Transcription-AustinPub Date : 2023-06-01Epub Date: 2024-01-23DOI: 10.1080/21541264.2023.2295044
Sandra María Fernández-Moya, Akshay Jaya Ganesh, Mireya Plass
{"title":"Neural cell diversity in the light of single-cell transcriptomics.","authors":"Sandra María Fernández-Moya, Akshay Jaya Ganesh, Mireya Plass","doi":"10.1080/21541264.2023.2295044","DOIUrl":"10.1080/21541264.2023.2295044","url":null,"abstract":"<p><p>The development of highly parallel and affordable high-throughput single-cell transcriptomics technologies has revolutionized our understanding of brain complexity. These methods have been used to build cellular maps of the brain, its different regions, and catalog the diversity of cells in each of them during development, aging and even in disease. Now we know that cellular diversity is way beyond what was previously thought. Single-cell transcriptomics analyses have revealed that cell types previously considered homogeneous based on imaging techniques differ depending on several factors including sex, age and location within the brain. The expression profiles of these cells have also been exploited to understand which are the regulatory programs behind cellular diversity and decipher the transcriptional pathways driving them. In this review, we summarize how single-cell transcriptomics have changed our view on the cellular diversity in the human brain, and how it could impact the way we study neurodegenerative diseases. Moreover, we describe the new computational approaches that can be used to study cellular differentiation and gain insight into the functions of individual cell populations under different conditions and their alterations in disease.</p>","PeriodicalId":47009,"journal":{"name":"Transcription-Austin","volume":" ","pages":"158-176"},"PeriodicalIF":3.6,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10807474/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139479182","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Transcription-AustinPub Date : 2023-06-01Epub Date: 2023-04-16DOI: 10.1080/21541264.2023.2200721
Pooja Sant, Karsten Rippe, Jan-Philipp Mallm
{"title":"Approaches for single-cell RNA sequencing across tissues and cell types.","authors":"Pooja Sant, Karsten Rippe, Jan-Philipp Mallm","doi":"10.1080/21541264.2023.2200721","DOIUrl":"10.1080/21541264.2023.2200721","url":null,"abstract":"<p><p>Single-cell sequencing of RNA (scRNA-seq) has advanced our understanding of cellular heterogeneity and signaling in developmental biology and disease. A large number of complementary assays have been developed to profile transcriptomes of individual cells, also in combination with other readouts, such as chromatin accessibility or antibody-based analysis of protein surface markers. As scRNA-seq technologies are advancing fast, it is challenging to establish robust workflows and up-to-date protocols that are best suited to address the large range of research questions. Here, we review scRNA-seq techniques from mRNA end-counting to total RNA in relation to their specific features and outline the necessary sample preparation steps and quality control measures. Based on our experience in dealing with the continuously growing portfolio from the perspective of a central single-cell facility, we aim to provide guidance on how workflows can be best automatized and share our experience in coping with the continuous expansion of scRNA-seq techniques.</p>","PeriodicalId":47009,"journal":{"name":"Transcription-Austin","volume":" ","pages":"127-145"},"PeriodicalIF":3.6,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10807473/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9761929","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Transcription-AustinPub Date : 2023-06-01Epub Date: 2023-03-16DOI: 10.1080/21541264.2023.2190296
Rituparna Das, Anusree Dey, Sheetal Uppal
{"title":"A method for in situ visualization of Protein-Nascent RNA interactions in single cell using Proximity Ligation Assay (IPNR-PLA) in mammalian cells.","authors":"Rituparna Das, Anusree Dey, Sheetal Uppal","doi":"10.1080/21541264.2023.2190296","DOIUrl":"10.1080/21541264.2023.2190296","url":null,"abstract":"<p><p>Proximity ligation assay (PLA) is an immunofluorescence assay, which determines in situ interaction of two biomolecules present within 40 nm close proximity. Here, we describe a modification of PLA for visual detection of in situ protein interactions with nascent RNA in a single cell (IPNR-PLA). In IPNR-PLA, nascent RNA is labeled by incorporating 5-fluorouridine (FU), a uridine nucleotide analogue, followed by covalent cross-linking of the interacting partners in proximity to newly synthesized RNA. By using combination of anti-BrdU antibody, which specifically binds to FU, and primary antibody against a protein of interest, the IPNR reaction results in fluorescent puncta as a positive signal, only if the candidate proteins are in proximity to nascent RNA. We have validated this method by demonstrating known CDK9 and elongating RNA pol II interaction with nascent RNA. Finally, we used this method to test for the presence of DNA double strand breaks as well as Poly (ADP-ribose) polymerase 1 (PARP1), an RNA binding protein, in the vicinity of nascent RNA in cancer cells. The capability of performing parallel IF labeling and quantifiable multiparameter measurements within heterogeneous cell populations makes IPNR-PLA very attractive for use in biological studies. Overall, we have developed the IPNR-PLA method for analysis of protein association with nascent RNA with single-cell resolution, which is highly sensitive, quantitative, efficient, and requires little starting experimental material.</p>","PeriodicalId":47009,"journal":{"name":"Transcription-Austin","volume":" ","pages":"146-157"},"PeriodicalIF":3.6,"publicationDate":"2023-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10807467/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9124822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Transcription-AustinPub Date : 2022-08-01Epub Date: 2022-09-25DOI: 10.1080/21541264.2022.2127602
Zachary F Mandell, Dani Zemba, Paul Babitzke
{"title":"Factor-stimulated intrinsic termination: getting by with a little help from some friends.","authors":"Zachary F Mandell, Dani Zemba, Paul Babitzke","doi":"10.1080/21541264.2022.2127602","DOIUrl":"10.1080/21541264.2022.2127602","url":null,"abstract":"<p><p>Transcription termination is known to occur via two mechanisms in bacteria, intrinsic termination (also frequently referred to as Rho-independent or factor-independent termination) and Rho-dependent termination. Based primarily on <i>in vitro</i> studies using <i>Escherichia coli</i> RNA polymerase, it was generally assumed that intrinsic termination and Rho-dependent termination are distinct mechanisms and that the signals required for intrinsic termination are present primarily within the nucleic acids. In this review, we detail recent findings from studies in <i>Bacillus subtilis</i> showing that intrinsic termination in this organism is highly stimulated by NusA, NusG, and even Rho. In NusA-stimulated intrinsic termination, NusA facilitates the formation of weak terminator hairpins and compensates for distal U-rich tract interruptions. In NusG-stimulated intrinsic termination, NusG stabilizes a sequence-dependent pause at the point of termination, which extends the time frame for RNA hairpins with weak terminal base pairs to form in either a NusA-stimulated or a NusA-independent fashion. In Rho-stimulated intrinsic termination, Rho prevents the formation of antiterminator-like RNA structures that could otherwise compete with the terminator hairpin. Combined, NusA, NusG, and Rho stimulate approximately 97% of all intrinsic terminators in <i>B. subtilis</i>. Thus, the general view that intrinsic termination is primarily a factor-independent process needs to be revised to account for recent findings. Moreover, the historical distinction between Rho-dependent and intrinsic termination is overly simplistic and needs to be modernized.</p>","PeriodicalId":47009,"journal":{"name":"Transcription-Austin","volume":"13 4-5","pages":"96-108"},"PeriodicalIF":3.6,"publicationDate":"2022-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9715273/pdf/KTRN_13_2127602.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10516566","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Alternative polyadenylation regulation: insights from sequential polyadenylation.","authors":"Peng Tang, Yu Zhou","doi":"10.1080/21541264.2022.2114776","DOIUrl":"https://doi.org/10.1080/21541264.2022.2114776","url":null,"abstract":"<p><p>The processing of the proximal and distal poly(A) sites in alternative polyadenylation (APA) has long been thought to independently occur on pre-mRNAs during transcription. However, a recent study by our groups demonstrated that the proximal sites for many genes could be activated sequentially following the distal ones, suggesting a multi-cleavage-same-transcript mode beyond the canonical one-cleavage-per-transcript view. Here, we review the established mechanisms for APA regulation and then discuss the additional insights into APA regulation from the perspective of sequential polyadenylation, resulting in a unified leverage model for understanding the mechanisms of regulated APA.</p>","PeriodicalId":47009,"journal":{"name":"Transcription-Austin","volume":"13 4-5","pages":"89-95"},"PeriodicalIF":3.6,"publicationDate":"2022-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9715272/pdf/KTRN_13_2114776.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10063203","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Function and dynamics of the Mediator complex: novel insights and new frontiers","authors":"R. Morse","doi":"10.1080/21541264.2022.2085502","DOIUrl":"https://doi.org/10.1080/21541264.2022.2085502","url":null,"abstract":"ABSTRACT The Mediator complex was discovered in the early 1990s as a biochemically fractionated factor from yeast extracts that was necessary for activator-stimulated transcriptional activation to be observed in in vitro transcription assays. The structure of this large, multi-protein complex is now understood in great detail, and novel genetic approaches have provided rich insights into its dynamics during transcriptional activation and the mechanism by which it facilitates activated transcription. Here I review recent findings and unanswered questions regarding Mediator dynamics, the roles of individual subunits, and differences between its function in yeast and metazoan cells.","PeriodicalId":47009,"journal":{"name":"Transcription-Austin","volume":"9 1","pages":"39 - 52"},"PeriodicalIF":3.6,"publicationDate":"2022-05-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"80975616","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"A BEN-domain protein and polycomb complex work coordinately to regulate transcription.","authors":"Fredy Kurniawan, Supriya G Prasanth","doi":"10.1080/21541264.2022.2105128","DOIUrl":"https://doi.org/10.1080/21541264.2022.2105128","url":null,"abstract":"<p><p>Transcription regulation is an important mechanism that controls pluripotency and differentiation. Transcription factors dictate cell fate decisions by functioning cooperatively with chromatin regulators. We have recently demonstrated that BEND3 (BANP, E5R and Nac1 domain) protein regulates the expression of differentiation-associated genes by modulating the chromatin architecture at promoters. We highlight the collaboration of BEND3 with the polycomb repressive complex in coordinating transcription repression and propose a model highlighting the relevance of the BEND3-PRC2 axis in gene regulation and chromatin organization.<b>Abbreviations:</b> BEND3, BANP, E5R and Nac1 domain; rDNA, ribosomal DNA; PRC2, <i>Polycomb</i> Repressive Complex 2; H3K27me3, Histone H3 Lysine 27 methylation; PcG, Polycomb group.</p>","PeriodicalId":47009,"journal":{"name":"Transcription-Austin","volume":"13 1-3","pages":"82-87"},"PeriodicalIF":3.6,"publicationDate":"2022-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9467525/pdf/KTRN_13_2105128.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9888680","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"The histone chaperone FACT: a guardian of chromatin structure integrity.","authors":"Célia Jeronimo, François Robert","doi":"10.1080/21541264.2022.2069995","DOIUrl":"https://doi.org/10.1080/21541264.2022.2069995","url":null,"abstract":"<p><p>The identification of FACT as a histone chaperone enabling transcription through chromatin in vitro has strongly shaped how its roles are envisioned. However, FACT has been implicated in essentially all aspects of chromatin biology, from transcription to DNA replication, DNA repair, and chromosome segregation. In this review, we focus on recent literature describing the role and mechanisms of FACT during transcription. We highlight the prime importance of FACT in preserving chromatin integrity during transcription and challenge its role as an elongation factor. We also review evidence for FACT's role as a cell-type/gene-specific regulator of gene expression and briefly summarize current efforts at using FACT inhibition as an anti-cancer strategy.</p>","PeriodicalId":47009,"journal":{"name":"Transcription-Austin","volume":"13 1-3","pages":"16-38"},"PeriodicalIF":3.6,"publicationDate":"2022-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9467567/pdf/KTRN_13_2069995.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9360692","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Meghan M Conner, Haley V Parker, Daniela R Falcone, Gehoon Chung, Christine E Schaner Tooley
{"title":"Novel regulation of the transcription factor ZHX2 by N-terminal methylation.","authors":"Meghan M Conner, Haley V Parker, Daniela R Falcone, Gehoon Chung, Christine E Schaner Tooley","doi":"10.1080/21541264.2022.2079184","DOIUrl":"https://doi.org/10.1080/21541264.2022.2079184","url":null,"abstract":"<p><p>N-terminal methylation (Nα-methylation) by the methyltransferase NRMT1 is an important post-translational modification that regulates protein-DNA interactions. Accordingly, its loss impairs functions that are reliant on such interactions, including DNA repair and transcriptional regulation. The global loss of Nα-methylation results in severe developmental and premature aging phenotypes, but given over 300 predicted substrates, it is hard to discern which physiological substrates contribute to each phenotype. One of the most striking phenotypes in NRMT1 knockout (<i>Nrmt1<sup>-/-</sup></i>) mice is early liver degeneration. To identify the disrupted signaling pathways leading to this phenotype and the NRMT1 substrates involved, we performed RNA-sequencing analysis of control and <i>Nrmt1<sup>-/-</sup></i> adult mouse livers. We found both a significant upregulation of transcripts in the cytochrome P450 (CYP) family and downregulation of transcripts in the major urinary protein (MUP) family. Interestingly, transcription of both families is inversely regulated by the transcription factor zinc fingers and homeoboxes 2 (ZHX2). ZHX2 contains a non-canonical NRMT1 consensus sequence, indicating that its function could be directly regulated by Nα-methylation. We confirmed misregulation of CYP and MUP mRNA and protein levels in <i>Nrmt1<sup>-/-</sup></i> livers and verified NRMT1 can methylate ZHX2 <i>in vitro</i>. In addition, we used a mutant of ZHX2 that cannot be methylated to directly demonstrate Nα-methylation promotes ZHX2 transcription factor activity and target promoter occupancy. Finally, we show <i>Nrmt1<sup>-/-</sup></i> mice also exhibit early postnatal de-repression of ZHX2 targets involved in fetal liver development. Taken together, these data implicate ZHX2 misregulation as a driving force behind the liver phenotype seen in <i>Nrmt1<sup>-/-</sup></i> mice.</p>","PeriodicalId":47009,"journal":{"name":"Transcription-Austin","volume":"13 1-3","pages":"1-15"},"PeriodicalIF":3.6,"publicationDate":"2022-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9467584/pdf/KTRN_13_2079184.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9518748","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Transcription-AustinPub Date : 2022-02-01Epub Date: 2022-09-01DOI: 10.1080/21541264.2022.2108302
Elisabeth Altendorfer, Yelizaveta Mochalova, Andreas Mayer
{"title":"BRD4: a general regulator of transcription elongation.","authors":"Elisabeth Altendorfer, Yelizaveta Mochalova, Andreas Mayer","doi":"10.1080/21541264.2022.2108302","DOIUrl":"https://doi.org/10.1080/21541264.2022.2108302","url":null,"abstract":"<p><p>Transcription elongation by RNA polymerase II (Pol II) has emerged as a regulatory hub in gene expression. A key control point occurs during early transcription elongation when Pol II pauses in the promoter-proximal region at the majority of genes in mammalian cells and at a large set of genes in <i>Drosophila</i>. An increasing number of <i>trans</i>-acting factors have been linked to promoter-proximal pausing. Some factors help to establish the pause, whereas others are required for the release of Pol II into productive elongation. A dysfunction of this elongation control point leads to aberrant gene expression and can contribute to disease development. The BET bromodomain protein BRD4 has been implicated in elongation control. However, only recently direct BRD4-specific functions in Pol II transcription elongation have been uncovered. This mainly became possible with technological advances that allow selective and rapid ablation of BRD4 in cells along with the availability of approaches that capture the immediate consequences on nascent transcription. This review sheds light on the experimental breakthroughs that led to the emerging view of BRD4 as a general regulator of transcription elongation.</p>","PeriodicalId":47009,"journal":{"name":"Transcription-Austin","volume":" ","pages":"70-81"},"PeriodicalIF":3.6,"publicationDate":"2022-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9467588/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40337396","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}