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1. Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator

   https://doi.org/10.7554/ELIFE.68068  

   Sanborn, Adrian L; Yeh, Benjamin T; Feigerle, Jordan T; Hao, Cynthia V; Townshend, Raphael JL; Lieberman Aiden, Erez; Dror, Ron O; Kornberg, Roger D (April 2021, eLife)

   null (Ed.)

   Cells adapt and respond to changes by regulating the activity of their genes. To turn genes on or off, they use a family of proteins called transcription factors. Transcription factors influence specific but overlapping groups of genes, so that each gene is controlled by several transcription factors that act together like a dimmer switch to regulate gene activity. The presence of transcription factors attracts proteins such as the Mediator complex, which activates genes by gathering the protein machines that read the genes. The more transcription factors are found near a specific gene, the more strongly they attract Mediator and the more active the gene is. A specific region on the transcription factor called the activation domain is necessary for this process. The biochemical sequences of these domains vary greatly between species, yet activation domains from, for example, yeast and human proteins are often interchangeable. To understand why this is the case, Sanborn et al. analyzed the genome of baker’s yeast and identified 150 activation domains, each very different in sequence. Three-quarters of them bound to a subunit of the Mediator complex called Med15. Sanborn et al. then developed a machine learning algorithm to predict activation domains in both yeast and humans. This algorithm also showed that negatively charged and greasy regions on the activation domains were essential to be activated by the Mediator complex. Further analyses revealed that activation domains used different poses to bind multiple sites on Med15, a behavior known as ‘fuzzy’ binding. This creates a high overall affinity even though the binding strength at each individual site is low, enabling the protein complexes to remain dynamic. These weak interactions together permit fine control over the activity of several genes, allowing cells to respond quickly and precisely to many changes. The computer algorithm used here provides a new way to identify activation domains across species and could improve our understanding of how living things grow, adapt and evolve. It could also give new insights into mechanisms of disease, particularly cancer, where transcription factors are often faulty. 

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2. Gene regulatory network reconstruction using single-cell RNA sequencing of barcoded genotypes in diverse environments

   https://doi.org/10.7554/eLife.51254  

   Jackson, Christopher A; Castro, Dayanne M; Saldi, Giuseppe-Antonio; Bonneau, Richard; Gresham, David (January 2020, eLife)

   Organisms switch their genes on and off to adapt to changing environments. This takes place thanks to complex networks of regulators that control which genes are actively ‘read’ by the cell to create the RNA molecules that are needed at the time. Piecing together these networks is key to fully understand the inner workings of living organisms, and how to potentially modify or artificially create them. Single-cell RNA sequencing is a powerful new tool that can measure which genes are turned on (or ‘expressed’) in an individual cell. Datasets with millions of gene expression profiles for individual cells now exist for organisms such as mice or humans. Yet, it is difficult to use these data to reconstruct networks of regulators; this is partly because scientists are not sure if the computational methods normally used to build these networks also work for single-cell RNA sequencing data. One way to check if this is the case is to use the methods on single-cell datasets from organisms where the networks of regulators are already known, and check whether the computational tools help to reach the same conclusion. Unfortunately, the regulatory networks in the organisms for which scientists have a lot of single-cell RNA sequencing data are still poorly known. There are living beings in which the networks are well characterised – such as yeast – but it has been difficult to do single-cell sequencing in them at the scale seen in other organisms. Jackson, Castro et al. first adapted a system for single-cell sequencing so that it would work in yeast. This generated a gene expression dataset of over 40,000 yeast cells. They then used a computational method (called the Inferelator) on these data to construct networks of regulators, and the results showed that the method performed well. This allowed Jackson, Castro et al. to start mapping how different networks connect, for example those that control the response to the environment and cell division. This is one of the benefits of single-cell RNA methods: cell division for example is not a process that can be examined at the level of a population, since the cells may all be at different life stages. In the future, the dataset will also be useful to scientists to benchmark a variety of single cell computational tools. 

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3. Single-cell nascent RNA sequencing unveils coordinated global transcription

   https://doi.org/10.1038/s41586-024-07517-7  

   Mahat, Dig B; Tippens, Nathaniel D; Martin-Rufino, Jorge D; Waterton, Sean K; Fu, Jiayu; Blatt, Sarah E; Sharp, Phillip A (July 2024, Nature)

   Abstract Transcription is the primary regulatory step in gene expression. Divergent transcription initiation from promoters and enhancers produces stable RNAs from genes and unstable RNAs from enhancers^1,2. Nascent RNA capture and sequencing assays simultaneously measure gene and enhancer activity in cell populations³. However, fundamental questions about the temporal regulation of transcription and enhancer–gene coordination remain unanswered, primarily because of the absence of a single-cell perspective on active transcription. In this study, we present scGRO–seq—a new single-cell nascent RNA sequencing assay that uses click chemistry—and unveil coordinated transcription throughout the genome. We demonstrate the episodic nature of transcription and the co-transcription of functionally related genes. scGRO–seq can estimate burst size and frequency by directly quantifying transcribing RNA polymerases in individual cells and can leverage replication-dependent non-polyadenylated histone gene transcription to elucidate cell cycle dynamics. The single-nucleotide spatial and temporal resolution of scGRO–seq enables the identification of networks of enhancers and genes. Our results suggest that the bursting of transcription at super-enhancers precedes bursting from associated genes. By imparting insights into the dynamic nature of global transcription and the origin and propagation of transcription signals, we demonstrate the ability of scGRO–seq to investigate the mechanisms of transcription regulation and the role of enhancers in gene expression. 

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4. A new Bayesian factor analysis method improves detection of genes and biological processes affected by perturbations in single-cell CRISPR screening

   https://doi.org/10.1038/s41592-023-02017-4  

   Zhou, Yifan; Luo, Kaixuan; Liang, Lifan; Chen, Mengjie; He, Xin (September 2023, Nature Methods)

   Abstract Clustered regularly interspaced short palindromic repeats (CRISPR) screening coupled with single-cell RNA sequencing has emerged as a powerful tool to characterize the effects of genetic perturbations on the whole transcriptome at a single-cell level. However, due to its sparsity and complex structure, analysis of single-cell CRISPR screening data is challenging. In particular, standard differential expression analysis methods are often underpowered to detect genes affected by CRISPR perturbations. We developed a statistical method for such data, called guided sparse factor analysis (GSFA). GSFA infers latent factors that represent coregulated genes or gene modules; by borrowing information from these factors, it infers the effects of genetic perturbations on individual genes. We demonstrated through extensive simulation studies that GSFA detects perturbation effects with much higher power than state-of-the-art methods. Using single-cell CRISPR data from human CD8⁺T cells and neural progenitor cells, we showed that GSFA identified biologically relevant gene modules and specific genes affected by CRISPR perturbations, many of which were missed by existing methods, providing new insights into the functions of genes involved in T cell activation and neurodevelopment. 

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5. The conformation of the histone H3 tail inhibits association of the BPTF PHD finger with the nucleosome

   https://doi.org/10.7554/eLife.31481  

   Morrison, Emma A; Bowerman, Samuel; Sylvers, Kelli L; Wereszczynski, Jeff; Musselman, Catherine A (April 2018, eLife)

   The human genome contains all the instructions needed to build the human body. However, each human cell does not read all of these instructions, which come in the form of genes encoded in the DNA. Instead, different subsets of genes are switched on in each type of cell, while the rest of the genes are switched off. DNA within human cells is wrapped around proteins called histones, to form hundreds of thousands of structures called nucleosomes. If the DNA that encodes a gene contains a lot of nucleosomes, the DNA is not very accessible and the gene will generally be off; removing the histones or rearranging the nucleosomes can turn the gene on. Each histone contains a region called a tail – because it protrudes like the tail of a cat – that can be chemically modified in dozens of different ways. Particular combinations of histone modifications are thought to signal how the nucleosomes should be arranged so that each gene is properly regulated. However, it is unclear how these combinations of modifications actually work because, historically, it has been difficult to study tails in the context of a nucleosome. Instead most studies had looked at tails that had been removed from the nucleosome. Now, Morrison et al. set out to investigate how one protein, called BPTF, recognizes a specific chemical modification on the tail of a histone, referred to as H3K4me3, in the context of a human nucleosome. Unexpectedly, the experiments showed that the histone-binding domain of BPTF, which binds to H3K4me3, was impeded when the tail was attached to the nucleosome but not when it was removed from the nucleosome. Morrison et al. went on to show that this was because the histone tail is tucked onto the rest of the nucleosome and not easily accessible. Further experiments revealed that additional chemical modifications made the tail more accessible, making it easier for the histone-binding domain to bind. Together these findings show that a combination of histone modifications acts to positively regulate the binding of a regulatory protein to H3K4me3 in the context of the nucleosome by actually regulating the nucleosome itself. The disruption of the histone signals is known to lead to a number of diseases, including cancer, autoimmune disease, and neurological disorders, and these findings could guide further research that may lead to new treatments. Yet first, much more work is needed to investigate how other histone modifications are recognized in the context of the nucleosome, and how the large number of possible combinations of histone signals affects this process. 

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