Widespread Changes In Gene Expression Research Articles

Abstract PR006: ARID1A/B mutations retarget mSWI/SNF chromatin remodeler activity and define a spectrum of dedifferentiation in endometrial carcinoma

Abstract Dedifferentiated/undifferentiated endometrial carcinoma (DEC/UEC) is a highly aggressive histologic subtype of uterine cancer with a higher incidence among non-Hispanic Black women. Recent exome-wide sequencing studies have revealed frequent and often concomitant mutations in the ARID1A and ARID1B genes in over 50% of DEC/UEC cases and 30% of all endometrial carcinomas. The mSWI/SNF family of chromatin remodelers are critical regulators of cell type-specific chromatin accessibility and gene expression, existing in three distinct forms: canonical BAF (cBAF), Poly bromodomain-associated BAF (PBAF), and non-canonical BAF (ncBAF). The paralog subunits, ARID1A and ARID1B, specifically nucleate the assembly of cBAF complexes. However, the precise role ARID1A/ARID1B mutations (cBAF loss) play in the development of dedifferentiated endometrial carcinoma remains unclear. In this study, we define an expression signature associated with endometrial “dedifferentiation” by profiling the well-differentiated and dedifferentiated components of cBAF loss DEC cases through laser capture microdissection followed by RNA sequencing. Introduction of ARID1A or ARID1B in cBAF loss DEC cell lines demonstrated the restoration of cBAF assembly, confirmed by glycerol gradient and IP mass spectrometry. Upon the expression of ARID1A or ARID1B, we observed widespread chromatin occupancy and DNA accessibility at distinct paralog-specific sites, along with common sites associated with epithelial lineage differentiation, estrogen receptor, and PI3K/AKT pathway signaling. These findings align with the differentiation signature, defined in DEC cases demonstrating downregulation of genes associated with Myc targets, cell cycle checkpoint pathways, and DNA repair, suggesting these pathways represent potential therapeutic avenues for targeting DEC/UEC. Collectively, these data establish that well-differentiated endometrial carcinoma and DEC/UEC with mSWI/SNF mutations represent a spectrum of cBAF loss, defining the unique roles of ARID1A and ARID1B in endometrial carcinoma development. Furthermore, these findings imply that cBAF loss drives the widespread changes in DEC morphology, gene expression, and clinical behavior, enhancing our understanding of the etiology and therapeutic options for these cancers. Citation Format: Jessica D. St. Laurent, Grace Xu, Kasey Cervantas, Ajinkya Patil, Akshay Sankar, Shary Chen, David Kolin, Yemin Wang, David G. Huntsman, Cigall Kadoch. ARID1A/B mutations retarget mSWI/SNF chromatin remodeler activity and define a spectrum of dedifferentiation in endometrial carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR006.

Age-related changes in human skeletal muscle transcriptome and proteome are more affected by chronic inflammation and physical inactivity than primary aging.

Evaluation of the influence of primary and secondary aging on the manifestation of molecular and cellular hallmarks of aging is a challenging and currently unresolved issue. Our study represents the first demonstration of the distinct role of primary aging and chronic inflammation/physical inactivity - the most important drivers of secondary aging, in the regulation of transcriptomic and proteomic profiles in human skeletal muscle. To achieve this purpose, young healthy people (n = 15), young (n = 8) and older (n = 37) patients with knee/hip osteoarthritis, a model to study the effect of long-term inactivity and chronic inflammation on the vastus lateralis muscle, were included in the study. It was revealed that widespread and substantial age-related changes in gene expression in older patients relative to young healthy people (~4000 genes regulating mitochondrial function, proteostasis, cell membrane, secretory and immune response) were related to the long-term physical inactivity and chronic inflammation rather than primary aging. Primary aging contributed mainly to the regulation of genes (~200) encoding nuclear proteins (regulators of DNA repair, RNA processing, and transcription), mitochondrial proteins (genes encoding respiratory enzymes, mitochondrial complex assembly factors, regulators of cristae formation and mitochondrial reactive oxygen species production), as well as regulators of proteostasis. It was found that proteins associated with aging were regulated mainly at the post-transcriptional level. The set of putative primary aging genes and their potential transcriptional regulators can be used as a resource for further targeted studies investigating the role of individual genes and related transcription factors in the emergence of a senescent cell phenotype.

Heterozygous deletion of the autism-associated gene CHD8 impairs synaptic function through widespread changes in gene expression and chromatin compaction

Whole-exome sequencing of autism spectrum disorder (ASD) probands and unaffected family members has identified many genes harboring de novo variants suspected to play a causal role in the disorder. Of these, chromodomain helicase DNA-binding protein 8 (CHD8) is the most recurrently mutated. Despite the prevalence of CHD8 mutations, we have little insight into how CHD8 loss affects genome organization or the functional consequences of these molecular alterations in neurons. Here, we engineered two isogenic human embryonic stem cell lines with CHD8 loss-of-function mutations and characterized differences in differentiated human cortical neurons. We identified hundreds of genes with altered expression, including many involved in neural development and excitatory synaptic transmission. Field recordings and single-cell electrophysiology revealed a 3-fold decrease in firing rates and synaptic activity in CHD8+/- neurons, as well as a similar firing-rate deficit in primary cortical neurons from Chd8+/- mice. These alterations in neuron and synapse function can be reversed by CHD8 overexpression. Moreover, CHD8+/- neurons displayed a large increase in open chromatin across the genome, where the greatest change in compaction was near autism susceptibility candidate 2 (AUTS2), which encodes a transcriptional regulator implicated in ASD. Genes with changes in chromatin accessibility and expression in CHD8+/- neurons have significant overlap with genes mutated in probands for ASD, intellectual disability, and schizophrenia but not with genes mutated in healthy controls or other disease cohorts. Overall, this study characterizes key molecular alterations in genome structure and expression in CHD8+/- neurons and links these changes to impaired neuronal and synaptic function.

Modeling of pigmentation disorders associated with MITF mutation in Waardenburg syndrome revealed an impaired melanogenesis pathway in iPS-derived melanocytes.

Waardenburg Syndrome (WS) is a rare genetic disorder that leads to congenital hearing loss and pigmentation defects. Microphthalmia-associated transcription factor (MITF) is one of its significant pathogenic genes. Despite the comprehensive investigation in animal models, the pathogenic mechanism is still poorly described in humans due to difficulties accessing embryonic tissues. In this work, we used induced pluripotent stem cells derived from a WS patient carrying a heterozygous mutation in the MITF gene c.626A>T (p.His209Leu), and differentiated toward melanocyte lineage, which is the most affected cell type involved in WS. Compared with the wild-type cell line, the MITFmut cell line showed a reduced expression of the characteristic melanocyte-related genes and a lesser proportion of mature, fully pigmented melanosomes. The transcriptome analysis also revealed widespread gene expression changes at the melanocyte stage in the MITFmut cell line. The differentially expressed genes were enriched in melanogenesis and cell proliferation-related pathways. Interestingly, ion transport-related genes also showed a significant difference in MITFmut -induced melanocytes, indicating that the MITF mutant may lead to the dysfunction of potassium channels and transporters produced by intermediate cells in the cochlea, further causing the associated phenotype of deafness. Altogether, our study provides valuable insights into how MITF mutation affects WS patients, which might result in defective melanocyte development and the related phenotype based on the patient-derived iPSC model.

The transcriptome of HTLV-1-infected primary cells following reactivation reveals changes to host gene expression central to the proviral life cycle.

Infections by Human T cell Leukaemia Virus type 1 (HTLV-1) persist for the lifetime of the host by integrating into the genome of CD4+ T cells. Proviral gene expression is essential for proviral survival and the maintenance of the proviral load, through the pro-proliferative changes it induces in infected cells. Despite their role in HTLV-1 infection and a persistent cytotoxic T lymphocyte response raised against the virus, proviral transcripts from the sense-strand are rarely detected in fresh cells extracted from the peripheral blood, and have recently been found to be expressed intermittently by a small subset of cells at a given time. Ex vivo culture of infected cells prompts synchronised proviral expression in infected cells from peripheral blood, allowing the study of factors involved in reactivation in primary cells. Here, we used bulk RNA-seq to examine the host transcriptome over six days in vitro, following proviral reactivation in primary peripheral CD4+ T cells isolated from subjects with non-malignant HTLV-1 infection. Infected cells displayed a conserved response to reactivation, characterised by discrete stages of gene expression, cell division and subsequently horizontal transmission of the virus. We observed widespread changes in Polycomb gene expression following reactivation, including an increase in PRC2 transcript levels and diverse changes in the expression of PRC1 components. We hypothesize that these transcriptional changes constitute a negative feedback loop that maintains proviral latency by re-deposition of H2AK119ub1 following the end of proviral expression. Using RNAi, we found that certain deubiquitinases, BAP1, USP14 and OTUD5 each promote proviral transcription. These data demonstrate the detailed trajectory of HTLV-1 proviral reactivation in primary HTLV-1-carrier lymphocytes and the impact on the host cell.

Circular RNA circPLOD2 regulates pericyte function by targeting the transcription factor KLF4

Circular RNAs are generated by backsplicing and control cellular signaling and phenotypes. Pericytes stabilize capillary structures and play important roles in the formation and maintenance of blood vessels. Here, we characterize hypoxia-regulated circular RNAs (circRNAs) in human pericytes and show that the circular RNA of procollagen-lysine,2-oxoglutarate 5-dioxygenase-2 (circPLOD2) is induced by hypoxia and regulates pericyte functions. Silencing of circPLOD2 affects pericytes and increases proliferation, migration, and secretion of soluble angiogenic proteins, thereby enhancing endothelial migration and network capability. Transcriptional and epigenomic profiling of circPLOD2-depleted cells reveals widespread changes in gene expression and identifies the transcription factor krüppel-like factor 4 (KLF4) as a key effector of the circPLOD2-mediated changes. KLF4 depletion mimics circPLOD2 silencing, whereas KLF4 overexpression reverses the effects of circPLOD2 depletion on proliferation and endothelial-pericyte interactions. Together, these data reveal an important function of circPLOD2 in controlling pericyte proliferation and capillary formation and show that the circPLOD2-mediated regulation of KLF4 significantly contributes to the transcriptional response to hypoxia.

Adaptive structural and functional evolution of the placenta protects fetal growth in high-elevation deer mice

Environmental hypoxia challenges female reproductive physiology in placental mammals, increasing rates of gestational complications. Adaptation to high elevation has limited many of these effects in humans and other mammals, offering potential insight into the developmental processes that lead to and protect against hypoxia-related gestational complications. However, our understanding of these adaptations has been hampered by a lack of experimental work linking the functional, regulatory, and genetic underpinnings of gestational development in locally adapted populations. Here, we dissect high-elevation adaptation in the reproductive physiology of deer mice (Peromyscus maniculatus), a rodent species with an exceptionally broad elevational distribution that has emerged as a model for hypoxia adaptation. Using experimental acclimations, we show that lowland mice experience pronounced fetal growth restriction when challenged with gestational hypoxia, while highland mice maintain normal growth by expanding the compartment of the placenta that facilitates nutrient and gas exchange between gestational parent and fetus. We then use compartment-specific transcriptome analyses to show that adaptive structural remodeling of the placenta is coincident with widespread changes in gene expression within this same compartment. Genes associated with fetal growth in deer mice significantly overlap with genes involved in human placental development, pointing to conserved or convergent pathways underlying these processes. Finally, we overlay our results with genetic data from natural populations to identify candidate genes and genomic features that contribute to these placental adaptations. Collectively, these experiments advance our understanding of adaptation to hypoxic environments by revealing physiological and genetic mechanisms that shape fetal growth trajectories under maternal hypoxia.

N-methyl-D-aspartate receptor regulates the circadian clock in megakaryocytic cells and impacts cell proliferation through BMAL1

Peripheral circadian clocks control cell proliferation and survival, but little is known about their role and regulation in megakaryocytic cells. N-methyl-D-aspartate receptor (NMDAR) regulates the central clock in the brain. The purpose of this study was to determine whether NMDAR regulates the megakaryocytic cell clock and whether the megakaryocytic clock regulates cell proliferation and cell death. We found that both the Meg-01 megakaryocytic cell line and native murine megakaryocytes expressed circadian clock genes. Megakaryocyte-directed deletion of Grin1 in mice caused significant disruption of the circadian rhythm pathway at the transcriptional level and increased expression of BMAL1 at the protein level. Similarly, both pharmacological (MK-801) and genetic (GRIN–/–) inhibition of NMDAR in Meg-01 cells in vitro resulted in widespread changes in clock gene expression including increased expression of BMAL1, the core clock transcription factor. BMAL1 overexpression reduced Meg-01 cell proliferation and altered the time-dependent expression of the cell cycle regulators MYC and WEE1, whereas BMAL1 knockdown led to increased cell death in Meg-01-GRIN1–/– cells. Our results demonstrate that NMDAR regulates the circadian clock in megakaryocytic cells and that the circadian clock component BMAL1 contributes to the control of Meg-01 cell proliferation and survival.

Hypoxia-mediated regulation of DDX5 through decreased chromatin accessibility and post-translational targeting restricts R-loop accumulation.

Local hypoxia occurs in most solid tumors and is associated with aggressive disease and therapy resistance. Widespread changes in gene expression play a critical role in the biological response to hypoxia. However, most research has focused on hypoxia-inducible genes as opposed to those which are decreased in hypoxia. We demonstrate that chromatin accessibility is decreased in hypoxia, predominantly at gene promoters and specific pathways are impacted including DNA repair, splicing and the R-loop interactome. One of the genes with decreased chromatin accessibility in hypoxia was DDX5, encoding the RNA helicase, DDX5, which showed reduced expression in various cancer cell lines in hypoxic conditions, tumor xenografts and in patient samples with hypoxic tumors. Most interestingly, we found that when DDX5 is rescued in hypoxia, replication stress and R-loop levels accumulate further, demonstrating that hypoxia-mediated repression of DDX5 restricts R-loop accumulation. Together these data support the hypothesis that a critical part of the biological response to hypoxia is the repression of multiple R-loop processing factors, however, as shown for DDX5, their role is specific and distinct.

Angiotensin II Increases Oxidative Stress and Inflammation in Female, But Not Male, Endothelial Cells.

Women are at elevated risk for certain cardiovascular diseases, including pulmonary arterial hypertension, Alzheimer's disease, and vascular complications of diabetes. Angiotensin II (AngII), a circulating stress hormone, is elevated in cardiovascular disease; however, our knowledge of sex differences in the vascular effects of AngII are limited. We therefore analyzed sex differences in human endothelial cell response to AngII treatment. Male and female endothelial cells were treated with AngII for 24h and analyzed by RNA sequencing. We then used endothelial and mesenchymal markers, inflammation assays, and oxidative stress indicators to measure female and male endothelial cell functional changes in response to AngII. Our data show that female and male endothelial cells are transcriptomically distinct. Female endothelial cells treated with AngII had widespread gene expression changes related to inflammatory and oxidative stress pathways, while male endothelial cells had few gene expression changes. While both female and male endothelial cells maintained their endothelial phenotype with AngII treatment, female endothelial cells showed increased release of the inflammatory cytokine interleukin-6 and increased white blood cell adhesion following AngII treatment concurrent with a second inflammatory cytokine. Additionally, female endothelial cells had elevated reactive oxygen species production compared to male endothelial cells after AngII treatment, which may be partially due to nicotinamide adenine dinucleotide phosphate oxidase-2 (NOX2) escape from X-chromosome inactivation. These data suggest that endothelial cells have sexually dimorphic responses to AngII, which could contribute to increased prevalence of some cardiovascular diseases in women. The online version contains supplementary material available at 10.1007/s12195-023-00762-2.

APOE4 impairs myelination via cholesterol dysregulation in oligodendrocytes.

APOE4 is the strongest genetic risk factor for Alzheimer's disease1-3. However, the effects of APOE4 on the human brain are not fully understood, limiting opportunities to develop targeted therapeutics for individuals carrying APOE4 and other risk factors for Alzheimer's disease4-8. Here, to gain more comprehensive insights into the impact of APOE4 on the human brain, we performed single-cell transcriptomics profiling of post-mortem human brains from APOE4 carriers compared with non-carriers. This revealed that APOE4 is associated with widespread gene expression changes across all cell types of the human brain. Consistent with the biological function of APOE2-6, APOE4 significantly altered signalling pathways associated with cholesterol homeostasis and transport. Confirming these findings with histological and lipidomic analysis of the post-mortem human brain, induced pluripotent stem-cell-derived cells and targeted-replacement mice, we show that cholesterol is aberrantly deposited in oligodendrocytes-myelinating cells that are responsible for insulating and promoting the electrical activity of neurons. We show that altered cholesterol localization in the APOE4 brain coincides with reduced myelination. Pharmacologically facilitating cholesterol transport increases axonal myelination and improves learning and memory in APOE4 mice. We provide a single-cell atlas describing the transcriptional effects of APOE4 on the aging human brain and establish a functional link between APOE4, cholesterol, myelination and memory, offering therapeutic opportunities for Alzheimer's disease.

Transcriptome alterations in chicken HD11 cells with steady knockdown and overexpression of RIPK2 gene

Receptor interacting protein kinase 2 (RIPK2) is involved in a variety of signaling pathway to produce a series of inflammatory cytokines in response to a diverse of bacterial, viral and protozoal pathogens. However, the underlying regulating of RIPK2 remain unknown. Transcriptome alterations in chicken HD11 cells following RIPK2 overexpression or silencing by shRNA were analyzed by next-generation sequencing. Both overexpression and knockdown of the RIPK2 gene caused wide-spread changes in gene expression in chicken HD11 cells. Differentially expressed genes (DEGs) caused by altered RIPK2 gene expression were associated with multiple biological processes linked with biological regulation, response to stimulus, cell communication, and signal transduction etc. KEGG analysis revealed that many of the DEGs were enriched in VEGF signaling pathway, ECM-receptor interaction, Focal adhesion, TGF-beta signaling pathway etc. Moreover, we show that initiation genes, TGFB1 and TGFB3, in the TGF-beta signaling pathway are biological targets regulated by RIPK2 in chicken HD11 cells. This is the first transcriptome-wide study in which RIPK2-regulated genes in chicken cells have been screened. Our findings elucidate the molecular events associated with RIPK2 in chicken HD11 cells.

Mode and Tempo of 3D Genome Evolution in Drosophila.

Topologically associating domains (TADs) are thought to play an important role in preventing gene misexpression by spatially constraining enhancer-promoter contacts. The deleterious nature of gene misexpression implies that TADs should, therefore, be conserved among related species. Several early studies comparing chromosome conformation between species reported high levels of TAD conservation; however, more recent studies have questioned these results. Furthermore, recent work suggests that TAD reorganization is not associated with extensive changes in gene expression. Here, we investigate the evolutionary conservation of TADs among 11 species of Drosophila. We use Hi-C data to identify TADs in each species and employ a comparative phylogenetic approach to derive empirical estimates of the rate of TAD evolution. Surprisingly, we find that TADs evolve rapidly. However, we also find that the rate of evolution depends on the chromatin state of the TAD, with TADs enriched for developmentally regulated chromatin evolving significantly slower than TADs enriched for broadly expressed, active chromatin. We also find that, after controlling for differences in chromatin state, highly conserved TADs do not exhibit higher levels of gene expression constraint. These results suggest that, in general, most TADs evolve rapidly and their divergence is not associated with widespread changes in gene expression. However, higher levels of evolutionary conservation and gene expression constraints in TADs enriched for developmentally regulated chromatin suggest that these TAD subtypes may be more important for regulating gene expression, likely due to the larger number of long-distance enhancer-promoter contacts associated with developmental genes.

Abstract IA024: The role of the epigenome and phenotypic plasticity in colorectal cancer evolution

Abstract The evolutionary dynamics of colorectal metastasis and the role of the epigenome in disease progression remain to be fully determined. We have used multi-region, multi-omic profiling (DNA, RNA and chromatin), together with computational modelling, of a prospectively collected cohort of primary CRCs (pCRCs) and DNA and RNA analysis of a retrospective series of metastatic CRCs (mCRCs) to define pCRC biology and track clonal evolution from primary tumour to metastasis. Computational analysis of multi-region sequencing data provided a sensitive test confirming subclonal selection is rare in pCRCs. We detected substantial epigenetic rewiring at the outset of pCRC formation, implicating changes in chromatin accessibility in the initial genesis of CRCs. Using DNA to trace clonal ancestry and overlaying RNA to measure gene expression phenotypes, we observe widespread gene expression changes without underlying clonal selection, indicative that pCRC cells are phenotypically plastic. In mCRCs, we observe that the karyotype of the primary tumour is often not substantially modified in metastasis, across multiple organs, through treatment and over substantial periods of time. This is suggestive that stabilising selection, acting on copy number alterations, constrains karyotype evolution. Paired analysis of DNA and RNA in metastasis indicate that the phenotypic plasticity persists during metastatic spread. Epigenetic rewiring and phenotypic plasticity play potentially causative roles in the genesis and progression of colorectal cancer. Citation Format: Trevor A. Graham. The role of the epigenome and phenotypic plasticity in colorectal cancer evolution [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr IA024.

Bromodomain Inhibitors Modulate FcγR-Mediated Mononuclear Phagocyte Activation and Chemotaxis.

IgG antibodies form immune complexes (IC) that propagate inflammation and tissue damage in autoimmune diseases such as systemic lupus erythematosus. IgG IC engage Fcγ receptors (FcγR) on mononuclear phagocytes (MNP), leading to widespread changes in gene expression that mediate antibody effector function. Bromodomain and extra-terminal domain (BET) proteins are involved in governing gene transcription. We investigated the capacity of BET protein inhibitors (iBET) to alter IgG FcγR-mediated MNP activation. We found that iBET dampened IgG IC-induced pro-inflammatory gene expression and decreased activating FcγR expression on MNPs, reducing their ability to respond to IgG IC. Despite FcγR downregulation, iBET-treated macrophages demonstrated increased phagocytosis of protein antigen, IgG IC, and apoptotic cells. iBET also altered cell morphology, generating more amoeboid MNPs with reduced adhesion. iBET treatment impaired chemotaxis towards a CCL19 gradient in IC-stimulated dendritic cells (DC) in vitro, and inhibited IC-induced DC migration to draining lymph nodes in vivo, in a DC-intrinsic manner. Altogether, our data show that iBET modulates FcγR-mediated MNP activation and migration, revealing the therapeutic potential of BET protein inhibition in antibody-mediated diseases.

DNA Methylation in Degenerating Human Intervertebral Discs

Intervertebral disc (IVD) degeneration is a major cause of low back pain (LBP) and is associated with widespread changes in gene expression, extracellular matrix (ECM) degradation, inflammation, and innervation. Epigenetic modifications, including DNA methylation, can drive changes in gene expression without modifying DNA sequences. To date, little is known about epigenetic dysregulation in degenerated IVDs. The objective of this study was to investigate changes in DNA methylation in degenerating human IVDs from chronic LBP patients. Human lumbar IVD segments at L4/5 level including control (n=4) and degenerating (n=6) were homogenized in liquid nitrogen with a mortar and pestle and genomic DNA was extracted using the Qiagen DNeasy Blood and Tissue kit. Epigenome-wide 5mC mapping was performed following bisulfite sequencing using the Infinium MethylationEPIC BeadChip 850k Arrays. All analysis was performed in the R package RnBeads 2.0. Principal component analysis and hierarchical clustering analysis revealed group differences between healthy and degenerating IVDs. Pathway analysis revealed enrichment in genes related to ECM organization, signaling by receptor tyrosine kinases and signaling by interleukins. Further analysis at the single gene promoter level showed hypermethylation in ECM-related genes linked to IVD degeneration including sparc and col2a1 and hypomethylation of pro-inflammatory cytokines associated with IVD degeneration including IL-1b and IL-8. These changes in methylation would be expected to result in loss of ECM and increased inflammation. Persistent reprogramming of gene expression by DNA methylation could drive IVD degeneration and inflammation. Understanding the role of DNA methylation in IVD pathology may provide a scientific framework for the development of new therapeutic approaches.

Rat Spinal Cord Injury Associated with Spasticity Leads to Widespread Changes in the Regulation of Retained Introns.

To determine molecular changes that correlate with long-term physiological changes after spinal cord injury associated with spasticity, we used a complete transection model with an injury at sacral spinal level S2, wherein tail spasms develop in rats weeks to months post-injury. Using Illumina and nanopore sequencing, we found that from 12,266 expressed genes roughly 11% (1,342) change expression levels in the rats with spasticity. The transcription factor PU.1 (Spi-1 proto-oncogene) and several of its known regulated genes were upregulated during injury, possibly reflecting changes in cellular composition. In contrast to widespread changes in gene expression, only a few changes in alternative exon usage could be detected because of injury. There were more than 1,000 changes in retained intron usage, however. Unexpectedly, most of these retained introns have not been described yet but could be validated using direct RNA nanopore sequencing. In addition to changes from injury, our model allowed regional analysis of gene expression. Comparing the segments rostral and caudal to the injury site in naïve animals showed 525 differentially regulated genes and differential regional use of retained introns. We did not detect changes in the serotonin receptor 2C editing that were implicated previously in this spinal cord injury model. Our data suggest that regulation of intron retention of polyadenylated pre-mRNA is an important regulatory mechanism in the spinal cord under both physiological and pathophysiological conditions.

Epigenetic Regulation During Hypoxia and Its Implications in Cancer.

Hypoxia is defined as a cellular stress condition caused by a decrease in oxygen below physiologically normal levels. Cells in the core of a rapidly growing solid tumor are faced with the challenge of inadequate supply of oxygen through the blood, owing to improper vasculature inside the tumor. This hypoxic microenvironment inside the tumor initiates a gene expression program that alters numerous signaling pathways, allowing the cancer cell to eventually evade adverse conditions and attain a more aggressive phenotype. A multitude of studies covering diverse aspects of gene regulation has tried to uncover the mechanisms involved in hypoxia-induced tumorigenesis. The role of epigenetics in executing widespread and dynamic changes in gene expression under hypoxia has been gaining an increasing amount of support in recent years. This chapter discusses, in detail, various epigenetic mechanisms driving the cellular response to hypoxia in cancer.

Single-cell immunophenotyping of the fetal immune response to maternal SARS-CoV-2 infection in late gestation.

BackgroundDuring the COVID-19 pandemic, thousands of pregnant women have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The implications of maternal SARS-CoV-2 infection on fetal and childhood well-being need to be characterized. We aimed to characterize the fetal immune response to maternal SARS-CoV-2 infection.MethodsWe performed single-cell RNA-sequencing and T cell receptor sequencing on cord blood mononuclear cells (CBMCs) from newborns of mothers infected with SARS-CoV-2 in the third trimester (cases) or without SARS-CoV-2 infection (controls).ResultsWe identified widespread gene expression changes in CBMCs from cases, including upregulation of interferon-stimulated genes and major histocompatibility complex genes in CD14+ monocytes, transcriptional changes suggestive of activation of plasmacytoid dendritic cells, and activation and exhaustion of natural killer cells. Lastly, we observed fetal T cell clonal expansion in cases compared to controls.ConclusionsAs none of the infants were infected with SARS-CoV-2, our results suggest that maternal SARS-CoV-2 infection might modulate the fetal immune system in the absence of vertical transmission.Impact The implications of maternal SARS-CoV-2 infection in the absence of vertical transmission on fetal and childhood well-being are poorly understood.Maternal SARS-CoV-2 infection might modulate the fetal immune system in the absence of vertical transmission.This study raises important questions about the untoward effects of maternal SARS-CoV-2 on the fetus, even in the absence of vertical transmission.

Visualizing formation of the active site in the mitochondrial ribosome.

Ribosome assembly is an essential and conserved process that is regulated at each step by specific factors. Using cryo-electron microscopy (cryo-EM), we visualize the formation of the conserved peptidyl transferase center (PTC) of the human mitochondrial ribosome. The conserved GTPase GTPBP7 regulates the correct folding of 16S ribosomal RNA (rRNA) helices and ensures 2'-O-methylation of the PTC base U3039. GTPBP7 binds the RNA methyltransferase NSUN4 and MTERF4, which sequester H68-71 of the 16S rRNA and allow biogenesis factors to access the maturing PTC. Mutations that disrupt binding of their Caenorhabditis elegans orthologs to the large subunit potently activate mitochondrial stress and cause viability, development, and sterility defects. Next-generation RNA sequencing reveals widespread gene expression changes in these mutant animals that are indicative of mitochondrial stress response activation. We also answer the long-standing question of why NSUN4, but not its enzymatic activity, is indispensable for mitochondrial protein synthesis.