InteractiveFly: GeneBrief

TATA box binding protein-related factor 2: Biological Overview | References

Gene name - TATA box binding protein-related factor 2

Synonyms -

Cytological map position - 7E7-7E9

Function - general RNA polymerase II transcription factor

Keywords - RNA polymerase and general transcription factors, regulation the TATA-less promoters

Symbol - Trf2

FlyBase ID: FBgn0261793

Genetic map position - X: 8,317,598..8,324,233 [+]

Classification - TBP-like factor, ATPase containing von Willebrand factor type A domain

Cellular location - nuclear

NCBI links: EntrezGene

trf2 Orthologs: Biolitmine
Recent literature
Chowdhury, Z. S., Sato, K. and Yamamoto, D. (2017). The core-promoter factor TRF2 mediates a Fruitless action to masculinize neurobehavioral traits in Drosophila. Nat Commun 8(1): 1480. PubMed ID: 29133872
In fruit flies, the male-specific fruitless (fru) gene product FruBM plays a central role in establishing the neural circuitry for male courtship behavior by orchestrating the transcription of genes required for the male-type specification of individual neurons. This study identified the core promoter recognition factor gene Trf2 as a dominant modifier of fru actions. Trf2 knockdown in the sexually dimorphic mAL neurons leads to the loss of a male-specific neurite and a reduction in male courtship vigor. TRF2 forms a repressor complex with FruBM, strongly enhancing the repressor activity of FruBM at the promoter region of the robo1 gene, whose function is required for inhibiting the male-specific neurite formation. In females that lack FruBM, TRF2 stimulates robo1 transcription. These results suggest that TRF2 switches its own role from an activator to a repressor of transcription upon binding to FruBM, thereby enabling the ipsilateral neurite formation only in males.
Neves, A. and Eisenman, R. N. (2019). Distinct gene-selective roles for a network of core promoter factors in Drosophila neural stem cell identity. Biol Open 8(4). PubMed ID: 30948355
The transcriptional mechanisms that allow neural stem cells (NSC) to balance self-renewal with differentiation are not well understood. Employing an in vivo RNAi screen this study identified NSC-TAFs, a subset of nine TATA-binding protein associated factors (TAFs), as NSC identity genes in Drosophila. Depletion of NSC-TAFs results in decreased NSC clone size, reduced proliferation, defective cell polarity and increased hypersensitivity to cell cycle perturbation, without affecting NSC survival. Integrated gene expression and genomic binding analyses revealed that NSC-TAFs function with both TBP and TRF2, and that NSC-TAF-TBP and NSC-TAF-TRF2 shared target genes encode different subsets of transcription factors and RNA-binding proteins with established or emerging roles in NSC identity and brain development. Taken together, these results demonstrate that core promoter factors are selectively required for NSC identity in vivo by promoting cell cycle progression and NSC cell polarity. Because pathogenic variants in a subset of TAFs have all been linked to human neurological disorders, this work may stimulate and inform future animal models of TAF-linked neurological disorders.
Cherezov, R. O., Vorontsova, J. E. and Simonova, O. B. (2020). TBP-Related Factor 2 as a Trigger for Robertsonian Translocations and Speciation. Int J Mol Sci 21(22). PubMed ID: 33238614
Robertsonian (centric-fusion) translocation is the form of chromosomal translocation in which two long arms of acrocentric chromosomes are fused to form one metacentric. These translocations reduce the number of chromosomes while preserving existing genes and are considered to contribute to speciation. This study asked whether hypomorphic mutations in genes that disrupt the formation of pericentromeric regions could lead to centric fusion. TBP-related factor 2 (Trf2) encodes an alternative general transcription factor. A decrease of TRF2 expression disrupts the structure of the pericentromeric regions and prevents their association into chromocenter. This study revealed several centric fusions in two lines of Drosophila melanogaster with weak Trf2 alleles in genetic experiments. An RNAi-mediated knock-down of Trf2 was performed in Drosophila and S2 cells; Trf2 was shown to upregulate expression of D1-one of the major genes responsible for chromocenter formation and nuclear integrity in Drosophila. These data indicate that Trf2 may be involved in transcription program responsible for structuring of pericentromeric regions and may contribute to new karyotypes formation in particular by promoting centric fusion.

The 100 copies of tandemly arrayed Drosophila linker (H1) and core (H2A/B and H3/H4) histone gene cluster are coordinately regulated during the cell cycle. However, the molecular mechanisms that must allow differential transcription of linker versus core histones prevalent during development remain elusive. This study used fluorescence imaging, biochemistry, and genetics to show that TBP (TATA-box-binding protein)-related factor 2 (TRF2) selectively regulates the TATA-less Histone H1 gene promoter, while TBP/TFIID targets core histone transcription. Importantly, TRF2-depleted polytene chromosomes display severe chromosomal structural defects. This selective usage of TRF2 and TBP provides a novel mechanism to differentially direct transcription within the histone cluster. Moreover, genome-wide chromatin immunoprecipitation (ChIP)-on-chip analyses coupled with RNA interference (RNAi)-mediated functional studies revealed that TRF2 targets several classes of TATA-less promoters of >1000 genes including those driving transcription of essential chromatin organization and protein synthesis genes. These studies establish that TRF2 promoter recognition complexes play a significantly more central role in governing metazoan transcription than previously appreciated (Isogai, 2007).

Core promoters serve as the platform for the assembly of transcription initiation complexes critical for specifying accurate and regulated RNA synthesis. The eukaryotic cellular RNA polymerase II (Pol II) machinery has evolved to recognize multiple core-promoter elements such as the TATA box, Initiator, and DPE (Smale, 2003). Indeed, studies of metazoan core promoters revealed considerably greater cis-element diversification than previously expected. For example, TATA boxes, which were thought to be the most widely distributed prototypic core-promoter element recognized by the general transcription factor TBP (TATA-box-binding protein)/TFIID (consisting of TBP and TBP-associated factors, TAFs), are found in <20%-30% of annotated promoters in Drosophila and human. Instead, the majority of core promoters fall into various distinct TATA-less categories. Consistent with diversified core-promoter structures, recent studies identified a family of TBP-related factors (TRFs), but their potential core-promoter recognition functions have remained elusive (Isogai, 2007).

Metazoan cells have been found to use a diversified set of TBP-related molecules that display altered DNA-binding specificities (Hochheimer. 2003). In Drosophila, TRFs have been implicated in promoter-selective transcription for both Pol II and Pol III gene promoters. However, a comprehensive analysis of TRFs in promoter-selective recognition of Pol II core promoters has not been performed. Earlier studies found that a multisubunit TRF2-containing complex includes the transcription factor DREF and is involved in targeting a subset of promoters containing the DNA replication-related element (DRE) (Hochheimer, 2002). The PCNA gene promoter contains such a DRE (Hirose, 1993) and represents a novel tandem core-promoter class composed of two distinct transcriptional start sites, each of which appears to be subject to regulation either by the TRF2/DREF complex or TBP/TAFs. While TRF2 recruitment to the core promoter via DREF may account for a subset of TRF2-dependent promoters, TRF2 is also found in complexes lacking DREF. For example, TRF2 and DREF display only a limited set of overlapping sites in Drosophila Schneider cells visualized by immunofluorescence staining, suggesting that TRF2 may be playing multiple roles -- some in conjunction with DREF and others independent of DREF. It was therefore surmised that there may be additional important TRF2 target promoters that remained uncharacterized (Isogai, 2007).

In order to gain a more comprehensive map of potential TRF2-dependent promoters, a genome-wide analysis of TRF2 recognition sites was conducted both by polytene chromosome staining as well as chromatin immunoprecipitation (ChIP) coupled with high-density tiling microarray detection (ChIP-on-chip). These approaches have revealed several important target genes that illustrate how TRF2 is used as an alternative core-promoter recognition factor. First, biochemical and genetic evidence is provided that two distinct sets of core-promoter recognition factors are responsible for directing transcription of the nucleosome core histone genes (H2A/B and H3/H4) and the linker histone H1. Genome-wide ChIP-on-chip analysis revealed that TRF2 recognizes and binds in vivo to a large number of TATA-less core promoters. Importantly, a majority of these TATA-less promoters are selectively recognized by TRF2, but not by TBP. Moreover, with salivary gland-specific depletion of TRF2, it was found that TRF2 participates in regulation of chromatin organization and cell growth, by controlling Histone H1 and ribosomal protein gene expression. Taken together, these data establish that TRF2 is responsible for differentially recognizing and regulating a subset of TATA-less promoters that have shed the requirement for TBP through the usage of novel core-promoter structures. Remarkably, even coordinately expressed gene clusters such as the histone complex have evolved mechanisms to be differentially regulated by alternative core-promoter recognition machinery (Isogai, 2007).

In Drosophila, the five histone genes are found in a cluster that is tandemly amplified ~100 times. Despite the need to coordinate histone gene expression during the cell cycle, the ratio of linker and core histones can vary dramatically within each cell, among different tissues, and during embryonic development. This observation suggested that Histone H1 gene expression may be differentially regulated relative to the patterns of core histone gene transcription. A genome-wide survey of TRF2 target sites uncovered the finding that the histone gene cluster contains both TBP and TRF2 recognition sites. Most strikingly, these two core-promoter recognition factors are segregated within the histone cluster with TBP targeted to the core histone (H2A/B, H3, and H4) promoters, while TRF2 selectively directs transcription of the linker histone H1. This finding reveals a novel mechanism in which Histone H1 gene expression may be differentially regulated relative to the patterns of core histone gene transcription (Isogai, 2007).

The finding that a TRF2-containing preinitiation complex is responsible for Histone H1 expression while the prototypic TBP/TFIID complex directs transcription of the core histones suggests that the expression of the linker histone H1 and core histones must be uncoupled under certain circumstances, possibly in a developmental-specific and cell type-specific manner. The analysis of TRF2-depleted salivary gland polytene chromosomes suggests that this is indeed the case. Remarkably, the polytene chromosomes in TRF2-deficient cells exhibited severe defects in chromosome organization and structure reminiscent of the failure to form 30-nm fibers in H1-depleted chromatin. Given that the Drosophila genome encodes only one H1 subtype compared with five to six in mammals, it is interesting that the H1 knockdown via TRF2 depletion resulted in a severely altered chromatin structure, which represents another in vivo evidence that histone H1 is indeed linked to organization of chromatin structure. Importantly, these TRF2-depleted cells appear to specifically down-regulate Histone H1 mRNA while leaving core histone transcripts intact. These findings suggest that TRF2 must serve as a key component of the transcriptional initiation complex evolved to differentially control linker histone versus core histone expression (Isogai, 2007).

Transcription of nonpolyadenylated histone genes appears to be associated with a specific nuclear body, the histone locus body (HLB), through a physical coupling between the HLB and the histone gene cluster locus (Liu, 2006). The HLB is loaded with RNA synthesis and processing machinery, possibly serving as a "factory" for histone mRNA production. Thus, in order to rapidly produce histone transcripts during embryogenesis, Drosophila appears to have adapted an elegant strategy that involves tandemly amplified gene cassettes sequestered within a distinct nuclear address (the HLB). Interestingly, it appears that only specific subsets of transcription factors are deposited in the HLB. For example, among the three TBP paralogs in Drosophila (TBP, TRF1, and TRF2), only TRF2 and TBP that are used for linker and core histone transcription, in addition to Pol II, are 'preloaded' within the HLB, perhaps to facilitate rapid as well as differential linker versus core histone transcript production. Therefore, the histone gene cluster presents an important paradigm wherein a distinct nuclear body loaded with specific transcriptional as well as post-transcriptional machinery becomes dedicated to the purpose of coordinately and differentially regulating five essential genes (Isogai, 2007).

High-resolution genome mapping of TRF2 recognition sites using the ChIP-on-chip platform has revealed >1000 novel binding sites, with 80% distinct from and 20% overlapping with TBP-binding sites. These results suggest that the TRF2-dependent and TBP-independent Histone H1 promoter is not an exception. Indeed, the H1 case may represent a more general case for how TRF2 can serve as an alternative core-promoter recognition factor at many Pol II genes. A comprehensive and detailed sequence motif analysis of the Drosophila genome revealed that TRF2-bound promoters significantly lack TATA boxes, while the TATA box is tightly correlated with TBP-binding sites. Instead, TRF2 appears to selectively recognize promoters containing other distinct core-promoter elements such as Motif 1, DRE, and Motif 7 (Ohler, 2002). In addition, functional analysis of transcripts derived from TRF2-depleted salivary glands confirmed that TRF2 activity is indeed required for directing these TRF2 target promoters. Thus, the genome-wide analysis significantly strengthens the emerging picture that TRF2 likely evolved to recognize and regulate a large class of TATA-less core promoters (Isogai, 2007).

One question concerning TRF2 function in promoter recognition is whether TRF2, like TBP, can directly recognize and bind to a distinct core-promoter element. TRF2 is likely to possess very different DNA-binding specificities from TBP since the amino acid residues critical for TATA-box recognition have been altered in TRF2 (Dantonel, 1999; Ohbayashi, 1999; Rabenstein, 1999). However, all attempts to experimentally identify a direct TRF2-binding sequence have thus far failed. Similarly, the most recent computational efforts using TRF2 ChIP-on-chip data sets failed to identify any strong consensus core-promoter motifs comparable with the prototypic TATA box with its approximately minus 30-bp location relative to the start of transcription. Instead, motifs such as the DRE and other uncharacterized elements were identified with no set common position relative to the transcriptional start site. These findings are, however, consistent with previous studies (Hochheimer, 2002) in which TRF2 failed to bind the core promoter by itself. Instead, it appears that TRF2 recruitment to at least a subset of core promoters relies on specific interactions between TRF2 and various other sequence-specific DNA-binding proteins, such as DREF. However, unlike previous studies, the genome-wide survey of TRF2- and TBP-binding sites in Drosophila revealed a considerably more comprehensive picture of how TRF2 may be used as an alternative core-promoter recognition factor. Importantly, mixing and matching various enhancer-binding factors (i.e., sequence-specific DNA-binding factors) and alternative core-promoter recognition factors (i.e., TFIID vs. TRF2) appears to be a powerful and perhaps common strategy for metazoan organisms to diversify transcriptional outputs (Isogai, 2007).

The genome-wide ChIP-on-chip analysis also provides strong evidence that metazoan organisms make much more use of tandem core promoters containing both TFIID and TRF2 recognition sites than might have been anticipated. Whether or not this type of dual core-promoter structure represents a case of redundant pathways or is subject to selective and differential regulation of downstream targets remains unclear. Interestingly, two previously characterized TRF2 targets (PCNA and DNApolα180) appear unaffected when TRF2 is depleted in salivary glands, possibly due to the ability of such dual core promoters to use alternative transcription complexes. Thus, the possibility that TRF2 may be used in lieu of TBP/TFIID to diversify transcriptional outputs in response to specific signals cannot be ruled out. It would be of interest for future studies to determine how these two distinct core-promoter recognition factors TBP/TRF2 operating at dual tandem promoters may be coordinated. Are these core-promoter recognition complexes at tandem core promoters recruited by common or distinct activator proteins? Since salivary gland depletion of TRF2 protein resulted in developmental defects, TRF2 may be necessary to selectively up-regulate genes required for specific developmental pathways (Isogai, 2007).

The identification of direct TRF2 target genes in the present study has revealed a striking link between TRF2 and specific biological processes such as chromatin organization and protein synthesis. Since TRF2 is conserved among many metazoan organisms, its role in various model organisms has been of considerable interest. Several studies found that inactivating TRF2 in nematode, fly, fish, and frog all resulted in lethality due to a block in embryogenesis (Dantonel, 2000; Kaltenbach, 2000; Veenstra, 2000; Muller, 2001; Kopytova, 2006). In contrast, germ cell-specific functions of TRF2 have also been reported for Drosophila and mice (Martianov, 2001; Zhang, 2001; Kopytova, 2006). In particular, while TRF2-null mice appear to display a modest non-Mendelian ratio of inheritance, the major defect manifests as a lack of spermiogenesis (Martianov, 2001; Zhang, 2001). Although these studies revealed that TRF2 provides nonredundant functions during development, these genetic studies were unable to link TRF2 to selective core-promoter recognition functions in vivo. For instance, direct TRF2 target genes responsible for these previously observed phenotypes have not been identified or characterized. The identification of histone H1 and ribosomal proteins as key gene products misregulated in TRF2-depleted Drosophila organs not only provides candidate TRF2 target genes responsible for the chromatin defects observed in TRF2-depleted Drosophila germ cells (Kopytova, 2006), but also underscores the potential role of TRF2 in other organisms. For example, TRF2-null mice display a major defect in chromocenter formation in spermatids (Martianov, 2002). This suggests that, consistent with TRF2-mediated H1 regulation in Drosophila somatic cells, TRF2 may also target genes that are essential for chromatin structure in mammalian gonads. However, the precise molecular targets and mechanisms of TRF2 action may differ. Indeed, a recent report points to the involvement of a human DREF homolog in regulating transcription from a TATA-box-containing histone H1 promoter in human cells (Ohshima, 2003). In contrast, in Drosophila, it was found that the H1 gene is TATA-less and does not appear to be regulated by DREF (Isogai, 2007).

In addition, the finding that Drosophila TRF2 directs the expression of a large number of gene products critical for essential cell function such as growth (i.e., ribosomal subunits and histones) would be consistent with the lethality associated with the loss of TRF2 in most organisms. These findings also suggest that in mammals TRF2 may play an important role regulating essential cell functions in tissues other than testis. The biological context of TRF2 usage as an alternative core-promoter recognition factor may well be more universal than had been anticipated (Isogai, 2007).

A sequence-specific core promoter-binding transcription factor recruits TRF2 to coordinately transcribe ribosomal protein genes

Ribosomal protein (RP) genes must be coordinately expressed for proper assembly of the ribosome yet the mechanisms that control expression of RP genes in metazoans are poorly understood. Recently, TATA-binding protein-related factor 2 (TRF2) rather than the TATA-binding protein (TBP) was found to function in transcription of RP genes in Drosophila. Unlike TBP, TRF2 lacks sequence-specific DNA binding activity, so the mechanism by which TRF2 is recruited to promoters is unclear. This study shows that the transcription factor M1BP, which associates with the core promoter region, activates transcription of RP genes. Moreover, M1BP directly interacts with TRF2 to recruit it to the RP gene promoter. High resolution ChIP-exo was used to analyze in vivo the association of M1BP, TRF2 and TFIID subunit, TAF1. Despite recent work suggesting that TFIID does not associate with RP genes in Drosophila, it was found that TAF1 is present at RP gene promoters and that its interaction might also be directed by M1BP. Although M1BP associates with thousands of genes, its colocalization with TRF2 is largely restricted to RP genes, suggesting that this combination is key to coordinately regulating transcription of the majority of RP genes in Drosophila (Baumann, 2017).

This study shows that M1BP activates transcription of RP genes in Drosophila and that it can do so by recruiting TRF2 to RP gene promoters in cells. These conclusions are based on the demonstration that M1BP is detected in the core promoter region of the majority of RP genes in cells and that mutation of Motif 1 diminished the level of expression from RP reporter genes. Additionally, it was demonstrated that M1BP activates transcription of RP gene promoters in nuclear extracts. Also, M1BP was shown to recruit TRF2 to promoter DNA in vitro and that M1BP and TRF2 colocalize on the RP gene promoters in cells. M1BP, therefore, is the first sequence-specific DNA-binding protein that has been directly shown to activate RP gene transcription in metazoans. DREF is possibly the only other protein, but it remains to be determined if it activates RP genes in vitro. Since these transcription factors associate with a broad spectrum of genes, loss of function assays in cells must be viewed with caution as it is difficult to distinguish between direct and indirect effects regardless of whether the protein can be detected at a particular genez (Baumann, 2017).

Mechanisms by which TRF2 associates with promoters are not well understood. DREF was purified in a complex with TRF2 but no direct measurement of TRF2 recruitment to DNA by this complex was provided. An uncharacterized TRF2 complex associates with promoters bearing the DPE and canonical initiator, but RP genes lack both of these DNA elements. This study provides a direct mechanism that involves M1BP associating with its cognate-binding site and interacting directly with TRF2. Since there is little overlap between M1BP and TRF2 outside of RP gene promoters, it follows that additional cis-elements are required for TRF2's association with M1BP. It is suspected that the TCT motif, along with M1BP and DREF, may be an additional key contributor to TRF2's association with gene promoters. Additionally, ChIP-exo data reveals that TAF1 is present at virtually all promoters that are associated with Pol II including most promoters that associate with TRF2. Therefore, since TAF1 recognizes sequences at the initiator and the DPE, TAF1 could be part of the currently uncharacterized TRF2-containing complex that selectively binds the initiator-DPE-containing promoters (Baumann, 2017).

The total number of TRF2 peaks that were observed is considerably lower than that reported previously. There could be a couple reasons for this discrepancy. First, the previous study used 2~4 h embryos, whereas this study used S2R+ cells. It is possible that TRF2 functions at a broader spectrum of developmentally regulated genes in the early embryo than in S2R+ cells. Additionally, the difference could be due to the increased signal to noise ratio afforded by ChIP-exo which results in more reliable peak detection (Baumann, 2017).

Detection of TAF1 on RP gene promoters was unexpected because TAF1 is best known for being a subunit of the general transcription factor, TFIID and biochemical evidence argued against TFIID being involved in RP gene transcription. Moreover, previous analysis of the PCNA promoter showed that immunodepletion of TFIID with TAF1 antibody from a Drosophila transcription reaction did not inhibit transcription of a TRF2-dependent promoter. ChIP-exo data provide evidence for M1BP being in close proximity to and potentially interacting with, TRF2 and TAF1 on RP gene promoters. The ChIP-exo data showed a peak of M1BP contacts downstream from the TSS yet Motif 1 that binds M1BP typically resides upstream from the TSS. Since the ChIP-exo data for M1BP and TAF1 display overlapping peaks in the +30 to +50 region, it is proposed that M1BP is in contact with TAF1 and that the ChIP-exo signal for M1BP in this region is a consequence of M1BP crosslinking to TAF1 and TAF1 in turn crosslinking to the +30 to +50 region. In contrast, the ChIP exo signals for M1BP and TRF2 are shifted relative to each other by ∼10 nts suggesting that M1BP might position TRF2 on the DNA adjacent to M1BP (Baumann, 2017).

A unique feature of the RP gene promoters in Drosophila and humans is the presence of the TCT motif located at the TSS. What recognizes this motif is currently not known. Since TAF1 is known to recognize the canonical initiator element, its presence at RP gene promoters raises the possibility that this TAF also recognizes the TCT motif. DNAse I footprinting analysis of TFIID binding to RP gene promoters indicated that binding was extremely weak. However, close inspection of the DNase I cutting patterns in the absence and presence of TFIID reveals the appearance of weak hypersensitive cut sites near the TCT initiator. One possibility is that M1BP together with TRF2 enhance the affinity of TAF1 for the RP gene initiator (Baumann, 2017).

It is proposed that M1BP functions as a hub to recruit TRF2 and TAF1. Since the only known TAF1-containing complex in metazoans is TFIID, it is proposed that TFIID still binds to RP gene promoters along with TRF2. One possibility is that TRF2 displaces TBP at the RP gene promoter. A recent model of TFIID bound to promoter DNA indicates that TFIIA is involved in connecting TBP to TAF1. Since TRF2 associates with TFIIA , displacement of TBP from TAF1 by TRF2 is tenable (Baumann, 2017).

This analysis of STARR-seq data indicates that RP gene promoters can act as enhancers and that they are selective in activating housekeeping gene core promoters and not core promoters of developmental and stress activated genes. The RP gene promoters more strongly activated the candidate RP gene promoter over all the other tested candidates. This selectivity could establish a network in which active RP genes and other housekeeping genes act reciprocally to activate each other. In addition, the selectivity of the enhancer activity of these RP promoters would prevent them from inadvertently activating nearby developmentally regulated genes (Baumann, 2017).

Drosophila TRF2 and TAF9 regulate lipid droplet size and phospholipid fatty acid composition

The general transcription factor TBP (TATA-box binding protein) and its associated factors (TAFs) together form the TFIID complex, which directs transcription initiation. Through RNAi and mutant analysis, this study identified a specific TBP family protein, TRF2, and a set of TAFs that regulate lipid droplet (LD) size in the Drosophila larval fat body. Among the three Drosophila TBP genes, trf2, tbp and trf1, only loss of function of trf2 results in increased LD size. Moreover, TRF2 and TAF9 regulate fatty acid composition of several classes of phospholipids. Through RNA profiling, TRF2 and TAF9 were found to affect the transcription of a common set of genes, including peroxisomal fatty acid beta-oxidation-related genes that affect phospholipid fatty acid composition. Knockdown of several TRF2 and TAF9 target genes results in large LDs, a phenotype which is similar to that of trf2 mutants. Together, these findings provide new insights into the specific role of the general transcription machinery in lipid homeostasis (Fan, 2017).

This study reveals a rather specific role of TRF2 and TAFs, which are general transcription factors, in regulating LD size. In addition, TRF2 and TAF9 affect phospholipid fatty acid composition, most likely through ACOX genes which mediate peroxisomal fatty acid β-oxidation (Fan, 2017).

By binding to their responsive elements in target genes, specific transcription factors like SREBP (see Drosophila Srebp), PPARs and NHR49, play important roles in lipid metabolism. It is interesting to find that the general transcription machineries, in this case TRF2 and core TAFs, also exhibit specificity in regulating lipid metabolism. In the Drosophila late 3rd instar larval fat body, defects in trf2 cause increased LD size, whereas mutation of the other two homologous genes, tbp and trf1, have no obvious effects on lipid storage. Inactivation of taf genes causes a similar phenotype to trf2 mutation, suggesting that TRF2 may associate with these TAF proteins to direct transcription of specific target genes. Moreover, trf2 mutants have large LDs at both 2nd and early 3rd instar larval stages, suggesting that general transcription factors are also required at early developmental stages for LD size regulation. Interestingly, taf9 mutants have no obvious phenotype at these stages. It is possible that TAF9 may act as an accessory factor compared to promoter-binding TRF2. This is consistent with the fact that less genes are affected in taf9 mutants than trf2 mutants in RNA-seq analysis. It was also found that knockdown of trf2 in larval and adult fat body leads to different LD phenotype. This may be due to different lipid storage status or different LD size regulatory mechanisms between larval and adult stages (Fan, 2017).

The finding of this study adds to the growing evidence supporting a specific role of general transcription factors in lipid homeostasis. For example, knockdown of RNA Pol II subunits such as RpII140 and RpII33 leads to small and dispersed LDs in Drosophila S2 cells. Mutation in DNA polymerase δ (POLD1) leads to lipodystrophy with a progressive loss of subcutaneous fat. Furthermore, TAF8 and TAF7L were reported to be involved in adipocyte differentiation. Moreover, previous studies showed that several subunits of the Mediator complex interact with specific transcription factors and play important roles in lipid metabolism. Added together, these lines of evidence strongly support essential and specific roles of the core/basal transcriptional machinery components in lipid metabolism (Fan, 2017).

Using RNA-seq analysis, rescue experiments and ChIP-qPCR, identified several target genes regulated by TRF2 and TAF9. It is possible that other genes may regulate LD size but were missed in the RNA-seq analysis and RNAi screening assay because of either insufficient alterations in genes expression (lower than the twofold threshold) or low efficiency of RNAi. Among all the verified target genes of TRF2 and TAF9,CG10315, which strongly rescues the trf2G0071 mutant phenotype when overexpressed and encodes the eukaryotic translation initiation factor eIF2B-δ, may be a good candidate for further study. Although they are best known for their molecular functions in mRNA translation regulation, eIFs have been implicated in several other processes, including cancer and metabolism. For example, in yeast, eIF2B physically interacts with the VLCFA synthesis enzyme YBR159W. In adipocytes, eIF2α activity is correlated with the anti-lipolytic and adipogenesis inhibitory effects of the AMPK activator AICAR. In addition, given the evidence that some eIFs, such as eIF4G and eIF-4a, localize on LDsand knockdown of some eIFs, including eIF-1A, eIF-2β, eIF3ga, eIF3-S8 and eIF3-S9, results in large LDs in Drosophila S2 cells, it is important to further explore the specific mechanisms of these eIFs in LD size regulation (Fan, 2017).

Although TRF2 exists widely in metazoans and shares sequence homology in its core domain with TBP, it recognizes sequence elements distinct from the TATA-box. A previous study has investigated TRF2- and TBP-bound promoters throughout the Drosophila genome in S2 cells and revealed that some sequence elements, such as DRE, are strongly associated with TRF2 occupancy while the TATA-box is strongly associated with TBP occupancy (Isogai, 2007). This study also identified that DRE is significantly enriched in extended promoters of the 181 target genes. The distribution of TATA-boxes in the core promoters of the 181 target genes compared with all genes was further explored, and it was found that the TATA-box is not enriched in the core promoters of TRF2 target genes. The proportion of TATA-box is 0.155 (75 of 484 isoforms) for the 181 target genes while the proportion is 0.217 (7849 of 36099 isoforms) for all genes as the background. These results suggest that TRF2 and TAF9 may regulate the expression of a subset of genes by recognizing specific sequence elements such as DRE but not the TATA-box (Fan, 2017).

This study shows that expression of peroxisomal fatty acid β-oxidation pathway genes, including two acyl-CoA oxidase (ACOX) genes, CG4586 and CG9527, the β-ketoacyl-CoA thiolase gene CG9149, and the enoyl-CoA hydratase gene CG9577, is regulated by TRF2 and TAF9. Lipidomic analysis indicates that in the fat body of trf2 and taf9 RNAi, many phospholipids, such as PA, PC, PG and PI, contain more long chain fatty acids. Furthermore, knockdown of CG4586 and CG9527 in the fat body also causes similar changes.

These results coincide with the function of ACOX, which is implicated in the peroxisomal fatty acid β-oxidation pathway for catabolizing very long chain fatty acids and some long chain fatty acids. Similar to these findings, a previous study found that defective peroxisomal fatty acid β-oxidation resulted in enlarged LDs in C. elegans and blocked catabolism of LCFAs, such as vaccenic acid, which probably contributed to LD expansion in mutant worms. Since overexpressing CG4586 or CG9527 only marginally rescues the enlarged LD phenotype of trf2 mutants, it remains to be determined whether the increased level of long chain fatty acid-containing phospholipids contributes to LD size. Regarding the regulation of fatty acid chain length in phospholipids, a recent study reported that there was increased acyl chain length in phospholipids of lung squamous cell carcinoma accompanied by significant changes in the expression of fatty acid elongases (ELOVLs) compared to matched normal tissues. A functional screen followed by phospholipidomic analysis revealed that ELOVL6 is mainly responsible for phospholipid acyl chain elongation in cancer cells. The current findings provide new clues about the regulation of fatty acid chain length in phospholipids. ELOVL and the peroxisomal fatty acid β-oxidation pathway may represent two opposing regulators in determining fatty acid chain length in vivo (Fan, 2017).

Previous studies have shown that TRF2 is involved in specific biological processes including embryonic development, metamorphosis, germ cell differentiation and spermiogenesis. The current results reveal a novel function of TRF2 in the regulation of specialized transcriptional programs involved in LD size control and phospholipid fatty acid composition. Since TRF2 is conserved among metazoans, its role in the regulation of lipid metabolism may be of considerable relevance to various organisms including mammals. These findings may provide new insights into both the regulation of lipid metabolism and the physiological functions of TRF2 (Fan, 2017).

A heterochromatin-dependent transcription machinery drives piRNA expression

Nuclear small RNA pathways safeguard genome integrity by establishing transcription-repressing heterochromatin at transposable elements. This inevitably also targets the transposon-rich source loci of the small RNAs themselves. How small RNA source loci are efficiently transcribed while transposon promoters are potently silenced is not understood. This study show that, in Drosophila, transcription of PIWI-interacting RNA (piRNA) clusters-small RNA source loci in animal gonads-is enforced through RNA polymerase II pre-initiation complex formation within repressive heterochromatin. This is accomplished through Moonshiner, a paralogue of a basal transcription factor IIA (TFIIA) subunit, which is recruited to piRNA clusters via the heterochromatin protein-1 variant Rhino. Moonshiner triggers transcription initiation within piRNA clusters by recruiting the TATA-box binding protein (TBP)-related factor TRF2, an animal TFIID core variant. Thus, transcription of heterochromatic small RNA source loci relies on direct recruitment of the core transcriptional machinery to DNA via histone marks rather than sequence motifs, a concept that is a recurring theme in evolution (Andersen, 2017).

This study has identified a heterochromatin-dependent transcription machinery in Drosophila that allows piRNA precursor production despite potent silencing of transposon-encoded promoters and enhancers. Moonshiner-dependent transcription, which cannot rely on recognition of DNA motifs because of their inaccessibility in heterochromatin, achieves locus specificity through Rhino, an HP1 variant that binds H3K9me3 marks at piRNA clusters. Thereby the cell allows transcription of transposon-rich loci into piRNA precursors while transcription of the same loci into functional transposon mRNAs is suppressed via heterochromatin-mediated exclusion of sequence-specific transcription factors (Andersen, 2017).

Small RNA source loci embedded in heterochromatin and transcribed on both genomic strands are also a hallmark of genome defence pathways in plants and fungi. In fission yeast, a 'passive' mode of small RNA expression has been proposed, where Pol II transcribes small RNA precursors from pericentromeric regions during G1/S phases when heterochromatin is less condensed. In contrast, an active recruitment mode with conceptual similarities to the Moonshiner pathway occurs in plants. Here SHH1, a reader of H3K9me marks, recruits the plant-specific RNA polymerase IV to heterochromatin to transcribe small RNA precursors. Although SHH1 and Rhino both bind H3K9me residues, the two proteins are unrelated, suggesting that specification of small RNA source locus transcription via heterochromatin readers has evolved independently in animals and plants. Also in plants, small RNA precursor transcription initiates at 'YR' initiator sites dispersed on both genomic strands. Whether Moonshiner-mediated transcription, like that of plant Pol IV, depends on collaboration with nucleosome remodellers to access heterochromatic target loci is unclear. The reported interaction of TRF2 with the NURF chromatin remodelling complex supports this possibility. The recurring evolution of small RNA source locus transcription specified by chromatin marks rather than DNA sequence suggests that this constitutes a common alternative mode of transcriptional activation. The DNA inaccessibility of heterochromatin is thereby transformed into a specificity mark for non-canonical transcription activation. It is noted that the major Drosophila somatic piRNA cluster, flamenco, is transcribed from a single defined enhancer-driven promoter and avoids piRNA-mediated silencing because of the antisense orientation of the vast majority of the contained transposons. The production of plant siRNAs from Pol IV transcripts initiates a positive feedback loop: siRNA-mediated targeting leads to DNA methylation, which in turn increases H3K9 methylation, thereby bringing in SHH1 and Pol IV. In a similar fashion, production of Moonshiner-dependent piRNA precursors leads to generation of Piwi-bound piRNAs, which in turn guide H3K9 methylation and thereby Rhino recruitment. This explains how Piwi-mediated transcriptional silencing 'transforms' active transposon insertions into heterochromatic piRNA source loci with bidirectional transcription (Andersen, 2017).

Rhino and the associated factors Deadlock and Cutoff are required for transcription of dual-strand piRNA clusters. Owing to its ability to inhibit co-transcriptional processes such as splicing and transcription termination, Cutoff has been suggested to be the main effector of this complex. Such an inhibition of termination is supported by data on cluster38C1, where transcription from defined promoters results in 10-15 kb transcripts in a Rhino-, Deadlock-, and Cutoff-dependent manner. Cutoff also interacts with the transcription/export (TREX) complex, which orchestrates several co-transcriptional processes and which is required for transcription of Rhino-dependent piRNA source loci. Together with the identification of Moonshiner/TRF2 as piRNA cluster transcription initiation factors, this suggests that Rhino acts as a molecular hub for several effector proteins that stimulate different (co)-transcriptional processes. Though Rhino is not conserved outside drosophilids, data from mouse studies support a conserved role of TRF2 in transcription of germline heterochromatin. In summary, this study has uncovered the molecular mechanism by which heterochromatic piRNA loci are transcribed in Drosophila and propose that the identified coupling of chromatin readers to basal transcription factors is a recurring theme in eukaryotic heterochromatin biology (Andersen, 2017).

Functional Comparison of Short and Long Isoforms of the TRF2 Protein in Drosophila melanogaster

TRF2 protein (TBP-related factor 2) can substitute for TBP forming alternative transcription initiation complexes on TATA-less promoters, including the promoters of histone H1 and piRNA clusters required for transposon repression. The Drosophila trf2 gene codes for two isoforms: a 'short' and a 'long' one, in which the same short TRF2 sequence is preceded by a long N-terminal domain. This study demonstrates that the long TFR2 isoform has a greater functional activity than the short isoform by expressing each of them at a reduced rate under the endogenous promoters. Expression of the long isoform alone affects neither the flies' viability nor the sex ratio. Expression of the short isoform alone leads to the phenotype described for the trf2 gene insufficiency and derepression of transposable elements, that is, decreased viability, disturbance of homologous chromosome pairing and segregation, and apparent female-biased sex ratio (Osadchiy, 2019).

Adaptive evolution targets a piRNA precursor transcription network

In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are bound by the Rhino-Deadlock-Cutoff complex. The HP1 homolog Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription from both genomic strands. Cuff function is less well understood, but this Rai1 homolog shows hallmarks of adaptive evolution, which can remodel functional interactions within host defense systems. Supporting this hypothesis, Drosophila simulans Cutoff is a dominant-negative allele when expressed in Drosophila melanogaster, in which it traps Deadlock, TRF2, and the conserved transcriptional co-repressor CtBP in stable complexes. Cutoff functions with Rhino and Deadlock to drive non-canonical transcription. In contrast, CtBP suppresses canonical transcription of transposons and promoters flanking the major germline clusters, and canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution thus targets interactions among Cutoff, TRF2, and CtBP that balance canonical and non-canonical piRNA precursor transcription (Parhad, 2020).

Transposable elements (TEs) are major genome components that can induce mutations and facilitate ectopic recombination, but transposons have also been co-opted for normal cellular functions, and transposon mobilization has rewired transcriptional networks to drive evolution. Species survival may therefore depend on a balance of transposon silencing and activation. The Piwi interacting RNA (piRNA) pathway transcriptionally and post-transcriptionally silences transposons in the germline. However, how this pathway is regulated is not completely understood (Parhad, 2020).

In Drosophila, piRNAs are produced from heterochromatic clusters composed of complex arrays of nested transposon fragments, which appear to provide genetic memory of past genome invaders. Adaptation to new genome invaders is proposed to involve transposition into a cluster, which leads to sequence incorporation into precursors that are processed into trans-silencing anti-sense piRNAs. However, a subset of isolated transposon insertions also produce sense and anti-sense piRNAs, providing an independent adaptation mechanism and epigenetic memory of genome invaders. Expression of piRNAs from these loci is disrupted by piwi mutations, but Piwi-bound piRNAs map to all insertions, not just the subset that function in piRNA biogenesis. The mechanism that defines these 'mini-cluster' thus remains to be determined (Parhad, 2020).

In Drosophila, germline piRNA clusters and transposon mini-clusters are bound by the RDC complex, which is composed of the HP1 homolog Rhino (Rhi), which co-localizes with the linker protein Deadlock (Del) and the Rai1 homolog Cutoff (Cuff). The three components of the RDC are co-dependent for localization to clusters and essential to germline piRNA production. Rhi is composed of chromo, hinge, and shadow domains. The chromo domain binds to H3K9me3-modified histones, and the shadow domain binds Del, which recruits Moonshiner (Moon) and TATA box binding protein-related factor 2 (TRF2), promoting 'non-canonical' transcription from both genomic strands (Parhad, 2020).

The third RDC component, Cuff, was identified in a screen for female sterile mutations and found to encode a homolog of the decapping exonuclease Rai1 required for transposon silencing and piRNA biogenesis. Critical residues in the catalytic pocket of Rai1 are not conserved in Cuff, but sidechains that bind the RNA backbone are retained, suggesting that Cuff may be an RNA 5' end-binding protein. Intriguingly, germline piRNAs in Drosophila are preferentially produced from unspliced transcripts, and cuff mutations significantly increase piRNA precursor splicing, and 5' cap binding by the nuclear cap binding complex (CBC) promotes splicing. Together, these findings suggest that that Cuff competes with the CBC for binding to capped cluster transcripts, suppressing splicing and promoting piRNA biogenesis. However, tethering Cuff to a reporter transcript increases read-through transcription, consistent with suppression of transcription termination. The molecular function for Cuff in piRNA biogenesis thus remains enigmatic (Parhad, 2020).

All three RDC genes are rapidly evolving under positive selection, suggesting that adaptive evolution of the complex is driven by a genetic conflict with the transposons the piRNA pathway silences, but other mechanisms are possible. Previous work found that rapid evolution has modified the Rhi-Del interface, producing orthologs that function as mutant alleles when moved across species. Analysis of these cross-species incompatibilities defined an interaction between the Rhi shadow domain and Del that prevents ectopic assembly of piRNA cluster chromatin. Crosses between Drosophila melanogaster and Drosophila simulans, which are sibling species, lead to hybrid sterility and are important model for genetic control of reproductive isolation. Significantly, sterile hybrids between these species phenocopy piRNA pathway mutations. Adaptive evolution of piRNA pathway genes may therefore contribute to reproductive isolation and speciation (Parhad, 2020).

These findings also suggest that cross-species analysis of rapidly evolving genes may provide a powerful genetic approach to structure-function analysis. This study applies this approach to cuff. These studies indicate that adaptive evolution has targeted direct or indirect interactions among Cuff, the Del-TRF2 non-canonical transcriptional complex, and the transcriptional co-repressor C-terminal binding protein (CtBP). CtBP was first identified as a host binding partner of Adenovirus E1A and subsequently implicated in diverse developmental pathways and cancer. This study shows that Drosophila CtBP suppresses canonical transcription from promoters in transposon terminal repeats and from promoters flanking two major germline piRNA clusters. Significantly, in both contexts, activation of canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution has therefore targeted interactions between Cuff and two transcription regulators, which coordinately control germline piRNA expression (Parhad, 2020).

Adaptive evolution is a hallmark of genes engaged in a genetic conflict, which typically leads to co-evolution of host-pathogen gene pairs that encode interacting proteins. However, pathogens can also produce mimics that target interactions within host defense systems, raising the possibility that adaptation can also remodel interaction between host proteins. Supporting the possibility, adaptive evolution has remodeled an interface between the Rhi and Del, which are core components of the host transposon defense machinery. These adaptive changes prevent gene function across closely relates species and define an interaction that is required to restrict the RDC to piRNA clusters, which defines the specificity of the transposon silencing machinery. These findings suggest that adaptive evolution targets important functional domains, which can be functionally analyzed using cross-species complementation. This approach is applied to the third RDC component, cuff and shows that adaptive evolution targets interactions between this Rai1 homolog and proteins that coordinate canonical and non-canonical piRNA cluster transcription and piRNA biogenesis (Parhad, 2020).

Transposon silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, and Cuff co-localizes with Rhi and Del at these piRNA source loci. Rhi binds to H3K9me3 marks and recruits Del. Del, in turn, binds Moon, which recruits TRF2 to initiate non-canonical transcription from both genomic strands. In contrast, the current data suggest that Cuff coordinates canonical and non-canonical cluster expression. D. simulans cuff ortholog fails to rescue D. melanogaster cuff mutations and leads to dominant sterility when overexpressed in wild-type flies. Significantly, these phenotypes are associated with stable binding to Del, TRF2, and CtBP. As noted above, Del and TRF2 function in non-canonical transcription of piRNA clusters. CtBP is a conserved transcriptional co-repressor, first identified as a host factor that binds to Adenovirus E1a, and subsequently shown to function in numerous developmental pathways. CtBP does not directly interact with DNA, but binds sequence specific transcription factors and recruits histone-modifying enzymes. This study shows that CtBP-kd activates canonical promoters linked to piRNA source loci. Adaptive evolution has therefore remodeled interactions between Cuff and factors that control both canonical and non-canonical transcription of piRNA precursors loci (Parhad, 2020).

Dominant phenotypes can result from mutations that produce new interactions or functions (neomorphic mutations) and assembly of complexes that are not formed by wild-type proteins. However, the current findings, with previous studies, suggest that substitutions in sim-Cuff stabilize normally transient complexes with both TRF2 and CtBP. In D. melanogaster, Cuff and Del do not co-precipitate, but the proteins co-localize to nuclear foci, interact in two-hybrid assays, and are co-dependent for association with piRNA clusters (Mohn, 2014). Del, in turn, co-precipitates with TRF2 and Moon, and all three proteins are required for non-canonical cluster transcription (Andersen, 2017), but TRF2 does not normally accumulate at clusters. In contrast, overexpression of sim-Cuff drives TRF2 co-localization with the RDC. Similarly, ChIP-seq shows that Cuff and Del localize to canonical promoters that are suppressed by CtBP, but CtBP does not accumulate at these promoters. Substitutions in the sim-Cuff ortholog thus appear to stabilize normally transient associations with Del and TRF2 and with CtBP (Parhad, 2020).

The majority of Drosophila germline clusters are transcribed from internal non-canonical initiation sites and do not have flanking canonical promoters. CtBP-kd does not significantly alter long RNA or piRNA expression from these loci. However, canonical promoters flank the right side of the 42AB cluster and both ends of the 38C cluster, and CtBP-kd increases transcription from these canonical promoters, which is associated with reduced transcription and piRNA production from downstream regions]. It has not been possible directly assay non-canonical transcription at most transposon insertions that produce piRNAs, as the inserted sequences are repeated, but CtBP-kd increases canonical transcription of transposons and is linked to collapse of piRNAs mapping to sequences flanking these insertions. In addition, deletion of the promoters flanking 42AB and 38C leads to spreading of piRNA production into flanking domains (Andersen, 2017). Together, these findings indicate that canonical transcription directly or indirectly represses non-canonical transcription and piRNA production (Parhad, 2020).

On the basis of these findings, it is proposed that Cuff coordinates canonical and non-canonical piRNA precursor transcription. By stabilizing Rhi, Del, Moon and TRF2, Cuff promotes non-canonical transcription. By contrast, Cuff appears to function with CtBP to control canonical transcription. Rescue of cuff mutants with sim-Cuff, which shows enhanced binding to CtBP, is phenocopied by CtBP-kd: both lead to increased canonical transcription. Formation of stable complexes with sim-Cuff thus appears to inhibit CtBP, activating canonical transcription and reducing downstream non-canonical transcription. Normally, the interaction between Cuff and CtBP is weak and free CtBP suppresses canonical promoters, while Cuff functions with Del-TRF2 to drive of non-canonical transcription. It is speculated that this balance may be altered in response to stress or environmental signals, which can activate transposons. Intriguingly, CtBP is also an NADH/NAD binding protein, suggesting that the balance between canonical and non-canonical piRNA precursor transcriptions may be regulated in response to metabolic state (Parhad, 2020).

The RDC proteins Moon and TRF2 are required for piRNA precursor transcription, and all of these factors are rapidly evolving. By contrast, CtBP is conserved from flies to humans, and a putative human oncogene. The data presented here, with earlier analysis of Rhi and Del (Parhad, 2017), indicate that rapid evolution has modified multiple interactions between rapidly evolving proteins in the piRNA biogenesis, and association of these proteins with a highly conserved transcriptional co-repressor. Rapidly evolving genes with specialized functions are frequently the most accessible to phenotype-based forward genetic approaches in model systems, and linking these specialized genes to conserved pathways can be a challenge. The studies reported in this study indicate that cross-species studies can help define these links, bridging the gap between genetically tractable model organisms and human biology (Parhad, 2020).

Expression of the core promoter factors TBP and TRF2 in Drosophila germ cells and their distinct functions in germline development

In Drosophila, the expression of germline genes is initiated in primordial germ cells (PGCs) and its expression is known to be associated with germline establishment. However, the transcriptional regulation of germline genes remains elusive. Previous studies found that the BTB/POZ-Zn-finger protein, Mamo, is necessary for the expression of the germline gene, vasa, in PGCs. Moreover, the truncated Mamo lacking the BTB/POZ domain (MamoAF) is a potent vasa activator. This study investigated the genetic interaction between MamoAF and specific transcriptional regulators to gain insight into the transcriptional regulation of germline development. A general transcription factor, TATA box binding protein (TBP)-associated factor 3 (TAF3/BIP2), and a member of the TBP-like proteins, TBP-related factor 2 (TRF2), were found as new genetic modifiers of MamoAF. In contrast to TRF2, TBP was found to show no genetic interaction with MamoAF, suggesting that Trf2 has a selective function. Therefore, this study focused on Trf2 expression and investigated its function in germ cells. The Trf2 mRNA, rather than the Tbp mRNA, was found to be preferentially expressed in PGCs during embryogenesis. The depletion of TRF2 in PGCs resulted in decreased mRNA expression of vasa. RNA interference-mediated knockdown showed that while Trf2 is required for the maintenance of germ cells, Tbp is needed for their differentiation during oogenesis. Therefore, these results suggest that Trf2 and Tbp expression is differentially regulated in germ cells, and that these factors have distinct functions in Drosophila germline development (Nakamura, 2020).

During early embryogenesis in animals, primordial germ cells (PGCs) are specified, and the expression of germline genes is initiated in these cells. The vasa (vas) gene encodes a highly conserved DEAD-box RNA helicase, and its expression is a marker of germline establishment in many animal species. Additionally, recent transcriptome analyses have revealed more novel germline genes that are preferentially expressed in PGCs. Expression of germline genes implies that a network of transcriptional regulators exists in these germline cells. However, the underlying mechanism of transcriptional regulators that control germline gene expression remains elusive (Nakamura, 2020).

In Drosophila, maternal factors localized in the germ plasm are necessary for germ cell establishment. It was assumed that the regulation of germline gene expression requires the presence of maternal transcriptional regulators in the germ plasm. However, RNA interference (RNAi)-mediated knockdown experiments of germ plasm-enriched maternal mRNAs have demonstrated that several transcriptional regulators are required for the expression of germline genes in PGCs. Among these, the transcriptional activator, OvoB, is predominantly expressed in PGCs, and its function is essential for germline development (Nakamura, 2020).

Previous work has shown that Mamo, a PGC-enriched maternal factor, is necessary for the expression of vas in PGCs (Mukai, 2007). Mamo protein has been identified as a zinc-finger protein that contains both a BTB/POZ domain and C2H2 Zn-finger domains (Mukai, 2007). Subsequent biochemical analyses demonstrated that the C2H2 Zn-finger domains of Mamo directly bind to a specific DNA sequence in the first intron of the vas gene. Overexpression of N-terminal truncated Mamo (MamoAF) lacking the BTB/POZ domain, but having the C2H2 Zn-finger domains, strongly promotes vas expression. Furthermore, Mamo mRNA encoding a truncated Mamo isoform, which is similar to MamoAF, is predominantly expressed in PGCs (Nakamura, 2019). Therefore, these results suggest that the short Mamo lacking the BTB/POZ domain is a potent vas activator. Moreover, this study found that MamoAF collaborates with both an epigenetic regulator, CREB-binding protein (CBP), and the germline transcriptional activator, OvoB, to activate vas expression (Nakamura, 2019). These observations imply that MamoAF acts as a hub molecule to interact with transcriptional regulators (Nakamura, 2020).

Recently, core promoter factors and their homologs have been found to selectively regulate gene expression and control specific developmental processes. The TATA box-binding protein (TBP), which is a subunit of the TFIID complex, recognizes core promoters containing the TATA box to regulate gene expression (Haberle, 2018). TBP-associated factor 3 (TAF3/BIP2) is a general transcription factor, but exhibits selective physical interactions with transcriptional regulators such as Antennapedia, BAB1, and BAB2. TBP-related factor 2 (TRF2), a member of the TBP-like proteins, selectively regulates the expression of a subset of genes that differ from those regulated by TBP. TRF2 interacts with the transcriptional regulator, Fruitless, to masculinize neurobehavioral traits in Drosophila. Therefore, core promoter factors may mediate a new layer of transcriptional regulation that controls specific developmental processes (Nakamura, 2020).

In this study, a genetic experiment was conducted to identify the cofactors of MamoAF to provide insights into the role of transcriptional regulators in germ cells. The Drosophila compound eye is a precise structure that sensitively reflects genetic interactions. Genetic modifier screening using the compound eyes has been performed to identify cofactors for many genes. Genetic modifier screening has also succeeded in identifying the cofactors that play a role in germ cells. Therefore, a genetic experiment was conducted on compound eyes overexpressing MamoAF to identify the genetic modifiers of MamoAF. The core promoter factors, TAF3/BIP2 and TRF2, were identified as the genetic modifiers of MamoAF. Trf2 mRNA, rather than Tbp mRNA, was preferentially expressed in PGCs. Although Trf2 is required for maintenance of germ cells, Tbp is necessary for their differentiation during oogenesis. Therefore, these results suggest that the expression of Trf2 and Tbp is differentially regulated in germ cells and these factors have distinct functions in germline development in Drosophila (Nakamura, 2020).

Previous transcriptome analyses revealed that the genes encoding these Mamo interactors are expressed in the adult heads or the cell line derived from eye-antenna discs. Recently, Mamo has also been shown to play a role in neuronal fate specification and maintenance during brain development. Therefore, the transcriptional regulators that collaborate with Mamo can be expressed in neural cells. Moreover, genetic screening using adult compound eyes may be useful for the identification of Mamo interactors that act on germ cells (Nakamura, 2020).

As several regulators of histone modifications are essential for germline development, this study found that both CBP and HDAC1 preferentially interacted with MamoAF. Additionally, since CBP is responsible for H3K27ac and HDAC1 is required for the deacetylation of H3K27ac, it can be said that proper regulation of H3K27ac levels may play an essential role in germline development. Therefore, future studies will focus on identifying the target genes that are epigenetically regulated by H3K27ac in germ cells (Nakamura, 2020).

Some TFIID subunits vary with target genes and cell types. The Drosophila TAF-TRF2 complex has been proposed to perform distinct functions in regulating neural stem cell identity (Neves, 2019). Moreover, previous biochemical studies have shown that both TAF3/BIP2 and TRF2 interact with TAF6 (Weake, 2009). This study also found that MamoAF preferentially interacted with both Taf3 and Trf2. Therefore, future studies investigating the physical interaction between Mamo and these general transcription factors will provide insights into the mechanism through which Mamo collaborates with the complex containing both TAF3/BIP2 and TRF2 (Nakamura, 2020).

Core promoter factors are considered to be universally required in all eukaryotic cells. However, recent studies suggested that some core promoter factors are developmentally regulated. This study showed that Trf2 and Tbp expression was differentially regulated in germ cells. A previous report showed that TBP protein is highly stable in insect TN-368 cells. Consistent with this report, TBP protein appeared to be stable in these experiments, as it was detected in PGCs and germline cysts where Tbp mRNA was not detected. Therefore, the stability of TBP protein may ensure the robustness of transcriptional regulation. In contrast, Trf2 mRNA was continuously expressed in germline cells, and TRF2 protein in PGCs was found to be sensitive to Trf2-RNAi. Therefore, as compared with Tbp, Trf2 activity can be transcriptionally regulated in germline cells (Nakamura, 2020).

Trf2 may regulate the expression of target genes that differ from those of Tbp during germline development. TRF2, rather than TBP, specifically mediates the transcription of ribosomal protein genes. TRF2 selectively regulates the TATA-less histone H1 gene promoter. Moreover, TRF2 is required for piRNA expression. Combined with the current data, TRF2 may selectively support the transcription of target genes in order to maintain germline cells. This study also found that the Trf2 RNAi (BL64561)-mediated knockdown affected the formation of egg chambers. Therefore, the expression of the genes that control germ cell differentiation may be regulated by TRF2 (Nakamura, 2020).

Drosophila Trf2 encodes TRF2S and TRF2L. TRF2S is conserved between Drosophila and mammals because TRF2S appears to be more closely related to the human TRF2 protein, which lacks the long N-terminal extension present in TRF2L. TRF2S is known to act on the DNA replication-related element, downstream promoter element (DPE), and TCT motifs. In this study, Trf2 activity was found to be required for vas expression in PGCs. The vas gene contains a DPE-like motif (+25 to +29) near the transcription start site. Whether TRF2S acts on this DPE motif is unclear, but it supports the idea that TRF2S is involved in the transcriptional regulation of vas. In contrast to Trf2 RNAi (V10443), the overexpression of TbpRNAi did not decrease vas expression. However, as it was not possible to knock down TBP in PGCs, it could not be definitively excluded that TBP mediates the transcription of vas. TBP binds to the TATA box in promoters to regulate TATA-dependent transcription. However, the vas promoter does not appear to contain a TATA box near the transcription start site. Therefore, it is concluded that vas transcription is regulated by TRF2S rather than by TBP. TRF2L may also regulate vas transcription because TRF2L can activate both DPE-dependent and TATA-dependent promoters (Nakamura, 2020).

Previous studies using a weak Trf2 allele, polyhomeoticP1 Trf2P1, in which TRF2 expression in germ cells is decreased but TRF2 is not depleted from germ cells, have shown that Trf2 is required for germ cell differentiation and that TRF2L and TRF2S can rescue the mutant phenotypes individually. This shows that TRF2S and TRF2L have redundant functions in germ cell differentiation. This study also showed that Trf2 RNAi (V10443)-mediated knockdown resulted in an agametic phenotype and that the overexpression of Trf2L rescued the agametic phenotype, thereby suggesting that germ cell maintenance may require a TRF2L function. The Trf2L cDNA used in the rescue experiment may produce both TRF2S and TRF2L because of the presence of the IRES sequence in the Trf2L cDNA. However, this study found that Trf2S expression alone did not rescue the agametic phenotype induced by Trf2 RNAi, thereby confirming that TRF2L is required for germ cell maintenance (Nakamura, 2020).

Previous reporter assays have shown that TRF2S preferentially activates DPE-dependent promoters, whereas TRF2L activates both DPE-dependent and TATA-dependent promoters TRF2L may regulate the expression of distinct target genes to maintain germ cells. Previous biochemical studies have shown that both TRF2S and TRF2L are present in the same protein complex that contains ISWI ATPase. However, some TRF2L proteins exhibit different chromatographic properties from those of TRF2S, thereby suggesting that TRF2L cofactors may differ from those of TRF2S. Moreover, the coiled-coil domains in the long N-terminal domains of TRF2L may mediate their interaction. Therefore, future studies on the identification of TRF2L target genes and TRF2L cofactors may provide insights into the mechanisms through which TRF2L regulates germ cell maintenance (Nakamura, 2020).

The core promoter factors that mediate a new layer of transcriptional regulation may be involved in germline development in Drosophila. Moreover, the homologs of core promoter factors have been found to selectively regulate transcriptional programs and control specific developmental processes in Drosophila and mouse models. This study found that TRF2 and TBP had distinct functions in germline development. As Trf2 is conserved in bilaterian organisms and is required for spermiogenesis in mice, it is speculated that Trf2 might play a role in germline development in other animals as well. Hence, it is expected that the current results will facilitate the understanding of the transcriptional regulation network that controls germline development in animals (Nakamura, 2020).

Drosophila TRF2 is a preferential core promoter regulator

Transcription of protein-coding genes is highly dependent on the RNA polymerase II core promoter. Core promoters, generally defined as the regions that direct transcription initiation, consist of functional core promoter motifs (such as the TATA-box, initiator [Inr], and downstream core promoter element [DPE]) that confer specific properties to the core promoter. The known basal transcription factors that support TATA-dependent transcription are insufficient for in vitro transcription of DPE-dependent promoters. In search of a transcription factor that supports DPE-dependent transcription, a biochemical complementation approach was used to identify the Drosophila TBP (TATA-box-binding protein)-related factor 2 (TRF2) as an enriched factor in the fractions that support DPE-dependent transcription. The short TRF2 isoform was demonstrated to preferentially activate DPE-dependent promoters. DNA microarray analysis reveals the enrichment of DPE promoters among short TRF2 upregulated genes. Using primer extension analysis and reporter assays, the importance of the DPE in transcriptional regulation of TRF2 target genes was demonstrated. It has been shown that, unlike TBP, TRF2 fails to bind DNA containing TATA-boxes. Using microfluidic affinity analysis, short TRF2-bound DNA oligos were found to be enriched for Inr and DPE motifs. Taken together, these findings highlight the role of short TRF2 as a preferential core promoter regulator (Kedmi, 2014).

Transcription of protein-coding genes is highly dependent on the RNA polymerase II (Pol II) core promoter. Core promoters, generally defined as the regions that direct transcription initiation, span from -40 to +40 relative to the +1 transcription start site (TSS). Core promoters contain functional subregions (termed core promoter elements or motifs, such as the TATA-box, TFIIB recognition elements [BREu and BREd], initiator [Inr], TCT motif, motif 10 element [MTE], and downstream core promoter element [DPE]) that confer specific properties to the core promoter. A synthetic core promoter (termed the super core promoter [SCP]) that contains a TATA, Inr, MTE, and DPE in a single promoter has been shown to yield high levels of transcription, implying that gene expression levels can be modulated via the core promoter (Juven-Gershon 2006) (Kedmi, 2014).

The set of basal transcription factors has been defined using TATA-dependent promoters. Transcription of DPE-dependent genes, however, is fundamentally different from transcription of TATA-dependent genes. First and foremost, the set of basal transcription factors that is necessary to transcribe TATA-dependent promoters in vitro is insufficient to transcribe DPE-dependent promoters. Moreover, enhancers with a preference for DPE-containing promoters or TATA-containing promoters have been discovered. Furthermore, TBP, which is necessary for TATA-dependent transcription, down-regulates DPE-dependent transcription. Additionally, NC2 and MOT1, which are positive regulators of DPE-dependent transcription, act by counteracting TBP, thus relieving its inhibition of DPE transcription. DPE-dependent promoters can be transcribed using Drosophila high-salt nuclear extracts (Kedmi, 2014).

To identify the factors that support DPE-dependent transcription, a biochemical complementation approach was used; high-salt nuclear extracts were fractionated in search of proteins that would support DPE transcription. TBP (TATA-box-binding protein)-related factor 2 (TRF2) was found to be enriched in the fractions supporting DPE transcription. Drosophila trf2 encodes two protein isoforms that show similarity to the core domain of TBP: a 632-amino-acid protein (the 'short TRF2,' which has been extensively studied) and a 1715-amino-acid protein in which the same short TRF2 sequence is preceded by a long N-terminal domain. This study explored the functions of TRF2, which is the TRF with the least similarity to TBP, in transcriptional regulation; short TRF2 was found to preferentially bind and activate DPE-containing promoters. This study highlights the role of short TRF2 as a preferential core promoter regulator and provides insights into the complexity of transcription initiation (Kedmi, 2014).

This study demonstrated that the conserved short TRF2 protein preferentially activates DPE-dependent promoters. Using biochemical complementation, TRF2 was found to be enriched in Drosophila embryo protein fractions that activate transcription from a DPE-dependent core promoter. Furthermore, DNA affinity chromatography demonstrated that TRF2 is enriched in protein fractions that bind DPE-containing promoter DNA. By luciferase reporter assays in transiently transfected Drosophila S2R+ cells, it was shown that short TRF2, as opposed to the long TRF2, preferentially activates the DPE-dependent ftz and gt reporters. Microarray analysis was used in search of novel TRF2 targets, and the enrichment of Inr- and DPE-containing promoters was found among short TRF2 upregulated genes. MEME analysis has not identified overrepresented motifs in the long TRF2 upregulated genes. RNAi followed by microarray analysis has revealed the enrichment of the DPE motif in TRF2 downregulated genes. Notably, the microarray analyses identified both direct and indirect TRF2 targets. In the future, it will be interesting to test specific targets and distinguish between direct and indirect TRF2 targets (Kedmi, 2014).

The human TRF2 protein is 186 amino acids long, of which amino acids 7­186 constitute the TBP core domain. The short Drosophila TRF2 isoform, which constitutes the C terminus of the long Drosophila TRF2 isoform, is homologous to TBP and the human TRF2 as well as TRF2 from other species. The short TRF2 protein isoform is 632 amino acids long and has a theoretical pI of 8.0, whereas the long TRF2 isoform, which may be unique to Drosophila, is 1715 amino acids long and has a theoretical pI of 4.65. The long TRF2 isoform contains an aspartic-rich domain, a serine-rich domain, and a glutamine-rich helical domain that is typical to certain histone deacetylases. The functions of the long TRF2 (and whether these sequence domains contribute to its activity) remain to be discovered (Kedmi, 2014).

Although the core domain of Drosophila TRF2 is 39% identical to the core domain of Drosophila TBP, the TBP residues required for TATA-box binding are not conserved, and, indeed, the short TRF2 does not bind TATA-containing promoters. A long-standing question has been: What are the DNA elements that TRF2 binds? Using microfluidics, this study now identified protein-­DNA interactions of TRF2-containing complexes. Furthermore, the affinities were calculated of interactions between TRF2-containingcomplexes and DNA in vitro. Mutation in the DPE of the four short TRF2 target promoters that were tested significantly reduces DNA binding. Interestingly, the long TRF2- containing complexes bind promoter DNA with high affinity, albeit without any preference for a wild-type promoter as compared with a mDPE-containing promoter DNA. Importantly, TRF2 has recently been demonstrated to mediate the transcription of ribosomal protein genes via the TCT motif and not the TATA-box (Kedmi, 2014).

ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) analysis of Drosophila embryos using anti-TRF2 antibodies indicate a peak of TRF2 occupancy in the vicinity of the TSS. This ChIP-seq data set was analyzed and it was discovered that, remarkably, the most enriched motif in the top TRF2-bound genes (quantile 0.9) is the DPE motif. MEME analysis of the least TRF2-bound genes (quantile 0.1) has not identified any enriched motifs. This suggests that DPE-containing promoters are direct targets of TRF2. Taken together, this study discovered that TRF2 is a unique core promoter factor that is involved in DNA binding of DPE-containing promoters (Kedmi, 2014).

Using microfluidics, this study demonstrated sequence-specific DNA-binding of TRF2-containing complexes. Whether TRF2 binds DNA directly remains to be determined. It is likely that there are TRF2-associated factors that assist TRF2 in core promoter DNA-binding in a manner similar to that of TBP-associated factors that, together with TBP, constitute the TFIID complex (Kedmi, 2014).

Transcriptional regulation is achieved by diversity in enhancers, core promoter composition, and the basal transcription machinery. An example for such diversity is the existence of a TRF family, which includes TRF1, TRF2, and TRF3. In this study, a connection was discovered between core promoter composition and the basal transcription factor TRF2. Remarkably, a significant reduction was observed of protein–DNA interaction when the DPE motif was mutated. It is of great interest that not only do the core promoters of many (although not all) short TRF2 target genes lack a TATA-box, they share Inr and DPE motifs. Moreover, it was noted that nucleotides at specific positions located between the Inr and DPE, such as +17, +19, and +24 relative to the A+1, are overrepresented in the Inr- and DPE-containing short TRF2 targets (Kedmi, 2014).

These positions were previously recognized as overrepresented in DPE-containing genes. This study further demonstrated that the core promoters of four short TRF2 target genes are functionally DPE-dependent and are activated by short TRF2. In a sense, TRF2 is acting in a manner antagonistic to TBP, which has been shown to down-regulate DPE transcription (Kedmi, 2014).

This study discovered that TRF2 mediates transcription of DPE-containing promoters. TRF2 has been shown recently to mediate the transcription of ribosomal protein genes via the TCT motif (Wang, 2014). Furthermore, TRF2 has been shown previously to play an important role in RNA Pol II transcription and has been shown to activate the TATA-less histone H1 promoter. Hence, it is likely that TRF2 is important for multiple transcription systems that mediate important biological processes and pathways. Importantly, these findings support the notion that the basal transcription factors are not necessarily general transcription factors. Rather, they are active components that act in conjunction with the core promoters in the complex regulation of gene expression (Kedmi, 2014).

TRF2 associates with DREF and Iswi and regulates the PCNA promoter

Drosophila TATA-box-binding protein (TBP)-related factor 2 is a member of a family of TBP-related factors present in metazoan organisms. Recent evidence suggests that TRF2s are required for proper embryonic development and differentiation. However, true target promoters and the mechanisms by which TRF2 operates to control transcription remain elusive. A Drosophila TRF2-containing complex has been purified by antibody affinity; this complex contains components of the nucleosome remodelling factor (NURF) chromatin remodelling complex as well as the DNA replication-related element (DRE)-binding factor DREF. This latter finding leads to potential target genes containing TRF2-responsive promoters. A combination of in vitro and in vivo assays has been used to show that the DREF-containing TRF2 complex directs core promoter recognition of the proliferating cell nuclear antigen (PCNA) gene. Additional TRF2-responsive target genes involved in DNA replication and cell proliferation have also been identified. These data suggest that TRF2 functions as a core promoter-selectivity factor responsible for coordinating transcription of a subset of genes in Drosophila (Hochheimer, 2002).

Metazoan organisms have evolved diverse mechanisms to control the spatial and temporal patterns of gene expression during growth, differentiation and development. It has become increasingly evident that cell-type-specific components of the general transcriptional apparatus, for example the mammalian TFIID component TAFII105 or the Drosophila TAFII80 homolog Cannonball contribute significantly to tissue-specific and gene-selective transcriptional regulation in metazoan organisms. Recent studies have also established that TBP-related factors like Drosophila TRF1 can direct transcription from an alternative core promoter and a TRF1:BRF complex is required for RNA polymerase III transcription of transfer RNA genes. TRF2 is a third member of the TBP family in Drosophila and, like TBP and TRF1, TRF2 interacts with the basal transcription factors TFIIA and TFIIB10. However, the primary amino acid sequence of the putative TRF2 DNA-binding domain has diverged from TBP and TRF1 and, not surprisingly, TRF2 fails to bind to DNA containing canonical TATA boxes. But TRF2 is associated with loci on Drosophila chromosomes that are distinct from TBP and TRF1. This suggested that TRF2 may direct promoter specificity and perhaps coordinate a subset of target genes. Size exclusion chromatography indicated that Drosophila TRF2 is likely to be part of a macromolecular complex. Unlike TRF1, Drosophila TRF2 has amino-terminal and carboxy-terminal extensions flanking the putative DNA-binding core domain. This suggested that TRF2 may be associated with a set of proteins that are distinct from TBP- and TRF1-associated factors. It was reasoned that the purification and identification of TRF2-associated factors might enable the identification of TRF2-specific promoters and reveal how TRF2 operates to execute transcriptional specificity (Hochheimer, 2002).

In the absence of a functional assay allowing conventional purification of TRF2-associated factors a panel of monoclonal antibodies was generated directed against several domains of TRF2 to affinity purify TRF2 and its putative associated subunits. Approximately 3,500 hybridomas were screened and a clone was isolated that efficiently immunoprecipitated TRF2 and its associated factors from Drosophila embryo nuclear extract. A Sarkosyl-eluted complex containing TRF2 was analysed by SDS-polyacrylamide gradient gel electrophoresis (PAGE) that revealed a set of 18 polypeptides with relative molecular mass (Mr) ranging from 300K to 29K that co-immunoprecipitated consistently with TRF2 even under very stringent conditions (Hochheimer, 2002).

An 80K protein associated with TRF2 is identical to DREF1. DREF and its corresponding response element DRE have been well documented to be important for the regulation of cell-cycle and cell-proliferation genes in Drosophila (that is, genes for PCNA and the 180K and 73K subunits of DNA polymerase). The identification of the promoter-selective DNA-binding protein DREF was intriguing because Drosophila TRF2 thus far had failed to bind to canonical TATA-box elements, which suggests that TRF2 may cooperate with DREF to execute promoter specificity and perhaps operate like a metazoan sigma factor (Hochheimer, 2002).

The 140K protein associated with TRF2 is identical to Drosophila Iswi, which is the catalytic ATPase subunit of NURF, ACF and CHRAC chromatin remodelling complexes. Moreover, the 55K and 38K proteins associated with TRF2 turned out to be NURF-55/CAF-1 and NURF-38/inorganic pyrophosphatase, respectively. Notably, the peptide sequences obtained from the three largest (300K, 250K and 230K) proteins associated with TRF2 do not match NURF-301, suggesting that the presence of some NURF subunits is not merely a result of contaminating NURF in the TRF2 complex. However, analysis of the cDNAs encoding the 190K and 160K proteins associated with TRF2 revealed that both proteins contain conserved sequence motifs for 11 and 5 zinc finger motifs (C2H2), respectively; these smaller proteins thus resemble factors like the CCCTC-binding factor CTCF that has been implicated in mediating chromatin-dependent processes such as the regulation of insulator function. It is therefore possible that the TRF2 complex encompasses both promoter-selectivity functions and NURF-like components as well as other activities with distinct subunits and specificity (Hochheimer, 2002).

Sequence analysis of the cDNA encoding the 65K protein revealed a significant similarity to the RNA-binding protein Rap55 isolated from Pleurodeles waltl and Xenopus laevis, whereas sequence analysis of the 70K, 116K and 118K proteins showed no significant similarity to known proteins in the databases. The functional relevance of ß-tubulin in the TRF2 complex is at present unclear and the 47K and the 29K polypeptides associated with TRF2 are yet to be characterized (Hochheimer, 2002).

Having identified DREF as a tightly associated component of the TRF2 complex, it was next asked whether TRF2 can function as a true core promoter recognition factor and selectively initiate transcription at a promoter that is documented to be stimulated by the DRE/DREF system. The DREF-responsive PCNA promoter, which contains at least three promoter-proximal regulatory elements including an upstream regulatory element URE, DRE and two E2F recognition sites located within 200 bp upstream of the start site, was chosen (Hochheimer, 2002).

To test the responsiveness of the PCNA promoter in vitro and to map the transcription start site(s), a -580 PCNA (-580 to +56) promoter fragment, which contains all known regulatory elements, and a -64 PCNA (-64 to +56) promoter fragment, which lacks all regulatory elements except for the E2F-binding sites were used as DNA templates for in vitro transcription. Increasing amounts of a partially purified Drosophila embryo nuclear extract (H.4) that contains all the necessary basal factors were added, as well as both the TRF2 complex and limiting amounts of TFIID to the transcription reaction. Using the -580 PCNA template two distinct transcription start sites separated by 63 nucleotides were detected. Promoter 1 (with start site at position +1) was stimulated with increasing amounts of H.4 supplemented with TFIID whereas promoter 2 (with start site at position -63) was detected only with the lowest amounts of H.4 added. Using the truncated -64 PCNA, template, transcription from promoter 2 was essentially abolished, whereas a weak activity could be detected from promoter 1 by adding the maximum amount of H.4 + TFIID. In vitro and in vivo results suggest that promoter 2 might be TRF2- and DRE-dependent, whereas promoter 1 appears to be mediated by TFIID (Hochheimer, 2002).

It was next asked whether TRF2 can contribute to the enhancer-dependent activated transcription of the PCNA promoters by E2F and DP, which cooperate in DNA-binding and transcriptional activation. The co-expression of just the trancriptional activators E2F and DP in the absence of exogenous TRF2/DREF results in a substantial transcriptional activation of the PCNA reporter. It is likely that this activation by E2F/DP is mediated by endogenous TRF2. This activation is abolished with the -64 PCNA reporter, which lacks the DRE-binding sites but still contains the E2F-binding sites. As expected, inducing the co-expression of all three promoter recognition factors -- TRF2, DREF and E2F -- results in a strong synergistic activation of the PCNA promoters (80-fold) in a DRE-dependent fashion. These results suggest that in SL2 cells TRF2 and DREF can work together to stimulate the PCNA reporter in a DRE-dependent fashion. This is consistent with the finding in vitro that the TRF2 complex can selectively initiate transcription from promoter 2 of the PCNA gene in a DRE-dependent manner (Hochheimer, 2002).

To investigate whether TRF2 is involved in the coordinate regulation of other DNA replication and cell cycle genes in the Drosophila genome, oligonucleotide-based microarrays representing 13,500 Drosophila genes were hybridized with RNA probes isolated at different time points after induction of TRF2 expression in SL2 cells. The microarray analysis revealed that only 1.9% of all genes analysed were upregulated more than 2-fold and only 1.6% downregulated by more than 2-fold. These biochemical studies and cell based assays suggested that TRF2 functions as a core promoter-selectivity factor that collaborates with DREF. It was therefore asked whether there are other TRF2-responsive genes that also contain a promoter-proximal DRE. An analysis of the distribution of DREs in the Drosophila genome revealed that about 100 genes bear a consensus DRE within 1 kb of the predicted promoter region. Microarray analysis revealed that 38 of these DRE-containing genes were also responsive to TRF2 overexpression. For example, genes encoding PCNA, the 180K subunit of DNA polymerase, the α-subunit of mitochondrial DNA polymerase, and E2F were all found to be upregulated 2-5-fold by TRF2 in the microarray analysis and confirmed by RNase protection assays. Three additional DRE-regulated genes encoding TBP, the 73K subunit of DNA polymerase, and the 50K subunit of DNA polymerase were found to be downregulated (Hochheimer, 2002).

These data suggest that in addition to the PCNA gene a number of other Drosophila DRE-containing genes may also be regulated by TRF2 and further support the model that TRF2 can function as a core promoter-selectivity factor that governs a restricted subset of genes that are coordinately regulated. A recent bioinformatics study of core promoter sequences in the Drosophila genome identified a consensus DRE as the second most frequent control core element (other than TATA and INR) providing independent evidence for DRE as a core promoter element (Hochheimer, 2002).

Because these studies have relied largely on 'gain of function' assays double stranded RNA interference (dsRNAi) was imployed in flies and SL2 cells25 to determine the consequences of ablating TRF2/DREF on transcription of putative target genes such as PCNA and DNApol 180 in cultured cells. SL2 cells were treated with in vitro synthesized dsRNA and the depletion of TRF2 and DREF proteins was monitored by immunoblot analysis. After 2 days of incubation the specifically targeted TRF2 and DREF proteins were severely depleted. After 48 h of dsRNA treatment, a significant number of cells sloughed off the plate and died. This is in accordance with previous finding that TRF2 RNAi in Drosophila embryos is lethal for embryos (Hochheimer, 2002).

However, between 24 and 48 h of dsRNAi treatment it was possible to reproducably measure the activity of transiently transfected luciferase reporters fused to either the PCNA or DNApol 180 promoters and compare them to an internal control reporter gene. The activities from both the PCNA (-580 to +56) and DNApol 180 (-620 to +20) promoters were significantly reduced in TRF2-depleted cells (4.2-fold and 3-fold, respectively) relative to a Renilla luciferase control reporter driven by the HSV TK promoter. Likewise, these two target gene promoters were downregulated in DREF-depleted cells (3.5-fold and 5-fold, respectively). These depletion experiments using dsRNAi thus support the findings that TRF2 and DREF participate in directing transcription of a select subset of genes that include PCNA and DNApol 180 (Hochheimer, 2002).

Although the dsRNAi studies provide an independent line of evidence to support the notion that TRF2/DREF play a role in promoter selectivity in vivo, they fail to provide a direct mechanistic link between TRF2/DREF and the PCNA and DNApol 180 promoters. Chromatin immunoprecipitation (ChIP) experiments were carried to determine the occupancy of TRF2/DREF at the PCNA and DNApol 180 promoters in formaldehyde-treated SL2 cells using antibodies raised against TRF2, DREF and TBP. The precipitated DNA fragments with an average length of 500-1,000 base pairs (bp) were analysed directly by polymerase chain reaction (PCR). The PCNA promoter region was specifically precipitated by anti-TRF2 and anti-DREF and to a lesser extent by anti-TBP; this is consistent with previous findings and confirms that TRF2, DREF and the TFIID subunit TBP can co-localize and directly interact with the PCNA promoter region in living cells. As further evidence for the targeting of specific promoters by TRF2 and DREF, an analysis was carried out of the DRE-containing DNApol 180 promoter region, which is selectively precipitated by the TRF2-, DREF- and TBP-specific antibodies. These ChIP experiments strongly support the in vitro transcription results as well as the cell-based dsRNAi transcription assays in establishing a core promoter selectivity for the TRF2/DREF complex (Hochheimer, 2002).

Two isoforms of Drosophila TRF2 are involved in embryonic development, premeiotic chromatin condensation, and proper differentiation of germ cells of both sexes

The Drosophila TATA box-binding protein (TBP)-related factor 2 (TRF2 or TLF) was shown to control a subset of genes different from that controlled by TBP. This study investigated the structure and functions of the trf2 gene. It encodes two protein isoforms: the previously described 75-kDa TRF2 and a newly identified 175-kDa version in which the same sequence is preceded by a long N-terminal domain with coiled-coil motifs. Chromatography of Drosophila embryo extracts revealed that the long TRF2 is part of a multiprotein complex also containing ISWI. Both TRF2 forms are detected at the same sites on polytene chromosomes and have the same expression patterns, suggesting that they fulfill similar functions. A study of the manifestations of the trf2 mutation suggests an essential role of TRF2 during embryonic Drosophila development. The trf2 gene is strongly expressed in germ line cells of adult flies. High levels of TRF2 are found in nuclei of primary spermatocytes and trophocytes with intense transcription. In ovaries, TRF2 is present both in actively transcribing nurse cells and in the transcriptionally inactive oocyte nuclei. Moreover, TRF2 is essential for premeiotic chromatin condensation and proper differentiation of germ cells of both sexes (Kopytova, 2007; full text of article).

Three major forms of the trf2 mRNAs were detected at different stages of fly development. However, all of them contain ORFs encoding a 175-kDa protein. The data suggest that p75 is the result of translation initiation from an internal ribosome entry site (IRES) present in the trf2 mRNA. Several cell mRNAs have been shown to contain IRES elements. Most of the cell IRES elements described so far are localized to the 5' UTR of the mRNA. In the case of p75, however, the IRES is present in the coding region of the trf2 mRNA (Kopytova, 2007).

The p175 protein is present in the extracts from Drosophila embryos as a component of a multiprotein complex of about 1 MDa, which shows a high affinity for heparin, implying DNA-binding properties. The polytene chromosome staining with antibodies specific to p175 confirms this inference and suggests that the p175-containing complex is functional. The experiments demonstrate that this complex also contains the ISWI ATPase, which was previously found in the TRF2-DREF complex (Hochheimer, 2002). A certain amount of p75 cofractionates with p175, which makes one suppose that these proteins are components of the same complex (Kopytova, 2007).

Analysis of the expression of the two TRF2 polypeptides argues in favor of their similar functions. The fact that the antibodies recognizing only p175 did not reveal any specific sites on polytene chromosomes suggests that both isoforms are involved in regulation of the same target genes. The rescue data show that the two isoforms of TRF2 may functionally replace each other in vivo to rectify the defects in the P-element insertion mutants (Kopytova, 2007).

A significant amount of p75 does not cofractionate with p175. The different chromatographic properties of TRF2 isoforms may be explained by supposing that the extensive N-terminal part of the longer form, with coiled-coil domains, may be involved in interaction with several other accessory proteins. Partial dissociation of the 1-MDa complex during fractionation is also not excluded (Kopytova, 2007).

TLF/TRF2 in different metazoan organisms is required for proper embryonic development and differentiation. The high mortality of homozygous phP1 trf2P1 embryos (phP1 is a mutation in polyhomeotic that causes no mutant phenotype, represses the transcription of P-element-induced alleles and, thus, strongly enhances the manifestation of mutations caused by P-element insertion) suggests a vital role of TRF2 at this stage. The results show that TRF2 plays an important role in establishing the body plan during embryonic development. This study also demonstrates that TRF2 is important for development of the CNS and Peripheral nervous system. Many genes that are putative targets of the TRF2-containing complex have been implicated in DNA replication and cell proliferation (Hochheimer, 2002). In particular, the selenophosphate synthetase 1 gene regulated by TRF2 (Jin, 2004) is involved in imaginal disc morphogenesis, brain development, and cell proliferation. In line with these findings, this study demonstrates that reduced TRF2 expression in the phP1 trf2P1 mutant leads to a broad range of developmental defects in antenna, wings, legs, eyes, and bristles (Kopytova, 2007).

Previously, mutation of an unidentified gene (the lawcP1) displaying a phenotype similar to trf2P1 was cytologically mapped to the same region (see Zorin, 1999). The recessive lawcP1 mutation did not complement trfP1, whereas both transgenes expressing TRF2 isoforms corrected the mutant lawcP1 phenotype, suggesting that lawcP1 is an allele of the trf2 gene. The lawcP1 was characterized as a trx-like mutation (Zorin, 1999). However, this study did not find genetic interactions between trfP1 and either trx (trxb11 and brm2) or Pc [Pc1, Pc3, Su(z)2, and Psc1] mutations. It is possible that lawcP1 and trf2P1 have different effects on trf2 expression. This possibility is also supported by the observation that lawcP1 interacts genetically with mod(mdg4) (Zorin, 1999), whose protein product is the component of the gypsy insulator. It was shown that the lawcP1 mutation caused alterations in the punctuated distribution of the Mod(mdg4) protein within the nucleus (Zorin, 1999). In contrast, neither phP1 trf2P1 nor any of lethal trf2 mutations obtained from Bloomington changed the distribution of Mod(mdg4) or Su(Hw) proteins within the nucleus (Kopytova, 2007).

This study demonstrated the crucial role of TRF2 in oogenesis. TRF2 expression is detected in the female germ line stem cells and at subsequent stages. High expression is observed in trophocytes (nurse cells) and in the oocyte nucleus. In developing cysts, the oocyte is arrested at prophase I of meiosis (G2) until a late stage of oogenesis, while trophocytes actively synthesize multiple mRNAs and proteins that are further loaded to the oocyte cytoplasm. Some of these factors are essential for meiotic cleavage, while others are essential for the development of the future embryo after fertilization (Kopytova, 2007).

Further, the results show that, like mammalian TLF, the Drosophila TRF2 is essential for male germ cell differentiation; i.e., the function of TRF2 in spermatogenesis is conserved in evolution. The beginning of TRF2 accumulation in male germ cell nuclei coincides with the onset of the G2 phase of meiosis; its content rises as the primary spermatocytes grow. Many genes required for further meiotic cell cycle progression, spermatid differentiation, and elongation are expressed at this stage. Contrary to TRF2, TBP expression is largely restricted to mitotically dividing gonial cells and drops below the detection limit in primary spermatocytes; thus, TRF2 may be a major transcription-initiating factor at this stage of differentiation of male germ cells (Kopytova, 2007).

The abundance of TRF2 in both types of germ cells at G2 of meiosis implies that it may be involved in the control of expression of genes responsible for the meiotic progression. The abnormalities observed in germ cell differentiation in mutated flies support this suggestion (Kopytova, 2007).

In gonads of both sexes, TRF2 exhibits a cell- and tissue-specific expression pattern. It is present in germ line cells but is undetectable in the somatic cells of gonads. It is not found in mitotically dividing spermatogonia but is abundant in primary spermatocytes. Interestingly, like the Drosophila TRF2, the murine TLF was not detected in mitotically dividing spermatogonia. TLF expression was observed in late-meiotic and postmeiotic cells (Martianov, 2002), being highest in late pachytene spermatocytes deploying an extensive tissue-specific transcription program (Kopytova, 2007).

Recent data suggest that an alternative form of transcription initiation machinery controls the expression of a set of genes required for spermatid differentiation in primary spermatocytes. Several TAFs have tissue-specific homologues expressed at this stage; similarly to TRF2, the onset of their expression coincides with the transition from mitosis to meiosis. Future study should clarify whether TRF2 is involved in maintenance of this cell-type-specific transcription program (Kopytova, 2007).

The TRF2 expression pattern does not always coincide with active transcription. TRF2 is abundant in oocytes, which are held to be nearly transcriptionally silent, with highly condensed chromatin. The lack of appreciable transcription is corroborated by the observation that the oocyte nucleus is virtually devoid of TBP. Much TRF2 is detected in late primary spermatocytes that exit the growth and transcription program, their chromatin rapidly condensing in preparation to meiosis. These data suggest that TRF2 may have some functions besides mediating transcription initiation. Particularly, at these stages of development, TRF2/TLF may act as an inhibitor of transcription by blocking the access of the basal transcription machinery to promoters as it has been described at very early stages of C. elegans development (Kaltenbach, 2000). Alternatively, TRF2/TLF may play a role in chromosome condensation at these transcriptionally inactive states, as suggested by (Martianov, 2002; Kopytova, 2007 and references therein).

The data also support the participation of TRF2 in chromatin organization. Both in oocytes and in primary spermatocytes, the trf2 mutation causes defects in meiotic chromosome condensation. The observed colocalization of TRF2 with condensed chromatin is consistent with the idea that the TRF2-containing complexes may be directly involved in maintaining the chromosome structure. Note that mouse TLF has been shown to participate in the organization of the chromocenter (condensed structure formed by association of centromeric heterochromatin in round spermatids) (Martianov, 2002) and is generally supposed to play a dual role of a classical transcription factor and a structural factor (Martianov, 2001). These results indicate that the function of TLF/TRF2 has been preserved during evolution (Kopytova, 2007).

putzig is required for cell proliferation and regulates notch activity in Drosophila

The gene putzig (pzg) is a key regulator of cell proliferation and of Notch signaling in Drosophila. pzg encodes a Zn-finger protein that exists within a macromolecular complex, including TATA-binding protein-related factor 2 (TRF2)/DNA replication-related element factor (DREF). This complex is involved in core promoter selection, where DREF functions as a transcriptional activator of replication-related genes. This study provides in vivo evidence that pzg is required for the expression of cell cycle and replication-related genes, and hence for normal developmental growth. Independent of its role in the TRF2/DREF complex, pzg acts as a positive regulator of Notch signaling that may occur by chromatin activation. Down-regulation of pzg activity inhibits Notch target gene activation, whereas Hedgehog (Hh) signal transduction and growth regulation are unaffected. These findings uncover different modes of operation of pzg during imaginal development of Drosophila, and they provide a novel mechanism of Notch regulation (Kugler, 2007; full text of article).

In a developing organism, cell proliferation and apoptosis must be strictly coordinated with patterning processes to correctly shape the organs. Thus, it is not surprising that all major morphogenetic and developmental signaling pathways have been involved in the regulation of cell proliferation and apoptosis and that they have been linked to numerous cases of cancer formation in mammals. In Drosophila, a large body of work shows that several of these pathways act in concert in the coordination of cell survival and death. For example, overexpression of Notch causes vast overgrowth, whereas inhibition of Notch activity by overexpression of its antagonist Hairless results in tissue loss and apoptosis. Indeed, the combined activity of Hedgehog (Hh), Decapentaplegic (Dpp), and Notch is required to promote reentry into the cell cycle after a developmentally regulated G1 arrest in the eye anlagen of Drosophila larvae. Moreover, it was shown that Hh signaling promotes cellular growth by transcriptional activation of G1 cyclins Cyclin D and Cyclin E. However, to this end, the understanding of the molecular principles that connect these pathways to either control of cell cycle or apoptosis remains largely fragmentary (Kugler, 2007).

Cell cycle entry requires the activity of G1-S cyclins that eventually activate dE2F1, a transcription factor that induces transcription of downstream genes required, e.g., for DNA replication. In Drosophila, transcriptional activation of replication-related genes encoding, for example, proliferating cell nuclear antigen (PCNA) or DNA-polymerase alpha subunit involves also DNA replication-related element factor (DREF) that recognizes DNA replication-related element (DRE) response elements. DREF can be part of a macromolecular complex including TRF2, a TATA-binding protein-related factor that binds to a subset of selected promoters, including one promoter in the PCNA gene. TRF2 has been isolated from several different organisms, where it is required for transcription of replication-related genes and key developmental genes as well. The TRF2/DREF complex consists of more than a dozen proteins, including several known chromatin-remodeling components. Three of them confer chromatin activation, whereas two others, including p160, resemble regulators of insulator function. Interestingly, p160 was recently found to enhance position effect variegation and hence chromatin silencing and to be associated with interband regions of polytene chromosomes (Eggert, 2004). To this end, the biochemical activity and functional specificity of most of the proteins within the TRF2-complex, i.e., their role in transcriptional activation or in chromatin remodeling, however, remain elusive (Kugler, 2007).

This study isolated the Zn-finger protein p160 as a genetic suppressor of Hairless activity, prompting an interest in its role during Drosophila development and especially during Notch signaling. In vivo RNA interference resulted in tiny larvae and developmental delay, which is why the corresponding gene was named putzig (pzg). This study presents in vivo evidence that pzg is essential for fly survival by regulating cell cycle entry and progression. In addition, pzg encodes a key regulator of the Notch signaling pathway and that it is involved in histone modification and chromatin activation. Interestingly, this activity is independent of DREF, suggesting context dependence of Pzg activity (Kugler, 2007).

EP756 was identified in a genetic screen as suppressor of tissue loss caused by an overexpression of the Notch antagonist Hairless (H) during eye development. This positive effect was not restricted to the eye, because it was likewise observed during wing development. Moreover, cell growth and proliferation induced by an enforced Notch signal was significantly enhanced (~20%) by a combined overexpression with EP756. Tissue specific overexpression of EP756 caused a very mild enlargement of the respective tissues on its own. These data suggest a more general role of EP756 in the control of cell proliferation as well as an intimate interaction with Notch signaling (Kugler, 2007).

Pzg is one component of a multiprotein complex that contains TRF2 and DREF. TRF2 allows transcription initiation from selected promoters independently of TFIID. DREF is a positive transcriptional regulator of cell cycle and replication-related genes, and it may guide TRF2 to the PCNA and DNA-polymerase alpha promoters. Assuming promoter recognition or binding requires Pzg contained within the TRF2/DREF complex, depletion of Pzg might destroy the complex or reduce its activity, easily explaining the dramatic proliferation defects. However, it is noted that only a subset of promoters containing DREF binding sites involves activation through TRF2, suggesting that DREF can act independently of TRF2. Moreover, Pzg activity is found independently of DREF, indicating that TRF2/DREF complex components can act either alone or in conjunction with other factors (Kugler, 2007).

The TRF2/DREF complex contains several proteins involved in chromatin remodeling. Notably, Pzg and one other TRF2/DREF component p190 are reminiscent of factors implicated in insulator function. In accordance, Pzg activity has been associated with position effect variegation and chromatin silencing. In contrast, assays reveal an essential function of Pzg in retaining robust K4-trimethylation of histone H3, which is directly associated with open chromatin structures. In accordance with these findings, EP756 was recently identified as a suppressor of the cut allele ctK. This cut mutation is caused by the insulator activity of a gypsy retrotransposon, which can be relieved by EP756 overexpression. EP756 is shown in this stody to drive Pzg expression, in support of the notion that Pzg's epigenetic activity overcomes gypsy insulator function (Kugler, 2007).

Three of the proteins found in the TRF2/DREF complex have been identified previously in the nucleosome-remodeling factor NURF (see NURF301), which consists in total of four subunits. NURF is associated with chromatin activation by facilitating transcription of chromatin in vivo. In fact, mutations in Drosophila ISWI, the catalytic subunit of NURF, and other nucleosome remodeling complexes caused phenotypes that are very reminiscent of pzg-RNAi-induced defects. Because DREF down-regulation has no effect on trimethylation of H3K4, it seems unlikely that the TRF2/DREF complex as a whole is involved in chromatin activation. Instead, Pzg may be part of a NURF-like chromatin-remodeling complex, depending on the developmental context (Kugler, 2007).

Apart from a role in proliferation, this study has uncovered an important role for Pzg as positive regulator of Notch signaling. Interestingly, it was found that Pzg binds to chromatin in the regulatory region of the Notch target genes E(spl)m8 and vg. This regulation is independent of DREF: albeit DREF binding sites are common to Drosophila promoters, neither Notch nor Notch target genes that were investigated are transcriptional targets of DREF. Thus, reduced transcriptional activity of Notch target genes in pzg-RNAi mutant cells is due to a DREF-independent role of Pzg. Alternatively, Pzg could facilitate formation of the transcriptional activator complex that is assembled on Notch target promoters involving intracellular Notch itself. By using the yeast two-hybrid system, several Notch pathway members were tested; however, no binding to Pzg was detected. It is proposed that Pzg has a dual function that is effected differently. On one hand, it activates proliferation-related genes in conjunction with TRF2/DREF, and on the other hand, it activates Notch signaling by chromatin activation independently of DREF (Kugler, 2007).

Several lines of evidence support the idea that Notch signaling is particularly susceptible to chromatin remodeling. For example, Notch transcriptional activity requires the histone-modifying enzyme dBre1 that is indirectly required for K4-methylation of histone H3. Moreover, chromatin-modifiers were also shown to potentiate Notch activity during Drosophila wing development. Finally, general transcriptional regulators and chromatin remodeling factors were found in several independent genetic screens to influence Notch signaling, indicating to a role of pzg in linking Notch to chromatin remodeling. The bimodal activity of Pzg onto both cell cycle genes and Notch signaling provides further insight into the complex interplay between cell proliferation and differentiation in the fly (Kugler, 2007).


Search PubMed for articles about Drosophila Trf2

Andersen, P. R., Tirian, L., Vunjak, M. and Brennecke, J. (2017). A heterochromatin-dependent transcription machinery drives piRNA expression. Nature 549(7670): 54-59. PubMed ID: 28847004

Andersen, P. R., Tirian, L., Vunjak, M. and Brennecke, J. (2017). A heterochromatin-dependent transcription machinery drives piRNA expression. Nature 549(7670): 54-59. PubMed ID: 28847004

Mohn, F., Sienski, G., Handler, D. and Brennecke, J. (2014). The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157(6): 1364-1379. PubMed ID: 24906153

Parhad, S. S., Tu, S., Weng, Z. and Theurkauf, W. E. (2017). Adaptive evolution leads to cross-species incompatibility in the piRNA transposon silencing machinery. Dev Cell 43(1): 60-70 e65. PubMed ID: 28919205

Parhad, S. S., Yu, T., Zhang, G., Rice, N. P., Weng, Z. and Theurkauf, W. E. (2020). Adaptive evolution targets a piRNA precursor transcription network. Cell Rep 30(8): 2672-2685. PubMed ID: 32101744

Baumann, D. G. and Gilmour, D. S. (2017). A sequence-specific core promoter-binding transcription factor recruits TRF2 to coordinately transcribe ribosomal protein genes. Nucleic Acids Res 45(18): 10481-10491. PubMed ID: 28977400

Dantonel, J. C., Wurtz, J. M., Poch, O., Moras, D., and Tora, L. (1999). The TBP-like factor: An alternative transcription factor in metazoa? Trends Biochem. Sci. 24: 335-339. PubMed ID: 10470030

Fan, W., Lam, S. M., Xin, J., Yang, X., Liu, Z., Liu, Y., Wang, Y., Shui, G. and Huang, X. (2017). Drosophila TRF2 and TAF9 regulate lipid droplet size and phospholipid fatty acid composition. PLoS Genet 13(3): e1006664. PubMed ID: 28273089

Haberle, V. and Stark, A. (2018). Eukaryotic core promoters and the functional basis of transcription initiation. Nat Rev Mol Cell Biol 19(10): 621-637. PubMed ID: 29946135

Hirose, F., Yamaguchi, M., Handa, H., Inomata, Y., and Matsukage, A. (1993). Novel 8-base pair sequence (Drosophila DNA replication-related element) and specific binding factor involved in the expression of Drosophila genes for DNA polymerase α and proliferating cell nuclear antigen. J. Biol. Chem. 268: 2092-2099. PubMed ID: 8093616

Hochheimer, A., et al. (2002). TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila. Nature 420(6914): 439-45. PubMed ID: 12459787

Hochheimer, A. and Tjian, R. (2003). Diversified transcription initiation complexes expand promoter selectivity and tissue-specific gene expression. Genes Dev. 17: 1309-1320. PubMed ID: 12782648

Isogai, Y, Keles S, Prestel M, Hochheimer A, Tjian R. (2007). Transcription of histone gene cluster by differential core-promoter factors. Genes Dev. 21(22): 2936-49. PubMed ID: 17978101

Jin, J. S., et al. (2004). A DNA replication-related element downstream from the initiation site of Drosophila selenophosphate synthetase 2 gene is essential for its transcription. Nucleic Acids Res. 32: 2482-2493. PubMed ID: 15121905

Kaltenbach, L., Horner, M. A., Rothman, J. H., and Mango, S. E. (2000). The TBP-like factor CeTLF is required to activate RNA polymerase II transcription during C. elegans embryogenesis. Mol. Cell 6: 705-713. PubMed ID: 11030349

Kedmi, A., Zehavi, Y., Glick, Y., Orenstein, Y., Ideses, D., Wachtel, C., Doniger, T., Waldman Ben-Asher, H., Munster, N., Thompson, J., Anderson, S., Avrahami, D., Yates, J. R., Shamir, R., Gerber, D., and Juven-Gershon, T. (2014). Drosophila TRF2 is a preferential core promoter regulator. Genes Dev 28(19): 2163-74.. PubMed ID: 25223897

Kopytova, D. V., et al. (2006). Two isoforms of Drosophila TRF2 are involved in embryonic development, premeiotic chromatin condensation, and proper differentiation of germ cells of both sexes. Mol. Cell. Biol. 26: 7492-7505. PubMed ID: 17015475

Kugler, S. J. and Nagel, A. C. (2007). putzig is required for cell proliferation and regulates notch activity in Drosophila. Mol. Biol. Cell 18(10): 3733-40. PubMed ID: 17634285

Liu, J. L., Murphy, C., Buszczak, M., Clatterbuck, S., Goodman, R. and Gall, J. G. (2006). The Drosophila melanogaster Cajal body. J. Cell Biol. 172: 875-884. PubMed ID: 16533947

Martianov, I., et al. (2001). Late arrest of spermiogenesis and germ cell apoptosis in mice lacking the TBP-like TLF/TRF2 gene. Mol. Cell 7: 509-515. PubMed ID: 11463376

Martianov, I., et al. (2002). Distinct functions of TBP and TLF/TRF2 during spermatogenesis: requirement of TLF for heterochromatic chromocenter formation in haploid round spermatids. Development 129: 945-955. PubMed ID: 11861477

Mohn, F., Sienski, G., Handler, D. and Brennecke, J. (2014). The rhino-deadlock-cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila. Cell 157(6): 1364-1379. PubMed ID: 24906153

Mukai, M., Hayashi, Y., Kitadate, Y., Shigenobu, S., Arita, K. and Kobayashi, S. (2007). MAMO, a maternal BTB/POZ-Zn-finger protein enriched in germline progenitors is required for the production of functional eggs in Drosophila. Mech Dev 124(7-8): 570-583. PubMed ID: 17600690

Muller, F., Lakatos, L., Dantonel, J., Strahle, U., and Tora, L. (2001). TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish. Curr. Biol. 11: 282-287. PubMed ID: 11250159

Nakamura, S., Hira, S., Fujiwara, M., Miyagata, N., Tsuji, T., Kondo, A., Kimura, H., Shinozuka, Y., Hayashi, M., Kobayashi, S. and Mukai, M. (2019). A truncated form of a transcription factor Mamo activates vasa in Drosophila embryos. Commun Biol 2: 422. PubMed ID: 31799425

Nakamura, S., Hira, S., Kojima, M., Kondo, A. and Mukai, M. (2020). Expression of the core promoter factors TBP and TRF2 in Drosophila germ cells and their distinct functions in germline development. Dev Growth Differ. PubMed ID: 33219538

Neves, A. and Eisenman, R. N. (2019). Distinct gene-selective roles for a network of core promoter factors in Drosophila neural stem cell identity. Biol Open 8(4). PubMed ID: 30948355

Parhad, S. S., Tu, S., Weng, Z. and Theurkauf, W. E. (2017). Adaptive evolution leads to cross-species incompatibility in the piRNA transposon silencing machinery. Dev Cell 43(1): 60-70 e65. PubMed ID: 28919205

Parhad, S. S., Yu, T., Zhang, G., Rice, N. P., Weng, Z. and Theurkauf, W. E. (2020). Adaptive evolution targets a piRNA precursor transcription network. Cell Rep 30(8): 2672-2685. PubMed ID: 32101744

Ohbayashi, T., Makino, Y. and Tamura, T. A. (1999). Identification of a mouse TBP-like protein (TLP) distantly related to the Drosophila TBP-related factor. Nucleic Acids Res. 27: 750-755. PubMed ID: 9889269

Ohler, U., Liao, G. C., Niemann, H., and Rubin, G. M. (2002). Computational analysis of core promoters in the Drosophila genome. Genome Biol. 3: research0087.1-0087.12. PubMed ID: PubMed ID; Online text

Ohshima, N., Takahashi, M. and Hirose, F. (2003). Identification of a human homologue of the DREF transcription factor with a potential role in regulation of the histone H1 gene. J. Biol. Chem. 278: 22928-22938. PubMed ID: 12663651

Osadchiy, I. S., Georgiev, P. G. and Maksimenko, O. G. (2019). Functional Comparison of Short and Long Isoforms of the TRF2 Protein in Drosophila melanogaster. Dokl Biochem Biophys 486(1): 224-228. PubMed ID: 31367827

Rabenstein, M.D., Zhou, S., Lis, J.T., and Tjian, R. 1999. TATA box-binding protein (TBP)-related factor 2 (TRF2), a third member of the TBP family. Proc. Natl. Acad. Sci. 96: 4791-4796. PubMed ID: 10220372

Smale, S. T. and Kadonaga, J. T. (2003). The RNA polymerase II core promoter. Annu. Rev. Biochem. 72: 449-479. PubMed ID: 12651739

Veenstra, G. J., Weeks, D. L. and Wolffe, A. P. (2000). Distinct roles for TBP and TBP-like factor in early embryonic gene transcription in Xenopus. Science 290: 2312-2315. PubMed ID: 11125147

Wang, Y. L., Duttke, S. H., Chen, K., Johnston, J., Kassavetis, G. A., Zeitlinger, J. and Kadonaga, J. T. (2014). TRF2, but not TBP, mediates the transcription of ribosomal protein genes. Genes Dev 28: 1550-1555. PubMed ID: 24958592

Weake, V. M., et al. (2009). A novel histone fold domain-containing protein that replaces TAF6 in Drosophila SAGA is required for SAGA-dependent gene expression. Genes Dev. 23: 2818-2823. PubMed ID: 20008933

Zhang, D., Penttila, T. L., Morris, P. L., Teichmann, M., and Roeder, R. G. (2001). Spermiogenesis deficiency in mice lacking the Trf2 gene. Science 292: 1153-1155. PubMed ID: 11352070

Zorin, I. D., Gerasimova, T. I. and Corces, V. G. (1999). The lawc gene is a new member of the trithorax-group that affects the function of the gypsy insulator of Drosophila. Genetics 152: 1045-1055. PubMed ID: 10388823

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date revised: 13 April 2021

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