Explore the vast range of titles available.

The cell division cycle 25A (CDC25A) phosphatase is a key regulator of cell cycle progression that acts on the phosphorylation status of Cyclin–Cyclin-dependent kinase complexes, with an emergent role in the DNA damage response and cell survival control. The regulation of CDC25A activity and its protein level is essential to control the cell cycle and maintain genomic integrity. Here we describe a novel ubiquitin/proteasome-mediated pathway negatively regulating CDC25A stability, dependent on its phosphorylation by the serine/threonine kinase DYRK2. DYRK2 phosphorylates CDC25A on at least 7 residues, resulting in its degradation independent of the known CDC25A E3 ubiquitin ligases. CDC25A in turn is able to control the phosphorylation of DYRK2 at several residues outside from its activation loop, thus affecting DYRK2 localization and activity. An inverse correlation between DYRK2 and CDC25A protein amounts was observed during cell cycle progression and in response to DNA damage, with CDC25A accumulation responding to the manipulation of DYRK2 levels or activity in either physiological scenario. Functional data show that the pro-survival activity of CDC25A and the pro-apoptotic activity of DYRK2 could be partly explained by the mutual regulation between both proteins. Moreover, DYRK2 modulation of CDC25A expression and/or activity contributes to the DYRK2 role in cell cycle regulation. Altogether, we provide evidence suggesting that DYRK2 and CDC25A mutually control their activity and stability by a feedback regulatory loop, with a relevant effect on the genotoxic stress pathway, apoptosis, and cell cycle regulation.

The genome of Hepatitis B virus (HBV) persists in infected hepatocytes as a nuclear episome (cccDNA) that is responsible for the transcription of viral genes and viral rebound, following antiviral treatment arrest in chronically infected patients. There is currently no clinically approved therapeutic strategy able to efficiently target cccDNA (Lucifora J 2016). The development of alternative strategies aiming at permanently abrogating HBV RNA production requires a thorough understanding of cccDNA transcriptional and post-transcriptional regulation. In a previous study, we discovered that 1C8, a compound that inhibits the phosphorylation of some cellular RNA-binding proteins, could decrease the level of HBV RNAs. Here, we aimed at identifying kinases responsible for this effect. Among the kinases targeted by 1C8, we focused on DYRK1A, a dual-specificity kinase that controls the transcription of cellular genes by phosphorylating transcription factors, histones, chromatin regulators as well as RNA polymerase II. The results of a combination of genetic and chemical approaches using HBV-infected hepatocytes, indicated that DYRK1A positively regulates the production of HBV RNAs. In addition, we found that DYRK1A associates with cccDNA, and stimulates the production of HBV nascent RNAs. Finally, reporter gene assays showed that DYRK1A up-regulates the activity of the HBV enhancer 1/X promoter in a sequence-dependent manner. Altogether, these results indicate that DYRK1A is a proviral factor that may participate in the HBV life cycle by stimulating the production of HBx, a viral factor absolutely required to trigger the complete cccDNA transcriptional program.

Dysregulation of the DYRK1A protein kinase has been associated with human disease. On the one hand, its overexpression in trisomy 21 has been linked to certain pathological traits of Down syndrome, while on the other, inactivating mutations in just one allele are responsible for a distinct yet rare clinical syndrome, DYRK1A haploinsufficiency. Moreover, altered expression of this kinase may also provoke other human pathologies, including cancer and diabetes. Although a few DYRK1A substrates have been described, its upstream regulators and downstream targets are still poorly understood, an information that could shed light on the functions of DYRK1A in the cell. Here, we carried out a proteomic screen using antibody-based affinity purification coupled to mass spectrometry to identify proteins that directly or indirectly bind to endogenous DYRK1A. We show that the use of a cell line not expressing DYRK1A, generated by CRISPR/Cas9 technology, was needed in order to discriminate between true positives and non-specific interactions. Most of the proteins identified in the screen are novel candidate DYRK1A interactors linked to a variety of activities in the cell. The in-depth characterization of DYRK1A’s functional interaction with one of them, the E3 ubiquitin ligase RNF169, revealed a role for this kinase in the DNA damage response. We found that RNF169 is a DYRK1A substrate and we identified several of its phosphorylation sites. In particular, one of these sites appears to modify the ability of RNF169 to displace 53BP1 from sites of DNA damage. Indeed, DYRK1A depletion increases cell sensitivity to ionizing irradiation. Therefore, our unbiased proteomic screen has revealed a novel activity of DYRK1A, expanding the complex role of this kinase in controlling cell homeostasis.

Regulation of histone acetylation dictates patterns of gene expression and hence cell identity. Due to their clinical relevance in cancer biology, understanding how human embryonic stem cells (hESCs) regulate their genomic patterns of histone acetylation is critical, but it remains largely to be investigated. Here, we provide evidence that acetylation of histone H3 lysine-18 (H3K18ac) and lysine-27 (H3K27ac) is only partially established by p300 in stem cells, while it represents the main histone acetyltransferase (HAT) for these marks in somatic cells. Our analysis reveals that whereas p300 marginally associated with H3K18ac and H3K27ac in hESCs, it largely overlapped with these histone marks upon differentiation. Interestingly, we show that H3K18ac is found at “stemness” genes enriched in RNA polymerase III transcription factor C (TFIIIC) in hESCs, whilst lacking p300. Moreover, TFIIIC was also found in the vicinity of genes involved in neuronal biology, although devoid of H3K18ac. Our data suggest a more complex pattern of HATs responsible for histone acetylations in hESCs than previously considered, suggesting a putative role for H3K18ac and TFIIIC in regulating “stemness” genes as well as genes associated with neuronal differentiation of hESCs. The results break ground for possible new paradigms for genome acetylation in hESCs that could lead to new avenues for therapeutic intervention in cancer and developmental diseases.

Ribosomal proteins (RPs) are evolutionary conserved proteins that are essential for protein translation. RP expression must be tightly regulated to ensure the appropriate assembly of ribosomes and to respond to the growth demands of cells. The elements regulating the transcription of RP genes (RPGs) have been characterized in yeast and Drosophila, yet how cells regulate the production of RPs in mammals is less well understood. Here, we show that a subset of RPG promoters is characterized by the presence of the palindromic TCTCGCGAGA motif and marked by the recruitment of the protein kinase DYRK1A. The presence of DYRK1A at these promoters is associated with the enhanced binding of the TATA-binding protein, TBP, and it is negatively correlated with the binding of the GABP transcription factor, establishing at least two clusters of RPGs that could be coordinately regulated. However, DYRK1A silencing leads to a global reduction in RPGs mRNAs, pointing at DYRK1A activities beyond those dependent on its chromatin association. Significantly, cells in which DYRK1A is depleted have reduced RP levels, fewer ribosomes, reduced global protein synthesis and a smaller size. We therefore propose a novel role for DYRK1A in coordinating the expression of genes encoding RPs, thereby controlling cell growth in mammals.

Transcription factors (TFs) bind DNA in a sequence-specific manner and are generally cell type-specific factors and/or developmental master regulators. In contrast, general TFs (GTFs) are part of very large protein complexes and serve for RNA polymerases’ recruitment to promoter sequences, generally in a cell type-independent manner. Whereas, several TFs have been proven to serve as anchors for the 3D genome organization, the role of GTFs in genome architecture have not been carefully explored. Here, we used ChIP-seq and Hi-C data to depict the role of TFIIIC, one of the RNA polymerase III GTFs, in 3D genome organization. We find that TFIIIC genome occupancy mainly occurs at specific regions, which largely correspond to Alu elements; other characteristic classes of repetitive elements (REs) such as MIR, FLAM-C and ALR/alpha are also found depending on the cell’s developmental origin. The analysis also shows that TFIIIC-enriched regions are involved in cell type-specific DNA looping, which does not depend on colocalization with the master architectural protein CTCF. This work extends previous knowledge on the role of TFIIIC as a bona fide genome organizer whose action participates in cell type-dependent 3D genome looping via binding to REs.

The mammalian genome is shaped by the expansion of repetitive elements that provide new regulatory networks for coordinated control of gene expression and genome folding. Alu elements (AEs) are selectively retained close to the transcription start site of genes, show proto-enhancer functions, correlate with the level of chromatin interactions and are recognized by transcription factor III (TFIIIC), but the relevance of all this is not clear. Here we report regulatory mechanisms that unveil a central role of AEs and TFIIIC in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of pre-marked AEs near cell cycle genes recruit TFIIIC to alter their chromatin accessibility via TFIIIC-mediated acetylation of histone H3 Lysine-18 (H3K18). This facilitates AEs contact with distant CTCF sites near other cell cycle genes promoters, which also become hyperacetylated at H3K18. These changes ensure basal transcription of crucial cell cycle genes, and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor adjusts 3D genome folding and gene expression. We anticipate that expansion of several families of repetitive elements during evolution might have served to generate new genomic cis-regulatory circuits enabling the coordinated regulation of a large set of genes relevant for cellular stress survival. As growth factor withdrawal is a situation relevant in cancer biology, our study identifies TFIIIC as a new potential target for clinical intervention. Footnotes * The manuscript has gone through another deep revision and formatted to a new more coincides version compared to the second act.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a desmoplastic, immunosuppressive stroma dominated by cancer-associated fibroblasts (CAFs). Paracrine signals from cancer cells and CAFs shape the tumor microenvironment (TME), facilitating interactions that drive tumor growth and immune evasion, yet the molecular regulators of this crosstalk remain incompletely understood. Here, we show that Dual-specificity Tyrosine-Regulated Kinase 1A (DYRK1A), previously shown to promote PDAC growth by stabilizing c-MET in cancer cells, is also expressed in CAFs at levels comparable to those in cancer cells. Secretome profiling of DYRK1A-depleted cancer cells and CAFs by quantitative mass spectrometry revealed DYRK1A-dependent factors in each cell type associated with terms related to cell migration, while combining the DYRK1A-regulated proteins from both compartments highlighted additional categories associated with immune infiltration and cell-cell interactions in the TME. Genetic or pharmacological inhibition of DYRK1A reduced CCL2, CCL5 and CSF-1 secretion in cancer cells, and CXCL12 in CAFs, linking DYRK1A activity to immunosuppressive paracrine signaling. Conditioned media from DYRK1A-depleted cancer cells or CAFs impaired the migration of monocytes and myeloid-derived suppressor cells, indicating that DYRK1A remodels the PDAC secretome to recruit immunosuppressive myeloid populations. These findings extend the established role of DYRK1A beyond cancer cell-intrinsic signaling, highlighting its role as a regulator of secreted factors that shape the PDAC TME, and position this kinase as a potential target to reduce immunosuppressive signaling. DYRK1A regulates chemokine and cytokine secretion in PDAC cancer cells and CAFs, promoting recruitment of immunosuppressive myeloid populations, and revealing a previously unrecognized role for this kinase in shaping the TME.

The genome of Hepatitis B virus (HBV) persists in infected hepatocytes as a nuclear episome (cccDNA) that is responsible for the transcription of viral genes and viral rebound, following antiviral treatment arrest in chronically infected patients. Beside pegylated interferon-alpha, there is currently no clinically approved therapeutic protocol able to target cccDNA [1]. The development of alternative strategies aiming at permanently abrogating HBV RNA production requires a thorough understanding of cccDNA transcriptional and post-transcriptional regulation. In a previous study, we discovered that 1C8, a compound that inhibits the phosphorylation of some cellular RNA-binding proteins (RBPs), could decrease the level of HBV RNAs. Here, we aimed at identifying kinases responsible for this effect. Among the kinases targeted by 1C8, we focused on DYRK1A, a dual-specificity kinase that controls the transcription of cellular genes by phosphorylating transcription factors, histones, chromatin regulators as well as RNA polymerase II. The results of a combination of genetic and chemical approaches using HBV-infected hepatocytes, indicated that DYRK1A positively regulates the production of HBV RNAs. In addition, we found that DYRK1A associates with cccDNA, and stimulates the production of HBV nascent RNAs. Finally, reporter gene assays showed that DYRK1A up-regulates the activity of the HBV enhancer 1/X promoter in a sequence-dependent manner. Altogether, these results indicate that DYRK1A is a proviral factor that may participate in the the HBV life cycle by stimulating the production of HBx, a viral factor absolutely required to trigger the complete cccDNA transcriptional program.

Ribosomal proteins (RPs) are evolutionary conserved proteins that are essential for protein translation. RP expression must be tightly regulated to ensure the appropriate assembly of ribosomes and to respond to the growth demands of cells. The elements regulating the transcription of RP genes (RPGs) have been characterized in yeast and Drosophila, yet how cells regulate the production of RPs in mammals is less well understood. Here, we show that a subset of RPG promoters is characterized by the presence of the palindromic TCTCGCGAGA motif and marked by the recruitment of the protein kinase DYRK1A. The presence of DYRK1A at these promoters is associated with enhanced binding of the TATA-binding protein, TBP, and it is negatively correlated with the binding of the GABP transcription factor, establishing at least two clusters of RPGs that could be coordinately regulated. However, DYRK1A silencing leads to a global reduction of RPGs, mRNA pointing at DYRK1A activities beyond those dependent on its chromatin association. Significantly, cells in which DYRK1A is depleted have reduced RP levels, fewer ribosomes, reduced global protein synthesis and smaller size. We therefore propose a novel role for DYRK1A in coordinating the expression of genes encoding RPs, thereby controlling cell growth in mammals.