Epigenetic factors in imprinting diseases

Natalia Cucu, PhD, Assisstant Prof, University of Bucharest

Gabriela Anton, PhD, Senior Res, Institute of Virology”Stefan Nicolau” Bucharest

Maria Puiu, PhD, Professor , UMF Timisoara

Objectives and motivation

• Genetic alterations claimed for cca 80% of the rare diseases are transmitted in non-Mendelian way; it is established presently that such process involves also an additional hereditary information which is not linked with genetic information encoded in well known DNA (nucleotide) sequences. That is why it has been named epi-genetic information: it is encoded in specific chromatin structures which are no more static, but rather very dynamic and depend on DNA and histone covalent modifications mediated by both the enzyme activities and specific DNA-DNA-protein and protein-protein interactions

• Epigenetic domain emerged recently from genetics, developmental and cell biology and of course, biochemistry. It explained the natural, phisiological activity of endogenous enzymes not only upon DNA but also histones during vital developmental steps (cytodifferentiation, gametogenesis, embryogenesis, aging, stem cells capacity for tissue renewal etc). Now it is well established that alterations in epigenetic level of hereditary information lead to pathological states (sporadic cancer, aging related disorders, rare diseases).

• Rare diseases are generally determined by alterations in imprinting process.This is defined by the establishment of certain molecular imprints or marks on a specific parental allele, which determines the so-called “parental-specific gene expression in diploid cells”. Thus diploid cells containing 2 parental copies of all genes will express only one parental copy in the case of the so - called imprinted genes.In contrast, the rest of genome, containing NG (nonimprinted genes), will express both parental alleles. Usualy, the subset of IG largely code for factors regulating embryonic and neonatal growth.

• IG usually are located in clusters on specific chromosomes: for example, a cluster of critical IG is located on chr15 ; alteration in their imprinting is associated with PW and A syndromes.

• A description of the molecular factors involved in the establishment of the imprinting marks during specific reproduction steps (gametogenesis and embryogenesis) is the aim of this presentation. The directions envisaged in their investigations and the approached methods will be described.

DNA molecule is not free: eukaryote genetic material is represented by chromatin, a nucleoprotein complex

• The key epigenetic concept is chromatin conformation dynamics.

• DNA molecule is not free, as it has been long time supposed, during its interaction with transcription machinery. The complexation with additional informational macromolecules, specific basic proteins, named histones, is crucial not only for its compaction into the same chromosomal structures, in general independently of the genetic information or DNA sequence, but especially for its subtle controlled decondensation for the genetic information to perform its decoding action and expression into needed proteins. This controlled process is enabled by an interesting dynamics of the chromatin conformation, determined by molecular interactions between certain nonhistone proteins (histone acetylases, deacetylases, phosphorylases, ubiquitylases, sumoylases, biotinylases, ADP ribosylases and also histone and DNA methylases) and the chromatin components (DNA and histones)

(Richards and Elgin, 2002; Bird,2002).

Key molecule of hereditary information is not free DNA, but rather a complex between DNA and histones called chromatin

• Steps of chromatin compaction; nucleosomal structures highlighted

• Two main epigenetic codes control these processes:

-DNA methylation

-Histone code

Relationship between the chromatin structure and the epigenetic (DNA and histone) codes

DNA methylation

• Covalent modification of DNA by addition of methyl groups on specific nucleotide residues (cytidine)

• Key molecules in DNA methylation process:

• Cytosine (major base)• 5-Methylcytosine

(minor base)

DNA methylation in CpG repetitive sequences

Types of methylated deDNA and specific DNA methyltransferases

DNA methylation enzymology depends on SAM/SAH ratio and on the exogenous methyl pool provided by

folates

The main biological role of DNA methylation-transcriptional repression

Key points of the histone code represented on the

nucleosomal core histones

Specific epigenotypes for active and repressed (silenced) chromatin

Imprinting process and epigenetic factors

• DNA methylation dynamics during development

DNA dynamics during reproduction steps (gametogenesis and embryogenesis) is critical for correct imprinting establishment and

maintenance

Critical stages of DNA methylation process during developmental steps

DNA methylation during the imprinting process is controlled by a complex enzymology:

there is an interplay between at least known 3 enzymes: DNMT3a/DNMT3b and DNMT3L

• Erasure and reset of imprints by DNA and histone methylation depends on specific gametofenesis and emvryogenesis steps and hence the activity of corresponding epigenetic effectors (DNA methylases, DNA demethylase, histone methylase, histone demethylases)

Erasure and reset of DNA methylation pattern are key processes in correct imprinting process

Genes on critical region of Chr15 involved in altered imprinting for PW and A syndromes

Genes from 15 chr associated with PWS and AS

For AS it is well established the cause linked with the loss of function by mutation or DNA methylation in UBE3A gene (ubiquitin E3 ligase gene) encoding the E6-associated protein A (E6-AP). Normally it is expressed only from the maternal allele. Paternal UPD or maternal deletions or DNA methylation in 15q11-q13 region of chr 15 leads to loss of expression and hence the maternal contribution.

.

For PWS there are several candidate imprinting genes that are only expressed normally from paternal allele; however it is not clear which of these genes is contributing to PWS phenotype. There are two classes of such genes-

• protein coding genes: the best candidates are (i) SNURF-SNPRN (SNPRN:small nuclear riboprotein that function in regulation of splicing; and SNURF:“SNPRN upstream reading frame”) and (ii) NDN (necdin)

SNURF-SNPRN has its major transcriptional site at IC which is the major site of imprinting defects (disruption in SNURF leads to altered imprinting in SNPRN and other imprionted genes from critical region)

• noncoding snoRNA : loss of one or more such genes possibly in combination with loss of other 1 class genes may contribute to PWS phenotypes

Classes of genetic and epigenetic alterations in PW and A syndromes

Control of allele specific expression is mediated by epigenetic factors (DNA methylation)

The influence of the DNA methylation status on the transcriptional activity of the gene affected in the promoter sequences and the proximal exons

Alteration of specific allele DNA methylation in PW and A syndromes

Methods for epigenetic monitoring

• Epigenotype modifications for specific sequences (promoters of genes involved in certain cromosome regions) may be monitored by specific antibody based (ChIP) methods, MS-PCR with specific primers or mass spectrometry as well as HPLC.

• Immunohistochemical and RT-PCR methods tackle the gene expression variation

• Cytoimmunofluorescence methods may be used coupled with flow cytometry to track specific epigenetic modifications at the level of cells, nuclei or chromosomes during specific cell cycle points.

• DNase I hypersensibility coupled with MNase analysis coupled with Southern hybridisation are currently used methods coupled with flow cytometry for demonstration of the accessibility of the chromatin conformations linked with the active/repressed gene status.

• Array based chromatin immunoprecipitation (ChIP on a chip) methods are presently developing for integrating the genetic and epigenetic observation of the gene expression status at the key genes promoters.

Experimental models used so far in our network of epigenetics and nutrigenomics

• The methylation status of parental allele DNA has been estimated by MSPCR with specific primers for U and M sequences

• Western blotting for immunochemical monitoring the histone modifications

Principle of the MS-PCR assay for the estimation of the DNAmethylation status in

promoter gene regions

Normal, AS and PWS electrophoretic patterns after MS-PCR

PWS

AS

Preliminary results in validating PW cases by MSPCR method

• A family of a PW case presented the following pattern of PCR amplification mediated by bisulphite mutagenesis:

• -normal –mother/father/paternal grandmother (2 bands)

• PW case (child) one specific band which represents the amplicon 313pb of the PCR of the paternal allele methylated SNPRN promoter

METABOLOMIC APPROACH

Conclusions

• Inspite of the enormous effort invested in genetic causes of numerous diseases, especially those named “rare diseases”, clinical strategies still proved poor efficiency from both diagnosis and therapeutic points of view. The newly emerged epigenetics domain introduced an additional level of gene expression control, to the genetic (mutations) one, which is more efficiently linking the external environmental factors, endogenous developmental ones and the gene sequence. The chromatin dynamics is a central key explanation of such a new approach of gene expression which may be tackled by monitoring the reversible biochemical modifications of both DNA and histones around the target gene promoter sequence.

• Tackling chromatin components (remodelling factors such as enzyme effectors, covalently modified histones and DNA) by specific integrative methods (MSPCR, RTPCR, immunohistochemistry, ChIP, Western blotting, HPLC) is possible presently to monitor the chromatin structure dynamics linked with nornal/aberrant gene expression linked with health status

• A Romanian epigenetics and nutrigenomics network has been established in 2006 on the occasion of approval of the first epigenetic approach of breast cancer as a national research program (VIASAN) project. Presently its work envisages presentlz epigenetic factors in gynecological cancers, but also rare diseases, represented by Prader Willi and Angelman syndromes.

Perspectives of the imprinting diseases epigenomicsenvisage

1. continuing to implement the epigenetic methods for validating the genetic analyses;2.deciphering pathological epigenetic mechanisms for preventive and therapeutical strategies

• A feature of the epigenetic modifications is presently focussing the interest of those part of the medicine dealing with early detection, prevention and more efficient treatment of diseases, especially, cancer for somatic cell transfromation and rare diseases for reproductive cells (gametes and embryons). This feature is linked with the reversible characteristics of the covalent epigenetic modifications at the level of DNA and histones, as compared with the “fixed”, hardly manageable mutations.

• Having in mind the environmental effects upon the versatile enzymatic action of nonhistone on DNA and histone substrates, it is increasingly apparent that the early detection of the epigenetic chromatin modifications with specific power of oncogenic action or imprinting process alteration may be in due time corrected by a proper diet and life style and also by new (epigenetic) drugs (DNA and histone methyltrnsferase and HDAC inhibitors) in order a reestablished gene expression pattern encoded by a specific chromatin conformation to be transmitted in descendants.

• Transgenerational effects are now considered by ART (assissted reproductive and therapeutic cloning techniques), gathering beneficies from such approaches of therapeutics in cancer and other diseases. The newly emerging drugs in this concerns are represented mainly by specific inhibitors of chromatin remodelling enzymes, inducing silencing processes in tumor suppressor genes, such as DNA and histone methylases and histone deacetylases.