Chromatin signatures at transcriptional start sites separate two equally populated yet distinct classes of intergenic long noncoding RNAs

Background: Mammalian transcriptomes contain thousands of long noncoding RNAs (lncRNAs). Some lncRNAs originate from intragenic enhancers which, when active, behave as alternative promoters producing transcripts that are processed using the canonical signals of their host gene. We have followed up this observation by analyzing intergenic lncRNAs to determine the extent to which they might also originate from intergenic enhancers. Results: We integrated high-resolution maps of transcriptional initiation and transcription to annotate a conservative set of intergenic lncRNAs expressed in mouse erythroblasts. We subclassified intergenic lncRNAs according to chromatin status at transcriptional initiation regions, defined by relative levels of histone H3K4 mono- and trimethylation. These transcripts are almost evenly divided between those arising from enhancer-associated (elncRNA) or promoter-associated (plncRNA) elements. These two classes of 5′ capped and polyadenylated RNA transcripts are indistinguishable with regard to their length, number of exons or transcriptional orientation relative to their closest neighboring gene. Nevertheless, elncRNAs are more tissue-restricted, less highly expressed and less well conserved during evolution. Of considerable interest, we found that expression of elncRNAs, but not plncRNAs, is associated with enhanced expression of neighboring protein-coding genes during erythropoiesis. Conclusions: We have determined globally the sites of initiation of intergenic lncRNAs in erythroid cells, allowing us to distinguish two similarly abundant classes of transcripts. Different correlations between the levels of elncRNAs, plncRNAs and expression of neighboring genes suggest that functional lncRNAs from the two classes may play contrasting roles in regulating the transcript abundance of local or distal loci

The MicroRNA-132 and MicroRNA-212 Cluster Regulates Hematopoietic Stem Cell Maintenance and Survival with Age by Buffering FOXO3 Expression

MicroRNAs are critical post-transcriptional regulators of hematopoietic cell-fate decisions, though little remains known about their role in aging hematopoietic stem cells (HSCs). We found that the microRNA-212/132 cluster (Mirc19) is enriched in HSCs and is upregulated during aging. Both overexpression and deletion of microRNAs in this cluster leads to inappropriate hematopoiesis with age. Enforced expression of miR-132 in the bone marrow of mice led to rapid HSC cycling and depletion. A genetic deletion of Mirc19 in mice resulted in HSCs that had altered cycling, function, and survival in response to growth factor starvation. We found that miR-132 exerted its effect on aging HSCs by targeting the transcription factor FOXO3, a known aging associated gene. Our data demonstrate that Mirc19 plays a role in maintaining balanced hematopoietic output by buffering FOXO3 expression. We have thus identified it as a potential target that might play a role in age-related hematopoietic defects

Core Circadian Clock Genes Regulate Leukemia Stem Cells in AML

Leukemia stem cells (LSCs) have the capacity to self-renew and propagate disease upon serial transplantation in animal models, and elimination of this cell population is required for curative therapies. Here, we describe a series of pooled, in vivo RNAi screens to identify essential transcription factors (TFs) in a murine model of acute myeloid leukemia (AML) with genetically and phenotypically defined LSCs. These screens reveal the heterodimeric, circadian rhythm TFs Clock and Bmal1 as genes required for the growth of AML cells in vitro and in vivo. Disruption of canonical circadian pathway components produces anti-leukemic effects, including impaired proliferation, enhanced myeloid differentiation, and depletion of LSCs. We find that both normal and malignant hematopoietic cells harbor an intact clock with robust circadian oscillations, and genetic knockout models reveal a leukemia-specific dependence on the pathway. Our findings establish a role for the core circadian clock genes in AML.National Institutes of Health (U.S.) (Grant P01 CA066996)National Institutes of Health (U.S.) (Grant R01 HL082945)National Cancer Institute (U.S.) (Grant P30-CA14051

A Distinct Gene Module for Dysfunction Uncoupled from Activation in Tumor-Infiltrating T Cells

Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8+tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and we use CRISPR-Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8+TILs. Our results open novel avenues for targeting dysfunctional T cell states while leaving activation programs intact

Heterogeneous Responses of Hematopoietic Stem Cells to Inflammatory Stimuli are Altered with Age

Long-term hematopoietic stem cells (LT-HSCs) maintain hematopoietic output throughout an animal’s lifespan. However, with age, the balance is disrupted, and LT-HSCs produce a myeloid-biased output, resulting in poor immune responses to infectious challenge and the development of myeloid leukemias. Here, we show that young and aged LT-HSCs respond differently to inflammatory stress, such that aged LT-HSCs produce a cell-intrinsic, myeloid-biased expression program. Using single-cell RNA sequencing (scRNA-seq), we identify a myeloid-biased subset within the LT-HSC population (mLT-HSCs) that is prevalent among aged LT-HSCs. We identify CD61 as a marker of mLT-HSCs and show that CD61-high LT-HSCs are uniquely primed to respond to acute inflammatory challenge. We predict that several transcription factors regulate the mLT-HSCs gene program and show that Klf5, Ikzf1, and Stat3 play an important role in age-related inflammatory myeloid bias. We have therefore identified and isolated an LT-HSC subset that regulates myeloid versus lymphoid balance under inflammatory challenge and with age

The neuropeptide NMU amplifies ILC2-driven allergic lung inflammation

Type 2 innate lymphoid cells (ILC2s) both contribute to mucosal homeostasis and initiate pathologic inflammation in allergic asthma. However, the signals that direct ILC2s to promote homeostasis versus inflammation are unclear. To identify such molecular cues, we profiled mouse lung-resident ILCs using single-cell RNA sequencing at steady state and after in vivo stimulation with the alarmin cytokines IL-25 and IL-33. ILC2s were transcriptionally heterogeneous after activation, with subpopulations distinguished by expression of proliferative, homeostatic and effector genes. The neuropeptide receptor Nmur1 was preferentially expressed by ILC2s at steady state and after IL-25 stimulation. Neuromedin U (NMU), the ligand of NMUR1, activated ILC2s in vitro, and in vivo co-administration of NMU with IL-25 strongly amplified allergic inflammation. Loss of NMU-NMUR1 signalling reduced ILC2 frequency and effector function, and altered transcriptional programs following allergen challenge in vivo. Thus, NMUR1 signalling promotes inflammatory ILC2 responses, highlighting the importance of neuro-immune crosstalk in allergic inflammation at mucosal surfaces

Interleukin-1beta Promoter (−31T/C and −511C/T) Polymorphisms in Major Recurrent Depression

To elucidate a genetic predisposition to major depressive disorder, we investigated two polymorphisms (−31T/C and −511C/T) in the interleukin-1beta promoter region in patients who suffered from major recurrent depression. The aim of the current work was to compare alleles and genotype layout between patients with major recurrent depression and healthy people. We would like to indicate such combination of genotypes which corresponds with major recurrent depression. Correlations between genotypes for analyzed polymorphisms and number of episodes, number of points in Hamilton Depression Rating Scale, and age of onset were investigated as well. The study group consisted of 94 patients diagnosed with major recurrent depression. The control group included 206 healthy individuals. Both groups involved representatives of Caucasian population. Genotyping of polymorphisms was performed by using PCR-RFLP technique. A specific haplotype, composed of the C allele at −31 and the T allele at −511, has a tendency to have a statistically significant difference (p = 0.064) between patients and control group. Correspondence analysis revealed that genotype T/T at −31 and genotype C/C at −511 are associated with major recurrent depression. No association was found between genotypes for studied polymorphic sites and number of episodes, number of points in Hamilton Depression Rating Scale, and age of onset

Induction and transcriptional regulation of the co-inhibitory gene module in T cells

Expression of co-inhibitory receptors, such as CTLA-4 and PD-1, on effector T cells is a key mechanism for ensuring immune homeostasis. Dysregulated co-inhibitory receptor expression on CD4+ T cells promotes autoimmunity while sustained overexpression on CD8+ T cells promotes T cell dysfunction or exhaustion, leading to impaired ability to clear chronic viral infections and cancer1,2. Here, we used RNA and protein expression profiling at single-cell resolution to identify a module of co-inhibitory receptors that includes not only several known co-inhibitory receptors (PD-1, Tim-3, Lag-3, and TIGIT), but also a number of novel surface receptors. We functionally validated two novel co-inhibitory receptors, Activated protein C receptor (Procr) and Podoplanin (Pdpn). The module of co-inhibitory receptors is co-expressed in both CD4+ and CD8+ T cells and is part of a larger co-inhibitory gene program that is shared by non-responsive T cells in multiple physiological contexts and is driven by the immunoregulatory cytokine IL-27. Computational analysis identified the transcription factors Prdm1 and c-Maf as cooperative regulators of the co-inhibitory module, which we validated experimentally. This molecular circuit underlies the co-expression of co-inhibitory receptors in T cells and identifies novel regulators of T cell function with the potential to regulate autoimmunity and tumor immunity