Transcriptional Changes Research Articles

Th1 polarization in Bordetella pertussis vaccine responses is maintained through a positive feedback loop.

Outbreaks of Bordetella pertussis (BP), the causative agent of whooping cough, continue despite broad vaccination coverage and have been increasing since vaccination switched from whole-BP (wP) to acellular BP (aP) vaccines. wP vaccination has been associated with more durable protective immunity and an induced Th1 polarized memory T cell response. Here, a multi-omics approach was applied to profile the immune response of 30 wP and 31 aP-primed individuals and identify correlates of T cell polarization before and after Tdap booster vaccination. We found that transcriptional changes indicating an interferon response on day 1 post-booster along with elevated plasma concentrations of IFN-γ and interferon-induced chemokines that peaked at day 1-3 post-booster correlated best with the Th1 polarization of the vaccine-induced memory T cell response on day 28. Our studies suggest that wP-primed individuals maintain their Th1 polarization through this early memory interferon response. This suggests that stimulating the interferon pathway during vaccination could be an effective strategy to elicit a predominant Th1 response in aP-primed individuals that protects better against infection.

HIV-1 Vpr-induced DNA damage activates NF-κB through ATM-NEMO independent of cell cycle arrest.

Lentiviruses encode a number of multi-functional accessory proteins, however, the primary role of the accessory protein Vpr remains unclear. As Vpr engages the host DNA damage response (DDR) at multiple steps, modulation of the DDR is considered central to the function(s) of Vpr. Vpr activates ataxia telangiectasia and Rad3 (ATR)-mediated DDR signaling, resulting in cell cycle arrest. However, the cellular consequences of Vpr-induced DNA damage, and the connection of Vpr-induced DNA damage to other Vpr functions, are unknown. Here, we determined that HIV-1 Vpr-induced DNA damage activates the ATM-NF-κB essential modulator (NEMO) pathway and alters cellular transcription via NF-κB/RelA. Through RNA-sequencing (RNA-seq) of cells expressing Vpr or mutants that separate the ability of Vpr to induce DNA damage from other DDR phenotypes, we identified that Vpr alters the transcriptome independent of cell cycle arrest. In tissue-cultured U2OS cells and primary human monocyte-derived macrophages (MDMs), we showed Vpr activates both ataxia telangiectasia mutated (ATM) and NF-κB/RelA signaling cascades. While inhibition of NEMO did not affect Vpr-induced DNA damage, it prevented NF-κB activation by Vpr, highlighting the importance of NEMO in Vpr-mediated transcriptional reprogramming. Virion-delivered Vpr was sufficient to induce DNA damage and activate ATM-NEMO dependent NF-κB transcription, suggesting that engagement of the DDR and transcriptional changes can occur early during viral replication. Together, our data uncover cellular consequences of Vpr-induced DNA damage and provide a mechanism for how Vpr activates NF-κB through DNA damage and ATM-NEMO signaling, which occur independent of cell cycle arrest. We propose this is essential to overcoming restrictive environments, such as in macrophages, to enhance viral replication.IMPORTANCEThe HIV accessory protein Vpr is multi-functional and required for viral replication in vivo, yet how Vpr enhances viral replication is unknown. Emerging literature suggests that a conserved function of Vpr is the engagement of the host DNA damage response (DDR). For example, Vpr activates DDR signaling, causes DDR-dependent cell cycle arrest, promotes degradation of various DDR proteins, and alters cellular consequences of DDR activation. However, a central understanding of how these phenotypes connect and how they affect HIV-infected cells remains unknown. Here, we found that Vpr-induced DNA damage alters the host transcriptome by activating an essential transcription pathway, NF-κB. This occurs early during the infection of primary human immune cells, suggesting NF-κB activation and transcriptome remodeling are important for establishing productive HIV-1 infection. Together, our study provides novel insights into how Vpr alters the host environment through the DDR, and what roles Vpr and the DDR play to enhance HIV replication.

Single-Base Methylome Analysis of Sweet Cherry (Prunus avium L.) on Dwarfing Rootstocks Reveals Epigenomic Differences Associated with Scion Dwarfing Conferred by Grafting

Plant grafting using dwarfing rootstocks is one of the important cultivation measures in the sweet cherry (Prunus avium) industry. In this work, we aimed to explore the effects of the dwarfing rootstock “Pd1” (Prunus tomentosa) on sweet cherry ‘Shuguang2’ scions by performing morphological observations using the paraffin slice technique, detecting GA (gibberellin) and IAA (auxin) contents using UPLC-QTRAP-MS (ultra-performance liquid chromatography coupled with a hybrid triple quadrupole-linear ion trap mass spectrometer), and implementing integration analyses of the epigenome and transcriptome using whole-genome bisulfite sequencing and transcriptome sequencing. Anatomical analysis indicated that the cell division ability of the SAM (shoot apical meristem) in dwarfing plants was reduced. Pd1 rootstock significantly decreased the levels of GAs and IAA in sweet cherry scions. Methylome analysis showed that the sweet cherry genome presented 15.2~18.6%, 59.88~61.55%, 28.09~33.78%, and 2.99~5.28% methylation at total C, CG, CHG, and CHH sites, respectively. Shoot tips from dwarfing plants exhibited a hypermethylated pattern mostly due to increased CHH methylation, while leaves exhibited a hypomethylated pattern. According to GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, DMGs (differentially methylated genes) and DEGs (differentially expressed genes) were enriched in hormone-related GO terms and KEGG pathways. Global correlation analysis between methylation and transcription revealed that mCpG in the gene body region enhanced gene expression and mCHH in the region near the TSS (transcription start site) was positively correlated with gene expression. Next, we found some hormone-related genes and TFs with significant changes in methylation and transcription, including SAURs, ARF, GA2ox, ABS1, bZIP, MYB, and NAC. This study presents a methylome map of the sweet cherry genome, revealed widespread DNA methylation alterations in scions caused by dwarfing rootstock, and obtained abundant genes with methylation and transcription alterations that are potentially involved in rootstock-induced growth changes in sweet cherry scions. Our findings can lay a good basis for further epigenetic studies on sweet cherry dwarfing and provide valuable new insight into understanding rootstock–scion interactions.

Transcriptome analysis reveals role of transcription factor WRKY70 in early N-hydroxy-pipecolic acid signaling.

N-hydroxy-pipecolic acid (NHP) is a mobile metabolite essential for inducing and amplifying systemic acquired resistance (SAR) following pathogen attack. Early phases of NHP signaling leading to immunity have remained elusive. Here, we report the early transcriptional changes mediated by NHP and the role salicylic acid (SA) plays during this response in Arabidopsis (Arabidopsis thaliana). We show that distinct waves of expression within minutes to hours of NHP treatment include increased expression of WRKY transcription factor genes as the primary transcriptional response, followed by the induction of WRKY-regulated defense genes as the secondary response. Most genes induced by NHP within minutes were SA-dependent, whereas those induced within hours were SA-independent. These data suggest that NHP induces the primary transcriptional response under basal levels of SA and that new SA biosynthesis via ISOCHORISMATE SYNTHASE 1/SA-INDUCTION DEFICIT 2 (ICS1/SID2) is dispensable for inducing the secondary transcriptional response. We demonstrate that WRKY70 is required for the induced expression of a set of genes defining some of the secondary transcriptional response, SAR protection, and NHP-dependent enhancement of ROS production in response to flagellin treatment. Our study highlights the key genes and pathways defining early NHP responses and the role of WRKY70 in regulating NHP-dependent transcription.

Integration of host gene regulation and oral microbiome reveals the influences of smoking during the development of oral squamous cell carcinoma.

This study aims to investigate the regulation of host gene transcription and microbial changes during the development of oral squamous cell carcinoma (OSCC) associated with smoking. The OSCC mouse model and smoking mouse model were established using 200 μg/mL 4-nitroquinoline-1-oxide (4NQO) in drinking water and exposure to cigarette smoke (four cigarettes per session, once a day, 5 days a week). Tongue tissues were harvested at 4 weeks and 16 weeks. Histopathological changes were evaluated using hematoxylin and eosin staining and Ki67 staining. RNA sequencing was performed on the mouse tongue tissues to identify differentially expressed genes (DEGs), and the results were validated by RT-PCR and immunohistochemistry. 16S rDNA sequencing was used to analyze changes in the oral microbiota during the early development of OSCC, identifying differentially abundant taxa associated with smoking. Finally, associations between the relative abundances of the oral microbiome and host gene expression were modeled using the Origin software. DEGs associated with smoking during the development of OSCC were identified. There were 12 upregulated genes, including NR4A3 and PPP1R3C, and 23 downregulated genes, including CD74 and ANKRD1. These genes were enriched in functions related to the signal transduction of cellular processes such as inflammation, differentiation, immunity, and PI3K/AKT, NF-κB signaling pathways. 4NQO and smoking treatment decreased oral microbial diversity and reduced the abundance of Bacteroidetes, Proteobacteria, and Lactobacillus but increased the abundance of Staphylococcus. Integrative analysis showed that the expression of CD74 was positively correlated with the relative abundance of Lactobacillus, while PPP1R3C was negatively correlated with Bacteroidota. In addition to characterizing host gene expression and the oral microbiome, our study explored the potential role of host-microbiome interactions in the development of OSCC. These findings enhance our understanding of smoking-related OSCC occurrence and development, providing new insights for its prevention.

Transcriptional changes of genes encoding sarcoplasmic reticulum calcium binding and up-taking proteins in normal and Duchenne muscular dystrophy dogs

BackgroundCytosolic calcium overload contributes to muscle degradation in Duchenne muscular dystrophy (DMD). The sarcoplasmic reticulum (SR) is the primary calcium storage organelle in muscle. The sarco-endoplasmic reticulum ATPase (SERCA) pumps cytosolic calcium to the SR during muscle relaxation. Calcium is kept in the SR by calcium-binding proteins.MethodsGiven the importance of the canine DMD model in translational studies, we examined transcriptional changes of SERCA (SERCA1 and SERCA2a), SERCA regulators (phospholamban, sarcolipin, myoregulin, and dwarf open reading frame), and SR calcium-binding proteins (calreticulin, calsequestrin 1, calsequestrin 2, and sarcalumenin) in skeletal muscle (diaphragm and extensor carpi ulnaris) and heart (left ventricle) in normal and affected male dogs by droplet digital PCR before the onset (≤ 2-m-old), at the active stage (8 to 16-m-old), and at the terminal stage (30 to 50-m-old) of the disease. Since many of these proteins are expressed in a fiber type-specific manner, we also evaluated fiber type composition in skeletal muscle.ResultsIn affected dog skeletal muscle, SERCA and its regulators were down-regulated at the active stage, but calcium-binding proteins (except for calsequestrin 1) were upregulated at the terminal stage. Surprisingly, nominal differences were detected in the heart. We also examined whether there exists sex-biased expression in 8 to 16-m-old dogs. Multiple transcripts were significantly downregulated in the heart and extensor carpi ulnaris muscle of female dogs. In fiber type analysis, we found significantly more type I fiber in the diaphragm of 8 to 16-m-old affected dogs, and significantly more type II fibers in the extensor carpi ulnaris of 30 to 50-m-old affected dogs. However, no difference was detected between male and female dogs.ConclusionsOur study adds new knowledge to the understanding of muscle calcium regulation in normal and dystrophic canines.

Schizophrenia-associated changes in neuronal subpopulations in the human midbrain.

Dysfunctional GABAergic and dopaminergic neurons are thought to exist in the ventral midbrain of patients with schizophrenia, yet transcriptional changes underpinning these abnormalities have not yet been localized to specific neuronal subsets. In the ventral midbrain, control over dopaminergic activity is maintained by both excitatory (glutamate) and inhibitory (GABA) input neurons. To further elucidate neuron pathology at the single-cell level, we characterized the transcriptional diversity of distinct NEUN+ populations in the human ventral midbrain and then tested for schizophrenia-associated changes in neuronal subset proportions and gene activity changes within neuronal subsets. Combining single nucleus RNA-sequencing with fluorescence-activated sorting of NEUN+ nuclei, we analysed 31,669 nuclei. Initially, we detected 18 transcriptionally distinct neuronal populations in the human ventral midbrain, including 2 "mixed" populations. The presence of neuronal populations in the midbrain was orthogonally validated with immunohistochemical stainings. "Mixed" populations contained nuclei expressing transcripts for vesicular glutamate transporter 2 (SLC17A6) and Glutamate Decarboxylase 2 (GAD2), but these transcripts were not typically co-expressed by the same nucleus. Upon more fine-grained subclustering of the 2 "mixed" populations, 16 additional subpopulations were identified that were transcriptionally classified as excitatory or inhibitory. In the midbrains of individuals with schizophrenia, we observed potential differences in the proportions of two (sub)populations of excitatory neurons, two subpopulations of inhibitory neurons, one "mixed" subpopulation, and one subpopulation of TH-expressing neurons. This may suggest that transcriptional changes associated with schizophrenia broadly affect excitatory, inhibitory, and dopamine neurons. We detected 99 genes differentially expressed in schizophrenia compared to controls within neuronal subpopulations identified from the 2 "mixed" populations, with the majority (67) of changes within small GABAergic neuronal subpopulations. Overall, single-nucleus transcriptomic analyses profiled a high diversity of GABAergic neurons in the human ventral midbrain, identified putative shifts in the proportion of neuronal subpopulations, and suggested dysfunction of specific GABAergic subpopulations in schizophrenia, providing directions for future research.

Isotretinoin promotes elimination of translation-competent HIV latent reservoirs in CD4T cells.

Development of novel therapeutic strategies that reactivate latent HIV and sensitize reactivated cells to apoptosis is crucial towards elimination of the latent viral reservoir. Among the clinically relevant latency reversing agents (LRA) under investigation, the γc-cytokine IL-15 and the superagonist N-803 have been shown to reactivate latent HIV ex vivo and in vivo. However, their clinical benefit can be hindered by IL-15 promoting survival of infected cells. We previously identified a small molecule, HODHBt, that sensitizes latently infected cells to death upon reactivation with γc-cytokines through a STAT-dependent pathway. In here, we aimed to identify and evaluate FDA-approved compounds that could also sensitize HIV-infected cells to apoptosis. Using the Connectivity Map (CMap), we identified the retinol derivative 13-cis-retinoic acid (Isotretinoin) causes similar transcriptional changes as HODHBt. Isotretinoin enhances IL-15-mediated latency reversal without inducing proliferation of memory CD4 T cells. Ex vivo analysis of PBMCs from ACTG A5325, where Isotretinoin was administered to ART-suppressed people with HIV, showed that Isotretinoin treatment enhances IL-15-mediated latency reversal. Furthermore, we showed that a combination of IL-15 with Isotretinoin promotes the reduction of translation-competent reservoirs ex vivo. Mechanistically, combination of IL-15 and Isotretinoin increases caspase-3 activation specifically in HIV-infected cells but not uninfected cells. Our results suggest that Isotretinoin can be a novel approach to target and eliminate translation-competent HIV reservoirs.

Integrated transcriptional and biochemical profiling suggests mechanisms associated with rapid cold hardening in adult Liriomyza trifolii (Burgess)

The leafminer Liriomyza trifolii causes severe economic damage on ornamental and horticultural crops in China. Rapid cold hardening (RCH) is a phenomenon where cold tolerance in insects can be significantly enhanced after a short-term acclimation to low temperatures. In this study, the regulation of transcription in response to cold hardening was investigated in L. trifolii adults, and fatty acids and cryoprotectant levels were measured. The composition of fatty acids changed after RCH treatment, and glucose and trehalose levels showed significant accumulation after acclimation, thus indicating that changes in fatty acids and cryoprotectants contribute to RCH in L. trifolii. RNA-seq was used to analyze transcriptional regulation after a 4 h hardening period and showed that differentially expressed genes clustered in multiple metabolic pathways, which indicates the importance of transcriptional regulation in RCH. This study expands our knowledge of biochemical and transcriptional changes in L. trifolii during cold hardening and provides a basis for further investigations aimed at understanding thermal adaptation in insects.

NAD+-boosting agent nicotinamide mononucleotide potently improves mitochondria stress response in Alzheimer’s disease via ATF4-dependent mitochondrial UPR

Extensive studies indicate that mitochondria dysfunction is pivotal for Alzheimer’s disease (AD) pathogenesis; while cumulative evidence suggests that increased mitochondrial stress response (MSR) may mitigate neurodegeneration in AD, explorations to develop a MSR-targeted therapeutic strategy against AD are scarce. We combined cell biology, molecular biology, and pharmacological approaches to unravel a novel molecular pathway by which NAD+-boosting agent nicotinamide mononucleotide (NMN) regulates MSR in AD models. Here, we report dyshomeostasis plasma UPRmt-mitophagy-mediated MSR profiles in AD patient samples. NMN restores NAD+ metabolic profiles and improves MSR through the ATF4-dependent UPRmt pathway in AD-related cross-species models. At the organismal level, NAD+ repletion with NMN supplementation ameliorates mitochondrial proteotoxicity, decreases hippocampal synaptic disruption, decreases neuronal loss, and brain atrophy in mice model of AD. Remarkably, omics features of the hippocampus with NMN show that NMN leads to transcriptional changes of genes and proteins involved in MSR characteristics, principally within the astrocyte unit rather than microglia and oligodendrocytes. In brief, our work provides evidence that MSR has an active role in the pathogenesis of AD, as reducing mitochondrial homeostasis via atf4 depletion in AD mice aggravates the hallmarks of the disease; conversely, bolstering mitochondrial proteostasis by NMN decreases protein aggregation, restores memory performance, and delays disease progression, ultimately translating to increased healthspan.

Single-Cell Transcriptomics Reveals Cellular Heterogeneity and Drivers in Serrated Pathway-Driven Colorectal Cancer Progression

Serrated lesions are common precancerous pathways in colorectal cancer (CRC), but the process by which they progress to malignancy remains unclear. We aimed to elucidate this progression through a single-cell RNA landscape. We conducted single-cell RNA sequencing on three normal colonic tissues and fifteen SLs (including HPs, SSLs, SSLD, and TSAs) and integrated these data with datasets containing tumor samples. We identified three invasive malignant epithelial cell subtypes related to CRC progression: SLC1, SLC2, and tumor cell. SLC1, specific to SSLs, is involved in cell proliferation and shows a continuum of malignancy in gene expression. TSA-specific SLC2 exhibited FOXQ1 upregulation and active EMT, indicating invasiveness. The trajectory analysis showed that HPs do not progress to cancer, and different SL types are linked to the MSI status of advanced CRCs. We validated molecular drivers in premalignant lesions and later carcinogenesis. In the tumor microenvironment, CAF and pre-CAF fibroblast subtypes associated with progression were identified. During the premalignant stage, SLC1 triggered CD8+ T cell responses, while at the advanced stage, CAFs promoted tumor invasion and metastasis via FN1-CD44, influencing tumor progression and the treatment response. Our findings highlight transcriptional changes across serrated pathway stages, aiding in early CRC diagnosis and treatment.

(+) Anatoxin-a elicits differential survival, photolocomotor behavior, and gene expression in two alternative vertebrate models

Anatoxin-a is a globally occurring, yet understudied, chiral cyanobacterial toxin that threatens public health and the environment. It has led to numerous dog. livestock and bird poisonings and although it has been studied in rodent models, comparatively little research has occurred in aquatic species. To advance a comparative toxicology understanding of this toxin in alternative vertebrate models, developing zebrafish and fathead minnow were exposed to environmentally relevant and elevated levels (13–4400 μg/L) of (+) anatoxin-a to examine potential mortality and sublethal effects, including photolocomotor behavior and gene expression responses. We observed significantly higher mortality (p < 0.05) in fathead minnows exposed to ≥ 1400 μg/L (65 – 83 % survival versus 97 % in controls). Locomotor response profiles for zebrafish typically displayed hypoactivity after exposure to (+) anatoxin-a in both light and dark periods, while hyperactivity of fathead minnows was observed at the lowest treatment level, but only in light conditions. Gene expression in zebrafish was significantly (p < 0.05) downregulated for mbp, which is associated with myelin sheath formation, and elavl3, which is involved in neurogenesis, along with cyp3a65 and gst, two genes related to phase I and II metabolism. However, no significant (p > 0.05) transcriptional changes were observed in the fathead minnow model. These differential responses between commonly employed species employed as alternative vertebrate models in toxicology research and chemicals risk assessments highlight the need for more comparative studies to understand sensitivities and variations in organismal response. Furthermore, we identified higher mortality, refractory behavioral effects, and gene expression in (+) anatoxin-a exposed fish when compared to previously reported (±) anatoxin-a (racemic 50:50 enantiomer mixture) studies, which is frequently used as a surrogate chemical for experimental work. Our findings identify the importance of understanding species and enantiomer specific effects of natural toxins.

Multi-omics profiling of DNA methylation and gene expression alterations in human cocaine use disorder

Structural and functional changes of the brain are assumed to contribute to excessive cocaine intake, craving, and relapse in cocaine use disorder (CUD). Epigenetic and transcriptional changes were hypothesized as a molecular basis for CUD-associated brain alterations. Here we performed a multi-omics study of CUD by integrating epigenome-wide methylomic (N = 42) and transcriptomic (N = 25) data from the same individuals using postmortem brain tissue of Brodmann Area 9 (BA9). Of the N = 1 057 differentially expressed genes (p < 0.05), one gene, ZFAND2A, was significantly upregulated in CUD at transcriptome-wide significance (q < 0.05). Differential alternative splicing (AS) analysis revealed N = 98 alternatively spliced transcripts enriched in axon and dendrite extension pathways. Strong convergent overlap in CUD-associated expression deregulation was found between our BA9 cohort and independent replication datasets. Epigenomic, transcriptomic, and AS changes in BA9 converged at two genes, ZBTB4 and INPP5E. In pathway analyses, synaptic signaling, neuron morphogenesis, and fatty acid metabolism emerged as the most prominently deregulated biological processes. Drug repositioning analysis revealed glucocorticoid receptor targeting drugs as most potent in reversing the CUD expression profile. Our study highlights the value of multi-omics approaches for an in-depth molecular characterization and provides insights into the relationship between CUD-associated epigenomic and transcriptomic signatures in the human prefrontal cortex.

DNA Methylation Negatively Regulates Gene Expression of Key Cytokines Secreted by BMMCs Recognizing FMDV-VLPs

Virus-like particles (VLPs) have been studied and used as vaccines to control foot-and-mouth disease (FMD). Mast cells (MCs) express various pattern recognition receptors that recognize pathogens and secrete numerous cytokines to initiate and modulate immune responses. Our previous study showed that bone marrow-derived mast cells (BMMCs) can recognize foot-and-mouth disease virus-like particles (FMDV-VLPs) to differentially express various cytokines and that histone acetylation can regulate the cytokines secreted during BMMC recognition of FMDV-VLPs. To demonstrate the role of DNA methylation in this response process, BMMCs that recognize FMDV-VLPs were treated with azacytidine (5-AZA), an inhibitor of DNA methylation transferase. We prepared FMDV-VLPs as described previously and cultured the BMMCs. The transcription and expression of key cytokines and transcription factors were determined using real-time quantitative PCR (RT-qPCR) and Western blotting. Results showed that pre-treatment with AZA resulted in the increased transcription and expression of tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-13, and IL-10, while the changes in IL-13 transcription and IL-6 expression were irrelevant to mannose receptors (MRs). Furthermore, analysis of the transcription factors indicated that both the transcription and expression of nuclear factor-kappa B (NF-κB) increased significantly in the AZA pre-treated group, indicating that DNA methylation may also regulate NF-κB expression to modulate TNF-α, IL-13, and IL-6. However, pre-treatment with AZA did not alter the expression of microphthalmia-associated transcription factor (MITF) or GATA-2. All the data demonstrate that DNA methylation negatively regulates the transcription and expression of TNF-α, IL-13, IL-10, and IL-6 secreted by recognizing FMDV-VLPs. These results provide new ideas for the mast cell-based design of more effective vaccine adjuvants and targeted therapies in the future.

Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y chromosome segment lengths in phenotypic males with 46,XX testicular disorder/difference of sex development.

46,XX testicular disorder/difference of sex development (46,XX DSD) is a rare congenital condition, characterized by a combination of the typical female sex chromosome constitution, 46,XX, and a variable male phenotype. In the majority of individuals with 46,XX DSD, a Y chromosome segment containing the sex-determining region gene (SRY) has been translocated to the paternal X chromosome. However, the precise genomic content of the translocated segment and the genome-wide effects remain elusive. We performed long-read DNA sequencing, RNA sequencing and DNA methylation analyses on blood samples from 46,XX DSD (n = 11), male controls (46,XY; variable cohort sizes) and female controls (46,XX; variable cohort sizes), in addition to RNA sequencing and DNA methylation analysis on blood samples from males with Klinefelter syndrome (47,XXY, n = 22). We also performed clinical measurements on all 46,XX DSD and a subset of 46,XY (n = 10). We identified variation in the translocated Y chromosome segments, enabling subcategorization into 46,XX DSD (1) lacking Y chromosome material (n = 1), (2) with short Yp arms (breakpoint at 2.7-2.8Mb, n = 2), (3) with medium Yp arms (breakpoint at 7.3Mb, n = 1), and (4) with long Yp arms (n = 7), including deletions of AMELY, TBLY1 and in some cases PRKY. We also identified variable expression of the X-Y homologues PRKY and PRKX. The Y-chromosomal transcriptome and methylome reflected the Y chromosome segment lengths, while changes to autosomal and X-chromosomal regions indicated global effects. Furthermore, transcriptional changes tentatively correlated with phenotypic traits of 46,XX DSD, including reduced height, lean mass and testicular size. This study refines our understanding of the genetic composition in 46,XX DSD, describing the translocated Y chromosome segment in more detail than previously and linking variability herein to genome-wide changes in the transcriptome and methylome.

Hepatocyte differentiation requires anisotropic expansion of bile canaliculi.

During liver development, bipotential progenitor cells called hepatoblasts differentiate into hepatocytes or cholangiocytes. Hepatocyte differentiation is uniquely associated with multi-axial polarity, enabling the anisotropic expansion of apical lumina between adjacent cells and formation of a three-dimensional network of bile canaliculi (BC). Cholangiocytes, the cells forming the bile ducts, exhibit the vectorial polarity characteristic of epithelial cells. Whether cell polarization feeds back on the gene regulatory pathways governing hepatoblast differentiation is unknown. Here, we used primary hepatoblasts to investigate the contribution of anisotropic apical expansion to hepatocyte differentiation. Silencing of the small GTPase Rab35 caused isotropic lumen expansion and formation of multicellular cysts with the vectorial polarity of cholangiocytes. Gene expression profiling revealed that these cells express reduced levels of hepatocyte markers and upregulate genes associated with cholangiocyte identity. Time-course RNA sequencing demonstrated that loss of lumen anisotropy precedes these transcriptional changes. Independent alterations in apical lumen morphology induced either by modulation of the subapical actomyosin cortex or increased intraluminal pressure caused similar transcriptional changes. These findings suggest that cell polarity and lumen morphogenesis feedback to hepatoblast-to-hepatocyte differentiation.

8320 Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y-chromosome segment lengths in phenotypic males with 46,XX testicular disorder of sex development

Abstract Disclosure: E.B. Johannsen: None. A. Berglund: None. A. Skakkebæk: None. S. Chang: None. J. Rohayem: None. S. Laurentino: None. A. Hørlyck: None. S.O. Drue: None. E.N. Bak: None. J. Fedder: None. F. Tuttelmann: None. J. Gromoll: None. J. Just: None. C.H. Gravholt: None. Background: 46,XX testicular disorder of sex development is a rare congenital condition affecting approximately three per 100,000 newborn males. It is characterized by a combination of the typical female sex chromosome constitution, 46,XX, and a variable male phenotype. In the majority of XX males, a Y chromosome segment containing the sex-determining region gene (SRY) has been translocated to the paternal X chromosome. However, the precise genomic content of the translocated segment, breakpoints, genome-wide effects, and the effect on other biological pathways essential for the phenotype remain unknown. Methods: We performed long-read DNA sequencing (Oxford Nanopore), RNA sequencing (paired-end, 100 bp) and DNA methylation (Infinium EPIC) analysis on blood samples from males with 46,XX testicular disorder of sex development (XX males; n = 11), male controls (46,XY; n = 12 for long-read DNA sequencing, n = 26 for RNA sequencing and DNA methylation) and female controls (46,XX; n = 12 for long-read DNA sequencing, n = 32 for RNA sequencing and DNA methylation), in addition to RNA sequencing and DNA methylation analysis on blood samples from males with Klinefelter syndrome (47,XXY, n = 22). We also performed clinical measures on all XX males and a subset of male controls (n = 10). Results: We identified variation in the translocated Y-chromosome segment of our XX male cohort, allowing for subcategorization into 1) XX males without Y chromosome material (n = 1), 2) those with short Yp arms (breakpoint at 2.7-2.8 Mbp, n = 2), 3) those with medium Yp arms (breakpoint at 7.3 Mbp, n = 1) and 4) those with long Yp arms, including AMELY, TBLY1 deletions, and in some cases PRKY deletions (n = 7). We also identified variable expression of the X-Y homologues PRKY and PRKX, appeared to have balanced expression level resulting in overall equilibrium. The Y-chromosomal transcriptome and methylome were affected, reflecting the Y-chromosome segment length. Genomic changes induced by the translocated Yp segment were evident in the transcriptome and methylome of autosomal and X-chromosomal regions, indicating global effects. Furthermore, the transcriptional changes in XX males correlated with phenotypic traits of the XX males, including lower height, decreased lean mass and smaller testicular size. Conclusion: Integration of long-read DNA sequencing, DNA methylation, and RNA sequencing analysis allowed for the identification of a heterogenous translocation process in XX males that exerted widespread effects on methylation and transcription. Presentation: 6/2/2024

Heterochromatin dynamics during the initial stages of sexual development in Plasmodium falciparum

Asexual replication of Plasmodium falciparum in the human blood results in exponential parasite growth and causes all clinical symptoms of malaria. However, at each round of the replicative cycle, some parasites convert into sexual precursors called gametocytes, which develop through different stages until they become infective to mosquito vectors. The genome-wide distribution of heterochromatin, a type of chromatin generally refractory to gene expression, is identical at all asexual blood stages, but is altered in stage II/III and more mature gametocytes. However, it is not known if these changes occur concomitantly with sexual conversion or at a later time during gametocyte development. Using a transgenic line in which massive sexual conversion can be conditionally induced, we show that the genome-wide distribution of heterochromatin at the initial stages of sexual development (i.e., sexual rings and stage I gametocytes) is almost identical to asexual blood stages, and major changes do not occur until stage II/III. However, we found that at loci with heterochromatin alterations, transcriptional changes associated with sexual development typically precede, rather than follow, changes in heterochromatin occupancy.

8320 Integration of long-read sequencing, DNA methylation and gene expression reveals heterogeneity in Y-chromosome segment lengths in phenotypic males with 46,XX testicular disorder of sex development

Abstract Disclosure: E.B. Johannsen: None. A. Berglund: None. A. Skakkebæk: None. S. Chang: None. J. Rohayem: None. S. Laurentino: None. A. Hørlyck: None. S.O. Drue: None. E.N. Bak: None. J. Fedder: None. F. Tuttelmann: None. J. Gromoll: None. J. Just: None. C.H. Gravholt: None. Background: 46,XX testicular disorder of sex development is a rare congenital condition affecting approximately three per 100,000 newborn males. It is characterized by a combination of the typical female sex chromosome constitution, 46,XX, and a variable male phenotype. In the majority of XX males, a Y chromosome segment containing the sex-determining region gene (SRY) has been translocated to the paternal X chromosome. However, the precise genomic content of the translocated segment, breakpoints, genome-wide effects, and the effect on other biological pathways essential for the phenotype remain unknown. Methods: We performed long-read DNA sequencing (Oxford Nanopore), RNA sequencing (paired-end, 100 bp) and DNA methylation (Infinium EPIC) analysis on blood samples from males with 46,XX testicular disorder of sex development (XX males; n = 11), male controls (46,XY; n = 12 for long-read DNA sequencing, n = 26 for RNA sequencing and DNA methylation) and female controls (46,XX; n = 12 for long-read DNA sequencing, n = 32 for RNA sequencing and DNA methylation), in addition to RNA sequencing and DNA methylation analysis on blood samples from males with Klinefelter syndrome (47,XXY, n = 22). We also performed clinical measures on all XX males and a subset of male controls (n = 10). Results: We identified variation in the translocated Y-chromosome segment of our XX male cohort, allowing for subcategorization into 1) XX males without Y chromosome material (n = 1), 2) those with short Yp arms (breakpoint at 2.7-2.8 Mbp, n = 2), 3) those with medium Yp arms (breakpoint at 7.3 Mbp, n = 1) and 4) those with long Yp arms, including AMELY, TBLY1 deletions, and in some cases PRKY deletions (n = 7). We also identified variable expression of the X-Y homologues PRKY and PRKX, appeared to have balanced expression level resulting in overall equilibrium. The Y-chromosomal transcriptome and methylome were affected, reflecting the Y-chromosome segment length. Genomic changes induced by the translocated Yp segment were evident in the transcriptome and methylome of autosomal and X-chromosomal regions, indicating global effects. Furthermore, the transcriptional changes in XX males correlated with phenotypic traits of the XX males, including lower height, decreased lean mass and smaller testicular size. Conclusion: Integration of long-read DNA sequencing, DNA methylation, and RNA sequencing analysis allowed for the identification of a heterogenous translocation process in XX males that exerted widespread effects on methylation and transcription. Presentation: 6/2/2024

Chromatin Accessibility and Gene Expression Vary Between a New and Evolved Autopolyploid of Arabidopsis arenosa.

Polyploids arise from whole-genome duplication (WGD) events, which have played important roles in genome evolution across eukaryotes. WGD can increase genome complexity, yield phenotypic novelty, and influence adaptation. Neo-polyploids have been reported to often show seemingly stochastic epigenetic and transcriptional changes, but this leaves open the question whether these changes persist in evolved polyploids. A powerful approach to address this is to compare diploids, neo-polyploids, and evolved polyploids of the same species. Arabidopsis arenosa is a species that allows us to do this-natural diploid and autotetraploid populations exist, while neo-tetraploids can be artificially generated. Here, we use ATAC-seq to assay local chromatin accessibility, and RNA-seq to study gene expression on matched leaf and petal samples from diploid, neo-tetraploid and evolved tetraploid A. arenosa. We found over 8,000 differentially accessible chromatin regions across all samples. These are largely tissue specific and show distinct trends across cytotypes, with roughly 70% arising upon WGD. Interestingly, only a small proportion is associated with expression changes in nearby genes. However, accessibility variation across cytotypes associates strongly with the number of nearby transposable elements. Relatively few genes were differentially expressed upon genome duplication, and ∼60% of these reverted to near-diploid levels in the evolved tetraploid, suggesting that most initial perturbations do not last. Our results provide new insights into how epigenomic and transcriptional mechanisms jointly respond to genome duplication and subsequent evolution of autopolyploids, and importantly, show that one cannot be directly predicted from the other.