27 research outputs found
Histone H3 clipping in human embryonic stem cells: in pursuit of an epigenetic outcast
Epigenetic mechanisms such as posttranslational histone modifications (PTM) regulate gene expression (variations) without altering the DNA sequence itself. Human embryonic stem cells (hESC) are a relevant model to study these mechanisms and are hallmarked by self-renewal and pluripotency. Epigenetics plays a key role in regulating the balance between pluripotency and differentiation, and the accompanying developmental processes.
Upon hESC differentiation, the epigenetic signature and thus transcriptional activity, alters drastically. Those gene expression variations can be monitored via RT-qPCR. To correct for technical variability introduced along the experiment, RT-qPCR data can be normalized to several reference genes selected according to the experimental set-up. As such, B2M, RPL13A and Alu repeats appeared the most stable references for hESC undergoing retinoic acid-induced differentiation and normalization based on this set yielded a significantly different result compared to the use of classic references such as GAPDH.
A whole range of PTM are already described and one particularly interesting is histone clipping, i.e. the removal of a series of amino acids from a histone tail. Based on an earlier publication describing N-terminal histone H3 clipping catalyzed by cathepsin L in differentiating mouse ESC, we showed for the first time that this event also occurs in hESC. However, in this setting H3 clipping occurs in both pluripotent and differentiating hESC, thus a direct link with the differentiation process was not found. We identified three possible cleavage sites and found that the reaction was catalyzed by a serine protease. Histone clipping can severely disturb the epigenetic pattern as it removes existing PTM, disturbs chromatin stability, abolishes the binding of interacting non-histone proteins or conversely creates new target sites. More research is needed to fully uncover the purpose of clipping, but its epigenetic potential is yet obviously clear
Embryonic Stem Cells: Keeping Track of the Pluripotent Status
Embryonic stem cells are defined by their pluripotent status, which allows them to differentiate toward all cell types of an adult organism. This pluripotency can be characterized through many parameters, ranging from morphological traits, over certain enzymatic activities, to the expression of specific pluripotency factors, taken into account that these parameters may vary depending on the pluripotent stem cell type. As such, considerable differences are seen between human and mouse embryonic stem cell (ESC), or more generally stated, between primed and naïve pluripotent stem cells. This chapter offers an overview of the markers involved and the molecular biology techniques to monitor them during both ESC culture maintenance or differentiation experiments
Reference loci for RT-qPCR analysis of differentiating human embryonic stem cells
Background: Selecting stably expressed reference genes is essential for proper reverse transcription quantitative polymerase chain reaction gene expression analysis. However, this choice is not always straightforward. In the case of differentiating human embryonic stem (hES) cells, differentiation itself introduces changes whereby reference gene stability may be influenced.
Results: In this study, we evaluated the stability of various references during retinoic acid-induced (2 microM) differentiation of hES cells. Out of 12 candidate references, beta-2-microglobulin, ribosomal protein L13A and Alu repeats are found to be the most stable for this experimental set-up.
Conclusions: Our results show that some of the commonly used reference genes are actually not amongst the most stable loci during hES cell differentiation promoted by retinoic acid. Moreover, a novel normalization strategy based on expressed Alu repeats is validated for use in hES cell experiments
Comparison of fractionation proteomics for local SWATH library building
For data-independent acquisition by means of sequential window acquisition of all theoretical fragment ion spectra (SWATH), a reference library of data-dependent acquisition (DDA) runs is typically used to correlate the quantitative data from the fragment ion spectra with peptide identifications. The quality and coverage of such a reference library is therefore essential when processing SWATH data. In general, library sizes can be increased by reducing the impact of DDA precursor selection with replicate runs or fractionation. However, these strategies can affect the match between the library and SWATH measurement, and thus larger library sizes do not necessarily correspond to improved SWATH quantification. Here, three fractionation strategies to increase local library size were compared to standard library building using replicate DDA injection: protein SDS-PAGE fractionation, peptide high-pH RP-HPLC fractionation and MS-acquisition gas phase fractionation. The impact of these libraries on SWATH performance was evaluated in terms of the number of extracted peptides and proteins, the match quality of the peptides and the extraction reproducibility of the transitions. These analyses were conducted using the hydrophilic proteome of differentiating human embryonic stem cells. Our results show that SWATH quantitative results and interpretations are affected by choice of fractionation technique. Data are available via ProteomeXchange with identifier PXD006190
Extracting histones for the specific purpose of label-free MS
Extracting histones from cells is the first step in studies that aim to characterize histones and their post-translational modifications (hPTMs) with MS. In the last decade, label-free quantification is more frequently being used for MS-based histone characterization. However, many histone extraction protocols were not specifically designed for label-free MS. While label-free quantification has its advantages, it is also very susceptible to technical variation. Here, we adjust an established histone extraction protocol according to general label-free MS guidelines with a specific focus on minimizing sample handling. These protocols are first evaluated using SDS-PAGE. Hereafter, a selection of extraction protocols was used in a complete histone workflow for label-free MS. All protocols display nearly identical relative quantification of hPTMs. We thus show that, depending on the cell type under investigation and at the cost of some additional contaminating proteins, minimizing sample handling can be done during histone isolation. This allows analyzing bigger sample batches, leads to reduced technical variation and minimizes the chance of in vitro alterations to the hPTM snapshot. Overall, these results allow researchers to determine the best protocol depending on the resources and goal of their specific study. Data are available via ProteomeXchange with identifier PXD002885
Neutrophil elastase in the capacity of the 'H2A-specific protease'
The amino-terminal tail of histones and the carboxy-tail of histone H2A protrude from the nucleosome and can become modified by many different posttranslational modifications (PTM). During a mass spectrometric proteome analysis on haematopoietic cells we encountered a histone PTM that has received only little attention since its discovery over 35 years ago: truncation of the histone H2A C-tail at V-114 which is mediated by the "H2A specific protease" (H2Asp). This enzyme is still referenced today but it was never identified. We first developed a sensitive AQUA approach for specific quantitation of the H2AV(114) clipping. This clipping was found only in myeloid cells and further cellular fractionation lead to the annotation of the H2Asp as Neutrophil Elastase (NE). Ultimate proof was provided by NE incubation experiments and by studying histone extracts from NE Null mice. The annotation of the H2Asp not only is an indispensable first step in elucidating the potential biological role of this enzymatic interaction but equally provides the necessary background to critically revise earlier reports of H2A clipping
Quantitative proteomics to characterize specific histone H2A proteolysis in chronic lymphocytic leukemia and the myeloid THP-1 cell line
Proteome studies on hematological malignancies contribute to the understanding of the disease mechanism and to the identification of new biomarker candidates. With the isobaric tag for relative and absolute quantitation (iTRAQ) method we analyzed the protein expression between B-cells of healthy people and chronic lymphocytic leukemia (CLL) B-cells. CLL is the most common lymphoid cancer of the blood and is characterized by a variable clinical course. By comparing samples of patients with an aggressive vs. indolent disease, we identified a limited list of differentially regulated proteins. The enhanced sensitivity attributed to the iTRAQ labels led to the discovery of a previously reported but still not clarified proteolytic product of histone H2A (cH2A) which we further investigated in light of the suggested functional properties of this modification. In the exploratory proteome study the Histone H2A peptide was up-regulated in CLL samples but a more specific and sensitive screening of a larger patient cohort indicated that cH2A is of myeloid origin. Our subsequent quantitative analysis led to a more profound characterization of the clipping in acute monocytic leukemia THP-1 cells subjected to induced differentiation
Influence of activin A supplementation during human embryonic stem cell derivation on germ cell differentiation potential
Human embryonic stem cells (hESCs) are more similar to primed mouse epiblast stem cells (mEpiSCs). mEpiSCs, which are derived in Activin A, show an increased propensity to form primordial germ cell (PGC)-like cells in response to bone morphogenic protein 4 (BMP4). Hence, we hypothesized that hESCs derived in the presence of Activin A may be more competent in differentiating towards PGC-like cells after supplementation with BMP4 compared to standard hESC lines. We were able to successfully derive two hESC lines in the presence of Activin A, which were pluripotent and showed higher base levels of STELLA and cKIT compared to standard hESC lines derived without Activin A addition. Furthermore, upon differentiation as embryoid bodies in the presence of BMP4, we observed upregulation of VASA at day 7, both at the transcript and protein level compared to standard hESC lines, which appeared to take longer time for PGC specification. Unlike other hESC lines, nuclear pSMAD2/3 presence confirmed that Activin signalling was switched on in Activin A-derived hESC lines. They were also responsive to BMP4 based on nuclear detection of pSMAD1/5/8 and showed endodermal differentiation as a result of GATA-6 expression. Hence, our results provide novel insights into the impact of hESC derivation in the presence of Activin A and its subsequent influence on germ cell differentiation potential in vitro
Use of amplicon-based sequencing for testing fetal identity and monogenic traits with Single Circulating Trophoblast (SCT) as one form of cell-based NIPT
A major challenge for cell-based non-invasive prenatal testing (NIPT) is to distinguish individual presumptive fetal cells from maternal cells in female pregnancies. We have sought a rapid, robust, versatile, and low-cost next-generation sequencing method to facilitate this process. Toward this goal, single isolated cells underwent whole genome amplification prior to genotyping. Multiple highly polymorphic genomic regions (including HLA-A and HLA-B) with 10-20 very informative single nucleotide polymorphisms (SNPs) within a 200 bp interval were amplified with a modified method based on other publications. To enhance the power of cell identification, approximately 40 Human Identification SNP (Applied Biosystems) test amplicons were also utilized. Using SNP results to compare to sex chromosome data from NGS as a reliable standard, the true positive rate for genotyping was 83.4%, true negative 6.6%, false positive 3.3%, and false negative 6.6%. These results would not be sufficient for clinical diagnosis, but they demonstrate the general validity of the approach and suggest that deeper genotyping of single cells could be completely reliable. A paternal DNA sample is not required using this method. The assay also successfully detected pathogenic variants causing Tay Sachs disease, cystic fibrosis, and hemoglobinopathies in single lymphoblastoid cells, and disease-causing variants in three cell-based NIPT cases. This method could be applicable for any monogenic diagnosis
Detailed method description for noninvasive monitoring of differentiation status of human embryonic stem cells
The (non)differentiation status of human embryonic stem cells (hESCs) is usually analyzed by determination of key pluripotency defining markers (e.g., OCT4, Nanog, SOX2) by means of reverse transcription quantitative polymerase chain reaction (RT-qPCR), flow cytometry (FC), and immunostaining. Despite proven usefulness of these techniques, their destructive nature makes it impossible to follow up on the same hESC colonies for several days, leading to a loss of information. In 2003, an OCT4-eGFP knock-in hESC line to monitor OCT4 expression was developed and commercialized. However, to the best of our knowledge, the use of fluorescence microscopy (FM) for monitoring the OCT4-eGFP expression of these cells without sacrificing them has not been described to date. Here, we describe such a method in detail, emphasizing both its resolving power and its complementary nature to FC as well as the potential pitfalls in standardizing the output of the FM measurements. The potential of the method is demonstrated by comparison of hESCs cultured in several conditions, both feeder free (vitronectin, VN) and grown on feeder cells (mouse embryonic fibroblasts, MEFs)