Mitogen-activated protein (MAP) kinases such as extracellular signal-regulated kinase (ERK) are important signaling proteins that phosphorylate (S/T)P sites in many different protein substrates. ERK binding to substrate proteins is mediated by docking sites including the FXFP motif and the D-domain. We characterized the sequence of amino acids that can constitute the FXFP motif using peptide and protein substrates. Substitutions of the phenylalanines at positions 1 and 3 had significant effects, indicating that these phenylalanines provide substantial binding affinity, whereas substitutions of the residues at positions 2 and 4 had less effect. The FXFP and D-domain docking sites were analyzed in a variety of positions and arrangements in the proteins ELK-1 and KSR-1. Our results indicate that the FXFP and D-domain docking sites form a flexible, modular system that has two functions. First, the affinity of a substrate for ERK can be regulated by the number, type, position, and arrangement of docking sites. Second, in substrates with multiple potential phosphorylation sites, docking sites can direct phosphorylation of specific (S/T)P residues. In particular, the FQFP motif of ELK-1 is necessary and sufficient to direct phosphorylation of serine 383, whereas the D-domain directs phosphorylation of other (S/T)P sites in ELK-1. The MAP1 kinase superfamily is composed of several subfamilies including ERK, c-Jun N-terminal kinase, and p38 (1, 2). These MAP kinases can be activated by a remarkably diverse set of stimuli that function through a variety of signaling pathways. MAP kinase activation is regulated by two upstream protein kinases; a MAP kinase kinase kinase, such as Raf, phosphorylates and thereby activates a MAP kinase kinase (3, 4). The MAP kinase kinase that regulates ERK is called MEK (MAP kinase kinase or ERK kinase). MEK is a dual specificity protein kinase that phosphorylates a threonine and tyrosine in a TXY motif of ERK, resulting in a significant increase in ERK kinase activity. ERK is inactivated by dual specificity phosphatases that dephosphorylate the TXY motif. Many different stimuli can activate the protein kinase cascade that activates ERK. One extensively characterized activation pathway is initiated by a secreted growth factor, such as epidermal growth factor, that leads to the activation of a receptor tyrosine kinase, Ras, and Raf (5).Signaling pathways that include ERK mediate a remarkably diverse set of responses during the development and homeostasis of organisms such as Caenorhabditis elegans, Drosophila, and vertebrates. The mechanisms that enable highly conserved signaling pathways to elicit cell type-specific responses are being actively investigated, yet remain poorly understood. In principle, any protein in a signaling cascade could function differently in different cell types and, thus, contribute to a specific response. However, ERK is likely to play an important role in generating cell type-specific responses (6). By contrast to Raf and MEK, whose only well documented physiologic...

H1t is an H1 histone variant unique to late spermatocytes and early spermatids. Using gene targeting and embryonic stem cell technologies, we have produced mice with a disrupted H1t gene. Homozygous H1t-null mice have normal fertility and show no obvious phenotypic consequence due to the lack of this histone. Biochemical and immunohistochemical approaches were used to show that normal changes in chromosomal proteins occurred during spermatid development, including the appearance and disappearance of transition proteins 1 and 2. Both protamines 1 and 2 are present in normal amounts in sonication-resistant spermatid nuclei from H1t-null mice. Analysis of H1 histones by quantitative gel electrophoresis in enriched populations of pachytene spermatocytes and round spermatids showed that the lack of H1t is only partially compensated for by somatic H1s, so that the chromatin of these cells is H1 deficient. Because H1t is thought to create a less tightly compacted chromatin environment, it may be that H1-deficient chromatin is functionally similar to chromatin with H1t present, at least with respect to permitting spermatogenesis to proceed.

H1t is a testis-specific histone 1 variant restricted to the male germ line and expressed only in pachytene spermatocytes. Understanding the regulation of the H1t gene is an interesting challenge as its promoter shares all of the recognized control elements of standard somatic H1 genes, yet H1t is not expressed in somatic or in early spermatogenic cells. To investigate the mechanism of this apparent repression, we exchanged three promoter subregions between H1t and a major somatic H1 gene (H1d) by introduction of suitable restriction sites just 5 of the TATA box and 3 of the conserved H1 AC box. Hybrid promoters were joined to a lacZ reporter gene and assayed by transient transfection in NIH3T3 fibroblasts. In this system the wild type H1d promoter was 20-fold stronger than the H1t promoter. Much of this difference in activity was traced to inhibitory sequences immediately downstream of the TATA box in H1t, although sequences upstream of the H1t AC box and within the H1t 5-untranslated region played some role as well. A series of deletions and short oligonucleotide mutations scanned across the region between the TATA box and cap site identified two tracts of C (GC box 2) as the inhibitory sequences. While both Sp1 and Sp3 bind to this region weakly in vitro, they are unlikely to be responsible for the inhibitory effect of GC box 2, and additional binding proteins (CTB-4 and CTB-5) were identified by electrophoretic mobility shift assays as better candidates for mediating the repressive effect. When repression of the H1t promoter was relieved by mutation of GC box 2, additional mutations introduced into GC box 1 upstream of the CAAT box led to a large decrease in activity, indicating that these two G/C-rich elements have opposite effects on promoter activity.Spermatogenesis is the only example of cellular development in mammals that involves expression of tissue-specific histone variants (1-3). H1t 1 is a testis-specific linker histone variant, appearing late in the prophase of meiosis I in pachytene spermatocytes, retained in early haploid cells, and lost from the nucleus prior to release of mature sperm (3-5). While the amino acid sequence of H1t has a number of novel features (6, 7), it is clearly related to the standard somatic H1 histone family (8). Isolation of the H1t gene from several mammals (9 -12) revealed that the promoter region also shows a surprising similarity to those of standard somatic H1 variants (13,14). Homologies include a TATA box, a CAAT box, a GC box, and an H1-specific AC motif within 100 nucleotides of the cap site as well as an inverted AC motif located farther upstream (15). Despite these shared regulatory elements, H1t expression differs almost completely from that of the common somatic H1 variants. Common variants are produced during S phase of the cell cycle to accommodate the duplication of the chromosomes (14), whereas H1t is expressed only in pachytene spermatocytes, well after completion of replicative DNA synthesis (4,5,16,17). As the presence of H1t mRNA correlates with H1t ...

Histone H1t is synthesized only in male germ cells during the late pachytene stage of meiosis and is retained in spermatids until the nucleus elongates. Transgenic experiments suggest that spermatocyte-directing sequences lie within 140 base pairs of the cap site. To study the mechanism of this specificity we compared the DNase I footprints made on the immediate promoter regions of H1t and H1d (a typical somatic H1) by testis and liver extracts and observed both common and differentially protected regions. The common footprints of H1t included an Sp1 consensus (GC box 1) and a CCAAT motif. Electrophoretic mobility shift assays (EMSA) identified ubiquitous binding factors for GC box 1 and a binding factor for the CCAAT element that we identified immunologically as H1TF2. H1t-specific footprints occurred over the palindrome CCTAGG and a GC-rich sequence downstream of the TATA box (GC box 2). EMSA analysis of the palindrome identified testis-specific as well as ubiquitous binding factors. UV irradiation of a palindrome-binding reaction generated a cross-linked doublet of about 50 kDa from both testis and liver. Protein factors that bound to the GC box 2 sequence were similar from testis and liver, and GC box 1 and an Sp1 consensus competed for them. In vitro transcription directed by H1t occurred at comparable levels in testis and liver extracts. The importance of both GC box 1 and CCAAT elements was demonstrated by deletion analysis and by oligonucleotide competition. No dependence on the H1t palindrome was observed for in vitro transcription.

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 LIN-1 ETS transcription factor plays a pivotal role in controlling cell fate decisions during development of the Caenorhabditis elegans vulva. Prior to activation of the RTK/Ras/ERK-signaling pathway, LIN-1 functions as a SUMOylated transcriptional repressor that inhibits vulval cell fate. Here we demonstrate using the yeast two-hybrid system that SUMOylation of LIN-1 mediates interactions with a protein predicted to be involved in transcriptional repression: the RAD-26 Mi-2β/CHD4 component of the nucleosome remodeling and histone deacetylation (NuRD) transcriptional repression complex. Genetic studies indicated that rad-26 functions to inhibit vulval cell fates in worms. Using the yeast two-hybrid system, we showed that the EGL-27/MTA1 component of the NuRD complex binds the carboxy-terminus of LIN-1 independently of LIN-1 SUMOylation. EGL-27 also binds UBC-9, an enzyme involved in SUMOylation, and MEP-1, a zinc-finger protein previously shown to bind LIN-1. Genetic studies indicate that egl-27 inhibits vulval cell fates in worms. These results suggest that LIN-1 recruits multiple proteins that repress transcription via both the SUMOylated amino-terminus and the unSUMOylated carboxy-terminus. Assays in cultured cells showed that the carboxy-terminus of LIN-1 was converted to a potent transcriptional activator in response to active ERK. We propose a model in which LIN-1 recruits multiple transcriptional repressors to inhibit the 1° vulval cell fate, and phosphorylation by ERK converts LIN-1 to a transcriptional activator that promotes the 1° vulval cell fate.

Expression of the diphtheria toxin A-chain gene was directed to the male germ line by fusion to 1 kilobase of the 5'-flanking DNA of the rat histone H1t gene. Two independent lines of mice were established that expressed the toxic transgene. Female carriers were fertile; males were sterile although otherwise apparently normal. Adult transgenic males had very small testes that were virtually devoid of germ cells. A developmental study showed that germ cells survived until late fetal life but that testes of 3-day-old transgenic mice were severely depleted of prospermatogonia. During postnatal development of transgenic animals, remaining germ cells progressed to the pachytene stage of meiosis in 10% to 30% of tubular cross sections but degenerated before the completion of meiosis. By 3 mo of age the residual germ cells had almost completely disappeared. These transgenic lines demonstrate the complete tissue specificity of the H1t promoter and reveal a period of its activity just prior to formation of the definitive adult spermatogonial stem cell population. Whereas full expression of H1t occurs only in mid to late pachytene spermatocytes, one or more of the factors that impart tissue specificity to its expression must be transiently activated in the neonatal germ line. This report discusses the possibility that this genetic technique for eliminating germ cells may have practical application in making recipients for spermatogonial stem cell transplantation.

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