Cell Type-specific Transcription Research Articles

Definition of regulatory elements and transcription factors controlling porcine immune cell gene expression at single cell resolution using single nucleus ATAC-seq

The transcriptome of porcine peripheral blood mononuclear cells (PBMC) at single cell (sc) resolution is well described, but little is understood about the cis-regulatory mechanism behind scPBMC gene expression. Here, we profiled the open chromatin landscape of porcine PBMC that define cis-regulatory elements and mechanism contributing to the transcription using single nucleus ATAC sequencing (snATAC-seq). Approximately 22 % of the identified peaks overlapped with annotated transcription start sites (TSS). Using clustering based on open chromatin pattern similarity, we demonstrate that cell type annotations using snATAC-seq are highly concordant to that reported for sc RNA sequencing (scRNA-seq). The differentially accessible peaks (DAPs) for each cell type were characterized and the pattern of accessibility of the DAPs near cell type markers across cell types was similar to that of the average gene expression level of corresponding marker genes. Additionally, we found that peaks identified in snATAC-seq have the potential power to predict the cell type specific transcription starting site (TSS). We identified both transcription factors (TFs) whose binding motif were enriched in cell type DAPs of multiple cell types and cell type specific TFs by conducting transcription factor binding motif (TFBM) analysis. Furthermore, we identified the putative enhancer or promoter regions bound by TFs for each differentially expressed gene (DEG) with a DAP that overlapped with its TSS by generating cis-co-accessibility networks (CCAN). To predict the regulators of such DEGs, TFBM analysis was performed for each CCAN. The regulator TF-target DEG pairs predicted in this way were largely consistent with the results reported in the ENCODE Transcription Factor Targets Dataset (TFTD). This snATAC-seq approach provides insights into the regulation of chromatin accessibility landscape of porcine PBMCs and enables discovery of TFs predicted to control DEG through binding regulatory elements whose chromatin accessibility correlates with the DEG promoter region.

Evaluating Methods for the Prediction of Cell Type-Specific Enhancers in the Mammalian Cortex.

Identifying cell type-specific enhancers in the brain is critical to building genetic tools for investigating the mammalian brain. Computational methods for functional enhancer prediction have been proposed and validated in the fruit fly and not yet the mammalian brain. We organized the 'Brain Initiative Cell Census Network (BICCN) Challenge: Predicting Functional Cell Type-Specific Enhancers from Cross-Species Multi-Omics' to assess machine learning and feature-based methods designed to nominate enhancer DNA sequences to target cell types in the mouse cortex. Methods were evaluated based on in vivo validation data from hundreds of cortical cell type-specific enhancers that were previously packaged into individual AAV vectors and retro-orbitally injected into mice. We find that open chromatin was a key predictor of functional enhancers, and sequence models improved prediction of non-functional enhancers that can be deprioritized as opposed to pursued for in vivo testing. Sequence models also identified cell type-specific transcription factor codes that can guide designs of in silico enhancers. This community challenge establishes a benchmark for enhancer prioritization algorithms and reveals computational approaches and molecular information that are crucial for the identification of functional enhancers for mammalian cortical cell types. The results of this challenge bring us closer to understanding the complex gene regulatory landscape of the mammalian brain and help us design more efficient genetic tools and potential gene therapies for human neurological diseases.

Establishment of single-cell transcriptional states during seed germination

Germination involves highly dynamic transcriptional programs as the cells of seeds reactivate and express the functions necessary for establishment in the environment. Individual cell types have distinct roles within the embryo, so must therefore have cell type-specific gene expression and gene regulatory networks. We can better understand how the functions of different cell types are established and contribute to the embryo by determining how cell type-specific transcription begins and changes through germination. Here we describe a temporal analysis of the germinating Arabidopsis thaliana embryo at single-cell resolution. We define the highly dynamic cell type-specific patterns of gene expression and how these relate to changing cellular function as germination progresses. Underlying these are unique gene regulatory networks and transcription factor activity. We unexpectedly discover that most embryo cells transition through the same initial transcriptional state early in germination, even though cell identity has already been established during embryogenesis. Cells later transition to cell type-specific gene expression patterns. Furthermore, our analyses support previous findings that the earliest events leading to the induction of seed germination take place in the vasculature. Overall, our study constitutes a general framework with which to characterize Arabidopsis cell transcriptional states through seed germination, allowing investigation of different genotypes and other plant species whose seed strategies may differ.

CDK9 phosphorylates RUNX1 to promote megakaryocytic fate in megakaryocytic-erythroid progenitors

AbstractThe specification of megakaryocytic (Mk) or erythroid (E) lineages from primary human megakaryocytic-erythroid progenitors (MEPs) is crucial for hematopoietic homeostasis, yet the underlying mechanisms regulating fate specification remain elusive. In this study, we identify RUNX1 as a key modulator of gene expression during MEP fate specification. Overexpression of RUNX1 in primary human MEPs promotes Mk specification, whereas pan-RUNX inhibition favors E specification. Although total RUNX1 levels do not differ between Mk progenitors (MkPs) and E progenitors (ErPs), there are higher levels of serine-phosphorylated RUNX1 in MkPs than ErPs, and mutant RUNX1 with phosphorylated-serine/threonine mimetic mutations (RUNX1-4D) significantly enhances the functional efficacy of RUNX1. To model the effects of RUNX1 variants, we use human erythroleukemia (HEL) cell lines expressing wild-type (WT), phosphomimetic (RUNX1-4D), and nonphosphorylatable (RUNX1-4A) mutants showing that the 3 forms of RUNX1 differentially regulate expression of 2625 genes. Both WT and RUNX1-4D variants increase expression in 40%, and decrease expression in another 40%, with lesser effects of RUNX1-4A. We find a significant overlap between the upregulated genes in WT and RUNX1-4D–expressing HEL cells and those upregulated in primary human MkPs vs MEPs. Although inhibition of known RUNX1 serine/threonine kinases does not affect phosphoserine RUNX1 levels in primary MEPs, specific inhibition of cyclin dependent kinase 9 (CDK9) in MEPs leads to both decreased RUNX1 phosphorylation and increased E commitment. Collectively, our findings show that serine/threonine phosphorylation of RUNX1 promotes Mk fate specification and introduce a novel kinase for RUNX1 linking the fundamental transcriptional machinery with activation of a cell type–specific transcription factor.

Identifying cell type-specific transcription factor-mediated activity immune modules reveal implications for immunotherapy and molecular classification of pan-cancer.

Systematic investigation of tumor-infiltrating immune (TII) cells is important to the development of immunotherapies, and the clinical response prediction in cancers. There exists complex transcriptional regulation within TII cells, and different immune cell types display specific regulation patterns. To dissect transcriptional regulation in TII cells, we first integrated the gene expression profiles from single-cell datasets, and proposed a computational pipeline to identify TII cell type-specific transcription factor (TF) mediated activity immune modules (TF-AIMs). Our analysis revealed key TFs, such as BACH2 and NFKB1 play important roles in B and NK cells, respectively. We also found some of these TF-AIMs may contribute to tumor pathogenesis. Based on TII cell type-specific TF-AIMs, we identified eight CD8+ T cell subtypes. In particular, we found the PD1 + CD8+ T cell subset and its specific TF-AIMs associated with immunotherapy response. Furthermore, the TII cell type-specific TF-AIMs displayed the potential to be used as predictive markers for immunotherapy response of cancer patients. At the pan-cancer level, we also identified and characterized six molecular subtypes across 9680 samples based on the activation status of TII cell type-specific TF-AIMs. Finally, we constructed a user-friendly web interface CellTF-AIMs (http://bio-bigdata.hrbmu.edu.cn/CellTF-AIMs/) for exploring transcriptional regulatory pattern in various TII cell types. Our study provides valuable implications and a rich resource for understanding the mechanisms involved in cancer microenvironment and immunotherapy.

Non-coding variants impact cis-regulatory coordination in a cell type-specific manner

BackgroundInteractions among cis-regulatory elements (CREs) play a crucial role in gene regulation. Various approaches have been developed to map these interactions genome-wide, including those relying on interindividual epigenomic variation to identify groups of covariable regulatory elements, referred to as chromatin modules (CMs). While CM mapping allows to investigate the relationship between chromatin modularity and gene expression, the computational principles used for CM identification vary in their application and outcomes.ResultsWe comprehensively evaluate and streamline existing CM mapping tools and present guidelines for optimal utilization of epigenome data from a diverse population of individuals to assess regulatory coordination across the human genome. We showcase the effectiveness of our recommended practices by analyzing distinct cell types and demonstrate cell type specificity of CRE interactions in CMs and their relevance for gene expression. Integration of genotype information revealed that many non-coding disease-associated variants affect the activity of CMs in a cell type-specific manner by affecting the binding of cell type-specific transcription factors. We provide example cases that illustrate in detail how CMs can be used to deconstruct GWAS loci, assess variable expression of cell surface receptors in immune cells, and reveal how genetic variation can impact the expression of prognostic markers in chronic lymphocytic leukemia.ConclusionsOur study presents an optimal strategy for CM mapping and reveals how CMs capture the coordination of CREs and its impact on gene expression. Non-coding genetic variants can disrupt this coordination, and we highlight how this may lead to disease predisposition in a cell type-specific manner.

GET: a foundation model of transcription across human cell types.

Transcriptional regulation, involving the complex interplay between regulatory sequences and proteins, directs all biological processes. Computational models of transcription lack generalizability to accurately extrapolate in unseen cell types and conditions. Here, we introduce GET, an interpretable foundation model designed to uncover regulatory grammars across 213 human fetal and adult cell types. Relying exclusively on chromatin accessibility data and sequence information, GET achieves experimental-level accuracy in predicting gene expression even in previously unseen cell types. GET showcases remarkable adaptability across new sequencing platforms and assays, enabling regulatory inference across a broad range of cell types and conditions, and uncovering universal and cell type specific transcription factor interaction networks. We evaluated its performance on prediction of regulatory activity, inference of regulatory elements and regulators, and identification of physical interactions between transcription factors. Specifically, we show GET outperforms current models in predicting lentivirus-based massive parallel reporter assay readout with reduced input data. In fetal erythroblasts, we identify distal (>1Mbp) regulatory regions that were missed by previous models. In B cells, we identified a lymphocyte-specific transcription factor-transcription factor interaction that explains the functional significance of a leukemia-risk predisposing germline mutation. In sum, we provide a generalizable and accurate model for transcription together with catalogs of gene regulation and transcription factor interactions, all with cell type specificity.

Mapping Single-Cell Transcriptomes of Endometrium Reveals Potential Biomarkers in Endometrial Cancer.

The heterogeneity and dynamic changes of endometrial cells have a significant impact on health as they determine the normal function of the endometrium during the menstrual cycle. Dysfunction of the endometrium can lead to the occurrence of various gynecological diseases. Therefore, deconvolution of immune microenvironment that drives transcriptional programs throughout the menstrual cycle is key to understand regulatory biology of endometrium. Herein, we comprehensively analyzed single-cell transcriptome of 59,397 cells across ten human endometrium samples and revealed the dynamic cellular heterogeneity throughout the menstrual cycle. We identified two perivascular cell subtypes, four epithelial subtypes and four fibroblast cell types in endometrium. Moreover, we inferred the cell type-specific transcription factor (TF) activities and linked critical TFs to transcriptional output of diverse immune cell types, highlighting the importance of transcriptional regulation in endometrium. Dynamic interactions between various types of cells in endometrium contribute to a range of biological pathways regulating differentiation of secretory. Integration of the molecular biomarkers identified in endometrium and bulk transcriptome of 535 endometrial cancers (EC), we revealed five RNA-based molecular subtypes of EC with highly intratumoral heterogeneity and different clinical manifestations. Mechanism analysis uncovered clinically relevant pathways for pathogenesis of EC. In summary, our results revealed the dynamic immune microenvironment of endometrium and provided novel insights into future development of RNA-based treatments for endometriosis and endometrial carcinoma.

Single-cell insights: pioneering an integrated atlas of chromatin accessibility and transcriptomic landscapes in diabetic cardiomyopathy

BackgroundDiabetic cardiomyopathy (DCM) poses a growing health threat, elevating heart failure risk in diabetic individuals. Understanding DCM is crucial, with fibroblasts and endothelial cells playing pivotal roles in driving myocardial fibrosis and contributing to cardiac dysfunction. Advances in Multimodal single-cell profiling, such as scRNA-seq and scATAC-seq, provide deeper insights into DCM’s unique cell states and molecular landscape for targeted therapeutic interventions.MethodsSingle-cell RNA and ATAC data from 10x Multiome libraries were processed using Cell Ranger ARC v2.0.1. Gene expression and ATAC data underwent Seurat and Signac filtration. Differential gene expression and accessible chromatin regions were identified. Transcription factor activity was estimated with chromVAR, and Cis-coaccessibility networks were calculated using Cicero. Coaccessibility connections were compared to the GeneHancer database. Gene Ontology analysis, biological process scoring, cell-cell communication analysis, and gene-motif correlation was performed to reveal intricate molecular changes. Immunofluorescent staining utilized various antibodies on paraffin-embedded tissues to verify the findings.ResultsThis study integrated scRNA-seq and scATAC-seq data obtained from hearts of WT and DCM mice, elucidating molecular changes at the single-cell level throughout the diabetic cardiomyopathy progression. Robust and accurate clustering analysis of the integrated data revealed altered cell proportions, showcasing decreased endothelial cells and macrophages, coupled with increased fibroblasts and myocardial cells in the DCM group, indicating enhanced fibrosis and endothelial damage. Chromatin accessibility analysis unveiled unique patterns in cell types, with heightened transcriptional activity in myocardial cells. Subpopulation analysis highlighted distinct changes in cardiomyocytes and fibroblasts, emphasizing pathways related to fatty acid metabolism and cardiac contraction. Fibroblast-centered communication analysis identified interactions with endothelial cells, implicating VEGF receptors. Endothelial cell subpopulations exhibited altered gene expressions, emphasizing contraction and growth-related pathways. Candidate regulators, including Tcf21, Arnt, Stat5a, and Stat5b, were identified, suggesting their pivotal roles in DCM development. Immunofluorescence staining validated marker genes of cell subpopulations, confirming PDK4, PPARγ and Tpm1 as markers for metabolic pattern-altered cardiomyocytes, activated fibroblasts and endothelial cells with compromised proliferation.ConclusionOur integrated scRNA-seq and scATAC-seq analysis unveils intricate cell states and molecular alterations in diabetic cardiomyopathy. Identified cell type-specific changes, transcription factors, and marker genes offer valuable insights. The study sheds light on potential therapeutic targets for DCM.

Abstract P32: Cell Type-specific Genetic Therapy of Cancer, Virus Infection and Mitochondrial Disease

Abstract Cell type-specific gene delivery which restricts gene transfer to therapy-relevant cells, hence reducing unwanted side effects caused by the ectopic expression of transgenes, remains one of the biggest challenges. To mitigate potential off-target effects, we developed a programmable cell type-specific gene therapy using an RNA trans-splicing-based approach for gene replacement therapy or suicide gene therapy that can selectively express therapeutic proteins or suicide signals based on the cell’s endogenous pre-mRNA expression profile. Using the liver as a medical target organ, we developed a hepatocellular carcinoma (HCC)-specific trans-splicing based Herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene therapy approach. We rationally designed suicide RNAs that can target multiple HCC-related pre-mRNA biomarkers thereby increasing the overall concentration of targetable pre-mRNA. For delivery, non-viral redosable dumbbell-shaped DNA vectors were designed, manufactured, and tested in tissue culture cells, in 3D patient-derived xenograft spheroids and in mouse model. For cell-type specific delivery, the conjugation of dumbbell vectors with GalNAc3 residues enabled targeted delivery into hepatoblastoma-derived human tissue culture cells. To deliver nucleic acids into the mitochondria of human cells, we studied the long non-coding β2.7 RNA of the human cytomegalovirus which was previously reported to colocalise with mitochondrial complex I. Using thermodynamic profiling, we identified four thermodynamically conserved structural subdomains within the β2.7 RNA that are responsible for its mitochondrial targeting activity. A vector comprising eight mitochondrial targeting domains exhibited a three times higher mitochondrial targeting activity as compared with the parental β2.7 RNA. The amalgamation of mitochondrial targeting RNA and trans-splicing allows cell type-specific transcription of mRNA which can be delivered into mitochondria for mitochondrial gene therapy. Trans-splicing based gene therapy represent a technology that is highly translational offering promising perspectives towards suicide gene therapy of any cancer types or incurable infections with integrating viruses and may enable cell type-specific mitochondrial gene therapy. Citation Format: Pei She Loh (Rachel), Sushmita Poddar, Kaustav Chatterjee, Chiu Jing Wen, Derrick Wee Zan Liang, Fritz Lai, Chen Qingfeng, Volker Patzel. Cell Type-specific Genetic Therapy of Cancer, Virus Infection and Mitochondrial Disease [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P32.

Evolution of the enhancer-rich regulatory region of the gene for the cell-type specific transcription factor POU1F1

Precise spatio-temporal expression of genes in organogenesis is regulated by the coordinated interplay of DNA elements such as promoter and enhancers present in the regulatory region of a given locus. POU1F1 transcription factor plays a crucial role in the development of somatotrophs, lactotrophs and thyrotrophs in the anterior pituitary gland, and in maintaining high expression of growth hormone, prolactin and TSH. In mouse, expression of POU1F1 is controlled by a region fenced by two CTCF sites, containing 5 upstream enhancer elements, designated E-A (5′ to 3′). Elements C, B and A correspond to elements shown previously to play a role in pituitary development and hormonal expression; functional roles for elements E and D have not been reported. We performed comparative sequence analysis of this regulatory region and discovered that three elements, B, C and E, are present in all vertebrate groups except Agnatha. One very long (>2 kb) element (A) is unique to mammals suggesting a specific change in regulation of the gene in this group. Using DNA accessibility assay (ATAC-seq) we showed that conserved elements in anterior pituitary of four non-mammals are open, suggesting functionality as regulatory elements. We showed that, in many non-mammalian vertebrates, an additional upstream exon closely follows element E, leading to alternatively spliced transcripts. Here, element E functions as an alternative promoter, but in mammals this feature is lost, suggesting conversion of alternative promoter to enhancer. Our work shows that regulation of POU1F1 changed markedly during the course of vertebrate evolution, use of a low number of enhancer elements combined with alternative promoters in non-mammalian vertebrates being replaced by use of a unique combination of regulatory units in mammals. Most importantly, our work suggests that evolutionary conversion of alternate promoter to enhancer could be one of the evolutionary mechanisms of enhancer birth.

Abstract 1686: Dissecting the 3D enhancer-promoter hubs in prostate tumorigenesis

Abstract The transcription process is controlled by regulatory elements that are often located in non-coding regions. Among regulatory elements, activities of enhancers that are bound by cell-type specific transcription factors (TFs) are tightly linked to cell identity. Enhancers loop to interact with the promoters of target genes at a distance to drive different cell fates. Evidently, transcriptional dysregulation linked to enhancers that are differentially activated in cancer is an underlying factor in tumorigenesis. To identify enhancers linked to prostate tumorigenesis, we analyzed hundreds of in-house and public H3K27ac ChIP-seq data generated from normal prostate and prostate cancer cell lines and tissues. We identified thousands of prostate cancer-specific enhancers (PSEs) that were activated exclusively in cancer conditions. To further identify the target genes of PSEs, we performed 3C-derived assays, such as Hi-C and Micro-C, and mapped genome-wide interactions. However, they were limited by the resolution to detect enhancer-promoter interactions. The recent development of Region Capture Micro-C (RCMC), a hybridization-based capture Micro-C method, allowed us to deeply map the highly nested interactions between regulatory elements. By utilizing RCMC, we generated high-resolution, focal contact maps that revealed comprehensive enhancer-promoter interactions near PSEs in prostate cancer cells. Interestingly, we found that PSEs loop to other PSEs clustered in the proximal genomic region, in addition to promoters of significantly upregulated genes in cancer compared to normal cells. We termed these regions as multi-connected "3D enhancer-promoter hubs”. To mechanistically dissect these hubs, we deleted 5+ PSEs one by one using the CRISPR/Cas9 system and performed multi-omics analyses, including RNA-seq and ChIP-seq. We found that single deletions of a subset of PSEs led to the downregulation of multiple genes looped to the deleted PSE as well as to other PSEs. Moreover, we found that deletion reduced the activity of other connected PSEs. When we further evaluated the chromatin contacts of regulatory elements in the PSE-deleted cells, we observed that many chromatin contacts remained unaffected. Finally, we identified TFs that bind to PSEs, and we are currently in the process of investigating enhancer-promoter hub connectivity by performing knockdown experiments on these TFs. Furthermore, we are using molecular assays to identify PSEs that could exert a phenotypic shift in cancer cell identity to drive prostate tumorigenesis. Overall, our study not only provides mechanistic insights into the spatial regulation of 3D enhancer-promoter hubs but also identifies new non-coding drivers in prostate tumorigenesis. Citation Format: Huan Cao, Zexun Wu, Leonardo Gonzalez-Smith, Seolyn Yang, Suhn K. Rhie. Dissecting the 3D enhancer-promoter hubs in prostate tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1686.

Deciphering the spatiotemporal transcriptional and chromatin accessibility of human retinal organoid development at the single-cell level

Molecular information on the early stages of human retinal development remains scarce due to limitations in obtaining early human eye samples. Pluripotent stem cell-derived retinal organoids (ROs) provide an unprecedented opportunity for studying early retinogenesis. Using a combination of single cell RNA-seq and spatial transcriptomics we present for the first-time a single cell spatiotemporal transcriptome of RO development. Our data demonstrate that ROs recapitulate key events of retinogenesis including optic vesicle/cup formation, presence of a putative ciliary margin zone, emergence of retinal progenitor cells and their orderly differentiation to retinal neurons. Combining the scRNA- with scATAC-seq data, we were able to reveal cell-type specific transcription factor binding motifs on accessible chromatin at each stage of organoid development, and to show that chromatin accessibility is highly correlated to the developing human retina, but with some differences in the temporal emergence and abundance of some of the retinal neurons.

The chromatin accessibility dynamics during cell fate specifications in zebrafish early embryogenesis.

Chromatin accessibility plays a critical role in the regulation of cell fate decisions. Although gene expression changes have been extensively profiled at the single-cell level during early embryogenesis, the dynamics of chromatin accessibility at cis-regulatory elements remain poorly studied. Here, we used a plate-based single-cell ATAC-seq method to profile the chromatin accessibility dynamics of over 10000 nuclei from zebrafish embryos. We investigated several important time points immediately after zygotic genome activation (ZGA), covering key developmental stages up to dome. The results revealed key chromatin signatures in the first cell fate specifications when cells start to differentiate into enveloping layer (EVL) and yolk syncytial layer (YSL) cells. Finally, we uncovered many potential cell-type specific enhancers and transcription factor motifs that are important for the cell fate specifications.

Cell-type specific circadian transcription factor BMAL1 roles in excitotoxic hippocampal lesions to enhance neurogenesis

Circadian clocks, generating daily rhythms in biological processes, maintain homeostasis in physiology, so clock alterations are considered detrimental. Studies in brain pathology support this by reporting abnormal circadian phenotypes in patients, but restoring the abnormalities by light therapy shows no dramatic effects. Recent studies on glial clocks report the complex effects of altered clocks by showing their beneficial effects on brain repairs. However, how neuronal clocks respond to brain pathology is elusive. This study shows that neuronal BMAL1, a core of circadian clocks, reduces its expression levels in neurodegenerative excitotoxicity. In the dentate gyrus of excitotoxic hippocampal lesions, reduced BMAL1 in granule cells precedes apoptosis. This subsequently reduces BMAL1 levels in neighbor neural stem cells and progenitors in the subgranular zone, enhancing proliferation. This shows the various BMAL1 roles depending on cell types, and its alterations can benefit brain repair. Thus, cell-type-specific BMAL1 targeting is necessary to treat brain pathology.

Single Cell RNASeq Analysis of AD and Control Post Mortem Brain Samples

Alzheimer’s disease (AD) is a devastative late life disease, molecularly characterized by biochemical lesions of amyloid β plaques and Tau tangles. the 2nd most frequent one worldwide with no early detection methods/cure. I have applied single cell sequencing and analysed post mortem brain samples (using a Chromium machine) from 2 AD and 2 control frontal cortex samples. All the samples had ethics approval, and the RNA was tested for quality. The samples were obtained from the Edenborough MRC brain bank. Very few genes have been linked to far to AD (mainly through GWAS studies [1]). Overall, I quantified about 24,000 nuclei. Using statistical, bioinformatics and computational analyses I have detected significant differential expression changes in astrocyte, neuronal and microglial marker genes. I also conducted network analyses. The percentage of reads passing filter was slightly low (68%). Overall output was a bit lower than optimal. There was still over 1.6Bn clusters generated though, which is within the specification for an S1 flow-cell, and base call quality (quality control) was excellent: 97% >Q30. The read counts mapping to genome occupancy rate was also fairy low (63-81%). The number of barcodes is as follows: sample 1 (AD, Braak I-II) - 6,223 sample 2 (control) 6,262 sample 3 (AD Braak I-II) 5,332 sample 4 (AD, Braak I-II) 5,332 as well. The data is available for download under the GEO (accession number GSE175814). to assess global and cell-type specific gene expression impacts in Alzheimer’s versus control brain samples, we performed a holistic comparison of matching cell populations in AD and controls with the software cell Harmony. To identify possible gene regulatory network underlying transcriptional regulation in these distinct populations, we further explored prior evidenced transcription factor targets relationships from cell Harmony. Importantly, we observe both common and cell-type specific core transcription factors regulators, with both enriched downstream targets and regulated transcription factors. Upregulated networks in Astrocytes were associated with broad regulation by HIF1A, SOX2, NRF1, and RB1. the proportion of endothelial cells was higher in the Alzheimer’s disease samples than the neurological control samples. results reveal that the cell type-specific transcriptomic changes in Alzheimer’s disease are associated with 4 molecular pathways: angiogenesis in endothelial cells, immune response in endothelial cells and microglia, myelination in oligodendrocytes, and synaptic signalling in astrocytes and neurons. I also compared the results with microarray data from large cohort studies that examined samples from the prefrontal cortex (Alzheimer’s disease: n = 310; Neurological controls: n = 157) or temporal cortex (Alzheimer’s disease: n = 106; Neurological controls: n = 135). Among the DEGs identified in our snRNA-seq analysis, 1,113 and 764 genes were significantly differentially expressed in the microarray data from the prefrontal cortex and temporal cortex, respectively. subcluster analysis of microglia identified 13 subpopulations; VGF signalling was also found as changed. In the future, imaging of post mortem Alzheimer’s patients may verify brain cellular morphology/cell volume of vascular changes in the Alzheimer’s compared to control samples. My study findings [2] may open new venues to our understanding of the disease underlying molecular processes and cellular signaling. Regarding the glial cell changes, our main conclusion is that the glial cells may be reactive in the disease and not causal.

Interconnections between DDX41-Dependent Post-Transcriptional and GATA Factor-Instigated Transcriptional Mechanisms That Govern Hematopoiesis

Heterozygous DDX41 germline mutations occur in familial myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) and acute erythroid leukemia (AEL). DDX41 encodes an RNA helicase that regulates RNA splicing, cGAS-Sting signaling in innate immunity, and maintains genome stability in erythropoiesis. We described the innovation of a facile genetic rescue system to compare RNA-regulatory activities of wild type and disease-associated DDX41 variants in engineered HoxB8-immortalized (hi) murine myeloid progenitor cells (hi-Ddx41+/+ and Ddx41+/- cells) (Kim et al., Leukemia 2023). We identified DDX41-regulated transcripts and membrane proteins as quantitative metrics of DDX41 activity that discriminate benign from pathogenic DDX41 alleles. As these transcripts and the cognate proteins were not known to be components of GATA2 genetic networks that govern hematopoietic stem/progenitor cell (HSPC) transitions, we hypothesized that DDX41 mechanisms in progenitors operate in parallel with or independent of GATA2 mechanisms. Considering the biological and pathological importance of DDX41 and many unanswered questions on how clinical variants impact DDX41 activity, we instituted studies to discover DDX41-regulated transcript isoforms. The datasets are being used to unveil how DDX41 controls cellular functions, how clinical variants derail mechanisms, and to establish interconnections or independence with GATA factor mechanisms. To develop a broader perspective, we extended the myeloid data by innovating a rescue system to analyze DDX41 structure/function in G1E-ER-GATA1 erythroblasts that express a conditionally active ER-GATA1 allele. We used CRISPR/Cas9 to generate Ddx41 +/- clonal lines that expressed ~50% lower levels of DDX41 protein. A comparable system is being generated in human HUDEP2 erythroid precursor cells. We compared DDX41 with two disease-associated germline variants: G173R and K331deletion. G1E-ER-GATA1- Ddx41 +/-cells were infected with retrovirus expressing GFP, or GFP and DDX41 (WT, G173R, or K331del), GFP + cells were sorted, and total RNA was analyzed by RNA-seq. Differential expression analysis revealed 138 Ddx41-regulated transcripts (P < 0.05), with 63 elevated and 75 decreased. Disease-associated variants failed to regulate these transcripts. To establish relationships between DDX41-regulated and GATA1-regulated transcripts in erythroblasts, we compared DDX41-regulated transcripts with our GATA1 transcriptomic data (Tanimura et al., EMBO Rep. 2016; Dev. Cell 2018; Liao et al., Cell Rep. 2020), which revealed 78 transcripts co-regulated by DDX41 and GATA1. The RNA analysis is being extended to the proteome. To investigate similarities between DDX41-dependent post-transcriptional and GATA1-dependent transcriptional mechanisms, we analyzed the genes encoding the 78 co-regulated transcripts using gene ontology and pathway analyses. The gene signatures included downregulation of apoptosis (p=0.0006). We are testing the hypothesis that DDX41 and GATA1 operate additively or synergistically to control levels of transcripts encoding apoptotic regulators in developing erythroblasts, thus establishing a balance between pro-apoptotic and anti-apoptotic factors. We compared myeloid and erythroid DDX41-regulated transcript datasets to identify common and cell type-specific DDX41-regulated transcripts. This analysis unveiled 11 transcripts that are DDX41-regulated in both systems. One transcript encodes CLK3, a member of Cdc2-like kinase family that phosphorylates splicing factors. DDX41 increased intron 3 retention in Clk3 mRNA in both systems. By quantifying exon-intron junctions, we demonstrated that DDX41 promoted intron 3 retention in hi- Ddx41 +/- cells 1.7-fold (P = 0.0002) and in G1E-ER-GATA1- Ddx41 +/- cells 3.3-fold (P = 0.0002). We are testing models to explain the functional significance of DDX41-regulated Clk3 intron retention and discovered DDX41 regulates intron retention at other loci. We are assessing how DDX41 controls alternative splicing globally in the erythroid and myeloid systems and elucidating the biological and pathological consequences of corrupting these mechanisms. In aggregate, these studies established a system to discriminate pathogenic from benign DDX41 variants, and mechanistic insights that provide the basis for new myeloid and erythroid paradigms.

LINE-1 retrotransposons drive human neuronal transcriptome complexity and functional diversification.

The genetic mechanisms underlying the expansion in size and complexity of the human brain remain poorly understood. Long interspersed nuclear element-1 (L1) retrotransposons are a source of divergent genetic information in hominoid genomes, but their importance in physiological functions and their contribution to human brain evolution are largely unknown. Using multiomics profiling, we here demonstrate that L1 promoters are dynamically active in the developing and the adult human brain. L1s generate hundreds of developmentally regulated and cell type-specific transcripts, many that are co-opted as chimeric transcripts or regulatory RNAs. One L1-derived long noncoding RNA, LINC01876, is a human-specific transcript expressed exclusively during brain development. CRISPR interference silencing of LINC01876 results in reduced size of cerebral organoids and premature differentiation of neural progenitors, implicating L1s in human-specific developmental processes. In summary, our results demonstrate that L1-derived transcripts provide a previously undescribed layer of primate- and human-specific transcriptome complexity that contributes to the functional diversification of the human brain.

Identification of cell-type specific alternative transcripts in the multicellular alga Volvox carteri

BackgroundCell type specialization is a hallmark of complex multicellular organisms and is usually established through implementation of cell-type-specific gene expression programs. The multicellular green alga Volvox carteri has just two cell types, germ and soma, that have previously been shown to have very different transcriptome compositions which match their specialized roles. Here we interrogated another potential mechanism for differentiation in V. carteri, cell type specific alternative transcript isoforms (CTSAI).MethodsWe used pre-existing predictions of alternative transcripts and de novo transcript assembly with HISAT2 and Ballgown software to compile a list of loci with two or more transcript isoforms, identified a small subset that were candidates for CTSAI, and manually curated this subset of genes to remove false positives. We experimentally verified three candidates using semi-quantitative RT-PCR to assess relative isoform abundance in each cell type.ResultsOf the 1978 loci with two or more predicted transcript isoforms 67 of these also showed cell type isoform expression biases. After curation 15 strong candidates for CTSAI were identified, three of which were experimentally verified, and their predicted gene product functions were evaluated in light of potential cell type specific roles. A comparison of genes with predicted alternative splicing from Chlamydomonas reinhardtii, a unicellular relative of V. carteri, identified little overlap between ortholog pairs with alternative splicing in both species. Finally, we interrogated cell type expression patterns of 126 V. carteri predicted RNA binding protein (RBP) encoding genes and found 40 that showed either somatic or germ cell expression bias. These RBPs are potential mediators of CTSAI in V. carteri and suggest possible pre-adaptation for cell type specific RNA processing and a potential path for generating CTSAI in the early ancestors of metazoans and plants.ConclusionsWe predicted numerous instances of alternative transcript isoforms in Volvox, only a small subset of which showed cell type specific isoform expression bias. However, the validated examples of CTSAI supported existing hypotheses about cell type specialization in V. carteri, and also suggested new hypotheses about mechanisms of functional specialization for their gene products. Our data imply that CTSAI operates as a minor but important component of V. carteri cellular differentiation and could be used as a model for how alternative isoforms emerge and co-evolve with cell type specialization.

Exploring intrinsic variability between cultured nasal and bronchial epithelia in cystic fibrosis

The nasal and bronchial epithelium are unified parts of the respiratory tract that are affected in the monogenic disorder cystic fibrosis (CF). Recent studies have uncovered that nasal and bronchial tissues exhibit intrinsic variability, including differences in mucociliary cell composition and expression of unique transcriptional regulatory proteins which relate to germ layer origin. In the present study, we explored whether intrinsic differences between nasal and bronchial epithelial cells persist in cell cultures and affect epithelial cell functioning in CF. Comparison of air–liquid interface (ALI) differentiated epithelial cells from subjects with CF revealed distinct mucociliary differentiation states of nasal and bronchial cultures. Moreover, using RNA sequencing we identified cell type-specific signature transcription factors in differentiated nasal and bronchial epithelial cells, some of which were already poised for expression in basal progenitor cells as evidenced by ATAC sequencing. Analysis of differentiated nasal and bronchial epithelial 3D organoids revealed distinct capacities for fluid secretion, which was linked to differences in ciliated cell differentiation. In conclusion, we show that unique phenotypical and functional features of nasal and bronchial epithelial cells persist in cell culture models, which can be further used to investigate the effects of tissue-specific features on upper and lower respiratory disease development in CF.