TTF1 control of LncRNA synthesis delineates a tumor suppressor pathway directly regulating the ribosomal RNA genes.

The tumor suppressor p14/19ARF regulates ribosomal RNA (rRNA) synthesis by controlling the nucleolar localization of Transcription Termination Factor 1 (TTF1). However, the role played by TTF1 in regulating the rRNA genes and in potentially controlling growth has remained unclear. We now show that TTF1 expression regulates cell growth by determining the cellular complement of ribosomes.

Transcription factor UBF depletion in mouse cells results in downregulation of both downstream and upstream elements of the rRNA transcription network.

Transcription/processing of the ribosomal RNA (rRNA) precursor, as part of ribosome biosynthesis, is intensively studied and characterized in eukaryotic cells. Here, we constructed shRNA-based mouse cell lines partially silenced for the Upstream Binding Factor UBF, the master regulator of rRNA transcription and organizer of open rDNA chromatin. Full Ubf silencing in vivo is not viable, and these new tools allow further characterization of rRNA transcription and its coordination with cellular signaling.

HMG-boxes, ribosomopathies and neurodegenerative disease.

The UBTF E210K neuroregression syndrome is a predominantly neurological disorder caused by recurrent dominant variants in Upstream Binding Factor, that is, essential for transcription of the ribosomal RNA genes. This unusual form of ribosomopathy is characterized by a slow decline in cognition, behavior, and sensorimotor functioning during the critical period of development. UBTF (or UBF) is a multi-HMGB-box protein that acts both as an epigenetic factor to establish "open" chromatin on the ribosomal genes and as a basal transcription factor in their RNA Polymerase I transcription.

The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans.

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity.

Control of ribosomal RNA synthesis by hematopoietic transcription factors.

Ribosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2,200 ChIP-seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA and identified robust binding of multiple TF families to canonical TF motifs on rDNA.

Liquid-Liquid Phase Separation in Cancer Signaling, Metabolism and Anticancer Therapy.

The cancer state is thought to be maintained by genetic and epigenetic changes that drive a cancer-promoting gene expression program. However, recent results show that cellular states can be also stably maintained by the reorganization of cell structure leading to the formation of biological condensates via the process of liquid-liquid phase separation. Here, we review the data showing cancer-specific biological condensates initiated by mutant oncoproteins, RNA-binding proteins, or lincRNAs that regulate oncogenic gene expression programs and cancer metabolism.

Ribosomal DNA promoter recognition is determined in vivo by cooperation between UBTF1 and SL1 and is compromised in the UBTF-E210K neuroregression syndrome.

Transcription of the ~200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15.

The chemotherapeutic agent CX-5461 irreversibly blocks RNA polymerase I initiation and promoter release to cause nucleolar disruption, DNA damage and cell inviability.

In the search for drugs to effectively treat cancer, the last 10 years have seen a resurgence of interest in targeting ribosome biogenesis. CX-5461 is a potential inhibitor of ribosomal RNA synthesis that is now showing promise in phase I trials as a chemotherapeutic agent for a range of malignancies. Here, we show that CX-5461 irreversibly inhibits ribosomal RNA transcription by arresting RNA polymerase I (RPI/Pol1/PolR1) in a transcription initiation complex.

The Short N-Terminal Repeats of Transcription Termination Factor 1 Contain Semi-Redundant Nucleolar Localization Signals and P19-ARF Tumor Suppressor Binding Sites.

The p14/p19 (ARF) tumor suppressor provides an important link in the activation of p53 (TP53) by inhibiting its targeted degradation via the E3 ligases MDM2/HDM2. However, ARF also limits tumor growth by directly inhibiting ribosomal RNA synthesis and processing. Initial studies of the ARF tumor suppressor were compounded by overlap between the INK4A and ARF genes encoded by the CDKN2A locus, but mouse models of pure ARF-loss and its inactivation in human cancers identified it as a distinct tumor suppressor even in the absence of p53.

Mutation Interrupts Nucleolin-mTOR-P70S6K Signaling in Rett Syndrome Patients.

Rett syndrome (RTT) is a severe and rare neurological disorder that is caused by mutations in the X-linked (methyl CpG-binding protein 2) gene. MeCP2 protein is an important epigenetic factor in the brain and in neurons. In -deficient neurons, nucleoli structures are compromised.

The chromatin landscape of the ribosomal RNA genes in mouse and human.

The rRNA genes of mouse and human encode the three major RNAs of the ribosome and as such are essential for growth and development. These genes are present in high copy numbers and arranged as direct repeats at the Nucleolar Organizer Regions on multiple chromosomes. Not all the rRNA genes are transcriptionally active, but the molecular mechanisms that determine activity are complex and still poorly understood.

A recurrent de novo missense mutation in UBTF causes developmental neuroregression.

UBTF (upstream binding transcription factor) exists as two isoforms; UBTF1 regulates rRNA transcription by RNA polymerase 1, whereas UBTF2 regulates mRNA transcription by RNA polymerase 2. Herein, we describe 4 patients with very similar patterns of neuroregression due to recurrent de novo mutations in UBTF (GRCh37/hg19, NC_000017.10: g.

A Deconvolution Protocol for ChIP-Seq Reveals Analogous Enhancer Structures on the Mouse and Human Ribosomal RNA Genes.

The combination of Chromatin Immunoprecipitation and Massively Parallel Sequencing, or ChIP-Seq, has greatly advanced our genome-wide understanding of chromatin and enhancer structures. However, its resolution at any given genetic locus is limited by several factors. In applying ChIP-Seq to the study of the ribosomal RNA genes, we found that a major limitation to resolution was imposed by the underlying variability in sequence coverage that very often dominates the protein-DNA interaction profiles.

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance.

Disruption of the UBF gene induces aberrant somatic nucleolar bodies and disrupts embryo nucleolar precursor bodies.

The nucleolus is the site of ribosome biogenesis and forms around the actively transcribed ribosomal RNA (rRNA) genes. However, the nucleolus is also implicated in cell cycle regulation, tumour suppression and chromosome segregation and nucleolar disfunction is linked to a wide range of human diseases. Interestingly, the nucleolus is also required for genome reprogramming and the establishment of heterochromatin in the mammalian embryo.

Metabolic Labeling in the Study of Mammalian Ribosomal RNA Synthesis.

RNA metabolic labeling is a method of choice in the study of dynamic changes in the rate of gene transcription and RNA processing. It is particularly applicable to transcription of the ribosomal RNA genes and their processing products due to the very high levels of ribosomal RNA synthesis. Metabolic labeling can detect changes in ribosomal RNA transcription that occur within a few minutes as opposed to the still widely used RT-PCR or Northern blot procedures that measure RNA pool sizes and at best are able to detect changes occurring over several hours or several days.

Extended-Synaptotagmins (E-Syts); the extended story.

The Extended-Synaptotagmin (E-Syt) membrane proteins were only recently discovered, but have already been implicated in a range of interrelated cellular functions, including calcium and receptor signaling, and membrane lipid transport. However, despite their evolutionary conservation and detailed studies of their molecular actions, we still have little idea of how and when these proteins are required in cellular and organism physiology. Here we review our present understanding of the E-Syts and discuss the molecular functions and in vivo requirements for these proteins.

The Cruciform DNA Mobility Shift Assay: A Tool to Study Proteins That Recognize Bent DNA.

So-called architectural DNA-binding proteins such as those of the HMGB-box family induce DNA bending and kinking. However, these proteins often display only a weak sequence preference, making the analysis of their DNA-binding characteristics difficult if not impossible in a standard electrophoretic mobility shift assay (EMSA). In contrast, such proteins often bind prebent DNAs with high affinity and specificity.

Depletion of the cisplatin targeted HMGB-box factor UBF selectively induces p53-independent apoptotic death in transformed cells.

Cisplatin-DNA adducts act as strong decoys for the Upstream Binding Factor UBF (UBTF) and have been shown to inhibit transcription of the ribosomal RNA genes by RNA polymerase I. However, it is unclear if this plays a significant role in the chemotherapeutic activity of cis- or carboplatin. We find that cisplatin in fact induces a very rapid displacement of UBF from the ribosomal RNA genes and strong inhibition of ribosomal RNA synthesis, consistent with this being an important factor in its cytotoxicity.

Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity.

We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca(2+) dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain.

Loss of Extended Synaptotagmins ESyt2 and ESyt3 does not affect mouse development or viability, but in vitro cell migration and survival under stress are affected.

The Extended Synaptotagmins (Esyts) are a family of multi-C2 domain membrane proteins with orthologs in organisms from yeast to human. Three Esyt genes exist in mouse and human and these have most recently been implicated in the formation of junctions between endoplasmic reticulum and plasma membrane, as well as the Ca(2+) dependent replenishment of membrane phospholipids. The data are consistent with a function in extracellular signal transduction and cell adhesion, and indeed Esyt2 was previously implicated in both these functions in Xenopus.

Conditional inactivation of Upstream Binding Factor reveals its epigenetic functions and the existence of a somatic nucleolar precursor body.

Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription.