Fiber-seq Applied to Brain Cells Enhances Neuro Research
- Stuart P. Atkinson
Fiber-seq Provides a More Complete View of the Human Brain Epigenome
Fiber-seq: A Solution to Uncover Unmapped Epigenomic Regions
Chromatin profiling assays employed to detect accessible chromatin regions represent a widely used means of generating genome-scale maps of nucleosome positioning and chromatin occupancy along chromatin fibers in complex tissues such as the brain.1 Analysis of this type of data has enabled a deeper understanding of active gene regulation networks in the brain by highlighting sites where transcription factors and other chromatin regulatory proteins bind. When applied to brain cells and tissues, these sites can include enhancer elements that regulate the expression of genes, often located at distant loci, implicated in neurodevelopmental and neurodegenerative diseases.
Importantly, conventional approaches to this type of analysis, such as ATAC-seq, are constrained by technical challenges that limit the depth of insight we can achieve, ultimately leaving a considerable part of the epigenome “in the dark”.2 This problem relates to the use of short-read (100-150 bp) DNA sequencing, which generally excludes genomic regions not uniquely spanned by overlapping short reads, leaving the epigenomic composition of over 50% of the human genome unmapped. Also of significant importance, these regions often harbor regulatory features with huge relevance to neurodegenerative disease and disease risk.
To solve this problem and provide a clearer, more complete view of the brain epigenome, researchers have now applied a single-molecule chromatin fiber sequencing technique that adds a readout of accessible chromatin to existing long-read sequencing workflows to nuclei isolated from human brain tissue. “Fiber-seq” – an innovative multiomic method with advantages over existing approaches – can identify cell-specific regulatory elements, map regulatory elements within complex genomic loci, resolve haplotype-specific regulatory mechanisms, and footprint transcription factor binding.
Can this exciting multiomic technique give your neuroscience research a boost and help you gain a clearer, more complete view of the epigenome?
Applying Fiber-seq to Isolated Cells from Human Brain Tissues
The multiomic long-read sequencing assay CUTANA™ Fiber-seq simultaneously profiles chromatin accessibility, DNA methylation, protein footprints, and genetic variants at single-molecule resolution even within highly repetitive and structurally complex genomic regions. EpiCypher pioneers complete solutions for long-read sequencing-based epigenomics, starting with Fiber-seq Kits and also Modular Fiber-seq Services. While first used in fly cells and human fibroblasts3, researchers from the lab of Dr. Schahram Akbarian (Icahn School of Medicine at Mount Sinai) applied Fiber-seq to sorted nuclei from neuronal and non-neuronal cells of the adult human prefrontal cortex. This approach, which does not require PCR-mediated amplification, enabled the precise mapping of accessible chromatin regions and multi-kilobase nucleosome positions at single-fiber resolution on a genome-wide scale. Overall, this Cell Reports Methods paper positions Fiber-seq as an innovative multiomic method with crucial advantages over existing approaches for the resolution of cell-selective promoters/enhancers in the human brain.4
In brief, Fiber-seq involves the Hia5-mediated enzymatic methylation of adenines in accessible chromatin regions to create N6-methyladenine (6mA) followed by detection via native/direct long-read sequencing, allowing researchers to study accessible DNA on individual fibers genome-wide by haplotype or reference genome. Fiber-seq can resolve individual nucleosomes (unlike conventional short-read methods), allowing the definition of truly nucleosome-depleted regions rather than just broadly accessible chromatin (as detected by conventional ATAC-seq).
By applying Fiber-seq, the researchers were able to:
- Identify cell-specific promoter and enhancer elements on single fibers (including transcription factor footprinting)
- Define the positioning of nucleosomes near transcription start sites and regulatory motifs
- Uncover regulatory features – haplotype-specific chromatin patterning and accessible chromatin sites – of sequences that had remained “unmappable” using previous short-read epigenomic sequencing-based approaches
Fiber-seq Applied to Cells of the Human Brain: All the Details and The Potential Applications
In general, Fiber-seq helped to uncover previously undetectable details regarding gene regulation, yielding important insights at the genome-wide, haplotype, and locus-specific levels.
At the genome-wide level, the initial reference maps developed following this protocol revealed cell-specific nucleosome positioning in isolated neuronal and non-neuronal nuclei of the human brain at single-fiber resolution and identified accessible chromatin regions with much sharper resolution than conventional chromatin assays employing short-read sequencing. The sensitive nature of Fiber-seq allowed the authors to resolve individual nucleosomes and internucleosomal linker DNA on single chromatin fibers, confirming principles of nucleosomal organization known from studies in lower organisms (such as the sequence-specific positioning of nucleosomes at transcription start sites of active promoters). Such insights had remained unconfirmed in higher eukaryotes due to technical limitations associated with conventional epigenetic assays and short-read sequencing.
Importantly, this long-read epigenomic profiling approach also identified ~20,000 accessible chromatin regions that short-read sequencing-based accessibility mapping could not define (having left ~60% of accessible chromatin regions unexplored). These regions lie within repetitive regions of the genome, whose characteristics create a barrier to mapping with traditional epigenetic assays based on short-read sequencing. The analysis of these regions by Fiber-seq represents an important advance; rather than representing epigenomic “noise”, repetitive regions contain regulatory features relevant to neurodegenerative disease and disease risk. Examples of such regions include mammalian interspersed repeats, an ancient short interspersed nuclear element type that makes up 2.5% of the human genome, spans short regions of ~250 bp, and serves as RNA polymerase docking sites relevant to neurodegenerative disease.5,6
Haplotypes – the combination of specific alleles along individual chromosomes – reflect shared ancestry and gene associations; as such, epigenomic differences at allele-specific sites may affect non-coding DNA regulatory regions and gene expression. At the haplotype level, Fiber-seq identified over 3,000 haplotype-specific accessible chromatin regions in non-neuronal cells; this included the promoter of the primate-specific zinc finger gene ZNF343, which codes a protein strongly expressed in the human brain that may bind genome-wide to hundreds of promoters7 and, as such, hold relevance to neurodevelopment and neurodegenerative disease.
Importantly, Fiber-seq also captured co-accessible regions (co-regulated promoters and enhancers lying on the same single fiber that function together in cis to regulate target gene expression) along individual long reads and linked risk loci to adjacent co-regulated elements. Importantly, the median genomic distance between enhancer elements and target promoters (13–16 kb) makes exploring co-regulation a hugely challenging (if not impossible) task; however, the long-read sequencing approach used by Fiber-seq overcomes these problems.
When focusing on locus-specific analysis, Fiber-seq had the ability to i) define narrower accessible chromatin regions that display enrichment for brain tissue-specific transcription factor binding motifs, ii) detect protein footprints within DNA at accessible chromatin sites at single-molecule resolution, and iii) reveal protein-bound regulatory elements, highlighting candidate transcription factors that may drive disease-relevant gene dysregulation. As an example of this type of analysis, Fiber-seq in non-neuronal cells precisely identified binding sites for NFY/SP1 transcription factors (critical for oligodendrocyte survival8) at promoter sequences and the chromosomal loop organizer, CTCF, and astrocyte/microglial regulators, SOX9/NEUROD1, at enhancers and promoters within accessible chromatin regions.
Next Steps: Toward a Clearer, More Complete View of the Diseased Human Brain Epigenome
Fiber-seq has significant advantages over existing epigenetic analyses using short-read sequencing when aiming to identify cell-specific promoters/enhancers, map regulatory elements within complex genomic loci, explore haplotype-specific regulatory mechanisms, and support transcription factor footprinting, as shown here in sorted human brain nuclei. The authors hope to next compare data from normal and diseased human brain samples, which will likely describe genome-wide, haplotype-specific, and locus-specific alterations in the epigenome of neuronal and non-neuronal cells. Could this boost our knowledge of neurodevelopmental and neurodegenerative diseases (and more) and help guide the development of novel therapeutic strategies?
Check out the EpiCypher website for more information on Fiber-seq Kits, which provide the reagents and comprehensive manual required to perform Fiber-seq. EpiCypher also offers Modular Fiber-seq Services, providing unique access to our genomic expertise and multiomic capabilities, enabling diverse applications across biomedical research, including neuroscience and drug development.
References
- Minnoye, L., Marinov, G.K., Krausgruber, T. et al. Chromatin accessibility profiling methods. Nat Rev Methods Primers 1, 10 (2021). https://doi.org/10.1038/s43586-020-00008-9
- Treangen, T.J. & Salzberg, S.L. Repetitive DNA and next-generation sequencing: computational challenges and solutions. Nat Rev Genet 13, 36-46 (2012). https://doi.org/10.1038/nrg3117
- Stergachis, A.B., Debo, B.M., Haugen, E., Churchman, L.S. & Stamatoyannopoulos, J.A. Single-molecule regulatory architectures captured by chromatin fiber sequencing. Science 368, 1449-1454 (2020). https://doi.org/10.1126/science.aaz1646
- Peter, C.J., Agarwal, A., Watanabe, R. et al. Single chromatin fiber profiling and nucleosome position mapping in the human brain. Cell Reports Methods 4, 100911 (2024). https://doi.org/10.1016/j.crmeth.2024.100911
- Simone, R. et al. MIR-NATs repress MAPT translation and aid proteostasis in neurodegeneration. Nature 594, 117-123 (2021). https://doi.org/10.1038/s41586-021-03556-6
- Carnevali, D., Conti, A., Pellegrini, M. & Dieci, G. Whole-genome expression analysis of mammalian-wide interspersed repeat elements in human cell lines. DNA Research 24, 59-69 (2017). https://doi.org/10.1093/dnares/dsw048
- Farmiloe, G., Lodewijk, G.A., Robben, S.F., van Bree, E.J. & Jacobs, F.M.J. Widespread correlation of KRAB zinc finger protein binding with brain-developmental gene expression patterns. Philos Trans R Soc Lond B Biol Sci 375, 20190333 (2020). https://doi.org/10.1098/rstb.2019.0333
- Begum, G., Otsu, M., Ahmed, U., Ahmed, Z., Stevens, A. & Fulton, D. NF-Y-dependent regulation of glutamate receptor 4 expression and cell survival in cells of the oligodendrocyte lineage. GLIA (2018). https://doi.org/10.1002/glia.23446