lysis and digestion of scarce cells

Uma abordagem abrangente para estudar o interactoma do promotor em células escassas

Temporal gene expression drives cell differentiation and, ultimately, organism development, and its alteration is closely related to a wide plethora of diseases1,2,3,4,5. Gene transcription is finely regulated by the action of regulatory elements, which can be classified as proximal (i.e., gene promoters) and distal (e.g., enhancers or silencers), the latter of which are frequently located afar from their target genes and physically interact with them through chromatin looping to modulate gene expression6,7,8.
The identification of distal regulatory regions in the genome is a matter which is widely agreed upon, since these regions harbor specific histone modifications9,10,11 and contain specific transcription factor recognition motifs, acting as recruiting platforms for them12,13,14. Besides, in the case of enhancers and super-enhancers15,16, they also have low-nucleosome occupancy17,18 and are transcribed into non-coding eRNAs19,20.
Nonetheless, each distal regulatory element&#39;s target genes are more difficult to predict. More often than not, interactions between distal regulatory elements and their targets are cell-type and stimulus specific21,22, span hundreds of kilobases, bridging over other genes in any direction23,24,25, and can even be located inside intronic regions of their target gene or other non-intervening genes26,27. Furthermore, distal regulatory elements can also control more than one gene at the same time, and vice versa28,29. This positional complexity hinders pinpointing regulatory associations between them, and therefore, most of each regulatory element&#39;s targets in every cell type remain unknown.
During recent years, there has been a significant boom in the development of chromosome conformation capture (3C) techniques for studying chromatin interactions. The most widely used of them, Hi-C, allows to generate a map of all the interactions between every fragment of a cell&#39;s genome30. However, to detect significant interactions at the restriction fragment level, Hi-C relies on ultra-deep sequencing, prohibiting its use to routinely study the regulatory landscape of individual genes. To overcome this economic limitation, several enrichment-based 3C techniques have emerged, such as ChIA-PET31, HiChIP32, and its low-input counterpart HiCuT33. These techniques depend on the use of antibodies to enrich for genome-wide interactions mediated by a specific protein. Nonetheless, the unique feature of these 3C techniques is also the bane of their application; users count on the availability of high-quality antibodies for the protein of interest and cannot compare conditions in which the binding of the protein is dynamic.
Promoter Capture Hi-C (PCHi-C) is another enrichment-based 3C technique that circumvents these limitations34,35. By employing a biotinylated RNA probe enrichment system, PCHi-C is able to generate genome-wide high-resolution libraries of genomic regions interacting with 28,650 human- or 27,595 mouse-annotated gene promoters, also known as the promoter interactome. This approach allows one to detect significant long-range interactions at the restriction fragment level resolution of both active and inactive promoters, and robustly compare promoter interactomes between any condition independently of the dynamics of histone modifications or protein binding. PCHi-C has been widely used over recent years to identify promoter interactome reorganizations during cell differentiation36,37, identify the mechanism of action of transcription factors38,39, and discover new potential genes and pathways deregulated in disease by non-coding variants40,41,42,43,44,45,46,47,48, alongside new driver non-coding mutations49,50. Besides, by just modifying the capture system, this technique can be customized according to the biological question to interrogate any interactome (e.g., the enhancer interactome51 or the interactome of a collection of non-coding alterations41,52).
However, PCHi-C relies on a minimum of 20 million cells to perform the technique, which prevents the study of scarce cell populations such as the ones often used in developmental biology and clinical applications. For this reason, we have developed low input Capture Hi-C (liCHi-C), a new cost-effective and customizable method based on the experimental framework of PCHi-C to generate high-resolution promoter interactomes with low-cell input. By performing the experiment with minimal tube changes, swapping or eliminating steps from the original PCHi-C protocol, drastically reducing reaction volumes, and modifying reagent concentrations, library complexity is maximized and it is possible to generate high-quality libraries with as little as 50,000 cells53.
Low input Capture Hi-C (liCHi-C) has been benchmarked against PCHi-C and used to elucidate promoter interactome rewiring during human hematopoietic cell differentiation, discover potential new disease-associated genes and pathways deregulated by non-coding alterations, and detect chromosomal abnormalities53. The step-by-step protocol and the different quality controls through the technique are detailed here until the final generation of the libraries and their computational analysis.

Autor em destaque: um fluxo de trabalho integrado para estudar a arquitetura do genoma espaço-temporal centrado no promotor em populações de células escassas  

Um fluxo de trabalho integrado para estudar a arquitetura do genoma espaço-temporal centrada no promotor em populações celulares escassas

With liCHi-C, the main question that we are trying to answer is how the chromatin interacts in the nucleus, and therefore, to understand a new layer of gene regulation. We have proven with liCHi-C that we can explore the genome organization of a given sample, link the non-coding mutation associated with disease with potential genes affected, and detect chromosomal rearrangements, such as translocation, for example, in tumor samples. Now we can explore the 3D chromatin organization of scar cell types, such as primary stem cells or relevant clinical samples.Our protocol reduces the input materials and the time and cost needed to obtain quality data to explore the genome organization at the restriction fragment resolution. liCHi-C is expanding the reach of the 3D chromatin organization field, allowing us to explore new cell types previously impossible to analyze. Thanks to liCHi-C, the scientific community can explore the special regulation of gene expression.For example, in malignant transformation only in a specific clonal sub-population inside a tumor.

Watch this Scientific Journal Video about Lysis and Digestion of Scarce Cells at JoVE.com

Lysis and Digestion of Scarce Cells  

Lise e digestão de células escassas

Para começar, pegue uma alíquota de células escassas congeladas e adicione 1 mililitro de tampão de lise fria para romper a membrana celular. Incubar as células no gelo por 30 minutos, misturando por inversão a cada cinco minutos. Centrifugar os núcleos por 10 minutos a 1.000 G.Retire o sobrenadante e ressuspenda os núcleos em 500 microlitros de tampão de restrição 2 a frio de 1,25X.Em seguida, adicione 5,5 microlitros de dodecil sulfato de sódio a 10%, misture por vórtice e incube em um bloco térmico. Em seguida, apague o SDS adicionando 37,5 microlitros de 10% Triton X-100. Misture e incube a amostra conforme demonstrado anteriormente.Adicione 7,5 microlitros de Hind III para digerir a cromatina, misture a solução e incube durante a noite a 37 graus Celsius.

Research

JoVE Journal

Genetics

Lysis and Digestion of Scarce Cells

212 visualizações.  Josep Carreras Leukaemia Research Institute. Para começar, pegue uma alíquota de células escassas congeladas e adicione 1 mililitro de tampão de lise fria para romper a membrana celular. Incubar as células no gelo por 30 minutos, misturando por inversão a cada cinco minutos. Centrifugar os núcleos por 10 minutos a 1.000 G.Retire o sobrenadante e ressuspenda os núcleos em 500 microlitros de tampão de restrição 2 a frio de 1,25X.Em seguida, adicione 5,5 microlitros de dodecil sulfato de sódio a 10%, misture por vórtice...