This is a presentation I made for my PhD coursework back in 2011

1. Supratim Ghatak
Post M.Sc., Biophysical Sciences (2010-11)
Under the guidance of Prof. Sanghamitra Raha
Crystallography and Molecular Biology Division
Saha Institute Of Nuclear Physics

2.  Histones and Nucleosome
 Key Histone Modifications
 Reported Cancer links of these modifications
 Effect of Plant derived chemicals obtained from diet

3. Genetic Change
Epigenetic Change
Normal Cell Transformed Cell
Tumor
Malignant
Transformation
Metastasis to other
tissues

4. Modes of Epigenetic Changes
DNA methylation Histone
modifications
Non-coding
RNA effects
CH3
miRNAmRNA

5. Histone & Nucleosome

6. Histone Modifications
Acetylation Lys (K)-ac
Methylation Lys (K)-me, Arg (R)-me
Phosphorylation Ser (S)-ph, Thr (T)-ph
Ubiquitylation Lys (K)-ub
SUMOylation Lys (K)-su
ADP ribosylation Glu (E)-ar
Deimination Arg (R)  Cit
Isomerization Pro (P)-cis Pro (P)-trans

7. Histone Acetylation

8. HAT
Type B
Type A
free cytosolic histones
 HAT1 H4 (K5, K12)
Chromatin histones
 Gcn5 H3 (K14)
H4 (K8, K16)
 p300/CBP H3 (K14, K18)
H4 (K5, K8)
H2A (K5)
H2B (K12, K15)
 TAFII250 H3, H4

9. Histone Decetylation

10. HDAC
SIRTUINS CLASSICAL
Class I Class II Class IV
 Yeast Homolog : Sir2
 SIRT1-7
 Yeast Homolog : Rpd3  Yeast Homolog : Hda1
 HDAC1
 HDAC2
 HDAC3
 HDAC8
 HDAC4
 HDAC5
 HDAC7
 HDAC9
 HDAC6
 HDAC10
 HDAC11

11. Histone Phosphorylation
Ser (S)-P , Thr (T)-P
Prime target Histone H3
 Aurora  H3 (S10)
 MSK1/2 H3 (S28)
 CKII H4 (S1)
 MST1 H2B (S14)
 Haspin H3 (T3)

12. Histone Methylation
Arg(R)-Me , Lys (L)-Me
Mono Di Tri
MLL1-5 H3 (K4)
NSD1 H3 (K36)
DOT1 H3 (K79)
SUV39H1/2 H3 (K9)
EZH2 H3 (K27)
SUV420H1/2 H3 (K3)
PRMT4 H4 (R3)

13. Cancer Link of Histone Modifications and
Modifying enzymes
Alteration of histone modifying enzymes
Alteration of histone modification marks
Alteration of gene expression profile
Gain of oncogene function and/ or loss of
tumor suppressor gene function
CANCER

14.  Genes encoding HATs often mutated/ overexpressed/ translocated/ amplified
in multiple malignancies
 work on mice showed CBP deletion leads to leukemia.
 Rubenstein-Tyabi patients with loss of CBP functions prone to cancers
 loss of heterozygocity of p300 found in human gastric & colon cancers
 In multiple malignancies tumor suppressor p21 is silenced by the abrupt
action of HDAC or synergistic action of DNMT & HDAC
[Gibbons RJ (2005)]
[Zhang K, Dent SYR (2005)]

15. Overexpression of Aurora kinase found to have link with mitotic mis-
segregation and aneuploidy
Loss of histone H2B phosphorylation found in overgrowing cell mass
 H2A variant gH2AX involved in double strand break repair & genetic
recombination and its mutation is often linked to B-cell lymphoma
[Jia J et. al (2003)]
[Bassing CH et. al (2003)]

16. EZH2 often overexpressed in many malignancies including lymphoma,
breast cancer, metastatic prostate cancer
RIZ1 (H3K9) HMT inactivation is linked to human carcinomas of lung, liver,
breast, colon etc
SMYD3 is overexpressed in colorectal and hepatocellular cancers
[Gibbons RJ (2005)]
[Varambally S et. al.(2005)]

17. Effect of natural Phytochemicals
Ester of Epigallocatechin & Gallic acid found in green tea
Potential HAT inhibitor
Order of specificity against HATs : p300>CBP>Tip60>PCAF
 acetylation crucial proteins such as p65 , NF-kB etc
inhibited by EGCC Good anticancer agent
[Hauser AT et. al.(2008)]

18. Polyphenolic compound found in roots Turmeric
Inhibits p300/CBP.
No effect on Gcn5, PCAF etc.
Inhibition of p53 acetylation is reported (by p300 inhibition)
Caspase 3 dependent apoptosis of glioma cells, cervical cancer cells
HDAC1,3,8 inhibition is reported and apoptosis of B cell lymphoma
[Link A et. al.(2010)]
[Balasubramanyam Ket. al.(2010)]

19. Chiefly obtained from cashew nuts
 inhibits the binding of cofactor with HATs namely p300n PCAF, Tip60
Interferes NF-kB related pathways
AA IkBa Kinase IkBa NFkB
 Anacardic Acid
Tissue invasion Proliferation Angiogenesis
Bcl2, Bcl-xL MMP-9 Cyclin D1 VEGF
Apoptosis
downregulates
[Sung B et. al.(2007)]

20.  lipid soluble organosulfur compound from garlic
 Potent HDAC inhibitor
 chiefly H3 and H4 histone acetylation enhanced
 P21 expression enhanced at mRNA and protein level
 Enhances expression of proapoptotic potein such as Bax
 cell cycle arrest at G2/M checkpoint [Druesne N et. al.(2004)]

21. Kind of isothiocyanate compound Broccoli
 Potent HDAC inhibitor
 chiefly H3 and H4 histone acetylation enhanced
 P21 expression enhanced at mRNA and protein level
 Enhances expression of proapoptotic potein such as Bax
 cell cycle arrest at G2/M checkpoint
 metabolite of of SFN; SFN-Cys is the inhibitory principle
 Enhances expression of RARb gene in esophagal cancer cells
[Link A et. al.(2010)]

22.  polyphenolic compound from grapes, red wine
 trans isoform is more stable and active
 activates Class III HDAC SIRT1
 downregulates anti apoptotic protein survivin by active SIRT1
(SIRT1 deacetylates H3K9ac on survivin gene promoter)
 effective on multiple cancers such as breast cancer
[Link A et. al.(2010)]
[Wang RH et. al.(2008)]

24.  CML is a myeloproliferative clonal disorder of bone marrow stem cells
 Presence of “Philadelphia Chromosome” & Bcr-Abl fusion Tyrosine Kinase
 Conventional Therapy with Imatinib Mesylate (Gleevec)
 Reports include CML resitance against Gleevec
 Resveratrol (polyphenolic) inhibits proliferation, induces apoptosis in
cancer cells
[Gorre ME et. al.(2001)]
 HSPs (eg Hsp70) are over expressed in may cancers
Anti apoptotic effect  contribute to tumorigenesis
Hsp 70 (target to kill CML k562 cell)
[Takayama S et. al.(2003)]

25. KC KVC KR24 KR48
KC= K562 control KVC= K562 treated with DMSO
KR24= K562 treated with resveratrol fro 24hrs
KR48= K562 treated with resveratrol for 48hrs
Western Blot for Hsp70

26. Future Works…
 Repeating the western blot analysis of cellular Hsp70 level
 Analysis on HSF1/2 upon resveratrol treatment
 Effect of resveratrol on proteins that signs apoptosis (apocaspase-3
activation, PARP cleavage etc)

27. Acknowledgement
The Director, Saha Institute of Nuclear Physics
Head, Crystallography & Molecular Biology Division
Convener, Post-M.Sc Biophysical Sciences
My Project Guide
My Lab mates
My Parents