IL-27-BST2 signaling pathway activation promotes cutaneous inflammation and itch, both leading into an aggravation of AD. This finding might help us to better understand the immune-neuro-modulatory mechanism in itch circuits

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: primary human keratinocytes were stimulated with IL-27 (100 ng/ml) or BST2 (250 ng/ml) or vehicle control; primary mouse trigeminal ganglion neurons were stimulated with IL-27 (100 ng/ml) or BST2 (500 ng/ml) or vehicle control.  gene expression of these cells were investigated utilizing MGISEQ-2000.

first: Jianghui

last: Meng


title: Histone lysine demethylase 4 family proteins maintain the transcriptional program and adrenergic cellular state of MYCN-amplified neuroblastoma [RNA-seq]

summary: Neuroblastoma with MYCN amplification (MNA) is a high-risk disease that requires long-term intensive multimodal therapies. Less than 50% survival rate of high-risk patients has prompted active and extensive studies to seek more effective therapies against MNA neuroblastomas but with few successes. We show that MYCN transdifferentiates the neuroblastoma cells from mesenchymal state to adrenergic state accompanied with induction of histone lysine demethylase 4 family members (KDM4A-C), all of which act in concert to control the expression of MYCN and adrenergic core regulatory transcription factors (CRC TF). Pharmacologic inhibition of KDM4 blocks expression of MYCN and adrenergic CRC transcriptome with genome-wide induction of transcriptionally repressive H3K9me3, resulting in potent anticancer activity against MNA neuroblastomas by inducing differentiation, apoptosis and type I interferon response. KDM4 inhibition in combination with chemotherapy leads to complete tumor response of MNA xenografts, without overt toxicity in animals. Thus, KDM4 blockade may be a transformative strategy to target the dependency of adrenergic CRC TFs in MNA neuroblastomas.

overall_design: In order to verify MYCN alters the mesenchymal state of neuroblastoma cells to a more adrenergic state, we generated MYCN inducible GIMEN cell line. Then, we performed gene profiling analysis with RNA-seq data. For exploring the KDM4 protein role in the neuroblastoma cells, we treated GIMEN, SH_SY5Y with QC6352, also generated QC6352-resistant BE2C cells. We investigated the tumor samples from TH-MYCN/ALKF1178L mouse model with and without QC6352 treatment. We compare gene profiles changes with or without QC6352 treatment or QC6352 resistant.

first: Abu-Zaid

last: Ahmed

first: Singh

last: Shivendra


title: RNA-seq of KDM4A, B and C siRNA knockdown in NB cells

overall_design: Examination of gene expression profile changes with KDM4A, B and C siRNAs knockdown.

last: Abuzaid

first: Jie

last: Fang

first: Jun

last: Yang


title: Effect of stimulation with lipopolysaccharide (LPS) at 0,30, and 60 minutes on gene expression in immortalized mouse macrophages (IMM).

first: Matthew

middle: James

last: Marino

address: 9000 Rockville Pike

department: NIAID

institute: NIH

summary: The interactions between RNA and protein within cellular signaling pathways, known as the interactome, have modulatory effects on RNA binding proteins (RBP’s) effector functions, which contribute to post-transcriptional control of inflammatory cytokine release in the innate immune response. Post-translational modifications (PTMs) have a regulatory effect on a protein’s effector function. Thus, investigating the effect of RNA-protein interactions can answer questions central to our understanding of cellular signaling. Global RNA sequencing was used to investigate the differentially expressed (DE) genes from baseline following induction with LPS.  Shotgun proteomics along with SILAC labeling was used to quantify relative abundance and differential protein expression in response to LPS.  To assess the proteins interacting with RNA after LPS induction, total RNA associated protein purification was utilized. Using the tandem MS approach and methodology mentioned above, we were able to identify a list of candidate RNA binding proteins. Global RNA sequencing provided a baseline transcriptomic profile as well as quantification of DE genes across a stimulation time course (0, 30, 60 minutes). We then handpicked potential RBPs of immunological interest (Il1a, MARCKS, and Acod1). Interestingly, Il1a, MARCKS, and Acod1 were all highly DE compared to baseline in the RNA Seq results and displayed a tendency to disassociate from RNA as the LPS time course pursued. Il1a, MARCKS, and Acod1 can all directly regulate pro-inflammatory gene expression in macrophages. These RBPs could offer a new regulatory mechanism to tune their effector function, which could lead to new therapeutic innovations for disease pathologies these proteins are implicated in. This research was supported by the Intramural Research Program of NIAID, NIH.

overall_design: To invstigate changes in the transcriptome following stimulation with LPS, we examined 3 biological replicates per time point, where 0 minute was the control and 30 and 60 minutes correspond to the treated samples.

first: Rathore

last: Deepali

first: Marino

last: Matthew

first: Nita-Lazar

last: Aleksandra


title: CUT&Tag of KDM4A, B and C in BE2C cells

first: Hongjian

last: Jin

summary: CUT&Tag of KDM4A, B and C in BE2C cells

overall_design: To investigate interactions between KDM4A, B and C proteins and DNA and to identify DNA binding sites.


title: circSHAPE-MaP of EPIC

first: Li

overall_design:
To exclude the effect of LNP encapsulation and biotin/Cy5 labeling on EPIC structure, we perform circSHAPE-MaP of LNP encapsulated EPIC, biotin/Cy5 labeled EPIC and EPIC among 3 biological replicates. Each samples has 2 techonical repeats.
To understand interacting region of EPIC when binding with PKR, we perform circSHAPE-MaP of EPIC with or without PKR.

first: Si-Kun

last: Guo

first: Chu-Xiao

last: Liu

first: Yi-Feng

last: Xu

first: Fang

first: Ling-Ling

last: Chen


title: RNA Sequencing of npr-15(tm12539) and  wild type (WT) N2 C elegans infected with Staphylococcus aureus for 8 hours

first: Benson

last: Otarigho

state: Texas

address: 6767 Bertner Avenue

department: Department of Genetics

institute: The University of Texas MD Anderson Cancer Center

summary:
Purpose: To uncover immune genes and pathways that are modulated by the GPCR/NPR-15 during S. aureus infection
Methods: RNA was extracted from synchronized L4 stage npr-15(tm12539) and WT animals grown at 20 C and infected with S. aureus for 8 hours, followed by Qiagen extraction kits and following standard methods
Results: RNA seq analyses shows enriched and signficant upregulated immune, neuropeptide, synaptic signaling and metabolism genes and pathways that are dependent on NPR-15
Conclusions: Our study uncovered NPR-15 to be modulator of the innate immunity in C. elegans under infection

overall_design: RNA seq  profiles of L4-stage npr-15(tm12539) and wild type (WT) C elegans animals were generated by deep sequencing, in triplicate, using Illumina NovaSeq 6000 platform.

first: Alejandro

last: Aballay


title: Ribo– RNA-seq of psoriasis mouse model

overall_design: To evaluate the circRNA-PKR axis in psoriasis , we established a psoriasis mouse (C57BL/6) model via IMQ (imiquimod) treatment at day (D) 0, 1, 2, 3, 5. Then we conducted total RNA sequencing of mice spleens at day 0,1,2,5 following IMQ treatment. Of note, each RNA sequencing sample comprised total RNA extracted from two spleens of two mice. Each examined time point has two repeats, and each repeat includes 2 biologically independent mice.


title: Effect of CD19-CD28 and glofitamab treatments in vivo on the RNA-Seq profile in tumor tissue from humanized mice over time

status: Public on Mar 03 2024

first: Daniel

last: Marbach

address: Grenzacherstrasse 124

department: pRED Pharmaceutical Sciences

institute: F. Hoffmann - La Roche Ltd

summary: The study's objective is to investigate the mode of action of a bispecific CD19-targeted CD28 agonist (RG6333, CD19-CD28) in combination with the glofitamab through bulk RNA sequencing of tumor tissue from humanized mice over time. This analysis aims to describe the molecular and immunological changes induced by the combination therapy, which is designed to potentiate T cell-mediated antitumor activity. Glofitamab, a T cell bispecific antibody, targets CD20-expressing malignant B cells and engages CD3ε to deliver a robust TCR signal. To enhance this effect, CD19-CD28 was developed to provide a necessary costimulatory signal by binding to CD19 on tumor-infiltrating T cells. This agent is engineered to require concurrent TCR signaling and CD19 target presence, avoiding the issues associated with previous superagonistic antibodies. In this study, we explore the synergistic potential of CD19-CD28 with glofitamab in activating T cell responses and inducing tumor regression in humanized mouse models. The RNA-Seq analysis identified gene expression signatures and immune pathways activated by the treatment. These findings underscore the promise of CD19-CD28 as an effective combination partner to glofitamab.

overall_design: In the study, humanized mice were subcutaneously implanted with human tumor cells, OCY-Ly18, to establish a model for aggressive lymphomas. Upon tumor engraftment, the study included three treatment groups: one group received monotherapy with glofitamab, another group was treated with the combination therapy of the bispecific CD19-targeted CD28 agonist (CD19-CD28) and glofitamab, and a control group was administered a vehicle solution. Tumor tissues were harvested from 3 to 5 (up to 11 at termination) mice per treatment condition at day 19, 21 or 29 (termination) time points. This approach was intended to capture the temporal changes in gene expression induced by the therapeutic agents. The harvested tumors were then processed for bulk RNA sequencing to analyze the gene expression profiles and to elucidate the immunological mechanisms activated by the combination therapy.

first: Johannes

last: Sam