Louis S. Ramagli

Conserved DNA methylation patterns in healthy blood cells and extensive changes in leukemia measured by a new quantitative technique

Genome wide analysis of DNA methylation provides important information in a variety of diseases, including cancer. Here, we describe a simple method, Digital Restriction Enzyme Analysis of Methylation (DREAM), based on next generation sequencing analysis of methylation-specific signatures created by sequential digestion of genomic DNA with SmaI and XmaI enzymes. DREAM provides information on 150,000 unique CpG sites, of which 39,000 are in CpG islands and 30,000 are at transcription start sites of 13,000 RefSeq genes. We analyzed DNA methylation in healthy white blood cells and found methylation patterns to be remarkably uniform. Inter individual differences > 30% were observed only at 227 of 28,331 (0.8%) of autosomal CpG sites. Similarly, > 30% differences were observed at only 59 sites when we comparing the cord and adult blood. These conserved methylation patterns contrasted with extensive changes affecting 18–40% of CpG sites in a patient with acute myeloid leukemia and in two leukemia cell lines. The method is cost effective, quantitative (r2 = 0.93 when compared with bisulfite pyrosequencing) and reproducible (r2 = 0.997). Using 100-fold coverage, DREAM can detect differences in methylation greater than 10% or 30% with a false positive rate below 0.05 or 0.001, respectively. DREAM can be useful in quantifying epigenetic effects of environment and nutrition, correlating developmental epigenetic variation with phenotypes, understanding epigenetics of cancer and chronic diseases, measuring the effects of drugs on DNA methylation or deriving new biological insights into mammalian genomes.

The epigenome of AML stem and progenitor cells

Acute myeloid leukemia (AML) is sustained by a population of cancer stem cells (CSCs or cancer-initiating cell). The mechanisms underlying switches from CSCs to non-CSCs in vivo remain to be understood. We address this issue in AML from the aspect of epigenetics using genome-wide screening for DNA methylation and selected histone modifications. We found no major differences in DNA methylation, especially in promoter CpG islands, between CSCs and non-CSCs. By contrast, we found thousands of genes that change H3K4me3 and/or H3K27me3 status between stem and progenitor cells as well as between progenitor and mature cells. Stem cell related pathways and proliferation or metabolism related pathways characterize genes differentially enriched for H3K4me3/H3K27me3 in stem and progenitor populations. Bivalent genes in stem cells are more plastic during differentiation and are more likely to lose H3K4me3 than to lose H3K27me3, consistent with increasingly closed chromatin state with differentiation. Our data indicates that histone modifications but not promoter DNA methylation are involved in switches from CSCs to non-CSCs in AML.

HRAD17, a structural homolog of the Schizosaccharomyces pombe RAD17 cell cycle checkpoint gene, stimulates p53 accumulation

The RAD17 gene product of S. Pombe is an essential component of the checkpoint control pathway which responds to both DNA damage and disruption of replication. We have identified a human cDNA that encodes a polypeptide which is structurally conserved with the S. Pombe Rad17 protein. The human gene, designated hRAD17, predicts an encoded protein of 590 amino acids and a molecular weight of 69 kD. Amino acid sequence alignment revealed that hRad17 has 28.3% and 52.5% similarity with the S. Pombe Rad17 protein, and 21.8% identity and 45.8% similarity to the budding yeast cell cycle checkpoint protein, Rad 24. When introduced into the S. Pombe rad17 mutant, hRAD17 was able to partially revert its hydroxyurea and ionizing radiation hypersensitivity, but not its UV hypersensitivity. Permanent overexpression of the hRAD17 gene in human fibrosarcoma cells resulted in p53 activation and a significant reduction of S- and G2/M-phase cells accompanied by an accumulation of the G1-phase population, suggesting that hRAD17 may have a role in cell cycle checkpoint control. Immunostaining of HT-1080 cells transiently transfected with a hRAD17 construct confirmed the nuclear accumulation of p53, which mimics the induction caused by DNA damage. Using FISH analysis, we have mapped the hRAD17 locus to human chromosome 5q11.2.

Heat-stress proteins of rat lung endothelial and mammary adenocarcinoma cells.

A rat mammary adenocarcinoma cell clone, MTC, and a rat lung endothelial cell clone, RLE cl.4, both syngeneic to the Fisher 344 rat, were compared for proteins synthesized at 37 degrees C and after a 1-h, 42 degrees C heat dose. The heat stress-induced or -enhanced synthesis of a series of molecular mass groups and isoelectric point species (isomers) was observed in both equilibrium and nonequilibrium two-dimensional gel electrophoresis. Tumor and endothelial cell heat-stress proteins (hsp) were strikingly similar with most hsp in 11 or 13 molecular mass groups having from 1 to 12 major isomers. In comparing the two cell types, 6 of about 23 major hsp isomers appeared different in equilibrium pH gels, with tumor cells seemingly exhibiting less synthesis of these 6 isomers. Four additional endothelial cell hsp isomers were apparent in nonequilibrium pH gels. Since two of these later hsp can be found at higher heat doses in tumor cells, some of these apparent differences between tumor and endothelial cells may be attributable to different dose ranges for induction of hsp. Fluorograms and silver-stained gels showed that several hsp were being synthesized at appreciable levels in unheated cells. However, there were hsp whose synthesis appeared to be de novo rather than representing enhanced synthesis of existing proteins. These last two observations were made in both tumor and normal cells. The constitutive levels of hsp synthesis appeared to be generally similar in unheated tumor and normal cells in vitro with few exceptions. These results indicate the presence of few unique hsp in syngeneic tumor and normal cells in vitro. However, focusing subsequent studies on the few differences may lead to insights concerning hyperthermic biology of tumor and normal cells, phenotypic differences between these cells, and roles of some hsp.

Microsatellite instability in the peripheral blood leukocytes of HNPCC patients

Most hereditary nonpolyposis colorectal cancer (HNPCC) patients inherit a defective allele of a mismatch repair (MMR) gene, usually MLH1 or MSH2, resulting in high levels of microsatellite instability (MSI‐H) in the tumors. Presence of MSI in the normal tissues of mutation carriers has been controversial. Here we directly compare MSI in the peripheral blood leukocyte (PBL) DNA of seven HNPCC patients carrying different types of pathogenic MMR mutations in MLH1 and MSH2 genes with the PBL DNA of normal age‐matched controls and of patients with sporadic colorectal cancer (SCRC). Small pool PCR (SP‐PCR) was used studying three microsatellite loci for at least 100 alleles each in most samples. The average frequencies of mutant microsatellite fragments in each HNPCC patient (0.04–0.24) were significantly higher (p<0.01) relative to their age‐matched normal controls with mutant frequencies (MF) from 0.00 to 0.06, or SCRC patients (MF from 0.01–0.03). The data support the conclusions that higher MF in the PBL DNA of HNPCC patients is real and reproducible, may vary in extent according to the type of germline MMR mutation and the age of the individual, and provide a possible genetic explanation for anticipation in HNPCC families. Hum Mutat 31:317–324, 2010.

Genetic Analysis of Nonhistone Chromosomal Protein Inheritance in Recombinant Inbred Mouse Strains Using Two-Dimensional Electrophoresis

Analysis of hepatic nonhistone chromosomal protein (NHCP) expression in male mice from progenitor strains (C3H/HeN, C57BL/6N), their F1 hybrid (B6C3), and seven recombinant inbred strains (RIs) (B6N×C3N) by high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) detected 16 NHCPs whose expression in RIs could be correlated to each other and with strain distribution patterns (SDP) of 20 genetic markers differing in the progenitors. Of the 400+ NHCP spots detected in RI 2D-PAGE maps, 172 were common to progenitors and all RIs. There was a characteristic absence of five NHCPs in one RI, Y. Ten C3H-specific and six C57-specific NHCP inherited in B6C3 also appeared in RIs. The SDP of C3H-specific NHCP 2 matched the SDP of beta-glucuronidase on chromosome 5 and carbonic anhydrase on chromosome 3, and C57-specific NHCP 5 SDP corresponded to that for nonagouti trait on chromosome 2. These 16 NHCP genetic marker inheritance differences detected in RIs add to the 23 previously established genetic marker differences between the progenitors.

Abstract 3004: Low levels of methylation in normal DNA mark a large subset of genes primed for hypermethylation in cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Cytosine methylation of DNA is a vital component of epigenetic memory. Complex changes of DNA methylation in cancer permanently disturb epigenetic regulation and participate in neoplastic development. To characterize changes in methylome in myeloid neoplasms, we analyzed DNA methylation in 8 patients with myelodysplastic syndrome, 2 patients with acute myeloid leukemia (MDS/AML) and 3 healthy controls. Using Digital Restriction Enzyme Analysis of Methylation (DREAM), we performed quantitative mapping of DNA methylation status at 40,000 autosomal CpG sites. To measure methylation, we first create distinct DNA signatures at unmethylated or methylated CCCGGG sites by sequential restriction digests of gDNA with the SmaI and XmaI endonucleases and then resolve these signatures by massively parallel sequencing. The sequences are mapped back to the genome and methylation levels for individual CpG sites are calculated based on the numbers of sequencing reads with unmethylated or methylated signatures. We found that DNA methylation status had a binomial distribution with the majority of CpG sites showing either very low or very high methylation. CpG islands (CGI) were generally unmethylated, while non-CGI were predominantly methylated with the exception of gene transcription start sites (TSS). When we compared average methylation in MDS/AML with average methylation in controls, the picture was different in CGI and in non-CGI. DNA methylation was increased in MDS/AML in CGI at all distances from gene transcription start sites (TSS) by 2-3%. In contrast, methylation in non-CGI was decreased in MDS/AML by approximately 3% with the exception of regions within 1 kb from TSS. Interestingly, low levels of CGI methylation detectable in blood DNA from healthy controls were associated with a propensity for hypermethylation in leukemia. Methylation levels 0-1% in controls were observed in 5,680 genes. Of those, 51 genes (1%) showed average methylation >20% in MDS/AML. Methylation 1-5% in controls was seen in 1,912 genes. Of those, 192 genes (10%) showed average methylation >20% in MDS/AML. Methylation 5-10% affected 364 genes in controls, while 131 (36%) of these genes had >20% methylation in MDS/AML (P=8.3E-240). The same pattern was observed when we restricted our analysis to methylation in CGI close to TSS (−1 kb to +1 kb). Methylation 0-1% in controls was found in 4,743 genes. Of those, only 24 genes (0.5%) showed average methylation >20% in MDS/AML. Methylation 1-5% in controls was detected in 1,204 genes. Of those, 94 genes (8%) showed average methylation >20% in MDS/AML. Methylation 5-10% was observed in 158 genes in controls, while 55 (35%) of these had >20% methylation in MDS/AML (P=7.7E-172). We conclude that low levels of methylation in CGI present in DNA of healthy individuals mark a large subset of genes destined for hypermethylation in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3004. doi:10.1158/1538-7445.AM2011-3004