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The TCTCGCGAGA Sequence Is a Regulatory Motif in DYRK1A Target Promoters (A) Web logo of the significantly enriched motif identified in DYRK1A targets. (B and C) Percentage of DYRK1A targets that contain the TCTCGCGAGA motif, classified by their genomic annotation (B) or by the number of motifs within the DYRK1A peaks (C). (D) Distribution of the TCTCGCGAGA motif in the DYRK1A ChIP-seq peak regions. Peaks were centered on the x axis = 0. See also Figure S4A. (E) EMSA performed using increasing amounts of nuclear extracts from HeLa cells and an oligonucleotide probe (30-mer) containing the motif from the RPS11 promoter region. See also Figure S4B. (F) Nuclear extracts were preincubated with control IgGs or two different DYRK1A antibodies prior to EMSA (m: Abnova mouse monoclonal clone 7D10; r: Abcam rabbit antibody). The asterisk indicates a supershift and/or complex II. See also Figures S4C-S4F. (G) Reporter assays in which luciferase expression is driven by the promoter region of RPS11 (À450 to +25) containing two consensus sequences (white boxes in the scheme) or with the RPS11 promoter region with the two motif sequences deleted (mut). The data represent the mean (±SD) of three independent biological replicates. (H) Transfection of T98G cells infected with a lentivirus expressing a shRNA control or in cells depleted of DYRK1A with two different shRNAs. The values are expressed as the fold change over a pGL2-basic reporter in each of the shRNA-expressing cell pools. See also Figure S4G. (I) The effect of the wild-type (WT) or a kinase dead (KR) DYRK1A was assayed by co-transfecting the corresponding expression plasmids with the reporters indicated. The data are represented as the fold change over the values of each reporter without any DYRK1A expression. In (H) and (I), a representative experiment of three performed is shown.
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DYRK1A is a dosage-sensitive protein kinase that fulfills key roles during development and in tissue homeostasis, and its dysregulation results in human pathologies. DYRK1A is present in both the nucleus and cytoplasm of mammalian cells, although its nuclear function remains unclear. Genome-wide analysis of DYRK1A-associated loci reveals that the k...
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... determine whether the chromatin targeting of DYRK1A occurs by a mechanism involving transcription factors, we assessed whether DYRK1A-occupied genomic sequences were enriched for certain DNA motifs. De novo MEME analysis on DYRK1A ChIP-seq regions revealed significant enrichment of only one motif, corresponding to the palindromic sequence TCTCGC GAGA (p value < 2.0 3 10 À431 ; Figure 4A). This motif was not distributed uniformly within the genomic categories identified in the DYRK1A ChIP-seq since more than 80% of the DYRK1A-associated promoters contained the consensus sequence, but only 6% of the intergenic regions had it (Fig- ure 4B), suggesting a regulatory role for this palindrome. ...
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... novo MEME analysis on DYRK1A ChIP-seq regions revealed significant enrichment of only one motif, corresponding to the palindromic sequence TCTCGC GAGA (p value < 2.0 3 10 À431 ; Figure 4A). This motif was not distributed uniformly within the genomic categories identified in the DYRK1A ChIP-seq since more than 80% of the DYRK1A-associated promoters contained the consensus sequence, but only 6% of the intergenic regions had it (Fig- ure 4B), suggesting a regulatory role for this palindrome. Most of the DYRK1A peaks contain more than one consensus motif ( Figure 4C), and this palindromic motif was highly conserved in placental mammals ( Figure S4A), suggesting a high degree of functional conservation. ...
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... motif was not distributed uniformly within the genomic categories identified in the DYRK1A ChIP-seq since more than 80% of the DYRK1A-associated promoters contained the consensus sequence, but only 6% of the intergenic regions had it (Fig- ure 4B), suggesting a regulatory role for this palindrome. Most of the DYRK1A peaks contain more than one consensus motif ( Figure 4C), and this palindromic motif was highly conserved in placental mammals ( Figure S4A), suggesting a high degree of functional conservation. In addition, we observed a precise positioning of the motif around the center of the DYRK1A peaks ( Figure 4D), an indication of a role in DYRK1A recruitment. ...
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... motif was not distributed uniformly within the genomic categories identified in the DYRK1A ChIP-seq since more than 80% of the DYRK1A-associated promoters contained the consensus sequence, but only 6% of the intergenic regions had it (Fig- ure 4B), suggesting a regulatory role for this palindrome. Most of the DYRK1A peaks contain more than one consensus motif ( Figure 4C), and this palindromic motif was highly conserved in placental mammals ( Figure S4A), suggesting a high degree of functional conservation. In addition, we observed a precise positioning of the motif around the center of the DYRK1A peaks ( Figure 4D), an indication of a role in DYRK1A recruitment. ...
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... of the DYRK1A peaks contain more than one consensus motif ( Figure 4C), and this palindromic motif was highly conserved in placental mammals ( Figure S4A), suggesting a high degree of functional conservation. In addition, we observed a precise positioning of the motif around the center of the DYRK1A peaks ( Figure 4D), an indication of a role in DYRK1A recruitment. ...
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... assess the functional role of this putative DYRK1A binding site, we tested the ability of the TCTCGCGAGA motif to interact with proteins in HeLa cell nuclear extracts using electropho- retic mobility shift assays (EMSAs). A double-strand oligonucle- otide containing the palindromic consensus sequence formed two different DNA-protein complexes ( Figure 4E) that were competed out by excess unlabeled wild-type probe, yet not by a non-consensus probe ( Figure S4B). Pre-incubation of the nuclear extracts with two different antibodies that immunopre- cipitate DYRK1A ( Figure S4C) either prevented the formation of the DNA-protein complexes (mouse antibody) or enhanced the intensity of the shifted band and induced the appearance of a retarded band (as a result of antibody-mediated supershift and/or stabilization of complex II) ( Figure 4F). ...
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... assess the functional role of this putative DYRK1A binding site, we tested the ability of the TCTCGCGAGA motif to interact with proteins in HeLa cell nuclear extracts using electropho- retic mobility shift assays (EMSAs). A double-strand oligonucle- otide containing the palindromic consensus sequence formed two different DNA-protein complexes ( Figure 4E) that were competed out by excess unlabeled wild-type probe, yet not by a non-consensus probe ( Figure S4B). Pre-incubation of the nuclear extracts with two different antibodies that immunopre- cipitate DYRK1A ( Figure S4C) either prevented the formation of the DNA-protein complexes (mouse antibody) or enhanced the intensity of the shifted band and induced the appearance of a retarded band (as a result of antibody-mediated supershift and/or stabilization of complex II) ( Figure 4F). ...
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... double-strand oligonucle- otide containing the palindromic consensus sequence formed two different DNA-protein complexes ( Figure 4E) that were competed out by excess unlabeled wild-type probe, yet not by a non-consensus probe ( Figure S4B). Pre-incubation of the nuclear extracts with two different antibodies that immunopre- cipitate DYRK1A ( Figure S4C) either prevented the formation of the DNA-protein complexes (mouse antibody) or enhanced the intensity of the shifted band and induced the appearance of a retarded band (as a result of antibody-mediated supershift and/or stabilization of complex II) ( Figure 4F). Concurring with the behavior in the EMSA experiments, the mouse anti-DYRK1A antibody did not work in ChIP experiments ( Figures S4D-S4F). ...
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... double-strand oligonucle- otide containing the palindromic consensus sequence formed two different DNA-protein complexes ( Figure 4E) that were competed out by excess unlabeled wild-type probe, yet not by a non-consensus probe ( Figure S4B). Pre-incubation of the nuclear extracts with two different antibodies that immunopre- cipitate DYRK1A ( Figure S4C) either prevented the formation of the DNA-protein complexes (mouse antibody) or enhanced the intensity of the shifted band and induced the appearance of a retarded band (as a result of antibody-mediated supershift and/or stabilization of complex II) ( Figure 4F). Concurring with the behavior in the EMSA experiments, the mouse anti-DYRK1A antibody did not work in ChIP experiments ( Figures S4D-S4F). ...
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... of the nuclear extracts with two different antibodies that immunopre- cipitate DYRK1A ( Figure S4C) either prevented the formation of the DNA-protein complexes (mouse antibody) or enhanced the intensity of the shifted band and induced the appearance of a retarded band (as a result of antibody-mediated supershift and/or stabilization of complex II) ( Figure 4F). Concurring with the behavior in the EMSA experiments, the mouse anti-DYRK1A antibody did not work in ChIP experiments ( Figures S4D-S4F). Thus, we conclude that DYRK1A participates in protein com- plexes formed at the TCTCGCGAGA sequence. ...
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... examined the relevance of the palindromic sequence in DYRK1A-mediated transcriptional activity in vivo using a cell- based luciferase assay in which the putative regulatory region of the RPS11 gene (À450 to +25) that harbors two TCTCGCG AGA motifs was cloned upstream of the luciferase gene (Fig- ure 4G). While this genomic fragment drove luciferase expres- sion, removal of the consensus sequences reduced the tran- scriptional activity of the reporter ( Figure 4G), indicating a functional role for the palindrome in transcriptional activation. ...
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... examined the relevance of the palindromic sequence in DYRK1A-mediated transcriptional activity in vivo using a cell- based luciferase assay in which the putative regulatory region of the RPS11 gene (À450 to +25) that harbors two TCTCGCG AGA motifs was cloned upstream of the luciferase gene (Fig- ure 4G). While this genomic fragment drove luciferase expres- sion, removal of the consensus sequences reduced the tran- scriptional activity of the reporter ( Figure 4G), indicating a functional role for the palindrome in transcriptional activation. In fact, the consensus sequence alone was able to drive expres- sion in luciferase reporter assays ( Figure S4G). ...
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... this genomic fragment drove luciferase expres- sion, removal of the consensus sequences reduced the tran- scriptional activity of the reporter ( Figure 4G), indicating a functional role for the palindrome in transcriptional activation. In fact, the consensus sequence alone was able to drive expres- sion in luciferase reporter assays ( Figure S4G). To provide exper- imental evidence of a link between the TCTCGCGAGA motif and DYRK1A regulation, we tested the activation of the RPS11 re- porter in cells depleted of DYRK1A. ...
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... provide exper- imental evidence of a link between the TCTCGCGAGA motif and DYRK1A regulation, we tested the activation of the RPS11 re- porter in cells depleted of DYRK1A. Luciferase levels were reduced in parallel with the loss of DYRK1A, whereas there was little or no change when the mutant reporter without the consensus sequence was used ( Figures 4H and S4G). When the impact of DYRK1A kinase activity was assessed in overex- pression experiments, luciferase activity was enhanced when the WT version of DYRK1A was overexpressed, whereas there was no effect or even a reduction when the reporter was co- transfected with a kinase-inactive construct ( Figure 4I). ...
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... levels were reduced in parallel with the loss of DYRK1A, whereas there was little or no change when the mutant reporter without the consensus sequence was used ( Figures 4H and S4G). When the impact of DYRK1A kinase activity was assessed in overex- pression experiments, luciferase activity was enhanced when the WT version of DYRK1A was overexpressed, whereas there was no effect or even a reduction when the reporter was co- transfected with a kinase-inactive construct ( Figure 4I). Consis- tent with the DYRK1A depletion assays, none of these effects were observed when the mutant reporter was used ( Figure 4I). ...
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... the impact of DYRK1A kinase activity was assessed in overex- pression experiments, luciferase activity was enhanced when the WT version of DYRK1A was overexpressed, whereas there was no effect or even a reduction when the reporter was co- transfected with a kinase-inactive construct ( Figure 4I). Consis- tent with the DYRK1A depletion assays, none of these effects were observed when the mutant reporter was used ( Figure 4I). Taken together, these results indicate that the TCTCGCGAGA consensus sequence is necessary to drive DYRK1A-dependent transcriptional activation. ...
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... can interact with KAISO and its p120-catenin partner when overexpressed, allowing p120-cate- nin to relieve KAISO-mediated repression of Wnt target genes (that do not have the TCTCGCGAGA motif) ( Hong et al., 2012). While we did detect the interaction of DYRK1A with p120-cate- nin in nuclear extracts, KAISO was not detected in the DYRK1A immunoprecipitates ( Figure S4H), indicating that KAISO and DYRK1A do not interact in nuclear extracts under the conditions tested. ...
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Citations
... Activation of DYRK is obtained by phosphorylation of a tyrosine residue in the activation loop of the kinase (for review see [128]). Following activation, DYRK1A was reported to regulate various signaling pathways by activating or inactivating transcription and translation factors (RNA polymerase II CTD [129], Sprouty2 [130], DREAM complex [131], cAMP response element-binding protein (CREB) [132], and other proteins such as caspase-9 [133,134], Notch [135], and glycogen synthase [136]. DYRK1A was reported to be expressed in beta-cells [137][138][139]. ...
The prevalence of diabetes is increasing worldwide. Massive death of pancreatic beta-cells causes type 1 diabetes. Progressive loss of beta-cell function and mass characterizes type 2 diabetes. To date, none of the available antidiabetic drugs promotes the maintenance of a functional mass of endogenous beta-cells, revealing an unmet medical need. Dysfunction and apoptotic death of beta-cells occur, in particular, through the activation of intracellular protein kinases. In recent years, protein kinases have become highly studied targets of the pharmaceutical industry for drug development. A number of drugs that inhibit protein kinases have been approved for the treatment of cancers. The question of whether safe drugs that inhibit protein kinase activity can be developed and used to protect the function and survival of beta-cells in diabetes is still unresolved. This review presents arguments suggesting that several protein kinases in beta-cells may represent targets of interest for the development of drugs to treat diabetes.
... The DYRK family of proteins are key regulators of cell growth, apoptosis, and differentiation, with DYRK1A and DYRK1B being the best characterized among the five mammalian DYRKs [12]. The DYRK1A protein is active in the nucleus and cytoplasm, and regulates cellular quiescence, and the proliferation and differentiation of neuronal progenitor cells, while also modulating signaling pathways including Sonic Hedgehog (SHH) [13][14][15][16][17]. DYRK1A is considered a significant gene in the Down syndrome phenotype and has been implicated in other neurological malformations, some of which are seen in congenital CMV infection [18]. ...
Human cytomegalovirus (CMV) infection is the leading non-genetic cause of congenital malformation in developed countries, causing significant fetal injury, and in some cases fetal death. The pathogenetic mechanisms through which this host-specific virus infects then damages both the placenta and the fetal brain are currently ill-defined. We investigated the CMV modulation of key signaling pathway proteins for these organs including dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) and Sonic Hedgehog (SHH) pathway proteins using human first trimester placental trophoblast (TEV-1) cells, primary human astrocyte (NHA) brain cells, and CMV-infected human placental tissue. Immunofluorescence demonstrated the accumulation and re-localization of SHH proteins in CMV-infected TEV-1 cells with Gli2, Ulk3, and Shh re-localizing to the CMV cytoplasmic virion assembly complex (VAC). In CMV-infected NHA cells, DYRK1A re-localized to the VAC and DYRK1B re-localized to the CMV nuclear replication compartments, and the SHH proteins re-localized with a similar pattern as was observed in TEV-1 cells. Western blot analysis in CMV-infected TEV-1 cells showed the upregulated expression of Rb, Ulk3, and Shh, but not Gli2. In CMV-infected NHA cells, there was an upregulation of DYRK1A, DYRK1B, Gli2, Rb, Ulk3, and Shh. These in vitro monoculture findings are consistent with patterns of protein upregulation and re-localization observed in naturally infected placental tissue and CMV-infected ex vivo placental explant histocultures. This study reveals CMV-induced changes in proteins critical for fetal development, and identifies new potential targets for CMV therapeutic development.
... The DNA damage response category overlaps with that of p53 but is nonetheless distinct and includes genes encoding the ATM and CHK2 kinases (Blackford and Jackson, 2017) and the FOXM1 transcription factor that activates DDR gene expression networks (Zona et al., 2014). This category also includes two depleted genes, encoding FBXL5-which antagonizes ATM signaling -and DYRK1A-a kinase involved in the DDR (Laham et al., 2021), DREAM complex activation (Litovchick et al., 2011), and RPG transcription (Di Vona et al., 2015). The involvement of this category of enriched genes is intriguing, given the lack of ɣ-H2AX accumulation in non-apoptotic cells (Figure 3-figure supplement 4), and warrants further investigation in the future. ...
The chromatin-associated protein WD Repeat Domain 5 (WDR5) is a promising target for cancer drug discovery, with most efforts blocking an arginine-binding cavity on the protein called the ‘WIN’ site that tethers WDR5 to chromatin. WIN site inhibitors (WINi) are active against multiple cancer cell types in vitro, the most notable of which are those derived from MLL-rearranged (MLLr) leukemias. Peptidomimetic WINi were originally proposed to inhibit MLLr cells via dysregulation of genes connected to hematopoietic stem cell expansion. Our discovery and interrogation of small-molecule WINi, however, revealed that they act in MLLr cell lines to suppress ribosome protein gene (RPG) transcription, induce nucleolar stress, and activate p53. Because there is no precedent for an anticancer strategy that specifically targets RPG expression, we took an integrated multi-omics approach to further interrogate the mechanism of action of WINi in human MLLr cancer cells. We show that WINi induce depletion of the stock of ribosomes, accompanied by a broad yet modest translational choke and changes in alternative mRNA splicing that inactivate the p53 antagonist MDM4. We also show that WINi are synergistic with agents including venetoclax and BET-bromodomain inhibitors. Together, these studies reinforce the concept that WINi are a novel type of ribosome-directed anticancer therapy and provide a resource to support their clinical implementation in MLLr leukemias and other malignancies.
... It can interact with other transcription factors (RAD54L2 or FOXO1) to enhance the transcription of target genes (29,41). DYRK1A can also modulate the activity of p300/CBP or phosphorylates the C-terminal domain of RNAPII to activate transcription (42,43). Our data suggest that DYRK1A is complexed with TRAF3 and RAD54L2, and the autophosphorylation of DYRK1A is important for the expression of ACE2 (Fig. 4). ...
Angiotensin converting enzyme 2 (ACE2), the host receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, is differentially expressed in a wide variety of tissues and cell types. The expression of ACE2 is under tight regulation, but the mechanisms regulating ACE2 expression have not yet been well defined. Through a genome-wide CRISPR knockout screen, we discovered that host factors TRAF3, DYRK1A, and RAD54L2 (TDR) form a complex to regulate the expression of ACE2. Knockout of TRAF3, DYRK1A, or RAD54L2 reduces the mRNA levels of ACE2 and inhibits the cellular entry of SARS-CoV-2. On the other hand, SARS-CoV-2 continuously evolves by genetic mutations for the adaption to the host. We have identified mutations in spike (S) (P1079T) and nucleocapsid (N) (S194L) that enhance the replication of SARS-CoV-2 in cells that express ACE2 at a low level. Our results have revealed the mechanisms for the transcriptional regulation of ACE2 and the adaption of SARS-CoV-2.
IMPORTANCE
The expression of ACE2 is essential for the entry of SARS-CoV-2 into host cells. We identify a new complex—the TDR complex—that acts to maintain the abundance of ACE2 in host cells. The identification and characterization of the TDR complex provide new targets for the development of therapeutics against SARS-CoV-2 infection. By analysis of SARS-CoV-2 virus replicating in cells expressing low levels of ACE2, we identified mutations in spike (P1079T) and nucleocapsid (S194L) that overcome the restriction of limited ACE2. Functional analysis of these key amino acids in S and N extends our knowledge of the impact of SARS-CoV-2 variants on virus infection and transmission.
... However, whether Dyrk1a regulates one or more signaling pathways in the developing face needs further exploration. DYRK1A has also been reported to regulate transcription by its ability to directly phosphorylate RNA polymerase II and histone components and therefore it could also be necessary for the function of transcriptional machinery (Di Vona et al., 2015;Jang et al., 2014;Yu et al., 2019). Our results warrant further experiments to decipher if and how Dyrk1a could be regulating gene expression during craniofacial development. ...
Loss of function mutations in the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) gene are associated with craniofacial malformations in humans. Here we characterized the effects of deficient DYRK1A in craniofacial development using a developmental model, Xenopus laevis. Dyrk1a mRNA and protein was expressed throughout the developing head and was enriched in the branchial arches which contribute to the face and jaw. Consistently, reduced Dyrk1a function, using dyrk1a morpholinos and pharmacological inhibitors, resulted in orofacial malformations including hypotelorism, altered mouth shape, slanted eyes, and narrower face accompanied by smaller jaw cartilage and muscle. Inhibition of Dyrk1a function resulted in misexpression of key craniofacial regulators including transcription factors and members of the retinoic acid signaling pathway. Two such regulators, sox9 and pax3 are required for neural crest development and their decreased expression corresponds with smaller neural crest domains within the branchial arches. Finally, we determined that the smaller size of the faces, jaw elements and neural crest domains in embryos deficient in Dyrk1a could be explained by increased cell death and decreased proliferation. This study is the first to provide insight into why craniofacial birth defects might arise in humans with DYRK1A mutations.
... Hence, RPG expression could be regulated by specific combinations of TFs in different organisms and/or physiological conditions. The M4 motif matches the palindromic sequence that is bound by the dual-specificity tyrosine-regulated kinase 1A (DYRK1A) protein kinase [20]. DYRK1A fulfills many diverse functions by phosphorylating a wide range of substrates [21][22][23], and it is a kinase with exquisite gene-dosage dependency. ...
... DYRK1A has also been proposed as a pharmacological target for neurodegenerative disorders, diabetes and cancer [22,23,28,29]. We have shown that DYRK1A is a transcriptional activator when recruited to proximal promoter regions of a subset of genes that are enriched for the palindromic motif TCTCGCGAGA [20]. DYRK1A phosphorylates serine residues 2, 5 and 7 within the C-terminal domain (CTD) of the catalytic subunit of Pol II [20]. ...
... We have shown that DYRK1A is a transcriptional activator when recruited to proximal promoter regions of a subset of genes that are enriched for the palindromic motif TCTCGCGAGA [20]. DYRK1A phosphorylates serine residues 2, 5 and 7 within the C-terminal domain (CTD) of the catalytic subunit of Pol II [20]. This activity takes over that of the general TF p-TEFb at gene loci involved in myogenic differentiation [30]. ...
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.
... Dyrk1a (dual specificity tyrosine phosphorylation-regulated kinase 1A) is a gene encoding a member of the dual-specificity tyrosine phosphorylation-regulated kinase family, which is expressed in developing and mature nervous systems (25). It raised a lot of concerns for relating to learning disorders caused by Down syndrome (26) and RNA polymerase II (27,28). However, Dyrk1a is rarely studied in TE specification or placental development. ...
Trophectoderm (TE) and the inner cell mass are the first two lineages in murine embryogenesis and cannot naturally transit to each other. The barriers between them are unclear and fascinating. Embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) retain the identities of inner cell mass and TE, respectively, and, thus, are ideal platforms to investigate these lineages in vitro. Here, we develop a loss-of-function genetic screening in haploid ESCs and reveal many mutations involved in the conversion of TSCs. The disruption of either Catip or Dyrk1a (candidates) in ESCs facilitates the conversion of TSCs. According to transcriptome analysis, we find that the repression of Dyrk1a activates totipotency, which is a possible reason for TE specification. Dyrk1a -null ESCs can contribute to embryonic and extraembryonic tissues in chimeras and can efficiently form blastocyst-like structures, indicating their totipotent developmental abilities. These findings provide insights into the mechanisms underlying cell fate alternation in embryogenesis.
... Our study revealed that DYRK1A KO can inhibit viral RNA synthesis by suppressing DMV formation independent of its catalytic activity, which has not yet been reported. DYRK1A primarily localizes in the nucleus, yet it acts as a potent host factor with wide-ranging transcriptional regulatory effects, capable of modulating proteins in both the nucleus and cytoplasm (54)(55)(56)(64)(65)(66)(67). Our RNA sequencing also demonstrated that DYRK1A influences a diverse array of host factors that are involved in multiple pathways (Table S1). ...
Coronaviruses (CoVs) pose a major threat to human and animal health worldwide, which complete viral replication by hijacking host factors. Identifying host factors essential for the viral life cycle can deepen our understanding of the mechanisms of virus–host interactions. Based on our previous genome-wide CRISPR screen of α-CoV transmissible gastroenteritis virus (TGEV), we identified the host factor dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), but not DYRK1B, as a critical factor in TGEV replication. Rescue assays and kinase inhibitor experiments revealed that the effect of DYRK1A on viral replication is independent of its kinase activity. Nuclear localization signal modification experiments showed that nuclear DYRK1A facilitated virus replication. Furthermore, DYRK1A knockout significantly downregulated the expression of the TGEV receptor aminopeptidase N (ANPEP) and inhibited viral entry. Notably, we also demonstrated that DYRK1A is essential for the early stage of TGEV replication. Transmission electron microscopy results indicated that DYRK1A contributes to the formation of double-membrane vesicles in a kinase-independent manner. Finally, we validated that DYRK1A is also a proviral factor for mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. In conclusion, our work demonstrated that DYRK1A is an essential host factor for the replication of multiple viruses, providing new insights into the mechanism of virus–host interactions and facilitating the development of new broad-spectrum antiviral drugs.
IMPORTANCE
Coronaviruses, like other positive-sense RNA viruses, can remodel the host membrane to form double-membrane vesicles (DMVs) as their replication organelles. Currently, host factors involved in DMV formation are not well defined. In this study, we used transmissible gastroenteritis virus (TGEV) as a virus model to investigate the regulatory mechanism of dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) on coronavirus. Results showed that DYRK1A significantly inhibited TGEV replication in a kinase-independent manner. DYRK1A knockout (KO) can regulate the expression of receptor aminopeptidase N (ANPEP) and endocytic-related genes to inhibit virus entry. More importantly, our results revealed that DYRK1A KO notably inhibited the formation of DMV to regulate the virus replication. Further data proved that DYRK1A is also essential in the replication of mouse hepatitis virus, porcine deltacoronavirus, and porcine sapelovirus. Taken together, our findings demonstrated that DYRK1A is a conserved factor for positive-sense RNA viruses and provided new insights into its transcriptional regulation activity, revealing its potential as a candidate target for therapeutic design.
... Many transcription factors, including NFAT, FOXO1, and STAT3, are controlled by DYRK1A-dependent phosphorylation in the nucleus (Arron et al, 2006;Gwack et al, 2006;Bhansali et al, 2021). DYRK1A interacts with RNA polymerase II in the nucleus and promotes its hyperphosphorylation in the C-terminal domain repeats through a phase separation mechanism (Di Vona et al, 2015;Lu et al, 2018;Yu et al, 2019). In addition, DYRK1A directly binds to chromatin regulatory regions to control gene expression (Di Vona et al, 2015;Li et al, 2018;Yu et al, 2019). ...
... DYRK1A interacts with RNA polymerase II in the nucleus and promotes its hyperphosphorylation in the C-terminal domain repeats through a phase separation mechanism (Di Vona et al, 2015;Lu et al, 2018;Yu et al, 2019). In addition, DYRK1A directly binds to chromatin regulatory regions to control gene expression (Di Vona et al, 2015;Li et al, 2018;Yu et al, 2019). These previous reports indicate that DYRK1A functions in the cell nucleus. ...
The protein kinase DYRK1A encoded in human chromosome 21 is the major contributor to the multiple symptoms observed in Down syndrome patients. In addition, DYRK1A malfunction is associated with various other neurodevelopmental disorders such as autism spectrum disorder. Here, we identified FAM53C with no hitherto known biological function as a novel suppressive binding partner of DYRK1A. FAM53C is bound to the catalytic protein kinase domain of DYRK1A, whereas DCAF7/WDR68, the major DYRK1A-binding protein, binds to the N-terminal domain of DYRK1A. The binding of FAM53C inhibited autophosphorylation activity of DYRK1A and its kinase activity to an exogenous substrate, MAPT/Tau. FAM53C did not bind directly to DCAF7/WDR68, whereas DYRK1A tethered FAM53C and DCAF7/WDR68 by binding concurrently to both of them, forming a tri-protein complex. DYRK1A possesses an NLS and accumulates in the nucleus when overexpressed in cells. Co-expression of FAM53C induced cytoplasmic re-localization of DYRK1A, revealing the cytoplasmic anchoring function of FAM53C to DYRK1A. Moreover, the binding of FAM53C to DYRK1A suppressed the DYRK1A-dependent nuclear localization of DCAF7/WDR68. All the results show that FAM53C binds to DYRK1A, suppresses its kinase activity, and anchors it in the cytoplasm. In addition, FAM53C is bound to the DYRK1A-related kinase DYRK1B with an Hsp90/Cdc37-independent manner. The results explain for the first time why endogenous DYRK1A is distributed in the cytoplasm in normal brain tissue. FAM53C-dependent regulation of the kinase activity and intracellular localization of DYRK1A may play a significant role in gene expression regulation caused by normal and aberrant levels of DYRK1A.
... The reduction of phosphorylation in both Ser2 and Ser5 upon DYRK1A knockdown was previously reported. 20 To investigate the possibility of alternative sites being phosphorylated with different sequence contexts, we systematically characterized DYRK1A's specificity using biochemical product profiling with high-resolution mass spectrometric characterization ( Figure 2). With the 7 th residue in the previous heptad iScience Article identified as key to DYRK1A ( Figure 1E), we systematically replaced the preceding 7 th residue and characterized the product phosphorylation sites ( Figure 2). ...
... Our detailed investigation explained why DYRK1A exhibits both Ser2 and Ser5 activity in human cells, in which the 7 th residue diverges widely from the consensus. 20 Most relevant to our interactome study, the analysis shows that DYRK1A produces the product with exclusive Ser2 phosphorylation if we use consensus sequence as substrate. ...
During eukaryotic transcription, RNA polymerase II undergoes dynamic post-translational modifications on the C-terminal domain (CTD) of the largest subunit, generating an information-rich PTM landscape that transcriptional regulators bind. The phosphorylation of Ser5 and Ser2 of CTD heptad occurs spatiotemporally with the transcriptional stages, recruiting different transcriptional regulators to Pol II. To delineate the protein interactomes at different transcriptional stages, we reconstructed phosphorylation patterns of the CTD at Ser5 and Ser2 in vitro. Our results showed that distinct protein interactomes are recruited to RNA polymerase II at different stages of transcription by the phosphorylation of Ser2 and Ser5 of the CTD heptads. In particular, we characterized calcium homeostasis endoplasmic reticulum protein (CHERP) as a regulator bound by phospho-Ser2 heptad. Pol II association with CHERP recruits an accessory splicing complex whose loss results in broad changes in alternative splicing events. Our results shed light on the PTM-coded recruitment process that coordinates transcription.
