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.
Knowledge of both the global chromatin structure and the gene expression programs of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) should provide a robust means to assess whether the genomes of these cells have similar pluripotent states. Recent studies have suggested that ESCs and iPSCs represent different pluripotent states with substantially different gene expression profiles. We describe here a comparison of global chromatin structure and gene expression data for a panel of human ESCs and iPSCs. Genome-wide maps of nucleosomes with histone H3K4me3 and H3K27me3 modifications indicate that there is little difference between ESCs and iPSCs with respect to these marks. Gene expression profiles confirm that the transcriptional programs of ESCs and iPSCs show very few consistent differences. Although some variation in chromatin structure and gene expression was observed in these cell lines, these variations did not serve to distinguish ESCs from iPSCs.
Polycomb proteins maintain cell identity by repressing the expression of developmental regulators specific for other cell types. Polycomb repressive complex-2 (PRC2) catalyses trimethylation of histone H3 lysine-27 (H3K27me3). Although repressed, PRC2 targets are generally associated with the transcriptional initiation marker H3K4me3 but the significance of this remains unclear. Here, we identify a new class of short RNAs, ~50-200 nucleotides in length, transcribed from the 5′-end of polycomb target genes in primary T-cells and embryonic stem cells. Short RNA transcription is associated with RNA polymerase II and H3K4me3, occurs in the absence of mRNA transcription and is independent of polycomb activity. Short RNAs form stem-loop structures resembling PRC2 binding sites in Xist, interact with PRC2 through SUZ12, cause gene repression in cis and are depleted from polycomb target genes activated during cell differentiation. We propose that short RNAs play a role in the association of PRC2 with its target genes.
Recruitment of the RNA Polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA binding transcription factors is well recognized as a key regulatory step in gene expression. We report here that promoter-proximal pausing is a general feature of transcription by Pol II in mammalian cells, and thus an additional step where regulation of gene expression occurs. This suggests that some transcription factors recruit the transcription apparatus to promoters, while others effect promoter-proximal pause release. Indeed, we find that the transcription factor c-Myc, a key regulator of cellular proliferation, plays a major role in Pol II pause release rather than Pol II recruitment at its target genes. We discuss the implications of these results for the role of c-Myc amplification in human cancer.
Habitat destruction and predation by invasive alien species has led to the disappearance of several island populations of Darwin's finches but to date none of the 13 recognized species have gone extinct. However, driven by rapid economic growth in the Galápagos, the effects of introduced species have accelerated and severely threatened these iconic birds. The critically endangered mangrove finch (Camarhynchus heliobates) is now confined to three small mangroves on Isabela Island. During 2006–2009, we assessed its population status and monitored nesting success, both before and after rat poisoning. Population size was estimated at around only 100 birds for the two main breeding sites, with possibly 5–10 birds surviving at a third mangrove. Before rat control, 54 per cent of nests during incubation phase were predated with only 18 per cent of nests producing fledglings. Post-rat control, nest predation during the incubation phase fell to 30 per cent with 37 per cent of nests producing fledglings. During the nestling phase, infestation by larvae of the introduced parasitic fly (Philornis downsi) caused 14 per cent additional mortality. Using population viability analysis, we simulated the probability of population persistence under various scenarios of control and showed that with effective management of these invasive species, mangrove finch populations should start to recover.
Camarhynchus heliobates; Galápagos; Philornis; population monitoring; population viability analysis; rat control
Transcription factors control cell specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. We report here that Mediator and Cohesin physically and functionally connect the enhancers and core promoters of active genes in embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with Cohesin, which can form rings that connect two DNA segments. The Cohesin loading factor Nipbl is associated with Mediator/Cohesin complexes, providing a means to load Cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by Mediator and Cohesin. Mediator and Cohesin occupy different promoters in different cells, thus generating cell-type specific DNA loops linked to the gene expression program of each cell.