BIOLOGICAL OVERVIEW

Mi-2 and ISWI, two members of the Snf2 superfamily of ATPases, reside in separate ATP-dependent chromatin remodelling complexes. These complexes differ in their biochemical properties and are believed to perform distinct functions in the cell. Both Mi-2 and ISWI are nucleosome-stimulated ATPases and promote nucleosome mobilization. However, Mi-2 and ISWI differ in their interaction with nucleosome core particles, in their substrate requirements and in the direction of nucleosome mobilization. The complex containing Mi-2 and the RPD3 histone deacetylase shares the nucleosome-stimulated ATPase and nucleosome mobilization properties of recombinant Mi-2, demonstrating that these activities are maintained in a physiological context. Its functional properties distinguish Mi-2 from both SWI2/SNF2 and ISWI, defining a new family of ATP-dependent remodelling machines (Brehm, 2000).

Molecular machines that carry out the fundamental processes of DNA replication, recombination, repair and transcription need to recognize and bind to their DNA substrate. The packaging of DNA into chromatin, however, hinders their access to DNA. Chromatin remodelling factors facilitate DNA binding by creating a more dynamic chromatin structure. Two broad classes of remodelling activities have received much attention in recent years. The first class is comprised of ATP-dependent chromatin remodelling complexes containing ATPases related to SWI2/SNF2 or ISWI. These remodel interactions between histones and DNA in a poorly defined reaction requiring ATP hydrolysis. The second class consists of complexes that acetylate or deacetylate specific lysine residues in the N-terminal tails of histones. Acetylation is believed to lead to a more open chromatin structure by changing the nucleosome-nucleosome interactions involved in the folding of the nucleosomal fiber. Alternatively, acetylated lysine tails could serve as recognition sites for chromatin binding factors, which in turn elicit changes in chromatin structure (Brehm, 2000 and references therein).

Before discussing Drosophila Mi-2 in detail, there will be presented a short review of chromatin remodeling machines and Mi-2 containing complexes in particular. The view that ATP-dependent chromatin remodelling and histone acetylation/deacetylation are carried out by separate entities has recently been challenged. Complexes containing the ATPases Mi-2alpha and/or Mi-2ß as well as histone deacetylases HDAC1 and HDAC2 have been identified in human and frog. These complexes combine ATP-dependent chromatin remodelling and histone deacetylase (HDAC) activities, and have accordingly been named NuRD or NRD [nucleosome remodelling and deacetylation (Tong, 1998; Xue, 1998; Zhang, 1998). The Mi-2 subunits of these complexes belong to the CHD (chromo-helicase/ATPase-DNA binding) family of proteins (Woodage, 1997). Four CHD proteins have been identified in vertebrates: CHD1, CHD2, CHD3 (Mi-2alpha) and CHD4 (Mi-2ß). CHD proteins share two conserved chromo domains and one ATPase domain. In addition, CHD1 and CHD2 have a DNA-binding domain whereas Mi-2alpha and Mi-2ß contain two PHD fingers. The physiological function of CHD proteins is not clear. CHD1 has been localized to decompacted interphase chromosomes in mammalian cells and to regions of high transcriptional activity on Drosophila polytene chromosomes, implicating this family member in transcriptional control (Brehm, 2000 and references therein).

Histone deacetylation of nucleosomal substrates by Mi-2 complexes is stimulated by ATP hydrolysis, arguing that energy-dependent remodelling of the nucleosome is required for the deacetylases to gain access to histone tails. The Mi-2 complexes are believed to repress transcription through their remodelling and deacetylation activities in a targeted manner. Two ways of recruiting Mi-2 complexes to promoter regions have been proposed. (1) Targeting could be mediated via the interaction between the Mi-2 complex and DNA-bound transcriptional repressors. Indeed, the Mi-2 complex copurifies with the Ikaros repressor and functionally interacts with thyroid hormone receptor in vitro (Xue, 1998; Kim, 1999). Furthermore, the Drosophila homolog of Mi-2 physically and genetically interacts with the Hunchback repressor (Kehle, 1998). (2) The Mi-2 complex could be targeted to methylated DNA either directly via its MBD3 subunit or indirectly via association with the MBD2A-methylated DNA-binding protein (Wade, 1999; Zhang, 1999; Brehm, 2000 and references therein).

Several observations suggest that Mi-2 complexes are involved in cell cycle regulation and that their deregulation contributes to cancer. Mi-2 itself was originally identified as an autoimmune antigen in patients suffering from dermatomyositis. These patients suffer an increased risk of developing cancer. Furthermore, Mi-2 is targeted by the E7 oncoprotein of human papilloma virus 16 (Brehm, 1999). Finally, the Mi-2 complex subunit MTA2 is closely related to MTA1, which has been found to be overexpressed in cancer cells with a high potential for metastasis (Brehm, 2000 and references therein).

Mi-2 and other CHD proteins are members of the growing SNF2 superfamily of ATPases. The founding member of this family, yeast SWI2/SNF2, is a well characterized ATP-dependent chromatin remodelling enzyme. However, since the similarity between members of the Snf2 superfamily is restricted to the ATPase domain it is unclear whether all family members are nucleosome remodelling factors. SWI/SNF, Sth1 and ISWI complexes can facilitate factor binding to chromatin and activate transcription from a chromatin template in vitro. Furthermore, both SWI/SNF and ISWI complexes are able to promote movement of a nucleosome along DNA. Both types of ATPase also display a series of distinct biochemical properties. Whereas BRG1 and hBRM ATPase activities are stimulated to the same extent by nucleosomes and naked DNA, the ISWI ATPase is preferentially stimulated by nucleosomes. ATPase activity and nucleosome mobilization by ISWI depends on an intact histone H4 tail. In contrast, remodelling of nucleosomal arrays by SWI/SNF is variably affected by the removal of all four histone tails depending on the precise assay conditions. These findings show that SWI/SNF and ISWI complexes differ in the way they interact with the nucleosome to promote chromatin remodelling (Brehm, 2000 and references therein).

The two unrelated recombinant chromatin remodelling ATPases, Mi-2 and ISWI were compared in tests of nucleosome binding, nucleosome mobilization and ATPase assays. These studies suggest that Mi-2 and ISWI recognize different features of the nucleosome. The activity of the better characterized ATPases ISWI and SWI2/SNF2 depends, to a varying degree, on the presence of histone N-terminal domains. In order to monitor the effect of N-terminal tail sequences, nucleosomes were reconstituted from recombinant Xenopus histones lacking N-termini, and intact and tailless nucleosomes were compared for stimulation of the ATPase activities of recombinant ISWI and Mi-2. The amounts of ISWI and Mi-2 used in this experiment were chosen to give comparable activation by intact nucleosomes. The ATPase activity of ISWI is strongly stimulated by intact nucleosomes reconstituted from recombinant histones in agreement with previous findings (Corona, 1999). Nucleosomes lacking all four histone tails do not stimulate ISWI beyond the levels observed with naked DNA. No significant activation of the ISWI ATPase could be detected by tailless nucleosomes. In sharp contrast to ISWI, Mi-2 is equally well stimulated by intact and tailless nucleosomes, indicating that nucleosomal activation of the Mi-2 ATPase is histone tail independent (Brehm, 2000).

The observations that Mi-2 ATPase activity is stimulated by nucleosomes suggest that Mi-2 can physically associate with nucleosomes. To address this question biotinylated DNA was immobilized on streptavidin-coated paramagnetic beads, and nucleosomes were reconstituted on this DNA. To test for Mi-2 binding, the nucleosomes on beads were incubated with recombinant Mi-2 and then stringently washed to remove loosely bound proteins. Western analysis reveals that Mi-2 binds strongly to nucleosomal arrays assembled from either purified or recombinant histones. No binding to naked DNA is detected under these conditions. It is concluded that Mi-2 binds preferentially to nucleosomal arrays over naked DNA. Mi-2 can also associate with mononucleosomes to form a complex that is sufficiently stable to withstand electrophoresis through polyacrylamide gels (Brehm, 2000).

The abilities of recombinant Mi-2 and ISWI to bind two different mononucleosome species were tested in a bandshift assay. Binding to a mononucleosome (core particle) containing 146 bp of DNA was compared with binding to a nucleosome assembled on the 248 bp fragment. The primary difference between these substrates is that in the 146 bp nucleosome all DNA is entirely in contact with the histone octamer, whereas the 248 bp nucleosome contains ~100 bp of naked DNA extending from the octamer. Incubation with increasing amounts of Mi-2 results in the formation of a Mi-2-core particle complex or a complex of Mi-2 with the 248 bp nucleosome. In contrast, stable ISWI-core particle complexes could not be detected, even if higher amounts of ISWI were used. Incubation of increasing amounts of ISWI with the 248 bp nucleosome produces up to three ISWI-nucleosome complexes, suggesting that more than one ISWI molecule can simultaneously associate with this substrate (Brehm, 2000).

It is concluded that Mi-2 can stably associate with a mononucleosomal substrate lacking free DNA, in agreement with the finding that free DNA does not stimulate the Mi-2 ATPase. In marked contrast, ISWI only binds the nucleosome when free DNA is protruding from the histone octamer. These results strongly suggest that Mi-2 and ISWI recognize different features of the nucleosome (Brehm, 2000).

ATP hydrolysis by Mi-2 is believed to drive remodelling of chromatin by the Mi-2 complex. In order to characterize nucleosome remodelling directly, use was made of a recently established 'nucleosome sliding' assay. The CHRAC complex and recombinant ISWI can mobilize mononucleosomes to change their position on short DNA fragments (Längst, 1999). Nucleosome reconstitution on a 248 bp fragment derived from the mouse rDNA promoter results in the formation of two main species, which differ in the position of the nucleosome relative to the DNA ends and which can be separated by gel electrophoresis under non-denaturing conditions (Längst, 1999). Mononucleosomes positioned near the ends and at the center of the DNA fragment were used as substrates for mobilization by Mi-2 and ISWI. To avoid loss of labelled nucleosomes due to formation of stable enzyme-substrate complexes (as in the bandshift assay) the reaction was stopped by addition of excess unlabelled nucleosomes. Recombinant ISWI moves the centrally positioned nucleosome to the ends of the fragment but not vice versa (Längst, 1999). Remarkably, recombinant Mi-2 behaves in exactly the opposite way: Mi-2 fails to move the central nucleosome but mobilizes the end-positioned nucleosome. Mi-2-promoted nucleosome mobilization is ATP dependent. The effect of removal of individual histone tails on nucleosome mobilization by Mi-2 was also tested. Mi-2 is able to mobilize nucleosomes lacking individual histone N-termini. It is concluded that histone tails are dispensable for stimulation of the Mi-2 ATPase (Brehm, 2000).

The presence of the CHRAC subunits affects the direction of nucleosome sliding by ISWI. Like recombinant Mi-2, the CHRAC complex moves the mononucleosome from the end towards the center of the DNA fragment but fails to mobilize the nucleosome in the opposite direction. In order to investigate whether the presence of Mi-2-associated proteins affects the direction of Mi-2- mediated nucleosome mobilization, immunoprecipitated Mi-2 complexes were tested in the mobilization assay. The alphadMi-2-C antiserum precipitates an activity that mobilizes the end-positioned nucleosome but not the centrally positioned nucleosome. This mobilization activity is ATP dependent. Immunoprecipitated Mi-2 displays the same nucleosome mobilization activity as recombinant Mi-2. Taken together, this analysis of ATPase and nucleosome mobilization activity suggests that the properties of recombinant Mi-2 are fully preserved in a native Mi-2 complex (Brehm, 2000).

Therefore, this comparative analysis has uncovered a number of fundamental differences between these two remodelling enzymes. Both recombinant ATPases can be stimulated by nucleosomes assembled from recombinant histones. Whereas the Mi-2 ATPase does not respond to free DNA, the ISWI ATPase is stimulated by free DNA to some extent. Free DNA also appears to play a role in the interaction of ISWI with the nucleosome: ISWI binds to a nucleosome reconstituted on 248 bp of DNA, which displays free DNA, but not to the core particle consisting entirely of 146 bp of nucleosomal DNA under the stringent conditions of this bandshift assay. In striking contrast, Mi-2 interacts with both nucleosomes equally well, demonstrating that it does not require free DNA for interaction. Taken together, these observations suggest that ISWI, but not Mi-2, recognizes its chromatin substrate in part through an interaction with free DNA (Brehm, 2000).

The ability to promote the movement of a nucleosome along DNA has been demonstrated for the ISWI-containing NURF and CHRAC complexes, recombinant ISWI and the SWI/SNF complex (Hamiche, 1999; Längst, 1999; Whitehouse, 1999). It is conceivable that all remodelling ATPases mobilize nucleosomes in the same way, simply reflecting a common chromatin remodelling activity. Indeed, ISWI and Mi-2 share the intrinsic capacity to promote nucleosome mobilization in an ATP-dependent manner. Surprisingly, however, ISWI and Mi-2 move the nucleosome in opposite directions in these assays. ISWI moves nucleosomes prepositioned at the center to the ends of a DNA fragment but is not able to mobilize end-positioned nucleosomes (Längst, 1999). Mi-2 moves nucleosomes positioned at the end of the DNA fragment to a central position but fails to mobilize the central mononucleosome. Whether this observed directionality of nucleosome movement translates to a regulatory difference in a physiological chromatin context is unclear at present. Nevertheless, the observed difference in the direction of nucleosome movement within the constraints of this assay system suggests that ISWI and Mi-2 interact differently with nucleosomes and that they employ different mechanisms to mobilize them. At present the mechanisms of nucleosome mobilization are not understood. ISWI only moves the central nucleosome, which is flanked on either side by free DNA. Given that free DNA plays a role in substrate recognition by ISWI, it is speculated that ISWI needs to interact with two DNA segments extending from the nucleosome in order to mobilize the nucleosome. This scenario would predict that at least two ISWI molecules simultaneously interact with the central mononucleosome. In agreement with this hypothesis, the formation of multiple ISWI-nucleosome complexes is observed in the bandshift assay, most likely reflecting the binding of multiple ISWI molecules to the 248 bp nucleosome. In contrast, Mi-2 nucleosome binding and ATPase activity is not influenced by free DNA, pointing to a different mechanism of nucleosome mobilization. Interestingly, nucleosome mobilization by Mi-2 in this assay is similar to that of ISWI in the context of the CHRAC complex (Längst, 1999). Identification of the principle that modulates ISWI activity to change the direction of nucleosome mobilization should shed light on the mechanism of nucleosome mobilization by Mi-2 as well (Brehm, 2000).

Guschin (2000) has reported that the highly purified Xenopus Mi-2 complex can redistribute a nucleosome positioned near the end of a fragment derived from the Xenopus thyroid hormone receptor ßA gene towards more central positions in an ATP-dependent manner. It therefore appears that the directionality of Mi2-mediated nucleosome mobilization has been conserved across species (Brehm, 2000).

Nucleosomal stimulation of ISWI ATPase activity and nucleosome mobilization by ISWI are sensitive to the removal of specific histone tails. In contrast, Mi-2 ATPase activity and nucleosome mobilization by Mi-2 are unaffected, suggesting that these are tail-independent processes. The substrate requirements for ATPase and chromatin remodelling activity of Mi-2 reside within the globular domain of the core nucleosome. The differences in dependence on the histone N-termini reinforce the notion that ISWI and Mi-2 approach the nucleosome in fundamentally different ways (Brehm, 2000).

A corresponding comparison between SWI2/SNF2 or Sth1 ATPases and Mi-2 has not been carried out to date. It is not known in which way SWI2/SNF2 and Sth1 ATPases mobilize nucleosomes and whether or not histone tails are required for this activity. It is clear, however, that important differences exist between SWI2/SNF2 or Sth1 and Mi-2 activities. SWI2/SNF2 and Sth1 ATPases are both activated to the same extent by nucleosomes and free DNA. ISWI is preferentially stimulated by nucleosomes but also shows some activation by naked DNA (Corona, 1999). This contrasts sharply with the exclusive stimulation of recombinant Mi-2 and the Mi-2 complex by nucleosomes. In agreement with these results, ATPase activity of the Xenopus Mi-2 complex is stimulated by chicken erythrocyte mononucleosomes but not by salmon sperm DNA (Wade, 1998). In contrast, the ATPase activity of the human NuRD complex is strongly stimulated by naked DNA (Zhang, 1998). It is possible that this discrepancy is due to species-specific differences between different Mi-2 complexes or different experimental setups (Brehm, 2000 and references therein).

Based on this biochemical analysis it is proposed that Mi-2 defines a new class of nucleosome remodelling ATPases. It will be important to determine the molecular basis for the observed differences between Mi-2 and other ATPases in order to gain a better understanding of how chromatin remodelling machines work (Brehm, 2000).

Mi-2 resides in a large complex (~1.0 MDa) that also contains the HDAC RPD3. The estimated size of the complex suggests that it contains further subunits in addition to Mi-2 and RPD3. Most likely some of the additional Mi-2-associated subunits will correspond to proteins identified in vertebrate Mi-2 complexes. Indeed, several of these homologous sequences can be found in Drosophila EST databases (Wade, 1999). The vertebrate Mi-2 complexes have been implicated in mediating transcriptional repression by binding to methylated DNA. In contrast to its vertebrate counterparts, the Drosophila genome is not methylated to any appreciable extent. It will be fascinating to determine whether the Mi-2 complex contains subunits related to the methylated DNA-binding proteins present in the vertebrate complexes and what their role might be in the absence of DNA methylation. A major challenge now is to understand how the different enzymatic subunits of the Mi-2 complex cooperate to regulate chromatin (Brehm, 2000).

Blocking promiscuous activation at cryptic promoters directs cell type-specific gene expression

To selectively express cell type-specific transcripts during development, it is critical to maintain genes required for other lineages in a silent state. This study shows in the Drosophila male germline stem cell lineage that a spermatocyte-specific zinc finger protein, Kumgang (Kmg; CG5204), working with the chromatin remodeler dMi-2 prevents transcription of genes normally expressed only in somatic lineages. By blocking transcription from normally cryptic promoters, Kmg restricts activation by Aly, a component of the testis-meiotic arrest complex, to transcripts for male germ cell differentiation. These results suggest that as new regions of the genome become open for transcription during terminal differentiation, blocking the action of a promiscuous activator on cryptic promoters is a critical mechanism for specifying precise gene activation (Kim, 2017).

Highly specialized cell types such as red blood cells, intestinal epithelium, and spermatozoa are produced throughout life from adult stem cells. In such lineages, mitotically dividing precursors commonly stop proliferation and initiate a cell type-specific transcription program that sets up terminal differentiation of the specialized cell type. In the Drosophila male germ line, stem cells at the apical tip of the testis self-renew and produce daughter cells that each undergo four rounds of spermatogonial mitotic transit amplifying (TA) divisions, after which the germ cells execute a final round of DNA synthesis (premeiotic S-phase) and initiate terminal differentiation as spermatocytes. Transition to the spermatocyte state is accompanied by transcriptional activation of more than 1500 genes, many of which are expressed only in male germ cells. Expression of two-thirds of these depends both on a testis-specific version of the MMB (Myb-Muv B)/dREAM (Drosophila RBF, dE2F2, and dMyb-interacting proteins) complex termed the testis meiotic arrest complex (tMAC) and on testis-specific paralogs of TATA-binding protein-associated factors (tTAFs). Although this is one of the most dramatic changes in gene expression in Drosophila, it is not yet understood how the testis-specific transcripts are selectively activated during the 3-day spermatocyte period (Kim, 2017).

To identify the first transcripts up-regulated at onset of spermatocyte differentiation, germ cells were genetically manipulated to synchronously differentiate from spermatogonia to spermatocytes in vivo using bam^-/- testes, which contain large numbers of overproliferating spermatogonia. Brief restoration of Bam expression under heat shock control in hs-bam;bam^-/- flies induced synchronous differentiation of bam^-/- spermatogonia, resulting in completion of a final mitosis, premeiotic DNA synthesis, and onset of spermatocyte differentiation by 24 hours after Bam expression, eventually leading to production of functional sperm. Comparison by means of microarray of transcripts expressed before versus 24 hours after heat shock of hs-bam;bam^-/- testes identified 27 early transcripts that were significantly up-regulated more than twofold in testes from hs-bam;bam^-/- but not from bam^-/- flies subjected to the same heat shock regime. Among these was the early spermatocyte marker RNA binding protein 4 (Rbp4). At this early time point, the transcript for CG5204 - now named kumgang (kmg), from the Korean name of mythological guardians at the gate of Buddhist temples - had the greatest increase among all 754 Drosophila predicted transcription factors (Kim, 2017).

Kumgang (CG5204) encodes a 747-amino acid protein with six canonical C2H2-type zinc finger domains expressed in testes but not in ovary or carcass. Kmg protein was expressed independently from the tMAC component Always early (Aly) or the tTAF Spermatocyte Arrest (Sa), and both kmg mRNA and protein were up-regulated before Topi, another component of tMAC. Immunofluorescence staining of wild-type testes revealed Kmg protein expressed specifically in differentiating spermatocytes, where it was nuclear and enriched on the partially condensed bivalent chromosomes. Consistent with dramatic up-regulation of kmg mRNA after the switch from spermatogonia to spermatocyte, expression of Kmg was first detected with immunofluorescence staining after completion of premeiotic S-phase marked by down-regulation of Bam, coinciding with expression of Rbp4 protein (Kim, 2017).

Function of Kmg in spermatocytes was required for male germ cell differentiation. Reducing function of Kmg in spermatocytes-either by means of cell type-specific RNA interference (RNAi) knockdown (KD) or in flies trans-heterozygous for a CRISPR (clustered regularly interspaced short palindromic repeats)-induced kmg frameshift mutant and a chromosomal deficiency (kmg^Δ7/Df)-resulted in accumulation of mature primary spermatocytes arrested just before the G₂/M transition for meiosis I and lack of spermatid differentiation. A 4.3-kb genomic rescue transgene containing the 2.3-kb kmg open reading frame fully rescued the differentiation defects and sterility of kmg^Δ7/Df flies, confirming that the meiotic arrest phenotype was due to loss of function of Kmg. In both kmg KD and kmg^Δ7/Df, Kmg protein levels were less than 5% that of wild type. kmg^Δ7/Df mutant animals were adult-viable and female-fertile but male-sterile, which is consistent with the testis-specific expression (Kim, 2017).

Function of Kmg was required in germ cells for repression of more than 400 genes not normally expressed in wild-type spermatocytes. Although the differentiation defects caused by loss of function of kmg appeared, by means of phase contrast microscopy, to be similar to the meiotic arrest phenotype of testis-specific tMAC component mutants, analysis of gene expression in kmg KD testes showed that many Aly (tMAC)-dependent spermatid differentiation genes were expressed, although some at a lower level than that in wild type. Among the 652 genes with more than 99% lower expression in aly^-/- mutant as compared with wild-type testes, only four showed similar reduced expression in kmg KD as compared with that of sibling control (no Gal4 driver) testes. In contrast, transcripts from more than 500 genes were strongly up-regulated in kmg KD testes, with almost no detectable expression in testes from sibling control males. Hierarchical clustering identified 440 genes specifically up-regulated in kmg KD testes compared with testes from wild-type, bam^-/-, aly^-/-, or sa^-/- mutant flies. These 440 genes were significantly associated with Gene Ontology terms such as 'substrate specific channel activity' or 'detection of visible light' that appeared more applicable to non-germ cell types, such as neurons. Analysis of published transcript expression data for a variety of Drosophila tissues revealed that the 440 were normally not expressed or extremely low in wild-type adult testes, but many were expressed in specific differentiated somatic tissues such as eye, brain, or gut. Confirming misexpression of neuronal genes at the protein level, immunofluorescence staining revealed that the neuronal transcription factor Prospero (Pros), normally not detected in male germ cells, was expressed in clones of spermatocytes that are homozygous mutant for kmg induced by Flp-FRT-mediated mitotic recombination. The misexpression of Pros was cell-autonomous, occurring only in mutant germ cells. Mid-stage to mature spermatocytes homozygous mutant for kmg misexpressed Pros, but mutant early spermatocytes did not, indicating that the abnormal up-regulation of Pros occurred only after spermatocytes had reached a specific stage in their differentiation program (Kim, 2017).

A small-scale cell type-specific RNAi screen of chromatin regulators revealed that KD of dMi-2 in late TA cells and spermatocytes resulted in meiotic arrest, similar to loss of function of kmg. Immunofluorescence analysis of testes from a protein trap line in which an endogenous allele of dMi-2 was tagged by green fluorescent protein (GFP) revealed that dMi-2-GFP, like the untagged endogenous protein, was expressed and nuclear in progenitor cells and spermatocytes, as well as in somatic hub and cyst cells. dMi-2-GFP colocalized to chromatin with Kmg in spermatocytes, and the level of dMi-2 protein appeared lower and less concentrated on chromatin in nuclei of kmg^-/- spermatocytes than in neighboring kmg^+/+ or kmg^+/- spermatocytes, suggesting that Kmg may at least partially help recruit dMi-2 to chromatin in spermatocytes. Furthermore, in testis extracts Kmg coimmunoprecipitated with dMi-2 and vice versa, suggesting that Kmg and dMi-2 form a protein complex in spermatocytes. Comparison of microarray data revealed that most of the 440 transcripts up-regulated in testes upon loss of function of kmg were also abnormally up-regulated in dMi-2 KD testes, suggesting that Kmg and dMi-2 may function together to repress expression of the same set of normally somatic transcripts in spermatocytes (Kim, 2017).

Chromatin immunoprecipitation followed by sequencing (ChIP-seq) revealed that Kmg protein localized along the bodies of genes actively transcribed in the testis. ChIP-seq with antibody to Kmg identified 798 genomic regions strongly enriched by immunoprecipitation of Kmg from wild-type but not from kmg KD testes. Of the 798 robust Kmg ChIP-seq peaks, 698 overlapped with exonic regions of 680 different genes actively transcribed in testes. The enrichment was often strongest just downstream of the transcription start site (TSS), but with substantial enrichment along the gene body as well (Kim, 2017).

ChIP-seq with antibody to dMi-2 also showed enrichment along the gene bodies of the same 680 genes bound by Kmg, with a similar bias just downstream of the TSS. The dMi-2 ChIP signal along these genes was partially reduced in kmg KD testes, suggesting that Kmg may recruit dMi-2 to the bodies of genes actively transcribed in the testis (Kim, 2017).

RNA-seq analysis revealed that the 680 genes bound by Kmg were strongly expressed in testes and most strongly enriched in the GO term categories 'spermatogenesis' and 'male gamete generation'. One-third of the genes bound by Kmg were robustly activated as spermatogonia differentiate into spermatocytes and were much more highly expressed in the testes than in other tissues. The median levels of transcript expression of most of the 680 Kmg bound genes did not show appreciable change upon loss of Kmg (Kim, 2017).

Genes that are normally transcribed in somatic cells that became up-regulated upon loss of Kmg function in spermatocytes for the most part did not appear to be bound by Kmg. Only 3 of the 440 genes up-regulated in kmg KD overlapped with the 680 genes with robust Kmg peaks, suggesting that Kmg may prevent misexpression of normally somatic transcripts either indirectly or by acting at a distance (Kim, 2017).

Inspection of RNA-seq reads from kmg and dMi-2 KD testes mapped onto the genome showed that ~80% of the transcripts that were detected with microarray analysis as misexpressed in KD as compared with wild-type testes did not initiate from the promoters used in the somatic tissues in which the genes are normally expressed. Metagene analysis, as well as visualization of RNA expression centered on the TSSs annotated in the Ensembl database, showed that most of the 143 genes that are normally expressed in wild-type heads but not in wild-type testes were misexpressed in kmg or dMi-2 KD testes from a start site different from the annotated TSS used in heads. Transcript assembly from RNA-seq data by using Cufflinks for the 143 genes also showed that the transcripts that are misexpressed in kmg or dMi-2 KD testes most often initiate from different TSSs than the transcripts from the same gene assembled from wild-type heads (Kim, 2017).

Of the 440 genes scored via microarray as derepressed in kmg KD testes, 346 could be assigned with TSSs in kmg KD testes based on visual inspection of the RNA-seq data mapped onto the genome browser. Of these, only 67 produced transcripts in kmg KD testes that started within 100 base pairs (bp) of the TSS annotated in the Ensembl database, based on the tissue(s) in which the gene was normally expressed. In contrast, for the rest of the 346 genes, the transcripts expressed in kmg KD testes started from either a TSS upstream (131 of 346) or downstream (148 of 346) of the annotated TSSs. Of the 346 genes, 262 were misexpressed starting from nearly identical positions in dMi-2 KD as in kmg KD testes, suggesting that Kmg and dMi-2 function together to prevent misexpression from cryptic promoters (Kim, 2017).

Many of the ectopic promoters from which the misexpressed transcripts originated appeared to be bound by Aly, a component of tMAC, in kmg KD testes. ChIP for Aly was performed by using antibody to hemagglutinin (HA) on testis extracts from flies bearing an Aly-HA genomic transgene able to fully rescue the aly^-/- phenotype. Of 346 genes with new TSSs assigned via visual inspection, 181 had a region of significant enrichment for Aly as detected with ChIP, with its peak summit located within 100 bp of the cryptic promoter. Motif analysis by means of MEME revealed that these regions were enriched for the DNA sequence motif (AGYWGGC). This motif was not significantly enriched in the set of 165 cryptic promoters at which Aly was not detected in kmg KD testes. Enrichment of Aly at the cryptic promoters was much stronger in kmg KD as compared with wild-type testes, suggesting that in the absence of Kmg, Aly may bind to and activate misexpression from cryptic promoters (Kim, 2017).

Genetic tests revealed that the misexpression of somatic transcripts in kmg KD spermatocytes indeed required function of Aly. The neuronal transcription factor Pros, abnormally up-regulated in kmg KD or mutant spermatocytes, was no longer misexpressed if the kmg KD spermatocytes were also mutant for aly, even though germ cells in kmg KD;aly^-/- testes appear to reach the differentiation stage at which Pros turned on in the kmg KD germ cells. Assessment by means of quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed that misexpression of five out of five transcripts in kmg KD testes also required function of Aly. Global transcriptome analysis via microarray of kmg KD versus kmg KD;aly^-/- testes showed that the majority of the 440 genes that were derepressed because of loss of function of kmg in spermatocytes were no longer abnormally up-regulated in kmg KD;aly^-/- testes. Even genes without noticeable binding of Aly at their cryptic promoters were suppressed in kmg KD;aly^-/-, suggesting that Aly may regulate this group of genes indirectly (Kim, 2017).

Together, the ChIP and RNA-seq data show that Kmg and dMi-2 bind actively transcribed genes but are required to block expression of aberrant transcripts from other genes that are normally silent in testes. The mammalian ortholog of dMi-2, CHD4 (Mi-2β), has been shown to bind active genes in mouse embryonic stem cells or T lymphocyte precursors but also plays a role in ensuring lineage-specific gene expression in other contexts. It cannot be ruled out that Kmg and dMi-2 might also act directly at the cryptic promoter sites but that the ChIP conditions did not capture their transient or dynamic binding because several chromatin remodelers or transcription factors, such as the thyroid hormone receptor, have been difficult to detect with ChIP. Kmg and dMi-2 may repress misexpression from cryptic promoters indirectly by activating as-yet-unidentified repressor proteins. However, it is also possible that Kmg and dMi-2 act at a distance by modulating chromatin structure or confining transcriptional initiation or elongation licensing machinery to normally active genes (Kim, 2017).

Changes in the genomic localization of Aly protein in wild-type versus kmg KD testes raised the possibility that Kmg may in part prevent misexpression from cryptic promoters by concentrating Aly at active genes. Of the 1903 Aly peaks identified with ChIP from wild-type testes, the 248 Aly peaks that overlapped with strong Kmg peaks showed via ChIP an overall reduction in enrichment of Aly from kmg KD testes as compared with wild type. In contrast, the Aly peaks at cryptic promoters were more robust in kmg KD testes than in wild type. In general, over the genome 4129 new Aly peaks were identified by means of ChIP from kmg KD testes that were absent or did not pass the statistical cutoff in wild-type testes. More than 30% of the genomic regions with new Aly peaks in kmg KD showed elevated levels of RNA expression starting at or near the Aly peak in kmg KD but not in wild-type testes, suggesting that misexpression of transcripts from normally silent promoters in kmg KD testes is more widespread than initially assessed with microarray. Together, these findings raise the possibility that Kmg may prevent misexpression of aberrant transcript by concentrating Aly to active target genes in wild-type testes, preventing binding and action of Aly at cryptic promoter sites (Kim, 2017).

The results suggest that selective gene activation is not always mediated by a precise transcriptional activator but can instead be directed by combination of a promiscuous activator and a gene-selective licensing mechanism. Cryptic promoters may become accessible as chromatin organization is reshaped to allow expression of terminal differentiation transcripts that were tightly repressed in the progenitor state. It is posited that this chromatin organization makes a number of sites that are accessible for transcription dependent on the testis-specific tMAC complex component Aly. In this context, activity of Kmg and dMi-2 is required to prevent productive transcript formation from unwanted initiation sites, potentially by confining Aly to genes actively transcribed in the testis and limiting the amount of Aly protein acting at cryptic promoters (Kim, 2017).

The initiation of transcripts from cryptic promoters is reminiscent of loss of function of Ikaros, a critical regulator of T and B cell differentiation and a tumor suppressor in the lymphocyte lineage. Like Kmg, Ikaros is a multiple-zinc finger protein associated with Mi-2β, which binds to active genes in T and B cell precursors. In T cell lineage acute lymphoblastic leukemia (T-ALL) associated with loss of function of Ikaros, cryptic intragenic promoters were activated, leading to expression of ligand-independent Notch1 protein, contributing to leukemogenesis. Thus, in addition to being detrimental for proper differentiation, firing of abnormal transcripts from normally cryptic promoters because of defects in chromatin regulators may contribute to tumorigenesis through generation of oncogenic proteins (Kim, 2017).

GENE STRUCTURE

cDNA clone length - 6475

Bases in 5' UTR - 131

Bases in 3' UTR - 395

PROTEIN STRUCTURE

Amino Acids - 1982

Structural Domains

Mi-2 contains five conserved sequence motifs that are also present in the two human Mi-2 proteins and in two Caenorhabditis elegans ORFs: two chromodomains, a DNA-stimulated adenosine triphosphatase (ATPase) domain, two PHD finger motifs, a truncated helix-turn-helix motif resembling the DNA-binding domain of c-myb, and a motif with similarity to the first two helices of an HMG domain (Kehle, 1998). \

Drosophila Mi-2 (dMi-2) is the ATPase subunit of a complex combining ATP-dependent nucleosome remodelling and histone deacetylase activities. dMi-2 contains an HMG box-like region, two PHD fingers, two chromodomains and a SNF2-type ATPase domain. It is not known which of these domains contribute to nucleosome remodelling. A panel of dMi-2 deletion mutants was tested in ATPase, nucleosome mobilization and nucleosome binding assays. Deletion of the chromodomains impairs all three activities. A dMi-2 mutant lacking the chromodomains is incorporated into a functional histone deacetylase complex in vivo but has lost nucleosome-stimulated ATPase activity. In contrast to Drosophila HP1, dMi-2 does not bind methylated histone H3 tails and does not require histone tails for nucleosome binding. Instead, the dMi-2 chromodomains display DNA binding activity that is not shared by other chromodomains. These results suggest that the chromodomains act at an early step of the remodelling process to bind the nucleosome substrate predominantly via protein-DNA interactions. Furthermore, this study identifies DNA binding as a novel chromodomain-associated activity (Bouazoune, 2002).

Fusion of the chromodomain region and the ATPase domain is sufficient for DNA and nucleosome binding, but not for nucleosome-stimulated ATPase and nucleosome mobilization activities. It follows that additional regions outside these domains make critical contributions to nucleosome remodelling. Conversion of the chromo domain-ATPase domain fusion (dMi-2 484-1271) to an active nucleosome remodeller can be achieved by addition of the remainder of the N-terminal region (NTR) or by addition of the C-terminal region (CTR). This implies that both NTR and CTR provide activities that are redundant in the assay. The CTR binds the repression domains of Hunchback and Tramtrack 69. The C-terminus of mammalian Mi2ß interacts with the KAP-1 co-repressor. The results suggest that CTR function is not restricted to transcription factor binding. Instead, it plays an active role in ATPase regulation: although deletion of the CTR does not affect nucleosome mobilization it makes the ATPase responsive to DNA. In this respect, the dMi-2 1-1271 mutant resembles ATPases of the SWI/SNF subgroup. This observation suggests that the CTR is directly involved in regulation of the ATPase domain: it is required to suppress activity in presence of the 'wrong' effector (DNA), when no remodelling substrate (nucleosome) is available. It is conceivable that the CTR might undergo a change in conformation following nucleosome recognition, which then allows the ATPase domain to function (Bouazoune, 2002).

date revised: 23 June 2023

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