Emma K. Baker

Hyperglycemia Induces a Dynamic Cooperativity of Histone Methylase and Demethylase Enzymes Associated With Gene-Activating Epigenetic Marks That Coexist on the Lysine Tail

OBJECTIVE Results from the Diabetes Control Complications Trial (DCCT) and the subsequent Epidemiology of Diabetes Interventions and Complications (EDIC) Study and more recently from the U.K. Prospective Diabetes Study (UKPDS) have revealed that the deleterious end-organ effects that occurred in both conventional and more aggressively treated subjects continued to operate >5 years after the patients had returned to usual glycemic control and is interpreted as a legacy of past glycemia known as “hyperglycemic memory.” We have hypothesized that transient hyperglycemia mediates persistent gene-activating events attributed to changes in epigenetic information. RESEARCH DESIGN AND METHODS Models of transient hyperglycemia were used to link NFκB-p65 gene expression with H3K4 and H3K9 modifications mediated by the histone methyltransferases (Set7 and SuV39h1) and the lysine-specific demethylase (LSD1) by the immunopurification of soluble NFκB-p65 chromatin. RESULTS The sustained upregulation of the NFκB-p65 gene as a result of ambient or prior hyperglycemia was associated with increased H3K4m1 but not H3K4m2 or H3K4m3. Furthermore, glucose was shown to have other epigenetic effects, including the suppression of H3K9m2 and H3K9m3 methylation on the p65 promoter. Finally, there was increased recruitment of the recently identified histone demethylase LSD1 to the p65 promoter as a result of prior hyperglycemia. CONCLUSIONS These studies indicate that the active transcriptional state of the NFκB-p65 gene is linked with persisting epigenetic marks such as enhanced H3K4 and reduced H3K9 methylation, which appear to occur as a result of effects of the methyl-writing and methyl-erasing histone enzymes.

Brahma links the SWI/SNF chromatin-remodeling complex with MeCP2-dependent transcriptional silencing

Transcriptional repression of methylated genes can be mediated by the methyl-CpG binding protein MeCP2. Here we show that human Brahma (Brm), a catalytic component of the SWI/SNF-related chromatin-remodeling complex, associates with MeCP2 in vivo and is functionally linked with repression. We used a number of different molecular approaches and chromatin immunoprecipitation strategies to show a unique cooperation between Brm, BAF57 and MeCP2. We show that Brm and MeCP2 assembly on chromatin occurs on methylated genes in cancer and the gene FMR1 in fragile X syndrome. These experimental findings identify a new role for SWI/SNF in gene repression by MeCP2.

Epigenetic changes to the MDR1 locus in response to chemotherapeutic drugs

The mechanism of action of chemotherapeutic drugs and their ability to induce multidrug resistance (MDR) are of relevance to cancer treatment. Overexpression of P-glycoprotein (Pgp) encoded by the MDR1 gene following chemotherapy can severely limit the efficacy of anticancer agents; however, the manner by which cells acquire high levels of Pgp has not been defined. Herein, we demonstrate that chemotherapeutic drugs induce specific epigenetic modifications at the MDR1 locus, concomitant with MDR1 upregulation mediated by transcriptional activation, and a potential post-transcriptional component. We have established that the mechanisms are not mutually exclusive and are dependent on the methylation state of the MDR1 promoter. MDR1 upregulation did not result in further changes to the CpG methylation profile. However, dramatic changes in the temporal and spatial patterning of histone modifications occurred within the 5′ hypomethylated region of MDR1, directly correlating with MDR1 upregulation. Specifically, drug-induced upregulation of MDR1 was associated with increases in H3 acetylation and induction of methylated H3K4 within discrete regions of the MDR1 locus. Our results demonstrate that chemotherapeutic drugs can actively induce epigenetic changes within the MDR1 promoter, and enhance the MDR phenotype.

The rise of DNA methylation and the importance of chromatin on multidrug resistance in cancer

In recent years, the different classes of drugs and regimens used clinically have provided an improvement in tumour management. However, treatment is often palliative for the majority of cancer patients. Transformed cells respond poorly to chemotherapy mainly due to the development of the multidrug resistance (MDR) phenotype. Response to treatment does not generally result in complete remission and disease cure is uncommon for patients presenting with advanced stage cancer. Successful treatment of cancer requires a clearer understanding of chemotherapeutic resistance. Here, we examine what is known of one of the most extensively studied mechanisms of cellular drug resistance. The human multidrug resistance gene 1 (MDR1) is associated with expression of p-glycoprotein (Pgp). A transmembrane protein, Pgp acts as an efflux pump and reduces intracellular drug levels and thus its effectiveness as an antitumor agent. The precise mechanism of transcriptional regulation has been unclear due to the complex regulatory nature of the gene. It has become increasingly apparent that trans-activation or genetic amplification is by no means the only mechanism of activation. Consequently, alternative pathways have received more attention in the area of epigenetics to help explain transcriptional competence at a higher level of organization. The goal of this article is to highlight important findings in the field of methylation and explain how they impinge on MDR1 gene regulation. In this review, we cover the current information and postulate that epigenetic modification of MDR1 chromatin influences gene transcription in leukaemia. Finally, we explore transcriptional regulation and highlight recent progress with engineered ZFPs (zinc finger proteins).

EphrinB2/EphB4 inhibition in the osteoblast lineage modifies the anabolic response to parathyroid hormone.

Previous reports indicate that ephrinB2 expression by osteoblasts is stimulated by parathyroid hormone (PTH) and its related protein (PTHrP) and that ephrinB2/EphB4 signaling between osteoblasts and osteoclasts stimulates osteoblast differentiation while inhibiting osteoclast differentiation. To determine the role of the ephrinB2/EphB4 interaction in the skeleton, we used a specific inhibitor, soluble EphB4 (sEphB4), in vitro and in vivo. sEphB4 treatment of cultured osteoblasts specifically inhibited EphB4 and ephrinB2 phosphorylation and reduced mRNA levels of late markers of osteoblast/osteocyte differentiation (osteocalcin, dentin matrix protein‐1 [DMP‐1], sclerostin, matrix‐extracellular phosphoglycoprotein [MEPE]), while substantially increasing RANKL. sEphB4 treatment in vivo in the presence and absence of PTH increased osteoblast formation and mRNA levels of early osteoblast markers (Runx2, alkaline phosphatase, Collagen 1α1, and PTH receptor [PTHR1]), but despite a substantial increase in osteoblast numbers, there was no significant change in bone formation rate or in late markers of osteoblast/osteocyte differentiation. Rather, in the presence of PTH, sEphB4 treatment significantly increased osteoclast formation, an effect that prevented the anabolic effect of PTH, causing instead a decrease in trabecular number. This enhancement of osteoclastogenesis by sEphB4 was reproduced in vitro but only in the presence of osteoblasts. These data indicate that ephrinB2/EphB4 signaling within the osteoblast lineage is required for late stages of osteoblast differentiation and, further, restricts the ability of osteoblasts to support osteoclast formation, at least in part by limiting RANKL production. This indicates a key role for the ephrinB2/EphB4 interaction within the osteoblast lineage in osteoblast differentiation and support of osteoclastogenesis.

MDR1, Chemotherapy and Chromatin Remodeling

The development of multidrug resistance (MDR) in cancer can severely impede the efficacy of chemotherapy treatment. P-glycoprotein (Pgp) overexpression, encoded by the MDR1 gene, is a well-established mediator of MDR. MDR1 expression is rapidly upregulated by chemotherapeutic drugs and a number of other exogenous stimuli, however the mechanisms underlying its transcriptional regulation remain unclear. In recent years, research has indicated that chromatin accessibility, or epigenetic modifications, will play a large role in controlling the endogenous MDR1 expression state, and its response to activation stimuli. This review examines some of these studies, and discusses how new developments from the greatly expanding epigenetics field may extend to MDR1 transcriptional research.

BET inhibitors induce apoptosis through a MYC independent mechanism and synergise with CDK inhibitors to kill osteosarcoma cells

Osteosarcoma (OS) survival rates have plateaued in part due to a lack of new therapeutic options. Here we demonstrate that bromodomain inhibitors (BETi), JQ1, I-BET151, I-BET762, exert potent anti-tumour activity against primary and established OS cell lines, mediated by inhibition of BRD4. Strikingly, unlike previous observations in long-term established human OS cell lines, the antiproliferative activity of JQ1 in primary OS cells was driven by the induction of apoptosis, not cell cycle arrest. In further contrast, JQ1 activity in OS was mediated independently of MYC downregulation. We identified that JQ1 suppresses the transcription factor FOSL1 by displacement of BRD4 from its locus. Loss of FOSL1 phenocopied the antiproliferative effects of JQ1, identifying FOSL1 suppression as a potential novel therapeutic approach for OS. As a monotherapy JQ1 demonstrated significant anti-tumour activity in vivo in an OS graft model. Further, combinatorial treatment approaches showed that JQ1 increased the sensitivity of OS cells to doxorubicin and induced potent synergistic activity when rationally combined with CDK inhibitors. The greater level of activity achieved with the combination of BETi with CDK inhibitors demonstrates the efficacy of this combination therapy. Taken together, our studies show that BET inhibitors are a promising new therapeutic for OS.

Modeling distinct osteosarcoma subtypes in vivo using Cre:lox and lineage-restricted transgenic shRNA

Osteosarcoma is the most common primary cancer of bone and one that predominantly affects children and adolescents. Osteoblastic osteosarcoma represents the major subtype of this tumor, with approximately equal representation of fibroblastic and chondroblastic subtypes. We and others have previously described murine models of osteosarcoma based on osteoblast-restricted Cre:lox deletion of Trp53 (p53) and Rb1 (Rb), resulting in a phenotype most similar to fibroblastic osteosarcoma in humans. We now report a model of the most prevalent form of human osteosarcoma, the osteoblastic subtype. In contrast to other osteosarcoma models that have used Cre:lox mediated gene deletion, this model was generated through shRNA-based knockdown of p53. As is the case with the human disease the shRNA tumors most frequently present in the long bones and preferentially disseminate to the lungs; feature less consistently modeled using Cre:lox approaches. Our approach allowed direct comparison of the in vivo consequences of targeting the same genetic drivers using two different technologies, Cre:lox and shRNA. This demonstrated that the effects of Cre:lox and shRNA mediated knock-down are qualitatively different, at least in the context of osteosarcoma, and yielded distinct subtypes of osteosarcoma. Through the use of complementary genetic modification strategies we have established a model of the most common clinical subtype of osteosarcoma that was not previously represented and more fully recapitulated the clinical spectrum of this cancer.

Testing for association between MeCP2 and the brahma-associated SWI/SNF chromatin-remodeling complex

Reply to “Testing for association between MeCP2 and the brahma-associated SWI/SNF chromatin-remodeling complex”

Epigenetic Modification of the Norepinephrine Transporter Gene in Postural Tachycardia Syndrome

Objective—The postural tachycardia syndrome (POTS) has multiple symptoms, chief among which are tachycardia, weakness, and recurrent blackouts while standing. Previous research has implicated dysfunction of the norepinephrine transporter. A coding mutation in the norepinephrine transporter gene (SLC6A2) sequence has been reported in 1 family kindred only. The goal of the present study was to further characterize the role and regulation of the SLC6A2 gene in POTS. Methods and Results—Sympathetic nervous system responses to head-up tilt were examined by combining norepinephrine plasma kinetics measurements and muscle sympathetic nerve activity recordings in patients with POTS compared with that in controls. The SLC6A2 gene sequence was investigated in leukocytes from POTS patients and healthy controls using single nucleotide polymorphisms genotyping, bisulphite sequencing, and chromatin immunoprecipitation assays for histone modifications and binding of the transcriptional regulatory complex, methyl-CpG binding protein 2. The expression of norepinephrine transporter was lower in POTS patients compared with healthy volunteers. In the absence of altered SLC6A2 gene sequence or promoter methylation, this reduced expression was directly correlated with chromatin modifications. Conclusion—We propose that chromatin-modifying events associated with SLC6A2 gene suppression may constitute a mechanism of POTS.