The Evolutionary Duplication and Probable Demise of an Endodermal GATA Factor in Caenorhabditis elegans

Abstract We describe the elt-4 gene from the nematode Caenorhabditis elegans. elt-4 is predicted to encode a very small (72 residues, 8.1 kD) GATA-type zinc finger transcription factor. The elt-4 gene is located ∼5 kb upstream of the C. elegans elt-2 gene, which also encodes a GATA-type transcription factor; the zinc finger DNA-binding domains are highly conserved (24/25 residues) between the two proteins. The elt-2 gene is expressed only in the intestine and is essential for normal intestinal development. This article explores whether elt-4 also has a role in intestinal development. Reporter fusions to the elt-4 promoter or reporter insertions into the elt-4 coding regions show that elt-4 is indeed expressed in the intestine, beginning at the 1.5-fold stage of embryogenesis and continuing into adulthood. elt-4 reporter fusions are also expressed in nine cells of the posterior pharynx. Ectopic expression of elt-4 cDNA within the embryo does not cause detectable ectopic expression of biochemical markers of gut differentiation; furthermore, ectopic elt-4 expression neither inhibits nor enhances the ectopic marker expression caused by ectopic elt-2 expression. A deletion allele of elt-4 was isolated but no obvious phenotype could be detected, either in the gut or elsewhere; brood sizes, hatching efficiencies, and growth rates were indistinguishable from wild type. We found no evidence that elt-4 provided backup functions for elt-2. We used microarray analysis to search for genes that might be differentially expressed between L1 larvae of the elt-4 deletion strain and wild-type worms. Paired hybridizations were repeated seven times, allowing us to conclude, with some confidence, that no candidate target transcript could be identified as significantly up- or downregulated by loss of elt-4 function. In vitro binding experiments could not detect specific binding of ELT-4 protein to candidate binding sites (double-stranded oligonucleotides containing single or multiple WGATAR sequences); ELT-4 protein neither enhanced nor inhibited the strong sequence-specific binding of the ELT-2 protein. Whereas ELT-2 protein is a strong transcriptional activator in yeast, ELT-4 protein has no such activity under similar conditions, nor does it influence the transcriptional activity of coexpressed ELT-2 protein. Although an elt-2 homolog was easily identified in the genomic sequence of the related nematode C. briggsae, no elt-4 homolog could be identified. Analysis of the changes in silent third codon positions within the DNA-binding domains indicates that elt-4 arose as a duplication of elt-2, some 25–55 MYA. Thus, elt-4 has survived far longer than the average duplicated gene in C. elegans, even though no obvious biological function could be detected. elt-4 provides an interesting example of a tandemly duplicated gene that may originally have been the same size as elt-2 but has gradually been whittled down to its present size of little more than a zinc finger. Although elt-4 must confer (or must have conferred) some selective advantage to C. elegans, we suggest that its ultimate evolutionary fate will be disappearance from the C. elegans genome.

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bZIP transcription factor zip-2 mediates an early response to Pseudomonas aeruginosa infection in Caenorhabditis elegans

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0914643107 ◽

2010 ◽

Vol 107 (5) ◽

pp. 2153-2158 ◽

Cited By ~ 93

Author(s):

Kathleen A. Estes ◽

Tiffany L. Dunbar ◽

Jennifer R. Powell ◽

Frederick M. Ausubel ◽

Emily R. Troemel

Keyword(s):

Pseudomonas Aeruginosa ◽

Transcription Factor ◽

Caenorhabditis Elegans ◽

Bacterial Pathogen ◽

Early Response ◽

Bzip Transcription Factor ◽

Wild Type ◽

Response Gene ◽

C Elegans ◽

Infection Response

Very little is known about how animals discriminate pathogens from innocuous microbes. To address this question, we examined infection-response gene induction in the nematode Caenorhabditis elegans. We focused on genes that are induced in C. elegans by infection with the bacterial pathogen Pseudomonas aeruginosa, but are not induced by an isogenic attenuated gacA mutant. Most of these genes are induced independently of known immunity pathways. We generated a GFP reporter for one of these genes, infection response gene 1 (irg-1), which is induced strongly by wild-type P. aeruginosa strain PA14, but not by other C. elegans pathogens or by other wild-type P. aeruginosa strains that are weakly pathogenic to C. elegans. To identify components of the pathway that induces irg-1 in response to infection, we performed an RNA interference screen of C. elegans transcription factors. This screen identified zip-2, a bZIP transcription factor that is required for inducing irg-1, as well as several other genes, and is important for defense against infection by P. aeruginosa. These data indicate that zip-2 is part of a specialized pathogen response pathway that is induced by virulent strains of P. aeruginosa and provides defense against this pathogen.

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The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3.

Molecular and Cellular Biology ◽

10.1128/mcb.15.3.1522 ◽

1995 ◽

Vol 15 (3) ◽

pp. 1522-1535 ◽

Cited By ~ 217

Author(s):

W J Fredericks ◽

N Galili ◽

S Mukhopadhyay ◽

G Rovera ◽

J Bennicelli ◽

...

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Fusion Protein ◽

Target Genes ◽

Transcriptional Activator ◽

Wild Type ◽

Dna Binding Domains ◽

Binding Domains ◽

Pediatric Solid Tumors ◽

Single Polypeptide

Alveolar rhabdomyosarcomas are pediatric solid tumors with a hallmark cytogenetic abnormality: translocation of chromosomes 2 and 13 [t(2;13) (q35;q14)]. The genes on each chromosome involved in this translocation have been identified as the transcription factor-encoding genes PAX3 and FKHR. The NH2-terminal paired box and homeodomain DNA-binding domains of PAX3 are fused in frame to COOH-terminal regions of the chromosome 13-derived FKHR gene, a novel member of the forkhead DNA-binding domain family. To determine the role of the fusion protein in transcriptional regulation and oncogenesis, we identified the PAX3-FKHR fusion protein and characterized its function(s) as a transcription factor relative to wild-type PAX3. Antisera specific to PAX3 and FKHR were developed and used to examine PAX3 and PAX3-FKHR expression in tumor cell lines. Sequential immunoprecipitations with anti-PAX3 and anti-FKHR sera demonstrated expression of a 97-kDa PAX3-FKHR fusion protein in the t(2;13)-positive rhabdomyosarcoma Rh30 cell line and verified that a single polypeptide contains epitopes derived from each protein. The PAX3-FKHR protein was localized to the nucleus in Rh30 cells, as was wild-type PAX3, in t(2;13)-negative A673 cells. In gel shift assays using a canonical PAX binding site (e5 sequence), we found that DNA binding of PAX3-FKHR was significantly impaired relative to that of PAX3 despite the two proteins having identical PAX DNA-binding domains. However, the PAX3-FKHR fusion protein was a much more potent transcriptional activator than PAX3 as determined by transient cotransfection assays using e5-CAT reporter plasmids. The PAX3-FKHR protein may function as an oncogenic transcription factor by enhanced activation of normal PAX3 target genes.

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Evaluation of Novel Design Strategies for Developing Zinc Finger Nucleases Tools for Treating Human Diseases

Biotechnology Research International ◽

10.1155/2014/970595 ◽

2014 ◽

Vol 2014 ◽

pp. 1-27 ◽

Cited By ~ 2

Author(s):

Christian Bach ◽

William Sherman ◽

Jani Pallis ◽

Prabir Patra ◽

Hassan Bajwa

Keyword(s):

Zinc Finger ◽

Dna Sequences ◽

Specific Binding ◽

Zinc Finger Nucleases ◽

Design Strategies ◽

Binding Ability ◽

Dna Binding Domains ◽

Binding Domains ◽

Evolutionary Success ◽

Novel Design

Zinc finger nucleases (ZFNs) are associated with cell death and apoptosis by binding at countless undesired locations. This cytotoxicity is associated with the binding ability of engineered zinc finger domains to bind dissimilar DNA sequences with high affinity. In general, binding preferences of transcription factors are associated with significant degenerated diversity and complexity which convolutes the design and engineering of precise DNA binding domains. Evolutionary success of natural zinc finger proteins, however, evinces that nature created specific evolutionary traits and strategies, such as modularity and rank-specific recognition to cope with binding complexity that are critical for creating clinical viable tools to precisely modify the human genome. Our findings indicate preservation of general modularity and significant alteration of the rank-specific binding preferences of the three-finger binding domain of transcription factor SP1 when exchanging amino acids in the 2nd finger.

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Mapping and analysis of Caenorhabditis elegans transcription factor sequence specificities

eLife ◽

10.7554/elife.06967 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 41

Author(s):

Kamesh Narasimhan ◽

Samuel A Lambert ◽

Ally WH Yang ◽

Jeremy Riddell ◽

Sanie Mnaimneh ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Dna Binding ◽

Regulatory Elements ◽

Nuclear Hormone Receptor ◽

Binding Motifs ◽

T Box ◽

C Elegans ◽

Dna Binding Domains ◽

Binding Domains ◽

Protein Binding Microarray

Caenorhabditis elegans is a powerful model for studying gene regulation, as it has a compact genome and a wealth of genomic tools. However, identification of regulatory elements has been limited, as DNA-binding motifs are known for only 71 of the estimated 763 sequence-specific transcription factors (TFs). To address this problem, we performed protein binding microarray experiments on representatives of canonical TF families in C. elegans, obtaining motifs for 129 TFs. Additionally, we predict motifs for many TFs that have DNA-binding domains similar to those already characterized, increasing coverage of binding specificities to 292 C. elegans TFs (∼40%). These data highlight the diversification of binding motifs for the nuclear hormone receptor and C2H2 zinc finger families and reveal unexpected diversity of motifs for T-box and DM families. Motif enrichment in promoters of functionally related genes is consistent with known biology and also identifies putative regulatory roles for unstudied TFs.

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elt-1, an embryonically expressed Caenorhabditis elegans gene homologous to the GATA transcription factor family.

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4651 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4651-4659 ◽

Cited By ~ 49

Author(s):

J Spieth ◽

Y H Shim ◽

K Lea ◽

R Conrad ◽

T Blumenthal

Keyword(s):

Transcription Factor ◽

Caenorhabditis Elegans ◽

Dna Binding ◽

Single Copy ◽

Amino Acid Sequences ◽

Recognition Sequence ◽

Sequence Element ◽

C Elegans ◽

Binding Domains ◽

Degenerate Oligonucleotide

The short, asymmetrical DNA sequence to which the vertebrate GATA family of transcription factors binds is present in some Caenorhabditis elegans gene regulatory regions: it is required for activation of the vitellogenin genes and is also found just 5' of the TATA boxes of tra-2 and the msp genes. In vertebrates GATA-1 is specific to erythroid lineages, whereas GATA-2 and GATA-3 are present in multiple tissues. In an effort to identify the trans-acting factors that may recognize this sequence element in C. elegans, we used a degenerate oligonucleotide to clone a C. elegans homolog to this gene. We call this gene elt-1 (erythrocytelike transcription factor). It is single copy and specifies a 1.75-kb mRNA that is present predominantly, if not exclusively, in embryos. The region of elt-1 encoding two zinc fingers is remarkably similar to the DNA-binding domain of the vertebrate GATA-binding proteins. However, outside of the DNA-binding domains the amino acid sequences are quite divergent. Nevertheless, introns are located at identical or nearly identical positions in elt-1 and the mouse GATA-1 gene. In addition, elt-1 mRNA is trans-spliced to the 22-base untranslated leader, SL1. The DNA upstream of the elt-1 TATA box contains eight copies of the GATA recognition sequence within the first 300 bp, suggesting that elt-1 may be autogenously regulated. Our results suggest that the specialized role of GATA-1 in erythroid gene expression was derived after separation of the nematodes and the line that led to the vertebrates, since C. elegans lacks an erythroid lineage.

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elt-1, an embryonically expressed Caenorhabditis elegans gene homologous to the GATA transcription factor family

Molecular and Cellular Biology ◽

10.1128/mcb.11.9.4651-4659.1991 ◽

1991 ◽

Vol 11 (9) ◽

pp. 4651-4659

Author(s):

J Spieth ◽

Y H Shim ◽

K Lea ◽

R Conrad ◽

T Blumenthal

Keyword(s):

Transcription Factor ◽

Caenorhabditis Elegans ◽

Dna Binding ◽

Single Copy ◽

Amino Acid Sequences ◽

Recognition Sequence ◽

Sequence Element ◽

C Elegans ◽

Binding Domains ◽

Degenerate Oligonucleotide

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The Caenorhabditis elegans gene lin-26 is required to specify the fates of hypodermal cells and encodes a presumptive zinc-finger transcription factor

Development ◽

10.1242/dev.120.9.2359 ◽

1994 ◽

Vol 120 (9) ◽

pp. 2359-2368 ◽

Cited By ~ 8

Author(s):

M. Labouesse ◽

S. Sookhareea ◽

H.R. Horvitz

Keyword(s):

Transcription Factor ◽

Caenorhabditis Elegans ◽

Zinc Finger ◽

Precursor Cells ◽

Embryonic Lethality ◽

Zinc Finger Transcription Factor ◽

C Elegans ◽

Neural Fate ◽

Vulval Precursor Cells

The mutation lin-26(n156) prevents vulva formation in C. elegans by transforming the vulval precursor cells into neurons or neuroblasts. We have isolated and characterized three new lin-26 alleles, which result in embryonic lethality. These mutations cause a few other hypodermal cells to express a neural fate and most hypodermal cells to degenerate. lin-26 encodes a presumptive zinc-finger transcription factor. Our data indicate that lin-26 is required for cells to acquire the hypodermal fate.

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The lin-11 LIM domain transcription factor is necessary for morphogenesis of C. elegans uterine cells

Development ◽

10.1242/dev.126.23.5319 ◽

1999 ◽

Vol 126 (23) ◽

pp. 5319-5326 ◽

Cited By ~ 3

Author(s):

A.P. Newman ◽

G.Z. Acton ◽

E. Hartwieg ◽

H.R. Horvitz ◽

P.W. Sternberg

Keyword(s):

Transcription Factor ◽

Caenorhabditis Elegans ◽

Egg Laying ◽

Wild Type ◽

Lim Domain ◽

C Elegans ◽

Expression Studies ◽

Vulval Precursor Cells ◽

Cell Induction ◽

Anchor Cell

The Caenorhabditis elegans hermaphrodite egg-laying system comprises several tissues, including the uterus and vulva. lin-11 encodes a LIM domain transcription factor needed for certain vulval precursor cells to divide asymmetrically. Based on lin-11 expression studies and the lin-11 mutant phenotype, we find that lin-11 is also required for C. elegans uterine morphogenesis. Specifically, lin-11 is expressed in the ventral uterine intermediate precursor (pi) cells and their progeny (the utse and uv1 cells), which connect the uterus to the vulva. Like (pi) cell induction, the uterine lin-11 expression responds to the uterine anchor cell and the lin-12-encoded receptor. In wild type animals, the utse, which forms the planar process at the uterine-vulval interface, fuses with the anchor cell. We found that, in lin-11 mutants, utse differentiation was abnormal, the utse failed to fuse with the anchor cell and a functional uterine-vulval connection was not made. These findings indicate that lin-11 is essential for uterine-vulval morphogenesis.

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Characterization of the DNA-binding properties of the myeloid zinc finger protein MZF1: two independent DNA-binding domains recognize two DNA consensus sequences with a common G-rich core.

Molecular and Cellular Biology ◽

10.1128/mcb.14.3.1786 ◽

1994 ◽

Vol 14 (3) ◽

pp. 1786-1795 ◽

Cited By ~ 138

Author(s):

J F Morris ◽

R Hromas ◽

F J Rauscher

Keyword(s):

Transcription Factor ◽

Dna Binding ◽

Zinc Finger ◽

Binding Sites ◽

Zinc Fingers ◽

Consensus Sequences ◽

Dna Binding Domains ◽

Binding Domains ◽

Gel Shift ◽

Myeloid Zinc Finger

The myeloid zinc finger gene 1, MZF1, encodes a transcription factor which is expressed in hematopoietic progenitor cells that are committed to myeloid lineage differentiation. MZF1 contains 13 C2H2 zinc fingers arranged in two domains which are separated by a short glycine- and proline-rich sequence. The first domain consists of zinc fingers 1 to 4, and the second domain is formed by zinc fingers 5 to 13. We have determined that both sets of zinc finger domains bind DNA. Purified, recombinant MZF1 proteins containing either the first set of zinc fingers or the second set were prepared and used to affinity select DNA sequences from a library of degenerate oligonucleotides by using successive rounds of gel shift followed by PCR amplification. Surprisingly, both DNA-binding domains of MZF1 selected similar DNA-binding consensus sequences containing a core of four or five guanine residues, reminiscent of an NF-kappa B half-site: 1-4, 5'-AGTGGGGA-3'; 5-13, 5'-CGGGnGAGGGGGAA-3'. The full-length MZF1 protein containing both sets of zinc finger DNA-binding domains recognizes synthetic oligonucleotides containing either the 1-4 or 5-13 consensus binding sites in gel shift assays. Thus, we have identified the core DNA consensus binding sites for each of the two DNA-binding domains of a myeloid-specific zinc finger transcription factor. Identification of these DNA-binding sites will allow us to identify target genes regulated by MZF1 and to assess the role of MZF1 as a transcriptional regulator of hematopoiesis.

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