Insulator Mechanism of Imprinting : (e.g. H19 & IGF2 genes)

The H19 and IGF2 genes, the essential regulatory elements coordinating their imprinted expression have been well characterized. Shared enhancers that regulate H19 and IGF2 in endodermal and mesodermal tissues have been delimited. An imprinting control region (ICR) has been well defined and consists of a CG-rich differentially methylated domain (DMD), located 22 to 24 kb relative to the H19 transcription start site. The human H19 DMD encompasses seven different binding sites for CTCF. Only the sixth CTCF binding site has been reported to act as a key regulatory domain for switching between H19 and IGF2 expression, whereas the other sites appear to be hypermethylated in a study by Takai et al.

This DMD is essential for establishing the pattern of imprinting by which H19, a non-coding RNA, is exclusively expressed from the maternal chromosome and IGF2, a fetal growth factor, is only active paternally.

DMD has at least two distinct regulatory roles on each of the parental chromosomes. The activity of the DMD is determined by its methylation status. On the maternal chromosome, the DMD is hypomethylated and binds the protein CTCF via four highly conserved CG-rich repetitive sites. The binding of CTCF creates a physical boundary on the maternal allele and inhibits interaction of downstream enhancers with IGF2 promoters.

Maternal binding of CTCF to the four sites within the DMD has been further hypothesized to protect actively the region from becoming methylated during oogenesis and development. The DMD is also essential for optimal H19 and IGF2 expression and is required on the maternal allele for the initiation of H19 expression. On the other hand, the paternal H19 DMD is hypermethylated and hence the CTCF is unable to bind to it, resulting in expression of IGF2 while H19 is silenced.

DNA Methylation & Gene Expression

In mammals DNA methylation occurs after DNA replication and involves the transfer of a methyl group from S-adenosyl-methionine (SAM) to the 5’ – position of cytosine residues, in a reaction catalysed by the enzyme DNA methyltransferase (Dnmt). Three enzyme families associated with DNA, identified are as follows :

DNA methyltransferase I   : primarily mediates maintenance of methyl    transferase  activity during the  S phase.

DNA methyltransferase II       :  plays a role in some aspects of  centromere function.

DNA methyltransferase III           :    plays an essential role in the de novo

[ Dnmt3a and Dnmt3b ]                    methylation in vivo.

DNA Methylation & Gene Expression :

CpG island is usually the site for DNA methylation. CpG islands are genomic regions that contain a high frequency of CG nucleotides. They are regions with at least 200 bp and with a GC percentage that is greater than 50% and with an observed/expected CpG ratio that is greater than 60%. CpG islands typically occur at or near the transcription start site of genes, particularly housekeeping genes. They act as strong promoters and have also been proposed to function as replication origins. It is estimated that CpG islands are associated with about half of all mammalian genes. Unmethylated CpG islands are associated house keeping genes, while the CpG islands of many tissue-specific genes are methylated, except in the tissues where they are expressed. Interactions between proteins and DNA are changed by methylation, leading to alterations in chromatin structure and a transcription rate change. Thus DNA methylation mechanisms for gene regulation can be as follows :

·         Preventing the binding of transcription factors to their target sequences via proteins that bind preferentially to methylated promoters.

·         Interfering with the binding of transcription factors to the methylated cytosine.

·         Altering the chromatin structure leading to a change in the rate of transcription.

The best-characterized cluster that follows a strict insulator model for imprinted expression is the cluster containing maternally expressed H19 and paternally expressed insulin-like growth factor 2 (IGF2). This cluster resides at 11p15.5 in humans and is found in conserved synteny on distal chromosome 7 in mice.

Human chromosome 11p15.5 contains a large cluster of imprinted genes spanning a region of 1-Mb. This cluster can be divided into two independent imprinting sub-domains: one containing H19, IGF2 and INS2 (H19 domain) and other containing CDKN1C, KCNQ1, ASCL2 and TSSC3 (KCNQ1 domain). These two domains contain several genes that play a role in embryo growth. Regulatory disturbances of a number of genes in this cluster result in imprinting disorder like Beckwith-Wiedemann Syndrome (BWS), Silver Russell Syndrome (SRS) and increased predisposition to development of specific embryonic tumors, like Wilm’s Tumor (Verona et al., 2003).

While most studies on H19 and IGF2 have been performed in the mouse, many attributes of these genes including their expression profile and regulatory mechanisms are similar in humans. In both mouse and human, H19 and IGF2 are widely expressed during embryonic development and postnatally down regulated in most tissues.

H19 encodes a fully processed 2.3 kb non-coding RNA and was initially implicated as a tumor suppressor. However, it has also been shown to have oncogenic properties. IGF2 encodes a protein that plays a major role in promoting embryonic and placental growth and development.

As with all imprinted clusters, imprinted expression of H19 and IGF2 is regulated by Imprinting Control Region [designated imprinting center 1 (IC1) in humans and ICR or differentially methylated domain (DMD) in mouse] located between the two genes. This region is approximately 5 kb and 2 kb long in humans and mice, respectively.

The H19 and IGF2 genes are co-coordinately regulated, most probably through interaction of either promoter with a common ‘enhancer element’ and the action of a further insulator.

Insulator proteins, such as CTCF (a zinc finger CCCTC-binding factor) and BORIS have been demonstrated to bind to specific DNA elements and prevent promoter and enhancer interaction. CTCF is present in both somatic and germ cells, whereas BORIS is expressed specifically in the male germ line and has been suggested to be associated with both demethylases mediating erasure of imprinting marks and methylases mediating de novo methylation.

Epigenetic Regulation of Imprinting

Imprinting is regulated by the interplay of different epigenetic components which are as follows :


• DNA methylation.
• Chromatin structure.
• Degree of histone acetylation.


Among all these, one of the key factors that play a major role in the epigenetic modification of the genome is DNA methylation.

Life Cycle of an Imprint

Life cycle of an imprint is a dynamic process, which involves erasure, establishment, maintenance and implementation of the imprint markings.


Erasure and Establishment :


Each gamete carries sex specific imprint markings that are required for normal development. Upon fertilization, the paternal pronucleus is rapidly and actively demethylated within the zygote prior to first cellular division. In contrast, the maternal genome becomes demethylated in a passive manner during subsequent divisions, presumably due to lack of Dnmt – 1.

Erasure of the paternal methylation profile by the oocyte is a potentially powerful mechanism by which maternal factors modify chromatin structure to regulate the paternal genome. Demethylation at this stage is proposed to be required for the activation of genes necessary for the early embryonic growth. However imprint methylation marks present on both the paternal and maternal genomes are maintained despite this global demethylation event.


Another reprogramming event occurs later, within the primordial germ cells of the developing fetus. After the demethylation in the primordial germ cells, parental specific methylation is re-established during gametogenesis. This occurs in sperm post-natally within diploid genocytes prior to meiosis and within oocytes arrested at the diplotene stage of meiosis.


In the oocyte, the Dnmt 3 family of methyltransferases is required to set maternal specific methylation patterns for imprinted genes. Dnmt 3a and Dnmt 3b play an important role in establishing methylation patterns in oocytes.

Characteristics of Imprinted Genes

Monoallelic expression :


This refers to the transcription of a gene from a single parental allele. Some imprinted genes are monoallelically expressed only in particular organ or tissue, and can be expressed from both parental alleles in other parts of the body.


Clustering of Imprinted genes into evolutionary conserved domains :
One of the hallmarks of imprinted genes is that many are evolutionary conserved domains are found in clusters throughout the genome. These clusters contain two or more imprinted genes over a region that can span 1 Mb or more. The genes in the clusters, which can be either maternally or paternally expressed, are jointly regulated through an imprinting control region (ICR). The ICRs exhibit parental-specific epigenetic modifications (DNA methylation and histone modifications) that govern their activity.


Parental – allele – specific methylation :
DNA can be modified by methyl groups that attach to cytosine residues within specific CpG (cytosine phosphate diester guanine) dinucleotide pairs. Methylation of a single parental allele is a hallmark of imprinted genes. This usually occurs across the CpG islands (generally at the 5’- end of the genes), and methylation of an allele usually correlates with its transcriptional inactivation.

Genomic Imprinting

Genomic Imprinting is an example of epigenetic inheritance in eutherian mammals in which the gene expression depends on the parental origin of the allele, i.e. a particular gene will be expressed only from one specific parental allele, thereby leading to monoallelic expression.


The parental specific expression of genes is due to the epigenetic modification that differentially marks the parental alleles during gametogenesis. Most of the genes involved in embryo development are imprinted. (IGF2, H19, etc).

Epigenetic changes are alterations in gene expression without affecting the primary DNA sequence thereby modulating gene function. The epigenetic modifications involve DNA methylation, chromatin remodeling through histone modifications, etc. The best characterized epigenetic mark for gene imprinting is DNA methylation at specific regions known as differentially methylated regions (DMR).

Genetics versus Epigenetics

Gene expression can be regulated at multiple levels, i.e. DNA structure, gene transcription, and/or posttranscriptional modifications. Four genetic mechanisms (DNA point mutation, deletion, rearrangement, and amplification) and two epigenetic mechanisms (DNA methylation and the preservation of DNA-protein complexes) can account for the majority of enduring changes observed in cultured cells.


Genetic alterations in DNA sequence appear to be largely responsible for altered growth regulation associated with transformation, but there is also evidence to suggest that epigenetic mechanisms play a role in transformation. Differentiation of cultured cells is often associated with lack of growth, and has been ascribed in part to epigenetic mechanisms. However, differentiation and transformation are not mutually exclusive but may be regulated by parallel multistep pathways.

EPIGENETICS refers to heritable changes in phenotype or gene expression caused by mechanisms other than changes in the underlying DNA sequence.

Genomic Imprinting (Overview):

Each somatic cell of the human body contains 46 chromosomes consisting of two sets of 23; one inherited from each parent. These chromosomes can be categorized as 22 pairs of autosomes and two sex chromosomes. Similarly at the molecular level, two copies of each autosomal gene exist, one copy derived from each parent.


Until the mid-1980s, it was assumed that each copy of an autosome or gene was functionally equivalent, irrespective of which parent it was derived from. However it is now clear from classical experiments in mice and from examples of human genetic diseases that this is not the case.


The functional activity of some genes or chromosomal regions is unequal, and dependent on whether they have been inherited maternally or paternally. This phenomenon is termed ‘GENOMIC IMPRINTING’ and the activity or silence of an imprinted gene or chromosomal region is set during gametogenesis.


Genomic Imprinting involving the autosomes appears to be restricted to eutherian mammals. When normal pattern of imprinting is disrupted, the phenotypes observed in humans and mice are generally associated with abnormal fetal growth, development and behaviour, illustrating its importance for a normal intrauterine environment.