GATA BS point mutation

does not significantly affect

bas-1::gfp expression in the

wild type background (in any CRM context). However, it synergises with ast-1 mutant background leading to a com- plete loss of GFP expression. These results unravel a direct role for GATA sites in bas-1 gene expression and synergy between egl-18 and ast-1.

C

D

bas-1prom1::gfp (-1492/-4) bas-1prom18::gfp (-1250/-1133) bas-1 HSN cis-regulatory analysis

HSN %

bas-1prom18::gfp (-1250/-1133)

Lines

2/4

bas-1prom65::gfp SPALT MUT bas-1prom71::gfp POU MUT

GATA MUT bas-1prom78::gfp 3/3 3/3 3/3 2/2

bas-1prom73::gfp ETS MUT

+ − + +/− − A B POU BS (UNC-86) ETS BS

(AST-1) SPALT BS(SEM-4) HLH BS(HLH-3) INSM BS(EGL-46) GATA BS(EGL-18)

GFP GFP GFP GFP GFP GFP bas-1prom18::gfp (-1250/-1133) bas-1prom73::gfp GATA MUT

GFP GFP 5 5 3 58 60 38 43 63 83 20 30 32 0 0 0 GFP GFP GFP bas-1prom13::gfp (-1510/-1183) bas-1prom77::gfp HLH MUT bas-1prom76::gfp INSM MUT

bas-1prom84::gfp GATA MUT2

bas-1prom83::gfp GATA MUT1

bas-1prom86::gfp 3X GATA MUT

GFP GFP 90 91 68 73 12 22 + 2/2 +/− 2/2 − 1/2 + 3/3 73 87 87 + + 2/2 82 92 3/3 88 93 95 GFP GFP 1.5 kb 117 bp HSN expression

wild type ast-1

+ (100 %) + (100 %) + (83 %) +/− (66 %)

HSN expression

wild type ast-1

+ (83 %) +/− (66 %) + (59 %) − (0 %)

150 151 the regulatory regions of the 5-HT pathway genes.

Further supporting these results, in the next sec- tion we will describe how UNC-86, AST-1 and EGL- 18 bind to serotonergic CRMs in vitro. Our extensive analysis provides us with additional information about how the individual roles of each TF depend on specific DNA contexts. For instance, we detect- ed several examples of genetic redundancy that provide robustness of expression to the system and that can be unravelled in the context of small- er CRMs or mutant backgrounds. Notably, redun- dancy is specific to the CRM architecture as two TFs can act redundantly in one CRM but not in oth- ers. In addition, we have observed clear examples of genetic enhancement between TFs, suggesting that they act as a regulatory code (HSN regulatory code). Moreover, each CRM has a different dispo- sition of TFBS arrangements supporting a flexible function of these TFs in the HSN. Finally, we also found that short HSN CRMs that lack TFBSs for some HSN TF collective members can drive par- tially penetrant HSN expression, while longer CRMs with functional BSs for all HSN TF collective mem- bers drive more robust expression.

UNC-86, AST-1 and EGL-18 directly bind to the regulatory regions of the serotonin pathway genes in vitro

In order to validate the previously inferred direct binding of the HSN regulatory code to the 5-HT pathway genes, Electrophoretic Mobility Shift Assays (EMSA) were performed. DNA probes for

tph-1, cat-1 and bas-1 genes were labelled with the

radioactive isotope phosphorous-32 (32P_{) and incu-}

bated with the purified proteins of some members of the HSN code. One or two p robes targeting previ- ously identified functional BSs for every member in the regulatory regions of the genes were designed. EMSA experiments reveal that UNC-86 is able to bind to the tph-1, cat-1 and bas-1 probes in vitro

→Figure 3.2.12-A. We had previously shown UNC-

86 binding to tph-1 and bas-1 (Zhang et al. 2014) but not to cat-1. The binding is also dose dependent because we observe a stronger band in the gel as we add increasing concentrations of the probe. To test for POU BS specificity, the EMSA experiments were repeated using probes with mutations in the functional POU sites determined from our cis-reg-

ulatory analysis. UNC-86 binding is absent in these conditions → Figure 3.2.12-A, indicating that UNC-

86 specifically and directly binds to the DNA at this specific location.

In addition, AST-1 is able to bind in vitro to cat-1 and bas-1 in a specific manner but we did not observe binding to tph-1 regulatory regions

→Figure 3.2.12-B. In the same way as with UNC-

86, binding is dose dependent and deletion of the ETS site that is required for reporter gene expres- sion in vivo resulted in the loss of AST-1 binding in

vitro → Figure 3.2.12-B.

We also detected in vitro binding of EGL-18 to the cat-1 probe, but no interaction was seen with two different tph-1 probes (tph-1.1 and tph-1.2;

→ Figure 3.2.12-C). EGL-18 binding to cat-1 was

specific as we only observed a supershift in the EGL-18 band when EGL-18-His tagged protein was incubated with the anti-6xhistag antibody and not when it was incubated with an anti-GFP antibody

→Figure 3.2.12-C. As a positive control the 3’ en-

hancer region of the Wilms Tumour 1 gene (WT1) was used. GATA2 (closest human orthologue to EGL-18) has been shown to bind to this region in several solid tumour cell lines and to be critical in the expression of WT1 (Furuhata et al. 2009). Moreover, the cat-1 DNA probe was modified in order to truncate both GATA BSs that had been shown to reduce expres- sion in the HSN in vivo when simultaneously delet- ed → Figure 3.2.9-A. As with UNC-86 and AST-1,

the band was lost, indicating that EGL-18 binds to

cat-1 regulatory regions in vitro, through GATA BSs.

Unfortunately, no binding corresponding to SEM-4, HLH-3 or EGL-46 was detected under these exper- imental conditions. Our results show that, at least, AST-1, UNC-86 and EGL-18 are able to bind in vitro to the in vivo determined CRMs. A summary of posi- tive EMSA assays is shown in → Figure 3.2.13.

The HSN regulatory code is expressed in the HSN

So far, we have described that our six candidate TFs are required for proper HSN terminal differ- entiation. In order to confirm the cell autonomous actions of the proteins, and to start studying their inter-relationships, we analysed their expression in the HSN, as it had only been partially assessed. All strains and the corresponding genotypes used are listed in → Table 2.21.

When possible, integrated fosmid reporter strains were used. Unlike transcriptional reporters, which consist on a DNA fragment of a few kilobases imme- diately 5’ upstream of the start codon of the gene of interest, fosmids cover large genomic regions (around 40 kb) and in C. elegans are considered a good approach for endogenous gene expression assessment, as they usually contain all regulato- ry information (Tursun et al. 2009). Fortunately, in

C. elegans a fosmid library that covers 80% of the

genome and 90% of the worm genes is available (http://www.sourcebioscience.com). As it shows 5.74 X clone coverage of the genome, one can usu- ally find a fosmid where the gene of interest is close to the centre, with at least 2–3 additional genet- ic loci on either side. Engineering fluorescently la- belled genes of interest in a genomic clone context allows for evaluation of the expression pattern and functionality of the tagged gene. Previous to the appearance of CRISPR-Cas9 technology, insert- ing tags at the target gene locus contained within these fosmids by homologous recombination (also called ‘recombineering’) represented the most ac- curate method to generate reporters that recapit- ulate full endogenous expression of a given gene. Starting with UNC-86, it is well-known when and where this TF is expressed. Using rabbit antisera against UNC-86 protein, expression is detect- ed in the embryonic Q lineage and is maintained in the adult, including in the HSN neuron. UNC-86 is first detected in the postmitotic HSN in the em-

Figure 3.2.11

Conservation of the putative transcription factor binding sites of the six candidate regulators of the HSN in

tph-1, cat-1 and bas-1 CRMs

Conservation between six

Caenorhabditis species (C. brenneri, C. brigssae, C. japonica, C. remanei, C. sp. 5 ju800, C. tropicalis)

was assessed generating multiple alignments using the Multiz and PhyloP tools from UCSC Genome Browser. TFBSs belonging to different families are represented with different coloured boxes. Numbers below TFBS indicate the number of species in which is conserved. tph-1prom2::gfp (-378/-1) 1 2 5 6 6 6 5 0 2 6 6 6 0 0 conservation in # / 6 GFP cat-1prom14::gfp (-1088/-566) 0 0 0 0 0 0 1 0 0 0 6 6 6 0 6 conservation in # / 6 GFP bas-1prom13::gfp (-1510/-1183) 0 0 0 0 0 0 0 0 0 0 0 0 0 conservation in # / 6 GFP

153 AST-1 Probe ø ø bas-1 wt mut ø ø cat-1 wt mut ø