Mapping 3D genome organization relative to nuclear compartments using TSA-Seq as a cytological ruler.

   December 6th, 2018 at 8:03pm



While nuclear compartmentalization is an essential feature of three-dimensional genome organization, no genomic method exists for measuring chromosome distances to defined nuclear structures. In this study, we describe TSA-Seq, a new mapping method capable of providing a "cytological ruler" for estimating mean chromosomal distances from nuclear speckles genome-wide and for predicting several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results in K562 cells reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple spatially separated nuclear domains including two types of transcription "hot zones." Transcription hot zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, housekeeping genes, genes with low transcriptional pausing, and super-enhancers. Our results demonstrate the capability of TSA-Seq for genome-wide mapping of nuclear structure and suggest a new model for spatial organization of transcription and gene expression.


Chen Y  •  Zhang Y  •  Wang Y  •  Zhang L  •  Brinkman EK  •  Adam SA  •  Goldman R  •  van Steensel B  •  Ma J  •  Belmont AS



The Journal of cell biology



November 5th, 2018

About TSA-seq

TSA sequencing (TSA-Seq), is a genomic method that allows the estimation of cytological distances of chromosome loci genome-wide relative to a particular nuclear compartment and can be used to infer chromosome trajectories from one compartment to another.

TSA uses an antibody-coupled HRP in conjunction with an antibody targeting a specific protein or compartment to catalyze the formation of diffusible biotin-tyramide free radicals and create a free radical concentration gradient centered at the target in fixed nuclei. The TSA reaction is followed by the reversal of formaldehyde cross-linking, DNA isolation, pulldown of biotinylated DNA, and high-throughput sequencing. TSA labels DNA directly and the steady-state concentration of tyramide free radicals during the TSA reaction can be modeled by a simple exponential decay proportional to the distance from the target.

TSA enrichment genomic maps are generated by plotting the log2 ratio of the normalized pulldown read density versus the normalized read density of input DNA over a 20-kbp sliding window and can be visualized as normalized counts in 1D bigwig tracks.

About this study

This publication describes the technology development of the TSA-seq method and it's application in K562 cells to examine the distribution of genome loci relative to nuclear speckles and nuclear lamina and well as proximity to markers of active domains of transcription.

The publication discusses the relationship of previously identified chromatin compartments and TSA-seq signal and distance estimations. Several datasets using this TSA-seq version 1 method were produced for this publication and are shown in the Experiments Summary tab.

In addition Supplementary Table 4 from this publication which is an excel workbook that lists external datasets used in this analysis as confirmative and comparative data is available at this link. NOTE: The analysis presented in this paper and the files referenced in this table are done on human assembly hg19 while the processed files available on the 4DN data portal have been reanalyzed on the hg38 assembly.

TSA-seq comparing speckle/active regions and lamin-associated regions

The top 2 tracks show TSA-seq using independent markers of nuclear speckles: SON and phosphorylated SC35. The middle track uses an antibody that detects a poised version of RNA Pol II subunit as a marker of actively transcribed regions. While the bottom 2 tracks are for Lamins markers of the nuclear lamina. The display below highlights the finding that actively transcribing regions and nuclear speckles tend to have similar TSA-seq profiles that are generally inverse to profiles using markers for the more inactive lamina regions.

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