Clonal analysis of differentiating embryonic stem cells reveals a hematopoietic progenitor with primitive erythroid and adult lymphoid-myeloid potential.

Embryonic stem (ES) cells differentiate into multiple hematopoietic lineages during embryoid body formation in vitro, but to date, an ES-derived hematopoietic stem cell has not been identified and subjected to clonal analysis in a manner comparable with hematopoietic stem cells from adult bone marrow. As the chronic myeloid leukemia-associated BCR/ABL oncogene endows the adult hematopoietic stem cell with clonal dominance without inhibiting pluripotent lymphoid and myeloid differentiation, we have used BCR/ABL as a tool to enable engraftment and clonal analysis. We show that embryoid body-derived hematopoietic progenitors expressing BCR/ABL maintain a primitive hematopoietic blast stage of differentiation and generate only primitive erythroid cell types in vitro. These cells can be cloned, and when injected into irradiated adult mice, they differentiate into multiple myeloid cell types as well as T and B lymphocytes. While the injected cells express embryonic (beta-H1) globin, donor-derived erythroid cells in the recipient express only adult (beta-major) globin, suggesting that these cells undergo globin gene switching and developmental maturation in vivo. These data demonstrate that an embryonic hematopoietic stem cell arises in vitro during ES cell differentiation that constitutes a common progenitor for embryonic erythroid and definitive lymphoid-myeloid hematopoiesis.

Tantalus, a novel ASX-interacting protein with tissue-specific functions.

The Drosophila trithorax- and Polycomb-group (trxG and PcG) proteins maintain activated and repressed transcriptional states at specific target gene loci. The Additional sex combs (Asx) gene is of particular interest as it appears to function in both protein complexes and yet its effects on target genes are more restricted. A novel protein, Tantalus (TAN), was identified in a yeast two-hybrid screen for ASX-interacting proteins that might confer tissue-specific ASX functions. TAN contains consensus nuclear localization sites and binds DNA in vitro. However, its subcellular localization varies in a tissue-specific fashion. In salivary glands, TAN is predominantly nuclear and associates with 66 euchromatic sites on polytene chromosomes, more than half of which overlap with ASX. These loci do not include the homeotic genes of the ANT and BX complexes bound by other PcG and trxG proteins. Rather, tan mutant defects are restricted to sensory organs. We show that one of these defects, shared by Asx, is genetically enhanced by Asx. Taken together, the data suggest that TAN is a tissue-specific cofactor for ASX, and that its activity may be partially controlled by subcellular trafficking.

The combgap locus encodes a zinc-finger protein that regulates cubitus interruptus during limb development in Drosophila melanogaster.

The combgap locus, first described by C. B. Bridges in 1925, is a gene required for proper anteroposterior pattern formation in the limbs of Drosophila melanogaster. The development of the anteroposterior axis of fly limbs is initiated by hedgehog signaling from cells of the posterior half to cells of the anterior half of the limb primordium. Hedgehog signaling requires the anterior-specific expression of the gene cubitus interruptus to establish posterior-specific hedgehog secretion and anterior-specific competence to respond to hedgehog. We have cloned combgap and find that it encodes a chromosomal protein with 11 C(2)H(2) zinc fingers. Limb defects found in combgap mutants consist of either loss or duplication of pattern elements in the anteroposterior axis and can be explained through the inappropriate expression of cubitus interruptus and its downstream target genes. In combgap mutants, cubitus interruptus is ectopically expressed in the posterior compartments of wing imaginal discs and is downregulated in the anterior compartment of legs, wings and antennae. We are able to rescue anterior compartment combgap phenotypes by expressing additional cubitus interruptus using the Gal4/UAS system. Dominant alleles of cubitus interruptus, which result in posterior expression, phenocopy combgap posterior compartment phenotypes. Finally, we find that the combgap protein binds to polytene chromosomes at many sites including the cubitus interruptus locus, suggesting that it could be a direct regulator of cubitus interruptus transcription.

A novel member of murine Polycomb-group proteins, Sex comb on midleg homolog protein, is highly conserved, and interacts with RAE28/mph1 in vitro.

The Polycomb group of (PcG) genes were originally described in Drosophila, but many PcG genes have mammalian homologs. Genetic studies in flies and mice show that mutations in PcG genes cause posterior transformations caused by failure to maintain repression of homeotic loci, suggesting that PcG proteins have conserved functions. The Drosophila gene Sex comb on midleg (Scm) encodes an unusual PcG protein that shares motifs with the PcG protein polyhomeotic, and with a Drosophila tumor suppressor, lethal(3)malignant brain tumor (l(3)mbt). Expressed sequence tag (EST) databases were searched to recover putative mammalian Scm homologs, which were used to screen murine cDNA libraries. The recovered cDNA encodes two mbt repeats and the SPM domain that characterize Scm, but lacks the cysteine clusters and the serine/threonine-rich region found at the amino terminus of Scm. Accordingly, we have named the gene Sex comb on midleg homolog 1 (Scmh1). Like their Drosophila counterparts, Scmh1 and the mammalian polyhomeotic homolog RAE28/mph1 interact in vitro via their SPM domains. We analyzed the expression of Scmh1 and rae28/mph1 using northern analysis of embryos and adult tissues, and in situ hybridization to embryos. The expression of Scmh1 and rae28/mph1 is well correlated in most tissues of embryos. However, in adults, Scmh1 expression was detected in most tissues, whereas mph1/rae28 expression was restricted to the gonads. Scmh1 is strongly induced by retinoic acid in F9 and P19 embryonal carcinoma cells. Scmh1 maps to 4D1-D2.1 in mice. These data suggest that Scmh1 will have an important role in regulation of homeotic genes in embryogenesis and that the interaction with RAE28/mph1 is important in vivo.

The Additional sex combs gene of Drosophila encodes a chromatin protein that binds to shared and unique Polycomb group sites on polytene chromosomes.

The Additional sex combs (Asx) gene of Drosophila is a member of the Polycomb group of genes, which are required for maintenance of stable repression of homeotic and other loci. Asx is unusual among the Polycomb group because: (1) one Asx allele exhibits both anterior and posterior transformations; (2) Asx mutations enhance anterior transformations of trx mutations; (3) Asx mutations exhibit segmentation phenotypes in addition to homeotic phenotypes; (4) Asx is an Enhancer of position-effect variegation and (5) Asx displays tissue-specific derepression of target genes. Asx was cloned by transposon tagging and encodes a protein of 1668 amino acids containing an unusual cysteine cluster at the carboxy terminus. The protein is ubiquitously expressed during development. We show that Asx is required in the central nervous system to regulate Ultrabithorax. ASX binds to multiple sites on polytene chromosomes, 70% of which overlap those of Polycomb, polyhomeotic and Polycomblike, and 30% of which are unique. The differences in target site recognition may account for some of the differences in Asx phenotypes relative to other members of the Polycomb group.

The Drosophila polycomb group protein Psc contacts ph and Pc through specific conserved domains.

The Polycomb group proteins are transcriptional repressors that are thought to act through multimeric nuclear complexes. We show that ph and Psc coprecipitate with Pc from nuclear extracts. We have analyzed the domains required for the association of Psc with ph and Pc by using the yeast two-hybrid system and an in vitro protein-binding assay. Psc and ph interact through regions of sequence conservation with mammalian homologs, i.e., the H1 domain of ph (amino acids 1297 to 1418) and the helix-turn-helix-containing region of Psc (amino acids 336 to 473). Psc contacts Pc primarily at the helix-turn-helix-containing region of Psc (amino acids 336 to 473), but also at the ring finger (amino acids 250 to 335). The Pc chromobox is not required for this interaction. We discuss the implication of these results for the nature of the complexes formed by Polycomb group proteins.

RAE28, BMI1, and M33 are members of heterogeneous multimeric mammalian Polycomb group complexes.

The Polycomb group loci in Drosophila encode chromatin proteins required for repression of homeotic loci in embryonic development. We show that mouse Polycomb group homologues, RAE28, BMI1 and M33, have overlapping but not identical expression patterns during embryogenesis and in adult tissues. These three proteins coimmunoprecipitate from embryonic nuclear extracts. Gel filtration analysis of embryonic extracts indicates that RAE28, BMI1 and M33 exist in large multimeric complexes. M33 and RAE28 coimmunoprecipitate and copurify as members of large complexes from F9 cells, which express BMI1 at very low levels, suggesting that different Polycomb group complexes can form in different cells. RAE28, BMI1 and M33 interact homotypically, and both RAE28 and M33 interact with BMI1, but not with each other. The domains required for interaction were localized. Together, these studies indicate that murine Polycomb group proteins are developmentally regulated and function as members of multiple, heterogeneous complexes.

The SAM domain of polyhomeotic, RAE28, and scm mediates specific interactions through conserved residues.

The SAM (sterile alpha motif) domain is a 65- to 70-amino acid sequence found in many diverse proteins whose functions range from signal transduction to transcriptional repression. We show that the SAM domain of the Drosophila Polycomb group protein, polyhomeotic (ph), is capable of binding to itself in vitro. We test a number of near relatives of the ph SAM domain from fruit fly, mouse, and yeast and show that all are capable of self-binding. Heterologous interactions are seen among a subset of SAM domains, including ph, Scm, and RAE28. Several conserved amino acid residues were mutated in the ph SAM domain, and the effects on self-binding and heterologous association were demonstrated. L33, L41, and 162 are shown to be important determinants of the binding interface, while W1 and G50 are likely essential for the structure of the domain.

A domain shared by the Polycomb group proteins Scm and ph mediates heterotypic and homotypic interactions.

The Sex comb on midleg (Scm) and polyhomeotic (ph) proteins are members of the Polycomb group (PcG) of transcriptional repressors. PcG proteins maintain differential patterns of homeotic gene expression during development in Drosophila flies. The Scm and ph proteins share a homology domain with 38% identity over a length of 65 amino acids, termed the SPM domain, that is located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, we show that the SPM domain mediates direct interaction between Scm and ph. Binding studies with isolated SPM domains from Scm and ph show that the domain is sufficient for these protein interactions. These studies also show that the Scm-ph and Scm-Scm domain interactions are much stronger than the ph-ph domain interaction, indicating that the isolated domain has intrinsic binding specificity determinants. Analysis of site-directed point mutations identifies residues that are important for SPM domain function. These binding properties, predicted alpha-helical secondary structure, and conservation of hydrophobic residues prompt comparisons of the SPM domain to the helix-loop-helix and leucine zipper domains used for homotypic and heterotypic protein interactions in other transcriptional regulators. In addition to in vitro studies, we show colocalization of the Scm and ph proteins at polytene chromosome sites in vivo. We discuss the possible roles of the SPM domain in the assembly or function of molecular complexes of PcG proteins.

The polyhomeotic locus of Drosophila melanogaster is transcriptionally and post-transcriptionally regulated during embryogenesis.

The polyhomeotic (ph) locus of Drosophila is a complex locus essential for the maintenance of segmental identity. Genetic analysis suggested that two independent units contribute to ph function. Comparison of genomic sequence shows that the ph locus has been duplicated, and that it contains proximal and distal transcription units. The proximal transcription unit encodes two embryonic mRNAs of 6.4 and 6.1 kb, and the distal unit encodes a 6.4-kb embryonic mRNA. The proximal and distal transcription units are differentially regulated at the mRNA level during development as shown by developmental Northern analysis. The distal protein is very similar to the proximal product, except for the absence of an amino terminal region, and a small region near the carboxy terminus. The long open reading frame in the distal cDNA does not begin with an ATG codon, and an internal ATG is used for a start codon. We show that the proximal protein occurs in two forms that are developmentally regulated, and that probably arise from use of two different initiator methionine codons. We find no evidence for differential binding of proximal and distal products to polytene chromosomes. Nevertheless, we show that mutations in the proximal and distal proteins have differing effects on regulation of a reporter under the control of a regulatory region from bithoraxoid, suggesting that ph proximal and distal proteins have different functions. These results show that the ph locus undergoes complex developmental regulation, and suggest that Polycomb group regulation may be more dynamic than anticipated.