Ugrás a tartalomhoz

Transdifferentiation and regenerative medicine

Dr. Péter Balogh, Dr. Péter Engelmann (2011)

University of Pécs

Epigenetic factors in transdifferentiation

Epigenetic factors in transdifferentiation

Epigenetics refers to the non-sequence based structural changes of chromosomal regions that can alter the gene expression activities in response to the external signals. Major types of epigenetic modifications include DNA methylation, histone modifications, chromatin remodeling and noncoding RNAs. Distinct histone modifications which carry regulatory information were dubbed ‘histone code’, based on the analogy with the ‘genetic code’ of genomic DNA sequence. Histones are the subjects of over 8 major distinct classes of modifications with dynamic natures and consequences. Such covalent modifications play fundamental roles in chromatin condensation, replication, DNA repair, and transcriptional regulation. Among them, methylation and acetylation are the most studied and best understood histone modifications. Histone acetylation is generally associated with actively transcribed genomic domains and the degree of modification correlates with the level of transcription, whereas histone methylation can have different effects on gene expression depending on which residue is modified. Among the diverse histone modifications, methylations in histone 3 lysine 4 and lysine 27 are of particular importance. These modifications are catalyzed by Trithorax (TrxG) and Polycomb (PcG) group complexes respectively, which establish developmental decisions and mediate the expression programs of lineage specific genes. Lysine 4 methylation positively regulates gene expression by serving as a binding platform to recruit nucleosome remodeling enzymes, whereas lysine 27 methylation negatively regulates gene expression by generating a compact chromatin domain. Similarly, H3K36me3 is associated with actively transcribed regions whereas H3K9me3 is involved in inactive genomic domains. Particularly, H3K4me3 is regarded as a hallmark for the promoters of actively transcribed genes and H3K36me3 is an indicator of transcription elongation. The different preferences of these histone modification occupancies on genome-wide landscape could be used to define transcription units.

The mechanism adopted by histone modifications to regulate transcription and chromatin organization is not clearly defined. An attractive hypothesis is that epigenetic factors, including modifying enzymes or remodeling factors, could potentially tether chromatins to mediate cis and trans interactions. Such interactions presumably cause the structural changes of underlying DNA and chromatins. Conformation changes could even be mediated by specific protein complexes recruited by these modifications. These interactions can alter the physical property of the chromatin and affect its high order structures.

Figure III-4: Epigenetic gene regulation of stem cell genome

In mammalian cells, both specific histone modification and DNA methylation are involved in chromatin silencing. DNA methylation and histone modification are believed to be interdependent processes. Recent studies suggest that a combination of histone acetylation and DNA demethylation induces neuronal stem cells (NSC) to trans-differentiate into hematopoietic cells.

In ES cells, the overall nuclear architecture is globally decondensed and condensation reoccurs during differentiation. Specific changes in histone modifications have been shown to accompany ES cell differentiation and mammalian development.

The establishment of symmetric DNA methylation patterns could be prevented passively during replication by the steric hindrance of Dnmt1 due to the stochastic binding of the reprogramming factors to target sites or by inhibiting Dnmt1 function indirectly. Hemimethylation of the DNA would result in a progressive loss of methylation upon further rounds of cell division. Alternatively, DNA methylation could be actively removed by the recruitment of a demethylating enzyme.

Figure III-5: DNA methylation in stem cells

A new integrated global regulatory network is currently emerging based on the dynamic interplay of chromatin remodeling components, TFs, and small ncRNAs. These three mechanisms synergize to choreograph stem cell self-renewal and the generation of cell diversity. Mammalian cells harbor numerous

small ncRNAs, including small nucleolar RNAs, (snoRNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs) and small double-stranded RNAs, which regulate gene expression at many levels including chromatin architecture.

Most show distinctive temporal- and tissue-specific expression patterns in different tissues, including embryonic (ESC) stem cells and the brain, and some are imprinted.

Many miRNAs are specifically expressed during ESC differentiation, embrygenesis, neuronal differentiation and hematopoetic lineage commitments.

Several miRNAs, including ESC miRNAs, Myc-induced miRNAs, miR-92b, and the miR-520 cluster, have been shown to positively regulate the self-renewal and pluripotency of ES cells. Among these, only ESC miRNAs have been tested for their ability to promote reprogramming. Additionally, a number of tissue-specific miRNAs, such as let-7, miR-134, miR-470, miR-296, and miR-145, have been shown to interfere with the self-renewal and pluripotency of ES cells. However, with the exception of let-7, the effects of inhibiting the activity of these miRNAs on reprogramming are not known. Recent study demonstrated that inhibition of let-7 activity promotes reprogramming. miR-125, which inhibits the expression of Lin28, is also expected to positively influence reprogramming.

Additionally, miRNAs that target specific signaling pathways (e.g. TFG-beta signaling) and epigenetic processes (e.g. DNA methylation) can also be tested for their ability to promote reprogramming. miRNAs encoded by Dlk1–Dio3 gene cluster are also attractive candidates for promoting reprogramming because activation of imprinted Dlk1–Dio3 gene cluster is essential for generating fully reprogrammed iPS cells, which are functionally equivalent to ES cells.

Figure III-6: miRNA and stem cell differentiation