AHEAD(the Alliance for Human Epigenomics and Disease)

I found from last paper that Epigenome Project is continued by AHEAD. In this post, I will introduce the article about AHEAD which is published in Nature.  Ah.. the funny thing is.. when I was googling about AHEAD, I found that  Prof, Young-Joon Kim presented his research in AHEAD conference in last year.


The title of article which will be introduced is "Moving AHEAD with an international human epigenome project". This was published on August in 2008.

A plan to 'genomicize' epigenomics research and pave the way for breakthroughs in the prevention, diagnosis and treatment of human disease.


here we discuss the benefits of the AHEAD frame work to coordinate and plan an international Human Epigenome Project.

Epigenetic mechanisms : histone modification, positioning of histone variants, nucleosome remodelling, DNA methylation, small and non-coding RNAs. These things interact with transcription factor or other protein to regulate target gene expression.
epigenetic mechanisms are recognized as being involved disease.
although this mechanisms have heritable characteristics, drug can reverse them.
So more comprehensive characterization of them is needed to maximize utility of them in treatment of disease.
the goal of AHEAD project is provide high-resolution reference epigenome maps.
an international project would provide the bioinformatics tool.

-Early steps- 

About from 2004 global movements were appeared to organize the international community.

In Europe, strong tradition for epigenetic study supported by European Union funding programmes or individual national initiatives. more than US $79M was supported to DNA methylation(HEP, Human Epigenome Project), chromatin profiling(HEROIC,High-Throughput Epigenetic Regulatory Organization In Chromatin) and treatment of neoplastic disease(EPITRON, EPIgenetic TReatment Of Neoplastic disease). The special function is provided by the NoE(Epigenome Network of Excellence).
In U.S. 2004 NCI(international cancer institute)-sponsored Epigenetic Mechanisms in Cancer Think Tank, 2005 NCI workshop, AACR(The Ametican Association for Cancer Research) organized a Human Epigenome Workshop in 2005, 2006.
On the heels of these workshop AACR Human Epigenome Task Force was formed to design strategy and develop a timetable for the implementation of an international Human Epigenome Project. This task force recommended the formation of AHEAD to coordinate a transdisciplinary, international project.


In summary, each country (EU, US) organize and develop their own community and project. And after Human peigenome workshop from AACR, AACR Human Epigenome Task Force was formed and they made AHEAD, internation project to map a defined subject of robust epigenetic makers and support bioinformatics infrastructure.


-The scope of AHEAD-


1.provide complete epigenome maps at very high resolution for important histone modifications across diverse cellular states in both human and mouse
2.complete and catalogue epigenome maps of model yeasts, plants, and animals
3.deliver a high resolution DNA methylation map of the entire human genome in defined cell types and a landmark map for transcription start sites of all protein coding genes and a representative number of other features throughout the genome
4.define non-coding and small RNAs
5.establish a bioinformatics platform including a relational database, website and suite of analytic tools to organize, intergrate and display whole epigenomic data on model organisms and humans.
AHEAD differ from and complement ENCODE(ENCyclopedia Of DNA Elements).
ENCODE is focused on defining the functional sequences in the genome.
AHEAD define the patterns of epigenetic regulation at that sequences.


-Reference epignomes-

The criteria for selection of the reference system:
1.cells should be easy to sample in a reporoducible fashion.
2.cell numbers should be sufficient for analyses of DNA methylation and chromatin modifications.
3.cell progenitors should be identified that can be suitably manipulated and harvested in a pure state.
4.where possible, cells should be amenable to tissue reconstruction and three-dimensional model systems
5.systems must provide insight into key differentiation and related disease states.

-Advances in technology-
there is nothing new, so I just skip this part

-Model organisms-



-Computational challenges-
A central relational database and a web interface which include analytic and statistical tools to present and visualize data are needed.

pseudo gene

I felt the need to clarify about pseudo gene while I organized an annotation of zymo. Therefore in this post I will review about pseudo gene, what it is and how it was happened.
All information comes from  http://en.wikipedia.org/wiki/Pseudogene


Pseudogene : non-expressed or defunctional relatives of known gene (homology to a known gene, nonfunctionality).
1.homology to a known gene : usually sequence identity is between 40% and 100%.
2.nonfunctionality : if any one of the common processes, which are needed in making functional product from DNA, such as transcription and pre-mRNA processing fails, this will be nonfunctional. In high-throughput psudogene identification, modification of stop codon and frameshifts are the most common factors.


Types and origin of pseudogenes


1. Processed (retrptransposed) pseudogenes : retrotransposition event is common in mammals and in case of human 30%~44% of genome is composed of this. In retrotransposition, mRNA transcript of a gene is reverse transcribed back into DNA become pseudogene. This kind of pseudogene have features of cDNA like poly-A tail, introns spliced out and lack of promoter.

2. Non-processed pseudogenes : Through gene duplication A copy of gene is made and subsequent mutation make them as pseudogene. This kind of pseudogene have the most feature of genes.


3. Disable genes (unitary pseudogene) : same mechanism (i.e. mutation) which make non-processed pseudogene happened to genes without duplication and became fixed in population.

DNA methylation profiling of human chromosomes 6, 20 and 22

This paper was found while I have been searching Epignome Project. This is the last paper which was published from Epigenome Project, and I decided to present this on next journal club meeting. This paper have been cited by 317 articles.


The really interesting discovery from introduction of this paper, Epigenome project was dubbed to AHEAD (the Alliance for Human Epigenomics and Disease). And there are some clue (http://www.nature.com/nature/journal/v454/n7205/full/454711a.html, http://www.aacr.org/home/scientists/working-groups--task-forces/task-forces/human-epigenome-task-force.aspx). I will summarize this on next time.


Result

In this paper, they report the methylation profiling of human chromosomes 6, 20 and 22 in 43 samples derived from 12 different normal tissues. They controlled age and sex, which can affect the methylation state, in each samples. 2,524 amplicons with 1.88 M CpG sites on chromosome 6, 20 and 22 that are associated with 873 genes were sequenced by sanger sequencer.

Distribution of methylation
bimodal distribution of amplicon's methylation state, 27.4% unmethylated(<20% methylation), 42.2 hyper-methylated(>80% methylation) and 30.2 heterogeneously methylated(20%~80%). Only 9.2 of CGIs were hyper-methylated.

By distinction  mosaicism versus imprinting among heterogeneously methylated amplicons, they confirmed the most of them were by mosaicism.

Comethylation (relationship between the degree of methylation over distance) was quite strong over short distances(<1000 bp). This fact showed that in normal the range of comethylation was shorter than in specific diseases. Absolute difference in methylation betweens tissues were shown in figure. Sperm stood out, and related tissue showed low level of difference.




Promoter methylation

They divide promoter region in three types. 5' UTR, putative TSSs, Sp1(transcription factor) binding site. And 5' UTR are sub-divided according existence of CGIs, and gene type (novel CDS, novel transcript, pseudo gene).
The most CGIs in 5' UTR are unmethylated, while non CGI 5' UTRs are hyper-methylated. There is no big difference between CDS, transcript, pseudo in exonic region, but in 5' UTR the most amplicon which belong to CDS are unmethylated.

TSSs showed unmethylated core region of about 1,000 bp.

Age- and sex-dependent DNA methylation


They compared methylation state according to age (26+-4 age, 68+-8 age) and sex, but couldn't find any significant difference.


Differential methylation


T-DMRs (tissue-specific differentially methylated regions) was confirmed by hierarchical clustering which showed biological replicate of each tissue grouped together. 22 % of amplicons were T-DMRs. The frequency of T-DMRs which belong to CGIs was low.

They found that T-DMRs in 5' UTRs which are without CGIs could affect mRNA expression. T-DMRs located in gene have little effect on mRNA expression.


Conservation of DNA methylation

To check conservation of methylation across species, they compare 59 orthologous amplicons between human and mouse. The majority of profiles were conserved. They extrapolate that 70  % of orthologous loci between human and mouse may have conserved methylation profiles.








Like most papers in early stage of high-resolution sequencing for methylation state, This paper also just showed landscape of methylation state over large region in chromosomes. There were just arbitrary classification of methylation state and correlation this classification with some specific conditions. There is no absolute rule.




I think that It will be fun that finding orthologous genes which show different methylation state between human and mouse. Are these genes species-specific genes?