Enhancer RNA Expression in Response to Glucocorticoid Treatment in Murine Macrophages

Glucocorticoids are potent anti-inflammatory drugs; however, their molecular mode of action remains complex and elusive. They bind to the glucocorticoid receptor (GR), a nuclear receptor that controls gene expression in almost all tissues in a cell type-specific manner. While GR’s transcriptional targets mediate beneficial reactions in immune cells, they also harbor the potential of adverse metabolic effects in other cell types such as hepatocytes. Here, we have profiled nascent transcription upon glucocorticoid stimulation in LPS-activated primary murine macrophages using 4sU-seq. We compared our results to publicly available nascent transcriptomics data from murine liver and bioinformatically identified non-coding RNAs transcribed from intergenic GR binding sites in a tissue-specific fashion. These tissue-specific enhancer RNAs (eRNAs) correlate with target gene expression, reflecting cell type-specific glucocorticoid responses. We further associate GR-mediated eRNA expression with changes in H3K27 acetylation and BRD4 recruitment in inflammatory macrophages upon glucocorticoid treatment. In summary, we propose a common mechanism by which GR-bound enhancers regulate target gene expression by changes in histone acetylation, BRD4 recruitment and eRNA expression. We argue that local eRNAs are potential therapeutic targets downstream of GR signaling which may modulate glucocorticoid response in a cell type-specific way.

Structure-guided approach to relieving transcriptional repression inResistance to Thyroid Hormone α

Mutations in thyroid hormone receptor α (TRα), a ligand-inducible transcription factor, cause Resistance to Thyroid Hormone α (RTHα). This disorder is characterised by tissue-specific hormone refractoriness and hypothyroidism, due to inhibition of target gene expression by mutant TRα-corepressor complexes. Using biophysical approaches, we show that RTHα-associated TRα mutants devoid of ligand-dependent transcription activation function, unexpectedly retain the ability to bind thyroid hormone. Visualisation of ligand (T3) within the crystal structure of a prototypic TRα mutant, validates this notion. This finding prompted synthesis of different thyroid hormone analogues, identifying a lead compound (ES08) which dissociates corepressor from mutant human TRα more efficaciously than T3. ES08 rescues developmental anomalies in a zebrafish model of RTHα and induces target gene expression in TRα mutation-containing cells from an RTHα patient, more effectively than T3. Our observations provide proof-of-principle for developing synthetic ligands that can relieve transcriptional repression by the mutant TRα-corepressor complex, for treatment of RTHα.

pVHL regulates protein stability of TCF/LEF transcription factor family via ubiquitin-independent proteasomal degradation

The Wnt/β-catenin signaling pathway plays key roles in development and adult tissue homeostasis by controlling cell proliferation and cell fate decisions. In this pathway, transcription factors TCF/LEFs are the key components to repress target gene expression by recruiting co-repressors or to activate target gene expression by recruiting β-catenin when the Wnt signals are absent or present, respectively. While progress has been made in our understanding of Wnt signaling regulation, the underlying mechanism that regulates the protein stability of the TCF/LEF family is far less clear. Here, we show that von Hippel-Lindau protein (pVHL), which is the substrate recognition component in an E3 ubiquitin ligase complex, controls TCF/LEF protein stability. Unexpectedly, pVHL directly binds to TCF/LEFs and promotes their proteasomal degradation independent of E3 ubiquitin ligase activity. Knockout of vhl in zebrafish embryos leads to a reduction of dorsal habenular neurons and this effect is upstream of dorsal habenular neurons phenotype in tcf7l2-null mutants. Our study uncovers a previously unknown mechanism for the protein stability regulation of the TCF/LEF transcription factors and demonstrates that pVHL contains a 26S proteasome binding domain that drives ubiquitin-independent proteasomal degradation. These findings provide new insights into the ubiquitin-independent actions of pVHL and uncover novel mechanistical regulation of Wnt/β-catenin signaling.

Mapping the evolving landscape of super-enhancers during cell differentiation

Background Super-enhancers are clusters of enhancer elements that play critical roles in the maintenance of cell identity. Current investigations on super-enhancers are centered on the established ones in static cell types. How super-enhancers are established during cell differentiation remains obscure. Results Here, by developing an unbiased approach to systematically analyze the evolving landscape of super-enhancers during cell differentiation in multiple lineages, we discover a general trend where super-enhancers emerge through three distinct temporal patterns: conserved, temporally hierarchical, and de novo. The three types of super-enhancers differ further in association patterns in target gene expression, functional enrichment, and 3D chromatin organization, suggesting they may represent distinct structural and functional subtypes. Furthermore, we dissect the enhancer repertoire within temporally hierarchical super-enhancers, and find enhancers that emerge at early and late stages are enriched with distinct transcription factors, suggesting that the temporal order of establishment of elements within super-enhancers may be directed by underlying DNA sequence. CRISPR-mediated deletion of individual enhancers in differentiated cells shows that both the early- and late-emerged enhancers are indispensable for target gene expression, while in undifferentiated cells early enhancers are involved in the regulation of target genes. Conclusions In summary, our analysis highlights the heterogeneity of the super-enhancer population and provides new insights to enhancer functions within super-enhancers.

Highly tunable TetR-dependent target gene expression in the acetic acid bacterium Gluconobacter oxydans

For the acetic acid bacterium (AAB) Gluconobacter oxydans only recently the first tight system for regulatable target gene expression became available based on the heterologous repressor-activator protein AraC from Escherichia coli and the target promoter ParaBAD. In this study, we tested pure repressor-based TetR- and LacI-dependent target gene expression in G. oxydans by applying the same plasmid backbone and construction principles that we have used successfully for the araC-ParaBAD system. When using a pBBR1MCS-5-based plasmid, the non-induced basal expression of the Tn10-based TetR-dependent expression system was extremely low. This allowed calculated induction ratios of up to more than 3500-fold with the fluorescence reporter protein mNeonGreen (mNG). The induction was highly homogeneous and tunable by varying the anhydrotetracycline concentration from 10 to 200 ng/mL. The already strong reporter gene expression could be doubled by inserting the ribosome binding site AGGAGA into the 3’ region of the Ptet sequence upstream from mNG. Alternative plasmid constructs used as controls revealed a strong influence of transcription terminators and antibiotics resistance gene of the plasmid backbone on the resulting expression performance. In contrast to the TetR-Ptet-system, pBBR1MCS-5-based LacI-dependent expression from PlacUV5 always exhibited some non-induced basal reporter expression and was therefore tunable only up to 40-fold induction by IPTG. The leakiness of PlacUV5 when not induced was independent of potential read-through from the lacI promoter. Protein-DNA binding simulations for pH 7, 6, 5, and 4 by computational modeling of LacI, TetR, and AraC with DNA suggested a decreased DNA binding of LacI when pH is below 6, the latter possibly causing the leakiness of LacI-dependent systems hitherto tested in AAB. In summary, the expression performance of the pBBR1MCS-5-based TetR-Ptet system makes this system highly suitable for applications in G. oxydans and possibly in other AAB. Key Points • A pBBR1MCS-5-based TetR-Ptet system was tunable up to more than 3500-fold induction. • A pBBR1MCS-5-based LacI-PlacUV5 system was leaky and tunable only up to 40-fold. • Modeling of protein-DNA binding suggested decreased DNA binding of LacI at pH < 6.