Interaction between the Drosophila CAF-1 and ASF1 chromatin assembly factors.

The assembly of newly synthesized DNA into chromatin is essential for normal growth, development, and differentiation. To gain a better understanding of the assembly of chromatin during DNA synthesis, we identified, cloned, and characterized the 180- and 105-kDa polypeptides of Drosophila chromatin assembly factor 1 (dCAF-1). The purified recombinant p180+p105+p55 dCAF-1 complex is active for DNA replication-coupled chromatin assembly. Furthermore, we have established that the putative 75-kDa polypeptide of dCAF-1 is a C-terminally truncated form of p105 that does not coexist in dCAF-1 complexes containing the p105 subunit. The analysis of native and recombinant dCAF-1 revealed an interaction between dCAF-1 and the Drosophila anti-silencing function 1 (dASF1) component of replication-coupling assembly factor (RCAF). The binding of dASF1 to dCAF-1 is mediated through the p105 subunit of dCAF-1. Consistent with the interaction between dCAF-1 p105 and dASF1 in vitro, we observed that dASF1 and dCAF-1 p105 colocalized in vivo in Drosophila polytene chromosomes. This interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin.

Localization of endogenous and recombinant Na(+)-driven anion exchanger protein NDAE1 from Drosophila melanogaster.

Na(+)-dependent Cl(-)/HCO exchange activity helps maintain intracellular pH (pH(i)) homeostasis in many invertebrate and vertebrate cell types. Our laboratory cloned and characterized a Na(+)-dependent Cl(-)/HCO exchanger (NDAE1) from Drosophila melanogaster (Romero MF, Henry D, Nelson S, Harte PJ, and Sciortino CM. J Biol Chem 275: 24552--24559, 2000). In the present study we used immunohistochemical and Western blot techniques to characterize the developmental expression, subcellular localization, and tissue distribution of NDAE1 protein in D. melanogaster. We have shown that a polyclonal antibody raised against the NH(2) terminus of NDAE1 (alpha CWR57) recognizes NDAE1 electrophysiologically characterized in Xenopus oocytes. Moreover, our results begin to delineate the NDAE1 topology, i.e., both the NH(2) and COOH termini are intracellular. NDAE1 is expressed throughout Drosophila development in the central and peripheral nervous systems, sensilla, and the alimentary tract (Malpighian tubules, gut, and salivary glands). Coimmunolabeling of larval tissues with NDAE1 antibody and a monoclonal antibody to the Na(+)-K(+)-ATPase alpha-subunit revealed that the majority of NDAE1 is located at the basolateral membranes of Malpighian tubule cells. These results suggest that NDAE1 may be a key pH(i) regulatory protein and may contribute to basolateral ion transport in epithelia and nervous system of Drosophila.

The Drosophila Polycomb Group proteins ESC and E(Z) are present in a complex containing the histone-binding protein p55 and the histone deacetylase RPD3.

The Drosophila Polycomb Group (PcG) proteins are required for stable long term transcriptional silencing of the homeotic genes. Among PcG genes, esc is unique in being critically required for establishment of PcG-mediated silencing during early embryogenesis, but not for its subsequent maintenance throughout development. We previously showed that ESC is physically associated in vivo with the PcG protein E(Z). We report here that ESC, together with E(Z), is present in a 600 kDa complex that is distinct from complexes containing other PcG proteins. We have purified this ESC complex and show that it also contains the histone deacetylase RPD3 and the histone-binding protein p55, which is also a component of the chromatin remodeling complex NURF and the chromatin assembly complex CAF-1. The association of ESC and E(Z) with p55 and RPD3 is conserved in mammals. We show that RPD3 is required for silencing mediated by a Polycomb response element (PRE) in vivo and that E(Z) and RPD3 are bound to the Ubx PRE in vivo, suggesting that they act directly at the PRE. We propose that histone deacetylation by this complex is a prerequisite for establishment of stable long-term silencing by other continuously required PcG complexes.

Cloning and characterization of a Na+-driven anion exchanger (NDAE1). A new bicarbonate transporter.

Regulation of intra- and extracellular ion activities (e.g. H(+), Cl(-), Na(+)) is key to normal function of the central nervous system, digestive tract, respiratory tract, and urinary system. With our cloning of an electrogenic Na(+)/HCO(3)(-) cotransporter (NBC), we found that NBC and the anion exchangers form a bicarbonate transporter superfamily. Functionally three other HCO(3)(-) transporters are known: a neutral Na(+)/ HCO(3)(-) cotransporter, a K(+)/ HCO(3)(-) cotransporter, and a Na(+)-dependent Cl(-)-HCO(3)(-) exchanger. We report the cloning and characterization of a Na(+)-coupled Cl(-)-HCO(3)(-) exchanger and a physiologically unique bicarbonate transporter superfamily member. This Drosophila cDNA encodes a 1030-amino acid membrane protein with both sequence homology and predicted topology similar to the anion exchangers and NBCs. The mRNA is expressed throughout Drosophila development and is prominent in the central nervous system. When expressed in Xenopus oocytes, this membrane protein mediates the transport of Cl(-), Na(+), H(+), and HCO(3)(-) but does not require HCO(3)(-). Transport is blocked by the stilbene 4,4'-diisothiocyanodihydrostilbene- 2, 2'-disulfonates and may not be strictly electroneutral. Our functional data suggest this Na(+) driven anion exchanger (NDAE1) is responsible for the Na(+)-dependent Cl(-)-HCO(3)(-) exchange activity characterized in neurons, kidney, and fibroblasts. NDAE1 may be generally important for fly development, because disruption of this gene is apparently lethal to the Drosophila larva.

Mouse homolog of the Drosophila Pc-G gene esc exerts a dominant negative effect in Drosophila.

The Polycomb group genes are involved in maintaining long term transcriptional repression of the homeotic genes in both Drosophila and mammals. The mouse eed locus encodes the highly conserved ortholog of the Drosophila ESC protein. To test the functional conservation between the two genes, eed was introduced into the fly to determine whether it could rescue the esc mutant phenotype. eed exerted a dominant negative effect on the leg transformation phenotype associated with the esc mutation. This result is interpreted in light of in vitro protein-protein binding data and in vivo polytene chromosome staining indicating the lack of significant interaction between Eed and fly E(Z), a molecular partner of ESC. genesis 26:67-76, 2000

Assignment of a novel bifurcated SET domain gene, SETDB1, to human chromosome band 1q21 by in situ hybridization and radiation hybrids.

We have identified a human gene encoding an unusual bifurcated SET domain protein containing a large "insertion" between the most highly conserved parts of the SET domain. The existence of an evolutionarily related C. elegans gene encoding a similarly bifurcated SET domain suggests that SET domains may generally be composed of two functionally distinct subdomains. We mapped this gene, called SETDB1, to human chromosome 1q21. This region is targeted by a large number of recurrent translocations, suggesting that like the SET domain protein MLL, mutant forms of SETDB1 may be associated with human neoplasias.

Binding of trithorax and Polycomb proteins to the bithorax complex: dynamic changes during early Drosophila embryogenesis.

In Drosophila, the maintenance of developmentally important transcription patterns is controlled at the level of chromatin structure. The Polycomb group (PcG) and trithorax group (trxG) genes encode proteins involved in chromatin remodelling. PcG genes have been proposed to act by packaging transcriptional repressed chromosomal domains into condensed heterochromatin-like structures. Some of the trxG proteins characterized so far are members of chromatin opening complexes (e.g. SWI/SNF and GAGA/NURF) which facilitate binding of transcription factors and components of the basal transcriptional machinery. Genetic and biochemical data suggest that these two groups of regulatory factors may act through a common set of DNA elements. In the present study, we have investigated the binding of Trithorax (TRX) and Polycomb (PC) protein in the bithorax complex (BX-C) during embryogenesis. In addition, we have identified the minimal fragments from the Ultrabithorax (Ubx) regulatory region that are capable of recruiting TRX to chromosomal sites containing them. Comparative analysis of the binding of the two proteins shows that TRX and PC bind target sequences (PcG-regulated elements, PREs) by cellular blastoderm, when BX-C transcription begins. At the same stage, TRX but not PC is strongly associated with core promoters. Later, at germ band extension, the time of derepression in Polycomb mutants, PC binding is also detected outside core PREs and additionally binds to the fragments containing promoters.

The Drosophila Polycomb Group proteins ESC and E(Z) bind directly to each other and co-localize at multiple chromosomal sites.

The Polycomb Group gene esc encodes an evolutionarily conserved protein required for transcriptional silencing of the homeotic genes. Unlike other Polycomb Group genes, esc is expressed and apparently required only during early embryogenesis, suggesting it is required for the initial establishment of silencing but not for its subsequent maintenance. We present evidence that the ESC protein interacts directly with E(Z), another Polycomb Group protein required for silencing of the homeotic genes. We show that the most highly conserved region of ESC, containing seven WD motifs that are predicted to fold into a beta-propeller structure, mediate its binding to a conserved N-terminal region of E(Z). Mutations in the WD region that perturb ESC silencing function in vivo also perturb binding to E(Z) in vitro. The entire WD region forms a trypsin-resistant structure, like known beta -propeller domains, and mutations that would affect the predicted ESC beta-propeller perturb its trypsin-resistance, while a putative structure-conserving mutation does not. We show by co-immunoprecipitation that ESC and E(Z) are directly associated in vivo and that they also co-localize at many chromosomal binding sites. Since E(Z) is required for binding of other Polycomb Group proteins to chromosomes, these results suggest that formation of an E(Z):ESC complex at Polycomb Response Elements may be an essential prerequisite for the establishment of silencing.

The extra sex combs protein is highly conserved between Drosophila virilis and Drosophila melanogaster.

Extra sex combs (esc) is one of the Polycomb Group genes, whose products are required for long term maintenance of the spatially restricted domains of homeotic gene expression initially established by the products of the segmentation genes. We recently showed that the esc protein contains five copies of the WD motif, which in other proteins has been directly implicated in protein-protein interactions. Mutations affecting the WD repeats of the esc protein indicate that they are essential for its function as a repressor of the homeotic genes. We proposed that they may mediate interactions between esc and other Polycomb Group proteins, recruiting them to their target genes, perhaps by additional interactions with transiently expressed repressors such as hunchback. To further investigate the functional importance of the WD motifs and identify other functionally important regions of the esc protein, we have begun to determine its evolutionary conservation by characterizing the esc gene from Drosophila virilis, a distantly related Drosophila species. We show that the esc protein is highly conserved between these species, particularly its WD motifs. Their high degree of conservation, particularly at positions which are not conserved in the WD consensus derived from alignment of all known WD motifs, suggests that each of the WD repeats in the esc protein is functionally specialized and that this specialization has been highly conserved during evolution. Its highly charged N-terminus exhibits the greatest divergence, but even these differences are conservative of its predicted physical properties. These observations suggest that the esc protein is functionally compact, nearly every residue making an important contribution to its function.

The Drosophila trithorax proteins contain a novel variant of the nuclear receptor type DNA binding domain and an ancient conserved motif found in other chromosomal proteins.

The products of the trithorax gene are required to stably maintain homeotic gene expression patterns established during embryo-genesis by the action of the transiently expressed segmentation genes. We have determined the intron/exon structure of the trx gene and the large alternatively spliced trx RNAs, which are capable of encoding only two protein isoforms. These very large trx proteins differ only in a long Ser- and Gly-rich N-terminal extension, encoded by exon II, which is present only in the larger trx isoform. We have identified a novel variant of the highly conserved nuclear receptor type of DNA binding domain. We have found that the previously identified Cys-rich central region contains multiple novel zinc finger motifs which are also present in the Polycomb-like protein and RBP2, a retinoblastoma binding protein. The trx proteins terminate with another novel conserved domain which we have identified in proteins from three kingdoms, including plants and fungi, indicating that has an ancient origin. Many of these proteins are chromosomally associated, suggesting that this domain may be involved in interactions between trx and other highly conserved components of chromatin involved in transcription regulation. The sequence alterations of trx mutations identify the highly conserved regions of trx as critical for the function of these large proteins. We show that zygotically expressed trx RNAs encoding the larger protein isoform are initially expressed in a spatially restricted pattern which overlaps the expression domains of the BX-C genes Ubx, abd-A and Abd-B. This pattern is transient and evolves into a broader expression domain encompassing the entire germ band during the extended germ band stage.

The Drosophila extra sex combs protein contains WD motifs essential for its function as a repressor of homeotic genes.

Extra sex combs is a member of the Polycomb Group genes, whose products are required for stable long term transcriptional repression of the homeotic genes of the Bithorax and Antennapedia complexes. The Pc-G proteins are required to maintain the spatially restricted domains of homeotic gene expression established by the transiently expressed repressors, e.g., hunchback, but are not required for the functioning of these early repressors. This implies two distinct modes of repression and raises the question: how does the transition from early transient repression to stable Pc-G-mediated repression occur? While other Pc-G proteins are required continuously throughout development, the esc RNA is only present transiently in early embryos, suggesting that esc may play a role in mediating this transition to stable long term Pc-G-mediated repression. The predicted esc protein contains multiple copies of the WD motif, found in G-protein beta subunits as well as non-G proteins involved in diverse cellular functions, including transcriptional repression. The sequence alterations of a number of esc mutations cause amino acid substitutions within the WD repeats, identifying them as essential for the function of the esc protein as a repressor of homeotic gene expression. Other WD proteins are components of reversible macromolecular assemblies and the WD motif has recently been directly implicated in mediating interactions with other proteins in such complexes. We propose that the esc protein is similarly involved in the initial recruitment of Pc-G repressors to the homeotic genes to establish their stable long term repression.

Trithorax is required to maintain engrailed expression in a subset of engrailed-expressing cells.

We show that maintenance but not initiation of engrailed (en) gene expression in the Drosophila embryo requires trithorax (trx), which is also required to maintain stable long-term expression of the homeotic genes throughout the development. Like the homeotic genes, en expression is dependent on trx in only a subset of embryonic cells normally expressing en, including specific cells in the nervous system and the dorsal fat body cells surrounding the gonad. Loss of en expression in the dorsal fat body is correlated with the sterility of en females which also carry trx mutations. In addition, trx is required for normal en expression in the posterior compartment of the developing wing, reflected in enhancement of en phenotypes in en adults which also carry trx mutations. trx appears to be dispensable for maintenance of en expression in other embryonic cells. The trx protein binds to the region of the polytene chromosomes which contains the en gene, suggesting that trx regulates en expression directly by binding to the en regulatory region.

The Drosophila trithorax protein binds to specific chromosomal sites and is co-localized with Polycomb at many sites.

trithorax is required to stably maintain homeotic gene expression patterns established during embryogenesis by the action of the transiently expressed products of the segmentation genes. The large trithorax proteins contain a number of highly conserved novel motifs, some of which have been hypothesized to interact directly with specific DNA sequences in their target genes. Using antibodies directed against trithorax proteins, we show that they are bound to 63 specific sites on the polytene chromosomes of the larval salivary gland. trithorax binding is detected at the sites of its known targets, the Bithorax and Antennapedia complexes, despite the transcriptionally repressed state of these loci in the salivary gland. A temperature-sensitive trithorax mutation greatly reduces the number of binding sites. Simultaneous localization of trithorax and Polycomb indicates that many of their chromosomal binding sites coincide. We localized one trithorax binding site within a portion of the large 5' regulatory region of the Ubx gene, to an interval which also contains binding sites for Polycomb group proteins. These results suggest that trithorax exerts its effects by binding directly or indirectly to specific DNA sequences in its target genes. Co-localization with Polycomb also suggests that interactions between these activators and repressors of the homeotic genes may be a significant feature of their mode of action.

Trithorax regulates multiple homeotic genes in the bithorax and Antennapedia complexes and exerts different tissue-specific, parasegment-specific and promoter-specific effects on each.

The trithorax (trx) gene is required for normal development of the body plan in Drosophila embryos and adults. Mutations in trx cause homeotic transformations throughout the body. Genetic studies suggest that trx encodes a positive regulatory factor required throughout development for normal expression of multiple homeotic genes of the bithorax and Antennapedia complexes (BX-C and ANT-C). To determine how trx influences homeotic gene expression, we examined the expression of the BX-C genes Ultrabithorax, abdominal-A, Abdominal-B and the ANT-C genes Antennapedia, Sex combs reduced and Deformed in trx embryos. We show that trx does indeed exert its effects by positively regulating homeotic gene expression and that its effects on expression of individual homeotic genes are complex: each of the BX-C and ANT-C genes examined exhibits different tissue-specific, parasegment-specific and promoter-specific reductions in their expression. This implies that each of these genes have different requirements for trx in different spatial contexts in order to achieve normal expression levels, presumably depending on the promoters involved and the other regulatory factors bound at each of their multiple tissue- and parasegment-specific cis-regulatory sites in different regions of the embryo. These results also imply that those components of homeotic gene expression patterns for which trx is dispensable, require other factors, possibly those encoded by other trithorax-like genes.

Molecular characterization of the trithorax gene, a positive regulator of homeotic gene expression in Drosophila.

The Drosophila gene trithorax (trx) is required for the normal expression of a number of the homeotic genes in the bithorax complex (BX-C) and the Antennapedia complex (ANT-C). Flies homozygous for trx mutations exhibit segmental identity transformations similar to those caused by loss-of-function mutations in the homeotic genes Sex combs reduced (Scr), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B). We present a molecular characterization of the trx locus and show that it is necessary for normal levels of Antennapedia (Antp), Ubx, and abd-A protein accumulation. Interestingly, the loss of trx function differentially affects the expression these proteins; Ubx protein levels are greatly reduced, abd-A protein levels are reduced to a lesser extent, and Antp protein levels are only slightly reduced. P-element mediated transformation using 34 kb of genomic DNA containing the 25 kb trx transcription unit identifies all sequences necessary for normal trx function and limits the 5' and 3' flanking sequences that could be used in a regulatory capacity to relatively small regions. The primary transcription unit is differentially spliced to produce two large transcripts of 12 and 15 kb that have different developmental profiles.

Structure and expression of a family of Ultrabithorax mRNAs generated by alternative splicing and polyadenylation in Drosophila.

The 77-kb primary transcript of the homeotic Ultrabithorax (Ubx) gene is alternatively spliced to yield at least five different coding regions. Each is restricted to either a 3.2- or a 4.3-kb size class generated by alternative polyadenylation. The pathways for splicing and polyadenylation are therefore coordinately regulated, and because the relative abundance of the respective mRNAs varies throughout development, these pathways also appear to be developmentally regulated. Translation of these mRNAs yields a family of Ubx proteins characterized by constant amino- and carboxy-proximal regions of 247 and 99 amino acid residues, respectively. Members of this family are distinguished by a short variable region that links the constant regions and consists of different combinations of three optional elements of 9, 17, and 17 residues. Only four amino acid residues separate this variable region from the 60-residue homeo domain of the carboxy-terminal constant region. This proximity suggests that functional differences among the Ubx proteins derive from the differential effects of their variable regions on the DNA-binding capacity of the homeo domain. An argument is made that these functional differences are tissue specific.

Suppurative inflammation of vas deferens: an unusual groin mass.

We report a case of suppurative vasitis that presented as an inguinal mass in a young man. Inflammation of the vas deferens may occur as a manifestation of genital tuberculosis or other genitourinary tract infection. A granulomatous vasitis, vasitis nodosa, is usually associated with previous trauma or surgery. Suppuration is rare. We report a case that represents a rare cause of an inguinal mass.

Paratesticular leiomyosarcoma: an ultrastructural study.

We report a case of paratesticular leiomyosarcoma in an 84-year-old man. The tumor was firm and nodular, and attached to the upper pole of the left testis. Light microscopy showed a sarcoma with numerous mitoses. On electron microscopy tumor cells contained 7 nm. microfilaments, with regular dense bodies, micropinocytotic vesicles and the other hallmarks of smooth muscle differentiation, in addition to prominent pools of glycogen. This case illustrates the value of electron microscopy in the investigation of unusual urological tumors.