Mark N. Cruickshank

Neonatal DNA methylation profile in human twins is specified by a complex interplay between intrauterine environmental and genetic factors, subject to tissue-specific influence

Comparison between groups of monozygotic (MZ) and dizygotic (DZ) twins enables an estimation of the relative contribution of genetic and shared and nonshared environmental factors to phenotypic variability. Using DNA methylation profiling of ∼20,000 CpG sites as a phenotype, we have examined discordance levels in three neonatal tissues from 22 MZ and 12 DZ twin pairs. MZ twins exhibit a wide range of within-pair differences at birth, but show discordance levels generally lower than DZ pairs. Within-pair methylation discordance was lowest in CpG islands in all twins and increased as a function of distance from islands. Variance component decomposition analysis of DNA methylation in MZ and DZ pairs revealed a low mean heritability across all tissues, although a wide range of heritabilities was detected for specific genomic CpG sites. The largest component of variation was attributed to the combined effects of nonshared intrauterine environment and stochastic factors. Regression analysis of methylation on birth weight revealed a general association between methylation of genes involved in metabolism and biosynthesis, providing further support for epigenetic change in the previously described link between low birth weight and increasing risk for cardiovascular, metabolic, and other complex diseases. Finally, comparison of our data with that of several older twins revealed little evidence for genome-wide epigenetic drift with increasing age. This is the first study to analyze DNA methylation on a genome scale in twins at birth, further highlighting the importance of the intrauterine environment on shaping the neonatal epigenome.

Longitudinal, genome-scale analysis of DNA methylation in twins from birth to 18 months of age reveals rapid epigenetic change in early life and pair-specific effects of discordance

BackgroundThe extent to which development- and age-associated epigenetic changes are influenced by genetic, environmental and stochastic factors remains to be discovered. Twins provide an ideal model with which to investigate these influences but previous cross-sectional twin studies provide contradictory evidence of within-pair epigenetic drift over time. Longitudinal twin studies can potentially address this discrepancy.ResultsIn a pilot, genome-scale study of DNA from buccal epithelium, a relatively homogeneous tissue, we show that one-third of the CpGs assayed show dynamic methylation between birth and 18 months. Although all classes of annotated genomic regions assessed show an increase in DNA methylation over time, probes located in intragenic regions, enhancers and low-density CpG promoters are significantly over-represented, while CpG islands and high-CpG density promoters are depleted among the most dynamic probes. Comparison of co-twins demonstrated that within-pair drift in DNA methylation in our cohort is specific to a subset of pairs, who show more differences at 18 months. The rest of the pairs show either minimal change in methylation discordance, or more similar, converging methylation profiles at 18 months. As with age-associated regions, sites that change in their level of within-pair discordance between birth and 18 months are enriched in genes involved in development, but the average magnitude of change is smaller than for longitudinal change.ConclusionsOur findings suggest that DNA methylation in buccal epithelium is influenced by non-shared stochastic and environmental factors that could reflect a degree of epigenetic plasticity within an otherwise constrained developmental program.

A critical assessment of the factors affecting reporter gene assays for promoter SNP function : a reassessment of -308 TNF polymorphism function using a novel integrated reporter system

One of the greatest challenges facing genetics is the development of strategies to identify functionally relevant genetic variation. The most common test of function is the reporter gene assay, in which allelic regulatory regions are used to drive the expression of a reporter gene, and differences in expression in a cell line after transient transfection are taken to be a reflection of the polymorphism. Many studies have reported small differences in single nucleotide polymorphism (SNP)-specific reporter activity, including the tumor necrosis factor (TNF) G−308A polymorphism. However, we have established that many variables inherent in the reporter gene approach can account for the reported allelic differences. Variables, such as the amount of DNA used in transfection, the amount of transfection control vector used, the method of transfection, the growth history of the host cells, and the quality and purity of DNA used, all influence TNF −308 SNP-specific transient reporter gene assays and serve as a caution for those researchers who apply this method to the functional assessment of polymorphic promoter sequences. We have developed an integrated reporter system that obviates some of these problems and shows that the TNF G−308A polymorphism is functionally relevant in this improved assay, thus confirming that the −308A allele expresses at a higher level compared with the −308G allele.

The impact of histone post-translational modifications on developmental gene regulation

Eukaryotic genomic DNA is orderly compacted to fit into the nucleus and to inhibit accessibility of specific sequences. DNA is manipulated in many different ways by bound RNA and proteins within the composite material known as chromatin. All of the biological processes that require access to genomic DNA (such as replication, recombination and transcription) therefore are dependent on the precise characteristics of chromatin in eukaryotes. This distinction underlies a fundamental property of eukaryotic versus prokaryotic gene regulation such that chromatin structure must be regulated to precisely repress or relieve repression of particular regions of the genome in an appropriate spatio-temporal manner. As well as playing a key role in structuring genomic DNA, histones are subject to site-specific modifications that can influence the organization of chromatin structure. This review examines the molecular processes regulating site-specific histone acetylation, methylation and phosphorylation with an emphasis on how these processes underpin differentiation-regulated transcription.

Quantitative differences in chromatin accessibility across regulatory regions can be directly compared in distinct cell-types

Transcriptional activation in eukaryotes is often accompanied by alterations to chromatin structure at specific regulatory sites while other genomic regions may remain unchanged. In this study, we have examined the correlation between expression and chromatin accessibility of the human CR2 gene in a panel of cell lines (U937, REH, Ramos, and Raji) using the CHART-PCR assay with the accessibility agent micrococcal nuclease (MNase). To validate the use of this assay for comparing multiple cell-types, we first tested a series of genomic regions to determine if we could observe consistent, site-specific levels of MNase chromatin accessibility. Promoter regions of the ubiquitously expressed genes GAPDH and beta-actin were similar and showed high accessibility to MNase digestion in each of the cell lines, while on the other hand, promoter regions of developmentally restricted genes PAX-7 and SP-A2 showed consistently reduced chromatin accessibility. Since CHART-PCR detected site-specific differences in chromatin accessibility in a manner that could be compared between cell-types, we next examined chromatin accessibility over the CR2 core promoter in the panel of cell lines representing either CR2 expressing or CR2 non-expressing cell-types. Our data revealed significantly enhanced accessibility over the -289 to -101 and the -115 to -12 regions of the CR2 promoter in expressing B-cells (Ramos, Raji) compared to non-expressing cells (U937, REH). Thus, CHART-PCR assays detected a correlation between chromatin accessibility and expression of the human CR2 gene, while the accessibility of other genomic regions was site-specific, but not altered between cell-types.

Human active X-specific DNA methylation events showing stability across time and tissues

The phenomenon of X chromosome inactivation in female mammals is well characterised and remains the archetypal example of dosage compensation via monoallelic expression. The temporal series of events that culminates in inactive X-specific gene silencing by DNA methylation has revealed a ‘patchwork’ of gene inactivation along the chromosome, with approximately 15% of genes escaping. Such genes are therefore potentially subject to sex-specific imbalance between males and females. Aside from XIST, the non-coding RNA on the X chromosome destined to be inactivated, very little is known about the extent of loci that may be selectively silenced on the active X chromosome (Xa). Using longitudinal array-based DNA methylation profiling of two human tissues, we have identified specific and widespread active X-specific DNA methylation showing stability over time and across tissues of disparate origin. Our panel of X-chromosome loci subject to methylation on Xa reflects a potentially novel mechanism for controlling female-specific X inactivation and sex-specific dimorphisms in humans. Further work is needed to investigate these phenomena.

The role of notch signaling in the development of a normal B-cell repertoire

The notch signaling pathway is evolutionarily conserved across the animal kingdom and regulates developmental ‘decisions’, such as cell fate commitment, differentiation, proliferation and apoptosis. In the mammalian immune system, notch signaling events have been extensively studied during T lymphopoiesis, and have a role both during early development, as well as differentiation into discreet effector cell compartments. In contrast, the impact of notch signaling in the B‐cell compartment is less obvious. It is clear that notch signaling is crucial to generate the marginal zone B‐cell population located within the spleen; however, the full effects of notch signaling during normal B‐cell development remain unresolved. Nevertheless, there is compelling evidence that notch signaling regulates multiple stages of B‐cell differentiation and in shaping the antibody repertoire; however, the molecular details have not been elucidated. This review explores the relationship between notch signaling and B‐cell development with attention to how these processes contribute to a normal B‐cell repertoire.

Cell- and stage-specific chromatin structure across the Complement receptor 2 (CR2/CD21) promoter coincide with CBF1 and C/EBP-β binding in B cells

Stringent developmental transcription requires multiple transcription factor (TF) binding sites, cell-specific expression of signaling molecules, TFs and co-regulators and appropriate chromatin structure. During B-lymphopoiesis, human Complement receptor 2 (CR2/CD21) is detected on immature and mature B cells but not on B cell precursors and plasma cells. We examined cell- and stage-specific human CR2 gene regulation using cell lines modeling B-lymphopoiesis. Chromatin accessibility assays revealed a region between -409 and -262 with enhanced accessibility in mature B cells and pre-B cells, compared to either non-lymphoid or plasma cell-types, however, accessibility near the transcription start site (TSS) was elevated only in CR2-expressing B cells. A correlation between histone acetylation and CR2 expression was observed, while histone H3K4 dimethylation was enriched near the TSS in both CR2-expressing B cells and non-expressing pre-B cells. Candidate sites within the CR2 promoter were identified which could regulate chromatin, including a matrix attachment region associated with CDP, SATB1/BRIGHT and CEBP-beta sites as well as two CBF1 sites. ChIP assays verified that both CBF1 and C/EBP-beta bind the CR2 promoter in B cells raising the possibility that these factors facilitate or respond to alterations in chromatin structure to control the timing and/or level of CR2 transcription.

Analysis of tandem E-box motifs within human Complement receptor 2 (CR2/CD21) promoter reveals cell specific roles for RP58, E2A, USF and localized chromatin accessibility.

Complement receptor 2 (CR2/CD21) plays an important role in the generation of normal B cell immune responses. As transcription appears to be the prime mechanism via which surface CR2/CD21 expression is controlled, understanding transcriptional regulation of this gene will have broader implications to B cell biology. Here we report opposing, cell-context specific control of CR2/CD21 promoter activity by tandem E-box elements, spaced 22 bp apart and within 70 bp of the transcription initiation site. We have identified E2A and USF transcription factors as binding to the distal and proximal E-box sites respectively in CR2-positive B-cells, at a site that is hypersensitive to restriction enzyme digestion compared to non-expressing K562 cells. However, additional unidentified proteins have also been found to bind these functionally important elements. By utilizing a proteomics approach we have identified a repressor protein, RP58, binding the distal E-box motif. Co-transfection experiments using RP58 overexpression constructs demonstrated a specific 10-fold repression of CR2/CD21 transcriptional activity mediated through the distal E-box repressor element. Taken together, our results indicate that repression of the CR2/CD21 promoter can occur through one of the E-box motifs via recruitment of RP58 and other factors to bring about a silenced chromatin context within CR2/CD21 non-expressing cells.

Tumor penetrating peptides inhibiting MYC as a potent targeted therapeutic strategy for triple-negative breast cancers

Overexpression of MYC oncogene is highly prevalent in many malignancies such as aggressive triple-negative breast cancers (TNBCs) and it is associated with very poor outcome. Despite decades of research, attempts to effectively inhibit MYC, particularly with small molecules, still remain challenging due to the featureless nature of its protein structure. Herein, we describe the engineering of the dominant-negative MYC peptide (OmoMYC) linked to a functional penetrating ‘Phylomer’ peptide (FPPa) as a therapeutic strategy to inhibit MYC in TNBC. We found FPPa-OmoMYC to be a potent inducer of apoptosis (with IC50 from 1–2 µM) in TNBC cells with negligible effects in non-tumorigenic cells. Transcriptome analysis of FPPa-OmoMYC-treated cells indicated that the fusion protein inhibited MYC-dependent networks, inducing dynamic changes in transcriptional, metabolic, and apoptotic processes. We demonstrated the efficacy of FPPa-OmoMYC in inhibiting breast cancer growth when injected orthotopically in TNBC allografts. Lastly, we identified strong pharmacological synergisms between FPPa-OmoMYC and chemotherapeutic agents. This study highlights a novel therapeutic approach to target highly aggressive and chemoresistant MYC-activated cancers.