Cytoplasmic dynein is an essential microtubule motor protein that powers organelle transport and mitotic spindle assembly. Its activity depends on dynein-dynactin-cargo adaptor complexes, such as dynein-dynactin-BicD2, which typically function with two dynein motors. We show that mechanical tension recruits a third dynein motor via an auxiliary BicD2 adaptor binding the light intermediate chain of the third dynein, stabilizing multidynein assemblies and enhancing force generation. Lis1 prevents dynein from transitioning into a force-limiting phi-like conformation, allowing single-dynein dynein-dynactin-BicD2 to sustain forces up to approximately 4.5 pN, whereas force generation often ends at about 2.5 pN without Lis1. Complexes with two or three dyneins generate 7 pN and 9 pN, respectively, consistent with a staggered motor arrangement that enhances collective output. Under load, dynein-dynactin-BicD2 primarily takes 8-nm steps, challenging existing dynein coordination models. These findings reveal adaptive mechanisms that enable robust intracellular transport under varying mechanical demands.

Cytoplasmic dynein is an essential microtubule motor protein that powers organelle transport and mitotic spindle assembly. Its activity depends on dynein-dynactin-cargo adaptor complexes, such as dynein-dynactin-BicD2 (DDB), which typically function with two dynein motors. We show that mechanical tension recruits a third dynein motor via an auxiliary BicD adaptor binding the light intermediate chain of the third dynein, stabilizing multi-dynein assemblies and enhancing force generation. Lis1 prevents dynein from transitioning into a force-limiting phi-like conformation, allowing single-dynein DDB to sustain forces up to ~4.5 pN, whereas force generation often ends at ~2.5 pN without Lis1. Complexes with two or three dyneins generate ~7 pN and ~9 pN, respectively, consistent with a staggered motor arrangement that enhances collective output. Under load, DDB primarily takes ~8 nm steps, challenging existing dynein coordination models. These findings reveal adaptive mechanisms that enable robust intracellular transport under varying mechanical demands.

Gene transcription by RNA polymerase II (Pol II) is under control of promoters and distal regulatory elements known as enhancers. Enhancers are themselves transcribed by Pol II correlating with their activity. How enhancer transcription is regulated and coordinated with transcription at target genes has remained unclear. Here, we developed a high-sensitive native elongating transcript sequencing approach, called HiS-NET-seq, to provide an extended high-resolution view on transcription, especially at lowly transcribed regions such as enhancers. HiS-NET-seq uncovers new transcribed enhancers in human cells. A multi-omics analysis shows that genome-wide enhancer transcription depends on the BET family protein BRD4. Specifically, BRD4 co-localizes to enhancer and promoter-proximal gene regions, and is required for elongation activation at enhancers and their genes. BRD4 keeps a set of enhancers and genes in proximity through long-range contacts. From these studies BRD4 emerges as a general regulator of enhancer transcription that may link transcription at enhancers and genes.

Cell type-specific gene expression is coordinated by DNA-encoded enhancers and the transcription factors (TFs) that bind to them in a sequence-specific manner. As such, these enhancers and TFs are critical mediators of normal development and altered enhancer or TF function is associated with the development of diseases such as cancer. While initially defined by their ability to activate gene transcription in reporter assays, putative enhancer elements are now frequently defined by their unique chromatin features including DNase hypersensitivity and transposase accessibility, bidirectional enhancer RNA (eRNA) transcription, CpG hypomethylation, high H3K27ac and H3K4me1, sequence-specific transcription factor binding, and co-factor recruitment. Identification of these chromatin features through sequencing-based assays has revolutionized our ability to identify enhancer elements on a genome-wide scale, and genome-wide functional assays are now capitalizing on this information to greatly expand our understanding of how enhancers function to provide spatiotemporal coordination of gene expression programs. Here, we highlight recent technological advances that are providing new insights into the molecular mechanisms by which these critical cis-regulatory elements function in gene control. We pay particular attention to advances in our understanding of enhancer transcription, enhancer-promoter syntax, 3D organization and biomolecular condensates, transcription factor and co-factor dependencies, and the development of genome-wide functional enhancer screens.

Transcription by RNA polymerase II (Pol II) gives rise to all nuclear protein-coding and a large set of non-coding RNAs, and is strictly regulated and coordinated with RNA processing. Bromodomain and extraterminal (BET) family proteins including BRD2, BRD3, and BRD4 have been implicated in the regulation of Pol II transcription in mammalian cells. However, only recent technological advances have allowed the analysis of direct functions of individual BET proteins with high precision in cells. These studies shed new light on the molecular mechanisms of transcription control by BET proteins challenging previous longstanding views. The most studied BET protein, BRD4, emerges as a master regulator of transcription elongation with roles also in coupling nascent transcription with RNA processing. In contrast, BRD2 is globally required for the formation of transcriptional boundaries to restrict enhancer activity to nearby genes. Although these recent findings suggest non-redundant functions of BRD4 and BRD2 in Pol II transcription, more research is needed for further clarification. Little is known about the roles of BRD3. Here, we illuminate experimental work that has initially linked BET proteins to Pol II transcription in mammalian cells, outline main methodological breakthroughs that have strongly advanced the understanding of BET protein functions, and discuss emerging roles of individual BET proteins in transcription and transcription-coupled RNA processing. Finally, we propose an updated model for the function of BRD4 in transcription and co-transcriptional RNA maturation. This article is categorized under: RNA Processing > 3' End Processing RNA Processing > Splicing Regulation/Alternative Splicing.

Female mammals achieve dosage compensation by inactivating one of their two X chromosomes during development, a process entirely dependent on Xist, an X-linked long non-coding RNA (lncRNA). At the onset of X chromosome inactivation (XCI), Xist is up-regulated and spreads along the future inactive X chromosome. Contextually, it recruits repressive histone and DNA modifiers that transcriptionally silence the X chromosome. Xist regulation is tightly coupled to differentiation and its expression is under the control of both pluripotency and epigenetic factors. Recent evidence has suggested that chromatin remodelers accumulate at the X Inactivation Center (XIC) and here we demonstrate a new role for Chd8 in Xist regulation in differentiating ES cells, linked to its control and prevention of spurious transcription factor interactions occurring within Xist regulatory regions. Our findings have a broader relevance, in the context of complex, developmentally-regulated gene expression.

Transcription elongation has emerged as a regulatory hub in gene expression of metazoans. A major control point occurs during early elongation before RNA polymerase II (Pol II) is released into productive elongation. Prior research has linked BRD4 with transcription elongation. Here, we use rapid BET protein and BRD4-selective degradation along with quantitative genome-wide approaches to investigate direct functions of BRD4 in Pol II transcription regulation. Notably, as an immediate consequence of acute BRD4 loss, promoter-proximal pause release is impaired, and transcriptionally engaged Pol II past this checkpoint undergoes readthrough transcription. An integrated proteome-wide analysis uncovers elongation and 3'-RNA processing factors as core BRD4 interactors. BRD4 ablation disrupts the recruitment of general 3'-RNA processing factors at the 5'-control region, which correlates with RNA cleavage and termination defects. These studies, performed in human cells, reveal a BRD4-mediated checkpoint and begin to establish a molecular link between 5'-elongation control and 3'-RNA processing.

Enhancers are transcribed by RNA polymerase II (Pol II). In order to study the regulation of enhancer transcription and its function in target gene control, methods are required that track genome transcription with high precision in vivo. Here, we provide step-by-step guidance for performing native elongating transcript sequencing (NET-Seq) in mammalian cells. NET-Seq allows quantitative measurements of transcription genome-wide, including enhancer transcription, with single-nucleotide and DNA strand resolution. The approach consists of capturing and efficiently converting the 3'-ends of the nascent RNA into a sequencing library followed by next-generation sequencing and computational data analysis. The protocol includes quality control measurements to monitor the success of the main steps. Following this protocol, a NET-Seq library is obtained within 5 days.