Pseudogenes have long been labeled as ''junk'' DNA, failed copies of genes that arise during the evolution of genomes. However, recent results are challenging this moniker; indeed, some pseudogenes appear to harbor the potential to regulate their protein-coding cousins. Far from being silent relics, many pseudogenes are transcribed into RNA, some exhibiting a tissuespecific pattern of activation. Pseudogene transcripts can be processed into short interfering RNAs that regulate coding genes through the RNAi pathway. In another remarkable discovery, it has been shown that pseudogenes are capable of regulating tumor suppressors and oncogenes by acting as microRNA decoys. The finding that pseudogenes are often deregulated during cancer progression warrants further investigation into the true extent of pseudogene function. In this review, we describe the ways in which pseudogenes exert their effect on coding genes and explore the role of pseudogenes in the increasingly complex web of noncoding RNA that contributes to normal cellular regulation.

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Bone marrow (BM)-derived classical monocytes are critical to wound repair, where they differentiate into macrophages and purge foreign materials and dead cells while also laying the framework for tissue repair and regeneration. A subset of this recruited population persists in the wound and acquires alternative activation states to promote cell proliferation and matrix remodeling. In diabetes, this phenotypic switch is impaired and inflammation persists in an elevated state, contributing to delayed wound healing. Long-term tissue-resident macrophages can also play a key role in the resolution of inflammation to varying degrees across different organs. In this study, we investigated different macrophage subpopulations in nondiabetic and diabetic wounds over time using Cx3CR1 eGFP transgenic mice and BM transplants. We show Cx3CR1 eGFP-hi macrophages in skin wounds are derived from long-term tissue-resident macrophages and predominantly exhibit an alternative activation state, whereas cells expressing low-intermediate Cx3CR1 eGFP are derived from the BM, contribute to both early and later stages of wound healing, and show both classical and alternative activation states. Diabetic mice showed significant differences in the dynamics of these subpopulations, which likely contribute to elevated and persisting inflammatory states over time. In particular, failure of Cx3CR1 int macrophages to mature into Cx3CR1 hi links maturation to resolution of inflammation. Thus strategies to promote macrophage maturation may be effective therapeutic tools in chronic inflammatory environments. ImmunoHorizons, 2019, 3: 262-273.

OBJECTIVEInactivating mutations in glucokinase (GCK) cause mild fasting hyperglycemia. Identification of a GCK mutation has implications for treatment and prognosis; therefore, it is important to identify these individuals. A significant number of patients have a phenotype suggesting a defect in glucokinase but no abnormality of GCK. We hypothesized that the GCK β-cell promoter region, which currently is not routinely screened, could contain pathogenic mutations; therefore, we sequenced this region in 60 such probands.RESEARCH DESIGN AND METHODSThe β-cell GCK promoter was sequenced in patient DNA. The effect of the identified novel mutation on GCK promoter activity was assessed using a luciferase reporter gene expression system. Electrophoretic mobility shift assays (EMSAs) were used to determine the impact of the mutation on Sp1 binding.RESULTSA novel −71G>C mutation was identified in a nonconserved region of the human promoter sequence in six apparently unrelated probands. Family testing established cosegregation with fasting hyperglycemia (≥5.5 mmol/l) in 39 affected individuals. Haplotype analysis in the U.K. family and four of the Slovakian families demonstrated that the mutation had arisen independently. The mutation maps to a potential transcriptional activator binding site for Sp1. Reporter assays demonstrated that the mutation reduces promoter activity by up to fourfold. EMSAs demonstrated a dramatic reduction in Sp1 binding to the promoter sequence corresponding to the mutant allele.CONCLUSIONSA novel β-cell GCK promoter mutation was identified that significantly reduces gene expression in vitro through loss of regulation by Sp1. To ensure correct diagnosis of potential GCK-MODY (maturity-onset diabetes of the young) cases, analysis of the β-cell GCK promoter should be included.

The TNF locus on chromosome 6p21 encodes a family of proteins with key roles in the immune response whose dysregulation leads to severe disease. Transcriptional regulation is important, with cell type and stimulus-specific enhancer complexes involving the proximal TNF promoter. We show how quantitative chromatin profiling across a 34 kb region spanning the TNF locus has allowed us to identify a number of novel DNase hypersensitive sites and characterize more distant regulatory elements. We demonstrate DNase hypersensitive sites corresponding to the lymphotoxin alpha (LTA) and tumour necrosis factor (TNF) promoter regions, a CpG island in exon 4 of lymphotoxin beta (LTB), the 3′ end of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like 1 (NFKBIL1) and 3.4 kb upstream of LTA. These sites co-localize to highly conserved DNA sequences and show evidence of cell type specificity when lymphoblastoid, Jurkat, U937, HeLa and HEK293T cell lines are analysed using Southern blotting. For Jurkat T cells, we define histone modifications across the locus. Peaks of acetylated histone H3 and H4, together with tri-methyl K4 of histone H3, correspond to hypersensitive sites, notably in exon 4 of LTB. We provide evidence of a functional role for an intergenic DNase I hypersensitive site distal to LTA in Jurkat cells based on reporter gene analysis, with evidence of recruitment of upstream stimulatory factors (USF) transcription factors.

Aims: Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells.Results: Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces vascular endothelial growth factor receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA, and antibody-based receptor inhibition, we demonstrate that the activation of NF-κB and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays in vivo using NOX2−/− mice.Innovation: For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex.Conclusions: This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and upregulation of VEGFR2. Antioxid. Redox Signal. 28, 110–130.

Recruitment of innate immune cells from the bone marrow (BM) to an injury site is required for effective repair. In diabetes, this process is altered, leading to excessive recruitment and retention of dysfunctional myeloid cells that fail to promote angiogenesis, prolong inflammation, and block healing. The aberrant myeloid phenotype is partially mediated by stable intrinsic changes to developing cells in the BM that are induced by the diabetic (db) environment, but the exact mechanisms remain largely unknown. Here, we show that the db-derived Gr-1+CD11b+ immature myeloid population has widespread misexpression of chromatin-remodeling enzymes and myeloid differentiation factors. Crucially, diabetes represses transcription of the key myeloid transcription factor CEBPA via diminished H3 Lys 27 promoter acetylation, leading to a failure in monocyte and granulocyte maturation. Restoring Cebpa expression by granulocyte colony-stimulating factor reverses the db phenotype and rescues myeloid maturation. Importantly, our data demonstrate a possible link between myeloid cell maturation and chronic inflammation.

Chronic inflammation is a hallmark of impaired healing in a plethora of tissues, including skin, and is associated with aging and diseases such as diabetes. Diabetic chronic skin wounds are characterized by excessive myeloid cells that display an aberrant phenotype, partially mediated by stable intrinsic changes induced during hematopoietic development. However, the relative contribution of myeloid celleintrinsic factors to chronic inflammation versus aberrant signals from the local environmental was unknown. Moreover, identification of myeloid cell intrinsic factors that contribute to chronic inflammation in diabetic wounds remained elusive. Here we show that Gr-1 þ CD11b þ myeloid cells are retained specifically within the presumptive granulation tissue region of the wound at a higher density in diabetic mice and associate with endothelial cells at the site of injury with a higher frequency than in nondiabetic mice. Adoptive transfer of myeloid cells demonstrated that aberrant wound retention is due to myeloid cell intrinsic factors and not the local environment. RNA sequencing of bone marrow and wound-derived myeloid cells identified Selplg as a myeloid cell intrinsic factor that is deregulated in chronic wounds. In vivo blockade of this protein significantly accelerated wound healing in diabetic mice and may be a potential therapeutic target in chronic wounds and other chronic inflammatory diseases.