Bromodomain Proteins Epigenetically Regulate the Mitotically Associated lncRNA MANCR in Triple Negative Breast Cancer Cells.

Long non-coding RNA (lncRNA)-mediated control of gene expression contributes to regulation of biological processes that include proliferation and phenotype, as well as compromised expression of genes that are functionally linked to cancer initiation and tumor progression. lncRNAs have emerged as novel targets and biomarkers in breast cancer. We have shown that mitotically associated lncRNA MANCR is expressed in triple-negative breast cancer (TNBC) cells and that it serves a critical role in promoting genome stability and survival in aggressive breast cancer cells. Using an siRNA strategy, we selectively depleted BRD2, BRD3, and BRD4, singly and in combination, to establish which bromodomain proteins regulate MANCR expression in TNBC cells. Our findings were confirmed by using in situ hybridization combined with immunofluorescence analysis that revealed BRD4, either alone or with BRD2 and BRD3, can support MANCR regulation of TNBC cells. Here we provide evidence for MANCR-responsive epigenetic control of super enhancers by histone modifications that are required for gene transcription to support cell survival and expression of the epithelial tumor phenotype in triple negative breast cancer cells.

Multispectral Imaging of Metabolic Fluorophores: Comparing In Vivo and Fresh Ex Vivo Tissue.

The enhanced uptake of glucose by cancer cells via aerobic glycolysis occurs when the lactic acid pathway is favored over the citric acid cycle. The lactic acid cycle in cancer cells influences the cytosolic concentration of metabolic fluorophores including NADH (the reduced form of nicotinamide adenine dinucleotide) and flavin adenine dinucleotide (FAD). In particular, the literature has shown that breast cancer influences the relative magnitude of fluorescence from NADH and FAD. A multispectral imaging system has been developed for rapid non-destructive imaging of intrinsic fluorescence in tissue. This paper compares in vivo data to fresh ex vivo data gathered as a function of time in mouse models. The data indicate that, if measured within 30 min of excision, a cancer diagnosis in fresh ex vivo tissue correlates with a cancer diagnosis in in vivo tissue. These results justify a plan to evaluate fresh ex vivo human tissue to quantify the sensitivity and specificity of the multispectral system.

LINC01638 sustains human mesenchymal stem cell self-renewal and competency for osteogenic cell fate.

The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.

LINC01638 Sustains Human Mesenchymal Stem Cell Self-Renewal and Competency for Osteogenic Cell Fate.

The skeleton forms from multipotent human mesenchymal stem cells (hMSCs) competent to commit to specific lineages. Long noncoding RNAs (lncRNAs) have been identified as key epigenetic regulators of tissue development. However, regulation of osteogenesis by lncRNAs as mediators of commitment to the bone phenotype is largely unexplored. We focused on LINC01638, which is highly expressed in hMSCs and has been studied in cancers, but not in regulating osteogenesis. We demonstrated that LINC01638 promotes initiation of the osteoblast phenotype. Our findings reveal that LINC01638 is present at low levels during the induction of osteoblast differentiation. CRISPRi knockdown of LINC01638 in MSCs prevents osteogenesis and alkaline phosphatase expression, inhibiting osteoblast differentiation. This resulted in decreased MSC cell growth rate, accompanied by double-strand breaks, DNA damage, and cell senescence. Transcriptome profiling of control and LINC01638-depleted hMSCs identified > 2,000 differentially expressed mRNAs related to cell cycle, cell division, spindle formation, DNA repair, and osteogenesis. Using ChIRP-qPCR, molecular mechanisms of chromatin interactions revealed the LINC01638 locus (Chr 22) includes many lncRNAs and bone-related genes. These novel findings identify the obligatory role for LINC01638 to sustain MSC pluripotency regulating osteoblast commitment and growth, as well as for physiological remodeling of bone tissue.

Spatiotemporal Epigenetic Control of the Histone Gene Chromatin Landscape during the Cell Cycle.

Higher-order genomic organization supports the activation of histone genes in response to cell cycle regulatory cues that epigenetically mediates stringent control of transcription at the G1/S-phase transition. Histone locus bodies (HLBs) are dynamic, non-membranous, phase-separated nuclear domains where the regulatory machinery for histone gene expression is organized and assembled to support spatiotemporal epigenetic control of histone genes. HLBs provide molecular hubs that support synthesis and processing of DNA replication-dependent histone mRNAs. These regulatory microenvironments support long-range genomic interactions among non-contiguous histone genes within a single topologically associating domain (TAD). HLBs respond to activation of the cyclin E/CDK2/NPAT/HINFP pathway at the G1/S transition. HINFP and its coactivator NPAT form a complex within HLBs that controls histone mRNA transcription to support histone protein synthesis and packaging of newly replicated DNA. Loss of HINFP compromises H4 gene expression and chromatin formation, which may result in DNA damage and impede cell cycle progression. HLBs provide a paradigm for higher-order genomic organization of a subnuclear domain that executes an obligatory cell cycle-controlled function in response to cyclin E/CDK2 signaling. Understanding the coordinately and spatiotemporally organized regulatory programs in focally defined nuclear domains provides insight into molecular infrastructure for responsiveness to cell signaling pathways that mediate biological control of growth, differentiation phenotype, and are compromised in cancer.

Spatiotemporal higher-order chromatin landscape of human histone gene clusters at histone locus bodies during the cell cycle in breast cancer progression.

Human Histone Locus Bodies (HLBs) are nuclear subdomains comprised of clustered histone genes that are coordinately regulated throughout the cell cycle. We addressed temporal-spatial higher-order genome organization for time-dependent chromatin remodeling at HLBs that supports control of cell proliferation. Proximity distances of specific genomic contacts within histone gene clusters exhibit subtle changes during the G1 phase in MCF10 breast cancer progression model cell lines. This approach directly demonstrates that the two principal histone gene regulatory proteins, HINFP (H4 gene regulator) and NPAT, localize at chromatin loop anchor-points, denoted by CTCF binding, supporting the stringent requirement for histone biosynthesis to package newly replicated DNA as chromatin. We identified a novel enhancer region located âˆ¼ 2 MB distal to histone gene sub-clusters on chromosome 6 that consistently makes genomic contacts with HLB chromatin and is bound by NPAT. During G1 progression the first DNA loops form between one of three histone gene sub-clusters bound by HINFP and the distal enhancer region. Our findings are consistent with a model that the HINFP/NPAT complex controls the formation and dynamic remodeling of higher-order genomic organization of histone gene clusters at HLBs in early to late G1 phase to support transcription of histone mRNAs in S phase.

Identification of molecularly unique tumor-associated mesenchymal stromal cells in breast cancer patients.

The tumor microenvironment is a complex mixture of cell types that bi-directionally interact and influence tumor initiation, progression, recurrence, and patient survival. Mesenchymal stromal cells (MSCs) of the tumor microenvironment engage in crosstalk with cancer cells to mediate epigenetic control of gene expression. We identified CD90+ MSCs residing in the tumor microenvironment of patients with invasive breast cancer that exhibit a unique gene expression signature. Single-cell transcriptional analysis of these MSCs in tumor-associated stroma identified a distinct subpopulation characterized by increased expression of genes functionally related to extracellular matrix signaling. Blocking the TGFß pathway reveals that these cells directly contribute to cancer cell proliferation. Our findings provide novel insight into communication between breast cancer cells and MSCs that are consistent with an epithelial to mesenchymal transition and acquisition of competency for compromised control of proliferation, mobility, motility, and phenotype.

Epigenetic-Mediated Regulation of Gene Expression for Biological Control and Cancer: Fidelity of Mechanisms Governing the Cell Cycle.

The cell cycle is governed by stringent epigenetic mechanisms that, in response to intrinsic and extrinsic regulatory cues, support fidelity of DNA replication and cell division. We will focus on (1) the complex and interdependent processes that are obligatory for control of proliferation and compromised in cancer, (2) epigenetic and topological domains that are associated with distinct phases of the cell cycle that may be altered in cancer initiation and progression, and (3) the requirement for mitotic bookmarking to maintain intranuclear localization of transcriptional regulatory machinery to reinforce cell identity throughout the cell cycle to prevent malignant transformation.

Epigenetic-Mediated Regulation of Gene Expression for Biological Control and Cancer: Cell and Tissue Structure, Function, and Phenotype.

Epigenetic gene regulatory mechanisms play a central role in the biological control of cell and tissue structure, function, and phenotype. Identification of epigenetic dysregulation in cancer provides mechanistic into tumor initiation and progression and may prove valuable for a variety of clinical applications. We present an overview of epigenetically driven mechanisms that are obligatory for physiological regulation and parameters of epigenetic control that are modified in tumor cells. The interrelationship between nuclear structure and function is not mutually exclusive but synergistic. We explore concepts influencing the maintenance of chromatin structures, including phase separation, recognition signals, factors that mediate enhancer-promoter looping, and insulation and how these are altered during the cell cycle and in cancer. Understanding how these processes are altered in cancer provides a potential for advancing capabilities for the diagnosis and identification of novel therapeutic targets.

The Shared Core Resource as a Partner in Innovative Scientific Research: Illustration from an Academic Microscopy Imaging Center.

Core facilities have a ubiquitous and increasingly valuable presence at research institutions. Although many shared cores were originally created to provide routine services and access to complex and expensive instrumentation for the research community, they are frequently called upon by investigators to design protocols and procedures to help answer complex research questions. For instance, shared microscopy resources are evolving from providing access to and training on complex imaging instruments to developing detailed innovative protocols and experimental strategies, including sample preparation techniques, staining, complex imaging parameters, and high-level image analyses. These approaches require close intellectual collaboration between core staff and research investigators to formulate and coordinate plans for protocol development suited to the research question. Herein, we provide an example of such coordinated collaboration between a shared microscopy facility and a team of scientists and clinician-investigators to approach a complex multiprobe immunostaining, imaging, and image analysis project investigating the tumor microenvironment from human breast cancer samples. Our hope is that this example may be used to convey to institute administrators the critical importance of the intellectual contributions of the scientific staff in core facilities to research endeavors.

LncMIR181A1HG is a novel chromatin-bound epigenetic suppressor of early stage osteogenic lineage commitment.

Bone formation requires osteogenic differentiation of multipotent mesenchymal stromal cells (MSCs) and lineage progression of committed osteoblast precursors. Osteogenic phenotype commitment is epigenetically controlled by genomic (chromatin) and non-genomic (non-coding RNA) mechanisms. Control of osteogenesis by long non-coding RNAs remains a largely unexplored molecular frontier. Here, we performed comprehensive transcriptome analysis at early stages of osteogenic cell fate determination in human MSCs, focusing on expression of lncRNAs. We identified a chromatin-bound lncRNA (MIR181A1HG) that is highly expressed in self-renewing MSCs. MIR181A1HG is down-regulated when MSCs become osteogenic lineage committed and is retained during adipogenic differentiation, suggesting lineage-related molecular functions. Consistent with a key role in human MSC proliferation and survival, we demonstrate that knockdown of MIR181A1HG in the absence of osteogenic stimuli impedes cell cycle progression. Loss of MIR181A1HG enhances differentiation into osteo-chondroprogenitors that produce multiple extracellular matrix proteins. RNA-seq analysis shows that loss of chromatin-bound MIR181A1HG alters expression and BMP2 responsiveness of skeletal gene networks (e.g., SOX5 and DLX5). We propose that MIR181A1HG is a novel epigenetic regulator of early stages of mesenchymal lineage commitment towards osteo-chondroprogenitors. This discovery permits consideration of MIR181A1HG and its associated regulatory pathways as targets for promoting new bone formation in skeletal disorders.

Hinfp is a guardian of the somatic genome by repressing transposable elements.

Germ cells possess the Piwi-interacting RNA pathway to repress transposable elements and maintain genome stability across generations. Transposable element mobilization in somatic cells does not affect future generations, but nonetheless can lead to pathological outcomes in host tissues. We show here that loss of function of the conserved zinc-finger transcription factor Hinfp causes dysregulation of many host genes and derepression of most transposable elements. There is also substantial DNA damage in somatic tissues of Drosophila after loss of Hinfp. Interference of transposable element mobilization by reverse-transcriptase inhibitors can suppress some of the DNA damage phenotypes. The key cell-autonomous target of Hinfp in this process is Histone1, which encodes linker histones essential for higher-order chromatin assembly. Transgenic expression of Hinfp or Histone1, but not Histone4 of core nucleosome, is sufficient to rescue the defects in repressing transposable elements and host genes. Loss of Hinfp enhances Ras-induced tissue growth and aging-related phenotypes. Therefore, Hinfp is a physiological regulator of Histone1-dependent silencing of most transposable elements, as well as many host genes, and serves as a venue for studying genome instability, cancer progression, neurodegeneration, and aging.

Higher order genomic organization and regulatory compartmentalization for cell cycle control at the G1/S-phase transition.

Fidelity of histone gene regulation, and ultimately of histone protein biosynthesis, is obligatory for packaging of newly replicated DNA into chromatin. Control of histone gene expression within the 3-dimensional context of nuclear organization is reflected by two well documented observations. DNA replication-dependent histone mRNAs are synthesized at specialized subnuclear domains designated histone locus bodies (HLBs), in response to activation of the growth factor dependent Cyclin E/CDK2/HINFP/NPAT pathway at the G1/S transition in mammalian cells. Complete loss of the histone gene regulatory factors HINFP or NPAT disrupts HLB integrity that is necessary for coordinate control of DNA replication and histone gene transcription. Here we review the molecular histone-related requirements for G1/S-phase progression during the cell cycle. Recently developed experimental strategies, now enable us to explore mechanisms involved in dynamic control of histone gene expression in the context of the temporal (cell cycle) and spatial (HLBs) remodeling of the histone gene loci.

Mitotically-Associated lncRNA (MANCR) Affects Genomic Stability and Cell Division in Aggressive Breast Cancer.

Aggressive breast cancer is difficult to treat as it is unresponsive to many hormone-based therapies; therefore, it is imperative to identify novel, targetable regulators of progression. Long non-coding RNAs (lncRNA) are important regulators in breast cancer and have great potential as therapeutic targets; however, little is known about how the majority of lncRNAs function within breast cancer. This study characterizes a novel lncRNA, MANCR (mitotically-associated long noncoding RNA; LINC00704), which is upregulated in breast cancer patient specimens and cells. Depletion of MANCR in triple-negative breast cancer cells significantly decreases cell proliferation and viability, with concomitant increases in DNA damage. Transcriptome analysis, based on RNA sequencing, following MANCR knockdown reveals significant differences in the expression of >2,000 transcripts, and gene set enrichment analysis identifies changes in multiple categories related to cell-cycle regulation. Furthermore, MANCR expression is highest in mitotic cells by both RT-qPCR and RNA in situ hybridization. Consistent with a role in cell-cycle regulation, MANCR-depleted cells have a lower mitotic index and higher incidences of defective cytokinesis and cell death. Taken together, these data reveal a role for the novel lncRNA, MANCR, in genomic stability of aggressive breast cancer, and identify it as a potential therapeutic target.Implications: The novel lncRNA, MANCR (LINC00704), is upregulated in breast cancer and is functionally linked with cell proliferation, viability, and genomic stability. Mol Cancer Res; 16(4); 587-98. ©2018 AACR.

Intranuclear and higher-order chromatin organization of the major histone gene cluster in breast cancer.

Alterations in nuclear morphology are common in cancer progression. However, the degree to which gross morphological abnormalities translate into compromised higher-order chromatin organization is poorly understood. To explore the functional links between gene expression and chromatin structure in breast cancer, we performed RNA-seq gene expression analysis on the basal breast cancer progression model based on human MCF10A cells. Positional gene enrichment identified the major histone gene cluster at chromosome 6p22 as one of the most significantly upregulated (and not amplified) clusters of genes from the normal-like MCF10A to premalignant MCF10AT1 and metastatic MCF10CA1a cells. This cluster is subdivided into three sub-clusters of histone genes that are organized into hierarchical topologically associating domains (TADs). Interestingly, the sub-clusters of histone genes are located at TAD boundaries and interact more frequently with each other than the regions in-between them, suggesting that the histone sub-clusters form an active chromatin hub. The anchor sites of loops within this hub are occupied by CTCF, a known chromatin organizer. These histone genes are transcribed and processed at a specific sub-nuclear microenvironment termed the major histone locus body (HLB). While the overall chromatin structure of the major HLB is maintained across breast cancer progression, we detected alterations in its structure that may relate to gene expression. Importantly, breast tumor specimens also exhibit a coordinate pattern of upregulation across the major histone gene cluster. Our results provide a novel insight into the connection between the higher-order chromatin organization of the major HLB and its regulation during breast cancer progression.

Precocious Phenotypic Transcription-Factor Expression During Early Development.

A novel role for phenotypic transcription factors in very early differentiation was recently observed and merits further study to elucidate what role this precocious expression may have in development. The RUNX1 transcription factor exhibits selective and transient upregulation during early mesenchymal differentiation. In contrast to phenotype-associated transcriptional control of gene expression to establish and sustain hematopoietic/myeloid lineage identity, precocious expression of RUNX1 is functionally linked to control of an epithelial to mesenchymal transition that is obligatory for development. This early RUNX1 expression spike provides a paradigm for precocious expression of a phenotypic transcription factor that invites detailed mechanistic study to fully understand its biological importance. J. Cell. Biochem. 118: 953-958, 2017. © 2016 Wiley Periodicals, Inc.

Unique Regulatory Mechanisms for the Human Embryonic Stem Cell Cycle.

The cell cycle in pluripotent human embryonic stem cells is governed by unique mechanisms that support unrestricted proliferation and competency for endodermal, mesodermal, and ectodermal differentiation. The abbreviated G1 period with retention of uncompromised fidelity for genetic and epigenetic mechanisms operative in control of proliferation support competency for expansion of the pluripotent cell population that is fundamental for initial stages of development. Regulatory events during the G1 period of the pluripotent cell cycle are decisive for the transition from pluripotency to lineage commitment. Recent findings indicate that a G2 cell cycle pause is present in both endodermal and mesodermal lineage cells, and is obligatory for differentiation to endoderm. J. Cell. Physiol. 232: 1254-1257, 2017. © 2016 Wiley Periodicals, Inc.

Transient RUNX1 Expression during Early Mesendodermal Differentiation of hESCs Promotes Epithelial to Mesenchymal Transition through TGFB2 Signaling.

The transition of human embryonic stem cells (hESCs) from pluripotency to lineage commitment is not fully understood, and a role for phenotypic transcription factors in the initial stages of hESC differentiation remains to be explored. From a screen of candidate factors, we found that RUNX1 is selectively and transiently upregulated early in hESC differentiation to mesendodermal lineages. Transcriptome profiling and functional analyses upon RUNX1 depletion established a role for RUNX1 in promoting cell motility. In parallel, we discovered a loss of repression for several epithelial genes, indicating that loss of RUNX1 impaired an epithelial to mesenchymal transition during differentiation. Cell biological and biochemical approaches revealed that RUNX1 depletion specifically compromised TGFB2 signaling. Both the decrease in motility and deregulated epithelial marker expression upon RUNX1 depletion were rescued by reintroduction of TGFB2, but not TGFB1. These findings identify roles for RUNX1-TGFB2 signaling in early events of mesendodermal lineage commitment.

Maternal expression and early induction of histone gene transcription factor Hinfp sustains development in pre-implantation embryos.

Fidelity of histone gene expression is important for normal cell growth and differentiation that is stringently controlled during development but is compromised during tumorigenesis. Efficient production of histones for packaging newly replicated DNA is particularly important for proper cell division and epigenetic control during the initial pre-implantation stages of embryonic development. Here, we addressed the unresolved question of when the machinery for histone gene transcription is activated in the developing zygote to accommodate temporal demands for histone gene expression. We examined induction of Histone Nuclear Factor P (HINFP), the only known transcription factor required for histone H4 gene expression, that binds directly to a unique H4 promoter-specific element to regulate histone H4 transcription. We show that Hinfp gene transcripts are stored in oocytes and maternally transmitted to the zygote. Transcripts from the paternal Hinfp gene, which reflect induction of zygotic gene expression, are apparent at the 4- to 8-cell stage, when most maternal mRNA pools are depleted. Loss of Hinfp expression due to gene ablation reduces cell numbers in E3.5 stage embryos and compromises implantation. Reduced cell proliferation is attributable to severe reduction in histone mRNA levels accompanied by reduced cell survival and genomic damage as measured by cleaved Caspase 3 and phospho-H2AX staining, respectively. We conclude that transmission of maternal Hinfp transcripts and zygotic activation of the Hinfp gene together are necessary to control H4 gene expression in early pre-implantation embryos in order to support normal embryonic development.