KAP1 Is a Chromatin Reader that Couples Steps of RNA Polymerase II Transcription to Sustain Oncogenic Programs.

Precise control of the RNA polymerase II (RNA Pol II) cycle, including pausing and pause release, maintains transcriptional homeostasis and organismal functions. Despite previous work to understand individual transcription steps, we reveal a mechanism that integrates RNA Pol II cycle transitions. Surprisingly, KAP1/TRIM28 uses a previously uncharacterized chromatin reader cassette to bind hypo-acetylated histone 4 tails at promoters, guaranteeing continuous progression of RNA Pol II entry to and exit from the pause state. Upon chromatin docking, KAP1 first associates with RNA Pol II and then recruits a pathway-specific transcription factor (SMAD2) in response to cognate ligands, enabling gene-selective CDK9-dependent pause release. This coupling mechanism is exploited by tumor cells to aberrantly sustain transcriptional programs commonly dysregulated in cancer patients. The discovery of a factor integrating transcription steps expands the functional repertoire by which chromatin readers operate and provides mechanistic understanding of transcription regulation, offering alternative therapeutic opportunities to target transcriptional dysregulation.

Transcription factor bHLH121 regulates root cortical aerenchyma formation in maize.

Root anatomical phenotypes present a promising yet underexploited avenue to deliver major improvements in yield and climate resilience of crops by improving water and nutrient uptake. For instance, the formation of root cortical aerenchyma (RCA) significantly increases soil exploration and resource capture by reducing the metabolic costs of root tissue. A key bottleneck in studying such phenotypes has been the lack of robust high-throughput anatomical phenotyping platforms. We exploited a phenotyping approach based on laser ablation tomography, termed Anatomics, to quantify variation in RCA formation of 436 diverse maize lines in the field. Results revealed a significant and heritable variation for RCA formation. Genome-wide association studies identified a single-nucleotide polymorphism mapping to a root cortex-expressed gene-encoding transcription factor bHLH121. Functional studies identified that the bHLH121 Mu transposon mutant line and CRISPR/Cas9 loss-of-function mutant line showed reduced RCA formation, whereas an overexpression line exhibited significantly greater RCA formation when compared to the wild-type line. Characterization of these lines under suboptimal water and nitrogen availability in multiple soil environments revealed that bHLH121 is required for RCA formation developmentally as well as under studied abiotic stress. Overall functional validation of the bHLH121 gene's importance in RCA formation provides a functional marker to select varieties with improved soil exploration and thus yield under suboptimal conditions.

Root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus antigravitropic offset (AGO) mechanisms. Here we report a root angle regulatory gene termed ENHANCED GRAVITROPISM1 (EGT1) that encodes a putative AGO component, whose loss-of-function enhances root gravitropism. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes an F-box and Tubby domain-containing protein that is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wild-type. Transcript profiling revealed Hvegt1 roots deregulate reactive oxygen species (ROS) homeostasis and cell wall-loosening enzymes and cofactors. ROS imaging shows that Hvegt1 root basal meristem and elongation zone tissues have reduced levels. Atomic force microscopy measurements detected elongating Hvegt1 root cortical cell walls are significantly less stiff than wild-type. In situ analysis identified HvEGT1 is expressed in elongating cortical and stele tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. We propose that EGT1 controls root angle by regulating cell wall stiffness in elongating root cortical tissue, counteracting the gravitropic machinery's known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

The auxin efflux carrier PIN1a regulates vascular patterning in cereal roots.

Barley (Hordeum vulgare) is an important global cereal crop and a model in genetic studies. Despite advances in characterising barley genomic resources, few mutant studies have identified genes controlling root architecture and anatomy, which plays a critical role in capturing soil resources. Our phenotypic screening of a TILLING mutant collection identified line TM5992 exhibiting a short-root phenotype compared with wild-type (WT) Morex background. Outcrossing TM5992 with barley variety Proctor and subsequent SNP array-based bulk segregant analysis, fine mapped the mutation to a cM scale. Exome sequencing pinpointed a mutation in the candidate gene HvPIN1a, further confirming this by analysing independent mutant alleles. Detailed analysis of root growth and anatomy in Hvpin1a mutant alleles exhibited a slower growth rate, shorter apical meristem and striking vascular patterning defects compared to WT. Expression and mutant analyses of PIN1 members in the closely related cereal brachypodium (Brachypodium distachyon) revealed that BdPIN1a and BdPIN1b were redundantly expressed in root vascular tissues but only Bdpin1a mutant allele displayed root vascular defects similar to Hvpin1a. We conclude that barley PIN1 genes have sub-functionalised in cereals, compared to Arabidopsis (Arabidopsis thaliana), where PIN1a sequences control root vascular patterning.

Root angle in maize influences nitrogen capture and is regulated by calcineurin B-like protein (CBL)-interacting serine/threonine-protein kinase 15 (ZmCIPK15).

Crops with reduced nutrient and water requirements are urgently needed in global agriculture. Root growth angle plays an important role in nutrient and water acquisition. A maize diversity panel of 481 genotypes was screened for variation in root angle employing a high-throughput field phenotyping platform. Genome-wide association mapping identified several single nucleotide polymorphisms (SNPs) associated with root angle, including one located in the root expressed CBL-interacting serine/threonine-protein kinase 15 (ZmCIPK15) gene (LOC100285495). Reverse genetic studies validated the functional importance of ZmCIPK15, causing a approximately 10° change in root angle in specific nodal positions. A steeper root growth angle improved nitrogen capture in silico and in the field. OpenSimRoot simulations predicted at 40 days of growth that this change in angle would improve nitrogen uptake by 11% and plant biomass by 4% in low nitrogen conditions. In field studies under suboptimal N availability, the cipk15 mutant with steeper growth angles had 18% greater shoot biomass and 29% greater shoot nitrogen accumulation compared to the wild type after 70 days of growth. We propose that a steeper root growth angle modulated by ZmCIPK15 will facilitate efforts to develop new crop varieties with optimal root architecture for improved performance under edaphic stress.

Structure of a low-population binding intermediate in protein-RNA recognition.

The interaction of the HIV-1 protein transactivator of transcription (Tat) and its cognate transactivation response element (TAR) RNA transactivates viral transcription and represents a paradigm for the widespread occurrence of conformational rearrangements in protein-RNA recognition. Although the structures of free and bound forms of TAR are well characterized, the conformations of the intermediates in the binding process are still unknown. By determining the free energy landscape of the complex using NMR residual dipolar couplings in replica-averaged metadynamics simulations, we observe two low-population intermediates. We then rationally design two mutants, one in the protein and another in the RNA, that weaken specific nonnative interactions that stabilize one of the intermediates. By using surface plasmon resonance, we show that these mutations lower the release rate of Tat, as predicted. These results identify the structure of an intermediate for RNA-protein binding and illustrate a general strategy to achieve this goal with high resolution.

Simultaneous NMR characterisation of multiple minima in the free energy landscape of an RNA UUCG tetraloop.

RNA molecules in solution tend to undergo structural fluctuations of relatively large amplitude and to populate a range of different conformations some of which with low populations. It is still very challenging, however, to characterise the structures of these low populated states and to understand their functional roles. In the present study, we address this problem by using NMR residual dipolar couplings (RDCs) as structural restraints in replica-averaged metadynamics (RAM) simulations. By applying this approach to a 14-mer RNA hairpin containing the prototypical UUCG tetraloop motif, we show that it is possible to construct the free energy landscape of this RNA molecule. This free energy landscapes reveals the surprisingly rich dynamics of the UUCG tetraloop and identifies the multiple substates that exist in equilibrium owing to thermal fluctuations. The approach that we present is general and can be applied to the study of the free energy landscapes of other RNA or RNA-protein systems.

Integrating cryo-OrbiSIMS with computational modelling and metadynamics simulations enhances RNA structure prediction at atomic resolution.

The 3D architecture of RNAs governs their molecular interactions, chemical reactions, and biological functions. However, a large number of RNAs and their protein complexes remain poorly understood due to the limitations of conventional structural biology techniques in deciphering their complex structures and dynamic interactions. To address this limitation, we have benchmarked an integrated approach that combines cryogenic OrbiSIMS, a state-of-the-art solid-state mass spectrometry technique, with computational methods for modelling RNA structures at atomic resolution with enhanced precision. Furthermore, using 7SK RNP as a test case, we have successfully determined the full 3D structure of a native RNA in its apo, native and disease-remodelled states, which offers insights into the structural interactions and plasticity of the 7SK complex within these states. Overall, our study establishes cryo-OrbiSIMS as a valuable tool in the field of RNA structural biology as it enables the study of challenging, native RNA systems.

A method of determining RNA conformational ensembles using structure-based calculations of residual dipolar couplings.

We describe a method of determining the conformational fluctuations of RNA based on the incorporation of nuclear magnetic resonance (NMR) residual dipolar couplings (RDCs) as replica-averaged structural restraints in molecular dynamics simulations. In this approach, the alignment tensor required to calculate the RDCs corresponding to a given conformation is estimated from its shape, and multiple replicas of the RNA molecule are simulated simultaneously to reproduce in silico the ensemble-averaging procedure performed in the NMR measurements. We provide initial evidence that with this approach it is possible to determine accurately structural ensembles representing the conformational fluctuations of RNA by applying the reference ensemble test to the trans-activation response element of the human immunodeficiency virus type 1.

Identification and characterisation of a rare MTTP variant underlying hereditary non-alcoholic fatty liver disease.

Background & Aims: Non-alcoholic fatty liver disease (NAFLD) is a complex trait with an estimated prevalence of 25% globally. We aimed to identify the genetic variant underlying a four-generation family with progressive NAFLD leading to cirrhosis, decompensation, and development of hepatocellular carcinoma in the absence of common risk factors such as obesity and type 2 diabetes. Methods: Exome sequencing and genome comparisons were used to identify the likely causal variant. We extensively characterised the clinical phenotype and post-prandial metabolic responses of family members with the identified novel variant in comparison with healthy non-carriers and wild-type patients with NAFLD. Variant-expressing hepatocyte-like cells (HLCs) were derived from human-induced pluripotent stem cells generated from homozygous donor skin fibroblasts and restored to wild-type using CRISPR-Cas9. The phenotype was assessed using imaging, targeted RNA analysis, and molecular expression arrays. Results: We identified a rare causal variant c.1691T>C p.I564T (rs745447480) in MTTP, encoding microsomal triglyceride transfer protein (MTP), associated with progressive NAFLD, unrelated to metabolic syndrome and without characteristic features of abetalipoproteinaemia. HLCs derived from a homozygote donor had significantly lower MTP activity and lower lipoprotein ApoB secretion than wild-type cells, while having similar levels of MTP mRNA and protein. Cytoplasmic triglyceride accumulation in HLCs triggered endoplasmic reticulum stress, secretion of pro-inflammatory mediators, and production of reactive oxygen species. Conclusions: We have identified and characterised a rare causal variant in MTTP, and homozygosity for MTTP p.I564T is associated with progressive NAFLD without any other manifestations of abetalipoproteinaemia. Our findings provide insights into mechanisms driving progressive NAFLD. Impact and Implications: A rare genetic variant in the gene MTTP has been identified as responsible for the development of severe non-alcoholic fatty liver disease in a four-generation family with no typical disease risk factors. A cell line culture created harbouring this variant gene was characterised to understand how this genetic variation leads to a defect in liver cells, which results in accumulation of fat and processes that promote disease. This is now a useful model for studying the disease pathways and to discover new ways to treat common types of fatty liver disease.

Crystal structure of the C-terminal domain of DENR.

The density regulated protein (DENR) forms a stable heterodimer with malignant T-cell-amplified sequence 1 (MCT-1). DENR-MCT-1 heterodimer then participates in regulation of non-canonical translation initiation and ribosomal recycling. The N-terminal domain of DENR interacts with MCT-1 and carries a classical tetrahedral zinc ion-binding site, which is crucial for the dimerization. DENR-MCT-1 binds the small (40S) ribosomal subunit through interactions between MCT-1 and helix h24 of the 18S rRNA, and through interactions between the C-terminal domain of DENR and helix h44 of the 18S rRNA. This later interaction occurs in the vicinity of the P site that is also the binding site for canonical translation initiation factor eIF1, which plays the key role in initiation codon selection and scanning. Sequence homology modeling and a low-resolution crystal structure of the DENR-MCT-1 complex with the human 40S subunit suggests that the C-terminal domain of DENR and eIF1 adopt a similar fold. Here we present the crystal structure of the C-terminal domain of DENR determined at 1.74 Å resolution, which confirms its resemblance to eIF1 and advances our understanding of the mechanism by which DENR-MCT-1 regulates non-canonical translation initiation and ribosomal recycling.