Neuronal and Muscle Differentiation of Mammalian Cells Is Accompanied by a Change in PHF10 Isoform Expression.

The PBAF chromatin remodeling complex of the SWI/SNF family plays a critical role in the regulation of gene expression during tissue differentiation and organism development. The subunits of the PBAF complex have domains responsible for binding to N-terminal histone sequences. It determines the specificity of binding of the complex to chromatin. PHF10, a specific subunit of the PBAF complex, contains a DPF domain, which is a unique chromatin interaction domain. A PHF10 isoform that lacks the DPF domain is also present in vertebrate cells. This work shows that during neuronal and muscle differentiation of human and mouse cells, the expression of PHF10 isoforms changes: the form that does not have DPF replaces the form in which it is present. Replacement of PHF10 isoforms in the PBAF complex may affect its selectivity in the regulation of genes in differentiating cells.

Chromatin Remodeling Complex PBAF Activates and Represses Inflammatory Genes.

The PBAF chromatin remodeling complex regulates chromatin state and gene transcription in higher eukaryotes. In this work, we studied the role of PBAF in the regulation of NF-κB-and JAK/STAT-dependent activation of inflammatory genes. We performed knockdown of specific module subunit BAF200, which resulted in destruction of the entire PBAF specific module and changed the level of the genes transcription of both pathways. PBAF can be both an activator and a repressor of inflammatory genes. Thus, PBAF is an important regulator of inflammatory gene expression.

Contact Inhibition of Proliferation Is Accompanied by Expression of the PHF10D Subunit of the Chromatin Remodeling Complex PBAF in Mouse and Human Cell Lines.

PHF10 is a subunit of the PBAF complex, which regulates the expression of many genes in developing and maturing organisms. PHF10 has four isoforms that differ in domain structure. The PHF10A isoform, containing a DPF domain at the C-terminus and 46 amino acids at the N-terminus, is necessary for the expression of proliferation genes; the functions of the other isoforms are less studied. In this work, we have established that, upon contact inhibition of mouse and human cell proliferation caused by the establishment of a tight junction and adherence junction between cells, the expression of the PHF10A isoform stops and instead the PHF10D isoform is expressed, which does not contain DPF-domain and N-terminal sequence. The function of the PHF10D isoform may be associated with the establishment of intercellular contacts.

[Role of the SWI/SNF Chromatin Remodeling Complex in Regulation of Inflammation Gene Expression].

The process of inflammation is the body's natural defense response to the penetration of foreign substances and molecules from the outside. Many proteins, signaling cascades, and transcription factors are involved in the activation of inflammation genes. Their coordinated activity leads to a change in the expression of proinflammatory genes. The chromatin state of genes responding to the inflammation stimulus is the main factor determining the binding of transcriptional activators to the gene regulatory elements and a key mechanism in the induction of inflammatory genes. The rapid change in the state of chromatin, the creation of an open structure and the removal of the "nucleosome barrier" facilitates the binding of transcription factors and the initiation of transcription. This process is realized by attracting complexes to the gene that modify and remodel chromatin. One of the most important complexes restructuring the chromatin structure during gene activation is the SWI/SNF multisubunit complex. SWI/SNF regulates the expression of inflammation genes through interaction with transcription factors, including factors of the NF-κВ signaling pathway. The variability of the subunits of this complex determines the specificity of its binding to the chromatin and various transcriptional activators. This review considers the role of SWI/SNF in the regulation of inflammation genes, describes its interactions with chromatin, and the molecular mechanisms of its recruitment to the promoters.

[PHF10, a Subunit of the PBAF Chromatin Remodeling Complex, Changes Its Localization and Interacts with c-FOS during the Initiation of Long-Term Potentiation in Neuronal Culture].

The PBAF chromatin remodeling complex interacts with many transcriptional activators and is recruited to target chromatin regions. PBAF plays an important role in maintaining and modifying the chromatin structure in mammalian cells. A subunit of the PBAF complex, the PHF10 transcription factor, is required for proliferation of neuronal precursors in the early stages of mouse brain development and gene expression in differentiated neurons. We showed that PHF10 interacts with the protein product of the early response gene c-FOS, the c-FOS transcriptional activator, which is expressed in response to the induction of long-term potentiation (LTP). LTP induction triggers the transcription of genes and the synthesis of proteins that provide changes that lead to the establishment of long-term contacts between neurons. We showed that in cells in differentiated neuronal culture, after the induction of LTP, expression of c-FOS, which is initially localized in the cytoplasm and then moves to the nucleus, begins. PHF10 is expressed in neuronal cells prior to LTP induction and has nuclear localization. However, 1 h after LTP induction, PHF10 is detected in the cytoplasm together with c-FOS, and then moves into the nucleus with it. Importantly, this behavior of PHF10 in response to KC1 stimulation is specific for neuronal cultures. It is assumed that during LTP, PHF10 together with c-FOS participates in the activation of secondary response genes that regulate the maintenance of plastic modifications and homeostasis of neuronal synapses. The PHF10 export from the nucleus and its rapid return together with c-FOS to the nucleus is possibly necessary for the rapid modulation of expression of target secondary response genes during LTP.

Inhibition of the c-Myc Oncogene by the Aureolic Acid Group Antibiotics.

GC-rich stretches in the DNA minor groove are the established intracellular targets for the aureolic acid group of antibiotics such as olivomycin A and its semisynthetic analogue olivamide. We demonstrated here that both antibiotics at nanomolar concentrations inhibited transcription of the c-Myc oncogene in cultured human tumor cells. The mechanism of transcriptional inhibition did not require the full-length binding site for Sp1, a GC-dependent transcriptional factor. GC quartets with the nucleotide sequences optimal for drug binding are sufficient for c-Myc transcriptional block by the aureolic acid derivatives.

PHF10 subunit of PBAF complex mediates transcriptional activation by MYC.

The PBAF complex, a member of SWI/SNF family of chromatin remodelers, plays an essential role in transcriptional regulation. We revealed a disease progression associated elevation of PHF10 subunit of PBAF in clinical melanoma samples. In melanoma cell lines, PHF10 interacts with MYC and facilitates the recruitment of PBAF complex to target gene promoters, therefore, augmenting MYC transcriptional activation of genes involved in the cell cycle progression. Depletion of either PHF10 or MYC induced G1 accumulation and a senescence-like phenotype. Our data identify PHF10 as a pro-oncogenic mechanism and an essential novel link between chromatin remodeling and MYC-dependent gene transcription.

The DPF Domain As a Unique Structural Unit Participating in Transcriptional Activation, Cell Differentiation, and Malignant Transformation.

The DPF (double PHD finger) domain consists of two PHD fingers organized in tandem. The two PHD-finger domains within a DPF form a single structure that interacts with the modification of the N-terminal histone fragment in a way different from that for single PHD fingers. Several histone modifications interacting with the DPF domain have already been identified. They include acetylation of H3K14 and H3K9, as well as crotonylation of H3K14. These modifications are found predominantly in transcriptionally active chromatin. Proteins containing DPF belong to two classes of protein complexes, which are the transcriptional coactivators involved in the regulation of the chromatin structure. These are the histone acetyltransferase complex belonging to the MYST family and the SWI/SNF chromatin-remodeling complex. The DPF domain is responsible for the specificity of the interactions between these complexes and chromatin. Proteins containing DPF play a crucial role in the activation of the transcription of a number of genes expressed during the development of an organism. These genes are important in the differentiation and malignant transformation of mammalian cells.

Oncogene c-MYC Controls the Expression of PHF10 Subunit of PBAF Chromatin Remodeling Complex in SW620 Cell Line.

The PBAF(SWI/SNF) multiprotein complex, which changes the chromatin structure, is widely involved in the regulation of eukaryotic gene expression. A specific component of this complex is the PHF10 protein, which is involved in recruiting this complex to chromatin. We showed that the PHF10 expression in cells of different lines is activated by the c-MYC oncogene. Since PHF10 stimulates cell proliferation, its c-MYC-dependent activation in cancer cells should lead to an increase in their proliferation rate.

An optimized permeabilization step for flow cytometry analysis of nuclear proteins in myeloid differentiation of blood cells into neutrophils.

Polymorphonuclear leukocytes (PMNLs) or neutrophils play an important role in the innate immune response. Working with human neutrophils is challenging because these cells are sensitive to changes in the surrounding media and quickly become apoptotic. Meanwhile the experiments with mature neutrophils may be very important for studies of blood function. In this paper we propose an improved technique of flow cytometry nuclear protein analysis with double antibody labeling, which allows direct comparison of protein quantity (overlay histograms) in the primary cells (neutrophils) and progenitor cell lines (line HL-60), to study differentiation process and for other research purposes. We suggest improved technique to analyze and compare nuclear proteins levels in the myeloid differentiation model system (HL-60 cell line) and / or primary human neutrophils. This method was justified with measurement of GFI1 protein expression level, as well-known transcription factor, typical and essential for mature neutrophils. The key protocol features are as follows: •Suggested protocol allows simply, direct and correct visual comparison of flow cytometry data in overlay diagrams for myeloid blood cells on various stages of differentiation.•70% ethanol permeabilization of neutrophils and HL-60 cells results in lower background fluorescence and better peak resolution than MeOH and Saponin permeabilization.•Non-specific antibody binding in neutrophils can be efficiently blocked by using 1% BSA and non-immune goat serum.

Stability of Chromatin Remodeling Complex Subunits Is Determined by Their Phosphorylation Status.

It was found that, in the differentiated cells of mouse brain, the level of core (Brg1 and BAF155) and specific (BRD7, BAF180, and PHF10) subunits of the chromatin-remodeling complex PBAF is reduced compared to the undifferentiated proliferating cells. Phosphorylation of PBAF complex subunits is required for maintaining their stability in differentiated brain cells.

Zinc Finger Protein CG9890 - New Component of ENY2-Containing Complexes of Drosophila.

In previous studies, we showed that the insulator protein Su(Hw) containing zinc finger domains interacts with the ENY2 protein and recruits the ENY2-containing complexes on Su(Hw)-dependent insulators, participating in the regulation of transcription and in the positioning of replication origins. Here, we found interaction between ENY2 and CG9890 protein, which also contains zinc finger domains. The interaction between ENY2 and CG9890 was confirmed. It was established that CG9890 protein is localized in the nucleus and interacts with the SAGA, ORC, dSWI/SNF, TFIID, and THO protein complexes.

[Ratio of transcription factor PHF10 splice variants in lymphocytes as a molecular marker of Parkinson's disease].

Parkinson's disease (PD) is the second most common neurodegenerative disorder and causes degeneration of dopaminergic neurons in the nigrostriatal system of the brain. PHF10 is one of the most important regulatory subunits of the SWI/SNF chromatin-remodeling protein complex, which controls the gene function and chromatin state in neurons. Two alternative RHF10 isoforms, PHF10-P and PHF10-S, replace each other in the complex to change the target gene pattern. Expression of the PHF10-P and PHF10-S transcripts in the nigrostriatal system and their ratio in blood lymphocytes were found to change in a mouse model of early clinical stage of PD as compared with control mice. Changes in PHF10-S level were also observed in peripheral blood lymphocytes from patients with early clinical stage of PD. A ratio of the PHF10-P and PHF10-S transcripts in peripheral blood cells was assumed to provide a potential marker of early stage PD.

[PHF10 isoforms are phosphorylated in the PBAF mammalian chromatin remodeling complex].

Chromatin remodeling complex PBAF(SWI/SNF) alters the structure of chromatin and controls gene expression. PHF10 is a specific subunit of PBAF complex and is expressed as four isoforms in mammalian cells. We demonstrated that all isoforms are expressed in various human cell types of different histological origins. All four isoforms are extensively phosphorylated and their phosphorylation level is depended on the cell type. Phosphorylation of PHF10 isoforms occurs while they are incorporated as a subunit of the PBAF complex, and therefore phosphorylation of PHF10 isoforms may play an essential role in regulation of PBAF complex's function and mechanism of action.

The level of the Phf10 protein, a PBAF chromatin-remodeling complex subunit, correlates with the Mts1/S100A4 expression in human cancer cell lines.

Mts1 (S100A4) protein is a marker of metastatic tumor cells, which is associated with a poor diagnostic prognosis for cancer progression. Therefore, it is important to study the S100A4 gene expression. According to our preliminary data, PHF10, a PBAF remodeling complex component, can play an important role in the transcription regulation of the S100A4 gene. We studied the expression of S100A4 and the total PHF10 protein in some cell lines. We have found that, in the cell lines studied, the PHF10 expression is correlated with the S100A4 expression.

[Novel conservative domain of SAYP coactivator mediates TFIID and Brahma transcriptional complexes interaction].

In the S2 cell system of Drosophila melanogaster a key protein domain mediating the interaction of TFIID and Brahma transcriptional complexes into the BTFly supercomplex has been shown to be an evolutionary conserved SAY domain of the SAYP. TFIID and Brahma coactivators participated in the reporter gene activation induced by the SAY domain in cellular nuclei. The TFIID and Brahma components directly interacting with the SAY domain were identified.

[SAYP--a novel regulator of metazoan development].

SAYP is a dual-function transcriptional coactivator of RNA polymerase II. It is a metazoan-specific factor involved in different signaling pathways that control normal development. In Drosophila, SAYP is present in the organism from the early stages of development and participates in cell cycle synchronization at the blastoderm stage. SAYP is abundant in many embryonic cells and in imaginal discs of larvae and is crucial for oogenesis in adults. At the molecular level, SAYP serves as a basis for assembling the BTFly nuclear supercomplex consising of the Brahma and TFIID coactivators. We suppose that BTFly and other similar nuclear supercomplexes play an important role in ontogenesis.

[Conservative E(y)2/Sus1 protein interacts with the Su(Hw)-dependent insulators in Drosophila].

Insulators are regulatory elements having two properties. First, they are able to disturb the interaction between promoters and enhancers/silencers. Second, they are able to block distribution of the heterochromatin. The best-studied are the Su(Hw)-dependent insulators of Drosophila melanogaster, activity of which is determined by the Su(Hw) protein. In this study it was demonstrated that novel, evolutionary conservative transcription factor E(y)2/Sus1 interacted with the Su(Hw) zinc-finger domain and was present in the protein complex, associated with the Su(Hw)-dependent insulators.

[Novel complex, formed by transcription coactivator SAYP].

The multisubunit complex which contains the novel evolutionarily conservative transcription factor SAYP was isolated and the protein composition of the complex was determined. It was shown that SAYP unites two complexes with different functions in transcription activation: the chromatin - remodeling complex PBAP (SWI/SNF) and the main component of preinitiation complex of Pol II, general transcription factor TFIID. The isolated super-complex contained the full set of PBAP and TFIID subunits. All components of supercomplex (SAYP, TFIID and PBAP) are essential for its effective interaction with promoters of SAYP-dependent genes.