Epigenetic Loss of MLH1 Expression in Normal Human Hematopoietic Stem Cell Clones is Defined by the Promoter CpG Methylation Pattern Observed by High-Throughput Methylation Specific Sequencing

Abstract

Normal human hematopoietic stem and progenitor cells (HPC) lose expression of MLH1, an important mismatch repair (MMR) pathway gene, with age. Loss of MMR leads to replication dependent mutational events and microsatellite instability observed in secondary acute myelogenous leukemia and other hematologic malignancies. Epigenetic CpG methylationThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. *Corresponding author: Stanton L. Gerson, Seidman Cancer Center, University Hospitals of Cleveland, Cleveland, OH, USA, Tel: 1-216-844-8562, stanton.gerson@case.edu. Author Contributions: JK designed the study, performed research, data analysis, and wrote the manuscript. GN helped design the study, performed research, and data analysis. YQ assisted with study design and data analysis. GS-G and ED performed research and assisted in manuscript preparation. PF, SS, and XB assisted in study design and data analysis. DW assisted the study design and provided essential reagents. EA helped design the study and provided essential reagents. SLG helped design the study, assisted in manuscript preparation, and provided essential reagents. Conflicts of Interest:The authors have no competing financial interests in relation to the work described in this manuscript. HHS Public AccessAuthor manuscript Int J Stem Cell Res Ther. Author manuscript; available in PMC 2016 August 24. Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript upstream of the MLH1 promoter is a contributing factor to acquired loss of MLH1 expression in tumors of the epithelia and proximal mucosa. Using single molecule high-throughput bisulfite sequencing we have characterized the CpG methylation landscape from −938 to −337 bp upstream of the MLH1 transcriptional start site (position +0), from 30 hematopoietic colony forming cell clones (CFC) either expressing or not expressing MLH1. We identify a correlation between MLH1 promoter methylation and loss of MLH1 expression. Additionally, using the CpG site methylation frequencies obtained in this study we were able to generate a classification algorithm capable of sorting the expressing and non-expressing CFC. Thus, as has been previously described for many tumor cell types, we report for the first time a correlation between the loss of MLH1 expression and increased MLH1 promoter methylation in CFC derived from CD34 + selected hematopoietic stem and progenitor cells.

MMR Deficiency Does Not Sensitize or Compromise the Function of Hematopoietic Stem Cells to Low and High LET Radiation

“…Exposure of MMR‐deficient mice to gamma radiation results in hypermutability compared to wild‐type mice, and much of this hypermutability can be attributed to induced instability of simple sequence repeats . Interestingly, recent results from our laboratory have demonstrated that acquired MMR deficiency and increased MSI in human hematopoietic stem and progenitor cells is age‐related, attributable to progressive loss of Mlh1 through promoter hypermethylation . The data show that as many as 30% of HSCs in healthy individuals have lost MLH1 by 45 years of age, which is well within the age range of current and former astronauts.…”

RNA‐Seq analysis of differentially expressed genes relevant to mismatch repair in aging hematopoietic stem‐progenitor cells

“…11 Recent results from Welford SM' laboratory have demonstrated that acquired MMR deficiency and increased microsatellite instability, a commonly observed marker of DNA MMR deficiency in human hematopoietic stem and progenitor cells is agerelated, attributable to progressive loss of Mlh1 through promoter hypermethylation. 5,6 The data show that as many as 30% of HSCs in healthy individuals have lost MLH1 by 45 years of age. 7 It was suggested from this study that not only the reported widely genes of MMR such as MSH2, MSH3, MSH6, MLH1, PMS1, and PMS2 were significantly downregulated in aging HSPC, but also more than 20 MMR/MMR complex-related genes in GO and KEGG databases such as Pcna, Exo1, Rpa1, Rpa2, Rpa3, and other genes were significantly downregulated.…”

“…3 However, studies on the relationship between MMR and HSC aging are not so much. [4][5][6][7] In this paper, we used RNA-Seq technology and an old hematopoietic stem and progenitor cells (HSPCs) model in vitro to analyze differential expressions of MMR-related genes in aged HSPCs, so as to explore the mechanism of DNA MMR injury in HSC aging.…”

Mlh1 deficiency increases the risk of hematopoietic malignancy after simulated space radiation exposure

“…A recent study by our group demonstrated that MSI accumulates in human HSCs as a function of age, with loss of MLH1 by promoter hypermethylation 23, 24 . Given that upper end of astronauts are ~46 years old, HSCs with deficient MMR function will likely be exposed to space radiation.…”