about omics group · chromatin affinity purification with mass spectrometry . needle in a haystack...
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About OMICS Group
OMICS Group International is an amalgamation of Open Access publications and worldwide international science conferences and events. Established in the year 2007 with the sole aim of making the information on Sciences and technology ‘Open Access’, OMICS Group publishes 400 online open access scholarly journals in all aspects of Science, Engineering, Management and Technology journals. OMICS Group has been instrumental in taking the knowledge on Science & technology to the doorsteps of ordinary men and women. Research Scholars, Students, Libraries, Educational Institutions, Research centers and the industry are main stakeholders that benefitted greatly from this knowledge dissemination. OMICS Group also organizes 300 International conferences annually across the globe, where knowledge transfer takes place through debates, round table discussions, poster presentations, workshops, symposia and exhibitions.
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About OMICS Group Conferences
OMICS Group International is a pioneer and leading science event organizer, which publishes around 400 open access journals and conducts over 300 Medical, Clinical, Engineering, Life Sciences, Pharma scientific conferences all over the globe annually with the support of more than 1000 scientific associations and 30,000 editorial board members and 3.5 million followers to its credit.
OMICS Group has organized 500 conferences, workshops and national symposiums across the major cities including San Francisco, Las Vegas, San Antonio, Omaha, Orlando, Raleigh, Santa Clara, Chicago, Philadelphia, Baltimore, United Kingdom, Valencia, Dubai, Beijing, Hyderabad, Bengaluru and Mumbai.
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Proteomic Approaches for Epiproteome Analysis
Alan Tackett, PhD
Univ of Arkansas for Medical Sciences Dept Biochemistry & Molecular Biology
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Epigenetics
Science 29 March 2013: Vol. 339 no. 6127 pp. 1567-1570
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Epigenetic Reprogramming in Cancer
Science 29 March 2013: Vol. 339 no. 6127 pp. 1567-1570
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Source: Cell , Volume 150, Issue 1, Pages 12-27 (DOI:10.1016/j.cell.2012.06.013)
Epigenetic Inhibitors as Cancer Therapies
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Histone epigenetics
H3
H4
H2A
H2B
Luger et al., Nature 1997
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Histone Code Hypothesis
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Gene Transcription in the Context of Chromatin
http://www.nature.com/npp/journal/v33/n1/images/1301544f3.jpg
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://en
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Numerous Histone Epigenetic Targets
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Source: Cell , Volume 150, Issue 1, Pages 12-27 (DOI:10.1016/j.cell.2012.06.013)
Cancer Mutations Affecting Epigenetic Regulators Involved in Histone Acetylation
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Source: Cell , Volume 150, Issue 1, Pages 12-27 (DOI:10.1016/j.cell.2012.06.013)
Cancer Mutations Affecting Epigenetic Regulators Involved in Histone Methylation
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Local Epiproteome
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Methods for locus-specific epiproteome measurements
1) Target one protein/PTM at a time 2) Co-localization of proteins/PTMs can only be inferred 3) No information of the combinatorial set of PTMs on single
histone molecules 4) You have to know what you are targeting
Limitations with Standard ChIP
ChIP
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Chromatin Affinity Purification with Mass Spectrometry
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Needle in a Haystack (1:3,000,000 – that’s a lot of straw)
Let’s start with budding yeast 1:12,000
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Preventing Histone Exchange During Chromatin Isolation
Transient I-DIRT (isotopic differentiation of interactions as random or targeted)
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Solubilizing and Purifying Cognate Chromatin Sections
FA (%)
No Sonication Sonication
2000 1650
1000 850
500
Genomic DNA
bp
0 0.05 0.25 1.25 0 0.05 0.25 1.25
} H3/H2B/H2A/H4
H2B-TAP
0 0.05 0.25 1.25
36-
22-
16-
FA (%)
kDa
6-
Efficient purification of chromatin sections requires optimized in vivo cross-linking
S1 AP
0
0.05
0.25
1.25
FA (%
)
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Cross-linking is Necessary to Prevent Histone Exchange
FA (%)
Isot
opic
ally
ligh
t pep
tides
(%)
40
50
60
70
80
90
100
0 0.05 0.25 1.25
H2B-TAP
H2A
H3
H4
Non-specific
1.25% formaldehyde cross-linking is necessary to prevent histone exchange in yeast synthetic media – level is dependent on reactive content of media
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ChAP Technology
Byrum et al (2011) JIOMICS Byrum et al (2013) Nuc Acids Res Byrum et al (2011) J Clin Bioinfor Byrum et al (2014) Meth Mol Biol Byrum et al (2012) Cell Reports US Patent 61/726,936 Waldrip et al (2014) Epigenetics R01DA025755 & R01GM106024 (AJT)
Three Platforms for Single Locus Purification
1st Generation
ChAP-MS
2nd Generation
TAL-ChAP-MS
3rd Generation
CRISPR-ChAP-MS
Gene X Gene Y Gene X Gene Y
Genomic Engineering of a LexA binding site
LexA
PrA
Gene X Gene Y
• No genomic engineering • Transcription activator-like
effector nuclease (TALEN) • 18 tandem domains of 34 amino
acids each recognize single nucleotides
• Targets 18mer
PrA-Cas9
• No genomic engineering • Clustered regularly
interspaced short palindromic repeats (CRISPR)
• RNA-guided nuclease • Catalytically dead nuclease
available • Targets 20mer
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4336–4343 Nucleic Acids Research, 2013, Vol. 41, No. 7
CRISPR-ChAP-MS
X X
PrA
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Measurement of Epiproteome at GAL1 Promoter Chromatin Using CRISPR-ChAP-MS
.... .... .... .... GAL1
PrA-Cas9
Cross-link Shear Chromatin
Affinity Enrich
MS/MS
m/z
inte
nsity
.... .... .... ....
PrA-Cas9
• Glucose sets an inactive chromatin state
• Galactose sets
transcriptionally active
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0
250
500
750
1000
- gRNA +gRNA
Rela
tive
GAL
1 Tr
ansc
riptio
n (G
alac
tose
/Glu
cose
)
+ − + − + + + +
pPrA-Cas9
pgRNA-GAL1
PrA-Cas9
Histone H4
Galactose Glucose
Expression of the PrA-Cas9/gRNA
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0 10 20 30 40 50 60 70 80 90
100
2000 up GAL1 2000 down
2000 up GAL1 2000 down
OT1 OT2 OT3 OT4
Rela
tive
Enric
hmen
t of T
arge
t pPrA-Cas9
pPrA-Cas9 pgRNA-GAL1
Galactose Glucose
+ PAM motif - PAM motif
4.9-fold 4.4-fold 3.2-fold
70-fold
*
*
* *
Chromatin Enrichment by PrA-Cas9/gRNA
GAL1
PrA-Cas9
GAL10
GAL1 primers
2000 down primers
2000 up primers
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Proteomic Analysis of GAL1 Promoter Chromatin
• Chromatin isolated from 109 cells
• 1832 proteins identified
• Spectral count comparison revealed 86 specific
• 11 of the 86 had annotated functions on chromatin
• H3K23ac and H3K14ac
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0
2
4
6
8
10
12
Pob3 Rsc6 Rsc7 Spt5 H3K14ac
Rela
tive
Enric
hmen
t at G
AL1
Glucose
Galactose
ChIP Validation of CRISPR-ChAP-MS Results
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Chromatin Upstream of GAL1
Silent
GAL1
H3K36me3
GAL1
Active
Galactose Hyper-Ac RNA pol & yFACT
Gal3 } }
viewing window
viewing window
Rsc
Spt5
All Three ChAP-MS Platforms
Hyper-Ac Histones
H3K14ac
H3K23ac
H3K9acK14ac
H3K18acK23ac
H4K5acK8ac
H4K12acK16ac
} Only approach to obtain combinatorial info
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• Currently targeting promoter chromatin of EZH2 RUNX3 E-cadherin DAB2IP BCL2 • WM115 vs WM266-4 melanoma cell lines
• FFPE patient melanoma Fourth Generation ChAP-MS
Application of ChAP-MS to Melanoma Cells
rTAL-ChAP-MS
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Tackett Lab Stephanie Byrum, PhD
Marie Burdine, PhD Nathan Avaritt
Linley Moreland, MS Deepa Sengupta
Zach Waldrip Kirk West
Lauren Davis Lisa Orr
UAMS Proteomics Facility Rick Edmondson, PhD Sam Mackintosh, PhD Linley Moreland, MS
Lisa Orr
Tackett Lab Funding NIH R01DA025755 (AJT) NIH R01GM106024 (AJT) NIH R33CA173264(AJT)
People Responsible…..
Sean Taverna’s Lab at Johns Hopkins Medical School
UAMS Pathology Dept Sara Shalin, MD/PhD
Jennifer Hunt, MD
Kevin Raney’s Lab UAMS
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Let Us Meet Again
We welcome you all to our future conferences of OMICS Group
International
Please Visit: www.omicsgroup.com
www.conferenceseries.com www.proteomicsconference.com