BIOLOGICAL OVERVIEW

Regulatory biology is currently mesmerized (one might say the field is going gaga). The fascination is with molecular machines: protein complexes that change the conformation of chromatin, making its constituent DNA accessable to transcription factors. Iswi is a component of one of these machines. Iswi was isolated on the basis of homology to yeast transcriptional activator SWI2, also known as SNF2. Iswi differs from the yeast protein in its lack of a bromodomain (Elfring, 1994). The Iswi shares with the yeast protein a presumably non functional DNA dependent helicase domain. (Helicases unwrap the helical structure of DNA).

Iswi appears to be part of a multiprotein complex consisting of four subunits. This complex, termed NURF (nucleosome remodeling factor), is able to activate the Drosophila Heat-shock-protein-70 (Hsp70) promoter along with the GAGA transcription factor (Tsukiyama, 1995a). Iswi, the 140 kDa subunit of NURF, is localized to the cell nucleus and expressed throughout Drosophila development (Tsukiyama, 1995b).

The different structures of Brahma protein and Iswi point to the possible existence of more than one chromatin remodeling system. The Iswi component of NURF is homologous to yeast Iswi, a member of the SWI2/SNF2 family of ATPases, a transcriptional activator that counters the repressive effects on nucleosomal histones. Iswi is plentiful in Drosophila, on the order of 100,000 molecules per cell.

The ATPase activity of NURF is stimulated by chromatin (Tsukiyama, 1995b). However, the ATPase activity of a second family of activators in yeast and Drosophila, called SWI/SNF, is stimulated by naked DNA (DNA free of chromatin). The concentration of SWI/SNF in yeast is on the order of 100 molecules per cell, much lower than that of Iswi in Drosophila (Tsukiyama, 1995a). Brahma is a component of the SWI/SNF complex, and contains a bromodomain unlike Iswi. This suggests the existence of two families of ATP dependent activators; one (SWI/SNF) dependent on DNA and the another (NURF) dependent on chromatin (DNA wrapped with histones). These molecular transcription activating machines work in conjunction with transcription factors to activate or silent genes, or maintain activity of already active genes.

A chromatin-accessibility complex (CHRAC), distinct from NURF but containing Iswi, has been identified that uses energy to increase the general accessibility of DNA in chromatin. Unlike other known chromatin remodelling factors, CHRAC can also function during chromatin assembly: it uses ATP to convert irregular chromatin into a regular array of nucleosomes with even spacing. CHRAC combines enzymes that modulate nucleosome structure and DNA topology. Using mass spectrometry, two of the five CHRAC subunits have been identified as the ATPase Iswi, which is also part of NURF, and topoisomerase II. Topoisomerase II normally functions in the ATP dependent relaxation of negative supercoils, but the topoisomerase activity does not seem to be involved in CHRAC function. Topo II exists as a dimer in CHRAC. Therefore the function of Topo II in CHRAC is not yet known. No other subunit is shared by the two complexes. Two hSNF2L (the human Iswi homolog) containing complexes of 700K and 450K have been identified, corresponding to the Mr determined here for CHRAC as well as that of NURF, suggesting the existence of human counterparts for both Iswi-containing complexes. CHRAC and NURF increase the accessibility of chromatin by different mechanisms. CHRAC and NURF preparations have different activities when compared in accessibility assays: CHRAC is less effective in the GAGA-factor-dependent nucleosome remodelling assay than NURF; conversely, NURF does not stimulate the sensitivity to restriction endonucleases. NURF, but not CHRAC, perturbs histone-DNA interactions in monosomes when added at a stoichiometry of one NURF per 20 nucleosome core particles. The most striking difference between CHRAC and NURF is in their effect on nucleosomal arrays. Addition of CHRAC to chromatin assembled in the absence of ATP (and therefore lacking regular nucleosome spacing) results in an ATP-dependent alignment of nucleosomes. NURF has the opposite effect: addition of large amounts of NURF to a nucleosomal array perturbs the regular repeat pattern. The presence of Iswi in different contexts suggests that chromatin remodelling machines have a modular nature and that Iswi has a central role in different chromatin remodelling reactions (Varga-Weisz, 1997).

NURF catalyzes the bidirectional redistribution of mononucleosomes reconstituted on hsp70 promoter DNA. Within minutes, in the presence of NURF, nucleosomes adopt one predominant position from an ensemble of possible locations. Movements occur in cis, with no transfer to competing DNA. Migrating intermediates trapped by Exo III digestion reveal progressive nucleosome motion in increments of several base pairs. All four core histones are retained quantitatively during this process, indicating that the general integrity of the histone octamer is maintained. It is suggested that NURF remodels nucleosomes by transiently decreasing the activation energy for short-range sliding of the histone octamer (Hamiche, 1999).

Mononucleosome reconstitution by salt gradient dialysis on a 359 bp fragment of the Drosophila hsp 70 promoter generates a mixed nucleosome population distributed over several translational positions, whose locations are dependent on the underlying DNA sequence. These nucleosomes can be fractionated by native polyacrylamide gel electrophoresis on the basis of differences in length and spatial orientation of the linker DNA. Such position-dependent electrophoretic mobility has been described for DNA-bending proteins such as CAP. The native gel mobility is minimal for DNA molecules with centrally positioned nucleosomes; mobility increases linearly, as the nucleosome location approaches the end of the fragment. The nucleosome positions were mapped precisely by gel elution of each of the four species, termed N1-N4, followed by Exonuclease III (Exo III) digestion. The pause positions of Exo III define nucleosome boundaries, which can be displayed relative to the hsp70 promoter elements. Thus, nucleosomes N1 and N2 overlap and are located over GAGA elements of the hsp70 promoter. The N3 position is located further upstream from, and partially overlaps, N1 and N2. As is generally observed when nucleosomes are reconstituted on linear fragments, there appears to be a greater preference for nucleosome location at either fragment end (N4 and N4'). Native gel electrophoresis reveals that when NURF is added to the mixed population of N1-N4 nucleosomes in the presence of ATP, there is a clear increase in the N3 species and a corresponding loss of the N1 and N2 nucleosome positions. A subtle change in the electrophoretic mobility of the N4 species can also be observed (Hamiche, 1999).

To illustrate the effects of NURF on a single positioned species, the N1 nucleosome was purifed by native gel electrophoresis and it was eluted and incubated with NURF and ATP. Nucleosomes at the N1 position moved primarily to the N3 position, but a small, clearly defined signal is also observed at N2, and between the N3 and N4 positions.The results demonstrate that nucleosome redistribution mediated by NURF is bidirectional, repositioning nucleosomes from N1 to either upstream (N3) or downstream (N2) positions on the same DNA fragment. This can be further illustrated by incubation of gel-purified N3 nucleosomes with NURF and ATP. A fraction of the N3 species is relocated to the positions corresponding to N1, N2, N4, and between N3 and N4 nucleosomes. However, most of the nucleosomes remain at N3, confirming this location as the favored one in the ensemble of equilibrium positions. When the reaction time is shortened in a similar experiment, the presence of migrating intermediates trapped by Exo III digestion at increments of a few base pairs suggests the presence of migration intermediates. The existence of such migration intermediates suggests that migration of the histone octamer is not entirely smooth, occurring in small processive steps, rather than large jumps on DNA (Hamiche, 1999).

Remodeling mediated by NURF does not proceed by the complete dissociation of the histone octamer from DNA. Since the remodeling reactions were performed in a vast excess of carrier DNA, nucleosome transfer to an unlinked DNA fragment would readily have been detected by the production of free labeled DNA. No increase in the free 359 bp DNA is observed after remodeling by NURF. Furthermore, it is argued that NURF acts catalytically; remodeling reactions were performed at a ratio of 1 NURF:50-100 nucleosomes (Hamiche, 1999).

In principle, a histone octamer could be translocated in several ways. The octamer could be made to jump by complete detachment from one position and reassociate at a new location. The octamer could undergo displacement transfer in which a stretch of free DNA could displace another stretch of nucleosomal DNA from its interaction with the histone octamer. The nucleosome could also be relocated by a rolling or sliding of the histone octamer relative to DNA, without dissociation. Sliding is favored as a means of nucleosome mobilization by NURF because it would require only the transient alteration of individual weak interactions between histones and DNA, whereas jumping would entail the energetically unfavorable disruption of all histone octamer-DNA contacts simultaneously. The crystal structure of the histone octamer complexed with DNA indicates sites of interaction between the core histones and DNA approximately every ten base pairs over the entire 146 bp length of nucleosomal DNA, with the notable insertion of an arginine residue in the minor groove at every site. To maintain critical minor groove contacts during translational sliding, a rotation of the double helix as in the advance of a screw in its screwthread would be required (Hamiche, 1999 and references).

The experimental data obtained in this study suggest that NURF promotes nucleosome sliding. (1) Migration of the core histone octamer on the same DNA fragment is observed without displacement in trans, even in the presence of a 3000-fold excess of exogenous carrier DNA. This concentration of competing DNA is 3-fold greater than that which is effective for capturing histone octamers displaced from the 5S RNA gene by the SP6 RNA polymerase. (2) At early times in the reaction with NURF, migration intermediates trapped by Exo III digestion are observed at increments of a few base pairs. The presence of these migration intermediates is not consistent with a jumping or displacement transfer process, except over a distance of a few base pairs. Sliding has long been known to be an intrinsic property of nucleosomes. However, this passive and spontaneous sliding of the histone octamer, which occurs over a period of hours, is readily distinguished from the rapid sliding that is facilitated by NURF. Moreover, while spontaneous sliding is enhanced by elevated temperatures and ionic strengths and is suppressed by 2 mM MgCl2, sliding faciliated by NURF can occur at room temperature and in moderate concentrations of both monovalent and divalent cations (Hamiche, 1999).

ISWI regulates higher-order chromatin structure and histone H1 assembly in vivo

Imitation SWI (ISWI) and other ATP-dependent chromatin-remodeling factors play key roles in transcription and other processes by altering the structure and positioning of nucleosomes. Recent studies have also implicated ISWI in the regulation of higher-order chromatin structure, but its role in this process remains poorly understood. To clarify the role of ISWI in vivo, defects in chromosome structure and gene expression were examined resulting from the loss of Iswi function in Drosophila. Consistent with a broad role in transcriptional regulation, the expression of a large number of genes is altered in Iswi mutant larvae. The expression of a dominant-negative form of ISWI leads to dramatic alterations in higher-order chromatin structure, including the apparent decondensation of both mitotic and polytene chromosomes. The loss of ISWI function does not cause obvious defects in nucleosome assembly, but results in a significant reduction in the level of histone H1 associated with chromatin in vivo. These findings suggest that ISWI plays a global role in chromatin compaction in vivo by promoting the association of the linker histone H1 with chromatin (Corona, 2007; full text of article).

Most studies of ISWI complexes in Drosophila and other organisms have focused on their ability to alter the structure or spacing of nucleosomes, the fundamental unit of chromatin structure. These findings reveal that ISWI also plays a global role in the regulation of higher-order chromatin structure. The Iswi mutations used in this study eliminate the function of multiple chromatin-remodeling complexes, including ACF, NURF, and CHRAC. Which of these complexes are required for the formation of higher-order chromatin structure? Loss of function mutations in Acf1 -- which encodes a subunit protein shared by ACF and CHRAC -- do not cause obvious defects in higher-order chromatin structure. By contrast, loss of function mutations in E(bx) -- which encodes a subunit specific to NURF -- cause male X chromosome defects similar to those observed in Iswi mutants. These findings suggest that ISWI modulates higher-order chromatin structure within the context of NURF, as opposed to ACF or CHRAC (Corona, 2007).

A striking correlation was observed between the severity of the chromosome defects resulting from the loss of ISWI function and the loss of the linker histone H1. This correlation suggests that ISWI regulates higher-order chromatin structure by promoting the association of histone H1 with chromatin. Histone H1 and other linker histones influence higher-order chromatin structure in vitro by stabilizing interactions between nucleosomes and chromatin fibers. Although the ability of histone H1 to promote chromatin compaction in vitro is well established, its function in vivo has been a topic of considerable debate. A protein with biochemical properties reminiscent of linker histones -- HHO1 -- is present in budding yeast; surprisingly, HHO1 is not essential for viability in yeast, and hho1 mutations have little effect on either gene expression or chromatin structure. Genetic studies in Tetrahymena have suggested roles for linker histones in chromatin condensation and gene expression, but the relevance of these studies to histone H1 function in higher eukaryotes remains unclear. Studies of histone H1 function in higher eukaryotes have been complicated by the presence of redundant genes encoding histone H1 or histone H1 subtypes. In spite of these difficulties, recent studies have revealed important roles for histone H1 in chromosome compaction in Xenopus and mice. Thus, the chromosome defects observed in Iswi mutants could easily result from inefficient incorporation of histone H1 into chromatin (Corona, 2007).

How might ISWI promote the association of histone H1 with chromatin? Since ISWI is not required for histone H1 synthesis, ISWI may directly promote the assembly of chromatin containing histone H1 following DNA replication. Recent biochemical studies provide support for this possibility: ACF promotes the ATP-dependent assembly of H1-containing chromatin in vitro. Loss of ACF1 function does not cause obvious changes in chromosome structure, however, suggesting that ACF either does not regulate higher-order chromatin structure in vivo or plays a redundant role in this process. It remains possible that ISWI promotes the assembly of histone-H1-containing chromatin within the context of NURF or another chromatin-remodeling complex (Corona, 2007).

The ability to promote histone H1 assembly is not a common property of all chromatin-remodeling factors, as illustrated by recent biochemical studies of CHD1. Like ACF and other ISWI complexes, the CHD1 ATPase promotes the assembly of regularly spaced nucleosomes in vitro. By contrast, CHD1 does not promote the incorporation of histone H1 during chromatin assembly in vitro. These biochemical studies provide a plausible explanation for why the loss of ISWI function leads to the loss of histone H1 without causing dramatic changes in nucleosome assembly in vivo (Corona, 2007).

In other organisms, depletion of histone H1 leads to a significant decrease in the nucleosome repeat length, presumably because of the failure to efficiently incorporate histone H1 during replication-coupled chromatin assembly. By contrast, the loss of ISWI function in salivary gland nuclei leads to a decrease in the amount of histone H1 associated with chromatin without causing dramatic changes in nucleosome repeat length. It is therefore tempting to speculate that ISWI promotes histone H1 incorporation via a replication-independent process. The association of histone H1 with chromatin is far less stable than that of core histones; histone H1 undergoes dynamic, global exchange throughout the cell cycle. Photobleaching experiments in Tetrahymena and vertebrates have suggested that the majority of histone H1 molecules associated with chromatin are exchanged every few minutes, but little is known about the factors that regulate this process. Based on the current findings, ISWI is an excellent candidate for a factor that regulates the dynamic exchange of histone H1 in vivo. Further work will be necessary to determine whether ISWI promotes histone H1 incorporation via replication-dependent or -independent mechanisms (Corona, 2007).

These findings suggest that acetylation of H4K16 may regulate the association of linker histones with chromatin in vivo. The histone H4 tail is required for the nucleosome-stimulated ATPase activity of ISWI, and for its ability to slide nucleosomes and alter their spacing in vitro. The region of the H4 tail that is critical for ISWI function in vitro is a DNA-bound basic patch (R₁₇H₁₈R₁₉) adjacent to H4K16, the residue that is acetylated by the MOF histone acetyltransferase. The acetylation of H4K16 interferes with the ability of ISWI to interact with the histone H4 tail and alter the spacing of nucleosome arrays in vitro. Consistent with these findings, dosage compensation is necessary and sufficient for the decondensation of the X chromosome in Iswi mutant larvae, and genetic studies have revealed a strong functional antagonism between ISWI and MOF. Thus, H4K16 acetylation may function as a switch that regulates the histone H1 assembly mediated by ISWI (Corona, 2007).

Microarray studies revealed that ISWI is required for the proper expression of a large number of genes. These findings are consistent with numerous studies implicating ISWI in transcriptional regulation in vitro and in vivo. Does ISWI modulate transcription by altering higher-order chromatin structure? It is suspected that ISWI regulates transcription and higher-order chromatin structure via distinct mechanisms, since no obvious correlation is observed between the magnitude of the changes in gene expression and chromosome structure observed in Iswi mutant larvae. This is consistent with genetic studies in other organisms that have revealed that the loss of histone H1 does not cause dramatic changes in gene expression. No correlation was observed between the magnitude of transcriptional derepression and gene size in Iswi mutant larvae, as would be expected if ISWI relieved a general block to transcriptional elongation by Pol II. It should be noted, however, that relatively subtle, but biologically important, changes in gene expression may have escaped detection in the microarray studies. Further work will be necessary to clarify this issue and to determine whether ISWI regulates transcription and higher-order chromatin structure via distinct or related mechanisms (Corona, 2007).

Identification and characterization of ToRC, a novel ISWI-containing ATP-dependent chromatin assembly complex

SNF2-like motor proteins, such as ISWI, cooperate with histone chaperones in the assembly and remodeling of chromatin. This study describes a novel, evolutionarily conserved, ISWI-containing complex termed ToRC (Toutatis-containing chromatin remodeling complex). ToRC comprises ISWI, Toutatis/TIP5 (TTF-I-interacting protein 5), and the transcriptional corepressor CtBP (C-terminal-binding protein). ToRC facilitates ATP-dependent nucleosome assembly in vitro. All three subunits are required for its maximal biochemical activity. The toutatis gene exhibits strong synthetic lethal interactions with CtBP. Thus, ToRC mediates, at least in part, biological activities of CtBP and Toutatis. ToRC subunits colocalize in euchromatic arms of polytene chromosomes. Furthermore, nuclear localization and precise distribution of ToRC in chromosomes are dependent on CtBP. ToRC is involved in CtBP-mediated regulation of transcription by RNA polymerase II in vivo. For instance, both Toutatis and CtBP are required for repression of genes of a proneural gene cluster, achaete-scute complex (AS-C), in Drosophila larvae. Intriguingly, native C-terminally truncated Toutatis isoforms do not associate with CtBP and localize predominantly to the nucleolus. Thus, Toutatis forms two alternative complexes that have differential distribution and can participate in distinct aspects of nuclear DNA metabolism (Emelyanov, 2012).

Mounting genetic and molecular evidence suggests that Toutatis, the Drosophila ortholog of mammalian TIP5, plays important roles in the regulation of transcription by RNA polymerase II. In fact, tou was originally characterized as a suppressor of a Polycomb group gene polyhomeotic (Fauvarque, 2001). Furthermore, tou mutants exhibit wing defects, suggesting that Tou may be involved in the regulation of wing development (Emelyanov, 2012).

Data from a recent report (Liu, 2008) indicate that ISWI and CtBP colocalize at certain loci in the fly genome. For instance, a wingless target gene, hth, contains several sites where CtBP colocalizes with ISWI (Liu, 2008). Furthermore, hth is regulated by CtBP and ISWI in Kc cells and the developing wing (Liu, 2008). Thus, it is possible that Tou, ISWI, and CtBP may share genomic targets in vivo in Drosophila, where polymerase II-mediated transcription can be regulated by remodeling of chromatin structure by ToRC (Emelyanov, 2012).

The expression pattern of AS-C genes in the wing imaginal disc is maintained through antagonistic actions of transcriptional activators, such as GATA factor Pannier, and transcriptional repressors, such as zinc finger protein U-shaped, at the 5.7-kb DC enhancer element. Long-distance enhancer-promoter communications in AS-C are further facilitated by a LIM domain-binding protein, Chip. Tou has been previously shown to be involved in AS-C regulation (Vanolst, 2005). tou genetically interacts with pnr and Chip, and Tou protein may physically associate with Pannier and Chip. Accordingly, it has been proposed that tou, Iswi, pnr, and Chip cooperate to establish long-distance enhancer-promoter interactions in AS-C, possibly through chromatin remodeling (Vanolst, 2005). Consistent with biochemical and genetic interactions of Tou and CtBP reported in this study, loss-of-function mutants of CtBP exhibit similar genetic interactions with pnr and ac, as does tou, and CtBP physically interacts with Pannier and U-shaped (Emelyanov, 2012).

This study demonstrates that Tou and CtBP function as repressors of genes of AS-C in vivo. The data provide evidence that CtBP tethers Tou to regulatory regions of ac and sc and that Tou is required for the corepressor function of CtBP. In the future, it will be interesting to examine whether particular sequence-specific transcription factors such as U-shaped exhibit physical and genetic interactions with Tou. Furthermore, it will be important to understand how biochemical activities of ToRC mediate remodeling of nucleosome structure in AS-C and affect transcriptional regulation of ac and sc (Emelyanov, 2012).

In previous studies, it has been assumed that mammalian TIP5 is localized exclusively to the nucleolus, where it regulates transcription by RNA polymerase I (Nemeth, 2004). NoRC is localized to the nucleolus by a complex mechanism that involves direct physical interactions of TIP5 with short RNA originating from the intergenic spacer in the rRNA gene cluster (IGS transcripts) and with TTF-I, a general transcription factor of RNA polymerase I. Mammalian NoRC can also regulate replication of rDNA in cultured cells through mechanisms that involve histone and DNA methylation and IGS transcripts (Emelyanov, 2012).

Immunoprecipitation experiments indicate that in HeLa cells, human TIP5 associates with CtBP, which is recruited to and regulates genes that are transcribed by polymerase II. Thus, the existence of an alternative TIP5/Tou-containing complex (ToRC) is conserved in evolution from flies to mammals. Since NIH-3T3 cells exhibit exclusive nucleolus-specific IF staining with anti-TIP5 antibody, it is possible that these cells predominantly express the truncated form of TIP5 or limiting amounts of CtBP, which would largely abolish the formation of ToRC and tethering of TIP5 to sites outside of the nucleolus (Emelyanov, 2012).

This study has discovered that Tou/TIP5 is localized at multiple genomic sites, where it is tethered by a polymerase II-specific corepressor, CtBP. Based on molecular and genetic interaction data, the stable complex of Tou, ISWI, and CtBP mediates at least some aspects of the regulatory function of CtBP. In contrast, C-terminally truncated polypeptide isoforms of Tou fail to interact with CtBP and are instead recruited to nucleoli. Thus, depending on the primary structure and interaction partners, TIP5/Tou targets ATP-dependent nucleosome assembly/remodeling activity of ISWI to alternative genomic sites that undergo transcription by RNA polymerase I or II. In the future, it will be interesting to examine how the primary structure of Tou isoforms and, potentially, their association with CtBP affect the ability of Tou to interact with TTF-I and IGS transcripts and vice versa. These analyses will help to understand the differential distribution of NoRC/ToRC and the dual role of TIP5/Tou in regulation of transcription by RNA polymerases I and II (Emelyanov, 2012).

Loss of ISWI function in Drosophila nuclear bodies drives cytoplasmic redistribution of Drosophila TDP-43

Over the past decade, evidence has identified a link between protein aggregation, RNA biology, and a subset of degenerative diseases. An important feature of these disorders is the cytoplasmic or nuclear aggregation of RNA-binding proteins (RBPs). Redistribution of RBPs, such as the human TAR DNA-binding 43 protein (TDP-43) from the nucleus to cytoplasmic inclusions is a pathological feature of several diseases. Indeed, sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration share as hallmarks ubiquitin-positive inclusions. Recently, the wide spectrum of neurodegenerative diseases characterized by RBPs functions' alteration and loss was collectively named proteinopathies. This study shows that TBPH (TAR DNA-binding protein-43 homolog), the Drosophila ortholog of human TDP-43 TAR DNA-binding protein-43, interacts with the 'architectural RNA' (arcRNA) hsromega and with hsromega-associated hnRNPs. Additionally, it was found that the loss of the omega speckles remodeler ISWI (Imitation SWI) changes the TBPH sub-cellular localization to drive a TBPH cytoplasmic accumulation. These results, hence, identify TBPH as a new component of omega speckles and highlight a role of chromatin remodelers in hnRNPs nuclear compartmentalization (Lo Piccolo, 2018).

To confirm these interactions, a co-immunoprecipitation assay was conducted using anti-TBPH antibody starting from wild-type (WT) fresh larval nuclear extracts. Squid and Hrb87F are enriched in TBPH pulled-down fractions (Lo Piccolo, 2018).

Several in vitro experiments through proteomic studies and co-immunoprecipitation assay in HEK293 cells showed that in human cells TDP-43 interacts with the Drosophila orthologs of Squid and Hrb87F hnRNPs. This study confirmed these results in vivo, showing that in Drosophila tissue TBPH also interacts with Squid and Hrb87F hnRNP (Lo Piccolo, 2018).

Considering the co-localization between TBPH and Squid/Hrb87F hnRNPs, it was asked whether TBPH and hsrω could physically interact as well. To answer this question, an immunofluorescence and fluorescence RNA in situ hybridization was conducted. A physical interaction was observed between TBPH and the architectural RNA (arcRNA) hsrω in Malpighian tubules (MT) in vivo (Lo Piccolo, 2018).

As hnRNPs are known to shuttle between nucleus and cytoplasm, Western blot analysis was performed using a previously described method to produce in a single experiment nuclear and cytoplasmic protein fractions (NF and CF). These experiments were performed using MT and brain cells (BCs). Omega speckles are present in all the larval and adult Drosophila cell-type tissues but cells from Malpighian tubules were used for their large nuclear size, which allow a better understanding of nuclear bodies' distribution, as well as the eventual hsrω-interacting protein subcellular localization. BCs were characterized, as TBPH is largely expressed and has fundamental roles in the brain (Lo Piccolo, 2018).

The localization of TBPH in BCs is predominantly localized in the nucleus, but in BCs there is also a fraction of TBPH protein in the cytoplasm (Lo Piccolo, 2018b).

To rule out that the physical association observed between TBPH and hsrω was due to fortuitous interactions occurring during nuclear extract preparation, a cross-linking RNA-immuno-precipitation (CLIP-RIP) biochemical assay was performed using the anti-TBPH antibody on fixed larval nuclear extracts from brain cells. The CLIP-RIP data confirmed the specific interaction between TBPH and hsrω in the nuclear extract from the brain cells (+3.05-fold), compared to Rox1 (+0.77-fold) and U4 (+1.1-fold), two other abundant nuclear non-coding RNAs (Lo Piccolo, 2018).

It was thus demonstrated that TBPH, as previously reported for Hrb87F and Squid, is able to bind the hsrω RNA in vitro. Furthermore, using a gel shift assay employing an hsrω-n repeat unit (280b) transcribed in vitro and a full-length recombinant TBPH, it was shown that TBPH effectively retards hsrω RNA gel mobility. Finally, as seen for Hrb87F and Squid hnRNPs, the addition of ISWI protein in the reaction is a strong modulator of the interaction between TBPH-hsrω, changing the gel shift delay (Lo Piccolo, 2018).

In conclusion, these experiments confirmed the interaction of TBPH with hsrω arcRNA, Squid and Hrb87F hnRNPs in the omega speckles context. These results strongly suggest that, like Hrb87F and Squid, TBPH is another hnRNP belonging to the omega speckles complexes. Moreover, as shown for Squid and Hrb87F hnRNPs, ISWI function is essential for the modulation of TBPH/hsrω interaction (Lo Piccolo, 2018).

The chromatin remodeler ISWI is essential for a correct organization of the nucleoplasmic omega speckles (Oranati, 2011). Indeed, the organization and distribution of omega speckles are profoundly altered in ISWI null mutants when compared to wild-type cells. Omega speckles lose their dot shape and assume a trail shape distribution in the nucleus, suggesting a severe defect in their maturation or organization. Squid and Hrb87F hnRNPs also form trail-like structures in the nucleus of ISWI null mutants, showing that not only the distribution of the hsrω arcRNA, but also that of omega speckle-associated hnRNPs is compromised (Lo Piccolo, 2018).

Therefore, the distribution of TBPH protein was analyzed in ISWI null mutants to check if loss of ISWI could influence TBPH organization in omega speckles NBs as for Hrb87F and Squid hnRNPs. Remarkably, it was found that compared to wild-type cells, loss of ISWI function changes TBPH distribution in the context of omega speckles, inducing a dramatic alteration of TBPH sub-cellular localization. While in WT MT TBPH immunoreactive spots are nucleus limited, in ISWI null mutants' MT cytoplasmic TBPH-positive spots and trails were detected. Of note, these cytoplasmic spots show to be organized in different shapes, as indicated by arrows and arrowheads (Lo Piccolo, 2018).

These data were confirmed in vitro by Western blot of nuclear and cytoplasmic fractions in WT versus ISWI null mutant MT and BCs. In detail, this study showed that in ISWI null mutant the TBPH protein in MT disappears from the nucleus while moving to the cytoplasm, where the TBPH abundance increased compared to the WT NF (+2.37-fold). It was also observed a similar phenomenon in BCs where it was found that in ISWI null mutant TBPH disappears from the NF while its cytoplasmic fraction (CF) is not significantly changed compared to WT (Lo Piccolo, 2018).

Analyzing in detail the ventral ganglion of WT and ISWI null larvae, it was observed that the mean intensity of TBPH in motoneuron nuclei of ISWI null mutants is reduced compared to WT (Lo Piccolo, 2018).

Interestingly, loss of Squid in the Squid- null mutant also affects the cellular distribution of TBPH and causes its aberrant cytoplasmic localization in MT. While in WT MT TBPH immunoreactive spots are nucleus-limited, TBPH-positive cytoplasmic spots and trails were detected in MT of Squid null mutants. These data were confirmed in vitro by performing Western blots of nuclear and cytoplasmic fractions in WT versus Squid null mutant MT and BCs. In detail, it was shown that in Squid null mutant the TBPH protein in MT disappears from the nucleus while moving to the cytoplasm, where the TBPH abundance increased compared to the WT nuclear fraction (Lo Piccolo, 2018).

The human orthologs of Squid and Hrb87F proteins interact with TDP-43 to function cooperatively in RNA metabolism regulation. This study addressed whether TBPH interacts with Hrb87F and Squid in Drosophila cells as well. Double-immunofluorescence was conducted for TBPH/Hrb87F and TBPH/Squid and it was found that TBPH co-localizes in Malpighian tubules with Squid and Hrb87F hnRNPs in vivo (Lo Piccolo, 2018).

A similar phenomenon was observed in BCs where it was found that in Squid null mutant TBPH disappear from the NF while its cytoplasmic fraction is quite similar compared to the WT (Lo Piccolo, 2018).

Unlike Squid, Hrb87F does not affect TBPH subcellular distribution. To explain this result, the existence of a hierarchical order was hypothesized in omega speckles assembling, and it was speculated that Squid together with ISWI could be master regulators in the formation of physiologically functional hnRNP-hsrω complexes. In this case it could be hypothesized that the loss of Squid protein forces TBPH protein to escape the nucleus as a consequence of incorrect interaction among all omega speckle-associated hnRNPs (Lo Piccolo, 2018).

All these data collectively suggest that, in the Drosophila cells, the disorganization of omega speckles' compartments caused by loss of ISWI's role lead to a redistribution of TBPH protein from the nucleus to the cytoplasm. This could be a very important observation, considering that intracellular deposition of aggregated and ubiquitinated proteins are a prominent cyto-pathological feature of most neurodegenerative disorders frequently correlated with neural cell death (Lo Piccolo, 2018).

To explain all the results presented, it is hypothesized that loss of ISWI's function may indirectly affect TBPH distribution as a consequence of incorrect interaction among the omega speckle-associated hnRNPs and hsrω arcRNA. Indeed, while Squid and Hrb87F in ISWI null mutants are disorganized in their structure, but remain in the nucleus, TBPH seems to be more affected and to escape from the nucleus to the cytoplasm (Lo Piccolo, 2018).

For instance, the data reinforce the role of the chromatin remodeler ISWI in the modulation of the cellular localization of aggregation-prone proteins and show that the correct nuclear compartmentalization of TBPH hnRNP is dependent on nuclear body maintenance regulated by the chromatin remodeler. Finally, the data are in line with the recent findings showing that TDP-43-dependent reduction of the chromatin remodeler Chd1's recruitment to chromatin affects the induction of several key stress genes necessary to protect from diseases like ALS and FTD (Frontotemporal Dementia) (Lo Piccolo, 2018).

Developmental and housekeeping transcriptional programs in Drosophila require distinct chromatin remodelers

Gene transcription is a highly regulated process in all animals. In Drosophila, two major transcriptional programs, housekeeping and developmental, have promoters with distinct regulatory compatibilities and nucleosome organization. However, it remains unclear how the differences in chromatin structure relate to the distinct regulatory properties and which chromatin remodelers are required for these programs. Using rapid degradation of core remodeler subunits in Drosophila melanogaster S2 cells, this study demonstrates that developmental gene transcription requires SWI/SNF-type complexes, primarily to maintain distal enhancer accessibility. In contrast, wild-type-level housekeeping gene transcription requires the Iswi and Ino80 remodelers to maintain nucleosome positioning and phasing at promoters. These differential remodeler dependencies relate to different DNA-sequence-intrinsic nucleosome affinities, which favor a default ON state for housekeeping but a default OFF state for developmental gene transcription. Overall, these results demonstrate how different transcription-regulatory strategies are implemented by DNA sequence, chromatin structure, and remodeler activity (Hendy, 2022).

The differential transcription of protein-coding genes by RNA polymerase II (RNA Pol II) is a major determinant of cell types in multicellular organisms. In Drosophila, two major classes of genes, the ubiquitously expressed housekeeping genes and highly cell-type-specific developmental genes, have characteristically distinct promoters. These not only differ in the occurrence of core-promoter sequence elements such as the Ohler 1 motifs (Ohler, 2002) or the TATA box, respectively, but also display different regulatory properties and differentially respond to distinct enhancer types and cofactors. In addition to these differences in sequence and function, the chromatin structure is also noticeably different. Although promoters in general display characteristic nucleosome positioning and phasing, housekeeping promoters have a more stereotypical nucleosome arrangement with a well-defined nucleosome-free region (NFR) and a strongly positioned +1 nucleosome, whereas developmental promoters have a less pronounced NFR and +1 nucleosome, and much weaker downstream nucleosome phasing. Nucleosomes prevent DNA access of regulatory proteins such as transcription factors or the transcription machinery, but it remains unclear whether and how these differences in chromatin structure at promoters relate to the distinct regulatory properties of developmental versus housekeeping gene transcription (Hendy, 2022).

Chromatin remodelers facilitate nucleosome assembly, repositioning, and removal to shape chromatin structure and are therefore likely key factors for establishing the nucleosome environment of developmental and housekeeping genes. Remodelers have been classified into four sub-families that share the same core ATPase domain but differ in domain organization and complex composition: SWI/SNF, Iswi, Ino80, and Chd). In Drosophila, there are two SWI/SNF-type complexes, BAP and PBAP, which are orthologous to yeast SWI/SNF and mammalian BAF/PBAF. Both Drosophila SWI/SNF-type complexes share the ATPase Brahma (also called Brm) and core subunits such as Snr1 and mediate nucleosome removal to grant accessibility of activators to DNA but differ in some peripheral subunits. The Iswi ATPase is found in three remodeling complexes: NURF, ACF, and CHRAC. Iswi complexes can assemble histones onto DNA and slide nucleosomes to create NFRs as well as regularly spaced nucleosome arrays. Ino80 and Swr1 comprise the Ino80 family remodelers and have been shown to be important for exchange of the histone variant H2AZ at promoters. Finally, the Chd family remodelers are a family of at least 5 chromodomain containing remodelers that have diverse functions in transcription, replication, and DNA repair (Hendy, 2022).

In yeast, specialized roles for these remodelers have been well characterized through reconstitution and deletion/depletion experiments: SWI/SNF establishes DNA accessibility at the promoter and enhances activator binding, whereas other remodelers such as Iswi and Chd1 set the appropriate downstream nucleosome positioning and spacing. However, the direct roles of individual remodelers in multicellular organisms with complex regulatory landscapes and distinct promoter types have received less attention. In Drosophila, loss of these remodelers is associated with early developmental lethality, suggesting a critical role in early development or even basic cellular functions; however, the direct gene-regulatory changes underlying these defects cannot be determined from stable mutations. The potential for regulatory specialization of various essential regulators is supported by previous studies in humans and flies, which have shown that certain promoter and enhancer types can depend on different cofactors, including Mediator and/or P300. Additionally, recent studies have found that DNA binding of various factors, including CTCF and Oct4, differentially depend on the SWI/SNF and Iswi remodelers. However, whether such dependencies on chromatin remodelers affect nucleosome organization and transcription at different promoter types and how this relates to the promoters' regulatory properties have remained unclear (Hendy, 2022).

This study combined rapid inducible protein depletion with nascent transcriptomics to uncover the genome-wide transcriptional dependencies on the four main classes of chromatin remodelers in Drosophila S2 cells. This study demonstrates that the transcription of developmental genes specifically depends on the SWI/SNF-type complexes BAP/PBAP (hereafter referred to collectively as SWI/SNF) primarily because SWI/SNF is required to maintain the DNA accessibility of their enhancers. In contrast, housekeeping transcription is independent of SWI/SNF but modulated by Iswi and Ino80, which maintain these promoters' highly stereotypical nucleosome arrangement. The differences in chromatin structure and remodeler dependencies relate to distinct DNA-sequence-encoded nucleosome affinities, which are low at promoter-proximal housekeeping enhancers but high at promoter-distal developmental enhancers. Together, these results show how different chromatin structures and remodelers support the highly cell-type-specific versus broad nature of developmental versus housekeeping gene expression, respectively (Hendy, 2022).

The two main transcriptional programs of Drosophila driving expression of housekeeping and developmental genes, respectively, have fundamentally different promoter and enhancer types that differ in regulatory properties, characteristic sequence motifs, and chromatin structure. This study demonstrates that these programs differentially depend on different ATPase chromatin remodelers. Developmental gene transcription is shown to depend on SWI/SNF, whereas housekeeping gene transcription is largely remodeler independent but fine-tuned by Iswi and Ino80 (Hendy, 2022).

The apparent differences in remodeler dependencies of the developmental and housekeeping regulatory regions could result from these regions' different intrinsic nucleosome favorability. Distal developmental enhancers have a high affinity for nucleosomes and therefore require the action of SWI/SNF to create accessible DNA. Given that these regulatory regions control genes that are meant to be expressed in a highly cell-type-specific manner, this intrinsically inhibited, or 'default OFF,' state is appropriate. In contrast, housekeeping gene promoters and their enhancers, which often overlap the core promoter, have a low affinity for nucleosomes, which might explain their lack of dependence on SWI/SNF. This study only observed moderate effects on housekeeping transcription, and many genes were not significantly changed after depletion of the tested remodelers. The 'default ON' state of these promoters is appropriate, given that the downstream genes are meant to be broadly expressed. This limited effect may be due to remodeler redundancy or requirement of another chromatin remodeler that was not tested in this study. It cannot be ruled out that these sequences require remodeler activity to be opened initially but then are resistant to closing after acute depletion of a given remodeler. Alternatively, accessibility may be maintained independently of chromatin remodeler activity through a combination of the intrinsically low nucleosome affinity and the presence of many binding sites for sequence-specific DNA binders such as BEAF-32, Dref, and M1BP (Hendy, 2022).

In addition to nucleosome affinity based on DNA sequence, the results also reveal two mechanisms that explain the dependency of developmental genes on SWI/SNF. First, SWI/SNF preferentially localizes to developmental regulatory regions, particularly distal enhancers, consistent with the strong loss of enhancer accessibility after SWI/SNF depletion. Second, the observation that SWI/SNF is compatible with developmental but not housekeeping promoters also suggests a role of this remodeler at the promoter, consistent with its described in vitro function. The specific loss of accessibility at enhancers, but not promoters, may arise from greater robustness of promoters due to their low nucleosome affinity as well as the many factors that strongly bind this region, including transcription factors (TFs), general transcription factors (GTFs), and Pol II. The enhancer-specific loss of DNA accessibility is consistent with SWI/SNF's requirement in mammals: loss of core SWI/SNF subunits results in loss of enhancer accessibility and of active marks such as H3K27ac but few changes at promoters. The requirement of SWI/SNF to maintain accessibility of such distal regulatory regions and transcription factor binding sites is consistent with their high sequence intrinsic nucleosome favorability, pointing to a conserved regulatory role in metazoans. Interestingly, some promoter-distal regions such as CTCF-binding sites have been shown to be SWI/SNF independent but Iswi dependent, suggesting that remodelers are also used distinctly at different types of regulatory elements in mammals (Hendy, 2022).

Although the accessibility of housekeeping-type promoters was generally unperturbed after depletion of any of the remodelers, changes were observed in downstream nucleosome positioning that could explain the observed changes in transcription. Loss of Iswi caused +1 nucleosome encroachment on the promoter, whereas Ino80 had the opposite effect, and these effects correlated with changes in transcription. Although the effects on nucleosome positioning could be a consequence of the transcriptional changes (for example, in the absence of Ino80 increased passage of Pol II may shift the +1 nucleosome further downstream), the established roles of Iswi and Ino80 as chromatin remodelers make it more likely that Iswi and Ino80 depletion leads to changes in nucleosome positioning, which in turn affects transcription. Consistent with previous studies, the data support a model in which encroachment of the +1 nucleosome of the promoter may interfere with pre-initiation-complex binding through steric occlusion. In the same vein, movement of the +1 nucleosome away from the promoter may augment transcriptional output of the promoter by increasing accessibility and/or revealing previously inaccessible TSSs, a phenomenon also seen in yeast. Interestingly, some of the TSSs most affected by Iswi depletion were ribosomal protein genes, which have focused initiation patterns, an atypical feature of housekeeping promoters. It is speculated that the focused nature of these promoters makes them susceptible to +1 nucleosome encroachment, which could prevent initiation at the native site; in contrast, most housekeeping promoters have dispersed initiation patterns which could provide robustness against nucleosome encroachment. Similarly, Inr-containing promoters were downregulated after Ino80 depletion, suggesting that they might be sensitive to precise TSS to +1 nucleosome spacing for pre-initiation complex (PIC) assembly (Hendy, 2022).

In contrast to its role in flies, Iswi has been shown in yeast and mammals to act as a 'puller,' moving nucleosomes toward promoters and other boundaries; for example, in S. cerevisiae, Iswi counteracts opening of the promoter by RSC, and when depleted, it causes expansion of the nucleosome-free region (NFR) at the promoter. In addition, Iswi has been shown to be directly recruited by sequence-specific factors, such as RSC, in yeast. A recent study noted that Drosophila Iswi has lost critical acidic residues required for activator interaction, which may help explain the species-specific differences in chromatin positioning and accessibility after Iswi loss. The broad localization of Iswi seen in this study shows that it indeed may lack mechanisms of direct recruitment to specific cis-regulatory elements (Hendy, 2022).

Overall, the demonstration that distinct transcription-regulatory programs depend on different chromatin remodelers and are affected distinctly after remodeler depletion is intriguing. It illustrates how promoter DNA sequences and chromatin structure can evolve to adopt distinct default regulatory states that reflect the promoters' functions and shows that chromatin remodelers are both functionally specialized and differentially employed during gene regulation. The results also assign regulatory significance to the stereotypical nucleosome arrangement at housekeeping promoters, a decade-old observation whose functional implication has remained unclear. Together, these insights have important implications for understanding of broad and cell-type-specific gene-regulatory programs and their implementation at the DNA sequence and chromatin level in animals (Hendy, 2022).

This study has tested the hypothesis that the characteristically different chromatin structure at different types of promoters, particularly developmental versus housekeeping promoters, implies promoter-class-specific remodeler functions and dependencies. To achieve this, this study has selected representative remodelers for rapid AID and assessed direct transcriptional changes. Therefore, thie study has several limitations inherent to the experimental design (Hendy, 2022).

As expected for an approach based on protein depletion, depleting remodelers with partially or entirely redundant functions may not result in transcriptional changes. This could be redundancies within a remodeler family, which is presumably the case for Chd1, or due to compensation from other remodelers, as might be partially the case for Iswi at housekeeping promoters. These limitations stemming from remodeler redundancies could be overcome by the combined depletion of multiple redundantly acting proteins (Hendy, 2022).

Certain remodelers may also have other context-specific functions in other cell types where different transcription factors are expressed that could not be assess in this study. For example, Ino80 is known to act in concert with Polycomb during development, which may extend its regulatory role from the housekeeping role that was observe in a differentiated cell type (Hendy, 2022).

Given the aim and to avoid redundancies between different subcomplexes as much as possible, this study targeted core components (typically the catalytic subunits) shared between entire remodeler families rather than accessory subunits that are unique to one or some family members. This study therefore cannot assess differences between family members, and future work will be needed to determine the potentially more fine-grained regulatory roles within each remodeler family. For example, differences between the distinct Iswi containing complexes NURF, ACF, and CHRAC might explain how depletion of the Iswi ATPase resulted in downregulation of housekeeping promoters and upregulation of developmental promoters. Moreover, as this study attempted inactivate remodelers to study the direct transcriptional consequences, the approach could not assess more detailed aspects of remodeler complex assembly or disassembly beyond the stability of individual subunits (Hendy, 2022).

GENE STRUCTURE

Bases in 5' UTR - 326

Bases in 3' UTR - 309

PROTEIN STRUCTURE

Amino Acids - 1027

Structural Domains

The 1,027-residue Iswi protein contains the DNA-dependent ATPase domain characteristic of the SWI2 protein family but lacks the three other domains common to Brahma and SWI2, including the bromodomain. In contrast, the Iswi protein is highly related (70% identical) to the human hSNF2L protein over its entire length, suggesting that they may be functional homologs (Elfring, 1994).

The 140 kDa subunit of NURF has been identified as Iswi, highly related to yeast SWI2/SNF2. The relationship extends only to the ATPase domain, as Iswi lacks a bromodomain (Tsukiyama, 1995b).

Energy-dependent nucleosome remodeling emerges as a key process endowing chromatin with dynamic properties. However, the principles by which remodeling ATPases interact with their nucleosome substrate to alter histone-DNA interactions are only poorly understood. A substrate recognition domain has been identified in the C-terminal half of the remodeling ATPase Iswi and its structure has been determined by X-ray crystallography. The structure comprises three domains, a four-helix domain with a novel fold and two alpha-helical domains related to the modules of c-Myb, SANT and SLIDE, which are linked by a long helix. An integrated structural and functional analysis of these domains provides insight into how Iswi interacts with the nucleosomal substrate (Grüne, 2003).

Because Iswi-C can bind to nucleosomes, attempts were made to model such a complex using the available nucleosome structure. Since the SLIDE domain is most important for DNA binding, the structure of Iswi-C was modeled to bind a DNA duplex by superimposing SLIDE with c-Myb repeat R3 bound to DNA. Subsequently, this complex was positioned onto the nucleosomal DNA. Most potential DNA binding sites on the nucleosome are excluded because either the major groove of the nucleosomal DNA is inaccessible or because steric clashes occur between Iswi-C and the nucleosome. However, at seven different positions at the top and bottom of the nucleosomal disk, the major groove is exposed and Iswi-C can be positioned without steric clashes and without assuming any deviations from the crystal structure. At these positions, Iswi-C can be clamped onto the nucleosome with the central cylinder functioning as a molecular ruler of about 50 Å spanning the width of the nucleosomal disk. One jaw of the clamp is formed by the recognition helix of the SLIDE domain inserted into the major groove of DNA, and the other jaw is formed by the HAND domain protruding from the opposite side of the cylinder. In these models conserved basic residues of the SLIDE domain, the spacer helix, and the HAND domain would point toward the nucleosome, whereas the negatively charged SANT domain surface would point away from it (Grüne, 2003).

date revised: 5 August 2023

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