Single Cell Regulatory Variation Clayton Barham

Goals of Studying Single Cell Regulatory Variation Discover the mechanisms that cause different cells to have different chromatin structures and different expressions of regulatory elements Determine how those differences affect cell phenotype and cell behavior

Applications Better understand how stem cells differentiate and change their DNA regulation to adapt to their new specialization Better understand the regulatory diversity within tumors, for more effective diagnosis and treatment Better understanding how individual cells adapt their transcriptome in the face of exposure to chemicals may help better understand and counteract drug resistance

First Paper Single-cell chromatin accessibility reveals principles of regulatory variation Published in Nature, 2015, by Buenrostro et al. Hypothesis: phenotypic diversity between individual cells stems from differences in chromatin accessibility between cells Goal: develop a method, single cell ATAC-seq (scATAC-seq), to map the chromatin regions that are accessible in individual cells, which can be used to investigate specific problems

Background: ATAC-seq scATAC-seq adapts ATAC-seq for use on individual cells ATAC-seq is an ensemble method, exposing populations of cells to Tn5 transposase to tag exposed regions of chromatin

scATAC-seq Assay cells using Fluidigm microfluidics environment Use PCR to amplify collected libraries Pool and sequence single cell libraries

Measuring Regulatory Variation

Experimental Validation of scATAC-seq Measure variability of multiple features between cells Inhibit proteins associated with significant variability of specific features and observe results on scATAC-seq data Measure sets of peaks that co-vary within individual cells, to reveal long range dependency in regulation

Results: Variability of Features Between Cells

Results: Inhibition of Factors Associated with Variability

Results: Co-dependent Peaks

Second Paper Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation Published in Cell, 2018, by Buenrostro et al. Measure regulatory variation in differentiating adult stem cells

Background: Hematopoiesis Human hematopoiesis involves differentiation of pluripotent adult stem cells Process defined by Transcription Factors and their influence on chromatin

scATAC-seq Pipeline to Study Stem Cells

Experiments Used scATAC-seq to analyze 10 populations of adult stem cells from blood and bone marrow Combine scATAC-seq and RNA-seq to analyze the epigenome and transcriptome of Granulocyte-Macrophage Progenitor (GMP) cells Integrate scATAC-seq and RNA-seq data to study how changes in regulatory elements affects chromatin accessibility and gene expression

Results: Regulatory Variation in 10 Adult Stem Cell Populations

Results: Transcriptome of GMP Cells

Results: Regulatory Elements and Motif Expression

References J. D. Buenrostro et al., “Single-cell chromatin accessibility reveals principles of regulatory variation,” Nature, vol 523, pp. 486–490, July 23, 2015 J. D. Buenrostro et al., “Integrated Single-Cell Analysis Maps the Continuous Regulatory Landscape of Human Hematopoietic Differentiation,” vol. 173.6, pp. 1535-1548.E16, May 31, 2018