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.

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.

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.

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.

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.

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.

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.

PBAF lacking PHD domains maintains transcription in human neutrophils.

The myeloid precursor cell differentiation requires an extensive chromatin remodeling. We show that the level of the PBAF chromatin remodeling complex decreases following the start of differentiation of myeloid precursors, becoming very low in the terminally differentiated peripheral blood (PB) neutrophils where it co-localizes with Pol II on the transcriptionally active chromatin. Previously, we have shown that the PHF10 subunit of the PBAF signature module has four isoforms, two of them (PHF10-P) contain a tandem of C-terminal PHD domains. We found that out of four PHF10 isoforms present in the myeloid precursor cells, only the PHF10-Ss isoform lacking PHD domains, is actively expressed in the PB neutrophils. In particular, the longest of the PHF10 isoforms (PHF10-Pl), which is essential for proliferation, completely disappears in PB neutrophils. In addition, in the myeloid precursors, promoters of neutrophil-specific genes are associated with the PHD-containing isoforms, together with PBAF and Pol II, when these genes are inactive and only during their activation stage. However, at the later stages of differentiation, when neutrophil-specific genes are actively transcribed, PHF10-P isoforms on their promoters are replaced by the PHF10-S isoforms. Evidently, PHD domains of PHF10 are essential for active chromatin remodeling during transcription activation, but are dispensable for the constantly transcribed genes.

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.