BIOLOGICAL OVERVIEW

The Sex combs on midleg (Scm) gene (Jürgens, 1985; Breen, 1986) encodes one of the Polycomb group (PcG) repressors (McKeon, 1991; Simon, 1992). Embryos lacking both maternal and zygotic Scm product die with most segments transformed into copies of the eighth abdominal segment (Breen, 1986). This null phenotype, which is among the strongest seen in single PcG mutants, shows that the Scm product is a central component in PcG repression. Scm protein represses multiple homeotic genes during embryonic stages (Breen, 1986; McKeon 1991, and Simon, 1992). Analysis of pupal lethal Scm alleles (Wu, 1989) shows that Scm is also required postembryonically. These genetic data and the continuous developmental expression of SCM mRNA imply a long-term role for Scm product in homeotic repression, like most other PcG products, (Bornemann, 1996).

Although Scm has been most well-characterized in terms of homeotic gene control, it is also likely to be involved in other processes, as are many of the PcG proteins. Scm is a regulator of the segmentation gene engrailed (Moazed, 1992) and genetic studies suggest a role in dorsal-ventral development (Adler, 1991). The suppression of zeste 1 eye color by Scm mutations may reflect an Scm role in white gene expression. This suppression does not require unusual Scm alleles, since it occurs when there is a deficiency at the Scm locus and in apparent Scm null mutations. Although the mechanism of zeste 1 suppression is unclear, it is intriguing that a subset of PcG products, including Scm, Enhancer of zeste and Posterior sexcombs, share the zeste interaction. Investigation of the physical interactions between Scm protein and its PcG cohorts should help define how transcription is modulated at homeotic loci and at other loci under PcG control (Bornemann, 1996 and references).

The Scm and Polyhomeotic proteins have in common the same domain (termed the SPM domain) located at their respective C termini. Using the yeast two-hybrid system and in vitro protein-binding assays, it has been shown 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, predict an alpha-helical secondary structure, and conservation of hydrophobic residues has prompted 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. Scm and Ph proteins co-localize at polytene chromosome sites in vivo (Peterson, 1997).

To begin to investigate the mechanism and specific residues used for SPM domain protein contact, the effects of site-directed mutations in either the Scm or Ph domains on in vitro binding were tested. The mutations were targeted to residues that are highly conserved in alignments of proteins with similar domains. The point mutations fall into two classes: those that target conserved residues in the extended SAM domain family and those that target residues conserved only in the high-homology SPM subgroup. Three site-directed mutations have been generated in the SPM domain of Scm. The G31S mutation alters a residue that is absolutely conserved in all 23 compiled versions in the extended domain family. This mutant was tested in the context of radiolabelled full-length Scm protein for binding to the minimal Scm and Ph domains. Both Scm-Scm and Scm-Ph interactions are greatly reduced in vitro. Consistent with the residual binding activity seen in the G31S mutant, G31S is found to mediate a reduced but still detectable interaction in the two-hybrid system (Peterson, 1997).

The L35S;L36S double mutation and the K49A mutation affect residues conserved in the high-homology subgroup but not in the extended domain family. Substantial self- and cross-binding activity is retained with these mutant proteins. The only reduction seen with these two mutants is a modest effect of the L35S:L36S double substitution upon the Scm-Scm interaction. This mutant causes a several-fold loss in Scm-Scm binding but retains Scm-Ph cross-binding activity comparable to that of the wild type (Peterson, 1997).

Five site-directed mutations were generated in the SPM domain of Polyhomeotic. All five mutations alter residues that are highly conserved in the extended domain family. These mutations were inserted into the context of the minimal GSTph1511-1576 fusion protein and then tested for binding to the minimal Scm radiolabelled domain. W1A and G51A ph mutations cause significant reductions in binding activity to Scm. In contrast, mutations in the conserved hydrophobic residues (L34A, L42A, and I63D) have little effect on in vitro Scm-Ph interaction (Peterson, 1997).

The two-hybrid and GST pulldown assays show that the Scm and Ph proteins can bind each other directly and that their respective SPM domains mediate qualitatively strong interactions. However, these experiments do not address whether the Scm and Ph proteins are partners at sites of action in vivo. To assess association in vivo, the Scm and Ph distributions were compared on wild-type polytene chromosomes. In addition, colocalization was tested for at an engineered chromosomal site containing an isolated segment of homeotic gene regulatory DNA. Polytene chromosome immunostaining experiments have shown that Ph protein accumulates at its two most well-characterized target loci, the Antennapedia (Ant-C) and bithorax (BX-C) homeotic gene complexes. In addition, Ph protein is associated with approximately 100 other sites in the genome. Ph protein immunolocalizes at the BX-C site as well as at five flanking sites on chromosome 3R. The same section of chromosome stains with antibody against Scm protein. There is strong signal at the BX-C locus, and the Scm distribution on flanking sites is identical to the Ph distribution. The Ph and Scm protein distributions in the Ant-C region are also identical. Since the antibodies used in these studies are both rabbit polyclonal antibodies, double-staining experiments to determine if all the approximately 100 Ph and Scm sites are identical could not be performed. However, comparison of the Scm sites on the five major chromosome arms with the Ph sites indicates that there is at least 90% overlap in the distributions of these two proteins on polytene chromosomes (Peterson, 1997).

To compare Ph and Scm association with an additional site of action in vivo, colocalization was tested at a site containing regulatory DNA isolated from a homeotic gene. The germ line transformant, 85-39, contains a 14-kb segment from the bxd regulatory region of the BX-C complex inserted near the tip of chromosome 3L at cytological location 62A. Previous work has shown that this transformed DNA segment creates a novel site of Ph protein accumulation and that expression programmed by this 14-kb DNA segment is regulated by Ph and Scm in vivo. Scm protein accumulates at the insertion site of this bxd regulatory DNA. Thus, Scm and Ph proteins are both recruited to an engineered chromosomal site containing an in vivo regulatory target. This result, together with the coincidence of the Ph and Scm proteins at many wild-type chromosomal sites, provides evidence for association of these proteins in vivo (Peterson, 1997).

Sex comb on midleg (Scm) is a functional link between PcG-repressive complexes in Drosophila

The Polycomb group (PcG) proteins are key regulators of development in Drosophila and are strongly implicated in human health and disease. How PcG complexes form repressive chromatin domains remains unclear. Using cross-linked affinity purifications of BioTAP-Polycomb (Pc) or BioTAP-Enhancer of zeste [E(z)], this study captured all PcG-repressive complex 1 (PRC1) or PRC2 core components and Sex comb on midleg (Scm) as the only protein strongly enriched with both complexes. Although previously not linked to PRC2, direct binding of Scm and PRC2 was confirmed using recombinant protein expression and colocalization of Scm with PRC1, PRC2, and H3K27me3 in embryos and cultured cells using ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing). Furthermore, it was found that RNAi knockdown of Scm and overexpression of the dominant-negative Scm-SAM (sterile α motif) domain both affected the binding pattern of E(z) on polytene chromosomes. Aberrant localization of the Scm-SAM domain in long contiguous regions on polytene chromosomes revealed its independent ability to spread on chromatin, consistent with its previously described ability to oligomerize in vitro. Pull-downs of BioTAP-Scm captured PRC1 and PRC2 and additional repressive complexes, including PhoRC, LINT, and CtBP. It is proposed that Scm is a key mediator connecting PRC1, PRC2, and transcriptional silencing. Combined with previous structural and genetic analyses, these results strongly suggest that Scm coordinates PcG complexes and polymerizes to produce broad domains of PcG silencing (Kang, 2015).

One of the most interesting properties of chromatin modification is the ability, under certain circumstances, to propagate in cis independent of sequence. This ability to 'spread' may be important for the inheritance of chromatin states initially established through interactions at nucleation sites such as PREs. SAM domain-mediated polymerization is therefore an attractive model to explain the propagation of PcG silencing. From that perspective, Ph, one of the core components of PRC1, may be responsible for the spreading of PRC1 through Ph-SAM polymerization. The compact chromatin environment formed by PRC1 spreading may improve the enzymatic activity of PRC2, and the capacity of the Pc chromodomain to interact with H3K27me3 may also contribute to the synergistic spreading of PcG silencing. However, consistent with the identification of PRC1 and PRC2 as distinct complexes that purify independently, E(z) RNAi does not significantly affect binding patterns of Pc on polytene chromosomes, and E(z) binding is likewise still detected after Pc RNAi. This study found that Scm directly interacts with the PRC2 complex and colocalizes with H3K27me3 in genome-wide analyses. Scm RNAi results in the loss of major sites of E(z) binding and the redistribution of H3K27me3 on polytene chromosomes. In addition, overexpression of the Scm-SAM domain interferes with binding of endogenous Scm to chromosomes and appears to self-polymerize for long distances on polytene chromosomes independently of PRC1 and PRC2. Taken together, it is suggested that the interaction of Scm with PRC2 and polymerization by the Scm-SAM domain may be key factors contributing to PRC2 and H3K27me3 spreading (Kang, 2015).

The strong interaction that discovered between Scm and the G9a SET domain protein, an H3K9 methyltransferase, suggests a new link between H3K27 and H3K9 methylation in Drosophila. These classical histone marks, associated with silent chromatin, were once thought to be largely distinct but are now proposed to have a functional relationship in PcG silencing in mammals, most notably in X inactivation. There was also evidence for colocalization of these two marks in early ChIP analyses at the HOX gene Ubx in imaginal discs. G9a may also play a role in regulation of H3K27 methylation (Mozzetta, 2014). Alternatively, the abundance of G9a may reflect a key role for the CtBP corepressor complex in PcG function rather than for G9a itself, which is a nonessential gene. Genome-wide binding profile analyses have shown that the components of the CtBP complex such as Su(var)3-3 (LSD1) and Rpd3 (HDAC1) are mainly enriched on active genes rather than repressed genes in human cells, and L(3)mbt, one component of the LINT complex, colocalizes with insulator proteins, including CP190 and mod(mdg4), rather than with PcG proteins in Drosophila. Therefore, the possibility that the interactions of Scm with CtBP or LINT repressor complexes occur as independent complexes irrelevant to PcG silencing cannot be excluded. However, considering that PcG silencing could require dynamic interactions during development, components of these repressor complexes may not be permanently stationed in PcG silenced domains but rather participate in PcG silencing transiently. Furthermore, some of the CtBP subunits were copurified in Pc and E(z) affinity purifications, and previous studies reported that CtBP complex components can contribute to PcG silencing in Drosophila. For example, CtBP mutation causes the loss of Pc recruitment to many PREs. Furthermore, Rpd3 deacetylates H3K27ac, which is mutually exclusive with H3K27me3, and Su(var)3-3 demethylates H3K4me1 and H3K4me2, which are active marks linked to Trithorax (Trx) activity and H3K27ac (Kang, 2015).

How PcG complexes find PREs and spread to create repressive domains is not known on a mechanistic level. Perhaps repressor complexes such as CtBP help remove active chromatin marks to attract the PcG initially or enable cycles of spreading to maintain those domains. Future analyses will entail dissecting the direct interactions of Scm, including nucleosomes and their post-translational modifications. Furthermore, through iterative use of BioTAP-XL, the wealth of additional candidates in the Pc, E(z), and Scm pull-downs featured in this study will be invaluable in extending understanding of chromatin-based PcG repression (Kang, 2015).

Differentiating Drosophila female germ cells initiate Polycomb silencing by regulating PRC2-interacting proteins

Polycomb silencing represses gene expression and provides a molecular memory of chromatin state that is essential for animal development. This study shows that Drosophila female germline stem cells (GSCs) provide a powerful system for studying Polycomb silencing. GSCs have a non-canonical distribution of PRC2 activity and lack silenced chromatin like embryonic progenitors. As GSC daughters differentiate into nurse cells and oocytes, nurse cells, like embryonic somatic cells, silence genes in traditional Polycomb domains and in generally inactive chromatin. Developmentally controlled expression of two Polycomb repressive complex 2 (PRC2)-interacting proteins, Pcl and Scm, initiate silencing during differentiation. In GSCs, abundant Pcl inhibits PRC2-dependent silencing globally, while in nurse cells Pcl declines and newly induced Scm concentrates PRC2 activity on traditional Polycomb domains. These results suggest that PRC2-dependent silencing is developmentally regulated by accessory proteins that either increase the concentration of PRC2 at target sites or inhibit the rate that PRC2 samples chromatin (DeLuca, 2020).

The work described here shows that the Drosophila female germline has multiple advantages for studying the developmental regulation of chromatin silencing both before and during differentiation. Female GSCs continuously divide to produce new undifferentiated progenitors, which expand and differentiate into nurse cells or oocytes, generating large amounts of a much simpler tissue than a developing embryo. Additionally, an inducible reporter assay compatible with the female germline was developed that sensitively responds to developmental changes in local chromatin repression in individual cells. In contrast to RNAseq, which measures steady state RNA levels, or ChIPseq, which correlates chromatin epitopes with their perceived function on gene expression, the reporters directly test how local chromatin influences the inducibility of surrounding genes, and are easily combined with tissue specific knockdowns to identify trans-acting factors contributing to reporter inducibility. Finally, a genetic engineering approach allows any construct (not just hsGFP) to be efficiently integrated into many pre-existing 'donor' sites, including those used previously with other reporters, or sites heavily silenced by repressive chromatin in differentiated cells. Although the number of different donor sites in certain types of chromatin is currently limited, new sites continue to be generated using CRISPR/Cas9 targeting and the method can ultimately be applied virtually anywhere in the genome (DeLuca, 2020).

Analysis of Polycomb repression with reporters, ChIP, and PcG-gene knockdowns provided numerous insights into how chromatin affects gene expression and female germline development in Drosophila. GSCs, the precursors of oocytes and nurse cells, contain a non-canonical, binary distribution of moderate H3K27me3 enrichment on all transcriptionally inactive loci and very low enrichment on active chromatin. A similar non-canonical H3K27me3 distribution was observed in early fly embryos, suggesting that noncanonical chromatin represents a 'ground state' for progenitors that will propagate future generations of undifferentiated germ cells or somatic cells that differentiate into specialized tissues. Such non-canonical chromatin was first identified in mouse oocytes and preimplantation embryos. If non-canonical H3K27me3 chromatin is a characteristic of undifferentiated, totipotent cells, what function might it confer to account for its conservation (DeLuca, 2020)?

Experiments confirmed previous workshowing that chromatin modified by PRC2 is essential for the Drosophila female germ cell cycle. Germline cysts lacking PRC2 are unable to stably generate oocytes, and E(z) GermLine-specific RNAi Knock Down (GLKD) nurse cells mis-express multiple genes and degenerate at about stage 5. In contrast, GSCs lacking PRC2 properly populate their niche, divide, and produce daughters that interact with female follicle cells and begin nurse cell differentiation. Removing PRC2 activity from GSCs did not generally increase the steady state abundance of genes or the inducibility of reporters in H3K27me3-enriched inactive or PcG domains. These results suggest that PRC2 and non-canonical chromatin lack vital functions in undifferentiated germline progenitors but are critical for repressing genes upon differentiation. However, a requirement for PRC2 or non-canonical chromatin under stress conditions or prolonged aging cannot be dismissed. For example, PRC2 could promote the long-term maintenance of female GSCs, similarly to how it maintains male germline progenitors in flies and mice (DeLuca, 2020).

Germline cysts and nurse cells are found in diverse animal species across the entire phylogenetic spectrum, but their function has been well studied mostly in insects such as Drosophila where they persist throughout most of oogenesis. While nurse cells have traditionally been considered germ cells rather than late-differentiating somatic cells, this study shows that that Drosophila nurse cells initiate Polycomb silencing and enrich PRC2 activity on a nearly identical collection of PcG domains as somatic cells. In more distant species, such as mice, nurse cells initially develop in a similar manner within germline cysts and contribute their cytoplasm to oocytes, but undergo programmed cell death before the vast majority of oocyte growth. Consequently, it remains an open question whether somatic differentiation plays a role in nurse cell function in mammals and many other groups (DeLuca, 2020).

The size and composition of oocyte cytoplasm are uniquely tailored to promote optimal fecundity and meet the demands of early development. In some species, including flies, nurse cells synthesize large amounts of specialized ooplasm to rapidly produce multitudes of large, pre-patterned embryos. In others, including mammals, oocytes more slowly synthesize the majority of ooplasm. Interestingly, both ooplasm synthesis strategies apparently require Polycomb silencing. However, the nurse cell-based strategy in flies primarily requires PRC2 but not PRC1 to silence hundreds of somatic genes, while the oocyte-based strategy in mice requires PRC1 but not PRC2 (DeLuca, 2020).

Different strategies of ooplasm synthesis may have evolved to be compatible with noncanonical germ cell chromatin. Staining experiments show that Drosophila oocytes maintain a widely distributed, non-canonical H3K27me3 distribution similar to pre-meiotic precursors or mouse oocytes, suggesting that non-canonical chromatin is conserved and maintained throughout the germ cell cycle. Similar to mouse oocytes, Drosophila spermatocytes also contain non-canonical chromatin and autonomously synthesize large amounts of cytoplasm by deploying PRC1 but not PRC2. Thus, three different types of germ cells are filled with large amounts of differentiated cytoplasm that requires Polycomb silencing for its synthesis, but nevertheless maintain a non-canonical, silencing-deficient PRC2 activity (DeLuca, 2020).

The conservation of undifferentiated, non-canonical chromatin despite a strong selection for Polycomb silencing during ooplasm synthesis argues that non-canonical chromatin must have a presently unappreciated fundamental purpose in germ cells. Noncanonical chromatin could regulate multigenerational processes like mutation, recombination, or transposition, that are not easily assayed in sterile individuals. Tests of these ideas will require a better understanding of how non-canonical chromatin is regulated and methods to disrupt non-canonical chromatin without disrupting other functions required for germline viability. Additionally, non-canonical chromatin could simply result from the silencing- incompetent PRC2 that was observed in progenitors (DeLuca, 2020).

Pcl was uncovered as both an inhibitor of PRC2 silencing and promoter of non-canonical chromatin in GSCs. Pcl^GLKD dramatically altered the footprint of PRC2 activity in GSCs. Pcl^GLKD favored H3K27me3 enrichment on PREs versus inactive domains, and increased the total amount of H3K27me1 by 13-fold and H3K27me2 by 1.4-fold and decreased the total amount of H3K27me3 by 1.8 fold. By binding DNA through its winged-helix domain, Pcl triples PRC2's residence time on chromatin and promotes higher states of H3K27 methylation in vitro. In GSCs, Pcl could simply change the result of each PRC2-chromatin binding event from H3K27me1 to me3. However, it is hard to imagine how an equivalent number of nucleosomes bearing a higher H3K27 methylation state could explain how Pcl inhibits silencing. Instead, it is proposed that Pcl inhibits silencing by reducing the number of PRC2-chomatin binding events per unit time by increasing the residence time of PRC2 on chromatin with each binding event. In this model, Pcl^GLKD would not only convert many H3K27me3 nucleosomes into H3K27me1 nucleosomes, it would also convert many unmethylated nucleosomes into H3K27me1 nucleosomes. Pcl^GLKD would more subtly affect H3K27me2 abundance because it simultaneously increases the number of PRC2-chromatin binding events while reducing the probability of each binding event leading to H3K27me2 versus me1 (DeLuca, 2020).

By reducing the number of PRC2 binding events, Pcl could increase the abundance of unmethylated H3K27 residues available for acetylation - a transcription promoting modification. In both flies and mammals, PRC2 transiently associates with chromatin to mono- and dimethylate H3K27 outside of traditional PcG domains, blocking H3K27 acetylation and antagonizing transcription. This study similarly found strong and widespread PRC2-dependent silencing in H3K27me1/2 enriched chromatin in nurse cells. Because inactive domain silencing was not affected by depletion of Pcl, Jarid2, or H3K27me3, it is proposed that core-PRC2, but not Pcl-PRC2 or H3K27me3, primarily silences inactive chromatin (DeLuca, 2020).

In GSCs, abundant Pcl could saturate PRC2, effectively depleting faster-sampling core-PRC2 complexes in favor of slower sampling Pcl-PRC2. In somatic embryonic cells, Pcl is present in a small fraction of PRC2 complexes. Compared to other fly tissues, Pcl mRNA is most abundant in the ovary, and within the ovary, Pcl protein is much more abundant in GSCs and nurse cell precursors than differentiated nurse cells and somatic cells. Within each differentiated germline cyst, Pcl mRNA is depleted from nurse cells and enriched in oocytes, suggesting that Pcl protein levels may be regulated by an mRNA transport mechanism induced in region 2 that also triggers the differentiation of oocytes from nurse cells (DeLuca, 2020).

Pcl, and a second PRC2-interacting protein, Scm, regulate the transition from noncanonical to canonical chromatin and initiate Polycomb repression. During nurse cell differentiation, it is proposed that Pcl depletion frees core-PRC2 to rapidly sample and silence inactive domains, while Scm (which is absent from the GSC) induction recruits high levels of PRC1 and PRC2 activity around PREs. ScmGLKD nurse cell chromatin retained a noncanonical H3K27me3 pattern characteristic of GSCs, as if differentiation at PcG domains had not occurred. In mice, Scm homologue, Scml2, similarly associates with PcG domains to recruit PRC1/2 and silence PcG targets during male germline development. However, unlike its fly orthologue in female GSCs, Scml2 is expressed in male germline precursors. This difference could explain why mammalian PGCs partially enrich PRC2 activity on CGIs while fly female GSCs do not enrich PRC2 on specific sites. While PcG domain-associated Scm is sufficient to enrich PRC2 activity above background levels found throughout inactive chromatin, a second PRC2 interacting protein, Pcl, is additionally required to promote full PRC2 and H3K27me3 enrichment on PcG domains. Because Scm oligomerizes and interacts with PRC2 in vitro, it could form an array of PRC2 binding sites anchored to PREs through Sfmbt. It is proposed that two cooperative interactions, PRC2 with PRE-tethered Scm, and Pcl with DNA, preferentially concentrate H3K27me3-generating Pcl-PRC2 versus H3K27me1/2-generating core-PRC2 on PcG domains (Figure 6D). H3K27me3 could then be further enriched by H3K27me3-induced allosteric PRC2 activation through the Esc subunit (DeLuca, 2020).

By promoting PRC1 and PRC2 concentration on PcG domains, Scm enhances silencing on PcG-localized reporters. While Scm-depleted nurse cells completed oogenesis, a subset of PcG domain-localized PcG including chinmo and the posterior Hox gene, Abd-b, escaped repression and were potentially loaded into embryos. Eggs derived from Scm^GLKD nurse cells failed to hatch, and mis-expressed Abd-b in anterior segments following germ band elongation. This defect more closely resembled maternal plus zygotic than maternal-only Scm mutants, suggesting that Scm^GLKD may deplete both maternal and zygotic Scm (DeLuca, 2020).

However, the additional possibility that mis-regulation of maternal Polycomb targets like chinmo contribute to the subtle embryonic defects observed in maternal-only Scm mutant clones cannot be excluded (DeLuca, 2020).

Further study of the Polycomb-mediated repression described in this study will help define the gene regulation program of Drosophila nurse cells and its contribution to oocyte growth. Additional characterization and perturbation of non-canonical chromatin throughout the germ cell cycle will yield further insights into its function in development. Finally, incorporating studies of other chromatin modifications, including H3K9me3-based repression, during germ cell development will contribute to a fuller understanding of how chromatin contributes to an immortal cell lineage (DeLuca, 2020).

GENE STRUCTURE

Bases in 5' UTR - 434

Bases in 3' UTR - 676 and 979

PROTEIN STRUCTURE

Amino Acids - 877

Structural Domains

The predicted Scm protein is 877 amino acids long with a relative molecular mass of 94,000 Da and a pI of 9.4. A potential nuclear localization signal (RQRGRPAKR) starts at amino acid 52. Restriction mapping and sequence analysis have shown that the difference between the 4.1 and 3.8 kb cDNAs results from use of alternative polyadenylation sites. The poly(A) tail of the longer cDNA begins at position 4046 whereas the poly(A) tail of the shorter cDNA begins at position 3744. There is a consensus poly(A) addition signal located 25 bp upstream of the 3.8 kb cDNA poly(A) tail. There are only imperfect matches to the poly(A) addition signal in the region immediately upstream of the 4.1 kb cDNA poly(A) tail (Bornemann, 1996).

The Scm and Polyhomeotic (DeCamillis, 1992) proteins share the presence of a homologous region with regard to the SPM domain. This domain is 38% identical between the two proteins, over a length of 65 amino acids. Each protein has an SPM domain located at its respective C termini. This domain is predicted to be largely alpha-helical. Besides these proteins, there are numerous proteins that contain a related domain with much lower overall identity (Alkema, 1997 and Ponting, 1995). These more distantly related proteins include members of the Ets family of transcription factors and yeast proteins required for mating. The high-homology domain subgroup that includes the Scm and Ph versions are referred to as the SPM domain, and the extended domain family is referred to as the SAM domain (Ponting, 1995). One of the more well-characterized SAM domains is present in the human TEL oncoprotein, an Ets class transcription factor, where the SAM has been referred to as a helix-loop-helix (HLH) domain. Recent studies have shown that this domain mediates self-binding and oligomerization of TEL protein and of TEL fusion protein derivatives (Peterson, 1997).

Besides sharing homology to other proteins in the SPM domain, Scm is even more similar to another fly protein, the product of the tumor suppressor gene lethal (3) malignant brain tumor ([l(3)mbt]; Wismar, 1995). The Scm and L(3)mbt proteins share zinc fingers, the SPM domain, and a third domain consisting of 100-amino-acid long repeats. These repeats, termed mbt repeats (Wismar, 1995 and Bornemann, 1996), are present in two tandem copies in SCM and three copies in L(3)Mbt. The biochemical role of mbt repeats is not known (Peterson, 1997).

Polyhomeotic, Rae-28 and l(3)mbt all contain putative Cys2-Cys2 zinc fingers that define a distinct zinc finger subclass, which is marked by identical spacing between the cysteine pairs and conservation of residues that flank the cysteines. There are two such zinc fingers located near the N terminus of Scm protein and a single finger in the Ph, Rae-28 and L(3)mbt proteins. The spacing between cysteines is distinct from the Cys2-Cys2 fingers of known DNA-binding proteins such as the nuclear hormone receptors. Scm also contains a third potential zinc-binding region (Zn3), which differs from the N-terminal fingers and is not shared in Ph, Rae-28 or L(3)mbt. This third region can be arranged as a Cys2-Cys2 finger, but the presence of additional cysteine and histidine residues between the outer cysteine pairs may reflect alternative forms of a zinc-binding domain (Bornemann, 1996).

Scm protein also contains a region with a high density of alanine residues. Starting at amino acid position 748, there is a stretch of 29 residues comprised of 52% alanine. Alanine-rich regions have been associated with transcriptional repression domains in the Drosophila Engrailed, Even-skipped and Krüppel proteins (Bornemann, 1996).

date revised: 13 Sept 99

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