Oliver G. McDonald

Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition

Epithelial-to-mesenchymal transition (EMT) is an extreme example of cell plasticity that is important for normal development, injury repair and malignant progression. Widespread epigenetic reprogramming occurs during stem cell differentiation and malignant transformation, but EMT-related epigenetic reprogramming is poorly understood. Here we investigated epigenetic modifications during EMT mediated by transforming growth factor beta. Although DNA methylation was unchanged during EMT, we found a global reduction in the heterochromatin mark H3 Lys9 dimethylation (H3K9Me2), an increase in the euchromatin mark H3 Lys4 trimethylation (H3K4Me3) and an increase in the transcriptional mark H3 Lys36 trimethylation (H3K36Me3). These changes depended largely on lysine-specific demethylase-1 (Lsd1), and loss of Lsd1 function had marked effects on EMT-driven cell migration and chemoresistance. Genome-scale mapping showed that chromatin changes were mainly specific to large organized heterochromatin K9 modifications (LOCKs), which suggests that EMT is characterized by reprogramming of specific chromatin domains across the genome.

Control of SRF binding to CArG box chromatin regulates smooth muscle gene expression in vivo

Precise control of SMC transcription plays a major role in vascular development and pathophysiology. Serum response factor (SRF) controls SMC gene transcription via binding to CArG box DNA sequences found within genes that exhibit SMC-restricted expression. However, the mechanisms that regulate SRF association with CArG box DNA within native chromatin of these genes are unknown. Here we report that SMC-restricted binding of SRF to murine SMC gene CArG box chromatin is associated with patterns of posttranslational histone modifications within this chromatin that are specific to the SMC lineage in culture and in vivo, including methylation and acetylation to histone H3 and H4 residues. We found that the promyogenic SRF coactivator myocardin increased SRF association with methylated histones and CArG box chromatin during activation of SMC gene expression. In contrast, the myogenic repressor Kruppel-like factor 4 recruited histone H4 deacetylase activity to SMC genes and blocked SRF association with methylated histones and CArG box chromatin during repression of SMC gene expression. Finally, we observed deacetylation of histone H4 coupled with loss of SRF binding during suppression of SMC differentiation in response to vascular injury. Taken together, these findings provide novel evidence that SMC-selective epigenetic control of SRF binding to chromatin plays a key role in regulation of SMC gene expression in response to pathophysiological stimuli in vivo.

Concise Review: Epigenetic Mechanisms Contribute to Pluripotency and Cell Lineage Determination of Embryonic Stem Cells

Epigenetic mechanisms, such as histone modifications and DNA methylation, have been shown to play a key role in the regulation of gene transcription. Results of recent studies indicate that a novel “bivalent” chromatin structure marks key developmental genes in embryonic stem cells (ESCs), wherein a number of untranscribed lineage‐control genes, such as Sox1, Nkx2‐2, Msx1, Irx3, and Pax3, are epigenetically modified with a unique combination of activating and repressive histone modifications that prime them for potential activation (or repression) upon cell lineage induction and differentiation. However, results of these studies also showed that a subset of lineage‐control genes, such as Myf5 and Mash1, were not marked by these histone modifications, suggesting that distinct epigenetic mechanisms might exist for lineage‐control genes in ESCs. In this review article, we summarize evidence regarding possible mechanisms that control these unique histone modifications at lineage‐control gene loci in ESCs and consider their possible contribution to ESC pluripotency. In addition, we propose a novel “histone modification pulsing” model wherein individual pluripotent stem cells within the inner cell mass of blastocysts undergo transient asynchronous histone modifications at these developmental gene loci, thereby conferring differential responsiveness to environmental cues and morphogenic gradients important for cell lineage determination. Finally, we consider how these rapid histone modification exchanges become progressively more stable as ESCs undergo differentiation and maturation into specialized cell lineages.

L-type Voltage-Gated Ca2+ Channels Modulate Expression of Smooth Muscle Differentiation Marker Genes via a Rho Kinase/Myocardin/SRF–Dependent Mechanism

Vascular smooth muscle cell (SMC) contraction is mediated in part by calcium influx through L-type voltage-gated Ca2+ channels (VGCC) and activation of the RhoA/Rho kinase (ROK) signaling cascade. We tested the hypothesis that Ca2+ influx through VGCCs regulates SMC differentiation marker expression and that these effects are dependent on RhoA/ROK signaling. Depolarization-induced activation of VGCCs resulted in a nifedipine-sensitive increase in endogenous smooth muscle myosin heavy chain (SMMHC) and SM &agr;-actin expression and CArG-dependent promoter activity, as well as c-fos promoter activity. The ROK inhibitor, Y-27632, prevented depolarization-induced increase in SMMHC/SM &agr;-actin but had no effect on c-fos expression. Conversely, the Ca2+/calmodulin-dependent kinase inhibitor, KN93, prevented depolarization-induced increases in c-fos expression with no effect on SMMHC/SM &agr;-actin. Depolarization increased expression of myocardin, a coactivator of SRF that mediates CArG-dependent transcription of SMC marker gene promoters containing paired CArG cis regulatory elements (SMMHC/SM &agr;-actin). Both nifedipine and Y-27632 prevented the depolarization-induced increase in myocardin expression. Moreover, short interfering RNA (siRNA) specific for myocardin attenuated depolarization-induced SMMHC/SM &agr;-actin transcription. Chromatin immunoprecipitation (ChIP) assays revealed that depolarization increased SRF enrichment of the CArG regions in the SMMHC, SM &agr;-actin, and c-fos promoters in intact chromatin. Whereas Y-27632 decreased basal and depolarization-induced SRF enrichment in the SMMHC/SM &agr;-actin promoter regions, it had no effect of SRF enrichment of c-fos. Taken together, these results provide evidence for a novel mechanism whereby Ca2+ influx via VGCCs stimulates expression of SMC differentiation marker genes through mechanisms that are dependent on ROK, myocardin, and increased binding of SRF to CArG cis regulatory elements.

A G/C Element Mediates Repression of the SM22α Promoter Within Phenotypically Modulated Smooth Muscle Cells in Experimental Atherosclerosis

A hallmark of smooth muscle cell (SMC) phenotypic switching in atherosclerotic lesions is suppression of SMC differentiation marker gene expression. Yet little is known regarding the molecular mechanisms that control this process. Here we show that transcription of the SMC differentiation marker gene SM22&agr; is reduced in atherosclerotic lesions and identify a cis regulatory element in the SM22&agr; promoter required for this process. Transgenic mice carrying the SM22&agr; promoter–&bgr;-galactosidase (&bgr;-gal) reporter transgene were crossed to apolipoprotein E (ApoE)−/− mice. Cells of the fibrous cap, intima, and underlying media showed complete loss of &bgr;-gal activity in advanced atherosclerotic lesions. Of major significance, mutation of a G/C-rich cis element in the SM22&agr; promoter prevented the decrease in SM22&agr; promoter–&bgr;-gal reporter transgene expression, including in cells that compose the fibrous cap of the lesion and in medial cells in proximity to the lesion. To begin to assess mechanisms whereby the G/C repressor element mediates suppression of SM22&agr; in atherosclerosis, we tested the hypothesis that effects may be mediated by platelet-derived growth factor (PDGF)-BB–induced increases in the G/C binding transcription factor Sp1. Consistent with this hypothesis, results of studies in cultured SMCs showed that: (1) PDGF-BB increased expression of Sp1; (2) PDGF-BB and Sp1 profoundly suppressed SM22&agr; promoter activity as well as smooth muscle myosin heavy chain promoter activity through mechanisms that were at least partially dependent on the G/C cis element; and (3) a short interfering RNA to Sp1 increased basal expression and attenuated PDGF-BB induced suppression of SM22&agr;. Together, these results support a model whereby a G/C repressor element within the SM22&agr; promoter mediates transcriptional repression of this gene within phenotypically modulated SMCs in experimental atherosclerosis and provide indirect evidence implicating PDGF-BB and Sp1 as possible mediators of these effects.

PRISM/PRDM6, a Transcriptional Repressor That Promotes the Proliferative Gene Program in Smooth Muscle Cells

ABSTRACT Smooth muscle cells (SMCs) display remarkable phenotypic diversity and plasticity and can readily switch between proliferative and differentiated states in response to extracellular cues. In an effort to identify novel transcriptional regulators of smooth muscle phenotypes, we compared the gene expression profiles of arterial and venous SMCs by microarray-based transcriptional profiling. Among numerous genes displaying distinct expression patterns in these two SMC types, we discovered an expressed sequence tag encoding a previously uncharacterized zinc finger protein belonging to the PRDM (PRDI-BF1 and RIZ homology domain) family of chromatin-remodeling proteins and named it PRISM (PR domain in smooth muscle). PRISM is expressed in a variety of smooth muscle-containing tissues and displays especially robust expression in the cardiac outflow tract and descending aorta during embryogenesis. PRISM is localized to the nucleus and contains an amino-terminal PR domain and four Krüppel-like zinc fingers at the carboxy terminus. We show that PRISM acts as a transcriptional repressor by interacting with class I histone deacetylases and the G9a histone methyltransferase, thereby identifying PRISM as a novel SMC-restricted epigenetic regulator. Overexpression of PRISM in cultured primary SMCs induces genes associated with the proliferative smooth muscle phenotype while repressing regulators of differentiation, including myocardin and GATA-6. Conversely, small interfering RNA-mediated knockdown of PRISM slows cell growth and induces myocardin, GATA-6, and markers of SMC differentiation. We conclude that PRISM acts as a novel epigenetic regulator of SMC phenotypic plasticity by suppressing differentiation and maintaining the proliferative potential of vascular SMCs.

5′ CArG degeneracy in smooth muscle α-actin is required for injury-induced gene suppression in vivo

CC(A/T)6GG-dependent (CArG-dependent) and serum response factor-dependent (SRF-dependent) mechanisms are required for gene expression in smooth muscle cells (SMCs). However, an unusual feature of many SMC-selective promoter CArG elements is that they contain a conserved single G or C substitution in their central A/T-rich region, which reduces binding affinity for ubiquitously expressed SRF. We hypothesized that this CArG degeneracy contributes to cell-specific expression of smooth muscle alpha-actin in vivo, since substitution of c-fos consensus CArGs for the degenerate CArGs resulted in relaxed specificity in cultured cells. Surprisingly, our present results show that these substitutions have no effect on smooth muscle-specific transgene expression during normal development and maturation in transgenic mice. However, these substitutions significantly attenuated injury-induced downregulation of the mutant transgene under conditions where SRF expression was increased but expression of myocardin, a smooth muscle-selective SRF coactivator, was decreased. Finally, chromatin immunoprecipitation analyses, together with cell culture studies, suggested that myocardin selectively enhanced SRF binding to degenerate versus consensus CArG elements. Our results indicate that reductions in myocardin expression and the degeneracy of CArG elements within smooth muscle promoters play a key role in phenotypic switching of smooth muscle cells in vivo, as well as in mediating responses of CArG-dependent smooth muscle genes and growth regulatory genes under conditions in which these 2 classes of genes are differentially expressed.

Epigenomic reprogramming during pancreatic cancer progression links anabolic glucose metabolism to distant metastasis

During the progression of pancreatic ductal adenocarcinoma (PDAC), heterogeneous subclonal populations emerge that drive primary tumor growth, regional spread, distant metastasis, and patient death. However, the genetics of metastases largely reflects that of the primary tumor in untreated patients, and PDAC driver mutations are shared by all subclones. This raises the possibility that an epigenetic process might operate during metastasis. Here we report large-scale reprogramming of chromatin modifications during the natural evolution of distant metastasis. Changes were targeted to thousands of large chromatin domains across the genome that collectively specified malignant traits, including euchromatin and large organized chromatin histone H3 lysine 9 (H3K9)-modified (LOCK) heterochromatin. Remarkably, distant metastases co-evolved a dependence on the oxidative branch of the pentose phosphate pathway (oxPPP), and oxPPP inhibition selectively reversed reprogrammed chromatin, malignant gene expression programs, and tumorigenesis. These findings suggest a model whereby linked metabolic–epigenetic programs are selected for enhanced tumorigenic fitness during the evolution of distant metastasis.

Molecular haplotyping of genomic DNA for multiple single-nucleotide polymorphisms located kilobases apart using long-range polymerase chain reaction and intramolecular ligation.

Genetic polymorphisms are well-recognized causes of interindividual differences in disease risk and treatment response in humans. For genes containing multiple single-nucleotide polymorphisms (SNPs), haplotype structure is often the principal determinant of phenotypic consequences, and haplotype distribution represents the best approach for assessing patterns of linkage disequilibrium. To permit more widespread molecular determination of haplotypes, we developed a simple yet robust method to determine haplotype structure for multiple SNPs located up to 30 kb apart in genomic DNA using long-range polymerase chain reaction (LR-PCR) and intramolecular ligation. Complete concordance was shown between the new method and conventional approaches, such as family pedigree analysis or cloning and sequencing. The availability of a simple method to directly determine haplotype structure using genomic DNA, without family pedigree analysis, cloning or complex instrumentation, provides an important new tool for elucidating the genetic determinants of drug disposition and effects, disease risk, and molecular evolution.

Large hypomethylated blocks as a universal defining epigenetic alteration in human solid tumors

BackgroundOne of the most provocative recent observations in cancer epigenetics is the discovery of large hypomethylated blocks, including single copy genes, in colorectal cancer, that correspond in location to heterochromatic LOCKs (large organized chromatin lysine-modifications) and LADs (lamin-associated domains).MethodsHere we performed a comprehensive genome-scale analysis of 10 breast, 28 colon, nine lung, 38 thyroid, 18 pancreas cancers, and five pancreas neuroendocrine tumors as well as matched normal tissue from most of these cases, as well as 51 premalignant lesions. We used a new statistical approach that allows the identification of large hypomethylated blocks on the Illumina HumanMethylation450 BeadChip platform.ResultsWe find that hypomethylated blocks are a universal feature of common solid human cancer, and that they occur at the earliest stage of premalignant tumors and progress through clinical stages of thyroid and colon cancer development. We also find that the disrupted CpG islands widely reported previously, including hypermethylated island bodies and hypomethylated shores, are enriched in hypomethylated blocks, with flattening of the methylation signal within and flanking the islands. Finally, we found that genes showing higher between individual gene expression variability are enriched within these hypomethylated blocks.ConclusionThus hypomethylated blocks appear to be a universal defining epigenetic alteration in human cancer, at least for common solid tumors.