Cell Type-specific Transcription Research Articles

Exon Shuffling Played a Decisive Role in the Evolution of the Genetic Toolkit for the Multicellular Body Plan of Metazoa.

Division of labor and establishment of the spatial pattern of different cell types of multicellular organisms require cell type-specific transcription factor modules that control cellular phenotypes and proteins that mediate the interactions of cells with other cells. Recent studies indicate that, although constituent protein domains of numerous components of the genetic toolkit of the multicellular body plan of Metazoa were present in the unicellular ancestor of animals, the repertoire of multidomain proteins that are indispensable for the arrangement of distinct body parts in a reproducible manner evolved only in Metazoa. We have shown that the majority of the multidomain proteins involved in cell–cell and cell–matrix interactions of Metazoa have been assembled by exon shuffling, but there is no evidence for a similar role of exon shuffling in the evolution of proteins of metazoan transcription factor modules. A possible explanation for this difference in the intracellular and intercellular toolkits is that evolution of the transcription factor modules preceded the burst of exon shuffling that led to the creation of the proteins controlling spatial patterning in Metazoa. This explanation is in harmony with the temporal-to-spatial transition hypothesis of multicellularity that proposes that cell differentiation may have predated spatial segregation of cell types in animal ancestors.

Differential transcriptional regulation of the NANOG gene in chicken primordial germ cells and embryonic stem cells

BackgroundNANOG is a core transcription factor (TF) in embryonic stem cells (ESCs) and primordial germ cells (PGCs). Regulation of the NANOG gene by TFs, epigenetic factors, and autoregulatory factors is well characterized in ESCs, and transcriptional regulation of NANOG is well established in these cells. Although NANOG plays a key role in germ cells, the molecular mechanism underlying its transcriptional regulation in PGCs has not been studied. Therefore, we investigated the mechanism that regulates transcription of the chicken NANOG (cNANOG) gene in PGCs and ESCs.ResultsWe first identified the transcription start site of cNANOG by 5′-rapid amplification of cDNA ends PCR analysis. Then, we measured the promoter activity of various 5′ flanking regions of cNANOG in chicken PGCs and ESCs using the luciferase reporter assay. cNANOG expression required transcriptional regulatory elements, which were positively regulated by POU5F3 (OCT4) and SOX2 and negatively regulated by TP53 in PGCs. The proximal region of the cNANOG promoter contains a positive transcriptional regulatory element (CCAAT/enhancer-binding protein (CEBP)-binding site) in ESCs. Furthermore, small interfering RNA-mediated knockdown demonstrated that POU5F3, SOX2, and CEBP played a role in cell type-specific transcription of cNANOG.ConclusionsWe show for the first time that different trans-regulatory elements control transcription of cNANOG in a cell type-specific manner. This finding might help to elucidate the mechanism that regulates cNANOG expression in PGCs and ESCs.

From chemoproteomic-detected amino acids to genomic coordinates: insights into precise multi-omic data integration.

The integration of proteomic, transcriptomic, and genetic variant annotation data will improve our understanding of genotype–phenotype associations. Due, in part, to challenges associated with accurate inter‐database mapping, such multi‐omic studies have not extended to chemoproteomics, a method that measures the intrinsic reactivity and potential “druggability” of nucleophilic amino acid side chains. Here, we evaluated mapping approaches to match chemoproteomic‐detected cysteine and lysine residues with their genetic coordinates. Our analysis revealed that database update cycles and reliance on stable identifiers can lead to pervasive misidentification of labeled residues. Enabled by this examination of mapping strategies, we then integrated our chemoproteomics data with computational methods for predicting genetic variant pathogenicity, which revealed that codons of highly reactive cysteines are enriched for genetic variants that are predicted to be more deleterious and allowed us to identify and functionally characterize a new damaging residue in the cysteine protease caspase‐8. Our study provides a roadmap for more precise inter‐database mapping and points to untapped opportunities to improve the predictive power of pathogenicity scores and to advance prioritization of putative druggable sites.

Defining the functionally sufficient regulatory region and liver-specific roles of the erythropoietin gene by transgene complementation

BackgroundErythropoietin (EPO) is an essential growth factor for erythroid cells and is mainly secreted from the kidneys and subsidiarily from the livers of adult mammals in an anemia/hypoxia-inducible manner. Aim and methodTo elucidate the regulatory mechanisms of stress-inducible and cell type-specific Epo gene transcription, the rate-limiting step of EPO production, we investigated the sufficiency of a 180-kb genomic fragment flanking the mouse Epo gene locus for recapitulating endogenous Epo gene function by a transgene complementation strategy. Key findingsWhile Epo gene-deficient mice exhibited lethal anemia in utero with defects in erythroblast proliferation and maturation, Epo-knockout mice integrated with the 180-kb Epo transgene showed normal erythropoiesis throughout life. In the transgene-rescued mice, liver-specific deletion of the transgene by the Cre-loxP recombination system caused neonatal anemia with erythropoietic defects in the liver but not in the spleen, indicating the essential function of hepatic EPO on normal erythropoiesis in the liver, which is the major erythropoietic site in late embryonic and neonatal stages. SignificanceThese results demonstrate that the 180 kb Epo gene flanking region contains the fully functional Epo gene unit and that EPO from the liver dominantly stimulates hepatic erythropoiesis but contributes less to erythropoiesis in other organs.

Gene Regulatory Networks Controlling Temporal Patterning, Neurogenesis, and Cell Fate Specification in the Mammalian Retina

Gene regulatory networks (GRNs), consisting of transcription factors and their target cis-regulatory sequences, control neurogenesis and cell fate specification in the developing central nervous system, but their organization is poorly characterized. In this study, we performed integrated single-cell RNA- and scATAC-seq analysis in both mouse and human retina to profile dynamic changes in gene expression, chromatin accessibility and transcription factor footprinting during retinal neurogenesis. We identified multiple interconnected, evolutionarily-conserved GRNs consisting of cell type-specific transcription factors that both activate expression of genes within their own network and often inhibit expression of genes in other networks. These GRNs control state transitions within primary retinal progenitors that underlie temporal patterning, regulate the transition from primary to neurogenic progenitors, and drive specification of each major retinal cell type. We confirmed the prediction of this analysis that the NFI transcription factors Nfia, Nfib, and Nfix selectively activate expression of genes that promote late-stage temporal identity in primary retinal progenitors. We also used GRNs to identify additional transcription factors that promote (Insm1/2) and inhibit (Tbx3 and Tcf7l1/2) rod photoreceptor specification in postnatal retina. This study provides an inventory of cis- and trans-acting factors that control retinal development, identifies transcription factors that control the temporal identity of retinal progenitors and cell fate specification, and will potentially guide cell-based therapies aimed at replacing retinal neurons lost due to disease.

Identification of <i>Cis</i>-Regulatory Modules for Aav-Based Cell Type-Specific Targeting in the Retina and Brain

Adeno Associated Viruses (AAVs) targeting specific cell types are powerful tools for studying distinct cell types in the central nervous system (CNS). Cis-regulatory modules (CRMs), e.g., enhancers, are highly cell type-specific and can be integrated into AAVs to render cell type specificity. Chromatin accessibility has been commonly used to nominate CRMs, which were then incorporated into AAVs and tested for cell type-specificity in CNS. However, chromatin accessibility data alone cannot accurately annotate active CRMs. Many of the chromatin accessible CRMs are not active and fail to drive gene expression in vivo. Using available large-scale datasets on chromatin accessibility, such as those published by the ENCODE project, we explored strategies that can increase the efficiency of identifying active CRMs for AAV-based cell type-specific labeling and manipulation. We found that pre-screening of chromatin accessible putative CRMs based on the density of cell type-specific transcription factor binding sites (TFBSs) can significantly increase the efficiency of identifying active CRMs. In addition, generation of synthetic CRMs by stitching chromatin accessible regions flanking cell type-specific genes can render cell type-specificity in AAVs in many cases. Both strategies utilize available genomic datasets and can be employed to generate cell type-specific AAVs in CNS without conducting comprehensive screening and sequencing experiments. Using these straightforward strategies, we generated AAVs that can target the extensively studied interneuron and glial cell types in the retina and brain.

Large-scale meta-analysis across East Asian and European populations updated genetic architecture and variant-driven biology of rheumatoid arthritis, identifying 11 novel susceptibility loci

ObjectivesNearly 110 susceptibility loci for rheumatoid arthritis (RA) with modest effect sizes have been identified by population-based genetic association studies, suggesting a large number of undiscovered variants behind a highly...

Mediator's Kinase Module: A Modular Regulator of Cell Fate.

Selective gene expression is crucial in maintaining the self-renewing and multipotent properties of stem cells. Mediator is a large, evolutionarily conserved, multi-subunit protein complex that modulates gene expression by relaying signals from cell type-specific transcription factors to RNA polymerase II. In humans, this complex consists of 30 subunits arranged in four modules. One critical module of the Mediator complex is the kinase module consisting of four subunits: MED12, MED13, CDK8, and CCNC. The kinase module exists in variable association with the 26-subunit Mediator core and affects transcription through phosphorylation of transcription factors and by controlling Mediator structure and function. Many studies have shown the kinase module to be a key player in the maintenance of stem cells that is distinct from a general role in transcription. Genetic studies have revealed that dysregulation of this kinase subunit contributes to the development of many human diseases. In this review, we discuss the importance of the Mediator kinase module by examining how this module functions with the more recently identified transcriptional super-enhancers, how changes in the kinase module and its activity can lead to the development of human disease, and the role of this unique module in directing and maintaining cell state. As we look to use stem cells to understand human development and treat human disease through both cell-based therapies and tissue engineering, we need to remain aware of the on-going research and address critical gaps in knowledge related to the molecular mechanisms that control cell fate.

DIANA-miRGen v4: indexing promoters and regulators for more than 1500 microRNAs.

Deregulation of microRNA (miRNA) expression plays a critical role in the transition from a physiological to a pathological state. The accurate miRNA promoter identification in multiple cell types is a fundamental endeavor towards understanding and characterizing the underlying mechanisms of both physiological as well as pathological conditions. DIANA-miRGen v4 (www.microrna.gr/mirgenv4) provides cell type specific miRNA transcription start sites (TSSs) for over 1500 miRNAs retrieved from the analysis of >1000 cap analysis of gene expression (CAGE) samples corresponding to 133 tissues, cell lines and primary cells available in FANTOM repository. MiRNA TSS locations were associated with transcription factor binding site (TFBSs) annotation, for >280 TFs, derived from analyzing the majority of ENCODE ChIP-Seq datasets. For the first time, clusters of cell types having common miRNA TSSs are characterized and provided through a user friendly interface with multiple layers of customization. DIANA-miRGen v4 significantly improves our understanding of miRNA biogenesis regulation at the transcriptional level by providing a unique integration of high-quality annotations for hundreds of cell specific miRNA promoters with experimentally derived TFBSs.

Regulation of Ribosomal RNA Synthesis in Myeloid Progenitors By Cell Type Specific Transcription Factors

Haematopoiesis relies on the ability of hematopoietic stem cells to progress through a systematic hierarchy to produce lineage-restricted progenitors that terminally differentiate into phenotypically distinct types of mature hematopoietic cells. This process is precisely coordinated by the combinatorial activity of lineage-specifying transcription factors (TFs). Indeed, the critical transcriptional program of every hematopoietic cell type, and indeed of all cell types throughout the body, requires a set of core TFs for its proper execution. A frequently-overlooked component of the cellular transcriptional program is the transcription of ribosomal RNA (rRNA), the major component of ribosomes. Ribosomal RNA comprises 90% of total cellular RNA, and its transcription from hundreds of rDNA genes by RNA polymerase I is one of the most intense transcriptional processes in the cell. Different progenitor and mature cell types in the hematopoietic tree have different sizes, ribosome abundances, and rates of protein synthesis. Tight control of ribosome abundance is essential for a normal cellular proteome, and different cell types within the hematopoietic tree have varied rRNA transcription rates. However, there has been limited study of the molecular basis of lineage-specific regulation of rDNA transcription in hematopoiesis. We explored the binding to rDNA of over twenty key hematopoietic TFs that were determined by the Broad Institute DepMap database to be crucial for survival of hematopoietic cell lines. Using a customized bioinformatics pipeline, we mapped over three hundred ChIP-Seq datasets for these factors (generated by us as well as publicly available from ENCODE and GEO) to mouse and human rDNA assemblies, and found that several essential hematopoietic TFs such as MYC, MYB, RUNX1, PU.1, CEBPA and others bind to rDNA at conserved sites and motifs (Fig A). MYC is well-known as a master regulator of rDNA, and RUNX1 was recently reported to bind rDNA, but most of the others have never been linked to rDNA, and their functional roles in regulating rDNA transcription have not been explored. We picked for further study CEBPA, a crucial TF required for specification of granulocyte-monocyte progenitors (GMPs). For our experiments, we used the mouse HOXA9-ER cell line, which mimics GMPs. We used CRISPR/Cas9 and homologous recombination to fuse FKBPV degron into bi-allelic endogenous loci of the Cebpa gene in HOXA9-ER cells, and, upon addition of dTAG-13 (the ligand for FKBPV), the CEBPA-FKBPV fusion protein could be rapidly degraded within 2 hours (Fig B, C), providing us an experimental system to study the immediate consequences of CEBPA loss. In order to quantify the rate of rRNA transcription, we devised an assay titled "47S FISH-Flow" that combined fluorescent in-situ hybridization (FISH) using probes against nascent 47S rRNA with flow cytometry (Fig D, E). This assay not only allows us to quantify the rate of rRNA transcription on a per-cell basis in millions of cells, but also allows us to separately gate and quantify rRNA transcription in different stages of the cell cycle, eliminating a major confounder in bulk cell studies - cell cycle distribution. Using 47S FISH-Flow, we observed that degradation of CEBPA in the HOXA9-ER mouse GMP cell line led to decrease in synthesis of 47S rRNA within hours (Fig F) before any change in cell cycle or growth kinetics, and was followed by growth arrest in 24 hours. In summary, we show that several critical hematopoietic TFs show abundant, conserved binding to rDNA, and the depletion of CEBPA rapidly reduces nascent rRNA, indicating that it directly promotes rRNA transcription. Our results, and the tools and experimental systems we have developed, shed light on an important and largely unexplored aspect of hematopoietic biology: the regulation of rRNA transcription by a wide range of lineage-specific hematopoietic TFs. Figure Disclosures No relevant conflicts of interest to declare.

MicroRNAs and Ascl1 facilitate direct conversion of porcine fibroblasts into induced neurons.

Direct neuronal conversion describes the process of generating induced neurons from somatic cells such as fibroblasts by overexpressing cell type-specific transcription factors, microRNAs or by culturing in the presence of small molecules. This was first achieved by expressing Brn2, Ascl1 and Myt1L in mouse fibroblasts, and was later achieved in human cells by the inclusion of additional factors such as NeuroD1. Here, we present the first protocol for directly converting porcine fibroblasts into induced neurons. We used lentivirus-mediated delivery of previously identified neuron-specifying transcription factors and microRNAs and evaluated morphology and neuron marker expression after ten days of conversion. We found that Ascl1 and microRNAs, miR-9/9* and miR-124 together generated more neuronal cells than other conditions tested. The porcine induced neurons expressed common mature markers such as MAP2 and Synaptophysin after four weeks of conversion. Transcriptomic analysis revealed that fibroblast-specific signatures were silenced early in the conversion process, while the neuron-specific genes became more abundant during conversion. We generated a heterogeneous population of glutamatergic and GABAergic neurons.

The iNs and Outs of Direct Reprogramming to Induced Neurons.

Understanding of cell-type specific transcription factors has promoted progress in methods for cellular reprogramming, such as directly reprogramming somatic cells to induced neurons (iN). Methods for direct reprogramming require neuronal-fate determining gene activation via neuron-specific microRNAs, chemical modulation of key neuronal signaling pathways or overexpression via viral vectors, with some reprogramming strategies requiring a combination of these methods to induce the neuronal-cell fate. These methods have been employed in a multitude of cell types, including fibroblasts, hepatocytes, peripheral blood mononuclear, and T cells. The ability to create iN from skin biopsies and blood samples coupled with recent advancements in artificially inducing age- and disease-associated phenotypes are accelerating the development of disease models for late-onset neurodegenerative disorders. Here, we review how activation of the neuronal transcriptome alters the epigenetic landscape of the donor cell to facilitate reprogramming to neurons. We also discuss the advantages of using DNA binding domains such as CRISPR/dCas9 to overcome epigenetic barriers to induce neuronal-cell fate by activating endogenous neuronal cell-fate determining genes.

Epigenetic regulator function through mouse gastrulation.

Summary paragraph:During ontogeny, proliferating cells become restricted in their fate through the combined action of cell-type specific transcription factors and ubiquitous epigenetic machinery, which recognize universally available histone residues or nucleotides but are nonetheless deployed in a highly context-dependent manner1,2. The molecular functions of these regulators are generally well understood, but assigning direct developmental roles is hampered by complex mutant phenotypes that often emerge following gastrulation3,4. Recently, single-cell RNA sequencing (scRNA-seq) and analytical approaches have explored this highly conserved process across numerous model organisms5–8, including mouse9–18. To elaborate on these strategies, we investigated a panel of ten essential regulators using a combined zygotic perturbation, scRNA-seq platform where many mutant embryos can be assayed simultaneously to recover robust transcriptional and morphological information. Deeper analysis of central Polycomb Repressive Complex (PRC) 1 and 2 members indicate substantial cooperativity, but distinguishes a PRC2-dominant role in restricting the germline that emerges from gross molecular changes within the initial conceptus. We believe our experimental framework will eventually allow for a fully quantitative view of how cellular diversity emerges using an identical genetic template and from a single totipotent cell.

Single Nuclei Chromatin Profiles of Ventral Midbrain Reveal Cell Identity Transcription Factors and Cell Type-Specific Gene Regulatory Variation

Cell types in ventral midbrain are involved in diseases with variable genetic susceptibility such as Parkinson's disease and schizophrenia. Many genetic variants affect regulatory regions and alter gene expression. We report 20 658 single nuclei chromatin accessibility profiles of ventral midbrain from two genetically and phenotypically distinct mouse strains. We distinguish ten cell types based on chromatin profiles and analysis of accessible regions controlling cell identity genes highlights cell type-specific key transcription factors. Regulatory variation segregating the mouse strains manifests more on transcriptome than chromatin level. However, cell type-level data reveals changes not captured at tissue level. To discover the scope and cell-type specificity of cis-acting variation in midbrain gene expression, we identify putative regulatory variants and show them to be enriched at differentially expressed loci. Finally, we find TCF7L2 to mediate trans-acting variation selectively in midbrain neurons. Our dataset provides an extensive resource to study gene regulation in mesencephalon.

A Generative Neural Network for Maximizing Fitness and Diversity of Synthetic DNA and Protein Sequences.

SUMMARYEngineering gene and protein sequences with defined functional properties is a major goal of synthetic biology. Deep neural network models, together with gradient ascent-style optimization, show promise for sequence design. The generated sequences can however get stuck in local minima and often have low diversity. Here, we develop deep exploration networks (DENs), a class of activation-maximizing generative models, which minimize the cost of a neural network fitness predictor by gradient descent. By penalizing any two generated patterns on the basis of a similarity metric, DENs explicitly maximize sequence diversity. To avoid drifting into low-confidence regions of the predictor, we incorporate variational autoencoders to maintain the likelihood ratio of generated sequences. Using DENs, we engineered polyadenylation signals with more than 10-fold higher selection odds than the best gradient ascent-generated patterns, identified splice regulatory sequences predicted to result in highly differential splicing between cell lines, and improved on state-of-the-art results for protein design tasks.

Corrigendum: Developmental regulation of cell type-specific transcription by novel promoter-proximal sequence elements.

Corrigendum: Developmental regulation of cell type-specific transcription by novel promoter-proximal sequence elements.

Intrinsic and extrinsic mechanisms regulating neuronal dendrite morphogenesis

Neurons are the structural and functional unit of the nervous system. Precisely regulated dendrite morphogenesis is the basis of neural circuit assembly. Numerous studies have been conducted to explore the regulatory mechanisms of dendritic morphogenesis. According to their action regions, we divide them into two categories: the intrinsic and extrinsic regulators of neuronal dendritic morphogenesis. Intrinsic factors are cell type-specific transcription factors, actin polymerization or depolymerization regulators and regulators of the secretion or endocytic pathways. These intrinsic factors are produced by neuron itself and play an important role in regulating the development of dendrites. The extrinsic regulators are either secreted proteins or transmembrane domain containing cell adhesion molecules. They often form receptor-ligand pairs to mediate attractive or repulsive dendritic guidance. In this review, we summarize recent findings on the intrinsic and external molecular mechanisms of dendrite morphogenesis from multiple model organisms, including Caenorhabditis elegans, Drosophila and mice. These studies will provide a better understanding on how defective dendrite development and maintenance are associated with neurological diseases.

Quantitative Proteomics of All 14 Renal Tubule Segments in Rat.

Previous research has used RNA sequencing in microdissected kidney tubules or single cells isolated from the kidney to profile gene expression in each type of kidney tubule epithelial cell. However, because proteins, not mRNA molecules, mediate most cellular functions, it is desirable to know the identity and amounts of each protein species to understand function. Recent improvements in the sensitivity of mass spectrometers offered us the ability to quantify the proteins expressed in each of 14 different renal tubule segments from rat. We manually dissected kidney tubules from rat kidneys and subjected samples to protein mass spectrometry. We used the "proteomic ruler" technique to estimate the number of molecules of each protein per cell. Over the 44 samples analyzed, the average number of quantified proteins per segment was 4234, accounting for at least 99% of protein molecules in each cell. We have made the data publicly available online at the Kidney Tubule Expression Atlas website (https://esbl.nhlbi.nih.gov/KTEA/). Protein abundance along the renal tubule for many commonly studied water and solute transport proteins and metabolic enzymes matched expectations from prior localization studies, demonstrating the overall reliability of the data. The site features a "correlated protein" function, which we used to identify cell type-specific transcription factors expressed along the renal tubule. We identified and quantified proteins expressed in each of the 14 segments of rat kidney tubules and used the proteomic data that we obtained to create an online information resource, the Kidney Tubule Expression Atlas. This resource will allow users throughout the world to browse segment-specific protein expression data and download them for their own research.

SMART-Q: An Integrative Pipeline Quantifying Cell Type-Specific RNA Transcription.

Accurate RNA quantification at the single-cell level is critical for understanding the dynamics of gene expression and regulation across space and time. Single molecule FISH (smFISH), such as RNAscope, provides spatial and quantitative measurements of individual transcripts, therefore, can be used to explore differential gene expression among a heterogeneous cell population if combined with cell identify information. However, such analysis is not straightforward, and existing image analysis pipelines cannot integrate both RNA transcripts and cellular staining information to automatically output cell type-specific gene expression. We developed an efficient and customizable analysis method, Single-Molecule Automatic RNA Transcription Quantification (SMART-Q), to enable the analysis of gene transcripts in a cell type-specific manner. SMART-Q efficiently infers cell identity information from multiplexed immuno-staining and quantifies cell type-specific transcripts using a 3D Gaussian fitting algorithm. Furthermore, we have optimized SMART-Q for user experiences, such as flexible parameters specification, batch data outputs, and visualization of analysis results. SMART-Q meets the demands for efficient quantification of single-molecule RNA and can be widely used for cell type-specific RNA transcript analysis.

Developmental regulation of cell type-specific transcription by novel promoter-proximal sequence elements.

Cell type-specific transcriptional programs that drive differentiation of specialized cell types are key players in development and tissue regeneration. One of the most dramatic changes in the transcription program in Drosophila occurs with the transition from proliferating spermatogonia to differentiating spermatocytes, with >3000 genes either newly expressed or expressed from new alternative promoters in spermatocytes. Here we show that opening of these promoters from their closed state in precursor cells requires function of the spermatocyte-specific tMAC complex, localized at the promoters. The spermatocyte-specific promoters lack the previously identified canonical core promoter elements except for the Inr. Instead, these promoters are enriched for the binding site for the TALE-class homeodomain transcription factors Achi/Vis and for a motif originally identified under tMAC ChIP-seq peaks. The tMAC motif resembles part of the previously identified 14-bp β2UE1 element critical for spermatocyte-specific expression. Analysis of downstream sequences relative to transcription start site usage suggested that ACA and CNAAATT motifs at specific positions can help promote efficient transcription initiation. Our results reveal how promoter-proximal sequence elements that recruit and are acted upon by cell type-specific chromatin binding complexes help establish a robust, cell type-specific transcription program for terminal differentiation.