1. By Wenfei Jin Presenter: Peter Kyesmu
Genome-wide Detection of DNase I Hypersensitive Sites in Single Cells and FFPE Samples
By Wenfei Jin
Presenter: Peter Kyesmu

2. Introduction to DNase I hypersensitive sites (DHS )
DNase I hypersensitive sites (DHSs)
Contain valuable information regarding the presence of transcriptional regulatory elements.
transcriptional regulatory elements = promoters and enhancers

3. Transcriptional Regulatory Elements
These DHSs sites are known to provide important information on transcriptional regulatory elements (promoters and enhancers).
Enzyme must recognize a region called the promoter on the DNA sequence that has the gene to be expressed.
Promoters are known to provide position for initial binding of RNA polymerase.

4. Biological Significance of these sites
These regions become free due to prospector proteins that are nucleosome remodelers that push nucleosomes off DNA that frees areas.
DHS and genomic seq shows there are sites free of nuclei found adjacent to transcription start site corresponding to enhancers
Promotors and enhancer have been detected for this enzyme and importantly can be detected in just single cells.

5. Bases to Chromosome
The main focus of this upcoming section will be focused on the strands in in which is utilized to identify DHS sites along with the histone and their modifications

6. DNase I hypersensitive sites (DHSs) Fall in two main categories
Intro to DHS
DNase I hypersensitive sites (DHSs) Fall in two main categories
1. Promoters
2. Enhancers
Transcription, the first stage of gene expression involves three stages:
Initiation
Elongation
Termination
Main Focus - Initiation

7. Pico-Seq
Pico-Seq detects a much larger number of DHSs per cell, which provides information on cell-to-cell variations of individual DHSs. 
Pico-Seq is expected to find its use in multiple settings,
analysis of rare cell populations during lineage development
the study of clinical samples with extremely small number of cells such as circulating tumor cells, laser-captured cells, core biopsy or fine needle aspiration samples. 
Pico-Seq detects a much larger number of DHSs per cell, which provides information on cell-to-cell variations of individual DHSs. 
Fine-needle aspiration (FNA) is a diagnostic procedure used to investigate lumps or masses. 

8. Pico-Seq
Pico-Seq can reliably detect DHSs in single cells as compared to the conventional method require millions of cells.
This greatly extends the range of DHS analysis for both basic and translational research and may provide critical information for personalized medicine combating health issues.

9. Notable Mention of Significance
The most significant difference in Wenfei Jin et al 2016 contribution is the ability to detect DHSs in single cells as against the conventional approach that will require millions of cells to detect DHSs.
Through their single–cell DHSs technique, the were able to predict enhancers that can regulate cell specific gene expression
They were also able to use the technique to detect tumor–specific DHSs that are critically involved in cancer development.
This can facilitate the detection of early cancer development in patients.

10. Histone Modifications
DNase I hypersensitive sites (DHSs) are associated with multiple histone modifications.
Distal DHSs in single cells clearly enriched in the modification
Non - Repressive
Repressive
H3K4me1
H3K9me2
H3K4me3
H3K27me3
H3K9ac
H3K27ac
H2A.Z
H3K36me3

11. Suppress gene expression
Histone mod have high pico tag density than non repressive meaning that the non repressive histone mod will be open for gen expression.
Histone modifications are associated with higher Pico-Seq tag density than the repressive H3K27me3 and H3K9me2 modifications in single cells.

12. If + correlation, the mod in the dna sequence with the histones are open for gene expression, start of gene expresion is transcript that ends with transl
The dna with those histones there are available for transcription
The Pico-Seq density in cell #1 correlated with the number of histone active modifications.

13. Histone Modifications and Correlation To DHS’s
The tag densities of each active histone modification peak were determined and whether the peak was a DHS in each single cell
This can identify if the density of an active histone mark is related to the number of cells with DHS at the same locus. 
Pico-Seq is utilized to examine the DHS site to determine whether they occur in correlation to the histone modifications which allow or inhibit expression.
Among different single cells, highly expressed gene promoters and the enhancers associated with multiple active histone modifications display constitutive DHS (accurately repeated and low variance) while chromatin regions with fewer histone modifications exhibit high variation not validated by repeats of DHS.
Furthermore, the single-cell DHSs predict enhancers that regulate cell-specific gene expression programs and the cell-to-cell variations of DHS are predictive of gene expression.. 

14. Medical Significance of DHS normal and tumorous cells in Patient
Despite the small fraction of normal specific and tumor specific DHSs in the total DHS, the ability to analyze and locate these regions is a clinical advantage.
Possible tagging of Histones to help identify proteins of a tumor-specific DHS region is a technique that can be used in detecting cancer in a patient predisposed.
 Normal-specific and tumor-specific DHSs account a small fraction of the total DHS

15. Utilizing Pico-Seq in FTC patients to recognize tumor related DHS sites
Pico-Seq is applied to pools of tumor cells and pools of normal cells from formalin- fixed paraffin-embedded (FFPE) tissue slides from thyroid cancer patients, and detect thousands of tumor-specific DHSs.
Many of these DHSs are associated with promoters and enhancers critically involved in cancer development.
Analysis of the DHS sequences uncovers one single-nucleotide variant in the tumor cells of a follicular thyroid carcinoma (FTC) patient, which affects the binding of the tumor suppressor protein p53 and correlates with decreased expression of its target gene TXNL1.
After dhs is identified

16. Correlation of Tumor DHS sites and sample specific DHS sites
Comparison of the tumor-specific DHSs identified in the three FTC samples revealed very few shared DHSs among all three FTC samples 
#440, #797, #957
The vast majority of DHSs are unique to each individual tumor case.
applied Pico-Seq to cells dissected from follicular thyroid carcinoma (FTC) sample fixed on FFPE slides

17. Observance of tumor specific DHS in FTC sample #440
There are genes associated with the tumor-specific DHSs such as TIAM1 and PIP4K2A.
applied Pico-Seq to cells dissected from follicular thyroid carcinoma (FTC) sample fixed on FFPE slides
a. Genome Browser image showing the increased chromatin accessibility of two tumor-specific DHS regions at the PIP4K2A gene locus (left panel). The ENCODE H3K4me1 and H3K4me3 peaks are shown at the bottom of the panel. The PIP4K2A mRNA levels in normal and tumor cells, respectively, determined by quantitative RT-PCR and normalized to GAPDH (Right panel).
b, Genome Browser image showing the increased chromatin accessibility of the TIAM1 promoter in thyroid tumor cells (left panel). TIAM1 mRNA levels in normal and tumor cells, respectively, determined by quantitative RT-PCR and normalized to GAPDH (Right panel).

18. Observance of tumor specific DHS in FTC samples #440, #957, and #797
Genome Browser image showing the two tumor- specific DHSs at the HMGA2 locus in three FTC patients

19. HMGA2 Promoter variance in FTC samples #440, #957, and #797
The HMGA2 promoter exhibited a strong DHS in the tumor cells but not in their neighboring normal cells in #440, #797, #957
These results suggest that the mis-regulation of HMGA2 in the tumor cells may be attributed to different regulatory elements in different patients
Indicating that the large majority of DHSs are patient-specific, meaning that these tumors may likely progress through different mechanisms in different patients.

20. Conclusion
Cancer tumors require proteins and to synthesize them within promotors and enhancers, they require special markers.
Using tumor sites can allow the identification of proteins associated with tumor cells. This technique can be used to detect early cancer.
Providing an innovative clinical advantage through sequencing, this is a method that can be used in the future for various disease detection and treatments.
DMEM formula contains 1000 mg/L of glucose and was first reported for culturing embryonic mouse cells