title: Study of the cellular component-dependent functions of Xrn1

city: Solna

first: Vicent

last: Pelechano

address: Nobels väg 16

department: MTC

country: Sweden

institute: ScilifeLab - Karolinska Institutet

summary: The main cytoplasmic mRNA decay pathway in yeast  uses the 5’-3’ exonuclease Xrn1 (Parker and Song, Nat Struct Mol Biol. 2004 ). This protein shuttles from the cytoplasm to the nucleus, where it has a role as transcription factor (Haimovich et al. Cell 2013). In this work, we find that most of the global phenotypes of an xrn1 mutant are partially complemented by a cytoplasmic version of the paralogous 5’-3’exonuclease Rat1 (cRat1) indicating that this 5’-3’-exonuclease has a similar enzymatic capacity as Xrn1. The lack of a cytoplasmatic 5’-3’-exoribonuclease is the cause of the physiological defects of an xrn1 mutant. The capacity of cRat1 to perform co-translational decay is, however, very limited. The comparison with the strain having a NLS1∆-NLS2∆-Xrn1 version shows that it is slightly deficient in 5’→3’-co-translational decay but much more efficient than cRat1. In both strains, cRat1 and -Xrn1-NLS1&2, the lack of nuclear Xrn1 has a very minor influence on cell growth.

overall_design: We performed HT-5PSeq experiments on 2 sets of strains to measure if cytoplasmic Rat1 was able to perform ribosome-associated co-translational decay : set #1: BY4741 (wt) and xrn1∆ transformed, or not, with Rat1-∆NLS-3xFlag. This set includes BY4741, xrn1Δ, BY4741 + Rat1-∆NLS-3xFlag (wt+cRat1), xrn1Δ+Rat1-∆NLS-3xFlag (xrn1+cRat1). Set #2)  BY4741 (wt) and xrn1∆ transformed with a plasmid containing the Rat1-∆NLS version without the FLAG epitopes, the same Rat1-∆NLS plus the Xrn1 C-termainal domain or with the empty vector. These three plasmids are: YCpLac33 (URA3, CEN4ARS1, empty vector), pBBM3 (YCpLac33+Rat1∆NLS) and pLCM9 (YCpLac33+ Rat1∆NLS-Xrn1-Cterm-3xFLAG). No transformation of pLCM9 in BY4741 was done. Additionally we analyzed Set #3 of strains to explore whether co-translational decay was dependent on nucleo-cytoplasmic shuttling of Xrn1. We performed HT-5PSeq on Xrn1 with a version lacking one or both NLS sequences (Xrn1-NLS1&2). This set includes Xrn1-ΔNLS1, Xrn1-ΔNLS2, Xrn1-ΔNLS1&NLS2. All the three sets of strains were done in duplicate or in triplicate samples for each strain grown in YPD to middle exponential phase (0.5 OD600).

first: José

last: Pérez-Ortín

first: Yujie

last: Zhang


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

status: Public on Oct 23 2024

city: Grand Rapids

first: Scott

last: Rothbart

state: MI

address: 333 Bostwick Avenue NE

country: USA

institute: Van Andel Research Institute

type: Methylation profiling by genome tiling array

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

overall_design: Genomic DNA was isolated from HCT116 cells lines modified with stable integration of a doxycycline-inducible shRNA targeting the 3’UTR of either UHRF1 or DNMT1. Bulk (shDNMT1 = 2, shUHRF1 = 1) and clonally expanded (shDNMT1 = 2 clones, shUHRF1 = 3 clones) cell populations were used. In all cases, Baseline (NoDox) samples were grown in culture for 2 weeks in the absence of doxycycline, and serve as the baseline methylome control for Knockdown and Recovery samples. Knockdown samples were treated with doxycycline (10 ng/mL) for two weeks to induce expression of the shRNA. Recovery samples were treated with doxycycline for two weeks (to induce knockdown of the target protein), and then doxycycline was removed for an additional two weeks to measure recovery methylation dynamics with re-expression of the either UHRF1 or DNMT1. Multiple time-points were gathered for the shUHRF1 Cl3 and Cl6 throughout the Knockdown and Recovery phases. Transgenes for WT and mutant UHRF1 and DNMT1 were stably introduced into the clonally expanded Cl9 shUHRF1 and Cl3 shDNMT1 cell lines, respectively. Doxycycline treatment (10 ng/mL) over 2 weeks depleted the endogenous enzyme in the presence of the transgene protein cover.

first: Rochelle

middle: L

last: Tiedemann

first: Joel

last: Hrit

first: Qian

last: Du

first: Ashley

middle: K

last: Wiseman

first: Hope

middle: E

last: Eden

first: Bradley

middle: M

last: Dickson

first: Xiangqian

last: Kong

first: Alison

middle: A

last: Chomiak

first: Robert

last: Vaughan

first: Jakob

last: Hebert

first: Yael

last: David

first: Wanding

last: Zhou

first: Stephen

middle: B

last: Baylin

first: Peter

last: Jones

first: Susan

middle: J

last: Clark


title: Host immune responses associated with SARS-CoV-2 Omicron infection result in protection or pathology during reinfection depending on mouse genetic background

city: La Jolla

first: Lars

last: Pache

state: CA

address: 10901 N Torrey Pines Rd

institute: Sanford Burnham Prebys Medical Discovery Institute

type: Expression profiling by high throughput sequencing

summary: Rapid emergence of antigenic distinct SARS-CoV-2 variants implies a greater risk of reinfection as viruses can escape neutralizing antibodies induced by vaccination or previous viral exposure. Disease severity during COVID-19 depends on many variables such as age-related comorbidities, host immune status and genetic variation. The host immune response during infection with SARS-CoV-2 may contribute to disease severity, which can range from asymptomatic to severe with fatal outcome. Furthermore, the extent of host immune response activation may rely on underlying genetic predisposition for disease or protection. To address these questions, we performed immune profiling studies in mice with different genetic backgrounds - transgenic K18-hACE2 and wild-type 129S1 mice – subjected to reinfection with the severe disease-causing SARS-CoV-2 B.1.351 variant, 30 days after experimental milder BA.1 infection. BA.1 preinfection conferred protection against B.1.351-induced morbidity in K18-hACE2 mice but aggravated disease in 129S1 mice. We found that he cytokine/chemokine profile in B.1.351 re-infected 129S1mice is similar to that during severe SARS-CoV-2 infection in humans and is characterized by a much higher level of IL-10, IL-1β, IL-18 and IFN-γ , whereas in B.1.351 re-infected K18-hACE2 mice, the cytokine profile echoes the signature of naïve mice undergoing viral infection for the first time. Interestingly, the enhanced pathology observed in 129S1 mice upon reinfection cannot be attributed to a less efficient induction of adaptive immune responses to the initial BA.1 infection, as both K18-hACE2 and 129S1 mice exhibited similar B and T cell responses at 30 DPI against BA.1, with similar anti-BA.1 or B.1.351 spike-specific ELISA binding titers, levels of germinal center B-cells, and SARS-CoV-2-Spike specific tissue-resident T-cells. Long-term effects of BA.1 infection are associated with differential transcriptional changes in bronchoalveolar lavage-derived CD11c+ immune cells from K18-hACE2 and 129S1, with K18-hACE2 CD11c+ cells showing a strong antiviral defense gene expression profile whereas 129S1 CD11c+ cells showed a more pro-inflammatory response. In conclusion, initial infection with BA.1 induces cross-reactive adaptive immune responses in both K18-hACE2 and 129S1 mice, however the different disease outcome of reinfection seems to be driven by differential responses of CD11c+ cells in the alveolar space.

overall_design: To invesitgate bronchoalveolar lavage-derived CD11c+ immune cells from K18-hACE2 and 129S1 in protection against re-infection against SARS-CoV-2 varinats

first: Gagandeep

last: Singh

first: Adolfo

last: Garcia-Sastre

first: Michael

last: Schotsaert


title: BiP/GRP78 is a pro-viral factor for diverse dsDNA viruses that promotes the survival and proliferation of cells upon KSHV infection

status: Public on Oct 22 2024

city: San Francisco

first: Leah

last: Dorman

address: 499 Illinois St

department: Data Science

institute: CZBiohub

summary: The Endoplasmic Reticulum (ER)-resident HSP70 chaperone BiP (HSPA5) plays a crucial role in maintaining and restoring protein folding homeostasis in the ER. BiP’s function is often dysregulated in cancer and virus-infected cells, conferring pro-oncogenic and pro-viral advantages. We explored BiP's functions during infection by the Kaposi's sarcoma-associated herpesvirus (KSHV), an oncogenic gamma-herpesvirus associated with cancers of immunocompromised patients. Our findings reveal that BiP protein levels are upregulated in infected epithelial cells during the lytic phase of KSHV infection. This upregulation occurs independently of the unfolded protein response (UPR), a major signaling pathway that regulates BiP availability. Genetic and pharmacological inhibition of BiP halts KSHV viral replication and reduces the proliferation and survival of KSHV-infected cells. Notably, inhibition of BiP limits the spread of other alpha- and beta-herpesviruses and poxviruses with minimal toxicity for normal cells. Our work suggests that BiP is a potential target for developing broad-spectrum antiviral therapies against double-stranded DNA viruses and a promising candidate for therapeutic intervention in KSHV-related malignancies.

overall_design: Exogenous RTA expression was induced in iSLK.219 cells by treatment with 1 μg/mL of doxycycline (Fisher Scientific, Waltham, MA, USA). To prevent viral DNA replication, these cells were induced with Dox in the presence of phosphonoformate (PFA) 100µM (Sigma Aldrich P6801). Viral reactivation was evaluated by microscopy detection of the PAN-RFP reporter and immunoblot for viral proteins. Total cellular and viral RNA was isolated from iSLK.219 cells at 72h post reactivation in the presence or absence of 10µM HA15 (Selleckchem S8299), using the RNAeasy Plus Mini kit (QIAGEN 74134) following manufacturers' recommendations, including a DNAse treatment step. RNA sequencing libraries were generated using the NEBNext Ultra II RNA Library Prep Kit (New England BioLabs E7760) and sequenced using a 150bp paired-end protocol on an Illumina NextSeq550 instrument. Following demultiplexing, the sequenced reads were aligned to the human genome GRcHg38 version 41 and the KSHV genome GQ994935.1. Read abundance and differential gene expression were determined using the DESeq2 package.

first: Guillermo

last: Najarro

first: Kevin

last: Brackett

first: Hunter

last: Woosley

middle: C

first: Vincent

last: Turon-Lagot

first: Sudip

last: Khadka

first: Catya

last: Faeldonea

first: Osvaldo

last: Moreno

first: Adriana

middle: R

last: Negron

first: Christina

last: Love

first: Ryan

last: Ward

first: Charles

last: Langelier

first: Frank

last: McCarthy

first: Carlos

last: Gonzalez

first: Joshua

last: Elias

first: Brooke

last: Gardner

first: Carolina

last: Arias


title: Identification of pathological pathways centered on circRNA dysregulation associated with irreversible progression of Alzheimer’s disease [circGigyf2KD]

city: Atlanta

first: Bing

last: Yao

address: 615 Michael Street

department: Department of Human Genetics

institute: Emory University

summary: Background Circular RNAs (circRNAs) are highly stable regulatory molecules, often accumulated in the mammalian brain and thought to serve as “memory molecules” that govern the long process of aging. Mounting evidence demonstrated circRNA dysregulation in postmortem cortical regions of Alzheimer’s disease (AD) patients. However, whether and how circRNA dysregulation underlies AD progression remains unexplored. Methods Poly(A)-tailing/RNase R digestion was coupled with our recently published computational algorithm CARP to identify genome-wide dysregulation of circRNAs and their downstream pathways in the 5xFAD mouse cerebral cortex between 5- and 7-month of age, a critical window that marks the transition from reversible to irreversible pathogenic progression. Irreversibly dysregulated circRNAs and pathways in 5xFAD cortex were systematically compared with a large human AD cohort study. A top ranked circRNA conserved and commonly affected in AD patients and 5xFAD mice was depleted in cultured cells to confirm its causal roles in AD-relevant molecular and cellular changes. Results We discovered genome-wide circRNA alterations specifically in the 5xFAD cortex associated with AD progression and found numerous circRNAs also consistently affected in AD patient postmortem cortical areas. Among such circRNAs, circGigyf2 showed the highest net reduction starting from 7-month of age, which is highly conserved and negatively correlated with human AD severity. Mechanistically, we found multiple AD-affected splicing factors that are essential for circGigyf2 biogenesis. Functionally, we identified and experimentally validated the conserved roles of circGigyf2 in sponging AD-relevant miRNAs and AD-associated RNA binding proteins (RBPs) including the cleavage and polyadenylation factor 6 (CPSF6). CircGigyf2 downregulation in AD exert silencing activities of its sponged miRNAs and aberrantly enhanced the polyadenylation efficiency of many CPSF6 targets. These post-transcriptionally tuned mRNAs by circGigyf2 were enriched in apoptotic pathways. Furthermore, circGigyf2 depletion in a mouse immortalized neuronal cell line dysregulated circGigyf2-miRNA and circGigyf2-CPSF6 axes and potentiated neuronal responses to apoptotic insults, which strongly support the causative role of circGigyf2 deficiency in AD disease progression. Conclusions Together, our results unveiled pathological alterations of the brain circRNA landscape associated with irreversible disease stage in an AD mouse model and identified novel molecular mechanisms underlying dysregulation of conserved circRNA pathways that contribute to AD pathogenesis.

overall_design: 7 month 5xFAD mouse hippocampus were used for rRNAdepleted RNA-seq and A-tailing RNaseR treated RNAseq. circGigyf2 is knockdown in CAD cells for rRNA depleted RNAseq.

first: Feng

last: Wang

first: Yangping

last: Li

first: Yue

last: Feng


title: A STAG2-PAXIP1/PAGR1 axis suppresses lung tumorigenesis

first: Jesse

last: Dixon

address: 10010 N. Torrey Pines Rd.

institute: Salk Institute for Biological Studies

summary: The cohesin complex is a critical regulator of gene expression. STAG2 is the most frequently mutated cohesin subunit across several cancer types and is a key tumor suppressor in lung cancer. Here, we coupled somatic CRISPR-Cas9 genome editing and tumor barcoding with an autochthonous oncogenic KRAS-driven lung cancer model and show that STAG2 is uniquely tumor suppressive among all core and auxiliary cohesin components. The heterodimeric complex components PAXIP1 and PAGR1 have highly correlated effects with STAG2 in human lung cancer cell lines, are tumor suppressors in vivo, and are epistatic to STAG2 in oncogenic KRAS-driven lung tumorigenesis in vivo. STAG2 inactivation elicits changes in gene expression, chromatin accessibility, and 3D genome confirmation that impact cancer cell state. Gene expression and chromatin accessibility similarities between STAG2- and PAXIP1-deficient neoplastic cells further relates STAG2-cohesin to PAXIP1/PAGR1. These findings reveal a STAG2-PAXIP1/PAGR1 tumor-suppressive axis and uncover novel PAXIP1-dependent and PAXIP1-independent STAG2-cohesin mediated mechanisms of lung tumor suppression.

overall_design: Using Hi-C experiments to investigate the 3D chromatin architecture of FACS-sorted neoplastic cells from mice with Stag2WT and Stag2Flox background.

first: Dixon

last: Jesse

first: Xu

last: Zhichao

first: Ashkin

last: Emily


title: Exploring Immune Microenvironment of Colorectal Cancer Patients after Renal Transplantation

city: fukukoka

first: Jinghui

last: Zhang

address: 3-1-1 maidashi fukuoka

country: Japan

institute: kyushu university

summary: The present data characterizing the colorectal cancer tumor microenvironment influenced by immunosuppressants.

overall_design: We performed scRNA-seq analysis from 4 colorectal cancers and 3 adjacent normal colon mucosae.  After surgical resection, fresh tissue specimens of tumor lesions and normal colon tissues (>5 cm from the edge of the tumor) were collected and immediately transferred for scRNA-seq.

first: Kyoko

last: Hisano

first: Yusuke

last: Mizuuchi

first: Kenoki

last: Ohuchida


title: Identification of pathological pathways centered on circRNA dysregulation associated with irreversible progression of Alzheimer’s disease [5xFADHippocampus]

type: Non-coding RNA profiling by high throughput sequencing


title: Single-cell gene expression profie of developing photoreceptor cells in larval zebrafish

city: Saint Louis

first: Joseph

middle: C.

last: Corbo

state: MO

address: 660 S. Euclid

department: Pathology and Immunology

institute: Washington University School of Medicine

summary: Molecular underpinnings of vertebrate retinal differentiation and maturation are poorly understood, particularly for non-mammalian species. We generated single-cell transcriptome data from the larval zebrafish retina and characterized gene expression diversity among photoreceptor subtypes and their progenitors.

overall_design: GFP-positive differentiating photoreceptor cells and bipolar cells were collected from 4-dpf larval transgenic zebrafish, Tg(crx:EGFP)stl887, using fluorescence-activated cell sorting.

first: Yohei

last: Ogawa


title: Host-microbiota interaction is regulated by skin autonomous production of antibodies

city: BETHESDA

first: Cihan

last: Oguz

state: MD

address: NIHBC 29B BG RM 1NN20, 9000 ROCKVILLE PIKE

institute: NIH

summary: The microbiota colonizes each barrier site and broadly controls host physiology. However, when uncontrolled, microbial colonists can also promote inflammation and induce systemic infection. The unique strategies employed at each barrier tissue to control the coexistence of the host with its microbiota remain largely elusive. Here we uncover that, within the skin, host-microbiota symbiosis depends on the remarkable ability of the skin to act as an autonomous lymphoid organ. Notably, an encounter with a new skin commensal promotes two parallel responses, both under the control of Langerhans cells. On one hand, skin commensals induce the formation of classical germinal centers within secondary lymphoid organs associated with IgG1 and IgG3 antibody responses. On the other hand, microbial colonization also leads to the development of tertiary lymphoid organs within the skin itself that can locally sustain and mature IgG2b and IgG2c responses. These phenomena are supported, at least in part, by the ability of the skin regulatory T cells to convert into T follicular helper cells that accumulate within organized tertiary lymphoid organs within the dermis. Skin autonomous production of antibodies is sufficient to control local commensal colonization as well as subsequent systemic infection with the same microbe. Collectively, these results reveal a striking compartmentalization of humoral responses to the microbiota. Further, this work uncovers a previously unappreciated function for the skin as a compartment able to develop, in the absence of inflammation, powerful and long-lived antibody responses independently of secondary lymphoid organs.

overall_design: WT CD4 T cells were sorted by FACS from the skin 7 days post topical association (TA) with S. epidermidies and 5' single cell RNA-seq was performed with HTO hashtags.  Single-cell RNA sequencing (scRNAseq) of FACS-sorted CD19+B220+ B cells from Ltα-/- mice at 14 days post-TA

first: Inta

last: Gribonika

first: Verena

last: Link

first: Yasmine

last: Belkaid

first: Eduard

last: Ansaldo

first: Djenet

last: Bousbaine

first: Michael

last: Fischbach

first: Liang

last: Chi

first: Paula

last: Perez-Chaparro

first: Victor

last: Band


title: Derivation of transcriptomics-based points of departure for twenty per- or polyfluoroalkyl substances (PFAS) using a larval fathead minnow (Pimephales promelas) reduced transcriptome assay.

city: Duluth

first: Daniel

middle: L.

last: Villeneuve

state: MN

address: 6201 Congdon Blvd

department: Mid-Continent Ecology Division

institute: US EPA

summary: Traditional toxicity testing has been unable to keep pace with the introduction of new chemicals into commerce. Consequently, there are limited or no toxicity data upon which to base a risk assessment for many chemicals to which fish and wildlife may be exposed. Per- and polyfluoroalkyl substances (PFAS) are emblematic of this issue in that most the ecological hazards of most PFAS remain uncharacterized. The present study employed a high throughput assay to identify the concentration at which 20 PFAS, with diverse properties, elicited a concerted gene expression response in larval fathead minnows (Pimephales promelas, 5-6 days post-fertilization) exposed for 24 h. Based on a reduced transcriptome approach that measured whole body expression of 1832 genes, the median transcriptomic point of departure (tPOD) for the 20 PFAS tested was 10 µM. Longer chain carboxylic acids (12-13 C-F) and an eight C-F di-alcohol, N-alkyl sulfonamide, and telomer sulfonic acid were among the most potent PFAS, eliciting gene expression responses at concentrations below 1 µM. With a few exceptions, larval fathead minnow tPODs were concordant with those based on whole transcriptome response in human cell lines. However, larval fathead minnow tPODs were often greater than those for Daphnia magna exposed to the same PFAS. The tPODs overlapped concentrations at which other sub-lethal effects have been reported in fish (available for 10 PFAS; including a range of species, life stages, and study designs). Nonetheless, fathead minnow tPODs were all orders of magnitude higher than aqueous PFAS concentrations detected in tributaries of the North American Great Lakes suggesting a substantial margin of safety in those systems, even for PFAS with significant potential for bioaccumulation. Overall, results broadly support the use of a fathead minnow larval transcriptomics assay to derive screening level potency estimates for use in ecological risk-based prioritization. Traditional toxicity testing has been unable to keep pace with the introduction of new chemicals into commerce. Consequently, there are limited or no toxicity data upon which to base a risk assessment for many chemicals to which fish and wildlife may be exposed. Per- and polyfluoroalkyl substances (PFAS) are emblematic of this issue in that most the ecological hazards of most PFAS remain uncharacterized. The present study employed a high throughput assay to identify the concentration at which 20 PFAS, with diverse properties, elicited a concerted gene expression response in larval fathead minnows (Pimephales promelas, 5-6 days post-fertilization) exposed for 24 h. Based on a reduced transcriptome approach that measured whole body expression of 1832 genes, the median transcriptomic point of departure (tPOD) for the 20 PFAS tested was 10 µM. Longer chain carboxylic acids (12-13 C-F) and an eight C-F di-alcohol, N-alkyl sulfonamide, and telomer sulfonic acid were among the most potent PFAS, eliciting gene expression responses at concentrations below 1 µM. With a few exceptions, larval fathead minnow tPODs were concordant with those based on whole transcriptome response in human cell lines. However, larval fathead minnow tPODs were often greater than those for Daphnia magna exposed to the same PFAS. The tPODs overlapped concentrations at which other sub-lethal effects have been reported in fish (available for 10 PFAS; including a range of species, life stages, and study designs). Nonetheless, fathead minnow tPODs were all orders of magnitude higher than aqueous PFAS concentrations detected in tributaries of the North American Great Lakes suggesting a substantial margin of safety in those systems, even for PFAS with significant potential for bioaccumulation. Overall, results broadly support the use of a fathead minnow larval transcriptomics assay to derive screening level potency estimates for use in ecological risk-based prioritization.

overall_design: Larval fathead minnows (Pimephales promelas; 5-6 days post-fertilization) were exposed to each of the 20 PFAS, in concentration response, for 24 h in 96-well plate format. For each assay, 30 mM and 9.5 mM stock solutions in DMSO were used to prepare a dilution series spanning from 30 mM to 0.0095 mM (1/2 log spacing between concentrations) in DMSO. Solutions in DMSO were then diluted 300-fold in sand filtered, UV-treated Lake Superior water (LSW) to generate a series of aqueous nominal concentrations ranging from 100 µM to 0.03 µM (1/2 log-spacing between concentrations), each, including control treatments, containing 0.33% DMSO. Larval fathead minnows (five days post fertilization) were transferred to wells of three 96-well (1 ml wells; NEST), one animal per well. The volume (variable from organism loading) in each well of the three plates was reduced to a uniform volume of 50 µl using a Biomek i5 liquid handler (Beckman), and then 550 µl of dosing solution was added to each well, bringing the total volume to 600 µl. On each of the three replicate plates five individual fish were exposed to each concentration of two PFAS test chemicals, eight individuals were exposed to control water, and the remaining eight wells were treated with reference chemicals (150 µg CuSO4/L). In total, 15 fish (distributed across 3 plates) were exposed to each PFAS treatment and 24 fish (distributed across 3 plates) were exposed to control water. Following the addition of the exposure solutions, plates were incubated at 25C for 24 h. At the end of each exposure period, each well was inspected under a dissecting microscope. Mortalities were enumerated wells containing dead animals noted. Any non-lethal phenotypic abnormalities such as edema, spinal malformations, or unusual behaviors were noted. A Biomek i5 liquid handler was used to transfer 100 µl of exposure water from one of the plates (Plate A) to be used for exposure verification (below) to a new 96 well plate. All wells from plates B and C were de-watered and frozen at -80 until homogenized. For homogenization, 75 µl of Biospyder homogenization buffer and a 2.3 mm stainless steel bead added. The fish were then homogenized in 96 well plate format using a bead mixer mill (Omni-Bead Ruptor; 30 Hz for 2 min). Homogenates corresponding to the same well position on each of two replicate plates (both from the same treatment) were pooled together (except in the case of mortality on one or both plates). This resulted in five replicate pools (independent biological replicates) per treatment, each representing the mean of two individuals. For controls there were eight replicate pools, each representing the mean of two individuals, however, the controls were shared for both PFAS tested on the same plate. PFAS concentrations in the exposure medium (collected from plate A) were measured at the end of each 24 h exposure.

first: Kendra

last: Bush

first: Monique

last: Hazemi

first: John

middle: X

last: Hoang

first: Michelle

last: Le

first: Brett

last: Blackwell

first: Emma

last: Stacy

first: Kevin

last: Flynn


title: Increased translation driven by a non-canonical EZH2 cistrome creates a synthetic vulnerability in enzalutamide-resistant prostate cancer (CUT&RUN) II

city: New York

first: Jorge

last: Moscat

state: NY

address: 1188 York Avenue

department: Pathology and Laboratory Medicine

institute: WEILL CORNELL MEDICINE

type: Genome binding/occupancy profiling by high throughput sequencing

summary: Resistance to therapy is a major challenge in castration-resistant prostate cancer (CRPC). Lineage plasticity towards a neuroendocrine phenotype enables CRPC to adapt and survive targeted-therapies. However, the molecular mechanisms of epigenetic reprogramming during this process are still poorly understood. Here we show that the protein kinase PKCl/i phosphorylates the epigenetic regulator enhancer of zeste homologue 2 (EZH2) to regulate its proteasomal degradation and maintain EZH2 as part of the canonical polycomb repressive complex (PRC2). Loss of PKCl/i promotes a switch during enzalutamide treatment to a non-canonical EZH2 cistrome that triggers the transcriptional activation of the translational machinery to induce a transforming growth factor b (TGFb) resistance program. The increased reliance on protein synthesis creates a synthetic vulnerability in PKCl/i-deficient CRPC.

overall_design: RNA-seq of LNCaP sgPRKCI and sgC treated with Enzalutamide

first: Shankha

middle: S

last: Chatterjee

first: Juan

middle: F

last: Linares

first: Tania

last: Cid-Diaz

first: Angeles

last: Duran

first: Mohammad

middle: I

last: Khan

first: Marta

last: Osrodek

first: Nicholas

last: Brady

first: Miguel

last: Reina-Campos

first: Antonio

last: Marzio

first: Varadha

last: Venkadaksrishnan

first: Martin

last: Bakht

first: Francesca

last: Khani

first: Jenna

last: Moyer

first: Olivier

last: Elemento

first: David

last: Rickman

first: Himisha

last: Beltran

first: Maria

middle: T

last: Diaz-Meco


title: Tet3 regulates terminal cell differentiation at the metabolic level

city: Valencia

first: Natalia

last: Tapia

state: València

address: Jaime Roig 11

country: Spain

institute: Spanish National Research Council

type: Methylation profiling by high throughput sequencing

summary: To define TET3 function in cell differentiation, we profiled the intestinal epithelium at the single-cell level from wild-type and Tet3 knockout mice. Our works shows that, in the absence of TET3, enterocytes exhibit an aberrant differentiation trajectory and do not acquire a physiological cell identity due to an impairment in oxidative phosphorylation, specifically due to the reduced expression of genes encoding for ATP synthase membrane domain subunits. To assess whether the downregulation of ATP synthase subunits upon TET3 loss was due to a TET3 catalytic or non-catalytic activity, we examined global DNA methylation patterns in wild-type and Tet3 knockout intestinal epithelium by whole-genome bisulfite sequencing (WGBS). Our results show that these critical ATP synthase subunits coding-genes did not show any hyperDMR in their regulatory regions, excluding TET3 catalytic activity as the mechanism underlying their decreased gene expression and, pointing thus to TET3 non-catalytic activity.

overall_design: Whole genome bisulfite sequencing (WGBS) from E18.5 wild-type and Tet3 knockout intestinal epithelium.


title: Uptake of alpha-synuclein preformed fibrils is suppressed by inflammation and induces an aberrant phenotype in human microglia

city: Kuopio

first: Jonna

middle: Elisa

last: Niskanen

address: Neulaniementie 2

department: A.I.Virtanen Institute of Molecular Medicine

country: Finland

institute: University of Eastern Finland

summary: To detect the microglial mRNA signature whilst phagocytosing alphasynuclein preformed fibrils (αSyn PFFs) with or without inflammatory stimulus, we treated the healthy control microglia with IFNγ (20ng/ml) 28h and αSyn PFFs (0.5µM) 4h

overall_design: Comparative gene expression analysis of RNA-seq data for human iPSC derived microglia treated with IFNγ 28h and αSyn PFFs 4h (n= 5-7 replicates/treatment, 1 male line, 1 female line)

first: Luca

last: Giudice

first: Feroze

last: Fazaludeen


title: Cell polarity proteins promote macropinocytosis in response to metabolic stress

first: Cosimo

last: Commisso

address: 10901 N. Torrey Pines Road

department: Cancer Metabolism and Microenvironment Program

summary: Macropinocytosis has emerged as a nutrient-scavenging pathway that cancer cells exploit to survive the nutrient-deprived conditions of the tumor microenvironment. Cancer cells are especially reliant on glutamine for their survival, and in pancreatic ductal adenocarcinoma (PDAC) cells, glutamine deficiency can enhance the stimulation of macropinocytosis, allowing the cells to escape metabolic stress through the production of extracellular-protein-derived amino acids. Here, we identify the atypical protein kinase C (aPKC) enzymes, PKC and PKCas novel regulators of macropinocytosis. In normal epithelial cells, aPKCs are known to regulate cell polarity in association with the scaffold proteins Par3 and Par6, controlling the function of several targets, including the Par1 kinases. In PDAC cells, we identify that each of these cell polarity proteins are required for glutamine stress-induced macropinocytosis. Mechanistically, we find that the aPKCs are regulated by EGFR signaling or by the transcription factor CREM to promote the relocation of Par3 to microtubules, facilitating macropinocytosis in a dynein-dependent manner. Importantly, we determine that cell fitness impairment caused by aPKC depletion is rescued by the restoration of macropinocytosis and that aPKCs support PDAC growth in vivo. These results identify a previously unappreciated role for cell polarity proteins in the regulation of macropinocytosis and provide a better understanding of the mechanistic underpinnings that control macropinocytic uptake in the context of metabolic stress.

overall_design: To decipher which genes are upregulated when AsPC-1 cells (human pancreatic cancer cell line)  were treated with 6-Diazo-5-oxo-l-norleucine (DON), a glutamine analog that blocks glutamine metabolism

first: Guillem

last: Lambies

first: Szu-Wei

last: Lee

first: Karen

last: Duong-Polk


title: Increased translation driven by a non-canonical EZH2 cistrome creates a synthetic vulnerability in enzalutamide-resistant prostate cancer (scRNA-Seq)

first: Jorge

last: Moscat

first: Shankha

last: Subhra Chatterjee

first: Juan

last: Linares

first: Tania

last: Cid-Diaz

first: Angeles

last: Duran

first: Mohammad

last: Khalil Khan

first: Marta

last: Osrodek

first: Nicholas

last: Brady

first: Miguel

last: Reina-Campos

first: Antonio

last: Marzio


title: Impact of molecular classification on prognosis in children and adolescents with spinal ependymoma: Results from the HIT-MED database

city: Graz

first: Lara

last: Engertsberger

address: Auenbruggerplatz 34/2

department: Division of Pediatric Hemato-Oncology

country: Austria

institute: Medical University of Graz

type:
Third-party reanalysis
Methylation profiling by array

summary: Ependymomas of the spinal cord are rare among children and adolescents, and the individual risk of disease progression is difficult to predict. This study aims at evaluating the prognostic impact of molecular typing of pediatric spinal cord ependymomas. Eighty-three patients with spinal ependymomas ≦ 22 years registered in the HIT-MED database between 1992 and 2022 were included. Forty-seven tumors were analyzed by DNA methylation array profiling. In six cases, HOXB13 and MYCN proteins were detected as surrogate markers for specific methylation classes. With a median follow-up time of 4.9 years (y), 5y- and 10y-overall survival (OS) were 100% and 86%, while 5y- and 10y-progression-free survival (PFS) were 65% and 54%. Myxopapillary ependymoma (SP-MPE, n=32, 63%) was the most common molecular type followed by spinal ependymoma (SP-EPN, n=17, 33%) and MYCN-amplified ependymoma (n=2, 4%). One case could not be molecularly classified, and one was reclassified as anaplastic pilocytic astrocytoma. 5y-PFS did not significantly differ between SP-MPE and SP-EPN (65% versus 78%, p=0.64). MYCN-amplification was associated with early relapses (<2.3y) in both cases and death in one patient. Patients with SP-MPE subtype B (n=9) showed a non-significant trend for better 5y-PFS compared to subtype A (n=18; 86% versus 56%, p=0.15). The extent of resection and WHO tumor grades significantly influenced PFS in a uni- and multivariate analysis.

overall_design: 47 specicmens were analyzed using the Illumina Infinium HumanMethylation450 or the MethylationEPIC BeadChip.

first: Ulrich

last: Schüller


title: Increased translation driven by a non-canonical EZH2 cistrome creates a synthetic vulnerability in enzalutamide-resistant prostate cancer (RNA-Seq organoids)

last: Chatterjee

first: Mohd. Imran K.

last: Khan

first: Varadha Balaji

last: Venkadaksrishnan

first: Martin

last: Bakht

first: Francesca

last: Khani

first: Jenna

last: Moyer

first: Olivier

last: Elemento

first: David

last: Rickman

first: Juan Miguel

last: Mosquera

first: Andrew

last: Hsieh

middle: W

last: Goodrich

first: Brian

middle: D

last: Robinson

first: Himisha

last: Beltran

first: Maria T.

last: Diaz-Meco


title: Increased translation driven by a non-canonical EZH2 cistrome creates a synthetic vulnerability in enzalutamide-resistant prostate cancer (RNA-Seq LNCaP)

first: Jorge

last: Moscat

first: Shankha

first: Juan

last: Linares

first: Tania

last: Cid-Diaz

first: Angeles

last: Duran

first: Marta

last: Osrodek

first: Nicholas

last: Brady

last: Reina-Campos

last: Marzio

middle: K


title: ZEB2 drives intra-tumor heterogeneity and skin squamous cell carcinoma formation with distinct EMP transition states.

city: Zwijnaarde

first: Jordy

last: De Coninck

address: Technologiepark 71

department: Department of Biomedical Molecular Biology

country: Belgium

institute: University Gent

summary: Poor prognosis in cutaneous squamous cell carcinoma (cSCC) stems from poor differentiation, invasion and metastasis, all of which are linked to the process of epithelial-to-mesenchymal plasticity (EMP). Here we showed that the EMP-associated transcription factor ZEB2 drives cSCC heterogeneity which resembles biphasic carcinosarcoma-like tumors. Single cell RNA sequencing revealed different subpopulations ranging from fully epithelial (E) to intermediate (EM) to fully mesenchymal (M) which was associated with the graded loss of cell surface markers EPCAM, CDH1, ITGB4 and CD200. Mesenchymal features were associated with a higher metastatic capacity and anoikis resistance, yet this comes with a sensitivity towards TNF-induced cell death. Altogether we provide insights in cSCC heterogeneity and modes to target mesenchymal- metastasis inducing cells

overall_design: Krt14CreTg/+ -Rosa26-Zeb2Tg/+/Trp53Fl/Fl mice develop skin tumors that show a mixed epithelial-mesenchymal phenotype or fully mesenchymal-like tumors. These mice express Zeb2 as a bicistronic transcript with GFP. We performed single cell RNA sequencing on GFP+DAPI- sorted cells from a mixed epithelial-mesenchymal primary tumor and a fully mesenchymal primary tumor. We also performed single cell RNA sequencing on an early passage cell culture derived from a tumor with a mixed epithelial/mesenchymal phenotype.

first: Jeroen

last: Verstappe

first: Nicolas

last: Skrypek

first: Steven

last: Goossens

first: Niels

last: Vandamme

first: Geert

last: Berx

first: Bram

last: De Craene

first: Bieke

last: Soen


title: Increased translation driven by a non-canonical EZH2 cistrome creates a synthetic vulnerability in enzalutamide-resistant prostate cancer (CUT&RUN)

last: Subhra Chatterjee

first: Mohd. Imran K.

last: Khan

first: Miguel

first: Antonio

first: Varadha Balaji

last: Venkadaksrishnan

first: Martin

last: Bakht

first: Francesca

last: Khani

first: Jenna

last: Moyer

first: Olivier

last: Elemento

first: David

last: Rickman

first: Himisha

last: Beltran

first: Andrew

last: Hsieh

last: Goodrich

first: Juan Miguel

last: Mosquera

first: Brian

last: Robinson

last: Diaz-Meco


title: Increased translation driven by a non-canonical EZH2 cistrome creates a synthetic vulnerability in enzalutamide-resistant prostate cancer

first: Jorge

last: Moscat

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

sample_organism:
Homo sapiens
Mus musculus

platform_organism:
Homo sapiens
Mus musculus

overall_design: Refer to individual Series

first: Shankha

last: Chatterjee

first: Juan

last: Linares

first: Tania

last: Cid-Diaz

first: Angeles

last: Duran

first: Marta

last: Osrodek

first: Nicholas

last: Brady

last: Reina-Campos

last: Marzio

last: Venkadakrishnan

first: Maria T.


title: Identification of pathological pathways centered on circRNA dysregulation initiated at a critical window of Alzheimer’s disease progression

first: Bing

last: Yao

summary: Although circRNA abnormalities are found in Alzheimer’s disease (AD) postmortem brains, whether and how altered circRNA landscape lead to pathological gene network changes during AD progression remain undefined. Employing our recently published experimental and computational approaches, we found genome-wide dysregulated circRNA expression and disrupted functional cooperation of circRNAs in sponging microRNAs (miRNAs) and RNA-binding proteins (RBPs) during early pathogenic progression in a mouse AD model. In addition, we identified AD progression-associated mouse circRNAs that are conserved and affected in AD patients, which underlie malfunction of downstream microRNAs and RBPs in AD brains. An exemplar circRNA is circGigyf2, which progressively declines along with AD pathogenic severity. Furthermore, we identified AD pathological pathways centered on circGigyf2, including upstream RBPs that control circGigyf2 biogenesis and downstream miRNA-mRNA and RBP-mRNA axis regulated by circGigyf2, all affected in AD patients. Our discoveries provide strong evidence suggesting the contribution of circRNA abnormality in early AD pathogenesis.

overall_design: 5 month and 7 month 5xFAD mouse with their littermate controls were subjected for RNA-seq with or without A-tailing/Rnase R treatment

first: Feng

last: Wang

first: Yangping

last: Li

first: Yue

last: Feng


title: Spatial-Mux-seq: Multiplexed spatial mapping of chromatin features, transcriptome, and proteins at tissue scale

city: Philadelphia

first: Liran

last: Mao

state: PA

address: 422 Curie Boulevard

department: Perelman School of Medicine

institute: University of Pennsylvania

summary: we present a multi-modal spatial technology that allows simultaneous profiling of four different modalities, including multi epigenomic modalities (any combination of two histone modifications and open chromatin at the same time), whole transcriptome, and a panel of proteins at tissue scale and cellular level in a spatially resolved manner.

overall_design: Spatially resolved multiple modalities sequencing in mouse tissues.

first: Yanxiang

last: Deng

first: Pengfei

last: Guo


title: An integrated neuromuscular training intervention applied in primary school induces epigenetic modifications in disease-related genes: A genome-wide DNA methylation study

status: Public on Oct 21 2024

city: Girona

first: Anna

last: Prats-Puig

state: Girona

address: Carrer del President Francesc Macià, 65, 17190 Salt, Girona

institute: University School of Health and Sport

summary: Physical exercise has been shown to induce epigenetic modifications with various health implications, and directly affect DNA methylation. The objectives of the present work were: 1) to identify differently methylated genes (DMGs) in saliva of school-aged children following a 3-month integrated neuromuscular training (INT); and 2) to explore if any of these genes are considered core-genes, i.e., genes of essential importance within the human genome. An additional goal was to locate the methylation changes in those genes in relation to the transcription start site (TSS) and the CpG site. Forty children (17 boys and 23 girls) were recruited from schools in Girona, Spain and allocated into control (N=20) or INT (N=20) group. Genome-wide DNA methylation analysis was performed with the Illumina 900K microarray. Core-genes were identified based on their participation in the enriched pathways, high connectivity (≥10) and as target-genes of key-transcription factors, using the bioinformatics software Metascape and Cytoscape, and the ENCODE dataset. There were 1200 DMGs in the control group and 414 DMGs in the INT group (p<0.05). After screening among the DMGs, no core-genes were identified in the control group, but 17 core-genes were identified in the INT group. Remarkably, the 3-month-INT epigenetically modified 17 genes that are of essential importance within the human genome, related to inflammation, fat metabolism, protein metabolism, ATP binding and growth, as well as to metabolic, cardiovascular, neurodegenerative diseases, musculoskeletal disorders, cancer and various syndromes (e.g., Noonan syndrome, Perry syndrome). Moreover, 88% of the methylation changes were close to the TSS, and 59% were in CpG islands. A 3-month-INT induced substantial epigenetic modifications with potential implications for disease prevention and health promotion in school-aged children.

overall_design: We aimed to evaluate DNA methylation changes after a 3-month intergrated neuromuscular training intrevention applied in primary school. To identify differently methylated genes we used the Infinum Methylation Epic Array in a cohort of 40 children (20 controls and 20 intervention). The children from the control group had traditional physical education classes, whereas the children form the intervention group performed the integrated neuromuscular training as a warm-up activity during the physical education classes.The integrated neuromuscular training consisted of 24 sessions of progressively structured exercises organized in circuits and games, focusing on strength, coordination, dynamic stabilization, plyometrics, speed and agility.

middle: P

last: Puig


title: Transcriptomic Landscape of Hyperthyroidism in Mice Overexpressing Thyroid Stimulating Hormone

city: Kyoto

first: Ichiro

last: Yamauchi

address: 54 Kawaharacho, Shogoin, Sakyo-ku

department: Department of Diabetes, Endocrinology and Nutrition

institute: Kyoto University Graduate School of Medicine

summary: Activation of thyroid stimulating hormone receptor (TSHR) fundamentally leads to hyperthyroidism. To elucidate TSHR signaling, we conducted transcriptome analyses for hyperthyroid mice that we generated by overexpressing TSH. TSH overexpression drastically changed their thyroid transcriptome. In particular, enrichment analyses identified the cell cycle, phosphatidylinositol-3 kinase/Akt pathway, and Ras-related protein 1 pathway as possibly associated with goiter development. Regarding hyperthyroidism, Slc26a4 was exclusively upregulated with TSH overexpression among genes crucial to thyroid hormone secretion. To verify its significance, we overexpressed TSH in Slc26a4 knockout mice. TSH overexpression caused hyperthyroidism in Slc26a4 knockout mice, equivalent to that in control mice. Thus, we did not observe significant changes in known genes and pathways involved in thyroid hormone secretion with TSH overexpression. Our datasets might include candidate genes that have not yet been identified as regulators of thyroid function. Our transcriptome datasets regarding hyperthyroidism can contribute to future research on TSHR signaling.

overall_design:
Our hyperthyroid mice exhibited excessive secretion of thyroid hormones and goiter development through the effects of TSH overexpression. Using this model, we subsequently obtained data on the thyroid transcriptome using RNA-seq.
Empty = mice injected with pLIVE-Empty vector
TSH = mice overexpressing TSH by injecting with pLIVE-TSHB and pLIVE-CGA vectors
TSH-MMI = mice overexpressing TSH that were treated with thiamazole (MMI)
Empty-MMI = mice injected with pLIVE-Empty vector that were treated with MMI
more details in bioRxiv 2023.10.27.564354.


title: Effect of myricetin treatment on Roseovarius tolerans EL-164 during exponential and early stationary phase growth.

city: Princeton

first: Jack

last: Ganley

state: NJ

address: Frick Chemistry Laboratory, 333, Washington Road

institute: Princeton University

summary: Discovery of cryptic natural products can be expedited by the use of high throughput elicitor screening (HiTES). In this study, we discover that catechol-containing flavonoids elicit the production of a new group of ceramide lipids in the marine bacterium Roseovarius tolerans EL-164. To investigate the mechanism of elicitation of myricetin, a model catechol flavonoid, we used RNA-seq to determine the molecular response. The transcriptomic dataset illustrated that iron-starvation genes, as well as the import and catabolism of carbon sources, specifically branched chain amino acids, were upregulated upon myricetin treatment. Simultaneously, complex IV of the electron transport chain was universally downregulated as were oxidative stress responses. Together, along with further follow up studies, we were able to support the model that myricetin reduces the bioavailability of iron and reduced oxidative phosphorylation and the proton motive force. Our data suggests that the hampering of the proton motive force is what initiates the upregulation of the ceramide lipids.

overall_design: R. tolerans starter cultures were used to inoculate 25 mL of ½ YTSS liquid media in 125 mL flasks at a starting OD600 of 0.01 containing either 12.5 or 0 µM myricetin (1% DMSO for all) and grown at 30 ºC and 40 RPM. Growth curves were monitored by OD600 and 3 mL samples were collected in Rnase-free centrifuge tubes when cultures reached an OD600 of ~0.3 and ~0.75. This was completed in biological triplicate. The samples were centrifuged, and the pellets were resuspended in 1 mL of RNAlaterTM stabilization solution (Thermo) and stored at 4 ºC for 24 hours. To isolate total RNA for RNA-seq, the samples were centrifuged, the RNAlaterTM solution was discarded, and total RNA was isolated using the Rneasy kit (QIAGEN). Any contaminating genomic DNA was removed by treating the total RNA with Dnase (TURBO Dnase Kit), per manufacturer’s instructions. RNA integrity was confirmed through gel electrophoresis

middle: G

first: Mohammad

last: Seyedsayamdost


title: DNA methylation constrains nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos

city: Basel

first: Antoine

last: Peters

address: Maulbeerstrasse 66

country: Switzerland

institute: Friedrich Miescher Institute


title: MatP local enrichment delays segregation independently of tetramer formation and septal anchoring in Vibrio cholerae [3C]

city: Gif sur Yvette

first: Elena

last: ESPINOSA

address: 1, Avenue de la Terrasse

country: France

institute: CNRS

summary: Vibrio cholerae harbours a primary chromosome derived from the monochromosomal ancestor of the Vibrionales (ChrI) and a secondary chromosome derived from a megaplasmid (ChrII). Both carry a single origin of replication with replication terminating in a diametrically opposite zone, TerI and TerII, respectively. The choreography of TerI and TerII segregation determines when and where cell division occurs. Using a combination of deep sequencing and fluorescence microscopy techniques, we show that TerI and TerII both carry a very high density of MatP binding sites, are both structured into macrodomains, and are both targeted to mid-cell during replication by MatP, demonstrating that ChrII behaves as a bona fide chromosome. We further show that MatP-mediated cohesion and cellular positioning are independent of cell division and tetramer formation, but are linked to the local density of matS sites, in apparent contradiction to the mechanism of action of MatP in Escherichia coli.

overall_design: 3C experiments were made to decipher the effect of matp deletion in Vibrio cholerae chormosome conformation

last: Espinosa

first: Jihane

last: Challita

first: Jean-Michel

last: Desfontaines

first: Christophe

last: Possoz

first: Marie-Eve

last: Val

first: Didier

last: Mazel

first: Martial

last: Marbouty

first: Romain

last: Kozul

first: Elisa

last: Galli

first: Francois-Xavier

last: Barre


title: Cell type mapping reveals tissue niches and interactions in subcortical multiple sclerosis lesions [spatial transcriptomics]

city: Mannheim

first: Lucas

last: Schirmer

address: Theodor-Kutzer-Ufer 1-3

country: Germany

institute: University of Heidelberg

summary: We generated a paired snRNA-seq  (n= 15) and spatial transcriptomics (n=19) dataset from subcortical chronic active and chronic inactive MS lesions, identifying spatial niches and key cell interactions driving inflammation and disease progression at the lesion rim. This repository offers access to all the trancriptomics data that was used in the paper. It includes, all FASTQ files for both transcriptomics, along with the necessary files for running spatial transcriptomic samples (H&E images and JSON files), as well as the curated atlas, all derived cell subtype atlases from the main atlas and all curated ST slides.

overall_design: Paired single-nuclei RNA-sequencing and spatial transcriptomics data from multiple sclerosis (MS) subcortical white matter lesions from 7 controls and 12 MS human donors.

first: Celia

last: Lerma-Martin

first: Pau

last: Badia-i-Mompel

first: Julio

last: Saez-Rodriguez


title: MatP local enrichment delays segregation independently of tetramer formation and septal anchoring in Vibrio cholerae [HiSC2]

overall_design: HiSC2 was used to monitor the frequency of sister chromosomes contacts after replication (Cre-loxP) or before cell division (XerCD-dif) in wt and matP mutants in V. cholerae


title: Cell type mapping reveals tissue niches and interactions in subcortical multiple sclerosis lesions.


title: Concerted neuron-specific splicing events restrict nucleosome engagement of the LSD1 histone demethylase complex

city: Ann Arbor

first: Shigeki

last: Iwase

state: Michigan

address: 1241 E. Catherine St.

department: Human Genetics

institute: University of Michigan

summary: How cell-type-specific chromatin landscapes emerge and progress during metazoan ontogenesis remains an important question. Transcription factors are expressed in a cell type-specific manner and recruit chromatin-regulatory machinery to specific genomic loci. In contrast, most chromatin-regulatory proteins are expressed broadly and are assumed to exert the same intrinsic function across cell types. However, human genetics studies have revealed an unexpected vulnerability of neurodevelopment to chromatin factor mutations with unknown mechanisms. Here, we report that 13 chromatin regulators undergo evolutionary-conserved neuron-specific splicing events involving microexons. Two of these 13 factors are integral components of a histone H3K4 demethylase complex; the catalytic subunit LSD1 and an H3K4me0-reader protein PHF21A. We found that canonical PHF21A (PHF21A-c) binds to DNA by AT-hook motif, and the neuronal counterpart PHF21A-n lacks this DNA-binding function. PHF21A-n showed significantly weaker affinity to the histone H3 tail compared to PHF21A-c. Furthermore, neuronal LSD1 splicing led to reduced affinity of LSD1 to the nucleosome. In-vitro reconstitution of the canonical and neuronal PHF21A-LSD1 complexes, combined with in-vivo methylation mapping, identified the neuronal complex as a hypomorphic H3K4 demethylating machinery with reduced nucleosome engagement. The neuronal PHF21A, albeit its weaker nucleosome binding, is necessary for normal gene expression and H3K4 landscape in developing neurons. Thus, ubiquitously expressed chromatin regulatory complexes can exert neuron-specific functions via alternative splicing of their subunits.

overall_design: CUT&RUN was performed using H3K4me2, H3K9me2, H4K20me2 and IgG antibodies on mouse embryonic day 16.5 cortex. 2 biological replicates per genotype (both wild-type and Phf21a homozygous knockout). RNA-seq was performed using RNAs from E16 cortices, 3 embryos per genotype (WT or Phf21a-/-, the sex was balanced between genotypes),

first: Robert

last: Porter

first: Sojin

last: An

first: Maria

last: Gavilan

first: Masayoshi

last: Nagai

first: Yumie

last: Murata-Nakamura

first: Bo

last: Zhou

first: Katherine

middle: M

last: Bonefas

first: Olivier

last: Dionne

first: Jeru

last: Manuel

first: Joannie

last: St-Germain

first: Suzanne

last: Gascon

first: Jacqueline

last: Kim

first: Liam

last: Browning

first: Benoit

last: Laurent

first: Uhn-Soo

last: Cho


title: HIRA protects telomeres against R-loop-induced instability in ALT cancer cells [ATAC-seq]

city: Pittsburgh

first: Sarah

last: Hainer

address: 4249 Fifth Ave

department: Biological Sciences

institute: University of Pittsburgh

summary: Alterations to chromatin modifiers, such as the inactivating mutations in the ATRX-DAXX chromatin remodeling/histone H3.3 deposition complex, are implicated as drivers of the cancer-specific Alternative Lengthening of Telomeres (ALT) pathway. Prior studies revealed that HIRA adapts to compensate for ATRX-DAXX loss to sustain ALT cancer cell survival. However, the specific mechanisms underlying HIRA’s ability to rescue telomeres from the consequences of ATRX-DAXX loss remain unclear. Here, using ATAC-seq and CUT&RUN, we demonstrate that HIRA-mediated deposition of new H3.3 is essential to maintain chromatin accessibility and to prevent the detrimental accumulation of nucleosome-free single-stranded DNA (ssDNA) at telomeres in ATRX-deficient ALT cancer cells. We provide evidence that the timely deposition of new H3.3 by HIRA and interacting partners UBN1 and UBN2 is crucial to prevent unwarranted TERRA R-loop formation and transcription-replication conflicts (TRCs) at telomeres. Furthermore, we determined that the delivery of H3.3 to telomeric chromatin by HIRA may link the phosphorylation of an H3.3-specific amino acid, serine 31, by Chk1 with mechanisms that promote productive ALT. Therefore, these studies identify a role for HIRA-mediated histone H3.3 deposition in TERRA R-loop homeostasis that we propose is essential for ensuring the survival of ALT cancer cells where the ATRX-DAXX complex is activated.

overall_design: ATAC-seq samples from human IMR90 WT (CTRL) or ATRX KO (ALT) cells treated with HIRA siRNA or control siRNA treatment. Sets of samples in biological triplicate (n=3).

first: Michelle

middle: L

last: Lynskey

first: Emily

middle: E

last: Brown

first: Santana

last: Lardo

middle: J

first: Roderick

last: O'Sullivan


title: Cell type mapping reveals tissue niches and interactions in subcortical multiple sclerosis lesions [snRNA-seq]

first: Lucas

last: Schirmer

first: Celia

last: Lerma-Martin

first: Pau

last: Badia-i-Mompel

first: Julio

last: Saez-Rodriguez


title: HIRA protects telomeres against R-loop-induced instability in ALT cancer cells [CUT&RUN]

overall_design: CUT&RUN samples from human IMR90 WT (CTRL) or ATRX KO (ALT) cells treated with HIRA siRNA or control siRNA treatment. Sets of samples in biological triplicate (n=3).


title: Gut-Lung Microbiota Dynamics in Mice Exposed to Nanoplastics

city: Florence

first: Sara

last: Bertorello

address: Viale Gaetano Pieraccini 6

country: Italy

institute: University of Florence

summary: The objective was to evaluate the impact of nanoplastics on various niches of the microbiome in a mouse model

overall_design: We conducted a sub-chronic exposure study involving 12 male mice, divided into two groups: a control group (n=6) and a PET-NPs exposure group (n=6). PET-NPs, administered by oral gavage at a dose of 0.5 mg/day in a fixed volume of 0.1 ml/mice, were given daily for 28 days. Microbiota analyses were performed on lung, colon, oral cavity, and stool samples using 16S rRNA sequencing. Additionally, fecal short and medium-chain fatty acids were analysed by GC/MS.

first: Nur

last: Kaluç

first: Oğuz

last: Tombul

first: Giulia

last: Nannini

first: Simone

last: Baldi

first: Gianluca

last: Bartolucci

first: Elena

last: Niccolai

first: Amedeo

last: Amedei


title: Single-Cell Transcriptomics Reveals the Molecular Basis of Human iPS Cell Differentiation into Ectodermal Ocular Lineages

city: Cardiff

first: Laura

last: Howard

address: Maindy Road

country: United Kingdom

institute: Cardiff University

summary: The generation of a self-formed, ectodermal, autonomous multi-zone (SEAM) from human induced pluripotent stem cells (hiPSCs) offers a unique perspective to study the dynamics of ocular cell differentiation over time. Here, by utilising single-cell transcriptomics, we have (i) identified, (ii) molecularly characterised and (iii) ascertained the developmental trajectories of ectodermally-derived ocular cell populations which emerge within SEAMs as they form. Our analysis reveals interdependency between tissues of the early eye and delineates the sequential formation and maturation of distinct cell types over a 12-week period. We demonstrate a progression from pluripotency through to tissue specification and differentiation which encompasses both surface ectodermal and neuroectodermal ocular lineages and the generation of iPSC-derived components of the developing cornea, conjunctiva, lens, and retina.  Our findings not only advance the understanding of ocular development in a stem cell-based system of human origin, but also establish a robust methodological paradigm for exploring cellular and molecular dynamics during SEAM formation at single-cell resolution and highlight the potential of hiPSC-derived systems as powerful platforms for modelling human eye development and disease.

overall_design: Human iPSC cells (clone 201B7, RIKEN BioResource Center, Tsukuba, Japan) were maintained on LN511E8 (Nippi, Tokyo, Japan) coated culture dishes in serum-free StemFit medium (Ajinomoto, Tokyo, Japan) for at least 2 cell passages for stabilisation. Differentiation was initiated by culture in differentiation medium (DM) following established protocols. After culture for 4 weeks, DM was substituted for corneal DM (CDM) containing growth factors (KGF, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and the ROCK inhibitor Y-27623 (FUJIFILM Wako Pure Chemical Corporation) to promote epithelial cell growth and survival.  After an additional 4 weeks of CDM culture (8 weeks in total), the medium was changed to corneal epithelial maintenance medium (CEM) consisting of DMEM/F12 (1:1; Life Technologies, Carlsbad, CA, USA) containing KGF, Y-27623 and B-27 supplement (Life Technologies). Cells were harvested at successive timepoints spanning 0 – 12 weeks and single cell suspensions were prepared according to current 10x Genomics guidelines.

first: Yuki

last: Ishikawa

first: Tomohiko

last: Katayama

first: Sung-Joon

last: Park

first: Matthew

last: Hill

first: Derek

last: Blake

first: Kohji

last: Nishida

first: Ryuhei

last: Hayashi

first: Andrew

last: Quantock


title: Xenogeneic-free culture of human intestinal stem cells on functional polymer-coated substrates for scalable, clinical-grade stem cell therapy

city: Daejeon

first: Sung Gap

last: Im

country: South Korea

institute: Korea Advanced Institute of Science and Technology

summary: Inevitable need for basement membrane extract (BME) with undefined constituents, such as Matrigel, for intestinal stem cell (ISC) culture in traditional system poses a significant barrier that must be overcome for the development of clinical-grade, scalable, ready-to-use ISCs. Here, we propose a functional polymer-based xenogeneic-free dish for the culture of intestinal stem cells (XF-DISC), ensuring substantially prolonged maintenance of ISCs derived from 3-dimensional human intestinal organoid (ISCs3D-hIO). XF-DISC enables remarkable expandability, exhibiting a 24-fold amplification in cell number within 30 days, with long-term maintenance of ISCs3D-hIO through consecutive passaging more than 30 times (> 210 days), and is fully compatible with a cell banking system. Notably, the ISCs3D-hIO cultured on XF-DISC are successfully transplanted into the intestinal injury and inflammation models, leading to full regeneration of damaged intestinal epithelium. XF-DISC, an extraordinary reliable and scalable xenogeneic-free ISC3D-hIO culture method exhibits significant promise in the development of regenerative ISC therapy for human intestinal diseases.

overall_design: Bulk RNA-sequencing of human intestinal stem cells (ISCs) on the Matrigel-coated dishes or XF-DISC dishes

first: Sung-Gap

first: Seonghyeon


title: Charting the transcriptomic landscape of primary and metastatic cancers in relation to their origin and target normal tissues

city: Rome

first: Stefano

last: Scalera

address: Via Elio Chianesi 53

institute: IRCCS Regina Elena National Cancer Institute

summary: Most cancer deaths are caused by metastasis but understanding how metastatic cancers adapt from their origin tissues to their target tissues remains a fundamental challenge. Here, we ask if the gene expression of primary and metastatic tumors is more similar to their tissue of origin or to their target tissue. We analyzed the gene expression profiles of both paired and unpaired primary and metastatic tumor patient cohorts across multiple cancer types, studying single-cell and bulk RNA-seq data. To quantify the similarities between tumor samples and their normal, noncancerous origin and targeted tissues, we computed the Euclidean distance between their expression profiles, termed their transcriptomic distances, at the whole-genome and pathway levels. While the expression of most primary tumors is more similar to origin than to their target tissues, the expression patterns of metastases shift towards their target tissue. In specific cases, we chart the alterations occurring at the pathway level, pointing to key adaptive transcriptomic changes. Notably, our analysis indicates that primary cancers across several cancer types are primed for metastasis before the process begins. We further reproduce our findings using a new, unpublished RNA-seq data from colorectal cancer patients with liver metastases This first of its kind systematic analysis of the expression landscape of primary tumors and metastases with respect to their normal origin and target tissues provides a transcriptomic-wide view of cancer tumors adaptations to their metastatic niches

overall_design: RNA profiles of primary and metastatic cancer were generated by deep sequencing using Illumina. We analyzed three biological replicates for each conditions. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA) and TopHat followed by Cufflinks. RT–qPCR validation was performed using SYBR Green assays

first: Nishant

last: Nair

first: Neel

last: Sanghvi

first: Eytan

last: Ruppin

first: Giovanni

last: Blandino


title: Elderly lung mucosa impact on Mycobacterium tuberculosis adaptation during infection of alveolar epithelial cells

city: San Antonio

first: Anna

last: Allue Guardia

state: Texas

address: 8715 Military Dr W

institute: Texas Biomedical Research Institute

summary: Tuberculosis (TB) is one of the leading causes of death due to a single infectious agent and is considered a global threat killing over 4,300 people every day. Upon infection, Mycobacterium tuberculosis (M.tb) deposits in the alveoli and encounters the lung mucosa or alveolar lining fluid (ALF), whose primary function is to keep the lung’s health intact, dictating host-cell interactions. Age-associated changes to innate soluble components in the ALF lead to increased susceptibility of the elderly population to respiratory infectious diseases such as TB. We previously determined that increased M.tb replication in human macrophages and alveolar epithelial cells (ATs) is mediated by age-associated changes in human ALF. In this study, we determine the transcriptional profile of M.tb when exposed to healthy human ALF from adult (A-ALF) or elderly (E-ALF) individuals prior and during intracellular replication in ATs, using A549 cells as a model. Prior to infection, exposure of M.tb to E-ALF upregulates several genes associated with the ESX-4 secretion system as well as immunomodulatory proteins linked to the ESX-5 secretion system. It additionally increases the expression of genes related to phospholipases, phosphatases, and proteases, which are important virulence factors in the establishment of M.tb infection. Importantly, we present the first transcriptomic analysis of the impact of the elderly lung mucosa on M.tb pathogenesis during intracellular replication in ATs. Interestingly, exposure to E-ALF altered expression of key genes related to the ESX-5 secretion system during infection. Additionally, compared to the A-ALF group, there was enhanced expression of genes associated with PDIMs biosynthesis and translocation to the cell surface. Lastly, E-ALF-exposed M.tb exhibits upregulation of genes linked to ROS defense mechanisms during ATs infection. Overall, these findings demonstrate how altered ALF functions in old age can impact the M.tb metabolic status, enabling greater adaptation and increased survival in the host cells.

overall_design: RNAseq data of M.tb Erdman exposed to human alveolar lining fluid (ALF) from adult or elderly individuals before infection and during ATs infection at 72hpi, 3 replicates each (except E-ALF-exposed M.tb with 2 replicates). Control sample is unexposed bacteria prior to infection. Total: 14 samples

first: Angélica

last: Olmo-Fontánez

last: Allué-Guardia

first: Jeremy

last: Glenn

first: Shu-Hua

last: Wang

first: Robert

last: Merrit

first: Yufeng

first: Joanne

last: Turner

first: Larry

last: Schlesinger

first: Jordi

middle: B

last: Torrelles


title: RNAseq of SMAD2/3DKO and Smad4KO in pluripotent mESC and EBs

city: Shanghai

first: yanhua

last: du

address: 227 South Chongqing Road, Huangpu District

department: Shanghai Institute of Immunology

country: China

institute: Shanghai Jiaotong University School of Medicine

summary: To dissected the roles of SMAD2/3 and SMAD4 in mouse embryonic stem cells (mESC),  knockouts of SMAD2/3 (SMAD2 and SMAD3 double knockout, S2/3DKO) and SMAD4 (SMAD4 knockout, S4KO) were used to conduct RNAseq in ES and EB conditions.

overall_design: WT, S2/3DKO and S4KO cells were collected at ES (D0) and  embryoid body (EB) Day 4 conditions. Each sample were duplicated for RNAseq analysis.

first: Bingnan

last: Zhao

first: Jintong

last: Shi

first: Yanhua

last: Du

first: Qiong


title: The binding sites of SMAD2/3, Dnmt3a and Dnmt3b  in pluripotent mESC, EBs and ME when Smad4 is lost with or without Dnmt3b knockdown

summary: To understand the genome wide binding profile of SMAD2/3 when SMAD4 is lost, Smad2/3 ChIPseq was performed in SMAD4 knockout (S4KO) mouse embryonic stem cells (mESC) or in S4KO embryonic bodies (EBs). To figure out the effect of Dnmt3b on SMAD2/3 binding ability in the absence of SMAD4, we further performed SMAD2/3 CUT&Tag in EBs of S4KO and S4KO with Dnmt3b konckdown, as well as in mesendoderm (ME) stage of WT and S4KO. The binding sites of Dnmt3a and Dnmt3b were explored by Dnmt3a or Dnmt3b ChIP-seq and Dnmt3b CUT&Tag in WT EBs and S4KO EBs. WT with Dnmt3b knockdown and S4KO with Dnmt3b knockdown were used to as a control for the Dnmt3b CUT&Tag. Besides, we inserted a 3xFlag-tag at the C-terminus of Dnmt3a or Dnmt3b and then carried out Flag CUT&Tag in EBs.

overall_design: For Smad2/3 ChIP-seq, S4KO cells were collected at ES (D0),  embryoid body (EB) Day 3.5 treated with Activin (AC) or with SB505124(SB). For SMAD2/3 CUT&Tag, S4KO cells and S4KO with Dnmt3b-shRNA knockdown cells were harvested at EB Day 3.5 treated with AC. WT and S4KO were collected after mesendoderm (ME) induction for SMAD2/3 CUT&Tag. WT and S4KO cells on EB Day 3.5 after AC treatment were used for Dnmt3a or Dnmt3b ChIP-seq. Dnmt3b and Flag CUT&Tag were carried out in D3.5 EBs of WT, WT with Dnmt3b-shRNA knockdown, S4KO, S4KO with Dnmt3b-shRNA knockdown cells or Flag-tagged cells, respectively.


title: DNA methylation constrains nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos [ChIP-seq 2]

first: Antoine

last: Peters

summary: DNA methylation serves a stable gene regulatory function in mature somatic cells. In the germ line and during early embryogenesis, however, DNA methylation undergoes global erasure and re-establishment to support germ cell and embryonic development. While de novo DNA methylation during male germ cell development is essential for setting genomic imprints, possible other intergenerational roles for paternal DNA methylation following fertilization are unknown. To address this question, we reduced the level of DNA methylation in developing male germ cells through conditional gene deletion of the de novo DNA methyltransferases DNMT3A and DNMT3B in undifferentiated spermatogonia. Mutant male germ cells nevertheless completed their differentiation to sperm. We observed that DNMT3A serves a largely maintenance-like methylation function at many intragenic sites in undifferentiated spermatogonia while DNMT3B catalyzes de novo methylation during spermatogonial differentiation. In spermatogonia, the acquisition of DNA methylation and deposition of H3K4me3 occur mutually exclusive. Failing de novo DNA methylation in spermatogonia leads to increased nucleosome occupancy in mature sperm at sites with high CpG content, reinforcing the model that DNA methylation constrains nucleosome retention in sperm. To assess the impact of altered sperm chromatin in the formation of embryonic chromatin, we measured H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a highly sensitive transposon-based tagging assay for modified chromatin. Our data show that reduced DNA methylation in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently from paternal inheritance of sperm born H3K4me3. Together, this study provides first evidence that paternally inherited DNA methylation directs chromatin formation during early embryonic development.

overall_design: To assess the role of these proteins during adult spermatogenesis, we generated conditional deletion models in which excision of floxed alleles of Dnmt3a and Dnmt3b was driven by the improved iCre recombinase transgene under the control of the Stra8 promoter, which is active in postnatal undifferentiated and differentiating spermatogonia. We investigated the chromatin status of control and mutant undifferentiated spermatogonia, differentiated spermatogonia by ChIP sequencing.

first: Grigorios

last: Fanourgakis


title: MatP local enrichment delays segregation independently of tetramer formation and septal anchoring in Vibrio cholerae

first: Elena

last: ESPINOSA

overall_design: ChIP-seq was used to decipher MatP binding sites in Vibrio cholerae

last: Espinosa

first: Jihane

last: Challita

first: Jean-Michel

last: Desfontaines

first: Christophe

last: Possoz

first: Marie-Eve

last: Val

first: Didier

last: Mazel

first: Martial

last: Marbouty

first: Romain

last: Kozul

first: Elisa

last: Galli

first: Francois-Xavier

last: Barre


title: The Fork Protection Complex generates DNA topological stress induced DNA damage while ensuring full and faithful genome duplication

city: Brighton

first: Andrea

last: Keszthelyi

address: University of Sussex, Sussex House, Falmer

department: Genome Damage and Stability Centre

institute: University of Sussex

summary: This SuperSeries is composed of the SubSeries listed below (GSE239965, GSE239966).

overall_design: Chromatin immunoprecipitation of H2AS129P or Rfa1, followed by next generation sequencing (ChIP-SEQ) in Saccharomyces cerevisiae (W303), in wt and tof1∆, mrc1∆, tof1997, tof1627, ars501∆ and tof1∆ars501∆ cells, in exponential or M phase (GSE239965).  Transferase-Activated End Ligation sequencing (TrAEL-seq) in Saccharomyces cerevisiae (W303) in exponential phase wt∆, and tof1∆ cells (GSE239966).

first: Sahar

last: Mansoubi

first: Alex

last: Whale

first: Jonathan

last: Houseley

last: Baxter


title: DNA methylation constrains nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos [RRBS]

last: Peters

overall_design: To assess the role of these proteins during adult spermatogenesis, we generated conditional deletion models in which excision of floxed alleles of Dnmt3a and Dnmt3b was driven by the improved iCre recombinase transgene under the control of the Stra8 promoter, which is active in postnatal undifferentiated and differentiating spermatogonia. We investigated the methylation status of control and mutant undifferentiated spermatogonia, differentiated spermatogonia and sperm by Reduced Representation Bisulphite Sequencing (RRBS).

last: Fanourgakis

first: Laura

last: Toneu


title: DNMT3b mediates TGFβ-Smad2/3 action to maintain Epiblast homeostasis upon Smad4 Loss

first: yanhua

last: du

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

overall_design: Refer to individual Series


title: Increased translation driven by a non-canonical EZH2 cistrome creates a synthetic vulnerability in enzalutamide-resistant prostate cancer (RNA-Seq polysome)

city: New York

first: Jorge

last: Moscat

address: 1188 York Avenue

institute: WEILL CORNELL MEDICINE

summary: Resistance to therapy is a major challenge in castration-resistant prostate cancer (CRPC). Lineage plasticity towards a neuroendocrine phenotype enables CRPC to adapt and survive targeted-therapies. However, the molecular mechanisms of epigenetic reprogramming during this process are still poorly understood. Here we show that the protein kinase PKCl/i phosphorylates the epigenetic regulator enhancer of zeste homologue 2 (EZH2) to regulate its proteasomal degradation and maintain EZH2 as part of the canonical polycomb repressive complex (PRC2). Loss of PKCl/i promotes a switch during enzalutamide treatment to a non-canonical EZH2 cistrome that triggers the transcriptional activation of the translational machinery to induce a transforming growth factor b (TGFb) resistance program. The increased reliance on protein synthesis creates a synthetic vulnerability in PKCl/i-deficient CRPC.

overall_design: RNA-seq of LNCaP sgPRKCI and sgC treated with Enzalutamide

first: Shankha

last: Chatterjee

first: Juan

last: Linares

first: Tania

last: Cid-Diaz

first: Angeles

last: Duran

first: Mohd. Imran K.

last: Khan

first: Marta

last: Osrodek

first: Nicholas

last: Brady

first: Miguel

last: Reina-Campos

first: Antonio

last: Marzio

first: Varadha Balaji

last: Venkadaksrishnan

first: Martin

last: Bakht

first: Francesca

last: Khani

first: Juan Miguel

last: Mosquera

first: Brian

last: Robinson

first: Jenna

last: Moyer

first: Olivier

last: Elemento

first: Andrew

middle: C

last: Hsieh

first: David

last: Goodrich

last: Rickman

first: Himisha

last: Beltran

first: Maria T.

last: Diaz-Meco


title: DNA methylation constrains nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos [ChIP-seq]

first: Antoine

last: Peters

overall_design: To assess the role of these proteins during adult spermatogenesis, we generated conditional deletion models in which excision of floxed alleles of Dnmt3a and Dnmt3b was driven by the improved iCre recombinase transgene under the control of the Stra8 promoter, which is active in postnatal undifferentiated and differentiating spermatogonia. We investigated the chromatin status of control and mutant sperm by ChIP sequencing.

first: Grigorios

last: Fanourgakis

first: Laura

last: Toneu


title: DNA methylation constrains nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos [RNA-seq]

overall_design: To assess the role of these proteins during adult spermatogenesis, we generated conditional deletion models in which excision of floxed alleles of Dnmt3a and Dnmt3b was driven by the improved iCre recombinase transgene under the control of the Stra8 promoter, which is active in postnatal undifferentiated and differentiating spermatogonia. We investigated the transcriptome status of control and mutant undifferentiated spermatogonia, differentiated spermatogonia and sperm by total RNA sequencing.


title: DNA methylation constrains nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos [EM-seq]

overall_design: To assess the role of these proteins during adult spermatogenesis, we generated conditional deletion models in which excision of floxed alleles of Dnmt3a and Dnmt3b was driven by the improved iCre recombinase transgene under the control of the Stra8 promoter, which is active in postnatal undifferentiated and differentiating spermatogonia. We investigated the methylation status of control and mutant sperm by whole genome Enzymatic Methyl-seq.


title: DNA methylation constrains nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos [ATATA]

overall_design: To assess the role of these proteins during adult spermatogenesis and early embryogenesis, we generated conditional deletion models in which excision of floxed alleles of Dnmt3a and Dnmt3b was driven by the improved iCre recombinase transgene under the control of the Stra8 promoter, which is active in postnatal undifferentiated and differentiating spermatogonia. We investigated the chromatin status of control and mutant sperm derived early 2 ell stage embryos by Antibody Targeted Tagmentation (ATATA) sequencing.


title: DNA methylation constrains nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos [RNA-seq 2]

overall_design: To assess the role of these proteins during adult spermatogenesis and early embryogenesis, we generated conditional deletion models in which excision of floxed alleles of Dnmt3a and Dnmt3b was driven by the improved iCre recombinase transgene under the control of the Stra8 promoter, which is active in postnatal undifferentiated and differentiating spermatogonia. We investigated the transcriptome of control and mutant sperm derived 4 cell stage embryos by SmartSeq2 RNA sequencing.


title: Focal deletions of a promoter tether activate the IRX3 oncogene in T-cell acute lymphoblastic leukemia [UMI4C]

status: Public on Oct 20 2024

city: London

first: Sunniyat

last: Rahman

address: 72 Huntley Street

institute: UCL

summary: Oncogenes can be activated in cis through multiple mechanisms including enhancer hijacking events and noncoding mutations that create enhancers or promoters de novo. These paradigms have helped parse somatic variation of noncoding cancer genomes, thereby providing a rationale to identify noncanonical mechanisms of gene activation. Here we describe a novel mechanism of oncogene activation whereby focal copy number loss of an intronic element within the FTO gene leads to aberrant expression of IRX3, an oncogene in T cell acute lymphoblastic leukemia (T-ALL). Loss of this CTCF bound element downstream to IRX3 (+224 kb) leads to enhancer hijack of an upstream developmentally active super-enhancer of the CRNDE long noncoding RNA (-644 kb). Unexpectedly, the CRNDE super-enhancer interacts with the IRX3 promoter with no transcriptional output until it is untethered from the FTO intronic site. We propose that 'promoter tethering' of oncogenes to inert regions of the genome is a previously unappreciated biological mechanism preventing tumorigenesis.

overall_design: HiChIP, ATAC-seq, and 4C from T cell leukaemia (T-ALL cell lines)

first: Gianna

last: Bloye

first: Nadine

last: Farah

first: Jonas

last: Demeulemeester

first: Joana

middle: R

last: Costa

last: O'Connor

first: Rachael

last: Pocock

first: Tanya

last: Rapoz-D'Silva

first: Adam

last: Turna

first: Lingyi

last: Wang

first: SooWah

last: Lee

first: Adele

last: Fielding

first: Juliette

last: Roels

first: Roman

last: Jaksik

first: Małgorzata

last: Dawidowska

first: Pieter

last: Van Vlierberghe

first: Suzana

last: Hadjur

first: Jim

last: Hughes

first: James

last: Davies

first: Alejandro

last: Gutierrez

first: Michelle

last: Kelliher

first: Peter

last: Van Loo

first: Mark

middle: A

last: Dawson

first: Marc

last: Mansour


title: Focal deletions of a promoter tether activate the IRX3 oncogene in T-cell acute lymphoblastic leukemia [ATAC-Seq]

first: Sunniyat

last: Rahman

first: Gianna

last: Bloye

first: Nadine

last: Farah

first: Jonas

last: Demeulemeester

first: Joana

last: Costa

first: David

last: O'Connor

first: Rachael

last: Pocock

first: Tanya

last: Rapoz-D'Silva


title: Focal deletions of a promoter tether activate the IRX3 oncogene in T-cell acute lymphoblastic leukemia [HiChIP]

first: Adam

last: Turna

first: Lingyi

last: Wang

first: SooWah

last: Lee

first: Adele

last: Fielding

first: Juliette

last: Roels

first: Roman

last: Jaksik

first: Małgorzata

last: Dawidowska

first: Pieter

last: Van Vlierberghe


title: Single-cell transcriptomic mapping of immune cells in mouse bladders during urinary tract infection

city: Jinan

first: Zizhuo

last: Yang

state: Shandong

address: 44 Wenhua West Road, Lixia District

institute: Shandong University

summary: To explore the classification and functional roles of bladder immune cells during urinary tract infection (UTI), we performed scRNA-seq analysis of immune cells extracted from mouse bladders.

overall_design: Mice were intravesically infected with uropathogenic Escherichia coli(UPEC) CFT073, and bladder immune cells were isolated using flow cytometry 8 hours post-infection.

first: Benkang

last: Shi

first: Yan

last: Li


title: Single-nuclear multiome (RNA & ATAC) profiling of Pax3-Cre lineage traced endothelium from E9.5 mouse embryos and bulk ATAC-seq from E13.5 mouse embryos

status: Public on Oct 19 2024

city: Oxford

first: Oliver

middle: Andrew

last: Stone

state: Oxfordshire

address: IDRM, Old Road Campus, Roosevelt Drive

department: Physiology, Anatomy and Genetics

institute: University of Oxford

summary:
Background: The formation of the lymphatic system has been a subject of controversy for over a century. Although the accepted view is that lymphatic endothelial cells originate exclusively from veins, there have been reports of non-venous derived lymphatics in multiple tissues.
Methods: We performed single-nuclear multiome sequencing analyses on 3,801 individual nuclei isolated from Pax3Cre/+;Rosa26tdTomato mouse embryos at E9.5, the temporal window in which lymphatic endothelium initially emerges. Additionally, we performed bulk ATAC-seq analyses following the OmniATAC protocol on FACS-sorted ECs from Pax3Cre/+;Rosa26tdTomato embryos at E13.5.
Results: These analyses revealed the existence of angioblasts which arise from paraxial mesoderm and directly differentiate into lymphatic endothelium. Our findings support the notion that lymphatic endothelial cells also have non-venous origins and explain the discrepancies between previous studies.

overall_design:
Single-nuclear multiome (RNA & ATAC) profiling, endothelial progenitors and endothelial cells (ECs) were isolated from Pax3Cre/+;Rosa26tdTomato embryos at ~E9.5, to define the transcriptional and chromatin accessibility landscape of paraxial mesoderm as it differentiates into angioblasts and endothelial cells. Single nuclear multiome-seq was performed using the 10xChromium Next GEM Single Cell Multiome ATAC + Gene Expression kit.

For bulk ATAC-seq, ECs were FACS sorted from Pax3Cre/+;Rosa26tdTomato embryos at E13.5 and divided into tdTomato positive (spos) and negative blood ECs (dneg), and tdTomato positive lymphatic ECs (dpos) using podoplanin (PDPN) as a marker.

first: Irina-Elena

last: Lupu

last: Grainger


title: A ONECUT1 regulatory, non-coding region in pancreatic development and diabetes [RNA-seq]

city: Ulm

first: Sarah

last: Merz

address: Albert-Einstein-Allee 23

department: Institute of Molecular Oncology and Stem Cell Biology

institute: Ulm University Hospital

summary: In a patient with permanent neonatal syndromic diabetes clinically similar to cases with ONECUT1 biallelic mutations, we identified a disease-causing deletion located upstream of ONECUT1. Through genetic, genomic and functional studies we identified a crucial regulatory region acting as an enhancer of ONECUT1 specifically during pancreatic development. This enhancer region contains a low-frequency variant showing strong association with type 2 diabetes and other glycemic traits, thus extending the contribution of this region to common forms of diabetes. Clinical relevance is provided by experimentally tailored therapy options for patients carrying ONECUT1 coding or regulatory mutations.

overall_design: RNA-sequencing (RNA-seq) of pancreatic progenitors (PP) differentiated from hESCs (HUES8). The following genotypes were generated with CRISPR/Cas9 gene editing and used for RNA-seq: Wildtype (WT), ONECUT1  knockouts (heterozygous and homozygous), 108 kb deletion of Patient 1 (Pat1-del; heterozygous and homozygous), heterozygous Pat1-del in heterozygous ONECUT1 deleted clones (orientation of deletions in cis or trans).

first: Cécile

last: Julier

first: Sandra

last: Heller

first: Alexander

last: Kleger


title: Heparan sulfate regulates the fate decisions of human pluripotent stem cells

city: Cambridge

first: Deepsing

last: Syangtan

state: MA

address: 77 Massachusetts Ave.

department: Chemistry

institute: MIT

summary: Heparan sulfate (HS) is an anionic polysaccharide generated by all animal cells. Studies in model organisms have revealed some of its critical developmental roles, though HS role in human pluripotent stem cell (hPSC) self-renewal and differentiation is poorly understood. We, therefore, generated HS-deficient hPSCs by disrupting EXT1 glycosyltransferase. The depletion of heparan sulfate resulted in impaired differentiation of EXT1-/- cells to the mesendoderm lineage. To elucidate the underlying mechanisms behind defective ME differentiation, we further assessed the global transcriptomic profiles of wildtype (WT) and EXT1-/- hPSCs following directed differentiation. The hierarchical gene clustering showed that the upregulated genes were mainly associated with neuronal development, whereas the downregulated genes were associated with extracellular matrix organization and regulation of signal transduction. Consistent with these findings, EXT1-/- hPSCs failed to activate FGF and Nodal pathways during mesendoderm induction. These results underscore the previously unexplored roles of heparan sulfate in the Activin/Nodal pathway. Taken together, our study provides insight into the mechanistic roles of HS in hPSC fate decisions.

overall_design: To investigate how HS regulates cell fate decisions in hPSCs, we generated a cell line that lacks HS. The EXT1 locus was targeted using CRISPR/Cas9. Wildtype H9 cells and EXT1-/- hPCS were subjected to chemically defined conditions that induce mesendoderm differentiation. 28 hours after induction, both cell types were collected for RNA-seq analysis. Comparative gene expression profiling was performed using data obtained from RNA-seq of quadrupliate samples each of H9 and EXT1-/- cells.

first: Sayaka

last: Masuko

first: Qiao

first: Laura

last: Kiessling


title: Basal/squamous and mixed subtype bladder cancers present poor outcomes after neoadjuvant chemotherapy in the VESPER trial

city: Paris

first: Clarice

last: Groeneveld

address: 11 rue de Pierre et Marie Curie

institute: Institut Curie

summary: Background: Neoadjuvant chemotherapy (NAC) is the standard treatment for muscle-invasive bladder cancer (MIBC), yet 40% of patients progress, emphasizing the need for biomarkers predictive for response or chemoresistance. Gene expression-based subtypes may serve as biomarkers, though which subtypes will respond, notably when it comes to the basal subtype, remains contentious. Patients and methods: This post hoc study analyzed 300 NAC-treated patients enrolled in the GETUG/AFU VESPER trial, with transurethral diagnostic formalin-ﬁxed parafﬁn-embedded which underwent pathological review before being sequenced. ‘Mixed’ subtype was deﬁned for tumors displaying at least two different Consensus molecular subtypes in separate regions. We evaluated the association between molecular subtypes and outcome after NAC. Tumors with remaining tissue at cystectomy (n ¼ 83) were compared with pre-treatment tumors. Results: Cases were classiﬁed basal/squamous (Ba/Sq) (n ¼ 84), luminal unstable (n = 57), stroma-rich (n = 53), mixed (n = 48), luminal papillary (n ¼ 39), luminal non-speciﬁc (n = 18), and neuroendocrine-like (n ¼ 1), with 30/48 mixed cases including a Ba/Sq component. Compared with other molecular subtypes in a multivariate Cox model, Ba/Sq (pure or mixed) patients had an increased hazard ratio (HR) of progression-free survival [HR 2.0, 95% conﬁdence interval (CI) 1.36-3.0]. Mixed tumors were associated with decreased metabolic activity that could account for chemoresistance. Ba/Sq and mixed non-responders mostly maintained their subtype at cystectomy and have fewer myeloid dendritic cells after NAC. Tumors classiﬁed luminal papillary at transurethral resection of the urinary bladder tumor exhibited an increase in T CD4þ and macrophage signatures after NAC. Other subtypes did not show signiﬁcant immune changes after NAC. Our study design relied on detailed pathological review, which precluded evaluating the mixed subtype in published datasets. Furthermore, the sample size for post-NAC analyses constrained the statistical power of these ﬁndings. Conclusions: Our ﬁndings underscore the importance of recognizing intra-tumor heterogeneity in MIBC and its role in chemoresistance associated with Ba/Sq subtype, and provide valuable insights that could help future treatment development and improve patient outcomes.

overall_design: Samples from TURBT and cystectomy were collected from FFPE tissue of patients with muscle-invasive bladder cancer (MIBC) enrolled in the VESPER trial. Samples were macrodissected to enrich for tumor content. Multiple samples were collected when tissue heterogeneity was detected.

middle: S

first: Christian

last: Pfister

first: Stephane

last: Culine

first: Valentin

last: Harter

first: Jacqueline

last: Fontugne

first: Victoria

last: Dixon

first: Nanor

last: Sirab

first: Isabelle

last: Bernard-Pierrot

first: A

last: de Reyniès

first: Francois

last: Radvanyi

first: Yves

last: Allory


title: A ONECUT1 regulatory, non-coding region in pancreatic development and diabetes [Nanopore-seq]

first: Sarah

last: Merz

overall_design: Nanopore sequencing with adaptive sampling and 5hmC and 5mC methylation calling was conducted. Variant pahsing was performed with hESC (HUES) wildtype and in two knockout lines. The gene-edited hESCs consisted of a heterozygous 108kb deletion of Patient 1 (Pat1-del) and ONECUT1 heterozgyous knockout on the same allel (cis) or on different allels (trans). Methylation analysis was performed for HUES8 wildtype cells at hESC and differentiated pancreatic progenitors (PP).

first: Sandra

first: Cécile

last: Julier


title: The immune landscape of murine skeletal muscle regeneration and aging

status: Public on Oct 18 2024

city: Lisboa

first: Pedro

last: Sousa-Victor

address: Avenida Professor Egas Moniz

country: Portugal

institute: Instituto de Medicina Molecular (iMM)

summary: Age-related alterations in the immune system are starting to emerge as key contributors to impairments found in aged organs. A decline in regenerative capacity is a hallmark of tissue aging, however the contribution of immune aging to regenerative failure is just starting to be explored. Here, we applied a strategy combining single-cell RNA-sequencing with flow cytometry and functional assays to perform a complete analysis of the immune environment in the aged regenerating skeletal muscle, with time and single cell resolution. Our results identified previously undescribed immune cell types in the skeletal muscle and revealed an unanticipated complexity and functional heterogeneity in immune populations, that have been regarded as homogeneous. Furthermore, we uncovered a profound remodeling of both myeloid and lymphoid compartments in aging. These discoveries challenge established notions on immune regulation of skeletal muscle regeneration, providing a new set of potential targets to improve skeletal muscle health and regenerative capacity in aging.

overall_design: The CD45+ population (all immune cells) were fluorescence-activated cell sorting (FACS)-purified from non-injured and injured SkM at 1, 3 and 5 days post-injury from yg (3mo) and aged (25mo) mice. These samples were used to prepare scRNAseq libraries using 10× Chromium technology.

first: Neuza

last: Sousa

first: Marta

last: Bica

first: Nuno

last: Barbosa-Morais

first: Joana

last: Neves


title: Role of IL-33/ILC2 in bladder infection

first: Zizhuo

last: Yang

summary: The impact of IL-33 on urinary tract infections (UTI) was assessed by comparing Il33KO mice with wild-type (WT) mice within the bladder;The effect of ILC2 on UTI was assessed by comparing Il5cre+/DTA+ mice with WT mice within the bladder.

overall_design: Mouse were infected intravesically with uropathogenic Escherichia coli(UPEC) CFT073 and bladder samples were collected after 8 hours.

first: Benkang

last: Shi

first: Yan

last: Li