Uncovering the distinct properties of a bacterial type I-E CRISPR activation system

ABSTRACTSynthetic gene regulators based upon CRISPR-Cas systems offer highly programmable technologies to control gene expression in bacteria. Bacterial CRISPR activators (CRISPRa) have been developed that use engineered type II CRISPR-dCas9 to localize transcription activation domains near promoter elements to activate transcription. However, several reports have demonstrated distance-dependent requirements and periodical activation patterns that overall limit the flexibility of these systems. Here, we demonstrate the potential of using an alternative type I-E CRISPR-Cas system to create a CRISPRa with distinct and expanded regulatory properties. We create the first bacterial CRISPRa system based upon a type I-E CRISPR-Cas, and demonstrate differences in the activation range of this system compared to type II CRISPRa systems. Furthermore, we characterize the distance-dependent activation patterns of type I-E CRISPRa to reveal a distinct and more frequent periodicity of activation.

Modular and Molecular Optimization of a LOV (Light–Oxygen–Voltage)-Based Optogenetic Switch in Yeast

Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light–oxygen–voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call “HAP-LOV”, displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

Relationship between the structure and function of the transcriptional regulator E2A

E proteins are transcriptional regulators that regulate many developmental processes in animals and lymphocytosis and leukemia in Homo sapiens. In particular, E2A, a member of the E protein family, plays a major role in the transcriptional regulatory network that promotes the differentiation and development of B and T lymphocytes. E2A-mediated transcriptional regulation usually requires the formation of E2A dimers, which then bind to coregulators. In this review, we summarize the mechanisms by which E2A participates in transcriptional regulation from a structural perspective. More specifically, the C-terminal helix-loop-helix (HLH) region of the basic HLH (bHLH) domain first dimerizes, and then the activation domains of E2A bind to different coactivators or corepressors in different cell contexts, resulting in histone acetylation or deacetylation, respectively. Then, the N-terminal basic region (b) of the bHLH domain binds to or dissociates from a specific DNA motif (E-box sequence). Last, trans-activation or trans-repression occurs. We also summarize the properties of these E2A domains and their interactions with the domains of other proteins. The feasibility of developing drugs based on these domains is discussed.

Target Gene Identification and sgRNA Design for Waterlogging Tolerance in Foxtail Millet via CRISPR-based Transcriptional Activation

Background: CRISPR activation (CRISPRa) uses non-functional Cas9 endonuclease (dCas9) but retains the genome targeting ability through its single guide RNAs (sgRNAs). CRISPRa is widely utilized as a gene activation system exploiting its ability to recruit various transcriptional activation domains (TADs) to enhance the expression of the target gene(s). Drought tolerant and resource-efficient crops like millets can mitigate the effects of climate change and strengthen food security. Objective: This study aimed to use the Setaria italica (foxtail millet) genome sequence to identify a target gene and the subsequent generation of sgRNAs for use in CRISPRa for conferring waterlogging tolerance that will benefit the future expansion of its cultivation area. Methods and Results: Leveraging existing RNA-seq data and information on functional studies in model plants and from other cereal species, maize and barley, have enabled the identification of candidate ERFVII from the foxtail millet genome sequence in the attempt to engineer waterlogging tolerance. The study provides a step-by-step example for using publicly accessible databases and bioinformatics tools from NCBI and Phytozome to identify and characterize the ortholog from Setaria italica. Softberry was used for promoter annotation to obtain the transcription start site (TSS). Subsequently, CRISP-P 2.0 design tools were employed to generate and select a few efficient sgRNAs for CRISPRa that minimize potentially deleterious off-target binding. Conclusion: The study is a helpful example of how to advance in genomics research, including the revolutionizing CRISPR technology in Setaria italica, which can be adopted in other plant species by utilizing the available genome sequence.

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

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity ('fuzzy' binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

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

SUMMARYGene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity (“fuzzy” binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

Human transcriptional activation domains require hydrophobic and acidic residues

Transcription factors activate gene expression with separable DNA binding domains and activation domains (Latchman, 2008). High-throughput studies have uncovered rules for how DNA binding domains recognize their cognate DNA motifs, but the design principles of activation domains remain opaque. For over thirty years it has been a mystery why activation domains are acidic and unstructured (Sigler, 1988). Activation domains require hydrophobic motifs to bind coactivators and join transcriptional condensates, but low evolutionary conservation and intrinsic disorder have made it difficult to identify the design principles that govern the sequence to function relationship (Boija et al., 2018; Chong et al., 2018; Cress and Triezenberg, 1991; Dyson and Wright, 2016). Consequently, activation domains cannot be predicted from amino acid sequence (Finn et al., 2016). Here, we resolve the functional roles of acidity and disorder in activation domains and use these insights to build a new predictor. We designed sequence variants in seven acidic activation domains and measured their activities in parallel with a high-throughput assay in human cell culture. Our results support a flexible model in which acidic residues solubilize hydrophobic motifs so that they can interact with coactivators. This model accurately predicts activation domains in the human proteome. We identify three general rules for activation domain function: hydrophobic motifs must be balanced by acidic residues; acidic residues make large contributions to activity when they are adjacent to motifs; and within motifs, the presence of aromatic or leucine residues reflects the structural constraints of coactivator interactions. We anticipate these design principles will aid efforts to predict activations from amino acid sequence and to engineer new domains.

Automated CUT&Tag profiling of chromatin heterogeneity in mixed-lineage leukemia

Acute myeloid and lymphoid leukemias often harbor chromosomal translocations involving the Mixed Lineage Leukemia-1 gene, which encodes the KMT2A lysine methyltransferase. The most common translocations produce in-frame fusions of KMT2A to trans-activation domains of chromatin regulatory proteins. Here we develop a strategy to map the genome-wide occupancy of oncogenic KMT2A fusion proteins in primary patient samples regardless of fusion partner. By modifying the versatile CUT&Tag method for full automation we identify common and tumor-specific patterns of aberrant chromatin regulation induced by different KMT2A fusion proteins. Integration of automated and single-cell CUT&Tag uncovers lineage heterogeneity within patient samples and provides an attractive avenue for future diagnostics.