PCID2 Subunit of the Drosophila TREX-2 Complex Has Two RNA-Binding Regions.

Drosophila PCID2 is a subunit of the TREX-2 mRNA nuclear export complex. Although the complex has long been studied in eukaryotes, it is still unclear how TREX-2 interacts with mRNA in multicellular organisms. Here, the interaction between Drosophila PCID2 and the ras2 RNA was studied by EMSA. We show that the C-terminal region of the WH domain of PCID2 specifically binds the 3'-noncoding region of the ras2 RNA. While the same region of PCID2 interacts with the Xmas-2 subunit of the TREX-2 complex, PCID2 interacts with RNA independently of Xmas-2. An additional RNA-binding region (M region) was identified in the N-terminal part of the PCI domain and found to bind RNA nonspecifically. Point mutations of evolutionarily conserved amino acid residues in this region completely abolish the PCID2-RNA interaction, while a deletion of the C-terminal domain only partly decreases it. Thus, the specific interaction of PCID2 with RNA requires nonspecific PCID2-RNA binding.

New Approach for Studying of Isoforms and High-Homology Proteins in Mammalian Cells.

In mammals, a large number of proteins are expressed as more than one isoform, resulting in the increased diversity of their proteome. Understanding the functions of isoforms is very important, since individual isoforms of the same protein can have oncogenic or pathogenic properties, or serve as disease markers. The high homology of isoforms with ubiquitous expression makes it difficult to study them. In this work, we propose a new approach for the study of protein isoforms in mammalian cells, which makes it possible to individually detect and investigate the functions of an individual isoform. The approach was developed to study the functions of isoforms of the PHF10 protein, a chromatin subunit of the PBAF remodeling complex. We demonstrated the possibility of induced simultaneous suppression of all endogenous PHF10 isoforms and the expression of a single recombinant FLAG-tagged isoform. For this purpose, we created constructs based on the pSLIK plasmid with a cloned cassette containing the recombinant gene of interest and miR30 with the corresponding shRNAs. The doxycycline-induced activation of the cassette allows on and off switching. Using this construct, we achieved the preferential expression of only one recombinant PHF10 isoform with a simultaneously reduced number of all endogenous isoforms. Our approach can be used to study the role of point mutations, the functions of individual domains and important sites, or to individually detect untagged isoforms with knockdown of all endogenous isoforms.

Conserved Structure and Evolution of DPF Domain of PHF10-The Specific Subunit of PBAF Chromatin Remodeling Complex.

Transcription activation factors and multisubunit coactivator complexes get recruited at specific chromatin sites via protein domains that recognize histone modifications. Single PHDs (plant homeodomains) interact with differentially modified H3 histone tails. Double PHD finger (DPF) domains possess a unique structure different from PHD and are found in six proteins: histone acetyltransferases MOZ and MORF; chromatin remodeling complex BAF (DPF1-3); and chromatin remodeling complex PBAF (PHF10). Among them, PHF10 stands out due to the DPF sequence, structure, and functions. PHF10 is ubiquitously expressed in developing and adult organisms as four isoforms differing in structure (the presence or absence of DPF) and transcription regulation functions. Despite the importance of the DPF domain of PHF10 for transcription activation, its structure remains undetermined. We performed homology modeling of the human PHF10 DPF domain and determined common and distinct features in structure and histone modifications recognition capabilities, which can affect PBAF complex chromatin recruitment. We also traced the evolution of DPF1-3 and PHF10 genes from unicellular to vertebrate organisms. The data reviewed suggest that the DPF domain of PHF10 plays an important role in SWI/SNF-dependent chromatin remodeling during transcription activation.

TRF4, the novel TBP-related protein of Drosophila melanogaster, is concentrated at the endoplasmic reticulum and copurifies with proteins participating in the processes associated with endoplasmic reticulum.

Understanding the functions of TBP-related factors is essential for studying chromatin assembly and transcription regulation in higher eukaryotes. The novel TBP-related protein-coding gene, trf4, was described in Drosophila melanogaster. trf4 is found only in Drosophila and has likely originated in Drosophila common ancestor. TRF4 protein has a distant homology with TBP and TRF2 in the region of TBP-like domain and is evolutionarily conserved among distinct Drosophila species, which indicates its functional significance. TRF4 is widely expressed in D. melanogaster with high levels of its expression being observed in testes. Interestingly enough, TRF4 has become a cytoplasmic protein having lost nuclear localization signal sequence. TRF4 is concentrated at the endoplasmic reticulum (ER) and copurifies with the proteins participating in the ER-associated processes. We suggest that trf4 gene is an example of homolog neofunctionalization by protein subcellular relocalization pathway, where the subcellular relocalization of gene product of duplicated gene leads to the new functions in ER-associated processes.

Different N-terminal isoforms of Oct-1 control expression of distinct sets of genes and their high levels in Namalwa Burkitt's lymphoma cells affect a wide range of cellular processes.

Oct-1 transcription factor has various functions in gene regulation. Its expression level is increased in several types of cancer and is associated with poor survival prognosis. Here we identified distinct Oct-1 protein isoforms in human cells and compared gene expression patterns and functions for Oct-1A, Oct-1L, and Oct-1X isoforms that differ by their N-terminal sequences. The longest isoform, Oct-1A, is abundantly expressed and is the main Oct-1 isoform in most of human tissues. The Oct-1L and the weakly expressed Oct-1X regulate the majority of Oct-1A targets as well as additional sets of genes. Oct-1X controls genes involved in DNA replication, DNA repair, RNA processing, and cellular response to stress. The high level of Oct-1 isoforms upregulates genes related to cell cycle progression and activates proliferation both in Namalwa Burkitt's lymphoma cells and primary human fibroblasts. It downregulates expression of genes related to antigen processing and presentation, cytokine-cytokine receptor interaction, oxidative metabolism, and cell adhesion, thus facilitating pro-oncogenic processes.

Multifunctional factor ENY2 is associated with the THO complex and promotes its recruitment onto nascent mRNA.

Metazoan E(y)2/ENY2 is a multifunctional protein important for transcription activation and mRNA export, being a component of SAGA/TFTC and the mRNA export complex AMEX. Here, we show that ENY2 in Drosophila is also stably associated with THO, the complex involved in mRNP biogenesis. The ENY2-THO complex is required for normal Drosophila development, functioning independently on SAGA and AMEX. ENY2 and THO arrive on the transcribed region of the hsp70 gene after its activation, and ENY2 plays an important role in THO recruitment. ENY2 and THO show no direct association with elongating RNA polymerase II. Recruitment of ENY2 and THO occurs by their loading onto nascent mRNA, apparently immediately after its synthesis, while the AMEX component Xmas-2 is loaded onto mRNA at a later stage. Knockdown of either ENY2 or THO, but not SAGA or AMEX, affects the processing of the transcript's 3' end. Thus, ENY2, as a shared subunit of several protein complexes governing the sequential steps of gene expression, plays an important role in the coordination of these steps.

Telomeric repeat silencing in germ cells is essential for early development in Drosophila.

The germline-specific role of telomeres consists of chromosome end elongation and proper chromosome segregation during early developmental stages. Despite the crucial role of telomeres in germ cells, little is known about telomere biology in the germline. We analyzed telomere homeostasis in the Drosophila female germline and early embryos. A novel germline-specific function of deadenylase complex Ccr4-Not in the telomeric transcript surveillance mechanism is reported. Depletion of Ccr4-Not complex components causes strong derepression of the telomeric retroelement HeT-A in the germ cells, accompanied by elongation of the HeT-A poly(A) tail. Dysfunction of transcription factors Woc and Trf2, as well as RNA-binding protein Ars2, also results in the accumulation of excessively polyadenylated HeT-A transcripts in ovaries. Germline knockdowns of Ccr4-Not components, Woc, Trf2 and Ars2, lead to abnormal mitosis in early embryos, characterized by chromosome missegregation, centrosome dysfunction and spindle multipolarity. Moreover, the observed phenotype is accompanied by the accumulation of HeT-A transcripts around the centrosomes in early embryos, suggesting the putative relationship between overexpression of telomeric transcripts and mitotic defects. Our data demonstrate that Ccr4-Not, Woc, Trf2 and Ars2, components of different regulatory pathways, are required for telomere protection in the germline in order to guarantee normal development.

Insulator protein Su(Hw) recruits SAGA and Brahma complexes and constitutes part of Origin Recognition Complex-binding sites in the Drosophila genome.

Despite increasing data on the properties of replication origins, molecular mechanisms underlying origin recognition complex (ORC) positioning in the genome are still poorly understood. The Su(Hw) protein accounts for the activity of best-studied Drosophila insulators. Here, we show that Su(Hw) recruits the histone acetyltransferase complex SAGA and chromatin remodeler Brahma to Su(Hw)-dependent insulators, which gives rise to regions with low nucleosome density and creates conditions for ORC binding. Depletion in Su(Hw) leads to a dramatic drop in the levels of SAGA, Brahma and ORC subunits and a significant increase in nucleosome density on Su(Hw)-dependent insulators, whereas artificial Su(Hw) recruitment itself is sufficient for subsequent SAGA, Brahma and ORC binding. In contrast to the majority of replication origins that associate with promoters of active genes, Su(Hw)-binding sites constitute a small proportion (6%) of ORC-binding sites that are localized preferentially in transcriptionally inactive chromatin regions termed BLACK and BLUE chromatin. We suggest that the key determinants of ORC positioning in the genome are DNA-binding proteins that constitute different DNA regulatory elements, including insulators, promoters and enhancers. Su(Hw) is the first example of such a protein.

Transcription co-activator SAYP mediates the action of STAT activator.

Jak/STAT is an important signaling pathway mediating multiple events in development. We describe participation of metazoan co-activator SAYP/PHF10 in this pathway downstream of STAT. The latter, via its activation domain, interacts with the conserved core of SAYP. STAT is associated with the SAYP-containing co-activator complex BTFly and recruits BTFly onto genes. SAYP is necessary for stimulating STAT-driven transcription of numerous genes. Mutation of SAYP leads to maldevelopments similar to those observed in STAT mutants. Thus, SAYP is a novel co-activator mediating the action of STAT.

SAYP and Brahma are important for 'repressive' and 'transient' Pol II pausing.

Drosophila SAYP, a homologue of human PHF10/BAF45a, is a metazoan coactivator associated with Brahma and essential for its recruitment on the promoter. The role of SAYP in DHR3 activator-driven transcription of the ftz-f1 gene, a member of the ecdysone cascade was studied. In the repressed state of ftz-f1 in the presence of DHR3, the Pol II complex is pre-recruited on the promoter; Pol II starts transcription but is paused 1.5 kb downstream of the promoter, with SAYP and Brahma forming a 'nucleosomal barrier' (a region of high nucleosome density) ahead of paused Pol II. SAYP depletion leads to the removal of Brahma, thereby eliminating the nucleosomal barrier. During active transcription, Pol II pausing at the same point correlates with Pol II CTD Ser2 phosphorylation. SAYP is essential for Ser2 phosphorylation and transcription elongation. Thus, SAYP as part of the Brahma complex participates in both 'repressive' and 'transient' Pol II pausing.

The alternative protein isoform NK2B, encoded by the vnd/NK-2 proneural gene, directly activates transcription and is expressed following the start of cells differentiation.

NK-2 is a homeodomain protein essential for the development of the central nervous system in the Drosophila embryo. Here, we show that the vnd/NK-2 gene encodes an additional protein isoform (NK-2B) that differs from the known one (NK-2A) in its N-terminal domain. While NK-2A is a transcription repressor, NK-2B directly activates transcription from promoters containing NK-2 binding sites, with its N-terminal domain possessing a strong transcription activation potency. The transcription of NK-2B starts at the onset of metamorphosis. Its expression is observed in precursors of differentiating photoreceptors and in photoreceptors of the adult eye. Both NK-2B and NK-2A are expressed in the lamina. However, the expression of NK-2A is mostly associated with the undifferentiated state of nervous cells.

A novel chromatin-remodeling complex variant, dcPBAF, is involved in maintaining transcription in differentiated neurons.

The Polybromo-associated BAF (BRG1- or BRM-associated factors) (PBAF) chromatin-remodeling complex is essential for transcription in mammalian cells. In this study, we describe a novel variant of the PBAF complex from differentiated neuronal cells, called dcPBAF, that differs from the canonical PBAF existing in proliferating neuroblasts. We describe that in differentiated adult neurons, a specific subunit of PBAF, PHF10, is replaced by a PHF10 isoform that lacks N- and C-terminal domains (called PHF10D). In addition, dcPBAF does not contain the canonical BRD7 subunit. dcPBAF binds promoters of the actively transcribed neuron-specific and housekeeping genes in terminally differentiated neurons of adult mice. Furthermore, in differentiated human neuronal cells, PHF10D-containing dcPBAF maintains a high transcriptional level at several neuron-specific genes.

Transcription coactivator SAYP combines chromatin remodeler Brahma and transcription initiation factor TFIID into a single supercomplex.

Transcription activation by RNA polymerase II is a complicated process driven by combined, precisely coordinated action of a wide array of coactivator complexes, which carry out chromatin-directed activities and nucleate the assembly of the preinitiation complex on the promoter. Using various techniques, we have shown the existence of a stable coactivator supercomplex consisting of the chromatin-remodeling factor Brahma (SWI/SNF) and the transcription initiation factor TFIID, named BTFly (Brahma and TFIID in one assembly). The coupling of Brahma and TFIID is mediated by the SAYP factor, whose evolutionarily conserved activation domain SAY can directly bind to both BAP170 subunit of Brahma and TAF5 subunit of TFIID. The integrity of BTFly is crucial for its ability to activate transcription. BTFly is distributed genome-wide and appears to be a means of effective transcription activation.

Triple-negative and triple-positive breast cancer cells reciprocally control their growth and migration via the S100A4 pathway.

The study's aim was to investigate the S100A4-mediated mechanisms of the regulation of tumor cell proliferation and migration in the human triple-positive breast carcinoma cell line MCF-7 (TPBC) and triple-negative breast carcinoma cell line MDA-MB-231 (TNBC). The proliferative activity of TNBC more than doubled during the incubation in the conditioned medium of TPBC. Extracellular S100A4 dose-dependently decreased the proliferative response of TPBC. TPBC negatively impacted the growth of TNBCs during their co-culturing. TPBC significantly decreased the migration activity of the TNBC cells while the S100A4 intracellular level in the TNBC was also decreasing. The decrease in the S100A4 intracellular level occurred due to the protein's monomeric form while the contribution of the dimeric form into the overall S100A4 concentration in TNBC cells increased 1.5-2-fold. The S100A4 pathway in the intercellular communication between TNBC and TPBCs also included the dexamethasone-sensitive mechanisms of S100A4 intra- and extracellular pools regulation.

Suppression of OCT-1 in Metastatic Breast Cancer Cells Reduces Tumor Metastatic Potential, Hypoxia Resistance, and Drug Resistance.

OCT-1/POU2F1 is a ubiquitously expressed transcription factor. Its expression starts at the earliest stage of embryonic development. OCT-1 controls genes involved in the regulation of differentiation, proliferation, cell metabolism, and aging. High levels of OCT-1 transcription factor in tumor cells correlate with tumor malignancy and resistance to antitumor therapy. Here, we report that suppression of OCT-1 in breast cancer cells reduces their metastatic potential and drug resistance. OCT-1 knockdown in the MDA-MB231 breast cancer cells leads to a fivefold decrease (p < 0.01) in cell migration rates in the Boyden chamber. A decrease in the transcription levels of human invasion signature (HIS) genes (ARHGDIB, CAPZA2, PHACTR2, CDC42, XRCC5, and CAV1) has been also demonstrated by real-time PCR, with high expression of these genes being a hallmark of actively metastasizing breast cancer cells. Transcriptional activity of ATF6 response elements is significantly reduced in the cell lines with decreased OCT-1 expression, which results in lower levels of adaptive EPR stress response. OCT-1 knockdown more than two times increases the MDA-MB231 cell death rate in hypoxia and significantly increases the doxorubicin or docetaxel-treated MDA-MB231 cell death rate. Our findings indicate that OCT-1 may be an important therapeutic target and its selective inhibition may have significant therapeutic effects and may improve prognosis in breast cancer patients.

SAGA and a novel Drosophila export complex anchor efficient transcription and mRNA export to NPC.

SAGA/TFTC-type multiprotein complexes play important roles in the regulation of transcription. We have investigated the importance of the nuclear positioning of a gene, its transcription and the consequent export of the nascent mRNA. We show that E(y)2 is a subunit of the SAGA/TFTC-type histone acetyl transferase complex in Drosophila and that E(y)2 concentrates at the nuclear periphery. We demonstrate an interaction between E(y)2 and the nuclear pore complex (NPC) and show that SAGA/TFTC also contacts the NPC at the nuclear periphery. E(y)2 forms also a complex with X-linked male sterile 2 (Xmas-2) to regulate mRNA transport both in normal conditions and after heat shock. Importantly, E(y)2 and Xmas-2 knockdown decreases the contact between the heat-shock protein 70 (hsp70) gene loci and the nuclear envelope before and after activation and interferes with transcription. Thus, E(y)2 and Xmas-2 together with SAGA/TFTC function in the anchoring of a subset of transcription sites to the NPCs to achieve efficient transcription and mRNA export.

Primate-specific stress-induced transcription factor POU2F1Z protects human neuronal cells from stress.

The emergence of new primate-specific genes is an essential factor in human and primate brain development and functioning. POU2F1/Oct-1 is a transcription regulator in higher eukaryotes which is involved in the regulation of development, differentiation, stress response, and other processes. We have demonstrated that the Tigger2 transposon insertion into the POU2F1 gene which occurred in the primate lineage led to the formation of an additional exon (designated the Z-exon). Z-exon-containing primate-specific Oct-1Z transcript includes a short upstream ORF (uORF) located at its 5'-end and the main ORF encoding the Oct-1Z protein isoform (Pou2F1 isoform 3, P14859-3), which differs from other Oct-1 isoforms by its N-terminal peptide. The Oct-1Z-encoding transcript is expressed mainly in human brain cortex. Under normal conditions, the translation of the ORF coding for the Oct-1Z isoform is repressed by uORF. Under various stress conditions, uORF enables a strong increase in the translation of the Oct-1Z-encoding ORF. Increased Oct-1Z expression levels in differentiating human neuroblasts activate genes controlling stress response, neural cell differentiation, brain formation, and organogenesis. We have shown that the Oct-1Z isoform of the POU2F1/Oct-1 transcription factor is an example of a primate-specific genomic element contributing to brain development and cellular stress defense.

The sequential phosphorylation of PHF10 subunit of the PBAF chromatin-remodeling complex determines different properties of the PHF10 isoforms.

The mammalian PBAF subfamily of SWI/SNF chromatin remodeling complexes plays a wide role in the regulation of gene expression. PHF10 is a subunit of the signature module of PBAF, responsible for its interaction with chromatin. PHF10 is represented by four different isoforms, which are alternatively incorporated in the complex. Two of PHF10 isoforms lacking C-terminal PHD domains contain a cluster of phosphorylated serine residues, designated as X-cluster. In the present study, we explore the phosphorylation of the X-cluster in detail. We identified additional phosphorylated serine residues and designated them as either frequently or rarely phosphorylated. The X-cluster consists of two independently phosphorylated subclusters. Phosphorylation of the second subcluster depends on phosphorylation of a primary serine 327. These two subclusters surround a sequence, which is predicted to be a nuclear localization sequence (NLS3). The NLS3 does not affect localization of PHF10 isoforms. However, it is essential for X-cluster phosphorylation and increased stability of isoforms that lack PHD. Conversely, the presence of NLS3 signal in isoforms that contain C-terminal PHD domains reduces their stability. Thus, phosphorylation of PHF10 isoforms regulates their cell level, determining the rate of incorporation in PBAF. This may alter the pattern of PBAF regulated genes.

Two isoforms of Drosophila TRF2 are involved in embryonic development, premeiotic chromatin condensation, and proper differentiation of germ cells of both sexes.

The Drosophila TATA box-binding protein (TBP)-related factor 2 (TRF2 or TLF) was shown to control a subset of genes different from that controlled by TBP. Here, we have investigated the structure and functions of the trf2 gene. We demonstrate that it encodes two protein isoforms: the previously described 75-kDa TRF2 and a newly identified 175-kDa version in which the same sequence is preceded by a long N-terminal domain with coiled-coil motifs. Chromatography of Drosophila embryo extracts revealed that the long TRF2 is part of a multiprotein complex also containing ISWI. Both TRF2 forms are detected at the same sites on polytene chromosomes and have the same expression patterns, suggesting that they fulfill similar functions. A study of the manifestations of the trf2 mutation suggests an essential role of TRF2 during embryonic Drosophila development. The trf2 gene is strongly expressed in germ line cells of adult flies. High levels of TRF2 are found in nuclei of primary spermatocytes and trophocytes with intense transcription. In ovaries, TRF2 is present both in actively transcribing nurse cells and in the transcriptionally inactive oocyte nuclei. Moreover, TRF2 is essential for premeiotic chromatin condensation and proper differentiation of germ cells of both sexes.