A non-canonical function of Arabidopsis ERECTA proteins and a role of the SWI3B subunit of the SWI/SNF chromatin remodeling complex in gibberellin signaling.

The Arabidopsis ERECTA family (ERf) of leucine-rich repeat receptor-like kinases (LRR-RLKs) comprising ERECTA (ER), ERECTA-LIKE 1 (ERL1), and ERECTA-LIKE 2 (ERL2) controls epidermal patterning, inflorescence architecture, and stomata development and patterning. These proteins are reported to be plasma membrane associated. Here we show that the er/erl1/erl2 mutant exhibits impaired gibberellin (GA) biosynthesis and perception alongside broad transcriptional changes. The ERf kinase domains were found to localize to the nucleus where they interact with the SWI3B subunit of the SWI/SNF chromatin remodeling complex (CRCs). The er/erl1/erl2 mutant exhibits reduced SWI3B protein level and affected nucleosomal chromatin structure. Similar to swi3c and brm plants with inactivated subunits of SWI/SNF CRCs, it also does not accumulate DELLA RGA and GAI proteins. The ER kinase phosphorylates SWI3B in vitro, and the inactivation of all ERf proteins leads to the decreased phosphorylation of SWI3B protein in vivo. The identified correlation between DELLA overaccumulation and SWI3B proteasomal degradation, and the physical interaction of SWI3B with DELLA proteins indicate an important role of SWI3B-containing SWI/SNF CRCs in gibberellin signaling. Co-localization of ER and SWI3B on GID1 (GIBBERELLIN INSENSITIVE DWARF 1) DELLA target gene promoter regions and abolished SWI3B binding to GID1 promoters in er/erl1/erl2 plants supports the conclusion that ERf-SWI/SNF CRC interaction is important for transcriptional control of GA receptors. Thus, the involvement of ERf proteins in the transcriptional control of gene expression, and observed similar features for human HER2 (epidermal growth family receptor member), indicate an exciting target for further studies of evolutionarily conserved non-canonical functions of eukaryotic membrane receptors.

Common low complexity regions for SARS-CoV-2 and human proteomes as potential multidirectional risk factor in vaccine development.

BACKGROUND: The rapid spread of the COVID-19 demands immediate response from the scientific communities. Appropriate countermeasures mean thoughtful and educated choice of viral targets (epitopes). There are several articles that discuss such choices in the SARS-CoV-2 proteome, other focus on phylogenetic traits and history of the Coronaviridae genome/proteome. However none consider viral protein low complexity regions (LCRs). Recently we created the first methods that are able to compare such fragments. RESULTS: We show that five low complexity regions (LCRs) in three proteins (nsp3, S and N) encoded by the SARS-CoV-2 genome are highly similar to regions from human proteome. As many as 21 predicted T-cell epitopes and 27 predicted B-cell epitopes overlap with the five SARS-CoV-2 LCRs similar to human proteins. Interestingly, replication proteins encoded in the central part of viral RNA are devoid of LCRs. CONCLUSIONS: Similarity of SARS-CoV-2 LCRs to human proteins may have implications on the ability of the virus to counteract immune defenses. The vaccine targeted LCRs may potentially be ineffective or alternatively lead to autoimmune diseases development. These findings are crucial to the process of selection of new epitopes for drugs or vaccines which should omit such regions.

Succinate Dehydrogenase-Deficient Renal Cancer Featuring Fructose-1,6-Biphosphatase Loss, Pyruvate Kinase M2 Overexpression, and SWI/SNF Chromatin Remodeling Complex Aberrations: A Rare Case Report.

Succinate dehydrogenase (SDH)-deficient renal cancer is a rare renal cancer subtype recently accepted by the World Health Organization as a unique subtype of renal cell carcinoma (RCC). Here we report a case of 17-year-old man. The detailed evaluation indicated occurrence of the SDHB-deficient RCC. The genetic testing revealed no germline mutation in SDH genes. Immunohistochemistry showed SDHB deficiency, overexpression of pyruvate kinase M2 and dramatic downregulation of fructose-1,6-bisphosphatase metabolic enzymes, and unaltered levels of phosphorylated AMP-activated protein kinase and mammalian target of rapamycin. Strong upregulation of INI1 and BRG1 and overexpression of BAF180, subunits of SWI/SNF ATP-dependent chromatin remodeling complex, were also found. The identified tumor pathologically did not resemble clear cell renal cell carcinoma (ccRCC), but some metabolic alterations are common for both cancer types. Thus, we postulate that the phenotypical differences between ccRCC and SDHB-deficient RCC may be related to distinct molecular and metabolic alterations. IMPLICATIONS FOR PRACTICE: Succinate dehydrogenase (SDH)-deficient renal cell carcinoma (RCC) is a rare renal tumor occurring even in young patients. Until now, in all described and genetically tested cases, mutations and deletions in SDH genes have been found. This article describes SDHB-deficient RCC without any germline mutations in SDH genes. Therefore, genetic analysis for germline mutations in SDH genes in SDH-deficient RCC, especially in young individuals, should be strongly recommended, although as of now it is not obligatory. This knowledge will allow improvement of patient monitoring including both disease recurrence and new cancer appearance.

Retinoid X receptor modulates vitellogenin gene expression in black tiger shrimp, Penaeus monodon.

Effective inducing of ovarian maturation in female shrimp broodstock is important for successful breeding programs. Vitellogenesis is a biochemical process during which a yolk protein precursor vitellogenin (Vg) is synthesized and thus, can be used to indicate ovarian maturation stage. In this study, transcriptional regulation of Vg synthesis in the black tiger shrimp, Penaeus monodon was investigated. Genome walking on 5' upstream sequence of Vg gene revealed several putative binding sites of lipophilic retinoic acid response elements (RARE), and nuclear hormone responsive elements. Deletion of RARE significantly reduced the promoter activity to drive the expression of luciferase reporter gene in Sf-9 cells. To validate the trans-factor that potentially controls Vg expression through RARE, a cDNA encoding retinoid X receptor (PmRXR), one of the RARE-bound transcription factors was cloned from P. monodon's ovary. PmRXR expression was detected in various shrimp tissues, and was up-regulated during ovary development in a similar way to Vg expression. The DNA-binding domain of PmRXR protein showed specific binding to RARE-containing region on Vg 5' upstream sequence as determined by Electrophoretic Mobility Shift Assay (EMSA). Furthermore, dsRNA-mediated PmRXR silencing in previtellogenic and vitellogenic shrimp revealed that suppression of PmRXR could reduce Vg transcript in both stages. Taken together, the results presented in this study indicate that RXR is possibly an activator protein that modulates Vg expression in shrimp ovary through the binding to RARE.

Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes.

To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLIN-DEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18 -StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies.

Cardiotoxicity danger in immunotherapy.

Immunotherapy based on immune checkpoint inhibitors (ICIs) is currently broadly used in the treatment of different types of cancer. The treatment targeting programmed cell death protein 1/programmed death-ligand 1 axis is already approved by Food and Drug Administration for numerous cancers. These kinds of therapy brought spectacular results in the treatment of non-small cell lung cancer where systemic therapy was ineffective. However, a wide range of applied therapies based on ICIs in the clinic have led to unexpected side effects, such as severe cardiotoxicity. It needs to be underlined that the molecular mechanism of myocarditis in response to ICIs is still not fully understood. Lack of sufficient knowledge, especially concerning the kind of risk factors increasing probability of myocarditis, poses currently a large clinical problem. Continuous cardiac monitoring of patients who undergo ICI treatment presents another problem as it is cost-ineffective for the healthcare system. Herein, we highlight the risks of use of anticancer therapy based on ICIs. We also stress that detailed monitoring of any event of cardiotoxicity following ICIs treatment should be carefully investigated and registered to give a global overview of the frequency of myocarditis occurrence. Moreover, we propose that the extension of molecular and systemic knowledge of etiology of myocarditis as a side effect, including the role of protein kinases, will be highly beneficial for the medical field. Last but not least, better understanding of mechanisms of cardiotoxicity induction will improve the safety of cancer patients and will help clinicians in prediction of unexpected side effect occurrence.

Human and Arabidopsis alpha-ketoglutarate-dependent dioxygenase homolog proteins-New players in important regulatory processes.

The family of AlkB homolog (ALKBH) proteins, the homologs of Escherichia coli AlkB 2-oxoglutarate (2OG), and Fe(II)-dependent dioxygenase are involved in a number of important regulatory processes in eukaryotic cells including repair of alkylation lesions in DNA, RNA, and nucleoprotein complexes. There are nine human and thirteen Arabidopsis thaliana ALKBH proteins described, which exhibit diversified functions. Among them, human ALKBH5 and FaT mass and Obesity-associated (FTO) protein and Arabidopsis ALKBH9B and ALKBH10B have been recognized as N6 methyladenine (N6 meA) demethylases, the most abundant posttranscriptional modification in mRNA. The FTO protein is reported to be associated with obesity and type 2 diabetes, and involved in multiple other processes, while ALKBH5 is induced by hypoxia. Arabidopsis ALKBH9B is an N6 meA demethylase influencing plant susceptibility to viral infections via m6 A/A ratio control in viral RNA. ALKBH10B has been discovered to be a functional Arabidopsis homolog of FTO; thus, it is also an RNA N6 meA demethylase involved in plant flowering and several other regulatory processes including control of metabolism. High-throughput mass spectrometry showed multiple sites of human ALKBH phosphorylation. In the case of FTO, the type of modified residue decides about the further processing of the protein. This modification may result in subsequent protein ubiquitination and proteolysis, or in the blocking of these processes. However, the impact of phosphorylation on the other ALKBH function and their downstream pathways remains nearly unexplored in both human and Arabidopsis. Therefore, the investigation of evolutionarily conserved functions of ALKBH proteins and their regulatory impact on important cellular processes is clearly called for.

Various forms of HIF-1α protein characterize the clear cell renal cell carcinoma cell lines.

Renal cell carcinoma (RCC) represents around 2-3% of all malignancies diagnosed in adult patients. Most frequent (around 70-80% cases) and the most aggressive subtype is clear cell RCC (ccRCC). Mutations in VHL (von Hippel Lindau) gene, characteristic for this cancer type, lead to altered activity of the trimeric VBC (pVHL-elongin B-C) complex and consequently to HIF-1α stabilization. In this study, we present results of exhaustive investigation of HIF-1α alternative transcript variants abundance in A498, CAKI-1, and 786-O ccRCC cell lines. We proved the existence of truncated HIF-1α protein form (HIF1A∆-6) in A498 and HIF1A gene rearrangements in 786-O cell lines. Subsequently, we found that HIF1A∆2-6 was more stable than the full-length HIF-1α. Moreover, the shorter HIF-1α was insensitive for hypoxia and was overaccumulated after proteasome inhibitor treatment indicative of potential diversified roles of full-length and truncated HIF-1α forms in the cell. We also showed that A498, CAKI-1, and 786-O exhibit differential expression of various regulatory genes involved in the control of metabolic processes, that is, glucose and lipid metabolism, and encoding subunits of such machineries like SWI/SNF chromatin remodeling complex. Furthermore, these cell lines exhibited differential responses to axitinib, everolimus, and sunitinib-anticancer drugs-in normoxia and hypoxia as well as various alterations in metabolism-related regulatory processes. Finally, we have shown that overexpression of truncated HIF1A∆2-6 form may affect the protein level of endogenous full-length HIF-1α protein. Thus, our study proves an important role of HIF-1α in the ccRCC development.

SWI/SNF chromatin remodeling complex and glucose metabolism are deregulated in advanced bladder cancer.

Bladder cancer (BC) is a frequently diagnosed malignancy affecting predominantly adult and elderly populations. It is expected that due to the longer life time, BC will become even more frequent in the future; thus in consequence, it will represent serious health problem of older society part. The treatment of advanced BC is mostly ineffective due to its very aggressive behavior. So far, no effective targeted therapy is used for BC treatment. Here, we found that BC is characterized by lower protein levels of BRM, INI1, and BAF155 main subunits of SWI/SNF chromatin remodeling complex (CRC) which is involved in global control of gene expression and influences various important cellular processes like: cell cycle control, apoptosis, DNA repair, etc. Moreover, the expression of SMARCA2, a BRM encoding gene, strongly correlated with BC metastasis and expression of such metabolic genes as PKM2 and PRKAA1. Furthermore, the analysis of T24 and 5637 commonly used BC cell lines revealed different expression levels of metabolic genes including FBP1 gene encoding Frutose-1,6-Bisphosphatase, an enzyme controlling glycolysis flux and gluconeogenesis. The tested BC cell lines exhibited various molecular and metabolic alterations as well as differential glucose uptake, growth rate, and migration potential. We have shown that BRM subunit is involved in the transcriptional control of genes encoding metabolic enzymes. Moreover, we found that the FBP1 expression level and the SWI/SNF CRCs may serve as markers of molecular subtypes of BC. Collectively, this study may provide a new knowledge about the molecular and metabolic BC subtypes which likely will be of high importance for the clinic in the future.

The SWI/SNF ATP-Dependent Chromatin Remodeling Complex in Arabidopsis Responds to Environmental Changes in Temperature-Dependent Manner.

SWI/SNF ATP-dependent chromatin remodeling complexes (CRCs) play important roles in the regulation of transcription, cell cycle, DNA replication, repair, and hormone signaling in eukaryotes. The core of SWI/SNF CRCs composed of a SWI2/SNF2 type ATPase, a SNF5 and two of SWI3 subunits is sufficient for execution of nucleosome remodeling in vitro. The Arabidopsis genome encodes four SWI2/SNF2 ATPases, four SWI3, a single SNF5 and two SWP73 subunits. Genes of the core SWI/SNF components have critical but not fully overlapping roles during plant growth, embryogenesis, and sporophyte development. Here we show that the Arabidopsis swi3c mutant exhibits a phenotypic reversion when grown at lower temperature resulting in partial restoration of its embryo, root development and fertility defects. Our data indicates that the swi3c mutation alters the expression of several genes engaged in low temperature responses. The location of SWI3C-containing SWI/SNF CRCs on the ICE1, MYB15 and CBF1 target genes depends on the temperature conditions, and the swi3c mutation thus also influences the transcription of several cold-responsive (COR) genes. These findings, together with genetic analysis of swi3c/ice1 double mutant and enhanced freezing tolerance of swi3c plants illustrate that SWI/SNF CRCs contribute to fine-tuning of plant growth responses to different temperature regimes.

[Mechanisms controlling chromatin structure]. / Mechanizmy kontrolujace strukture chromatyny.

Genomic DNA is highly packaged in eukaryotic cells and occurs in the form of nucleoprotein complex called chromatin. Although high DNA compaction allows to store large amount of genomic information in the cell nuclei, it also restricts the access to DNA regulatory sequences. Therefore, to overcome this issue, chromatin must be subjected to various alterations which are dependent on few interrelated factors: DNA modification, histones variants and modifications, ncRNA, chromatin remodeling complexes and chromatin architecture in nuclei. They allow to multilayer regulation of fragile balance between transcriptionally active euchromatin and inactive heterochromatin. The newest research describe new chromatin elements, e.g. half nucleosomes, bivalent chromatin marker and pointed to few intermediate states between euchromatin and heterochromatin. Variety and remarkable amount of chromatin modifications require existence of multiprotein complexes reading, editing and integrating genomic information. Some of them are able to remodel nucleosomes in order to control access to particular DNA sequence. Due to the complexity of chromatin structure regulation studies describing these mechanisms are fundamental to understanding the eukaryotes life.

SWP73 Subunits of Arabidopsis SWI/SNF Chromatin Remodeling Complexes Play Distinct Roles in Leaf and Flower Development.

Arabidopsis thaliana SWP73A and SWP73B are homologs of mammalian BRAHMA-associated factors (BAF60s) that tether SWITCH/SUCROSE NONFERMENTING chromatin remodeling complexes to transcription factors of genes regulating various cell differentiation pathways. Here, we show that Arabidopsis thaliana SWP73s modulate several important developmental pathways. While undergoing normal vegetative development, swp73a mutants display reduced expression of FLOWERING LOCUS C and early flowering in short days. By contrast, swp73b mutants are characterized by retarded growth, severe defects in leaf and flower development, delayed flowering, and male sterility. MNase-Seq, transcript profiling, and ChIP-Seq studies demonstrate that SWP73B binds the promoters of ASYMMETRIC LEAVES1 and 2, KANADI1 and 3, and YABBY2, 3, and 5 genes, which regulate leaf development and show coordinately altered transcription in swp73b plants. Lack of SWP73B alters the expression patterns of APETALA1, APETALA3, and the MADS box gene AGL24, whereas other floral organ identity genes show reduced expression correlating with defects in flower development. Consistently, SWP73B binds to the promoter regions of APETALA1 and 3, SEPALLATA3, LEAFY, UNUSUAL FLORAL ORGANS, TERMINAL FLOWER1, AGAMOUS-LIKE24, and SUPPRESSOR OF CONSTANS OVEREXPRESSION1 genes, and the swp73b mutation alters nucleosome occupancy on most of these loci. In conclusion, SWP73B acts as important modulator of major developmental pathways, while SWP73A functions in flowering time control.

DELLA-interacting SWI3C core subunit of switch/sucrose nonfermenting chromatin remodeling complex modulates gibberellin responses and hormonal cross talk in Arabidopsis.

Switch (SWI)/Sucrose Nonfermenting (SNF)-type chromatin-remodeling complexes (CRCs) are involved in regulation of transcription, DNA replication and repair, and cell cycle. Mutations of conserved subunits of plant CRCs severely impair growth and development; however, the underlying causes of these phenotypes are largely unknown. Here, we show that inactivation of SWI3C, the core component of Arabidopsis (Arabidopsis thaliana) SWI/SNF CRCs, interferes with normal functioning of several plant hormone pathways and alters transcriptional regulation of key genes of gibberellin (GA) biosynthesis. The resulting reduction of GA4 causes severe inhibition of hypocotyl and root elongation, which can be rescued by exogenous GA treatment. In addition, the swi3c mutation inhibits DELLA-dependent transcriptional activation of GIBBERELLIN-INSENSITIVE DWARF1 (GID1) GA receptor genes. Down-regulation of GID1a in parallel with the DELLA repressor gene REPRESSOR OF GA1-3 1 in swi3c indicates that lack of SWI3C also leads to defects in GA signaling. Together with the recent demonstration of function of SWI/SNF ATPase BRAHMA in the GA pathway, these results reveal a critical role of SWI/SNF CRC in the regulation of GA biosynthesis and signaling. Moreover, we demonstrate that SWI3C is capable of in vitro binding to, and shows in vivo bimolecular fluorescence complementation interaction in cell nuclei with, the DELLA proteins RGA-LIKE2 and RGA-LIKE3, which affect transcriptional activation of GID1 and GA3ox (GIBBERELLIN 3-OXIDASE) genes controlling GA perception and biosynthesis, respectively. Furthermore, we show that SWI3C also interacts with the O-GlcNAc (O-linked N-acetylglucosamine) transferase SPINDLY required for proper functioning of DELLAs and acts hypostatically to (SPINDLY) in the GA response pathway. These findings suggest that DELLA-mediated effects in GA signaling as well as their role as a hub in hormonal cross talk may be, at least in part, dependent on their direct physical interaction with complexes responsible for modulation of chromatin structure.

[AMP-activated protein kinase (AMPK) as therapeutic target]. / Kinaza bialkowa aktywowana przez AMP (AMPK) jako cel terapeutyczny.

AMP-activated protein kinase (AMPK) is one of the major energy sensor at both: cellular and whole body level. It exists as heterotrimer containing three subunits: the catalytic α subunit, ß and regulatory γ. AMPK is localized both in the cytoplasm and in the nucleus. It is activated by increasing concentrations of AMP during the energy shortage, causing activation of catabolic pathways and inhibition of energy consuming processes. AMPK activity can be regulated allosterically: by binding AMP to a regulatory γ subunit, as well as by phosphorylation on Thr172 of the catalytic α subunit by other kinases. Activated AMPK can effectively inhibit the mTOR pathway which is hyperactive in many types of cancer. On the other hand AMPK inactivation associates with the type II diabetes, diet-induced obesity, insulin resistance and the development of other metabolic disorders. The AMPK dysfunction is also observed in inflammation. It was discovered during last years that abnormalities in the AMPK function can induce the metabolic reprogramming in cancer cells known as the Warburg effect. Additionally, AMPK is activated during irradiation. Its activation leads to inhibition of growth. On the other hand, active AMPK enables cells to survive in difficult conditions such as hypoxia, or glucose deprivation. Because of its crucial role in maintaining of the energy homeostasis AMPK is an excellent therapeutic target. However, it still remains unknown what is better: to activate or inhibit the AMPK function.

RRM2 gene expression depends on BAF180 subunit of SWISNF chromatin remodeling complex and correlates with abundance of tumor infiltrating lymphocytes in ccRCC.

About 40% of clear cell renal cell carcinoma (ccRCC) cases carry the pbrm1 mutation inactivating BAF180 subunit of the SWI/SNF chromatin remodeling complex (CRC). Here we show that the majority of transcriptomic changes appear at the stage I of ccRCC development. By contrast, the stage II ccRCC exhibits hyperactivation of DNA replication demonstrated by the overexpression of several genes, e.g., RRM1 and RRM2 genes encoding subunits of ribonucleotide reductase (RNR) complex. We found that the degree of RRM1 and RRM2 upregulation in ccRCC patients depends on pbrm1 mutation. We show that the BAF180 protein product of the PBRM1 gene directly binds to RRM1 and RRM2 loci. The BAF180 binding regions are targeted by regulatory proteins previously reported as SWI/SNF CRC interacting partners. BAF180 binding to RRMs loci correlates with enrichment of H3K27me3 in case of RRM1 and H3K14Ac on RRM2, indicating the existence of differential regulatory mechanism controlling expression of these genes. We found that the strong overexpression of RRM2 in ccRCC patient samples correlates with T cell infiltration. Surprisingly, the majority of tumor infiltrating lymphocytes (TILs) consisted of CD4+ T cells. Furthermore, we show that exhausted CD4+ T cells induced the expression of the RRM2 gene in the primary ccRCC cell line. Collectively, our results provide the link between PBRM1 loss, RRM2 expression and T cell infiltration, which may lead to the establishment of new treatment of this disease.

PD-L1 Overexpression, SWI/SNF Complex Deregulation, and Profound Transcriptomic Changes Characterize Cancer-Dependent Exhaustion of Persistently Activated CD4+ T Cells.

Growing tumors avoid recognition and destruction by the immune system. During continuous stimulation of tumor-infiltrating lymphocytes (TILs) by tumors, TILs become functionally exhausted; thus, they become unable to kill tumor cells and to produce certain cytokines and lose their ability to proliferate. This collectively results in the immune escape of cancer cells. Here, we show that breast cancer cells expressing PD-L1 can accelerate exhaustion of persistently activated human effector CD4+ T cells, manifesting in high PD-1 and PD-L1 expression level son T cell surfaces, decreased glucose metabolism genes, strong downregulation of SWI/SNF chromatin remodeling complex subunits, and p21 cell cycle inhibitor upregulation. This results in inhibition of T cell proliferation and reduction of T cell numbers. The RNAseq analysis on exhausted CD4+ T cells indicated strong overexpression of IDO1 and genes encoding pro-inflammatory cytokines and chemokines. Some interleukins were also detected in media from CD4+ T cells co-cultured with cancer cells. The PD-L1 overexpression was also observed in CD4+ T cells after co-cultivation with other cell lines overexpressing PD-L1, which suggested the existence of a general mechanism of CD4+ T cell exhaustion induced by cancer cells. The ChIP analysis on the PD-L1 promoter region indicated that the BRM recruitment in control CD4+ T cells was replaced by BRG1 and EZH2 in CD4+ T cells strongly exhausted by cancer cells. These findings suggest that epi-drugs such as EZH2 inhibitors may be used as immunomodulators in cancer treatment.

Genetic analysis of functional redundancy of BRM ATPase and ATSWI3C subunits of Arabidopsis SWI/SNF chromatin remodelling complexes.

In yeast and mammals, ATP-dependent chromatin remodelling complexes of the SWI/SNF family play critical roles in the regulation of transcription, cell proliferation, differentiation and development. Homologues of conserved subunits of SWI/SNF-type complexes, including Snf2-type ATPases and SWI3-type proteins, participate in analogous processes in Arabidopsis. Recent studies indicate a remarkable similarity between phenotypic effects of mutations in the SWI3 homologue ATSWI3C and bromodomain-ATPase BRM genes. To verify the extent of functional similarity between BRM and ATSWI3C, we have constructed atswi3c brm double mutants and compared their phenotypic traits to those of simultaneously grown single atswi3c and brm mutants. In addition to inheritance of characteristic developmental abnormalities shared by atswi3c and brm mutants, some additive brm-specific traits were also observed in the atswi3c brm double mutants. Unlike atswi3c, the brm mutation results in the enhancement of abnormal carpel development and pollen abortion leading to complete male sterility. Despite the overall similarity of brm and atswi3c phenotypes, a critical requirement for BRM in the differentiation of reproductive organs suggests that its regulatory functions do not entirely overlap those of ATSWI3C. The detection of two different transcript isoforms indicates that BRM is regulated by alternative splicing that creates an in-frame premature translation stop codon in its SNF2-like ATPase coding domain. The analysis of Arabidopsis mutants in nonsense-mediated decay suggests an involvement of this pathway in the control of alternative BRM transcript level.

Hairpin structure within the 3'UTR of DNA polymerase beta mRNA acts as a post-transcriptional regulatory element and interacts with Hax-1.

Aberrant expression of DNA polymerase beta, a key enzyme involved in base excision repair, leads to genetic instability and carcinogenesis. Pol beta expression has been previously shown to be regulated at the level of transcription, but there is also evidence of post-transcriptional regulation, since rat transcripts undergo alternative polyadenylation, and the resulting 3'UTR contain at least one regulatory element. Data presented here indicate that RNA of the short 3'UTR folds to form a strong secondary structure (hairpin). Its regulatory role was established utilizing a luciferase-based reporter system. Further studies led to the identification of a protein factor, which binds to this element-the anti-apoptotic, cytoskeleton-related protein Hax-1. The results of in vitro binding analysis indicate that the formation of the RNA-protein complex is significantly impaired by disruption of the hairpin motif. We demonstrate that Hax-1 binds to Pol beta mRNA exclusively in the form of a dimer. Biochemical analysis revealed the presence of Hax-1 in mitochondria, but also in the nuclear matrix, which, along with its transcript-binding properties, suggests that Hax-1 plays a role in post-transcriptional regulation of expression of Pol beta.

BRM: the core ATPase subunit of SWI/SNF chromatin-remodelling complex-a tumour suppressor or tumour-promoting factor?

BRM (BRAHMA) is a core, SWI2/SNF2-type ATPase subunit of SWI/SNF chromatin-remodelling complex (CRC) involved in various important regulatory processes including development. Mutations in SMARCA2, a BRM-encoding gene as well as overexpression or epigenetic silencing were found in various human diseases including cancer. Missense mutations in SMARCA2 gene were recently connected with occurrence of Nicolaides-Baraitser genetics syndrome. By contrast, SMARCA2 duplication rather than mutations is characteristic for Coffin-Siris syndrome. It is believed that BRM usually acts as a tumour suppressor or a tumour susceptibility gene. However, other studies provided evidence that BRM function may differ depending on the cancer type and the disease stage, where BRM may play a role in the disease progression. The existence of alternative splicing forms of SMARCA2 gene, leading to appearance of truncated functional, loss of function or gain-of-function forms of BRM protein suggest a far more complicated mode of BRM-containing SWI/SNF CRCs actions. Therefore, the summary of recent knowledge regarding BRM alteration in various types of cancer and highlighting of differences and commonalities between BRM and BRG1, another SWI2/SNF2 type ATPase, will lead to better understanding of SWI/SNF CRCs function in cancer development/progression. BRM has been recently proposed as an attractive target for various anticancer therapies including the use of small molecule inhibitors, synthetic lethality induction or proteolysis-targeting chimera (PROTAC). However, such attempts have some limitations and may lead to severe side effects given the homology of BRM ATPase domain to other ATPases, as well as due to the tissue-specific appearance of BRM- and BRG1-containing SWI/SNF CRC classes. Thus, a better insight into BRM-containing SWI/SNF CRCs function in human tissues and cancers is clearly required to provide a solid basis for establishment of new safe anticancer therapies.

Advanced adenoid cystic carcinoma (ACC) is featured by SWI/SNF chromatin remodeling complex aberrations.

PURPOSE: Adenoid cystic carcinoma (ACC) is a rare neurotropic cancer with slow progression occurring in salivary glands and less frequently in other body parts. ACC is featured by hyperchromatic nuclei and various mutations in genes encoding chromatin-related machineries. The ACC treatment is mainly limited to the radical surgery and radiotherapy while the chemotherapy remains ineffective. As the knowledge about molecular basis of ACC development is limited, we investigated here the molecular features of this disease. PATIENTS AND METHODS: This study included 50 patients with ACC. Transcript profiling of available ACC samples vs normal salivary gland tissue, quantitative real-time PCR (qRT-PCR) transcript level measurements and the immunohistochemistry (IHC) for SWI/SNF chromatin remodeling complex (CRC) subunits and androgen receptor on surgery-derived paraffin-embedded samples were performed. RESULTS: Transcriptomic study followed by Gene Ontology classification indicated alteration of chromatin-related processes, including downregulated transcript levels of main SWI/SNF CRC subunits and elevated expression of BRM ATPase-coding SMARCA2 gene in ACC. Subsequent IHC indicated broad accumulation of BRM ATPase and several SWI/SNF subunits, suggesting affected control of their protein level in ACC. The IHC revealed ectopic, heterogeneous expression of androgen receptor (AR) in some ACC cells. CONCLUSIONS: Our study indicated that ACC features aberrant expression of genes controlling chromatin status and structure. We found that the balance between SWI/SNF classes is moved towards the BRM ATPase-containing complex in ACC. As BRM is known to be involved in chemoresistance in cancer cells, this observation may be the likely explanation for ACC chemoresistance.