Garrigues, Jacob et al. (2021) International Worm Meeting "C. elegans transposable elements harbor diverse transcription factor DNA-binding motifs"

Pasquinelli, Amy, Garrigues, Jacob

[International Worm Meeting, 2021]

Transposable elements (TEs) are powerful agents of evolution that can rewire transcriptional programs by mobilizing and distributing transcription factor (TF) DNA-binding motifs throughout genomes. To investigate the extent that TEs provide TF-binding motifs in C. elegans, we determined the genomic positions of DNA-binding motifs for over 200 different TFs (1). Surprisingly, we found that almost all of the examined TFs have binding motifs that reside within TEs, and all types of TEs have at least one instance of a TF motif, demonstrating that TEs provide previously unappreciated numbers of TF-binding motifs to the C. elegans genome. After determining the occurrence of TF motifs in TEs relative to the rest of the genome, we identified numerous TF-binding motifs that are highly enriched within TEs compared to what would be expected by chance. Consistent with potential functional roles for these TE-enriched TF-binding sequences, we found that significantly more TEs with TF motifs display evidence for selection compared to those lacking motifs through the use of publicly available genome variation data (2). We also compared the locations of TE-residing TF motifs to published ATAC-seq (3) and ChIP-seq (4) data, which identify regions of open chromatin associated with TF DNA binding and regions bound by TFs of interest, respectively. Strikingly, we found that all of the TF motif types that occur in TEs have instances of residing within accessible chromatin, and the overwhelming majority of TF-binding motifs located within TEs associate with their cognate TFs, suggesting extensive binding of TFs to sequences within TEs. Additionally, TEs with accessible or TF-bound motifs reside in the putative promoter regions of ~14% of all protein-coding genes, providing widespread possibilities for influencing gene expression. Taken together, our work shows that TE-provided TF-binding sites are ubiquitous in C. elegans and have broad potential to rewire gene expression. 1. Weirauch et al. (2014) Cell 158:1431-1443. 2. Cook et al. (2016) Nucleic Acids Research 45:D650-D657. 3. Daugherty et al. (2017) Genome Research 27:2096-2107. 4. Kudron et al. (2018) Genetics 208:937-949.

Lloret-Fernandez, Carla et al. (2017) International Worm Meeting "Conserved transcriptional regulatory rules define the serotonergic-neuron identity."

Maicas, Miren, Chirivella, Laura, Flames, Nuria, Lloret-Fernandez, Carla, Jimeno, Angela, Weinberg , Peter, Artacho, Alejandro, Mora, Carlos

[International Worm Meeting, 2017]

Cell differentiation is controlled by individual transcription factors (TFs) that together activate a selection of enhancers in specific cell types. How these combinations of TFs identify and activate their target sequences remains unknown. Here, we identify the cis-regulatory transcriptional code that controls the differentiation of serotonergic (5HT) HSN neurons in C. elegans. Loss of function mutant and cis-regulatory analyses reveal that direct activation of the HSN transcriptome is orchestrated by a collective of six TFs. This TF code is composed by AST-1 (ETS TF family), UNC-86 (POU TF family), SEM-4 (SPALT TF family), HLH-3 (bHLH TF family), EGL-46 (INSM TF family) and EGL-18 (GATA TF family). Bioinformatically identified binding site clusters for these six TFs are enriched in known HSN expressed genes compared to a random set of genes. Through in vivo reporter analysis, we demonstrate that the clustering of TF collective binding sites constitutes a regulatory signature that is sufficient for de novo identification of HSN neuron functional enhancers. This regulatory signature contains certain syntactic constrains that further improve the prediction of enhancer expression in the cell. Mouse orthologs of most members of this TF collective are known regulators of mammalian 5HT differentiation programs and can functionally substitute for their worm counterparts. Finally, Principal Coordinates Analysis suggests that, among C. elegans neurons, the HSN transcriptome most closely resembles that of mouse 5HT neurons, which reveals deep homology. Our results?identify rules governing the transcriptional regulatory code of a critically important neuronal type in two species separated by over 700 million years.

Reece-Hoyes, John S. et al. (2011) International Worm Meeting "A Core C. elegans Transcription Factor Regulatory Network."

Walhout, Albertha J.M., Shrestha, Shaleen, Kent, Amanda, Hill, David, Diallo, Alos, Reece-Hoyes, John S., Dricot, Amelie, Zhuang, Jiali, Weng, Zhiping, Myers, Chad

[International Worm Meeting, 2011]

Differential gene expression is controlled by a complex regulatory network of interactions between proteins, DNA, and microRNAs. Transcription factors (TFs) are primary modulators within this network, largely by binding to specific sites within a promoter and activating or repressing nearby genes. Some of these downstream genes encode TFs that subsequently also regulate a combination TFs and non-TFs. By focusing on TFs and their TF targets, the central core of the greater gene regulatory network will be revealed. Yeast one-hybrid (Y1H) assays provide a gene-centered approach for detecting interactions between genomic loci such as promoters and TF proteins. We comprehensively screened interactions between 650 TF-encoding gene promoters and 834 TF proteins using our novel enhanced Y1H (eY1H) pipeline that yields a quantitative readout of protein-DNA interactions. We will present this novel pipeline as well as insights we obtained from the core TF network that consists of more than 4500 interactions.

Reinke, V et al. (2017) International Worm Meeting "Model organism Encyclopedia of Regulatory Networks (modERN)."

Xu, J, Celniker, SE, Weiszmann, R, White, KP, Gewirtzman, L, Ma, L, Vafeados, D, Gerstein, M, Samanta, S, Fisher, WW, Yan, KK, Reinke, V, Patton, J, Victorsen, A, Hammonds, AS, Higdon, A, Han, M, Waterston, RH, Kudron, M

[International Worm Meeting, 2017]

The goal of the modERN consortium is to create comprehensive maps of transcription factor (TF) binding sites in C. elegans and D. melanogaster. We have developed an effective high throughput pipeline to create strains of flies and worms with GFP-tagged TFs, which we use to perform ChIP-seq experiments. We are targeting 691 sequence-specific TF genes in the worm and 708 TF genes in the fly. The modERN project has generated 420 worm strains and 330 fly strains to date. A subset of worm strains had tagged TFs that localized to the cytoplasm (54), were poorly expressed (23), or did not pass quality control measures for ChIP (37). Thus, including data generated previously for the modENCODE project, we have completed ~900 ChIP-seq experiments for 235 factors (219 TFs and 16 other regulatory proteins) in the worm and 287 TFs in the fly. We processed all data sets with a uniform peak-calling pipeline for worm and fly. The resultant inferred binding sites can be useful to predict gene expression, particularly using combinations of TF binding profiles. In the worm, for example, combinatorial TF binding patterns can suggest expression in muscle, intestine, hypodermis, pharynx and neurons. For a limited number of transcription factors, we are conducting RNA-seq of strains with knock down of specific transcription factors by deletion or RNAi to identify potential target genes of the given TF and to validate targets identified by the ChIP-seq experiments. To date, we have collected RNA-seq data from 30 worm TF knockdown lines and 12 TF fly knock down lines. RNA-seq data and ChIP-seq data are deposited to the ENCODE DCC for access by the research community through the ENCODE DCC (https://www.encodeproject.org), as well as through the UCSC genome browser. A dedicated track hub for the worm data at UCSC is available as well. TF tagged lines are available through the Caenorhabditis Genetics Center and Bloomington Drosophila Stock Center. Here, we will summarize progress, present future plans, and demonstrate how to access and use the data.

Zhiping Weng et al. (2005) International Worm Meeting "A C. elegans transcription regulatory network of the digestive tract."

Lynn Doucette-Stam, Zhiping Weng, Natalia Martinez, Wanyuan Ao, Ahmed Elewa, Christian Grove, Dirk McMurray, Bart Deplancke, Reynaldo Sequerra

[International Worm Meeting, 2005]

The control of gene expression is highly combinatorial: most transcription factors (TFs) regulate the expression of multiple genes, and most genes are themselves regulated by multiple TFs. Interactions between TFs and their target genes can be visualized in transcription regulatory networks. Such networks allow insights into higher order regulation of gene expression, for example by the identification and characterization of network <sym08>hubs<sym09> and network motifs. Here, we present a transcription regulatory network of the C. elegans digestive tract. To identify TF-target gene interactions in a high-throughput manner, we developed a Gateway-compatible yeast one-hybrid (Y1H) system. This Y1H system can be used to identify TFs that can bind to a gene promoter. Our Y1H system is compatible with ORFeome (for TF-encoding ORFs) and Promoterome (for target gene promoters) resources, and allows the identification of TF-target gene interactions from a <sym08>gene centered<sym09> perspective. Thus, this system provides information that is distinct from, but complementary to data obtained from <sym08>TF centered<sym09> chromatin-immunoprecipitation-based methods. We used our Y1H system to identify TF-promoter interactions for 180 genes involved in development of the worm digestive tract. We identified >300 TF-DNA interactions and visualized these into a transcription regulatory network. The resulting network is highly connected and contains several TF and promoter <sym08>hubs<sym09> (e.g. TFs that bind to many genes and vice versa). We characterized several TF hubs in more detail. For instance, we inspected the collection of DNA fragments they can interact with to predict consensus TF binding sites. We also identified several other network motifs, including feed forward loops, regulator chains and single- and multi-input motifs. We have begun to experimentally test several hypotheses derived from this network. Taken together, the Y1H system provides a powerful tool for the high-throughput mapping of transcription regulatory networks in metazoan systems.

Reece-Hoyes, John S. et al. (2009) International Worm Meeting "High-throughput transcription regulatory network mapping by yeast one-hybrid assays with high-density transcription factor arrays."

Reece-Hoyes, John S., Barrasa, M. Inmaculada, Walhout, A.J. Marian, Carraher, Ashley, Kent, Amanda, Hill, David E., Dricot, Amelie, Brown, Katie

[International Worm Meeting, 2009]

An organism''s development and response to the environment is governed by transcription regulatory networks that control the differential regulation of the genome. This process is largely driven by the activation or repression of transcription induced by the specific binding of a regulatory transcription factor (TF) within each locus, often within gene promoters. We aim to characterize the transcription regulatory networks of the model nematode Caenorhabditis elegans. The worm genome contains about 20,000 genes, of which 941 encode TFs. In contrast to other groups, we use gene-centred (''gene-to-protein''), rather than TF-centered (''protein-to-gene'') methods for the identification of promoter-TF interactions. We will use high-throughput yeast one-hybrid assays with our resource of 795 TF and 31 putative TF clones to screen the ~6000 cloned promoters of the Promoterome. We will describe improvements that utilize a Singer HDA RoToR robot that enables the interrogation of all TFs multiple times with only three plates. The data gained from this study will primarily elucidate the networks that underlie the process of gene regulation, but additionally can be used to identify individual DNA motifs bound by the TFs studied.

Rahe, D.P. et al. (2017) International Worm Meeting "usp-48 is a negative regulator of plasticity during development."

Hobert, O., Rahe, D.P.

[International Worm Meeting, 2017]

Both classic and more modern developmental biological experiments have shown that differentiation includes both the adoption of key cell fate markers as well as the restriction of developmental potential. The restriction of plasticity can be assayed by transcription factor (TF) overexpression experiments: the overexpression of a TF at an early stage of development often can induce the expression of its target genes, whereas overexpression in later stages has minimal to no effect, indicating a reduction in plasticity over developmental time. In order to understand this phenomenon, we generated mutants that lack the ability to restrict fate. In genetic screens we have isolated usp-48, a conserved, ubiquitous nuclear deubiquitinase, as a key regulator of cell fate in the hypodermis. In such mutants, the hypodermis adopts its cell fate normally, but no longer inhibits the ability of an overexpressed TF to induce target gene expression. This function is cell-autonomous and depends on the deubiquitinating activity of USP-48. Studies with a temperature-sensitive mutant reveal that loss of USP-48 at any stage results in the ability of an overexpressed TF to induce its target genes, indicating a continuous requirement for its function. Interestingly, in addition to TF overexpression now being able to induce target genes, the hypodermis loses several cell fate characteristics in usp-48 mutants upon TF overexpression. Using an improved INTACT protocol, we are generating and analyzing RNAseq profiles of wt and usp-48 hypodermal cells in both normal and TF overexpression conditions in order to reveal the extent and specificity of the changes in the usp-48 mutants upon TF overexpression. We have also begun to explore the mechanism of action of USP-48, which is likely affecting the nature of the chromatin landscape in the hypodermis during development. We have also isolated several new mutants with a similar phenotype as usp-48. Experiments to determine their role in relation to usp-48 are ongoing.

B Deplancke et al. (2004) East Coast Worm Meeting "Mapping transcription regulatory networks in C. elegans"

B Deplancke, M Vidal, AJ Marian Walhout, D Dupuy

[East Coast Worm Meeting, 2004]

Since the advent of microarrays, large amounts of gene expression data have become available for C. elegans . However, the transcription regulatory mechanisms that dictate the gene expression profiles observed frequently remain elusive. This is mainly because the identity of the regulatory transcription factors (TFs) that control differential gene expression are difficult to infer from such datasets. Regulatory TFs activate or repress transcription of their target genes by binding to cis -regulatory elements that are frequently located in or near a gene's promoter. Thus, in order to experimentally 'match' a regulatory TF with its target genes, assays are needed that demonstrate a physical interaction between regulatory TFs and the cis -regulatory elements or promoters they bind to. To facilitate the large-scale identification of TF-promoter interactions, we developed a Gateway-compatible version of the yeast one-hybrid (Y1H) system. This system enables the rapid identification of TF-DNA interactions using both small (i.e. repeats of DNA elements of interest) and large DNA fragments (i.e. gene promoters). The possibility to generate many Y1H DNA bait constructs simultaneously by Gateway cloning makes this system amenable to high-throughput settings. Here, we present Y1H data obtained using various DNA fragments as baits including four intact C. elegans promoters. These DNA baits were screened versus a Y1H-compatible worm cDNA library and a newly generated worm TF mini-library consisting of 675 predicted TF-encoding full-length open reading frames in the right orientation and frame, and in roughly equimolar amounts. The TF mini-library has a highly reduced complexity compared to the cDNA library, which increases both the throughput of the Y1H assay and the probability to detect TF-DNA interactions with TFs that are relatively underrepresented in cDNA libraries. Because of the ability to associate C. elegans TFs with their respective target genes, the high-throughput Y1H system will be a valuable tool to decipher the transcription regulatory networks that control differential gene expression during worm development.

Lloret, C et al. (2015) International Worm Meeting "C. elegans serotonergic neuron subtypes are regulated by different combinations of transcription factors."

Weinberg, P, Lloret, C, Flames, N, Chirivella, L, Maicas, M, Jimeno, A, Hobert, O

[International Worm Meeting, 2015]

Serotonin dysfunction has been linked to several mental disorders some of them with a clear genetic component. However, the precise mechanisms underlying these pathologies are not well understood. The study of serotonergic (5HT) neuron specification could help us understand the molecular bases of these diseases. 5HT neurons are present in all eumetazoan groups and all of them share the expression of a battery of genes, termed the '5HT pathway genes', that code for enzymes and transporters required for the synthesis, release and re-uptake of serotonin. Very little is known about the direct and coordinated regulation of the 5HT pathway gene expression in any organism. To address this question we take advantage of the amenability of the simple model organism C. elegans. The nematode hermaphrodites have three 5HT neuronal subclasses: the NSM, the HSN and the ADF neuron.To increase our understanding of 5HT terminal differentiation, we performed an in vivo cis-regulatory analysis of four 5HT pathway genes (tph-1, bas-1, cat-1 and cat-4). Our results show that independent cis-regulatory modules (CRM) are responsible for the expression of each gene in each serotonergic neuron subtype. This finding suggests that different transcription factors (TFs) regulate 5HT fate in each 5HT neuron subclass.We have focused our analysis on the HSN neuron and through a candidate approach we identified six different TFs that are required for the HSN terminal differentiation: unc-86 (a POU TF), ast-1 (an ETS TF), sem-4 (a Spalt TF), hlh-3 (a bHLH TF), egl-46 (an Insm TF) and egl-18 (a GATA TF). Moreover, we find functional binding sites for all these factors in the CRMs of the 5HT genes.Surprisingly, mouse homologs for four out of these six TFs are already known to be required in mammalian 5HT specification. We successfully carried out cell-specific rescue experiments using the corresponding mouse homolog of the worm TF mutant. Thus, our work offers a new strategy to identify new TFs that have a role in 5HT specification in superior organisms. We are currently exploring the possibility that vertebrate homologs of the two remaining C. elegans factors (UNC-86 and SEM-4) also play a role in 5HT terminal differentiation.

Ettorre, Victoria et al. (2015) International Worm Meeting "Mapping chromatin accessibility using ATAC-seq in C. elegans."

Ercan, Sevinc, Ettorre, Victoria

[International Worm Meeting, 2015]

Specificity of transcription factor (TF) targeting is at the heart of gene regulation, but it is a process that is not well understood. TF binding specificity is accomplished in part by the chromatin accessibility of the DNA sequence motifs to which the TF binds. To understand X-specific binding of the dosage compensation complex (DCC), we will map chromatin accessibility across the genome. This will allow us to test whether the 10 base pair DNA sequence motif that is important for DCC recruitment is more accessible on the X. We use transposase-accessible chromatin sequencing (ATAC-seq) to map chromatin accessibility. In this technique, transposases insert sequencing adaptors into regions of chromatin not occupied by nucleosomes. ATAC-seq has been developed by the Greenleaf lab using human lymphoblastoid cells. We have obtained preliminary data from embryo and L3 larval stage worms and naked DNA as a control. Our results indicate that the transposase has a sequence preference for insertion. In embyros, we do not see many distinct peaks, but we do see those in L3s. Currently, we are working to optimize ATAC-seq in C. elegans for embryo and larval stages. .