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In 2021, the World Health Organization reclassified glioblastoma, the most common form of adult brain cancer, into isocitrate dehydrogenase (IDH)-wild-type glioblastomas and grade IV IDH mutant (G4 IDHm) astrocytomas. For both tumor types, intratumoral heterogeneity is a key contributor to therapeutic failure. To better define this heterogeneity, genome-wide chromatin accessibility and transcription profiles of clinical samples of glioblastomas and G4 IDHm astrocytomas were analyzed at single-cell resolution. These profiles afforded resolution of intratumoral genetic heterogeneity, including delineation of cell-to-cell variations in distinct cell states, focal gene amplifications, as well as extrachromosomal circular DNAs. Despite differences in IDH mutation status and significant intratumoral heterogeneity, the profiled tumor cells shared a common chromatin structure defined by open regions enriched for nuclear factor 1 transcription factors (NFIA and NFIB). Silencing of NFIA or NFIB suppressed in vitro and in vivo growths of patient-derived glioblastomas and G4 IDHm astrocytoma models. These findings suggest that despite distinct genotypes and cell states, glioblastoma/G4 astrocytoma cells share dependency on core transcriptional programs, yielding an attractive platform for addressing therapeutic challenges associated with intratumoral heterogeneity.

Monocytes are progenitors to macrophages and a subclass of dendritic cells (monocyte-derived dendritic cells, MoDCs), but they also act as circulating sensors that respond to environmental changes and disease. Technological advances have defined the production of classical monocytes in the bone marrow through the identification of lineage-determining transcription factors (LDTFs) and have proposed alternative routes of differentiation. Monocytes released into the circulation can be recruited to tissues by specific chemoattractants where they respond to sequential niche-specific signals that determine their differentiation into terminal effector cells. New aspects of monocyte biology in the circulation are being revealed, exemplified by the influence of cancer on the systemic alteration of monocyte subset abundance and transcriptional profiles. These changes can act to enhance the metastatic spread of primary cancers and may offer therapeutic opportunities.

There is a growing body of evidence that cancer causes systemic changes. These influences are most evident in the bone marrow and the blood, particularly in the myeloid compartment. Here, we show that there is an increase in the number of bone marrow, circulating and splenic monocytes by using mouse models of breast cancer caused by the mammary epithelial expression of the polyoma middle T antigen. Cancer does not affect ratios of classical to non-classical populations of monocytes in the circulation nor does it affect their half-lives. Single cell RNA sequencing also indicates that cancer does not induce any new monocyte populations. Cancer does not change the monocytic progenitor number in the bone marrow, but the proliferation rate of monocytes is higher, thus providing an explanation for the expansion of the circulating numbers. Deep RNA sequencing of these monocytic populations reveals that cancer causes changes in the classical monocyte compartment, with changes evident in bone marrow monocytes and even more so in the blood, suggesting influences in both compartments, with the down-regulation of interferon type 1 signaling and antigen presentation being the most prominent of these. Consistent with this analysis, down-regulated genes are enriched with STAT1/STAT2 binding sites in their promoter, which are transcription factors required for type 1 interferon signaling. However, these transcriptome changes in mice did not replicate those found in patients with breast cancer. Consequently, this mouse model of breast cancer may be insufficient to study the systemic influences of human cancer.

Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese hamster genome sequence were computationally predicted and are frequently inaccurate. Here, we use nascent transcription start site sequencing methods to revise TSS annotations for 15 308 Chinese hamster genes and 3034 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. We further capture tens of thousands of putative transcribed enhancer regions with this method. Our revised TSSs improves upon the RefSeq annotation by revealing core sequence features of gene regulation such as the TATA box and the Initiator and, as exemplified by targeting the glycosyltransferase gene Mgat3, facilitate activating silent genes by CRISPRa. Together, we envision our revised annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

Author(s): Han, Claudia; Li, Rick; Hansen, Emily; Bennett, Hunter; Poirion, Olivier; Buchanan, Justin; Challacombe, Jean; Fixsen, Bethany; Trescott, Samantha; Schlachetzki, Johannes CM; Preissl, Sebastian; Wang, Allen; O’Connor, Carolyn; Warden, Anna; Shriram, Shreya; Kim, Roy; Nguyen, Celina; Schafer, Danielle; Ramirez, Gabriela; Anavim, Samuel; Johnson, Avalon; Sajti, Eniko; Gupta, Mihir; Levy, Michael; Ben-Haim, Sharona; Gonda, David; Laurent, Louise; Glass, Christopher; Coufal, Nicole | Abstract: The fetal period is a critical time for brain development, characterized by neurogenesis, neural migration, and synaptogenesis 1-3 . Microglia, the tissue resident macrophages of the brain, are observed as early as the fourth week of gestation 4 and are thought to engage in a variety of processes essential for brain development and homeostasis 5-11 . Conversely, microglia phenotypes are highly regulated by the brain environment 12-14 . Mechanisms by which human brain development influences the maturation of microglia and microglia potential contribution to neurodevelopmental disorders remain poorly understood. Here, we performed transcriptomic analysis of human fetal and postnatal microglia and corresponding cortical tissue to define age-specific brain environmental factors that may drive microglia phenotypes. Comparative analysis of open chromatin profiles using bulk and single-cell methods in conjunction with a new computational approach that integrates epigenomic and single-cell RNA-seq data allowed decoding of cellular heterogeneity with inference of subtype- and development stage-specific transcriptional regulators. Interrogation of in vivo and in vitro iPSC-derived microglia models provides evidence for roles of putative instructive signals and downstream gene regulatory networks which establish human-specific fetal and postnatal microglia gene expression programs and potentially contribute to neurodevelopmental disorders.

Chinese hamster ovary (CHO) cells, with their human-compatible glycosylation and high protein titers, are the most widely used cells for producing biopharmaceuticals. Engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the Chinese hamster genome were computationally predicted and are frequently inaccurate. Here, we revised TSS annotations for 15,308 Chinese hamster genes and 4,478 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. The experimental realignment and discovery of TSSs now expose previously hidden motifs, such as the TATA box. We further demonstrate, by targeting the glycosyltransferase geneMgat3, how accurate annotations readily facilitate activating silent genes by CRISPRa to obtain more human-like glycosylation. Together, we envision our annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.

Phagocytes express multiple phosphatidylserine (PtdSer) receptors that recognize apoptotic cells. It is unknown whether these receptors are interchangeable or if they play unique roles during cell clearance. Loss of the PtdSer receptor Mertk is associated with apoptotic corpse accumulation in the testes and degeneration of photoreceptors in the eye. Both phenotypes are linked to impaired phagocytosis by specialized phagocytes: Sertoli cells and the retinal pigmented epithelium (RPE). Here, we overexpressed the PtdSer receptor BAI1 in mice lacking MerTK (Mertk−/−Bai1 Tg ) to evaluate PtdSer receptor compensation in vivo. While Bai1 overexpression rescues clearance of apoptotic germ cells in the testes of Mertk−/− mice it fails to enhance RPE phagocytosis or prevent photoreceptor degeneration. To determine why MerTK is critical to RPE function, we examined visual cycle intermediates and performed unbiased RNAseq analysis of RPE from Mertk+/+ and Mertk−/− mice. Prior to the onset of photoreceptor degeneration, Mertk−/− mice had less accumulation of retinyl esters and dysregulation of a striking array of genes, including genes related to phagocytosis, metabolism, and retinal disease in humans. Collectively, these experiments establish that not all phagocytic receptors are functionally equal, and that compensation among specific engulfment receptors is context and tissue dependent.

Linking enhancers to disease Enhancers are genomic regions that regulate gene expression, sometimes in a cell-dependent manner. However, most of our knowledge of human brain cell–type enhancers derives from studies of bulk human brain tissue. Nott et al. examined chromatin and promoter activity in cell nuclei isolated from human brains. Genetic variants associated with brain traits and disease showed cell-specific patterns of enhancer enrichment. These data indicate that Alzheimer's disease is regulated by genetic variants within microglial cells, whereas psychiatric diseases tend to affect neurons. Science , this issue p. 1134

Unique cell type-specific patterns of activated enhancers can be leveraged to interpret non-coding genetic variation associated with complex traits and diseases such as neurological and psychiatric disorders. Here, we have defined active promoters and enhancers for major cell types of the human brain. Whereas psychiatric disorders were primarily associated with regulatory regions in neurons, idiopathic Alzheimer’s disease (AD) variants were largely confined to microglia enhancers. Interactome maps connecting GWAS variants in cell type-specific enhancers to gene promoters revealed an extended microglia gene network in AD. Deletion of a microglia-specific enhancer harboring AD-risk variants ablated BIN1 expression in microglia but not in neurons or astrocytes. These findings revise and expand the genes likely to be influenced by non-coding variants in AD and suggest the probable brain cell types in which they function.One Sentence SummaryIdentification of cell type-specific regulatory elements in the human brain enables interpretation of non-coding GWAS risk variants.

Macrophage-derived insulin-like growth factor enhances the uptake of microvesicles by non-professional phagocytes, such as airway epithelial cells and fibroblasts, thereby dampening tissue inflammation. Our bodies clear billions of cells every day through the actions of both tissue-resident and recruited 'professional' phagocytes such as macrophages, and neighbouring 'non-professional' phagocytes such as epithelial cells or fibroblasts. The relative contributions of each phagocyte type to phagocytosis and tissue homeostasis in general are not well understood. Kodi Ravichandran and colleagues provide evidence for a role of macrophage-derived insulin like growth factor in enhancing the uptake of microvesicles by non-professional phagocytes, such as airway epithelial cells and fibroblasts, thereby dampening tissue inflammation. Professional phagocytes (such as macrophages1) and non-professional phagocytes2,3,4,5,6,7,8 (such as epithelial cells) clear billions of apoptotic cells and particles on a daily basis9. Although professional and non-professional macrophages reside in proximity in most tissues, whether they communicate with each other during cell clearance, and how this might affect inflammation, is not known10. Here we show that macrophages, through the release of a soluble growth factor and microvesicles, alter the type of particles engulfed by non-professional phagocytes and influence their inflammatory response. During phagocytosis of apoptotic cells or in response to inflammation-associated cytokines, macrophages released insulin-like growth factor 1 (IGF-1). The binding of IGF-1 to its receptor on non-professional phagocytes redirected their phagocytosis, such that uptake of larger apoptotic cells was reduced whereas engulfment of microvesicles was increased. IGF-1 did not alter engulfment by macrophages. Macrophages also released microvesicles, whose uptake by epithelial cells was enhanced by IGF-1 and led to decreased inflammatory responses by epithelial cells. Consistent with these observations, deletion of IGF-1 receptor in airway epithelial cells led to exacerbated lung inflammation after allergen exposure. These genetic and functional studies reveal that IGF-1- and microvesicle-dependent communication between macrophages and epithelial cells can critically influence the magnitude of tissue inflammation in vivo.

iPSC-derived microglia offer a powerful tool to study microglial homeostasis and disease-associated inflammatory responses. Yet, microglia are highly sensitive to their environment, exhibiting transcriptomic deficiencies when kept in isolation from the brain. Furthermore, species-specific genetic variations demonstrate that rodent microglia fail to fully recapitulate the human condition. To address this, we developed an approach to study human microglia within a surrogate brain environment. Transplantation of iPSC-derived hematopoietic-progenitors into the postnatal brain of humanized, immune-deficient mice results in context-dependent differentiation into microglia and other CNS macrophages, acquisition of an ex vivo human microglial gene signature, and responsiveness to both acute and chronic insults. Most notably, transplanted microglia exhibit robust transcriptional responses to Aβ-plaques that only partially overlap with that of murine microglia, revealing new, human-specific Aβ-responsive genes. We therefore have demonstrated that this chimeric model provides a powerful new system to examine the in vivo function of patient-derived and genetically modified microglia.

Rapid and efficient removal of apoptotic cells by phagocytes plays a key role during development, tissue homeostasis, and in controlling immune responses1–5. An important feature of efficient clearance is the capacity of a single phagocyte to ingest multiple apoptotic cells successively, and to process the increased load of corpse-derived cellular material6–9. However, factors that influence sustained phagocytic capacity or how they in turn influence continued clearance by phagocytes are not known. Here we identify that the ability of a phagocyte to control its mitochondrial membrane potential is a critical factor in the capacity of a phagocyte to engulf apoptotic cells. Changing the phagocyte mitochondrial membrane potential (genetically or pharmacologically) significantly affected phagocytosis, with lower potential enhancing engulfment and higher membrane potential inhibiting uptake. We then identified that Ucp2, a mitochondrial membrane protein that acts to lower the mitochondrial membrane potential10–12, is upregulated in phagocytes engulfing apoptotic cells (but not synthetic targets, bacteria, or yeast). Loss of Ucp2 limited the capacity of phagocytes to continually ingest apoptotic cells, while overexpression of Ucp2 increased the capacity for engulfment and the ability to engulf multiple apoptotic cells. Mutational and pharmacological inhibition of Ucp2 uncoupling activity reversed the positive effect of Ucp2 on engulfment capacity, suggesting a direct role for Ucp2-mediated mitochondrial function in phagocytosis. Macrophages from Ucp2-deficient mice13, 14 were impaired in their capacity to engulf apoptotic cells in vitro, and Ucp2-deficient mice displayed profound in vivo defects in clearing dying cells in the thymus and the testes. Collectively, these data suggest that phagocytes alter the mitochondrial membrane potential during engulfment to regulate uptake of sequential apoptotic cells, and that Ucp2 is a key molecular determinant of this step in vivo. Since Ucp2 function has also been linked to metabolic diseases and atherosclerosis14–16, these data identifying a new role for Ucp2 in regulating apoptotic cell clearance may provide additional insights toward understanding the complex etiology and pathogenesis of these diseases.

The immune cell composition of the tumor microenvironment can be a decisive factor for tumor pathogenesis. Gliomas are tumors that develop from the glial cells of the brain and spinal cord and make up to 30% of all brain tumors. In gliomas, microglia and infiltrating macrophages can comprise up to 30 to 50% of total tumor-associated cells. Increased CD68 staining, a marker of microglia/macrophages, in adult gliomas is positively associated with histologic tumor grade. Despite the accumulated evidence substantiating a critical role for microglia and infiltrating macrophages in gliomagenesis, little is known is about the molecular mechanisms driving microglial contribution to tumor growth and whether microglia/macrophages are therapeutic targets in both low- and high-grade gliomas. Despite microglia sharing common properties with other tissue-resident macrophages, they express hundreds of genes at higher levels compared to other tissue-resident macrophages, many of which are influenced by the brain micro-environment. Additionally, engraftment of bone-marrow derived cells into the central nervous system fails to produce microglia identical to yolk sac-derived microglia at the transcriptional level. Hence, in any inflammatory context, including cancer, an interesting question arises: how does each population contribute to the pathogenesis and/or resolution of inflammation? To elucidate the role(s) of microglia/macrophages in gliomas, we isolated the myeloid fraction from primary pediatric and adult low-grade and high-grade gliomas using flow cytometry. By integrating bulk and single-cell transcriptome analysis, we find significant inter- and intratumoral heterogeneity within the myeloid population. Additionally, we find evidence for tumor environment-dependent gene change. In combination with ongoing comparative analysis of the corresponding epigenetic landscapes of the myeloid populations, we seek to decipher how the tumor microenvironment reprograms the transcription factor network in microglia/macrophages to generate tumor-promoting cells. Citation Format: Claudia Z. Han, Sascha H. Duttke, Zhengyu Ouyang, Sebastian Preissl, Johannes C.M. Schlachetzki, Alexander Nott, Conor Fitzpatrick, Carolyn O'Connor, Nicole G. Coufal, Mihir Gupta, David D. Gonda, Michael L. Levy, Ben-Haim Sharona, Barba David, Joseph D. Ciacci, Alexander A. Khalessi, Clark C. Chen, Bing Ren, Christopher K. Glass. Dissecting the myeloid lineage in human gliomas [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A078.

Apoptotic neurons generated during normal brain development or secondary to pathologic insults are efficiently cleared from the central nervous system. Several soluble factors, including nucleotides, cytokines, and chemokines are released from injured neurons, signaling microglia to find and clear debris. One such chemokine that serves as a neuronal-microglial communication factor is fractalkine, with roles demonstrated in several models of adult neurological disorders. Lacking, however, are studies investigating roles for fractalkine in perinatal brain injury, an important clinical problem with no effective therapies. We used a well-characterized mouse model of ethanol-induced apoptosis to assess the role of fractalkine in neuronal-microglial signaling. Quantification of apoptotic debris in fractalkine-knockout and CX3CR1-knockout mice following ethanol treatment revealed increased apoptotic bodies compared to wild type mice. Ethanol-induced injury led to release of soluble, extracellular fractalkine. The extracellular media harvested from apoptotic brains induces microglial migration in a fractalkine-dependent manner that is prevented by neutralization of fractalkine with a blocking antibody or by deficiency in the receptor, CX3CR1. This suggests fractalkine acts as a ‘find-me’ signal, recruiting microglial processes toward apoptotic cells to promote their clearance. Next, we aimed to determine whether there are downstream alterations in cytokine gene expression due to fractalkine signaling. We examined mRNA expression in fractalkine-knockout and CX3CR1-knockout mice after alcohol-induced apoptosis and found differences in cytokine production in the brains of these knockouts by 6 hours after ethanol treatment. Collectively, this suggests that fractalkine acts as a ‘find me’ signal released by apoptotic neurons, and subsequently plays a critical role in modulating both phagocytic clearance and inflammatory cytokine gene expression after ethanol-induced apoptosis.

Billions of cells die via apoptosis every day and are swiftly and efficiently removed. When a phagocyte engulfs an apoptotic cell, it essentially doubles its cellular contents, raising the question of how a phagocyte may manage the excess metabolic load. This review discusses phagocyte cellular metabolism, the digestion of the ingested apoptotic cell and the impact of these processes on engulfment.