The fusion of two haploid cells (a sperm and an egg) to produce the diploid
zygote heralds the development of a new organism. According to Mendel's laws of inheritance
the genetic information contributed by the sperm (from the father) and the egg (from the mother) is equal.
This would in effect mean that given the right conditions, fusion of two sperm nuclei or two egg nuclei should
lead to normal development of an organism. However, this was found not to be the case in mammals, some insects and plants.
Mouse embryos with two paternal copies (androgenetic) or two maternal copies (parthenogenetic) of the genome die very early
during embryonic development (Barton et al 1984 Role of paternal and maternal genomes in mouse development Nature 311(5984):374-6; Surani
et al 1984 Development of reconstituted mouse eggs suggests imprinting of the genome during gametogenesis. Nature 308(5959):548-50; McGrath
and Solter 1984 Completion of mouse embryogenesis requires both the maternal and paternal genomes. Cell 37(1):179-83). Therefore in mammals,
maternal and paternal contribution to the progeny is not equal and for normal development contribution from both parents is required. This
non-equivalence of the parental genomes is epigenetic in nature and forms the basis of the phenomenon known as genomic imprinting.
While genomic imprinting is a very unique and specialized example of epigenetic regulation, epigenetic modifications and their effect on gene
regulation are as important as the genetic information itself. Genetic makeup of an organism determines the various different functions it can
perform. However, the exact function of an individual cell is determined by the subset of genes that are expressed or repressed in that cell.
This in turn is controlled by chromatin organization at individual genetic loci, the specificity of which is controlled by epigenetic
modifications like DNA methylation, histone modifications and associated events. This unique epigenetic state of each cell type needs
to be maintained for normal development and well being of an organism. Our laboratory is interested in characterizing these cell-specific
epigenetic events and the associated epigenetic circuitry that modulates the genome to perform diverse functions in a multicellular organism.
Chromatin organisation and genomic imprinting
For imprinted genes one copy of a DNA molecule is transcribed and the other copy is silent. What distinguishes two molecules
of the same DNA sequence within the same nucleus? The answer lies not in the genetic information encoded by the DNA sequence
but in its epigenetic modifications. These modifications include differential modification of the DNA itself and differential
organisation of DNA within the chromatin scaffold. One important modification of DNA known in mammals is methylation of cytosine
residues in the CpG dinucleotides. However, methylation does not answer all mechanistic questions related to imprinted genes. If
one allele of a gene becomes methylated what prevents methylation of the other allele? Does the unmethylated allele carry another
epigenetic imprint? To investigate the mechanisms of genomic imprinting, it would therefore be essential to identify the alternative
imprint on the unmethylated allele.
It has been found that within the differentially methylated regions of some imprinted genes there are sub-regions that also show
differential organisation of chromatin. Interestingly, the regions which show differential chromatin organisation have also been genetically
defined as Imprinting Control Regions (ICRs) and shown to control the imprinting status of genes within that locus. In all the imprinted loci
analysed, specialised chromatin structures were found exclusively on the unmethylated allele. This suggests that for these regions, methylation
and specialised chromatin structures are mutually exclusive and reflects alternate epigenetic states. How is this mutual exclusiveness between
methylation and specialised chromatin structures maintained? Do the chromatin modifications on unmethylated allele prevent it from getting methylated?
More importantly, during development when are these chromatin structures established? Can such chromatin conformations behave as imprints marking the parental
alleles? The aim of the ongoing project is to answer these questions and identify the role of chromatin organisation in mechanisms underlying genomic
imprinting in particular and as a epigenetic regulator of gene expression in general (Feil and Khosla 1999, John et al 2001, Sowpati et al 2008)
The necessity of maintaining the correct epigenetic status of the genome is exemplified by studies on various cancers. To date, no one type of DNA mutation
(genetic) has been defined which can be correlated with all cancers, but in most cancers, aberrant methylation, an epigenetic modification, has been observed
(global hypomethylation accompanying region-specific hypermethylation). When hypermethylation events occur within the promoter of a tumor suppressor gene, this
can silence expression of the associated gene and provide the cell with a growth advantage in a manner akin to deletions or mutations. On the other hand,
hypomethylation can result in activation of proto-oncogenes. Moreover, DNA methylation changes occur early and these changes are present even in benign tumors.
Till recently the accepted model for cancer development proposed that cancer arises through a series of mutations and epigenetic changes occur secondary
to these mutations. However, the notion of genetic hits preceding epigenetic changes is being challenged by newer finding especially in solid tumors where
epigenetic changes arise very early in tumor development and sometimes even in normal tissue before tumor development. This has led to the proposal that
epigenetic aberrations probably are prelude to genetic mutations.
An important corollary to the epigenetic progenitor model is the role of nuclear reprogramming molecules like DNA methyltransferases, histone modifiers,
etc., which are the effectors for epigenetic changes. Any change in the epigenetic profile of a cell would have to be perpetuated through these molecules.
Our laboratory is examining the possibility of reprogramming genes being involved in the process of carcinogenesis (Gokul et al 2007, Gokul
et al 2009, Manderwad et al 2010).
Mapping the Human Epigenome
(a collaborative project with Dr. Rakesh Mishra of CCMB, as a part of DBT's Centre of Excellence in Epigenetics)
Identical DNA sequence of human genome generates >200 cell types that constitute the structures in the body. This variety in the genetic output in cell
type specific manner is determined by chromatin organization and epigenetic mechanisms. Epigenetic signature of a cell is crucial to decide its fate as the epigenetic
modifications assign active and inactive chromatin in the genome by DNA methylation and post-translational modification of histone proteins. Histone modifications include
acetylation, methylation, phosphorylation and ubiquitinylation whereas DNA is methylated at cytosine residue to control gene activity and nuclear architecture (reference epigenetics book).
Histone acetylation always correlates with active chromatin whereas methylations may lead to active or inactive chromatin depending on the site of lysine methylation. Tri methylation of H3K4,
H3K36 and H3K79 mark active chromatin whereas tri-methylation at H3K9, H3K27 and HK56 mark inactive chromatin. Cytosine methylation is one of the most widely studied epigenetic modifications.
CpG dinucleotides are characteristic of many promoters, which are the targets of methyltransferases. The unmethylated status of CpG island recruit transcription activators for gene activation
whereas methylation of CpG islands lead to inactivation. DNA methylation switch has been found to be associated with tissue, germ line and cancer specific genes. Histone hypoacetylation and hypermethylation
at specific residue of core histone protein is characteristics of DNA methylation. It shows that there is a relation between histone modifications and DNA methylation, which finally determine state
of chromatin. Histone modifications, together with DNA methylation, have a vital role in organizing nuclear architecture, which in turn, is involved in regulating transcription and other nuclear processes.
Clearly, global alterations of histone modification patterns have the potential to affect the structure and integrity of the genome and to disrupt normal patterns of gene expression. Having already mapped
H3K9ac3, H3K9me3, H3K27me3 histone modifications and CTCF a conserved mammalian insulator factor across the human Y-chromosome we have initiated studies which would allow us to interpret the genetic basis
or DNA sequence determinants of epigenetic marks that can then be linked to functional and evolutionary consequences (Singh et al 2011).