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NTT-Seq and Nano-CT: Nanobody-based Techniques that Deliver Epigenetic Profiles at the Single-cell Level

By Stuart P. Atkinson, Ph.D.

March 11, 2024

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Introduction: Pushing the Limits of Single-cell Epigenetic Analyses

Methodological advances have offered a means to evaluate a range of epigenetic modalities at single-cell resolution; however, data from conventional assays focusing on a solitary element may signify little regarding the functional definition of complex epigenetic states (Janssen and Lorincz). Instead, achieving this goal requires the integration of multiple histone modification profiles, DNA methylation patterns, transcription factor binding activities, and DNA accessibility data (and more!) at the level of single cells.

Reports by Ivan Raimondi (New York Genome Center/Weill Cornell Medicine) and by Marek Bartosovic and Gonçalo Castelo-Branco (Karolinska Institutet) recently described epigenetic profiling assays employing small single polypeptide chain antibodies or “nanobodies” (bind strongly to targets and display stability over a broad temperature/pH range). These techniques provide a means to evaluate multiple epigenetic modalities at single-cell resolution in a single assay; here, we briefly overview “nanobody-tethered transposition followed by sequencing” or NTT-Seq and “nano-CUT&Tag” or nano-CT.

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Section One: NTT-Seq Supports Multiplexed Histone Modification Profiling (and more) at Single-cell Resolution

Nanobody-Tethered Transposition Followed by Sequencing

A range of single-cell epigenetic profiling methods have employed a fusion of the Tn5 transposase with protein-A/G (Gopalan et al. and Meers et al., for example). In this approach, protein-A/G binding to IgG antibodies (directed against a histone modification, for example) guides the insertion of adapters by Tn5 for DNA sequencing. Unfortunately, the high-affinity binding of protein-A/G to IgG antibodies from distinct species makes the multiplexed profiling of histone modifications at the single-cell resolution with multiple antibodies more challenging.

As a solution, researchers headed by Ivan Raimondi replaced protein-A/G with a set of secondary nanobodies that specifically bind IgG from different species/IgG subtypes to support the application of a mixture of primary antibodies binding different epitopes in a single experiment (Pleiner et al.). In other words, their approach permits the implementation of multiple antibodies directed against distinct histone modifications to facilitate multiplexed epigenetic profiling at single-cell resolution. Their recent study now reports on the NTT-Seq technique, which employs nanobody–Tn5 fusion (nb–Tn5) proteins to profile up to three histone modifications (and protein-DNA binding) at single-cell resolution in a single assay run.

Validation and Bulk Analyses Prove a Point

The authors engineered four recombinant nb–Tn5 fusion proteins, confirmed specificity via “bulk” NTT-Seq experiments, and then evaluated the targeting specificity of nb–Tn5 fusion proteins by implementing antibodies against two mutually exclusive histone modifications - H3K27me3 and H3K27ac - in human peripheral blood mononuclear cells. Encouragingly, multiplexed NTT-Seq experiments provided almost identical H3K27me3 and H3K27ac profiles compared to individual NTT-Seq experiments, suggesting the highly accurate genome-wide profiling of histone modifications. A subsequent evaluation of multiplexed NTT-Seq employed three antibodies (H3K27me3, H3K27ac, and elongating RNAPII) – in K562 human leukemia cells; again, the multiplexed assays offered comparable results for each target to individual assays, thereby demonstrating the ability to efficiently profile three targets simultaneously.

Moving from Bulk Analyses to Single-cell Resolution

The application of NTT-Seq to characterize multimodal epigenetic states at single-cell resolution integrated a single-cell assay for transposase-accessible chromatin using sequencing (single-cell ATAC-Seq) kit and profiled H3K27me3, H3K27ac, and elongating RNAPII in a mixture of less than 10,000 K562 and HEK293 human embryonic kidney cells. The profiles for each target in multiplexed single-cell NTT-Seq assays displayed broad similarities to individual bulk assays - revealing the co-occupancy of RNAPII and H3K27ac in open chromatin and the mutual exclusivity of H3K27me3 – and provided evidence for this approach as an effective means to profile multiple epigenetic modalities at single-cell resolution.

The authors next assessed the ability of multiplexed single-cell NTT-Seq to simultaneously measure cell surface protein expression and multiple histone modifications at single-cell resolution. They built upon their single-cell CUT&Tag-pro method (Zhang et al.) to label peripheral blood mononuclear cells with an oligonucleotide-conjugated panel of antibodies targeting immune-relevant cell surface proteins before performing NTT-Seq for H3K27me3 and H3K27ac. The generated profiles revealed that multimodal histone modification data analysis faithfully resolved cell states and highlighted the ability of NTT-Seq to characterize heterogeneous tissues without the need for cell surface protein measurements.

Finally, the team applied multiplexed single-cell NTT-Seq experiments to profile H3K27me3 and H3K27ac in human bone marrow mononuclear cells as an example of a complex tissue containing differentiating cells to capture chromatin remodeling dynamics that shape cellular identity. Overall, the multimodal epigenetic data generated faithfully reflected the multimodal epigenetic state dynamics associated with the developmental trajectories of the B cell lineage.

The Huge Potential of Multiplexed Epigenetic Profiling at Single-cell Resolution

Previous multimodal epigenetic assays required intricate workflows not generally applicable to complex tissue samples (Gopalan et al. and Meers et al.) or had limited scope with regard to targets (Tedesco et al.). Here, the authors report the potential of their widely applicable NTT-Seq technique, the accuracy of resulting multiplexed epigenetic profiles (reflecting high-quality bulk ChIP-Seq data; The ENCODE Project Consortium), the compatibility with simultaneous cell surface protein expression measurement, and the applicability to human tissues. Of note, reagent development and protocol improvements may support the assay of more than three targets and expand the scope of assayable epigenetic modalities.

For more on how NTT-Seq generates multiplexed epigenetic profiles at single-cell resolution, see Nature Biotechnology, December 2022.

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Section Two: Nano-CT - Evaluating Multiple Histone Modifications and Chromatin Accessibility at Single-cell Resolution

Nano-Cleavage Under Targets and Tagmentation in Single Cells

The comparable nano-CT approach described by Marek Bartosovic and Gonçalo Castelo-Branco employs a similar nanobody-directed approach to generating multiplexed epigenetic profiles at single-cell resolution. Previous approaches, such as single-cell ATAC-Seq and single-cell cleavage under targets and tagmentation (single-cell CUT&Tag), had employed Tn5 transposase/fusion proteins to generate single-modality epigenetic data; however, with nano-CT, the duo hoped to move past limitations associated with the integration of distinct single-cell epigenetic profiles (Stuart et al. and Welch et al.) and gain deep insight by profiling multiple epigenetic modalities in the same cell. In nano-CT, nanobodies target mouse or rabbit primary antibodies to simultaneously profile two histone modifications, while combining nano-CT with single-cell ATAC-Seq integrates chromatin accessibility profiling.

Efficient Methodology and Modular Nanobody Design

The nano-CT protocol involves a newly developed antibody/nano-Tn5 incubation strategy, which reduced the number of steps required and increased the retention of nuclei for analysis; overall, 25,000 input nuclei sufficed to profile two histone modifications (without single-cell ATAC-Seq), although 50,000 input nuclei provided more complex data. Multiplexing two histone modifications with prior single-cell ATAC-Seq analysis increased the number of centrifugation and washing steps, which increased the input requirements to 200,000 nuclei. An improved tagmentation/nano-CT library preparation protocol for single cells involving deterministic tagmentation combined with the higher capture efficiency by nano-Tn5 fusion proteins supported higher sequencing depth (16-fold greater) from lower cell numbers (5-fold less) compared to related techniques. The authors also note that the modular design applied during the development of their nano-Tn5 fusion proteins should permit the application of this strategy to any combination of histone modifications, which may drive the widespread use of this approach to single-cell resolution multiplexed epigenetic profiling.

Analysis of the Mouse Brain and Oligodendrocyte Differentiation

To explore the potential of nano-CT in generating multiplexed epigenetic profiles at single-cell resolution, the team first assessed chromatin accessibility (via single-cell ATAC-Seq) and then simultaneously profiled H3K27ac and H3K27me3 in fresh brain tissue obtained from juvenile mice. The authors reported that nano-CT outperformed single-cell CUT&Tag (and related techniques) regarding multiple relevant parameters; furthermore, the data generated could efficiently cluster and classify cells into the major cell classes present in the brain. Analysis of individual epigenetic profiles revealed the expected patterns of histone modifications and chromatin accessibility according to marker gene expression in specific cells, which suggests that nano-CT can simultaneously provide robust and specific profiles of multiple histone modifications and chromatin accessibility at single-cell resolution.

Their subsequent analyses evaluated the dynamics of multiple histone modifications and chromatin accessibility in the same cell by assessing the differentiation of oligodendrocyte progenitor cells towards mature oligodendrocytes in the post-natal day juvenile mouse brain. Applying Nano-CT revealed that chromatin opening preceded the deposition of H3K27ac at loci to support gene expression and that H3K27me3 deposition occurred in two distinct waves during oligodendrocyte differentiation (which cannot be discriminated by analysis of transcriptomic data). The first wave occurred during early differentiation to repress neuronal gene expression, while the second later wave repressed genes associated with both the neuronal and oligodendrocyte progenitor cell lineage. Overall, these data describe how nano-CT can offer unique insight into the epigenetic regulation of critical biological processes at single-cell resolution.

Multiplexed Histone Modifications and Chromatin Accessibility Profiling at Single-cell Resolution

Overall, the findings of this exciting new paper suggest that the high resolution, versatility, and multiplexed nature of nano-CT help offer unique insight into the epigenetic regulation landscape of single cells. With a view to future research aims, the authors hoped that additional development to the nano-CT technique could support the evaluation of additional histone modifications, the expansion past two simultaneous histone modifications, and the measurement of various other chromatin-binding factors to gain mechanistic insights into processes such as the activation of enhancers and promoters, the initiation of transcription, and the dynamics of bivalent epigenetic states.

For more on how nano-CT can help to profile histone modifications and chromatin accessibility at single-cell resolution, see Nature Biotechnology, December 2022.

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About the author

Stuart P. Atkinson, Ph.D.

Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!

Contact Stuart on Twitter with any questions

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