Morphogenesis and metabolomics reveal the compatible relationship among Suillus bovinus, Phialocephala fortinii, and their co-host, Pinus massoniana.

Ectomycorrhizal (ECM) fungi and dark septate endophytes (DSEs) can both form a symbiotic relationship with the same host plant. However, the interactions that occur among these two types of fungi and their co-hosts are largely unknown. Here, we investigated interactions that occur among the ECM fungus Suillus bovinus, the DSE Phialocephala fortinii, and their co-host Pinus massoniana. We used both scanning electron microscopy and optical microscopy to characterize the morphogenesis of the two symbionts and employed the ultra-high-performance liquid chromatography-tandem mass spectrometry technique to assess the effects of fungal inoculation on the root metabolome. Under pure culture conditions, no synergistic or antagonistic effects were observed between Phi. fortinii and S. bovinus. Generally, S. bovinus and Phi. fortinii can simultaneously colonize P. massoniana roots without affecting each other's symbiotic processes. S. bovinus can colonize the root locus where Phi. fortinii has already invaded but not vice versa, which may be due to the physical barrier effect of the mantle. Both fungi can significantly promote the growth of P. massoniana, and they have a synergistic effect on host N and K uptake. Metabolite accumulation patterns in roots inoculated with Phi. fortinii and/or S. bovinus were greatly altered, especially with respect to organic acids, flavonoids, lipids, and phenolic acids. S. bovinus inoculation significantly enhanced root flavonoid biosynthesis, whereas Phi. fortinii and dual-inoculation treatments mainly induced phenylpropanoid biosynthesis. These findings reveal compatible relationships among P. massoniana, S. bovinus, and Phi. fortinii, and suggest a theoretical basis for ECM fungi and DSE co-application when cultivating seedlings. IMPORTANCE The prevalence of both ectomycorrhizal fungi and dark septate endophytes in the roots of a wide spectrum of tree species is well recognized. In this study, we investigated the interactions that occur among the ECM fungus S. bovinus, the DSE Phi. fortinii, and their co-host, P. massoniana. The two fungi can simultaneously colonize P. massoniana roots without affecting each other's symbiotic processes. S. bovinus appears to be superior to Phi. fortinii in microniche competition, which may be due to the physical barrier effect of the mantle. The two fungi have different effects on root metabolite accumulation patterns. S. bovinus inoculation significantly enhanced root flavonoid biosynthesis, whereas Phi. fortinii and dual-inoculation treatments mainly induced phenylpropanoid biosynthesis. This is the first study revealing the morphological and metabolic mechanisms that contribute to the compatible relationship among ECM fungi, DSEs, and their co-host.

Morphological and Transcriptional Characteristics of the Symbiotic Interaction between Pinus massoniana and Suillus bovinus.

Ectomycorrhiza (ECM) function has been well studied; however, there is little detailed information regarding the establishment of ECM symbioses. We investigated the morphological and transcriptional changes that occur during the establishment of the Pinus massoniana-Suillus bovinus ECM. S. bovinus promoted the growth of P. massoniana via the release of volatile organic compounds and exudates during the pre-symbiotic stage. Exudate-induced effects showed host plant specificity. At seven days post-inoculation (dpi), the mycelium started to penetrate P. massoniana roots. At 28 dpi, the Hartig net and mantle formed. At the pre-symbiotic stage, most differentially expressed genes in P. massoniana roots were mapped to the biosynthesis of secondary metabolites, signal transduction, and carbohydrate metabolism. At the symbiotic stage, S. bovinus colonization induced the reprogramming of pathways involved in genetic information processing in P. massoniana, particularly at the Hartig net and mantle formation stage. Phenylpropanoid biosynthesis was present at all stages and was regulated via S. bovinus colonization. Enzyme inhibitor tests suggested that hydroxycinnamoyl-CoA shikimate/quinate transferase is involved in the development of the Hartig net. Our findings outline the mechanism involved in the P. massoniana-S. bovinus ECM. Further studies are needed to clarify the role of phenylpropanoid biosynthesis in ECM formation.

Stimulation of Hyphal Ramification and Sporulation in Funneliformis mosseae by Root Extracts Is Host Phosphorous Status-Dependent.

A simulation of the environment inhabited by arbuscular mycorrhizal (AM) fungi could provide clues as to how to cultivate these obligate biotrophs axenically. Host intraradical and rhizospheric environments, root extracts and exudates in particular, would be crucial for AM fungi to complete their life cycles. In this study, we analyzed and compared the effects of root exudates (RE) and root extracts (RET) of white clover (Trifolium repens) on the asymbiotic growth of the AM fungus Funneliformis mosseae in vitro, and furtherly analyzed the chemical components of different RET with the LC-MS/MS technique in order to establish an asymbiotic cultivation system for this important and hardly domesticated AM fungus. RET is superior to RE in stimulating spore germination, hyphal elongation and branching, and secondary spore formation (p < 0.05). RET-induced effects were dependent on phosphate supplement levels, and the RET obtained following the treatment with low levels of phosphorus significantly promoted hyphal growth and sporulation (p < 0.05). A few newly formed secondary spores showed limited colonization of white clover roots. The low phosphorus-induced effects could be ascribed to the metabolic adjustment (mainly lipids and organic acids) of white clover roots under low phosphate conditions. Our findings demonstrate that the low phosphate-induced RET boosts the asymbiotic growth of AM fungus, and thus offers an alternative way to fulfill the life cycle of AM fungi asymbiotically.

Fungal diversity within the phyllosphere of Pinus massoniana and the possible involvement of phyllospheric fungi in litter decomposition.

Fungi play key roles in forest ecosystems and help to shape the forest's diverse functions. However, little is known about the diversity of phyllospheric fungi or their possible relationships with fungal communities residing in different micro-environments of Pinus massoniana forests. We investigated seven different sample types: mature needles (NM), dead needles (ND), needles falling as litter (L), fermenting needles (F), humus (H), top soil (0-20 cm) (TS), and secondary soil (20-40 cm) (SS). These seven fungal communities were examined and compared with ITS amplicons using a high-throughput sequencing technique. A total of 1213 fungal operational taxonomic units (OTUs) were obtained at a 97% sequence similarity level. Distinct fungal communities were associated with different sample types. A greater number of OTUs were present in both NM and F samples than those shared by both NM and TS samples, indicating that phyllospheric fungi may play crucial roles in litter decomposition. Sixty OTUs (the core microbiome) were found in all sample types, and they may probably play different ecological roles in different sample types. These findings extend our knowledge of the fungal diversity of the phyllosphere and its possible interactions with fungal communities found in distinct forest micro-habitats.

Comparative gene expression profile analysis of ovules provides insights into Jatropha curcas L. ovule development.

Jatropha curcas, an economically important biofuel feedstock with oil-rich seeds, has attracted considerable attention among researchers in recent years. Nevertheless, valuable information on the yield component of this plant, particularly regarding ovule development, remains scarce. In this study, transcriptome profiles of anther and ovule development were established to investigate the ovule development mechanism of J. curcas. In total, 64,325 unigenes with annotation were obtained, and 1723 differentially expressed genes (DEGs) were identified between different stages. The DEG analysis showed the participation of five transcription factor families (bHLH, WRKY, MYB, NAC and ERF), five hormone signaling pathways (auxin, gibberellic acid (GA), cytokinin, brassinosteroids (BR) and jasmonic acid (JA)), five MADS-box genes (AGAMOUS-2, AGAMOUS-1, AGL1, AGL11, and AGL14), SUP and SLK3 in ovule development. The role of GA and JA in ovule development was evident with increases in flower buds during ovule development: GA was increased approximately twofold, and JA was increased approximately sevenfold. In addition, the expression pattern analysis using qRT-PCR revealed that CRABS CLAW and AGAMOUS-2 were also involved in ovule development. The upregulation of BR signaling genes during ovule development might have been regulated by other phytohormone signaling pathways through crosstalk. This study provides a valuable framework for investigating the regulatory networks of ovule development in J. curcas.

Mirror Bisulfite Sequencing: A Method for Single-Base Resolution of Hydroxymethylcytosine.

Although the role of 5-methylcytosine has been well studied, the biological role of 5-hydroxymethylcytosine still remains unclear because of the limited methods available for single-base detection of 5-hydroxymethylcytosine (5hmC). Here, we present mirror bisulfite sequencing for 5hmC detection at a single CpG site by synthesizing a DNA strand to mirror the parental strand. This semiconservative duplex is sequentially treated with ß-glucosyltransferase and M.SssI methylase. The glucosyl-5hmCpG in the parental strand inhibits methylation of the mirroring CpG site, and after bisulfite conversion, a thymine in the mirroring strand indicates a 5hmCpG site in the parental strand, whereas a cytosine indicates a non-5hmC site. Using this method, the 5hmC levels of various human tissues and paired liver tissues were mapped genomewide.

Neuronal Expression of Opioid Gene is Controlled by Dual Epigenetic and Transcriptional Mechanism in Human Brain.

Molecular mechanisms that define patterns of neuropeptide expression are essential for the formation and rewiring of neural circuits. The prodynorphin gene (PDYN) gives rise to dynorphin opioid peptides mediating depression and substance dependence. We here demonstrated that PDYN is expressed in neurons in human dorsolateral prefrontal cortex (dlPFC), and identified neuronal differentially methylated region in PDYN locus framed by CCCTC-binding factor binding sites. A short, nucleosome size human-specific promoter CpG island (CGI), a core of this region may serve as a regulatory module, which is hypomethylated in neurons, enriched in 5-hydroxymethylcytosine, and targeted by USF2, a methylation-sensitive E-box transcription factor (TF). USF2 activates PDYN transcription in model systems, and binds to nonmethylated CGI in dlPFC. USF2 and PDYN expression is correlated, and USF2 and PDYN proteins are co-localized in dlPFC. Segregation of activatory TF and repressive CGI methylation may ensure contrasting PDYN expression in neurons and glia in human brain.

Transcriptome-Wide Detection of 5-Methylcytosine by Bisulfite Sequencing.

While low-throughput RNA bisulfite sequencing is the method of choice to assess the methylation status of specific cytosines in candidate RNAs, the combination of bisulfite treatment of RNA with today's high-throughput sequencing techniques opens the door to methylation studies at nucleotide resolution on a transcriptome-wide scale. Below we describe a protocol for the transcriptome-wide analysis of total or fractionated poly(A)RNA in cells and tissues. Although the nature of the bisulfite sequencing protocol makes it comparably easy to translate from a low to a high-throughput approach, several critical points require attention before starting such a project. We describe a step-by-step protocol for planning and performing the experiment and analyzing the data.

Reduced Representation Bisulfite Sequencing Determination of Distinctive DNA Hypermethylated Genes in the Progression to Colon Cancer in African Americans.

Background and Aims. Many studies have focused on the determination of methylated targets in colorectal cancer. However, few analyzed the progressive methylation in the sequence from normal to adenoma and ultimately to malignant tumors. This is of utmost importance especially in populations such as African Americans who generally display aggressive tumors at diagnosis and for whom markers of early neoplasia are needed. We aimed to determine methylated targets in the path to colon cancer in African American patients using Reduced Representation Bisulfite Sequencing (RRBS). Methods. Genomic DNA was isolated from fresh frozen tissues of patients with different colon lesions: normal, a tubular adenoma, a tubulovillous adenoma, and five cancers. RRBS was performed on these DNA samples to identify hypermethylation. Alignment, mapping, and confirmed CpG methylation analyses were performed. Preferential hypermethylated pathways were determined using Ingenuity Pathway Analysis (IPA). Results. We identified hypermethylated CpG sites in the following genes: L3MBTL1, NKX6-2, PREX1, TRAF7, PRDM14, and NEFM with the number of CpG sites being 14, 17, 10, 16, 6, and 6, respectively, after pairwise analysis of normal versus adenoma, adenoma versus cancer, and normal versus cancer. IPA mapped the above-mentioned hypermethylated genes to the Wnt/ß-catenin, PI3k/AKT, VEGF, and JAK/STAT3 signaling pathways. Conclusion. This work provides insight into novel differential CpGs hypermethylation sites in colorectal carcinogenesis. Functional analysis of the novel gene targets is needed to confirm their roles in their associated carcinogenic pathways.

Characterizing and handling different kinds of AM fungal spores in the rhizosphere.

Spores are important propagules as well as the most reliable species-distinguishing traits of arbuscular mycorrhizal (AM) fungi. During surveys of AM fungal communities, spore enumeration and spore identification are frequently conducted, but generally little attention is given to the age and viability of the spores. In this study, AM fungal spores in the rhizosphere were characterized as live or dead by vital staining and by performing a germination assay. A considerable proportion of the spores in the rhizosphere were dead despite their intact appearance. Furthermore, morphological and molecular analyses of spores to determine species identity revealed that both viable spores and dead spores with contents were identified. The accurate identification of spores at different developmental stages on the basis of morphology requires considerable experience. Our findings suggest that surveys of AM fungal communities based on spore enumeration and morphological and molecular identification are likely to be inaccurate, primarily because of the large proportion of dead spores in the rhizosphere. A viability check is recommended prior to spore molecular identification, and the use of trap cultures would give more reliable morphological identification results. We show that the abundance and activity of AM fungi in the rhizosphere can be determined by calculating the density of viable spores and the density of spores that could germinate. The adoption of these methods should provide a more reliable basis for further AM fungal community analysis.

Practical guidelines and consideration of using RRHP for 5hmC detection.

5-hydroxymethylcytosine (5hmC) is an epigenetic modification, which has been associated with gene expression in many biological contexts. Reduced representation hydroxymethylation profiling was developed as an enzymatic-based method for genome-wide 5hmC detection. It exploits ß-glucosyltransferase to inhibit enzymatic cleavage of adapters ligated to a genomic library, allowing only fragments with glucosylated 5hmC residues at adapter junctions to be amplified and sequenced. The simple workflow and high sensitivity make it an efficient assay for 5hmC mapping. In this review, we discuss some technical consideration in applying reduced representation hydroxymethylation profiling, such as the use of alternative restriction enzymes for increased genomic coverage in different species, application of control libraries and specifications for multiplexing, data processing and normalization.

The phosphate transporters LjPT4 and MtPT4 mediate early root responses to phosphate status in non mycorrhizal roots.

Arbuscular mycorrhizal (AM) symbiosis improves host plant phosphorous (P) status and elicits the expression of AM-inducible phosphate transporters (PTs) in arbuscule-containing cells, where they control arbuscule morphogenesis and P release. We confirmed such functions for LjPT4 in mycorrhizal Lotus japonicus. Promoter-GUS experiments showed LjPT4 transcription not only in arbusculated cells but also in root tips, in the absence of the fungus: here LjPT4 transcription profile depended on the phosphate level. In addition, quantitative RT-PCR confirmed the expression of Lotus and Medicago truncatula PT4 in the tips of non-mycorrhizal roots. Starting from these observations, we hypothesized that AM-inducible PTs may have a regulatory role in plant development, irrespective of the fungal presence. Firstly, we focused on root development responses to different phosphate treatments in both plants demonstrating that phosphate starvation induced a higher number of lateral roots. By contrast, Lotus PT4i plants and Medicago mtpt4 mutants did not show any differential response to phosphate levels, suggesting that PT4 genes affect early root branching. Phosphate starvation-induced genes and a key auxin receptor, MtTIR1, showed an impaired expression in mtpt4 plants. We suggest PT4 genes as novel components of the P-sensing machinery at the root tip level, independently of AM fungi.

H19 lncRNA alters DNA methylation genome wide by regulating S-adenosylhomocysteine hydrolase.

DNA methylation is essential for mammalian development and physiology. Here we report that the developmentally regulated H19 lncRNA binds to and inhibits S-adenosylhomocysteine hydrolase (SAHH), the only mammalian enzyme capable of hydrolysing S-adenosylhomocysteine (SAH). SAH is a potent feedback inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferases that methylate diverse cellular components, including DNA, RNA, proteins, lipids and neurotransmitters. We show that H19 knockdown activates SAHH, leading to increased DNMT3B-mediated methylation of an lncRNA-encoding gene Nctc1 within the Igf2-H19-Nctc1 locus. Genome-wide methylation profiling reveals methylation changes at numerous gene loci consistent with SAHH modulation by H19. Our results uncover an unanticipated regulatory circuit involving broad epigenetic alterations by a single abundantly expressed lncRNA that may underlie gene methylation dynamics of development and diseases and suggest that this mode of regulation may extend to other cellular components.

Effect of volatiles versus exudates released by germinating spores of Gigaspora margarita on lateral root formation.

Arbuscular mycorrhizal (AM) fungi influence the root system architecture of their hosts; however, the underlying mechanisms have not been fully elucidated. Ectomycorrhizal fungi influence root architecture via volatiles. To determine whether volatiles also play a role in root system changes in response to AM fungi, spores of the AM fungus Gigaspora margarita were inoculated on the same plate as either wild type (WT) Lotus japonicus, the L. japonicus mutant Ljcastor (which lacks the symbiotic cation channel CASTOR, which is required for inducing nuclear calcium spiking, which is necessary for symbiotic partner recognition), or Arabidopsis thaliana, separated by cellophane membranes (fungal exudates experiment), or on different media but with a shared head space (fungal volatiles experiment). Root development was monitored over time. Both germinating spore exudates (GSEs) and geminated-spore-emitted volatile organic compounds (GVCs) significantly promoted lateral root formation (LRF) in WT L. japonicus. LRF in Ljcastor was significantly enhanced in the presence of GVCs. GVCs stimulated LRF in A. thaliana, whereas GSEs showed an inhibitory effect. The expression profile of the genes involved in mycorrhizal establishment and root development were investigated using quantitative reverse transcription-PCR analysis. Only the expression of the LjCCD7 gene, an important component of the strigolactone synthesis pathway, was differentially expressed following exposure to GVCs. We conclude that volatile organic compounds released by the germinating AM fungal spores may stimulate LRF in a symbiosis signaling pathway (SYM)- and host-independent way, whereas GSEs stimulate LRF in a SYM- and host-dependent way.

RRHP: a tag-based approach for 5-hydroxymethylcytosine mapping at single-site resolution.

Current methods for genomic mapping of 5-hydroxymethylcytosine (5hmC) have been limited by either costly sequencing depth, high DNA input, or lack of single-base resolution. We present an approach called Reduced Representation 5-Hydroxymethylcytosine Profiling (RRHP) to map 5hmC sites at single-base resolution by exploiting the use of beta-glucosyltransferase to inhibit enzymatic digestion at the junction where adapters are ligated to a genomic library. Therefore, only library fragments presenting glucosylated 5hmC residues at the junction are sequenced. RRHP can detect sites with low 5hmC abundance, and when combined with RRBS data, 5-methylcytosine and 5-hydroxymethylcytosine can be compared at a specific site.

JBP1-seq: a fast and efficient method for genome-wide profiling of 5hmC.

We developed a novel approach, J-binding protein 1 sequencing (JBP1-seq), that combines the benefits of an improved recombinant JBP1 protein, Nextera-based library construction, and next-generation sequencing (NGS) for genome-wide profiling of 5-hydroxymethylcytosine (5hmC). Compared with the original JBP1, this new recombinant JBP1 was biotinylated in vivo and conjugated to magnetic beads via biotin-streptavidin interactions. These modifications allowed a more efficient and consistent pull-down of ß-glucosyl-5-hydroxymethylcytosine (ß-glu-5hmC), and sequence-ready libraries can be generated within 4.5h from DNA inputs as low as 50ng. 5hmC enrichment of human brain DNA using the new JBP1 resulted in over 25,000 peaks called, which is significantly higher than the 4003 peaks enriched using the old JBP1. Comparison of the technical duplicates and validations with other platforms indicated the results are reproducible and reliable. Thus, JBP1-seq provides a fast, efficient, and cost-effective method for accurate 5hmC genome-wide profiling.

MRTF-A and STAT3 synergistically promote breast cancer cell migration.

Breast cancer is the leading cause of cancer death in women worldwide which is closely related to metastasis. But the exact molecular mechanism on metastasis is still not fully understood; we now report that both MRTF-A and STAT3 play important role in breast cancer migration of MDA-MB-231 cells. Moreover, MRTF-A and STAT3 synergistically increased MDA-MB-231 cell migration by promoting the expression of migration markers Myl-9 and Cyr-61. Importantly, we identified a detailed molecular mechanism of MDA-MB-231 cell migration controlled via physical interaction between MRTF-A and STAT3, which synergistically promote the transactivity of the migration marker Myl-9 and Cyr-61 by CArG box binding. Interestingly, the two signaling pathways RhoA-MRTF-A and JAK-STAT3 across talk to regulate MDA-MB-231 cell migration. Our data thus provide important and novel insights into the roles of MRTF-A and STAT3 in regulating MDA-MB-231 cell migration.

Human chorionic gonadotropin decreases human breast cancer cell proliferation and promotes differentiation.

Human chorionic gonadotropin (hCG) is a glycoprotein produced by placental trophoblasts. Previous studies indicated that hCG could be responsible for the pregnancy-induced protection against breast cancer in women. It is reported that hCG decreases proliferation and invasion of breast cancer MCF-7 cells. Our research also demonstrates that hCG can reduce the proliferation of MCF-7 cells by downregulating the expression of proliferation markers, proliferating cell nuclear antigen (PCNA), and proliferation-related Ki-67 antigen (Ki-67). Interestingly, we find here that hCG elevates the state of cellular differentiation, as characterized by the upregulation of differentiation markers, ß-casein, cytokeratin-18 (CK-18), and E-cadherin. Inhibition of hCG secretion or luteinizing hormone/hCG receptors (LH/hCGRs) synthesis can weaken the effect of hCG on the induction of cell differentiation. Furthermore, hCG can suppress the expression of estrogen receptor alpha. hCG activated receptor-mediated cyclic adenosine monophosphate/protein kinase A signaling pathway. These findings indicated that a protective effect of hCG against breast cancer may be associated with its growth inhibitory and differentiation induction function in breast cancer cells.

Re-expression and epigenetic modification of maspin induced apoptosis in MCF-7 cells mediated by myocardin.

Breast cancer is the leading cause of cancer death in women worldwide. It is well known that oncogene activation and anti-oncogene inactivation affect the development and progression of breast cancer, but the role of oncogene activation and anti-oncogene inactivation in breast cancer is still not fully understood. We now report that maspin acts as a tumor suppressor gene to induce MCF-7 cell apoptosis. In addition, maspin promoter hypermethylation and histone hypoacetylation lead to silencing of maspin gene expression in MCF-7 cells. Moreover, DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-aza-dc) and/or the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) strongly up-regulated the expression of maspin in MCF-7 cells. Notably, myocardin can promote the re-expression of maspin in MCF-7 cells. Luciferase assay shows that myocardin activates the transcription of maspin promoter by CArG box. More importantly, 5-aza-dc/TSA and myocardin synergetically enhance re-expression of maspin and augment maspin-mediated apoptosis in MCF-7 cells. Thus, these data reveal the new insight that myocardin meditates apoptosis in breast cancer through affecting maspin re-expression and epigenetic modification to regulate the development of breast cancer, thereby raising the possibility of its use in breast cancer therapy.

DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia.

The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject's colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands-in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)-were significantly hypermethylated in tumor vs. normal tissues (P<0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-ß1 network-the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated genes, as well as hypomethylated LINE-1 sequences, which may serve as potential biomarkers for CRC in African Americans. Our discovered biomarkers were intimately linked to the insulin/TGF-B1 pathway, further strengthening the association of diabetic disorders with colon oncogenic transformation.