BackgroundTrypanosoma brucei is a unicellular parasite which multiplies in mammals (bloodstream form) and Tsetse flies (procyclic form). Trypanosome RNA polymerase II transcription is polycistronic, individual mRNAs being excised by trans splicing and polyadenylation. We previously made detailed measurements of mRNA half-lives in bloodstream and procyclic forms, and developed a mathematical model of gene expression for bloodstream forms. At the whole transcriptome level, many bloodstream-form mRNAs were less abundant than was predicted by the model.ResultsWe refined the published mathematical model and extended it to the procyclic form. We used the model, together with known mRNA half-lives, to predict the abundances of individual mRNAs, assuming rapid, unregulated mRNA processing; then we compared the results with measured mRNA abundances. Remarkably, the abundances of most mRNAs in procyclic forms are predicted quite well by the model, being largely explained by variations in mRNA decay rates and length. In bloodstream forms substantially more mRNAs are less abundant than predicted. We list mRNAs that are likely to show particularly slow or inefficient processing, either in both forms or with developmental regulation. We also measured ribosome occupancies of all mRNAs in trypanosomes grown in the same conditions as were used to measure mRNA turnover. In procyclic forms there was a weak positive correlation between ribosome density and mRNA half-life, suggesting cross-talk between translation and mRNA decay; ribosome density was related to the proportion of the mRNA on polysomes, indicating control of translation initiation. Ribosomal protein mRNAs in procyclics appeared to be exceptionally rapidly processed but poorly translated.ConclusionsLevels of mRNAs in procyclic form trypanosomes are determined mainly by length and mRNA decay, with some control of precursor processing. In bloodstream forms variations in nuclear events play a larger role in transcriptome regulation, suggesting aquisition of new control mechanisms during adaptation to mammalian parasitism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2624-3) contains supplementary material, which is available to authorized users.
Background: Histone H1 is the most mobile histone in the cell nucleus. Defining the positions of H1 on chromatin in situ, therefore, represents a challenge. Immunoprecipitation of formaldehyde-fixed and sonicated chromatin, followed by DNA sequencing (xChIP-seq), is traditionally the method for mapping histones onto DNA elements. But since sonication fragmentation precedes ChIP, there is a consequent loss of information about chromatin higherorder structure. Here, we present a new method, xxChIP-seq, employing antibody binding to fixed intact in situ chromatin, followed by extensive washing, a second fixation, sonication and immunoprecipitation. The second fixation is intended to prevent the loss of specifically bound antibody during washing and subsequent sonication and to prevent antibody shifting to epitopes revealed by the sonication process. In many respects, xxChIP-seq is comparable to immunostaining microscopy, which also involves interaction of the primary antibody with fixed and permeabilized intact cells. The only epitopes displayed after immunostaining are the "exposed" epitopes, not "hidden" by the fixation of chromatin higher-order structure. Comparison of immunoprecipitated fragments between xChIP-seq versus xxChIP-seq should indicate which epitopes become inaccessible with fixation and identify their associated DNA elements. Results: We determined the genomic distribution of histone variants H1.2 and H1.5 in human myeloid leukemia cells HL-60/S4 and compared their epitope exposure by both xChIP-seq and xxChIP-seq, as well as high-resolution microscopy, illustrating the influences of preserved chromatin higher-order structure in situ. We found that xChIP and xxChIP H1 signals are in general negatively correlated, with differences being more pronounced near active regulatory regions. Among the intriguing observations, we find that transcription-related regions and histone PTMs (i.e., enhancers, promoters, CpG islands, H3K4me1, H3K4me3, H3K9ac, H3K27ac and H3K36me3) exhibit significant deficiencies (depletions) in H1.2 and H1.5 xxChIP-seq reads, compared to xChIP-seq. These observations suggest the existence of in situ transcription-related chromatin higher-order structures stabilized by formaldehyde. Conclusion: Comparison of H1 xxChIP-seq to H1 xChIP-seq allows the development of hypotheses on the chromosomal localization of (stabilized) higher-order structure, indicated by the generation of "hidden" H1 epitopes following formaldehyde crosslinking. Changes in H1 epitope exposure surrounding averaged chromosomal binding sites or epigenetic modifications can also indicate whether these sites have chromatin higher-order structure. For example, comparison between averaged active or inactive promoter regions suggests that both regions can acquire stabilized
Deploying off-the-shelf segmentation networks on biomedical data has become common practice, yet if structures of interest in an image sequence are visible only temporarily, existing frame-by-frame methods fail. In this paper, we provide a solution to segmentation of imperfect data through time based on temporal propagation and uncertainty estimation. We integrate uncertainty estimation into Mask R-CNN network and propagate motion-corrected segmentation masks from frames with low uncertainty to those frames with high uncertainty to handle temporary loss of signal for segmentation. We demonstrate the value of this approach over frame-by-frame segmentation and regular temporal propagation on data from human embryonic kidney (HEK293T) cells transiently transfected with a fluorescent protein that moves in and out of the nucleus over time. The method presented here will empower microscopic experiments aimed at understanding molecular and cellular function.
In the rapidly expanding field of peptide therapeutics, the short in vivo half-life of peptides represents a considerable limitation for drug action. D-peptides, consisting entirely of the dextrorotatory enantiomers of naturally occurring levorotatory amino acids (AAs), do not suffer from these shortcomings as they are intrinsically resistant to proteolytic degradation, resulting in a favourable pharmacokinetic profile. To experimentally identify D-peptide binders to interesting therapeutic targets, so-called mirror-image phage display is typically performed, whereby the target is synthesized in D-form and L-peptide binders are screened as in conventional phage display. This technique is extremely powerful, but it requires the synthesis of the target in D-form, which is challenging for large proteins. Here we present finDr, a novel web server for the computational identification and optimization of D-peptide ligands to any protein structure ( https://findr.biologie.uni-freiburg.de/ ). finDr performs molecular docking to virtually screen a library of helical 12-mer peptides extracted from the RCSB Protein Data Bank (PDB) for their ability to bind to the target. In a separate, heuristic approach to search the chemical space of 12-mer peptides, finDr executes a customizable evolutionary algorithm (EA) for the de novo identification or optimization of D-peptide ligands. As a proof of principle, we demonstrate the validity of our approach to predict optimal binders to the pharmacologically relevant target phenol soluble modulin alpha 3 (PSMα3), a toxin of methicillin-resistant Staphylococcus aureus (MRSA). We validate the predictions using in vitro binding assays, supporting the success of this approach. Compared to conventional methods, finDr provides a low cost and easy-to-use alternative for the identification of D-peptide ligands against protein targets of choice without size limitation. We believe finDr will facilitate D-peptide discovery with implications in biotechnology and biomedicine.
Epigenomic regulation plays a vital role in cell differentiation. The leukemic HL-60/S4 [human myeloid leukemic cell line HL-60/S4 (ATCC CRL-3306)] promyelocytic cell can be easily differentiated from its undifferentiated promyelocyte state into neutrophil-and macrophage-like cell states. In this study, we present the underlying genome and epigenome architecture of HL-60/S4 through its differentiation. We performed whole-genome bisulphite sequencing of HL-60/S4 cells and their differentiated counterparts. With the support of karyotyping, we show that HL-60/S4 maintains a stable genome throughout differentiation. Analysis of differential Cytosine-phosphate-Guanine dinucleotide methylation reveals that most methylation changes occur in the macrophage-like state. Differential methylation of promoters was associated with immune-related terms. Key immune genes, CEBPA, GFI1, MAFB and GATA1 showed differential expression and methylation. However, we observed the strongest enrichment of methylation changes in enhancers and CTCF binding sites, implying that methylation plays a major role in large-scale transcriptional reprogramming and chromatin reorganisation during differentiation. Correlation of differential expression and distal methylation with support from chromatin capture experiments allowed us to identify putative proximal and long-range enhancers for a number of immune cell differentiation genes, including CEBPA and CCNF. Integrating expression data, we present a model of HL-60/S4 differentiation in relation to the wider scope of myeloid differentiation.
The optogenetic tool LEXY consists of the second light oxygen voltage (LOV) domain of Avena sativa phototropin 1 mutated to contain a nuclear export signal. It allows exporting from the nucleus with blue light proteins of interest (POIs) genetically fused to it. Mutations slowing the dark recovery rate of the LOV domain within LEXY were recently shown to allow for better depletion of some POIs from the nucleus in Drosophila embryos and for the usage of low light illumination regimes. We investigated these variants in mammalian cells and found they increase the cytoplasmic localization of the proteins we tested after illumination, but also during the dark phases, which corresponds to higher leakiness of the system. These data suggest that, when aiming to sequester into the nucleus a protein with a cytoplasmic function, the original LEXY is preferable. The iLEXY variants are, instead, advantageous when wanting to deplete the nucleus of the POI as much as possible.
In response to different stimuli many transcription factors (TFs) display different activation dynamics that trigger the expression of specific sets of target genes, suggesting that promoters have a way to decode dynamics. Here, we use optogenetics to directly manipulate the nuclear localization of a synthetic TF in mammalian cells without affecting other processes. We generate pulsatile or sustained TF dynamics and employ live cell microscopy and mathematical modelling to analyse the behaviour of a library of reporter constructs. We find decoding of TF dynamics occurs only when the coupling between TF binding and transcription pre-initiation complex formation is inefficient and that the ability of a promoter to decode TF dynamics gets amplified by inefficient translation initiation. Using the knowledge acquired, we build a synthetic circuit that allows obtaining two gene expression programs depending solely on TF dynamics. Finally, we show that some of the promoter features identified in our study can be used to distinguish natural promoters that have previously been experimentally characterized as responsive to either sustained or pulsatile p53 and NF-κB signals. These results help elucidate how gene expression is regulated in mammalian cells and open up the possibility to build complex synthetic circuits steered by TF dynamics.
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