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Methyl cytosine binding domain protein 2 (MBD2) has been shown to bind to and mediate repression of methylated tumor suppressor genes in cancer cells, where repatterning of CpG methylation and associated gene silencing is common. We have investigated the role of MBD2 in breast cancer cell growth and tumor suppressor gene expression. We show that stable short hairpin RNA (shRNA)-mediated knockdown of MBD2 leads to growth suppression of cultured human mammary epithelial cancer lines, SK-BR-3, MDA-MB-231, and MDA-MB-435. The peak antiproliferative occurs only after sustained, stable MBD2 knockdown. Once established, the growth inhibition persists over time and leads to a markedly decreased propensity for aggressive breast cancer cell lines to form in vivo xenograft tumors in Bagg Albino (BALB)/C nu/nu mice. The growth effects of MBD2 knockdown are accompanied by derepression of tumor suppressor genes, including DAPK1 and KLK10. Chromatin immunoprecipitation assays and bisulfite sequencing show MBD2 binding directly to the hyper methylated and CpG-rich promoters of both DAPK1 and KLK10. Remarkably, the promoter CpG island–associated methylation of these genes remained stable despite robust transcriptional activation in MBD2 knockdown cells. Expression of a shRNA-resistant MBD2 protein resulted in restoration of growth and resilencing of the MBD2-dependent tumor suppressor genes. Our data suggest that uncoupling CpG methylation from repressive chromatin remodeling and histone modifications by removing MBD2 is sufficient to initiate and maintain tumor suppressor gene transcription and suppress neoplastic cell growth. These results show a role for MBD2 in cancer progression and provide support for the prospect of targeting MBD2 therapeutically in aggressive breast cancers. Mol Cancer Res; 9(8); 1152–62. ©2011 AACR.

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Chromodomain helicase DNA-binding protein 4 (CHD4) is an ATPase that alters the phasing of nucleosomes on DNA and has recently been implicated in DNA double-stranded break (DSB) repair. Here, we show that depletion of CHD4 in acute myeloid leukemia (AML) blasts induces a global relaxation of chromatin that renders cells more susceptible to DSB formation, while concurrently impeding their repair. Furthermore, CHD4 depletion renders AML blasts more sensitive both in vitro and in vivo to genotoxic agents used in clinical therapy: daunorubicin (DNR) and cytarabine (ara-C). Sensitization to DNR and ara-C is mediated in part by activation of the ataxia-telangiectasia mutated pathway, which is preliminarily activated by a Tip60-dependent mechanism in response to chromatin relaxation and further activated by genotoxic agent-induced DSBs. This sensitization preferentially affects AML cells, as CHD4 depletion in normal CD34(+) hematopoietic progenitors does not increase their susceptibility to DNR or ara-C. Unexpectedly, we found that CHD4 is necessary for maintaining the tumor-forming behavior of AML cells, as CHD4 depletion severely restricted the ability of AML cells to form xenografts in mice and colonies in soft agar. Taken together, these results provide evidence for CHD4 as a novel therapeutic target whose inhibition has the potential to enhance the effectiveness of genotoxic agents used in AML therapy.

IFNs regulate most MHC class I genes by stimulating transcription initiation. As shown previously, IFN-γ controls HLA-A expression primarily at the posttranscriptional level. We have defined two 8-base sequences in a 39-nucleotide region in the 3′-transcribed region of the HLA-A gene that are required for the posttranscriptional response to IFN-γ. Stimulation of HLA-A expression by IFN-γ requires nuclear export of HLA-A mRNA by chromosome maintenance region 1 (CRM-1). Treatment of cells with leptomycin B, a specific inhibitor of CRM-1, completely inhibited IFN-γ induction of HLA-A. Expression of a truncated, dominant-negative form of the nucleoporin NUP214/CAN, ΔCAN, that specifically interacts with CRM-1, also prevented IFN-γ stimulation of HLA-A, providing confirmation of the role of CRM-1. Increased expression of HLA-A induced by IFN-γ also requires protein methylation, as shown by the fact that treatment of SK-N-MC cells or HeLa cells with the PRMT1 inhibitor 5′-methyl-5′-thioadenosine abolished the cellular response to IFN-γ. In contrast with HLA-A, IFN-γ-induced expression of the HLA class Ib gene, HLA-E, was not affected by either 5′-methyl-5′-thioadenosine or leptomycin B. These results provide proof of principle that it is possible to differentially modulate the IFN-γ-induced expression of the HLA-E and HLA-A genes, whose products often mediate opposing effects on cellular immunity to tumor cells, pathogens, and autoantigens.

The methylation pattern of a 248-base pair proximal transcribed region (rho248) of the avian embryonic rho-globin gene was found to correlate inversely with stage-specific expression in avian erythroid cells. In vitro methylation of the rho248 segment alone (in the absence of promoter methylation) resulted in a 5-fold inhibition of transcription in a transient transfection assay in primary erythroid cells in which the transfected gene is packaged into nucleosomal chromatin. This effect was observed if the rho248 segment was positioned adjacent to the promoter but not when it was located 2.7 kilobases downstream. Fully methylated but not unmethylated rho248 formed a novel cell type-specific methyl cytosine-binding protein complex (MeCPC) that contained methyl binding domain protein-2 (MBD-2) and histone deacetylase 1 proteins but differed from MeCP-1. The histone deacetylase inhibitor trichostatin A failed to relieve methylation-mediated repression of transcription from the rho-gene promoter, supporting the notion of the dominance of methylation over histone deacetylation in silencing through CpG-rich sequences at this locus. These data demonstrate that site-specific methylation of a vertebrate gene 5'-transcribed region alone at the exact CpGs that are methylated in vivo can suppress transcription in homologous primary cells and facilitate binding to a cell type-specific MeCPC.

We investigated a potential role for the soluble interleukin-6 receptor (sIL-6R) in modulating interleukin-6 (IL-6) function in the central nervous system by assessing IL-6 and sIL-6R effects on beta-amyloid precursor protein (beta-APP) transcription and expression in cells of human neuronal origin. Cells transfected with a luciferase reporter plasmid containing a 3.8 kb DNA fragment of the beta-APP promoter were shown to have inducible promoter activity when treated with phorbol ester or basic fibroblast growth factor, but not when treated with lipopolysaccharide or Il-6. PCR amplification analysis revealed the presence of mRNA encoding the signaling subunit of the Il-6 receptor complex, the gp130 subunit, at levels approximating that found in human cortical tissue. The mRNA encoding the IL-6 receptor, however, was poorly expressed and was detectable only at high amplification cycles. When purified sIL-6R protein was added together with IL-6, there was a rapid induction of promoter activity within 2 h of stimulation followed by elevations in protein levels of both cell-associated and secreted beta-APP. Analysis of mRNA transcripts from human cortical brain tissue and cell cultures derived from fetal human brain demonstrated the presence of an alternatively spliced secreted form of the IL-6 receptor mRNA, suggesting that cells of the central nervous system may themselves be a source of sIL-6R protein. The capacity for sIL-6R to enhance IL-6 function and broaden the IL-6 target cell population in the brain has implications for the regulation of beta-APP expression in disease states such as Alzheimer's disease where elevations in brain IL-6 levels have been reported.

The selectivity of heparin in inducing potentiation of binding of antagonist ligands to muscarinic receptors was investigated at the five known subtypes of muscarinic receptors. The effects of heparin on binding of [3H]N-methylscopolamine at equilibrium was studied in Chinese hamster ovary (CHO) cells which express each of the individual muscarinic receptor subtypes and in membranes prepared from these cells. Heparin markedly increased equilibrium binding of subsaturating concentrations of the ligand only in membranes of CHO cells which express muscarinic M2 receptors. These effects of heparin were qualitatively similar to those obtained in heart membranes. In contrast, heparin did not influence ligand binding to muscarinic M2 receptors in intact cells. The positive cooperative effects of heparin at muscarinic receptors were abolished following treatment of cells with pertussis toxin. The latter treatment by itself resulted in a significant increase in [3H]N-methylscopolamine binding. Taken together with previous reports of heparin-induced uncoupling of receptors and G-proteins, these data suggest that the effects of heparin on ligand binding to muscarinic M2 receptors might be due to disruption of receptor-G-protein interactions which results in enhancement of binding of antagonist ligands to the receptor.

Abstract 825 Epigenetics has emerged as a key regulator of the fetal to adult hemoglobin switch during development. Understanding the mechanisms of fetal 𝛄-globin gene silencing offers the promise of effective targeted therapy of β- thalassemia and sickle cell anemia. Previous studies carried out by our group using adult erythroid cells from mice transgenic for a yeast artificial chromosome containing the entire human β-globin locus (β-YAC) and primary human erythroid cells have shown that methyl-CpG binding protein 2 (MBD2) is critical for full silencing of the fetal 𝛄-globin gene. MBD2 binds to methylated CpG rich promoters and silences the associated target genes by recruiting the Nuclear Remodeling and Deacetylase (NuRD) co-repressor complex. Absent or ≥75% decreased expression of MBD2 has been shown to have a 10–50 fold stimulatory effect on the expression of the 𝛄-globin gene in adult β-YAC transgenic mice and a 5–6 fold effect in CD34+ progenitor derived human erythroid cells in culture. Mi2, a major component of the NuRD complex, is an ATP-dependent chromatin remodeler consisting of two isoforms, Mi2a (also known as CHD3) and Mi2β (also known as CHD4). We have observed that 80% knockdown of Mi2β leads to a significantly higher expression of the 𝛄-globin gene in βYAC containing murine adult hematopoietic CID cells (∼100 fold) when compared to equivalent knockdown of other components of the MBD2/NuRD complex (∼5-10 fold), including MBD2. Remarkably, in CD34+ progenitor derived adult human erythroid cells, as little as 40% knockdown of Mi2β resulted in a ∼30 fold increase in 𝛄/𝛄 + β mRNA levels. Moreover, simultaneous knockdown of MBD2 and Mi2β resulted in no greater 𝛄-globin gene expression than Mi2β knockdown alone. This suggests that Mi2β is acting independently of as well as through its role in the MBD2-NuRD complex to exert its silencing effect on the 𝛄-globin gene. Complete conditional knockout of Mi2β in transgenic mouse hematopoietic cells has been shown by others to result in arrest of erythroid differentiation. We observed that in human CD34+ progenitor cells ≥80% Mi2β knockdown also altered differentiation. In contrast, 40–50% knockdown of Mi2β does not affect erythroid differentiation compared to scramble shRNA controls and resulted in 25 – 40% 𝛄/𝛄 + β globin RNA expression versus 2–3% in scramble shRNA controls. To determine possible mechanisms of Mi2β-mediated silencing outside of its role in the MBD2-NuRD complex, we examined the effect of Mi2β knockdown on two key mediators of 𝛄-globin gene silencing, Bcl11A and KLF-1 (EKLF). Our results showed that RNA and protein levels of these proteins are diminished by 40% and 70% respectively in cells with 40–50% Mi2β knockdown. Knockdown of MBD2 caused no decrease in either Bcl11A or KLF-1 levels. Thus at least part of the effect of Mi2β on 𝛄-globin gene silencing is through its positive effects on KLF-1 and Bcl11A expression. In summary, we have shown that less than a 50% decrease in Mi2β expression in primary human adult erythroid cells resulted in high levels of 𝛄/𝛄 + β gene expression without altering erythroid differentiation. The silencing effects of Mi2β occur both through the MBD2-NuRD repressor complex and through positive regulation of the KLF-1 and Bcl11A genes. These results also suggest that partial inhibition of Mi2β function could be of therapeutic benefit in the treatment of β- thalassemia and sickle cell anemia. Disclosures: No relevant conflicts of interest to declare.

The first putative extracellular domains of both ml and ml muscarinic receptors contain a triplet of amino acid residues consisting of leucine (L), tyrosine (Y), and threonine (T).

Nucleosome remodeling complexes comprise several large families of chromatin modifiers that integrate multiple epigenetic control signals to play key roles in cell type-specific transcription regulation. We previously isolated a methyl-binding domain protein 2 (MBD2)-containing nucleosome remodeling and deacetylation (NuRD) complex from primary erythroid cells and showed that MBD2 contributes to DNA methylation-dependent embryonic and fetal β-type globin gene silencing during development in vivo. Here we present structural and biophysical details of the coiled-coil interaction between MBD2 and p66α, a critical component of the MBD2–NuRD complex. We show that enforced expression of the isolated p66α coiled-coil domain relieves MBD2-mediated globin gene silencing and that the expressed peptide interacts only with a subset of components of the MBD2–NuRD complex that does not include native p66α or Mi-2. These results demonstrate the central importance of the coiled-coil interaction and suggest that MBD2-dependent DNA methylation-driven gene silencing can be disrupted by selectively targeting this coiled-coil complex.

We compared the concentration of mRNA encoding the m1 and m2 muscarinic receptors in several brain regions obtained from young (5–8 months) and aged (24–28 months) male Fischer 344 rats. DNA-excess solution hybridization was employed as a quantitative measure of mRNA concentration. The results indicate the absence of changes in the m1 receptor message with aging in the cerebral cortex, hippocampus and striatum. While there was no statistically significant aging-associated alteration in the concentration of the message encoding the m2 receptor in the thalamus, midbrain, cerebellum and brainstem, there was a decrease in the message level in the hypothalamus.

Relationship between in vivo down-regulation of cardiac muscarinic receptors and changes in their encoding mRNA was investigated. Rats were treated either once or for ten days with an irreversible inhibitor of acetylcholinesterase, followed by measurements of cardiac acetylcholinesterase, the density and affinity of muscarinic receptors, and the concentration of mRNA coding for these receptors. mRNA was quantitated using the sensitive method of DNA-excess solution hybridization. Our data indicate that while short-term treatment resulted in a marked decrease in the density of cardiac muscarinic receptors by 34%, there was no accompanying significant change in the concentration of their mRNA. In contrast, long-term inhibition of acetylcholinesterase significantly decreased the concentration of both receptors and mRNA by 40% and 29%, respectively. These results are indicative of multiple mechanisms of down-regulation of cardiac muscarinic receptors, some of which might involve alterations at the transcriptional level.

The chicken embryonic β-type globin gene, ρ, is a member of a small group of vertebrate genes whose developmentally regulated expression is mediated by DNA methylation. Previously, we have shown that a methyl cytosine-binding complex binds to the methylated ρ-globin gene in vitro. We have now chromatographically purified and characterized this complex from adult chicken primary erythroid cells. Four components of the MeCP1 transcriptional repression complex were identified: MBD2, RBAP48, HDAC2, and MTA1. These 4 proteins, as well as the zinc-finger protein p66 and the chromatin remodeling factor Mi2, were found to coelute by gel-filtration analysis and pull-down assays. We conclude that these 6 proteins are components of the MeCPC. In adult erythrocytes, significant enrichment for MBD2 is seen at the inactive ρ-globin gene by chromatin immunoprecipitation assay, whereas no enrichment is observed at the active βA-globin gene, demonstrating MBD2 binds to the methylated and transcriptionally silent ρ-globin gene in vivo. Knock-down of MBD2 resulted in up-regulation of a methylated ρ-gene construct in mouse erythroleukemic (MEL)-ρ cells. These results represent the first purification of a MeCP1-like complex from a primary cell source and provide support for a role for MBD2 in developmental gene regulation.

The expression of several MHC class I genes is up-regulated at the transcriptional level by IFN-γ. Posttranscriptional mechanisms also have been implicated, but not well characterized. To investigate the mechanism of IFN-γ stimulation of the human MHC class I gene HLA-A2, several human tumor cell lines were transfected with reporter gene constructs driven by the HLA-A2 promoter. We have previously shown that the extended 525-bp HLA-A2 promoter alone, which includes a 5′ IFN-stimulated response element consensus sequence, is not sufficient for IFN-γ response in either K562 or Jurkat cells. In the current study, stable transfection of a genomic HLA-A2 gene construct, containing both 5′- and 3′-flanking sequences, resulted in stimulation of the gene by IFN-γ. Nuclear run-on assays revealed that, unlike other class I genes, IFN-γ stimulation of HLA-A mRNA accumulation occurs almost entirely through posttranscriptional mechanisms. RNA stability assays showed that the effect is not mediated by alteration of the half-life of the HLA-A2 mRNA. Formation of the 3′ end was unaffected by IFN-γ treatment. Sequences that mediate the majority of IFN-γ induction of HLA-A2 mRNA reside in a 127-bp 3′-transcribed region of the gene. This region contains the terminal splice site, the usage of which is not affected by IFN-γ treatment. These results demonstrate a novel posttranscriptional mechanism of regulation of MHC class I genes by IFN-γ.

We investigated the coupling of the M2 muscarinic acetylcholine receptors expressed in Chinese hamster ovary cells to activation of neuronal nitric oxide (NO) synthase. Stimulation of guanylate cyclase activity in detector neuroblastoma cells was used as an indirect measure of the generation of NO in Chinese hamster ovary cells. The muscarinic agonist carbachol induced marked time- and concentration-dependent enhancement of the activity of NO synthase. Activation of neuronal NO synthase by M2 muscarinic receptors was associated with a small increase in the concentration of intracellular Ca2+. These data suggest the presence of alternate mechanisms of activation of neuronal NO synthase which might be operative in the absence of large changes in the concentration of cellular Ca2+. These findings help to understand the mechanisms of activation of NO synthase.

The selectivity of coupling of m1, m3, and m5 muscarinic receptors to activation of the neuronal type of nitric oxide synthase was investigated. Stimulation with the agonist carbachol of all three receptor subtypes expressed in Chinese hamster ovary cells resulted in a rapid and transient activation of the enzyme, as measured by stimulation of guanylate cyclase in reporter neuroblastoma cells. Carbachol was more potent and efficacious at m5 receptors than at the other two receptor subtypes. Stimulation of all three muscarinic receptors resulted in an increased concentration of intracellular calcium, with a time course that preceded activation of nitric oxide synthase. At each receptor subtype, there was a close relationship between the magnitude of the maximal calcium response and that of enzyme activation.

The human MHC Class Ib gene, HLA-E, has been shown to inhibit both Natural Killer cells and a subset of CD8+ cytotoxic T-cells by engaging the CD94/NKG2A inhibitory receptor. IFN-γ induces the expression of HLA-E as well as Class Ia molecules, which are required for killing of target cells. Since HLA-E has negative effects on immune cell killing of target cells, we have sought to identify locus-specific mechanisms of IFN-γ induction in order to identify molecular targets for selective activation of Class Ia genes, but not HLA-E. We have previously identified a unique upstream IFN-γ response region in the HLA-E promoter and showed that GATA-1 is required for its function in the K562 leukemic cell line. We have now examined the effect of GATA family members on IFN-γ induction of HLA-E in other cell types. HLA-E CAT reporter gene assays demonstrated that only epithelial cells which express GATA factors, as determined by western blot and real-time PCR, mediate a 2-to-4 fold enhanced response to IFN-γ stimulation. Functional analysis of HLA-E CAT reporter gene constructs containing mutations of the core nucleotides in the GATA binding site resulted in a four-fold decreased response to IFN-γ. Tetracycline regulated siRNA knockdown of GATA 6 expression in the SKOV3 ovarian carcinoma cell line revealed a two-to-three fold decrease in IFN-γ response of both HLA-E promoter driven constructs and the endogenous HLA-E gene. We conclude that GATA factors play a tissue specific role in regulation of IFN-γ mediated HLA-E expression. This work was supported by National Institutes of Health Grant CA87496 and Immunology Training Grant 5T32AI007407-15.

Chinese hamster ovary (CHO) cells are commonly used for expression of the genes of cloned neurotransmitter receptors to study their pharmacology and coupling to signal transduction pathways. It is usually assumed that host cells do not endogenously express the specific receptor under consideration. We demonstrate in this report that CHO cells contain endogenous functional muscarinic acetylcholine receptors which, in some circumstances, might complicate interpretation of data related to the properties of exogenously expressed receptors.