Highly parallel assays of tissue-specific enhancers in whole Drosophila embryos

Transcriptional enhancers are a primary mechanism by which tissue-specific gene expression is achieved. Despite the importance of these regulatory elements in development, responses to environmental stresses, and disease, testing enhancer activity in animals remains tedious, with a minority of enhancers having been characterized. Here, we have developed ‘enhancer-FACS-Seq’ (eFS) technology for highly parallel identification of active, tissue-specific enhancers in Drosophila embryos. Analysis of enhancers identified by eFS to be active in mesodermal tissues revealed enriched DNA binding site motifs of known and putative, novel mesodermal transcription factors (TFs). Naïve Bayes classifiers using TF binding site motifs accurately predicted mesodermal enhancer activity. Application of eFS to other cell types and organisms should accelerate the cataloging of enhancers and understanding how transcriptional regulation is encoded within them

Contribution of Distinct Homeodomain DNA Binding Specificities to Drosophila Embryonic Mesodermal Cell-Specific Gene Expression Programs

Homeodomain (HD) proteins are a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, often acting within the same cell types, yet many members of this family paradoxically recognize similar DNA sequences. Thus, with multiple family members having the potential to recognize the same DNA sequences in cis-regulatory elements, it is difficult to ascertain the role of an individual HD or a subclass of HDs in mediating a particular developmental function. To investigate this problem, we focused our studies on the Drosophila embryonic mesoderm where HD TFs are required to establish not only segmental identities (such as the Hox TFs), but also tissue and cell fate specification and differentiation (such as the NK-2 HDs, Six HDs and identity HDs (I-HDs)). Here we utilized the complete spectrum of DNA binding specificities determined by protein binding microarrays (PBMs) for a diverse collection of HDs to modify the nucleotide sequences of numerous mesodermal enhancers to be recognized by either no or a single subclass of HDs, and subsequently assayed the consequences of these changes on enhancer function in transgenic reporter assays. These studies show that individual mesodermal enhancers receive separate transcriptional input from both I–HD and Hox subclasses of HDs. In addition, we demonstrate that enhancers regulating upstream components of the mesodermal regulatory network are targeted by the Six class of HDs. Finally, we establish the necessity of NK-2 HD binding sequences to activate gene expression in multiple mesodermal tissues, supporting a potential role for the NK-2 HD TF Tinman (Tin) as a pioneer factor that cooperates with other factors to regulate cell-specific gene expression programs. Collectively, these results underscore the critical role played by HDs of multiple subclasses in inducing the unique genetic programs of individual mesodermal cells, and in coordinating the gene regulatory networks directing mesoderm development.National Institutes of Health (U.S.) (Grant R01 HG005287

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Modulation of Translation Efficiency in S. cerevisiae by Codon Pairs and mRNA Structure

Thesis (Ph.D.)--University of Washington, 2016-06Synonymous codon choice modulates translation, but the properties of codons or codon combinations that result in impaired translation are not understood. We scored expression of 35,811 three-codon insertions in GFP in Saccharomyces cerevisiae and evaluated these variants for codon usage and RNA structure effects on GFP fluorescence levels. We have established that codon pairs affect translation elongation and efficiency in yeast in a manner distinct from the effects of individual codon tRNA abundance. Also, similar to previous studies in bacteria, we have found that the base-pairing status of nucleotides near the translation start site is likely to impair translation initiation. Both inhibitory codon pairs and 5’ mRNA structure can impose substantial limitations on translation efficiency through synonymous variation. For 17 inhibitory codon pairs, we show that it is the pair, rather than the dipeptide, the 6-base sequence, or the two individual codons, that is responsible for inhibition. Variants from the GFP insertion library that had an inhibitory pair had significantly lower expression than variants in which: the 6 base sequences were out of frame; the two codons were present but separated; or one of the codons of the pair was instead an optimal codon. We find that the inhibitory pairs act in translation, based on both suppression of inhibition by over expressed tRNA (11/12 tested) and the reduction in translation speeds relative to synonymous dipeptide sequences, as observed from ribosome occupancies along yeast transcripts. Furthermore, for 12 of the 17 pairs, preserving the order of codons in the pair was required for strong inhibitory effects. Thus the position of inhibitory pairs within the ribosome is likely a key factor in translation efficiency. Moreover, the identity of codons in inhibitory pairs is inconsistent with an inhibition mechanism governed primarily by limited tRNA supply. Rather, our data implicates wobble decoding and interactions between adjacent sites in the ribosome. The high-throughput experimental analysis described here has resulted in the direct and extensive identification of multiple inhibitory codon pairs, a quantitative analysis of their relative effects on translation in vivo, and tests of their activity as modulators of translation

Targeted mutagenesis of different classes of HD binding sites in the <i>ap</i> muscle enhancer.

<p>E-score (y-axis) binding profiles of the indicated HD TFs within a particular 22 base pair segment of the entire wild-type (WT) <i>Ndg</i> enhancer (A) and versions in which all I-HD plus Hox (“noHD”, B), all Hox (“noHox”, C), or all I-HD (“noI-HD”, D) binding sites are mutated. The mutant in which all NK-2 binding sites are mutated are wild-type for these other HD TFs (“noNK-2”, E). The horizontal black line represents a threshold E-score of 0.31 below which binding is not considered significant, and was chosen as described in the Materials and Methods [5]. The effects of E-score binding profiles for additional HD TFs, as well as additional mutants investigated in the current study, and the entirety of the <i>Ndg</i> enhancer are shown in Figures S1-S4. See Materials and Methods for details of mutagenesis design and Table S2 for the actual nucleotide sequences that were investigated.</p

Hox binding sites are required for the full activities of all tested mesodermal enhancers.

<p>(A) Loss of ßgal reporter (green) in the Lb-expressing SBM (magneta) driven by a version of the <i>lbl</i> enhancer in which the Hox binding sites are mutated (<i>lbl^noHox-lacZ</i>) in stage 14 embryos. Compare to the WT version of the <i>lbl</i> enhancer (<i>lbl^WT-lacZ</i>) in Figure 2A. (B) Loss of ßgal reporter (green) in the <i>ap</i> enhancer in which the Hox binding sites are mutated (<i>ap^noHox-lacZ</i>) in stage 14 embryos. Compare to the WT version of the <i>ap</i> enhancer (<i>ap^WT-lacZ</i>) in Figure 2C. (C) Attenuation of GFP (green) driven by a version of the <i>mib2</i> enhancer in which Hox binding sites are inactivated (<i>mib2^noHox-GFP</i>) as compared to ßgal (magneta) driven by a WT version of the <i>mib2</i> enhancer (<i>mib2^WT-lacZ</i>) in stage 12 embryos. Compare to WT versions of both GFP and lacZ reporters in Figure 2E. (D) Attenuation of GFP (green) driven by a version of the <i>Ndg</i> enhancer in which Hox binding sites are inactivated (<i>Ndg^noHox-GFP</i>) as compared to ßgal (magneta) driven by a WT version of the <i>Ndg</i> enhancer (<i>Ndg^WT-lacZ</i>) in stage 12 embryos. The ventral <i>Ndg</i> reporter-expressing cells are not in the indicated focal plane but do not express the GFP reporter (data not shown). Compare to WT versions of both GFP and lacZ reporters in Figure 2G.</p

Requirements for NK-2 binding sites for the full activities of multiple tested mesodermal enhancers.

<p>(A) Loss of ßgal reporter (green) in the Lb-expressing SBM (magneta) driven by a version of the <i>lbl</i> enhancer in which the Tin binding sites are inactivated (<i>lbl^noNK-2-lacZ</i>) in stage 14 embryos. Compare to the WT version of the <i>lbl</i> enhancer (<i>lbl^WT-lacZ</i>) in Figure 2A. (B) Normal GFP reporter (green) activity in the <i>ap</i> enhancer in which the Tin binding sites are mutated (<i>ap^noNK-2-GFP</i>) in stage 14 embryos. Compare to the WT version of the <i>ap</i> enhancer (<i>ap^WT-GFP</i>) in Figure 2C. (C) Attenuation of GFP (green) driven by a version of the <i>mib2</i> enhancer in which Tin binding sites are inactivated (<i>mib2^noNK-2-GFP</i>) as compared to ßgal (magneta) driven by a WT version of the <i>mib2</i> enhancer (<i>mib2^WT-lacZ</i>) in stage 12 embryos. Compare to WT versions of both GFP and lacZ reporters in Figure 2E. (D) Loss of GFP (green) driven by a version of the <i>Ndg</i> enhancer in which Tin binding sites are mutated (<i>Ndg^noNK-2-GFP</i>) as compared to ßgal (magneta) driven by a WT version of the <i>Ndg</i> enhancer (<i>Ndg^WT-lacZ</i>) in stage 12 embryos. The ventral <i>Ndg</i> reporter-expressing cells are not in this focal plane but do not express the GFP reporter (data not shown). Compare to WT versions of both GFP and lacZ reporters in Figure 2G.</p

I–HD binding sites are required for the full activities of all tested mesodermal enhancers.

<p>(A) Loss of ßgal reporter (green) in the Lb-expressing SBM (magneta) driven by a version of the <i>lbl</i> enhancer in which the I–HD binding sites are inactivated (<i>lbl^noI-HD-lacZ</i>) in stage 14 embryos. Compare to the WT version of the <i>lbl</i> enhancer (<i>lbl^WT-lacZ</i>) in Figure 2A. Asterix indicate ßgal-expressing myotube VT1. (B) Loss of ßgal reporter (green) in the <i>ap</i> enhancer in which the I–HD binding sites are mutated (<i>ap^noI-HD-lacZ</i>) in stage 14 embryos. Compare to the WT version of the <i>ap</i> enhancer (<i>ap^WT-lacZ</i>) in Figure 2C. (C) Attenuation of GFP (green) driven by a version of the <i>mib2</i> enhancer in which FCI-HD binding sites are inactivated (<i>mib2^noI-HD-GFP</i>) as compared to ßgal (magneta) driven by a WT version of the <i>mib2</i> enhancer (<i>mib2^WT-lacZ</i>) in stage 12 embryos. Compare to WT versions of both GFP and lacZ reporters in Figure 2E. (D) Loss of GFP (green) driven by a version of the <i>Ndg</i> enhancer in which I–HD binding sites are inactivated (<i>Ndg^noI-HD-GFP</i>) as compared to ßgal (magneta) driven by a WT version of the <i>Ndg</i> enhancer (<i>Ndg^WT-lacZ</i>) in stage 12 embryos. The ventral <i>Ndg</i> reporter-expressing cells are not present in the indicated focal plane but do not express the GFP reporter (data not shown). Compare to WT versions of both GFP and lacZ reporters in Figure 2G.</p

Requirements of Six binding sites for the activities of some but not all tested mesodermal enhancers.

<p>(A) Loss of GFP (green) reporter expression in the Lb-expressing SBM (magneta) driven by a version of the <i>lbl</i> enhancer in which the Six4 binding sites are inactivated (<i>lbl^noSix-GFP</i>) in stage 14 embryos. Compare to the WT version of the <i>lbl</i> enhancer (<i>lbl^WT-lacZ</i>) in Figure 2A. (B) The GFP (green) reporter driven by the WT <i>ap</i> enhancer (<i>ap^WT-GFP</i>) is active in a small subset of lateral Mef2-positive FCs (magenta) in stage 12 embryos. (C) De-repression of the GFP reporter (green) into additional Mef2-positive (magenta) mesodermal cells in a version of the <i>ap</i> enhancer in which the Six4 binding sites are mutated (<i>ap^noSix-GFP</i>) in stage 12 embryos. (D) The GFP (green) reporter driven by a version of the <i>mib2</i> enhancer in which Six4 binding sites are inactivated (<i>mib2^noSix-GFP</i>) co-expresses with ßgal (magneta) driven by a WT version of the <i>mib2</i> enhancer (<i>mib2^WT-lacZ</i>) in stage 12 embryos. Compare to WT versions of both GFP and lacZ reporters in Figure 2E. (E) The GFP (green) reporter driven by a version of the <i>Ndg</i> enhancer in which Six4 binding sites are mutated (<i>Ndg^noSix-GFP</i>) also co-expressed with ßgal (magneta) driven by a WT version of the <i>Ndg</i> enhancer (<i>Ndg^WT-lacZ</i>) in stage 12 embryos. The ventral <i>Ndg</i> reporter-expressing cells are not present in the indicated focal plane but do not express the GFP reporter (data not shown). Compare to WT versions of both GFP and lacZ reporters in Figure 2G.</p