Tumor progression and chromatin landscape of lung cancer are regulated by the lineage factor GATA6.

Lineage selective transcription factors (TFs) are important regulators of tumorigenesis, but their biological functions are often context dependent with undefined epigenetic mechanisms of action. In this study, we uncover a conditional role for the endodermal and pulmonary specifying TF GATA6 in lung adenocarcinoma (LUAD) progression. Impairing Gata6 in genetically engineered mouse models reduces the proliferation and increases the differentiation of Kras mutant LUAD tumors. These effects are influenced by the epithelial cell type that is targeted for transformation and genetic context of Kras-mediated tumor initiation. In LUAD cells derived from surfactant protein C expressing progenitors, we identify multiple genomic loci that are bound by GATA6. Moreover, suppression of Gata6 in these cells significantly alters chromatin accessibility, particularly at distal enhancer elements. Analogous to its paradoxical activity in lung development, GATA6 expression fluctuates during different stages of LUAD progression and can epigenetically control diverse transcriptional programs associated with bone morphogenetic protein signaling, alveolar specification, and tumor suppression. These findings reveal how GATA6 can modulate the chromatin landscape of lung cancer cells to control their proliferation and divergent lineage dependencies during tumor progression.

Transcriptomic Hallmarks of Tumor Plasticity and Stromal Interactions in Brain Metastasis.

The brain is a major site of relapse for several cancers, yet deciphering the mechanisms of brain metastasis remains a challenge because of the complexity of the brain tumor microenvironment (TME). To define the molecular landscape of brain metastasis from intact tissue in vivo, we employ an RNA-sequencing-based approach, which leverages the transcriptome of xenografts and distinguishes tumor cell and stromal gene expression with improved sensitivity and accuracy. Our data reveal shifts in epithelial and neuronal-like lineage programs in malignant cells as they adapt to the brain TME and the reciprocal neuroinflammatory response of the stroma. We identify several transcriptional hallmarks of metastasis that are specific to particular regions of the brain, induced across multiple tumor types, and confirmed in syngeneic models and patient biopsies. These data may serve as a resource for exploring mechanisms of TME co-adaptation within, as well as across, different subtypes of brain metastasis.

NG2 expression in NG2 glia is regulated by binding of SoxE and bHLH transcription factors to a Cspg4 intronic enhancer.

NG2 is a type 1 integral membrane glycoprotein encoded by the Cspg4 gene. It is expressed on glial progenitor cells known as NG2 glial cells or oligodendrocyte precursor cells that exist widely throughout the developing and mature central nervous system and vascular mural cells but not on mature oligodendrocytes, astrocytes, microglia, neurons, or neural stem cells. Hence NG2 is widely used as a marker for NG2 glia in the rodent and human. The regulatory elements of the mouse Cspg4 gene and its flanking sequences have been used successfully to target reporter and Cre recombinase to NG2 glia in transgenic mice when used in a large 200 kb bacterial artificial chromosome cassette containing the 38 kb Cspg4 gene in the center. Despite the tightly regulated cell type- and stage-specific expression of NG2 in the brain and spinal cord, the mechanisms that regulate its transcription have remained unknown. Here, we describe a 1.45 kb intronic enhancer of the mouse Cspg4 gene that directed transcription of EGFP reporter to NG2 glia but not to pericytes in vitro and in transgenic mice. The 1.45 kb enhancer contained binding sites for SoxE and basic helix-loop-helix transcription factors, and its enhancer activity was augmented cooperatively by these factors, whose respective binding elements were found in close proximity to each other. Mutations in these binding elements abrogated the enhancer activity when tested in the postnatal mouse brain.

Lineage, fate, and fate potential of NG2-glia.

NG2 cells represent a fourth major glial cell population in the mammalian central nervous system (CNS). They arise from discrete germinal zones in mid-gestation embryos and expand to occupy the entire CNS parenchyma. Genetic fate mapping studies have shown that oligodendrocytes and a subpopulation of ventral protoplasmic astrocytes arise from NG2 cells. This review describes recent findings on the fate and fate potential of NG2 cells under physiological and pathological conditions. We discuss age-dependent changes in the fate and fate potential of NG2 cells and possible mechanisms that could be involved in restricting their oligodendrocyte differentiation or fate plasticity. This article is part of a Special Issue entitled SI:NG2-glia(Invited only).

Modulation of oligodendrocyte generation during a critical temporal window after NG2 cell division.

Oligodendrocytes in the mammalian brain are continuously generated from NG2 cells throughout postnatal life. However, it is unclear when the decision is made for NG2 cells to self-renew or differentiate into oligodendrocytes after cell division. Using a combination of in vivo and ex vivo imaging and fate analysis of proliferated NG2 cells in fixed tissue, we demonstrate that in the postnatal developing mouse brain, the majority of divided NG2 cells differentiate into oligodendrocytes during a critical age-specific temporal window of 3-8 d. Notably, within this time period, damage to myelin and oligodendrocytes accelerated oligodendrocyte differentiation from divided cells, and whisker removal decreased the survival of divided cells in the deprived somatosensory cortex. These findings indicate that during the critical temporal window of plasticity, the fate of divided NG2 cells is sensitive to modulation by external signals.

Organotypic slice cultures to study oligodendrocyte dynamics and myelination.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

Disrupted in schizophrenia 1 modulates medial prefrontal cortex pyramidal neuron activity through cAMP regulation of transient receptor potential C and small-conductance K+ channels.

BACKGROUND: Disrupted in schizophrenia 1 (DISC1) is a protein implicated in schizophrenia, bipolar disorder, major depressive disorder, and autism. To date, most of research examining DISC1 function has focused on its role in neurodevelopment, despite its presence throughout life. DISC1 also regulates cyclic adenosine monophosphate (cAMP) signaling by increasing type 4 phosphodiesterase catabolism of cAMP when cAMP concentrations are high. In this study, we tested the hypothesis that DISC1, through its regulation of cAMP, modulates I-SK and I-TRPC channel-mediated ionic currents that we have shown previously to regulate the activity of mature prefrontal cortical pyramidal neurons. METHODS: We used patch-clamp recordings in prefrontal cortical slices from adult rats in which DISC1 function was reduced in vivo by short hairpin RNA viral knockdown or in vitro by dialysis of DISC1 antibodies. RESULTS: We found that DISC1 disruption resulted in an increase of metabotropic glutamate receptor-induced intracellular calcium (Ca2+) waves, small-conductance K+ (SK)-mediated hyperpolarization and a decrease of transient receptor potential C (TRPC)-mediated sustained depolarization. Consistent with a role for DISC1 in regulation of cAMP signaling, forskolin-induced cAMP production also increased intracellular Ca2+ waves, I-SK and decreased I-TRPC. Lastly, inhibiting cAMP generation with guanfacine, an α2A-noradrenergic agonist, normalized the function of SK and TRPC channels. CONCLUSIONS: Based on our findings, we propose that diminished DISC1 function, such as occurs in some mental disorders, can lead to the disruption of normal patterns of prefrontal cortex activity through the loss of cAMP regulation of metabotropic glutamate receptor-mediated intracellular Ca2+ waves, SK and TRPC channel activity.

NG2 cells in white matter but not gray matter proliferate in response to PDGF.

Glial cells that express the NG2 proteoglycan and the α receptor for PDGF (NG2 cells, polydendrocytes) make up the fifth major cell population that serves as oligodendrocyte progenitor cells in the postnatal CNS. Although recent studies have suggested differences in their proliferation and oligodendrocyte differentiation in gray and white matter, the mechanism underlying the observed differences has been unclear. Using organotypic slice cultures from the forebrain and cerebellum of early postnatal NG2creBAC:ZEG mice, we have compared basal and growth factor-induced proliferation of NG2 cells in gray and white matter. NG2 cells in white matter exhibited greater proliferative response to PDGF AA than those in gray matter. Heterotopic slice transplant and explant cultures suggested intrinsic mechanisms for the differential proliferative response of gray and white matter cells. Additionally, younger white matter NG2 cells showed a more robust proliferative response to PDGF. Basal and PDGF-induced proliferation of gray and white matter NG2 cells was largely dependent on Wnt/ß-catenin and phosphatidylinositol 3-kinase acting through the mammalian target of rapamycin pathway and not through ERK. These data uncover a previously unrecognized divergence between gray and white matter NG2 cells in the developing brain in their proliferative response to PDGF.