InteractiveFly: GeneBrief

Scm-related gene containing four mbt domains: Biological Overview | References

BIOLOGICAL OVERVIEW

Polycomb response elements (PREs) are specific cis-regulatory sequences needed for transcriptional repression of HOX and other target genes by Polycomb group (PcG) proteins. Among the many PcG proteins known in Drosophila, Pleiohomeotic (Pho) is the only sequence-specific DNA-binding protein. To gain insight into the function of Pho, Pho protein complexes were purified from Drosophila embryos and it was found that Pho exists in two distinct protein assemblies: a Pho-dINO80 complex containing the Drosophila INO80 nucleosome-remodeling complex, and a Pho-repressive complex (PhoRC) containing the uncharacterized gene product dSfmbt. Analysis of PhoRC reveals that dSfmbt is a novel PcG protein that is essential for HOX gene repression in Drosophila. PhoRC is bound at HOX gene PREs in vivo, and this targeting strictly depends on Pho-binding sites. Characterization of dSfmbt protein shows that its MBT repeats have unique discriminatory binding activity for methylated lysine residues in histones H3 and H4; the MBT repeats bind mono- and di-methylated H3-K9 and H4-K20 but fail to interact with these residues if they are unmodified or tri-methylated. These results establish PhoRC as a novel Drosophila PcG protein complex that combines DNA-targeting activity (Pho) with a unique modified histone-binding activity (dSfmbt). It is proposed that PRE-tethered PhoRC selectively interacts with methylated histones in the chromatin flanking PREs to maintain a Polycomb-repressed chromatin state (Klymenko, 2006).

The regulation of gene expression by Polycomb group (PcG) and trithorax group (trxG) proteins represents a paradigm for understanding the establishment and maintenance of heritable transcriptional states during development. PcG and trxG genes were first genetically identified as regulators that are required for the long-term maintenance of HOX gene expression patterns in Drosophila. PcG proteins keep HOX genes silenced in cells in which they must stay inactive, whereas trxG proteins maintain the active state of these genes in appropriate cells. This regulatory relationship is conserved in vertebrates, where PcG and trxG proteins also regulate HOX gene expression. In addition, mammalian PcG and trxG proteins have also been implicated in X-chromosome inactivation, hematopoietic development, control of cell proliferation, and oncogenic processes (Klymenko, 2006).

Drosophila HOX genes are among the best-studied target genes of the PcG/trxG system. Different studies have led to the identification of specific cis-regulatory sequences in HOX genes that are called Polycomb response elements (PREs) and are required for silencing by PcG proteins. PREs are typically several hundred base pairs in length, and they function as potent transcriptional silencer elements in the context of HOX reporter genes as well as in a variety of other reporter gene assays. This operational definition of PREs is complemented by their classification as DNA sequences to which PcG proteins bind, directly or indirectly. Among the 14 cloned Drosophila PcG genes, only Pleiohomeotic (Pho) and Pho-like (Phol) encode sequence-specific DNA-binding proteins. Pho and Phol bind the same DNA sequence, and while the two proteins act to a large extent redundantly, double mutants show severe loss of HOX gene silencing (Brown, 2003). DNA-binding sites for Pho and Phol are present in all PREs that have been characterized to date, and mutational analyses of these binding sites have shown that they are essential for silencing by PREs. In contrast, none of the other 12 characterized PcG proteins bind DNA in a sequence-specific manner. However, formaldehyde cross-linking studies showed that several of these proteins specifically associate with the chromatin of PREs in tissue culture cells and in developing embryos and larvae. Biochemical studies revealed that most of these non-DNA-binding PcG proteins are components of either PRC1 or PRC2, two distinct PcG protein complexes that have recently been purified and characterized. Specifically, PRC1 contains the PcG proteins Polycomb (Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), Sex combs extra/Ring (Sce/Ring), and Sex combs on midleg (Scm), whereas PRC2 contains the three PcG proteins Extra sex combs (Esc), Enhancer of zeste [E(z)], and Suppressor of zeste 12 [Su(z)12] (Klymenko, 2006).

What is the role of Pho and Phol at PREs? Biochemically purified PRC1 and PRC2 do not contain Pho or Phol. Several recent studies investigated possible physical interactions between Pho and PRC1 or PRC2 complex components. Based on coimmunoprecipitation and GST pull-down assays, it was proposed that Pho directly interacts with several different PRC1, PRC2, and SWI/SNF complex components. However, on polytene chromosomes of phol; pho double mutants, the binding of PRC1 and PRC2 to HOX genes and at most other loci is largely unperturbed (Brown, 2003), suggesting that, at least in this tissue, Pho and Phol are not strictly required for keeping PRC1 and PRC2 anchored to HOX genes (Klymenko, 2006).

To gain insight into the biological function of Pho, Pho-containing protein complexes were biochemically purified from Drosophila. The data show that Pho exists in two distinct multiprotein complexes that, contrary to expectation, do not contain any of the previously characterized PcG proteins. The functional analysis of one of these Pho complexes that was named PhoRC provides evidence that its binding to PREs is required for maintaining repressive HOX gene chromatin (Klymenko, 2006).

A tandem affinity purification (TAP) strategy was used to purify Pho protein complexes from Drosophila embryonic nuclear extracts. A transgene that expresses a TAP-tagged Pho fusion protein (Pho-TAP) was expressed under the control of the Drosophila alpha-tubulin promoter, and transgenic flies were generated. To test whether the Pho-TAP protein is functional, the transgene was introduced into the genetic background of animals homozygous for pho¹, a protein-negative allele of pho. pho¹ homozygotes die as pharate adults, but they are rescued into viable and fertile adults that can be maintained as a healthy strain if they carry one copy of the transgene expressing Pho-TAP. The Pho-TAP protein can thus substitute for the endogenous Pho protein, and this shows that the fusion protein is functional (Klymenko, 2006).

Proteins that are associated with the Pho-TAP protein were purified from embryonic nuclear extracts, following the TAP procedure. Seven different polypeptides that consistently copurified with the Pho-TAP bait protein in several independent purifications were identified through sequencing of peptides from individual protein bands by nanoelectrospray tandem mass spectrometry. In addition to Pho, the isolated protein assembly contains the product of CG31212, a protein that is most closely related to yeast INO80, the SWI/SNF2-like nucleosome-remodeling subunit in the yeast INO80 complex. The CG31212 locus as will therefore be referred to as dINO80. Five other subunits of the Pho complex were identified as Reptin (Rept), Pontin (Pon), Actin (Act), and the two actin-related proteins dArp5 and dArp8, which are encoded by CG7940 and CG7846, respectively. These five proteins represent the Drosophila homologs of five core subunits that assemble together with INO80 to form the yeast INO80 complex (Shen, 2000). Specifically, Rept and Pont are homologs of the yeast Rvb1 and Rvb2 AAA-ATPases that constitute a DNA helicase in the INO80 complex. Act, dArp5, and dArp8 are homologs of the Actin, Arp5, and Arp8 proteins, respectively, that are present in the yeast INO80 complex. Thus, it appears that a Drosophila dINO80 complex copurifies with Pho. In addition, the purified material also contained the product of CG16975, a protein that is not conserved in yeast but is closely related to the product of the murine Scm-related gene containing four mbt domains (Sfmbt) (Usui, 2000); the CG16975 gene is referred to as dSfmbt. The characteristic features of mammalian Sfmbt and the Drosophila dSfmbt protein are four malignant brain tumor (MBT) repeats and a sterile alpha motif (SAM) domain. The Drosophila genome encodes two other proteins that contain MBT repeats and show a similar domain architecture, l(3)mbt and the PcG repressor Scm. Taken together, these findings suggest that Pho exists in multiprotein assemblies that contain a dINO80 complex and dSfmbt but, unexpectedly, none of the previously characterized PcG proteins (Klymenko, 2006).

Since the yeast genome does not contain any dSfmbt-related protein, it was asked whether dSfmbt and dINO80 are part of distinct Pho protein complexes. To this end, crude embryonic nuclear extracts were fractionated by glycerol gradient sedimentation and individual fractions were probed by Western blotting with antibodies against Pho, Pho-like, dINO80, and dSfmbt. The results show that dINO80 and dSfmbt are present in separate fractions of the gradient but that Pho and Pho-like are present in both dINO80- and dSfmbt-containing fractions. dSfmbt and dINO80 thus exist in distinct protein complexes in embryonic nuclear extracts. It should be noted that Pho and Pho-like are also present in fractions that do not contain dINO80 or dSfmbt. This suggests that Pho and Pho-like also exists in soluble protein assemblies that are distinct from the complexes identified in this study, but that these assemblies are not stable enough to be isolated as complexes in the purification scheme (Klymenko, 2006).

It was asked whether components of the purified Pho complexes are associated with PREs in vivo. To this end, chromatin immunoprecipitation (X-ChIP) assays were performed. Drosophila embryos were treated with formaldehyde and DNA that was cross-linked to Pho, dSfmbt, dINO80, Reptin, Pontin, or Ph was immunoprecipitated with antibodies against these proteins. Real-time quantitative PCR was used to measure the abundance of the following endogenous and transgene PREs in the immunoprecipitates. The bxd and iab-7 PREs in the HOX genes Ultrabithorax (Ubx) and Abdominal-B (Abd-B), respectively, are well-characterized, and Pho binds to these PREs in vitro and in vivo. It has been reported that PRE_D, a 572-bp core fragment of the bxd PRE, silences a Ubx-LacZ reporter gene in imaginal discs and in embryos but that point mutations in all six Pho protein-binding sites in this fragment (PRE_{D pho mut}) completely abolish its silencing capacity (Fritsch, 1999). Therefore X-ChIP assays were performed in transformed embryos that carried either the wild-type PRE_D or the mutated PRE_{D pho mut} reporter gene; this allowed direct comparison of protein binding at the transgenic PRE with protein binding at the endogenous bxd and iab-7 PREs in the same preparation of chromatin. Specific PCR primer sets allowed X-ChIP signals at the reporter gene PRE to be distinguished from signals at the endogenous bxd PRE. It was found that Pho, Ph, and, importantly, also dSfmbt are specifically bound at the endogenous bxd and iab-7 PREs but not at sequences flanking those PREs. In contrast, binding of dINO80, Reptin, or Pontin at any of the sequences analyzed (data not shown). Pho, dSfmbt, and Ph are also bound at the PRE_D fragment in the transgene was not detected, but, strikingly, binding signals of Pho, dSfmbt, and Ph are severely reduced at the mutated PRE_{D pho mut} fragment. Taken together, these data show that Pho-dSfmbt complexes are bound at PREs in vivo and that binding of these complexes to PREs requires DNA-binding sites for Pho. Since association of dINO80 complex components with PREs was not detected in this assay, further analysis focused on the characterization of Pho-dSfmbt complexes (Klymenko, 2006).

Therefore, this study shows that the PcG protein Pho exists in two stable protein complexes, a Pho-dINO80 complex and PhoRC. Biochemical and genetic analyses identify PhoRC as a novel PcG protein complex that has a different subunit composition and molecular function than the previously described PcG complexes PRC1 and PRC2. The following conclusions can be drawn from these studies of PhoRC: (1) PhoRC contains Pho and dSfmbt, and these two proteins form a very stable complex that can be purified from embryos and reconstituted from recombinant proteins. (2) PhoRC is bound to PREs in vivo, and PRE-targeting of PhoRC requires intact Pho/Pho-like DNA-binding sites. (3) A dSfmbt knockout reveals that dSfmbt is a novel PcG protein that is critically needed for HOX gene silencing. (4) The MBT repeats of dSfmbt are a novel methyl-lysine-recognizing module that selectively binds to the N-terminal tails of histones H3 and H4 if they are mono- or di-methylated at H3-K9 or H4-K20, respectively. PhoRC thus contains sequence-specific DNA-binding activity via the Pho protein and methylated histone-binding activity via dSfmbt (Klymenko, 2006).

Pho and Pho-like are the only PcG proteins with sequence-specific DNA-binding activity. Therefore, it is likely that these factors might tether PRC1 or PRC2 to PREs. Unexpectedly, biochemical purification of Pho complexes revealed that Pho exists in stable assemblies with either the PcG protein dSfmbt or components of the Drosophila INO80 complex. However, native or recombinant Pho complexes that contain PRC1 or PRC2 components were not purified. Similarly, biochemically purified PRC1 and PRC2 also do not contain Pho. PhoRC, PRC1, and PRC2 thus seem to be separate biochemical entities (Klymenko, 2006).

Reconstitution of recombinant PhoRC shows that dSfmbt binds directly to Pho or to Pho-like to form stable dimeric complexes. Coimmunoprecipitation assays indicate that such interactions also take place in Drosophila, and it was found that dSfmbt is associated with Pho or Pho-like in vivo. Moreover, dSfmbt mutants and pho-like; pho double mutants show a comparable loss of HOX gene silencing with similar kinetics. These observations are consistent with dSfmbt being needed for repression by both Pho and Pho-like. Furthermore, the X-ChIP experiments show that Pho/Pho-like DNA-binding sites in PREs are critical for binding of both Pho and dSfmbt at PREs. These data thus suggest that PhoRC is tethered to PREs by Pho or Pho-like (Klymenko, 2006).

Binding of the PRC1 subunit Ph at the bxd PRE also depends on intact Pho protein-binding sites. Could dSfmbt in PRE-bound PhoRC interact with Scm or Ph, for example, through the C-terminal SAM domain and thereby tether PRC1 to PREs? In coimmunoprecipitation experiments, no association of dSfmbt with Ph or Scm was detected. These interactions, if they exist, might be either very weak or exist only transiently. Previous studies reported direct physical interactions between Pho and PRC1 or PRC2 subunits, respectively. A possible scenario could therefore be that multiple weak interactions between Pho and dSfmbt with PRC1 and/or with PRC2 subunits might help to stabilize the binding of these complexes to PREs. It is also possible that the lack of Ph binding to the PRE transgene with mutated Pho sites reflects an indirect role of PhoRC that does not involve direct physical interactions between PhoRC and PRC1. In this context, it is worth noting that, on polytene chromosomes, binding of Ph and other PRC1 components is largely unperturbed in animals that lack both Pho and Pho-like proteins (Klymenko, 2006).

Four consecutive MBT repeats are a key feature of the dSfmbt protein. Fluorescence polarization binding assays suggest that these MBT repeats selectively bind to the N-terminal tail of histones H3 and H4 if these are mono- or di-methylated, but not if the same sites are unmethylated or tri-methylated. This novel discriminatory methyl-lysine-binding activity of MBTs is in stark contrast to the well-documented preference of chromodomains for higher, i.e., tri-methylated, binding sites in histones and could constitute an important general function of chromatin-associated MBT-containing proteins. The dSfmbt methyl-lysine interaction seems to be specific for the H3K9 and H4K20 methylation sites since matched H3 peptides that are methylated at different lysine residues (i.e., H3-K4me instead of H3-K9me) or histone tail peptides in which the methylated lysine residue is embedded in the same amino acid sequence context (i.e., ARKmeS in H3-K27me instead of ARKmeS in H3-K9me) are bound with at least 20-fold lower affinity (Klymenko, 2006).

Since these results suggest that dSfmbt is targeted to HOX gene PREs primarily through interaction with Pho, it was reasoned that binding to methyl-lysine residues in histone tails is not a primary mechanism for targeting dSfmbt to HOX genes. Moreover, recent studies provide evidence that, in the PcG-repressed state, the silenced HOX gene Ubx is tri-methylated at H3-K9, H4-K20, and H3-K27 throughout the gene, whereas lower methylated states of these sites are largely absent. What, then, is the role of Sfmbt in binding histones that are mono- or di-methylated at H3-K9 and H4-K20 in silenced HOX genes? Mono- and di-methylation of H4-K20 are very abundant modifications in Drosophila chromatin, and mass spectroscopic analyses of histones in embryos imply that lower methylated forms of histone H4 (i.e., H4-K20me2) already exist prior to becoming incorporated into chromatin during S phase. It is therefore tempting to speculate that dSfmbt, tethered to PREs by Pho, scans the flanking HOX gene chromatin for nucleosomes that are only mono- or di-methylated at H3-K9 or H4-K20 and docks onto such nucleosomes through its MBT repeats. It is hypothesized that through this bridging interaction, nucleosomes of lower methylated states might be brought into proximity to PRE-bound PRC2 and other currently unknown HMTases that are responsible for local tri-methylation of H3-K9 and H4-K20 in silenced HOX genes. According to this model, PRE-bound PhoRC would act as a 'grappling hook' that tethers mono- and di-methylated histones in silenced HOX gene chromatin to PREs to ensure that they become hypermethylated to the tri-methylated state. Such a chromatin-scanning function might be particularly important during S phase, when newly incorporated histone octamers need to become fully tri-methylated in order to maintain silencing of HOX genes (Klymenko, 2006).

Search PubMed for articles about Drosophila Sfmbt

Brown, J. L., Fritsch, C., Muller, J. and Kassis, J. A. (2003). The Drosophila pho-like gene encodes a YY1-related DNA binding protein that is redundant with pleiohomeotic in homeotic gene silencing. Development 130: 285-294. PubMed ID: PubMed ID; Online text

Fritsch, C.., Brown, J. L., Kassis, J..A. and Muller, J. (1999). The DNA-binding Polycomb group protein pleiohomeotic mediates silencing of a Drosophila homeotic gene. Development 126: 3905-3913. PubMed ID: PubMed ID; Online text

Klymenko, T., Papp, B., Fischle, W., Köcher, T., Schelder, M., Fritsch, C., Wild, B., Wilm, M. and Müller, J. (2006). A Polycomb group protein complex with sequence-specific DNA-binding and selective methyl-lysine-binding activities. Genes Dev. 20: 1110-1122. PubMed ID: PubMed ID; Online text

Shen, X., Mizuguchi, G., Hamiche, A. and Wu, C. (2000). A chromatin remodelling complex involved in transcription and DNA processing. Nature 406: 541-544. PubMed ID: PubMed ID; Online text

Usui, H., Ichikawa, T., Kobayashi, K. and Kumanishi, T. (2000). Cloning of a novel murine gene Sfmbt, Scm-related gene containing four mbt domains, structurally belonging to the Polycomb group of genes. Gene 248: 127-135. PubMed ID: PubMed ID; Online text

date revised: 23 June 2023

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