He told that modifications in histone may be playing an important role in the regulation of cellular processes.
For example, lysine can be mono, di or tri methylated. Depending upon the degree of methylation, the expression of downstream genes is regulated.Catalytic domain is also known as writer domain.
non-histone proteins (HP1, polycomb)
BET family proteins are mainly involved in cell cycle regulation and transcriptional elongation.Sometimes, MYC also fail to respond to BET inhibition. The mode of action of this inhibition is the suppression of transcriptional elongation of the target gene.
Acetylation is not residue specific while methylation is.
LSD1 acts via amine oxidation pathway using FAD as cofactor. It requires a charged N terminal and hence can act on di-methylated or tri-methylated lysine only. JmjC acts via oxidation mechanism with the help of alpha ketoglutarate and donot require electron pair on N to initiate catalysis. LSD 1 as bivalent histone modifications.
Presented by -
K Harish (10035)
Rupam Ghosh (10075)
Sandeep Satapathy (10079)
Cancer Epigenetics: From
Mechanism to Therapy
Role of Histone modifications
1. Regulation of transcription
2. DNA template based processes (replication, regulation by miRNA, lncRNAs)
Hypothesized by Vincent Allfrey (1964)
Photo courtesy of the Rockefeller Archive Centre
Vincent Allfrey's Work on Histone Acetylation
G. Vidali, E. L. Gershey, V. G. Allfrey. Chemical
Studies of Histone Acetylation. The
Distribution of ϵ-N-Acetyl lysine in Calf Thymus
Histones J. Biol. Chem. 1968, 243, 6361–6366
Bivalent Histone domains
• The multiple coexisting histone modifications are
associated with activation, and repression. However,
these are not static entities but a dynamically changing
and complex landscape that evolves in a cell context-
• The combinatorial influence that one or more histone
modifications have on the deposition, interpretation,
or erasure of other histone modifications has been
broadly termed ‘‘histone crosstalk’’.
Epigenetics: heritable changes in a
cellular phenotype that were
independent of alterations in
the DNA sequence.
a consensus definition of
epigenetics remains both
contentious & ambiguous
of non cis- elements
of genes affecting
the expression levels.
A scaffold for recruitment
and binding of several
regulatory and enzymatic
Type of Epigenetic modifications
Can be at DNA
level or chromatin
1. Gene expression or
2. Binding affinity of
3. Expression of
Experimental approaches to epigenetics
• Next Gen Sequencing
• Chromatin Immuno Precipitation sequencing
• Deep sequencing
• Mass spectrometry
• SILC (specific Isotope labeled Lineage of cell )
- In sights into invitro gene expression patterns.
• Chromosomal/nucleosome context of gene expression – in
Regions of favorable epigenetic control
• The DNA had several regions of repeats like
centromeres, telomeres and other gene body
repeats as well as the intergenic sequences giving
rise to non coding RNAs are susceptible to
• There is an accumulation of somatic mutations in
genes over time, giving rise to different etiologies
of patho-histological cancers.
DNA hyper methylations and bivalent histone modifications are distinctive features of
Mostly CpG dinucleotide of promoter region gets hyper methylated.
follicular lymphoma contain
recurrent mutations of the
MLL2 in close to 90% of
Similarly, UTX, a histone
demethylase, is mutated in
up to 12 histologically
• Interestingly, many of these genes are targeted for
DNA methylation in cancer. Comparisons between
malignant and normal tissues from the same
• Demonstrate broad domains within the malignant
cells that contain significant alterations in DNA
• These regions appear to correlate with late-
replicating regions of the genome associated with
the nuclear lamina.
Mechanism to therapy
• Genetic lesions in chromatin modifiers and global
alterations in the epigenetic landscape also provide
potential targets for therapeutic intervention.
• A number of small-molecule inhibitors have already been
developed against chromatin regulators
• Few of these (targeting DNMTs, HDACs, and JAK2) have
already been granted approval by the US FDA.
• The reality is that the field of drug discovery had been somewhat held back due to
concerns over the pleiotropic effects of both the drugs and their targets.
Epigenetic Pathways of Cancer
• The methylation of the 5-carbon on cytosine residues (5mC) in
CpG dinucleotide is still important modification.
• Although global hypomethylation is commonly observed in
malignant cells, the methylation changes that occur within CpG
islands, which are present in 70% of all mammalian promoters.
• 5%–10% of normally unmethylated CpG promoter islands
become abnormally methylated in various cancer genomes.
• CpG hyper methylation of promoters not only affects the
expression of protein coding genes but also the expression of
various noncoding RNAs- role in malignancy.
• DNA methyltransferases (DNMTs) in higher eukaryotes.
• DNMT1 is a maintenance methyltransferase that
recognizes hemimethylated DNA generated during DNA
replication and then methylates newly synthesized CpG
• Conversely, DNMT3a and DNMT3b, although also capable
of methylating hemimethylated DNA, function primarily as
de novo methyltransferases to establish DNA methylation
• DNA methylation provides a platform for several methyl-
binding proteins like MBD1, MBD2, MBD3, and MeCP2.
Nature of mutations
• recently somatic mutations of the key genes in human
malignancies has been traced.
• Recurrent mutations in DNMT3A in up to 25% of patients
with acute myeloid leukemia (AML).
• These mutations are invariably heterozygous and are
predicted to disrupt the catalytic activity of the enzyme.
• Moreover, their presence appears to impact prognosis
• Hypomethylating agents – gained FDA approval
for routine clinical use .
• Azacitidine and decitabine have shown mixed
results in various solid malignancies, they have
found a therapeutic niche in the myelo-
dysplastic syndromes (MDS).
DNA Hydroxy Methylation and Its
• high-resolution genome-wide mapping of this modification in pluripotent and
differentiated cells has also confirmed the dynamic nature of DNA methylation.
• The ten-eleven translocation (TET 1–3) family of proteins are the mammalian
DNA hydroxylases responsible for catalytically converting 5mC to 5hmC. Iterative
oxidation of 5hmC by the TET family results in further oxidation
derivatives, including 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC).
• They are likely to be an essential intermediate in the process of both active and
passive DNA demethylation,
• they preclude or enhance the binding of several MBD proteins
• genome-wide mapping of 5hmC has identified a distinctive distribution of this
modification at both active and repressed genes, including its presence within
gene bodies and at the promoters.
• catalytic activity for the TET family of DNA hydroxylases, several reports
emerged describing recurrent mutations in TET2 in numerous
• TET2- deficient mice develop a chronic myelomonocytic leukemia
(CMML) phenotype, which is in keeping with the high prevalence of
• ET2 mutations appear to confer a poor prognosis.
• ET2-mediated oncogenesis have revealed that the patient-associated
mutations are largely loss-of-function mutations that consequently
result in decreased 5hmC levels and a reciprocal increase in 5mC levels
within the malignant cells .
Histone modifying enzymes
• Targets : histones and non histone
• Catalytic domain : catalyses the
chemical modification on target
• Reader domain : target specificity of
client proteins and respond to the
upstream signaling cascade
• Reader domain has two functionally
1. Binding domain : dictates the
2. Outside domain : dictates the
histone binding specificity
• N6 acetylation of lysine most
• Involved in
transcription, chromatin structure
modification and DNA repair
• Mostly Lys is modified in histone
tails ( rarely in the core) Why???
• Neutralizes Lys’s positive
charge, opening up chromatin
Type A (nuclear)
Acts on nucleosomal histones and
regulate gene expression
Type B (cytoplasmic in nature)- HAT 1
Acts on free histones
HATs in cancer
• CBP binds to viral oncoprotein
E1A and helps in neoplastic
transformation (Bannister and
• Non histone proteins like- MYC,
PTEN, p53 are also acetylated in
• HATs as therapeutic targets – HAT
inhibitors like curcumin, anacardic
acid and garcinol
Curcumin Anacardic acid
• Reversal of action of histone acetytransferases
and help to compact the chromatin packing
• Catalyzed by histone deacetylases (HDACs)
• Multi-enzyme complexes
• Targeted by transcriptional repressors
• Deacetylates histone tails
HDACs in cancer
• Chimeric fusion proteins (PML, AML1-ETO)
recruit HDACs for further oncogenic trait
• HDACs also interact with non chimeric
oncogenes like BCL6
• Drugs like Vorinostat and Romidepsin which
are HDAC inhibitors are effective in
suppressing the cancer cell phenotypes
Histone acetylation readers
• Family of readers like bromodomains containing
proteins (evolutionary conserved reading
• The question is :
Can these readers be used as targets for drugs ?
Because if we render these non functional, the
downstream histone modification will be hindered.
BET inhibition downregulated MYC expression.
• Many lysine residues can be
• Mainly on histone tails
(sometimes in core)
• Can be mono-, di-, or tri-
Mono methylation is related with
gene activation while tri
methylation is related with gene
Lysine Methyltransferases (KMTs)
• Target a certain lysine on a certain histone
• Put on mono, di, and/or tri methyl (me, me2, me3)
• Many contain SET domains (me-transferase)
• ‘Readout’ is very specific
• Ex. H3K4me1 vs. H3K4me3
Mutations in cancer involving histone
• Role of EZH2 in
• EZH2 has both
oncogenic and tumor
• It can methylate
H3K27-1me to H3K27-
2me or 3me which
helps in oncogenic
• LSD1: H3K4
• Jumonji family
Histone Demethylases in cancer
Mutations have been observed in the following
KDM5A (JARID1A), KDM5C (JARID1C), and KDM6A
(UTX) in solid and haematological malignancies
IDH1 and IDH2 : highly expressed in cancer like
glioblastoma and myeloma malignancies (AML)
CAN BE TARGETED FOR DRUG THERAPY!!!
Histone Methylation Readers
• Chromodomain (CHD ATPases, HP1, PC)
• Tudor (some histone demethylases)
• PhD (many chromatin regulators BPTF, ING2)
• MBT (in some polycomb proteins)
• WD-40 (WDR5)
In cancer, altered expression of chromodomain protein HP1 have
Mutations in PHD domain of a protein (like PHF23) or fusion of
PHD domain with NUP98 have been observed in some cancers.
Inhibitors, if target this protein- protein interaction, can prove
to be effective against cancer.
• JAK2 is amplified or mutated in hematological
malignancies which activates the expression of
oncogenes like Lmo2
• Phosphorylation of target proteins help in the
binding of other proteins (like 14-3-3 proteins).
Mutations in these proteins have been seen in
Targeting this interaction can be useful !!!!
Cancer mutations in Histone genes
• H3.3 and H3.1 genes are mutated and seen in
pediatric glioblastoma (amino acid
substitutions at K27M, H34R, H34V)
• Effect on chromatin structure and
List of selective histone H2B, H3 and H4 modifications. Covalent
histone modifications include methylation (M), acetylation
(Ac), phosphorylation (P) and ubiquitination (Ub)
Trends in Genetics: Volume 18, Issue 8, 1 August
2002, Pages 387-389:Asad U Khan, Michael
Modification of Histone Complexes
Chromatin Remodeling Complexes
These complexes are evolutionarily conserved, use ATP to evict,
modify and exchange histones. All this is done on the basis of
chromatin reader motifs which confer regional and contextual
specificity. Depending on their biochemical activity can be classified
• Switching Defective/ Sucrose Non fermenting family (SWI/SNF)
• Imitation SWI family
• Nucleosome remodeling and Deacetylation (NuRD)/
Chromodomain binding DNA Helicase family (CHD)
• Inositol requiring 80 family (INO80)
Role in Tumor Suppression
Possibly these Remodelers can be tumor suppressor genes as:
Mutated in Malignancies and hematological disorders
Development and maintenance of cancer
Mutations disrupt balance between self-renewal and
Regulate cell cycle
Nuclear hormone signaling
Small and Large ncRNAs
The small ones are highly conserved across species.
Larger ones are not conserved as compared to the smaller ones
but exhibit varied modes of action.
Long ones partake in chaperonic activity as well as acts as
scaffolds for regulators.
An Example Of HOTAIR and HOTTIP
Both are lncRNAs, expressed from mammalian clusters HOXC and HOXA
HOTAIR acts as Trans mediator and acts as scaffold to PRC2 and
LSD1 CoREST/REST containing complex.
Aberrant expression of HOTAIR is a key for identifying advanced breast
and colorectal cancers as PRC2’s modulation of chromatin is severely
HOTTIP acts as Cis mediator to activate 5’ HOXA genes which
would later recruit MLL1 complexes to regulate H3K4me3 and later
Haywired regulation of H3K4me3 causes spatial disorientation of
transcripted products and hence inability of 5’ HOXA to control
development and maintenance of cells.
Throwing Around Views:
1. Epigenetic pathways play an important role in oncogenesis.
2. How to target specific set of genes ubiquitously expressed serve as a
3. Cancer cells have an epigenetic vulnerability, i.e relying on a specific
pathway for deliverance of elements of interest, oncogenic addiction.
4. Malignancies can also be paradoxical, i.e. C-value paradox analogy.
5. Combinatorial therapeutic approaches might reduce the chance of
drug resistance and may improve synergistically chemotherapy.
Food For Thought
Questions to be addressed :
• Why do only these mutated histones get
incorporated in the nucleosome ?
• Are chromatin remodeling complexes playing
a silent role in this ?
• Are there any specific histone code in cancer?
Can these be interpreted? Are these codes
1. Epigenetic modifications like chromatin remodeling complexes and
histone mutations are one of the key components of cancer.
2. Hallmarks of cancers are profoundly influenced by epigenetic
modifiers and thus reflective of changes in epigenome.
3. These changes are mediated by a rather small set of genes rather
than a global genetic effect.
4. Changes like these are reflected dramatically by malignant cells while
normal cells are relatively unaltered.
5. Hematopoietic malignancies are vulnerable to epigenetic regulations
readily than solid malignancies.
6. Genetic lesions in epigenetic regulators may serve as drug targets
and thus overall cancer epigenetics is a field full of optimistic views.