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

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

Functional requirements for HD binding sites in all tested mesodermal enhancers.

<p>(A) ßgal (green) driven by the wild-type (WT) <i>lbl</i> enhancer (<i>lbl^WT-lacZ</i>) co-expresses with Lb protein (magenta) in the Lb-expressing SBM in stage 14 embryos. (B) Loss of ßgal reporter in the Lb-expressing SBM driven by a version of the <i>lbl</i> enhancer in which all I–HD plus Hox binding sites are selectively inactivated (<i>lbl^noHD-lacZ</i>) in stage 14 embryos. (C) The GFP (green) reporter driven by the WT <i>ap</i> enhancer (<i>ap^WT-GFP</i>) is active in stage 14 lateral transverse myotubes, two of which express Kr protein (magenta). (D) Loss of the GFP reporter in stage 14 lateral transverse myotubes by a version of the <i>ap</i> enhancer in which all I–HD plus Hox binding sites are inactivated (<i>ap^noHD-lacZ</i>). (E) GFP (green) and ßgal (magenta) are co-expressed in stage 12 embryos when driven by the WT <i>mib2</i> enhancer (<i>mib2</i>^<i>WT</i><i>-GFP</i> and <i>mib2</i>^<i>WT</i><i>-lacZ</i>, respectively). (F) GFP (green) expression driven by a version of the <i>mib2</i> enhancer in which all I–HD plus Hox binding sites are mutated (<i>mib2^noHD-GFP</i>) is significantly reduced compared to ßgal (magenta) driven by <i>mib2</i>^<i>WT</i><i>-lacZ</i> in stage 12 embryos. (G) GFP (green) and ßgal (magenta) are co-expressed when driven by the <i>Ndg</i> enhancer in stage 12 embryos (<i>Ndg</i>^<i>WT</i><i>-GFP</i> and <i>Ndg</i>^<i>WT</i><i>-lacZ</i>, respectively). The 1-2 non-co-expressing cells are due to ectopic reporter activity caused by the P-element insertion [22]. The ventral <i>Ndg</i> reporter-expressing cells are not present in the indicated focal plane. (H) Loss of GFP (green) driven by a version of the <i>Ndg</i> enhancer in which all I–HD plus Hox binding sites are mutated (<i>Ndg^noHD-GFP</i>) as compared to ßgal (magneta) driven by the WT <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).</p

Integrative analysis of the zinc finger transcription factor Lame duck in the Drosophila myogenic gene regulatory network

Contemporary high-throughput technologies permit the rapid identification of transcription factor (TF) target genes on a genome-wide scale, yet the functional significance of TFs requires knowledge of target gene expression patterns, cooperating TFs, and cis-regulatory element (CRE) structures. Here we investigated the myogenic regulatory network downstream of the Drosophila zinc finger TF Lame duck (Lmd) by combining both previously published and newly performed genomic data sets, including ChIP sequencing (ChIP-seq), genome-wide mRNA profiling, cell-specific expression patterns of putative transcriptional targets, analysis of histone mark signatures, studies of TF cooccupancy by additional mesodermal regulators, TF binding site determination using protein binding microarrays (PBMs), and machine learning of candidate CRE motif compositions. Our findings suggest that Lmd orchestrates an extensive myogenic regulatory network, a conclusion supported by the identification of Lmd-dependent genes, histone signatures of Lmd-bound genomic regions, and the relationship of these features to cell-specific gene expression patterns. The heterogeneous cooccupancy of Lmd-bound regions with additional mesodermal regulators revealed that different transcriptional inputs are used to mediate similar myogenic gene expression patterns. Machine learning further demonstrated diverse combinatorial motif patterns within tissue-specific Lmd-bound regions. PBM analysis established the complete spectrum of Lmd DNA binding specificities, and site-directed mutagenesis of Lmd and additional newly discovered motifs in known enhancers demonstrated the critical role of these TF binding sites in supporting full enhancer activity. Collectively, these findings provide insights into the transcriptional codes regulating muscle gene expression and offer a generalizable approach for similar studies in other systems.National Institutes of Health (U.S.) (Grant RO1 HG005287