fushi tarazu

Transcriptional Regulation

The ftz pattern is directed by at least two independent regulatory systems: first, stripe establishment is directed by regionally distributed factors that act differentially in individual stripes along both anterior-posterior and dorsal-ventral axes of the egg and, second, stripe refinement and maintenance are mediated by pair-rule gene products that interact with identified ftz regulatory elements.

The Tramtrack protein has been proposed as a maternally provided repressor of the fushi tarazu gene in Drosophila embryos at the preblastoderm stage. Consistent with this hypothesis, the TTK protein is present in preblastoderm embryos. This is followed by a complete decay upon formation of the cellular blastoderm when ftz striped expression is at its peak. In addition, the highly complex pattern of zygotic ttk expression suggests specific functions for ttk late in development that are separate from the regulation of ftz. Ectopic ttk causes complete or near-complete repression of the endogenous ftz gene, as well as significant repression of the pair-rule genes even skipped, odd skipped, hairy and runt (Brown, 1993).

Initiation of ftz transcription is a gradual process that is mediated by titration of TTK protein. When the local concentration of nuclei is experimentally altered, using a mutation that makes the migration of nuclei to the egg cortex uneven, ftz is activated only in nuclei that attain a high local density at the cortex (Pritchard, 1996).

The asymmetric distribution of the gap gene knirps (kni) in discrete expression domains is critical for striped patterns of pair-rule gene expression in the Drosophila embryo. To test whether these domains function as sources of morphogenetic activity, the stripe 2 enhancer of the pair-rule gene even-skipped was used to express kni in an ectopic position. Manipulating the stripe 2-kni expression constructs and examining transgenic lines with different insertion sites led to the establishment of a series of independent lines that display consistently different levels and developmental profiles of expression. Individual lines show specific disruptions in pair-rule patterning that are correlated with the level and timing of ectopic expression (Kosman, 1997).

It is likely the KNI functions as a repressor to set the posterior border of eve stripe three. To test whether the early repression of eve stripe 3 is mediated through the eve stripe three enhancer, stripe 2-kni constructs were crossed with a line carrying lacZ under the control of this enhancer. Ectopic kni specifically represses the stripe 3 enhancer in a dose-dependent manner. Stripe 2-kni causes disruption of runt stripes 2 and 3, but has no effect on stripe 1. The repression of stripe 3 increases in proportion to the level of ectopic kni, a response similar to that seen for eve stripe 3. Different levels of ectopic kni cause disruptions of fushi tarazu stripes 2 and 3, but have no effect on the expression of ftz stripe 1. It is possible that these effects are indirect and may be mediated through other segmentation genes but this possibility is made unlikely by the fact that hairy expression is virtually unaffected in stripe 2-kni embryos. These results suggest that the ectopic domain of kni acts as a source for morphogenetic activity that specifies regions in the embryo where pair-rule genes can be activated or repressed. Evidence is presented that the level and timing of expression, as well as protein diffusion, are important for determining the specific responses of target genes (Kosman, 1997).

The order of appearance of FTZ stripes is not inversely correlated with the order of appearance of Hairy stripes as would be expected if FTZ stripes were generated by H repression. Furthermore, the seven FTZ stripes are correctly established in embryos carrying mutations in h, eve or runt, with normal expression patterns decaying only after cellularization. Thus, the so called primary pair-rule genes are involved in the refinement rather than establishment of the FTZ stripes. Cis-acting regulatory elements, both the zebra and upstream elements, interact to generate seven correctly positioned stripes by the end of cellularization. However, stripe establishment is not correctly mimicked by any ftz/lac fusion gene: stripes arise in an order drastically different from the endogenous ftz gene suggesting the existence of ftz regulatory elements outside the 10-kb region examined to date (Yu, 1995).

Do Hairy and Runt repress target gene transcription independently of DNA binding, or as promoter bound regulators? Hairy-related transcriptional repressors show similar basic and HLH domains, and all terminate with an identical C-terminal tetrapeptide (WRPW), mutations of which largely or completely abolish repressor activity. It has proved difficult to define the precise molecular mechanism of Hairy action during segmentation. Although Hairy's embryonic patterning activity requires an intact basic (DNA binding) domain, none of the sequences in fushi tarazu promoter implicated in ftz repression by Hairy contain Hairy consensus binding sites. It is uncertain whether Runt acts primarily as a gene repressor or activator, as it behaves as a repressor of even-skipped and as an activator of fushi tarazu. In order to explore the ability of Hairy and Runt to act as promoter-bound transcriptional regulators, heterologous transcriptional activation domains (Act) were substituted for the WRPW repression domain (of Hairy) and the activation domain of Runt and the effects of such substitution were examined on presumed targets of Hairy and Runt. Expression of Hairy-Act during the blastoderm stage disrupts embryonic segmentation by driving ectopic expression of ftz, runt and odd-skipped. Activation depends on an intact basic domain, indicating that direct regulation occurs via sequence-specific binding to DNA. Expression of Runt-Act during the blastoderm stage likewise drives ectopic even-skipped, and shows that the normal apparent activation of fushi-tarazu by Runt is indirect, suggesting that Runt acts predominantly as a repressor. Hairy-Act has also been used to study sex determination. Ectopic Hairy mimics the activity of Deadpan in repressing early Sex-lethal transcription. Expression of Hairy-Act activates Sxl and causes male lethality, implying that Deadpan recognizes the Sxl promoter directly, and excludes models for Sxl regulation in which DPN functions as a passive repressor (Jiménez, 1996).

fushi tarazu autoregulates its own expression. In vivo activity of a ftz autoregulatory enhancer element is reduced by mutations of putative ftz-binding sites (Schier, 1992).

Although many of the genes that pattern the segmented body plan of the Drosophila embryo are known, there remains much to learn in terms of how these genes and their products interact with one another. Like many of these gene products, the protein encoded by the pair-rule gene odd-skipped (Odd) is a DNA-binding transcription factor. Genetic experiments have suggested several candidate target genes for Odd, all of which appear to be negatively regulated. Pulses of ectopic Odd expression have been used to test the response of these and other segmentation genes. Three different phenotypes are generated in embryos in which odd is expressed from a heat shock promoter: head defects only, a pair-rule phenotype and a pair-rule phenotype restricted to the dorsal half of the embryo. The head defects only phenotype prevails when Odd is induced between 2:10 and 2:30 hours after egg laying (AEL). The second phenotype is generated when Odd is induced between 2:30 and 2:50 AEL, while the third phenotype prevails when heat shocks are administered between 2:50 and 3:10 AEL. The results are complex, indicating that Odd is capable of repressing some genes wherever and whenever Odd is expressed, while the ability to repress others is temporally or spatially restricted (Dréan, 1998).

One target gene, fushi tarazu, is both repressed and activated by Odd, the outcome depending upon the stage of development. Rather than negatively regulating ftz, ectopic Odd causes a rapid expansion of all 7 ftz stripes. In some embryos, interstripe regions are difficult to discern. Since this activation is observed within 20-25 minutes of Odd induction, it likely reflects a direct interaction between the two genes. Consistent with this positive relationship, initiating ftz stripes are irregular in width and intensity in odd mutant embryos. Stripes 3-6 are the most strongly affected, particularly stripe 4. Odd does appear to be able to repress the ftz gene, but only in the middle region of the ftz parasegment, and only after gastrulation. When Odd switches from an activator to a repressor of ftz, its ability to repress ftz is excluded from the anterior-most cells of each ftz stripe. The inability of Odd to repress ftz in these cells indicates that either a necessary cofactor for Odd is missing in these cells, or that an overriding factor is present. Another possibility is that the levels of Odd required to repress ftz are higher than those that were induced (Dréan, 1998).

If the segmentation defects observed in embryos lacking maternal C-terminal binding protein are due to its interaction with Hairy, disruptions in patterning similar to those found in hairy mutations or loss of maternal Groucho are expected. In particular, the expression of the other primary pair-rule genes are expected to be disrupted and fushi tarazu expression to be derepressed. Consistent with this, Ftz stripes are found to be expanded in embryos lacking maternal dCtBP. However, this broad band of expression later resolves into stripe-specific ftz repression, with stripes 2, 4, 5 and 6 predominantly affected. Aberrant expression of the primary pair-rule gene proteins, Eve and Runt, as well as of Hairy itself are also observed. Since the primary pair-rule genes respond directly to gap gene cues, gap gene expression was examined in embryos lacking maternal dCtBP. Expression of the three gap genes examined, Hunchback, Krüppel and Knirps, appears normal in these embryos. In addition to its effects on anterior-posterior patterning, embryos lacking maternal dCtBP also show disruptions of dorsoventral patterning. Beginning with the expression of the pair-rule genes, a lack of segmentation gene expression is detected on the ventral surface (Poortinga, 1998).

The gene hopscotch (hop) is required maternally for the establishment of the normal array of embryonic segments. In hop embryos, although expression of the gap genes appears normal, there are defects in the expression patterns of the pair-rule genes even-skipped, runt, and fushi tarazu. The effect of hop on the expression of these genes is stripe-specific (Binari, 1994).

odd-skipped represses expression of fushi tarazu, a known activator of engrailed. naked prevents activation of engrailed by fushi tarazu, without affecting fushi tarazu expression, thus restricting engrailed expression to narrow stripes of cells at the anterior boundaries of parasegments (Mullen, 1995).

In the anterior domain Tailless exerts a repressive effect on the expression of fushi tarazu, hunchback, and Deformed (Reinitz, 1990).

The homoeodomain-containing protein encoded by caudal is a regulator of fushi tarazu. The cad gene product can increase the level of ftz transcription in the posterior half of the embryo by interacting with multiple copies of a TTTATG consensus sequence located in the zebra-stripe unit. This result demonstrates one pathway by which the product of a maternally expressed segmentation gene, expressed in an antero-posterior concentration gradient, can directly regulate the expression of a pair-rule gene (Dearolf, 1989).

Pair-rule gene expression is disrupted in Dichaete mutants. Expression of the gap genes Krüppel, knirps, and giant are normal, indicating that Dicaetae acts in parallel or downstream of these gap genes. the so-called primary pair-rule gene even-skipped, Hairy, and runt each show reductions in levels of expression in Dichaete mutants, with variable stripe specific effects on eve, fushi tarazu, hairy and runt. Since the stripes of pair rule genes generally occur in the correct anterior-posterior position in Dichaete mutants, the gene is unlikely to provide key positional information; it is more likely to be required in the maintainance or establishment of appropriate levels of pair-rule gene expression in the central region of the embryo (Russell, 1996 and Nambu, 1996).

Activation of fushi tarazu andeven-skipped expression in ganglion mother cells requires prospero function. Repression of deadpan and asense in ganglion mother cells requires prospero function (Doe, 1991).

A Drosophila Tbp interaction with transcription factors involves a coactivator of the transcription factor FTZ-F1. The coactivator, Multiprotein bridging factor 1 (MBF1), makes possible a connection, or bridges, the TATA box-binding protein (Tbp) and the nuclear hormone receptor FTZ-F1. MBF1 is functional in interactions with Tbp and a positive cofactor MBF2. MBF1 makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the binding of FTZ-F1 to its recognition site. The central region of MBF1 (residues 35-113) is essential for the binding of FTZ-F1, MBF2, and Tbp. MBF1, in the presence of MBF2, and FTZ622 bearing the FTZ-F1 DNA-binding domain, support selective transcriptional activation of the fushi tarazu gene. Mutations that disrupt the binding of FTZ622 to DNA or MBF1, or an MBF2 mutation that disrupts the binding to MBF1, all abolish the selective activation of transcription. These results suggest that tethering the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription (Takemaru, 1997).

A cell free system of Drosophila preblastoderm embryos was devised for the efficient assembly of cloned DNA into chromatin. The chromatin assembly system utilizes endogenous core histones and assembly factors and yield long arrays of regularly spaced nucleosomes with repeat length of 180 bp. Chromatin assembled with the preblastoderm embryo extract is deficient in histone H1, because of the absence of H1 in early embryos. Exogenous H1 can be incorporated during nucleosome assembly in vitro. When chromatin is reconstituted in the presence of H1, an increased nucleosome repeat length from 180 bp to about 197 bp is observed. The larger length is identical to the in vivo repeat length for postblastoderm chromatin. Regular spacing of nucleosomes with or without H1 is sufficient to maximally repress transcription from hsp70 and fushi tarazu gene promoters. There is a modest increase in the level of repression that is dependent on exogenous histone H1. These results show that optimal assembly or regularly spaced nucleosome cores is sufficient to maximally repress transcription in vitro, even in the absence of histone H1 (Becker, 1992).

Regulatory genes directing embryonic development are expressed in complex patterns. The Drosophila homeobox gene fushi tarazu (ftz) is expressed in a striped pattern that is controlled by several discrete and large cis- regulatory elements. One key cis-element is the ftz proximal enhancer, which is required for stripe establishment and which mediates autoregulation by direct binding of Ftz protein. To identify the trans-acting factors that regulate ftz expression and autoregulation, a modified yeast two hybrid screen, the Double Interaction Screen (DIS), was developed. The DIS was designed to isolate both DNA binding transcriptional regulators that interact with the proximal enhancer and proteins that interact with Ftz itself when it is bound to the enhancer. The screen identified two candidate Ftz protein cofactors as well as activators and repressors of ftz transcription that bind directly to the enhancer. One of these [Tramtrack (Ttk)] is known to bind to at least five sites in the proximal enhancer; genetic studies suggest that Ttk acts as a repressor of ftz in the embryo. In yeast cells, Ttk protein strongly activates transcription, suggesting that yeast may be missing a necessary co-repressor that is present in Drosophila embryos. Also characterized was the activity of a second candidate ftz repressor isolated in the screen: the product of the pair-rule gene sloppy paired, a member of the forkhead family. Slp1 is shown in this study to be a DNA binding protein. A high affinity binding site for Slp1 in the ftz proximal enhancer was identified. Slp1 represses transcription via this binding site in yeast cells, consistent with its role as a direct repressor of ftz stripes in interstripe regions during late stages of embryogenesis. The DIS should be a generally useful method used to identify DNA binding transcriptional regulators and protein partners of previously characterized DNA binding proteins (Yu, 1999).

Germ cells in embryos derived from nos mutant mothers do not migrate to the primitive gonad and prematurely express several germline-specific markers. These defects have been traced back to the syncytial blastoderm stage. Pole cells in nos minus embryos fail to establish/maintain transcriptional quiescence; the sex determination gene Sex-lethal (Sxl) and the segmentation genes fushi tarazu and even-skipped are ectopically activated in nos minus germ cells. nos minus germ cells are unable to attenuate the cell cycle and instead continue dividing. Unexpectedly, removal of the Sxl gene in the zygote mitigates both the migration and mitotic defects of nos minus germ cells. Supporting the conclusion that Sxl is an important target for nos repression, ectopic, premature expression of Sxl protein in germ cells disrupts migration and stimulates mitotic activity (Deshpande, 1999).

Soon after formation, wild-type pole cells in Drosophila downregulate RNA polymerase II transcription until they have been incorporated into the primitive gonad. The premature activation of these germline-specific genes is likely to reflect a more general defect in transcriptional regulation that arises early in embryogenesis, soon after the pole cells are formed. Instead of shutting off RNA polymerase II transcription, nos^- pole cells inappropriately transcribe several somatic genes. Why do nos^- germ cells fail to regulate RNA polymerase II transcription? The only known regulatory target for nos in the embryo is the hb transcription factor. Nos together with the Pumilio protein is thought to bind to maternally derived hb mRNA and block its translation. Since Hb protein is produced throughout much of the posterior in the absence of Nos, one possibility is that this gap gene protein activates transcription in the pole cells. However, this explanation does not seem likely. Although hb regulates eve and ftz in the soma, it is not clear that the ectopic expression of only the Hb protein would be sufficient to activate either of these genes in the absence of other factors. A more likely possibility is that nos^- germ cells have a defect in the system responsible for attenuating RNA polymerase II activity (Deshpande, 1999).

Given the potential role of lilliputian as a transcriptional regulator, an examination was performed to see whether the pair-rule phenotype in lilli germ-line clone (GLC) embryos corresponds to changes in the expression of early patterning genes. Examined was the spatiotemporal pattern of mRNA expression for several of these genes: the maternal coordinator gene bicoid; the gap gene hunchback; the pair-rule genes fushi tarazu, even-skipped, hairy, and runt; the segment polarity genes engrailed and wingless and the terminal gap genes tailless and huckebein. The expression patterns of bcd, hb, eve, h and run mRNA appear relatively normal. In contrast, levels of ftz mRNA were significantly lower in lilli GLC embryos than wild-type embryos at the end of cellularization. ftz expression appears normal prior to mid-cellularization, after which its distribution becomes diffuse and uniform, and it rarely accumulates in stripes. Using ftz-lacZ and hb-lacZ transgenes to determine the level of such regulation, it was found that expression of the ftz-lacZ transgene is markedly reduced in lilli GLC embryos, while that of the hb-lacZ transgene remains unimpaired. This suggests that Lilli regulates ftz gene expression at the transcriptional level. Since both ftz and lilli are required for even-numbered En stripes and odd-numbered segment formation, this disruption of ftz expression may account for the pair-rule phenotype observed in lilli GLC embryos (Tang, 2001).

To understand how loss of Grunge activity affects segmentation, the expression of hunchback (hb), Krüppel (Kr), knirps (kni) and fushi tarazu (ftz) was analyzed in embryos derived from Gug³⁵ germline clones fertilized by Gug³⁵ sperm. In wild-type embryos, the expression of these segmentation gene products localizes to discrete domains in the early embryo. In almost all of the expression domains, loss of Gug activity increases the number of cells expressing these segmentation genes, suggesting that Gug plays a role in their repression. Later the expression of ftz displays a more complex defective pattern with some stripes being broader, and others narrower, than wild type (Erkner, 2002).

Loss of Gug activity severely affects the process of segmentation and the expression of segmentation genes when missing from the female germ line. At the blastoderm stage, most of the expression domains of hb, Kr, kni and ftz genes are expanded compared with wild type. These observations indicate that maternal production of Gug is crucial for the repression of these genes to precise domains in the early embryo. Gap proteins, including Hb, Kr and Kni are known to be required to restrict each others domains of expression. It will be interesting to test if Gug acts with these proteins for these repression activities (Erkner, 2002).

Prospero, targeting eve and ftz, acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells

Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).

To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2-5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).

Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10-11 embryos, approximately 4-7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).

Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).

Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).

A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).

The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).

Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).

In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).

The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.

The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).

Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).

To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).

To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).

In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).

Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).

The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).

The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).

Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).

S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).

To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).

Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).

Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).

Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).

Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).

Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).

The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).

It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).

Establishment of cell fate during early Drosophila embryogenesis requires transcriptional Mediator subunit dMED31

During early Drosophila embryogenesis, formation of the anterior-posterior (A/P) axis depends on spatial gradients of maternal morphogens. It is well recognized that positional information is transmitted from these morphogens to the gap genes. However, how this information is being transmitted is largely unknown. The transcriptional Mediator complex is involved in the fine tuning of the signaling between chromatin status, transcription factors and the RNA polymerase II transcription machinery. This study found that a mutation in the conserved subunit of the Mediator complex, dMED31, hampers embryogenesis prior to gastrulation and leads to aberrant expression of the gap genes knirps and Krüppel and the pair-rule genes fushi tarazu and even-skipped along the A/P axis. Expression of the maternal morphogens dorsal and hunchback was not affected in dMED31 mutants. mRNA expression of dMED31 exactly peaks between the highest expression levels of the maternal genes and the gap genes. Together, these results point to a role for dMED31 in guiding maternal morphogen directed zygotic gap gene expression and provide the first in vivo evidence for a role of the Mediator complex in the establishment of cell fate during the cellular blastoderm stage of Drosophila (Bosveld, 2008).

Proper fine tuning of the eukaryotic transcriptome depends on numerous cis and trans acting factors that modulate the chromatin environment of genes and influence the RNA polymerase II (RNAPII) transcription machinery. The Mediator complex is a core processor in the signaling between RNAPII and transcription factors. This complex is an evolutionary conserved protein assembly of 25-30 subunits (Bosveld, 2008).

Support for a specific role during development of Mediator subunits is provided by several studies in Drosophila. These studies describe mostly functions of Mediator subunits during late developmental stages, but a role of the subunits during early embryonic development is largely unknown. The Mediator consists of more then 25 subunits, pointing to a multifaceted role of this complex during metazoa development. Understanding this complexity starts with the identification of the function of each subunit (Bosveld, 2008).

Drosophila MED31 was identified by bioinformatics analysis (Boube, 2000) and its presence in the Mediator complex was confirmed in purified complexes from embryos and cells (Park, 2001; Gu, 2002). In a pull down assay, the Mediator (containing dMED31) complex binds to the transcription factors Bicoid (Bcd), Krüppel (Kr), Fushi tarazu (Ftz), Dorsal (Dl) and HSF, but not Twist (Twi), Hunchback (Hb) and Even-skipped (Eve) (Park, 2001). Moreover, the Mediator complex is required for in vitro transcription from developmentally important promoters regulated by these transcription factors. Despite these in silico and in vitro results, to date the functional role of MED31 in eumetazoa remains elusive. This study reports the identification of the highly conserved Drosophila Mediator subunit dMED31 as a novel maternal-effect gene necessary for proper segment specification during early embryogenesis. dMED31 mutant females have fecundity defects and embryos deposited by homozygous mothers display severe defects along the anterior-posterior (A/P) axis when gastrulation is initiated. Whereas expression of maternal morphogens is not affected, alterations in gap and pair-rule gene expression during the proceeding blastoderm stage correlate with these defects observed in dMED31 mutant embryos. Remarkably, a small percentage of the progeny of homozygous mutant females escape from embryonic death and develop into adults. These escapers have defects in their abdominal segmentation pattern, a phenotype enhanced by mutations in dMED13. These findings provide the first in vivo evidence for a specific role of dMED31 in establishing cell fate in the cellular blastoderm and point to a role for the Mediator in guiding maternal morphogen directed zygotic gap gene expression (Bosveld, 2008).

These findings identify a component of the conserved eukaryotic transcriptional Mediator complex, dMED31, that is required for normal initiation of zygotic gene expression during the blastoderm stage of Drosophila embryogenesis. Female flies that carry a mutation in the dMED31 gene suffer from fecundity defects and the embryos deposited by these females display abnormal embryogenesis due to aberrant cell migration events upon gastrulation. Impaired embryogenesis coincided with changes in kni, Kr, ftz and eve expression along the A/P axis. Furthermore, adult flies derived from embryos that escaped from embryonic death displayed severe defects in their abdominal segmentation. Because mRNA production was hampered in dMED31^1/1, these abdominal defects were likely the result of abnormal maternal and zygotic dMED31 mRNA production. A mutation in the Mediator subunit dMED13 also caused segmentation defects and this mutant enhanced the dMED31 mutant maternal effect phenotype. Therefore, these data indicate that the Mediator complex directs zygotic gene expression upon egg deposition to establish cell fate in the embryonic blastoderm (Bosveld, 2008).

In order to accomplish cell fate determination, cells gain a transcriptional poised state during early embryogenesis that is maintained throughout development and requires many cis and trans acting factors that modulate the chromatin environment of the genes involved. In Drosophila, cell identity along the A/P is established in the blastoderm stage when the pair-rule genes are expressed. A/P polarity is controlled by the maternal morphogenes cad, nos, bcd and hb whose activity results in the spatio-temporal expression of the gap genes gt, kni, tll and Kr. These gap genes are the first genes expressed along the A/P axis and encode transcription factors that in turn govern patterned expression of the pair-rule genes. Pair-rule gene expression occurs in distinct stripes and is accompanied by cellularization. Thus when cellularization takes place, large clusters of cells gain an imprint that defines the primordial segments. Cell identity is fine tuned when expression of the segment polarity and Hox genes is activated. Although this cascade of maternal, gap, pair-rule and segment polarity genes is well studied, much remains unknown how the maternal morphogens regulate RNAPII activity at their cognate promoters in order to establish regional domains of gap gene expression (Bosveld, 2008).

Because segmentation defects in escaper flies derived from dMED31^1/1 mothers were restricted to the abdomen, it is possible that the bithorax complex (BX-C) is abnormally expressed. This complex contains the homeotic genes Ultrabithorax, abdominal A and Abdominal B, which control the identity of the posterior two-thirds of the fly. Mutations in hb, Kr, tll and kni affect expression of BX-C and result in homeotic transformation. No complete homeotic transformations of entire parasegments were observed, suggesting an indirect effect of dMED31¹ on Hox activation. Since segment identity is established during early embryogenesis, this implies that only groups of cells and not whole primordial segments gained abnormal imprinting. Regional errors in cellular imprinting are supported by the variety of the abdominal defects observed in adult flies. Moreover, defects in embryogenesis were accompanied by cell loss at the embryonic poles and aberrant migratory behavior of cells upon gastrulation, processes which occur prior to the activation of the segment polarity and the Hox genes. Finally, early developmental defects coincided with abnormal expression of the gap genes kni and Kr and subsequently the pair-rule genes ftz and eve. These genes are expressed prior to Hox gene expression and are required for activation/repression of the Hox cluster. Although it is possible that the abdominal region is preferentially sensitive for a mutation in dMED31, it is more likely that random defects during formation of the abdomen are tolerated, whereas defects in other regions of the embryo are incompatible with adult viability and these adults never eclose (Bosveld, 2008).

Several intriguing questions remain: why is the embryonic phenotype so variable (>90% of the mutant embryos die, while a small percentage of embryos is able to reach the adult stage), why are mainly embryos affected by a mutation in dMED31 and what is the primary embryonic defect caused by a mutation in dMED31? Answers to these questions can be derived from studies of the Mediator in yeast in combination with the current data. The budding yeast MED31 protein is part of the Mediator transcription initiation complex. Although a mutation in yeast MED31 affects gene expression, mutants display no sensitivity to transcriptional inhibition by 6-azauracil and MED31 is not essential for growth. However, yeast MED31 mutants have a synthetic growth defective phenotype when combined with mutations in genes encoding for the two largest subunits of RNAPII (RPB1, RPB2) and the transcription initiation factors TFIIB and TFIIS. As in yeast, depletion of dMED31 in Drosophila SL2 cells by RNAi does not interfere with the Mediator composition and no growth alterations have been reported. Thus, Drosophila MED31, like yeast MED31, might not be essential for RNAPII activity per se, but could be an auxiliary factor involved in the signaling between specific transcription factors and the RNAPII machinery. Together, these findings and the current data suggest that dMED31 is not required for transcription in general, but is merely required for the fine tuning of transcription of specific genes (Bosveld, 2008).

Based on studies in yeast, it has been proposed that the Mediator functions as a platform that allows rapid regulation of transcription at (re)initiation. Fast regulation and (re)initiation of transcription might be key during the interphase periods of the final syncytial cell cycles when zygotic transcription is initiated, while such large scale, strict and 'fast' control over transcription would not be essential during subsequent stages of development and thus may explain why dMED31 function is essential during early embryogenesis. The observation that a small percentage of embryos derived from dMED31^1/1 mothers is able to develop into an adult, while the majority of the embryos displayed severe defects during embryogenesis, might also be attributed to such auxiliary function(s) of the dMED31 protein. Minor differences in dMED31 protein levels, due to the hypomorphic dMED31¹ allele, may result in subtle changes in the expression of the gap and pair-rule genes and allow embryos to progress throughout embryogenesis, but with the formation of segmentation defects. On the other hand, in the majority of embryos, more severe changes in gap and pair-rule patterning occur, which results in embryonic death (Bosveld, 2008).

In summary, this study demonstrated that dMED31 is essential to establish regional domains of expression of cell fate determinants kni, Kr, ftz and eve. mRNA expression of dMED31 peaks exactly between maternal morphogen and gap gene expression and it has been demonstrated that the Mediator complex is able to bind to several maternal transcription factors. Together this indicates that the Mediator complex constitutes an interface between the maternal morphogens and the RNAPII machinery to guide zygotic gene expression of cell fate determinants that specify primordial segment identity. These findings provide the first in vivo evidence for a role of the Mediator complex in establishing cell fate during early embryogenesis and since MED31 resembles one of the most conserved subunits within the Mediator complex this protein could serve a crucial role in the control of RNAPII activity during early developmental processes in all higher eukaryotes (Bosveld, 2008).

Drosophila ptip is essential for anterior/posterior patterning in development and interacts with the PcG and trxG pathways

Development of the fruit fly Drosophila depends in part on epigenetic regulation carried out by the concerted actions of the Polycomb and Trithorax group of proteins, many of which are associated with histone methyltransferase activity. Mouse PTIP is part of a histone H3K4 methyltransferase complex and contains six BRCT domains and a glutamine-rich region. This study describes an essential role for the Drosophila ortholog of the mammalian Ptip (Paxip1) gene in early development and imaginal disc patterning. Both maternal and zygotic ptip are required for segmentation and axis patterning during larval development. Loss of ptip results in a decrease in global levels of H3K4 methylation and an increase in the levels of H3K27 methylation. In cell culture, Drosophila ptip is required to activate homeotic gene expression in response to the derepression of Polycomb group genes. Activation of developmental genes is coincident with PTIP protein binding to promoter sequences and increased H3K4 trimethylation. These data suggest a highly conserved function for ptip in epigenetic control of development and differentiation (Fang, 2009).

The establishment and maintenance of gene expression patterns in development is regulated in part at the level of chromatin modification through the concerted actions of the Polycomb and trithorax family of genes (PcG/trxG). In Drosophila, Polycomb and Trithorax response elements (PRE/TREs) are cis-acting DNA sequences that bind to Trithorax or Polycomb protein complexes and maintain active or silent states, presumably in a heritable manner. In mammalian cells however, such PRE/TREs have not been conclusively identified. Polycomb and Trithorax gene products function by methylating specific histone lysine residues, yet how these complexes recognize individual loci in a temporal and tissue specific manner during development is unclear. Recently, a novel protein, PTIP (also known as PAXIP1), was identified that is part of a histone H3K4 methyltransferase complex and binds to the Pax family of DNA-binding proteins (Patel, 2007). PTIP is essential for assembly of the histone methyltransferase (HMT) complex at a Pax DNA-binding site. These data suggest that Pax proteins, and other similar DNA-binding proteins, can provide the locus and tissue specificity for HMT complexes during mammalian development (Fang, 2009).

In mammals, the PTIP protein is found within an HMT complex that includes the SET domain proteins ALR (GFER) and MLL3, and the accessory proteins WDR5, RBBP5 and ASH2. This PTIP containing complex can methylate lysine 4 (K4) of histone H3, a modification implicated in epigenetic activation and maintenance of gene expression patterns. Furthermore, conventional Ptip^-/- mouse embryos and conditionally inactivated Ptip^-/- neural stem cell derivatives show a marked decrease in the levels of global H3K4 methylation, suggesting that PTIP is required for some subset of H3K4 methylation events (Patel, 2007). The PTIP protein contains six BRCT (BRCA1 carboxy terminal) domains that can bind to phosphorylated serine residues. This is consistent with the observation that PAX2 is serine-phosphorylated in response to inductive signals. In mammals, PAX2 specifies a region of mesoderm fated to become urogenital epithelia at a time when the mesoderm becomes compartmentalized into axial, intermediate and lateral plate. These data suggest that PTIP provides a link between tissue specific DNA-binding proteins that specify cell lineages and the H3K4 methylation machinery (Fang, 2009).

To extend these finding to a non-mammalian organism and address the evolutionary conservation of Ptip, it was asked whether a Drosophila ptip homolog could be identified and if so, whether it is also an essential developmental regulator and part of the epigenetic machinery. The mammalian Ptip gene encodes a novel nuclear protein with two amino-terminal and four carboxy-terminal BRCT domains, flanking a glutamine-rich sequence. Based on the number and position of the BRCT domains and the glutamine-rich domain, the Drosophila genome contains a single ptip homolog. To understand the function of Drosophila ptip in development, a ptip mutant allele was characterized that contained a piggyBac transposon insertion between BRCT domains three and four. Maternal and zygotic ptip mutant embryos exhibited severe patterning defects and developmental arrest, whereas zygotic null mutants developed to the third instar larval stage but also exhibited anterior/posterior (A/P) patterning defects. In cell culture, depletion of Polycomb-mediated repression activates developmental regulatory genes, such as the homeotic gene Ultrabithorax (Ubx). This derepression is dependent on trxG activity and also requires PTIP. Microarray analyses in cell culture of Polycomb and polyhomeotic target genes indicate that many, but not all, require PTIP for activation once repression is removed. The activation of PcG target genes is coincident with PTIP binding to promoter sequences and increased H3K4 trimethylation. These data argue for a conserved role for PTIP in Trithorax-mediated epigenetic imprinting during development (Fang, 2009).

Embryonic development requires epigenetic imprinting of active and inactive chromatin in a spatially and temporally regulated manner, such that correct gene expression patterns are established and maintained. This study shows that Drosophila ptip is essential for early embryonic development. In larval development, ptip coordinately regulates the methylation of histone H3K4 and demethylation of H3K27, consistent with the reports that mammalian PTIP complexes with HMT proteins ALR and MLL3, and the histone demethylase UTX. In wing discs, ptip is required for appropriate A/P patterning by affecting morphogenesis determinant genes, such as en and ci. These data demonstrate in vivo that dynamic histone modifications play crucial roles in animal development and PTIP might be necessary for coherent histone coding. In addition, ptip is required for the activation of a broad array of PcG target genes in response to derepression in cultured fly cells. These data are consistent with a role for ptip in trxG-mediated activation of gene expression patterns (Fang, 2009).

Early development requires ptip for the appropriate expression of the pair rule genes eve and ftz. The characteristic seven-stripe eve expression pattern is regulated by separate enhancer sequences, which are not all equally affected by the loss of ptip. The complete absence of en expression at the extended germband stage also indicates the dramatic effect of ptip mutations on transcription. The characteristic 14 stripes of en expression depends on the correct expression of pair rule genes, which are clearly affected in ptip mutants. However, the maintenance of en expression at later stages and in imaginal discs is regulated by PREs and PcG proteins. If ptip functions as a trxG cofactor, then expression of en along the entire A/P axis in the imaginal discs of ptip mutants might be due to the absence of a repressor. This might explain the surprising presence of ectopic en in the anterior halves of imaginal discs from zygotic ptip mutants. This ectopic en expression is likely to result in suppression of ci through a PcG-mediated mechanism. Yet, it is not clear how en is normally repressed in the anterior half, nor which genes are responsible for derepression of en in the ptip mutant wing and leg discs (Fang, 2009).

The direct interaction of PTIP protein with developmental regulatory genes is supported by ChIP studies in cell culture. Given the structural and functional conservation of mouse and fly PTIP, mPTIP was expressed in fly cells; it can localize to the 5' regulatory regions of many PcG target genes that are activated upon loss of PC and PH activity. Consistent with the interpretation that a PTIP trxG complex is necessary for activation of repressed genes, mPTIP only bound to DNA upon loss of Pc and ph function. In the Kc cells, suppression of both Pc and ph results in the activation of many important developmental regulators, including homeotic genes. A recent report details the genome-wide binding of PcG complexes at different developmental stages in Drosophila and reveals hundreds of PREs located near transcription start sites. Strikingly, most of the genes found to be activated in the Kc cells after PcG knockdown also contain PRE elements near the transcription start site (Fang, 2009).

In vertebrates, PTIP interacts with the Trithorax homologs ALR/MLL3 to promote assembly of an H3K4 methyltransferase complex. The tissue and locus specificity for assembly may be mediated by DNA-binding proteins such as PAX2 (Patel, 2007) or SMAD2 (Shimizu, 2001), which regulate cell fate and cell lineages in response to positional information in the embryo. In flies, recruitment of PcG or trxG complexes to specific sites also can require DNA-binding proteins such as Zeste, DSP1, Pleiohomeotic and Pipsqueak. Whereas PcG complexes have been purified and described in detail, much less is known about the Drosophila trxG complexes. Purification of a trxG complex capable of histone acetylation (TAC1) revealed the proteins CBP and SBF1 in addition to TRX. By contrast, the mammalian MLL/ALL proteins are components of large multi-protein complexes capable of histone H3K4 methylation. Although the mutant analysis, the reduction of H3K4 methylation and the dsRNA knockdowns in Kc cells all suggest that Drosophila ptip has trxG-like activity and hence might be a suppressor of PcG proteins, a more definitve biochemical analysis awaits the generation of antibodies and the delineation of in vivo DNA-binding sites for PTIP and its associated proteins at specific target genes (Fang, 2009).

Mammalian PTIP is also thought to play a role in the DNA damage response based on its ability to bind to phosphorylated p53BP1. PTIP also binds preferentially to the P-SQ motif, which is a good substrate for the ATR/ATM cell cycle checkpoint regulating kinases. Several reports demonstrate that PTIP is part of a RAD50/p53BP1 DNA damage response complex, which can be separated from the MLL2 histone H3K4 methyltransferase complex. Both budding and fission yeast contain multiple BRCT domain proteins that are involved in the DNA damage response, including Esc4, Crb2, Rad9 and Cut5. All of these yeast proteins have mammalian counterparts. However, neither the fission nor budding yeast genomes encodes a protein with six BRCT domains and a glutamine-rich region between domains two and three, whereas such characteristic PTIP proteins are found in Drosophila, the honey bee, C. elegans and all vertebrate genomes. These comparative genome analyses suggest that ptip evolved in metazoans, consistent with an important role in development and differentiation (Fang, 2009).

In summary, Drosophila ptip is an essential gene for early embryonic development and pattern formation. Maternal ptip null embryos show early patterning defects including altered and reduced levels of pair rule gene expression prior to gastrulation. In cultured cells PTIP activity is required for the activation of Polycomb target genes upon derepression, suggesting an important role for the PTIP protein in trxG-mediated activation of developmental regulatory genes. The conservation of gene structure and function, from flies to mammals, suggests an essential epigenetic role for ptip in metazoans that has remained unchanged (Fang, 2009).

Long- and short-range transcriptional repressors induce distinct chromatin states on repressed genes

Transcriptional repression is essential for establishing precise patterns of gene expression during development. Repressors governing early Drosophila segmentation can be classified as short- or long-range factors based on their ranges of action, acting either locally to quench adjacent activators or broadly to silence an entire locus. Paradoxically, these repressors recruit common corepressors, Groucho and CtBP, despite their different ranges of repression. To reveal the mechanisms underlying these two distinct modes of repression, chromatin analysis was performed using the prototypical long-range repressor Hairy and the short-range repressor Knirps. Chromatin immunoprecipitation and micrococcal nuclease mapping studies reveal that Knirps causes local changes of histone density and acetylation, and the inhibition of activator recruitment, without affecting the recruitment of basal transcriptional machinery. In contrast, Hairy induces widespread histone deacetylation and inhibits the recruitment of basal machinery without inducing chromatin compaction. This study provides detailed mechanistic insight into short- and long-range repression on selected endogenous target genes and suggests that the transcriptional corepressors can be differentially deployed to mediate chromatin changes in a context-dependent manner (Li, 2011).

To directly compare functional aspects of Hairy- and Knirps- mediated repression in the Drosophila embryo, these proteins’ interactions were studied with two segmentally expressed pair-rule genes. Hairy directly represses fushi tarazu (ftz), a secondary pair-rule gene expressed in the blastoderm embryo in a seven-stripe pattern. ftz is regulated by both regionally acting gap genes and the segmentally expressed hairy pair-rule gene. Chromatin immunoprecipitation (ChIP) experiments have revealed dense clusters of peaks around the ftz gene for key transcription factors active in the blastoderm embryo, including Caudal, Hunchback, Knirps, Giant, Huckebein, Krüppel, and Tailless. These transcription factors bind to the promoter-proximal Zebra element, the stripe 1+5 enhancer located 3' of ftz, and a presumptive 5' regulatory region located between 23 kbp and 28 kbp. Hairy has been found to bind in vivo to all of these regions. This repressor is expressed in a striped pattern in the blastoderm embryo; therefore, the ftz gene is active in some nuclei and repressed in others. In order to obtain a homogeneous population of nuclei for chromatin studies, Hairy protein was overexpressed in embryos using a heat-shock driver, which results in complete repression of ftz. This repression requires the recruitment of the Groucho corepressor, because a mutant version of Hairy that does not bind to Groucho fails to repress ftz (Li, 2011).

Interestingly, a titration of heat-shock induction resulted in a nonuniform, progressive loss of specific ftz stripes, with stripe 4 being the most sensitive and stripe 1+5 the least. This result points to the intriguing possibility that Hairy can act locally on specific enhancers, at least very transiently, although the end result of Hairy repression is complete silencing of all enhancer elements. The asynchronous repression of the ftz locus also suggests that Hairy-mediated long-range repression does not act solely by direct targeting the basal promoter, as suggested by a previous model for this class of repressor, because this mechanism should cause uniform inhibition of stripe elements. Similar to ftz, the pair-rule gene even skipped (eve) is also expressed in a seven-stripe pattern and is regulated by multiple modular enhancers. eve is a well-characterized target of the short-range repressor Knirps, which sets posterior boundaries of eve stripe 3 and 4 and anterior borders of eve stripe 6 and 7. After substantial overexpression of Knirps (20 min heat-shock induction), the repressor is able to repress all of the eve stripe enhancers except for the stripe 5 enhancer. When the induction is titrated, Knirps represses individual enhancers in a stepwise manner, with the most sensitive enhancers downregulated earliest, at a low dose of Knirps. Together, these experiments indicate that Hairy can initially act locally but ultimately acts in a globally dominant fashion, whereas Knirps acts in a restricted manner (Li, 2011).

To compare the effects of repression by Hairy and Knirps, chromatin changes associated with repression of ftz and eve were studied via ChIP. No significant change of histone H3 occupancy were detected at regions sampled throughout the ftz locus after Hairy overexpression (although some regions showed modest differences. In contrast, Knirps repression of eve resulted in significantly increased histone H3 density, particularly in two of the three regions corresponding to the Knirps-sensitive enhancers, namely stripe 4+6 and stripe 2. Little change was noted in the promoter region, transcribed region, or the stripe 1 and 5 enhancers, which are not readily repressed by Knirps. An apparent increase in histone H3 density on the repressed stripe 3+7 enhancer, although of low statistical significance, correlates with other alterations common to repressed enhancers, noted below (Li, 2011).

To provide a more detailed picture of chromatin structure, a micrococcal nuclease (MNase) mapping protocol used in yeast and cultured cells was adapted for Drosophila embryos. MNase mapping showed that Hairy repression had little effect on chromatin accessibility throughout the ftz locus, whereas Knirps induced a significant increase in MNase insensitivity specifically at the eve stripe 3+7, 2, and 4+6 enhancers and a minor increase in stripe 1 protection. The promoter and the eve stripe 5 enhancer were little changed, mirroring the patterns noted for overall histone H3 occupancy. The changes noted for the eve locus appear to be specific, because Knirps did not induce any change of a nontargeted intergenic site on the third chromosome. Hairy also had no effect at this locus. The similar results from overall histone H3 density and MNase mapping suggest that Hairy-mediated long-range repression does not involve a general compaction of chromatin on the ftz locus. In contrast, repression by Knirps is associated with an increase in the histone density of targeted enhancer regions, which may result either from Knirps recruitment of factors that mediate chromatin condensation or the blocking of proteins responsible for loosening of chromatin. Recruitment of Groucho by other repressor proteins is also associated with distinct effects: Runt-dependent repression of slp1 does not involve changes in H3 density, but Brinker repression of the vgQ enhancer does. The distance dependence of these repressors has not been established, but in light of the current results, it is apparent that the Groucho corepressor can be involved in distinct effects depending on the context of recruitment (Li, 2011).

Histone acetylation is dynamically regulated on transcribed genes in eukaryotes, with histone acetylation generally correlated with active loci. The histone deacetylase Rpd3 is a component of both Hairy and Knirps corepressor complexes; therefore, histone acetylation levels were assayed across the eve and ftz genes before and after repression. Hairy repression resulted in widespread histone H4 deacetylation throughout the ftz locus. The ectopically expressed Hairy protein itself was not observed to spread but remained restricted to regions of the gene previously observed to bind endogenous Hairy. Using anti-H3-acetylation antibodies, similar widespread H3 deacetylation was also noted. This distributed effect on the ftz locus correlates with prior observations that Hairy-mediated long-range repression might involve a Groucho-mediated 'spreading' mechanism. By this means, Rpd3 may be delivered to extensive areas of a gene. To test whether a spreading of histone deacetylation might correlate with the successive inhibition of ftz enhancers, histone acetylation levels were investigated across ftz after a brief 5 min heat shock followed by immediate fixing, before the entire complement of enhancers can be repressed. In this setting, deacetylation was mostly concentrated around the stripe 1+5 enhancer and the immediate 5' regulatory region, areas that show Hairy occupancy in vivo. More distal 5' regulatory regions and the transcription unit itself showed little initial change, consistent with a spreading action of this repressor during the more extensive repression period (Li, 2011).

A different picture emerged from studies of Knirps acting on eve. Here, repression led to selective decreases in H3 and H4 acetylation levels, concentrated over the eve stripe 4+6 and stripe 2 enhancers, with lesser decreases noted at stripe 3+7 and stripe 1 enhancers. A local change in acetylation was also noted near the transcriptional initiation site, but not immediately 5' and 3' of this area. The reductions in histone acetylation levels seen on both eve and ftz are consistent with Hairy and Knirps recruiting deacetylases to their target genes. However, it is striking that the broad deacetylation mediated by Hairy on ftz is not associated with dramatic changes in histone density or resistance to nuclease accessibility, whereas increased histone density and resistance to nuclease digestion are associated with Knirps repression on eve. It is possible that in addition to inducing deacetylation, Knirps triggers additional histone modifications or interacts with nucleosome-remodeling complexes to further alter chromatin at the enhancers. H3 lysine 27 methylation is one chromatin signature associated with silenced genes; however, no significant change in this modification was noted at ftz or eve upon repression (Li, 2011).

Previous studies indicated that Hairy can effectively repress a reporter gene without displacing the activators. Attempts were made to test whether this was the case on an endogenous gene, ftz, by examining occupancy by Caudal, a transcription factor that also activates eve. Caudal activates the posterior stripes of both ftz and eve, and it was found that Caudal binds the ftz 5' regulatory region and the promoter-proximal Zebra element. Repression of the locus by Hairy did not affect the Caudal binding pattern, similar to the results obtained with a Hairy-regulated reporter gene. In contrast, Knirps repression decreased Caudal occupancy specifically at the eve 3+7 and 4+6 enhancers, bringing overall protein occupancy down to near baseline levels. This decrease is not an effect of global decrease of Caudal occupancy, because the Caudal binding peak at the eve promoter was not affected. A similar decrease in Caudal occupancy was also observed on a hunchback enhancer after repression by Knirps. Interestingly, Bicoid occupancy of the eve stripe 2 and stripe 1 enhancers was not altered by Knirps, although these enhancers were repressed. Clearly, loss of transcription factor occupancy is not required for short-range repression of a cis-regulatory element. It is possible that different transcriptional activators exhibit differential sensitivity to chromatin changes induced during repression (Li, 2011).

New insights have suggested that many developmental genes, including those regulated by short-range repressors such as Snail, feature RNA polymerase paused in the promoter region even in their inactive state, suggesting postrecruitment levels of regulation. Components of the core machinery were analyzed before and after repression by Hairy and Knirps. Upon Hairy repression, a marked decrease of RNA polymerase II (Pol II) occupancy was observed at the ftz locus. The same trend was observed for the preinitiation, initiation, and elongation forms of Pol II. These results suggest that Hairy directly or indirectly blocks recruitment of Pol II. Similar decreases were noted with levels of TATA box-binding protein (TBP) at the promoter (Li, 2011).

In contrast, induction of Knirps did not change Pol II occupancy at the eve transcription unit, even under condition where most enhancers were repressed. (Under conditions tested in this study, over three-quarters of the embryos had shut down expression of all but stripe 1 and/or 5.) Similarly, TBP occupancy remained at a comparable level before and after Knirps repression. The constant level of RNA polymerase on the eve transcription unit was a surprise in light of the sharp reduction in mRNA production as measured by in situ hybridization. However, there is precedence for this effect: Runt repression of slp1 appears to act through elongation control, which causes no change of the concentration of Pol II on slp1. Knirps may produce a similar effect by inducing a slower transit rate of Pol II on the repressed eve locus. Similar observations have been made at the hsp70 gene upon depletion of elongation factors such as Spt6 or Paf1 (Li, 2011).

The differential distance dependence of short- and long- range repressors such as Hairy and Knirps has been observed in many contexts. However, the mechanisms by which these proteins function have been poorly understood. With the recent demonstration that transcriptional factors considered to be short- and long-range repressors utilize shared cofactors, namely CtBP and Groucho, there has been a question of whether long-range repression is actually functionally distinct from short-range repression (Payankaulam, 2009). The current study provides evidence that the chromatin states associated with long- and short-range repressors are distinct in several ways. It is not yet knowm whether the effects seen on ftz are observed for all Hairy targets, although the similarity of changes observed on the lacZ reporter subject to Hairy repression suggests that they are conserved (Martinez, 2008). Similarly, the reproducibility of Knirps-induced changes at different eve enhancers indicates that this protein can effect related chromatin changes on cis-regulatory modules bound by different activators. Snail, another short-range repressor, also appears to mediate localized deacetylation and activator displacement; thus, this mechanism may be a common feature of this entire class of repressors (Qi, 2009; Y. Nibu, personal communication to Li, 2011). It will be interesting to determine how general are the observations made in this study for long- and short-range repression, a question that can be approached using genome-wide methods. In any event, the highly divergent activities of Knirps and Hairy demonstrated in this study not only underscore the fact that these proteins can mediate biochemically divergent events but also raise interesting questions about how similar cofactors can participate in such distinct effects in a context-dependent manner. It is possible that the corepressors adopt distinct conformations when recruited by different repressors, or the corepressor may form distinct complexes with unique activities. In addition to determining how cis- and trans-acting factors affect repression pathways, these mechanistic insights will provide important contextual information for interpretation of genome-wide transcription factor binding and chromatin modifications and will inform quantitative modeling of cis-regulatory elements for the aim of understanding the activity and evolution of enhancers (Li, 2011).

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