Investigation of HDAC inhibitors action on metabolism using sci-Plex

da | Giu 26, 2020 | Biologia Molecolare

Abstract

Single-cell combinatorial indexing (sci-Plex) is an innovative sequencing technique to sequence transcriptomes from thousands of independently perturbated cells in a unique experiment. It revealed that HDAC inhibitors blocks the cell cycle inducing a metabolically depletion of acetyl-CoA precursors.

Sci-Plex combines nuclear hashing with sciRNA-seq in a single workflow and permits to profile information about thousands of independent perturbations at a single cell resolution. The technique exploits nuclear split-pool barcoding of cells using ssDNA and was illustrated in Science by Srivatsan et al. (1). Nuclei of permeabilized cells were barcoded in a 96wells plate using a specific strand of DNA, then pooled and subjected to a two-level sci-RNA-seq. The work-flow allowed the authors to perturb cells with a large number of drugs (188 in this experiment) at different doses in duplicate, obtaining hundreds of thousands of single-cell transcriptomes (about 650.000 in this experiment).

The advantage of sci-Plex is represented by the possibility to sequence all cells in a unique experiment at single-cell resolution thanks to the use of “nuclear hashing”, consisting in three consecutive pooling-split barcoding cycles (Figure 1).

In high throughput screens (HTS) it is impossible to obtain such a resolution unless you drill thousands of independent experiments, one for each drug-dose combination (2) (3). Authors demonstrated that sci-Plex can lead to collision events (two or more nuclei labeled with the same combination of barcodes by chance) but the rate of collision events can be reduced limiting the overloading by reducing the number of cells in each well of the plate.

Figure 1 – Schematic work flow of sci-Plex

Among all drugs, 17 HDAC inhibitors (HDACi) were tested.  To assess the similarity of the response among three cell lines (lung adenocarcinoma A549, chronic myelogenous leukemia K562, mammary adenocarcinoma MCF7), “pseudodose” trajectory was built using a mutual-nearest neighbor matching approach (4) (5).

This trajectory showed that some HDACi lead to a homogeneous response, with cells distributed in a narrow range of the pseudodose trajectory, whereas other drugs induced a much greater heterogeneity. One important pattern shared by every cell-line was the enrichment of genes and pathways indicative of cell-cycle arrest this common response also included an alteration of cellular metabolism genes expression.  

HDAC class I-, II- and IV- mediate the deacetylation of histone. The action of those enzymes is very important because acetate is the main precursor of acetyl-CoA, and it is required to chromatin acetylation. Those proteins have an important role in metabolic homeostasis (6) (7) (8) (9) (10) (11). The inhibition of HDAC enzymes leads to the sequestration of acetate on the chromatin in form of acetylated lysine resulting in the  limitation of the recycle of chromatin-bound acetyl groups for both catabolic and anabolic processes.  It was consequently shown an up-regulation of enzymes needed for cytoplasmatic acetyl-CoA synthesis from  citrate (ACLY) and acetate (ACSS2). Genes involved in citrate homeostasis (GLS, IDH1, ACO1), citrate cellular import (SLC13A3).

The transcriptional response observed by Srivatsan and coworkers suggest a metabolically consequential depletion of acetyl-CoA precursors in cells exposed to HDACi, while the conventional model suggested that HDAC inhibitors  act directly on chromatin regulation (9) (11) (12).

Sci-Plex permitted to obtain information about the distinct effects of drugs on single cells and to understand how the arrest of the cell cycle  obtained as a result of HDACi exposition can be strongly linked to alterations in acetyl-CoA metabolism. This alternative model does not exclude the conventional model but can explain how the alteration of acetate reservoirs can lead to an heterogenous transcriptional response to HDACi.  Further analysis are required but this work clearly illustrates the potentialities of sci-Plex as this technology permitted to sequence about 650.000 transcriptomes in a single experiment at single cell resolution.

In future single cell sequencing will become cheaper and analysis like this could be largely applicate in biomedicine and research.

References

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  2. Shaffer, S. M. et al. 2017, 546, 431–435.
  3. Spencer, S. L. et al. Nature . 2009, 459, 428–432.
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  5. Trapnell, C. et al. Nat. Biotechnol. 2014, 32, 381–386.
  6. Roe, J.-S. et al. Mol. Cell. 2015, 58, 1028–1039 .
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  11. Wellen, K. E. et al. Science . 2009, 324, 1076–1080.
  12. McBrian, M. A. et al. Mol. Cell . 2013, 49, 310–321.

Nicolò Bordignon

Master Industrial Biotechnology student

Luca Borio

Master Industrial Biotechnology student