Transcriptional activation of the interleukin-8 (IL-8) gene is restricted to distinct cell types, although the transcriptional regulatory proteins controlling IL-8 gene expression are ubiquitous. We show that cell-specific transcription of IL-8 is due to the distinct chromatin architecture on the enhancer/promoter before the arrival of the inducing signal. In expressing epithelial cells the enhancer/promoter is nucleosome-free, whereas in non-expressing B cells a nucleosome masks the entire regulatory region. The B-cell-specific nucleosome contains the histone variant macroH2A, which is responsible for preventing transcription factor binding. Recruitment of the repressive macroH2A nucleosome requires direct interactions between ATF-2 bound to the nearby AP1 site and macroH2A and it is regulated by DNA-induced protein allostery. siRNA against ATF-2 or macroH2A rescues IL-8 transcription in B cells. Thus, a transcription factor can work as a transcriptional repressor by orchestrating and maintaining the assembly of specialized local chromatin architectures.

Summary We present a strategy to examine the chromatin conformation of individual loci in specific cell types during Drosophila embryogenesis. Regulatory DNA is tagged with binding sites (lacO) for LacI, which is used to immunopreciptiate the tagged chromatin from specific cell types. We applied this approach to Distalless (Dll), a gene required for limb development in Drosophila. We show that the local chromatin conformation at Dll depends on the cell type: in cells that express Dll, the 5’ regulatory region is in close proximity to the Dll promoter. In Dll nonexpressing cells this DNA is in a more extended configuration. In addition, transcriptional activators and repressors are bound to Dll regulatory DNA in a cell type specific manner. The pattern of binding by GAGA factor and the variant histone H2Av suggest that they play a role in the regulation of Dll chromatin conformation in expressing and non-expressing cell types, respectively.

The histone variant macroH2A (mH2A) has been implicated in transcriptional repression, but the molecular mechanisms that contribute to global mH2A-dependent genome regulation remain elusive. Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with transcriptional profiling in mH2A knockdown cells, we demonstrate that singular mH2A nucleosomes occupy transcription start sites of subsets of both expressed and repressed genes, with opposing regulatory consequences. Specifically, mH2A nucleosomes mask repressor binding sites in expressed genes but activator binding sites in repressed genes, thus generating distinct chromatin landscapes that limit genetic or extracellular inductive signals. We show that composite nucleosomes containing mH2A and NRF-1 are stably positioned on gene regulatory regions and can buffer transcriptional noise associated with antiviral responses. In contrast, mH2A nucleosomes without NRF-1 bind promoters weakly and mark genes with noisier gene expression patterns. Thus, the strategic position and stabilization of mH2A nucleosomes in human promoters defines robust gene expression patterns.

Edited by Joel GottesfeldIL-2 is the first cytokine produced when naive T helper (Th) cells are activated and differentiate into dividing pre-Th0 proliferating precursors. IL-2 expression is blocked in naive, but not activated or memory, Th cells by the transcription factor Ets-2 that binds to the antigen receptor response element (ARRE)-2 of the proximal IL-2 promoter. Ets-2 acts as an independent preinduction repressor in naive Th cells and does not interact physically with the transcription factor NFAT (nuclear factor of activated T-cells) that binds to the ARRE-2 in activated Th cells. In naive Th cells, Ets-2 mRNA expression, Ets-2 protein levels, and Ets-2 binding to ARRE-2 decrease upon cell activation followed by the concomitant expression of IL-2. Cyclosporine A stabilizes Ets-2 mRNA and protein when the cells are activated. Ets-2 silences directly constitutive or induced IL-2 expression through the ARRE-2. Conversely, Ets-2 silencing allows for constitutive IL-2 expression in unstimulated cells. Ets-2 binding to ARRE-2 in chromatin is stronger in naive compared with activated or memory Th cells; in the latter, Ets-2 participates in a change of the IL-2 promoter architecture, possibly to facilitate a quick response when the cells re-encounter antigen. We propose that Ets-2 expression and protein binding to the ARRE-2 of the IL-2 promoter are part of a strictly regulated process that results in a physiological transition of naive Th cells to Th0 cells upon antigenic stimulation. Malfunction of such a repression mechanism at the molecular level could lead to a disturbance of later events in Th cell plasticity, leading to autoimmune diseases or other pathological conditions.

The generation of induced pluripotent stem cells (iPSCs) from somatic cells provides an excellent model to study mechanisms of transcription factor-induced global alterations of the epigenome and genome function. Here, we have investigated the early transcriptional events of cellular reprogramming triggered by the co-expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) in mouse embryonic fibroblasts (MEFs) and mouse hepatocytes (mHeps). In this analysis, we identified a gene regulatory network composed of nine transcriptional regulators (9TR; Cbfa2t3, Gli2, Irf6, Nanog, Ovol1, Rcan1, Taf1c, Tead4, and Tfap4), which are directly targeted by OSKM, in vivo. Functional studies using single and double shRNA knockdowns of any of these factors caused disruption of the network and dramatic reductions in reprogramming efficiency, indicating that this network is essential for the induction and establishment of pluripotency. We demonstrate that the stochastic co-expression of 9TR network components occurs in a remarkably small number of cells, approximating the percentage of terminally reprogrammed cells as a result of dynamic molecular events. Thus, the early DNA-binding patterns of OSKM and the subsequent probabilistic co-expression of essential 9TR components in subpopulations of cells undergoing reprogramming steer the reconstruction of a gene regulatory network marking the transition to pluripotency.