A Translational Approach to Cancer Research, Education and Training.

The demand for biomedical researchers and health science professionals has increased over the past several decades. This need is particularly acute in the fields of cancer research and oncology in which technological advances have fueled an unprecedented pace of laboratory discoveries and their applications in novel diagnostic and therapeutic strategies. Internships that expose undergraduate students to cancer research and patient care serve an important function in meeting this need by educating trainees about careers in this field and inspiring them to pursue these professional paths. Moreover, the translational impetus of cancer research incorporates research, regulatory, business, and clinical components, providing students with even more cancer-focused career options. With the goal of providing hands-on experiences in cancer research and oncology to undergraduate students who comprise the next generation of cancer physician-scientists and will fill this demand in our professional workforce, the Nathan Schnaper Intern Program in Translational Cancer Research (NSIP) has grown from a small laboratory-based local summer internship to a competitive national program. In this study, we evaluate three new modules of the NSIP research, education, and clinical components that have been implemented in the first 2 years of National Cancer Institute Cancer Research Education Grants Program funding. The impact of these modules on intern satisfaction, learning, and near-term career trajectory is assessed to identify the most effective approaches and key measures of program outcomes.

Insect Artifacts Are More than Just Altered Bloodstains.

The bases for forensic entomology are that insects and their arthropod relatives can serve as evidence in criminal, medical and civil legal matters. However, some of the very same species that provide utility to legal investigations can also complicate crime scenes by distorting existing body fluid evidence (e.g., bloodstains, semen, saliva) and/or depositing artifacts derived from the insect alimentary canal at primary or secondary crime scenes. The insect contaminants are referred to as insect stains, artifacts, specks or spots, and are most commonly associated with human bloodstains. This review will discuss the different types of insect artifacts that have been described from crime scenes and laboratory experiments, as well as examine insect contaminates (non-blood based artifacts, transfer patterns, meconium, and larval fluids) that have received little research or case attention. Methods currently used for distinguishing insect stains from human body fluids will also be discussed and compared to presumptive tests used for identification of human body fluids. Since all available methods have severe limitations, areas of new research will be identified for the purpose of development of diagnostic techniques for detection of insect artifacts.

Treatment of breast cancer cells with DNA demethylating agents leads to a release of Pol II stalling at genes with DNA-hypermethylated regions upstream of TSS.

Inactivation of tumor suppressor genes plays an important role in tumorigenesis, and epigenetic modifications such as DNA methylation are frequently associated with transcriptional repression. Here, we show that gene silencing at selected genes with signs of DNA hypermethylation in breast cancer cells involves Pol II stalling. We studied several repressed genes with DNA hypermethylation within a region 1-kb upstream of the transcriptional start site that were upregulated after treatment with DNA demethylating agents, such as Azacytidine and several natural products. All those selected genes had stalled Pol II at their transcriptional start site and showed enhanced ser2 phosphorylated Pol II and elevated transcripts after drug treatment indicating successful elongation. In addition, a decrease of the epigenetic regulator LSH in a breast cancer cell line by siRNA treatment reduced DNA methylation and overcame Pol II stalling, whereas overexpression of LSH in a normal breast epithelial cell line increased DNA methylation and resulted in repression. Decrease of LSH was associated with reduced DNMT3b binding to promoter sequences, and depletion of DNMT3b by siRNA could release Pol II suggesting that DNMT3b is functionally involved. The release of paused Pol II was accompanied by a dynamic switch from repressive to active chromatin marks. Thus release of Pol II stalling can act as a mechanism for gene reactivation at specific target genes after DNA demethylating treatment in cancer cells.

Lsh, chromatin remodeling family member, modulates genome-wide cytosine methylation patterns at nonrepeat sequences.

DNA methylation is critical for normal development and plays important roles in genome organization and transcriptional regulation. Although DNA methyltransferases have been identified, the factors that establish and contribute to genome-wide methylation patterns remain elusive. Here, we report a high-resolution cytosine methylation map of the murine genome modulated by Lsh, a chromatin remodeling family member that has previously been shown to regulate CpG methylation at repetitive sequences. We provide evidence that Lsh also controls genome-wide cytosine methylation at nonrepeat sequences and relate those changes to alterations in H4K4me3 modification and gene expression. Deletion of Lsh alters the allocation of cytosine methylation in chromosomal regions of 50 kb to 2 Mb and, in addition, leads to changes in the methylation profile at the 5' end of genes. Furthermore, we demonstrate that loss of Lsh promotes--as well as prevents--cytosine methylation. Our data indicate that Lsh is an epigenetic modulator that is critical for normal distribution of cytosine methylation throughout the murine genome.

Lymphoid-specific helicase (HELLS) is essential for meiotic progression in mouse spermatocytes.

Lymphoid-specific helicase (HELLS; also known as LSH) is a member of the SNF2 family of chromatin remodeling proteins. Because Hells-null mice die at birth, a phenotype in male meiosis cannot be studied in these animals. Allografting of testis tissue from Hells(-/-) to wild-type mice was employed to study postnatal germ cell differentiation. Testes harvested at Day 18.5 of gestation from Hells(-/-), Hells(+/-), and Hells(+/+) mice were grafted ectopically to immunodeficient mice. Bromodeoxyuridine incorporation at 1 wk postgrafting revealed fewer dividing germ cells in grafts from Hells(-/-) than from Hells(+/+) mice. Whereas spermatogenesis proceeded through meiosis with round spermatids in grafts from Hells heterozygote and wild-type donor testes, spermatogenesis arrested at stage IV, and midpachytene spermatocytes were the most advanced germ cell type in grafts from Hells(-/-) mice at 4, 6, and 8 wk after grafting. Analysis of meiotic configurations at 22 days posttransplantation revealed an increase in Hells(-/-) spermatocytes with abnormal chromosome synapsis. These results indicate that in the absence of HELLS, proliferation of spermatogonia is reduced and germ cell differentiation arrested at the midpachytene stage, implicating an essential role for HELLS during male meiosis. This study highlights the utility of testis tissue grafting to study spermatogenesis in animal models that cannot reach sexual maturity.

DNA methylation in early development.

Development from separate parental germ cells through fertilization and proceeding to a fully functioning adult animal occurs through an intricate program of transcriptional and chromatin changes. Epigenetic alterations such as DNA methylation are an important part of this process. This review looks at the role of DNA methylation in early embryonic development, as well as how this epigenetic mark affects stem cell differentiation and tissue-specific gene expression in somatic cells.

Lsh participates in DNA methylation and silencing of stem cell genes.

Transcriptional control of stem cell genes is a critical step in differentiation of embryonic stem cells and in reprogramming of somatic cells into stem cells. Here we report that Lsh, a regulator of repressive chromatin at retrotransposons, also plays an important role in silencing of stem cell-specific genes such as Oct4. We found that CpG methylation is gained during in vitro differentiation of several stem cell-specific genes (in 11 of 12 promoter regions) and thus appears to be a common epigenetic mark. Lsh depletion prevents complete silencing of stem cell gene expression and moreover promotes the maintenance of stem cell characteristics in culture. Lsh is required for establishment of DNA methylation patterns at stem cell genes during differentiation, in part by regulating access of Dnmt3b to its genomic targets. Our results indicate that Lsh is involved in the control of stem cell genes and suggest that Lsh is an important epigenetic modulator during early stem cell differentiation.

The see-saw of differentiation: tipping the chromatin balance.

Histone modifications, specifically, methylation of lysine residues in histone 3 (H3), determine chromatin structure and accessibility for transcription. A recent study by Bernstein and Lander identified a unique pattern of H3 methylation at the promoter region of developmental genes in pluripotent stem cells. The pattern consists of the simultaneous presence of a silencing mark [methylation of Lys(27) on H3 (H3K27me)] and an activation mark (H3K4me). This bivalent mark seems associated with a poised state of transcription and resolves upon differentiation largely into either H3K27me or H3K4me. Skin fibroblasts that can be reverted in culture to the pluripotent stage show re-establishment of the bivalent chromatin pattern. This switch in chromatin states may be key to our understanding of developmental and tissue specific regulation of gene expression and may be therapeutically useful in regenerative medicine.

Lsh controls Hox gene silencing during development.

Polycomb-mediated repression and DNA methylation are important epigenetic mechanisms of gene silencing. Recent evidence suggests a functional link between the polycomb repressive complex (PRC) and Dnmts in cancer cells. Here we provide evidence that Lsh, a regulator of DNA methylation, is also involved in normal control of PRC-mediated silencing during embryogenesis. We demonstrate that Lsh, a SNF2 homolog, can associate with some Hox genes and regulates Dnmt3b binding, DNA methylation, and silencing of Hox genes during development. Moreover, Lsh can associate with PRC1 components and influence PRC-mediated histone modifications. Thus Lsh is part of a physiological feedback loop that reinforces DNA methylation and silencing of PRC targets.

Lsh is involved in de novo methylation of DNA.

Deletion of Lsh perturbs DNA methylation patterns in mice yet it is unknown whether Lsh plays a direct role in the methylation process. Two types of methylation pathways have been distinguished: maintenance methylation by Dnmt1 occurring at the replication fork, and de novo methylation established by the methyltransferases Dnmt3a and Dnmt3b. Using an episomal vector in Lsh-/- embryonic fibroblasts, we demonstrate that the acquisition of DNA methylation depends on the presence of Lsh. In contrast, maintenance of previously methylated episomes does not require Lsh, implying a functional role for Lsh in the establishment of novel methylation patterns. Lsh affects Dnmt3a as well as Dnmt3b directed methylation suggesting that Lsh can cooperate with both enzymatic activities. Furthermore, we demonstrate that embryonic stem cells with reduced Lsh protein levels show a decreased ability to silence retroviral vector or to methylate endogenous genes. Finally, we demonstrate that Lsh associates with Dnmt3a or Dnmt3b but not with Dnmt1 in embryonic cells. These results suggest that the epigenetic regulator, Lsh, is directly involved in the control of de novo methylation of DNA.

Effects of chromatin structure on the enzymatic and DNA binding functions of DNA methyltransferases DNMT1 and Dnmt3a in vitro.

DNA methylation is an epigenetic modification of the genome critical for numerous processes, including transcriptional repression and maintenance of chromatin structure. Recent studies have revealed connections between DNA methylation and other epigenetic modifications such as ATP-dependent chromatin remodeling. It remains unclear, however, exactly how chromatin and epigenetic chromatin modifications affect the biological properties of the DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B). Using a highly purified system and the 5S rDNA gene as free DNA or assembled into a mononucleosome, we have compared the effects of chromatin structure on DNMT1 and Dnmt3a. The catalytic efficiency for both enzymes decreased on the mononucleosome, approximately 8-fold for DNMT1 and 17-fold for Dnmt3a. DNMT1 and Dnmt3a bound to DNA and mononucleosomal substrates in gel shift experiments with approximately equal affinity and in a cooperative manner. We also show that DNMT1 interacts with hSNF2H chromatin remodeling enzyme and that DNMT1 binds mononucleosomes with higher affinity in the presence of hSNF2H. These findings raise interesting implications about the interactions of mammalian DNA methyltransferases with chromatin and provide the first evidence that a chromatin remodeling enzyme can alter the biological properties of a DNMT.

Isolation and characterization of a novel DNA methyltransferase complex linking DNMT3B with components of the mitotic chromosome condensation machinery.

Proper patterns of genome-wide DNA methylation, mediated by DNA methyltransferases DNMT1, -3A and -3B, are essential for embryonic development and genomic stability in mammalian cells. The de novo DNA methyltransferase DNMT3B is of particular interest because it is frequently overexpressed in tumor cells and is mutated in immunodeficiency, centromere instability and facial anomalies (ICF) syndrome. In order to gain a better understanding of DNMT3B, in terms of the targeting of its methylation activity and its role in genome stability, we biochemically purified endogenous DNMT3B from HeLa cells. DNMT3B co-purifies and interacts, both in vivo and in vitro, with several components of the condensin complex (hCAP-C, hCAP-E and hCAP-G) and KIF4A. Condensin mediates genome-wide chromosome condensation at the onset of mitosis and is critical for proper segregation of sister chromatids. KIF4A is proposed to be a motor protein carrying DNA as cargo. DNMT3B also interacts with histone deacetylase 1 (HDAC1), the co-repressor SIN3A and the ATP-dependent chromatin remodeling enzyme hSNF2H. Further more, DNMT3B co-localizes with condensin and KIF4A on condensed chromosomes throughout mitosis. These studies therefore reveal the first direct link between the machineries regulating DNA methylation and mitotic chromosome condensation in mammalian cells.

DNMT3B interacts with hSNF2H chromatin remodeling enzyme, HDACs 1 and 2, and components of the histone methylation system.

The non-random pattern of genome-wide DNA methylation in mammalian cells is established and maintained by DNA methyltransferases DNMT1, 3A, and 3B. De novo DNA methyltransferase DNMT3B is critical for embryonic development and is mutated in ICF syndrome. Despite its importance in normal cellular functioning, little is known about how DNMT3B operates in the context of chromatin. Here we demonstrate that DNMT3B associates with four chromatin-associated enzymatic activities common to transcriptionally repressed, heterochromatic regions of the genome: DNA methyltransferase, histone deacetylase, ATPase, and histone methylase activities. By immunoprecipitation and GST pull-down, we show that DNMT3B interacts with HDAC1, HDAC2, HP1 proteins, Suv39h1, and the ATP-dependent chromatin remodeling enzyme hSNF2H. Endogenous hSNF2H is also associated with DNA methyltransferase activity. These proteins co-localize extensively with DNMT3B in heterochromatic regions. Our results therefore link DNMT3B to three other components of the epigenetic machinery and provide important insights into how DNA methylation patterns may be established within the chromatin environment.

Chromatin remodeling, histone modifications, and DNA methylation-how does it all fit together?

DNA methylation is important in the control of gene transcription and chromatin structure. The complexities of this process are just beginning to be elucidated in relationship to other epigenetic mechanisms. Exciting new research in the areas of histone methylation and chromatin remodeling make it clear just how important the connections between these various mechanisms and DNA methylation are for the control of chromosome structure and gene expression. Emerging evidence suggests that chromatin remodeling enzymes and histone methylation are essential for proper DNA methylation patterns. Other histone modifications, such as acetylation and phosphorylation, in turn, affect histone methylation and histone methylation also appears to be highly reliant on chromatin remodeling enzymes. This review will summarize what is likely only the beginning of a flood of new information that will ultimately link all epigenetic modifications of the mammalian genome. A model will also be put forth to account for how chromatin modifications lead to genomic DNA methylation patterns.

Defective de novo methylation of viral and cellular DNA sequences in ICF syndrome cells.

ICF syndrome (immunodeficiency, centromere instability and facial anomalies) is a recessive human genetic disorder resulting from mutations in the DNA methyltransferase 3B (DNMT3B) gene. Patients with this disease exhibit numerous chromosomal abnormalities, including anomalous decondensation, pairing, separation and breakage, primarily involving the pericentromeric regions of chromosomes 1 and 16. Global levels of DNA methylation in ICF cells are only slightly reduced; however, certain repetitive sequences and genes on the inactive X chromosome of female ICF patients are significantly hypomethylated. In the present report, we analyze the molecular defect of de novo methylation in ICF cells in greater detail by making use of a model Epstein-Barr virus (EBV)-based system and three members of the unique cellular cancer-testis (C-T) gene family. Results with the EBV-based system indicate that de novo methylation of newly introduced viral sequences is defective in ICF syndrome. Limited de novo methylation capacity is retained in ICF cells, indicating that the mutations in DNMT3B are not complete loss-of-function mutations or that other DNMTs cooperate with DNMT3B. Analysis of three C-T genes (two on the X chromosome and one autosomal) revealed that loss of methylation from cellular gene sequences is heterogeneous, with both autosomal and X chromosome-based genes demonstrating sensitivity to mutations in DNMT3B. Aberrant hypomethylation at a number of loci examined correlated with altered gene expression levels. Lastly, no consistent changes in the protein levels of the DNA methyltransferases were noted when normal and ICF cell lines were compared.