Repression of SMAD3 by STAT3 and c-Ski induces conventional dendritic cell differentiation.

A pleiotropic immunoregulatory cytokine, TGF-ß, signals via the receptor-regulated SMADs: SMAD2 and SMAD3, which are constitutively expressed in normal cells. Here, we show that selective repression of SMAD3 induces cDC differentiation from the CD115+ common DC progenitor (CDP). SMAD3 was expressed in haematopoietic cells including the macrophage DC progenitor. However, SMAD3 was specifically down-regulated in CD115+ CDPs, SiglecH- pre-DCs, and cDCs, whereas SMAD2 remained constitutive. SMAD3-deficient mice showed a significant increase in cDCs, SiglecH- pre-DCs, and CD115+ CDPs compared with the littermate control. SMAD3 repressed the mRNA expression of FLT3 and the cDC-related genes: IRF4 and ID2. We found that one of the SMAD transcriptional corepressors, c-SKI, cooperated with phosphorylated STAT3 at Y705 and S727 to repress the transcription of SMAD3 to induce cDC differentiation. These data indicate that STAT3 and c-Ski induce cDC differentiation by repressing SMAD3: the repressor of the cDC-related genes during the developmental stage between the macrophage DC progenitor and CD115+ CDP.

ID2 promotes tumor progression and metastasis in thyroid cancer.

BACKGROUND: Inhibitor of DNA Binding 2 (ID2) plays a crucial role in tumor cell proliferation, invasion, metastasis, and stemness. Aberrant ID2 expression is associated with poor prognosis in various cancers. However, the specific function of ID2 in thyroid cancer remain unclear. METHOD: The TCGA database were utilized to explore the clinical relevance of ID2 in cancer. GO, KEGG, and TIMER were employed to predict the potential roles of ID2 in cancer. Functional analysis, including CCK-8, colony formation, transwell, wound healing, and sphere formation experiments, were conducted to determine the biological functions of ID2 in human cancers. Western blot (WB), RT-qPCR, and immunohistochemical (IHC) analyses were used to investigate the relationship between ID2 and downstream targets. RESULTS: Our study revealed significant overexpression of ID2 in various malignant tumor cells. Knocking ID2 significantly inhibited cancer cell proliferation and invasion, while overexpressing ID2 enhanced these capabilities. Additionally, ID2 mediates resistance of cancer cells to protein kinase B (or Akt) inhibitions. Further WB and IHC experiments indicated that ID2 promotes the phosphorylation activation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, thereby upregulating the expression of downstream proliferation, epithelial-mesenchymal transition (EMT), and stemness-related markers. CONCLUSION: We found that ID2 significantly promotes thyroid cancer cell proliferation, migration, EMT, and stemness through the PI3K/Akt pathway. Moreover, ID2 plays a crucial role in regulating cancer immune responses. It may serve as a potential biomarker for enhancing the efficacy of chemotherapy, targeted therapy, and immunotherapy against cancer.

The RNA-binding protein Cpeb4 regulates splicing of the Id2 gene in osteoclast differentiation.

Cytoplasmic polyadenylation element-binding protein 4 (Cpeb4) is an RNA-binding protein that regulates posttranscriptional regulation, such as regulation of messenger RNA stability and translation. In the previous study, we reported that Cpeb4 localizes to nuclear bodies upon induction of osteoclast differentiation by RANKL. However, the mechanisms of the localization of Cpeb4 and osteoclastogenesis by Cpeb4 remain unknown. Here, we show that Cpeb4 localizes to the nuclear bodies by its RNA-binding ability and partially regulates normal splicing during osteoclast differentiation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with Phos-tag® revealed that the phosphorylation levels of Cpeb4 were already high in the RAW264.7 cells and were not altered by RANKL treatment. Immunofluorescence showed that exogenous Cpeb4 in HEK293T cells without RANKL stimulation localized to the same foci as shown in RANKL-stimulated RAW264.7 cells. Furthermore, when nuclear export was inhibited by leptomycin B treatment, Cpeb4 accumulated throughout the nucleus. Importantly, RNA recognition motif (RRM) 7 of Cpeb4 was essential for the localization. In contrast, the intrinsically disordered region, RRM1, and zinc finger domain CEBP_ZZ were not necessary for the localization. The mechanistic study showed that Cpeb4 co-localized and interacted with the splicing factors serine/arginine-rich splicing factor 5 (SRSF5) and SRSF6, suggesting that Cpeb4 may be involved in the splicing reaction. RNA-sequencing analysis revealed that the expression of genes related to cell proliferation processes, such as mitotic cell cycle and regulation of cell cycle processes, was elevated in osteoclasts depleted of Cpeb4. Interestingly, the splicing pattern of the inhibitor of DNA binding 2 (Id2) gene, which suppresses osteoclast differentiation, was altered by the depletion of Cpeb4. These results provide new insight into the role of Cpeb4 as a player of normal splicing of Id2 in osteoclast differentiation.

ID proteins promote the survival and primed-to-naive transition of human embryonic stem cells through TCF3-mediated transcription.

Inhibition of DNA binding proteins 1 and 3 (ID1 and ID3) are important downstream targets of BMP signalling that are necessary for embryonic development. However, their specific roles in regulating the pluripotency of human embryonic stem cells (hESCs) remain unclear. Here, we examined the roles of ID1 and ID3 in primed and naive-like hESCs and showed that ID1 and ID3 knockout lines (IDs KO) exhibited decreased survival in both primed and naive-like state. IDs KO lines in the primed state also tended to undergo pluripotent dissolution and ectodermal differentiation. IDs KO impeded the primed-to-naive transition (PNT) of hESCs, and overexpression of ID1 in primed hESCs promoted PNT. Furthermore, single-cell RNA sequencing demonstrated that ID1 and ID3 regulated the survival and pluripotency of hESCs through the AKT signalling pathway. Finally, we showed that TCF3 mediated transcriptional inhibition of MCL1 promotes AKT phosphorylation, which was confirmed by TCF3 knockdown in KO lines. Our study suggests that IDs/TCF3 acts through AKT signalling to promote survival and maintain pluripotency of both primed and naive-like hESCs.

The CDK1/TFCP2L1/ID2 cascade offers a novel combination therapy strategy in a preclinical model of bladder cancer.

Aberrant activation of embryogenesis-related molecular programs in urothelial bladder cancer (BC) is associated with stemness features related to oncogenic dedifferentiation and tumor metastasis. Recently, we reported that overexpression of transcription factor CP2-like protein-1 (TFCP2L1) and its phosphorylation at Thr177 by cyclin-dependent kinase-1 (CDK1) play key roles in regulating bladder carcinogenesis. However, the clinical relevance and therapeutic potential of this novel CDK1-TFCP2L1 molecular network remain elusive. Here, we demonstrated that inhibitor of DNA binding-2 (ID2) functions as a crucial mediator by acting as a direct repressive target of TFCP2L1 to modulate the stemness features and survival of BC cells. Low ID2 and high CDK1 expression were significantly associated with unfavorable clinical characteristics. TFCP2L1 downregulated ID2 by directly binding to its promoter region. Consistent with these findings, ectopic expression of ID2 or treatment with apigenin, a chemical activator of ID2, triggered apoptosis and impaired the proliferation, suppressed the stemness features, and reduced the invasive capacity of BC cells. Combination treatment with the specific CDK1 inhibitor RO-3306 and apigenin significantly suppressed tumor growth in an orthotopic BC xenograft animal model. This study demonstrates the biological role and clinical utility of ID2 as a direct target of the CDK1-TFCP2L1 pathway for modulating the stemness features of BC cells.

Temporal Expression of Transcription Factor ID2 Improves Natural Killer Cell Differentiation from Human Pluripotent Stem Cells.

Natural killer (NK) cells are one type of innate lymphoid cells, and NK cell-based immunotherapy serves as a potentially curative therapy for cancers. However, the lack of reliable resources for a large amount of NK cells required for clinical infusion has limited the broader application of NK cells in targeted immunotherapy. Substantial effort has thus been made to generate NK-like cells from human pluripotent stem cells (hPSCs), but detailed molecular mechanisms regulating NK cell differentiation remain elusive, preventing us from developing robust strategies for NK cell production. Here, we genetically engineered hPSCs with inducible overexpression of transcription factors NFIL3, ID2, or SPI1 via CRISPR/Cas9-mediated gene knock-in and investigated their temporal roles during NK cell differentiation. Our results demonstrated ID2 overexpression significantly promoted NK cell generation compared with NFIL3 and SPI1 overexpression under a chemically defined, feeder-free culture condition. The resulting ID2 hPSC-derived NK cells exhibited various mature NK-specific markers and displayed effective tumor-killing activities, comparable to NK cells derived from wildtype hPSCs. Our study provides a new platform for efficient NK cell production, serving as a realistic off-the-shelf cell source for targeted cancer immunotherapy.

KSHV RTA Induces Degradation of the Host Transcription Repressor ID2 To Promote the Viral Lytic Cycle.

The immediate early viral protein replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for activating the lytic cycle of KSHV. RTA induces the KSHV lytic cycle by several mechanisms, acting as a viral transcription factor that directly induces viral and host genes and acting as a viral E3 ubiquitin ligase by degrading host proteins that block viral lytic replication. Recently, we have characterized the global gene expression changes in primary effusion lymphoma (PEL) upon lytic reactivation of KSHV, which also led to the identification of rapidly downregulated genes such as ID2, an inhibitor of basic helix-loop-helix transcription factors. Here, we demonstrate that ID2 overexpression in PEL ablates KSHV lytic reactivation, indicating that ID2 inhibits the KSHV lytic cycle. Furthermore, we show that while ID2 is highly expressed during latency, its protein level is rapidly reduced by 4 h postinduction during lytic reactivation. Our results indicate that RTA binds to ID2 and induces its degradation during the KSHV lytic cycle by N-terminal ubiquitination through the ubiquitin-proteasome pathway. Importantly, we found that not only KSHV RTA but also its Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) homologs interact with ID2, and they can induce the degradation of all four members of the ID protein family, suggesting an evolutionarily conserved interplay between gammaherpesvirus RTAs and ID proteins. Taken together, we propose that ID2 acts as a repressor of the KSHV lytic cycle, which is counteracted by its RTA-mediated degradation. We also predict that ID proteins may act as restriction factors of the lytic phase of the other gammaherpesviruses as well. IMPORTANCE In addition to its transcription regulatory role, RTA is also known to have an E3 ubiquitin ligase activity, which RTA utilizes for inducing protein degradation. However, it is still largely unknown what host factors are downregulated during KSHV lytic reactivation by RTA-mediated protein degradation and what the biological significance of the degradation of these host factors is. In this study, we discovered that RTA employs N-terminal ubiquitination to induce degradation of ID2, a potent transcription repressor of host genes, via the ubiquitin-proteasome pathway to promote KSHV lytic reactivation in PEL cells. Furthermore, we found that not only KSHV RTA but also RTA of EBV and MHV68 gammaherpesviruses can induce the degradation of all four human ID proteins, indicating that the interplay between gammaherpesvirus RTAs and ID proteins is evolutionarily conserved.

Loss of E-cadherin leads to Id2-dependent inhibition of cell cycle progression in metastatic lobular breast cancer.

Invasive lobular breast carcinoma (ILC) is characterized by proliferative indolence and long-term latency relapses. This study aimed to identify how disseminating ILC cells control the balance between quiescence and cell cycle re-entry. In the absence of anchorage, ILC cells undergo a sustained cell cycle arrest in G0/G1 while maintaining viability. From the genes that are upregulated in anchorage independent ILC cells, we selected Inhibitor of DNA binding 2 (Id2), a mediator of cell cycle progression. Using loss-of-function experiments, we demonstrate that Id2 is essential for anchorage independent survival (anoikis resistance) in vitro and lung colonization in mice. Importantly, we find that under anchorage independent conditions, E-cadherin loss promotes expression of Id2 in multiple mouse and (organotypic) human models of ILC, an event that is caused by a direct p120-catenin/Kaiso-dependent transcriptional de-repression of the canonical Kaiso binding sequence TCCTGCNA. Conversely, stable inducible restoration of E-cadherin expression in the ILC cell line SUM44PE inhibits Id2 expression and anoikis resistance. We show evidence that Id2 accumulates in the cytosol, where it induces a sustained and CDK4/6-dependent G0/G1 cell cycle arrest through interaction with hypo-phosphorylated Rb. Finally, we find that Id2 is indeed enriched in ILC when compared to other breast cancers, and confirm cytosolic Id2 protein expression in primary ILC samples. In sum, we have linked mutational inactivation of E-cadherin to direct inhibition of cell cycle progression. Our work indicates that loss of E-cadherin and subsequent expression of Id2 drive indolence and dissemination of ILC. As such, E-cadherin and Id2 are promising candidates to stratify low and intermediate grade invasive breast cancers for the use of clinical cell cycle intervention drugs.

Inhibitor of DNA binding 2 (ID2) regulates the expression of developmental genes and tumorigenesis in ewing sarcoma.

Sarcomas are difficult to treat and the therapy, even when effective, is associated with long-term and life-threatening side effects. In addition, the treatment regimens for many sarcomas, including Ewing sarcoma, rhabdomyosarcoma, and osteosarcoma, are relatively unchanged over the past two decades, indicating a critical lack of progress. Although differentiation-based therapies are used for the treatment of some cancers, the application of this approach to sarcomas has proven challenging. Here, using a CRISPR-mediated gene knockout approach, we show that Inhibitor of DNA Binding 2 (ID2) is a critical regulator of developmental-related genes and tumor growth in vitro and in vivo in Ewing sarcoma tumors. We also identified that homoharringtonine, which is an inhibitor of protein translation and FDA-approved for the treatment of leukemia, decreases the level of the ID2 protein and significantly reduces tumor growth and prolongs mouse survival in an Ewing sarcoma xenograft model. Furthermore, in addition to targeting ID2, homoharringtonine also reduces the protein levels of ID1 and ID3, which are additional members of the ID family of proteins with well-described roles in tumorigenesis, in multiple types of cancer. Overall, these results provide insight into developmental regulation in Ewing sarcoma tumors and identify a novel, therapeutic approach to target the ID family of proteins using an FDA-approved drug.

Inhibitor of DNA binding 2 knockdown inhibits the growth and liver metastasis of colorectal cancer.

BACKGROUND: Liver metastasis of colorectal cancer (CRC) remains high mortality and the mechanism is still unknown. Here we investigated the effects of inhibitor of DNA binding 2 (Id2) on growth and liver metastasis of CRC. METHODS: qPCR and western blotting were used to demonstrate mRNA and protein expressions in Id2-knockdown HCT116 cells. Cell growth was observed by cell proliferation assay, colony formation assay and flow cytometry. Cell migration and invasion were observed with wound healing assay and transwell migration and invasion assay. The effects of Id2 knockdown on tumor growth and liver metastasis in vivo were evaluated respectively with subcutaneous tumor model and colorectal liver metastasis model by injecting HCT116 cells into the mesentery triangle of cecum in mice. RESULTS: Id2 overexpression was found in CRC cell lines. Id2 knockdown resulted in a reduction in the proliferation, colony formation, migration and invasion of HCT116 cells. The suppression of cell proliferation was accompanied by the cell cycle arrest in the G0/G1 phase with down-regulation of Cyclin D1, Cyclin E, p-Cdk2/3, Cdk6, p-p27 and up-regulation of p21 and p27. Id2 knockdown reversed epithelial-mesenchymal transition (EMT) through increasing E-Cadherin and inhibiting N-Cadherin, Vimentin, ß-catenin, Snail and Slug. Id2 was also found to inhibit CRC metastasis via MMP2, MMP9 and TIMP-1. Furthermore, Id2 knockdown suppressed CRC liver metastasis in vivo. CONCLUSION: Id2 promotes CRC growth through activation of the PI3K/AKT signaling pathway, and triggers EMT to enhance CRC migration and invasion.

ASCL1 phosphorylation and ID2 upregulation are roadblocks to glioblastoma stem cell differentiation.

The growth of glioblastoma (GBM), one of the deadliest adult cancers, is fuelled by a subpopulation of stem/progenitor cells, which are thought to be the source of resistance and relapse after treatment. Re-engagement of a latent capacity of these cells to re-enter a trajectory resulting in cell differentiation is a potential new therapeutic approach for this devastating disease. ASCL1, a proneural transcription factor, plays a key role in normal brain development and is also expressed in a subset of GBM cells, but fails to engage a full differentiation programme in this context. Here, we investigated the barriers to ASCL1-driven differentiation in GBM stem cells. We see that ASCL1 is highly phosphorylated in GBM stem cells where its expression is compatible with cell proliferation. However, overexpression of a form of ASCL1 that cannot be phosphorylated on Serine-Proline sites drives GBM cells down a neuronal lineage and out of cell cycle more efficiently than its wild-type counterpart, an effect further enhanced by deletion of the inhibitor of differentiation ID2, indicating mechanisms to reverse the block to GBM cell differentiation.

TOB1 Blocks Intestinal Mucosal Inflammation Through Inducing ID2-Mediated Suppression of Th1/Th17 Cell Immune Responses in IBD.

BACKGROUND & AIMS: TOB1 is an anti-proliferative protein of Tob/BTG family and typically involved in the tumorigenesis and T cell activation. Although TOB1 is associated with T helper 17 cell-related autoimmunity, its role in modulating T cell-mediated immune responses in IBD remains poorly understood. Here, we explored its expression and the underlying mechanisms involved in the pathogenesis of inflammatory bowel disease (IBD). METHODS: TOB1 and ID2 expression in IBD patients was examined by quantitative real time polymerase chain reaction and immunohistochemistry. IBD CD4+ T cells were transfected with lentivirus expressing TOB1, ID2, TOB1 short hairpin RNA and ID2 short hairpin RNA, respectively, and Tob1-/-CD4+ T cells were transfected with lentivirus expressing Id2. Experimental colitis was established in Tob1-/- mice by trinitrobenzene sulfonic acid enema and in Rag1-/- mice reconstituted with Tob1-/-CD45RBhighCD4+ T cells to further explore the role of Tob1 in intestinal mucosal inflammation. Splenic CD4+ T cells of Tob1-/- mice were sorted to determine transcriptome differences by RNA sequencing. RESULTS: TOB1 expression was decreased in inflamed mucosa and peripheral blood CD4+ T cells of IBD patients compared with healthy subjects. Overexpression of TOB1 downregulated IBD CD4+ T cells to differentiate into Th1/Th17 cells compared with control subjects. Severe colitis was observed in Tob1-/- mice through trinitrobenzene sulfonic acid enema or in Rag1-/- mice reconstituted with Tob1-/-CD45RBhighCD4+ T cells, compared with control animals. RNA sequencing analysis revealed ID2 as functional target of TOB1 to inhibit IBD CD4+ T cell differentiation into Th1/Th17 cells. Mechanistically, TOB1 was associated with Smad4/5 to induce ID2 expression and restrain Th1/Th17 cell differentiation. CONCLUSIONS: TOB1 restrains intestinal mucosal inflammation through suppressing Th1/Th17 cell-mediated immune responses via the Smad4/5-ID2 pathway. It may serve as a novel therapeutic target for treatment of human IBD.

Airway basal stem cells reutilize the embryonic proliferation regulator, Tgfß-Id2 axis, for tissue regeneration.

During development, quiescent airway basal stem cells are derived from proliferative primordial progenitors through the cell-cycle slowdown. In contrast, basal cells contribute to adult tissue regeneration by shifting from slow cycling to proliferating and subsequently back to slow cycling. Although sustained proliferation results in tumorigenesis, the molecular mechanisms regulating these transitions remain unknown. Using temporal single-cell transcriptomics of developing murine airway progenitors and genetic validation experiments, we found that TGF-ß signaling decelerated cell cycle by inhibiting Id2 and contributed to slow-cycling basal cell specification during development. In adult tissue regeneration, reduced TGF-ß signaling restored Id2 expression and initiated regeneration. Id2 overexpression and Tgfbr2 knockout enhanced epithelial proliferation; however, persistent Id2 expression drove basal cell hyperplasia that resembled a precancerous state. Together, the TGF-ß-Id2 axis commonly regulates the proliferation transitions in basal cells during development and regeneration, and its fine-tuning is critical for normal regeneration while avoiding basal cell hyperplasia.

Harmine targets inhibitor of DNA binding-2 and activator protein-1 to promote preosteoclast PDGF-BB production.

Osteoporosis is one of the most common metabolic bone diseases affecting millions of people. We previously found that harmine prevents bone loss in ovariectomized mice via increasing preosteoclast platelet-derived growth factor-BB (PDGF-BB) production and type H vessel formation. However, the molecular mechanisms by which harmine promotes preosteoclast PDGF-BB generation are still unclear. In this study, we revealed that inhibitor of DNA binding-2 (Id2) and activator protein-1 (AP-1) were important factors implicated in harmine-enhanced preosteoclast PDGF-BB production. Exposure of RANKL-induced Primary bone marrow macrophages (BMMs), isolated from tibiae and femora of mice, to harmine increased the protein levels of Id2 and AP-1. Knockdown of Id2 by Id2-siRNA reduced the number of preosteoclasts as well as secretion of PDGF-BB in RANKL-stimulated BMMs administrated with harmine. Inhibition of c-Fos or c-Jun (components of AP-1) both reversed the stimulatory effect of harmine on preosteoclast PDGF-BB production. Dual-luciferase reporter assay analyses determined that PDGF-BB was the direct target of AP-1 which was up-regulated by harmine treatment. In conclusion, our data demonstrated a novel mechanism involving in the production of PDGF-BB increased by harmine, which may provide potential therapeutic targets for bone loss diseases.

Inhibitor of DNA Binding 2 (ID2): A Novel Marker for Lymph Node Metastasis in Head and Neck Squamous Cell Carcinoma.

BACKGROUND: Although aggressive invasion and sequential lymph node metastasis (LNM) significantly affect the prognosis of patients with head and neck squamous cell carcinoma (HNSCC), studies on identifying the factors that regulate this process remain scarce. This study found an inhibitor of DNA binding 2 (ID2) as a novel molecule involved in the regulation of invasion and LNM of HNSCC and further verified its functional role. METHODS: The study examined the translational significance between ID2 expression levels and the presence of LNM as well as the prognosis for 119 patients with HNSCC after treatment. In addition, in vitro and in vivo experiments were performed using ID2 gene-modulated HNSCC cell lines to determine the functional role of ID2 in the invasion and LNM of HNSCC. RESULTS: Elevated levels of ID2 expression were closely associated with the presence of LNM in 119 patients with HNSCC, resulting in a poor prognosis. Overexpression of ID2-induced invasion and LNM of HNSCC cells was observed in vitro and in vivo. By contrast, knockdown of the ID2 gene diminished invasion and LNM of HNSCC cells. In addition, the ID2 expression level increased the expression level of matrix metalloproteinase 1 (MMP1), a molecule downstream to ID2. Furthermore, silencing of MMP1 in ID2-overexpressed HNSCC cells rescued the elevated invasion and LNM capabilities of these cells, suggesting that ID2 enhances invasion and LNM partly via MMP1 activation. CONCLUSION: In the invasion and LNM of HNSCC, ID2 plays an important role by modulating MMP1 expression, suggesting ID2-MMP1 axis to be a novel alternative therapeutic target for invasion and LNM of HNSCC.

Gut microbial metabolites facilitate anticancer therapy efficacy by modulating cytotoxic CD8+ T cell immunity.

Recent studies in both mice and humans have suggested that gut microbiota could modulate tumor responsiveness to chemo- or immunotherapies. However, the underlying mechanism is not clear yet. Here, we found that gut microbial metabolites, especially butyrate, could promote the efficacy of oxaliplatin by modulating CD8+ T cell function in the tumor microenvironment. Butyrate treatment directly boosted the antitumor cytotoxic CD8+ T cell responses both in vitro and in vivo in an ID2-dependent manner by promoting the IL-12 signaling pathway. In humans, the oxaliplatin responder cancer patients exhibited a higher amount of serum butyrate than did non-responders, which could also increase ID2 expression and function of human CD8+ T cells. Together, our findings suggest that the gut microbial metabolite butyrate could promote antitumor therapeutic efficacy through the ID2-dependent regulation of CD8+ T cell immunity, indicating that gut microbial metabolites could be effective as a part of cancer therapy.

RORα is a critical checkpoint for T cell and ILC2 commitment in the embryonic thymus.

Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 cells can arise in the embryonic thymus from shared T cell precursors, preceding the emergence of CD4+CD8+ (double-positive) T cells. Thymic ILC2 cells migrated to mucosal tissues, with colonization of the intestinal lamina propria. Expression of the transcription factor RORα repressed T cell development while promoting ILC2 development in the thymus. From RNA-seq, assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) data, we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 cells from common progenitors in the thymus. When Notch signaling is present, BCL11B dampens Nfil3 and Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORα overrides the repression of Nfil3 and Id2 repression, allowing ID2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.

MITF is a driver oncogene and potential therapeutic target in kidney angiomyolipoma tumors through transcriptional regulation of CYR61.

Tuberous sclerosis complex (TSC) is an autosomal dominant tumor suppressor syndrome, characterized by tumor development in multiple organs, including renal angiomyolipoma. Biallelic loss of TSC1 or TSC2 is a known genetic driver of angiomyolipoma development, however, whether an altered transcriptional repertoire contributes to TSC-associated tumorigenesis is unknown. RNA-seq analyses showed that MITF A isoform (MITF-A) was consistently highly expressed in angiomyolipoma, immunohistochemistry showed microphthalmia-associated transcription factor nuclear localization, and Chromatin immuno-Precipitation Sequencing analysis showed that the MITF-A transcriptional start site was highly enriched with H3K27ac marks. Using the angiomyolipoma cell line 621-101, MITF knockout (MITF.KO) and MITF-A overexpressing (MITF.OE) cell lines were generated. MITF.KO cells showed markedly reduced growth and invasion in vitro, and were unable to form xenografted tumors. In contrast, MITF.OE cells grew faster in vitro and as xenografted tumors compared to control cells. RNA-Seq analysis showed that both ID2 and Cysteine-rich angiogenic inducer 61 (CYR61) expression levels were increased in the MITF.OE cells and reduced in the MITF.KO cells, and luciferase assays showed this was due to transcriptional effects. Importantly, CYR61 overexpression rescued MITF.KO cell growth in vitro and tumor growth in vivo. These findings suggest that MITF-A is a transcriptional oncogenic driver of angiomyolipoma tumor development, acting through regulation of CYR61.

Effect of cAMP Signaling Regulation in Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells.

The successful implementation of adipose-derived mesenchymal stem cells (ADSCs) in bone regeneration depends on efficient osteogenic differentiation. However, a literature survey and our own experience demonstrated that current differentiation methods are not effective enough. Since the differentiation of mesenchymal stem cells (MSCs) into osteoblasts and adipocytes can be regulated by cyclic adenosine monophosphate (cAMP) signaling, we investigated the effects of cAMP activator, forskolin, and inhibitor, SQ 22,536, on the early and late osteogenic differentiation of ADSCs cultured in spheroids or in a monolayer. Intracellular cAMP concentration, protein kinase A (PKA) activity, and inhibitor of DNA binding 2 (ID2) expression examination confirmed cAMP up- and downregulation. cAMP upregulation inhibited the cell cycle and protected ADSCs from osteogenic medium (OM)-induced apoptosis. Surprisingly, the upregulation of cAMP level at the early stages of osteogenic differentiation downregulated the expression of osteogenic markers RUNX2, Osterix, and IBSP, which was more significant in spheroids, and it is used for the more efficient commitment of ADSCs into preosteoblasts, according to the previously reported protocol. However, cAMP upregulation in a culture of ADSCs in spheroids resulted in significantly increased osteocalcin production and mineralization. Thus, undifferentiated and predifferentiated ADSCs respond differently to cAMP pathway stimulation in terms of osteogenesis, which might explain the ambiguous results from the literature.

Macrophage development and activation involve coordinated intron retention in key inflammatory regulators.

Monocytes and macrophages are essential components of the innate immune system. Herein, we report that intron retention (IR) plays an important role in the development and function of these cells. Using Illumina mRNA sequencing, Nanopore direct cDNA sequencing and proteomics analysis, we identify IR events that affect the expression of key genes/proteins involved in macrophage development and function. We demonstrate that decreased IR in nuclear-detained mRNA is coupled with increased expression of genes encoding regulators of macrophage transcription, phagocytosis and inflammatory signalling, including ID2, IRF7, ENG and LAT. We further show that this dynamic IR program persists during the polarisation of resting macrophages into activated macrophages. In the presence of proinflammatory stimuli, intron-retaining CXCL2 and NFKBIZ transcripts are rapidly spliced, enabling timely expression of these key inflammatory regulators by macrophages. Our study provides novel insights into the molecular factors controlling vital regulators of the innate immune response.