BIOLOGICAL OVERVIEW

There are three GATA homologs in Drosophila: Serpent (dGATAb), found in the midgut and ovary (Lossky, 1995), Pannier (GATA-2), found in the dorsal epidermis, and dGATAc, found in the procephalic [Image] region, posterior spiracles, gut, and central nervous system.

Pannier acts as a local repressor of achaete and scute and is required for the normal pattern of sensory bristles in those parts of the epithelium where it is expressed. pannier mutation may take one of two forms: either an over- or under-expression of achaete-scute. Consequently the phenotype shows either an overabundance or a lack of bristles.

Studies suggest that Pannier's action on achaete-scute is negative. In this view, mutants with too few bristles would be due to a hyperactive enzyme, resulting in accentuated repressive activity. These mutations are dominant, affecting the primary structure of Pannier and suggest that mutant Pannier protein acts as a non-functional heterodimer with another transcription factor (Ramain, 1993).

Two other proteins act as repressors of achaete-scute: hairy and extramachrochaete. Loss of these two functions also results in overexpression of achaete-scute and ectopic bristles.

To identify genes involved in the patterning of adult structures, Gal4-UAS (upstream activating site) technology was used to visualize patterns of gene expression directly in living flies. The gene yellow (y) was made sensitive to Gal4 control and a Gal4-containing P element was inserted randomly into the fly genome. A large number of Gal4 insertion lines were generated and their expression patterns studied. In addition to identifying several characterized developmental genes, the approach revealed previously unsuspected genetic subdivisions of the thorax, which may control the disposition of pattern elements. For example, the pannier expression domain marks a dorsal band along the length of the body, from the occipital head region to the end of the abdomen but excluding the terminalia. In the notum (derived from the wing imaginal disc), the gene labels a territory extending from the dorsal midline laterally to a longitudinal (anterior to posterior) straight line defined by the dorsocentral bristles. Another insertion em462 shows y+ rescue in a territory adjacent to the pannier domain; it is also demarcated medially by the position of the dorsocentral bristles. The em462 domain extends laterally but does not reach the more lateral region of the notum. Possibly, the gene iroquois defines a distinct, more lateral domain.

The subdivision of the notum demarcated by the dorsocentral bristles may be significant because this same line also appears to demarcate the most medial expression border of the wingless gene. In the notum, wingless is expressed in a narrow stripe and the wingless domain is included within the em462 domain, extending laterally from the dorsocentral bristles. Apparently there is no overlap between pnr and wg in the scutum (the anterior part of the notum), but they do overlap in the more posterior scutellum. Genetic interaction experiments show that pannier acts as a negative regulator of wingless in the notum and suggest that some of the effects of pannier mutants are produced through an alteration of wingless function. Interestingly, the dorsocentral line is known not to function as a cell lineage border. In addition to bristles, another frequent pattern element in insects is pigmentation, often disposed in longitudial bands and used as a diagnostic criterion for the taxonomy of dipteran species. The medial boundary of a diagnostic pigment band is exactly delimited by the same longitudinal line straddling the dorsocentral bristles that in the Drosophila demarcates pannier, wingless and em462 (Calleja, 1996).

The pannier gene of Drosophila encodes a zinc-finger transcription factor of the GATA family and is involved in several developmental processes during embryonic and imaginal development. Novel aspects of the regulation and function of pnr during embryogenesis are reported in this study. Previous work has shown that pnr is activated by decapentaplegic (dpp) in early development, but it has been found that after stage 10, the roles are reversed and pnr becomes an upstream regulator of dpp. This function of pnr is necessary for the activation of the Dpp pathway in the epidermal cells implicated in dorsal closure and is not mediated by the JNK pathway, which is also necessary for Dpp activity in these cells. In addition, pnr behaves as a selector-like gene in generating morphological diversity in the dorsoventral body axis. It is responsible for maintaining a subdivision of the dorsal half of the embryo into two distinct, dorsomedial and dorsolateral, regions, and also specifies the identity of the dorsomedial region. These results, together with prior work on its function in adults, suggest that pnr is a major factor in the genetic subdivision of the body of Drosophila (Herranz, 2001).

In early development, pnr is activated in response to dpp activity in a broad dorsal domain, which extends from parasegments 2/3 to the border between 13/14, although the borders are not strictly parasegmental. The control by dpp is consistent with the effect of brk mutations on early pnr expression. The original expression domain is substantially modified during embryogenesis. By germ band extension (stage 10) pnr activity is limited dorsally by the border between the epidermis and the amnioserosa, and laterally by the dorsal border of iro. It is not known which factor(s) is responsible for the loss of expression in the amnioserosa, although likely candidates are several genes specifically active in this region, such as Race, zen, hindsight or serpent. In addition, it is not known how the late expression is regulated at the lateral border. It is not achieved by iro, since the loss of the entire Iroquois complex does not affect pnr expression (Herranz, 2001).

Another modification occurs between stages 10 and 11, and is the loss of expression in the A8 segment. Expectedly, it is under the control of Abd-B; in Abd-B mutants the gap in A8 does not appear. However, none of the known Abd-B target genes (sal, ems and grn) is involved in the regulation, since their mutations do not affect pnr expression. The finding that lin, which is considered as a co-factor of Abd-B, is involved, suggests that downregulation of pnr in the A8 segment is mediated either by an unknown Abd-B target or directly by interaction between the Abd-B and Lin products. It is not clear why pnr activity has to be eliminated precisely in the A8 segment. This segment gives rise to the spiracles, protruding structures that are very different from those differentiated by the other abdominal segments where pnr remains active. In fact, there are several Abd-B target genes specifically activated in the spiracles. It is possible that the formation of these structures demands that the pnr activity, which specifies larval epidermis of very different morphology, be turned off (Herranz, 2001).

Interestingly, whereas early pnr expression is under dpp control, the late expression is not. Late inactivation of the Dpp pathway, using a dominant negative form of thick veins, does not modify pnr expression. In addition, mutations at brk, which allow higher response levels to Dpp signaling fail to affect pnr expression in late development, although they affect early expression. This indicates that pnr expression is controlled independently in early and late development, and by different factors (Herranz, 2001).

There is already evidence that pnr has distinct functions during embryogenesis. Its activity in the dorsal epidermis is required for dorsal closure and it is also expressed in the dorsal mesoderm where it is involved in the specification of cardiac cells. Evidence is provided for another and more general function of pnr; it specifies the identity of a dorsomedial body region that spans from the labial segment to the end of the abdomen. This is clearly demonstrated by the effects seen in mutant embryos and after ectopic expression experiments. In pnr^VX6 embryos, the dorsomedial cuticle does not form, and there is an expansion of the dorsolateral epidermis, suggesting that the cells of the dorsomedial domain acquire a dorsolateral fate. The ectopic expression experiments also point to the same conclusion. In larvae ectopically expressing pnr (arm-Gal4/UAS-pnr) the entire larval epidermis acquires dorsomedial features, whereas using more restricted drivers (Ubx-Gal4, wg-Gal4) the transformation is limited to the region where the Pnr protein is present, suggesting that the effect of pnr is cell autonomous. Thus, the Pnr protein is able by itself to trigger a developmental pathway, a typical property of selector gene products. In addition, it induces a ventral to dorsal transformation, corresponding to each segment, indicating that it acts in combination with Hox genes. These observations indicate that selector genes in the AP and DV axes have to co-operate to determine the different spatial patterns (Herranz, 2001).

The transformation of ventral and dorsolateral epidermis towards dorsomedial observed after ectopic pnr expression is also reflected in the activity of marker genes of the distinct regions. Characteristic genes of the ventral neuroectoderm such as BP102 for the CNS or buttonhead are suppressed. In addition, pnr is able to suppress iro activity, a property that, as in the adult cells, is important to keep the dorsomedial and dorsolateral domains separate during embryogenesis (Herranz, 2001).

The developmental effects observed after either the loss or gain of pnr function in the larval epidermis resemble those reported for the adult cuticle. In the latter, it has been shown that the activity of pnr maintains the segregation of the dorsal cuticle into medial and lateral domains, and also specifies the identity of a medial domain. This indicates that pnr has a general function involved in the subdivision of the body along the DV axis. The longitudinal stripe of pnr expression established during embryogenesis is probably a major constituent of the body and represents a zone of common identity (Herranz, 2001).

In addition, Pnr has other more concrete functions connected with the specification of cardiac cells and embryonic dorsal closure. The involvement of pnr in dorsal closure is exerted through its activation of dpp in late embryogenesis, which is responsible for the formation of the Dpp stripe at the junction of the epidermis with the amnioserosa. Normal functioning of the Dpp pathway in this region is required for dorsal closure, suggesting that defects in dorsal closure observed in pnr mutant embryos is the result of the lack of the dorsal dpp stripe (Heranz, 2001).

There is evidence that this dpp expression requires function of the JNK kinase pathway, and it also requires pnr activity. The observation that in the absence of pnr activity the expression of puc, the end element of the JNK pathway is normal, indicates that in pnr mutants the JNK pathway is normally active. In turn, it shows that the activation of dpp in the dorsal stripe requires independent inputs from both the JNK pathway and pnr (Herranz, 2001).

One intriguing aspect of pnr function is that it is able to induce a developmental modification in all ectodermal structures along the DV body axis except in the amnioserosa, the most dorsal tissue. Even under conditions in which pnr is transcribed and translated in all the amnioserosa cells, it does not appear to elicit any developmental effect; none of the amnioserosa marker genes is affected by forcing pnr activity and the retraction of the germ band [a morphological indicator of the function of specific amnioserosa genes is also normal. Similarly, pnr is able to induce dpp activity all over the body except in the amnioserosa, where the presence of the Pnr protein appears to be inconsequential. This situation resembles the phenotypic suppression/posterior prevalence phenomenon discovered in the Hox genes specifying the AP body axis. It consists of a functional inactivation of a Hox protein by the presence of another normally expressed in a more posterior region of the body. It is conceivable that there might be a ‘dorsal prevalence’ in the DV axis, by which dorsal expressing genes are functionally dominant over the ventral expressing ones. It would be expected that genes specifying amnioserosa would be able to transform all structures since they would be ranking highest in the functional hierarchy (Herranz, 2001).

Dorsal eye selector pannier (pnr) suppresses the eye fate to define dorsal margin of the Drosophila eye

Axial patterning is crucial for organogenesis. During Drosophila eye development, dorso-ventral (DV) axis determination is the first lineage restriction event. The eye primordium begins with a default ventral fate, on which the dorsal eye fate is established by expression of the GATA-1 transcription factor pannier (pnr). Earlier, it was suggested that loss of pnr function induces enlargement in the dorsal eye due to ectopic equator formation. Interestingly, this study found that in addition to regulating DV patterning, pnr suppresses the eye fate by downregulating the core retinal determination genes eyes absent (eya), sine oculis (so) and dacshund (dac) to define the dorsal eye margin. pnr acts downstream of Ey and affects the retinal determination pathway by suppressing eya. Further analysis of the 'eye suppression' function of pnr revealed that this function is likely mediated through suppression of the homeotic gene teashirt (tsh) and is independent of homothorax (hth), a negative regulator of eye development. Pnr expression is restricted to the peripodial membrane on the dorsal eye margin, which gives rise to head structures around the eye, and pnr is not expressed in the eye disc proper that forms the retina. Thus, pnr has dual function, during early developmental stages pnr is involved in axial patterning whereas later it promotes the head specific fate. These studies will help in understanding the developmental regulation of boundary formation of the eye field on the dorsal eye margin (Oros, 2011).

This study has addressed a basic question pertaining to regulation of patterning, growth and differentiation of the developing eye field. The results provide an important insight into the role of pnr, a gene known to confer dorsal eye identity during axial patterning of the eye. The onset of pnr expression during early eye development results in the generation of dorsal lineage in the eye. It results in the formation of a DV boundary (equator), which triggers N signaling at the border of the dorsal and ventral compartments to initiate growth and differentiation (Oros, 2011).

The spatial as well as temporal requirement for the genes controlling ventral eye growth and development have been tested. During early eye development, prior to the onset of pnr expression in the dorsal eye, entire early eye primordium is ventral in fate. Removal of function of genes controlling ventral eye development prior to the onset of pnr expression results in complete elimination of the eye field whereas later when pnr starts expressing, the eye suppression phenotype gets restricted only to the ventral eye . These studies suggested that pnr plays an important role in dorso-ventral (axial) patterning. However, the role of dorsal selector pnr in retinal determination was unknown (Oros, 2011).

Loss-of-function clones of pnr in the dorsal eye exhibit eye enlargement. It was suggested that when pnr function is abolished in the dorsal eye using loss-of-function clones, it results in the change of dorsal eye fate to ventral. This results in generation of a de novo equator, the border between dorsal and ventral half of the eye, which triggers ectopic N signaling to promote growth and cell proliferation. The same premise has been used to explain the gain-of-function phenotype of pnr in the eye. Misexpression of pnr in the entire eye (ey > pnr^D4) generates a completely dorsalized eye field as pnr acts as the dorsal fate selector. The fully dorsalized eye lacks DV polarity (equator), resulting in the complete loss of eye field due to lack of N upregulation. This study addressed another possibility, to see if pnr suppresses the eye fate upon misexpression in the entire eye as evident from the 'no-eye' phenotype (Oros, 2011).

To test if pnr suppresses the eye fate, pnr was misexpressed both in the dorsal and ventral (DV) eye margins of the eye using a bi-Gal4 driver (bi > pnr^D4). The rationale was if pnr is only required to assign the dorsal eye fate, in that case pnr misexpression (bi > pnr^D4) will assign a dorsal fate on the margin of ventral eye. Thus, by this logic, it would result in generation of a de novo equator on the ventral eye margin, which should manifest as eye enlargements in the ventral eye. The argument was based on the similar premise that was employed to explain that loss-of-function clones of pnr in the dorsal eye generated a new equator and led to the dorsal eye enlargement. No ventral eye enlargements were observed in bi > pnr^D4 eye disc. Instead suppression of the eye was seen on both the dorsal as well as ventral margins. The suppression of eye fate on both the dorsal and the ventral margins suggests that pnr, irrespective of the domain where it is expressed, can suppress the eye fate (Oros, 2011).

Since pnr suppresses the eye fate, it is possible that it may be involved in regulation of expression of genes of the core retinal determination (RD) machinery. Loss-of-function of pnr in the dorsal eye clones results in the eye enlargements as evident from Elav positive cells but it does not induce ectopic Ey. Ey expression evolves during eye development and is localized anterior to the morphogenetic furrow in retinal precursor cells and is downregulated and degraded posterior to the furrow in differentiation retinal neurons. In the loss-of-function clones of pnr in the eye, the expression of retinal determination pathway members like Eya, So and Dac, which act downstream to Ey, are ectopically induced. In the converse situation, where pnr was misexpressed on both dorsal and ventral eye margins (bi > pnr^D4), ectopic induction of Ey was observed on both the dorsal and the ventral margins whereas the expression of Eya, So and Dac were suppressed. Thus, misexpression of pnr in the eye prevents the photoreceptor differentiation irrespective of the dorsal or ventral domain. Based on these results it is proposed that pnr suppresses the eye fate on the dorsal eye margin by downregulating RD genes like eya, so and dac (Oros, 2011).

Pnr suppresses the eye fate by downregulating tsh which results in suppression of retinal determination genes at the dorsal eye margin. GATA-1 transcription factor pnr, which is expressed in the peripodial membrane (PM) at the dorsal eye margin, suppresses the retinal determination. The suppression of retinal determination genes by pnr can be mediated by two possible ways: (1) pnr directly suppresses the retinal determination genes to suppress the eye. pnr may act downstream to ey and suppress the downstream retinal determination target eya and other downstream genes so and dac. During eye development, ey is required for eye specification and other downstream targets are required for retinal determination. These studies suggest that pnr acts on retinal determination process, which corresponds to the onset of pnr expression in the eye. (2) Alternatively, pnr suppresses the eye by downregulating homeotic gene teashirt (tsh) in the dorsal eye. Interestingly, the tsh gain-of-function in the dorsal eye is complementary to the loss-of-function of pnr in the dorsal eye. tsh is known to act upstream of eya, so and dac. Thus, the dorsal eye enlargement observed in pnr mutant is due to ectopic induction of tsh in the dorsal eye, which in turn can induce the RD genes. Lastly, Pnr mediated suppression of the eye fate is independent of Meis class of homeotic gene, homothorax (hth) function (Oros, 2011).

Since ey is responsible for the specification of the eye field and marks the retinal precursor cells, it suggests that pnr does not affect the eye field formation or specification. In fact pnr affects expression of RD genes eya, so and dac. These results suggest that pnr suppress retinal determination genes. Since endogenous expression of pnr is restricted to the peripodial membrane of the dorsal eye margin, it suggests that pnr may be involved in suppression of retinal determination on the dorsal peripodial membrane. Thus, the results suggest that pnr generally acts at the stage when photoreceptor differentiation is initiated with the formation of the morphogenetic furrow (MF) and promotes the dorsal head cuticle fate by suppressing retinal differentiation (Oros, 2011).

Based on these new findings, it is proposed that pnr may be required for two different functions during eye development: (1) axis determination during DV patterning and (2) suppression of the retinal determination process to define the dorsal eye field margin. These functions of pnr appear to be temporally controlled as DV axis determination takes place in late first- or early second- instar of eye development while suppression of the eye fate is evident in late second instar of larval development (Oros, 2011).

The axis determination function of pnr is required in the earlier time window. This is further validated by the loss-of-function clones of pnr of first category, which are bigger and exhibits non-autonomous dorsal eye enlargement phenotypes. These dorsal eye enlargements that are spanning both wild-type and pnr mutant cells in the eye disc conforms to the notion that when pnr is lost in a group of cells during early development, it fails to confer dorsal identity over the default ventral state. As a consequence de novo equator is generated which results in the ectopic dorsal eye enlargements. Since these clones are always bigger, suggesting that they might be formed earlier. The late function of pnr in suppression of retinal determination is validated both by the gain-of-function studies as well as the loss-of-function clones of second category, which are both bigger as well as smaller in size and are autonomous in nature. These clones have ectopic dorsal eyes, which are restricted within the clones, thereby suggesting that absence of pnr function promotes ectopic eye formation in the dorsal eye margin. Thus, during the early second instar of development, before the onset of retinal differentiation, pnr is required for defining the dorsal lineage by inducing Wg and members of the Iro-C complex. However, later during the late second-instar stage of eye development, when the morphogenetic furrow (MF) is initiated, pnr suppresses the photoreceptor differentiation at the dorsal eye margin. The endogenous expression of pnr in only the peripodial membrane of the dorsal eye margin further confirms this notion. Lack of phenotypes in pnr clones which are restricted to the disc proper (DP) alone verifies pnr localization. Thus, pnr defines the boundary between the eye field and the head cuticle on the dorsal margin. An interesting question will be to identify which gene is responsible for defining the ventral eye margin. In the ventral eye where pnr is not expressed, hth is known to suppress the eye fate. There is a strong possibility that hth may be involved in defining the boundary of eye field on the ventral eye margin between the disc proper and peripodial membrane (Oros, 2011).

Since pnr induces Wg, it is expected that pnr may suppress the eye by induction of Wg. Even though Wg signaling is responsible for suppression of photoreceptor differentiation on both dorsal and ventral eye margins, its regulation is different on both dorsal and ventral eye margins. In the ventral eye, wg is involved in a feedback loop with hth to suppress the eye fate. In the dorsal eye, Pnr induces Wg signaling, which in turn induces the members of Iro-C complex, and ultimately these signaling interactions define the dorsal eye fate. Interestingly, it seems Pnr is not the sole regulator of Wg in the dorsal eye. Since loss-of-function of hth does not exhibit phenotypes similar to the loss-of-function of pnr, it is expected that the positive feedback loop regulation of wg and hth as seen in the ventral eye margin does not hold true on the dorsal eye margin. This study found that hth is not affected in the pnr clones that exhibit dorsal eye enlargements. Furthermore, when clones of hth were made in pnr heterozygous condition, no dorsal eye enlargements were seen suggesting that hth and pnr do not interact. Thus the results also verified that hth is not involved in pnr mediated eye suppression on the dorsal eye margin (Oros, 2011).

It is known that the gain of function of tsh in the dorsal eye results in ectopic eye enlargement whereas gain of function of tsh in the ventral eye result in suppression of ventral eye. The dorsal eye enlargements seen in tsh gain-of-function is a phenotype similar to pnr loss-of-function in the dorsal eye. In pnr loss-of-function clones, it was found that tsh was ectopically induced. Furthermore, when pnr loss-of-function clones were generated in a heterozygous background of the tsh null allele tsh⁸/CyO, the dorsal eye enlargement phenotype was dramatically suppressed and no longer observed. Interestingly, this study found that dorsal enlargements were there but were not accompanied with ectopic eyes as evident from absence of Elav expression. All these dorsal enlargements were showing strong Ey expression. Among 500 flies counted only two flies were found that showed subtle dorsal eye enlargements. Interestingly, it was also found that in pnr loss-of-function clones the mini-white reporter gene under tsh was ectopically induced. The loss-of-function phenotypes of pnr were more pronounced when tsh was misexpressed in the clones. Thus, pnr expressed in the dorsal peripodial membrane may suppress tsh in the dorsal eye to suppress the eye fate. Interestingly, tsh is known to act upstream of eya, so and dac. Thus, the dorsal eye enlargement observed in the pnr mutant is due to ectopic induction of tsh in the dorsal eye, which in turn can induce the RD genes (Oros, 2011).

The Drosophila eye is similar to the vertebrate eye in several features: (1) the morphogenetic furrow in the fly eye is analogous to the wave of neurogenesis in the vertebrate eye, (2) As in Drosophila, in higher vertebrates dorsal eye genes like Bmp4 and Tbx5 act as 'dorsal selectors' and restrict the expression of ventral eye genes Vax2 and Pax2. These DV expression domains or developmental compartments lead to formation of DV lineage restriction as seen in the Drosophila eye, (3) The DV lineage in the vertebrate eye also develops from a ventral-equivalent initial state. The dorsal genes pnr and iro-C are highly conserved across the species, and are involved in organogenesis and neural development. Therefore, it would be interesting to see whether the dorsal selectors in the vertebrate eye play a role in defining the boundary of the eye by suppressing retinal differentiation (Oros, 2011).

Drosophila Mediator subunit Med1 is required for GATA-dependent developmental processes: divergent binding interfaces for conserved coactivator functions

DNA-bound transcription factors (TFs) governing developmental gene regulation have been proposed to recruit Polymerase II machinery at gene promoters through specific interactions with dedicated subunits of the evolutionarily-conserved Mediator complex (MED). However, whether such MED subunit specific functions and partnerships have been conserved during evolution has been poorly investigated. To address this issue, the first Drosophila loss-of-function mutants were generated for Med1, known as a specific cofactor for GATA TFs and hormone nuclear receptors in mammals. Med1 was shown to be required for cell proliferation and hematopoietic differentiation dependant on the GATA TF Serpent (Srp). Med1 binds Srp in cultured cells and in vitro through its conserved GATA Zinc Finger DNA-binding domain and the divergent Med1 C-terminal. Interestingly, GATA/Srp interaction occurs through the longest Med1 isoform, suggesting a functional diversity of MED complex populations. Furthermore, it was shown that Med1 acts as a coactivator for the GATA factor Pannier during thoracic development. In conclusion, the Med1 requirement for GATA-dependent regulatory processes is a common feature in insects and mammals, although binding interfaces have diverged. Further work in Drosophila should bring valuable insights to fully understand GATA-MED functional partnerships, which probably involve other MED subunits depending on the cellular context (Immarigeon, 2019).

Precise temporal and spatial regulation of gene transcription by RNA polymerase II (Pol II) is crucial to ensure the coordinated cell fate specification in multicellular organisms. To precisely control Pol II activity, metazoans have evolved an elaborate protein machinery, including the conserved multiprotein Mediator (MED) complex, which serves as a malleable interface between DNA-bound transcription factors (TFs) and the Pol II machinery. Dedicated MED subunits have been proposed to mediate specific TF activities. Whether these specific partnerships and binding interfaces have been conserved during evolution remains an open question (Immarigeon, 2019).

The MED complex, conserved from yeast to human, contains 25 to 30 subunits organized into the head, middle, and tail modules as well as a dissociable cyclin-dependent kinase 8 (CDK8) module. The core MED, interacting directly with Pol II and its associated general transcription factors, contains essential head and middle module subunits. Conversely, more peripheral MED subunits belonging to the tail (e.g., Med15), CDK8 (e.g., Med12), and middle (e.g., Med1) modules are not required for cell viability and display more specific functions during cell differentiation. It is generally assumed that MED subunit specificity comes from their ability to interact directly with specific TFs, allowing Mediator recruitment to gene regulatory elements. For example, it has been shown that Med12 interacts directly with Sox9 and Sox10, whereas Med15 binds SMADs (3) and Med19 binds HOX (4) TFs. Another example is Med1, identified for its role as a major cofactor of hormone nuclear receptors (NRs) that directly bind its LXXL domain. Mammalian Med1 also mediates transcriptional activity of the GATA zinc finger (ZF) TF family. Physically interacting with at least five of the six mammalian GATAs, Med1 is required for GATA1, GATA2, and GATA6 target gene expression in several developmental contexts, including erythropoiesis, and is recruited to specific GATA1 and GATA2 target genes. Whereas several MED subunit-TF partnerships have been characterized in mammals, it is not known to what extent these MED subunit-specific functions have been conserved in other species (Immarigeon, 2019).

Drosophila melanogaster is an ideal model to analyze MED subunit-specific functions given that homologs of the 33 human subunits are encoded by single-copy genes and that overall MED complex structure has been conserved during evolution. Furthermore, several transcription factor families are strongly conserved both structurally and functionally in Drosophila. A good example is the GATA zinc finger factor family. In mice and humans, the GATA1/2/3 subfamily is required for blood cell lineage differentiation, and the GATA4/5/6 subfamily is involved in the meso-endoderm lineage, notably in cardiac development. In Drosophila, the GATA factor Serpent (Srp) is a central regulator of hematopoietic cell differentiation, controlling the formation of the two embryonic populations of blood cells (plasmatocytes and crystal cells), and the GATA factor Pannier (Pnr) is involved in embryonic heart development, dorsal thoracic closure, and sensory organ precursor development, revealing a functional conservation during bilaterian evolution (Immarigeon, 2019).

Mammalian GATA factors generally contain two highly conserved Cys4-type ZFs. The C-terminal ZF (C-ZF) is both necessary and sufficient for sequence-specific DNA binding at [(A/T)GATA(G/A)] genomic sites, while the N-terminal ZF (N-ZF) appears only to modulate DNA binding affinity, notably at palindromic double sites. Whereas Drosophila Pnr also displays two ZFs, srp encodes different isoforms containing either only a C-ZF (SrpC) or both a C- and an N-ZF (SrpNC), with the N-finger stabilizing the interaction of Srp with palindromic GATA sites (Immarigeon, 2019).

The GATA N-ZF also mediates interactions with key coregulators, such as Friend-of-GATA (FOG) proteins, the LIM-only protein LMO2, and the basic helix-loop-helix (bHLH) factor SCL/TAL1. GATA1 forms a pentameric transactivation complex with LMO2, the LIM-binding protein Ldb1, and the bHLH factors SCL and E1A, binding a composite E box/GATA enhancer sequence to transactivate erythroid gene expression. An equivalent pentameric complex has been characterized during Drosophila sensory organ precursor development, where the Achaete (Ac) bHLH protein and its obligatory cofactor, Daughterless (Da), associate with GATA/Pnr, dLMO, and the Lbd protein Chip for ac gene autoregulation. Drosophila Srp also interacts with orthologues of mammalian GATA cofactors. Indeed, GATA/Srp associates with the RUNX cofactor Lozenge (Lz) or the FOG factor U-shaped (Ush) to induce or repress crystal cell differentiation, respectively (Immarigeon, 2019).

Thus, GATA factor functions, DNA binding interfaces, and transcriptional cofactors appear conserved in Drosophila, but less is known about how GATA factors contact the Mediator complex to activate their target genes. By a genome-wide RNA interference screen in cultured Drosophila blood cells, previous work has identified several MED subunits (including Med1, Med12, and Med13) as modulators of GATA/Srp-induced transactivation. It was further shown that Med12 and Med13 are indeed required in vivo for Srp-dependent crystal cell differentiation. Furthermore, a genome-wide expression profiling from Drosophila GATA/Srp- or Med12- or Med13-depleted cells revealed a significant overlap, notably concerning the innate immunity genes. Nevertheless, no direct physical interaction in vitro was detected between Srp and Med12 or Med13, suggesting that GATA/Srp recruits the MED complex by contacting another subunit (Immarigeon, 2019).

This work addresses the issue of the conservation of Mediator subunit-specific functions across bilaterian evolution using as a model the Drosophila Med1 subunit whose mammalian orthologue is known as a GATA and NR cofactor. The generation of the first Med1 mutants in an insect reveals defects in GATA/Srp-dependent embryonic hematopoiesis. it was further shown that Srp forms a complex with Med1's longest isoform in Drosophila cultured cells. Furthermore, the divergent, isoform-specific C terminus of Med1 interacts with the conserved zinc finger-containing domain of Srp in vitro. The generation of Med1 mutant clones indicates a Med1 requirement for cell proliferation control and for the expression of a GATA/Pnr target gene in larval imaginal tissues. Finally, this study shows a Med1 role in Pnr-dependent transactivation and a direct interaction between the GATA/Pnr ZF-containing domain and the Drosophila-specific Med1 C terminus. Taken together, these data reveal that the Med1 Mediator subunit has conserved GATA TF coactivator functions during bilaterian evolution through divergent binding interfaces (Immarigeon, 2019).

This work reported the first Med1 loss-of-function mutants in an insect. Med1 is shown to be an essential gene which is not strictly required for cell viability but is clearly involved in context-dependent proliferation or cell survival processes. As opposed to Drosophila, mammalian Med1 is not essential for cell viability given that primary embryonic fibroblasts can be derived from null Med1 mutants in mice. Nevertheless, mouse Med1 mutant cells display impaired cell cycle regulation, suggesting a conserved Med1 function in cell proliferation control that deserves to be analyzed in the future using Drosophila as a model (Immarigeon, 2019).

The take-home message of this work is the functional partnership between Drosophila Med1 and GATA transcription factors (see MED1-GATA molecular partnership). Med1 is shown to be involved in at least two developmental processes, embryonic crystal cell differentiation and larval thoracic development, depending on two GATA transcription factors, Serpent and Pannier, respectively. Med1 is required for the expression of at least one Srp and one Pnr target gene in vivo. The partial loss of crystal cells observed in Med1 zygotic mutant embryos could reflect a partial requirement for Med1 in this differentiation process or could be due to partial rescue by the Med1 maternal contributions. Furthermore, this study showed that Med1 interacts physically with Srp and Pnr both in vitro and in cultured cells. Taken together, these results reveal evolutionarily conserved functions of Drosophila Med1 as a GATA cofactor involving a divergent Med1 region binding the conserved GATA zinc fingers. This Med1-GATA partnership appears restricted to one Med1 isoform, suggesting a new layer of regulation by the Mediator complex through a diversity of MED populations. Finally, Med1 is not required for all GATA/Pannier activity, suggesting the use of alternative MED subunits depending on the cellular context (Immarigeon, 2019).

This study shows that Med1 activity as a GATA cofactor is not restricted to vertebrates, since it also acts in Drosophila for at least two different GATA factors, suggesting an ancient GATA-Med1 partnership among bilaterians. The MED-TF partnership conservation along evolution is not the rule, since the LXXL motif of mammalian Med1 interacting with hormone nuclear receptors is not present in Drosophila Med1, whereas it is in two other Drosophila Mediator subunits, Cdk8 and Med14, which have been shown to be necessary for ecdysone nuclear receptor activity (Immarigeon, 2019).

What about the conservation of binding interfaces between Med1 and GATA factors? The results indicate that two Drosophila GATAs, like mammalian GATA1, -2, -3, and -6 and chicken GATA1, -2, and -3, bind Med1 at least through their ZF-containing domains. Nevertheless, some discrepancies exist concerning N- or C-ZF involvement in mammals. In Drosophila, this study shows that either N-ZF or C-ZF interacts with Med1ex4 in vitro and that Srp N-ZF is dispensable for binding in cultured cells. More diversity is observed for GATA-interacting domains within the Med1 protein. Med1 primary sequence conservation lies essentially within the N-terminal part, corresponding to the entire yeast orthologue, whereas the long additional C-terminal part diverges in each metazoan species. In Drosophila, this study shows that both Srp and Pnr interact with the Med1 isoform A-specific domain lacking sequence homology with mammalian Med1. In mammals, GATA-interacting domains differ depending on the GATA paralogue considered and are distributed throughout the Med1 sequence. In conclusion, despite functional conservation of the Med1-GATA partnership, the GATA-binding interface within Med1 has not been fixed during bilaterian evolution, even among paralogs. Such versatility is clearly favored by the enrichment of intrinsically disordered regions (IDRs) within many metazoan MED subunits. Indeed, IDRs, which do not fold into stable three-dimensional globular structures, would be a natural way for the Mediator complex to adapt to the increasing diversity of transcriptional regulators during evolution. Shown to interact with many TFs, Med1 has the largest IDR among MED subunits. Interestingly, partially conserved IDRs lie within a specific fragment of the longest Drosophila isoform, which this study identified as the Srp and Pnr interacting domain. It has been proposed recently that mammalian Med1 IDRs can form phase-separated droplets that compartmentalize and concentrate the transcription apparatus at superenhancers to drive robust gene expression. Thus, it is proposed that GATA interaction with Med1 IDRs concentrates MED-Pol II clusters at GATA-bound enhancers to activate transcription (Immarigeon, 2019 and references therein).

This work reveals for the first time a TF partnership apparently restricted to one isoform of a Mediator subunit. Indeed, it was shown that (1) GATA/Srp only coprecipitates with the longest Med1A isoform in cultured cells, (2) a domain specific to isoform A is sufficient to bind both Srp and Pnr in vitro, and (3) a transgene ubiquitously expressing the Med1A isoform fully rescues the crystal cell differentiation defects of Med1-depleted embryos. More work is required to determine whether all of the GATA transactivation function of Med1 resides within the longest isoform or whether the shorter ones can partially fulfill this role or display antagonizing activity. With an antibody directed against a common protein portion, it was shown that all three Drosophila Med1 isoforms are incorporated into the MED complex (4) and that Med1 is ubiquitously expressed in imaginal tissues, but it was not possible to evaluate whether or not Med1 isoforms are differentially expressed. Nevertheless, this hypothesis is supported by the fact that the ratio between Med1 transcripts, as well as their relative levels, has been shown to change during development and in different cell lines. These data strongly suggest a physiological relevance of alternative transcript production for Med1 and pave the way for future research. If MED complexes containing different Med1 isoforms are functionally distinct, the view of MED as a unique ubiquitous entity would be challenged. Mediator would then be the name of a heterogeneous population of complexes with different regulatory specificities (Immarigeon, 2019).

Med1 is not an obligatory partner of GATA factors. Drosophila Med1 acts as a cofactor for the GATA factors Pnr and Srp, physically binding both TFs and GATA-type ZFs and mediating their target gene's transactivation. This suggests that Med1 is an obligatory partner of GATA factors necessary to recruit the Pol II transcription machinery to GATA-activated promoters, as was proposed in mammals in earlier studies. Nevertheless, it was found that Med1 is critical for achaete- but not for wingless-induced transactivation by Pnr. Similarly, it was shown that mammalian Med1 regulates only a limited subset of GATA1-dependent genes in erythroid cells and that GATA1 recruits Med1 at activated genes but not at repressed loci. In addition, the analysis of different blood cell types produced from conditional Med1 knockout mice showed that Med1 participates in GATA1-dependent erythropoiesis but is dispensable for other GATA-dependent processes. This paper suggested that GATA factors, despite binding Med1 in vitro, contact other MED subunits to regulate their target genes in vivo. Along these lines, Med14, Med17, and Med25 have also been proposed as GATA1 interactors (Immarigeon, 2019).

It is now clear that the view of MED action as a binary partnership, i.e., one subunit to one TF, is too simplistic. It has been postulated that MED subunits act in a concerted manner, in a positive or negative way, by interacting simultaneously with one or several transcription factors and cofactors bound at gene enhancers, as well as promoters, to finely regulate gene expression in response to TFs. This attractive view of MED action as an integrative molecular hub device, transforming complex combinatorial inputs (TFs, cofactors, chromatin modifiers, etc.) into a simple transcriptional output, has rarely been tackled experimentally, particularly in metazoans. The use of Drosophila and the GATA-MED paradigm should allow exploration of this view in vivo (Immarigeon, 2019).

Mediator complex subunit Med19 binds directly GATA transcription factors and is required with Med1 for GATA-driven gene regulation in vivo

The evolutionarily conserved multiprotein Mediator complex (MED) serves as an interface between DNA-bound transcription factors (TFs) and the RNA Pol II machinery. It has been proposed that each TF interacts with a dedicated MED subunit to induce specific transcriptional responses. But are these binary partnerships sufficient to mediate TF functions? Previous work established that the Med1 Mediator subunit serves as a cofactor of GATA TFs in Drosophila, as shown in mammals. This study observed mutant phenotype similarities between another subunit, Med19, and the Drosophila GATA TF Pannier (Pnr), suggesting functional interaction. It is further shown that Med19 physically interacts with the Drosophila GATA TFs, Pnr and Serpent (Srp), in vivo and in vitro through their conserved C-zinc finger domains. Moreover, Med19 loss of function experiments in vivo or in cellulo indicate that it is required for Pnr- and Srp- dependent gene expression, suggesting general GATA cofactor functions. Interestingly, Med19 but not Med1 is critical for the regulation of all tested GATA target genes, implying shared or differential use of MED subunits by GATAs depending on the target gene. Lastly, this study shows a direct interaction between Med19 and Med1 by GST-pull-down experiments indicating privileged contacts between these two subunits of the MED middle module. Together, these findings identify Med19/Med1 as a composite GATA TF interface and suggest that binary MED subunit - TF partnerships are probably oversimplified models. Several mechanisms are proposed to account for the transcriptional regulation of GATAs-targeted genes (Immarigeon, 2020).

Using molecular, cellular and genetic analyses this work establishes that Drosophila GATA factors' transcriptional activity depends on the Mediator complex subunit Med19, in addition to the previously characterized Med1 cofactor. Four main conclusions that are discussed below can be drawn from these results. (1) Med19 interacts with the GATA C-ZF domain which also serves as the GATA DNA- binding domain. (2) Med19 and GATA interacting domains are evolutionary conserved suggesting conserved Med19 cofactor functions in higher metazoans. (3) Comparative analysis of Med19 and Med1 depletion indicates that Med19 but not Med1 is systematically required for GATA target gene expression suggesting a differential use of MED subunits by GATAs depending on the target gene. (4) Med1 and Med19 interact in vitro. Taken together these data allow proposal of new models of Mediator complex mechanism of action (Immarigeon, 2020).

TFs minimally contain two domains: the DNA binding domains (DBD), which have been extensively studied and allowed to define different TF families, and transcriptional activation domains (TAD), which link TFs to the RNA polymerase II machinery, and whose structure and characteristics are less well defined. GATA TFs are characterised by the presence of two ZFs which were, so far, thought to play distinct roles. While the C-ZF appeared to be dedicated to DNA binding, the N-ZF was shown to bind co-activators such as dLMO and FOG. The present data show that Med19 interacts specifically with the Pnr C-ZF. Full interaction requires both the zinc finger and its adjacent basic tail which also contributes to DNA binding. It is the first evidence that the Drosophila GATA C-ZF may play a dual role, in DNA binding and as an interface with MED subunit(s). Interestingly, the analysis of GATA ZF evolutionary conservation indicates that N- and C-ZF domains comes from a duplication event of the C-ZF with its basic tail. Thus, this transactivation function of the GATA DBD might represent an ancestral GATA function allowing minimal primitive GATAs, essentially composed of the DBD, to connect the MED complex and thus recruit the transcriptional machinery to regulate its targetgenes. They provide rationale why slightly-extended GATA ZF domains are in some cases sufficient for transcriptional activities in vivo (Immarigeon, 2020).

This dual activity of DBD is not restricted to GATA factors. It was previously shown that HOX TFs also contact Med19 through their DNA-binding homeodomain (Boube, 2014). The current data also corroborate results from a recent high- throughput approach, looking for trans- activation domains of Drosophila transcription factors. This work shows that trans-activation domains of several zinc-finger- (ZF-) and basic Helix-Loop-Helix- (bHLH-) TFs overlap structured DNA-binding domains. Altogether, these results identify a novel class of TF characterized by overlapping TAD and DBD and suggest an emerging Med19 property as a dedicated cofactor directly connecting these TFs DNA-binding domains to the general PolII transcriptional machinery (Immarigeon, 2020).

How is this dual function of DBDs achieved? Do DNA binding and transactivation functions use distinct or shared molecular determinants? Recent improvements of electron microscopy analyses could allow characterizing GATA molecular residues involved in MED- versus DNA-binding to try to separate the GATA DNA binding- from GATA transactivation functions (Immarigeon, 2020).

While Med1 is a known GATA cofactor both in mammals and in Drosophila, the role of Med19 in mediating GATA transcription regulatory properties had never been investigated until now. This study shows that Drosophila Med19 binds GATA factors, via motifs lying within the evolutionary-conserved Med19 CORE and HIM domains. Both of these domains bind to the C-ZF domain of GATAs, which is a hallmark of GATA TF family suggesting that interaction with Med19 is likely to be conserved in mammals. Yet, Med1 depletion experiments in mammalian cultured cells induces defects in only a subset of GATA1-activated genes and does not prevent GATA1-dependent repression. Furthermore, in studies of the different blood cell types produced by conditional Med1 knock-out mice, Med1 appears to be critical for erythroid lineages which depend upon GATA1-function but is dispensable for hematopoietic stem cell production and T-cell development which require GATA2 and GATA3, respectively. Thus, despite being capable of binding all GATA factors in vitro, Med1 is not critical for all GATA functions, which suggests that (an) other MED subunit(s) also bind(s) GATAs to relay their regulatory signals to the PolII machinery. Considering the evolutionary-conservation of interaction motifs within both GATAs and Med19, it is argued that Med19 is a strong candidate as a GATA cofactor in mammals (Immarigeon, 2020).

This data show that most Drosophila GATA target genes require both Med19 and Med1. How does this work? Med19 only interacts with the C-ZF domain, but Med1 can bind both GATA zinc-finger domains, suggesting that Med1 and Med19 can simultaneously bind GATA factors. It is thus proposed that in the majority of cases where GATA-driven gene expression requires both Med19 and Med1, enhancer- bound GATAs must directly contact both Med1 and Med19 subunits to recruit the Mediator complex and thus the PolII machinery at GATA target genes. Some genes (e.g. wingless) require Med19 but not Med1. How does this kind of gene specificity occur mechanistically? It is hypothesized that for these Med1-independant genes, other transcription factors might be involved in recruiting the MED (through other subunits) and hence overcome the necessity for Med1-GATA interaction (Immarigeon, 2020).

A future challenge will be to test these models by site-directed mutagenesis in vivo to assess the functional contribution of each GATA- MED contact. Nevertheless, this task is complicated because of overlapping DNA- and MED- interacting domains within GATAs. On the other hand, Med19 CORE domain has also a dual function of MED anchorage and interaction with GATA. It thus requires prior structural analysis of molecular contacts to specifically target GATA-MED interaction without affecting essential DNA-binding activity of GATAs or Med19 ability to incorporate the MED-complex (Immarigeon, 2020).

Another interpretation of these results could be that other subunits necessary for GATA target gene expression fall off from the complex when Med19 or Med1 are deleted or knocked down. However, structural analyses of MED complexes from yeast and mammalian cells lacking Med19 or Med1 indicate that global MED organization is unchanged and it is therefore considered very unlikely. Other lines of evidence indicate that complexes missing only Med19 can be isolated from Med19 depleted mammalian or yeast cells. Altogether, these results suggest that Drosophila MED subunit loss is unlikely in Med19 or Med1 depletion conditions. Since a direct physical interaction was found between GATAs and Med1/Med19, the simplest explanation for these results is considered to be that the loss of either of these subunits is enough to abolish GATA regulatory signals (Immarigeon, 2020).

Previous models of core MED structure-function analysis suggested that the middle and head modules contact the PolII enzyme and associated general transcription factors (GTFs) while the tail module interacts with sequence- specific TFs. The data show that two MED subunits of the middle module, Med1 and Med19, are able to bind GATA factors and are required for their function. They emphasize that MED should be viewed as a much more complex interface using multiple MED subunits to contact different TF combinations thus mediating specific transcriptional responses (Immarigeon, 2020).

Modelization of MED spatial organization indicate that Med1 and Med19 are most likely located at two opposite ends of the middle module, Med1 near the tail module and Med19 within the so called 'hook' domain proposed to anchor the separable CDK8 module (CKM). Nevertheless, the data indicate that Med1 and Med19 interact in vitro. Furthermore, this interaction occurs via the highly-conserved, N-terminal, MED-addressing domain of Med1 suggesting an evolutionary conservation. How, then, is it possible to reconcile the proposed MED architecture with the results showing a direct interaction between Med1 and Med19 subunits in vitro (Immarigeon, 2020)?

Two non-exclusive hypotheses are proposed: first, MED complexes could adopt different conformations, which would differ from the 'canonical' architecture of the MED complex in isolation. This is supported by observations that the MED complex changes its overall shape when engaged in interactions with either TF, CKM or PolII. Perhaps when MED is recruited by GATA, Med1 - Med19 contacts within the MED complex could stabilize one of these 'alternative' conformations (Immarigeon, 2020).

A second possibility is that Med1-Med19 interactions do not occur within but between MED complexes and could thus stabilize 'multi-MED' structures. It has been shown that master TFs control gene expression programs by establishing clusters of enhancers called super-enhancers, at genes with prominent roles in cell identity. Recent studies have revealed that, at super-enhancers, master TFs and the Mediator coactivator form phase-separated condensates, which compartmentalize and concentrate the PolII machinery to specific nuclear foci, to ensure high level of transcription. Interestingly, mammalian Med1 can form such phase-separated droplets that concentrate the transcription machinery at super-enhancers. Bringing together several MED complexes associated with TFs via Med1-Med19 trans-interaction might thus help phase-separated droplet formation at clustered gene enhancers and ensure high transcriptional level (Immarigeon, 2020).

In conclusion, this work shows that 2 MED subunits physically bind GATAs and are required to relay the regulatory signals from common TFs. This argues against the generally admitted view of binary interaction between one MED subunit and one TF, which appears as an oversimplified model for MED action. The Mediator should be viewed as a complex interface allowing fine-tuned gene regulation by TFs through specific contacts with different MED subunit combinations. This study highlights the unexpected role of Drosophila Med19 as a GATA cofactor and Med1 interactor. This work sheds new light on the GATA-MED paradigm and suggests novel means by which several MED subunits might collaborate to regulate gene transcription (Immarigeon, 2020).

GENE STRUCTURE

Bases in 5' UTR - 689

Exons - four

Bases in 3'UTR - 688

PROTEIN STRUCTURE

Structural Domains

Pannier has two zinc fingers, a polyglutamine stretch and two helicies rich in hydrophobic amino acids (Ramain, 1993 and Winick, 1993).

date revised: 20 December 99

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