Here we report hematopoietic defects as well as erythroleukemia development in mice transplanted with Lsh−/− donor cells. Lsh depletion reduces Dnmt3b binding and DNA methylation at the PU.1 gene in hematopoietic stem cells. The rise in PU.1 transcripts and the slight rise in PU.1 protein level may then contribute to the development of erythroleukemia in the absence of Lsh.
Previous models of DNA hypomethylation and hematopoietic malignancies have utilized a hypomorphic Dnmt1 allele. Attenuation of Dnmt1 activity is sufficient to induce neoplasms, however, in contrast to our work these neoplasms are of T cell origin.9
By the time lymphomas were developing in Dnmt1 attenuated mice (4 to 8 months), most Lsh−/−
recipients had already died because of failed engraftment, reduced immune defense, or erythroleukemia, thus excluding an evaluation of Lsh deletion on lymphomagenesis. On the other hand, erythroleukemias were not observed in Dnmt1 attenuated mouse models, indicating significant differences in the molecular actions of Lsh and Dnmt1. Whereas Dnmt1 depletion leads to substantial alteration of imprints, loss of Lsh showed limited effects (so far only one imprinted locus was affected). While the Lsh−/−
model leads to a decrease in CpG methylation of about 50% resulting in retroviral activation, the hypomorphic Dnmt1 allele shows only moderate loss of methylation and as a consequence no enhanced retroviral activity.9
Furthermore, Dnmt1 shows multiple interactions with transcriptional repressors and tumor suppressor genes (e.g., Rb and HDAC) suggesting aberrant transcriptional regulation with Dnmt1 attenuation.13,15
Thus the exploration of molecular pathways using distinct genetic models is crucial for determining how genomic hypomethylation is involved in tumorigenesis.
Our data support a link between PU.1 increase and erythroleukemia development as has been previously demonstrated in the PU.1 transgenic animal model. It had been demonstrated that overexpression of PU.1 alone is sufficient to promote erythroleukemia.27,28
PU.1, an Ets family transcription factor, is expressed in hematopoietic lineages including erythroblasts.30
During normal hematopoiesis PU.1 is turned off in erythrocyte precursors. However, continuous overexpression of PU.1 blocks terminal differentiation and leads to an expansion of the erythrocyte precursor pool. This is consistent with our observation of a relative increase of erythrocyte precursors in the bone marrow of mice transplanted with Lsh−/−
progenitors. Subsequent mutational events are thought to be required for transformation of the expanded erythrocyte precursor pool.34
Based on the Friend virus model and transgenic mice data, PU.1 elevation in the absence of Lsh may play a role in leukemia development. However, additional mechanisms that are linked to DNA hypomethylation may also participate such as a failure to maintain all genomic imprints,35
chromosomal segregation defects due to perturbed centromeric heterochromatin,9,36
increases in mutations due to CpG hypomethylation37
and the transcriptional deregulation of other genes possibly involved in tumorigenesis.38–40
Lsh is involved in de novo methylation and can associate with Dnmt3b.22,39,40
Here we report binding of Lsh to retroviral elements located in the PU.1 gene and show that the presence of Lsh is important for association of Dnmt3b with these retroviral elements. This is consistent with the hypothesis that Lsh may regulate in part DNA methylation by controlling access of Dnmt3b to appropriate target sites.39
The two retroviral elements of the PU.1 gene, ERVL and MaLR, that showed differential methylation after Lsh depletion belong to the family of degenerate long-terminal repeat (LTR) transposons. 41,42
LTR elements make up 4–8% of the mammalian genome and are probably derived from past retroviruses. Many LTR elements lack a reverse transcriptase encoding region, have lost the ability to transpose, and are only 300 to 500 bp in size with a putative TATA box. The LTRs of the Moloney MuLV have been demonstrated to contain strong enhancer activity and insertion of this viral LTR up to 300 kb distal to the c-myc gene can lead to c-myc overexpression.42,43
DNA methylation is known to affect the LTR activity of retroviral elements in the mammalian genome.29,44
We observed de-methylation and transcriptional reactivation of the MaLR and ERVL elements at the PU.1 locus. This suggests the possibility that the re-activated LTR enhancers may also affect the PU.1 promoter. There are many examples for transcriptional control of mammalian genes by nearby integrated LTRs that are also controlled by DNA methylation such as the oncomodulin gene, the nocturnin gene and the agouti locus.44–47
Alternatively, the alteration in DNA methylation levels at the retroviral elements may subsequently affect other histone modifications and spread along the gene. Thus it may facilitate Pol II initiation at the promoter region or Pol II elongation through the gene body and therefore enhance PU.1 transcript levels.
In summary, genomic hypomethylation after Lsh deletion can cause a dual risk; on the one hand it may lead to severe hematopoietic defects with the danger of reduced immune responses and on the other hand, it may create an enhanced risk of developing hematopoietic neoplasm. Whether demethylating agents currently used in the clinic or developed in the future can reactivate silenced tumor suppressor genes and predispose patients to an additional cancer risk by oncogene activation is an important issue. Only future studies that address the contribution of epigenetics to tumorigenesis using suitable epigenetic models, will further clarify the risks and benefits of such treatments.