Gene regulation is orchestrated by the co-binding of proteins along chromosome-length chromatin fibers within single cells, yet the heterogeneity of this occupancy between haplotypes and cells remains poorly resolved in diploid organisms.
Gene regulation is orchestrated by the co-binding of proteins along chromosome-length chromatin fibers within single cells, yet the heterogeneity of this occupancy between haplotypes and cells remains poorly resolved in diploid organisms.
Here we present Deaminase-Assisted single-molecule chromatin Fiber sequencing , which enables single-molecule footprinting at near-nucleotide resolution while synchronously profiling single-molecule chromatin states and DNA sequence. DAF-seq illuminates cooperative protein occupancy at individual regulatory elements and resolves the functional impact of somatic variants and rare chromatin epialleles. Single-cell DAF-seq generates chromosome-length protein co-occupancy maps across 99% of each individual cell’s mappable genome. scDAF-seq uncovers extensive chromatin plasticity both within and between single diploid cells, with chromatin actuation diverging by 61% between haplotypes within a cell, and 63% between cells. Moreover, we find that regulatory elements are preferentially co-actuated along the same fiber in a distance-dependent manner that mirrors cohesin-mediated loops. Overall, DAF-seq enables the characterization of protein occupancy across entire chromosomes with single-nucleotide, single-molecule, single-haplotype and single-cell precision.Fig. 1: Deaminase-assisted single-molecule chromatin fiber sequencing .Fig. 5: Chromosome-scale genomic phasing in single cells.https://github.com/StergachisLab/DAF-seq-ManuscriptStergachis, A. B., Debo, B. M., Haugen, E., Churchman, L. S. & Stamatoyannopoulos, J. A. Single-molecule regulatory architectures captured by chromatin fiber sequencing.Google ScholarMok, B. Y. et al. CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA.Google ScholarIto, Y., Zhang, Y., Dangaria, S., Luan, X. & Diekwisch, T. G. H. NF-Y and USF1 transcription factor binding to CCAAT-box and E-box elements activates the CP27 promoter.Zhu, J., Giannola, D. M., Zhang, Y., Rivera, A. J. & Emerson, S. G. NF-Y cooperates with USF1/2 to induce the hematopoietic expression of HOXB4.Cheng, H., Concepcion, G. T., Feng, X., Zhang, H. & Li, H. Haplotype-resolved de novo assembly using phased assembly graphs with hifiasm.Monahan, K. et al. Role of CCCTC binding factor and cohesin in the generation of single-cell diversity of protocadherin-α gene expression.Rao, S. S. P. et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping.Google ScholarCheng, Y. H. H., Bohaczuk, S. C. & Stergachis, A. B. Functional categorization of gene regulatory variants that cause Mendelian conditions.We thank Northwest Genome Center and K. M. Munson for their assistance in PacBio sequencing, UW Mass Spectrometry Center for their assistance in mass spectrometry experiments, the Fowler lab and H. J. Kim for assistance with nuclei sorting, T. J. Bell and K. Leonard at The National Disease Research Interchange for primary frozen tissue and members of the UW-SCRI Somatic Mosaicism across Human Tissues Genome Characterization Center for generating the COLO829 cell mixture and Illumina sequencing data, and for their feedback and support. Funding: A.B.S. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund and is a Pew Biomedical Scholar. This research is supported by the National Institutes of Health Common Fund, through the Office of Strategic Coordination/Office of the NIH Director under award no. UM1DA058220 to A.B.S. This study was also supported by NIH grant no. 1DP5OD029630, CZIF2024-010284 from the Chan Zuckerberg Initiative and a UW ADRC Developmental Project to A.B.S. M.R.V. was supported by an NIH Pathway to Independence Award from NIGMS , and both M.R.V. and S.C.B. were supported by a training grant from the NIH . E.G.S. was supported by a Curci Fellowship, as well as a training grant from the NIH .Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA, USADivision of Medical Genetics, University of Washington School of Medicine, Seattle, WA, USAThe Edison Family Center for Genome Sciences and Systems Biology, School of Medicine, Washington University in St. Louis, Saint Louis, MO, USADepartment of Genetics, School of Medicine, Washington University in St. Louis, Saint Louis, MO, USACenter for Developmental Biology and Regenerative Medicine, Seattle Children’s Research Institute, Seattle, WA, USAA.B.S., E.G.S. and Y.M. conceived the overall method design. A.B.S. and E.G.S. wrote the manuscript with contributions from all authors. E.G.S. led the development of DAF-seq workflows, and performed DAF-seq experiments and data analysis. Y.M. cloned, produced and purified SsDddA, and performed all size-exclusion chromatography and mass spectrometry experiments. B.J.M. contributed to targeted DAF-seq PCR optimization and cell culture. M.R.V. contributed to the analysis of DAF-seq data, developed custom computational tools and advised on statistics. S.C.B. advised on experiments and developed the DAF-QC-SMK pipeline. C.B.O. contributed PCR primer design and targeted DAF-seq testing. C.B.O. and S.C.B. wrote the. B.A.C. and D.B.L. contributed thermodynamic modeling of transcription factor occupancy. J.R. contributed to cell culture and cell isolation. J.T.B. and N.L.P. produced the COLO829 cell mixture.A.B.S., E.G.S. and Y.M. are co-inventors on the US Provisional Patent Application 63/687,924 which includes discoveries described in this manuscript regarding ‘Chromatin Stenciling’™. The other authors declare no competing interests.) IGV browser displaying CpG methylation for 5mC negative control , 5mC positive control , and M.SssI plus SsDddA treated DNA . . Boxes represent the median and interquartile range and whiskers extend to the farthest samples within 1.5× the interquartile range of the median. Percentage of cytidine deamination by SsDddA grouped by CpG dinucleotides and all other cytidine bases. Data is shown for two targeted regions, thepromoter, excluding bases within TF footprint regions, for each DAF-seq library from GM12878 cells treated with a range of SsDddA enzyme concentrations and treatment times. Bases are colored by TpC and non-TpC dinucleotide context. . Boxes represent the median and interquartile range and whiskers extend to the farthest samples within 1.5× the interquartile range of the median.IGV browser displaying percent actuation of targeted DAF-seq Oxford Nanopore data , percent actuation of Fiber-seq PacBio HiFi data , and bulk ATAC-seq read coverage from GM12878 cells, K562 cells, and colon tissue, at each of 10 target regions.promoter, as well as a network diagram of elements 1, 2, and 3 with the edge weights corresponding to the strength of the codependency. are colored red, non-significant interactions are colored blue. Single-molecule conditional codependency with a bar graph showing the impact that removal of occupancy at elements 1, 2, or 3 has on the codependency score of the remaining elements within this cluster. A higher score indicates that an element is essential for a codependent network. Directed network diagram showing how elements 1, 2, or 3 go from the unbound to fully bound state. Individual nodes are colored based on their occupancy patterns and are sized based on the data from panel. The edges connecting the unbound and fully bound state are weighted based on the size of the smallest node through which they traverse during this path, and the translucency of each edge leaving the 1 bound state is dependent upon the data from panelisoform 4 TSS showing single-molecule protein occupancy at four binding elements. Bar graph showing single-molecule protein occupancy at each of the fourIGV browser displaying single-cell DAF-seq reads from one cell. Deamination events for raw reads are colored red for reads originating from the top strand and green for reads originating from the bottom strand. Consensus reads are grouped by strand and ordered by haplotype.) Scatter plot comparing sequenced bases with percent coverage of mappable GRCh38 for all consensus reads and phased consensus reads .) Joint violin and box plot showing the distribution of percent deamination of each cell . Boxes represent the median and interquartile range and whiskers extend to the farthest samples within 1.5× the interquartile range of the median. and promoter-proximal GM24385 Fiber-seq FIRE peaks. Autocorrelation plot of single-molecule deamination patterns in each cell showing a pattern consistent with nucleosomes being the predominant modulator of single-cell and single-molecule deamination by SsDddA. The vertical dashed line at 147 bp represents the theoretical nucleosome footprint size.) Single-cell codependent regulatory element actuation by binned log2 genomic distance. Average codependency scores between regulatory elements in each distance bin are displayed for single chromatin fibers in red, for opposite haplotypes within the same cell in yellow, and for chromatin fibers from different cells in blue. Percentage of raw scDAF-seq reads from each cell aligning to hg38 vs percent cytidine deamination within that cell. Mapping percentage is not significantly correlated with deamination rate Mean genome-wide CTCF occupancy within actuated regulatory elements from each cell vs percent cytidine deamination within that cell. CTCF occupancy is not significantly correlated with deamination rate . Boxes represent the median and interquartile range and whiskers extend to the farthest samples within 1.5× the interquartile range of the median. Stacked barplots showing the proportions of CTCF motif orientations contained within each loop anchor pair for each ChIA-PET bin and shuffled regions.
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