Mechanism Of Transcriptional Activation Research Articles

PXR interaction with p53: a meeting of two masters.

PXR interaction with p53: a meeting of two masters.

081 Resistant basal cell carcinomas require SRF/MRTF to maintain hedgehog pathway activation and tumor growth

080 Mutational landscape of MCPyV-positive and MCPyV-negative Merkel cell carcinomas with implications for immunotherapy T Walradt, V Makarov, R Doumani, N Riaz, K Stafstrom, A Moshiri, L Yelistratova, J Levinsohn, T Chan, P Nghiem, R Lifton and J Choi 1 Dermatology, Northwestern University, Chicago, IL, 2 Dermatology, Yale University, New Haven, CT, 3 Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, 4 Dermatology, University of Washington, Seattle, WA, 5 Genetics, Yale University, New Haven, CT and 6 Biochemistry and Molecular Genetics, Northwestern University, Chicago, IL Merkel cell carcinoma (MCC) is a rare but highly aggressive cutaneous neuroendocrine carcinoma, associated with the merkel cell polyomavirus (MCPyV) in 80% of cases. To define the genetic basis of MCCs, we performed exome sequencing of 49 MCCs. We show that MCPyV-negative MCCs have a high mutation burden (median of 1121 Somatic Single Nucleotide Variants (SSNVs) per-exome with frequent mutations in RB1 and TP53 and additional damaging mutations in genes in the chromatin modification (ASXL1, MLL2, and MLL3), JNK (MAP3K1 and TRAF7), and DNA-damage pathways (ATM, MSH2, and BRCA1). In contrast, MCPyV-positive MCCs harbor few SSNVs (median of 12.5 SSNVs/tumor) with none in the genes listed above. In both subgroups, there are rare cancer-promoting mutations predicted to activate the PI3K pathway (HRAS, KRAS, PIK3CA, PTEN, and TSC1) and to inactivate the NOTCH pathway (NOTCH1 and NOTCH2). Virus-negative MCCs harbor potentially targetable gain-of-function mutations in TP53 at p.R248 and p.P278. Moreover, TP53 mutational status predicts death in early stage MCC (5-year survival in TP53 mutant vs wild-type stage I and II MCCs is 20% vs. 92%, respectively; P1⁄40.0036). Lastly, we identified the tumor neoantigens in MCPyV-negative and MCPyV-positive MCCs. We found that virusnegative MCCs harbor more tumor neoantigens than melanomas or non-small cell lung cancers (median of 173, 65, and 111 neoantigens/sample, respectively), two cancers for which immune checkpoint blockade can produce durable clinical responses. Collectively, these data support the use of immunotherapies for virus-negative MCCs. 081 Resistant basal cell carcinomas require SRF/MRTF to maintain hedgehog pathway activation and tumor growth R Whitson, A Lee, N Urman, J Li, A Mirza, A Brown, C Yao, G Shankar, M Fry, SX Atwood, E Epstein, J Tang and AE Oro 1 Dermatology, Stanford, Stanford, CA, 2 Childrens Hospital Oakland Research Institute, Oakland, CA and 3 UC Irvine, Irvine, CA Basal cell carcinoma (BCC) and other hedgehog-dependent cancers commonly acquire drug resistance to the Smoothened (Smo) inhibitor vismodegib (Atwood et al., 2014; Sekulic et al., 2015). Resistant BCCs maintain activation of the hedgehog pathway suggesting the presence of mutations at or downstream of Smo. Variants in Smo are found in w50% of resistance cases (Atwood et al., 2015b), however, many variants are non-functional (Atwood et al., 2015a) leaving tumor escape mechanisms for a large proportion of patient tumors unexplored. We recently uncovered a novel resistance mechanism using multi-dimensional genomic analysis in human and mouse resistant BCCs to identify a non-canonical hedgehog activation pathway driven by the transcription factor, serum response factor (SRF). The majority of resistant human and mouse BCCs measured acquire the active nuclear form of SRF, which forms a novel protein complex and shares chromosomal occupancy with the hedgehog transcription factor Gli1. SRF is necessary for hedgehog pathway output, and activation by the SRF coactivator myocardin-related transcription factor (MRTF) is sufficient to potentiate hedgehog activation. Remarkably, pharmacological inhibitors targeting MRTF suppress tumor growth in mice harboring resistant BCCs. Our data uncovers a novel non-canonical mechanism for hedgehog transcriptional activation through SRF/MRTF which is activated in a majority of resistant BCCs. Combined with our in vivo data, we highlight SRF/MRTF-driven transcription as a promising therapeutic target to combat resistant BCC growth.

Understanding TERT Promoter Mutations: A Common Path to Immortality.

Telomerase (TERT) activation is a fundamental step in tumorigenesis. By maintaining telomere length, telomerase relieves a main barrier on cellular lifespan, enabling limitless proliferation driven by oncogenes. The recently discovered, highly recurrent mutations in the promoter of TERT are found in over 50 cancer types, and are the most common mutation in many cancers. Transcriptional activation of TERT, via promoter mutation or other mechanisms, is the rate-limiting step in production of active telomerase. Although TERT is expressed in stem cells, it is naturally silenced upon differentiation. Thus, the presence of TERT promoter mutations may shed light on whether a particular tumor arose from a stem cell or more differentiated cell type. It is becoming clear that TERT mutations occur early during cellular transformation, and activate the TERT promoter by recruiting transcription factors that do not normally regulate TERT gene expression. This review highlights the fundamental and widespread role of TERT promoter mutations in tumorigenesis, including recent progress on their mechanism of transcriptional activation. These somatic promoter mutations, along with germline variation in the TERT locus also appear to have significant value as biomarkers of patient outcome. Understanding the precise molecular mechanism of TERT activation by promoter mutation and germline variation may inspire novel cancer cell-specific targeted therapies for a large number of cancer patients.

‐Altered DNA‐Binding Specificity Transcription Activator Variant Provides Key To Unlocking Gene Activation Mechanism

Transcription activator proteins confer target selective, gene‐specific activation in response to appropriate stimuli. In order to dissect the molecular mechanisms of gene transcription activation, it is imperative to understand the role(s) of the transcription activator. Unfortunately, a subset of DNA‐binding transcriptional activator proteins are resistant to study by the conventional means of fusing putative Activation Domains (ADs) to heterologous DNA Binding Domains (DBDs) to identify key transcription factor functional elements. The budding yeast Saccharomyces cerevisiae Repressor Activator Protein 1 (Rap1) is one such activator protein. Rap1 specifically binds over 500 DNA sites in the yeast genome, serving key roles in multiple cellular processes including: (a) telomere stabilization; (b) transcription repression of silent mating type loci; and (c) activation of two of the most vigorously transcribed classes of yeast genes: the SAGA‐dominated glycolytic enzyme‐encoding genes (GEs) and the TFIID‐dominated ribosomal protein genes (RPGs). However, fusions of Rap1 or Rap1‐domains with heterologous DBDs reportedly fail to robustly drive reporter gene expression.To enable study of the role of Rap1 in activation, an altered DNA‐binding specificity Rap1 variant (Rap1 AS) was generated using a direct screening/selection strategy in yeast wherein activation of a selectable HIS3 reporter gene depended upon the ability of DBD‐mutagenized variant forms of Rap1 to efficiently drive expression from a mutant Rap1 enhancer (UASRAP1). Multiple candidate putative Rap1 AS variants scored in this screen. One has been characterized in detail. In both biochemical (DNA gel mobility shift) and functional (HIS3 reporter gene expression) assays, Rap1 AS preferentially bound the selecting mutant UASRAP1, displaying a significant discrimination for mutant vs. wild‐type (WT) UASRAP1 sequences. Yeast pseudodiploid for Rap1 (Rap1 WT and Rap1 AS) grow like WT, showing that Rap1 AS does not interfere with the many essential functions of Rap1 WT. Subsequent mutagenesis experiments with Rap1 AS identified a putative activation domain that mapped to a ~40 amino acid region within the C‐terminal 1/3 of Rap1. Systematic point mutagenesis of this mapped AD has identified eight amino acids that contribute critically to AD function.The collection of Rap1 AD mutants will be used to investigate the transcription activation mechanisms used to drive the TFIID‐dominated RPGs. Since we have previously shown that Rap1 interacts directly with TFIID, and is the only RPG activator absolutely required for RPG transcription, mechanistic analyses performed with the Rap1 AD mutants should unlock the key step(s) to RPG activation. Because few TFIID‐dependent activation mechanisms have been carefully dissected, these key steps will likely reveal general principles of TFIID‐dependent gene activation applicable to all organisms.Support or Funding InformationThis research was supported by the NIH Molecular Endocrinology Training Program (METP) training grant (T32 DK07563) and the NIH R01 grants GM52461 and GM115892.

Host Proteins Ku and HMGA1 As Participants of HIV-1 Transcription

Human immunodeficiency virus type 1 is known to use the transcriptional machinery of the host cell for viral gene transcription, and the only viral protein that partakes in this process is Tat, the viral trans-activator of transcription. During acute infection, the binding of Tat to the hairpin at the beginning of the transcribed viral RNA recruits the PTEFb complex, which in turn hyperphosphorylates RNA-polymerase II and stimulates transcription elongation. Along with acute infection, HIV-1 can also lead to latent infection that is characterized by a low level of viral transcription. During the maintenance and reversal of latency, there are no detectable amounts of Tat protein in the cell and the mechanism of transcription activation in the absence of Tat protein remains unclear. The latency maintenance is also a problematic question. It seems evident that cellular proteins with a yet unknown nature or role regulate both transcriptional repression in the latent phase and its activation during transition into the lytic phase. The present review discusses the role of cellular proteins Ku and HMGA1 in the initiation of transcription elongation of the HIV-1 provirus. The review presents data regarding Ku-mediated HIV-1 transcription and its dependence on the promoter structure and the shape of viral DNA. We also describe the differential influence of the HMGA1 protein on the induced and basal transcription of HIV-1. Finally, we offer possible mechanisms for Ku and HMGA1 proteins in the proviral transcription regulation.

Regulation of aryl hydrocarbon receptor-mediated transcription in human retinal pigmented epithelial cells

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with pleiotropic effects in normal physiology or vascular development, xenobiotic metabolism, and cancer. A previous study has reported that BRG1, a component of the SWI/SNF complex, is a coactivator for AHR and is recruited to the promoter region of the CYP1A1 gene in mouse hepatocytes. Recent data suggest that AHR is also expressed in human retinal pigment epithelial cells (ARPE-19), which play a crucial role in retinal physiology and the visual cycle. Multiple studies have shown that the AHR plays an important role in the pathogenesis of retinal diseases including age-related macular degeneration. However, the mechanism of AHR transcriptional activation in retinal pigment cells has not been reported. Here, we demonstrate that the AHR signaling pathway is active in ARPE-19 cells, as in hepatocytes, but with different target gene specificity. We also found that chromatin remodeling by the BRG1-containing SWI/SNF complex is required for the AHR-mediated expression of target genes in ARPE-19 cells. We identified a novel enhancer region (−12 kb) of the CYP1A1 gene in ARPE-19 cells, to which both AHR and BRG1 are recruited in a ligand-dependent manner. BRG1 is associated with the AHR in ARPE-19 cells, and the C-terminal activation domain of the AHR directly interacts with BRG1. Furthermore, depletion of BRG1 caused a reduction in chromatin accessibility at the CYP1A1 enhancer. These results suggest that ARPE-19 cells possess an AHR-mediated transcription pathway with different target gene specificity, and that BRG1 is required for AHR-mediated transcription in ARPE-19 cells.

SaRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription.

Small activating RNAs (saRNAs) targeting specific promoter regions are able to stimulate gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). It is known that RNAa depends on Ago2 and is associated with epigenetic changes at the target promoters. However, the precise molecular mechanism of RNAa remains elusive. Using human CDKN1A (p21) as a model gene, we characterized the molecular nature of RNAa. We show that saRNAs guide Ago2 to and associate with target promoters. saRNA-loaded Ago2 facilitates the assembly of an RNA-induced transcriptional activation (RITA) complex, which, in addition to saRNA-Ago2 complex, includes RHA and CTR9, the latter being a component of the PAF1 complex. RITA interacts with RNA polymerase II to stimulate transcription initiation and productive elongation, accompanied by monoubiquitination of histone 2B. Our results establish the existence of a cellular RNA-guided genome-targeting and transcriptional activation mechanism and provide important new mechanistic insights into the RNAa process.

Structural and functional analysis of RopB: a major virulence regulator in Streptococcus pyogenes.

SummaryGroup A Streptococcus (GAS) is an exclusive human pathogen that causes significant disease burden. Global regulator RopB of GAS controls the expression of several major virulence factors including secreted protease SpeB during high cell density. However, the molecular mechanism for RopB‐dependent speB expression remains unclear. To understand the mechanism of transcription activation by RopB, we determined the crystal structure of the C‐terminal domain of RopB. RopB‐CTD has the TPR motif, a signature motif involved in protein–peptide interactions and shares significant structural homology with the quorum sensing RRNPP family regulators. Characterization of the high cell density‐specific cell‐free growth medium demonstrated the presence of a low molecular weight proteinaceous secreted factor that upregulates RopB‐dependent speB expression. Together, these results suggest that RopB and its cognate peptide signals constitute an intercellular signalling machinery that controls the virulence gene expression in concert with population density. Structure‐guided mutational analyses of RopB dimer interface demonstrated that single alanine substitutions at this critical interface significantly altered RopB‐dependent speB expression and attenuated GAS virulence. Results presented here suggested that a properly aligned RopB dimer interface is important for GAS pathogenesis and highlighted the dimerization interactions as a plausible therapeutic target for the development of novel antimicrobials.

TFAM Regulates Mitochondrial Transcription through Sequence-Specific DNA Looping

Mitochondrial transcription factor A (TFAM) is an abundant human mitochondrial High Mobility Group Box (HMGB) protein. TFAM is an architectural protein that shapes mitochondrial DNA (mtDNA) and regulates mitochondrial transcription. Sequence-specific binding of TFAM to DNA upstream of the light-strand promoter (LSP) leads to bending that correlates with transactivation of this promoter. Here we use a dual promoter construct containing both LSP, HSP1 and the natural inter-promoter region (IPR) to understand how TFAM binding governs transcription activation. We show that the IPR contributes to TFAM transactivation of HSP1, while removal of the carboxyl-terminal tail of TFAM (TFAM-ΔCT26) leads to a complete loss of transactivation of HSP1, with only minimal effects on LSP, suggesting a different biophysical mechanism for TFAM-mediated transcription activation from HSP1. To understand the mechanism by which TFAM activates HSP1 transcription, we used atomic force microscopy (AFM) in liquid to probe the effect of TFAM on the IPR construct. We observe that at the low concentrations of TFAM where transcription activation is high, TFAM induces the formation of DNA loops in a region defined by the IPR, with TFAM present at the strand crossing point. Under the same conditions, TFAM-ΔCT26 fails to induce DNA looping. While random DNA also forms loops in the presence of TFAM, TFAM is twice as likely to mediate the loops when the IPR sequence is present. Optical tweezers experiments also demonstrate that TFAM stabilizes DNA loops, which require significant force to break. Taken together, our results are consistent with sequence-specific DNA looping contributing to TFAM transactivation of HSP1, suggesting that unique mechanisms are employed for TFAM-dependent transcription at LSP and HSP1.

Enhancement of human sodium iodide symporter gene therapy for breast cancer by HDAC inhibitor mediated transcriptional modulation

The aberrant expression of human sodium iodide symporter (NIS) in breast cancer (BC) has raised the possibility of using targeted radioiodide therapy. Here we investigate modulation of endogenous, functional NIS expression by histone deacetylase inhibitors (HDACi) in vitro and in vivo. Luciferase reporter based initial screening of six different HDACi shows 2–10 fold enhancement of NIS promoter activity in majority of the cell types tested. As a result of drug treatment, endogenous NIS transcript and protein shows profound induction in BC cells. To get an insight on the mechanism of such transcriptional activation, role of Stat4, CREB and other transcription factors are revealed by transcription factor profiling array. Further, NIS-mediated intracellular iodide uptake also enhances substantially (p < 0.05) signifying functional relevance of the transcriptional modulation strategy. Gamma camera imaging confirms 30% higher uptake in VPA or NaB treated BC tumor xenograft. Corroborating with such functional impact of NIS, significant reduction in cell survival (p < 0.005) is observed in VPA, NaB or CI994 drug and 131I combination treatment in vivo indicating effective radioablation. Thus, for the first time this study reveals the mechanistic basis and demonstrates functional relevance of HDACi pre-treatment strategy in elevating NIS gene therapy approach for BC management in clinic.

The Molecular Mechanism of Transcriptional Activation By MLL-AEP Fusion Proteins

Chromosomal translocations generate a variety of mixed lineage leukemia (MLL) fusion genes, which cause aggressive leukemia. Although >70 different fusion partners have been identified, the majority of the cases are caused by the chimeric genes of MLL and a component of the AEP co-activator complex (hereafter referred to as AEP), which comprises of AF4 family proteins (e.g. AF4, AF5Q31), ENL family proteins (e.g. ENL, AF9), and the P-TEFb elongation factor. MLL-AEP fusion proteins constitutively activate their target genes by recruiting AEP components to their target chromatin, whereas wild-type MLL recruits AEP in a context-dependent manner. In the hematopoietic lineage, MLL fusion proteins aberrantly activate a subset of genes implicated in the hematopoietic stem cell (HSC) program, such as HOXA9 and MEIS1. Constitutive expression of these HSC program genes in hematopoietic progenitors has been shown to induce leukemia in a mouse model. It has been speculated that MLL-AEP activates transcription of those HSC program genes by aberrantly activating transcription elongation. However, it is largely unclear how AEP activates transcription.Using an extensive structure/function analysis, we revealed that a serine-rich domain of the AF4 family proteins, termed pSER, is an essential functional component of MLL-AEP-dependent gene activation and leukemic transformation. Through biochemical purification, we have identified Selectivity Factor 1 (SL1) as a novel factor associated with the pSER domain. SL1 comprises TBP and four TBP-associated factors (TAF1A, TAF1B, TAF1C, TAF1D), and is known as a core component of the pre-initiation complex (PIC) of RNA polymerase I (RNAP1). In the presence of UBF, SL1 forms a PIC on the promoters of ribosomal RNA genes, to drive RNAP1-dependent transcription. However, its role in RNAP2-dependent transcription was unknown. The initiation of RNAP2-dependent transcription in eukaryotes occurs through the loading of TBP to the promoter, via a direct association with the TATA element or through as-yet-unidentified mechanisms. Our results demonstrate that AEP facilitates the initiation of RNAP2-dependent transcription via the loading of TBP onto the TATA element, through SL1 activity. MLL-AEP fusion proteins utilize this TBP-loading function to activate transcription initiation in leukemic transformation. The wild-type AEP complex activates gene expression in the same manner in the physiological conditions. Taken together, our results unveil a novel role of SL1 as a TBP-loading factor in RNAP2-dependent gene activation, and a previously unknown transcription initiation mechanism involving AEP, which is more important than its transcription elongation activities for leukemic transformation. These findings greatly advance our understanding of the molecular mechanism of MLL fusion-dependent leukemic transformation, which was previously interpreted simply as mis-regulated transcription elongation. DisclosuresYokoyama:Dainipon Sumitomo Pharma Co., Ltd.: Research Funding.

Abstract CN06-02: Regulation of AR variants by BET bromodomains in prostate cancer

Abstract Expression of androgen receptor (AR) variants (AR-Vs) lacking the AR ligand binding domain (LBD) in prostate cancer tissues or circulating tumor cells is associated with poor clinical outcomes. AR-Vs function as constitutively active transcription factors and represent a resistance mechanism whereby the growth of prostate cancer cells can remain AR-dependent, yet uncoupled from endocrine regulation. Tumors that have developed this mechanism of resistance are unlikely to respond to successive generations of endocrine therapies. The absence of a LBD presents a formidable challenge for direct inhibition of AR-Vs because alternative sites on the AR protein appropriate for specific and high-affinity drug targeting are not evident. An indirect approach may be through inhibition of AR-V expression and/or transcriptional activation mechanisms. To this end, we have found that treatment of prostate cancer cell lines with the bromodomain and extraterminal (BET) inhibitor JQ1 resulted in inhibition of AR and AR-V chromatin binding and impaired AR and AR-V driven prostate cancer cell growth in vitro and in vivo. These anti-AR activities were associated with JQ1-mediated reduction in binding of the BET family member BRD2 to a region near the AR transcription start-site featuring broad enrichment of histone H3 acetylated at lysine 27. Consistent with these chromatin effects at the AR locus, JQ1 treatment down-regulated AR and AR-V transcript and protein expression. Overall, these findings highlight regulation of AR and AR-V expression by BRD2, and provide pre-clinical rationale for BET inhibition as a strategy for overcoming resistance mediated by constitutively active AR-Vs in prostate cancer. Citation Format: Scott M. Dehm. Regulation of AR variants by BET bromodomains in prostate cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr CN06-02.

Abstract B84: Novel and selective inhibitors of histone deacetylases (HDAC) 1 and 2 significantly enhance the activity of the DNA methyltransferase inhibitor azacitidine in acute myeloid leukemia (AML)

Abstract AML is a heterogeneous group of hematopoietic stem cell disorders characterized by defects in myeloid differentiation and increased proliferation of neoplastic hematopoietic precursor cells. There is a great need for novel approaches to treat AML due to limited improvements in the treatment of patients over the past several decades. Aberrant epigenetic regulation plays an important role in the pathogenesis of AML, and the DNA methyltransferase inhibitor azacitidine is approved for the treatment of myelodysplastic (MDS), which frequently progresses to AML, and is commonly used in AML patients. Numerous clinical studies are ongoing to investigate the benefit of combining azacitidine with other investigational agents in AML. HDAC inhibitors are promising agents for the treatment of AML. Selective HDAC inhibitors are predicted to reduce combination drug toxicity and other side effects observed with pan-HDAC inhibitors, while still enabling therapeutic benefits. In this work, we demonstrate that potent and selective inhibitors of HDAC1/2 are highly efficacious both as single agents and in combination with azacitidine in preclinical models of AML, including established AML cell lines and bone marrow samples freshly derived from AML patients. Combination treatment in vivo demonstrated good tolerability, and ongoing efficacy studies in AML xenograft models will be discussed. At the molecular level, gene expression profiling and DNA methylation mapping were performed on MV4-11 cells treated with an HDAC1/2 inhibitor, azacitidine, or the combination. Molecular signatures were analyzed by GSEA. Significantly more genes and signatures were upregulated than downregulated by the single agent and combination treatments, consistent with the transcription activation mechanism of these compounds. Signatures and genes involved in myeloid cell differentiation, apoptosis and cell cycle arrest were identified as potential mediators of the combinatorial effects of HDAC1/2 inhibition with azacitidine. Together, these findings support the clinical evaluation of selective HDAC1/2 inhibitors in combination with azacitidine in AML and potentially MDS patients. Citation Format: Chengyin Min, Steven N. Quayle, Pengyu Huang, Jeffrey R. Shearstone, Simon S. Jones, Min Yang. Novel and selective inhibitors of histone deacetylases (HDAC) 1 and 2 significantly enhance the activity of the DNA methyltransferase inhibitor azacitidine in acute myeloid leukemia (AML). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B84.

Regulation of E2 Promoter Binding Factor 1 (E2F1) Transcriptional Activity through a Deubiquitinating Enzyme, UCH37

E2F1 is tightly controlled by multiple mechanisms, but whether ubiquitination regulates its transcriptional activity remains unknown. Here we identify UCH37 as the first, to our knowledge, deubiquitinating enzyme for E2F1. UCH37 does not deubiquitinate UbK48 chains or affect E2F1 protein stability. Instead, UCH37, but not a catalytically dead mutant, decreases the Lys-63-linked ubiquitination of E2F1 and activates its transcriptional activity. UCH37 depletion reduces the gene expression of both proliferative and pro-apoptotic E2F1 target genes. UCH37 depletion also decreases both cell proliferation and apoptosis induction in functional assays. Interestingly, UCH37 expression is induced by E2F1, and its level rises in G1/S transition and S phase, suggesting a positive feedback loop between UCH37 and E2F1. UCH37 protein and mRNA levels are also induced after DNA damage. UCH37 localizes to the promoters of E2F1 pro-apoptotic target genes such as caspase 3, caspase 7, PARP1, and Apaf-1 and activates their expression after DNA damage. Moreover, the expression of E2F1 proliferative and pro-apoptotic genes is correlated with the levels of UCH37 in many primary tumors. These results uncover a novel mechanism for E2F1 transcriptional activation through removal of its Lys-63-linked ubiquitination by UCH37.

How targets select activation or repression in response to Wnt.

In metazoans, the Wnt signaling pathway plays a key role in the regulation of binary decisions during development. During this process different sets of target genes are activated in cells where the Wnt pathway is active (classic target genes) versus cells where the pathway is inactive (opposite target genes). While the mechanism of transcriptional activation is well understood for classic target genes, how opposite target genes are activated in the absence of Wnt remains poorly characterized. Here we discuss how the key transcriptional mediator of the Wnt pathway, the TCF family member POP-1, regulates opposite target genes during C. elegans development. We examine recent findings suggesting that the direction of the transcriptional output (activation or repression) can be determined by the way TCF is recruited and physically interacts with its target gene.

Heat Stress-Induced Transcriptional Repression.

Heat stress is one of the most popular models for studying the regulation of gene expression. For decades, researchers' attention was focused on the study of the mechanisms of transcriptional activation of stress-induced genes. Although the phenomenon of heat stress-induced global transcriptional repression is known for a long time, the exact molecular mechanisms of such a repression are poorly explored. In this mini-review, we attempt to summarize the existing experimental data on heat stress-induced transcriptional repression.

Functional Characterization of the Mannitol Promoter of Pseudomonas fluorescens DSM 50106 and Its Application for a Mannitol-Inducible Expression System for Pseudomonas putida KT2440.

A new pBBR1MCS-2-derived vector containing the Pseudomonas fluorescens DSM10506 mannitol promoter PmtlE and mtlR encoding its AraC/XylS type transcriptional activator was constructed and optimized for low basal expression. Mannitol, arabitol, and glucitol-inducible gene expression was demonstrated with Pseudomonas putida and eGFP as reporter gene. The new vector was applied for functional characterization of PmtlE. Identification of the DNA binding site of MtlR was achieved by in vivo eGFP measurement with PmtlE wild type and mutants thereof. Moreover, purified MtlR was applied for detailed in vitro investigations using electrophoretic mobility shift assays and DNaseI footprinting experiments. The obtained data suggest that MtlR binds to PmtlE as a dimer. The proposed DNA binding site of MtlR is AGTGC-N5-AGTAT-N7-AGTGC-N5-AGGAT. The transcription activation mechanism includes two binding sites with different binding affinities, a strong upstream binding site and a weaker downstream binding site. The presence of the weak downstream binding site was shown to be necessary to sustain mannitol-inducibility of PmtlE. Two possible functions of mannitol are discussed; the effector might stabilize binding of the second monomer to the downstream half site or promote transcription activation by inducing a conformational change of the regulator that influences the contact to the RNA polymerase.

Jmjd2C increases MyoD transcriptional activity through inhibiting G9a-dependent MyoD degradation

Skeletal muscle cell differentiation requires a family of proteins called myogenic regulatory factors (MRFs) to which MyoD belongs. The activity of MyoD is under epigenetic regulation, however, the molecular mechanism by which histone KMTs and KDMs regulate MyoD transcriptional activity through methylation remains to be determined. Here we provide evidence for a unique regulatory mechanism of MyoD transcriptional activity through demethylation by Jmjd2C demethylase whose level increases during muscle differentiation. G9a decreases MyoD stability via methylation-dependent MyoD ubiquitination. Jmjd2C directly associates with MyoD in vitro and in vivo to demethylate and stabilize MyoD. The hypo-methylated MyoD due to Jmjd2C is significantly more stable than hyper-methylated MyoD by G9a. Cul4/Ddb1/Dcaf1 pathway is essential for the G9a-mediated MyoD degradation in myoblasts. By the stabilization of MyoD, Jmjd2C increases myogenic conversion of mouse embryonic fibroblasts and MyoD transcriptional activity with erasing repressive H3K9me3 level at the promoter of MyoD target genes. Collectively, Jmjd2C increases MyoD transcriptional activity to facilitate skeletal muscle differentiation by increasing MyoD stability through inhibiting G9a-dependent MyoD degradation.

Different Modes of Transactivation of Bacteriophage Mu Late Promoters by Transcription Factor C.

Transactivator protein C is required for the expression of bacteriophage Mu late genes from lys, I, P and mom promoters during lytic life cycle of the phage. The mechanism of transcription activation of mom gene by C protein is well understood. C activates transcription at Pmom by initial unwinding of the promoter DNA, thereby facilitating RNA polymerase (RNAP) recruitment. Subsequently, C interacts with the ß' subunit of RNAP to enhance promoter clearance. The mechanism by which C activates other late genes of the phage is not known. We carried out promoter-polymerase interaction studies with all the late gene promoters to determine the individual step of C mediated activation. Unlike at Pmom, at the other three promoters, RNAP recruitment and closed complex formation are not C dependent. Instead, the action of C at Plys, PI, and PP is during the isomerization from closed complex to open complex with no apparent effect at other steps of initiation pathway. The mechanism of transcription activation of mom and other late promoters by their common activator is different. This distinction in the mode of activation (promoter recruitment and escape versus isomerization) by the same activator at different promoters appears to be important for optimized expression of each of the late genes.

Upregulation of lncRNA MEG3 Promotes Osteogenic Differentiation of Mesenchymal Stem Cells From Multiple Myeloma Patients By Targeting BMP4 Transcription.

Multiple myeloma (MM) is characterized by the impaired osteogenic differentiation of mesenchymal stromal cells (MSCs). However, the underlying molecular mechanisms are still poorly understood. Long noncoding RNAs (lncRNAs) are emerging as important regulatory molecules in tumor-suppressor and oncogenic pathways. Here we showed that MSCs from MM expressed less lncRNA MEG3 relative to those from normal donors during osteogenic differentiation. To evaluate the effect of MEG3 on osteogenesis, bone marrow MSCs with enhanced or reduced MEG3 were prepared. We observed that MEG3 knockdown significantly reduced the expression of key osteogenic markers, including Runt-related transcription factor 2, osterix, and osteocalcin, while overexpression of MEG3 enhanced their expression. Additionally, MEG3 knockdown decreased BMP4 transcription. Here we showed that MEG3 was critical for SOX2 transcriptional repression of the BMP4. MEG3, which is located near the BMP4 gene, could dissociate the transcription factor SOX2 from the BMP4 promoter. A stable complex containing the MEG3, SOX2, and the SOX2 consensus site of BMP4 suggested that MEG3 activated transcriptional activity by directly influencing SOX2 activity. By using assays such as luciferase, chromatin immunoprecipitation, and RNA immunoprecipitation, we showed that MEG3 had a critical function in a mechanism of promoter-specific transcriptional activation. These results suggested that MEG3 played an essential role in osteogenic differentiation in bone marrow MSCs, partly by activating BMP4 transcription. Our data provided novel evidence for the biological and clinical significance of lncRNA MEG3 expression as a potential biomarker for identifying patients with MM and as a potential therapeutic target in MM.