multi sex combs: Biological Overview | References
Gene name - multi sex combs
Cytological map position - 8D2-8D2
Function - chromatin factor
Symbol - mxc
FlyBase ID: FBgn0260789
Genetic map position - chrX:9,236,801-9,243,467
Classification - a LisH domain and a novel SIF (Self Interaction Facilitator) domain
Cellular location - nuclear
|Tanabe, K., Awane, R., Shoda, T., Yamazoe, K. and Inoue, Y. H. (2019). Mutations in mxc tumor-suppressor gene induce chromosome instability in Drosophila male meiosis. Cell Struct Funct. PubMed ID: 31484839
Drosophila Mxc protein is a component of the histone locus body (HLB), which is required for the expression of canonical histone genes. A fraction of the spermatids in hypomorphic mxcG46 mutants contained extra micronuclei or abnormally sized nuclei. Lagging chromosomes were observed to retain between chromosomal masses separated toward spindle poles at telophase I. Time-lapse recordings show that micronuclei were generated from lagging chromosomes, and the abnormal chromosomes in mxcG46 mutants lacked centromeres. In normal spermatocyte nuclei, the histone locus body component FLASH, a protein that plays an essential role in 3' end processing of replication-dependent histone pre-mRNAs, colocalized with Mxc, whereas FLASH was dispersed in mxcG46 spermatocyte nuclei. Furthermore, genetic interactions were observed between Mxc and other histone locus body (HLB) components in meiotic chromosome segregation, suggesting that inhibition of HLB formation is responsible for aberrant chromosome segregation in mxcG46. Quantitative real-time PCR revealed that canonical histone mRNA levels were decreased in mxcG46. Lastly, similar meiotic phenotypes appeared in the spermatids of histone H4 mutants and in the spermatids in testes depleted for chromosome-construction factors. Considering these genetic data, it is proposed that abnormal chromosome segregation leading to CIN development results from a loss of chromosome integrity caused by diminished canonical histone levels in mxc mutants.
|Kurihara, M., Komatsu, K., Awane, R. and Inoue, Y. H. (2020). Loss of Histone Locus Bodies in the Mature Hemocytes of Larval Lymph Gland Result in Hyperplasia of the Tissue in mxc Mutants of Drosophila. Int J Mol Sci 21(5). PubMed ID: 32111032
Mutations in the multi sex combs (mxc) gene in Drosophila results in ma lignant hyperplasia in larval hematopoietic tissues, called lymph glands (LG). mxc encodes a component of the histone locus body (HLB) that is essential for cell cycle-dependent transcription and processing of histone mRNAs. The mammalian nuclear protein ataxia-telangiectasia (NPAT) gene, encoded by the responsible gene for ataxia telangiectasia, is a functional Mxc orthologue. However, their roles in tumorigenesis are unclear. Genetic analyses of the mxc mutants and larvae having LG-specific depletion revealed that a reduced activity of the gene resulted in the hyperplasia, which is caused by hyper-proliferation of immature LG cells. The depletion of mxc in mature hemocytes of the LG resulted in the hyperplasia. Furthermore, the inhibition of HLB formation was required for LG hyperplasia. In the mutant larvae, the total mRNA levels of the five canonical histones decreased, and abnormal forms of polyadenylated histone mRNAs, detected rarely in normal larvae, were generated. The ectopic expression of the polyadenylated mRNAs was sufficient for the reproduction of the hyperplasia. The loss of HLB function, especially 3-end processing of histone mRNAs, is critical for malignant LG hyperplasia in this leukemia model in Drosophila. It is proposed that mxc is involved in the activation to induce adenosine deaminase-related growth factor A (Adgf-A), which suppresses immature cell proliferation in LG.
|Hur, W., Kemp, J. P., Jr., Tarzia, M., Deneke, V. E., Marzluff, W. F., Duronio, R. J. and Di Talia, S. (2020). CDK-Regulated Phase Separation Seeded by Histone Genes Ensures Precise Growth and Function of Histone Locus Bodies. Dev Cell. PubMed ID: 32579968
Many membraneless organelles form through liquid-liquid phase separation, but how their size is controlled and whether size is linked to function remain poorly understood. The histone locus body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of histone mRNAs. This study shows that Drosophila HLBs form through phase separation. During embryogenesis, the size of HLBs is controlled in a precise and dynamic manner that is dependent on the cell cycle and zygotic histone gene activation. Control of HLB growth is achieved by a mechanism integrating nascent mRNAs at the histone locus, which facilitates phase separation, and the nuclear concentration of the scaffold protein Multi-sex combs (Mxc), which is controlled by the activity of cyclin-dependent kinases. Reduced Cdk2 activity results in smaller HLBs and the appearance of nascent, misprocessed histone mRNAs. Thus, these experiments identify a mechanism linking nuclear body growth and size with gene expression.
|Kemp, J. P., Jr., Yang, X. C., Dominski, Z., Marzluff, W. F. and Duronio, R. J. (2021). Superresolution light microscopy of the Drosophila histone locus body reveals a core-shell organization associated with expression of replication-dependent histone genes. Mol Biol Cell 32(9): 942-955. PubMed ID: 33788585
The histone locus body (HLB) is an evolutionarily conserved nuclear body that regulates the transcription and processing of replication-dependent (RD) histone mRNAs, which are the only eukaryotic mRNAs lacking a poly-A tail. Many nuclear bodies contain distinct domains, but how internal organization is related to nuclear body function is not fully understood. This study demonstrates, using structured illumination microscopy, that Drosophila HLBs have a "core-shell" organization in which the internal core contains transcriptionally active RD histone genes. The N-terminus of Mxc, which contains a domain required for Mxc oligomerization, HLB assembly, and RD histone gene expression, is enriched in the HLB core. In contrast, the C-terminus of Mxc is enriched in the HLB outer shell as is FLASH, a component of the active U7 snRNP that cotranscriptionally cleaves RD histone pre-mRNA. Consistent with these results, this study shows biochemically that FLASH binds directly to the Mxc C-terminal region. In the rapid S-M nuclear cycles of syncytial blastoderm Drosophila embryos, the HLB disassembles at mitosis and reassembles the core-shell arrangement as histone gene transcription is activated immediately after mitosis. Thus, the core-shell organization is coupled to zygotic histone gene transcription, revealing a link between HLB internal organization and RD histone gene expression.
|Sang, R., Wu, C., Xie, S., Xu, X., Lou, Y., Ge, W., Xi, Y. and Yang, X. (2022). Mxc, a Drosophila homolog of mental retardation-associated gene NPAT, maintains neural stem cell fate. Cell Biosci 12(1): 78. PubMed ID: 35642004
Mental retardation is a complex neurodevelopmental disorder. NPAT, a component of the histone locus body (HLB), has been implicated as a candidate gene for mental retardation. This study identified that multi sex combs (mxc), the Drosophila ortholog of NPAT, is required for the development of nervous system. Knockdown of mxc resulted in a massive loss of neurons and locomotion dysfunction in adult flies. In the mxc mutant or RNAi knockdown larval brains, the neuroblast (NB, also known as neural stem cell) cell fate is prematurely terminated and its proliferation potential is impeded concurrent with the blocking of the differentiation process of ganglion mother cells (GMCs). A reduction of transcription levels of histone genes was shown in mxc knockdown larval brains, accompanied by DNA double-strand breaks (DSBs). The subsidence of histone transcription levels leads to prematurely termination of NB cell fate and blockage of the GMC differentiation process. These data also show that the increase in autophagy induced by mxc knockdown in NBs could be a defense mechanism in response to abnormal HLB assembly and premature termination of NB cell fate.
|Takarada, K., Kinoshita, J. and Inoue, Y. H. (2023). Ectopic expression of matrix metalloproteinases and filopodia extension via JNK activation are involved in the invasion of blood tumor cells in Drosophila mxc mutant. Genes Cells. PubMed ID: 37615261
Drosophila mxc(mbn1) mutant exhibits severe hyperplasia in larval hematopoietic tissue called the lymph glands (LGs). However, the malignant nature of these cells remains unknown. This study aimed to identify if mxc(mbn1) LG cells behave as malignant tumor cells and uncover the mechanism(s) underlying the malignancy of the mutant hemocytes. When mutant LG cells were allografted into normal adult abdomens, they continued to proliferate; however, normal LG cells did not proliferate. Mutant circulating hemocytes also attached to the larval central nervous system (CNS), where the basement membrane was disrupted. The mutant hemocytes displayed higher expression of matrix metalloproteinase (MMP) 1 and MMP2 and higher activation of the c-Jun N-terminal kinase (JNK) pathway than normal hemocytes. Depletion of MMPs or JNK mRNAs in LGs resulted in reduced numbers of hemocytes attached to the CNS, suggesting that the invasive phenotype involved elevated expression of MMPs via hyperactivation of the JNK pathway. Moreover, hemocytes with elongated filopodia and extra lamellipodia were frequently observed in the mutant hemolymph, which also depended on JNK signaling. Thus, the MMP upregulation and overextension of actin-based cell protrusions were also involved in hemocyte invasion in mxc(mbn1) larvae. These findings contribute to the understanding of molecular mechanisms underlying mammalian leukemic invasion.
|Kinoshita, Y., Shiratsuchi, N., Araki, M. and Inoue, Y. H. (2023). Anti-Tumor Effect of Turandot Proteins Induced via the JAK/STAT Pathway in the mxc Hematopoietic Tumor Mutant in Drosophila. Cells 12(16). PubMed ID: 37626857
Several antimicrobial peptides supress the growth of lymph gland (LG) tumors in Drosophila multi sex comb (mxc) mutant larvae. The activity of another family of polypeptides, called Turandots, is also induced via the JAK/STAT pathway after bacterial infection; however, their influence on Drosophila tumors remains unclear. The JAK/STAT pathway was activated in LG tumors, fat body, and circulating hemocytes of mutant larvae. The mRNA levels of Turandot (Tot) genes increased markedly in the mutant fat body and declined upon silencing Stat92E in the fat body, indicating the involvement of the JAK/STAT pathway. Furthermore, significantly enhanced tumor growth upon a fat-body-specific silencing of the mRNAs demonstrated the antitumor effects of these proteins. The proteins were found to be incorporated into small vesicles in mutant circulating hemocytes (as previously reported for several antimicrobial peptides) but not normal cells. In addition, more hemocytes containing these proteins were found to be associated with tumors. The mutant LGs contained activated effector caspases, and a fat-body-specific silencing of Tots inhibited apoptosis and increased the number of mitotic cells in the LG, thereby suggesting that the proteins inhibited tumor cell proliferation. Thus, Tot proteins possibly exhibit antitumor effects via the induction of apoptosis and inhibition of cell proliferation.
Nuclear bodies (NBs) are structures that concentrate proteins, RNAs, and ribonucleoproteins that perform functions essential to gene expression. How NBs assemble is not well understood. Drosophila histone locus body (HLB), a NB that concentrates factors required for histone mRNA biosynthesis at the replication-dependent histone gene locus, was studied. Biochemical analysis was coupled with confocal imaging of both fixed and live tissues to demonstrate that the Drosophila Multi-Sex Combs (Mxc) protein contains multiple domains necessary for HLB assembly. An important feature of this assembly process is the self-interaction of Mxc via two conserved N-terminal domains: a LisH domain and a novel SIF (Self Interaction Facilitator) domain immediately downstream of the LisH domain. Molecular modeling suggests that the LisH and SIF domains directly interact, and mutation of either the LisH or SIF domains severely impairs Mxc function in vivo resulting in reduced histone mRNA accumulation. A region of Mxc between amino acids 721 and 1481 is also necessary for HLB assembly independent of the LisH and SIF domains. Lastly, the C-terminal 195 amino acids of Mxc are required for recruiting FLASH, an essential histone mRNA processing factor, to the HLB. It is concluded that multiple domains of the Mxc protein promote HLB assembly in order to concentrate factors required for histone mRNA biosynthesis (Terzo, 2015).
Numerous levels of molecular organization within the nucleus facilitate the highly regulated expression of the genome. One level of organization is the concentration of proteins, RNAs, and ribonucleoproteins into structures known as nuclear bodies (NBs) that are visible by light microscopy. NBs include well-known structures such as Cajal bodies and the nucleolus, and less well understood structures including PML bodies, speckles, paraspeckles and histone locus bodies (HLBs). An attractive hypothesis for NB function posits that NBs concentrate factors to accelerate reactions that would otherwise take longer if these factors were dispersed throughout the nucleus. This hypothesis has gained support from studies of vertebrate Cajal bodies, which promote efficient spliceosomal snRNP assembly. However, Drosophila snRNA modification by scaRNAs, which are localized to Cajal bodies, does not require Cajal body assembly. Thus, the general applicability and further tests of this hypothesis require additional study (Terzo, 2015).
An understanding of NB function requires detailed knowledge of NB composition and assembly. This issue has been explored by studying how HLB assembly contributes to the expression of replication-dependent histone genes, which encode the only known cellular mRNAs that are not polyadenylated. HLBs were defined by Gall and coworkers as a NB associated with the Drosophila histone gene locus that contained U7 snRNP (Liu, 2006), a factor essential for generating the unique histone mRNA 3' end. Similar factors necessary for histone transcription and pre-mRNA processing are found in both vertebrate and Drosophila HLBs, including human NPAT (Nuclear Protein mapped to the mutated Ataxia Telangectasia locus), which was identified as a Cyclin E/Cdk2 substrate essential for histone mRNA expression. The multi sex combs (mxc) locus encodes the Drosophila ortholog of NPAT. Mxc, like NPAT, is phosphorylated by Cyclin E/Cdk2, co-localizes with U7 snRNP at the histone locus, and is required for both HLB assembly and histone gene expression (White, 2011). Other known HLB components include FLASH and Mute. FLASH was identified in mammals as co-localizing with NPAT (Bongiorno-Borbone, 2008) and subsequently shown to interact with U7 snRNP and to be essential for histone pre-mRNA processing (Yang, 2009). Mute was identified as a Drosophila HLB component in a screen for factors required for muscle development, but its biochemical function is not known. Terzo, 2015 and references therein).
Previous experiments on Drosophila HLBs suggest that Mxc is critical for HLB assembly. Mxc and FLASH localize to the histone locus immediately before the beginning of histone gene expression in syncytial embryos, and prior to this time HLBs are not detected. Loss of Mxc results in a failure to localize other HLB components, including FLASH and U7 snRNP (White, 2011). The Drosophila HLB is present in all cells, independent of whether they are cycling. The 5 canonical Drosophila histone genes (H1, H2A, H2B, H3, and H4) are clustered together in a 5kb sequence that is repeated approximately 100 times at a single locus on chromosome 2. The 300 base pair bidirectional promoter of the H3-H4 gene pair within this cluster is necessary and sufficient for HLB assembly and is necessary for expression of the adjacent H2A-H2B gene pair (Salzler, 2013). This 300 bp sequence is also sufficient to recruit Mxc and FLASH, consistent with Mxc playing an integral role in HLB assembly and histone gene expression. How Mxc participates in coordinating these processes remains unclear. The mxc locus was originally described by an allelic series of mutations in which null alleles resulted in a failure of cell proliferation and lethality. Knocking out NPAT in cultured mammalian cells is similarly lethal (Ye, 2003). In contrast, viable, hypomorphic mxc alleles cause homeotic transformations in adult males (giving rise to the gene name) (Santamaria, 1995; Terzo, 2015 and references therein).
Whether there is any causal relationship between histone gene expression and the homeotic transformations observed in mxc hypomorphs is unknown. Two mxc hypomorphic alleles encode nonsense mutations at residues K1482 and Q1643 of the 1837-amino acid long Mxc protein (White, 2011). The resulting truncated mutant proteins support histone gene expression (Landais, 2014) while an amorphic mxc allele that does not produce Mxc protein does not (White, 2011). The Q1643 mutation (mxcG46) partially disrupts Mxc function resulting in replication stress and a persistent DNA damage response that contributes to the loss of germ line stem cells through mis-regulation of histone gene expression (Landais, 2014; Terzo, 2015).
Studies in human cell culture indicate that distinct domains of NPAT are required to activate histone gene expression and allow entry into S phase). These data suggest that Mxc/NPAT may contain multiple domains that organize HLB assembly and coordinate histone mRNA biosynthesis. Proteins harboring multiple protein-protein interaction domains are likely a critical feature of NBs. The focal organization provided by the multiple interaction domains could facilitate a more efficient and rapid physiological response to distinct stimuli. This study has identified functional domains of Mxc required for localization of Mxc to the HLB in the presence of full-length Mxc using cultured Drosophila S2 cells. mxc mutant animals expressing different Mxc mutant transgenic proteins were used to explore the function of Mxc in vivo, and time-lapse imaging of early embryos expressing GFP-Mxc to assess the dynamics of Mxc localization to the HLB. The data indicate that Mxc requires multiple domains for complete function in vivo, and that two self-interaction domains of Mxc are essential for HLB assembly, which in turn promotes histone mRNA biosynthesis (Terzo, 2015).
HLBs assemble at replication-dependent histone loci and provide a distinct compartment in the nucleus that promotes efficient transcription and processing of histone mRNA, likely by concentrating histone biosynthetic factors as well as excluding factors specifically required for polyadenylation. This study shows that multiple protein domains are necessary for Mxc to support HLB assembly and histone mRNA biosynthesis, and ultimately normal Drosophila development. Multiple domains of Mxc are required for HLB assembly Whether NBs form by an ordered assembly process, by random association of components, or by a combination of each of these processes is not clear for most NBs. In the case of the HLB, this study has demonstrated that hierarchical assembly contributes to NB formation, with Mxc and FLASH part of a complex that initially forms at a specific sequence at the histone locus (Salzler, 2013; White, 2011). This study defines two regions in the N-terminus of Mxc, the LisH domain and a novel domain that has been named the SIF domain, both of which are necessary for GFP-Mxc to concentrate in the HLB in the presence of endogenous Mxc and to support HLB assembly in the absence of endogenous Mxc (Terzo, 2015).
Although GFP-mxc1-354 and GFP-mxc1-721, which contain both LisH and SIF domains, are incorporated into the HLB in the presence of endogenous Mxc, they do not support formation of a complete HLB in the absence of endogenous Mxc and cannot rescue the lethality caused by an mxc null mutation. Thus sequences in addition to the LisH and SIF domains are required for HLB formation. Truncated Mxc proteins encoded by the viable, hypomorphic mxcG43 and mxcG46 alleles (1481 and 1642 amino acids, respectively) form nuclear bodies (HLBs) as judged by staining tissues with the MPM-2 antibody, which recognizes phosphorylated Mxc, and formation of nuclear foci by GFP-mxcG46 protein in the absence of endogenous Mxc. Thus, there is a region of Mxc between amino acids 721 and 1481 that together with the N-terminus is required for HLB formation. The larger Mxc proteins likely contain elements necessary for recruitment of Mxc to the H3-H4 intergenic region of the histone locus that is essential for HLB formation (Salzler, 2013). However, because maternal supplies of wild type Mxc in itially establish the HLB in the early embryo prior to the zygotic expression of mxcG43 and mxcG46, it is uncertain whether mxcG43 and mxcG46 proteins are capable of forming an HLB de novo. Finally, Mxc likely contains binding sites for other HLB components, such as FLASH, U7 snRNP or Mute, and is regulated by phosphorylation by Cyclin E/Cdk2. Self-interaction between different Mxc molecules is required for HLB assembly LisH domains are found in a variety of multi-protein complexes, and promote protein-protein interactions important for the assembly of these complexes. Some LisH domain proteins dimerize through their LisH domains, and a structure of a LisH domain homodimer has been solved. This study found that the Mxc N-terminus promotes interaction of two Mxc molecules, but that this interaction does not occur by LisH domain homodimerization. In Mxc there is a possible steric clash between His-7 of one LisH domain and Tyr-17 of a second LisH domain that may explain why the Mxc LisH domains do not homodimerize in a manner typical of other LisH domains. Instead, Mxc self-interaction requires a region downstream of the LisH domain between amino acids 39 and 185 (the SIF domain), and three amino acids (Leu52, Ile61, and Ile62) in this region conserved between flies and vertebrates are required for HLB assembly in vivo and for rescuing the lethality of an mxc null mutation. Furthermore, live imaging revealed dramatically reduced concentration of GFP-mxcLisH- AAA and GFP-mxcSIF-AAA in HLBs in the presence of endogenous Mxc, consistent with reduced binding affinity between the mutant and wild type Mxc molecules. Thus, the LisH domain of one molecule of Mxc binds the SIF domain (i.e. amino acids 39-185) of another molecule of Mxc. Molecular modeling suggests that this interaction may be mediated by direct binding between the LisH domain and the LxxII motif of the SIF domain. In addition to the LxxII motif, the SIF domain contains other amino acids that contribute to efficient Mxc self-interaction. These multiple interaction sites indicate that each Mxc molecule can potentially interact with at least two, and possibly more Mxc molecules, raising the possibility that the N-terminal region of Mxc can promote formation of a three-dimensional lattice that is likely an essential component of HLB structure. Similarly, an N-terminal domain of Coilin that mediates self interaction is necessary for Coilin accumulation in the CB, suggesting that oliogomerization is a common feature of NB formation (Terzo, 2015).
Many LisH domain-containing proteins also contain a CTLH domain (C-terminus to LisH) defined in both ProSite and SMART, which is often but not always immediately C-terminal to the LisH domain. Other than the prediction that this domain contains α-helical regions there is no structural information on the CTLH domain. The CTLH domains of several proteins have recently been shown to participate in protein-protein interactions important for the assembly of multi-protein complexes. The Mxc SIF domain that this study has identified functions similarly to the CTLH domain but is clearly distinct from the CTLH domain. The SMART and ProSite CTLH domain logos each contain a conserved glycine (G) at position 16, a conserved phenylalanine (F) at position 46, a conserved leucine (L) at position 48, a conserved glutamic acid (E) at position 55 (numbering of SMART logo), none of which are present in the SIF domain of Mxc. Thus the region in Mxc C-terminal to the LisH domain is distinct from the CTLH domain. Harper and colleagues previously demonstrated that human NPAT is essential for cell proliferation and histone gene expression, and that the NPAT LisH domain was necessary for stimulating His4 and H2B promoter activation in cell culture based transfection/reporter assays (Wei, 2003; Ye, 2003). They also reported that a LisH domain mutant NPAT protein could localize to Coilin-positive NBs (a subset of which are likely to be HLBs) (Wei et al., 2003). However these experiments were performed by transfecting RAT1 cells containing endogenous NPAT, and the role of the LisH domain in NB formation, cell proliferation and histone gene expression was not examined in the absence of endogenous NPAT. In addition, mutations of the NPAT SIF domain were not generated and analyzed in these previous studies. Based on the current results and the similarity between the N-termini of mammalian NPAT and Mxc, it is suspected that human NPAT LisH domain mutants can interact with endogenous NPAT via the SIF and/or other domains. It is proposed that the N-terminus of human NPAT promotes interaction between multiple NPAT molecules. Mxc's requirement for histone mRNA biosynthesis correlates with HLB assembly (Terzo, 2015).
Prior imaging of fixed embryos and live imaging reported in this study indicate that maternal Mxc and FLASH co-localize in nuclear foci prior to the initiation of zygotic histone gene transcription in the syncytial embryo (Salzler, 2013; White, 2011; White, 2007). Once histone transcription initiates these foci enlarge into mature HLBs as detected by increased intensity of both Mxc and FLASH staining as well as recruitment of other HLB components U7 snRNP and Mute. It has been previously reported that mxc null mutant 1st instar larvae fail to accumulate normal amounts of histone H3 mRNA, supporting a role for Mxc in histone gene expression (White, 2011). This study now shows that the maternal supply of Mxc (as determined by detection of HLBs by immunofluorescence) is depleted in most cells by 8 hrs of embryogenesis, and that this depletion is accompanied by a decrease in histone H3 transcript levels. In spite of reduced levels of histone mRNA, mxc null mutant embryos hatch. Thus as the maternal supply of Mxc is depleted in mxc mutant embryos, histone gene expression drops resulting in death in early larval stages. In contrast to the null allele, hypomorphic mxc mutant embryos (mxcG43 and mxcG46) develop to adults and hence are capable of supporting histone mRNA biosynthesis, consistent with previous observations (Landais, 2014). In ovaries the 1642 amino acid mxcG46 protein fails to recruit FLASH to HLBs (Rajendra, 2011), and results in accumulation of small amounts of misprocessed histone H3 mRNA. This study reports that unprocessed histone H3 RNA accumulates at the histone locus in mxcG43 and mxcG46 mutant embryos. This nascent, unprocessed H3 RNA was detected by in situ hybridization with a probe derived from sequence downstream of the normal H3 mRNA 3' end. These unprocessed RNAs were not detected in wild type embryos. Thus, loss of the last 195 amino acids from Mxc may reduce the efficiency of normal histone mRNA 3' end formation (Terzo, 2015).
Several lines of evidence suggest that proteins with multiple protein-protein interaction domains mediate the localized concentration of components that give rise to NBs. NB components can exchange with the nucleoplasm, suggesting there are multiple relatively weak protein- protein interactions between components of nuclear bodies, a property that is shared with other cellular bodies (e.g. P-bodies and stress granules in the cytoplasm). Together with previous work (Salzler, 2013; White, 2011), a model is proposed in which Mxc together with FLASH help drive formation of a large (i.e., visible by light microscopy) 3- dimensional lattice, the HLB, containing components necessary for efficient transcription and processing of histone mRNA. Gaining additional insight into the biogenesis of NBs will further understanding of the assembly and function of regulatory machineries required to effectively control gene expression, and is crucial to understand how these complex structures respond to diverse physiological stimuli during normal and pathological circumstances (Terzo, 2015).
Polycomb group (PcG) proteins establish and maintain genetic programs that regulate cell-fate decisions. Drosophila multi sex combs (mxc) was categorized as a PcG gene based on a classical Polycomb phenotype and genetic interactions; however, a mechanistic connection between Polycomb and Mxc has not been elucidated. Hypomorphic alleles of mxc are characterized by male and female sterility and ectopic sex combs. Mxc is an important regulator of histone synthesis, and this study found that increased levels of the core histone H3 in mxc mutants result in replicative stress and a persistent DNA damage response (DDR). Germline loss, ectopic sex combs and the DDR are suppressed by reducing H3 in mxc mutants. Conversely, mxc phenotypes are enhanced when the DDR is abrogated. Importantly, replicative stress induced by hydroxyurea treatment recapitulated mxc germline phenotypes. These data reveal how persistent replicative stress affects gene expression, tissue homeostasis, and maintenance of cellular identity in vivo (Landais, 2014).
The findings of this study reveal that sustained levels of replicative stress and an ongoing DNA damage response can interfere with maintenance of cell-fate decisions and tissue homeostasis. Defects in histone synthesis, resulting in higher histone levels, constitute a pernicious intracellular source of replicative stress: it persists while the cells attempt to repair DNA and will reoccur cyclically in subsequent S phases. Accordingly, an intense DDR is observed both in mxc mutant germline and somatic cells. Importantly, induction of replicative stress via another mechanism, i.e., continual exposure to hydroxyurea, recapitulated the mxc germline phenotypes, including loss of germ cells due to premature initiation of differentiation. Therefore, it is suggested that a widespread and persistent DDR contributes to the precocious initiation of differentiation in mxc mutant cells. Due to the degree of EdU incorporation in germ cells within mxc mutant testes, it is conclude that germ cells undergo a protracted S phase followed, ultimately, by germ cell loss. However, given the apparent DNA damage in mxc mutant cells, it is possible that germ cells are in G2 but continue to incorporate EdU as a consequence of DNA repair. Nonetheless, entry into mitosis is noticeably lacking in mxc mutant germ cells, as indicated by an absence of phosphorylated histone H3 (Landais, 2014).
One outstanding question is whether the mxc germline and hematopoietic defects described in this study truly reflect a loss of Polycomb function. Although a role for PcG in regulating stem cell behavior and maintenance of cell identity is well established, a bona fide PcG phenotype in the Drosophila male germline has not been described previously. Elegant experiments have demonstrated that several PRC1 components are recruited to the nucleolus in spermatocytes upon terminal differentiation, suggesting that PcG activity may be required in early germ cells to repress the expression of differentiation genes. Interestingly, loss of the Drosophila PRC1 members Psc [Mel18] and Su(z)2 [Bmi1] in germ cells did not result in loss of GSCs, which may suggest a different PRC1 composition in male germ cells. Moreover, the mammalian homolog of Mxc, NPAT, plays a role in DNA repair by regulating the expression of ATM (Ataxia telangiectasia mutated), in addition to H2 and H4 (Medina, 2007; White, 2011). Therefore, it is possible that mxc plays additional roles in mediating a DNA damage response, which would render mxc mutant cells more sensitive to replicative stress (Landais, 2014).
Although mutations in histones have previously been shown to reproduce or enhance Polycomb phenotypes, this study reports a sustained/hindered DDR enhancing a classical PcG phenotype in vivo. However, it remains unclear how replicative stress and a persistent DDR could influence Polycomb activity, leading to homeotic transformations such as those observed in mxc mutants. Recent evidence has implicated PcG proteins in DDR pathways; however, the precise role for PcG proteins in DNA repair has not yet been elucidated. One possibility is that PcG-mediated modification of histones is required for the change in chromatin conformation necessary to allow access of DNA repair machinery to the DNA. Alternatively, PcG activity could serve to repress transcription while repair is ongoing. Both histone H2A and H2Av are mono- and polyubiquitinylated during a DDR, including monoubiquitination by PRC1 at Lys118. Therefore, an incessant induction of a DDR, such as in the case of mxc mutation, could result in persistent, high levels of ubiquitination at sites of DNA repair, which may interfere with the normal dynamic of monoubiquitination/deubiquitination of H2Av on Lys118 necessary for PcG-mediated repression. In contrast, a persistent DDR could alter PcG activity by recruiting PcG proteins to sites of DNA damage and away from normal target genes that regulate cellular identity and cell-fate decisions. Regardless of whether PcG proteins play an active role in DNA repair, the current data provide evidence in vivo that Polycomb activity can be influenced by persistent DNA damage. Therefore, it is proposed that any moderate, continuous source of replicative stress during development and/or in adult stem cell lineages could trigger aberrant gene expression and alterations in cell-fate decisions (Landais, 2014).
Nuclear protein, ataxia-telangiectasia locus (NPAT) and FLICE-associated huge protein (FLASH) are two major components of discrete nuclear structures called histone locus bodies (HLBs). NPAT is a key co-activator of histone gene transcription, whereas FLASH through its N-terminal region functions in 3' end processing of histone primary transcripts. The C-terminal region of FLASH contains a highly conserved domain that is also present at the end of Yin Yang 1-associated protein-related protein (YARP) and its Drosophila homologue, Mute, previously shown to localize to HLBs in Drosophila cells. This study shows that the C-terminal domain of human FLASH and YARP interacts with the C-terminal region of NPAT and that this interaction is essential and sufficient to drive FLASH and YARP to HLBs in HeLa cells. Strikingly, only the last 16 amino acids of NPAT are sufficient for the interaction. This study also shows that the C-terminal domain of Mute interacts with a short region at the end of the Drosophila NPAT orthologue, multi sex combs (Mxc). Altogether, these data indicate that the conserved C-terminal domain shared by FLASH, YARP, and Mute recognizes the C-terminal sequence of NPAT orthologues, thus acting as a signal targeting proteins to HLBs. Finally, this study demonstrates that the C-terminal domain of human FLASH can be directly joined with its N-terminal region through alternative splicing. The resulting 190-amino acid MiniFLASH, despite lacking 90% of full-length FLASH, contains all regions necessary for 3' end processing of histone pre-mRNA in vitro and accumulates in HLBs (Yang, 2014).
Nuclear bodies are protein- and RNA-containing structures that participate in a wide range of processes critical to genome function. Molecular self-organization is thought to drive nuclear body formation, but whether this occurs stochastically or via an ordered, hierarchical process is not fully understood. This study addressed this question using RNAi and proteomic approaches in Drosophila melanogaster to identify and characterize novel components of the histone locus body (HLB), a nuclear body involved in the expression of replication-dependent histone genes. The transcription elongation factor suppressor of Ty 6 (Spt6) and a homologue of mammalian nuclear protein of the ataxia telangiectasia-mutated locus that is encoded by the homeotic gene multisex combs (mxc) were identified as novel HLB components. By combining genetic manipulation in both cell culture and embryos with cytological observations of Mxc, Spt6, and the known HLB components, FLICE-associated huge protein, Mute, U7 small nuclear ribonucleoprotein, and MPM-2 phosphoepitope, sequential recruitment and hierarchical dependency were demonstrated for localization of factors to HLBs during development, suggesting that ordered assembly can play a role in nuclear body formation (White, 2011).
The requirements for the multi sex combs (mxc) gene were examined during development to gain further insight into the mechanisms and developmental processes that depend on the important trans-regulators forming the Polycomb group (PcG) in Drosophila melanogaster. mxc is allelic with the tumor suppressor locus lethal (1) malignant blood neoplasm (l(1)mbn). This study showed that the mxc product is dramatically needed in most tissues because its loss leads to cell death after a few divisions. mxc has also a strong maternal effect. Hypomorphic mxc mutations enhance other PcG gene mutant phenotypes and cause ectopic expression of homeotic genes, confirming that PcG products are cooperatively involved in repression of selector genes outside their normal expression domains. It was also demonstrated that the mxc product is needed for imaginal head specification, through regulation of the ANT-C gene Deformed. This analysis reveals that mxc is involved in the maternal control of early zygotic gap gene expression previously reported for some PcG genes and suggests that the mechanism of this early PcG function could be different from the PcG-mediated regulation of homeotic selector genes later in development. This data is discuss in view of the numerous functions of PcG genes during development (Saget, 1998).
Genetic analysis of the 8D3;8D8-9 segment of the Drosophila melanogaster X chromosome has assigned seven complementation groups to this region, three of which are new. A Polycomb group (Pc-G) gene, multi sex combs (mxc), is characterized and mutant alleles are described. Besides common homeotic transformations characteristic of Pc-G mutants that mimic the ectopic gain of function of BX-C and ANT-C genes, mxc mutants show other phenotypes: they zygotically mimic, in males and females, the characteristic lack of germ line seen in progeny of some maternal effect mutants of the so-called posterior group (the grandchildless phenotype). Loss of normal mxc function can promote uncontrolled malignant growth which indicates a possible relationship between Pc-G genes and tumour suppressor genes. It is proposed that gain-of-function of genes normally repressed by the wild-type mxc product could, in mxc mutants, give rise to an incoherent signal which would be devoid of meaning in normal development. Such a signal could divert somatic and germ line development pathways, provoke the loss of cell affinities, but allow or promote growth (Santamaria, 1995).
Search PubMed for articles about Drosophila Mxc
Bongiorno-Borbone, L., De Cola, A., Vernole, P., Finos, L., Barcaroli, D., Knight, R. A., Melino, G. and De Laurenzi, V. (2008). FLASH and NPAT positive but not Coilin positive Cajal Bodies correlate with cell ploidy. Cell Cycle 7: 2357-2367. PubMed ID: 18677100
Landais, S., D'Alterio, C. and Jones, D. L. (2014). Persistent replicative stress alters polycomb phenotypes and tissue homeostasis in Drosophila melanogaster. Cell Rep 7: 859-870. PubMed ID: 24746823
Liu, J. L., Murphy, C., Buszczak, M., Clatterbuck, S., Goodman, R. and Gall, J. G. (2006). The Drosophila melanogaster Cajal body. J Cell Biol 172: 875-884. PubMed ID: 16533947
Medina, R., van der Deen, M., Miele-Chamberland, A., Xie, R. L., van Wijnen, A. J., Stein, J. L. and Stein, G. S. (2007). The HiNF-P/p220NPAT cell cycle signaling pathway controls nonhistone target genes. Cancer Res 67: 10334-10342. PubMed ID: 17974976
Rajendra, T. K., Praveen, K. and Matera, A. G. (2010). Genetic analysis of nuclear bodies: from nondeterministic chaos to deterministic order. Cold Spring Harb Symp Quant Biol 75: 365-374. PubMed ID: 21467138
Remillieux-Leschelle, N., Santamaria, P. and Randsholt, N. B. (2002). Regulation of larval hematopoiesis in Drosophila melanogaster: a role for the multi sex combs gene. Genetics 162: 1259-1274. PubMed ID: 12454071
Saget, O., Forquignon, F., Santamaria, P. and Randsholt, N. B. (1998). Needs and targets for the multi sex combs gene product in Drosophila melanogaster. Genetics 149: 1823-1838. PubMed ID: 9691040
Salzler, H. R., Tatomer, D. C., Malek, P. Y., McDaniel, S. L., Orlando, A. N., Marzluff, W. F. and Duronio, R. J. (2013). A sequence in the Drosophila H3-H4 Promoter triggers histone locus body assembly and biosynthesis of replication-coupled histone mRNAs. Dev Cell 24: 623-634. PubMed ID: 23537633
Santamaria, P. and Randsholt, N. B. (1995). Characterization of a region of the X chromosome of Drosophila including multi sex combs (mxc), a Polycomb group gene which also functions as a tumour suppressor. Mol Gen Genet 246: 282-290. PubMed ID: 7854313
Terzo, E. A., Lyons, S. M., Poulton, J. S., Temple, B. R., Marzluff, W. F. and Duronio, R. J. (2015). Distinct self-interaction domains promote Multi Sex Combs accumulation in and formation of the Drosophila histone locus body. Mol Biol Cell. PubMed ID: 25694448
Wei, Y., Jin, J. and Harper, J. W. (2003). The cyclin E/Cdk2 substrate and Cajal body component p220(NPAT) activates histone transcription through a novel LisH-like domain. Mol Cell Biol 23: 3669-3680. PubMed ID: 12724424
White, A. E., Leslie, M. E., Calvi, B. R., Marzluff, W. F. and Duronio, R. J. (2007). Developmental and cell cycle regulation of the Drosophila histone locus body. Mol Biol Cell 18: 2491-2502. PubMed ID: 17442888
White, A. E., Burch, B. D., Yang, X. C., Gasdaska, P. Y., Dominski, Z., Marzluff, W. F. and Duronio, R. J. (2011). Drosophila histone locus bodies form by hierarchical recruitment of components. J Cell Biol 193: 677-694. PubMed ID: 21576393
Yang, X. C., Sabath, I., Kunduru, L., van Wijnen, A. J., Marzluff, W. F. and Dominski, Z. (2014). A conserved interaction that is essential for the biogenesis of histone locus bodies. J Biol Chem 289: 33767-33782. PubMed ID: 25339177
Ye, X., Wei, Y., Nalepa, G. and Harper, J. W. (2003). The cyclin E/Cdk2 substrate p220(NPAT) is required for S-phase entry, histone gene expression, and Cajal body maintenance in human somatic cells. Mol Cell Biol 23: 8586-8600. PubMed ID: 14612403
date revised: 22 November 2022
Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.