Aim: Long noncoding RNAs serve critical regulatory functions highly specific for a tissue and its developmental stage. Antisense long ncRNA (AS-lncRNA) methylation changes in acute lymphoblastic leukemia (ALL) versus normal pre-B-cell lymphoblasts were evaluated to identify potential differential methylation in this group of genes. Materials & methods: The methylome of ALL and normal lymphoblasts was examined by the methylated CpG island recovery assay followed by NGS. Conclusion: The potential effect of trans regulation by AS-lncRNA through DNA/RNA binding is significant as sequence alignment analysis of the 25 most differentially methylated AS-lncRNAs revealed 368 genes containing highly similar sequences with a median nucleotide identity of 90.8% and binding span of 122 base pairs. Regulation of biological processes and anatomical structure development were over represented. ALL classification schemes based on AS-lncRNA methylation can provide new insights into its pathogenesis and treatment.

A complete understanding of the mechanisms involved in the development of pre-B ALL is lacking. In this study, we integrated DNA methylation data and gene expression data to elucidate the impact of aberrant intergenic DNA methylation on gene expression in pre-B ALL. We found a subset of differentially methylated intergenic loci that were associated with altered gene expression in pre-B ALL patients. Notably, 84% of these regions were also bound by transcription factors (TF) known to play roles in differentiation and B-cell development in a lymphoblastoid cell line. Further, an overall downregulation of eRNA transcripts was observed in pre-B ALL patients and these transcripts were associated with the downregulation of putative target genes involved in B-cell migration, proliferation, and apoptosis. The identification of novel putative regulatory regions highlights the significance of intergenic DNA sequences and may contribute to the identification of new therapeutic targets for the treatment of pre-B ALL.

Acute lymphoblastic leukemia (ALL) is the most common cancer diagnosed in children under the age of 15. In addition to genetic aberrations, epigenetic modifications such as DNA methylation are altered in cancer and impact gene expression. To identify epigenetic alterations in ALL, genome-wide methylation profiles were generated using the methylated CpG island recovery assay followed by next-generation sequencing. More than 25,000 differentially methylated regions (DMR) were observed in ALL patients with ∼ 90% present within intronic or intergenic regions. To determine the regulatory potential of the DMR, whole-transcriptome analysis was performed and integrated with methylation data. Aberrant promoter methylation was associated with the altered expression of genes involved in transcriptional regulation, apoptosis, and proliferation. Novel enhancer-like sequences were identified within intronic and intergenic DMR. Aberrant methylation in these regions was associated with the altered expression of neighboring genes involved in cell cycle processes, lymphocyte activation and apoptosis. These genes include potential epi-driver genes, such as SYNE1, PTPRS, PAWR, HDAC9, RGCC, MCOLN2, LYN, TRAF3, FLT1, and MELK, which may provide a selective advantage to leukemic cells. In addition, the differential expression of epigenetic modifier genes, pseudogenes, and non-coding RNAs was also observed accentuating the role of erroneous epigenetic gene regulation in ALL.

DNA methylation is responsible for regulating gene expression and cellular differentiation and for maintaining genomic stability during normal human development. Furthermore, it plays a significant role in the regulation of hematopoiesis. In order to elucidate the influence of DNA methylation during B-cell development, genome-wide DNA methylation status of pro-B, pre-BI, pre-BII, and naïve-B-cells isolated from human umbilical cord blood was determined using the methylated CpG island recovery assay followed by next generation sequencing. On average, 182 – 200 million sequences were generated for each precursor B-cell subset in 10 biological replicates. An overall decrease in methylation was observed during the transition from pro-B to pre-BI, whereas no differential methylation was observed in the pre-BI to pre-BII transition or in the pre-BII to naïve B-cell transition. Most of the methylated regions were located within intergenic and intronic regions not present in a CpG island context. Putative novel enhancers were identified in these regions that were differentially methylated between pro-B and pre-BI cells. The genome-wide methylation profiles are publically available and may be used to gain a better understanding of the involvement of atypical DNA methylation in the pathogenesis of malignancies associated with precursor B-cells.

Umbilical cord blood is highly enriched for hematopoietic progenitor cells at different lineage commitment stages. We have developed a protocol for isolating precursor B-cells at four different stages of differentiation. Because genes are expressed and epigenetic modifications occur in a tissue specific manner, it is vital to discriminate between tissues and cell types in order to be able to identify alterations in the genome and the epigenome that may lead to the development of disease. This method can be adapted to any type of cell present in umbilical cord blood at any stage of differentiation. This method comprises 4 main steps. First, mononuclear cells are separated by density centrifugation. Second, B-cells are enriched using biotin conjugated antibodies that recognize and remove non B-cells from the mononuclear cells. Third the B-cells are fluorescently labeled with cell surface protein antibodies specific to individual stages of B-cell development. Finally, the fluorescently labeled cells are sorted and individual populations are recovered. The recovered cells are of sufficient quantity and quality to be utilized in downstream nucleic acid assays.

Abstract Tissue specific DNA methylation is accountable for regulating gene expression and cellular differentiation during normal human development. Aberrant tissue specific gene regulation may lead to the development of many disease states including cancer. Genome-wide analysis of DNA methylation (methylome) in both healthy and diseased tissue is essential to understand the functional consequence of altered DNA methylation. Acute lymphoblastic leukemia (ALL) is a malignancy associated with precursor B-cells. In order to gain a better understanding of the role of altered DNA methylation in the pathogenesis of ALL, it is essential that we characterize the DNA methylation present in healthy precursor B-cells. Umbilical cord blood is enriched for precursor B-cells and is used as a source of hematopoietic stem cells in the treatment of blood related disorders and malignancies. To study the methylome in healthy precursor B-cells, we have optimized a protocol to isolate 4 subsets of precursor B-cells from umbilical cord blood, and to then generate MBD-seq libraries from the small amount of genomic DNA (< 100ng) isolated from those subsets of precursor B-cells. Subsets of precursor B-cells were isolated from cord blood based on the level of expression of cell surface antigen. Initially mononuclear cells were isolated by Ficoll-plaque followed by magnetic labeling and depletion of non B-cells. B-cells were labeled with antibodies against CD19, CD34 and CD45 surface antigens and sorted by flow cytometry. Based on expression level of surface antigen, four different subsets of precursor B-cells were sorted (CD19+/CD34+; CD19+/CD34-/CD45low; CD19+/CD34-/CD45med; and CD19+/CD34-/CD45high). Immediately after cell sorting, DNA was isolated by using a commercially available kit. The entire amount of DNA from CD19+/CD34+ and CD19+/CD34-/CD45low (<100ng) subsets, and 100ng of DNA from CD19+/CD34-/CD45med and CD19+/CD34-/CD45high subsets were fragmented by sonication using Diagenode Bioruptor. Sequencing libraries were generated using the Illumina Chip-seq protocol with modifications. Enrichment of methylated DNA was validated by PCR amplification of methylated (SLC25A37) and unmethylated (APC) regions. MBD-seq libraries were sequenced using Illumina next-generation sequencing technology and then sequenced data were analyzed by NextGENe® software. We found ∼45 million uniquely matched reads covering ∼700 million bases in the human genome for each precursor B-cell subset. Methylated peaks were identified using the Peak Identification tool in NextGENe. The results demonstrated a differential pattern of DNA methylation in the subsets of healthy precursor B-cells. Citation Format: Md Almamun, Kristen H. Taylor. Genome-wide DNA methylation analysis in subsets of precursor B-cells isolated from umbilical cord blood. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5365. doi:10.1158/1538-7445.AM2013-5365

Researchers whose experimental models are mammalian oocytes and preimplantation embryos are often limited by the yield of nucleic acids that can be isolated from such a small sample size. In addition, the limited number of cells from these types of samples makes the simultaneous recovery of RNA and DNA very difficult and often sample pooling is necessary to increase nucleic acid yield. Here we report a simple set of procedures using commercially available kits that results in consistent yield and quality of nucleic acids. After sample lysis, RNA is isolated and converted to a reusable cDNA library. Following RNA isolation, DNA is precipitated, isolated, and bisulfite converted for DNA methylation studies. Our results demonstrate the feasibility of isolating RNA and DNA from a small number of cells with repeatability of results.

The normal progression of acquisition of global DNA methylation in oocytes of adult female mice is not known. Assisted reproductive technologies (ART) are commonly used for the treatment of infertility problems. Several studies have shown that ART procedures alter the expression of genes, causing delayed embryonic development, increased abnormal blastocyst formation, pronounced fetal growth retardation and is associated with increased incidence of loss-of-imprinting syndromes. A previous study from our laboratory showed that the maternal pronuclei of one-cell mouse embryos formed after superovulation (SO) had 50% less methylation when compared to those formed after natural ovulation (NO). We hypothesize that oocytes in ovaries of females undergoing a SO scheme will have different levels of global DNA methylation than oocytes from naturally cycling animals. First, we determined the progression of global DNA methylation during oocyte growth in naturally cycling animals and then compared this to the levels of DNA methylation in oocytes from females undergoing a SO scheme. A group of 14 cyclic females were selected at diestrus and divided into seven groups. Two females were sacrificed and collected ovaries at diestrus (0h). Six females underwent a SO scheme while the remaining six did not. In brief, SO females were injected with 5 IU of eCG followed 44 h later by 5 IU of hCG. Ovaries were collected at 24- and 44 h post-eCG and 10 h post-hCG (data not discussed). Ovaries from their NO counterparts were collected at the same time as the SO females. Ovarian sections were stained with an antibody that recognizes methylated cytosines (5MeC). All oocytes in a section were photographed and the largest cross section was used for analysis. Measurements related to oocyte growth were; 1) oocyte size in micrometers, 2) area of germinal vesicle, 3) size and type of enclosing follicle. Global DNA methylation was determined using the background corrected density macro of ImageJ. The 5MeC staining intensity level of the GV was normalized to the staining of the surrounding granulosa cells. For analyses, correlations were made between oocyte size and level of methylation and GV size and level of methylation. These numbers were then compared between similar size oocytes from SO and NO females. Results show that at diestrus the level of methylation increases in a linear manner form 10 to 80 µm diameter. The curve becomes exponential at 24 and 44 h in the NO group but is sigmoid in both SO groups. Analysis of full-grown oocytes (>70 µm) show that global methylation increases from diestrus (0 h) to 24 h and again to 44 h in the NO group but not in the SO groups. Interestingly, the levels of methylation remain low until ~ 40 µm diameter and start increasing exponentially in oocytes > 41 µm. This is coincident with the initiation of the formation of the second layer of granulosa cells. At 44 h, levels of DNA methylation were different in full-grown oocytes of NO vs. SO groups (P < 0.02). These full-grown oocytes are expected to ovulate upon the LH surge. Our results show that acquisition of DNA methylation continues in full grown oocytes until the presumed LH surge and we speculated that this is necessary to attain full developmental competence. On the other hand, SO full-grown oocytes do not attained full methylation before ovulation and this may in part be responsible for the lower developmental competence observed in embryos produced from SO oocytes. (poster)

The effect of the extent of vascular perfusion of the wall of the preovulatory follicle on in vitro cleavage rate of the recovered oocyte and embryo development to > 8 cells was studied in 52 heifers. Heifers received a luteolytic dose of prostaglandin F2 alpha (PGF2 alpha) when the largest follicle was >= 11 mm. An ovulation-inducing injection of GnRH was given 36 h later (hour 0), and collection of follicular fluid and the oocyte was done at hour 26. Vascular perfusion of the follicular wall was assessed by colour Doppler ultrasonography at hours 0 and 26. Each of the recovered oocytes (41/52; 79%) was mature (extruded polar body). Cleavage and embryo development were assessed at 48 h and 120 h respectively, after in vitro fertilisation (IVF). The percentage of cleaved oocytes and > 8 cell embryos was 80% (31/39) and 55% (17/31) respectively. Vascular perfusion of the follicular wall was greater (lower pulsatility index; P < 0.001) for follicles that produced cleaved versus non-cleaved oocytes and greater (P < 0.04) for follicles that produced > 8 cell versus <= 8 cell embryos. Percentage of follicular wall with Doppler signals of blood flow was greater (P < 0.001) for > 8 cell versus <= 8 cell embryos. Follicular-fluid concentration of free IGF1 was lower for cleaved oocytes (P < 0.001) and > 8 cell embryos (P < 0.05), and oestradiol was lower (P < 0.05) for > 8 cell embryos. Results supported the hypothesis that greater vascular perfusion of the wall of the preovulatory follicle was positively associated with IVF and embryo development.

Color-Doppler ultrasonography was used to compare the vascularity of the preovulatory follicle between heifers that became pregnant compared with nonpregnant. Heifers (n=34) received a luteolytic dose of PGF2alpha when the diameter of the largest follicle of the second follicular wave reached > or =11 mm (actual diameter 11.63+/-0.06 mm). An ovulation-inducing injection of GnRH analogue was given 36 h after the PGF2alpha treatment. Artificial insemination (AI) was performed 26 h after GnRH treatment, and ovulation occurred in all heifers within 3h after AI. Follicle blood flow was assessed at Hour 0 (GnRH treatment) and at Hour 26 (AI). Follicle diameter at Hours 0 and 26 was greater (main effect of group; P<0.03) in the group that became pregnant (n=25) compared with nonpregnant (n=9). Percentage of follicle wall with power Doppler signals of blood flow showed an interaction of group-by-hour (P<0.03), reflecting greater blood flow in the pregnant group than in the nonpregnant group at Hour 26 but not at Hour 0. Spectral-Doppler evaluation of the resistance index for a vessel in the follicle wall resulted in a group-by-hour interaction (P<0.03) from a lower index in the pregnant group at Hour 26. The lower index indicated greater downstream vascular perfusion in the follicle wall. Results supported the hypothesis of a positive relationship between the extent of blood flow of the preovulatory follicle and successful establishment of pregnancy in cattle.

The mechanism for a reported temporal association between ovulation and a transient disruption in the periovulatory increase in LH concentrations was studied in nine mares treated with human chorionic gonadotropin when the preovulatory follicle was ≥32mm. Examinations for ovulation detection and blood collection were done at 2-h intervals and the results were retrospectively centralized to ovulation (Hour 0). Concentrations of LH began to increase (P<0.03) rapidly at Hour −18, decreased (P<0.04) between Hours 0 and 6, and again increased (P<0.0001) after Hour 12. A progressive decrease (P<0.0001) in estradiol between Hours −30 and 24 was interrupted temporarily by an increase (P<0.01) between Hours −2 and 0 and a decrease (P<0.007) between Hours 0 and 2. Results indicated that a disruption and depression in the periovulatory LH surge occurred at the time of detected ovulation in mares and was temporally associated with a transient increase in estradiol during an overall progressive decline. The disruption in LH concentrations is attributable to the estradiol increase, based on a reported negative effect of estradiol on LH. The source of the circulating estradiol was likely from the discharge of estradiol-laden follicular fluid into the abdomen during ovulation and rapid absorption of estradiol into the circulation.