Promoter Structure

bcd transcription is controlled by Serendipity delta (SRY delta), a zygotic-lethal zinc finger protein. Two overlapping sites binding the SRY delta protein were identified in the bcd promoter region, a few base pairs upstream of the putative TATA box. Mutating one site impairs bcd transcription in vivo, indicating that SRY delta acts directly upstream of bcd. The specific requirement of SRY delta for bcd transcription in the female germ line constitutes an unexpected link between a zygotic gene with pleiotropic functions and the establishment of coordinates of the Drosophila egg. It highlights the fundamental role of ubiquitous transcription factors in bringing about a specific developmental program (Payre, 1994).

Concentration of maternal Bicoid establishes the anterior pattern in the Drosophila embryo. Successive deletions in the bcd promoter allow the localization of an enhancer sequence in the 5'-UTR and a down-regulating element downstream of the ATG initiator codon, and the identification a 49 bp region sufficient to drive transcription of a reporter gene, specifically in nurse cells. This fragment contains two binding sites for the Serendipity (Sry) delta zinc finger activator that mediate its cooperative binding. Both sites (sdbs) are essential for bcd expression. Mutation of either of the two sites, termed sdbsA and sdbsB, results in the reduction of BCD transcript accumulation to undetectable levels, demonstrating that both Sry delta binding sites present in the proximal bcd promoter region are essential for bcd transcription. Together with the fact that Sry delta forms homodimers in solution, these in vivo results indicate that activation of bcd transcription requires cooperative binding of Sry delta homodimers mediated by the two sites present in the proximal promoter region. Further analysis has shown that the bcd promoter configuration is decisive for Sry delta activating function. Replacement of sdbs by binding sites for Sry beta, the Sry delta paralog, restores bcd transcription in sry delta mutant ovaries, demonstrating that the functional divergence between these two proteins during evolution was mainly driven by changes in their DNA-specific recognition properties, resulting in the control of separate developmental pathways (Ruez, 1998).

Transcriptional Regulation

Misexpression of Nanos protein at the anterior of the embryo prevents translation of the anterior morphogen Bicoid, suppressing head and thorax development (Gavis, 1994).

The Serendipity (Sry) delta zinc finger protein controls bicoid gene expression during Drosophila oogenesis. In addition, sry delta mutants display various zygotic phenotypes, ranging from abnormal embryogenesis to sex-biased adult lethality. Sry delta is a sequence-specific transcriptional activator. A single Sry delta consensus binding site (SDCS), in either orientation, is sufficient to promote transcription activation in cell culture, and multiple SDCSs mediate a strong synergistic activation, reflecting the cooperativity of Sry delta binding to DNA. Further, several lines of evidence strongly suggest that Sry delta binds to DNA as a dimer. While each of three point mutations located in the third zinc finger of Sry delta drastically reduces its DNA binding affinity, a fourth mutation, located in the N-terminal region of the protein, specifically affects the cooperativity of DNA binding. This mutation reveals the functional importance of a putative Cys2/Cys2 zinc finger motif of a novel type, located outside the DNA binding domain. A systematic deletion analysis shows that interaction between this proposed Cys2/Cys2 motif and a classical Cys2/His2 zinc finger mediates homodimerization, which is required for DNA binding cooperativity (Payre, 1997).

Susceptibility of bicoid to RNAi

Gene silencing by double-stranded RNA is a widespread phenomenon called RNAi, involving homology-dependent degradation of mRNAs. RNAi is established in the Drosophila female germ line. mRNA transcripts are translationally quiescent at the arrested oocyte stage and are insensitive to RNAi. Upon oocyte maturation, transcripts that are translated become sensitive to degradation while untranslated transcripts remain resistant. Mutations in aubergine and spindleE, members of the PIWI/PAZ and DE-H helicase gene families, respectively, block RNAi activation during egg maturation and perturb translation control during oogenesis, supporting a connection between gene silencing and translation in the oocyte (Kennerdell, 2002).

To analyze the effects of dsRNA on mRNA stability in Drosophila oocytes, dsRNAs corresponding to the maternally expressed genes bicoid and hunchback were used. These genes were chosen because their mRNAs are synthesized, processed, and localized to the cytoplasm of oocytes during mid- to late oogenesis. To test the sensitivity of bicoid and hunchback to RNAi, fertilized eggs were initially injected with dsRNA. bicoid dsRNA reduces the expression of Bicoid protein and induces a bicoid loss-of-function phenotype in which embryos have partial transformation of anterior structures to posterior identities. The effect is robust enough that dsRNA-coated gold particles randomly introduced into fertilized eggs by a gene gun generate mutant phenotypes. hunchback dsRNA induces phenotypes in which embryos are missing thoracic and head segments. These phenotypes resemble mutant embryos generated when maternal and zygotic hunchback gene activity is reduced. To determine if dsRNA injection causes mRNA degradation, endogenous mRNA levels were measured using a semiquantitative RT-PCR assay. The level of bicoid mRNA was reduced about fourfold 40 min after injection of bicoid dsRNA. Likewise, injection of hunchback dsRNA resulted in a reduction of hunchback mRNA levels. Coinjection of a pan-specific ribonuclease inhibitor, vanadyl-ribonucleoside, with bicoid dsRNA results in no reduction of bicoid mRNA, indicating the effect requires a ribonuclease activity (Kennerdell, 2002).

Whether and when transcripts become sensitive to dsRNAs during oogenesis was determined. dsRNA was injected into staged oocytes and their consequent levels of bicoid and hunchback mRNAs were examined. Although oocytes earlier than stage 14 could not be injected, stage 14 oocytes could be examined for RNAi activity. Levels of bicoid and hunchback mRNAs were unchanged in stage 14 oocytes after injection of dsRNA, indicating that oocytes at this stage are unable to carry out RNAi (Kennerdell, 2002).

Oocytes of most animals arrest at species-specific stages of meiosis while differentiation of the oocytes occurs. Drosophila oocytes arrest transiently in prophase I while the oocytes are loaded with RNAs and proteins. Some of these molecules are differentially localized within the oocyte, imparting positional information to be used for embryonic axis formation. When Drosophila oocytes reach stage 14, they undergo meiotic arrest once more, this time at metaphase I. These arrested oocytes remain translationally quiescent in the ovary, potentially for weeks. Arrest is relieved as in most animal eggs by the process of maturation or activation that precedes fertilization. In the case of Drosophila, it appears that ovulation triggers activation of the oocyte to resume meiosis. When oocytes are activated, meiosis is completed and translation of maternal RNAs is dramatically elevated. Shortly thereafter, the oocyte is fertilized as it passes into the uterus (Kennerdell, 2002).

RNAi-like effects are not detected in arrested stage 14 oocytes injected with dsRNA. Was this a general feature of the female germ line? To explore this issue, dsRNA was injected into mature activated oocytes. Injection of dsRNA causes reduction in bicoid and hunchback mRNA levels comparable to those seen in embryos. To confirm that mRNA sensitivity to dsRNA is strictly coincident with oocyte maturation, arrested stage 14 oocytes were isolated from dissected ovaries and the oocytes were activated in vitro. This maturation procedure reactivates meiosis, mRNA translation, and vitelline membrane cross-linking. After maturation, oocytes were injected with bicoid dsRNA and assayed for bicoid mRNA levels. These oocytes showed a decrease in bicoid mRNA. Thus, immature Drosophila oocytes that are coordinately blocked for meiosis and translation are resistant to RNAi, and the block to these processes can be released by maturation or activation of oocytes (Kennerdell, 2002).

There are several possible ways in which RNAi might be blocked in arrested oocytes. One possibility is that an essential component of the RNAi machinery might be missing at this stage. Oocyte maturation would then involve synthesis of the component. To address if synthesis of a missing component is responsible, oocytes were activated in the presence of the protein synthesis inhibitor cycloheximide. Arrested stage 14 oocytes were preincubated with cycloheximide and then activated in vitro in the presence of cycloheximide. This treatment inhibits >95% of the protein synthesis that occurs during maturation. These oocytes were injected with bicoid dsRNA and, strikingly, they showed a decrease in bicoid mRNA levels that was comparable to that of normal mature oocytes. RNAi is established during oocyte maturation even when protein synthesis is blocked. Thus, RNAi establishment during oocyte maturation does not likely occur by synthesis of an essential protein component of the RNAi machinery (Kennerdell, 2002).

The stage 14 oocyte is coordinately blocked in both translation and RNAi. The two processes are released near simultaneously from this block, suggesting perhaps that a shared mechanism links their regulation. To test this possibility, the effectiveness of dsRNA was examined against a transcript that is present but not translated after oocyte maturation. The alphaTubulin67C gene encodes one of three alpha-tubulin proteins synthesized during oogenesis and embryogenesis. Transcript accumulates and is actively translated in early immature oocytes. However, after oocyte maturation, no translation of alphaTubulin67C mRNA occurs, even though transcripts at this stage are associated with ribosomes and are competent to drive translation in vitro. The stable pool of alphaTubulin67C mRNA is comparable to levels of bicoid and hunchback mRNA in mature oocytes. When two nonoverlapping dsRNAs against alphaTubulin67C transcript were independently injected into mature activated oocytes, no destruction of mRNA was detected. This suggests that the ability of dsRNAs to destroy transcripts during oogenesis is coupled to the translation activity of the transcript. Successful translation of transcripts is perhaps necessary to link a transcript to dsRNA-triggered degradation (Kennerdell, 2002).

Several Drosophila genes have been identified that affect translation of maternal mRNAs during oogenesis. One of these genes, aubergine (aub), encodes a protein with a PIWI and PAZ domain. To determine whether Aub has any role for RNAi in oocytes, the effect of aub mutations on RNAi activity was examined. bicoid and hunchback dsRNAs were injected into aub mutant oocytes that were activated in vitro. Degradation of bicoid and hunchback mRNAs was not observed in aub mutants, indicating that Aub is necessary for germ-line RNAi. Two independent aub alleles in heteroallelic combination produced the same result, indicating that the effect was not due to the influence of linked modifiers (Kennerdell, 2002).

The aub gene is a member of a family of genes implicated in RNAi and PTGS. Indeed, aub has been implicated in PTGS regulation of the Stellate repeats and Su(Ste) genes on X and Y chromosomes. Another member of the family, piwi, has been implicated in PTGS within somatic cells. A third family member, Ago2, is a subunit of the mRNA-cleaving complex that mediates RNAi in Drosophila embryonic cells. Thus, several members of this gene family in Drosophila have been implicated in RNAi and PTGS at various steps (Kennerdell, 2002).

It was of interest to determine if other translational regulatory genes are involved in RNAi. To test this possibility, two genes that possibly act through interactions with RNA were examined. vasa and spindle-E (spn-E) encode DexH-box RNA helicases. When activated spn-E mutant oocytes were injected with bicoid or hunchback dsRNAs, no reduction in cognate mRNA levels occurred. In contrast, activated vasa mutant oocytes injected with bicoid dsRNA were found to show transcript degradation comparable to wild type. It is concluded that activation of RNAi in oocytes is dependent on the activity of Spn-E but not Vasa (Kennerdell, 2002).

Arrested Drosophila oocytes are unable to generate RNAi silencing of endogenous maternal mRNAs, but selectively establish this capability upon egg maturation. How is RNAi activated by egg maturation? It is argued that RNAi is linked in some way to translation of maternal mRNAs, which is also specifically activated by egg maturation. Establishment of RNAi is probably not caused by translation of a missing RNAi component. Rather, the complete RNAi apparatus may be present and poised for action but is unable to target homologous substrate mRNAs until egg maturation. Translational masking of mRNAs, a mechanism that operates on maternal Drosophila gene expression, may conceivably be one way in which mRNA is blocked from RNAi attack. Alternatively, targeting of mRNA might require transcripts be assembled onto active polysomes. This may be the case, because siRNA-containing RISC complexes physically fractionate with polysomes, and siRNAs associate with polysomes in Trypanosoma brucei. There is no evidence to indicate that dsRNA-targeting requires ribosome translocation on transcripts, because it is found that cycloheximide inhibition of ribosome translocation does not block RNAi activity in activated mature oocytes (Kennerdell, 2002).

Coupling RNAi to translated mRNA might facilitate base-pairing interactions between siRNAs and an unfolded mRNA target, or it might simply be a means to mark RNAs to be scanned for destruction. The key evidence suggesting that transcript translation is linked to transcript degradation by RNAi comes from experiments in which dsRNA against the alphaTubulin67C message was tested. dsRNA is ineffective against the untranslated alphaTubulin67C transcript in mature activated oocytes, which are nevertheless competent to carry out RNAi against translated bicoid and hunchback transcripts. Thus, there is a correlation between the ability of a transcript to be translated and its ability to be destroyed by dsRNA (Kennerdell, 2002).

Thus Aub and Spn-E are required for RNAi in Drosophila oocytes. Aub and Spn-E might play a specific role in gene silencing mechanisms, including RNAi, that nevertheless have a widespread impact on many features of development. Alternatively, Aub and Spn-E could be required for RNAi because they activate translation of germ-line transcripts including those for bicoid and hunchback. Although there is no evidence for translational control of bicoid mRNA in aub mutants, these mutants may perturb steps in the translation of transcripts that are essential for triggering RNAi. Future experiments should define the specific roles for Aub and Spn-E in dsRNA-mediated destruction and its relationship to translation control (Kennerdell, 2002).

Modulation of temporal dynamics of gene transcription by activator potency in the Drosophila embryo

The Drosophila embryo at the mid-blastula transition (MBT) experiences a concurrent receding of a first wave of zygotic transcription and surge of a massive second wave. It is not well understood how genes in the first wave become turned off transcriptionally and how their precise timing may impact embryonic development. This study perturbed the timing of the shutdown of Bicoid (Bcd)-dependent hunchback (hb) transcription in the embryo through the use of a Bcd mutant that has a heightened activating potency. A delayed shutdown increases specifically Bcd-activated hb levels that alter spatial characteristics of the patterning outcome and cause developmental defects. This study thus documents a specific participation of the maternal activator input strength in timing molecular events in precise accordance with the MBT morphological progression (Liu 2015).

A fundamental feature of animal development is the control of gene expression to achieve specific spatial and temporal patterns in a highly coordinated way. There are two aspects of the temporal dynamics of a gene's transcription in a developmental system, its onset and duration. Proper control of the temporal dynamics of transcription is particularly important for developmental systems that progress rapidly, such as the early Drosophila embryo. It is well known that alterations in transcription activation of early zygotic genes can cause morphological defects in the Drosophila embryo. The results show that the timing of hb transcription shutdown is associated with the MBT and can be perturbed specifically in embryos containing a Bcd mutant defective in sumoylation. A postponement of hb shutdown at nc14 can elongate the duration of active transcription, leading to increased levels of hb gene products and patterning defects. These results show that the precise timing in the shutdown phase of Bcd-activated hb transcription at nc14 is important for normal development. The effects of the Bcd sumoylation mutant on hb shutdown are highly specific, and they are restricted to the shutdown phase (without affecting the onset phase) only at nc14 and only on Bcd-activated hb transcription. It should be noted that the current results do not show that Bcd sumoylation is a temporally regulated event during the MBT, although it represents an attractive possibility that remains to be tested in the future. Temporally regulated Bcd sumoylation could directly account for the temporally restricted effects of the Bcd mutant, but 'constitutive' Bcd sumoylation can also exert time-dependent actions in association with the global events of the system (e.g., mitotic cycles and morphological progression of the MZT) (Liu 2015).

The results provide new insights into how Bcd-activated hb transcription becomes shut down at nc14. Evaluations of the bcd6-lacZ reporter gene demonstrate that neither the P2 promoter of hb nor any of its cis-regulatory elements is required for the shutdown of Bcd- activated transcription at nc14. Importantly, hb shutdown takes place at a time when the Bcd concentration gradient remains intact. It has been suggested that specific pathways can become activated at the MBT to cause a quick degradation of maternal proteins such as Twine. If hb shutdown were to merely reflect a decaying Bcd gradient at nc14, a shutdown process would be expected that initiates near mid-embryo (where Bcd concentration is low) and 'spread' toward the anterior (with increasing Bcd concentrations). But the results do not support this prediction. In addition, neither the length constant nor the amplitude of the Bcd gradient profile is affected by the Bcd mutation. Thus the results show that the timing of Bcd-activated transcription during nc14 does not require either a physical disappearance of this maternal activator or the accumulating activities of sequence-specific zygotic repressors. Instead it is the functional potency of the maternal activator Bcd that is a part of the mechanism in timing the molecular events in accordance with the MZT morphological progression. Importantly, the potency of Bcd activator can be either strengthened (this paper) or weakened to tune--in opposite directions--the hb shutdown timing and hb expression level. It is noted that the bcd6-lacZ reporter results do not formally exclude the possibility that the shutdown of Bcd-activated transcription at nc14 involves a zygotic repressor(s) that operates by competing with Bcd binding to its DNA sites. But this possibility is not favored because the position-independent and quick features of the shutdown event would likely require an unknown zygotic repressor(s) not only to have the same/overlapping Bcd binding specificity but also to accumulate in a spatially non-restricted (i.e., covering the entire hb expression domain) and temporally sudden way at nc14 (Liu 2015).

As part of the receding of the first wave of zygotic transcription in association with the MBT, hb is among a group of genes that exhibit a shutdown phase at nc14. These genes play key roles in different processes that are ongoing during the MBT, suggesting a possibility that a global or general mechanism may regulate the shutdown events of transcription in a coordinated manner. This study shows that the timing of the shutdown can be postponed by an elevated activating potency of Bcd, but only to a degree. For genes that are activated by combinatorial sets of maternal inputs, it remains to be determined whether it is also the activators' functions, as opposed to protein availability, that are regulated during transcription shutdown at the MBT. Genes that are transcriptionally active prior to the MBT tend to share promoter features that are distinct from those of the genes that become activated during the MBT. An intriguing possibility exists where the MBT might be associated with a systematic change in the composition of the transcription machinery. But the fact that a synthetic reporter containing a different core promoter also exhibits a shutdown phase at nc14 indicates that the hb P2 promoter is not required (Liu 2015).

A recent study reveals an interplay between zygotic transcription and DNA replication at the MBT. It has been proposed that euchromatin DNA is replicated within a few minutes into the nc14 interphase. Thus the DNA replication time coincides broadly with the time of hb shutdown, raising a question of whether DNA replication at the hb locus might trigger its transcription shutdown. A single embryo was captured in which nuclei contain more than two intron dots. The existence of nuclei with more than two dots is a positive indicator of DNA replication at the hb gene locus. The strong intron staining detectable at the anterior part of the embryo indicates that Bcd-activated hb transcription has not yet been turned off (nuclear height measurements suggest that this embryo belongs to time class t2). These results thus suggest that DNA replication at the hb locus does not directly trigger its transcription shutdown at nc14 (Liu 2015).

Sumoylation is a posttranslational modification that regulates a variety of biological processes through mechanisms that may involve protein-protein interactions, subcellular localization, and protein stability. From the perspective of developmental biology, many transcriptional activators with important developmental roles are substrates of sumoylation. It has been reported that sumoylation of Medea (Med), an intracellular transducer of Drosophila morphogen Decapentaplegic (Dpp), triggers Med nuclear export and therefore, restricts the range of the Dpp signaling. The lengthening of the duration of Bcd-activated hb transcription caused by the Bcd sumoylation mutation increases the amplitude of hb expression without extending its expression boundary. Thus, sumoylation of proteins involved in morphogen functions can alter either the action range (in space) or the output level (due to action time). In yeast, sumoylation has been suggested to play a role in terminating inducible activation events by evicting activator molecules from promoters. For example, disruption of Gcn4 sumoylation can extend its promoter association and increase the expression level of the target gene ARG1. Whether sumoylation of Bcd plays a mechanistically equivalent role in evicting Bcd molecules from the hb enhancer at nc14 remains an open question and speculative possibility (Liu 2015).

bicoid: Biological Overview | Evolutionary Homologs | Targets of Activity | Protein Interactions | Miscellaneous Interactions | Developmental Biology | Effects of Mutation | References

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