Masaru Miyano

Loss of silent-chromatin looping and impaired imprinting of DLX5 in Rett syndrome

Mutations in MECP2 are associated with Rett syndrome, an X-linked neurodevelopmental disorder. To identify genes targeted by Mecp2, we sequenced 100 in vivo Mecp2-binding sites in mouse brain. Several sequences mapped to an imprinted gene cluster on chromosome 6, including Dlx5 and Dlx6, whose transcription was roughly two times greater in brains of Mecp2-null mice compared with those of wild-type mice. The maternally expressed gene DLX5 showed a loss of imprinting in lymphoblastoid cells from individuals with Rett syndrome. Because Dlx5 regulates production of enzymes that synthesize γ-aminobutyric acid (GABA), loss of imprinting of Dlx5 may alter GABAergic neuron activity in individuals with Rett syndrome. In mouse brain, Dlx5 imprinting was relaxed, yet Mecp2-mediated silent-chromatin structure existed at the Dlx5-Dlx6 locus in brains of wild-type, but not Mecp2-null, mice. Mecp2 targeted histone deacetylase 1 to a sharply defined, ∼1-kb region at the Dlx5-Dlx6 locus and promoted repressive histone methylation at Lys9 at this site. Chromatin immunoprecipitation–combined loop assays showed that Mecp2 mediated the silent chromatin–derived 11-kb chromatin loop at the Dlx5-Dlx6 locus. This loop was absent in chromatin of brains of Mecp2-null mice, and Dlx5-Dlx6 interacted with far distant sequences, forming distinct active chromatin–associated loops. These results show that formation of a silent-chromatin loop is a new mechanism underlying gene regulation by Mecp2.

SATB1 targets chromatin remodelling to regulate genes over long distances.

Eukaryotic chromosomes are organized inside the nucleus in such a way that only a subset of the genome is expressed in any given cell type, but the details of this organization are largely unknown. SATB1 (‘special AT-rich sequence binding 1’), a protein found predominantly in thymocytes, regulates genes by folding chromatin into loop domains, tethering specialized DNA elements to an SATB1 network structure. Ablation of SATB1 by gene targeting results in temporal and spatial mis-expression of numerous genes and arrested T-cell development, suggesting that SATB1 is a cell-type specific global gene regulator. Here we show that SATB1 targets chromatin remodelling to the IL-2Rα (‘interleukin-2 receptor α’) gene, which is ectopically transcribed in SATB1 null thymocytes. SATB1 recruits the histone deacetylase contained in the NURD chromatin remodelling complex to a SATB1-bound site in the IL-2Rα locus, and mediates the specific deacetylation of histones in a large domain within the locus. SATB1 also targets ACF1 and ISWI, subunits of CHRAC and ACF nucleosome mobilizing complexes, to this specific site and regulates nucleosome positioning over seven kilobases. SATB1 defines a class of transcriptional regulators that function as a ‘landing platform’ for several chromatin remodelling enzymes and hence regulate large chromatin domains.

Disinfection of E. coli by nonthermal microplasma electrolysis in normal saline solution

We present a unique method to inactivate microorganisms in 0.9% NaCl solution (normal saline solution) by means of microplasmas. The device consists of a thin titanium wire covered by a glass tube for insulation except the tip and a ground electrode. Application of an asymmetric high-frequency, high voltage results in the formation of microbubbles at both electrodes. Repetitive light emission is observed in the vicinity of the powered electrode. We employed E. coli bacteria to investigate the disinfection efficiency of the device. More than 99.5% of E. coli were deactivated in 180 s. The survival curve showed biphasic behavior.

Age-related dysfunction in mechanotransduction impairs differentiation of human mammary epithelial progenitors.

Dysfunctional progenitor and luminal cells with acquired basal cell properties accumulate during human mammary epithelial aging for reasons not understood. Multipotent progenitors from women aged <30 years were exposed to a physiologically relevant range of matrix elastic modulus (stiffness). Increased stiffness causes a differentiation bias towards myoepithelial cells while reducing production of luminal cells and progenitor maintenance. Lineage representation in progenitors from women >55 years is unaffected by physiological stiffness changes. Efficient activation of Hippo pathway transducers YAP and TAZ is required for the modulus-dependent myoepithelial/basal bias in younger progenitors. In older progenitors, YAP and TAZ are activated only when stressed with extraphysiologically stiff matrices, which bias differentiation towards luminal-like phenotypes. In vivo YAP is primarily active in myoepithelia of younger breasts, but localization and activity increases in luminal cells with age. Thus, aging phenotypes of mammary epithelia may arise partly because alterations in Hippo pathway activation impair microenvironment-directed differentiation and lineage specificity.

DLX5 expression is monoallelic and Dlx5 is up-regulated in the Mecp2-null frontal cortex

There has been a controversy as to whether DLX5 is imprinted in human brain and lymphoblastoid cell lines (LCLs), and whether expression of Dlx5 and Dlx6 (Dlx5/6 exist as a bigene cluster [1]), is dysregulated in the Mecp2-null mouse frontal cortex. In the September issue of Am J Hum Genet. (Vol 81: 492–506, 2007) [2], Schüle et al. published a paper entitled ‘DLX5 and DLX6 expression is bi-allelic and not modulated by Mecp2 deficiency.’ This study concluded that DLX5 was not imprinted in normal human LCLs nor in human brain, contradicting the conclusions of Okita et al. (Genomics 81:556–559, 2003) [3] and Horike et al. (Nature Genetics 37:31–40, 2005) [4]. Schüle et al. also claimed that expression of Dlx5 and Dlx6 in mouse brain varies greatly among individual mice and is not necessarily up-regulated in the frontal cortex of Mecp2-null mice. These findings directly contradict those of Horike et al. We have now repeated and verified our previous experiments that are relevant to the points raised by Schüle et al. Further, we have also expanded our analyses to determine why Schüle et al., were not able to reproduce our findings. Here are our findings in response to their assertions. (1) Schüle et al. presented quantitative RT-PCR (qRT-PCR) data obtained from frontal cortex tissue samples of male wild-type and Mecp2-null mice from several litters. They reported that the expression levels of Dlx5, Dlx6 and the imprinted gene Peg3 were all highly variable among individual mice, independent of genotype (0.5–3.5-fold difference relative to a single wild-type mouse selected among several litters). Schüle et al. therefore concluded that the findings of Horike et al. – that there is a 2-fold increase in expression of Dlx5 and Dlx6 in Mecp2-null mice compared to wild type littermates – were the result of ‘biological noise.’ Considering that these genes are tightly regulated during development, we were surprised at their observations that their expression was completely random even among wild type animals from the same litter. We repeated these experiments using three sets of littermates of Mecp2-null and wild-type mice (42 or 51 days old) from an independent breeding colony of Mecp2-heterozygous mice (generated by Adrian Bird’s laboratory) [5] kindly provided by Masayuki Itoh (National Institute of Neuroscience, Japan), who carefully bred the Mecp2 heterozygous mice to retain the original phenotype. In all five Mecp2-null mice examined using Gapdh as a control, we were able to reproduce the ~2fold (1.7–2.6-fold) increase in expression of Dlx5 in Mecp2-null mice, compared with wild-type mice (Fig. 1A), just as originally reported by Horike et al. We similarly verified up-regulation of Dlx6 in the same brain subregions of the Mecp2-null mice tested (representative data are shown in Fig. 1B). We further examined expression levels of Peg3, which Schüle et al. also report as having high variability in expression. In contrast to their findings, we observed consistent expression levels with a small standard error of the mean (S.E.M.) between mice of the same genotype and saw similar levels in the frontal cortex between wild-type and Mecp2-null mice (Fig. 1C). Expression levels of genes in brains are best compared among littermates, since mice from different litters often show some variability in gene expression. Nonetheless, in our study, difference in expression levels of the control Gapdh typically do not exceed more than 20%, even among mice from different litters. Therefore, our results consistently show non-random expression of Dlx5 and Dlx6 relative to Gapdh among littermates, accurately reflecting the genotype of individual mice within each litter. In the frontal cortex of Mecp2-null mice, Dlx5 and Dlx6 are moderately up-regulated compared to wild-type mice, while there is no change in expression for Peg3. The differences between our results and those of Schüle et al. likely arose from differences in reproducibility in dissection of the frontal cortex and in the purity/quality of RNA; RNA degradation can dramatically alter qRT-PCR results. Because Dlx5 is expressed DLX5 expression is monoallelic and Dlx5 is up-regulated

Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16INK4A, or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

Characterizing cellular mechanical phenotypes with mechano-node-pore sensing

The mechanical properties of cells change with their differentiation, chronological age, and malignant progression. Consequently, these properties may be useful label-free biomarkers of various functional or clinically relevant cell states. Here, we demonstrate mechano-node-pore sensing (mechano-NPS), a multi-parametric single-cell-analysis method that utilizes a four-terminal measurement of the current across a microfluidic channel to quantify simultaneously cell diameter, resistance to compressive deformation, transverse deformation under constant strain, and recovery time after deformation. We define a new parameter, the whole-cell deformability index (wCDI), which provides a quantitative mechanical metric of the resistance to compressive deformation that can be used to discriminate among different cell types. The wCDI and the transverse deformation under constant strain show malignant MCF-7 and A549 cell lines are mechanically distinct from non-malignant, MCF-10A and BEAS-2B cell lines, and distinguishes between cells treated or untreated with cytoskeleton-perturbing small molecules. We categorize cell recovery time, ΔTr, as instantaneous (ΔTr~0 ms), transient (ΔTr⩽40 ms), or prolonged (ΔTr>40 ms), and show that the composition of recovery types, which is a consequence of changes in cytoskeletal organization, correlates with cellular transformation. Through the wCDI and cell-recovery time, mechano-NPS discriminates between sub-lineages of normal primary human mammary epithelial cells with accuracy comparable to flow cytometry, but without antibody labeling. Mechano-NPS identifies mechanical phenotypes that distinguishes lineage, chronological age, and stage of malignant progression in human epithelial cells. Supplementary information The online version of this article (doi:10.1038/micronano.2017.91) contains supplementary material, which is available to authorized users.

Age-related gene expression in luminal epithelial cells is driven by a microenvironment made from myoepithelial cells

Luminal epithelial cells in the breast gradually alter gene and protein expression with age, appearing to lose lineage-specificity by acquiring myoepithelial-like characteristics. We hypothesize that the luminal lineage is particularly sensitive to microenvironment changes, and age-related microenvironment changes cause altered luminal cell phenotypes. To evaluate the effects of different microenvironments on the fidelity of epigenetically regulated luminal and myoepithelial gene expression, we generated a set of lineage-specific probes for genes that are controlled through DNA methylation. Culturing primary luminal cells under conditions that favor myoepithelial propogation led to their reprogramming at the level of gene methylation, and to a more myoepithelial-like expression profile. Primary luminal cells’ lineage-specific gene expression could be maintained when they were cultured as bilayers with primary myoepithelial cells. Isogenic stromal fibroblast co-cultures were unable to maintain the luminal phenotype. Mixed-age luminal-myoepithelial bilayers revealed that luminal cells adopt transcription and methylation patterns consistent with the chronological age of the myoepithelial cells. We provide evidence that the luminal epithelial phenotype is exquisitely sensitive to microenvironment conditions, and that states of aging are cell non-autonomously communicated through microenvironment cues over at least one cell diameter.

Abstract A24: Age- and lineage-dependent gene expression is maintained by microenvironment imposed epigenetic states in human mammary epithelial cells

Normal healthy tissues show changing patterns of gene expression as a consequence of aging, and there is a functional cost to these changes that can be relevant to disease pathology. The most obvious age-related disease in breast is cancer, with the large majority of newly diagnosed breast cancer occuring in women over 50. We have shown that breast gene expression changes that occur with age have functional consequences in the epithelial progenitor and differentiated cells, i.e, a decline of the myoepithelial lineage, loss of luminal cell specificity, and accumulation of differentiation defective multipotent progenitor cells. We have hypothesized that these tissue-level changes make older epithelia more susceptible to transformation. Because gene expression patterns reflect the wiring and response diagrams of cells, it is of central importance to understand the origins of age-related transcriptomes. In our studies, early passage normal human mammary epithelial cells (HMEC) show age-dependent functional and molecular hallmarks consistent with in vivo, suggesting that the underlying gene expression patterns are metastable. DNA methylation is a stable, but malleable, form of epigenetic regulation that may underpin these biologically metastable states. Genome-wide analysis of primary epithelia was used to identify a set of genes that exhibit age- and lineage-specific expression that was inversely correlated with promoter CpG methylation. Chemical perturbation of DNA methylation in pre-menopausal HMEC resulted in a biochemical phenocopy of more advanced age. Tissue-mimetic cultures were used to demonstrate that lineage-specific gene expression and methylation in luminal cells were imposed by distinct microenvironments. Optimal maintenance of the luminal phenotype required direct contact with the apical surface of myoepithelial cells. Mimetic tissues built with HMEC that differed in chronological donor age revealed that age-dependent gene expression and methylation patterns are communicated between the two different lineages, as exposure of pre-menopausal luminal cells to a post-menopausal microenvironment imposed transcriptional patterns in luminal cells consistent with post-menopause. These data demonstrate that lineage- and age-dependent phenotypes in HMEC are maintained by microenvironment-imposed metastable epigenetic states. Note: This abstract was not presented at the conference. Citation Format: Masaru Miyano, Marcus Stoiber, Martha Stampfer, Ben Brown, Mark A. LaBarge. Age- and lineage-dependent gene expression is maintained by microenvironment imposed epigenetic states in human mammary epithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A24.

Abstract A112: The role of microenvironment in age-related breast cancers

Aside from being a woman, age is the greatest risk factor for developing breast cancer, but there is little understanding how the aging process modifies cancer susceptibility. Multipotent mammary epithelial progenitors are putative roots of some breast cancers, thus expansion combined with increased vulnerability of these cells to transformation could potentiate cancer progression. We have shown in humans that with age, the proportion of multipotent progenitors accumulates, whereas myoepithelial cells decrease, and that these shifts have a basis in altered progenitor function. Progenitors from post-menopausal women gave rise to incompletely differentiated myoepithelial cells and to basal-like luminal cells that expressed markers normally associated with myoepithelial cells in young women. Because epithelial progenitors are thought to be cells-of-origin for a number of breast cancers, and myoepithelial cells are thought to be tumor suppressive, we hypothesize that these tissue-level changes make post-menopausal women more vulnerable to malignant progression. Age-related tissue changes arise in parallel with age-dependent gene expression patterns that have unknown functional consequences and unknown origins. Here we demonstrate that utilizing biomimetic substrata with tuned physical and molecular properties to probe primary human mammary progenitors from women aged 16 to 91 years can reveal age-related functional consequences. As increased breast density is associated with greater breast cancer risk and stiff mechanical microenvironments are known to exacerbate malignant cell behavior, we examined how normal progenitors responded to changes in elastic modulus. Age-related changes in Rho and HIPPO pathway mechano-transduction and transcriptional regulation contributed to accumulation of progenitors by preventing normal differentiation in response to mechanical stimuli. Young progenitors gave rise to more luminal cells on compliant substrata and to more myoepithelial cells on stiffer substrata. However, post-menopausal progenitors were insensitive to physiological mechanical changes and differentiated stochastically instead. To better understand the genesis of the gene expression patterns underlying these age-related functional states, molecular analyses of co-cultures established with primary myoepithelial and luminal epithelial lineages from combinations of young and old individuals were preformed. Age-related gene expression states in luminal cells were imposed through heterotypic cell-cell interactions. Changes in gene expression were coincident with changes in DNA promoter methylation and expression of histone modifying enzymes. We propose that aging results in a continuum of microenvironmental changes that impose epigenetically regulated metastable gene expression states, which are the basis of age-related cell functions that ultimately lead to vulnerable tissue-level phenotypes. Citation Format: Fanny A. Pelissier, Masaru Miyano, James C. Garbe, Martha R. Stampfer, Mark A. LaBarge. The role of microenvironment in age-related breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A112.