The Augmented R-Loop Is a Unifying Mechanism for Myelodysplastic Syndromes Induced by High-Risk Splicing Factor Mutations.

Mutations in several general pre-mRNA splicing factors have been linked to myelodysplastic syndromes (MDSs) and solid tumors. These mutations have generally been assumed to cause disease by the resultant splicing defects, but different mutations appear to induce distinct splicing defects, raising the possibility that an alternative common mechanism is involved. Here we report a chain of events triggered by multiple splicing factor mutations, especially high-risk alleles in SRSF2 and U2AF1, including elevated R-loops, replication stress, and activation of the ataxia telangiectasia and Rad3-related protein (ATR)-Chk1 pathway. We further demonstrate that enhanced R-loops, opposite to the expectation from gained RNA binding with mutant SRSF2, result from impaired transcription pause release because the mutant protein loses its ability to extract the RNA polymerase II (Pol II) C-terminal domain (CTD) kinase-the positive transcription elongation factor complex (P-TEFb)-from the 7SK complex. Enhanced R-loops are linked to compromised proliferation of bone-marrow-derived blood progenitors, which can be partially rescued by RNase H overexpression, suggesting a direct contribution of augmented R-loops to the MDS phenotype.

R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters.

R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase-H-based approach; this reveals predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end; by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such a free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed.

Alternative polyadenylation dysregulation contributes to the differentiation block of acute myeloid leukemia.

Posttranscriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among posttranscriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in patients with acute myeloid leukemia (AML) by performing 3' region extraction and deep sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence lengthening due to differences in poly(A) site (PAS) usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS data set. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mechanistic target of rapamycin complex 1 (mTORC1) signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block in patients with AML.

Enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus.

The innate immune system limits viral replication via type I interferon and also induces the presentation of viral antigens to cells of the adaptive immune response. Using infection of mice with vesicular stomatitis virus, we analyzed how the innate immune system inhibits viral propagation but still allows the presentation of antigen to cells of the adaptive immune response. We found that expression of the gene encoding the inhibitory protein Usp18 in metallophilic macrophages led to lower type I interferon responsiveness, thereby allowing locally restricted replication of virus. This was essential for the induction of adaptive antiviral immune responses and, therefore, for preventing the fatal outcome of infection. In conclusion, we found that enforced viral replication in marginal zone macrophages was an immunological mechanism that ensured the production of sufficient antigen for effective activation of the adaptive immune response.

Hippo kinase loss contributes to del(20q) hematologic malignancies through chronic innate immune activation.

Heterozygous deletions within chromosome 20q, or del(20q), are frequent cytogenetic abnormalities detected in hematologic malignancies. To date, identification of genes in the del(20q) common deleted region that contribute to disease development have remained elusive. Through assessment of patient gene expression, we have identified STK4 (encoding Hippo kinase MST1) as a 20q gene that is downregulated below haploinsufficient amounts in myelodysplastic syndrome (MDS) and myeloproliferative neoplasm (MPN). Hematopoietic-specific gene inactivation in mice revealed Hippo kinase loss to induce splenomegaly, thrombocytopenia, megakaryocytic dysplasia, and a propensity for chronic granulocytosis; phenotypes that closely resemble those observed in patients harboring del(20q). In a JAK2-V617F model, heterozygous Hippo kinase inactivation led to accelerated development of lethal myelofibrosis, recapitulating adverse MPN disease progression and revealing a novel genetic interaction between these 2 molecular events. Quantitative serum protein profiling showed that myelofibrotic transformation in mice was associated with cooperative effects of JAK2-V617F and Hippo kinase inactivation on innate immune-associated proinflammatory cytokine production, including IL-1ß and IL-6. Mechanistically, MST1 interacted with IRAK1, and shRNA-mediated knockdown was sufficient to increase IRAK1-dependent innate immune activation of NF-κB in human myeloid cells. Consistent with this, treatment with a small molecule IRAK1/4 inhibitor rescued the aberrantly elevated IL-1ß production in the JAK2-V617F MPN model. This study identified Hippo kinase MST1 (STK4) as having a central role in the biology of del(20q)-associated hematologic malignancies and revealed a novel molecular basis of adverse MPN progression that may be therapeutically exploitable via IRAK1 inhibition.

SON controls cell-cycle progression by coordinated regulation of RNA splicing.

It has been suspected that cell-cycle progression might be functionally coupled with RNA processing. However, little is known about the role of the precise splicing control in cell-cycle progression. Here, we report that SON, a large Ser/Arg (SR)-related protein, is a splicing cofactor contributing to efficient splicing of cell-cycle regulators. Downregulation of SON leads to severe impairment of spindle pole separation, microtubule dynamics, and genome integrity. These molecular defects result from inadequate RNA splicing of a specific set of cell-cycle-related genes that possess weak splice sites. Furthermore, we show that SON facilitates the interaction of SR proteins with RNA polymerase II and other key spliceosome components, suggesting its function in efficient cotranscriptional RNA processing. These results reveal a mechanism for controlling cell-cycle progression through SON-dependent constitutive splicing at suboptimal splice sites, with strong implications for its role in cancer and other human diseases.

Distinct splicing signatures affect converged pathways in myelodysplastic syndrome patients carrying mutations in different splicing regulators.

Myelodysplastic syndromes (MDS) are heterogeneous myeloid disorders with prevalent mutations in several splicing factors, but the splicing programs linked to specific mutations or MDS in general remain to be systematically defined. We applied RASL-seq, a sensitive and cost-effective platform, to interrogate 5502 annotated splicing events in 169 samples from MDS patients or healthy individuals. We found that splicing signatures associated with normal hematopoietic lineages are largely related to cell signaling and differentiation programs, whereas MDS-linked signatures are primarily involved in cell cycle control and DNA damage responses. Despite the shared roles of affected splicing factors in the 3' splice site definition, mutations in U2AF1, SRSF2, and SF3B1 affect divergent splicing programs, and interestingly, the affected genes fall into converging cancer-related pathways. A risk score derived from 11 splicing events appears to be independently associated with an MDS prognosis and AML transformation, suggesting potential clinical relevance of altered splicing patterns in MDS.

Elevated Response to Type I IFN Enhances RANKL-Mediated Osteoclastogenesis in Usp18-Knockout Mice.

A balance between bone formation and bone resorption is critical for the maintenance of bone mass. In many pathological conditions, including chronic inflammation, uncontrolled activation of osteoclast differentiation often causes excessive bone resorption that results in osteoporosis. In this study, we identified the osteopenia phenotype of mice lacking Usp18 (also called Ubp43), which is a deISGylating enzyme and is known as a negative regulator of type I IFN signaling. The expression of Usp18 was induced in preosteoclasts upon receptor activator of NF-κB ligand (RANKL) treatment. In an in vitro osteoclast-differentiation assay, bone marrow macrophages from Usp18-deficient mice exhibited an enhanced differentiation to multinucleated cells, elevated activation of NFATc1, and an increased expression of osteoclast marker genes upon RANKL treatment. Furthermore, in vitro quantification of bone resorption revealed a great increase in osteoclastic activities in Usp18-deficient cells. Interestingly, proinflammatory cytokine genes, such as IP-10 (CXCL10), were highly expressed in Usp18-deficient bone marrow macrophages upon RANKL treatment compared with wild-type cells. In addition, serum cytokine levels, especially IP-10, were significantly high in Usp18-knockout mice. In sum, we suggest that, although type I IFN is known to restrict osteoclast differentiation, the exaggerated activation of the type I IFN response in Usp18-knockout mice causes an osteopenia phenotype in mice.

Type I IFN induces protein ISGylation to enhance cytokine expression and augments colonic inflammation.

Type I IFNs have broad activity in tissue inflammation and malignant progression that depends on the expression of IFN-stimulated genes (ISGs). ISG15, one such ISG, can form covalent conjugates to many cellular proteins, a process termed "protein ISGylation." Although type I IFNs are involved in multiple inflammatory disorders, the role of protein ISGylation during inflammation has not been evaluated. Here we report that protein ISGylation exacerbates intestinal inflammation and colitis-associated colon cancer in mice. Mechanistically, we demonstrate that protein ISGylation negatively regulates the ubiquitin-proteasome system, leading to increased production of IFN-induced reactive oxygen species (ROS). The increased cellular ROS then enhances LPS-induced activation of p38 MAP kinase and the expression of inflammation-related cytokines in macrophages. Thus our studies reveal a regulatory role for protein ISGylation in colonic inflammation and its related malignant progression, indicating that targeting ubiquitin-activating enzyme E1 homolog has therapeutic potential in treating inflammatory diseases.

Hmga2 is a direct target gene of RUNX1 and regulates expansion of myeloid progenitors in mice.

RUNX1 is a master transcription factor in hematopoiesis and mediates the specification and homeostasis of hematopoietic stem and progenitor cells (HSPCs). Disruptions in RUNX1 are well known to lead to hematologic disease. In this study, we sought to identify and characterize RUNX1 target genes in HSPCs by performing RUNX1 chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) using a murine HSPC line and complementing this data with our previously described gene expression profiling of primary wild-type and RUNX1-deficient HSPCs (Lineage(-)/cKit(+)/Sca1(+)). From this analysis, we identified and confirmed that Hmga2, a known oncogene, as a direct target of RUNX1. Hmga2 was strongly upregulated in RUNX1-deficient HSPCs, and the promoter of Hmga2 was responsive in a cell-type dependent manner upon coexpression of RUNX1. Conditional Runx1 knockout mice exhibit expansion of their HSPCs and myeloid progenitors as hallmark phenotypes. To further validate and establish that Hmga2 plays a role in inducing HSPC expansion, we generated mouse models of HMGA2 and RUNX1 deficiency. Although mice lacking both factors continued to display higher frequencies of HSPCs, the expansion of myeloid progenitors was effectively rescued. The data presented here establish Hmga2 as a transcriptional target of RUNX1 and a critical regulator of myeloid progenitor expansion.

Runx1 exon 6-related alternative splicing isoforms differentially regulate hematopoiesis in mice.

RUNX1 is an important transcription factor for hematopoiesis. There are multiple alternatively spliced isoforms of RUNX1. The best known isoforms are RUNX1a from use of exon 7A and RUNX1b and c from use of exon 7B. RUNX1a has unique functions due to its lack of C-terminal regions common to RUNX1b and c. Here, we report that the ortholog of human RUNX1a was only found in primates. Furthermore, we characterized 3 Runx1 isoforms generated by exon 6 alternative splicing. Runx1bEx6(-) (Runx1b without exon 6) and a unique mouse Runx1bEx6e showed higher colony-forming activity than the full-length Runx1b (Runx1bEx6(+)). They also facilitated the transactivation of Runx1bEx6(+). To gain insight into in vivo functions, we analyzed a knock-in (KI) mouse model that lacks isoforms Runx1b/cEx6(-) and Runx1bEx6e. KI mice had significantly fewer lineage-Sca1(+)c-Kit(+) cells, short-term hematopoietic stem cells (HSCs) and multipotent progenitors than controls. In vivo competitive repopulation assays demonstrated a sevenfold difference of functional HSCs between wild-type and KI mice. Together, our results show that Runx1 isoforms involving exon 6 support high self-renewal capacity in vitro, and their loss results in reduction of the HSC pool in vivo, which underscore the importance of fine-tuning RNA splicing in hematopoiesis.

Cooperation between RUNX1-ETO9a and novel transcriptional partner KLF6 in upregulation of Alox5 in acute myeloid leukemia.

Fusion protein RUNX1-ETO (AML1-ETO, RUNX1-RUNX1T1) is expressed as the result of the 8q22;21q22 translocation [t(8;21)], which is one of the most common chromosomal abnormalities found in acute myeloid leukemia. RUNX1-ETO is thought to promote leukemia development through the aberrant regulation of RUNX1 (AML1) target genes. Repression of these genes occurs via the recruitment of the corepressors N-COR and SMRT due to their interaction with ETO. Mechanisms of RUNX1-ETO target gene upregulation remain less well understood. Here we show that RUNX1-ETO9a, the leukemogenic alternatively spliced transcript expressed from t(8;21), upregulates target gene Alox5, which is a gene critically required for the promotion of chronic myeloid leukemia development by BCR-ABL. Loss of Alox5 expression reduces activity of RUNX1-ETO9a, MLL-AF9 and PML-RARα in vitro. However, Alox5 is not essential for the induction of leukemia by RUNX1-ETO9a in vivo. Finally, we demonstrate that the upregulation of Alox5 by RUNX1-ETO9a occurs via the C2H2 zinc finger transcription factor KLF6, a protein required for early hematopoiesis and yolk sac development. Furthermore, KLF6 is specifically upregulated by RUNX1-ETO in human leukemia cells. This identifies KLF6 as a novel mediator of t(8;21) target gene regulation, providing a new mechanism for RUNX1-ETO transcriptional control.

RUNX1 regulates the CD34 gene in haematopoietic stem cells by mediating interactions with a distal regulatory element.

The transcription factor RUNX1 is essential to establish the haematopoietic gene expression programme; however, the mechanism of how it activates transcription of haematopoietic stem cell (HSC) genes is still elusive. Here, we obtained novel insights into RUNX1 function by studying regulation of the human CD34 gene, which is expressed in HSCs. Using transgenic mice carrying human CD34 PAC constructs, we identified a novel downstream regulatory element (DRE), which is bound by RUNX1 and is necessary for human CD34 expression in long-term (LT)-HSCs. Conditional deletion of Runx1 in mice harbouring human CD34 promoter-DRE constructs abrogates human CD34 expression. We demonstrate by chromosome conformation capture assays in LT-HSCs that the DRE physically interacts with the human CD34 promoter. Targeted mutagenesis of RUNX binding sites leads to perturbation of this interaction and decreased human CD34 expression in LT-HSCs. Overall, our in vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34.

Usp18 driven enforced viral replication in dendritic cells contributes to break of immunological tolerance in autoimmune diabetes.

Infection with viruses carrying cross-reactive antigens is associated with break of immunological tolerance and induction of autoimmune disease. Dendritic cells play an important role in this process. However, it remains unclear why autoimmune-tolerance is broken during virus infection, but usually not during exposure to non-replicating cross-reactive antigens. Here we show that antigen derived from replicating virus but not from non-replicating sources undergoes a multiplication process in dendritic cells in spleen and lymph nodes. This enforced viral replication was dependent on Usp18 and was essential for expansion of autoreactive CD8⁺ T cells. Preventing enforced virus replication by depletion of CD11c⁺ cells, genetically deleting Usp18, or pharmacologically inhibiting of viral replication blunted the expansion of autoreactive CD8⁺ T cells and prevented autoimmune diabetes. In conclusion, Usp18-driven enforced viral replication in dendritic cells can break immunological tolerance and critically influences induction of autoimmunity.

Attenuation of AML1-ETO cellular dysregulation correlates with increased leukemogenic potential.

AML1-ETO (RUNX1-ETO) fusion proteins are generated by the 8;21 translocation, commonly found in acute myeloid leukemia, which fuses the AML1 (RUNX1) and ETO (MTG8, RUNX1T1) genes. Previous studies have shown that AML1-ETO interferes with AML1 function but requires additional cooperating mutations to induce leukemia development. In mouse models, AML1-ETO forms lacking the C-terminus have been shown to have greatly enhanced leukemogenic potential. Here, we investigate the role of 2 AML1-ETO C-terminal-interacting proteins, N-CoR, a transcriptional corepressor, and SON, a splicing/transcription factor required for cell cycle progression, in AML1-ETO-induced leukemia development. AML1-ETO-W692A loses N-CoR binding at NHR4, displays attenuated transcriptional repression ability and decreased cellular dysregulation, and promotes leukemia in vivo. These results support the importance of the degree of dysregulation by AML1-ETO in cellular transformation and demonstrate that AML1-ETO-W692A can be used as an effective experimental model for determining which factors compromise the leukemogenic potential of AML1-ETO.

RUNX1a enhances hematopoietic lineage commitment from human embryonic stem cells and inducible pluripotent stem cells.

Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo-expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with ß-globin production. Moreover, HPCs generated from RUNX1a EBs possess ≥9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs.

Activation of double-stranded RNA-activated protein kinase (PKR) by interferon-stimulated gene 15 (ISG15) modification down-regulates protein translation.

The ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly induced by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. However, how ISGylation contributes to innate immune responses is not clear. The dsRNA-dependent protein kinase (PKR) inhibits translation by phosphorylating eIF2α to exert its anti-viral effect. ISG15 and PKR are induced by interferon, suggesting that a relationship exists between ISGylation and translational regulation. Here, we report that PKR is ISGylated at lysines 69 and 159. ISG15-modified PKR is active in the absence of virus infection and phosphorylates eIF2α to down-regulate protein translation. The present study describes a novel pathway for the activation of PKR and the regulation of protein translation.

SON protein regulates GATA-2 through transcriptional control of the microRNA 23a~27a~24-2 cluster.

SON is a DNA- and RNA-binding protein localized in nuclear speckles. Although its function in RNA splicing for effective cell cycle progression and genome stability was recently unveiled, other mechanisms of SON functions remain unexplored. Here, we report that SON regulates GATA-2, a key transcription factor involved in hematopoietic stem cell maintenance and differentiation. SON is highly expressed in undifferentiated hematopoietic stem/progenitor cells and leukemic blasts. SON knockdown leads to significant depletion of GATA-2 protein with marginal down-regulation of GATA-2 mRNA. We show that miR-27a is up-regulated upon SON knockdown and targets the 3'-UTR of GATA-2 mRNA in hematopoietic cells. Up-regulation of miR-27a was due to activation of the promoter of the miR-23a∼27a∼24-2 cluster, suggesting that SON suppresses this promoter to lower the microRNAs from this cluster. Our data revealed a previously unidentified role of SON in microRNA production via regulating the transcription process, thereby modulating GATA-2 at the protein level during hematopoietic differentiation.

MicroRNA programs in normal and aberrant stem and progenitor cells.

Emerging evidence suggests that microRNAs (miRNAs), an abundant class of ∼22-nucleotide small regulatory RNAs, play key roles in controlling the post-transcriptional genetic programs in stem and progenitor cells. Here we systematically examined miRNA expression profiles in various adult tissue-specific stem cells and their differentiated counterparts. These analyses revealed miRNA programs that are common or unique to blood, muscle, and neural stem cell populations and miRNA signatures that mark the transitions from self-renewing and quiescent stem cells to proliferative and differentiating progenitor cells. Moreover, we identified a stem/progenitor transition miRNA (SPT-miRNA) signature that predicts the effects of genetic perturbations, such as loss of PTEN and the Rb family, AML1-ETO9a expression, and MLL-AF10 transformation, on self-renewal and proliferation potentials of mutant stem/progenitor cells. We showed that some of the SPT-miRNAs control the self-renewal of embryonic stem cells and the reconstitution potential of hematopoietic stem cells (HSCs). Finally, we demonstrated that SPT-miRNAs coordinately regulate genes that are known to play roles in controlling HSC self-renewal, such as Hoxb6 and Hoxa4. Together, these analyses reveal the miRNA programs that may control key processes in normal and aberrant stem and progenitor cells, setting the foundations for dissecting post-transcriptional regulatory networks in stem cells.

Negative effects of GM-CSF signaling in a murine model of t(8;21)-induced leukemia.

The t(8;21)(q22;q22) is common in adult acute myeloid leukemia (AML). The RUNX1-ETO fusion protein that is expressed by this translocation is poorly leukemogenic and requires additional mutations for transformation. Loss of sex chromosome (LOS) is frequently observed in t(8;21) AML. In the present study, to evaluate whether LOS cooperates with t(8;21) in leukemogenesis, we first used a retroviral transduction/transplantation model to express RUNX1-ETO in hematopoietic cells from XO mice. The low frequency of leukemia in these mice suggests that the potentially critical gene for suppression of t(8;21) leukemia in humans is not conserved on mouse sex chromosomes. The gene encoding the GM-CSF receptor α subunit (CSF2RA) is located on X and Y chromosomes in humans but on chromosome 19 in mice. GM-CSF promotes myeloid cell survival, proliferation, and differentiation. To determine whether GM-CSF signaling affects RUNX1-ETO leukemogenesis, hematopoietic stem/progenitor cells that lack GM-CSF signaling were used to express RUNX1-ETO and transplanted into lethally irradiated mice, and a high penetrance of AML was observed in recipients. Furthermore, GM-CSF reduced the replating ability of RUNX1-ETO-expressing cells. These results suggest a possible tumor-suppressor role of GM-CSF in RUNX1-ETO leukemia. Loss of the CSF2RA gene may be a critical mutation explaining the high incidence of LOS associated with the t(8;21)(q22;q22) translocation.