According to current models, once the cell has reached terminal differentiation, the enhancer repertoire is completely established and maintained by cooperatively acting lineage-specific transcription factors (TFs). TFs activated by extracellular stimuli operate within this predetermined repertoire, landing close to where master regulators are constitutively bound. Here, we describe latent enhancers, defined as regions of the genome that in terminally differentiated cells are unbound by TFs and lack the histone marks characteristic of enhancers but acquire these features in response to stimulation. Macrophage stimulation caused sequential binding of stimulus-activated and lineage-determining TFs to these regions, enabling deposition of enhancer marks. Once unveiled, many of these enhancers did not return to a latent state when stimulation ceased; instead, they persisted and mediated a faster and stronger response upon restimulation. We suggest that stimulus-specific expansion of the cis-regulatory repertoire provides an epigenomic memory of the exposure to environmental agents.

Macrophage stimulation with interferon-γ (IFN-γ) and interleukin 4 (IL-4) triggers distinct and opposing activation programs. During mixed infections or cancer macrophages are often exposed to both cytokines, but how these two programs influence each other remains unclear. We found that IFN-γ and IL-4 mutually inhibited epigenomic and transcriptional changes induced by each cytokine alone. Computational and functional analyses revealed the genomic bases for gene-specific cross-repression. For instance, while STAT1 and IRF1 motifs were associated with robust and IL-4-resistant responses to IFN-γ their coexistence with binding sites for auxiliary transcription factors such as AP-1, generated vulnerability to IL-4-mediated inhibition. These data provide a core mechanistic framework for the integration of signals that control macrophage activation in complex environmental conditions.

Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adaptor protein WDR82 and its associated complexes actively limit such non-coding transcription. WDR82 targets the SET1 H3K4 methyltransferases and the nuclear protein phosphatase 1 (PP1) complexes to the initiating Pol II. WDR82 and PP1 also interact with components of the transcriptional termination and RNA processing machineries. Depletion of WDR82, SET1, or the PP1 subunit required for its nuclear import caused distinct but overlapping transcription termination defects at highly expressed genes and active enhancers and promoters, thus enabling the increased synthesis of unusually long ncRNAs. These data indicate that transcription initiated from cis-regulatory elements is tightly coordinated with termination mechanisms that impose the synthesis of short RNAs.

Enhancers and promoters that control the transcriptional output of terminally differentiated cells include cell type-specific and broadly active housekeeping elements. Whether the high constitutive activity of these two groups of -regulatory elements relies on entirely distinct or instead also on shared regulators is unknown. By dissecting the-regulatory repertoire of macrophages, we found that the ELF subfamily of ETS proteins selectively bound within 60 base pairs (bp) from the transcription start sites of highly active housekeeping genes. ELFs also bound constitutively active, but not poised, macrophage-specific enhancers and promoters. The role of ELFs in promoting high-level constitutive transcription was suggested by multiple evidence: ELF sites enabled robust transcriptional activation by endogenous and minimal synthetic promoters, ELF recruitment was stabilized by the transcriptional machinery, and ELF proteins mediated recruitment of transcriptional and chromatin regulators to core promoters. These data suggest that the co-optation of a limited number of highly active transcription factors represents a broadly adopted strategy to equip both cell type-specific and housekeeping -regulatory elements with the ability to efficiently promote transcription.

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