Elevated expression of the c-Myc transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome. The effect of high levels of c-Myc on global gene regulation is poorly understood, but is widely thought to involve newly activated or repressed “Myc target genes”. We report here that in tumor cells expressing high levels of c-Myc, the transcription factor accumulates in the promoter regions of active genes and causes transcriptional amplification, producing increased levels of transcripts within the cell's gene expression program. Thus, rather than binding and regulating a new set of genes, c-Myc amplifies the output of the existing gene expression program. These results provide an explanation for the diverse effects of oncogenic c-Myc on gene expression in different tumor cells and suggest that transcriptional amplification reduces rate-limiting constraints for tumor cell growth and proliferation.
Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated athistoneH3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.
AL-38022A is a novel synthetic serotonergic (5-HT) ligand that exhibited high affinity for each of the 5-HT2 receptor subtypes (Ki ≤ 2.2 nM), but a significantly lower (>100-fold less) affinity for other 5-HT receptors. In addition, AL-38022A displayed a very low affinity for a broad array of other receptors, neurotransmitter transport sites, ion channels, and second messenger elements, making it a relatively selective agent. AL-38022A potently stimulated functional responses via native and cloned rat (EC50 range: 1.9 – 22.5 nM) and human (EC50 range: 0.5 – 2.2 nM) 5-HT2 receptor subtypes including [Ca2+]i mobilization and tissue contractions with apparently similar potencies and intrinsic activities and was a full agonist at all 5-HT2 receptor subtypes. The CNS activity of AL-38022A was assessed by evaluating its discriminative stimulus effects in both a rat and a monkey drug discrimination paradigm using DOM as the training drug. AL-38022A fully generalized to the DOM stimulus in each of these studies; in monkeys MDL 100907 antagonized both DOM and AL-38022A. The pharmacological profile of AL-38022A suggests that it could be a useful tool in defining 5-HT2 receptor signaling and receptor characterization where 5-HT may function as a neurotransmitter.
Drug discrimination; 5-HT2 receptor; R-DOI; DOM; MDL 100907; rat; monkey
Melanoma is a tumor of transformed melanocytes, which are derived from the embryonic neural crest. It is unknown to what extent the programs regulating neural crest development interact with mutations in the BRAF oncogene, the gene most commonly mutated in human melanoma1. We have utilized the zebrafish embryo to identify initiating transcriptional events upon BRAFV600E activation in the neural crest lineage. Transgenic mitf-BRAFV600E;p53-/- zebrafish embryos demonstrate a gene signature enriched for markers of multipotent neural crest cells, and exhibit a failure of terminal differentiation of neural crest progenitors. To determine if these early transcriptional events were important for melanoma pathogenesis, we performed a chemical genetic screen to identify small molecule suppressors of the neural crest lineage, which were then tested for effects in melanoma. One class of compounds, inhibitors of dihydroorotate dehydrogenase (DHODH) such as leflunomide, led to an almost complete abrogation of neural crest development in the zebrafish and a reduction in self-renewal of mammalian neural crest stem cells. Leflunomide exerts these effects by inhibiting transcriptional elongation of genes required for neural crest development and melanoma growth. When used alone or in combination with a specific inhibitor of the BRAFV600E oncogene, DHODH inhibition led to a marked decrease in melanoma growth both in vitro and in mouse xenograft studies. Taken together, these studies highlight developmental pathways in neural crest cells that have direct bearing upon subsequent melanoma formation.
The oncogenic transcription factor TAL1/SCL is aberrantly expressed in over 40% of cases of human T-cell acute lymphoblastic leukemia (T-ALL), emphasizing its importance in the molecular pathogenesis of T-ALL. Here we identify the core transcriptional regulatory circuit controlled by TAL1 and its regulatory partners HEB, E2A, LMO1/2, GATA3 and RUNX1. We show that TAL1 forms a positive interconnected auto-regulatory loop with GATA3 and RUNX1, and that the TAL1 complex directly activates the MYB oncogene, forming a positive feed-forward regulatory loop that reinforces and stabilizes the TAL1-regulated oncogenic program. One of the critical downstream targets in this circuitry is the TRIB2 gene, which is oppositely regulated by TAL1 and E2A/HEB and is essential for the survival of T-ALL cells.
Control of gene expression during development requires the concerted action of sequence-specific transcriptional regulators and epigenetic modifiers, which are spatially coordinated within the nucleus through mechanisms that are poorly understood. Here we show that transcriptional repression by the Msx1 homeoprotein in myoblast cells requires the recruitment of Polycomb to target genes located at the nuclear periphery. Target genes repressed by Msx1 display an Msx1-dependent enrichment of Polycomb-directed trimethylation of lysine 27 on histone H3 (H3K27me3). Association of Msx1 with the Polycomb complex is required for repression and regulation of myoblast differentiation. Furthermore, Msx1 promotes a dynamic spatial redistribution of the H3K27me3 repressive mark to the nuclear periphery in myoblast cells and the developing limb in vivo. Our findings illustrate a hitherto unappreciated spatial coordination of transcription factors with the Polycomb complex for appropriate regulation of gene expression programs during development.
HGF is a hypoxia-induced secreted protein that binds to cMET and regulates IL8 expression. We evaluated the role of circulating HGF and IL8 as prognostic and predictive factors for efficacy of tirapazamine (TPZ), a hypoxic cell cytotoxin.
Patients with Stage III–IV head and neck cancer were randomized to receive radiotherapy with cisplatin (CIS) or cisplatin plus TPZ (TPZ/CIS). Eligibility for the substudy included plasma sample availability for HGF and IL8 assay by ELISA and no major radiation deviations (N=498). Analyses included adjustment for major prognostic factors. p16INK4A staining (HPV surrogate) was performed on available tumors. 39 patients had hypoxia imaging with 18FAZA-PET.
Elevated IL8 level was associated with worse overall survival (OS) irrespective of treatment. There was an interaction between HGF and treatment arm (p=0.053): elevated HGF was associated with worse OS in the control but not in the TPZ/CIS arm. Similar trends were observed in analyses restricted to p16INK4A negative patients. Four subgroups defined by high and low HGF/IL8 levels were examined for TPZ effect; the test for interaction with arm was p=0.099. TPZ/CIS appeared to be beneficial for patients with high HGF and IL8, but adverse for low HGF and high IL8. Only HGF correlated with 18FAZA tumor SUV.
IL8 is an independent prognostic factor irrespective of treatment. There is an interaction between HGF and treatment arm. Certain subgroups based on IL8/HGF levels appeared to do better with TPZ/CIS while others do worse; highlighting the complexity of hypoxia targeting in unselected patients.
Hypoxia; head and neck cancer; Hepatocyte Growth Factor; Interleukin-8; Plasma
Most human genes are loaded with promoter proximally paused RNA polymerase II (Pol II) molecules that are poised for release into productive elongation by P-TEFb. We present evidence that Gdown1, a protein that renders Pol II responsive to mediator, is involved in Pol II elongation control. During in vitro transcription assays Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. Supporting these mechanistic insights, ChIP-Seq demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Our results establish that Gdown1 increases the stability of poised polymerases while maintaining their responsiveness to P-TEFb and suggest that mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function.
High plasma osteopontin (OPN) levels have been reported to be an adverse prognostic factor in HNSCC, correlate with tumor hypoxia and be predictive of benefit from hypoxia targeted therapy. We sought to confirm the prognostic and predictive significance of OPN in patients treated on a large international trial.
Patients with Stage III/IV HNSCC were randomized to receive definitive radiotherapy concurrently with cisplatin or cisplatin plus the hypoxic cell cytotoxin, tirapazamine (TPZ). Eligibility criteria for this prospective substudy included plasma sample availability for OPN assay by ELISA and absence of major radiation therapy deviations (N=578). OPN concentrations were analysed for overall survival (OS) and time to locoregional failure (TTLRF), adjusting for known prognostic factors. Additional analysis was performed in patients with available tumor p16 INK4A staining status.
The median OPN level was 544ng/mL (range: 7–2640). High OPN levels were not associated with worse OS (relative HR, 1.05 for highest tertile) or TTLRF (relative HR 0.90 for highest tertile). There was no interaction between OPN and treatment arm for OS or TTLRF (p=0.94 for OS; p=0.85 for TTLRF). For the highest tertile the 2-year OS was 66% on control arm and 67% on TPZ arm (HR=1.11, p=0.67). Similarly for p16 INK4A negative patients in the highest tertile, the 2-year OS was 61% on control arm and 63% on TPZ arm (HR=1.05, p=0.86).
We found no evidence that high plasma OPN levels were associated with an adverse prognosis in HNSCC, or were predictive of benefit with hypoxia targeting therapy.
“Osteopontin”; “Head and neck squamous cell carcinoma”; “prognostic”; “predictive”; “biomarker”
Transforming growth factor beta (TGF-β) signaling, mediated through the transcription factors Smad2 and Smad3 (Smad2/3), directs different responses in different cell types. Here we report that Smad3 co-occupies the genome with cell-type-specific master transcription factors. Thus, Smad3 occupies the genome with Oct4 in embryonic stem (ES) cells, Myod1 in myotubes, and PU.1 in pro-B cells. We find that these master transcription factors are required for Smad3 occupancy and that TGF-β signaling largely affects the genes bound by the master transcription factors. Furthermore, we show that induction of Myod1 in non-muscle cells is sufficient to re-direct Smad3 to Myod1 sites. We conclude that cell-type-specific master transcription factors determine the genes bound by Smad2/3 and are thus responsible for orchestrating the cell-type-specific effects of TGF-β signaling.
MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative co-activator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.
In mammalian embryonic stem cells, the acquisition of pluripotency is dependent upon Nanog, but the in vivo analysis of Nanog has been hampered by its requirement for early mouse development. In an effort to examine the role of Nanog in vivo, we identified a zebrafish Nanog ortholog, and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extra-embryonic yolk syncytial layer (YSL), which produces Nodal required for endoderm induction. We examined the genes that were regulated by Nanog-like, and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which in turn specifies the YSL lineage by directly activating YSL genes. Our study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extra-embryonic tissue required for endoderm induction.
Using mouse skin, where bountiful reservoirs of synchronized hair follicle stem cells (HF-SCs) fuel cycles of regeneration, we explore how adult SCs remodel chromatin in response to activating cues. By profiling global mRNA and chromatin changes in quiescent and activated HF-SCs and their committed, transit-amplifying (TA) progeny, we show that polycomb-group(PcG)-mediated H3K27-trimethylation features prominently in HF-lineage progression by mechanisms distinct from embryonic-SCs. In HF-SCs, PcG represses non-skin lineages and HF-differentiation. In TA-progeny, non-skin regulators remain PcG-repressed, HF-SC regulators acquire H3K27me3-marks and HF-lineage regulators lose them. Interestingly, genes poised in embryonic-SCs, active in HF-SCs and PcGrepressed in TA-progeny, encode not only key transcription factors, but also signaling regulators. We document their importance in balancing HF-SC quiescence, underscoring the power of chromatin mapping in dissecting SC behavior. Our findings explain how HF-SCs cycle through quiescent and activated states without losing stemness, and define roles for PcG-mediated repression in governing a fate switch irreversibly.
Study of developmentally regulated transcription factors by chromatin immunoprecipitation and sequencing (ChIP-seq) faces two major obstacles: availability of ChIP grade antibodies and access to sufficient number of cells. We describe a versatile method for genome-wide analysis of transcription factor binding sites by combining directed differentiation of embryonic stem cells and inducible expression of tagged proteins. We demonstrate its utility by mapping transcription factors involved in motor neuron specification.
The most common mutation in melanoma, BRAF(V600E), activates the BRAF serine/threonine kinase and causes excessive MAPK pathway activity1,2. BRAF(V600E)mutations are also present in benign melanocytic nevi3, highlighting the importance of additional genetic alterations in the genesis of malignant tumors. Such changes include recurrent copy number variations that result in the amplification of oncogenes4,5. For certain amplifications, the large number of genes in the interval has precluded an understanding of cooperating oncogenic events. Here, we have used a zebrafish melanoma model to test genes in a recurrently amplified region on chromosome 1 for the ability to cooperate with BRAF(V600E) and accelerate melanoma. SETDB1, an enzyme that methylates histone H3 on lysine 9 (H3K9), was found to significantly accelerate melanoma formation in the zebrafish. Chromatin immunoprecipitation coupled with massively parallel DNA sequencing (ChIP-Seq) and gene expression analyses revealed target genes, including Hox genes, that are transcriptionally dysregulated in response to elevated SETDB1. Our studies establish SETDB1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.
BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, while expression of the erythroid regulator GATA1 directs SMAD1 loss on non-erythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.
Embryonic stem cells and induced pluripotent stem cells, which can be propagated in culture in an undifferentiated state but induced to differentiate into specialized cell types, hold great promise for regenerative medicine. Moreover, these cells provide a powerful model system for studies of cellular identity and early mammalian development. Insights into the transcriptional control of embryonic stem cell state, including the regulatory circuitry underlying pluripotency and cellular reprogramming, have emerged from the study of these cells. These studies have also revealed fundamental mechanisms that control vertebrate gene expression, connect gene expression to chromosome structure, and contribute to human disease.
While many individual transcription factors are known to regulate hematopoietic differentiation, major aspects of the global architecture of hematopoiesis remain unknown. Here, we profiled gene expression in 38 distinct purified populations of human hematopoietic cells and used probabilistic models of gene expression and analysis of cis-elements in gene promoters to decipher the general organization of their regulatory circuitry. We identified modules of highly co-expressed genes, some of which are restricted to a single lineage, but most are expressed at variable levels across multiple lineages. We found densely interconnected cis-regulatory circuits and a large number of transcription factors that are differentially expressed across hematopoietic states. These findings suggest a more complex regulatory system for hematopoiesis than previously assumed.
Co-administration of the 5-HT1A serotonin receptor agonist (±)8-hydroxy-2-(N,N-di-n-propylamino) tetralin [(±)8-OH DPAT] enhances the discriminative stimulus effects of the classical hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM) in rats. In the present investigation, using Sprague-Dawley rats trained to discriminate DOM (1.0 mg/kg) from saline vehicle under a VI-15s schedule of reinforcement, it was shown that the stimulus-enhancing actions of 8-OH DPAT are related more to its R(+)-isomer than to its S(−)-enantiomer, and that the (±)- and R(+)8-OH DPAT-induced effects are antagonized by the 5-HT1A receptor antagonist NAN-190. (±)8-OH DPAT and its isomers substitute in rats trained to discriminate the designer drug N-methyl-1-(3,4-methylenedioxyphenyl)-2-aminopropane (MDMA; methylenedioxymethamphetamine) from vehicle indicating some similarity of effect. On this basis, it was hypothesized that MDMA might be capable of enhancing the DOM stimulus. Co-administration of MDMA with low (i.e., 0.1 and 0.3 mg/kg) doses of DOM resulted in greater DOM-appropriate responding than engendered by administration of DOM alone. As such, the present findings are the first to demonstrate an MDMA-induced enhancing effect on the discriminative stimulus actions of a classical hallucinogen. The results also suggest that a 5-HT1A serotonin receptor mechanism might contribute to this phenomenon.
DOM; MDMA; (±)8-OH DPAT; R(+)8-OH DPAT; S(−)8-OH DPAT; NAN-190; Methylenedioxymethamphetamine; Hallucinogens; Drug discrimination
Residency programs desire assessment tools for teaching and measuring resident attainment of the Accreditation Council for Graduate Medical Education competencies, including interpersonal and communication skills.
We sought to evaluate the use of emotional intelligence (EI) assessment and training tools in assessing and enhancing interpersonal and communication skills.
We used a quasi-experimental design, with an intervention and control group composed of 1 class each of family medicine residents. The intervention was EI coaching. The assessment used the Emotional and Social Competence Inventory, a 360-degree EI survey consisting of self and other (colleague) ratings for 12 EI competencies.
There were 21 participants in each of the 3 assessments (test, posttest, and control). Our EI coaching intervention had very limited participation due to a lack of protected time for EI coaching and residents' competing obligations. Return rates for self surveys were 86% to 91% and 66% to 68% for others. On all 3 trials, ratings by others were significantly higher than self ratings for every competence (range, P < .001–.045). None of the self ratings by the intervention group increased significantly for any of the competencies. None of the intervention group self ratings increased significantly on posttesting, whereas ratings by others increased significantly for coach/mentor (P < .001). The teamwork rating decreased significantly on both self and other ratings (P < .001). Achievement orientation was the highest intervention group posttest rating, and teamwork was the lowest.
EI is a necessary skill in today's health care environment, and our study found that a tool from another sector was useful in assessing resident EI skills. Because our EI coaching intervention was unsuccessful, the effects of coaching on interpersonal and communication skills could not be assessed.
We describe the results of a genome-wide analysis of human cells that suggests that most protein-coding genes, including most genes thought to be transcriptionally inactive, experience transcription initiation. We found that nucleosomes with H3K4me3 and H3K9,14Ac modifications, together with RNA polymerase II, occupy the promoters of most protein-coding genes in human embryonic stem cells. Only a subset of these genes produce detectable full-length transcripts and are occupied by nucleosomes with H3K36me3 modifications, a hallmark of elongation. The other genes experience transcription initiation but show no evidence of elongation, suggesting that they are predominantly regulated at post-initiation steps. Genes encoding most developmental regulators fall into this group. Our results also identify a class of genes that are excluded from experiencing transcription initiation, at which mechanisms that prevent initiation must predominate. These observations extend to differentiated cells, suggesting that transcription initiation at most genes is a general phenomenon in human cells.
To celebrate the first 10 years of Nature Reviews Genetics, we asked eight leading researchers for their views on the key developments in genetics and genomics in the past decade and the prospects for the future. Their responses highlight the incredible changes that the field has seen, from the explosion of genomic data and the many possibilities it has opened up to the ability to reprogramme adult cells to pluripotency. The way ahead looks similarly exciting as we address questions such as how cells function as systems and how complex interactions among genetics, epigenetics and the environment combine to shape phenotypes.
To determine the prognostic importance of p16 and human papillomavirus (HPV) in patients with oropharyngeal cancer treated on a phase III concurrent chemoradiotherapy trial.
Patients and Methods
Patients with stage III or IV head and neck squamous cell cancer were randomly assigned to concurrent radiotherapy and cisplatin with or without tirapazamine. In this substudy, analyses were restricted to patients with oropharyngeal cancer. p16 was detected by immunohistochemistry, and HPV was detected by in situ hybridization and polymerase chain reaction.
Slides were available for p16 assay in 206 of 465 patients, of which 185 were eligible, and p16 and HPV were evaluable in 172 patients. One hundred six (57%) of 185 were p16-positive, and in patients evaluable for both p16 and HPV, 88 (86%) of 102 p16-positive patients were also HPV-positive. Patients who were p16-positive had lower T and higher N categories and better Eastern Cooperative Oncology Group (ECOG) performance status. p16-positive tumors compared with p16-negative tumors were associated with better 2-year overall survival (91% v 74%; hazard ratio [HR], 0.36; 95% CI, 0.17 to 0.74; P = .004) and failure-free survival (87% v 72%; HR, 0.39; 95% CI, 0.20 to 0.74; P = .003). p16 was a significant prognostic factor on multivariable analysis (HR, 0.45; 95% CI, 0.21 to 0.96; P = .04). p16-positive patients had lower rates of locoregional failure and deaths due to other causes. There was a trend favoring the tirapazamine arm for improved locoregional control in p16-negative patients (HR, 0.33; 95% CI, 0.09 to 1.24; P = .13).
HPV-associated oropharyngeal cancer is a distinct entity with a favorable prognosis compared with HPV-negative oropharyngeal cancer when treated with cisplatin-based chemoradiotherapy.
A surprising portion of both mammalian and Drosophila genomes are transcriptionally paused, undergoing initiation without elongation. We tested the hypothesis that transcriptional pausing is an obligate transition state between definitive activation and silencing as human embryonic stem cells (hESCs) change state from pluripotency to mesoderm. Chromatin immunoprecipitation for trimethyl lysine 4 on histone H3 (ChIP-Chip) was used to analyze transcriptional initiation, and 3′ transcript arrays were used to determine transcript elongation. Pluripotent and mesodermal cells had equivalent fractions of the genome in active and paused transcriptional states (∼48% each), with ∼4% definitively silenced (neither initiation nor elongation). Differentiation to mesoderm changed the transcriptional state of 12% of the genome, with roughly equal numbers of genes moving toward activation or silencing. Interestingly, almost all loci (98–99%) changing transcriptional state do so either by entering or exiting the paused state. A majority of these transitions involve either loss of initiation, as genes specifying alternate lineages are archived, or gain of initiation, in anticipation of future full-length expression. The addition of chromatin dynamics permitted much earlier predictions of final cell fate compared to sole use of conventional transcript arrays. These findings indicate that the paused state may be the major transition state for genes changing expression during differentiation, and implicate control of transcriptional elongation as a key checkpoint in lineage specification.