Identification of RIOK2 as a master regulator of human blood cell development.

Anemia is a major comorbidity in aging, chronic kidney and inflammatory diseases, and hematologic malignancies. However, the transcriptomic networks governing hematopoietic differentiation in blood cell development remain incompletely defined. Here we report that the atypical kinase RIOK2 (right open reading frame kinase 2) is a master transcription factor (TF) that not only drives erythroid differentiation, but also simultaneously suppresses megakaryopoiesis and myelopoiesis in primary human stem and progenitor cells. Our study reveals the previously uncharacterized winged helix-turn-helix DNA-binding domain and two transactivation domains of RIOK2 that are critical to regulate key hematopoietic TFs GATA1, GATA2, SPI1, RUNX3 and KLF1. This establishes RIOK2 as an integral component of the transcriptional regulatory network governing human hematopoietic differentiation. Importantly, RIOK2 mRNA expression significantly correlates with these TFs and other hematopoietic genes in myelodysplastic syndromes, acute myeloid leukemia and chronic kidney disease. Further investigation of RIOK2-mediated transcriptional pathways should yield therapeutic approaches to correct defective hematopoiesis in hematologic disorders.

Intrinsic Dynamics of a Human Gene Reveal the Basis of Expression Heterogeneity.

Transcriptional regulation in metazoans occurs through long-range genomic contacts between enhancers and promoters, and most genes are transcribed in episodic "bursts" of RNA synthesis. To understand the relationship between these two phenomena and the dynamic regulation of genes in response to upstream signals, we describe the use of live-cell RNA imaging coupled with Hi-C measurements and dissect the endogenous regulation of the estrogen-responsive TFF1 gene. Although TFF1 is highly induced, we observe short active periods and variable inactive periods ranging from minutes to days. The heterogeneity in inactive times gives rise to the widely observed "noise" in human gene expression and explains the distribution of protein levels in human tissue. We derive a mathematical model of regulation that relates transcription, chromosome structure, and the cell's ability to sense changes in estrogen and predicts that hypervariability is largely dynamic and does not reflect a stable biological state.

Macrophage IRX3 promotes diet-induced obesity and metabolic inflammation.

Metabolic inflammation is closely linked to obesity, and is implicated in the pathogenesis of metabolic diseases. FTO harbors the strongest genetic association with polygenic obesity, and IRX3 mediates the effects of FTO on body weight. However, in what cells and how IRX3 carries out this control are poorly understood. Here we report that macrophage IRX3 promotes metabolic inflammation to accelerate the development of obesity and type 2 diabetes. Mice with myeloid-specific deletion of Irx3 were protected against diet-induced obesity and metabolic diseases via increasing adaptive thermogenesis. Mechanistically, macrophage IRX3 promoted proinflammatory cytokine transcription and thus repressed adipocyte adrenergic signaling, thereby inhibiting lipolysis and thermogenesis. JNK1/2 phosphorylated IRX3, leading to its dimerization and nuclear translocation for transcription. Further, lipopolysaccharide stimulation stabilized IRX3 by inhibiting its ubiquitination, which amplified the transcriptional capacity of IRX3. Together, our findings identify a new player, macrophage IRX3, in the control of body weight and metabolic inflammation, implicating IRX3 as a therapeutic target.

Non-catalytic ubiquitin binding by A20 prevents psoriatic arthritis-like disease and inflammation.

A20 is an anti-inflammatory protein that is strongly linked to human disease. Here, we find that mice expressing three distinct targeted mutations of A20's zinc finger 7 (ZF7) ubiquitin-binding motif uniformly developed digit arthritis with features common to psoriatic arthritis, while mice expressing point mutations in A20's OTU or ZF4 motifs did not exhibit this phenotype. Arthritis in A20ZF7 mice required T cells and MyD88, was exquisitely sensitive to tumor necrosis factor and interleukin-17A, and persisted in germ-free conditions. A20ZF7 cells exhibited prolonged IκB kinase activity that drove exaggerated transcription of late-phase nuclear factor-κB response genes in vitro and in prediseased mouse paws in vivo. In addition, mice expressing double-mutant A20 proteins in A20's ZF4 and ZF7 motifs died perinatally with multi-organ inflammation. Therefore, A20's ZF4 and ZF7 motifs synergistically prevent inflammatory disease in a non-catalytic manner.

The roles of histone variants in fine-tuning chromatin organization and function.

Histones serve to both package and organize DNA within the nucleus. In addition to histone post-translational modification and chromatin remodelling complexes, histone variants contribute to the complexity of epigenetic regulation of the genome. Histone variants are characterized by a distinct protein sequence and a selection of designated chaperone systems and chromatin remodelling complexes that regulate their localization in the genome. In addition, histone variants can be enriched with specific post-translational modifications, which in turn can provide a scaffold for recruitment of variant-specific interacting proteins to chromatin. Thus, through these properties, histone variants have the capacity to endow specific regions of chromatin with unique character and function in a regulated manner. In this Review, we provide an overview of recent advances in our understanding of the contribution of histone variants to chromatin function in mammalian systems. First, we discuss new molecular insights into chaperone-mediated histone variant deposition. Next, we discuss mechanisms by which histone variants influence chromatin properties such as nucleosome stability and the local chromatin environment both through histone variant sequence-specific effects and through their role in recruiting different chromatin-associated complexes. Finally, we focus on histone variant function in the context of both embryonic development and human disease, specifically developmental syndromes and cancer.

Biochemical and cellular properties of insulin receptor signalling.

The mechanism of insulin action is a central theme in biology and medicine. In addition to the rather rare condition of insulin deficiency caused by autoimmune destruction of pancreatic ß-cells, genetic and acquired abnormalities of insulin action underlie the far more common conditions of type 2 diabetes, obesity and insulin resistance. The latter predisposes to diseases ranging from hypertension to Alzheimer disease and cancer. Hence, understanding the biochemical and cellular properties of insulin receptor signalling is arguably a priority in biomedical research. In the past decade, major progress has led to the delineation of mechanisms of glucose transport, lipid synthesis, storage and mobilization. In addition to direct effects of insulin on signalling kinases and metabolic enzymes, the discovery of mechanisms of insulin-regulated gene transcription has led to a reassessment of the general principles of insulin action. These advances will accelerate the discovery of new treatment modalities for diabetes.

Single-cell transcriptional profiling: a window into embryonic cell-type specification.

During mammalian embryonic development, a single fertilized egg cell will proliferate and differentiate into all the cell lineages and cell types that eventually form the adult organism. Cell lineage diversification involves repeated cell fate choices that ultimately occur at the level of the individual cell rather than at the cell-population level. Improvements in single-cell technologies are transforming our understanding of mammalian development, not only by overcoming the limitations presented by the extremely low cell numbers of early embryos but also by enabling the study of cell fate specification in greater detail. In this Review, we first discuss recent advances in single-cell transcriptomics and imaging and provide a brief outline of current bioinformatics methods available to analyse the resulting data. We then discuss how these techniques have contributed to our understanding of pre-implantation and early post-implantation development and of in vitro pluripotency. Finally, we overview the current challenges facing single-cell research and highlight the latest advances and potential future avenues.

Eukaryotic core promoters and the functional basis of transcription initiation.

RNA polymerase II (Pol II) core promoters are specialized DNA sequences at transcription start sites of protein-coding and non-coding genes that support the assembly of the transcription machinery and transcription initiation. They enable the highly regulated transcription of genes by selectively integrating regulatory cues from distal enhancers and their associated regulatory proteins. In this Review, we discuss the defining properties of gene core promoters, including their sequence features, chromatin architecture and transcription initiation patterns. We provide an overview of molecular mechanisms underlying the function and regulation of core promoters and their emerging functional diversity, which defines distinct transcription programmes. On the basis of the established properties of gene core promoters, we discuss transcription start sites within enhancers and integrate recent results obtained from dedicated functional assays to propose a functional model of transcription initiation. This model can explain the nature and function of transcription initiation at gene starts and at enhancers and can explain the different roles of core promoters, of Pol II and its associated factors and of the activating cues provided by enhancers and the transcription factors and cofactors they recruit.

Nascent RNA antagonizes the interaction of a set of regulatory proteins with chromatin.

A number of regulatory factors are recruited to chromatin by specialized RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonized the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors, including BAF, NuRD, EHMT1, and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA, and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonizing the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.

A BRD4-mediated elongation control point primes transcribing RNA polymerase II for 3'-processing and termination.

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.

Transcription-mediated supercoiling regulates genome folding and loop formation.

The chromatin fiber folds into loops, but the mechanisms controlling loop extrusion are still poorly understood. Using super-resolution microscopy, we visualize that loops in intact nuclei are formed by a scaffold of cohesin complexes from which the DNA protrudes. RNA polymerase II decorates the top of the loops and is physically segregated from cohesin. Augmented looping upon increased loading of cohesin on chromosomes causes disruption of Lamin at the nuclear rim and chromatin blending, a homogeneous distribution of chromatin within the nucleus. Altering supercoiling via either transcription or topoisomerase inhibition counteracts chromatin blending, increases chromatin condensation, disrupts loop formation, and leads to altered cohesin distribution and mobility on chromatin. Overall, negative supercoiling generated by transcription is an important regulator of loop formation in vivo.

Native Chromatin Proteomics Reveals a Role for Specific Nucleoporins in Heterochromatin Organization and Maintenance.

Spatially and functionally distinct domains of heterochromatin and euchromatin play important roles in the maintenance of chromosome stability and regulation of gene expression, but a comprehensive knowledge of their composition is lacking. Here, we develop a strategy for the isolation of native Schizosaccharomyces pombe heterochromatin and euchromatin fragments and analyze their composition by using quantitative mass spectrometry. The shared and euchromatin-specific proteomes contain proteins involved in DNA and chromatin metabolism and in transcription, respectively. The heterochromatin-specific proteome includes all proteins with known roles in heterochromatin formation and, in addition, is enriched for subsets of nucleoporins and inner nuclear membrane (INM) proteins, which associate with different chromatin domains. While the INM proteins are required for the integrity of the nucleolus, containing ribosomal DNA repeats, the nucleoporins are required for aggregation of heterochromatic foci and epigenetic inheritance. The results provide a comprehensive picture of heterochromatin-associated proteins and suggest a role for specific nucleoporins in heterochromatin function.

An Early mtUPR: Redistribution of the Nuclear Transcription Factor Rox1 to Mitochondria Protects against Intramitochondrial Proteotoxic Aggregates.

The mitochondrial proteome is built mainly by import of nuclear-encoded precursors, which are targeted mostly by cleavable presequences. Presequence processing upon import is essential for proteostasis and survival, but the consequences of dysfunctional protein maturation are unknown. We find that impaired presequence processing causes accumulation of precursors inside mitochondria that form aggregates, which escape degradation and unexpectedly do not cause cell death. Instead, cells survive via activation of a mitochondrial unfolded protein response (mtUPR)-like pathway that is triggered very early after precursor accumulation. In contrast to classical stress pathways, this immediate response maintains mitochondrial protein import, membrane potential, and translation through translocation of the nuclear HMG-box transcription factor Rox1 to mitochondria. Rox1 binds mtDNA and performs a TFAM-like function pivotal for transcription and translation. Induction of early mtUPR provides a reversible stress model to mechanistically dissect the initial steps in mtUPR pathways with the stressTFAM Rox1 as the first line of defense.

Hap2-Ino80-facilitated transcription promotes de novo establishment of CENP-A chromatin.

Centromeres are maintained epigenetically by the presence of CENP-A, an evolutionarily conserved histone H3 variant, which directs kinetochore assembly and hence centromere function. To identify factors that promote assembly of CENP-A chromatin, we affinity-selected solubilized fission yeast CENP-ACnp1 chromatin. All subunits of the Ino80 complex were enriched, including the auxiliary subunit Hap2. Chromatin association of Hap2 is Ies4-dependent. In addition to a role in maintenance of CENP-ACnp1 chromatin integrity at endogenous centromeres, Hap2 is required for de novo assembly of CENP-ACnp1 chromatin on naïve centromere DNA and promotes H3 turnover on centromere regions and other loci prone to CENP-ACnp1 deposition. Prior to CENP-ACnp1 chromatin assembly, Hap2 facilitates transcription from centromere DNA. These analyses suggest that Hap2-Ino80 destabilizes H3 nucleosomes on centromere DNA through transcription-coupled histone H3 turnover, driving the replacement of resident H3 nucleosomes with CENP-ACnp1 nucleosomes. These inherent properties define centromere DNA by directing a program that mediates CENP-ACnp1 assembly on appropriate sequences.

Non-histone protein methylation as a regulator of cellular signalling and function.

Methylation of Lys and Arg residues on non-histone proteins has emerged as a prevalent post-translational modification and as an important regulator of cellular signal transduction mediated by the MAPK, WNT, BMP, Hippo and JAK-STAT signalling pathways. Crosstalk between methylation and other types of post-translational modifications, and between histone and non-histone protein methylation frequently occurs and affects cellular functions such as chromatin remodelling, gene transcription, protein synthesis, signal transduction and DNA repair. With recent advances in proteomic techniques, in particular mass spectrometry, the stage is now set to decode the methylproteome and define its functions in health and disease.

Dynamic Enhancer DNA Methylation as Basis for Transcriptional and Cellular Heterogeneity of ESCs.

Variable levels of DNA methylation have been reported at tissue-specific differential methylation regions (DMRs) overlapping enhancers, including super-enhancers (SEs) associated with key cell identity genes, but the mechanisms responsible for this intriguing behavior are not well understood. We used allele-specific reporters at the endogenous Sox2 and Mir290 SEs in embryonic stem cells and found that the allelic DNA methylation state is dynamically switching, resulting in cell-to-cell heterogeneity. Dynamic DNA methylation is driven by the balance between DNA methyltransferases and transcription factor binding on one side and co-regulated with the Mediator complex recruitment and H3K27ac level changes at regulatory elements on the other side. DNA methylation at the Sox2 and the Mir290 SEs is independently regulated and has distinct consequences on the cellular differentiation state. Dynamic allele-specific DNA methylation at the two SEs was also seen at different stages in preimplantation embryos, revealing that methylation heterogeneity occurs in vivo.

Transcription-mediated replication hindrance: a major driver of genome instability.

Genome replication involves dealing with obstacles that can result from DNA damage but also from chromatin alterations, topological stress, tightly bound proteins or non-B DNA structures such as R loops. Experimental evidence reveals that an engaged transcription machinery at the DNA can either enhance such obstacles or be an obstacle itself. Thus, transcription can become a potentially hazardous process promoting localized replication fork hindrance and stress, which would ultimately cause genome instability, a hallmark of cancer cells. Understanding the causes behind transcription-replication conflicts as well as how the cell resolves them to sustain genome integrity is the aim of this review.

The maintenance of chromosome structure: positioning and functioning of SMC complexes.

Structural maintenance of chromosomes (SMC) complexes, which in eukaryotic cells include cohesin, condensin and the Smc5/6 complex, are central regulators of chromosome dynamics and control sister chromatid cohesion, chromosome condensation, DNA replication, DNA repair and transcription. Even though the molecular mechanisms that lead to this large range of functions are still unclear, it has been established that the complexes execute their functions through their association with chromosomal DNA. A large set of data also indicates that SMC complexes work as intermolecular and intramolecular linkers of DNA. When combining these insights with results from ongoing analyses of their chromosomal binding, and how this interaction influences the structure and dynamics of chromosomes, a picture of how SMC complexes carry out their many functions starts to emerge.

Ligand Modulates Cross-Coupling between Riboswitch Folding and Transcriptional Pausing.

Numerous classes of riboswitches have been found to regulate bacterial gene expression in response to physiological cues, offering new paths to antibacterial drugs. As common studies of isolated riboswitches lack the functional context of the transcription machinery, we here combine single-molecule, biochemical, and simulation approaches to investigate the coupling between co-transcriptional folding of the pseudoknot-structured preQ1 riboswitch and RNA polymerase (RNAP) pausing. We show that pausing at a site immediately downstream of the riboswitch requires a ligand-free pseudoknot in the nascent RNA, a precisely spaced sequence resembling the pause consensus, and electrostatic and steric interactions with the RNAP exit channel. While interactions with RNAP stabilize the native fold of the riboswitch, binding of the ligand signals RNAP release from the pause. Our results demonstrate that the nascent riboswitch and its ligand actively modulate the function of RNAP and vice versa, a paradigm likely to apply to other cellular RNA transcripts.

Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein.

NusG/Spt5 proteins are the only transcription factors utilized by all cellular organisms. In enterobacteria, NusG antagonizes the transcription termination activity of Rho, a hexameric helicase, during the synthesis of ribosomal and actively translated mRNAs. Paradoxically, NusG helps Rho act on untranslated transcripts, including non-canonical antisense RNAs and those arising from translational stress; how NusG fulfills these disparate functions is unknown. Here, we demonstrate that NusG activates Rho by assisting helicase isomerization from an open-ring, RNA-loading state to a closed-ring, catalytically active translocase. A crystal structure of closed-ring Rho in complex with NusG reveals the physical basis for this activation and further explains how Rho is excluded from translationally competent RNAs. This study demonstrates how a universally conserved transcription factor acts to modulate the activity of a ring-shaped ATPase motor and establishes how the innate sequence bias of a termination factor can be modulated to silence pervasive, aberrant transcription.