Differential regulation of alternative promoters emerges from unified kinetics of enhancer-promoter interaction

Many eukaryotic genes contain alternative promoters with distinct expression patterns. How these promoters are differentially regulated remains elusive. Here, we apply single-molecule imaging to quantify the transcriptional regulation of two alternative promoters (P1 and P2) of the Bicoid (Bcd) target gene hunchback in syncytial blastoderm Drosophila embryos. Contrary to the previous notion that Bcd only activates P2, we find that Bcd activates both promoters via the same two enhancers. P1 activation is less frequent and requires binding of more Bcd molecules than P2 activation. Using a theoretical model to relate promoter activity to enhancer states, we show that the two promoters follow common transcription kinetics driven by sequential Bcd binding at the two enhancers. Bcd binding at either enhancer primarily activates P2, while P1 activation relies more on Bcd binding at both enhancers. These results provide a quantitative framework for understanding the dynamics of complex eukaryotic gene regulation.

Allosteric conformational ensembles have unlimited capacity for integrating information

Integration of binding information by macromolecular entities is fundamental to cellular functionality. Recent work has shown that such integration cannot be explained by pairwise cooperativities, in which binding is modulated by binding at another site. Higher-order cooperativities (HOCs), in which binding is collectively modulated by multiple other binding events, appear to be necessary but an appropriate mechanism has been lacking. We show here that HOCs arise through allostery, in which effective cooperativity emerges indirectly from an ensemble of dynamically interchanging conformations. Conformational ensembles play important roles in many cellular processes but their integrative capabilities remain poorly understood. We show that sufficiently complex ensembles can implement any form of information integration achievable without energy expenditure, including all patterns of HOCs. Our results provide a rigorous biophysical foundation for analysing the integration of binding information through allostery. We discuss the implications for eukaryotic gene regulation, where complex conformational dynamics accompanies widespread information integration.

Differential Bcd activation of two hunchback promoters emerges from unified kinetics of enhancer-promoter interaction

Many eukaryotic genes contain alternative promoters with distinct expression patterns. How these promoters are differentially regulated remains elusive. Here, we apply single-molecule imaging to quantify the transcriptional regulation of two alternative promoters (P1 and P2) of the Bicoid (Bcd) target gene hunchback in syncytial blastoderm Drosophila embryos. Contrary to the previous notion that Bcd only activates P2, we find that Bcd activates both promoters via the same two enhancers. P1 activation is less frequent and requires binding of more Bcd molecules than P2 activation. Using a theoretical model to relate promoter activity to enhancer states, we show that the two promoters follow common transcription kinetics driven by sequential Bcd binding at the two enhancers. Bcd binding at either enhancer primarily activates P2, while P1 activation relies more on Bcd binding at both enhancers. These results provide a quantitative framework for understanding the dynamics of complex eukaryotic gene regulation.

Allosteric conformational ensembles have unlimited capacity for integrating information

Integration of binding information by macromolecular entities is fundamental to cellular functionality. Recent work has shown that such integration cannot be explained by pairwise cooperativities, in which binding is modulated by binding at another site. Higher-order cooperativities (HOCs), in which binding is collectively modulated by multiple other binding events, appears to be necessary but an appropriate mechanism has been lacking. We show here that HOCs arise through allostery, in which effective cooperativity emerges indirectly from an ensemble of dynamically-interchanging conformations. Conformational ensembles play important roles in many cellular processes but their integrative capabilities remain poorly understood. We show that sufficiently complex ensembles can implement any form of information integration achievable without energy expenditure, including all HOCs. Our results provide a rigorous biophysical foundation for analysing the integration of binding information through allostery. We discuss the implications for eukaryotic gene regulation, where complex conformational dynamics accompanies widespread information integration.

Long noncoding RNAs in renal diseases

Long noncoding RNAs (lncRNAs) play critical roles in eukaryotic gene regulation and diseases, rather than being merely transcriptional “noise”. Over the past decade, the study of lncRNAs has emerged as a burgeoning field of research and expanded our knowledge of their functions and underlining mechanisms in both normal and malignant cells. However, lncRNAs are still one of the least understood groups of transcripts. Here, we review the classifications and functions of lncRNAs and their roles in renal diseases. This review will provide insights into the roles of lncRNAs in pathogenesis, diagnosis and therapeutics of renal diseases and indications of lncRNAs as potential targets for the treatment of kidney diseases.

An intron-derived motif strongly increases gene expression from transcribed sequences through a splicing independent mechanism in Arabidopsis thaliana

Certain introns significantly increase mRNA accumulation by a poorly understood mechanism. These introns have no effect when located upstream, or more than ~1 Kb downstream, of the start of transcription. We tested the ability of a formerly non-stimulating intron containing 11 copies of the sequence TTNGATYTG, which is over-represented in promoter-proximal introns in Arabidopsis thaliana, to affect expression from various positions. The activity profile of this intron at different locations was similar to that of a natural intron from the UBQ10 gene, suggesting that the motif increases mRNA accumulation by the same mechanism. A series of introns with different numbers of this motif revealed that the effect on expression is linearly dependent on motif copy number up to at least 20, with each copy adding another 1.5-fold increase in mRNA accumulation. Furthermore, 6 copies of the motif stimulated mRNA accumulation to a similar degree from within an intron or when introduced into the 5′-UTR and coding sequences of an intronless construct, demonstrating that splicing is not required for this sequence to boost expression. The ability of this motif to substantially elevate expression from several hundred nucleotides downstream of the transcription start site reveals a novel type of eukaryotic gene regulation.

Roles of the INO80 and SWR1 Chromatin Remodeling Complexes in Plants

Eukaryotic genes are packed into a dynamic but stable nucleoprotein structure called chromatin. Chromatin-remodeling and modifying complexes generate a dynamic chromatin environment that ensures appropriate DNA processing and metabolism in various processes such as gene expression, as well as DNA replication, repair, and recombination. The INO80 and SWR1 chromatin remodeling complexes (INO80-c and SWR1-c) are ATP-dependent complexes that modulate the incorporation of the histone variant H2A.Z into nucleosomes, which is a critical step in eukaryotic gene regulation. Although SWR1-c has been identified in plants, plant INO80-c has not been successfully isolated and characterized. In this review, we will focus on the functions of the SWR1-c and putative INO80-c (SWR1/INO80-c) multi-subunits and multifunctional complexes in Arabidopsis thaliana. We will describe the subunit compositions of the SWR1/INO80-c and the recent findings from the standpoint of each subunit and discuss their involvement in regulating development and environmental responses in Arabidopsis.

Automated in situ profiling of chromatin modifications resolves cell types and gene regulatory programs

Our understanding of eukaryotic gene regulation is limited by the complexity of protein-DNA interactions that comprise the chromatin landscape and by inefficient methods for characterizing these interactions. We recently introduced CUT&RUN, an antibody-targeted nuclease-cleavage method that profiles DNA-binding proteins, histones and chromatin modifying proteins in situ with exceptional sensitivity and resolution. Here we describe an automated CUT&RUN platform and apply it to characterize the chromatin landscapes of human cell lines. We find that CUT&RUN profiles of histone modifications crisply demarcate active and repressed chromatin regions, and we develop a continuous metric to identify cell-type specific promoter and enhancer activities. We test the ability of automated CUT&RUN to profile frozen tumor samples, and find that our method readily distinguishes two diffuse midline gliomas by their subtype-specific gene expression programs. The easy, cost-effective workflow makes automated CUT&RUN an attractive tool for high-throughput characterization of cell types and patient samples.