Mammalian brains have evolved a neocortex, which has diverged in size and morphology in different species over the course of evolution. In some mammals, a substantial increase in the number of neurons and glial cells resulted in the expansion and folding of the cerebrum, and it is believed that these evolutionary changes contributed to the acquisition of higher cognitive abilities in mammals. However, their underlying molecular and cellular mechanisms remain insufficiently elucidated. A major difficulty in addressing these mechanisms stemmed from the lack of appropriate animal models, as conventional experimental animals such as mice and rats have small brains without structurally obvious folds. Therefore, researchers including us have focused on using ferrets instead of mice and rats. Ferrets are domesticated carnivorous mammals with a gyrencephalic cerebrum, and, notably, they are amenable to genetic manipulations including in utero electroporation to knock out genes in the cerebrum. In this review, we highlight recent research into the mechanisms underlying the development and evolution of cortical folds using ferrets.

Uveal melanoma (UM) is the predominant form of eye cancer. The genes GNAQ and GNA11, encoding Gq and G11 respectively, are most frequently mutated in UM and are considered the major drivers of UM carcinogenesis by activating YAP. However, the mechanisms by which metastatic UM evades the immune system remain poorly understood. In this study, we found that oncogenic mutations of Gq/G11 promoted YAP and PD-L1 expression, modifying the tumor microenvironment and promoting immune evasion of UM. Consistently, the levels of GNAQ/GNA11 and YAP positively correlated to PD-L1 expression in UM patients. Furthermore, silencing YAP or treating with its inhibitor, Verteporfin, attenuated PD-L1 expression induced by Gq/G11 mutations, thereby enhancing T cell activation and T cell-mediated cytotoxicity. Collectively, this study reveals a potential role of Gq/G11 mutations on immune evasion of UM, a new mechanism of Gq/11 mutations-induced tumorigenesis, highlighting Gq/G11 and YAP as potential immunotherapeutic targets and suggesting Verteporfin as an adjuvant for immunotherapy of UM patients with GNAQ or GNA11 mutations.

Small cell lung carcinoma (SCLC) is a highly malignant cancer with early metastatic dissemination and poor clinical outcomes. Mutations in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway, including the frequently occurring rapamycin-insensitive protein (RICTOR) amplification, have been described in these tumours. Moreover, the associated mTOR hyperactivity could be exploited for personalised treatment. Our aim was to study mTOR activity, RICTOR amplification, and their role during SCLC progression. In situ mTOR activity and Rictor expression were characterised by immunohistochemistry in 50 primary and 50 brain metastatic tumours, and 14 paired SCLC patient samples. RICTOR copy number changes were analysed by fluorescence in situ hybridisation of the paired SCLC patient samples and in vivo experiments. Additionally, in vitro sensitivity to cisplatin and mTOR inhibitors was evaluated in SCLC cell lines harbouring RICTOR amplification and other mTOR pathway mutations. High Rictor expression and mTOR complex 2 (mTORC2) hyperactivity were significantly associated with brain metastases and worse overall survival. An increase in RICTOR copy number was observed in paired samples during progression. The importance of these alterations was confirmed both by the sensitising effect of vistusertib in vitro and the RICTOR copy number/expression changes in xenografts. Our study highlights the role of mTORC2 in SCLC progression. Early detection of RICTOR amplification in primary tumours and targeting mTORC2 in these cases may represent a promising novel strategy to develop personalised therapy for metastasis prevention.

Recent data shows that alterations in the expression and/or activation of the vascular endothelial growth factor receptor 2 (VEGFR2) in high grade serous ovarian cancer (HGSOC) modulate tumor progression. However, controversial results have been obtained, showing that in some cases VEGFR2 inhibition can promote tumorigenesis and metastasis. Thus, it is urgent to better define the role of the VEGF/VEGFR2 system to understand/predict the effects of its inhibitors administered as anti-angiogenic in HGSOC. Here, we modulated the expression levels of VEGFR2 and analyzed the effects in two cellular models of HGSOC. VEGFR2 silencing (or its pharmacological inhibition) promote the growth and invasive potential of OVCAR3 cells in vitro and in vivo. Consistent with this, the low levels of VEGFR2 in OV7 cells are associated with more pronounced proliferative and motile phenotypes when compared to OVCAR3 cells, and VEGFR2 overexpression in OV7 cells inhibits cell growth. In vitro data confirmed that VEGFR2 silencing in OVCAR3 cells favors the acquisition of an invasive phenotype by loosening cell-ECM contacts, reducing the size and the signaling of focal adhesion contacts (FAs). This is translated into a reduced FAK activity at FAs, ECM-dependent alterations of mechanical forces through FAs and YAP nuclear translocation. Together, the data show that low expression, silencing or inhibition of VEGFR2 in HGSOC cells alter mechanotransduction and lead to the acquisition of a pro-proliferative/invasive phenotype which explains the need for a more cautious use of anti-VEGFR2 drugs in ovarian cancer.

Cardiac muscle α-actin is a key protein of the thin filament in the muscle sarcomere that, together with myosin thick filaments, produce force and contraction important for normal heart function. Missense mutations in cardiac muscle α-actin can cause hypertrophic cardiomyopathy, a complex disorder of the heart characterized by hypercontractility at the molecular scale that leads to diverse clinical phenotypes. While the clinical aspects of hypertrophic cardiomyopathy have been extensively studied, the molecular mechanisms of missense mutations in cardiac muscle α-actin that cause the disease remain largely elusive. Here we used cryo-electron microscopy to reveal the structures of hypertrophic cardiomyopathy-associated actin mutations M305L and A331P in the filamentous state. We show that the mutations have subtle impacts on the overall architecture of the actin filament with mutation-specific changes in the nucleotide binding cleft active site, interprotomer interfaces, and local changes around the mutation site. This suggests that structural changes induced by M305L and A331P have implications for the positioning of the thin filament protein tropomyosin and the interaction with myosin motors. Overall, this study supports a structural model whereby altered interactions between thick and thin filament proteins contribute to disease mechanisms in hypertrophic cardiomyopathy.

During intracellular trafficking N-ethylmaleimide-sensitive-factor attachment receptor (SNARE) proteins catalyze the membrane fusion by assembling into a four-helix complex. In the mouse model, loss of the endosomal SNAREs vti1a and vti1b results in a perinatal lethal phenotype and neuronal defects including decreased neurite outgrowth in cultured primary neurons. We used a CRISPR/Cas9 system to generate a Vti1a Vti1b double knockout (DKO) in the neuroblastoma cell line N1E-115. Three different DKO cell lines were obtained and examined at genome and protein level. The double deficiency impaired proper differentiation based on lower levels of synaptic proteins as well as reduced neurite formation and elongation compared to wild type cells in differentiation medium. Neurite elongation can be induced by a variety of extracellular signals via different signaling cascades. Treatment with the Rho kinase inhibitor Y27632, which stimulates enlargeosome exocytosis, or with neurotrophic factors (BDNF, NGF and NT3) resulted in reduced stimulation of all DKO clones and in significantly shorter neurites compared to wild type cells. The loss of vti1a and vti1b disrupted Akt signaling during enlargeosome-mediated and Erk signaling during BDNF-induced neurite outgrowth.

Epigenetic editing is thriving as a robust tool for manipulating transcriptional regulation and cell fate. Despite its regulatory role in gene downregulation, epigenetic editing with histone deacetylation has been sparsely studied, especially in the context of cancer. In this current study, we have reconstructed a dCas9-HDAC8-EGFP fusion to perform histone deacetylation on the promoter of the ESR1, TERT and CDKN1C genes for the first time in breast cancer cell lines MCF-7 and MDA-MB-231 as well as in HEK293T cells. Our results demonstrated that dCas9-HDAC8-EGFP in combination with appropriate gRNAs were able to downregulate the expression of the ESR1, TERT and CDKN1C genes transcriptionally by specifically depleting the H3K9ac level on the recruitment loci. The addition of histone deacetylase inhibitors was found to neutralize the outcomes of dCas9-HDAC8-EGFP-induced epigenetic editing. Furthermore, we observed a significant downregulation of full length ERα expression in epigenetically edited MCF-7 cells with consequential alteration in cellular response toward estradiol and tamoxifen treatment due to dCas9-HDAC8-EGFP mediated epigenetic editing of the ESR1 gene. Overall, dCas9-HDAC8-EGFP is a novel circuit that enabled downregulation of crucial genes with cellular outcome in breast cancer cells by preferentially inducing H3K9 deacetylation of specific promoter regions.

Mesoderm induction is a crucial step for vascular cell specification, vascular development and vasculogenesis. However, the cellular and molecular mechanisms underlying mesoderm induction remain elusive. In the present study, a chemically-defined differentiation protocol was used to induce mesoderm formation and generate functional vascular cells including smooth muscle cells (SMCs) and endothelial cells (ECs) from human induced pluripotent stem cells (hiPSCs). Zebrafish larvae were used to detect an in vivo function of interleukin 10 (IL10) in mesoderm formation and vascular development. A three dimensional approach was used to create hiPSC-derived blood vessel organoid (BVO) and explore a potential impact of IL10 on BVO formation. A murine model hind limb ischemia was applied to investigate a therapeutic potential of hiPSC-derived cells treated with or without IL10 during differentiation. We found that IL10 was significantly and specifically up-regulated during mesoderm stage of vascular differentiation. IL10 addition in mesoderm induction media dramatically increased mesoderm induction and vascular cell generation from hiPSCs, whereas an opposite effect was observed with IL10 inhibition. Mechanistic studies revealed that IL10 promotes mesoderm formation and vascular cell differentiation by activating signal transducer and activator of transcription 3 signal pathway. Functional studies with an in vivo model system confirmed that knockdown of IL10 using morpholino antisense oligonucleotides in zebrafish larvae caused defective mesoderm formation, angiogenic sprouting and vascular development. Additionally, our data also show IL10 promotes blood vessel organoid development and enhances vasculogenesis and angiogenesis. Importantly, we demonstrate that IL10 treatment during mesoderm induction stage enhances blood flow perfusion recovery and increases vasculogenesis and therapeutic angiogenesis after hind limb ischemia. Our data, therefore, demonstrate a regulatory role for IL10 in mesoderm formation from hiPSCs and during zebrafish vascular development, providing novel insights into mesoderm induction and vascular cell specifications.

Human pluripotent stem cells (hPSCs) represent an unlimited source of β-like cells for both disease modeling and cellular therapy for diabetes. Numerous protocols have been published describing the differentiation of hPSCs into β-like cells that secret insulin in response to a glucose challenge. However, among the most widely used protocols it is not clear which yield the most functional cells with reproducible glucose-stimulated insulin-secretion (GSIS). Moreover, the technical challenges in culturing and differentiating hPSCs is a barrier for many researchers. In this study, we performed a side-by-side functional comparison based on three widely used methods to generate insulin expressing cells and identified optimal stages and conditions for cryopreserving and reconstituting stem cell (SC)-derived islets with a robust GSIS. Despite the fact that each protocol yields SC-islets consisting of insulin and glucagon-expressing cells, the SC-islets obtained from the two more recent revised protocols were more functional as measured by robust and reproducible GSIS. Moreover, we demonstrate that pancreatic progenitors and differentiated endocrine cells that have been cryopreserved for up to 10 months, can be reconstituted into glucose responsive SC-islets. These findings should enable the use of human PSC-derived β-like cells technologies even by groups with minimal PSC culture experience.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are prevalent inflammatory joint diseases characterized by synovitis, cartilage, and bone destruction. Fibroblast-like synoviocytes (FLSs) of the synovial membrane are a decisive factor in arthritis, making them a target for future therapies. Developing novel strategies targeting FLSs requires advanced in vitro joint models that accurately replicate non-diseased joint tissue. This study aims to identify a cell source reflecting physiological synovial fibroblasts. Therefore, we newly compared the phenotype and metabolism of "healthy" knee-derived FLSs from patients with ligament injuries (trauma-FLSs) to mesenchymal stromal cells (MSCs), their native precursors. We differentiated MSCs into fibroblasts using connective tissue growth factor (CTGF) and compared selected protein and gene expression patterns to those obtained from trauma-FLSs and OA-FLSs. Based on these findings, we explored the potential of an MSC-derived synovial tissue model to simulate a chronic inflammatory response akin to that seen in arthritis. We have identified MSCs as a suitable cell source for synovial tissue engineering because, despite metabolic differences, they closely resemble human trauma-derived FLSs. CTGF-mediated differentiation of MSCs increased HAS2 expression, essential for hyaluronan synthesis. It showed protein expression patterns akin to OA-FLSs, including markers of ECM components and fibrosis, and enzymes leading to a shift in metabolism towards increased fatty acid oxidation. In general, cytokine stimulation of MSCs in a synovial tissue model induced pro-inflammatory and pro-angiogenic gene expression, hyperproliferation, and increased glucose consumption, reflecting cellular response in human arthritis. We conclude that MSCs can serve as a proxy to study physiological synovial processes and inflammatory responses. In addition, CTGF-mediated mesenchymal-to-fibroblast transition resembles OA-FLSs. Thus, we emphasize MSCs as a valuable cell source for tools in preclinical drug screening and their application in tissue engineering.

Pancreatic ductal adenocarcinoma is an extremely incurable cancer type characterized by cells with highly proliferative capacity and resistance against the current therapeutic options. Our study reveals that IRS1 acts as a bridging molecule between EGFR and IGFR/InsR signalization providing a potential mechanism for the interplay between signaling pathways and bypassing EGFR-targeted or IGFR/InsR-targeted therapies. The analysis of IRS1 phosphorylation status in four pancreatic cell lines identified the impact of EGFR signaling on IRS1 activation in comparison with InsR/IGFR signaling. Significantly reduced viability was observed in IRS1-silenced cells even upon EGF stimulation showing the critical role of IRS1 in the EGFR signaling network in both malignant and normal pancreatic cells. This study also demonstrated that EGFR binds directly to IRS1 and at least on two tyrosine sites, Y612 and Y896, IRS1 becomes phosphorylated in response to EGF stimulation. Mechanistically, the EGFR-mediated phosphorylation of IRS1 can further activate the MAPK signaling pathway with the recruitment of GRB2 protein. Collectively, in this study, IRS1 was identified as a crucial regulator in the EGFR signaling suggesting IRS1 as a potential target for more durable responses to targeted PDAC therapy.

Our previous research revealed that androgen receptor (AR) activation reduces endothelial cell proliferation via non-genomic pathways. We hypothesized that AR activation might also affect endothelial cell migration, a critical step in angiogenesis. Our data demonstrates that treatment of human umbilical vein endothelial cells (HUVECs) with AR agonists, metribolone (R1881) or dihydrotestosterone (DHT), results in a dose-dependent reduction in migration, which can be reversed by AR antagonists or AR knockdown. Mechanistically, R1881 inhibits HUVEC migration by suppressing RhoA activity through the cSrc/FAK/paxillin pathway and promoting RhoA degradation via RhoA-p27 complex formation, ultimately resulting in RhoA ubiquitination. Transfection with constitutively active RhoA-V14 rescues the inhibitory effect of R1881 on HUVEC migration. Furthermore, R1881 elevates intracellular vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) levels but reduces VEGF secretion from HUVECs. This reduction is attributed to the formation of VEGF-CTGF complexes in the cytosol induced by R1881. Transfection with RhoA-V14 reduces CTGF levels and VEGF-CTGF complex formation, leading to enhanced VEGF secretion. Pre-treatment with WP631, a CTGF inhibitor, mitigates the R1881-induced reduction in VEGF secretion and HUVECs migration. In vivo assessments using zebrafish angiogenesis and mouse matrigel plug assays validate the anti-angiogenic effects of R1881. These findings provide insight into the molecular mechanisms through which AR activation modulates endothelial cell migration and angiogenesis.

In several solid tumors such as breast cancer, prostate cancer, colorectal cancer or melanoma, tumor draining lymph nodes are the earliest tissues where colonization by tumor cells is detected. Lymph nodes act as sentinels of metastatic dissemination, the deadliest phase of tumor progression. Besides hematogenous dissemination, lymphatic spread of tumor cells has been demonstrated, adding more complexity to the mechanisms involved in metastasis. A network of blood and lymphatic vessels surrounds tumors providing routes for tumor soluble factors to mediate regional and long-distance effects. Additionally, extracellular vesicles (EVs), particularly small EVs/exosomes, have been shown to circulate through the blood and lymph, favoring the formation of pre-metastatic niches in the tumor-draining lymph nodes (TDLNs) and distant organs. In this review, we present an overview of the relevance of lymph node metastasis, the structural and immune changes occurring in TDLNs during tumor progression, and how extracellular vesicles contribute to modulating some of these alterations while promoting the formation of lymph node pre-metastatic niches.

Mesenchymal Stem Cells (MSCs) derived from the embryonic mesoderm persist as a viable source of multipotent cells in adults and have a crucial role in tissue repair. One of the most promising aspects of MSCs is their ability to trans-differentiate into cell types outside of the mesodermal lineage, such as neurons. This characteristic positions MSCs as potential therapeutic tools for neurological disorders. However, the definition of a clear MSC signature is an ongoing topic of debate. Likewise, there is still a significant knowledge gap about functional alterations of MSCs during their transition to a neural fate. In this study, our focus is on the dynamic expression of RNA in MSCs as they undergo trans-differentiation compared to undifferentiated MSCs. To track and correlate changes in cellular signaling, we conducted high-throughput RNA expression profiling during the early time-course of human MSC neurogenic trans-differentiation. The expression of synapse maturation markers, including NLGN2 and NPTX1, increased during the first 24 h. The expression of neuron differentiation markers, such as GAP43 strongly increased during 48 h of trans-differentiation. Neural stem cell marker NES and neuron differentiation marker, including TUBB3 and ENO1, were highly expressed in mesenchymal stem cells and remained so during trans-differentiation. Pathways analyses revealed early changes in MSCs signaling that can be linked to the acquisition of neuronal features. Furthermore, we identified microRNAs (miRNAs) as potential drivers of the cellular trans-differentiation process. We also determined potential risk factors related to the neural trans-differentiation process. These factors include the persistence of stemness features and the expression of factors involved in neurofunctional abnormalities and tumorigenic processes. In conclusion, our findings contribute valuable insights into the intricate landscape of MSCs during neural trans-differentiation. These insights can pave the way for the development of safer treatments of neurological disorders.

Intracellular bacterial pathogens hijack the protein machinery of infected host cells to evade their defenses and cultivate a favorable intracellular niche. The intracellular pathogen Salmonella enterica subsp. Typhimurium (STm) achieves this by injecting a cocktail of effector proteins into host cells that modify the activity of target host proteins. Yet, proteome-wide approaches to systematically map changes in host protein function during infection have remained challenging. Here we adapted a functional proteomics approach - Thermal-Proteome Profiling (TPP) - to systematically assess proteome-wide changes in host protein abundance and thermal stability throughout an STm infection cycle. By comparing macrophages treated with live or heat-killed STm, we observed that most host protein abundance changes occur independently of STm viability. In contrast, a large portion of host protein thermal stability changes were specific to infection with live STm. This included pronounced thermal stability changes in proteins linked to mitochondrial function (Acod1/Irg1, Cox6c, Samm50, Vdac1, and mitochondrial respiratory chain complex proteins), as well as the interferon-inducible protein with tetratricopeptide repeats, Ifit1. Integration of our TPP data with a publicly available STm-host protein-protein interaction database led us to discover that the secreted STm effector kinase, SteC, thermally destabilizes and phosphorylates the ribosomal preservation factor Serbp1. In summary, this work emphasizes the utility of measuring protein thermal stability during infection to accelerate the discovery of novel molecular interactions at the host-pathogen interface.

Over the past decade, the induction protocols for the two types of kidney organoids (nephron organoids and ureteric bud organoids) from pluripotent stem cells (PSCs) have been established based on the knowledge gained in developmental nephrology. Kidney organoids are now used for disease modeling and drug screening, but they also have potential as tools for clinical transplantation therapy. One of the options to achieve this goal would be to assemble multiple renal progenitor cells (nephron progenitor, ureteric bud, stromal progenitor) to reproduce the organotypic kidney structure from PSCs. At least from mouse PSCs, all the three progenitors have been induced and assembled into such "higher order" kidney organoids. We will provide an overview of the developmental nephrology required for the induction of renal progenitors and discuss recent advances and remaining challenges of kidney organoids for clinical transplantation therapy.

Cardiac development requires precise gene expression programs at each developmental stage guided by multiple signaling pathways and transcription factors (TFs). MESP1 is transiently expressed in mesoderm, and is essential for subsequent cardiac development, while the precise mechanism regulating its own transcription and mesoderm cell fate is not fully understood. Therefore, we developed a high content screen assay to identify regulators of MESP1 expression in mesodermal cells differentiated from human pluripotent stem cells (hPSCs). The screen identified CYT387, a JAK1/JAK2 kinase inhibitor, as a potent activator of MESP1 expression, which was also found to promote cardiomyocyte differentiation in vitro. Mechanistic studies found that JAK inhibition promotes MESP1 expression by reducing cytoplasmic calcium concentration and subsequently activating canonical WNT signaling. Our study identified a role of JAK signaling in early mesodermal cells, and sheds light on the connection between the JAK-STAT pathway and transcriptional regulation of MESP1, which expands our understanding of mesoderm and cardiac development.

Human circulating monocytes are established targets for Zika virus (ZIKV) infection. Because of their important migratory properties toward any tissues, including the central nervous system (CNS), a better understanding of the mechanisms underlying monocyte transmigration upon ZIKV infection is required. Here, we monitored adhesion, migration and transmigration properties of monocytes exposed to ZIKV. We found that ZIKV enhanced monocyte adhesion on collagen compared to mock-exposed samples, and that pharmacological inhibition of mDia and Cdc42 function induced a significant decrease of adhesion in both mock- and ZIKV-exposed monocytes. In contrast, monocyte migration through collagen was inhibited by most of the tested small molecules targeting regulators of actin polymerization, including Rac1, ROCK, Cdc42, mDia, Arp2/3, Myosin-II and LFA-1. ZIKV-exposed monocyte migration showed a very similar profile to that of their mock-exposed counterparts. Finally, assessment of monocyte transmigration through human cerebral microvascular endothelial cells (hCMEC/D3) showed dependency on Rac1, ROCK, and Cdc42, independently of their infection status. In contrast, we identified that BIRT377, an antagonist of LFA-1, significantly inhibited transmigration of ZIKV-exposed but not mock-exposed monocytes. As BIRT377 increased adhesion of ZIKV-exposed monocytes, we propose that LFA-1 might be involved in a post-adhesion step to enhance viro-induced transmigration. These data suggest that ZIKV exposure triggers specific migratory properties of monocytes that are not exploited under physiological conditions. This work provides further insights on virus-host interactions important for viral neuroinvasion and offers novel targets to specifically inhibit the infiltration of infected cells to the CNS. SUMMARY SENTENCE: Monocyte transmigration involves massive actin cytoskeleton reorganization regulated by small Rho GTPases and integrins, which can be subverted by viruses.

The microenvironments of urinary systems play crucial roles in the development and metastasis of cancers due to their generation of complex temporal and spatial fluidic profiles. Because of their versatility in creating desired biomimetic flow, cone-and-plate bioreactors offer great potential for bladder cancer research. In this study, we construct a biocompatible cone-and-plate device coupled with a torque sensor, enabling the application and real-time monitoring of stable shear stress up to 50 dyne/cm². Under a stable shear stress stimulation at 12 dyne/cm, bladder cancer cell BFTC-905 is arrested at the G1 phase with decreased cell proliferation after 24-hour treatment. This effect is associated with increased cyclin-dependent kinase inhibitors p21 and p27, inhibiting cyclin D1/CDK4 complex with dephosphorylation of serine 608 on the retinoblastoma protein. Consequently, an increase in cyclin D3 and decreases in cyclin A2 and cyclin E2 are observed. Moreover, we demonstrate that the shear stress stimulation upregulates the expression of autophagy-related proteins Beclin-1, LC3B-I and LC3B-II, while caspase cleavages are not activated under the same condition. The design of this system and its application shed new light on flow-induced phenomena in the study of urothelial carcinomas.

CTCF is a key factor in three-dimensional chromatin folding and transcriptional control that was found to affect cancer cell migration by a mechanism that is still poorly understood. To identify this mechanism, we used mouse melanoma cells with a partial loss of function (pLoF) of CTCF. We found that CTCF pLoF inhibits cell migration rate while leading to an increase in the expression of multiple enzymes in the cholesterol biosynthesis pathway along with an elevation in the cellular cholesterol level. In agreement with the cholesterol change we detected altered membrane dynamics in CTCF pLoF cells as measured by reduced formation of migrasomes, extracellular vesicles formed at the rear side of migrating cells. Inhibition of cholesterol synthesis in CTCF pLoF cells restored the cellular migration rate and migrasome formation, suggesting that CTCF supports cell migration by suppressing cholesterol synthesis. Detailed analysis of the promoter of Hmgcs1, an early enzyme in the cholesterol synthesis pathway, revealed that CTCF prevents formation of a loop between that promoter and another promoter 200 kb away. CTCF also supports PRC2 recruitment to the promoter and deposition of H3K27me3. H3K27me3 at the promoter of Hmgcs1 prevents SREBP2 binding and activation of transcription. By this mechanism, CTCF fine-tunes cholesterol levels to support cell migration. Notably, genome wide association studies suggest a link between CTCF and cholesterol-associated diseases, thus CTCF emerges as a new regulator of cholesterol biosynthesis.