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Overarching goal of the lab:
• Understand how identical genotypes in identical environments give rise to different phenotypes The organization of the human genome in the nucleus is almost unbelievable
Image by Christian Bouvier
The functional organization of the human genome
Jonathan Dennis, FSU Biological Science
FSU College of Medicine, Grand Rounds
21 April 2011
Agenda
• Introduction to chromatin structure
• Techniques to assay chromatin structure
• RESULTS
– The molecular pharmacology of the anti‐
inflammatory butyrate
– Chromatin structural biomarkers of cancer tumor grade
Agenda
• Introduction to chromatin structure
• Techniques to assay chromatin structure
• RESULTS
– The molecular pharmacology of the anti‐
inflammatory butyrate
– Chromatin structural biomarkers of cancer tumor grade
We know surprisingly little about the organization of the human genome
Image by Christian Bouvier
The organization of the human genome must occur at multiple levels
Analogy to levels of protein structure
The organization of the human genome must occur at multiple levels
Analogy to levels of protein structure
We know surprisingly little about the organization of the human genome
PRIMARY STRUCTURE
Image by Christian Bouvier
We know the structure of the core particle
Crystal structure of the nucleosome core particle at 2.8 Å resolution
Karolin Luger, Armin W. Mäder, Robin K. Richmond, David F. Sargent and Timothy J. Richmond
Nature 389, 251‐260(18 September 1997)
The primary structure of chromatin is regulated multiple ways
Local chromatin architecture – How are nucleosomes distributed to give appropriate access to DNA sequence?
Two examples:
I. ATP‐dependent remodelers
II. Covalent modification of histones
The primary structure of chromatin is regulated multiple ways
Local chromatin architecture – How are nucleosomes distributed to give appropriate access to DNA sequence?
Two examples:
I. ATP‐dependent remodelers
II. Covalent modification of histones
The primary structure of chromatin is regulated multiple ways
I. ATP‐dependent remodelers
ATP-dep rem.
The primary structure of chromatin is regulated multiple ways
I. ATP‐dependent remodelers
ATP
ADP
ATP-dep rem.
The primary structure of chromatin is regulated multiple ways
I. ATP‐dependent remodelers
ATP dependent remodelers regulate the expression of several genes
Local chromatin architecture – How are nucleosomes distributed to give appropriate access to DNA sequence?
Ordered Recruitment
of Chromatin
Modifying and
General Transcription
Factors to the IFN-β
Promoter
Agalioti, Lomvardas, Parekh,Ye, Maniatis, and Thanos
Cell, Volume 103, Issue 4, 10 November 2000, Pages 667-678
The primary structure of chromatin is regulated multiple ways
Local chromatin architecture – How are nucleosomes distributed to give appropriate access to DNA sequence?
Two examples:
I. ATP‐dependent remodelers
II. Covalent modification of histones
Histones may be covalently modified by protein complexes
II. Covalent modification of histones
Histones may be covalently modified by protein complexes
II. Covalent modification of histones
Histones may be covalently modified by protein complexes
II. Covalent modification of histones
Histones may be covalently modified by protein complexes
II. Covalent modification of histones
•acetylation
•methylation
•ubiquitylation
•and many more…
Histones may be covalently modified by protein complexes
II. Covalent modification of histones
•acetylation
H3 K36: transcription
•methylation
H3 K4: gene activation
•ubiquitylation
H2A K119: Hox cellular memory
•and many more… and many combinations
We know surprisingly little about the organization of the human genome
SECONDARY STRUCTURE
Image by Christian Bouvier
Models of the chromatin fiber are controversial Robinson P J J et al. PNAS 2006;103:6506-6511
©2006 by National Academy of Sciences
We know surprisingly little about the organization of the human genome
TERTIARY STRUCTURE
Image by Christian Bouvier
Very little is known about the tertiary structure of chromosomes
Global chromatin architecture – How are chromosomes organized to give appropriate access to soluble regulatory factors?
LESS ACCESSIBLE
MORE ACCESSIBLE
Very little is known about the tertiary structure of chromosomes
Global chromatin architecture – How are chromosomes organized to give appropriate access to soluble regulatory factors?
CELL TYPE A
CELL TYPE B
The organization of the human genome must occur at multiple levels
Global chromatin architecture – How are chromosomes organized to give appropriate access to soluble regulatory factors?
We know surprisingly little about the organization of the human genome
QUATERNARY STRUCTURE
Image by Christian Bouvier
Chromosomes occupy fixed spaces in the human nucleus
Global chromatin architecture – How are chromosomes organized to interact with one another
Bolzer A, Kreth G, Solovei I, Koehler D, Saracoglu K, et al. 2005
Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes. PLoS Biol 3(5): e157.
A system for the simultaneous assessment of local and global chromatin architecture
MOTIVATIONS:
•What is the relationship between chromatin structure (both at the local and global level) and regulation of nuclear events?
•To what degree are local changes in nucleosome distribution, linked with global changes in chromatin accessibility? A system for the simultaneous assessment of local and global chromatin architecture
REQUIREMENTS:
•High‐throughput – Don’t just look at one cell type. Look at dozens of cell types
•Cost‐effective – The price must not be prohibitive
•Robust – The signal must be reliably interpretable
A system for the simultaneous assessment of local and global chromatin architecture
REQUIREMENTS:
•High‐throughput – Don’t just look at one cell type. Look at dozens of cell types (12 samples simultaneously)
•Cost‐effective – The price must not be prohibitive (~$200‐$300 per sample)
•Robust – The signal must be reliably interpretable (consistent with previous findings and between experiments)
Agenda
• Introduction to chromatin structure
• Techniques to assay chromatin structure
• RESULTS
– The molecular pharmacology of the anti‐
inflammatory butyrate
– Chromatin structural biomarkers of cancer tumor grade
Techniques to assay chromatin structure
We use a single nuclease (MNase) to assay
• Primary structure – nucleosome distribution
• Tertiary structure – chromosome accessibility
Techniques to assay chromatin structure
We use a single nuclease (MNase) to assay
• Primary structure – nucleosome distribution
• Tertiary structure – chromosome accessibility
Nuclease as a probe of nucleosome distribution–
Micrococcal nuclease is an internucleosomal cutter
Nuclease as a probe of nucleosome distribution –
Micrococcal nuclease is an internucleosomal cutter
Nuclease as a probe of nucleosome distribution –
Micrococcal nuclease is an internucleosomal cutter
[MNase]
4.3
kbp
2.0
kbp
7N
6N
5N
4N
3N
500 bp
2N
N
Brian Spetman
Nuclease as a probe of nucleosome distribution –
Micrococcal nuclease is an internucleosomal cutter
[MNase]
4.3
kbp
2.0
kbp
7N
6N
5N
4N
3N
500 bp
2N
N
Brian Spetman
Nuclease as a probe of nucleosome distribution –
Micrococcal nuclease is an internucleosomal cutter
[MNase]
4.3
kbp
2.0
kbp
7N
6N
5N
4N
3N
500 bp
2N
N
Brian Spetman
Nuclease as a probe of nucleosome distribution –
nucleosome distribution determined by microarray [MNase]
4.3
kbp
2.0
kbp
7N
6N
5N
4N
3N
500 bp
2N
N
Each “subarray” contains 135,000 probes that cover up to 500 TSS at 13bp resolution
~150 ~
bp DNA
nucleosome posi oning nucleosome posi oning Nuclease as a probe of nucleosome distribution –
readout = log (nucleosomal/bare genomic)
TSS ‐1 NFR +1 genomic posi on TSS ‐1 NFR +1 genomic posi on nucleosome posi oning nucleosome posi oning Nuclease as a probe of nucleosome distribution –
readout = log (nucleosomal/bare genomic)
TSS ‐1 NFR +1 genomic posi on IDEALIZED
TSS ‐1 NFR +1 genomic posi on CD69 TSS
U937 (human histiocytic lymphoma)
Brian Spetman
Nuclease as a probe of nucleosome distribution –
We can observe TSS poising or activation
Unstimulated U937
Imiquimod (viral) stimulated
Allows us to characterize the promoter architecture associated with specific classes of genes
Brittany Sexton
Techniques to assay chromatin structure
We use a single nuclease (MNase) to assay
• Primary structure – nucleosome distribution
• Tertiary structure – chromosome accessibility
Nuclease as a probe of chromatin accessibility
Nuclease as a probe of chromatin accessibility –
accessible areas are more readily cut
Nuclease as a probe of chromatin accessibility –
accessible areas are separable by electrophoresis
Nuclease as a probe of chromatin accessibility –
genomic coordinates of [in]accessible determined by microarray LESS ACCESSIBLE
Label with Cy5
MORE ACCESSIBLE
Label with Cy3
Nuclease as a probe of chromatin accessibility –
genomic coordinates of [in]accessible determined by microarray LESS ACCESSIBLE
Label with Cy5
MORE ACCESSIBLE
Label with Cy3
Nuclease as a probe of chromatin accessibility –
genomic coordinates of [in]accessible determined by microarray LESS ACCESSIBLE
Label with Cy5
MORE ACCESSIBLE
Label with Cy3
Each “subarray” contains 135,000 probes that cover the entire human genome at 12.5 kb resolution
Nuclease as a probe of chromatin accessibility ‐
readout = log (inaccessible/accessible)
log(inaccessible/accessible)
Whole human genome
Chr.
less accessible
more accessible
1
2
3
4
5
6
7
8 9 10
12
14
16
20
Jurkat ‐ T lymphocyte cells
Crystal Pickeral
Nuclease as a probe of chromatin accessibility ‐
readout = log (inaccessible/accessible)
log(inaccessible/accessible)
Whole human genome
Chr.
less accessible
more accessible
1
2
3
4
5
6
7
8 9 10
12
14
16
20
Jurkat ‐ T lymphocyte cells
Crystal Pickeral
Nuclease as a probe of chromatin accessibility ‐
readout = log (inaccessible/accessible)
log(inaccessible/accessible)
Chromosome 1
less accessible
more accessible
Jurkat ‐ T lymphocyte cells
Crystal Pickeral
CELLS
EXPRESSION
ACCESSIBILITY
NUCLEOSOME
Agenda
• Introduction to chromatin structure
• Techniques to assay chromatin structure
• RESULTS
– The molecular pharmacology of the anti‐
inflammatory butyrate
– Chromatin structural biomarkers of cancer tumor grade
Histone Deacetylase Inhibitors Prevent Nucleosome Redistributions at Transcription Start Sites
Crystal Pickeral
• FSU Undergraduate Senior (for about 10 more days)
• FSU College of Medicine Student (in about six weeks) Inflammatory Bowel disease is a prevalent autoimmune disorder with an unknown cause
genetic
immunological
IBD
dysbiosis
infectious
Chronic inflammation associated with IBD is caused by dysregulation of the mucosal immune system • Mucosal immune system – Depends on delicate balance of commensal
bacteria and harmful pathogens
– IBD patients have irregularities in their microflora
Frank DN, St. Amand AL, Feldman RA, Boedeker EC, Harpaz N, Pace NR. (2007) Molecular‐phylogenetic
characterization of microbial community imbalances in human inflammatory bowel diseases. Proc Natl Acad Sci
34:13780‐13785 One of the important roles of commensal bacteria is aiding in digestion
• Breaking down of complex carbohydrates into short chain fatty acids
• Butyrate is a short chain fatty acid that provides energy to intestinal epithelial cells
• Members of Firmicutes
phylum are the main butyrate producing bacteria
Frank DN, St. Amand AL, Feldman RA, Boedeker EC, Harpaz N, Pace NR. (2007) Molecular‐phylogenetic
characterization of microbial community imbalances in human inflammatory bowel diseases. Proc Natl Acad Sci
34:13780‐13785 Butyrate is a powerful histone deacetylase
inhibitor (HDACi)
• Histone tails can be modified by acetylation, methylation and phosphorylation
• These modifications affect the interactions between DNA and the histones that ultimately affect the organization of chromatin. • Butyrate is a powerful histone deacetylase inhibitor
Sparmann A, Lohuizen MV. 2006. Polycomb silencers control cell fate, development and cancer. Nature Reviews Cancer 6:846‐856
Butyrate blocks the removal of acetyl groups from histone tails leading to a hyperacetylated state. Johnstone RW. 2002. Histon‐deacetylase inhibitors: novel drugs for the treatment of cancer. Nature Reviews:Drug Discovery 1:287‐299
Butyrate provides a connection between chromatin structure and dysbiosis
Gut lumen
“good” bacteria
“bad” bacteria
Carbohydrates
butyrate
Epithelial Cells
butyrate
Macrophage
HDACi
AC
AC
AC
HDACi are classified according to structure Hydroxamate‐based HDACi
Kazantsev AG, Thompson LM. 2008. Therapuetic application of histone deacetylase
inhibitors for central nervous system disorders. Nature Reviews Drug Discovery 7:854‐868
Sodium butyrate Valproic acid (VPA)
• Hydroxamate‐based class 1 HDAC inhibitor
• Hydroxamate‐based class 1 HDAC inhibitor
• Butyrate is produced in the gut by commensal bacteria. • Used to treat certain types of seizures, mania in people with bipolar disorder and to prevent migraine headaches.
• Butyrates are common treatments for Inflammatory Bowel Disease (IBD)
• Being tested in treatments for inflammatory diseases.
HDACi have anti‐inflammatory properties • Butyrate decreases the production of TNF‐
and IL‐12
• Valproic acid decreases the production of INF‐
in a concentration dependent manner
Saemann MD, Bohmig GB, et. Al.2000. Anti‐inflammatory effects of sodium butyrate on human monocytes: potent inhibition of IL‐12 and up‐regulation of IL‐10 production. The FASEB journal 14:2380‐2382
HDACi have potent anti‐inflammatory properties • The mechanism behind these anti‐inflammatory properties is unknown
Predict that butyrate and VPA could operate via similar mechanisms • Compounds with similar structures
VPA: Butyrate:
• Both act by blocking the machinery that would remove acetyl groups from histones
• But are administered for very different reasons
• Investigating the effect of these drugs on chromatin structure will provide insight into the molecular pharmacology of these drugs.
Experimental goal
• The goal of this experiment is to understand the relationship between the anti‐
inflammatory properties of histone
deacetylase inhibitors and the changes they induce in chromatin structure in a resting state and during an immune insult.
Significance
• We propose that the study of the effect of class 1 HDAC inhibitor treatment on chromatin structure will illuminate the pathology of inflammatory disorders as well as provide insight into the mechanisms, behind how HDAC inhibitors are effective treatments. Treatment and harvesting of cells
PMA differentiated U937 cells 1x107/experiment
Time (hours)
Untreated
+1mM VPA
+2mM NAB
+LPS
4 hours
8 hours
+LPS
+LPS
4 hours
Untreated
NAB
NAB+LPS
VPA+LPS
VPA
LPS
LPS is a strong inducer of the inflammatory response
• Stimulating cells with LPS will allow us to challenge the anti‐
inflammatory effects of HDACi during an immune response
LPS
Pro‐inflammatory Response
LPS is a strong inducer of the inflammatory response
• Stimulating cells with LPS will allow us to challenge the anti‐
inflammatory effects of HDACi during an immune response
LPS
MAP Kinase
NF‐kB
TNF‐
IL‐6
ICAM‐1
Pro‐inflammatory Response
Experimental design UN
LPS
B
BL
V
VL
Statistical analysis allows objective determination of changes in nucleosomal
distributions
0.0
-1.0
-0.5
Signal
0.5
1.0
1.5
IL6
22765700
22766000
22766300
22766600
22766900
22767200
22767500
22767800
1.0
0.0
‐
-1.0
+
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
0
10
20
30
40
50
60
70
80
90
100
110
120
130
140
150
0.0 1.0 2.0
t-test
Correlation
Position
HDAC Inhibitors have limited effects on nucleosome redistributions at TSS HDACi
Number of genes with nuc. Redistributions Percent of genes with nuc. redistributions
Butyrate
23/505
4.55%
Valproic acid
22/505
4.3%
IL8
Log2 (mononucleosomal/bare genomic) CCL2
Log2 (mononucleosomal/bare genomic) HDAC Inhibitors induce modest changes in nucleosome distribution Genomic position (bp)
Genomic position (bp)
Nucleosomal redistributions that occur show HDACi specificity
NaB (23)
17
VPA (22)
NaB + VPA
6
16
Nucleosomal redistributions that occur show HDACi specificity
3
TMPRSS11D
butyrate valproic acid butyrate valproic acid -1.0
-1
-0.5
-0.5
0
0.0
0.0
1
0.5
0.5
2
1.0
1.0
1.5
butyrate valproic acid IL8
2
5
-1.0
Log2 (mononucleosomal/bare genomic) CCL2
Genomic position (bp)
HDACi’s decrease pro‐inflammatory protein expression
Cytokine produc on 80 70 pg/ml 60 50 40 IL6 30 TNF‐alpha 20 10 0 Un butyrate VPA Figure 6. Concentra ons of IL6 and TNF‐alpha secreted in media from untreated, 2mM butyrate, and 1mM valproic acid treatments as determined by ELISA. LPS serves as a reliable immune insult model
• Component of bacterial cell walls
• Strong inducer of the inflammatory response
• Stimulating cells with LPS will allow us to challenge the anti‐
inflammatory effects of HDACi during an immune response
LPS
MAP Kinase
NF‐kB
TNF‐
IL‐6
ICAM‐1
Pro‐inflammatory Response
HDACi’s limit the number of nucleosome
redistributions during an immune insult
Stimulus
Number of genes with nuc. Redistributions Percent of genes with nuc. redistributions
LPS
455/505
90.1%
Butyrate +LPS
65/505
12.8%
Valproic acid +LPS
70/505
13.8%
HDACi’s block nucleosome
redistributions induced by an immune insult
TREM1
1.0
0.5
0.0
-0.5
-1.0
-1.5
Log2(monoucleosomal/bare genomic)
1.5
Butyrate
Triggers release of pro‐inflammatory 500 TSS +500
chemokines and cytokines.
Genomic position (bp)
‐
TREM1
1.0
0.5
0.0
-0.5
-1.0
1.5
Log2(monoucleosomal/bare genomic)
1.5
Butyrate
LPS
Triggers release of pro‐inflammatory 500 TSS +500
chemokines and cytokines.
Genomic position (bp)
‐
TREM1
1.0
0.5
0.0
-0.5
-1.0
-1.5
Log2(monoucleosomal/bare genomic)
1.5
Butyrate
Butyrate+LPS
Triggers release of pro‐inflammatory 500 TSS +500
chemokines and cytokines.
Genomic position (bp)
‐
IL10
1.0
0.5
0.0
-0.5
-1.0
-1.5
Log2(monoucleosomal/bare genomic)
1.5
Butyrate
LPS
500 TSS +500
This cytokine has pleiotropic effects in Genomic position (bp)
immunoregulation and inflammation.
‐
IL10
1.0
0.5
0.0
-0.5
-1.0
-1.5
Log2(monoucleosomal/bare genomic)
1.5
Butyrate
Butyrate+LPS
500 TSS +500
This cytokine has pleiotropic effects in Genomic position (bp)
immunoregulation and inflammation.
‐
CX3CL1
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
Log2(monoucleosomal/bare genomic)
1.5
Butyrate
LPS
Chemotactic for T‐cells and monocytes.
‐
500 TSS +500
Genomic position (bp)
CX3CL1
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
Log2(monoucleosomal/bare genomic)
1.5
Butyrate
Butyrate+LPS
Chemotactic for T‐cells and monocytes.
‐
500 TSS +500
Genomic position (bp)
IL2RA
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
Log2(monoucleosomal/bare genomic)
1.5
Butyrate
LPS
Receptor for IL2 which is required for T‐
cell proliferation and other activities crucial to regulation of the immune response.
‐
500 TSS +500
Genomic position (bp)
IL2RA
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
Log2(monoucleosomal/bare genomic)
1.5
Butyrate
Butyrate+LPS
Receptor for IL2 which is required for T‐
cell proliferation and other activities crucial to regulation of the immune response.
‐
500 TSS +500
Genomic position (bp)
-0.5
0.0
0.5
1.0
1.5
Butyrate
LPS
-1.0
Log2(monoucleosomal/bare genomic)
IL6
‐
A cytokine that functions in inflammation.
500 TSS +500
Genomic position (bp)
-0.5
0.0
0.5
1.0
1.5
Butyrate
Butyrate+LPS
-1.0
Log2(monoucleosomal/bare genomic)
IL6
A cytokine that functions in inflammation.
‐
500 TSS +500
Genomic position (bp)
HDACi’s have cytokine specific effects
Cytokine produc on 1800 1600 1400 pg.ml 1200 1000 IL6 800 TNF‐alpha 600 400 200 0 LPS butyrate +LPS VPA +LPS Figure 8. Concentra ons of IL6 and TNF‐alpha secreted in media from LPS, butyrate + LPS, and valproic acid + LPS treatments, as determined by ELISA. HDACi “lock‐in” chromatin structural states at TSS
• 90% of genes had nucleosomal redistributions when stimulated with LPS.
• 13% of genes had nucleosomal redistributions with HDACi‐LPS co‐treatments.
• 70% of the loci showing LPS‐induced nucleosome redistributions were inhibited by HDACi pretreatment.
IL‐6 displays a canonical promoter architecture upon activation
nucleosome posi oning nucleosome posi oning LPS treated
TSS ‐1 NFR +1 +2
genomic posi on IDEALIZED
+3
‐1
NFR
+1
+2
+3
genomic posi on IL‐6 TSS
Figure 15. An idealized promoter structure shown in green consisting of a nucleosome free region (NFR) flanked by the ‐1 nucleosome upstream of the TSS and the +1 nucleosome downstream of the TSS. The promoter structure of activated IL‐6 is shown in red.
This canonical promoter structure is associated with activation
pg/ml
800
un
600
LPS
400
200
0
un
1.5
1.0
0.5
0.0
1000
-0.5
1200
Untreated
LPS
-1.0
IL‐6 Protein Expression
Log2(mononucleosomal/ bare genomic)
IL6
LPS
Genomic Position
Reduction in IL‐6 protein expression is linked to changes in chromatin structure
LPS
600
Butyrate +LPS
400
200
0
LPS
Butyrate +LPS
1.5
1.0
0.5
pg/ml
800
0.0
1000
-0.5
1200
LPS
Butyrate +LPS
-1.0
IL‐6 Protein Expression
Log2(mononucleosomal/ bare genomic)
IL6
Genomic Position
HDACi’s block LPS‐induced changes
pg/ml
un
30
Butyrate +LPS
20
10
0
un
Butyrate +LPS
1.5
1.0
0.5
40
0.0
50
-0.5
60
Untreated
Butyrate +LPS
-1.0
IL‐6 Protein Expression
Log2(mononucleosomal/ bare genomic)
IL6
Genomic Position
IL‐6 is a key pro‐inflammatory cytokine
• Activates acute phase proteins, which are key molecules in regulating the immune response
• Serve as effective markers of severity of disease state
• High expression of IL‐6 is seen in rheumatoid arthritis and IBD patients
HDACi’s block nucleosome redistributions induced by an immune insult
This is the first report linking the anti inflammatory properties of HDAC inhibitors to the alterations in the chromatin regulatory structural potentials.
HDACi’s block nucleosome redistributions induced by an immune insult
Further studies of this nature will help to illuminate the pathology of immune diseases and guide the identification of effective and targeted treatments.
Agenda
• Introduction to chromatin structure
• Techniques to assay chromatin structure
• RESULTS
– The molecular pharmacology of the anti‐
inflammatory butyrate
– Chromatin structural biomarkers of cancer tumor grade
Chromatin Structural Changes
Serve as Effective Biomarkers for Cancer Grade
Brooke Roberts Druliner
• FSU Biological Science Graduate Student
Tumor grades signify the differentiation status of tumor cells GRADE
DESCRIPTION
G1
low
Well‐differentiated
G2
intermediate
Moderately differentiated
G3
high
Poorly differentiated
G4
high
Undifferentiated
We obtained the following lung adenocarcinoma tumor samples with corresponding normal tissue Sample ID
Date
Organ
Cancer Type
Grade Age
Gender
0620T
1/10/07
Lung
Adenocarcinoma
1
55
Male
1357T
2/28/08
Lung
Adenocarcinoma
1
63
Female
1294T
1/10/08
Lung
Adenocarcinoma
2
67
Female
2123T
8/20/09
Lung
Adenocarcinoma
2
58
Female
0873T
5/24/07
Lung
Adenocarcinoma
3
51
Male
1831T
3/5/09
Lung
Adenocarcinoma
3
75
Male
TISSUE
EXPRESSION
ACCESSIBILITY
NUCLEOSOME
Low grade tumor samples show many changes in nucleosome distribution
TSS out of 415 showing changes in nucleosome distribution
(tumor compared to normal)
GRADE 1 GRADE 2 GRADE 3
400
350
337
300
250
200
150
180
100
61
50
35
0
0620_G1
1357_G1
1294_G2
26
2123_G2
0873_G3
Low grade tumor samples show many changes in nucleosome distribution
GRADE 1
GRADE 3
2
1
−1
−2
108092900
108093300
Black line= Normal
Red line = Tumor
−3
108092500
0
Signal
0
−3
−2
−1
Signal
1
2
3
chr11
3
chr11
108093700
108092500
Position
ATM
108094100
108094500
Black line= Normal
Red line = Tumor
108092900
108093300
108093700
108094100
108094500
Position
ATM
Ataxia telangiectasia mutated (ATM) gene. The protein made by the ATM gene functions to control the rate at which cells grow.
Low grade tumor samples show few changes in nuclease accessibility
GRADE 1
Chrom.
1
2
3
4
5
6
7
8 9 10 11 12 13 14
16 18 20
2
3
4
5
6
7
8 9 10 11 12 13 14
16 18 20
GRADE 1
Chrom.
1
Intermediate grade tumor samples show few changes in nuclease accessibility
GRADE 2
Chrom.
1
2
3
4
5
6
7
8 9 10 11 12 13 14
16 18 20
2
3
4
5
6
7
8 9 10 11 12 13 14
16 18 20
GRADE 2
Chrom.
1
High grade tumor sample shows major changes in nuclease accessibility
GRADE 3
Chrom.
1
2
3
4
5
6
7
8 9 10 11 12 13 14
16 18 20
High grade tumor sample shows major changes in nuclease accessibility
GRADE 3
Chrom.
1
2
3
4
5
6
7
8 9 10 11 12 13 14
16 18 20
2
3
4
5
6
7
8 9 10 11 12 13 14
16 18 20
GRADE 1
Chrom.
1
An initial model for chromatin structural biomarkers for tumor severity
GRADE
1
2
3
 Nucleosome distribution
 Chromosome accessibility
 gene expression
 cell type
Perhaps chromatin structural biomarkers may serve as indicators of cancer grade
• Independent of genotype
• Independent of gene expression
• Ability to differentiate specific grade subtypes
Thank you to the individuals who did the work and developed these techniques
Ph.D. Students
Brooke R. Druliner
Justin Fincher (CS)
Sarah Lueking
Brian Spetman
Undergraduates
Ely Gracia
Preston Hood
Josh Koerner
Tarreq Noori
Crystal Pickeral
Brittany Sexton
Postdoctoral Fellow
Parwez Alam
Immunology Collaborator
Marie Charrel Dennis
Funding
FSU Department of Biological Science
FSU CRC FYAP Program
Questions?
Thank you It will be interesting to understand the relationships between regulation at the gene locus level and regulation at the whole genome level that occurs upon HDACi treatment
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 x Untreated VPA Log2(Inacessible/accessible) Untreated
butyrate
0.0e+00
8.0e+08
1.2e+09
1.6e+09
2.0e+09
2.4e+09
0.0e+00
2.8e+09
1.6e+09
2.0e+09
2.4e+09
2.8e+09
Untreated VPA + LPS 4.0e+08
8.0e+08
1.2e+09
1.6e+09
2.0e+09
2.4e+09
2.8e+09
Log2(Inacessible/accessible)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 x ‐4 ‐2 0 2 Log2(Inacessible/accessible) C) 4.0e+08
1.2e+09
Log2(Inacessible/accessible) Untreated Butyrate + LPS 0.0e+00
8.0e+08
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 x 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 x ‐2 0 2 Log2(Inacessible/accessible) B) 4.0e+08
Untreated LPS Untreated LPS 0.0e+00
4.0e+08
8.0e+08
1.2e+09
1.6e+09
Genomic posi on 2.0e+09
2.4e+09
2.8e+09
0.0e+00
4.0e+08
8.0e+08
1.2e+09
1.6e+09
Genomic posi on 2.0e+09
2.4e+09
2.8e+09