InteractiveFly: GeneBrief

dre4 and Structure specific recognition protein: Biological Overview | References

Gene names - dre4 and Structure specific recognition protein

Synonyms - FACT (refers to both subunits), Spt16 (Dre4)

Cytological map positions- 62B7-62B7 and 60A2-60A2

Functions - miscellaneous transcription factors

Keywords - FACT (facilitates chromatin transcription), modulation of chromatin structure; position effect variegation

Symbols - dre4 and Ssrp

FlyBase IDs: dre4/Spt16: FBgn0002183 and Ssrp: FBgn0010278

Genetic map positions - 3L: 1,871,573..1,876,336 [-] and 2R:19,693,884..19,696,518 [-]

Classifications -
dre4: CDC68-like, SPT16, FACT complex subunit, Rtt106, Histone chaperone
Ssrp: Nucleosome-binding factor SPN, POB3 subunit

Cellular locations - nucleus

NCBI link for Dre4: EntrezGene
NCBI link for SSRP: EntrezGene

dre4 orthologs: Biolitmine
Ssrp orthologs: Biolitmine
Recent literature
Aoki, D., Awazu, A., Fujii, M., Uewaki, J. I., Hashimoto, M., Tochio, N., Umehara, T. and Tate, S. I. (2020). Ultrasensitive Change in Nucleosome Binding by Multiple Phosphorylations to the Intrinsically Disordered Region of the Histone Chaperone FACT. J Mol Biol 432(16): 4637-4657. PubMed ID: 32553729
Facilitates chromatin transcription (FACT) is a histone chaperone that functions as a nucleosome remodeler and a chaperone. The two subunits of FACT, Spt16 and SSRP1, mediate multiple interactions between the subunits and components of the nucleosome. Among the interactions, the role of the DNA-binding domain in SSRP1 has not been characterized. The DNA-binding domain in Drosophila SSRP1 (dSSRP1) has multiple casein kinase II phosphorylation sites; the DNA binding affinity of the domain changes sigmoidally in response to the degree of phosphorylation ("ultrasensitive response"). This report explored the molecular mechanisms for the ultrasensitive response of the DNA-binding domain in dSSRP1 using the shortest fragment (AB-HMG, residues 434-624) responsible for nucleosome binding. AB-HMG contains two intrinsically disordered (ID) regions: the N-terminal part rich in acidic residues (AID) and the C-terminal part rich in basic residues (BID) followed by the HMG box. NMR and coarse-grained molecular dynamics simulations revealed a phosphorylation-dependent change in intramolecular contacts between the AID and BID-HMG, which is mediated by a hinge bending motion of AB-HMG to enable the ultrasensitive response. Ultrasensitivity generates two distinct forms of dSSRP1, which are high- and low-affinity nucleosome-binding forms. Drosophila FACT (dFACT) switches function according to the degree of phosphorylation of the AID in dSSRP1. It is proposed that dFACT in various phosphorylation states functions cooperatively to facilitate gene regulation in the context of the chromatin.

Chromatin structure plays a critical role in the regulation of transcription. Drosophila GAGA factor directs chromatin remodeling to its binding sites. Drosophila FACT (facilitates chromatin transcription), a heterodimer of dSPT16 (Flybase name: Dre4) and SSRP1, is associated with GAGA factor through its dSSRP1 subunit, binds to a nucleosome, and facilitates GAGA factor-directed chromatin remodeling. Moreover, genetic interactions between Trithorax-like encoding GAGA factor and spt16 implicate the GAGA factor-FACT complex in expression of Hox genes Ultrabithorax, Sex combs reduced, and Abdominal-B. Chromatin immunoprecipitation experiments indicate the presence of the GAGA factor-FACT complex in the regulatory regions of Ultrabithorax and Abdominal-B. These data illustrate a crucial role of FACT in the modulation of chromatin structure for the regulation of gene expression (Shimojima, 2003).

GAGA factor-dFACT complex was identified by co-immunoprecipitation with epitope tagged GAGA factor. GST pull-down assays show that GAGA factor makes a direct contact with dFACT through its dSSRP1 subunit. Gel electrophoresis mobility shift assays reveal that dFACT binds to the nucleosome. Furthermore, dFACT stimulates GAGA factor-directed chromatin remodeling in the embryonic extract of Drosophila. Based on these data, the following model is proposed for GAGA factor-directed site-specific chromatin remodeling. The GAGA factor-dFACT complex binds to a GAGAG sequence on DNA. dFACT binds to nucleosome and stimulates chromatin remodeling. This allows remodeling in a GAGA factor binding site-dependent manner. Because human FACT binds to histones H2A and H2B (Orphanides, 1999), and the yeast SPN complex enhances DNase I sensitivity of nucleosome in a region where H2A and H2B contact the DNA (Formosa, 2001), it is most likely that FACT binds to DNA at the entry and exit site of the nucleosome through its HMG subunit SSRP1, and then acts to destabilize and remove the H2A/H2B dimers to facilitate chromatin remodeling. However, the H2A/H2B dimers remain associated with the FACT-nucleosome complex through SPT16 such that they can quickly rebind to the H3/H4 tetramer when required. In support of this model, an acidic amino acid stretch found in histone-interacting proteins such as nucleoplasmin and NAP1 is conserved in the C-terminal tail of SPT16. Furthermore, H2B (and probably H2A) has been shown to turn over more rapidly than H3 and H4 during transcription (Shimojima, 2003).

There are many ATP-dependent chromatin remodeling factors. Which factor is responsible for the GAGA factor-dFACT complex-induced chromatin remodeling? Because an antibody against ISWI abolishs the GAGA factor-induced chromatin remodeling in the embryonic extract (Okada, 1998), remodeling factors containing ISWI as the catalytic subunit must play a role. Among them, at least NURF appears to be involved in the remodeling, because GAGA factor interacts directly with NURF (Xiao, 2001; Shimojima, 2003).

Although ISWI is essential for the expression of en and Ubx in imaginal discs, it has been suggested that ISWI is mainly involved in transcription repression in vivo. Specific acetylation of histone H4at Lys 16 by MOF counteracts the action of ISWI and leads to derepression of chromatin transcription. Interestingly, the yeast SPT16-Pob3 complex interacts with Sas3, a yeast homolog of MOF (John, 2001). It is possible that dFACT may also recruit MOF to shut out ISWI and induce a change from repression to activation. Under such conditions, remodeling factor(s) other than NURF may cooperate with the GAGA factor-dFACT complex. Brahma (BRM) remodeling complex may be a candidate for the replacer, but no genetic interaction between Trl and brm has been demonstrated. CHD1 may be another candidate because mouse, Drosophila, and yeast CHD1 have been reported to interact with SSRP1 or its yeast counterpart POB3 (Shimojima, 2003).

The regions of GAGA factor, dSPT16, and dSSRP1 have been identified that are responsible for interactions of these proteins with GAGA factor. GAGA factor interacts with dSSRP1 through the region containing the Zn-finger domain and its flanking sequences. This finding is consistent with the observation that both BTB/POZ and Q-rich domains are not required for the GAGA factor-induced chromatin remodeling in the embryonic extract. It also suggests that GAGA factor can form an oligomer through its BTB/POZ and/or Q-rich domains and bind cooperatively to clusters of its binding sites, just as does the GAGA factor-dFACT complex. The cooperative and stable binding of GAGA factor-dFACT complex to chromatin may be important for the epigenetic maintenance of the active state (Shimojima, 2003).

The GAGA factor-interacting region of dSSRP1 contains the HMG box and its N-terminal flanking sequence that overlaps with the dSPT16-binding region of dSSRP1. However, the presence of equimolar complex of GAGA factor, dSSRP1, and dSPT16 in the embryonic nuclear extract indicates that the overlapped region in dSSRP1 does not interfere with the simultaneous binding of GAGA factor and dSPT16. Interestingly, dSSRP1 binds to naked DNA, but addition of increasing amounts of dSPT16 decreases the DNA binding of dSSRP1 in a dose-dependent manner. This observation suggests that dSPT16 suppresses the binding of dSSRP1 to naked DNA through its interaction with the HMG box region without affecting the affinity for nucleosome (Shimojima, 2003).

The dSSRP1-binding sequence of dSPT16 was defined as the C-terminal highly conserved region. This is in good agreement with the observation that expression of the corresponding region of yeast SPT16 is required to rescue yeast temperature-sensitive mutants of spt16 (Shimojima, 2003 and references therein).

The most interesting finding in this study is the involvement of the GAGA factor-dFACT complex in the regulation of gene expression. The anterior transformation of T3 and A6 in Deltaspt16 Trl double heterozygotes and the binding of the GAGA factor-dFACT complex to the bxd region of Ubx and the iab-6 element of Abd-B in vivo indicate that the complex contributes to the epigenetic maintenance of Hox gene expression. Based on these data, the following scheme is envisioned for the maintenance of the active state. The GAGA factor-dFACT complex induces chromatin remodeling in the regulatory regions of various GAGA factor-dependent genes and potentiates transcription. Whereas the expression of ftz and hsp70 is transient, the active state is maintained in Hox genes such as Ubx, Scr, and Abd-B with the aid of other trx group gene products (Shimojima, 2003).

What is the mechanism underlying the maintenance? Among trx group proteins, BRM constitutes an SWI/SNF-type chromatin remodeling complex. This type of chromatin remodeler possesses a unique ability to act on condensed mitotic chromatin. A sequence-specific regulator, Zeste, has been shown to recruit the BRM complex to its target sites. Functionally distinct chromatin remodeling induced by the GAGA-dFACT and Zeste-BRM complexes may be important to keep the active state through many rounds of cell cycle. In addition to the GAGA factor-dFACT and the BRM complexes, three trx group protein complexes have been identified to date. One is TAC1 consisting of Trx, dCBP, and Sbf1, which acetylates core histones in nucleosomes. Mutations in trx or nejire encoding dCBP have been shown to reduce the expression of Ubx. The others are ASH1 and ASH2 complexes. ASH1 also has been known to interact directly with dCBP. These data suggest that acetylation of core histones or other proteins plays a crucial role in the maintenance of the active state. In support of this hypothesis, hyper-acetylation of H4 has been shown to be a heritable epigenetic mark of the active state. The finding that a counteracting Pc group complex ESC/E(Z) contains histone deacetylase RPD3 is also consistent with this hypothesis. Chromatin remodeling induced by the GAGA factor-d-FACT and the Zeste-BRM complexes might be essential for maintenance of the hyperacetylated state of H4 (Shimojima, 2003).

Chromatin reassembly appears to be is facilitated by Spt6 stabilizing nucleosomes via interaction with H3 and H4 and by FACT maintaining a stable interaction with both the remodeled nucleosome and the displaced H2A-H2B dimer

RNA polymerase II (Pol II) transcription through nucleosomes is facilitated in vitro by the protein complex FACT (Facilitates Chromatin Transcription). This study shows that FACT is associated with actively transcribed Pol II genes on Drosophila polytene chromosomes. FACT displays kinetics of recruitment and of chromosome tracking in vivo similar to Pol II and elongation factors Spt5 and Spt6. Interestingly, FACT does not colocalize with Pol III-transcribed genes, which are known to undergo nucleosome transfer rather than disassembly in vitro. These observations are consistent with FACT being restricted to transcription that involves nucleosome disassembly mechanisms (Saunders, 2003).

Nucleosomes are inhibitory to transcription. One mechanism of inhibition is by blocking the path of the elongating RNA polymerase. In eukaryotes, three different RNA polymerases exist: Pol I, II, and III. Studies in vitro suggest that at least two polymerases, Pol III and II, have distinct mechanisms by which they transcribe through nucleosomes. Pol III is able to transcribe chromatin under physiological conditions in a purified system, whereas Pol II transcription requires nucleosome disassembly (Saunders, 2003).

The Drosophila heat shock gene hsp70 has promoter and leader regions that are free of nucleosomes, and a promoter-proximal, paused polymerase that prime it for rapid activation. Upon hsp70 gene induction by thermal stress, Pol II encounters nucleosomes downstream of +130. The machinery that enables Pol II to transcribe through these nucleosomes has yet to be characterized. The nucleosome remodeling complexes SWI/SNF and NURF do not appear to play a role during hsp70 transcription elongation. Prime candidates that remain include FACT (Facilitates Chromatin Transcription) and elongation factors Spt5 and Spt6. FACT is a complex that comprises Spt16 and SSRP1 (Orphanides, 1999) and facilitates Pol II elongation through chromatin in vitro (Orphanides, 1998). Spt6 has histone chaperone activity (Bortvin, 1996), and Spt5 and Spt6 show considerable colocalization with the elongating Pol II on Drosophila polytene chromosomes (Andrulis, 2000; Kaplan, 2000). Also Spt5, Spt6, and Spt16 have mutant phenotypes similar to those of histone genes (Saunders, 2003).

To examine whether FACT is positioned to regulate chromatin structure during transcription in vivo, comparative immunofluorescence analysis was performed on Drosophila polytene chromosomes. Immunofluorescence staining reveals the global distribution of FACT (Spt16) relative to the hyperphosphorylated, elongating Pol IIo. Antibodies against either FACT or Pol IIo label many sites on chromosomes prepared from unstressed, developing, third instar larvae, several of which correspond to major transcriptionally active loci (e.g., ecdysone puffs at 74E and 75B). Merging the FACT and Pol IIo images reveals a striking coincidence of FACT with Pol IIo labeled sites. FACT is also at the nucleolus, the site of Pol I transcription. In contrast, FACT is absent from the tandem cluster of 5S ribosomal RNA (rRNA) genes at 56F1-2 that are transcribed by Pol III and known to be strongly stained by antibodies to Pol III-specific transcription factors (Saunders, 2003).

Colocalization of FACT with many Pol II-transcribed genes suggests that FACT is intimately involved in Pol II transcription. This point is further strengthened by the observation that, after a 20-min heat shock, Pol IIo and FACT redistribute to and concentrate at heat shock loci. Pol II is recruited to heat shock loci within seconds, and the first Pol II molecule progresses through the gene in less than 2 min. If FACT facilitates Pol II transcription through nucleosomes at hsp70, then it must be recruited with corresponding rapidity. The fluorescence staining of FACT during a time course after heat shock was examined at the transgenic sites 9D and 61A, which contain just one copy of the hsp70 gene in a known orientation. The distribution was examined of the promoter-restricted heat shock transcription factor, HSF, relative to both FACT subunits, SSRP1 and Spt16, at 9D (the same results were obtained at 61A). HSF and FACT are strongly recruited to the transgenic loci within 2.5 min of heat shock. Even at this early time, FACT resolves from the promoter-associated HSF. The merged image shows a red edge of FACT staining emerging to the right of HSF, indicating that FACT localizes further downstream than HSF. Spt5 and Spt6, factors known to associate with the entire transcription unit, are also recruited within 2.5 min to these heat shock loci. In contrast to HSF, the staining of Spt6 and Spt5 completely overlaps with that of FACT. As activation continues, the chromatin at 9D decondenses further creating a chromosomal 'puff,' and the differential staining of HSF and FACT becomes more apparent, whereas Spt6 still completely overlaps with FACT, and Spt5 mostly overlaps with FACT. Thus, FACT, Spt6, and Spt5 are recruited rapidly to hsp70 upon heat shock, and they associate with the same decondensed regions of the puff (Saunders, 2003).

To examine at higher resolution the temporal and spatial distributions of Pol II, FACT, Spt6, and Spt5 on hsp70, cross-linking and chromatin immunoprecipitation (ChIP) experiments were performed at various times after an instantaneous heat shock. A short (1 min) cross-linking time provided snapshots of the process of transcription elongation. The rapid activation of hsp70 is evident from the detectable recruitment of Pol II to the leader region after a 75-s heat shock. FACT is rapidly recruited to and enriched in the first region of hsp70 that is packaged into nucleosomes at this early time. FACT associates with the 3' end of the gene after 150 s, the same time Pol II is first detected at the 3' region. After a 5-min heat shock, FACT is enriched in the open reading frame (ORF), and the level of FACT in the 3' region is at least eightfold higher than any level of FACT detected upstream (–154). This upstream region is constitutively nucleosome-free, and the level of FACT on it does not increase upon heat shock. These data are consistent with FACT and the Pol II elongation complex cotranslocating along hsp70, starting at the first nucleosome (Saunders, 2003).

Spt6, like FACT, is rapidly recruited to the leader and 5' regions of hsp70 and is first detectable at the 3' end 150 s after heat shock. The greater occupancy of Spt6 in the nucleosome-occupied coding region is consistent with Spt6 being involved in the modulation of the chromatin structure. Overall, the kinetic data place FACT and Spt6 at the correct time and place to contribute to cotranscriptional nucleosome disassembly on hsp70 in vivo. Although no reduction in hsp70 RNA was detected when FACT or Spt6 protein levels are depleted a few fold by RNA interference (RNAi) treatments of Kc cells, these proteins are abundant and may be in excess, especially during heat shock when the general reduction of transcription of most genes presumably increases the availability of elongation factors (Saunders, 2003).

Another factor implicated in the control of transcription through chromatin, Spt5, is also recruited to hsp70 upon heat shock and tracks along the gene with kinetics similar to Pol II, FACT, and Spt6. In contrast to FACT and Spt6, the level of Spt5 associated with hsp70 is less at the 3' end of the gene than at the leader region after a 5-min heat shock. Even before heat shock a strong signal for Spt5 can be detected in the region of the paused polymerase (leader region). Spt5 is known to have a role with the NELF complex in restricting Pol II's elongation early in the transcription cycle and in stimulating the mRNA 5'-capping machinery, activities that require its association with the leader region. Thus, in addition to a positive role in elongation, Spt5 appears to have a role that is both spatially and temporally separate from that of FACT and Spt6 (Saunders, 2003).

Coimmunoprecipitation in Drosophila nuclear extracts provides support for physical associations of Spt5, Spt6, FACT, and elongationally active Pol II. These results are consistent with those from yeast that support the idea that multiple Spt5 complexes exist, one of which is an elongation complex that includes Spt5, Spt6, and FACT (Lindstrom, 2003). Other elongation factors, the Paf1 complex and the chromodomain adenosine triphosphatase (ATPase), Chd1, also show physical and genetic interactions with FACT, indicating that transcription elongation through chromatin in vivo involves a sophisticated molecular machine (Saunders, 2003).

Nucleosome reassembly after transcription-induced disassembly is essential for the integrity of chromatin structure. A link has been established between Spt6 and nucleosome reassembly is known, and recent genetic evidence suggests a similar role for FACT. This study has demonstrated that upon hsp70 induction, FACT and Spt6 are strongly recruited to regions of hsp70 occupied by nucleosomes. Spt6 has been shown to interact with H3 and H4, and FACT with H2A and H2B. In an accompanying paper (Belotserkovskaya, 2003), it is shown that the SSRP1 subunit contacts the H3-H4 tetramer of the disassembled nucleosome, while Spt16 maintains interaction with the displaced H2A-H2B dimer. It is appealing to speculate that chromatin reassembly is facilitated by Spt6 stabilizing the nucleosomes via interaction with H3 and H4 and by FACT maintaining a stable interaction with both the remodeled nucleosome and the displaced H2A-H2B dimer. Whether disassembly or reassembly is the most critical function of FACT in vivo remains an intriguing question (Saunders, 2003).

Drosophila GAGA factor directs histone H3.3 replacement that prevents the heterochromatin spreading

Epigenetic maintenance of the expression state of the genome is critical for development. Drosophila GAGA factor interacts with FACT and modulates chromatin structure for the maintenance of gene expression. This study shows that the GAGA factor-FACT complex (Fact is a heterodimer of dSPT16 and dSSRP1; Shimojima, 2003) and its binding site just downstream from the white gene are crucial for position effect variegation. Interestingly there is a dip of histone H3 Lys 9 methylation and a peak of H3 Lys 4 methylation at this site. The GAGA factor and FACT direct replacement of histone H3 by H3.3 through association of HIRA at this site, and maintain white expression under the heterochromatin environment. Based on these findings it is proposed that the GAGA factor and FACT-dependent replacement of Lys 9-methylated histone H3 by H3.3 counteracts the spreading of silent chromatin (Nakayama, 2007).

This study shows that the GAGA factor-FACT complex is present on the GAGA factor-binding DNase-hypersensitive site d1, a site just downstream from w, and participates in PEV. d1 appears to be a peculiar site where histone H3 K4 methylation peaks and H3 K9 methylation dips, and necessary and sufficient to counteract the heterochromatin spreading. The GAGA factor and FACT contribute to replacement of histone H3 by H3.3 through association of histone H3.3 chaperone HIRA to d1, and maintains w expression under the heterochromatin environment. Based on these data, the following model is proposed for the maintenance of the active state against the spreading of silent chromatin. Heterochromatin is marked by K9-methylated histone H3 and its binding protein HP1, and has a tendency to spread into neighboring regions. Histone H3.3 replacement is thought to be achieved through either eviction of a nucleosome and deposition of a H3.3-containing nucleosome or stepwise disassembly-reassembly without eviction of a nucleosome. Since the GAGA factor-FACT complex facilitates chromatin remodeling and the GAGA factor is known to generate a nucleosome-free region around its binding site, it is most likely that eviction or disassembly of a nucleosome occurs at the DNase-hypersensitive site of d1. The GAGA factor and FACT participate association of HIRA to d1 and the histone replacement would be accomplished by subsequent deposition or reassembly of a H3.3-containing nucleosome. This process would be repeated constantly to eliminate K9-methylated histone H3 at d1 and counteract the spreading of silent chromatin (Nakayama, 2007).

It has been reported that histone H3.3 replacement is triggered by transcription elongation. However, genome-wide profiling has shown histone H3.3 replacement from upstream of to downstream from transcription units. Although some of the replacement may be explained by elongation during intergenic transcription, the histone H3.3 replacement at d1 appears to occur independent of transcription elongation. Thus, the present study indicates a distinct pathway for histone H3.3 replacement (Nakayama, 2007).

Transcription of the w adjacent gene CG32795 has been reported to start immediately after the GAGA factor-binding sequence of d1, suggesting that d1 is a part of the promoter region of CG32795. Therefore, the effect was examined of Trl and spt16 mutations on expression of CG32795. The reduction of a single dose of Trl or spt16 affect the CG32795 expression in the wm4 context but not in the w+ context. These data are consistent with the idea that d1 is a functional promoter element of CG32795 in the w+ context, although Trl and spt16 become haplo-insufficient only when the accessibility of the GAGA factor-FACT complex to d1 decreased under the heterochromatin environment. This raises the possibility that the protection from heterochromatin spreading by the GAGA factor and FACT at d1 is a consequence of their function within the CG32795 promoter. However, conventional promoters do not have a barrier function against heterochromatin silencing. For example, the presence of GAL4 (or E2F) on a promoter carrying GAL4 (or E2F)-binding site did not modify PEV of the attached reporter gene. Genome-wide profiling of H3.3 replacement in Drosophila has revealed the clear dip of H3.3-containing nucleosomes at immediately upstream of the transcription start sites of active genes. This is in sharp contrast with the case of d1, where peaks were observed of both the H3.3/H3 ratio and the actual H3.3 level, and illuminates the difference between d1 and ordinary promoters. Furthermore, the GAGA factor-dependent histone H3.3 replacement was detected also at the DNase HS1 in the Fab-7 boundary of Abd-B, where no promoter activity has been demonstrated. These findings indicate that the GAGA factor and FACT-dependent histone H3.3 replacement can occur without promoter functions. Nevertheless, the barrier function could be assisted by the putative promoter activity of d1 such as formation of a transcription initiation complex (Nakayama, 2007).

The GAGA factor-binding sequence at d1 consists of (GA)8. Since GAGA factor forms an oligomer through its BTB domain, the factor can make a cooperative and stable binding to closely spaced GAGAG elements. This is presumably the reason why d1 gave a prominent signal among the GAGAG sequences around w in the ChIP assay. Because the GAGA factor occupies many closely spaced GAGAG sequences within the Drosophila genome including the Polycomb/trithorax response elements of Hox genes, the proposed mechanism may operate not only in loci juxtaposed with heterochromatin but also in other loci such as the regulatory regions of Hox genes. Indeed GAGA factor and FACT-dependent histone H3.3 replacement were observed in the Fab-7 boundary of Abd-B. High levels of histone H3.3 have been also reported at the locus control region of the chicken folate receptor gene, suggesting that the barrier function against the chromatin silencing via histone H3.3 replacement may be evolutionarily conserved up to vertebrates (Nakayama, 2007).

Developmental arrest and ecdysteroid deficiency resulting from mutations at the dre4 locus of Drosophila

Loss-of-function mutations of the dre4 gene of Drosophila caused stage-specific developmental arrest, the stages of arrest coinciding with periods of ecdysteroid (molting hormone) regulated development. Nonconditional mutations resulted in the arrest of larval development in the first instar; embryogenesis was not impaired, and mutant larvae were behaviorally normal and long-lived. At 31 degrees the temperature-sensitive dre4e55 allele caused the arrest of larval development in the first or second instars. When upshifted to 31 degrees at various times during development, dre4e55 mutants exhibited nonpupariation of third-instar larvae, failure of pupal head eversion, failure of adult differentiation, or noneclosion of pharate adults. Under some temperature regimens second-instar larvae pupariated precociously without entering the normally intervening third-instar. Nonpupariation and defects in metamorphosis were associated with the reduction or elimination of ecdysteroid peaks normally associated with late-larval, prepupal, pupal and pharate adult development. Ecdysteroid production by larval ring glands from dre4e55 hemizygous larvae was suppressed after 2 hr of incubation in vitro at 31 degrees, indicating autonomous expression of the dre4 gene in the ring gland. It is postulated that the dre4 gene is required for ecdysteroid production at multiple stages of Drosophila development and that the pathologies observed in dre4 mutants reflect developmental consequences of ecdysteroid deficiency (Sliter, 1992; full text of article).


Andrulis, E. D., Guzman, E., Doring, P., Werner, J. and Lis, J. T. (2000). High-resolution localization of Drosophila Spt5 and Spt6 at heat shock genes in vivo: roles in promoter proximal pausing and transcription elongation. Genes Dev. 14: 2635-2649. PubMed ID: 11040217

Belotserkovskaya, R. et al. (2003). FACT facilitates transcription-dependent nucleosome alteration. Science 301: 1090-3. PubMed ID: 12934006

Bortvin, A. and Winston, F. (1996). Evidence that Spt6p controls chromatin structure by a direct interaction with histones. Science 272: 1473-6. PubMed ID: 8633238

Formosa, T., Eriksson, P., Wittmeyer, J., Ginn, J., Yu, Y., and Stillman, D.J. (2001). Spt16-Pob3 and the HMG protein Nhp6 combine to form the nucleosome-binding factor SPN. EMBO J. 20: 3506-3517. PubMed ID: 11432837

John, S., Howe, L., Tafrov, S. T., Grant, P. A. Sternglanz, R. and Workman, J. L. (2001). The something about silencing protein, Sas3, is the catalytic subunit of NuA3, a yTAFII30-containing HAT complex that interacts with the Spt16 subunit of the yeast CP (Cdc68/Pob3)-FACT complex. Genes Dev. 14: 1196-1208. PubMed ID: 10817755

Kaplan, C. D., Morris, J. R., Wu, C. and Winston, F. (2000). Spt5 and spt6 are associated with active transcription and have characteristics of general elongation factors in D. melanogaster. Genes Dev. 14: 2623-2634. PubMed ID: 11040216

Lindstrom, D. L., et al. (2003). Dual roles for Spt5 in pre-mRNA processing and transcription elongation revealed by identification of Spt5-associated proteins. Mol. Cell. Biol. 23(4): 1368-78. PubMed ID: 12556496

Nakayama, T., et al. (2007). Drosophila GAGA factor directs histone H3.3 replacement that prevents the heterochromatin spreading. Genes Dev. 21: 552-561. PubMed ID: 17344416

Okada, M. and Hirose, S. (1998). Chromatin remodeling mediated by Drosophila GAGA factor and ISWI activates fushi tarazu gene transcription in vitro. Mol. Cell. Biol. 18(5): 2455-2461. PubMed ID: 9566866

Orphanides, G. LeRoy, G., Chang, C., Luse, D. S. and Reinberg, D. (1998). FACT, a factor that facilitates transcript elongation through nucleosomes. Cell 92: 105-16. PubMed ID: 9489704

Orphanides, G., Wu, W.H., Lane, W.S., Hampsey, M., and Reinberg, D. (1999). The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins. Nature 400: 284-288. PubMed ID: 10421373

Shimojima, T., et al. (2003). Drosophila FACT contributes to Hox gene expression through physical and functional interactions with GAGA factor. Genes Dev. 17: 1605-1616. PubMed ID: 12815073

Sliter, T.J. and Gilbert, L.I. (1992). Developmental arrest and ecdysteroid deficiency resulting from mutations at the dre4 locus of Drosophila. Genetics 130: 555-568. PubMed ID: 1551577

Xiao, H., Sandaltzopoulos, R., Wang, H. M., Hamiche, A., Ranallo, R., Lee, K. M., Fu, D., and Wu, C. (2001). Dual functions of largest NURF subunit NURF301 in nucleosome sliding and transcription factor interactions. Mol. Cell 8: 531-543. PubMed ID: 11583616

Biological Overview

date revised: 13 December 2007

Home page: The Interactive Fly © 2009 Thomas Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.