Antibody-free profiling of transcription factor occupancy during early embryogenesis by FitCUT&RUN

2021 ◽  
pp. gr.275837.121
Author(s):  
Xiangxiu Wang ◽  
Wen Wang ◽  
Yiman Wang ◽  
Jia Chen ◽  
Guifen Liu ◽  
...  
Keyword(s):  
Significant Proportion ◽  
K562 Cells ◽  
Replication Timing ◽  
Early Embryogenesis ◽  
Clear Trend ◽  
Genome Wide ◽  
Sequential Binding ◽  
Reliability And Robustness ◽  
Genome Activation ◽  
Transcription Factor Occupancy

Key transcription factors (TFs) play critical roles in zygotic genome activation (ZGA) during early embryogenesis, while genome-wide occupancies of only a few factors have been profiled during ZGA due to the limitation of cell numbers or the lack of high-quality antibodies. Here, we present FitCUT&RUN, a modified CUT&RUN method, in which an Fc fragment of immunoglobulin G is used for tagging, to profile TF occupancy in an antibody-free manner and demonstrate its reliability and robustness using as few as five thousand K562 cells. We applied FitCUT&RUN to zebrafish undergoing embryogenesis to generate reliable occupancy profiles of three known activators of zebrafish ZGA: Nanog, Pou5f3 and Sox19b. By profiling the time-series occupancy of Nanog during zebrafish ZGA, we observed a clear trend toward a gradual increase in Nanog occupancy and found that Nanog occupancy prior to the major phase of ZGA is critical for the activation of a significant proportion of early transcribed genes. Our results further suggested that the sequential binding of Nanog may be controlled by replication timing and the presence of Nanog motifs.

scholarly journals Chromosomal coordination and differential structure of asynchronous replicating regions

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Britny Blumenfeld ◽  
Hagit Masika ◽  
Marganit Farago ◽  
Yishai Yehuda ◽  
Lamia Halaseh ◽  
...  
Keyword(s):  
Population Level ◽  
Replication Timing ◽  
Gene Families ◽  
Chromatin Accessibility ◽  
Early Embryogenesis ◽  
Epigenetic Mark ◽  
Parallel Orientation ◽  
Asynchronous Replication ◽  
Genome Wide ◽  
New Gene

AbstractStochastic asynchronous replication timing (AS-RT) is a phenomenon in which the time of replication of each allele is different, and the identity of the early allele varies between cells. By taking advantage of stable clonal pre-B cell populations derived from C57BL6/Castaneous mice, we have mapped the genome-wide AS-RT loci, independently of genetic differences. These regions are characterized by differential chromatin accessibility, mono-allelic expression and include new gene families involved in specifying cell identity. By combining population level mapping with single cell FISH, our data reveal the existence of a novel regulatory program that coordinates a fixed relationship between AS-RT regions on any given chromosome, with some loci set to replicate in a parallel and others set in the anti-parallel orientation. Our results show that AS-RT is a highly regulated epigenetic mark established during early embryogenesis that may be used for facilitating the programming of mono-allelic choice throughout development.

scholarly journals SPIN reveals genome-wide landscape of nuclear compartmentalization

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yuchuan Wang ◽  
Yang Zhang ◽  
Ruochi Zhang ◽  
Tom van Schaik ◽  
Liguo Zhang ◽  
...  
Keyword(s):  
Spatial Organization ◽  
K562 Cells ◽  
Replication Timing ◽  
Computational Method ◽  
Chromatin Interaction ◽  
Nuclear Speckles ◽  
Genome Spatial Organization ◽  
3D Genome ◽  
Genome Wide ◽  
Nuclear Compartmentalization

AbstractWe report SPIN, an integrative computational method to reveal genome-wide intranuclear chromosome positioning and nuclear compartmentalization relative to multiple nuclear structures, which are pivotal for modulating genome function. As a proof-of-principle, we use SPIN to integrate nuclear compartment mapping (TSA-seq and DamID) and chromatin interaction data (Hi-C) from K562 cells to identify 10 spatial compartmentalization states genome-wide relative to nuclear speckles, lamina, and putative associations with nucleoli. These SPIN states show novel patterns of genome spatial organization and their relation to other 3D genome features and genome function (transcription and replication timing). SPIN provides critical insights into nuclear spatial and functional compartmentalization.

scholarly journals SPIN reveals genome-wide landscape of nuclear compartmentalization

2020 ◽  
Author(s):  
Yuchuan Wang ◽  
Yang Zhang ◽  
Ruochi Zhang ◽  
Tom van Schaik ◽  
Liguo Zhang ◽  
...  
Keyword(s):  
Spatial Organization ◽  
K562 Cells ◽  
Replication Timing ◽  
Cell Types ◽  
Chromatin Interaction ◽  
Spin States ◽  
Nuclear Speckles ◽  
Genome Spatial Organization ◽  
Genome Wide ◽  
Nuclear Structures

AbstractChromosomes segregate differentially relative to distinct subnuclear structures, but this genome-wide compartmentalization, pivotal for modulating genome function, remains poorly understood. New genomic mapping methods can reveal chromosome positioning relative to specific nuclear structures. However, computational methods that integrate their results to identify overall intranuclear chromo-some positioning have not yet been developed. We report SPIN, a new method to identify genome-wide nuclear spatial localization patterns. As a proof-of-principle, we use SPIN to integrate nuclear compartment mapping (TSA-seq and DamID) and chromatin interaction data (Hi-C) from K562 cells to identify 10 spatial compartmentalization states genome-wide relative to nuclear speckles, lamina, and nucleoli. These SPIN states show novel patterns of genome spatial organization and their relation to genome function (transcription and replication timing). Comparisons of SPIN states with Hi-C sub-compartments and lamina-associated domains (LADs) from multiple cell types suggest constitutive compartmentalization patterns. By integrating different readouts of higher-order genome organization, SPIN provides critical insights into nuclear spatial and functional compartmentalization.

scholarly journals Distinctive types of postzygotic single-nucleotide mosaicisms in healthy individuals revealed by genome-wide profiling of multiple organs

2018 ◽  
Author(s):  
August Yue Huang ◽  
Xiaoxu Yang ◽  
Sheng Wang ◽  
Xianing Zheng ◽  
Qixi Wu ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Replication Timing ◽  
Clonal Expansion ◽  
Early Embryogenesis ◽  
Open Chromatin ◽  
Healthy Individuals ◽  
Single Nucleotide ◽  
Healthy Humans ◽  
Genome Wide ◽  
Healthy Donors

AbstractPostzygotic single-nucleotide mosaicisms (pSNMs) have been extensively studied in tumors and are known to play critical roles in tumorigenesis. However, the patterns and origin of pSNMs in normal organs of healthy humans remain largely unknown. Using whole-genome sequencing and ultra-deep amplicon re-sequencing, we identified and validated 164 pSNMs from 27 postmortem organ samples obtained from five healthy donors. The mutant allele fractions ranged from 1.0% to 29.7%. Inter- and intra-organ comparison revealed two distinctive types of pSNMs, with about half originating during early embryogenesis (embryonic pSNMs) and the remaining more likely to result from clonal expansion events that had occurred more recently (clonal expansion pSNMs). Compared to clonal expansion pSNMs, embryonic pSNMs had higher proportion of C>T mutations with elevated mutation rate at CpG sites. We observed differences in replication timing between these two types of pSNMs, with embryonic and clonal expansion pSNMs enriched in early- and late-replicating regions, respectively. An increased number of embryonic pSNMs were located in open chromatin states and topologically associating domains that transcribed embryonically. Our findings provide new insights into the origin and spatial distribution of postzygotic mosaicism during normal human development.Author SummaryGenomic mosaicism led by postzygotic mutation is the major cause of cancers and many non-cancer developmental disorders. Theoretically, postzygotic mutations should be accumulated during the developmental process of healthy individuals, but the genome-wide characterization of postzygotic mosaicisms across many organ types of the same individual remained limited. In this study, we identified and validated two types of postzygotic mosaicism from the whole-genomes of 27 organs obtained from five healthy donors. We further found that the postzygotic mosaicisms arising during early embryogenesis and later clonal expansion events show distinct genomic patterns in mutation spectrum, replication timing, and chromatin status.

CLAMP regulates Zygotic Genome Activation in Drosophila embryos

Genetics ◽  
2021 ◽  
Author(s):  
Megan M Colonnetta ◽  
Juan E Abrahante ◽  
Paul Schedl ◽  
Daryl M Gohl ◽  
Girish Deshpande
Keyword(s):  
Temporal Dynamics ◽  
Embryonic Patterning ◽  
Zygotic Genome Activation ◽  
Promoter Sequences ◽  
Genome Wide ◽  
Zygotic Transcription ◽  
Pioneer Factor ◽  
Genome Activation ◽  
Drosophila Embryos ◽  
Zygotic Genome

Abstract Embryonic patterning is critically dependent on zygotic genome activation (ZGA). In Drosophila melanogaster embryos, the pioneer factor Zelda directs ZGA, possibly in conjunction with other factors. Here we have explored novel involvement of Chromatin-Linked Adapter for MSL Proteins (CLAMP) during ZGA. CLAMP binds thousands of sites genome-wide throughout early embryogenesis. Interestingly, CLAMP relocates to target promoter sequences across the genome when ZGA is initiated. Although there is a considerable overlap between CLAMP and Zelda binding sites, the proteins display distinct temporal dynamics. To assess whether CLAMP occupancy affects gene expression, we analyzed transcriptomes of embryos zygotically compromised for either clamp or zelda and found that transcript levels of many zygotically-activated genes are similarly affected. Importantly, compromising either clamp or zelda disrupted the expression of critical segmentation and sex determination genes bound by CLAMP (and Zelda). Furthermore, clamp knockdown embryos recapitulate other phenotypes observed in Zelda-depleted embryos, including nuclear division defects, centrosome aberrations, and a disorganized actomyosin network. Based on these data, we propose that CLAMP acts in concert with Zelda to regulate early zygotic transcription.

scholarly journals Repli-seq: genome-wide analysis of replication timing by next-generation sequencing

2017 ◽  
Author(s):  
Claire Marchal ◽  
Takayo Sasaki ◽  
Daniel Vera ◽  
Korey Wilson ◽  
Jiao Sima ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Replication Timing ◽  
Nucleotide Polymorphisms ◽  
Robust Methods ◽  
Next Generation ◽  
Single Nucleotide ◽  
Cellular Processes ◽  
Genome Wide ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

ABSTRACTCycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in disease. Exploring mechanisms linking RT to other cellular processes in normal and diseased cells will be facilitated by rapid and robust methods with which to measure RT genome wide. Here, we describe a rapid, robust and relatively inexpensive protocol to analyze genome-wide RT by next-generation sequencing (NGS). This protocol yields highly reproducible results across laboratories and platforms. We also provide computational pipelines for analysis, parsing phased genomes using single nucleotide polymorphisms (SNP) for analyzing RT allelic asynchrony, and for direct comparison to Repli-chip data obtained by analyzing nascent DNA by microarrays.

scholarly journals CTCF looping is established during gastrulation in medaka embryos

2018 ◽  
Author(s):  
Ryohei Nakamura ◽  
Yuichi Motai ◽  
Masahiko Kumagai ◽  
Haruyo Nishiyama ◽  
Neva C. Durand ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Embryonic Development ◽  
Protein Binding ◽  
Early Development ◽  
Great Increase ◽  
Critical Role ◽  
Early Embryogenesis ◽  
Genome Architecture ◽  
Time Points ◽  
Genome Wide

Abstract:Genome architecture plays a critical role in gene regulation, but how the structures seen in mature cells emerge during embryonic development remains poorly understood. Here, we study early development in medaka (the Japanese killifish, Oryzias latipes) at 12 time points before, during, and after gastrulation which is the most dramatic event in early embryogenesis, and characterize transcription, protein binding, and genome architecture. We find that gastrulation is most associated with drastic changes in genome architecture, including the formation of the first loops between sites bound by the insulator protein CTCF and great increase in the size of contact domains. However, the position of CTCF is fixed throughout medaka embryogenesis. Interestingly, genome-wide transcription precedes the emergence of mature domains and CTCF-CTCF loops.

scholarly journals Genome-Wide Analysis of the Arabidopsis Replication Timing Program

2018 ◽  
Vol 176 (3) ◽  
pp. 2166-2185 ◽  
Author(s):  
Lorenzo Concia ◽  
Ashley M. Brooks ◽  
Emily Wheeler ◽  
Gregory J. Zynda ◽  
Emily E. Wear ◽  
...  
Keyword(s):  
Replication Timing ◽  
Genome Wide Analysis ◽  
Genome Wide

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

2018 ◽  
Vol 217 (11) ◽  
pp. 4025-4048 ◽  
Author(s):  
Yu Chen ◽  
Yang Zhang ◽  
Yuchuan Wang ◽  
Liguo Zhang ◽  
Eva K. Brinkman ◽  
...  
Keyword(s):  
Genome Organization ◽  
Spatial Organization ◽  
Essential Feature ◽  
K562 Cells ◽  
Housekeeping Genes ◽  
Mapping Method ◽  
Spatial Gradient ◽  
Nuclear Speckles ◽  
Nuclear Compartments ◽  
Genome Wide

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.

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