The Inducible Tissue-Specific Expression of the Human IL-3/GM-CSF Locus Is Controlled by a Complex Array of Developmentally Regulated Enhancers

Abstract

The closely linked human IL-3 and GM-CSF genes are tightly regulated and are expressed in activated T cells and mast cells. Here we used transgenic mice to study the developmental regulation of this locus and to identify DNA elements required for its correct activity in vivo. Because these two genes are separated by a CTCF-dependent insulator, and the GM-CSF gene is regulated primarily by its own upstream enhancer, the main aim was to identify regions of the locus required for correct IL-3 gene expression. We initially found that the previously identified proximal upstream IL-3 enhancers were insufficient to account for the in vivo activity of the IL-3 gene. However, an extended analysis of DNase I hypersensitive sites (DHSs) spanning the entire upstream IL-3 intergenic region revealed the existence of a complex cluster of both constitutive and inducible DHSs spanning the −34 to −40 kb region. The tissue specificity of these DHSs mirrored the activity of the IL-3 gene, and included a highly inducible CyclosporinA-sensitive enhancer at −37 kb which increased IL-3 promoter activity 40 fold. Significantly, inclusion of this region enabled correct in vivo regulation of IL-3 gene expression in T cells, mast cells and myeloid progenitor cells.

Inducible chromatin priming is associated with the establishment of immunological memory in T cells

“…In our previous studies of the human IL3 / CSF2 locus, we investigated the properties of regulatory elements that control the activation of these two highly inducible cytokine genes in T cells. Similar to the above studies, we identified two distinct classes of DHS that were acquired at different stages of T‐cell differentiation and activation (Mirabella et al , ; Baxter et al , ). One class existed as stably maintained DHSs that were absent in the thymus and in naïve T cells, and were formed during the process of T blast cell transformation, when T cells become activated via TCR signaling for the first time.…”

“…We have previously shown that the −4.1‐kb, −34‐kb, and −41‐kb pDHSs in the IL3 locus lack enhancer activity, whereas the −37‐kb iDHS is a powerful inducible enhancer (Hawwari et al , ; Baxter et al , ). We therefore hypothesized that the human IL3 −34‐kb and −41‐kb DHSs were representative of a distinct class of regulatory element that maintain stable zones of active chromatin, but lack classical enhancer activity (Baxter et al , ).…”

“…However, the function of these pDHSs remained unclear as they had no detectable enhancer activity when measured by transient transfection assays using reporter genes (Hawwari et al, 2002;Baxter et al, 2012). In addition to these pDHSs, we also identified several inducible DHSs (iDHSs) that appeared transiently in direct response to activation of TCR signaling (Cockerill, 2004;Baxter et al, 2012). Similar to iDHSs detected in other inducible genes in T cells, such as Il4 (Fields et al, 2004) and Il10 (Jones & Flavell, 2005), the iDHSs in the IL3/CSF2 locus were associated with strong inducible enhancer function in both transient transfection assays and transgenic mice (Cockerill et al, 1999;Baxter et al, 2012).…”

T Cell Receptor and Cytokine Signaling Can Function at Different Stages to Establish and Maintain Transcriptional Memory and Enable T Helper Cell Differentiation

“…However, in addition to differentiation-specific DHSs, CD4 T cells acquire many DHSs in genes such as IL4 in recently activated T cells prior to terminal differentiation (10, 42). These newly acquired DHSs represent the early stages of acquiring transcriptional competency prior to the activation of active transcription (18, 23, 45). …”

Prior epigenetic priming of cytokine genes in naive T cells is required for their subsequent activation by inducible enhancers

“…( 1 ) During blast cell transformation, the IL-3/GM-CSF locus acquires an extensive array of DNase I Hypersensitive Sites (DHSs) which are then maintained indefinitely for many cell cycles [1,2]. These primed DHSs are marked by me2K4 histone H3, and they also persist in non-dividing memory T cells in the peripheral blood [1].…”