title: Limosilactobacillus fermentum Limits Candida glabrata Growth by Ergosterol Depletion

city: Tulln an der Donau

first: Isabella

last: Zangl

address: Konrad Lorenz Str. 24

department: Insitute for Microbial Genetics

country: Austria

institute: University of Natural Resources and Life Sciences, Vienna

summary:
Candida glabrata is a human-associated opportunistic fungal pathogen. It
shares its niche with Lactobacillus spp. in the gastrointestinal and vaginal tract. In fact, Lactobacillus species are thought to competitively prevent Candida overgrowth. We investigated the molecular aspects of this antifungal effect by analyzing the interaction of C. glabrata strains with Limosilactobacillus fermentum. From a collection of clinical C. glabrata isolates, we identified strains with different sensitivities to L. fermentum in coculture. We analyzed the variation of their expression pattern to isolate the specific response to L. fermentum. C. glabrata-L. fermentum coculture induced genes associated with ergosterol biosynthesis, weak acid stress, and  drug/chemical stress. L. fermentum coculture depleted C. glabrata ergosterol. The reduction of ergosterol was dependent on the Lactobacillus species, even in coculture with different Candida species. We found a similar ergosterol-depleting effect with other lactobacillus strains (Lactobacillus crispatus and Lactobacillus rhamosus) on Candida albicans, Candida tropicalis, and Candida krusei. The addition of ergosterol improved C. glabrata growth in the coculture. Blocking ergosterol synthesis with fluconazole increased the susceptibility against L. fermentum, which was again mitigated by the addition of ergosterol. In accordance, a C. glabrata Derg11 mutant, defective in ergosterol biosynthesis, was highly sensitive to L. fermentum. In conclusion, our analysis indicates an unexpected direct function of ergosterol for C. glabrata proliferation in coculture with L. fermentum.

overall_design: Differential gene expression profiles for four clincal Candida glabrata isolates (at least duplicates) in co-culture together with L. fermentum.

first: Reinhard

last: Beyer

first: Arianna

last: Gattesco

first: Roman

last: Labuda

first: Ildiko-Julia

last: Pap

first: Joseph

last: Strauss

first: Christoph

last: Schüller


title: Next Generation Sequencing Facilitates Quantitative Analysis of TRAP from grafted human neural stem cells in stroke and naïve rat brains

status: Public on Feb 08 2023

city: Stanford

first: Ricardo

middle: Luiz

last: De Azevedo Pereira

state: CA

address: 1201 Welch Road

department: Neurosurgery

country: USA

institute: Stanford

type: Expression profiling by high throughput sequencing

summary:
Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to access grafted human neural stem cells and host tissue transcriptomes
Methods: Grafted mRNA profiles of 7 days transplanted hNSC  were generated by deep sequencing, in triplicate, using Illumina HiSeq 2500. The sequence reads that passed quality filters were analyzed at the transcript isoform level with Burrows–Wheeler Aligner (BWA) followed by RNA-Seq by Expectation Maximization (RSEM). qRT–PCR validation was performed using TaqMan and SYBR Green assays
Results: Using an optimized data analysis workflow, we mapped: 1) about 230 million sequence reads per sample to the human genome (build hg19) and identified 17,463 transcripts in the grafted hNSC; and 2) about 30 million sequence reads per sample to the rat genome (build rn5) and identified 13,4688 transcripts in the host tissues; using RSEM workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 4 housekeeping genes were validated with qPCR. RNA-seq data had a linear relationship with qPCR for more than four orders of magnitude. Approximately 14% of the transcripts showed differential expression between the grafted hNSC in naive and stroke; and approximately 41% of the transcripts showed differential expression between the naive and stroke tissues, with p value <0.05. Altered expression of over 10 genes was confirmed with qPCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to grafted hNSC and host function. Data analysis with TRAP and RSEM workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling.
Conclusions: Our study represents the first detailed analysis of grafted hNSC transcriptome, with biologic replicates, generated by TRAPseq technology. TRAPseq and RSEM could be applied to many xenograft cell transplantation paradigms. With this approach we can start to predict upstream regulators that signal between the host and graft, and to predict the downstream signaling pathways and biological processes these upstream regulators might affect.

sample_organism:
Homo sapiens
Rattus norvegicus

platform_organism:
Homo sapiens
Rattus norvegicus

overall_design: TRAPseq of grafted hNSC after 7 days of transplantation into naïve and stroke nude rats

first: R

middle: L

last: Azevedo-Pereira

first: T

middle: M

last: Bliss

first: G

middle: K

last: Steinberg


title: Effects of USB1 knockout on global miRNome in K562 cells

status: Public on Feb 07 2023

city: boulder

first: Roy

last: parker

address: colorado ave

institute: university of colorado boulder

type: Non-coding RNA profiling by high throughput sequencing

summary: USB1 mutations lead to poikiloderma with neutropenia. We investigated the effect of inactivating USB1 on the global miRome in K562 cells

overall_design: WT and USB1 KO K562 cells were harvested and total RNA was used for miRome sequencing

first: Hochang

last: Jeong

first: Siddharth

last: Shukla

first: Thao

last: Hyunh


title: Effects of USB1 mutation on global miRNome during hESC-derived definitive hematopoiesis

summary: USB1 mutations lead to poikiloderma with neutropenia. We investigated the effect of inactivating USB1 on the global miRome in undifferentiated and differentiated hematopoietic stem cells

overall_design: WT and USB1 mutant human stem cells were harvested at day 0, day 16 and day 30 after induction of differentiation into mature blood cells


title: Single cell RNA sequencing of distal thrombus tissue from CTEPH patients

city: Durham

first: Gayathri

last: Viswanathan

state: NC

address: 0150, Carl building, 130 Research Drive

department: Cardiology

institute: Duke University

summary: Our current understanding of CTEPH pathobiology is primarily derived from cell-based studies limited by the use of specific cell markers or phenotypic modulation in cell culture. Therefore, our main objective is to identify the multiple cell types that comprise the CTEPH thrombus and to study their dysfunction. Here we used single cell RNA sequencing (scRNAseq) of tissue removed at the time of pulmonary endarterectomy (PEA) surgery from five patients to identify the multiple cell types. Our scRNAseq identify the multiple cell types including macrophages, T cells, and smooth muscle cells, that comprise CTEPH thrombus. Notably, multiple macrophage subclusters were identified but broadly split into two categories, with the larger group characterized by an upregulation of inflammatory signaling predicted to promote pulmonary vascular remodeling. Both CD4+ and CD8+ T cells were identified and likely contribute to chronic inflammation in CTEPH. Smooth muscle cells were a heterogeneous population, with a cluster of myofibroblasts that express markers of fibrosis and are predicted to arise from other smooth muscle cell clusters based on pseudotime analysis. Lastly, our analysis identified protease-activated receptor 1 (PAR1) as a potential therapeutic target that links thrombosis to chronic PE in CTEPH

overall_design: The thrombus is processed for single cell RNA sequencing (scRNAseq). After rehydration of methanol-fixed cells, libraries were prepared using the 10X Genomics Single Cell Protocol through the Duke Human Vaccine Institute Genetics Analysis Core. The data was analyzed in collaboration with the Duke Center for Genome Biology.

first: Hélène

middle: F

last: Kirshner

first: Nour

last: Nazo

first: Saba

last: Ali

first: Asvin

last: Ganapathi

first: Ian

last: Cumming

first: Yonghua

last: Zhuang

first: Issac

last: Choi

first: Anmol

last: Warman

first: Susana

last: Almeida-Peters

first: John

last: Haney

first: David

last: Corcoran

first: Yen-Rei

last: Yu

first: Sudarshan

last: Rajagopal


title: Effect of USB1 mutation on global transcriptome during hESC-derived definitive hematopoiesis

first: Roy

last: parker

summary: USB1 mutations lead to poikiloderma with neutropenia. We investigated the effect of inactivating USB1 on the global transcriptome in undifferentiated hESCs and differentiated hematopoietic progeny

overall_design: WT and USB1 mutant hESCs were differentiated into mature blood cells and harvested at day 0, day 16 and day 30.

first: Ho-Chang

last: Jeong


title: Drug addiction unveils a repressive methylation ceiling in EZH2-mutant lymphoma

city: Cambridge

first: Brian

last: Liau

state: MA

address: 12 Oxford St.

department: Department of Chemistry and Chemical Biology

institute: Harvard University

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: Interleukin-27-dependent transcriptome signatures regulation of the transcriptome during neonatal sepsis

city: Morgantown

first: Gangqing

last: Hu

state: West Virginia

address: 64 Medical Center Drive

department: MicroBiology, Immunology, and Cell Biology

institute: West Virginia University


title: Interleukin-27-dependent transcriptome signatures regulation of the transcriptome  during neonatal sepsis [ATAC-seq]

summary: Human newborns exhibit increased vulnerability and risk of mortality from infection. Increased susceptibility to infection during the neonatal period reflects key differences in the innate and adaptive immune responses relative to those in adult cells. We have previously shown an increase in the pleiotropic immune suppressive cytokine, IL-27, in macrophages derived from human umbilical cord blood compared with those obtained from adult peripheral blood. Similar findings exist in the neonatal murine system, as well as elevated splenic transcripts and serum levels. Recently, we established a murine model of neonatal sepsis to explore the impact of early life IL-27 levels on the host response to infection. In this model, neonatal mice deficient in IL-27 signaling exhibit reduced mortality, increased weight gain, and better control of the bacterial burden with reduced systemic inflammation. These results suggest that there is a reprogramming of the host response in the presence of IL-27 signaling. To explore this hypothesis, we profiled the neonatal transcriptome of the spleen in the presence or absence of Escherichia coli-induced sepsis in wild-type (WT) and IL-27Rα-deficient mice.  The spleen, a known site of infection, exhibits increased IL-27 expression and bacterial burdens in WT pups that were significantly lower in IL-27Rα KO pups during infection. We identified 549 genes that were differentially expressed in WT neonates in response to E. coli infection. The upregulated genes were associated with inflammation, cytokine signaling, and G protein coupled receptor ligand binding and signaling. The expression level of these genes generally failed to increase in IL-27Rα KO mice upon the E. coli infection. We observed similar changes in gene expression, aligned with changes in chromatin accessibility, for macrophages isolated from the spleens of control and infected neonates with or without IL-27Rα KO, supporting macrophages as an innate myeloid population contributing to the inflammatory profile in septic WT pups.  Collectively, our findings highlight a less inflammatory environment in KO pups that is not expected given the anti-inflammatory/suppressive activity of IL-27. However, it is consistent with a lower bacterial burden. This suggests that improved bacterial clearance in the absence of IL-27 signaling does not require a heightened inflammatory state, and instead, there is a direct relationship between IL-27 signaling and bacterial killing. An improved response to infection that is not reliant upon heightened levels of inflammation offers new promise to the potential of antagonizing IL-27 as a host-directed therapy for neonates without concern for driving inflammatory responses that are pathological to host tissues.

overall_design: Four-day old C57BL/6 pups were sorted into four groups based on IL27RA status (KO or wild type) and based on infection status with Escherichia coli strain O1:K1:H7 infection (with or without infection; 24 hours). For each of the four group, PolyA RNA-seq were performed for spleen tissue and for myeloid cells enriched from the spleen. Additional ATAC-seq assay to measure chromatin accessibility was performed for myeloid cells from each group.

first: Jessica

last: Povroznik

first: Halima

last: Akhter

first: Jordan

last: Vance

first: Madhavi

last: Annamanedi

first: Sebastian

middle: A

last: Dziadowicz

first: Lei

last: Wang

first: Ashley

last: Divens

first: Cory

last: Robinson


title: Epigenome analysis of apoptotic and control cancer cell lines

city: Hall in Tirol

first: Charlotte

middle: Dafni

last: Vavourakis

state: Tirol

address: Milser Str. 10

department: Research Institute for Biomedical Aging Research

institute: University of Innsbruck

summary:
Genome wide DNA methylation profiling of four control and cancer cell lines where apoptosis was induced using BH3 mimetics. The Illumina Human MethylationEPIC methylation profiling array was used to obtain DNA methylation profiles across approximately 850,000 CpGs.  8 samples were from treated cells harvested at two different time points representing early and late apoptotic stages, and 4 samples were controls combined from the two timepoints. 4 additional samples was left over DNA from the Nalm6 cell line ran on the array to fill up the bead chips.

Data analysis will be presented in the manuscript "HPV-induced host epigenetic reprogramming is lost upon progression to high-grade cervical intraepithelial neoplasia" accepted for publication in the International Journal for Cancer.

overall_design: Bisulphite converted DNA from the 16 samples were hybridised to the Illumina Human MethylationEPIC methylation profiling array.

middle: D

first: Chiara

last: Herzog

first: Gerlinde

last: Karbon

first: Andreas

last: Villunger

first: Martin

last: Widschwendter


title: Bacterial pattern recognition in C. elegans by the nuclear hormone receptor NHR-86/HNF4

city: Worcester

first: Read

last: Pukkila-Worley

state: Massachusetts

address: 55 N Lake Ave

department: Medicine

institute: University of Massachusetts Medical School

summary: Pattern recognition of bacterial products by host receptors is essential for pathogen sensing in many metazoans. Caenorhabditis elegans, however, do not utilize canonical pattern recognition receptors to activate innate immunity toward bacterial pathogens. Whether other mechanisms evolved in nematodes to directly sense pathogens is not known. Here, we characterize the first bacterial pattern recognition receptor and its natural ligand in C. elegans. We show that the C. elegans nuclear hormone receptor NHR-86/HNF4 senses phenazine-1-carboxamide (PCN), a metabolite produced by pathogenic strains of Pseudomonas aeruginosa, to activate protective anti-pathogen defenses in the intestine. PCN binds to the ligand binding domain of NHR-86/HNF4, a ligand-gated transcription factor, which engages a transcriptional program in intestinal epithelial cells that promotes metabolism of toxic phenazines to provide protection against P. aeruginosa. These data de-orphan a nuclear hormone receptor and demonstrate that sensing a metabolite signal of bacterial virulence allows nematodes to detect pathogens in its environment that are poised to cause disease.

overall_design: RNA-seq of C. elegans wild-type (N2) animals exposed to Escherichia coli (OP50), Pseudomonas aeruginosa (PA14)-wt or PA14 Δphz (deletion of both phenazine producing operons) supplemented either with solvent control (DMSO) or PCN (25 μg/mL). RNA-seq of C. elegans wild-type (N2) grown on control RNAi or nhr-86(RNAi) and exposed at L4 stage to Escherichia coli (OP50) supplemented either with solvent control (DMSO) or PCN (25 μg/mL).

first: Nicholas

last: Peterson


title: VVC-associated strains of Candida albicans provoke more fungal shedding and exfoliation than colonizing strains in epithelial cell infections

city: Orono

first: Benjamin

last: King

state: ME

address: 5735 Hitchner Hall

department: Molecular and Biomedical Sciences

institute: University of Maine

summary: Vulvovaginal candidiasis (VVC) affects nearly ¾ of women during their lifetime and its symptoms seriously reduce quality of life. Although Candida albicans is a common commensal, it is unknown if VVC results from a commensal to pathogenic state switch, if only some strains can cause VVC, and/or if there is displacement of commensal strains with more pathogenic strains. We studied a set of VVC and Colonizing C. albicans strains to identify consistent in vitro phenotypes associated with one group or the other. We find that the strains do not differ in overall genetic profile or behavior in culture media (i.e. MLST profile, rate of growth, filamentation), but they show strikingly different behavior during their interactions with vaginal epithelial cells. Epithelial infections with VVC-derived strains yielded stronger fungal proliferation and shedding of fungi and epithelial cells. RNA-seq analysis of representative epithelial cell infections with selected pathogenic or commensal isolates identified several differentially activated epithelial signaling pathways, including the integrin, ferroptosis and type I interferon pathways; the latter has been implicated in damage protection. The type I interferon pathway is activated more by the Colonizing strains, while type I interferon inhibition selectively increases fungal shedding of only the colonizing fungi. These data suggest that VVC strains may intrinsically have more pathogenic potential via differential elicitation of epithelial responses, including type I interferon pathway. Therefore, it may be possible to evaluate pathogenic potential in vitro to refine VVC diagnosis.

overall_design: Comparative gene expression of a monolayer of A-431 vaginal epithelial cells challenged with a VVC strain (CA01887) or a colonized strain (CA14314).

first: Arianna

last: Sala

first: A

last: Ardizzoni

first: L

last: Spaggiari

first: N

last: Vaidya

first: J

last: van der Schaaf

first: C

last: Rizzato

last: Cermelli

first: S

last: Mogavero

first: T

last: Kruger

first: M

last: Himmel

first: O

last: Kniemeyer

last: Brakhage

first: B

last: Lupetti

last: Tavanti

first: E

last: Blasi

first: R

middle: T

last: Wheeler

last: Pericolini


title: Variant specific global loss of cellular m6A RNA methylation during SARS-CoV-2 infection

city: Gothenburg

first: Tanmoy

last: Mondal

state: Västra Götaland

address: Bruna Stråket 16

department: Department of Clinical Chemistry and Transfusion Medicine

country: Sweden

institute: University of Gothenburg

summary: COVID-19 pandemic caused by SARS-CoV-2 is far from getting over because of the emergence of different SARS-CoV-2 variants even though vaccination against SARS-CoV-2 is ongoing. Variant specific mechanistic understanding of host-viral interaction will be required to design novel anti-viral therapeutics. N6-methyladenosine modification (m6A) which is one of the abundant cellular RNA modifications regulates key processes in RNA metabolism during stress response.  We observed different SARS-CoV-2 variants during infection causes global loss of m6A in cellular RNA whereas m6A modification was detected abundantly in viral RNA. The loss of m6A in cellular RNAs was more drastic in B.1 and  B1.1.7 strains and the main m6A methyltransferase METTL3 shows cytoplasmic localization post-infection. In B1.351 variant effect on METTL3 localization and loss of m6A was less pronounced compared to B.1 and  B1.1.7  variants. Gene expression profile suggested changes in RNA catabolism-related pathways including key genes related to m6A readers and erasers during SARS-CoV-2 infection. Genes with m6A peaks were more susceptible to down-regulation during viral infection. Inhibition of export protein XPO1 during infection results in restoration of METTL3 nuclear localization and reduction in viral infection in cell culture suggesting XPO1 inhibition could be an effective therapeutic option for SARS-CoV-2.

sample_organism:
Homo sapiens
Chlorocebus sabaeus

platform_organism:
Homo sapiens
Chlorocebus sabaeus

overall_design: Examination of m6A RIP-seq and RNA-seq from Vero cells and Human Bronchial Epithelial cells infected with variuos SARS-CoV-2 variants.

first: Roshan

last: Vaid

first: Akram

last: Méndez


title: Effects of high cholesterol diet on intimal transcriptome

city: Riverside

first: Changcheng

last: Zhou

state: California

address: 900 University Avenue

department: Biomedical Sciences

institute: The University of California Riverside


title: Interleukin-27-dependent transcriptome signatures regulation of the transcriptome  during neonatal sepsis [RNA-seq]

first: Gangqing

last: Hu

overall_design: Four-day old C57BL/6 pups were sorted into four groups based on IL27RA status (KO or wild type) and infection status with Escherichia coli strain O1:K1:H7 infection (with or without infection; 24 hours). For each of the four group, PolyA RNA-seq were performed for spleen tissue and for myeloid cells enriched from the spleen. Additional ATAC-seq assay to measure chromatin accessibility was performed for myeloid cells from each group.

first: Jessica

last: Povroznik

first: Halima

last: Akhter

first: Jordan

last: Vance

first: Madhavi

last: Annamanedi

first: Sebastian

last: Dziadowicz

first: Lei

last: Wang

first: Ashley

last: Divens

first: Cory

last: Robinson


title: The histone demethylase KDM3 prevents auto-immune piRNAs production in Drosophila

city: PARIS

first: Antoine

last: Boivin

address: 9 quai St Bernard

department: IBPS

country: France

institute: Sorbonne Universite CNRS

summary: In animals, genome integrity of the germ line is protected from transposable element (TE) activity by small, non-coding, dedicated RNAs acting as an immune system against TEs, and called PIWI-interacting RNAs (piRNAs) (Czech et al. 2018, Ozata et al. 2018). In Drosophila, the production of piRNAs is initiated from heterochromatic loci containing remnants of TEs and enriched in histone H3 trimethylated on lysine 9 (H3K9me3) (Brennecke et al. 2007, Gunarwardane et al. 2007; Rangan et al. 2011). These loci, called piRNA clusters, constitute a memory of past TE invasions. Little is known about how piRNA clusters are genetically defined. Using a genetic screen combined with a bimodal epigenetic state piRNA cluster (BX2), we identified the splicing factor Half pint (Hfp) and the histone demethylase KDM3 as being able to prevent BX2 piRNA production. Furthermore, we showed that Hfp is needed to splice Kdm3 transcripts. Germline expression of Kdm3 coding sequence (splicing-independent) rescued the hfp germline knock-down (GLKD) effect demonstrating that Kdm3 is sufficient to prevent BX2 piRNA production. Our data revealed that in the absence of Kdm3, dozens of gene-containing regions become bona fide germinal dual strand piRNA clusters. Indeed, they produce piRNAs originating from both DNA strands, become transcribed in a Moonshiner-dependent manner and enriched in di-and tri-methylation of lysine 9 of histone H3 (H3K9me2/3) and in Rhino, an HP1-like protein. Eggs laid by Kdm3 GLKD females do not hatch and show developmental defects phenocopying loss of function of genes included into the new piRNA clusters, suggesting an inheritance of functional ovarian “auto-immune” piRNAs. Our results demonstrate that some gene-containing regions are actively prevented for piRNA production by proteins that counteract piRNA cluster emergence. Hence, a non-piRNA-producing state is therefore not a "by default" state but rather a cellular lock that is actively controlled for some genomic loci.

overall_design:
Samples are made from Drosophila ovaries. Biological replicates are indicated.

Additional samples are made from Drosophila 3-5h embryos. Biological replicates are indicated.

first: Karine

last: Casier

first: Julie

last: Autaa

first: Nathalie

last: Gueguen

first: Valérie

last: Delmarre

first: Marie

middle: P

last: Pauline

first: Stéphane

last: Ronsseray

first: Clément

last: Carré

first: Emilie

last: Brasset

first: Laure

last: Teysset


title: Functional role of HSPA5 in trophoblasts

city: Oulu

first: Antti

last: Haapalainen

address: Aapistie 5A

department: CRC

country: Finland

institute: University of Oulu

summary: Analysis of the effect of HSPA5 siRNA experiment

overall_design: RNA-seq analysis of silenced HSPA5 (n = 3) and negative control (n=3) samples

first: Heli

last: Tiensuu

first: Pinja

last: Tissarinen


title: The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting

city: Beijing

first: Jiansen

last: Lu

address: No. 5 Yiheyuan Road, Haidian District

department: Biomedical Pioneering Innovation Center (BIOPIC)

country: China

institute: Peking University

type: Methylation profiling by high throughput sequencing

summary: Pluripotent stem cells can be obtained from spermatogonial stem cells (SSCs) through spontaneous reprogramming without transgenic introduction, but this process is inefficient and lacks mechanism exploration. Here, we reconstructed the progression trajectory of mouse SSC reprogramming by single-cell RNA sequencing and identified a bona fide pluripotent route as the successful reprogramming branch. Notably, we developed five-chemicals combination which could boost the reprogramming efficiency nearly 1000 times than previous study. More importantly, chemical induced germline-derived pluripotent stem cells (5C-gPSCs) with embryonic stem cell-like imprinting status were characterized as fully pluripotent via tetraploid complementation assay. Mechanistically, not only the expression of canonical markers, but also the global DNA demethylation and re-methylation features from epiblast to primordial germ cells and subsequent spermatogonia were remarkably observed during SSC reprogramming; meanwhile, biallelic methylation of most imprinting control regions (ICRs) in SSCs had been switched to monoallelic methylation in gPSCs through such stepwise reprogramming process. Compared to the extremely hypomethylated ICRs in developmental deficient chemical induced pluripotent stem cells (CiPSCs), we demonstrated the highly correlation between proper methylation of ICRs and fully pluripotency. Therefore, our work sheds light on the unique genetic- and epigenetic regulatory network of SSC reprogramming, providing novel insights to understand generic mechanisms for cell fate determination and the epigenetic related developmental disorders in regenerative medicine.

overall_design: In this study, we established a highly efficient SSC reprogramming system to generate fully pluripotent gPSCs via chemical induction, moreover, with the aid of genetic- and epigenetic dissections using single-cell multi-omics (transcriptome and DNA methylome) sequencing, we explored the sophisticated regulation network of cell fate transition during this reprogramming process. SSCR: Spermatogonial stem cell reprogramming; gPSC: germline derived plueipotent stem cells; CiPS: chemical induced pluripotent stem cell.

first: Fuchou

last: Tang

first: Xiao-Yang

last: Zhao

first: Mei


title: Ectopic lymphoid structures in the aged lacrimal glands

status: Public on Feb 06 2023

city: Houston

first: Cintia

middle: S.

last: de Paiva

state: TX

address: 6565 Fannin Street NC505G

department: Ophthalmology

institute: Baylor College of Medicine

summary: To investigate the molecular pathways altered during aging of the lacrimal gland in C57BL/6J females, we compared the LG transcriptome at different time points.

overall_design: We sequenced the mRNA of lacrimal glands from C57BL/6J females mice at 2 months-old (M), 12 M and 24 M in triplicates.

first: Jeremias

middle: G

last: Galletti

first: Kaitlin

last: Scholand

first: Claudia

last: Trujillo-Vargas

first: Zhiyuan

last: Yu

first: Olivier

last: Mauduit

first: Vanessa

last: Delcroix

first: Helen

last: Makarenkova

middle: S


title: Negative regulation of TREM2-mediated C9orf72 poly-GA clearance by the NLRP3 inflammasome

city: Hangzhou

first: Chengyong

last: Shen

address: 268 Kaiquan Rd.

department: Institute of Translational medicine

institute: Zhejiang University

summary:
To examine the global responses to poly-GA aggregates in vivo, we used AAV to deliver the poly-GA DNA into mouse brains
We performed RNA sequencing of the cortex of control and poly-GA mice (5-month-old) to analyze the differential gene expression profile

overall_design: Comparative gene expression profiling analysis of RNA-seq data for AAV-GFP and AAV-GFP-GA injected cortex.


title: Biomarkers in semen/urine from non-obstructive azoospermia patients to predict the presence of testicular spermatozoa and spermatogonia

city: Brussel

first: Ellen

last: Goossens

address: Pleinlaan 2

country: Belgium

institute: Vrije Universiteit Brussel

summary: About half of testicular sperm extraction (TESE) procedures in men with non-obstructive azoospermia (NOA), including men with Klinefelter syndrome (KS), are unsuccessful. To avoid unnecessary invasive surgery, biomarkers for spermatozoa have been studied and identified. In addition, markers for spermatogonia in testis tissue have been researched. This study aimed to find biomarkers in semen and/or urine of NOA patients to predict the presence of spermatogonia in the testis. This would especially be interesting for young boys. Differentially expressed miRNAs were identified (1) between samples from patients with and those of patients without a positive TESE procedure as well as (2) between TESE negative patients with and those without spermatogonia. A total of ten up-regulated miRNAs (seven in seminal plasma and three in urine) were found in the TESE-negative/spermatogonia-positive group compared to the TESE-negative/spermatogonia-negative group. These miRNAs could become biomarkers for spermatogonia, however, more research is necessary.

overall_design: Differential miRNA expression analysis

first: Margo

last: Willems

first: Céline

last: Devriendt

first: Catharina

last: Olsen


title: Recycling of Histones H2A-H2B provides short-term memory of chromatin states [SCAR-seq II]

city: Copenhagen

first: Anja

last: Groth

address: Blegdamsvej 3B

country: Denmark

institute: Novo Nordisk Foundation Center for Protein Research

summary: Chromatin landscapes are disrupted during DNA replication and need to be restored faithfully to maintain appropriate gene expression, including post-translational modifications (PTMs) of newly deposited histones. Whether histones H2A-H2B are accurately recycled during DNA replication and the behaviour of their associated marks during and post replication is unknown. Here we comprehensively map key modifications on H2A-H2B including H2A.Z during DNA replication. We show that H2AK119ub, H2BK120ub, and H2A.Z are recycled quantitatively and accurately during DNA replication in a symmetrical manner. Recycling occurs independently from H3-H4 chaperone pathways apart from a minor role for Polymerase alpha. H2A-H2B modifications are restored rapidly post replication and are important for timely and accurate restoration of H3-H4 marks, such as H3K27me3. This work uncovers H3-H4 and H2A-H2B crosstalk in epigenome propagation across DNA replication and suggests a model where rapid short-term memory of recycled H2A-H2B marks facilitates reestablishment of slow, long-term memory chromatin states.

overall_design: SCAR-seq (Sister Chromatids After Replication) time-series measuring histone partition of H3K27me3, or  H4K20me0 in wildtype (WT), MCM2 histone-binding mutant (MCM2-2A), or Pole4 KO mouse embryonic stem cells (Parental WT cells, 2 MCM2-2A clones,  2 Pole4KO clones,  >2 replicates per cell line, stranded input controls). Note that the strandedInputs for Pole4KO cells are available in the accompanying Subseries SCAR-seq (GSE204984; GSM6203641-6).

first: Valentin

last: Flury

first: Nazaret

last: Reverón-Gómez

first: Nicolas

last: Alcaraz

first: Kathleen

last: Stewart-Morgan

first: Alice

last: Wenger

first: Robert

middle: J

last: Klose


title: RAS and PP2A activities converge on epigenetic gene regulation [RNA-Seq]

city: Tampere

first: Reetta

last: Nätkin

address: Arvo Ylpön katu 34

department: Faculty of Medicine and Health Technology, Prostate Cancer Research Center

institute: Tampere University and Tays Cancer Center

summary: RAS-mediated human cell transformation requires inhibition of the tumor suppressor Protein Phosphatase 2A (PP2A). Both RAS and PP2A mediate their effects by phosphoregulation, but phosphoprotein targets and cellular processes in which RAS and PP2A activities converge in human cancers have not been systematically analyzed. Here, based on mass spectrometry phosphoproteome data we discover that phosphosites co-regulated by RAS and PP2A are enriched on proteins involved in epigenetic gene regulation. As examples, RAS and PP2A co-regulate the same phosphorylation sites on HDAC1/2, KDM1A, MTA1/2, RNF168 and TP53BP1. Mechanistically, we validate co-regulation of NuRD chromatin repressor complex by RAS and PP2A. Consistent with their known synergistic effects in cancer, RAS activation and PP2A inhibition resulted in epigenetic reporter de-repression and activation of oncogenic transcription. Notably, transcriptional de-repression by PP2A inhibition was associated with increased euchromatin and decrease in global DNA methylation. Further, targeting of RAS- and PP2A-regulated epigenetic proteins decreased viability of KRAS-mutant human lung cancer cells. Collectively the results indicate that epigenetic protein complexes involved in oncogenic gene expression constitute a significant point of convergence for RAS hyperactivity and PP2A inhibition in cancer. Further, this work provides a resource for future studies focusing on phosphoregulation as a previously unappreciated layer of regulation of epigenetic gene regulation in cancer, and in other RAS/PP2A-regulated cellular processes.

overall_design: Characterization of global gene expression by RNA sequencing (RNA-seq), 72h post siRNA (Ctrl, PP2A and RAS) transfections.

first: Anna

last: Aakula

first: Mukund

last: Sharma

first: Francesco

last: Tabaro

first: Jesse

last: Kamila

first: Henrik

last: Honkanen

first: Matthieu

last: Schapira

first: Cheryl

last: Arrowsmith

first: Matti

last: Nykter

first: Jukka

last: Westermarck


title: probe based bacterial single-cell RNA sequencing predicts toxin regulation

city: Chapel Hill

first: Adam

last: Rosenthal

address: 125 Mason Farm Marsico Hall rm6201

department: Microbiology and Immunology

institute: UNC

summary: Clonal bacterial populations rely on transcriptional variation across individual cells to commit to specialized states that increase the population’s fitness. Such heterogeneous gene expression is implicated in fundamental microbial processes including sporulation, cell communication, detoxification, substrate utilization, competence, biofilm formation, and motility1. To identify specialized cell states and determine the processes by which they develop, isogenic bacterial populations need to be studied at the single cell level2,3. Here, we developed ProBac-seq a method that uses libraries of DNA probes and leverages an existing commercial microfluidic platform to conduct bacterial single cell RNA sequencing. We sequenced the transcriptome of thousands of individual bacterial cells per experiment, detecting several hundred transcripts per cell on average. When applying this method to the model organisms Bacillus subtilis and Escherichia coli, we correctly identify known cell states and uncover previously unreported transcriptional heterogeneity. In the context of bacterial pathogenesis, single cell RNA-seq of the pathogen Clostridium perfringens reveals that toxin is differentially expressed by a subpopulation of cells with a distinct transcriptional profile. We further show that the size of the toxin producing subpopulation and the secreted toxin levels can be downregulated by providing acetate, a short chain fatty acid highly prevalent in the gut. Overall, we demonstrate that our high throughput, highly resolved single cell transcriptomic platform can be broadly used to uncover heterogeneity in isogenic microbial populations and identify perturbations that can impact pathogenicity.

sample_organism:
Escherichia coli
Bacillus subtilis
Clostridium perfringens

overall_design: Before microfluidic encapsulation, bacteria were fixed in 1% paraformaldehyde to preserve transcripts and permeabilized by mild lysozyme treatment. Permeabilized bacteria were then incubated with the corresponding DNA probe library. Non-hybridized probes were removed with repeated washes . Next, the bacteria were run through a 10X Chromium Controller, where the DNA probes were captured and barcoded in a manner analogous to the barcoding of the transcriptome of eukaryotic cells

first: Ryan

last: McNulty

first: Duluxan

last: Sritharan

first: Seong

middle: H

last: Pahng

first: Jeffrey

last: Meisch

first: Shichen

last: Liu

first: Melanie

last: Brennan

first: Gerda

last: Saxer

first: Sahand

last: Hormoz

middle: Z


title: RAS and PP2A activities converge on epigenetic gene regulation [BS-Seq]

first: Reetta

last: Nätkin

overall_design: Characterization of global DNA methylation by Reduced Representation Bisulfite Sequencing (RRBS), 72h post siRNA (Ctrl and PP2A) transfections.

first: Anna

last: Aakula

first: Mukund

last: Sharma


title: RAS and PP2A activities converge on epigenetic gene regulation [ATAC-Seq]

overall_design: Characterization of chromatin accessibility by Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), 72h post siRNA (Ctrl and PP2A) transfections.

first: Francesco

last: Tabaro

first: Jesse

last: Kamila

first: Henrik

last: Honkanen


title: RAS and PP2A activities converge on epigenetic gene regulation

overall_design: Refer to individual Series


title: Quantitative profiling of pseudouridylation landscape in the human transcriptome

first: Jiang

last: Zhe

state: Beijing

department: School of Life Sciences

summary: Pseudouridine (Ψ) is an abundant post-transcriptional RNA modification in ncRNA and mRNA. However, transcriptome-wide measurement of individual Ψ sites remains unaddressed. Here, we develop “PRAISE”, via selective chemical labeling of Ψ by bisulfite to induce nucleotide deletion signature during reverse transcription, to realize quantitative assessment of the Ψ landscape in the human transcriptome. Unlike traditional RNA/DNA bisulfite treatment, our approach is based on quaternary base mapping and identifies 2,209 confident Ψ sites in HEK293T cells. By perturbing pseudouridine synthases, we obtained differential mRNA targets of PUS1, PUS7, TRUB1 and DKC1. In addition, we identified known and novel Ψ sites in mitochondrial mRNA, which are catalyzed by a mitochondria-localized isoform of PUS1. Collectively, we provide a reliable, sensitive and convenient method to quantify transcriptome-wide Ψ; we envision this approach would facilitate emerging efforts to elucidate the function and mechanism of mRNA pseudouridylation.

overall_design:
We developed a method named "PRAISE" to detect transcriptome-wide pseudouridine sites in human mRNA and ncRNA.
We first developed a tailored alignment approach and identified 18S, 28S, 5.8S rRNA sites using HEK293T total RNA (rRNA-WT_rep1,rRNA-WT_rep2,rRNA-WT_untreated), then we appiled PRAISE to characterize a quantitative Ψ landscape in the human nuclear-encoded and mitochondrial encoded transcriptome using HEK293T polyA+ RNA (PRAISE-WT-untreated_rep1, PRAISE-WT-untreated_rep2, PRAISE-WT_rep1,PRAISE-WT_rep2).
We also employed a recently established in vitro transcribed RNA (IVT RNA) library from HEK293T transcriptome as a negative control (PRAISE-IVT-untreated, PRAISE-IVT_rep1, PRAISE-IVT_rep2).
We next prepared 2nd and 3rd batches of biological replicates of HEK293T cells using the Takara v3 kit and the KAPA Strand RNA-Seq kit (PRAISE-WT-Takara-second-biological-repeats_rep1,PRAISE-WT-Takara-second-biological-repeats_rep2,PRAISE-WT-Takara-second-biological-repeats-untreated_rep1,PRAISE-WT-Takara-second-biological-repeats-untreated_rep2;PRAISE-WT-KAPA_rep1,PRAISE-WT-KAPA_rep2,PRAISE-WT-KAPA-untreated).
In addition, we applied PRAISE to the cellular polyA+ RNA of three KO cell lines and one stable knockdown cell line, dependent-Ψ sites were defined by comparing the Ψ profile between WT and KO (PRAISE-PUS1KO-untreated_rep1,PRAISE-PUS1KO-untreated_rep2, PRAISE-PUS1KO_rep1,PRAISE-PUS1KO_rep2,PRAISE-DKC1KD_rep1,PRAISE-DKC1KD_rep2,PRAISE-DKC1KD-untreated_rep1,PRAISE-DKC1NC_rep1,PRAISE-DKC1NC_rep2,PRAISE-DKC1NC-untreated_rep1,PRAISE-PUS7KO-untreated_rep1,PRAISE-PUS7KO-untreated_rep2, PRAISE-PUS7KO_rep1,PRAISE-PUS7KO_rep2,PRAISE-TRUB1KO-untreated_rep1,PRAISE-TRUB1KO-untreated_rep2, PRAISE-TRUB1KO_rep1,PRAISE-TRUB1KO_rep2).
We also applied the small RNA fraction of WT HEK293T to identify mitochondrial tRNA Ψ sites (PRAISE-WT-tRNA_rep1,PRAISE-WT-tRNA_rep2,PRAISE-WT-tRNA-untreated).

Please note that the GSM6513552, GSM7029102 records have been updated on Mar 25, 2023.

first: Meiling

last: Zhang

first: Zhe

last: Jiang

first: Yichen

last: Ma

first: Wenqing

first: Yuan

last: Zhuang

first: Bo

last: Lu

first: Kai

last: Li

first: Chengqi

last: Yi


title: SRSF1 regulates primordial follicle formation and number determination during meiotic prophase I

first: Jiali

last: Liu

address: 2 West Yuanmingyuan Road

institute: China Agricultural University

summary: Serine/arginine-rich splicing factor 1 (SRSF1; previously SF2/ASF) is a pivotal posttranscriptional regulator of gene expression in various biological processes. However, the physiological roles and mechanism of SRSF1 in mouse early-stage oocytes remain elusive. Here we show that SRSF1 is essential for primordial follicle formation and number determination during meiotic prophase I. The conditional knockout of Srsf1 in mouse oocytes impairs primordial follicle formation and leads to primary ovarian insufficiency (POI). Oocyte-specific genes (e.g., LHX8, NOBOX, SOHLH1, SOHLH2, FIGLA, KIT, JAGGED1, and RAC1) that regulate primordial follicle formation are suppressed in newborn Stra8-GFPCre Srsf1Fl/Fl (cKO) mouse ovaries. However, meiotic defects are the leading cause of abnormal primordial follicle formation. In cKO mouse ovaries, immunofluorescence results suggest that the failure of synapsis and the inability to undergo recombination result in fewer homologous DNA crossovers (COs). Moreover, SRSF1 directly binds and regulates the expression of the POI-related genes Six6os1 and Msh5 via alternative splicing to implement the meiotic prophase I program. Altogether, our data reveal a critical role of the SRSF1-mediated posttranscriptional regulatory mechanism in the mouse oocyte meiotic prophase I program, which provides a framework to elucidate molecular mechanisms of the posttranscriptional network underlying primordial follicle formation.

overall_design: 3 WT ovary samples and 3 SRSF1-cKO ovary samples


title: Effect of the gain-of-function mutation of CXCR4 on the molecular identity of murine Skeletal Stromal/Stem Cells (SSC) and OsteoProgenitor Cells  (OPC)

city: Paris

first: Zeina

last: Abou Nader

address: 1 AVENUE CLAUDE VELLEFAUX

institute: Institut de Recherche Saint Louis- Hopital Saint Louis

summary: In order to perform transcriptional analyses of two stromal subsets carrying the gain-of-function mutation of CXCR4, bulk RNA Sequencing (RNA-Seq) was conducted on SSC and OPC sorted from WT (Cxcr4+/+) , heterozygous (Cxcr4+/1013) and homozygous (Cxcr41013/1013) mutant mice for Cxcr4.

overall_design: Comparative analysis of gene expression profiling using the RNA-seq data for Cxcr4+/+, Cxcr4+/1013 and Cxcr41013/1013 SSC or OPC

first: Karl

last: BALABANIAN

last: ABOU NADER


title: Genetic Ablation of a Female-Specific Apetala 2 Transcription Factor Blocks Oocyst Shedding in Cryptosporidium parvum

city: Philadelphia

first: Katelyn

middle: Ann

last: Walzer

state: PA

address: 380 South University Avenue

department: Pathobiology

institute: University of Pennsylvania

summary: To enable functional studies of essential genes in the parasite Cryptosporidium, we developed and validated a rapamycin inducible Cre-recombinase system and used this approach to conditionally ablate the AP2-F transcription factor. Conditional knock out of AP2-F showed that this factor is dispensable for asexual growth and early female fate determination in vitro but that oocyst shedding is blocked in vivo. Transcriptional analyses conducted in the presence or absence of AP2-F revealed that this factor controls the transcription of genes encoding proteins that make up the crystalloid body, which are exclusively expressed in female gametes and make up a sporozoite-specific organelle. Additionally, treatment with rapamycin did not affect gene expression in wildtype bunchgrass parasites.

overall_design: Three biological replicates were analyzed per condition: wildtype bunchgrass control, wildtype bunchgrass rapamycin treatment, AP2-F DiCre control, AP2-F DiCre rapamycin treatment

first: Jayesh

last: Tandel

first: Jessica

last: Byerly

first: Brittain

last: Pinkston

first: Daniel

last: Beiting

first: Boris

last: Striepen


title: Gene expression in late gestation uterine layers in pregnant IL-33 KO mice

city: San Francisco

first: Johan

last: Siewiera

address: 513 Parnassus Avenue

institute: UCSF

summary: Our objective was to identify gene differentially expressed between B6 and IL-33 KO decidua, myometrium and mesometrial triangle one day prior to parturition.

overall_design: RNA was isolated from whole tissue decidua, myometrium and mesometrial of B6 control (n=5) and IL-33 KO (5) mice on gestation day 18.5, one day prior to parturition.One IL-33 KO decidua and one B6 mesometrial triangle were excluded due to failed QC.Next generation sequencing was performed using Illumina HiSeq 4000

first: Kelly

last: Cautivo

first: Karim

last: Mahiddine

first: Damon

last: Rideaux

first: Ari

middle: B

last: Molofsky

first: Adrian

last: Erlebacher


title: Recycling of H2A-H2B provides short-term memory of chromatin states

first: Anja

last: Groth

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus


title: Uncoupling from transcription protects polyadenylation site cleavage   from inhibition by DNA damage

city: Orsay

first: Céline

middle: M.

last: Labbé

address: Rue Henri Becquerel

institute: Institut Curie

summary: We studied the regulation of pre-messenger RNA 3'-end processing (i.e., cleavage at polyadenylation sites) in response to ultraviolet C irradiation in human cells.

overall_design: To analyze pre-messenger RNA cleavage at polyadenylation sites in response to ultraviolet C irradiation, we performed Total-RNA-seq analyses on irradiated and non-irradiated cells.

first: Rym

last: SFAXI

first: Biswendu

last: BISWAS

first: Galina

last: BOLDINA

first: Mandy

last: CADIX

first: Nicolas

last: SERVANT

first: Huimin

last: CHEN

middle: R

last: LARSON

first: Martin

last: DUTERTRE

first: Caroline

last: ROBERT

first: Stéphan

last: VAGNER


title: Recycling of H2A-H2B provides short-term memory of chromatin states [ChIP-Seq]

summary: Chromatin landscapes are disrupted during DNA replication and must be restored faithfully to maintain genome regulation and cell identity. The H3-H4 modification landscape is restored by parental histone recycling and post-replication modification of new histone H3-H4. How DNA replication impact on histone H2A-H2B is unknown. Here, we track H2A-H2B modifications and H2A.Z during DNA replication and across the cell cycle using quantitative genomics. We show that H2AK119ub, H2BK120ub, and H2A.Z are recycled quantitatively and accurately during DNA replication. H2A-H2B are recycled symmetrically to daughter strands largely independent of known H3-H4 recycling pathways. Post-replication, H2A-H2B modifications are rapidly restored, and the rapid wave of H2AK119ub supports accurate restoration of H3K27me3. This work reveals epigenetic transmission of H2A-H2B modification during DNA replication and identifies H3-H4 and H2A-H2B crosstalk in epigenome propagation. We propose that rapid short-term memory of recycled H2A-H2B modifications facilitates reestablishment of slow, long-term chromatin state memory.

overall_design: ChIP-Seq measuring chromatin occupancy for H3K27me3, H2AK119ub, H2A.Z, H2BK120ub, pan-H2A, pan-H3, JARID2, RING1B in wildtype mouse embryonic stem cells (WT cells)  or Ring1B-mAID BAP1dTAG Ring1AKO or  human HCT116 cells with corresponding Inputs in replicates.

first: Valentin

last: Flury

first: Nazaret

last: Reverón-Gómez

last: Alcaraz

first: Kathleen

last: Stewart-Morgan

first: Alice

last: Wenger

first: Robert

last: Klose


title: Genome-wide mapping of cleavage sites induced by topoisomerase IV in E. coli with a single-nucleotide resolution

status: Public on Feb 05 2023

city: Seattle

first: Dmitry

last: Sutormin

state: WA

address: 401 Terry Ave N

institute: Institute for Systems Biology

summary: Topo IV cleavage sites induced by ciprofloxacin were identified in the E. coli genome with a single-nucleotide resolution using Topo-Seq.

overall_design: To stabilizie cleavage complexes, exponentially growing E. coli DY330 parC-SPA or E. coli DY330 parC-SPA gyrA-S83L cells were treated with 10 mkM ciprofloxacin. Topo IV cleavage complexes with DNA were enriched by immunoprecipitation and obtained DNA fragments were sequenced. Non-treated samples and samples without the immunoprecipitation step served as controls. 3 biological replicates were processed and sequenced.


title: Tumor PD-L1 engages myeloid PD-1 to suppress type I interferon to impair cytotoxic T lymphocyte recruitment (single cells)

city: Augusta

first: Kebin

last: Liu

state: GA

address: 1410 Laney Walker Blvd

department: Biochemistry and Molecular Biology

institute: Augusta University

summary: A hallmark of PD-1/L1 blockade is long-term, sustained remission of metastatic disease. How the immune system coordinates the destruction of macro- and micro-metastases following checkpoint blockade, however, remains unclear. Here, we show that tumor-expressed PD-L1 (tPD-L1) enhanced metastasis in a mechanism distinct from and independent of its role in primary tumor growth. This difference in metastatic growth was mediated by cytotoxic T lymphocytes (CTLs), however, tPD-L1 did not promote effector CTL exhaustion or suppress lytic activity in vivo. Instead, single cell RNA sequencing revealed that tPD-L1 engaged macrophage-expressed PD-1 to antagonize type I interferon production and signaling, creating an immunologically ‘cold’ microenvironment. Loss of tPD-L1 eliminated metastases by driving interferon-mediated sensitization of tumor cells to CTL lysis and CTL recruitment.

overall_design: Balb/c mice were injected i.v. with 2.5*105 4T1 WT or PDL1-KO cells. After fourteen days, mice were sacrificed, lungs were perfused with PBS and harvested. Following collagenase digestion, leukocytes were positively selected using CD45 magnetic nanobeads (Biolegend). Cells were suspended in PBS (without Ca or Mg) with 0.04% BSA.  Cell viability was assessed using trypan blue and assured of ≥ 80% viable cells.  Cells were loaded at a concentration to capture approximately 2-3 x 103 targeted cells on a Chromium Chip B (10x Genomics).  scRNA-seq libraries were generated using the Chromium Single Cell 3’ Reagent Kit v3 (10x Genomics) according to the manufacturer’s instructions.  The libraries were sequenced on Illumina NextSeq 500 platform under the following sequencing protocol: 28 bp (Read 1), 8 bp (indexing Run), and 91 bp (RNA Read 2). Reads from the raw fastq files were mapped to mm10 Mouse Genome reference by STAR aligner in Cell Ranger 3.1.0 pipeline.  Alignment generated 30-39K  reads/cell with 75-102 million reads per sample at a target of 2.3-2.7 x 103 cells per sample identified at 88% in Q30 Bases in RNA read as well as greater than 91% genome-mapping rates along with approximately 1600 median genes per cell.

first: John

last: Klement


title: Transcriptomic analysis of liver mRNA from liver-specific Pck1-knockout mice fed with chow diet or NASH diet

city: chongqing

first: tang

last: ni

address: yixueyuan Road NO.1

institute: Chongqing Medical University

summary: To investigate the role of hepatic Pck1 in diet-induced nonalcoholic steatohepatitis (NASH),  liver-specific Pck1-knockout mice were developed with Alb-Cre. Wild-type (WT) and Pck1-cKO mice were fed a Chow diet (Research Diets, D12450J: 10% Kcal fat, with tap water) or NASH-inducing diet (Research Diets, D12492: 60% Kcal fat, with drinking water containig 23.1 g/L fructose and 18.9 g/L glucose) for 24 weeks. RNA was extracted from the livers of mice from all treatment groups and used for RNA seqencing. RNA-seq data demonstrated that gluconeogenic enzyme PCK1 deficiency played an important role in the development of NASH.

overall_design: Use RNA-sequencing to investigate the role of Pck1 in diet-induced NASH on a transcriptomic level, by comparing the livers of WT and Pck1-cKO mice fed a Chow or NASH diet for 24 weeks using Illumina technology.

first: Ni

last: Tang

first: Ai-Long

last: Huang

first: Kai

last: Wang

first: Qian

last: Ye

first: Yi

first: Gui-Ji

last: Zhang

first: Kang

last: Wu


title: Recycling of H2A-H2B provides short-term memory of chromatin states [ChOR-Seq]

first: Anja

last: Groth

overall_design: ChIP-Seq and ChOR-seq measuring chromatin occupancy for H3K27me3, H2AK119ub, H2A.Z, H2BK120ub, pan-H2A, pan-H3 in wildtype (WT) or Ring1B-mAID BAP1dTAG Ring1AKO mouse embryonic stem cells with corresponding clickedInputs controls in two biological replicates. CHOR-seq involves a 10min EdU labelling step, followed by a chase in medium without EdU (T15-T720; in min) or immediate harvesting (T0). Optional treatments are indicated. ClickedInputs are the corresponding inputs from the ChOR-seq time course where the EdU-labelled fraction has been clicked to Biotin, Streptavidin-purified and libraries generated.

first: Nicolas


title: A combinatorial panel for flow cytometry-based isolation of enteric nervous system cells from human intestine

city: Rotterdam

first: Maria

last: Alves

address: Doctor Molewaterplein 40

department: Clinical Genetics

country: Netherlands

institute: Erasmus MC

summary: This work describes flow-cytometry based enrichment of human enteric neurons and glia using a combination of cell surface markers (CD56/CD90/CD24) which enable studies of the human ENS.

overall_design: Pediatric small intestine and colon was enzymatically digested using Collagenase and Dispase to obtain a single cell suspension. Cells were stained with fluorescent antibodies (CD56/CD90/CD24). Enteric nervous system (ENS) cells were enriched by Fluorecence-activated cell sorting (FACS) according to the CD56+CD90+ gate. Sorted cells from 4 donors were processed with the 10X Gene expression profiling technology.

first: J

last: Windster

first: A

last: Sacchetti

first: G

last: Schaaf

first: E

last: Bindels

first: R

middle: M

last: Wijnen

first: C

middle: E

last: Sloots

first: M


title: Tumor PD-L1 engages myeloid PD-1 to suppress type I interferon to impair cytotoxic T lymphocyte recruitment

first: Kebin

last: Liu


title: PDGFRβ+ cells play a dual role as hematopoietic precursors and niche cells during mouse ontogeny

city: Edinburgh

first: Mihaela

last: Crisan

address: 5 Little France Drive

country: United Kingdom

institute: University of Edinburgh

summary: Hematopoietic stem cell (HSC) generation in the aorta-gonads-mesonephros region requires HSC specification signals from the surrounding microenvironment. In zebrafish, PDGF-B/PDGFRβ signaling controls hematopoietic stem/progenitor cell (HSPC) generation and is required in the HSC specification niche. Little is known about murine HSPC specification in vivo and whether PDGF-B/PDGFRβ is involved.  Here we show that PDGFRβ is expressed in distinct perivascular stromal cell layers surrounding the mid-gestation dorsal aorta, and its deletion impairs hematopoiesis. We demonstrate that PDGFRβ+ cells play a dual role in murine hematopoiesis. They act in the aortic niche to support HSPCs, and in addition, PDGFRβ+ embryonic precursors give rise to a subset of HSPCs that persist into adulthood. These findings provide crucial information for the controlled production of these clinically important cells in vitro.

overall_design: A C57/BL6 litter was analyzed (embryos pulled together in one sample)

first: D

last: Sá da Bandeira

last: Kilpatrick

last: Marques

last: Gomez-Salazar

first: T

last: Ventura

first: Z

middle: N

last: Gonzalez

last: Stefancova

first: F

last: Rossi

last: Vink

last: Beltran

first: N

middle: C

last: Henderson

first: B

last: Jung

last: van der Linden

first: H

last: van de Werken

first: W

middle: F

last: van IJcken

last: Betsholtz

first: S

last: Forbes

last: Cuervo


title: Longitudinal single-cell profiling of chemotherapy response in acute myeloid leukemia

city: Milano

first: Matteo

last: Barcella

address: Via Olgettina 60

country: Italy

institute: SR-TIGET


title: EPICARDIOID SINGLE-CELL GENOMICS UNCOVER PRINCIPLES OF HUMAN EPICARDIUM BIOLOGY IN HEART DEVELOPMENT AND DISEASE [scATAC-Seq]

city: Munich

first: Gianluca

last: Santamaria

state: Germany

address: Ismaninger Strasse 22

department: Department of Medicine I

country: Germany

institute: Klinikum rechts der Isar – Technical University Munich

summary: Native human epicardial precursors are virtually inaccessible, as they appear in the embryo less than four weeks post-conception, at which point pregnancy may not yet be detected. Protocols have been established to generate epicardial cells and their progeny from human pluripotent stem cells in vitro. However, no current model is amenable to studying the many facets of human epicardial biology, notably epicardium formation, lineage heterogeneity, and functional crosstalk with other cardiac cell types during organ development and disease. Here, we generated human pluripotent stem cell-derived 3D heart organoids showing retinoic acid-dependent self-organization of the epicardium and myocardium, which we called epicardioids. Time course single-cell RNA sequencing revealed that epicardioids are formed through the specification of first heart field progenitors, a subset of which closely correspond to juxta-cardiac field (JCF) cells that have recently been identified in the embryonic mouse heart as novel common progenitors of myocardium and epicardium and have not yet been described and studied in humans. Analysis of chromatin accessibility by single-cell ATAC-seq additionally revealed key transcriptional programs guiding fate decisions along the epicardial lineage tree. Human epicardioids provide a unique model to gain fundamental insights into human epicardium biology and function during heart development, homeostasis, and disease.

overall_design: single cell identification of accessible chromatin regions during timecourse differentiation (days 2,3,4,5,7,10,15) of hiPSC derived cardiac organoids (epicardioids)

first: Dorota

last: Zawada

first: Anna

last: Meier


title: EPICARDIOID SINGLE-CELL GENOMICS UNCOVER PRINCIPLES OF HUMAN EPICARDIUM BIOLOGY IN HEART DEVELOPMENT AND DISEASE

status: Public on Feb 04 2023


title: Activation of thermogenesis pathways in testis of diet-induced obesity mice

city: Not Hispanic or Latino

first: Sheng

last: Zhao

address: #87, Dingjiaqiao, Gulouqu

department: Biochemistry and Molecular biology

institute: Southeast University

summary: High-fat diet (HFD) induced obesity (DIO) has been shown impacts on metabolism, hormonal profile, male fertility, and spermatogenesis. We employed genome-wide transcriptional analysis on the testis of diet induced obesity (DIO) and normal chow (NC) C57BL/6 J male mice to search genes regulated by obesity in testis. Both blood glucose and lipids contents significantly increased in DIO mice after 8 weeks fat-rich feeding. RNA-seq analysis revealed 371 down-regulated and 460 up-regulated transcripts in DIO group comparing to NC group. Chromosome 3, 4, 9, 16, and 18 were significantly more active, while chromosome 5, 10, and 19 were significantly more inactive after 8-week fat-diet feeding. Wilcoxon enrichment analysis discovered that the thermogenesis pathway (KEGG) was significantly enriched in the testis of DIO group (with 8 enriched up-regulated genes: Smarca2, Adcy3, Atp5pb, Creb1, Gnas, Rps6kb2, Upcrc1 and Dpf1). Real-time PCR further confirmed that Smarca2 and Atp5pb were upregulated in the testis of DIO mice. These finding implied that diet-induced thermogenesis pathways could be altered in the testis of DIO mice.

overall_design: Testis mRNA profiles of 8 weeks high fat induced obesity mice and normal mice were generated by RNA-sequencing, in triplicate, using Illumina

first: Hucheng

last: Qing

first: Juan

last: Hu


title: FDPS-regulated transcriptome profile revealed its potential functions in osteoporosis as an RNA binding protein

city: Wuhan

first: Dong

last: Chen

state: Hubei

address: 388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone

department: Center for Genome Analysis

institute: ABLife, Inc.

summary: Farnesyl diphosphate synthase (FDPS), an essencial enzyme involved in mevalonate pathway, is implicated in cancers and osteoporosis by catalyzing head to tail condensation of two molecules of isopentenyl pyrophosphate with dimethylallyl pyrophosphate to form farnesyl pyrophosphate. It has also been identified as an RNA-binding protein (RBP). However, it’s exactly RNA-binding function in diseases, up to now, remain unknown. In the present study, the function of FDPS in HeLa cells was investigated with FDPS overexpression. The results showed that FDPS overexpression promoted proliferation in HeLa cells. FDPS overexpression extensively regulated the expression of genes in cell proliferation, cytokine-mediated signaling pathway, and extracellular matrix organization. In addition, FDPS extensively regulated the alternative splicing of numbers of genes related with osteoporosis, including NAB1, FGFR3, and ALPL, and FDPS-regulated DEGs and ASEs were highly validated by RT-qPCR. This is the first study to investigate the properties of FDPS as an RBP from a genome-wide perspective, our results suggested that FDPS acts as an RBP playing an important role in cancer progression and osteoporosis by altering gene expression and regulating alternative splicing, which contributes to a precise understanding of potentially FDPS-targeted therapies.

overall_design: transcrptional analysis of FDPS overexpression and control in Hela cells

first: Fengyun

last: Yu

first: Zhigang


title: ScRNA-seq of AD mouse hippocampus

status: Public on Feb 03 2023

city: Seoul

first: BYOUNGGOOK

last: CHO

address: 30 Pildong-ro 1-gil, Jung-gu

country: South Korea

institute: Dongguk university

summary: We describe a disease-associated subpopulation of oligodendrocytes that is present during progression of AD-like pathology in the maleAppNL-G-F

overall_design: Jongpil, Kim

first: Hanseul

last: Park

first: Byounggook

last: Cho


title: Effect of insulin and glucose supplementation during in vitro developpement of rabbit embryo

city: Jouy-en-josas

first: Sophie

last: Calderari

address: Allée de Vilvert

institute: INRAE UMR1198 BREED

summary: We investigate the Inner Cell mass and the Trophectoderm specific response to the presence of insulin, glucose or both in media culture from 1 cell to blatsocyst stage in rabbit embryo

overall_design: We then performed gene expression profiling analysis using data obtained from RNA-seq of ICM and TE of embryos developped in insulin, glucose or both insulin and glucose

first: Romina

middle: V

last: Rada

first: Nathalie

last: Daniel

first: Catherine

last: Archilla

first: Anne

last: Frambourg

first: Luc

last: Jouneau

first: Yan

last: Jaszczyszyn

first: Gilles

last: Charpigny

first: Véronique

last: Duranthon


title: Stepwise activation of p63 and EGFR-MEK/ERK pathway induces ARL4C expression to promote oral squamous cell carcinoma cell proliferation

city: Fukuoka

first: Kana

last: Hasegawa

state: Fukuoka

address: 3-1-1 Maidashi, Higashi-ku

country: Japan

institute: Kyushu University

summary: Carcinogenesis is a multistep process in which cells accumulate multiple genetic alterations, as they progress to a more malignant phenotype. It has been proposed that sequential accumulation of gene abnormalities in specific genes drives the transition from non-tumorous epithelia through preneoplastic lesion/benign tumor to cancer. Histologically, progression to invasive oral squamous cell carcinoma (OSCC) follows multistep ordered series beginning with mucosal epithelial cell hyperplasia, followed by dysplasia, carcinoma in situ and invasive carcinoma. It is therefore speculated that genetic alterations-mediated multistep carcinogenesis would be involved in OSCC development, but their detailed molecular mechanisms are unknown. Herein, we clarified the comprehensive gene expression patterns and carried out an enrichment analysis using DNA microarray data obtained from an OSCC pathological specimen, which consists of non-tumor region, carcinoma in situ lesion and invasive carcinoma lesion. Numerous numbers of gene expression and signal activation were altered in the OSCC development. Of them, the expression of p63 was increased and MEK/ERK-MAPK pathway was activated in carcinoma in situ lesion as well as in invasive carcinoma lesion. Immunohistochemical analyses revealed that p63 was initially upregulated in carcinoma in situ lesions and ERK was sequentially activated in invasive carcinoma lesions in OSCC specimens. ADP-ribosylation factor (ARF)-like 4c (ARL4C), the expression of which is reportedly induced by p63 and/or MEK/ERK-MAPK pathway in OSCC cells, has been shown to promote tumorigenesis. Immunohistochemically, in OSCC specimens, ARL4C was more frequently detected in tumor lesions, especially in invasive carcinoma lesions than in carcinoma in situ lesions. Additionally, ARL4C and phosphorylated ERK were merged at high frequency in invasive carcinoma lesions. Loss-of-function experiments using inhibitors and siRNAs revealed that p63 and EGFR-MEK/ERK-MAPK cooperatively induce ARL4C expression in OSCC cells. These results suggest that the stepwise activation of p63 and EGFR-MEK/ERK-MAPK contributes to OSCC tumor cell growth though regulation of ARL4C expression.

overall_design: A DNA microarray analysis was carried out in carcinoma in situ CIS lesion and/or invasive carcinoma lesion compared with non-tumor region. Compare1: CIS lesion was compared with non-tumor region. Compare2: Invasive carcinoma lesion was compared with  non-tumor region. Compare3: Invasive carcinoma lesion was compared with CIS lesion.

first: Dania

last: Alkhatib

first: Thinh

middle: T

last: Truong

first: Shinsuke

last: Fujii

first: Ryoko

last: Nagano

first: Yudai

last: Tajiri

first: Tamotsu

last: Kiyoshima


title: Nab6 and Mrn1 regulate the expression of cell wall mRNAs in budding yeast

first: Stefan

last: Bresson

state: Scotland

address: Max Born Crescent, Swann 5.1

department: Wellcome Trust Centre for Cell Biology

summary: The fungal cell wall provides protection and structure to cells, and serves as an important target for antifungal compounds. The cell wall integrity (CWI) signaling pathway regulates the transcriptional response to various cell wall stresses, but emerging evidence suggests posttranscriptional pathways play an important complementary role. Here, we show that the RNA-binding proteins (RBPs) Mrn1 and Nab6 specifically target the 3′ UTRs of a largely overlapping set of cell wall-associated mRNAs. These mRNAs are downregulated in the absence of Nab6, indicating that Nab6 directly or indirectly stabilizes its target mRNAs. We further show that Nab6 acts in parallel to conventional CWI signaling to maintain adequate expression of cell wall mRNAs. Cells lacking both pathways are hypersensitive to antifungal compounds targeting the cell wall, while deletion of MRN1 partially alleviates the growth and molecular phenotypes associated with the Dnab6 strain. Taken together, our results uncover a novel posttranscriptional pathway which mediates resistance to cell wall stress and antifungal compounds.

overall_design: There are 4 CRAC datasets and 50 RNAseq datasets. The CRAC datasets were generated using affinity tagged versions of Mrn1 and Nab6 under standard ('control') conditions in otherwise wild type cells. The RNAseq experiments were prepared from wild type, Dmrn1, Dnab6, Dslt2, and Dnab6Dslt2 cells before and after treatment with 2 µg/mL Congo Red (0, 1, 2, and 4hr). In addition, wild type, Dmrn1, Dnab6, and Dmrn1Dnab6 cells were treated with 60µg/mL Congo Red (0 and 2 hr).

first: Vadim

last: Shchepachev

first: David

last: Tollervey


title: Tumor PD-L1 engages myeloid PD-1 to suppress type I interferon to impair cytotoxic T lymphocyte recruitment (bulk)

first: Kebin

last: Liu

overall_design: Bulk RNA-sequencing: Balb/c mice were injected i.v. through the lateral tail vein with 2.5*105 4T1 WT or PDL1-KO cells. After seven days, mice were sacrificed, lungs were perfused with 20mL of PBS. Lungs were mechanically homogenized and RNA was extracted using Direct-zol RNA microprep kit according to manufacturer’s instructions (Zymo Research, Irvine CA). RNA was sequenced by Novogene. Reference genome (mm10) and gene model annotation files were downloaded from Ensembl directly. Indices were built and paired-end clean reads were aligned to the reference genome using STARv2.5. Reads were quantified using HTSeqv0.6.1.

first: John

middle: D

last: Klement


title: KLF10 deficiency in CD4+ T cells promotes atherosclerosis progression by altering macrophage dynamics

city: Valencia

first: Daniel

last: Perez-Cremades

state: Valencia

address: 15 Blasco Ibañez Avenue

department: Physiology Department

country: Spain

institute: University of Valencia

summary: Gene expression profiling of aortic tissue from WT and CD4-KLF10-KO mice injected once with the AAV-PCSK9D377Y gain-of-function transgene and placed on high cholesterol diet.

overall_design: 8 weeks old CD4-specific KLF10 knockout mice (TKO) and WT male mice were intraperitoneally injected once with the AAV-PCSK9D377Y gain-of-function transgene and placed on high cholesterol diet for 12 weeks ad libitum to develop advanced atherosclerotic plaques. Aortic tissue was harvested and RNA-Seq analysis was performed using Illumina HiSeq2500 V4 2x150 bp  (Genewiz, South Plainfield, NJ).

first: Akm

middle: K

last: Wara

first: Shruti

last: Rawal

first: Xilan

last: Yang

last: Pérez-Cremades

first: Jingshu

last: Chen

first: Mark

middle: W

last: Feinberg


title: SEACells: Inference of transcriptional and epigenomic cellular states from single-cell genomics data

first: Manu

last: Setty

address: 1100 Fairview Ave N

department: Basic Sciences Division

institute: Fred Hutchinson Cancer Center

summary: Metacells are cell groupings derived from single-cell sequencing data that represent highly granular, distinct cell states. Here, we present single-cell aggregation of cell-states (SEACells), an algorithm for identifying metacells; overcoming the sparsity of single-cell data, while retaining heterogeneity obscured by traditional cell clustering. SEACells outperforms existing algorithms in identifying accurate, compact, and well-separated metacells in both RNA and ATAC modalities across datasets with discrete cell types and continuous trajectories. We demonstrate the use of SEACells to improve gene-peak associations, compute ATAC gene scores and measure gene accessibility in each metacell. Metacell-level analysis scales to large datasets and are particularly well suited for patient cohorts, including facilitation of data integration. We use our metacells to reveal expression dynamics and gradual reconfiguration of the chromatin landscape during hematopoietic differentiation, and to uniquely identify CD4 T cell differentiation and activation states associated with disease onset and severity in a COVID-19 patient cohort.

overall_design: Cryopreserved bone marrow cells from healthy donor were purchased from AllCells, LLC and stored in vapor phase nitrogen. Each sample was processed and loaded into two 10X channels to increase the number of cells.

first: Sitara

last: Persad

first: Zi-Ning

last: Choo

first: Christine

last: Dien

first: Ignas

last: Masilionis

first: Ronan

last: Chaligne

first: Tal

last: Nawy

first: Chrysothemis

last: Brown

first: Itsik

last: Pe'er

first: Dana


title: Mitochondrial fitness and risk of pancreatic ductal adenocarcinoma

first: Priyankara

middle: J

last: Wickramasinghe

address: 3601 Spruce Street

department: Bioinformatics

institute: The Wistar Institute

summary: Changes in metabolism are a hallmark of cancer, but molecular signatures of altered bioenergetics to aid in clinical decision-making do not currently exist. We recently identified a group of human tumors with constitutively reduced expression of the mitochondrial structural protein, Mic60, also called mitofilin or inner membrane mitochondrial protein (IMMT). These Mic60-low tumors exhibit severe loss of mitochondrial fitness, paradoxically accompanied by increased metastatic propensity and upregulation of a unique transcriptome of Interferon (IFN) signaling and Senescence-Associated Secretory Phenotype (SASP). Here, we show that an optimized, 11-gene signature of Mic60-low tumors is differentially expressed in multiple malignancies, compared to normal tissues, and correlates with poor outcome. When analyzed in three independent patient cohorts of pancreatic ductal adenocarcinoma (PDAC), the Mic60-low gene signature was associated with aggressive molecular variants of PDAC, local inflammation, FOLFIRINOX failure and shortened survival, independently of age, gender, or stage. Therefore, the 11-gene Mic60-low signature may provide an easily accessible molecular tool to stratify patient risk in PDAC and potentially other malignancies.

overall_design: RNA-seq of Mic60 WT and KO cells.

first: Dario

last: Altieri

first: Andrew

last: Kossenkov


title: Spatially Resolved Transcriptomics Deconvolutes Histological Prognostic Subgroups in Patients with Colorectal Cancer and Synchronous Liver Metastases

status: Public on Feb 02 2023

city: Glasgow

first: Colin

last: Wood

address: 84 Castle Street

department: Academic Unit of Surgery

institute: University of Glasgow

summary: This bulk mRNA data was generated using the Nanostring ncounter platform using an io360 pancancer panel of 900 genes as part of a wider project which included spatial transcriptomic data generated using the Nanostring GeoMx platform.  In this study we compared tissue surgical resected from patients with colorectal cancer and liver metastases.

overall_design: We compered the matched primary and metastatic lesions.  The lesions were further annotated using histological scores to determine immune infiltration and stromal content, we further used these annotations to interrogate the mRNA data generated from nCounter

first: Kathryn

last: Pennel

first: Holly

last: Leslie

first: Assya

last: Legrini

last: Cameron

first: Lydia

last: Melissourgou-Syka

first: Jean

middle: A

last: Quinn

first: Hester

last: van Wyk

first: Jennifer

last: Hay

first: Antonia

last: Roseweir

last: Nixon

first: Campbell

middle: S

last: Roxburgh

first: Donald

last: McMillan

last: Biankin

first: Owen

last: Sansom

first: Paul

middle: G

last: Horgan

first: Joanne

last: Edwards

last: Steele

first: Nigel

last: Jamieson


title: Recycling of H2A-H2B provides short-term memory of chromatin states [SCAR-Seq]

city: Copenhagen

first: Anja

last: Groth

address: Blegdamsvej 3B

institute: Novo Nordisk Foundation Center for Protein Research

summary: Chromatin landscapes are disrupted during DNA replication and must be restored faithfully to maintain genome regulation and cell identity. The H3-H4 modification landscape is restored by parental histone recycling and post-replication modification of new histone H3-H4. How DNA replication impact on histone H2A-H2B is unknown. Here, we track H2A-H2B modifications and H2A.Z during DNA replication and across the cell cycle using quantitative genomics. We show that H2AK119ub, H2BK120ub, and H2A.Z are recycled quantitatively and accurately during DNA replication. H2A-H2B are recycled symmetrically to daughter strands largely independent of known H3-H4 recycling pathways. Post-replication, H2A-H2B modifications are rapidly restored, and the rapid wave of H2AK119ub supports accurate restoration of H3K27me3. This work reveals epigenetic transmission of H2A-H2B modification during DNA replication and identifies H3-H4 and H2A-H2B crosstalk in epigenome propagation. We propose that rapid short-term memory of recycled H2A-H2B modifications facilitates reestablishment of slow, long-term chromatin state memory.

overall_design: SCAR-seq (Sister Chromatids After Replication) time-series measuring histone partition of H3K27me3, H2AK119ub, H2A.Z and H4K20me0 in wildtype (WT), MCM2 histone-binding mutant (MCM2-2A), Pola1 histone-binding mutant (Pola1-3A), or Pole4 KO mouse embryonic stem cells (Parental WT cells, 2 MCM2-2A clones, 2 Pola1-3A clones, 2 Pole4KO clones,  >2 replicates per cell line, stranded input controls). SCAR-seq time-series measuring histone partition of H3K27me3 or JARID2 in Ring1B-mAID Ring1AKO MCM2-2A cells by either depleting H2AK119ub by the addition of indole-3-acetic acid (IAA) or corresponding DMSO treatment as a control.

first: Valentin

last: Flury

first: Nazaret

last: Reverón-Gómez

first: Nicolas

last: Alcaraz

first: Kathleen

last: Stewart-Morgan

first: Alice

last: Wenger

first: Robert

last: Klose


title: Lung epithelial cell-derived C3 protects against pneumonia-induced lung injury

first: Ruteja

last: Barve

state: MO

address: 4515 McKinley Ave

department: Genetics

institute: Washington University

summary: The complement component C3 is a fundamental plasma protein for host defense, produced largely by the liver. However, recent work has demonstrated the critical importance of tissue-specific C3 expression in cell survival.  Here we analyzed the effects of local versus peripheral sources of C3 expression in a model of bacterial pneumonia. While mice with global C3 deficiency had severe pneumonia-induced lung injury, those deficient in liver-deficient C3 remain protected, comparable to wildtype mice. Human lung transcriptome analysis showed that secretory epithelial cells, such as club cells, are a major source of C3.  Mice with a tamoxifen-induced C3 gene ablation from club cells in the lung had worse pulmonary injury compared to similarly treated controls, despite maintaining normal circulating C3 levels. Finally, in human cellular and mouse pneumonia models, we show that C3 reduces epithelial cell death mediated through the alternative pathway component Factor B, rather than the C3aR. Thus, our findings suggest that a locally-derived C3-Factor B pathway protects the lung mucosal barrier.

overall_design: BEAS-2B cells rendered deficient in Factor B using CRISPR (CFB-/-) were compared to their parent clones (CFB+/+) at 4 h after infection with Pseudomonas aeruginosa (Pa57-15, MOI 5). N=3 in each group.

first: Hrishikesh

last: Kulkarni

first: Sanjaya

last: Sahu

first: Devesha


title: DOT-1.1 (DOT1L) deficiency in C. elegans leads to small RNA-dependent gene activation [mRNA, embryos]

city: Boston

first: Alla

last: Grishok

address: 72 E Concord

institute: Boston University

summary: Methylation of histone H3 at lysine 79 (H3K79) is conserved from yeast to humans and is accomplished by Dot1 (disruptor of telomeric silencing-1) methyltransferases. The C. elegans enzyme DOT-1.1 and its interacting partners are similar to the mammalian DOT1L (Dot1-like) complex. The C. elegans DOT-1.1 complex has been functionally connected to RNA interference. Specifically, we have previously shown that embryonic and larval lethality of dot-1.1 mutant worms deficient in H3K79 methylation was suppressed by mutations in the RNAi pathway genes responsible for generation (rde-4) and function (rde-1) of primary small interfering RNAs (siRNAs). This suggests that dot-1.1 mutant lethality is dependent on the enhanced production of some siRNAs. We have also found that this lethality is suppressed by a loss-of-function of CED-3, a conserved apoptotic protease. Here, we describe a comparison of gene expression and primary siRNA production changes between control and dot-1.1 deletion mutant embryos. We found that elevated antisense siRNA production occurred more often at upregulated than downregulated genes. Importantly, gene expression changes were dependent on RDE-4 in both instances. Moreover, the upregulated group, which is potentially activated by ectopic siRNAs, was enriched in protease-coding genes. Our findings are consistent with a model where in the absence of H3K79 methylation there is a small RNA-dependent activation of protease genes, which leads to embryonic and larval lethality. DOT1 enzymes’ conservation suggests that the interplay between H3K79 methylation and small RNA pathways may exist in higher organisms.

overall_design: C. elegans dot-1.1(knu339); ced-3(n1286) mutants and ced-3(n1286) mutant controls (3 replicates each) were age-synchronized and their eggs were harvested via bleaching. RNA was extracted and mRNA and lncRNA sequencing was performed to find differences in gene expression.

first: Thomas

last: Liontis

first: Karisma

last: Verma


title: Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions

city: Parkville

first: Matthew

last: Ritchie

state: Victoria

address: 1G Royal Parade

country: Australia

institute: The Walter and Eliza Hall Institute of Medical Research

summary: Actinobacillus pleuropneumoniae is the cause of porcine pleuropneumonia, a severe respiratory tract infection that is responsible for major economic losses to the swine industry. Many host-adapted bacterial pathogens encode systems known as phasevarions (phase-variable regulons). Phasevarions result from variable expression of cytoplasmic DNA methyltransferases. Variable expression results in genome-wide methylation differences within a bacterial population, leading to altered expression of multiple genes via epigenetic mechanisms. Our examination of a diverse population of A. pleuropneumoniae strains determined that Type I and Type III DNA methyltransferases with the hallmarks of phase variation were present in this species. We demonstrate that phase variation is occurring in these methyltransferases, and show associations between particular Type III methyltransferase alleles and serovar. Using Pacific BioSciences Single-Molecule, Real-Time (SMRT) sequencing and Oxford Nanopore sequencing, we demonstrate the presence of the first ever characterised phase-variable, cytosine-specific Type III DNA methyltransferase. Phase variation of distinct Type III DNA methyltransferase in A. pleuropneumoniae results in the regulation of distinct phasevarions, and in multiple phenotypic differences relevant to pathobiology. Our characterisation of these newly described phasevarions in A. pleuropneumoniae will aid in the selection of stably expressed antigens, and direct and inform development of a rationally designed subunit vaccine against this major veterinary pathogen.

sample_organism:
Escherichia coli
Actinobacillus pleuropneumoniae

overall_design: Transposase (DNA sequencing)

first: Nusrat

last: Nahar

first: Greg

last: Tram

first: Freda

last: Jen

first: Zachary

last: Phillips

first: Lucy

last: Weinert

first: Janine

last: Bossé

first: Jafar

last: Jabbari

first: Quentin

last: Gouil

first: Mei

last: Du

first: Rory

last: Bowden

first: Paul

last: Langford

first: Alexander

last: Tucker

first: Michael

middle: P

last: Jennings

first: Conny

last: Turni

first: Patrick

last: Blackall

first: John

last: Atack


title: DOT-1.1 (DOT1L) deficiency in C. elegans leads to small RNA-dependent gene activation [small RNA, embryos]

first: Alla

last: Grishok

overall_design: Differential gene expression analysis of small RNA sequencing data in C. elegans embryos dot-1.1(knu339); ced-3(n1286) compared to ced-3(n1286) controls.

first: Thomas

last: Liontis

first: Karisma

last: Verma


title: DOT-1.1 (DOT1L) deficiency in C. elegans leads to small RNA-dependent gene activation

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

overall_design: Refer to individual Series


title: Interaction between tumor cell TNFR2 and monocyte membrane-bound TNFα triggers tumorigenic inflammation in neuroblastoma

city: HOUSTON

first: Leonid

last: Metelitsa

address: 3639 Skyline Dr1102 Bates Ave., Ste. 1570

department: Pediatrics

institute: BAYLOR COLLEGE OF MEDICINE/Texas Children's Hospital

summary: We found that NB expression of TNFR2 and monocyte mTNFα are required for monocyte activation and IL-6 production, while NB TNFR1 and monocyte sTNFα are required for NB NF-κB activation. Treatment of NB-monocyte co-cultures with clinical-grade etanercept completely abrogated release of IL-6, G-CSF, IL-1α, and IL-1β and eliminated monocyte-induced enhancement of NB cell proliferation in vitro. Furthermore, etanercept treatment inhibited tumor growth, ablated tumor angiogenesis, and suppressed oncogenic signaling in mice with subcutaneous NB/human monocyte xenografts. Finally, GSEA revealed significant enrichment for TNFα signaling in NB patients that relapsed.

overall_design: Comparative gene expression profiling analysis of RNA-seq data on etanercept-treated and control tumors

first: Chunchao

last: Zhang


title: Addition of losartan to FOLFIRINOX and chemoradiation reduces immunosuppression-associated genes, Tregs and FOXP3+ cancer cells in locally advanced pancreatic cancer

first: Sonu

last: Subudhi

state: MA

address: 100 Blossom St, Cox 7

department: Radiation Oncology

institute: Massachusetts General Hospital

summary: Purpose: Adding losartan (LOS) to FOLFIRINOX (FFX) chemotherapy followed by chemoradiation (CRT) resulted in 61% R0 surgical resection in our phase II trial in patients with locally advanced pancreatic cancer (LAPC). Here we identify potential mechanisms of benefit by assessing the effects of neoadjuvant losartan on the stromal tumor microenvironment.  Experimental Design: We performed a gene expression and immunofluorescence (IF) analysis using archived surgical samples from patients treated with LOS+FFX+CRT (NCT01821729), FFX+CRT (NCT01591733) or surgery upfront, without any neoadjuvant therapy. We also conducted a longitudinal analysis of multiple biomarkers in the plasma of treated patients.   Results: In comparison to FFX+CRT, LOS+FFX+CRT downregulated immunosuppression and pro-invasion genes. Overall survival (OS) was associated with DC and antigen presentation genes for patients treated with FFX+CRT, and with immunosuppression and invasion genes or DC– and blood vessel-related genes for those treated with LOS+FFX+CRT. Furthermore, losartan induced specific changes in circulating levels of IL-8, sTie2 and TGF-. IF revealed significantly less residual disease in lesions treated with LOS+FFX+CRT. Lastly, patients with a complete/near complete pathological response in the LOS+FFX+CRT-treated group had reduced CD4+FOXP3+ regulatory T cells (Tregs), fewer immunosuppressive FOXP3+ cancer cells (C-FOXP3) and increased CD8+ T cells in PDAC lesions.   Conclusions: Adding losartan to FFX+CRT reduced pro-invasion and immunosuppression related genes which were associated with improved survival in patients with LAPC. Lesions from responders in the LOS+FFX+CRT-treated group had reduced Tregs, decreased C-FOXP3 and increased CD8+ T cells. These findings suggest that losartan may potentiate the benefit of FFX+CRT by reducing immunosuppression.

overall_design: Analysis of gene expression in the immune TME in resected PDAC samples. To characterize the effects and identify potential molecular mechanisms of cytotoxic agents in combination with losartan on the immune TME, we used the nCounter PanCancer Immune Profiling Panel of 730 genes (NanoString) to analyze the RNA extracted from formalin-fixed paraffin embedded (FFPE) PDAC tissue sections.

first: Rakesh

last: Jain

first: Yves

last: Boucher

first: Jessica

last: Posada

first: Ashwin

last: Kumar


title: RNA sequencing on in vitro differentiated WT and GOT1-/- Th17 cells

city: Baltimore

first: Jonathan

last: Powell

state: MD

address: 1650 Orleans Street

institute: Johns Hopkins University

summary: Naive CD4+ T cells (CD4+CD25-CD44-CD62L+) were FACS sorted from WT or T cell conditional GOT1-/- C57BL/6 mouse spleen and lymph nodes. Sorted cells were differentiated into Th17 cells in the presence of TGFβ, IL1β, IL6, IL23, anti-IFNγ and anti-IL4. RNA sequencing was then performed on day4 differentiated TH17 cells.

overall_design: Expression profiling in triplicates by high throughput sequencing (Illumina Novaseq) on in vitro differentiated Th17 cells

first: Wei

last: Xu


title: scRNAseq RAT TA and EOMs WT and DMD 12months

city: CRETEIL

first: Valentina

last: TAGLIETTI

address: RUE GENERAL SERRAIL

institute: INSERM

summary: Thyroid Stimulating Hormone Receptor signalling restores skeletal muscle stem cell regeneration in Duchenne muscular dystrophy

overall_design: RAT Tibialis anterior and extraocular muscles WT and DMD 12months

last: Taglietti

first: Kaouthar

last: Kefi

first: Lea

last: Rivera

first: Oriane

last: Bergiers

first: Nastasia

last: Cardone

first: Fanny

last: Coulpier

first: Stamatia

last: Gioftsidi

first: Bernadette

last: Drayton-Libotte

first: Cyrielle

last: Hou

first: Francois-Jerome

last: Authier

first: Matthieu

last: Robert

first: Dominiqie

last: Bremond-Gignac

first: France

last: Pietri-Rouxel

first: Claudio

last: Bruno

first: Chiara

last: Fiorillo

first: Edoardo

last: Malfatti

first: Peggy

last: Lafuste

first: Laurent

last: Tiret

first: Frederic

last: Relaix


title: TRABID deubiquitinase overexpression enables synthetic lethality to PARP inhibitor via prolonged retention of 53BP1 at DSB sites

city: Xi'an

first: Jian

last: MA

state: Shan Xi

address: West Yanta road 227

institute: The First Affiliated Hospital of Xi'an Jiaotong University

summary: LSD1 and MBD3 CUT&Tag profiling in PC-3 cells treated with or without IR

overall_design: PC-3 cells were treated with or without IR and  harvested for cut tag sequencing

last: Ma

first: Lei

last: Li


title: Genome-wide transcriptomic profiling of human iPS-derived cardiomyocytes in response to ectopic expression of SARS-CoV-2 proteins

city: Clemson

first: Qing

last: Liu

state: SC

address: Jordan Hall

department: Biological Sciences

institute: Clemson University

summary: In this study, we investigated the effects of SARS-CoV-2 subunits on cardiomyocytes derived from human stem cells. The human induced pluripotent stem cells (hiPSCs) was differentiated into cardiomyocytes, and then were transfected with plasmids carrying different SARS-CoV-2 subunits. The cardiac functions, such as contractility and metabolic profiles, were investigated. We found the subunits 9B and S proteins has significantly adversed effects on the cardiac functions, compared to other groups. Thus, changes in transcriptome of cardiomyocytes (day30) after over expression of 9B, S for two days (on day 32) and two weeks (on day 45) were investigated by RNA-seq.

overall_design: The DNA sequences of SARS-CoV-2 (Wuhan-Hu-1 strain, GenBank: NC_045512.2) was used for DNA synthesis of each gene (General Bio, China). The viral genes were firstly cloned into pcDNA6B-Flag vector as described in our previous publication (PMID: 33203855). The constructs were further subcloned into a lentivirus transfer plasmid pCDH-CMV-MCS-EF1α-copGFP (System Biosciences, #CD511B-1, Palo Alto, CA, USA) with standard molecular cloning methods. An hiPSC line (generated from a health donor) obtained them from the Stanford Cardiovascular Institute (SCVI) Biobank was used for this study. The hiPSCs were differentiated into cardiomyocytes, and then were transfected with plasmids carrying SARS-CoV-2 subunits with a lentivirial transfection method.  After 4 days of treatment, the GFP expression was examined in the transfected cardiomyocytes. Cells after transfection with 9B, S, and control groups at day 32 and day 46 were harvested for RNA isolation and RNA-seq. The library preparation and RNA-sequencing were done by Novogene Corporation Inc (Sacramento, CA). The RNA-seq libraries were constructed using NEBNext UltraTM II RNA Library Prep Kit for Illumina and were sequenced by Novaseq 6000 PE150 system. Raw reads of FASTQ format were firstly processed through fastp, and clean data (clean reads) were obtained by removing reads containing adapter and poly-N sequences and reads with low quality from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the downstream analyses were based on the clean data with high quality.The human gene symbols and their raw counts were calculated using the HTSeq (version 0.6.1p1) package in Python with the hg19 Homo sapiens gtf file. Differential gene-expression analysis was performed using the edgeR package in R, and the normalization was performed using a trimmed mean of M-values (TMM) method across all samples.


title: Differential requirements for Gcn5 and NuA4 HAT activities in the starvation-induced versus basal transcriptomes

city: Bethesda

first: Alan

last: Hinnebusch

address: 6 Center Drive, 6/230

institute: NIH, NICHD

summary: The histone acetyltransferase (HAT) subunit of coactivator complex SAGA, Gcn5, is required for efficient eviction of promoter nucleosomes at certain highly expressed yeast genes, including those activated by transcription factor Gcn4 in amino acid-deprived cells; however, the importance of other HAT complexes in this process was poorly understood. Analyzing mutations that disrupt the integrity or activity of HAT complexes NuA4 or NuA3, or the HAT Rtt109, revealed that only NuA4 acts on par with Gcn5, and functions additively, in evicting and repositioning promoter nucleosomes and stimulating transcription of starvation-induced genes.  NuA4 is generally more important than Gcn5, however, in promoter nucleosome eviction, TBP recruitment, and transcription at most other genes expressed constitutively.  NuA4 also predominates over Gcn5 in stimulating TBP recruitment and transcription of genes categorized as principally dependent on the cofactor TFIID versus SAGA, except for the highly expressed subset encoding ribosomal proteins (RPs), where Gcn5 contributes strongly to PIC assembly and transcription. Both SAGA and NuA4 are recruited to promoter regions of SM-induced genes in a manner that appears to be feedback controlled by their HAT activities. Our findings reveal an intricate interplay between these two HATs in nucleosome eviction, PIC assembly, and transcription that differs between the starvation-induced and basal transcriptomes.

overall_design: Formaldehyde-fixed and sonicated DNA fragments were immunopurified and subjected to paired-end sequencing.

first: Qiaoyun

last: Zheng

first: Hongfang

last: Qiu

first: Hongen

last: Zhang


title: Regulation of Immunological Tolerance by the p53-Inhibitor iASPP [mNET-Seq]

city: Leicester

first: Michael

last: Tellier

state: Select State

address: Lancaster Road

department: Department of Molecular and Cell Biology

institute: University of Leicester

summary: Maintenance of immunological homeostasis between tolerance and autoimmunity is essential for the prevention of human diseases ranging from autoimmune disease to cancer.  Accumulating evidence suggests that p53 can mitigate phagocytosis-induced adjuvanticity thereby promoting immunological tolerance following programmed cell death.  Here we identify Inhibitor of Apoptosis Stimulating p53 Protein (iASPP), a negative regulator of p53 transcriptional activity, as a regulator of immunological tolerance.  iASPP-deficiency promoted lung adenocarcinoma and pancreatic cancer tumorigenesis, while iASPP-deficient mice were less susceptible to autoimmune disease.  Immune responses to iASPP-deficient tumors exhibited hallmarks of immunosuppression, including activated regulatory T cells and exhausted CD8+ T cells.  Interestingly, iASPP-deficient tumor cells and tumor-infiltrating myeloid cells, CD4+, and gamma-delta T cells expressed elevated levels of PD-1H, a recently identified transcriptional target of p53 that promotes tolerogenic phagocytosis.  Identification of an iASPP/p53 axis of immune homeostasis provides a therapeutic opportunity for both autoimmune disease and cancer.

overall_design: mNET-seq of total RNA polymerase II in A549 wild-type cells or A549 PPP1R13L knockdown cells.

first: Elliot

middle: H

last: Akama-Garren

first: Paul

last: Miller

first: Thomas

last: Carroll

first: Gopinath

last: Sutendra

first: Ludovico

last: Buti

first: Justyna

last: Zaborowska

first: Robert

last: Goldin

first: Elizabeth

last: Slee

first: Francis

last: Szele

first: Shona

last: Murphy

first: Xin

last: Lu