SUMMARY The remarkable capacity for pluripotency and self-renewal in embryonic stem cells (ESCs) requires a finely-tuned transcriptional circuitry wherein the pathways and genes that initiate differentiation are suppressed, but poised to respond rapidly to developmental signals. To elucidate transcriptional control in mouse ESCs in the naïve, ground state, we defined the distribution of engaged RNA polymerase II (Pol II) at high-resolution. We find that promoter-proximal pausing of Pol II is most enriched at genes regulating cell cycle and signal transduction, and not, as expected, at developmental or bivalent genes. Accordingly, ablation of the primary pause-inducing factor NELF does not increase expression of lineage markers, but instead causes proliferation defects, embryonic lethality and dysregulation of ESC signaling pathways. Indeed, ESCs lacking NELF have dramatically attenuated FGF/ERK activity, rendering them resistant to differentiation. This work thus uncovers a key role for NELF-mediated pausing in establishing the responsiveness of stem cells to developmental cues.

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8 lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.

A transient erythromyeloid wave of definitive hematopoietic progenitors (erythroid/ myeloid progenitors [EMPs]) emerges in the yolk sac beginning at embryonic day 8.25 (E8.25) and colonizes the liver by E10.5, before adult-repopulating hematopoietic stem cells. At E11.5, we observe all maturational stages of erythroid precursors in the liver and the first definitive erythrocytes in the circulation. These early fetal liver erythroblasts express predominantly adult ␤-globins and the definitive erythroid-specific transcriptional modifiers c-myb, Sox6, and Bcl11A. Surprisingly, they also express low levels of "embryonic" ␤H1-, but not y-, globin transcripts. Consistent with these results, RNA polymerase and highly modified histones are found associated with ␤H1-and adult globin, but not y-globin, genes. E11.5 definitive proerythroblasts from mice transgenic for the human ␤-globin locus, like human fetal erythroblasts, express predominately human ␥-, low ␤-, and no -globin transcripts. Significantly, E9.5 yolk sac-derived EMPs cultured in vitro have similar murine and human transgenic globin expression patterns. Later liver proerythroblasts express low levels of ␥-globin, while adult marrow proerythroblasts express only ␤-globin transcripts. We conclude that yolk sacderived EMPs, the first of 2 origins of definitive erythropoiesis, express a unique pattern of globin genes as they generate the first definitive erythrocytes in the liver of the mammalian embryo. IntroductionIn the adult, steady-state erythropoiesis begins with hematopoietic stem cells (HSCs) in the bone marrow (BM) that differentiate into lineage-restricted erythroid progenitors. These progenitors subsequently generate morphologically identifiable precursors that mature into enucleated red blood cells (RBCs). Embryonic erythropoiesis differs from the adult in 2 critical ways. First, it is not in steady state, as exponentially increasing numbers of RBCs must be generated to meet the needs of the rapidly growing embryo. 1 Second, RBCs are required for embryonic survival before transplantable HSCs are formed. In the mouse, circulating erythroid cells are required for normal development beyond embryonic day 9.5 (E9.5). 2 However, the first adult-repopulating HSCs are not observed until E10.5 and have just begun to colonize the site of fetal hematopoiesis, the forming liver, between E11.5 and E12.5. [3][4][5] One solution for this embryonic dilemma, the appearance of 2 distinct forms of erythroid cells in the bloodstream of mammalian embryos, was described over 100 years ago. 6 The first form consisted of "primitive" erythroid cells that emerge from the yolk sac and were characterized by their large size and nucleated state. Primitive erythroid cells were superseded in the circulation by a second erythroid cell population, which was termed "definitive," because of their resemblance to the smaller enucleated RBCs found in the adult. More recent studies have determined that the primitive erythroid lineage arises from a transient wave of primitive erythroid pr...

The expression of many metazoan genes is regulated through controlled release of RNA polymerase II (Pol II) that has paused during early transcription elongation. Pausing is highly enriched at genes in stimulus-responsive pathways, where it has been proposed to poise downstream targets for rapid gene activation. However, whether this represents the major function of pausing in these pathways remains to be determined. To address this question, we analyzed pausing within several stimulus-responsive networks in Drosophila and discovered that paused Pol II is much more prevalent at genes encoding components and regulators of signal transduction cascades than at inducible downstream targets. Within immune-responsive pathways, we found that pausing maintains basal expression of critical network hubs, including the key NF-kB transcription factor that triggers gene activation. Accordingly, loss of pausing through knockdown of the pause-inducing factor NELF leads to broadly attenuated immune gene activation. Investigation of murine embryonic stem cells revealed that pausing is similarly widespread at genes encoding signaling components that regulate self-renewal, particularly within the MAPK/ERK pathway. We conclude that the role of pausing goes well beyond poising-inducible genes for activation and propose that the primary function of paused Pol II is to establish basal activity of signal-responsive networks. All organisms have evolved strategies to facilitate rapid and balanced responses to environmental and developmental cues. One mechanism for achieving robust upregulation of transcription in response to the external environment is exemplified by the Drosophila heatshock (Hsp) genes, which possess preloaded RNA polymerase II (Pol II) on their promoters prior to induction (Lis 1998). This Pol II is engaged in early elongation and remains paused promoter-proximally, associated with a 20-to 60-nucleotide (nt) nascent RNA. Heat shock triggers nearly immediate release of paused Pol II into the Hsp genes, permitting the scaffold of general transcription factors left at the promoter to be reused by additional Pol II molecules that generate a dramatic induction of RNA levels within minutes of heat shock (Lis 1998;Zobeck et al. 2010).Pol II pausing has recently been identified as a widespread mechanism of transcriptional regulation in higher eukaryotes. Genome-wide localization of Pol II in Drosophila, mouse, and human cells showed that thousands of genes display an accumulation of Pol II just downstream from their promoters (Guenther et al. 2007;Muse et al. 2007;Zeitlinger et al. 2007;Core et al. 2008;Lee et al. 2008;Gilchrist et al. 2010; Rahl et al. 2010), and analyses of RNA confirm that this polymerase is predominantly in a transcriptionally engaged, but paused, state (Core et al. 2008;Nechaev et al. 2010;Min et al. 2011). Notably, in all cell types investigated, pausing is found to be highly enriched among genes in stimulusresponsive networks, such as those that sense environmental and developmental cues (Muse et al. 20...

Cigarette smoke (CS) induces abnormal and sustained lung inflammation; however, the molecular mechanism underlying sustained inflammation is not known. It is well known that activation of I kappaB kinase beta (IKK beta) leads to transient translocation of active NF-kappaB (RelA/p65-p50) in the nucleus and transcription of pro-inflammatory genes, whereas the role of IKK alpha in perpetuation of sustained inflammatory response is not known. We hypothesized that CS activates IKK alpha and causes histone acetylation on the promoters of pro-inflammatory genes, leading to sustained transcription of pro-inflammatory mediators in mouse lung in vivo and in human monocyte/macrophage cell line (MonoMac6) in vitro. CS exposure to C57BL/6J mice resulted in activation of IKK alpha, leading to phosphorylation of ser10 and acetylation of lys9 on histone H3 on the promoters of IL-6 and MIP-2 genes in mouse lung. The increased level of IKK alpha was associated with increased acetylation of lys310 RelA/p65 on pro-inflammatory gene promoters. The role of IKK alpha in CS-induced chromatin modification was confirmed by gain and loss of IKK alpha in MonoMac6 cells. Overexpression of IKK alpha was associated with augmentation of CS-induced pro-inflammatory effects, and phosphorylation of ser10 and acetylation of lys9 on histone H3, whereas transfection of IKK alpha dominant-negative mutants reduced CS-induced chromatin modification and pro-inflammatory cytokine release. Moreover, phosphorylation of ser276 and acetylation of lys310 of RelA/p65 was augmented in response to CS extract in MonoMac6 cells transfected with IKK alpha. Taken together, these data suggest that IKK alpha plays a key role in CS-induced pro-inflammatory gene transcription through phospho-acetylation of both RelA/p65 and histone H3.