Promoters and enhancers establish precise gene transcription patterns. The development of functional approaches for their identification in mammalian cells has been complicated by the size of these genomes. Here we report a new method called FIREWACh (Functional Identification of Regulatory Elements Within Accessible Chromatin), a high-throughput functional assay for directly identifying active promoter and enhancer elements. FIREWACh simultaneously assessed over 80,000 DNA fragments derived from “nucleosome-free regions” within embryonic stem cell (ESC) chromatin to identify 6,364 new active regulatory elements. Many FIREWACh DNAs represent newly discovered ESC-specific enhancers and their analyses identified enriched binding site motifs for ESC transcription factors including SOX2, OCT4 (POU5f1), and KLF4. Thus FIREWACh identifies endogenous regulators of gene expression and can be used for the discovery of key cell-specific transcription factors. The application of FIREWACh to additional cultured cell types will facilitate functional annotation of the genome and expand our view of transcriptional network dynamics.

Objective Diabetic subjects are at higher risk of ischemic peripheral vascular disease (PVD). We tested the hypothesis that advanced glycation end products (AGEs) and their receptor (RAGE) block neoangiogenesis and blood flow recovery after hind limb ischemia induced by femoral artery ligation (FAL) through modulation of immune/inflammatory mechanisms. Approach and Results Wild type (WT) mice rendered diabetic with streptozotocin and subjected to unilateral FAL displayed increased accumulation and expression of AGEs and RAGE in ischemic muscle. In diabetic WT mice, FAL attenuated neoangiogenesis and impaired blood flow recovery, in parallel with reduced macrophage content in ischemic muscle and suppression of early inflammatory gene expression, including chemokine (C-C motif) ligand 2 (Ccl2) and early growth response gene 1 (Egr1) versus non-diabetic mice. Deletion of Ager or transgenic expression of Glo1 (reduces AGEs) restored adaptive inflammation, neoangiogenesis and blood flow recovery in diabetic mice. In diabetes, deletion of Ager increased circulating Ly6Chi monocytes and augmented macrophage infiltration into ischemic muscle tissue after FAL. In vitro, macrophages grown in high glucose display inflammation that is skewed to expression of tissue damage versus tissue repair gene expression. Further, macrophages grown in high versus low glucose demonstrate blunted macrophage-endothelial cell interactions. In both settings, these adverse effects of high glucose were reversed by Ager deletion in macrophages. Conclusions These findings indicate that RAGE attenuates adaptive inflammation in hind limb ischemia; underscore microenvironment-specific functions for RAGE in inflammation in tissue repair versus damage; and illustrate that AGE/RAGE antagonism may fill a critical gap in diabetic PVD.

The four Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded interferon (IFN) regulatory factor homologues (vIRF1 to vIRF4) are used to counter innate immune defenses and suppress p53. The vIRF genes are arranged in tandem but differ in function and expression. In KSHV-infected effusion lymphoma lines, K10.5/vIRF3 and K11/vIRF2 mRNAs are readily detected during latency, whereas K9/vIRF1 and K10/vIRF4 mRNAs are upregulated during reactivation. Here we show that the K10/vIRF4 promoter responds to the lytic switch protein RTA in KSHV-infected cells but is essentially unresponsive in uninfected cells. Coexpression of RTA with vIRF4 is sufficient to restore regulation, a property not shared by other vIRFs. The K9/vIRF1 promoter behaves similarly, and production of infectious virus is enhanced by the presence of vIRF4. Synergy requires the DNAbinding domain (DBD) and C-terminal IRF homology regions of vIRF4. Mutations of arginine residues within the putative DNA recognition helix of vIRF4 or the invariant cysteines of the adjacent CxxC motif abolish cooperation with RTA, in the latter case by preventing self-association. The oligomerization and transactivation functions of RTA are also essential for synergy. The K10/ vIRF4 promoter contains two transcription start sites (TSSs), and a 105-bp fragment containing the proximal promoter is responsive to vIRF4/RTA. Binding of a cellular factor(s) to this fragment is altered when both viral proteins are present, suggesting a possible mechanism for transcriptional synergy. Reliance on coregulators encoded by either the host or viral genome provides an elegant strategy for expanding the regulatory potential of a master regulator, such as RTA.

Both pluripotent Embryonic Stem Cells (ESCs), established from preimplantation murine blastocysts, and Epiblast Stem cells (EpiSCs), established from postimplantation embryos, can self-renew in culture or differentiate into each of the primary germ layers. While the core transcription factors (TFs) OCT4, SOX2, and NANOG are expressed in both cell types, the gene expression profiles and other features suggest that ESCs and EpiSCs reflect distinct developmental maturation stages of the epiblast in vivo. Accordingly, ‘naïve’ or ‘ground state’ ESCs resemble cells of the ICM, whereas ‘primed’ EpiSCs resemble cells of the postimplantation egg cylinder. To gain insight into the relationship between naïve and primed pluripotent cells, and of each of these pluripotent states to that of non-pluripotent cells, we have used FAIRE-seq to generate a comparative atlas of the accessible chromatin regions within ESCs, EpiSCs, multipotent Neural Stem cells (NSCs) and Mouse Embryonic Fibroblasts (MEFs). We find a distinction between the accessible chromatin patterns of pluripotent and somatic cells that is consistent with the highly related phenotype of ESCs and EpiSCs. However, by defining cell-specific- and shared regions of open chromatin, and integrating these data with published gene expression- and ChIP analyses, we also illustrate unique features of the chromatin of naïve- and primed cells. Functional studies suggest that multiple stage-specific enhancers regulate ESC- or EpiSC- specific gene expression, and implicate auxiliary TFs as important modulators for stage-specific activation by the core TFs. Together these observations provide insights into the chromatin structure dynamics accompanying transitions between these pluripotent states.