Redox Signaling by the RNA Polymerase III TFIIB-Related Factor Brf2.

TFIIB-related factor 2 (Brf2) is a member of the family of TFIIB-like core transcription factors. Brf2 recruits RNA polymerase (Pol) III to type III gene-external promoters, including the U6 spliceosomal RNA and selenocysteine tRNA genes. Found only in vertebrates, Brf2 has been linked to tumorigenesis but the underlying mechanisms remain elusive. We have solved crystal structures of a human Brf2-TBP complex bound to natural promoters, obtaining a detailed view of the molecular interactions occurring at Brf2-dependent Pol III promoters and highlighting the general structural and functional conservation of human Pol II and Pol III pre-initiation complexes. Surprisingly, our structural and functional studies unravel a Brf2 redox-sensing module capable of specifically regulating Pol III transcriptional output in living cells. Furthermore, we establish Brf2 as a central redox-sensing transcription factor involved in the oxidative stress pathway and provide a mechanistic model for Brf2 genetic activation in lung and breast cancer.

Mechanism of selective recruitment of RNA polymerases II and III to snRNA gene promoters.

RNA polymerase II (Pol II) small nuclear RNA (snRNA) promoters and type 3 Pol III promoters have highly similar structures; both contain an interchangeable enhancer and "proximal sequence element" (PSE), which recruits the SNAP complex (SNAPc). The main distinguishing feature is the presence, in the type 3 promoters only, of a TATA box, which determines Pol III specificity. To understand the mechanism by which the absence or presence of a TATA box results in specific Pol recruitment, we examined how SNAPc and general transcription factors required for Pol II or Pol III transcription of SNAPc-dependent genes (i.e., TATA-box-binding protein [TBP], TFIIB, and TFIIA for Pol II transcription and TBP and BRF2 for Pol III transcription) assemble to ensure specific Pol recruitment. TFIIB and BRF2 could each, in a mutually exclusive fashion, be recruited to SNAPc. In contrast, TBP-TFIIB and TBP-BRF2 complexes were not recruited unless a TATA box was present, which allowed selective and efficient recruitment of the TBP-BRF2 complex. Thus, TBP both prevented BRF2 recruitment to Pol II promoters and enhanced BRF2 recruitment to Pol III promoters. On Pol II promoters, TBP recruitment was separate from TFIIB recruitment and enhanced by TFIIA. Our results provide a model for specific Pol recruitment at SNAPc-dependent promoters.

How to Recruit the Correct RNA Polymerase? Lessons from snRNA Genes.

Nuclear eukaryotic genomes are transcribed by three related RNA polymerases (Pol), which transcribe distinct gene sets. Specific Pol recruitment is achieved through selective core promoter recognition by basal transcription factors (TFs). Transcription by an inappropriate Pol appears to be rare and to generate mostly unstable products. A collection of short noncoding RNA genes [for example, small nuclear RNA (snRNA) or 7SK RNA genes], which play essential roles in processes such as maturation of RNA molecules or control of Pol II transcription elongation, possess highly similar core promoters, and yet are transcribed for some by Pol II and for others by Pol III as a result of small promoter differences. Here we discuss the mechanisms of selective Pol recruitment to such promoters.

HCF-2 inhibits cell proliferation and activates differentiation-gene expression programs.

HCF-2 is a member of the host-cell-factor protein family, which arose in early vertebrate evolution as a result of gene duplication. Whereas its paralog, HCF-1, is known to act as a versatile chromatin-associated protein required for cell proliferation and differentiation, much less is known about HCF-2. Here, we show that HCF-2 is broadly present in human and mouse cells, and possesses activities distinct from HCF-1. Unlike HCF-1, which is excluded from nucleoli, HCF-2 is nucleolar-an activity conferred by one and a half C-terminal Fibronectin type 3 repeats and inhibited by the HCF-1 nuclear localization signal. Elevated HCF-2 synthesis in HEK-293 cells results in phenotypes reminiscent of HCF-1-depleted cells, including inhibition of cell proliferation and mitotic defects. Furthermore, increased HCF-2 levels in HEK-293 cells lead to inhibition of cell proliferation and metabolism gene-expression programs with parallel activation of differentiation and morphogenesis gene-expression programs. Thus, the HCF ancestor appears to have evolved into a small two-member protein family possessing contrasting nuclear versus nucleolar localization, and cell proliferation and differentiation functions.

CTCF loss has limited effects on global genome architecture in Drosophila despite critical regulatory functions.

Vertebrate genomes are partitioned into contact domains defined by enhanced internal contact frequency and formed by two principal mechanisms: compartmentalization of transcriptionally active and inactive domains, and stalling of chromosomal loop-extruding cohesin by CTCF bound at domain boundaries. While Drosophila has widespread contact domains and CTCF, it is currently unclear whether CTCF-dependent domains exist in flies. We genetically ablate CTCF in Drosophila and examine impacts on genome folding and transcriptional regulation in the central nervous system. We find that CTCF is required to form a small fraction of all domain boundaries, while critically controlling expression patterns of certain genes and supporting nervous system function. We also find that CTCF recruits the pervasive boundary-associated factor Cp190 to CTCF-occupied boundaries and co-regulates a subset of genes near boundaries together with Cp190. These results highlight a profound difference in CTCF-requirement for genome folding in flies and vertebrates, in which a large fraction of boundaries are CTCF-dependent and suggest that CTCF has played mutable roles in genome architecture and direct gene expression control during metazoan evolution.

Microexon-based regulation of ITSN1 and Src SH3 domains specificity relies on introduction of charged amino acids into the interaction interface.

SH3 domains function as protein-protein interaction modules in assembly of signalling and endocytic protein complexes. Here we report investigations of the mechanism of regulation of the binding properties of the SH3 domains of intersectin (ITSN1) and Src kinase by alternative splicing. Comparative sequence analysis of ITSN1 and Src genes revealed the conservation of alternatively spliced microexons affecting the structure of the SH3 domains in vertebrates. We show that neuron-specific ITSN1 transcripts containing the microexon 20 that encodes five amino acid residues within the SH3A domain are expressed in zebrafish from the earliest stages of the development of the nervous system. Models of alternative isoforms of the ITSN1 SH3A domain revealed that the insertion encoded by the microexon is located at the beginning of the n-Src loop of this domain causing a shift of negatively charged amino acids towards the interaction interface. Mutational analysis confirmed the importance of translocation of these negatively charged amino acids for interaction with dynamin 1. We also identified a residue within the microexon-encoded insert in the SH3 domain of brain-specific variant of Src that abolishes interaction of the domain with dynamin 1. Thus microexons provide a mechanism for the control of tissue-specific interactions of ITSN1 and Src with their partners.

Intersectin 1 forms complexes with SGIP1 and Reps1 in clathrin-coated pits.

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein involved in clathrin-mediated endocytosis, cellular signaling and cytoskeleton rearrangement. ITSN1 gene is located on human chromosome 21 in Down syndrome critical region. Several studies confirmed role of ITSN1 in Down syndrome phenotype. Here we report the identification of novel interconnections in the interaction network of this endocytic adaptor. We show that the membrane-deforming protein SGIP1 (Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1) and the signaling adaptor Reps1 (RalBP associated Eps15-homology domain protein) interact with ITSN1 in vivo. Both interactions are mediated by the SH3 domains of ITSN1 and proline-rich motifs of protein partners. Moreover complexes comprising SGIP1, Reps1 and ITSN1 have been identified. We also identified new interactions between SGIP1, Reps1 and the BAR (Bin/amphiphysin/Rvs) domain-containing protein amphiphysin 1. Immunofluorescent data have demonstrated colocalization of ITSN1 with the newly identified protein partners in clathrin-coated pits. These findings expand the role of ITSN1 as a scaffolding molecule bringing together components of endocytic complexes.

Structural diversity and differential expression of novel human intersectin 1 isoforms.

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein that functions in clathrin-mediated endocytosis, cell signalling and cytoskeleton rearrangements. The ITSN1 gene encodes two main isoforms: a short form (ITSN1-s), which is ubiquitously expressed and consists of two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains, and a long form (ITSN1-l), which is predominantly expressed in the brain and contains three additional domains, a Dbl homology (DH) domain, a Pleckstrin homology (PH) domain and a C2 domain. Using computational analysis of the EST database and 3' RACE we determined the length of the 3' untranslated region of ITSN1-l and demonstrated that the polyadenylation site is located 11,559 nt downstream of the stop codon of the ITSN1-l mRNA. Recently, additional splicing events affecting ITSN1 transcripts were reported, but full-length transcriptional isoforms with different combinations of alternatively spliced exons remained unknown. Here we report the identification of fifteen novel transcriptional isoforms of the human ITSN1 gene with full-length coding sequences that are the result of different combinations of the alternatively spliced exons 5, 6/6', 20, 23, 25, 26, 26a and 35. The isoforms identified differ in domain organization and expression level in different tissues and more likely contribute to the modulation of many complex protein interactions in which ITSN1 participates.

CDK4 Regulates Lysosomal Function and mTORC1 Activation to Promote Cancer Cell Survival.

Cyclin-dependent kinase 4 (CDK4) is well-known for its role in regulating the cell cycle, however, its role in cancer metabolism, especially mTOR signaling, is undefined. In this study, we established a connection between CDK4 and lysosomes, an emerging metabolic organelle crucial for mTORC1 activation. On the one hand, CDK4 phosphorylated the tumor suppressor folliculin (FLCN), regulating mTORC1 recruitment to the lysosomal surface in response to amino acids. On the other hand, CDK4 directly regulated lysosomal function and was essential for lysosomal degradation, ultimately regulating mTORC1 activity. Pharmacologic inhibition or genetic inactivation of CDK4, other than retaining FLCN at the lysosomal surface, led to the accumulation of undigested material inside lysosomes, which impaired the autophagic flux and induced cancer cell senescence in vitro and in xenograft models. Importantly, the use of CDK4 inhibitors in therapy is known to cause senescence but not cell death. To overcome this phenomenon and based on our findings, we increased the autophagic flux in cancer cells by using an AMPK activator in combination with a CDK4 inhibitor. The cotreatment induced autophagy (AMPK activation) and impaired lysosomal function (CDK4 inhibition), resulting in cell death and tumor regression. Altogether, we uncovered a previously unknown role for CDK4 in lysosomal biology and propose a novel therapeutic strategy to target cancer cells. SIGNIFICANCE: These findings uncover a novel function of CDK4 in lysosomal biology, which promotes cancer progression by activating mTORC1; targeting this function offers a new therapeutic strategy for cancer treatment.

Alternative splicing affecting the SH3A domain controls the binding properties of intersectin 1 in neurons.

Intersectin 1 (ITSN1) is a conserved adaptor protein implicated in endocytosis, regulation of actin cytoskeleton rearrangements and mitogenic signaling. Its expression is characterized by multiple alternative splicing. Here we show neuron-specific expression of ITSN1 isoforms containing exon 20, which encodes five amino acid residues in the first SH3 domain (SH3A). In vitro binding experiments demonstrated that inclusion of exon 20 changes the binding properties of the SH3A domain. Endocytic proteins dynamin 1 and synaptojanin 1 as well as GTPase-activating protein CdGAP bound the neuron-specific variant of the SH3A domain with higher affinity than ubiquitously expressed SH3A. In contrast, SOS1, a guanine nucleotide exchange factor for Ras, and the ubiquitin ligase Cbl mainly interact with the ubiquitously expressed isoform. These results demonstrate that alternative splicing leads to the formation of two pools of ITSN1 with potentially different properties in neurons, affecting ITSN1 function as adaptor protein.

Ubiquitin-ligase AIP4 controls differential ubiquitination and stability of isoforms of the scaffold protein ITSN1.

At present, the role of ubiquitination of cargoes internalized from the plasma membrane is better understood than the consequences of ubiquitination of proteins comprising the endocytic machinery. Here, we show that the E3 ubiquitin ligase AIP4/ITCH contributes to the differential ubiquitination of isoforms of the endocytic scaffold protein intersectin1 (ITSN1). The major isoform ITSN1-s is monoubiquitinated, whereas the minor one, ITSN1-22a undergoes a combination of mono- and oligoubiquitination. The monoubiquitination is required for ITSN1-s stability, whereas the oligoubiquitination of ITSN1-22a causes its proteasomal degradation. This explains the observed low abundance of the minor isoform in cells. Thus, different modes of ubiquitination regulated by AIP4 have opposite effects on ITSN1 isoform stability.

Molecular mechanisms of Bdp1 in TFIIIB assembly and RNA polymerase III transcription initiation.

Initiation of gene transcription by RNA polymerase (Pol) III requires the activity of TFIIIB, a complex formed by Brf1 (or Brf2), TBP (TATA-binding protein), and Bdp1. TFIIIB is required for recruitment of Pol III and to promote the transition from a closed to an open Pol III pre-initiation complex, a process dependent on the activity of the Bdp1 subunit. Here, we present a crystal structure of a Brf2-TBP-Bdp1 complex bound to DNA at 2.7 Å resolution, integrated with single-molecule FRET analysis and in vitro biochemical assays. Our study provides a structural insight on how Bdp1 is assembled into TFIIIB complexes, reveals structural and functional similarities between Bdp1 and Pol II factors TFIIA and TFIIF, and unravels essential interactions with DNA and with the upstream factor SNAPc. Furthermore, our data support the idea of a concerted mechanism involving TFIIIB and RNA polymerase III subunits for the closed to open pre-initiation complex transition.Transcription initiation by RNA polymerase III requires TFIIIB, a complex formed by Brf1/Brf2, TBP and Bdp1. Here, the authors describe the crystal structure of a Brf2-TBP-Bdp1 complex bound to a DNA promoter and characterize the role of Bdp1 in TFIIIB assembly and pre-initiation complex formation.

The LMP2A protein of Epstein-Barr virus regulates phosphorylation of ITSN1 and Shb adaptors by tyrosine kinases.

Latent Membrane Protein 2A (LMP2A) is an Epstein-Barr virus-encoded protein that is important for the maintenance of latent infection. Its activity affects cellular differentiation, migration, proliferation and B cell survival. LMP2A resembles a constitutively activated B cell antigen receptor and exploits host kinases to activate a set of downstream signaling pathways. In the current study we demonstrate the interaction of LMP2A with intersectin 1 (ITSN1), a key endocytic adaptor protein. This interaction occurs via both the N- and C-tails of LMP2A and is mediated by the SH3 domains of ITSN1. Additionally, we identified the Shb adaptor and the Syk kinase as novel binding ligands of ITSN1. The Shb adaptor interacts simultaneously with the phosphorylated tyrosines of LMP2A and the SH3 domains of ITSN1 and mediates indirect interaction of ITSN1 to LMP2A. Syk kinase promotes phosphorylation of both ITSN1 and Shb adaptors in LMP2A-expressing cells. In contrast to ITSN1, Shb phosphorylation depends additionally on Lyn kinase activity. Considering that Shb and ITSN1 are implicated in various receptor tyrosine kinase signaling, our results indicate that LMP2A can affect a number of signaling pathways by regulating the phosphorylation of the ITSN1 and Shb adaptors.

Intersectin multidomain adaptor proteins: regulation of functional diversity.

Adaptor/scaffold proteins serve as platforms for the assembly of multiprotein complexes and regulate the efficiency and specificity of signalling cascades. Intersectins (ITSNs) are an evolutionarily conserved adaptor protein family engaged in endo- and exocytosis, actin cytoskeleton rearrangement and signal transduction. This review summarizes recent advances in the function of ITSNs in neuronal and non-neuronal cells, the role of alternative splicing and alternative transcription in regulating the structural and functional diversity of ITSNs, their expression patterns in different tissues and during development, their interactions with proteins, as well as the potential relevance of ITSNs for neurodegenerative diseases and cancer. The diversity of mechanisms in the regulation of ITSN expression and specificity in different cells emphasizes the important role of ITSN proteins in vesicle trafficking and signalling.

Identification and characterization of a novel mammalian isoform of the endocytic adaptor ITSN1.

Intersectin 1 (ITSN1) is an evolutionarily conserved adaptor protein engaged in clathrin-mediated endocytosis, cell signaling and actin cytoskeleton rearrangements. Two major ITSN1 isoforms were initially described, the ubiquitous short isoform (ITSN1-s) and the long isoform (ITSN1-l) expressed predominantly in neurons. Numerous alternative splicing events for ITSN1 pre-mRNA were later identified. Here we describe a novel isoform ITSN1-22a with an alternative C-terminus encoded by exon 22a. This exon is only found in placental mammals. The transcript of ITSN1-22a is detected in a wide range of human and mouse tissues. We show here that two alternative splicing events affect the coding sequence of the ITSN1-22a isoform. Moreover, alternative polyadenylation of these transcripts was demonstrated in human tissues. The protein encoded by the ITSN1-22a transcript possesses two EH domains, a coiled-coil region, an SH3A domain and a specific C-terminal domain (CTD) but lacks four SH3 domains in comparison with ITSN1-s. The level of ITSN1-22a protein varies in different mouse tissues and human cell lines. The highest amounts of this isoform occur in mouse brain, spleen, lung and the human B cell line DG75. ITSN1-22a binds via its CTD to the SH3 domain of the endocytic protein amphiphysin 1 and the SH3A domain of ITSN1. Furthermore association in vivo and codistribution of ITSN1-22a and ITSN1-s were demonstrated suggesting that these isoforms could function in concert. We have revealed differential binding of ITSN1-s and ITSN1-22a to the ubiquitin ligase Cbl. Both isoforms possess the SH3A domain capable of binding to Cbl; however ITSN1-22a in contrast to ITSN1-s did not interact with Cbl in vivo. In vitro binding experiments demonstrated that the CTD of ITSN1-22a negatively regulated its binding to Cbl; at the same time interaction with another partner, dynamin 1 was not affected by the presence of the CTD. These data suggest that intramolecular interaction within ITSN1-22a could specifically regulate its binding to protein partners. Thus, this novel mammalian ITSN1 isoform possesses a significantly altered domain structure and performs specific protein-protein interactions.