Identification of Residues of the Caenorhabditis elegans LIN-1 ETS Domain That Are Necessary for DNA Binding and Regulation of Vulval Cell Fates

Abstract

LIN-1 is an ETS domain protein. A receptor tyrosine kinase/Ras/mitogen-activated protein kinase signaling pathway regulates LIN-1 in the P6.p cell to induce the primary vulval cell fate during Caenorhabditis elegans development. We identified 23 lin-1 loss-of-function mutations by conducting several genetic screens. We characterized the molecular lesions in these lin-1 alleles and in several previously identified lin-1 alleles. Nine missense mutations and 10 nonsense mutations were identified. All of these lin-1 missense mutations affect highly conserved residues in the ETS domain. These missense mutations can be arranged in an allelic series; the strongest mutations eliminate most or all lin-1 functions, and the weakest mutation partially reduces lin-1 function. An electrophoretic mobility shift assay was used to demonstrate that purified LIN-1 protein has sequence-specific DNA-binding activity that required the core sequence GGAA. LIN-1 mutant proteins containing the missense substitutions had dramatically reduced DNA binding. These experiments identify eight highly conserved residues of the ETS domain that are necessary for DNA binding. The identification of multiple mutations that reduce the function of lin-1 as an inhibitor of the primary vulval cell fate and also reduce DNA binding suggest that DNA binding is essential for LIN-1 function in an animal. INTRACELLULAR signaling specifies many cell fates guanine nucleotide exchange factor. LET-341 is likely to cause LET-60 Ras to release GDP, resulting in GTP during development. The Caenorhabditis elegans vulva is a useful model system for understanding how signal binding and LET-60 activation. Activated LET-60 Ras can bind and activate the serine/threonine kinase LINtransduction cascades regulate cell fates. The vulva is a specialized epidermal structure used for egg laying and 45 Raf. Activated LIN-45 phosphorylates and thereby activates the MEK-2 mitogen-activated protein (MAP) sperm entry that is formed by the descendants of three kinase kinase. MEK-2 phosphorylates and thereby actiectodermal blast cells, P5.p, P6.p, and P7.p (Horvitz vates the MPK-1 extracellular signal-regulated kinase and Sternberg 1991). In wild-type hermaphrodites, the (ERK) MAP kinase. MPK-1 appears to phosphorylate anchor cell of the somatic gonad signals to P6.p using multiple target proteins, including the LIN-1 ETS tranthe LIN-3 epidermal growth factor-like ligand (reviewed scription factor, and these modifications cause P6.p to by Greenwald 1997; Kornfeld 1997; Sternberg and adopt the 1Њ vulval cell fate (eight descendants). When Han 1998). LIN-3 presumably binds to the LET-23 re-P6.p is induced to adopt the 1Њ vulval cell fate, it signals ceptor tyrosine kinase (RTK). This is likely to stimulate to P5.p and P7.p through the LIN-12 Notch receptor, receptor autophosphorylation and create docking sites causing these cells to adopt the 2Њ vulval cell fate (seven for the SEM-5 adaptor protein and the LET-341 Ras descendants

The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans

“…VPCrep represses this basal VPCact-driven transcription in VPCs other than P6.p, thereby restricting expression of the lag-2 minimal promoter to the 1°-fated VPC. VPCrep contains an Elk1 consensus site, which is bound by LIN-1 in vitro (Miley et al 2004), and requires lin-1/Ets for repression of transcription in VPCs other than P6.p (Zhang and Greenwald 2011). Our results indicate that cdk-8 is partially required for transcriptional repression of the lag-2 minimal promoter ( Figure 4D), suggesting that the CKM promotes LIN-1-mediated repression at VPCrep.…”

Spatial Regulation of lag-2 Transcription During Vulval Precursor Cell Fate Patterning in Caenorhabditis elegans lag-2

“…VPCrep conforms to the consensus Elk1 site; LIN-1, the C. elegans Elk1 ortholog (Beitel et al 1995), has been demonstrated to bind to this consensus sequence (Miley et al 2004), suggesting that LIN-1 may mediate negative regulation Figure 2 Deletion analysis of the lag-2 promoter identified a distal element conferring P6.p-specific expression. Strains carrying independent arrays formed from the different constructs were scored for expression as indicated.…”

“…LIN-1 binds to the core Ets binding consensus sequence and is a direct target of phosphorylation by MPK-1/MAPK (Jacobs et al 1998(Jacobs et al , 1999Miley et al 2004). In addition, if the MAPK phosphorylation site is disrupted, LIN-1 prevents P6.p from adopting the 1°fate (Jacobs et al 1998).…”

Sumoylation of LIN-1 promotes transcriptional repression and inhibition of vulval cell fates

“…Mutations in the ETS domain that abrogate DNA binding cause a strong Muv phenotype, demonstrating that DNA binding is necessary for LIN-1 to inhibit the 1°vulval cell fate (Miley et al, 2004). LIN-1 contains two docking sites for ERK, the D domain and FQFP motif, and 17 S/TP motifs that are potential ERK phosphorylation sites (Fig.…”