Artificial Gel-Based Organelles for Spatial Organization of Cell-Free Gene Expression Reactions

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

Download Full-text

Chromatin loop anchors contain core structural components of the gene expression machinery in maize

BMC Genomics ◽

10.1186/s12864-020-07324-0 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Stéphane Deschamps ◽

John A. Crow ◽

Nadia Chaidir ◽

Brooke Peterson-Burch ◽

Sunil Kumar ◽

...

Keyword(s):

Gene Expression ◽

High Resolution ◽

Transcriptional Activity ◽

Spatial Organization ◽

Regulatory Elements ◽

Open Chromatin ◽

Maize Genome ◽

Chromatin Loop ◽

Structural Components ◽

Chromatin Loops

Abstract Background Three-dimensional chromatin loop structures connect regulatory elements to their target genes in regions known as anchors. In complex plant genomes, such as maize, it has been proposed that loops span heterochromatic regions marked by higher repeat content, but little is known on their spatial organization and genome-wide occurrence in relation to transcriptional activity. Results Here, ultra-deep Hi-C sequencing of maize B73 leaf tissue was combined with gene expression and open chromatin sequencing for chromatin loop discovery and correlation with hierarchical topologically-associating domains (TADs) and transcriptional activity. A majority of all anchors are shared between multiple loops from previous public maize high-resolution interactome datasets, suggesting a highly dynamic environment, with a conserved set of anchors involved in multiple interaction networks. Chromatin loop interiors are marked by higher repeat contents than the anchors flanking them. A small fraction of high-resolution interaction anchors, fully embedded in larger chromatin loops, co-locate with active genes and putative protein-binding sites. Combinatorial analyses indicate that all anchors studied here co-locate with at least 81.5% of expressed genes and 74% of open chromatin regions. Approximately 38% of all Hi-C chromatin loops are fully embedded within hierarchical TAD-like domains, while the remaining ones share anchors with domain boundaries or with distinct domains. Those various loop types exhibit specific patterns of overlap for open chromatin regions and expressed genes, but no apparent pattern of gene expression. In addition, up to 63% of all unique variants derived from a prior public maize eQTL dataset overlap with Hi-C loop anchors. Anchor annotation suggests that < 7% of all loops detected here are potentially devoid of any genes or regulatory elements. The overall organization of chromatin loop anchors in the maize genome suggest a loop modeling system hypothesized to resemble phase separation of repeat-rich regions. Conclusions Sets of conserved chromatin loop anchors mapping to hierarchical domains contains core structural components of the gene expression machinery in maize. The data presented here will be a useful reference to further investigate their function in regard to the formation of transcriptional complexes and the regulation of transcriptional activity in the maize genome.

Download Full-text

Combinatorial control of the bradykinin B2 receptor promoter by p53, CREB, KLF-4, and CBP: implications for terminal nephron differentiation

AJP Renal Physiology ◽

10.1152/ajprenal.00370.2004 ◽

2005 ◽

Vol 288 (5) ◽

pp. F899-F909 ◽

Cited By ~ 26

Author(s):

Zubaida Saifudeen ◽

Susana Dipp ◽

Hao Fan ◽

Samir S. El-Dahr

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Dna Binding ◽

Spatial Organization ◽

Kidney Development ◽

Collecting Duct ◽

Proximal Promoter ◽

Bradykinin B2 Receptor ◽

Nephron Differentiation ◽

Terminal Nephron

Despite a wealth of knowledge regarding the early steps of epithelial differentiation, little is known about the mechanisms responsible for terminal nephron differentiation. The bradykinin B2 receptor (B2R) regulates renal function and integrity, and its expression is induced during terminal nephron differentiation. This study investigates the transcriptional regulation of the B2R during kidney development. The rat B2R 5′-flanking region has a highly conserved cis-acting enhancer in the proximal promoter consisting of contiguous binding sites for the transcription factors cAMP response element binding protein (CREB), p53, and Krüppel-like factor (KLF-4). The B2R enhancer drives reporter gene expression in inner medullary collecting duct-3 cells but is considerably weaker in other cell types. Site-directed mutagenesis and expression of dominant negative mutants demonstrated the requirement of CREB DNA binding and Ser-133 phosphorylation for optimal enhancer function. Moreover, helical phasing experiments showed that disruption of the spatial organization of the enhancer inhibits B2R promoter activity. Several lines of evidence indicate that cooperative interactions among the three transcription factors occur in vivo during terminal nephron differentiation: 1) CREB, p53, and KLF-4 are coexpressed in B2R-positive differentiating cells; 2) the maturational expression of B2R correlates with CREB/p53/KLF-4 DNA-binding activity; 3) assembly of CREB, p53, and KLF-4 on chromatin at the endogenous B2R promoter is developmentally regulated and is accompanied by CBP recruitment and histone hyperacetylation; and 4) CREB and p53 occupancy of the B2R enhancer is cooperative. These results demonstrate that combinatorial interactions among the transcription factors, CREB, p53, and KLF-4, and the coactivator CBP, may be critical for the regulation of B2R gene expression during terminal nephron differentiation.

Download Full-text

The Toolbox for Untangling Chromosome Architecture in Immune Cells

Frontiers in Immunology ◽

10.3389/fimmu.2021.670884 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shuai Liu ◽

Keji Zhao

Keyword(s):

Gene Expression ◽

Immune Cells ◽

Spatial Organization ◽

Chromosome Structure ◽

Immune Cell ◽

Genome Structure ◽

Chromosome Architecture ◽

The Past ◽

Important Player ◽

The Way

The code of life is not only encrypted in the sequence of DNA but also in the way it is organized into chromosomes. Chromosome architecture is gradually being recognized as an important player in regulating cell activities (e.g., controlling spatiotemporal gene expression). In the past decade, the toolbox for elucidating genome structure has been expanding, providing an opportunity to explore this under charted territory. In this review, we will introduce the recent advancements in approaches for mapping spatial organization of the genome, emphasizing applications of these techniques to immune cells, and trying to bridge chromosome structure with immune cell activities.

Download Full-text

Phosphorylation-dependent mitotic SUMOylation drives nuclear envelope–chromatin interactions

The Journal of Cell Biology ◽

10.1083/jcb.202103036 ◽

2021 ◽

Vol 220 (12) ◽

Cited By ~ 1

Author(s):

Christopher Ptak ◽

Natasha O. Saik ◽

Ashwini Premashankar ◽

Diego L. Lapetina ◽

John D. Aitchison ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Envelope ◽

Spatial Organization ◽

G1 Phase ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Chromatin Binding ◽

Chromatin Interactions ◽

Sumo Ligase ◽

Pore Complexes

In eukaryotes, chromatin binding to the inner nuclear membrane (INM) and nuclear pore complexes (NPCs) contributes to spatial organization of the genome and epigenetic programs important for gene expression. In mitosis, chromatin–nuclear envelope (NE) interactions are lost and then formed again as sister chromosomes segregate to postmitotic nuclei. Investigating these processes in S. cerevisiae, we identified temporally and spatially controlled phosphorylation-dependent SUMOylation events that positively regulate postmetaphase chromatin association with the NE. Our work establishes a phosphorylation-mediated targeting mechanism of the SUMO ligase Siz2 to the INM during mitosis, where Siz2 binds to and SUMOylates the VAP protein Scs2. The recruitment of Siz2 through Scs2 is further responsible for a wave of SUMOylation along the INM that supports the assembly and anchorage of subtelomeric chromatin at the INM and localization of an active gene (INO1) to NPCs during the later stages of mitosis and into G1-phase.

Download Full-text

A gene regulatory motif that generates oscillatory or multiway switch outputs

Journal of The Royal Society Interface ◽

10.1098/rsif.2012.0826 ◽

2013 ◽

Vol 10 (79) ◽

pp. 20120826 ◽

Cited By ~ 39

Author(s):

Jasmina Panovska-Griffiths ◽

Karen M. Page ◽

James Briscoe

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Spatial Organization ◽

Formation Mechanisms ◽

Regulatory Interactions ◽

Qualitative Dynamics ◽

Gene Regulatory ◽

Oscillatory Mechanisms ◽

Parameter Values ◽

Natural Way

The pattern of gene expression in a developing tissue determines the spatial organization of cell type generation. We previously defined regulatory interactions between a set of transcription factors that specify the pattern of gene expression in progenitors of different neuronal subtypes of the vertebrate neural tube. These transcription factors form a circuit that acts as a multistate switch, patterning the tissue in response to a gradient of Sonic Hedgehog. Here, by simplifying aspects of the regulatory interactions, we found that the topology of the circuit allows either switch-like or oscillatory behaviour depending on parameter values. The qualitative dynamics appear to be controlled by a simpler sub-circuit, which we term the AC–DC motif. We argue that its topology provides a natural way to implement a multistate gene expression switch and we show that the circuit is readily extendable to produce more distinct stripes of gene expression. Our analysis also suggests that AC–DC motifs could be deployed in tissues patterned by oscillatory mechanisms, thus blurring the distinction between pattern-formation mechanisms relying on temporal oscillations or graded signals. Furthermore, during evolution, mechanisms of gradient interpretation might have arisen from oscillatory circuits, or vice versa.

Download Full-text

Cell-Free Gene Expression from

Cell-Free Gene Expression - Methods in Molecular Biology ◽

10.1007/978-1-0716-1998-8_8 ◽

2022 ◽

pp. 135-149

Author(s):

Michael Levy ◽

Ohad Vonshak ◽

Yiftach Divon ◽

Ferdinand Greiss ◽

Noa Avidan ◽

...

Keyword(s):

Gene Expression ◽

Free Gene

Download Full-text

OnTAD: hierarchical domain structure reveals the divergence of activity among TADs and boundaries

Genome Biology ◽

10.1186/s13059-019-1893-y ◽

2019 ◽

Vol 20 (1) ◽

Cited By ~ 7

Author(s):

Lin An ◽

Tao Yang ◽

Jiahao Yang ◽

Johannes Nuebler ◽

Guanjue Xiang ◽

...

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Domain Structure ◽

High Frequency ◽

Spatial Organization ◽

Structural Units ◽

Regulatory Interactions ◽

Link Type ◽

Topologically Associating Domains

AbstractThe spatial organization of chromatin in the nucleus has been implicated in regulating gene expression. Maps of high-frequency interactions between different segments of chromatin have revealed topologically associating domains (TADs), within which most of the regulatory interactions are thought to occur. TADs are not homogeneous structural units but appear to be organized into a hierarchy. We present OnTAD, an optimized nested TAD caller from Hi-C data, to identify hierarchical TADs. OnTAD reveals new biological insights into the role of different TAD levels, boundary usage in gene regulation, the loop extrusion model, and compartmental domains. OnTAD is available at https://github.com/anlin00007/OnTAD.

Download Full-text