Transcriptional Regulatory Function Research Articles

Structural and functional characterization of the transcriptional regulator Rv3488 of Mycobacterium tuberculosis H37Rv.

Rv3488 of Mycobacterium tuberculosis H37Rv has been assigned to the phenolic acid decarboxylase repressor (PadR) family of transcriptional regulators that play key roles in multidrug resistance and virulence of prokaryotes. The binding of cadmium, zinc, and several other metals to Rv3488 was discovered and characterized by isothermal titration calorimetery to be an exothermic process. Crystal structures of apo-Rv3488 and Rv3488 in complex with cadmium or zinc ions were determined by X-ray crystallography. The structure of Rv3488 revealed a dimeric protein with N-terminal winged-helix-turn-helix DNA-binding domains composed of helices α1, α2, α3, and strands β1 and β2, with the dimerization interface being formed of helices α4 and α1. The overall fold of Rv3488 was similar to PadR-s2 and metal sensor transcriptional regulators. In the crystal structure of Rv3488-Cd complex, two octahedrally coordinated Cd2+ ions were present, one for each subunit. The same sites were occupied by zinc ions in the structure of Rv3488-Zn, with two additional zinc ions complexed in one monomer. EMSA studies showed specific binding of Rv3488 with its own 30-bp promoter DNA. The functional role of Rv3488 was characterized by expressing the rv3488 gene under the control of hsp60 promoter in Mycobacterium smegmatis Expression of Rv3488 increased the intracellular survival of recombinant M. smegmatis in murine macrophage cell line J774A.1 and also augmented its tolerance to Cd2+ ions. Overall, the studies show that Rv3488 may have transcription regulation and metal-detoxifying functions and its expression in M. smegmatis increases intracellular survival, perhaps by counteracting toxic metal stress.

PARP-1 inhibition protects the diabetic heart through activation of SIRT1-PGC-1α axis

Type 2 diabetes mellitus (DM2) follows impaired glucose tolerance in obesity and is frequently associated with hypertension, causing adverse myocardial remodelling and leading to heart failure. The DNA bound protein PARP (poly ADP ribose) polymerase catalyses a post translational modification (polymerization of negatively charged ADP-ribose chains) of nuclear proteins. PARP-1 activation is NAD+ dependent and takes part in DNA repair and in chromatin remodelling and has a function in transcriptional regulation, intracellular trafficking and energy metabolism. PARP-1 is activated in diabetic cardiomyopathy. We hypothesized that PARP-1 inhibition in diabetic mice may protect cardiomyocytes from inflammation and ROS production. MethodsObese Leptin resistant (db/db) mice suffering from DM2, were treated with angiotensin II (AT) for 4 weeks to enhance the development of cardiomyopathy. Mice were concomitantly treated with the PARP-1 inhibitor INO1001. Neonatal cardiomyocytes exposed to high levels of glucose (33 mM) with or without AT were treated with INO1001. or with SIRT inhibitor (EX-527) in the presence of INO1001 were tested in-vitro. ResultsThe in-vivo tests show that hearts from AT treated DM2 mice exhibited cardiac hypertrophy, fibrosis and an increase in the inflammatory marker TNFα. DM2 mice had an increased oxidative stress, concomitant with elevated PARP-1 activity and reduced Sirtuin-1 (SIRT1) expression. PARP-1 inhibition led to increased SIRT1 and Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) levels, attenuating oxidative stress, inflammation and fibrosis. In-vitro experiments demonstrated that inhibition of PARP-1 in cardiomyocytes exposed to high levels of glucose and AT led to a significant reduction in ROS (P < 0.01), which was abolished in the presence of the SIRT1 inhibitor together with increased protein expression of SIRT1 and PGC-1α. ConclusionPARP1 inhibitor INO1001 attenuated cardiomyopathic features in diabetic mice through the activation of SIRT1 and its downstream antioxidant defence mechanisms. The results of this study suggest a pivotal role of PARP-1 inhibition in treating diabetic and AT-induced cardiomyopathy.

Examining the contribution of smoking and HPV towards the etiology of oral cavity squamous cell carcinoma using high-throughput sequencing: A prospective observational study

Oral cavity Squamous Cell Carcinoma (OCSCC) is a common form of head and neck cancer throughout the developed and developing world. However, the etiology of OCSCC is still unclear.Here, we explored the extent to which tobacco use, Human Papillomavirus (HPV) infection and genetic and transcriptomic changes contributed to the oncogenesis of OCSCC. In a prospective observational study, we analysed fresh tissue biopsies from 45 OCSCC collected from 51 subjects presenting with OCSCC to the Brisbane Head and Neck Clinics between 2013 and 2015. Exploration of the genetic and transcriptomic landscape of the biopsies were performed using RNA sequencing (RNA-seq) and whole exome sequencing. HPV associated tumours were determined using p16 staining of histological sections and RNA sequencing. Patient demographics including tumor location within the oral cavity, and history of tobacco and alcohol use were correlated with genomic and transcriptomics analyses. About 4.5% of OCSCC were HPV associated. The most frequent mutations in the OCSCC samples were in the TP53 and CDKN2A genes, but no association of specific mutations with HPV or tobacco use was observed. Using weighted gene co-expression network analysis to explore the RNA-seq data, tumors from participants with a history of tobacco use showed a significant trend towards increased mammalian target of Rapamycin (mTOR) signaling and decreased mitochondrial respiration. In conclusion, HPV was shown to be an uncommon association with OCSCC and changes in TP53 transcriptional regulation, mTOR signaling and mitochondrial function were associated with a history of tobacco use. Larger data sets will be required to enable detection of differences which may help with development of personalized therapeutics in the future.

Ceramide Synthase 6: Comparative Analysis, Phylogeny and Evolution.

Ceramide synthase 6 (CerS6, also known as LASS6) is one of the six members of ceramide synthase gene family in humans. Comparisons of CerS6 amino acid sequences and structures as well as of CerS6 gene structures/locations were conducted using data from several vertebrate genome projects. A specific role for the CerS6 gene and protein has been identified as the endoplasmic reticulum C14- and C16-ceramide synthase. Mammalian CerS6 proteins share 90–100% similarity among different species, but are only 22–63% similar to other CerS family members, suggesting that CerS6 is a distinct gene family. Sequence alignments, predicted transmembrane, lumenal and cytoplasmic segments and N-glycosylation sites were also investigated, resulting in identification of the key conserved residues, including the active site as well as C-terminus acidic and serine residues. Mammalian CerS6 genes contain ten exons, are primarily located on the positive strands and transcribed as two major isoforms. The human CERS6 gene promoter harbors a large CpG island (94 CpGs) and multiple transcription factor binding sites (TFBS), which support precise transcriptional regulation and signaling functions. Additional regulation is conferred by 15 microRNA (miRNA) target sites identified in the CERS6 3′-UTR region. Phylogenetic analysis of the vertebrate CerS1–6 gene families relationships supports a major role for the CerS6 enzyme that is strongly conserved throughout vertebrate evolution.

Genomic characterization of Algerian Guelmoise cattle and their genetic relationship with other North African populations inferred from SNP genotyping arrays

Understanding between and within-breeds genetic variability is essential in the choice of conservation management decisions for threatened populations. In this study we assessed the genetic diversity of the Algerian Guelmoise cattle (GUE) by analyzing data on 24 GUE individuals genotyped for the Illumina BovineSNP50 BeadChipv2. We also provided a detailed description of the population structure of GUE using comparisons with 23 worldwide cattle populations, selected as being representative of African, South European and indicine populations, in addition to four North African populations. We show that GUE is an admixed population which has strong genetic similarity to Tunisian cattle owing both to a common ancestral origin and a continuous gene flow. GUE was found to be less subject to foreign breed introgression than native Tunisian cattle. We also found support for a past admixture between GUE and the Moroccan Oulmès Zaer breed. The observed heterozygosity of GUE population was similar to that observed for the South European breeds and the other Maghreb populations (Ho∼0.30). By investigating the occurrence of Runs of Homozysosity (ROHs) in a subset of nine populations, we found that GUE had the lowest cumulative ROH length per animal with the largest proportion of their genome covered with medium and long (> 5 Mb) ROHs. This likely indicates that GUE is derived from a large ancestral population which has subsequently undergone a recent increasing levels of inbreeding. ROH islands within GUE harbored genes enriched in immune defense and transcriptional regulation functions in addition to genes related to the Pregnancy-associated proteins family. Our study is the first of its kind providing insights into the genomic structure and the phylogenetic relationships of Guelmoise cattle population. It points out the possibility to establish a common genetic management program between Algeria and Tunisia aimed at safeguarding the endangered indigenous North African cattle.

Cartilaginous Fishes Provide Insights into the Origin, Diversification, and Sexually Dimorphic Expression of Vertebrate Estrogen Receptor Genes.

Vertebrate estrogen receptors (ERs) perform numerous cell signaling and transcriptional regulatory functions. ERɑ (Esr1) and ERβ (Esr2) likely evolved from an ancestral receptor that duplicated and diverged at the protein and cis-regulatory levels, but the evolutionary history of ERs, including the timing of proposed duplications, remains unresolved. Here we report on identification of two distinct ERs in cartilaginous fishes and demonstrate their orthology to ERα and ERβ. Phylogenetic analyses place the ERα/ERβ duplication near the base of crown gnathostomes (jawed vertebrates). We find that ERα and ERβ from little skate (Leucoraja erinacea) and mammals share key subtype-specific residues, indicating conserved protein evolution. In contrast, jawless fishes have multiple non-orthologous Esr genes that arose by parallel duplications. Esr1 and Esr2 are expressed in subtype-specific and sexually dimorphic patterns in skate embryos, suggesting that ERs might have functioned in sexually dimorphic development before the divergence of cartilaginous and bony fishes.

Imbalance of the reciprocally inhibitory loop between the ubiquitin-specific protease USP43 and EGFR/PI3K/AKT drives breast carcinogenesis.

Hyperactivation of EGFR/PI3K/AKT is a prominent feature of various human cancers. Thus, understanding how this molecular cascade is balanced is of great importance. We report here that the ubiquitin-specific protease USP43 is physically associated with the chromatin remodeling NuRD complex and catalyzes H2BK120 deubiquitination. Functionally this coordinates the NuRD complex to repress a cohort of genes, including EGFR, which are critically involved in cell proliferation and carcinogenesis. We show that USP43 strongly suppresses the growth and metastasis of breast cancer in vivo. Interestingly, USP43 also exists in the cytoplasm, where it is phosphorylated by AKT, enabling its binding to the 14-3-3β/ε heterodimer and sequestration in the cytoplasm. Significantly, hyperactivation of EGFR/PI3K/AKT in breast cancer is associated with the cytoplasmic retention of USP43 and thus, the inhibition of its transcriptional regulatory function. Moreover, cancer-associated mutations of USP43 affect its subcellular localization and/or epigenetic regulatory functions. Nuclear USP43 is significantly reduced in breast carcinomas and is associated with EGFR accumulation and AKT hyperactivation. A low level of nuclear USP43 correlates with higher histologic grades and poor prognosis. Our study identifies USP43 to be an H2BK120 deubiquitinase and a potential tumor suppressor and reveals a reciprocally inhibitory loop between USP43 and EGFR/PI3K/AKT, whose imbalance drives breast carcinogenesis.

Prediction of S-sulfenylation sites using mRMR feature selection and fuzzy support vector machine algorithm

Cysteine S-sulfenylation is an important protein post-translational modification, which plays a crucial role in transcriptional regulation, cell signaling, and protein functions. To better elucidate the molecular mechanism of S-sulfenylation, it is important to identify S-sulfenylated substrates and their corresponding S-sulfenylation sites accurately. In this study, a novel bioinformatics tool named Sulf_FSVM is proposed to predict S-sulfenylation sites by using multiple feature extraction and fuzzy support vector machine algorithm. On the one hand, amino acid factors, binary encoding, and the composition of k-spaced amino acid pairs features are incorporated to encode S-sulfenylation sites. And the maximum relevance minimum redundancy method are adopted to remove the redundant features. On the other hand, a fuzzy support vector machine algorithm is used to handle the class imbalance and noise problem in S-sulfenylation sites training dataset. As illustrated by 10-fold cross-validation, the performance of Sulf_FSVM achieves a satisfactory performance with a Sensitivity of 73.26%, a Specificity of 70.78%, an Accuracy of 71.07% and a Matthew's correlation coefficient of 0.2971. Independent tests also show that Sulf_FSVM significantly outperforms existing S-sulfenylation sites predictors. Therefore, Sulf_FSVM can be a useful tool for accurate prediction of protein S-sulfenylation sites.

Phase separation and gene control

Molecular Biology Many components of eukaryotic transcription machinery—such as transcription factors and cofactors including BRD4, subunits of the Mediator complex, and RNA polymerase II—contain intrinsically disordered low-complexity domains. Now a conceptual framework connecting the nature and behavior of their interactions to their functions in transcription regulation is emerging (see the Perspective by Plys and Kingston). Chong et al. found that low-complexity domains of transcription factors form concentrated hubs via functionally relevant dynamic, multivalent, and sequence-specific protein-protein interaction. These hubs have the potential to phase-separate at higher concentrations. Indeed, Sabari et al. showed that at super-enhancers, BRD4 and Mediator form liquid-like condensates that compartmentalize and concentrate the transcription apparatus to maintain expression of key cell-identity genes. Cho et al. further revealed the differential sensitivity of Mediator and RNA polymerase II condensates to selective transcription inhibitors and how their dynamic interactions might initiate transcription elongation. Science , this issue p. [eaar2555][1], p. [eaar3958][2], p. [412][3]; see also p. [329][4] [1]: /lookup/doi/10.1126/science.aar2555 [2]: /lookup/doi/10.1126/science.aar3958 [3]: /lookup/doi/10.1126/science.aar4199 [4]: /lookup/doi/10.1126/science.aau4795

Variants in genes encoding small GTPases and association with epithelial ovarian cancer susceptibility.

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer mortality in American women. Normal ovarian physiology is intricately connected to small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran) which govern processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. We hypothesized that common germline variation in genes encoding small GTPases is associated with EOC risk. We investigated 322 variants in 88 small GTPase genes in germline DNA of 18,736 EOC patients and 26,138 controls of European ancestry using a custom genotype array and logistic regression fitting log-additive models. Functional annotation was used to identify biofeatures and expression quantitative trait loci that intersect with risk variants. One variant, ARHGEF10L (Rho guanine nucleotide exchange factor 10 like) rs2256787, was associated with increased endometrioid EOC risk (OR = 1.33, p = 4.46 x 10−6). Other variants of interest included another in ARHGEF10L, rs10788679, which was associated with invasive serous EOC risk (OR = 1.07, p = 0.00026) and two variants in AKAP6 (A-kinase anchoring protein 6) which were associated with risk of invasive EOC (rs1955513, OR = 0.90, p = 0.00033; rs927062, OR = 0.94, p = 0.00059). Functional annotation revealed that the two ARHGEF10L variants were located in super-enhancer regions and that AKAP6 rs927062 was associated with expression of GTPase gene ARHGAP5 (Rho GTPase activating protein 5). Inherited variants in ARHGEF10L and AKAP6, with potential transcriptional regulatory function and association with EOC risk, warrant investigation in independent EOC study populations.

Abstract C47: Obesity enhances tumor colonization and promotes expression of the spliced variant MBD2_v2 and splicing factor SRSF2 in triple-negative breast cancer

Abstract Introduction: Triple-negative breast cancer (TNBC) is a molecular subtype that accounts for approximately 15-20% of invasive breast cancer diagnoses in the United States. TNBC occurs more prevalently in premenopausal African American women (AAW), and obesity coupled with chronic inflammation is a risk factor for both incidence and recurrence. TNBC is lacking therapeutic options and persons diagnosed with TNBC have the lowest 5-year survival rates among all breast cancer patients, with AAW disproportionally bearing this burden. In order to improve TNBC patient outcomes, etiologic factors driving disparities in disease progression need to be better understood. We previously showed in TNBC cell cultures that intrinsic oncogenic redox signaling promotes self-renewal capacity of cancer stem-like cells (CSC) by maintaining expression of an epigenetic reader protein and mRNA spliced variant, methyl binding domain protein 2 variant 2 (MBD2_v2). We hypothesized that obesity-induced chronic inflammation in vivo could promote a similar cancer phenotype. Methods: To study this potential phenomenon, we developed and characterized a diet-induced obesity mouse tumor xenograft (MTX) model of TNBC in C57BL/6J Rag1 (-/-) mice, a strain that maintains a functional innate immune system, to recapitulate the proinflammatory environment induced by increased adiposity. TNBC cell lines MDA-MB-468 and MDA-MB-231 were used to generate ectopic tumor xenografts in this model. Tumor colonization and growth were measured over 150 days. RT-PCR and microarray based assays were used to detect transcriptional changes in TNBC MTXs. Results: At 5 weeks post-diet consumption, mice fed a high-fat diet displayed a 5-10% increase in body weight and increased blood glucose concentration. By the end of the study high-fat fed mice displayed a 33-43% increase in body weight and appeared to have greater central adiposity post-mortem. In overweight mice relative to healthy weight controls, TNBC cell line tumor colonization rate and MBD2_v2 expression were significantly higher. Furthermore, staining for CSC marker EpCam was greater in overweight versus control MTXs, indicating that CSC self-renewal capacity could have been enhanced. Upon global gene expression analysis of overweight MTXs, we observed a significant upregulation of stem-related factors based on fold-change expression, as well as functional enrichment for RNA splicing, including SRSF2, a splicing factor that has been shown to drive MBD2_v2 expression in pluripotent stem cells. Knockdown of SRSF2 led to decreased expression of MBD2_v2 in MDA-MB-468 cells, and decreased CSC self-renewal capacity in vitro. Conclusions: Altogether these data support MBD2_v2 regulation by redox signaling, which may be driven by a proinflammatory environment in the presence of increased adiposity. Future studies will be conducted to confirm MBD2_v2's importance in obesity-related tumor colonization in in vivo overexpression and knockdown studies, and to determine its transcriptional regulatory functions in TNBC. Citation Format: Emily A. Girsch, Gregory Dyson, Cristina Mitrea, Bin Bao, Lisa Polin, Aliccia Bollig-Fischer. Obesity enhances tumor colonization and promotes expression of the spliced variant MBD2_v2 and splicing factor SRSF2 in triple-negative breast cancer [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr C47.

Abstract 2922: The utility of BET inhibition in the sensitization and re-sensitization of pancreatic cancer cells to paclitaxel

Abstract While the mortality rates of cancer are generally declining, pancreatic cancer persists to be an exception with a 5-year-survival rate of less than 7%. As late diagnosis and resistance to conventional therapies are major contributors to high mortality rates, novel treatment options are needed to improve the prognosis of pancreatic cancer patients. Recent findings showed that inhibition of the Bromodomain and Extraterminal Domain (BET) family of epigenetic reader proteins is effective, both alone and in combination with conventional chemotherapy, in decreasing pancreatic tumor growth in patient-derived xenografts. Thus, we aim to evaluate the potential role of and mechanisms of action of BET inhibitors (BETi) as an adjuvant therapy option in pancreatic ductal adenocarcinoma. We established L3.6 pancreatic cells that are resistant to paclitaxel by maintaining them in incrementally higher concentrations for 3 months. Paclitaxel-resistant cells showed a half maximal inhibitory concentration (IC50) 100-fold higher than that of parental cells. Intriguingly, we report that low, non-cytostatic concentrations of the BETi, JQ1, not only sensitized cells to paclitaxel, but also induced significant re-sensitization of chemoresistant cells. In order to elucidate the mechanism by which BETi induces chemo-sensitization, we investigated the differential gene expression profiles of resistant and sensitive cells. Thereby, we uncovered that BETi reverses the regulation of transcriptionally-activated genes in resistant cells. Interestingly, these genes showed a major tendency to gain BRD4 at putative enhancer regions. We anticipate that enhancer RNAs (eRNAs) transcribed at these particular enhancers may provide us with novel biomarkers which can be used to predict chemotherapeutic resistance and the possibility of re-sensitization by BETi. In conclusion, we provide evidence that BETi can potentially be used as adjuvant agents in pancreatic cancer. However, approaches may likely be largely independent of their anti-proliferative effects that require higher concentrations and possibly lead to intolerable adverse effects, but rather to their transcriptional regulatory functions that attenuate the activated programs in chemoresistant cancers. Citation Format: Feda H. Hamdan, Ana P. Kutschat, Madhobi Sen, Xin Wang, Steven A. Johnsen. The utility of BET inhibition in the sensitization and re-sensitization of pancreatic cancer cells to paclitaxel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2922.

Imaging dynamic and selective low-complexity domain interactions that control gene transcription.

Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity sequence domains (LCDs), but how these LCDs drive transactivation remains unclear. We used live-cell single-molecule imaging to reveal that TF LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the RNA Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for drugs targeting gene regulatory interactions implicated in disease.

Mediator and RNA polymerase II clusters associate in transcription-dependent condensates

Models of gene control have emerged from genetic and biochemical studies, with limited consideration of the spatial organization and dynamics of key components in living cells. We used live-cell superresolution and light-sheet imaging to study the organization and dynamics of the Mediator coactivator and RNA polymerase II (Pol II) directly. Mediator and Pol II each form small transient and large stable clusters in living embryonic stem cells. Mediator and Pol II are colocalized in the stable clusters, which associate with chromatin, have properties of phase-separated condensates, and are sensitive to transcriptional inhibitors. We suggest that large clusters of Mediator, recruited by transcription factors at large or clustered enhancer elements, interact with large Pol II clusters in transcriptional condensates in vivo.

Molecular and epigenetic regulations and functions of the LAFL transcriptional regulators that control seed development.

The LAFL (i.e. LEC1, ABI3, FUS3, and LEC2) master transcriptional regulators interact to form different complexes that induce embryo development and maturation, and inhibit seed germination and vegetative growth in Arabidopsis. Orthologous genes involved in similar regulatory processes have been described in various angiosperms including important crop species. Consistent with a prominent role of the LAFL regulators in triggering and maintaining embryonic cell fate, their expression appears finely tuned in different tissues during seed development and tightly repressed in vegetative tissues by a surprisingly high number of genetic and epigenetic factors. Partial functional redundancies and intricate feedback regulations of the LAFL have hampered the elucidation of the underpinning molecular mechanisms. Nevertheless, genetic, genomic, cellular, molecular, and biochemical analyses implemented during the last years have greatly improved our knowledge of the LALF network. Here we summarize and discuss recent progress, together with current issues required to gain a comprehensive insight into the network, including the emerging function of LEC1 and possibly LEC2 as pioneer transcription factors.

Functional Analysis of the Arabidopsis thaliana CDPK-Related Kinase Family: AtCRK1 Regulates Responses to Continuous Light.

The Calcium-Dependent Protein Kinase (CDPK)-Related Kinase family (CRKs) consists of eight members in Arabidopsis. Recently, AtCRK5 was shown to play a direct role in the regulation of root gravitropic response involving polar auxin transport (PAT). However, limited information is available about the function of the other AtCRK genes. Here, we report a comparative analysis of the Arabidopsis CRK genes, including transcription regulation, intracellular localization, and biological function. AtCRK transcripts were detectable in all organs tested and a considerable variation in transcript levels was detected among them. Most AtCRK proteins localized at the plasma membrane as revealed by microscopic analysis of 35S::cCRK-GFP (Green Fluorescence Protein) expressing plants or protoplasts. Interestingly, 35S::cCRK1-GFP and 35S::cCRK7-GFP had a dual localization pattern which was associated with plasma membrane and endomembrane structures, as well. Analysis of T-DNA insertion mutants revealed that AtCRK genes are important for root growth and control of gravitropic responses in roots and hypocotyls. While Atcrk mutants were indistinguishable from wild type plants in short days, Atcrk1-1 mutant had serious growth defects under continuous illumination. Semi-dwarf phenotype of Atcrk1-1 was accompanied with chlorophyll depletion, disturbed photosynthesis, accumulation of singlet oxygen, and enhanced cell death in photosynthetic tissues. AtCRK1 is therefore important to maintain cellular homeostasis during continuous illumination.

Selective recognition of N4-methylcytosine in DNA by engineered transcription-activator-like effectors.

The epigenetic DNA nucleobases 5-methylcytosine (5mC) and N4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'.

Functional domains of SP110 that modulate its transcriptional regulatory function and cellular translocation

BackgroundSP110, an interferon-induced nuclear protein, belongs to the SP100/SP140 protein family. Very recently, we showed that SP110b, an SP110 isoform, controls host innate immunity to Mycobacterium tuberculosis infection by regulating nuclear factor-κB (NF-κB) activity. However, it remains unclear how the structure of SP110 relates to its cellular functions. In this study, we provide experimental data illustrating the protein domains that are responsible for its functions.MethodsWe examined the effects of SP110 isoforms and a series of deletion mutants of SP110 on transcriptional regulation by luciferase reporter assays. We also employed confocal microscopy to determine the cellular distributions of enhanced green fluorescent protein-tagged SP110 isoforms and SP110 mutants. In addition, we performed immunoprecipitation and Western blotting analyses to identify the regions of SP110 that are responsible for protein interactions.ResultsUsing reporter assays, we first demonstrated that SP110 isoforms have different regulatory effects on NF-κB-mediated transcription, supporting the notion that SP110 isoforms may have distinct cellular functions. Analysis of deletion mutants of SP110 showed that the interaction of the N-terminal fragment (amino acids 1–276) of SP110 with p50, a subunit of NF-κB, in the cytoplasm plays a crucial role in the down-regulation of the p50-driven tumor necrosis factor-α (TNFα) promoter activity in the nucleus, while the middle and C-terminal regions of SP110 localize it to various cellular compartments. Surprisingly, a nucleolar localization signal (NoLS) that contains one monopartite nuclear localization signal (NLS) and one bipartite NLS was identified in the middle region of SP110. The identification of a cryptic NoLS in the SP110 suggests that although this protein forms nuclear speckles in the nucleoplasm, it may be directed into the nucleolus to carry out distinct functions under certain cellular conditions.ConclusionsThe findings from this study elucidating the multidomain structure of the SP110 not only identify functional domains of SP110 that are required for transcriptional regulation, cellular translocation, and protein interactions but also implicate that SP110 has additional functions through its unexpected activity in the nucleolus.

Troponin T3 associates with DNA consensus sequence that overlaps with p53 binding motifs

We recently reported that in addition to its classical cytoplasmic location, the fast skeletal muscle Troponin T3 (TnT3) shuttles to the nucleus, where it appears to perform nonclassical transcription regulatory functions. Importantly, changes in the composition of the nucleus-localized pool of TnT3 and its fragments contribute to age-dependent muscle damage and wasting. Here, using ChIP-Seq, we demonstrate that TnT3 associates with DNA consensus sequences including the TGCCT motif, which is required for p53 binding to the promoter area of p53-related genes. Gene set enrichment analysis further demonstrated that the p53 pathway was the most significantly enriched pathway among genes annotated to the TnT3 ChIP-Seq peaks. We further demonstrated a strong correlation (r = 0.78, P = 1 × 10−4) between the expression levels of TNNT3 and TP53-inducible ribonucleotide reductase regulatory subunit M2B (RRM2B) in skeletal muscle tissue of 21 lean non-diabetic human subjects and a significant (P < 0.05) reduction in the levels of both gene transcripts in the third age-tertile group [42.3–70 years of age (yoa)] as compared to the second age-tertile (31.3–42.3 yoa). Of note, both TNNT3 and RRM2B expression levels negatively associated with total body fat mass (each with r = 0.49, P < 0.05), whereas RRM2B positively correlated with pancreatic β cell function (rRRM2B~HOMA-B = 0.47, P = 0.047). This work suggests that reduced TNNT3 gene expression is another mechanism leading to reduced TnT3 and excitation-contraction coupling with aging. Consequently, TnT3 appears to contribute to age-related sarcopenia and possibly other age-related deficiencies such as muscle insulin resistance and β cell dysfunction by interacting with TnT3-binding sequences in the promoter area of p53-related genes, among others, and consequently modulating the transcriptional regulation of these target genes.

YY1 Positively Regulates Transcription by Targeting Promoters and Super-Enhancers through the BAF Complex in Embryonic Stem Cells.

SummaryYin Yang 1 (YY1) regulates early embryogenesis and adult tissue formation. However, the role of YY1 in stem cell regulation remains unclear. YY1 has a Polycomb group (PcG) protein-dependent role in mammalian cells. The PcG-independent functions of YY1 are also reported, although their underlying mechanism is still undefined. This paper reports the role of YY1 and BAF complex in the OCT4-mediated pluripotency network in mouse embryonic stem cells (mESCs). The interaction between YY1 and BAF complex promotes mESC proliferation and pluripotency. Knockdown of Yy1 or Smarca4, the core component of the BAF complex, downregulates pluripotency markers and upregulates several differentiation markers. Moreover, YY1 enriches at both promoter and super-enhancer regions to stimulate transcription. Thus, this study elucidates the role of YY1 in regulating pluripotency through its interaction with OCT4 and the BAF complex and the role of BAF complex in integrating YY1 into the core pluripotency network.