How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We traced nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter–enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream from the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, partial disruption of promoter–enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 transcription. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.

LUMAN/CREB3, originally identified through its interaction with a cell cycle regulator HCFC1, is a transcription factor involved in the unfolded protein response during endoplasmic reticulum stress. Previously using gene knockout mouse models, we have shown that LUMAN modulates the glucocorticoid (GC) response leading to enhanced glucocorticoid receptor (GR) activity and lower circulating GC levels. Consequently, the stress response is dysregulated, leading to a blunted stress response in the Luman-deficient mice. One question that remained was how LUMAN deficiency affected the stress response at the cellular level leading to the changes in the physiological stress response. Here, we found that LUMAN interacts with GR through a putative nuclear receptor box site and can activate GR in the absence of a ligand. Further investigation showed that, when activated, LUMAN binds to the glucocorticoid response element (GRE), increasing the activity of GR exponentially compared to GR-ligand binding alone. On the other hand, we also found that in the absence of LUMAN, cells were more sensitive to cellular stress, exhibiting decreased secretory capacity. Hence our current data suggest that LUMAN may function both as a transcriptional cofactor of GR and a hormone secretion regulator, and through this, plays a role in stress sensitivity and reactivity to stress.

Enhancers are cis-regulatory sequences located distally to target genes. These sequences consolidate developmental and environmental cues to coordinate gene expression in a tissue-specific manner. Enhancer function and tissue specificity depend on the expressed set of transcription factors, which recognize binding sites and recruit cofactors that regulate local chromatin organization and gene transcription. Unlike other genomic elements, enhancers are challenging to identify because they function independently of orientation, are often distant from their promoters, have poorly defined boundaries and display no reading frame. In addition, there are no defined genetic or epigenetic features that are unambiguously associated with enhancer activity. Over recent years there have been developments in both empirical assays and computational methods for enhancer prediction. We review genome-wide tools, CRISPR advancements and high-throughput screening approaches that have improved our ability to both observe and manipulate enhancers in vitro at the level of primary genetic sequences, chromatin states and spatial interactions. We also highlight contemporary animal models and their importance to enhancer validation. Together, these experimental systems and techniques complement one another and broaden our understanding of enhancer function in development, evolution, and disease.

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