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1.  Potential non-B DNA regions in the human genome are associated with higher rates of nucleotide mutation and expression variation 
Nucleic Acids Research  2014;42(20):12367-12379.
While individual non-B DNA structures have been shown to impact gene expression, their broad regulatory role remains elusive. We utilized genomic variants and expression quantitative trait loci (eQTL) data to analyze genome-wide variation propensities of potential non-B DNA regions and their relation to gene expression. Independent of genomic location, these regions were enriched in nucleotide variants. Our results are consistent with previously observed mutagenic properties of these regions and counter a previous study concluding that G-quadruplex regions have a reduced frequency of variants. While such mutagenicity might undermine functionality of these elements, we identified in potential non-B DNA regions a signature of negative selection. Yet, we found a depletion of eQTL-associated variants in potential non-B DNA regions, opposite to what might be expected from their proposed regulatory role. However, we also observed that genes downstream of potential non-B DNA regions showed higher expression variation between individuals. This coupling between mutagenicity and tolerance for expression variability of downstream genes may be a result of evolutionary adaptation, which allows reconciling mutagenicity of non-B DNA structures with their location in functionally important regions and their potential regulatory role.
PMCID: PMC4227770  PMID: 25336616
2.  FBP1 Is an Interacting Partner of Menin 
Multiple endocrine neoplasia type 1 (MEN1) is a syndrome characterized by tumors in multiple endocrine tissues such as the parathyroid glands, the pituitary gland, and the enteropancreatic neuroendocrine tissues. MEN1 is usually caused by mutations in the MEN1 gene that codes for the protein menin. Menin interacts with proteins that regulate transcription, DNA repair and processing, and maintenance of cytoskeletal structure. We describe the identification of FBP1 as an interacting partner of menin in a large-scale pull-down assay that also immunoprecipitated RBBP5, ASH2, and LEDGF, which are members of complex proteins associated with SET1 (COMPASS), a protein complex that methylates histone H3. This interaction was confirmed by coimmunoprecipitation and Flag-pull-down assays. Furthermore, menin localized to the FUSE site on the MYC promoter, a site that is transactivated by FBP1. This investigation therefore places menin in a pathway that regulates MYC gene expression and has important implications for the biological function of menin.
PMCID: PMC4123598  PMID: 25132853
3.  Notching up MYC gives a LIC 
Cell stem cell  2013;13(1):8-9.
King et al. (Cell, 2013) show that mutations in the ubiquitin ligase subcomponent FBXW7 increase MYC protein levels and expand the number of leukemia initiating cells (LIC) in Notch1-induced T-ALL. Genetic and pharmacological inhibition of Myc abolishes LIC activity with broader therapeutic implications.
PMCID: PMC3746020  PMID: 23827704
4.  DNA Break Mapping Reveals Topoisomerase II Activity Genome-Wide 
Genomic DNA is under constant assault by endogenous and exogenous DNA damaging agents. DNA breakage can represent a major threat to genome integrity but can also be necessary for genome function. Here we present approaches to map DNA double-strand breaks (DSBs) and single-strand breaks (SSBs) at the genome-wide scale by two methods called DSB- and SSB-Seq, respectively. We tested these methods in human colon cancer cells and validated the results using the Topoisomerase II (Top2)-poisoning agent etoposide (ETO). Our results show that the combination of ETO treatment with break-mapping techniques is a powerful method to elaborate the pattern of Top2 enzymatic activity across the genome.
PMCID: PMC4139894  PMID: 25056547
topoisomerases; DNA damage; transcription
5.  Global regulation of promoter melting in naïve lymphocytes 
Cell  2013;153(5):988-999.
Lymphocyte activation is initiated by a global increase in mRNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring ssDNA genome-wide, we show that the genome of naïve cells is poised for rapid activation. In G0, ~90% of promoters from genes to be expressed in cycling lymphocytes are polymerase-loaded but unmelted and support only basal transcription. Furthermore, the transition from abortive to productive elongation is kinetically limiting causing polymerases to accumulate nearer transcription start sites (TSSs). Resting lymphocytes also limit expression of the TFIIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our studies identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens.
PMCID: PMC3684982  PMID: 23706737
6.  H2A.Z Facilitates Access of Active and Repressive Complexes to Chromatin in Embryonic Stem Cell Self-renewal and Differentiation 
Cell stem cell  2012;12(2):180-192.
Chromatin modifications have been implicated in the self-renewal and differentiation of embryonic stem cells (ESCs). However, the function of histone variant H2A.Z in ESCs remains unclear. We show that H2A.Z is highly enriched at promoters and enhancers and is required for both efficient self-renewal and differentiation of murine ESCs. H2A.Z deposition leads to an abnormal nucleosome structure, decreased nucleosome occupancy and increased chromatin accessibility. In self-renewing ESCs, knockdown of H2A.Z compromises OCT4 binding to its target genes and leads to decreased binding of MLL complexes to active genes and of PRC2 complex to repressed genes. During differentiation of ESCs, inhibition of H2A.Z also compromises RA-induced RARα binding, activation of differentiation markers and the repression of pluripotency genes. We propose that H2A.Z mediates such contrasting activities by acting as a ‘general facilitator’ that generates access for a variety of complexes both activating and repressive.
PMCID: PMC3570599  PMID: 23260488
7.  Protein Expression Profiles Distinguish Between Experimental Invasive Pulmonary Aspergillosis and Pseudomonas Pneumonia 
Proteomics  2010;10(23):10.1002/pmic.200900768.
We hypothesized that invasive pulmonary aspergillosis (IPA) may generate a distinctive proteomic signature in plasma and bronchoalveolar lavage (BAL). Proteins in plasma and BAL from two neutropenic rabbit models of IPA and Pseudomonas pneumonia were analyzed by SELDI-TOF MS. Hierarchical clustering analysis of plasma time course spectra demonstrated two clusters of peaks that were differentially regulated between IPA and Pseudomonas pneumonia (57 and 34 peaks, respectively, p<0.001). PCA of plasma proteins demonstrated a time-dependent separation of the two infections. A random forest analysis that ranked the top 30 spectral points distinguished between late Aspergillus and Pseudomonas pneumonias with 100% sensitivity and specificity. Based on spectral data analysis, three proteins were identified using SDS-PAGE and LC/MS and quantified using reverse phase arrays. Differences in the temporal sequence of plasma haptoglobin (p <0.001), apolipoprotein A1 (p<0.001) and transthyretin (p<0.038) were observed between IPA and Pseudomonas pneumonia, as was C-reactive protein (p<0.001). In summary, proteomic analysis of plasma and BAL proteins of experimental Aspergillus and Pseudomonas pneumonias demonstrates unique protein profiles with principal components and spectral regions that are shared in early infection and diverge at later stages of infection. Haptoglobin, apolipoprotein A1, transthyretin and C-reactive protein are differentially expressed in these infections suggesting important contributions to host defense against IPA.
PMCID: PMC3859317  PMID: 21089047
proteomics; Aspergillus fumigatus; Pseudomonas aeruginosa; pneumonia; neutropenia
8.  Revisiting Global Gene Expression Analysis 
Cell  2012;151(3):476-482.
Gene expression analysis is a widely used and powerful method for investigating the transcriptional behavior of biological systems, for classifying cell states in disease and for many other purposes. Recent studies indicate that common assumptions currently embedded in experimental and analytical practices can lead to misinterpretation of global gene expression data. We discuss these assumptions and describe solutions that should minimize erroneous interpretation of gene expression data from multiple analysis platforms.
PMCID: PMC3505597  PMID: 23101621
9.  c-Myc is a universal amplifier of expressed genes in lymphocytes and embryonic stem cells 
Cell  2012;151(1):68-79.
The c-Myc HLH-bZIP protein has been implicated in physiological or pathological growth, proliferation, apoptosis, metabolism and differentiation at the cellular, tissue or organismal levels via regulation of numerous target genes. No principle yet unifies Myc action due partly to an incomplete inventory and functional accounting of Myc’s targets. To observe Myc target expression and function in a system where Myc is temporally and physiologically regulated, the transcriptomes and the genome-wide distributions of Myc, RNA polymerase II and chromatin modifications were compared during lymphocyte activation and in ES cells as well. A remarkably simple rule emerged from this quantitative analysis: Myc is not an on-off specifier of gene activity, but is a non-linear amplifier of expression, acting universally at active genes, except for immediate early genes that are strongly induced before Myc. This rule of Myc action explains the vast majority of Myc biology observed in literature.
PMCID: PMC3471363  PMID: 23021216
10.  Transcription dependent dynamic supercoiling is a short-range genomic force 
Transcription has the capacity to modify mechanically DNA topology, DNA structure, and nucleosome arrangement. Resulting from ongoing transcription, these modifications in turn, may provide instant feedback to the transcription machinery. To substantiate the connection between transcription and DNA dynamics, we charted an ENCODE map of transcription-dependent dynamic supercoiling in human Burkitt lymphoma cells using psoralen photobinding to probe DNA topology in vivo. Dynamic supercoils spread ~1.5 kb upstream of the start sites of active genes. Low and high output promoters handle this torsional stress differently as shown using inhibitors of transcription and topoisomerases, and by chromatin immunoprecipation of RNA polymerase and topoisomerases I and II. Whereas lower outputs are managed adequately by topoisomerase I, high output promoters additionally require topoisomerase II. The genome-wide coupling between transcription and DNA topology emphasizes the importance of dynamic supercoiling for gene regulation.
PMCID: PMC3594045  PMID: 23416947
11.  The importance of being supercoiled: how DNA mechanics regulate dynamic processes 
Biochimica et Biophysica Acta  2012;1819(7):632-638.
Through dynamic changes in structure resulting from DNA-protein interactions and constraints given by the structural features of the double helix, chromatin accommodates and regulates different DNA-dependent processes. All DNA transactions (such as transcription, DNA replication and chromosomal segregation) are necessarily linked to strong changes in the topological state of the double helix known as torsional stress or supercoiling. As virtually all DNA transactions are in turn affected by the torsional state of DNA, these changes have the potential to serve as regulatory signals detected by protein partners. This two-way relationship indicates that DNA dynamics may contribute to the regulation of many events occurring during cell life. In this review we will focus on the role of DNA supercoiling in the cellular processes, with particular emphasis on transcription. Besides giving an overview on the multiplicity of factors involved in the generation and dissipation of DNA torsional stress, we will discuss recent studies which give new insight into the way cells use DNA dynamics to perform functions otherwise not achievable.
PMCID: PMC3354648  PMID: 22233557
DNA supercoiling; DNA topology; Non-B DNA; Transcription
12.  Partition of Myc into Immobile vs. Mobile Complexes within Nuclei 
Scientific Reports  2013;3:1953.
Myc levels are highly regulated and usually low in vivo. Dimerized with Max, it regulates most expressed genes and so directly and indirectly controls most cellular processes. Intranuclear diffusion of a functional c-Myc-eGFP, expressed from its native locus in murine fibroblasts and 3T3 cells or by transient transfection, was monitored using Two Photon Fluorescence Correlation Spectroscopy, revealing concentration and size (mobility) of complexes. With increased c-Myc-eGFP, a very immobile pool saturates as a ‘mobile' pool increases. Both pools diffuse too slowly to be free Myc-Max dimers. Following serum stimulation, eGFP-c-Myc accumulated in the presence of the proteasome inhbitor MG132. Stimulating without MG132, Myc peaked at 2.5 hrs, and at steady was ~8 ± 1.3 nM. Inhbiting Myc-Max dimerization by Max-knockdown or drug treatment increased the ‘mobile' c-Myc pool size. These results indicate that Myc populates macromolecular complexes of widely heterogenous size and mobility in vivo.
PMCID: PMC3674427  PMID: 23739641
13.  The influence of DNA repair on neurological degeneration, cachexia, skin cancer and internal neoplasms: autopsy report of four xeroderma pigmentosum patients (XP-A, XP-C and XP-D) 
To investigate the association of DNA nucleotide excision repair (NER) defects with neurological degeneration, cachexia and cancer, we performed autopsies on 4 adult xeroderma pigmentosum (XP) patients with different clinical features and defects in NER complementation groups XP-A, XP-C or XP-D.
The XP-A (XP12BE) and XP-D (XP18BE) patients exhibited progressive neurological deterioration with sensorineural hearing loss. The clinical spectrum encompassed severe cachexia in the XP-A (XP12BE) patient, numerous skin cancers in the XP-A and two XP-C (XP24BE and XP1BE) patients and only few skin cancers in the XP-D patient. Two XP-C patients developed internal neoplasms including glioblastoma in XP24BE and uterine adenocarcinoma in XP1BE. At autopsy, the brains of the 44 yr XP-A and the 45 yr XP-D patients were profoundly atrophic and characterized microscopically by diffuse neuronal loss, myelin pallor and gliosis. Unlike the XP-A patient, the XP-D patient had a thickened calvarium, and the brain showed vacuolization of the neuropil in the cerebrum, cerebellum and brainstem, and patchy Purkinje cell loss. Axonal neuropathy and chronic denervation atrophy of the skeletal muscles were observed in the XP-A patient, but not in the XP-D patient.
These clinical manifestations and autopsy findings indicate advanced involvement of the central and peripheral nervous system. Despite similar defects in DNA repair, different clinicopathological phenotypes are seen in the four cases, and therefore distinct patterns of neurodegeneration characterize XP-D, XP-A and XP-C patients.
PMCID: PMC3776212  PMID: 24252196
DNA damage; DNA repair; Neurodegeneration; Glioblastoma; Cachexia
14.  The genome-wide distribution of non-B DNA motifs is shaped by operon structure and suggests the transcriptional importance of non-B DNA structures in Escherichia coli 
Nucleic Acids Research  2013;41(12):5965-5977.
Although the right-handed double helical B-form DNA is most common under physiological conditions, DNA is dynamic and can adopt a number of alternative structures, such as the four-stranded G-quadruplex, left-handed Z-DNA, cruciform and others. Active transcription necessitates strand separation and can induce such non-canonical forms at susceptible genomic sequences. Therefore, it has been speculated that these non-B DNA motifs can play regulatory roles in gene transcription. Such conjecture has been supported in higher eukaryotes by direct studies of several individual genes, as well as a number of large-scale analyses. However, the role of non-B DNA structures in many lower organisms, in particular proteobacteria, remains poorly understood and incompletely documented. In this study, we performed the first comprehensive study of the occurrence of B DNA–non-B DNA transition-susceptible sites (non-B DNA motifs) within the context of the operon structure of the Escherichia coli genome. We compared the distributions of non-B DNA motifs in the regulatory regions of operons with those from internal regions. We found an enrichment of some non-B DNA motifs in regulatory regions, and we show that this enrichment cannot be simply explained by base composition bias in these regions. We also showed that the distribution of several non-B DNA motifs within intergenic regions separating divergently oriented operons differs from the distribution found between convergent ones. In particular, we found a strong enrichment of cruciforms in the termination region of operons; this enrichment was observed for operons with Rho-dependent, as well as Rho-independent terminators. Finally, a preference for some non-B DNA motifs was observed near transcription factor-binding sites. Overall, the conspicuous enrichment of transition-susceptible sites in these specific regulatory regions suggests that non-B DNA structures may have roles in the transcriptional regulation of specific operons within the E. coli genome.
PMCID: PMC3695496  PMID: 23620297
15.  Thrombospondin-1 Signaling through CD47 Inhibits Self-renewal by Regulating c-Myc and Other Stem Cell Transcription Factors 
Scientific Reports  2013;3:1673.
Signaling through the thrombospondin-1 receptor CD47 broadly limits cell and tissue survival of stress, but the molecular mechanisms are incompletely understood. We now show that loss of CD47 permits sustained proliferation of primary murine endothelial cells, increases asymmetric division, and enables these cells to spontaneously reprogram to form multipotent embryoid body-like clusters. c-Myc, Klf4, Oct4, and Sox2 expression is elevated in CD47-null endothelial cells, in several tissues of CD47- and thrombospondin-1-null mice, and in a human T cell line lacking CD47. CD47 knockdown acutely increases mRNA levels of c-Myc and other stem cell transcription factors in cells and in vivo, whereas CD47 ligation by thrombospondin-1 suppresses c-Myc expression. The inhibitory effects of increasing CD47 levels can be overcome by maintaining c-Myc expression and are absent in cells with dysregulated c-Myc. Thus, CD47 antagonists enable cell self-renewal and reprogramming by overcoming negative regulation of c-Myc and other stem cell transcription factors.
PMCID: PMC3628113  PMID: 23591719
16.  Overexpression of the Far Upstream Element Binding Protein 1 in Hepatocellular Carcinoma Is Required for Tumor Growth 
Hepatology (Baltimore, Md.)  2009;50(4):1121-1129.
We identified the far upstream element binding protein 1 (FBP1), an activator of transcription of the proto-oncogene c-myc, in a functional yeast survival screen for tumor-related antiapoptotic proteins and demonstrated strong overexpression of FBP1 in human hepato-cellular carcinoma (HCC). Knockdown of the protein in HCC cells resulted in increased sensitivity to apoptotic stimuli, reduced cell proliferation, and impaired tumor formation in a mouse xenograft transplantation model. Interestingly, analysis of gene regulation in these cells revealed that c-myc levels were not influenced by FBP1 in HCC cells. Instead, we identified the cell cycle inhibitor p21 as a direct target gene repressed by FBP1, and in addition, expression levels of the proapoptotic genes tumor necrosis factor α, tumor necrosis factor–related apoptosis-inducing ligand, Noxa, and Bik were elevated in the absence of FBP1.
Our data establish FBP1 as an important oncoprotein overexpressed in HCC that induces tumor propagation through direct or indirect repression of cell cycle inhibitors and proapoptotic target genes.
PMCID: PMC3474328  PMID: 19637194
17.  Correction: Teasing Apart Translational and Transcriptional Components of Stochastic Variations in Eukaryotic Gene Expression 
PLoS Computational Biology  2012;8(10):10.1371/annotation/4498943d-525d-4ac1-8375-ae78e67ea0c8.
PMCID: PMC3476851
18.  Teasing Apart Translational and Transcriptional Components of Stochastic Variations in Eukaryotic Gene Expression 
PLoS Computational Biology  2012;8(8):e1002644.
The intrinsic stochasticity of gene expression leads to cell-to-cell variations, noise, in protein abundance. Several processes, including transcription, translation, and degradation of mRNA and proteins, can contribute to these variations. Recent single cell analyses of gene expression in yeast have uncovered a general trend where expression noise scales with protein abundance. This trend is consistent with a stochastic model of gene expression where mRNA copy number follows the random birth and death process. However, some deviations from this basic trend have also been observed, prompting questions about the contribution of gene-specific features to such deviations. For example, recent studies have pointed to the TATA box as a sequence feature that can influence expression noise by facilitating expression bursts. Transcription-originated noise can be potentially further amplified in translation. Therefore, we asked the question of to what extent sequence features known or postulated to accompany translation efficiency can also be associated with increase in noise strength and, on average, how such increase compares to the amplification associated with the TATA box. Untangling different components of expression noise is highly nontrivial, as they may be gene or gene-module specific. In particular, focusing on codon usage as one of the sequence features associated with efficient translation, we found that ribosomal genes display a different relationship between expression noise and codon usage as compared to other genes. Within nonribosomal genes we found that sequence high codon usage is correlated with increased noise relative to the average noise of proteins with the same abundance. Interestingly, by projecting the data on a theoretical model of gene expression, we found that the amplification of noise strength associated with codon usage is comparable to that of the TATA box, suggesting that the effect of translation on noise in eukaryotic gene expression might be more prominent than previously appreciated.
Author Summary
The stochastic nature of gene expression leads to cell-to-cell differences in protein level referred to as noise. Expression noise can be disadvantageous, by affecting the precision of biological functions, but it may also be advantageous by enabling heterogeneous stress-response programs to environmental changes. Therefore various genes and gene groups might display various levels of expression noise. Importantly, gene expression is a multi-step process and the stochasticity of its individual steps, including transcription and translation, contributes to the resulting variability. Recent single cell analyses of gene expression in yeast have confirmed the theoretically predicted general trend where expression noise scales with protein abundance. However, some deviations from this basic trend have also been observed, prompting questions about the contribution of gene-specific features to such deviations. Accounting for noise heterogeneity in different gene groups, we revealed a clear relationship between noise and translation-related genomic features, specifically codon usage and 5′ UTR secondary structure. Our results suggest that the effect of translation on these deviations might be more prominent than previously appreciated, and provide important clues towards understanding expression stochasticity in yeast.
PMCID: PMC3431295  PMID: 22956896
19.  Rapid genome-scale mapping of chromatin accessibility in tissue 
The challenge in extracting genome-wide chromatin features from limiting clinical samples poses a significant hurdle in identification of regulatory marks that impact the physiological or pathological state. Current methods that identify nuclease accessible chromatin are reliant on large amounts of purified nuclei as starting material. This complicates analysis of trace clinical tissue samples that are often stored frozen. We have developed an alternative nuclease based procedure to bypass nuclear preparation to interrogate nuclease accessible regions in frozen tissue samples.
Here we introduce a novel technique that specifically identifies Tissue Accessible Chromatin (TACh). The TACh method uses pulverized frozen tissue as starting material and employs one of the two robust endonucleases, Benzonase or Cyansase, which are fully active under a range of stringent conditions such as high levels of detergent and DTT. As a proof of principle we applied TACh to frozen mouse liver tissue. Combined with massive parallel sequencing TACh identifies accessible regions that are associated with euchromatic features and accessibility at transcriptional start sites correlates positively with levels of gene transcription. Accessible chromatin identified by TACh overlaps to a large extend with accessible chromatin identified by DNase I using nuclei purified from freshly isolated liver tissue as starting material. The similarities are most pronounced at highly accessible regions, whereas identification of less accessible regions tends to be more divergence between nucleases. Interestingly, we show that some of the differences between DNase I and Benzonase relate to their intrinsic sequence biases and accordingly accessibility of CpG islands is probed more efficiently using TACh.
The TACh methodology identifies accessible chromatin derived from frozen tissue samples. We propose that this simple, robust approach can be applied across a broad range of clinically relevant samples to allow demarcation of regulatory elements of considerable prognostic significance.
PMCID: PMC3464876  PMID: 22734930
Chromatin accessibility; Tissue; TACh; Benzonase; Cyanase; DNase I
20.  Cooperative Epigenetic Modulation by Cancer Amplicon Genes 
Cancer cell  2010;18(6):590-605.
Chromosome band 9p24 is frequently amplified in primary mediastinal B-cell lymphoma (PMBL) and Hodgkin lymphoma (HL). To identify oncogenes in this amplicon, we screened an RNA interference library targeting amplicon genes and thereby identified JAK2 and the histone demethylase JMJD2C as essential genes in these lymphomas. Inhibition of JAK2 and JMJD2C cooperated in killing these lymphomas by decreasing tyrosine 41 phosphorylation and increasing lysine 9 trimethylation of histone H3, promoting heterochromatin formation. MYC, a major target of JAK2-mediated histone phosphorylation, was silenced following JAK2 and JMJD2C inhibition, with a corresponding increase in repressive chromatin. Hence, JAK2 and JMJD2C cooperatively remodel the PMBL and HL epigenome, offering a mechanistic rationale for the development of JAK2 and JMJD2C inhibitors in these diseases.
PMCID: PMC3049192  PMID: 21156283
21.  You Don't Muck with MYC* 
Genes & cancer  2010;1(6):547-554.
MYC homeostasis is critical for major cellular and organismal processes. The physiological and pathologic patterns of c-myc transcription are programmed by a large number of cis-elements and transfactors (RNAs and proteins). These elements and factors receive inputs from a multitude of intracellular and extracellular pathways. Because c-myc regulation has customarily been dissected element by element and factor by factor, it has been difficult to appreciate how the c-myc promoter and regulatory sequences operate as a system. A full accounting of the regulation of c-myc transcription will require an understanding of the dynamic interplay of these factors and elements with one another, with chromatin, and with the changes in DNA structure and topology that are inevitably coupled with gene activity.
PMCID: PMC2946075  PMID: 20882108
c-myc promoter; noise; supercoiling; FUBP
22.  “You Don’t Muck with MYC”* 
Genes & Cancer  2010;1(6):547-554.
MYC homeostasis is critical for major cellular and organismal processes. The physiological and pathologic patterns of c-myc transcription are programmed by a large number of cis-elements and transfactors (RNAs and proteins). These elements and factors receive inputs from a multitude of intracellular and extracellular pathways. Because c-myc regulation has customarily been dissected element by element and factor by factor, it has been difficult to appreciate how the c-myc promoter and regulatory sequences operate as a system. A full accounting of the regulation of c-myc transcription will require an understanding of the dynamic interplay of these factors and elements with one another, with chromatin, and with the changes in DNA structure and topology that are inevitably coupled with gene activity.
PMCID: PMC2946075  PMID: 20882108
c-myc promoter; noise; supercoiling; FUBP
23.  How the c-myc Promoter Works and Why It Sometimes Does Not 
The c-myc promoter is regulated by scores of signals, transcription factors, and chromatin components. The logic integrating these multiple signals remains unexplored. Recent evidence suggests that activated MYC expression is regulated in several phases: 1) conventional transcription factors trigger transcription by the RNA polymerase II (pol II) paused within the proximal promoter region. Concurrently (and probably consequently), newly arrived chromatin-remodeling complexes mobilize a nucleo-some masking the far upstream element (FUSE), 1.7-kb upstream of the P2 start site; 2) binding by FUSE-binding proteins (first FBP3, then FBP); and 3) FBP-interacting repressor (FIR) binds FUSE and returns transcription to basal or steady-state levels. The recruitment and release of the FBPs and FIR is governed by FUSE-DNA conformation, itself controlled by dynamic supercoils propagated behind pol II. The organization and operation of the c-myc promoter make it difficult to inactivate, but sensitive to disturbances (translocations, viral insertions, amplification, and mutation) that disrupt the fine-tuning seen at its normal chromosomal context.
PMCID: PMC2683261  PMID: 18648001
24.  Dimerization of FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition 
The EMBO Journal  2007;27(1):277-289.
c-myc is essential for cell homeostasis and growth but lethal if improperly regulated. Transcription of this oncogene is governed by the counterbalancing forces of two proteins on TFIIH—the FUSE binding protein (FBP) and the FBP-interacting repressor (FIR). FBP and FIR recognize single-stranded DNA upstream of the P1 promoter, known as FUSE, and influence transcription by oppositely regulating TFIIH at the promoter site. Size exclusion chromatography coupled with light scattering reveals that an FIR dimer binds one molecule of single-stranded DNA. The crystal structure confirms that FIR binds FUSE as a dimer, and only the N-terminal RRM domain participates in nucleic acid recognition. Site-directed mutations of conserved residues in the first RRM domain reduce FIR's affinity for FUSE, while analogous mutations in the second RRM domain either destabilize the protein or have no effect on DNA binding. Oppositely oriented DNA on parallel binding sites of the FIR dimer results in spooling of a single strand of bound DNA, and suggests a mechanism for c-myc transcriptional control.
PMCID: PMC2206118  PMID: 18059478
c-myc; FBP interacting repressor; gene transcription; RRM domain; X-ray crystallography
25.  FBPs Are Calibrated Molecular Tools To Adjust Gene Expression†  
Molecular and Cellular Biology  2006;26(17):6584-6597.
The three far-upstream element (FUSE) binding protein (FBP) family members have been ascribed different functions in gene regulation. They were therefore examined with various biochemical, molecular biological, and cell biological tests to evaluate whether their sequence differences reflect functional customization or neutral changes at unselected residues. Each FBP displayed a characteristic profile of intrinsic transcription activation and repression, binding with protein partners, and subcellular trafficking. Although some differences, such as weakened FBP3 nuclear localization, were predictable from primary sequence differences, the unexpected failure of FBP3 to bind the FBP-interacting repressor (FIR) was traced to seemingly conservative substitutions within a small patch of an N-terminal α-helix. The transactivation strength and the FIR-binding strength of the FBPs were in the opposite order. Despite their distinguishing features and differential activities, the FBPs traffic to shared subnuclear sites and regulate many common target genes, including c-myc. Though a variety of functions have been attributed to the FBPs, based upon their panel of shared and unique features, we propose that they constitute a molecular regulatory kit that tunes the expression of shared targets through a common mechanism.
PMCID: PMC1592819  PMID: 16914741

Results 1-25 (28)