1. Chromatin accessibility by ATAC-seq
How to identify genome-wide open chromatin sites?
How to create gene regulatory network from ATAC-seq?
Clinical applications of ATAC-seq.
Discussion:
Limitations of motif based regulatory network prediction.

3. Limitations of current epigenomic methods
There are many key elements involved in gene regulation such as the position, arrangement, and chemical modifications of histones, DNA methylation, ncRNA expression, Insulator, transcription factors on enhancers and promoters, chromosome 3D structure etc. All these make the whole process extremely complicated. It is critical to understand how nuclreasomes are compacted. For chromosome positions with compact chromatin, open chromatin, and phased nucleosomes.
Consequences of these reqirements: forced to cell lines, expand cells ex vivo, or pool together many individuals.
Multiple steps include enrich for fragment, repair ends, ligate, purify.
Techniques (e.g., DNase-seq) for genome-wide chromatin measurement:
Require large number of cells (10-20 million)
Are time consuming (3-5 days); protocol > 40 steps

4. Assay of Transposase Accessible Chromatin
Tn5 Transposase
Closed chromatin
Transposase uses cut- and-paste mechanism to insert transposons. In ATAC-seq, Tn5 transposase integrates its adaptor payload into regions of accessible chromatin,
More open more likely to be cut and sequenced and higher signal. Greatly improves the sensitivity and speed.
Open chromatin
● 1 million fold improvement sensitivity, 100 fold improvement in speed.
Buenrostro et al., Nature Methods, 2013; Tn5 structure Davis et al., Science, 2000.

5. Identification of active regulatory elements
Since ATACseq uses few number of cells, the assay Make epigenomic-based diagnostic and drug repositioning possible

6. ATAC-seq reveals DNA-protein interactions
Genome-wide CTCF motifs
● Binding locations of hundreds of factors read out at once, in allele-specific fashion.
Bound
Unbound
ATAC-seq
p-value
ChIP-seq B A

7. Paired-end sequencing
ATAC-seq reveals higher order chromatin structure
Paired-end sequencing
Since ATACseq uses pairend sequencing without size selection, we are able to detect short and long DNA fragments
By looking at fragment size distribution, ATAC-seq is able to reveal higher order chromatin structure
Amplification of transposed DNA barcodes

8. ATAC insert size reveals chromatin structure
The insert size distribution of sequenced fragments from human chromatin had clear periodicity of approximately 200 bp, suggesting many fragments are protected by integer multiples of nucleosomes. This fragment size distribution also showed clear periodicity equal to the helical pitch of DNA.
Buenrostro et al., Nature Methods, 2013.

9. ATAC-seq reveals functional epigenomic states
By partitioning the insert size distribution according to functional classes of chromatin as defined by previous models and normalizing to the global insert distribution (Online Methods), we observed clear class-specific enrichments across this insert size distribution
Implication
Determination of epigenetic states in disease and impact of treatment on critical gene sets

10. We developed a rapid and efficient epigenomic profiling technology

11. ATACseq enables personal regulomes in real time
10.5 hours
Drug
selection
Because ATAC-seq is rapid, information rich and compatible with small numbers of cells, it may serve as a powerful tool to look at aspects of an individual’s epigenome on a clinical timescale
We applied ATAC-seq to assay the epigenome of a healthy volunteer’s T cells, and applied rapid T-cell activation protocols. ATACseq signal at 3 continuous days shows changes at IL2 locus. IL-2 is a key cytokine that drives T-cell growth and functions in inflammatory and autoimmune diseases.
Furthermore, ATACseq could be applied to drug selection. For example, distinct drugs inhibit the activities of different TFs that bind putative IL2 enhancers. One might wish to identify the TF pathway in order to rationally target inhibition without exposing the patient to drugs unlikely to serve the therapeutic goal of IL-2 blockade. ATAC-seq showed that in this condition, only nuclear factor of activated T cells (NFAT), but not two other drug targets, engaged IL2. Thus, ATAC-seq was able to provide clinically relevant information on the regulatory state of this individual.

12. Sen, D. R., et al. (2016). Science.
最近有两篇science文章，都是利用ATACseq，在PD-1基因Pdcd1基因上游23kb处，发现耗竭性T细胞特异性的调控位点，调节着PD-1基因的表达，并由此引起T细胞耗竭。这也说明了ATACseq技术的应用前景。
Fig. 3. PD-1 pathway blockade fails to restore memory-like recall capacity or reprogram the epigenetic landscape of TEX into TEFF or TMEM cells
Fig. 3. High-resolution functional mapping of an exhaustion-specific enhancer identifies minimal sequences that regulate PD-1.

13. Individuality and variation of T-cell regulome
Share some unpublished
First story. What is normal?
Diversity and fidelity of chromatin landscape over time in vivo not known.
This is a work in progress.
Qu et al, Cell Syst., 2015

14. Individuality and variation of T-cell regulome
Within over 60K open chromatin sites, there are about 10%
Each column is a sample. Each row is a regulatory element.
Color indicates how active the element is . There could be many reasons causes inter-individual variation
Intrinsic analysis
Gender shows the most striking difference, and some interindividual variants correlate with T cell subtypes.
Qu et al, Cell Syst., 2015

15. Gender and autoimmunity
● 80% of patients with autoimmunity are women
● Sex chromosome changes long postulated to be involved
Invernizzi et al., Autoimmunity, 2009.

16. X chromosome Inactivation
It is speculated that genes escape XCI is involved in auto-immune diseases.
In all mammalian females, one of two X chromosomes is made transcriptionally silent to equalize the dosage of X to autosome genes in males and females
The inactive X is permanently heterochromatinized: it is condensed (termed the Barr body), late replicating, and transcriptionally inactive.
Xist is 17kb ncRNA transcribed from inactive X and appears to coat the entire inactive X in cis.
Some genes escape X chromosome inactivation.

17. Gender specific regulome
● Personal regulome is sensitive and precise.
Qu et al, Cell Syst., 2015

18. FIRRE escapes X inactivation
Hacisuleyman et al., NSMB, 2014.

19. Gender-specific regulatory elements mediate XCI escape
● Regulome insight allows one to specifically control
male or female version of the same gene
Demonstrates Principle: When a gene is induced in a disease state, the regulatory input for its activation can be quite different to physiology. Targeting the gene product will affect both diseased and normal cells, causing side effects. Targeting regulatory input can specifically wipe out gene specifically in disease cells.
Qu et al, Cell Syst., 2015

20. Cutaneous T Cell Lymphoma
Neoplasm of skin resident T cells
Mycosis fungoides
Sezary syndrome
First approved indication for HDACi
Mechanism and dynamics of HDACi in patients unclear

21. Regulome dynamics of cutaneous T cell lymphoma
Qu et al, Cancer Cell, in press

22. Landscape of DNA accessibility in normal CD4+, CTCL leukemia and host cells
Qu et al, Cancer Cell, in press

23. Epigenomic signatures of CTCL leukemic and host cells
Qu et al, Cancer Cell, in press

24. DNA accessibility change is correlated with differential mRNA expression in CTCL
Qu et al, Cancer Cell, in press

25. TF occupancy networks in CTCL
Qu et al, Cancer Cell, in press

26. Genomic and epigenomic landscapes in CTCL
ATACseq也可以提供给我们一些遗传相关的信息。我们发现，和正常人相比，部分患者的染色体拷贝数存在差异，如染色体17p的loss，17q的gain，10q的loss，然而在患者的正常细胞和正常人样品中却没有发现类似的现象。有趣的是，我们发现在著名的抑癌基因TP53的promoter区域，有一个非常明显的调控位点。P11和P1424两位患者在接受药物治疗后，病情有所减轻，对药物有反应，可以看到TP53 promoter区域染色质逐渐变得更开放。相反，P20和P5两位患者，对药物没有反应，他们的在这个区域的染色质就没有逐渐开放的趋势。
Qu et al, Cancer Cell, in press

27. Personal chromatin dynamics with HDACi treatment
Qu et al, Cancer Cell, in press

28. Enhancer cytometry and cell-type specific response to HDACi therapy
Qu et al, Cancer Cell, in press

29. 正在开展的工作 已经收集了相当数量样品，并获得前期数据 为深入研究肿瘤与免疫疾病提供丰富的数据资源
自身免疫性疾病的表观遗传调控研究
已经收集了相当数量样品，并获得前期数据
为深入研究肿瘤与免疫疾病提供丰富的数据资源
提供多维度、多层次、系统性研究免疫疾病表观遗传机制的新方法
新技术的实现和生物信息分析系统的建立，将其他研究方向提供关键性技术支持和保障

32. Name: Date:
1. Name two technologies that measures chromatin accessibility genome-wide?
2. What are the main advantages of ATAC-seq?
3. Why do we care about chromatin accessibility?
4. What are the main limitations of the motif analysis from ATAC-seq?