BIOLOGICAL OVERVIEW

There are three GATA homologs in Drosophila: Serpent (dGATAb), found in the midgut and ovary; Pannier (dGATA-a), found in the dorsal epidermis, and dGATAc, found in the procephalic region, the posterior spiracles, the gut, and the central nervous system.

Serpent has a pivotal role in promoting morphogenesis of the anterior and posterior midgut, although it does not set the borders of early tissue movements during gastrulation. Very early in development it acts like an effector gene, responsive to inductive actions of the terminal system, specifically huckebein. In this way serpent is induced in the gut through the action of Huckebein. serpent is also expressed in the fat body and in ovaries where it regulates yolk proteins (Abel, 1993).

The fat body is a loose sheet of cells located between the gut and somatic musculature. It has a segmental origin; its organization is dependent upon the activity of genes of the bithorax complex. serpent is the earliest known gene to be expressed in the fat body; serpent targets genes expressed later, like Alcohol dehydrogenase ( Adh). The fat body is mesodermal in origin, but is derived from cells deeper in the mesoderm than myogenic cells identified by their expression of nautilus (Abel, 1993).

serpent expression is seen in a patch of cells in the anterior portion of the mesodermal primordium. These cells invaginate with the portion of the ventral furrow that is anterior to the cephalic furrow. Slightly later these cells differentiate into prohemocytes and subsequently, become distributed throughout the body and differentiate as mature hemocytes. It is proposed that the mesodermal patch of srp constitutes the hemocyte primordium at blastoderm stage. These findings imply involvement of GATA in blood cell development and in endodermal differentiation. Such an involvement reflects an early phenomenon of metazoan evolution and may be common to most higher animals (Rehorn, 1997).

The somatic muscles, the heart, the fat body, the somatic part of the gonad and most of the visceral muscles are derived from a series of segmentally repeated primordia in the Drosophila mesoderm. The early development of the fat body is described and its relationship to the gonadal mesoderm, as well as the genetic control of the development of these tissues. The first sign of fat body development is the expression of serpent in segmentally repeated clusters within the trunk mesoderm in parasegments 4-9. Segmentation and dorsoventral patterning genes define three regions in each parasegment in which fat body precursors can develop. The primary and secondary dorsolateral fat body primordia are formed ventral to the visceral muscle primoridium in each parasegment. The ventral secondary cluster forms more ventrally in the posterior portion of each parasegment. Fat body progenitors in these regions are specified by different genetic pathways. Two dorsolateral regions require engrailed and hedgehog (within the even-skipped domain) for their development while the ventral secondary cluster is controlled by wingless. Ubiquitous mesodermal en expression leads to an expansion of the primary clusters into the sloppy-paired domain, resulting in a continuous band of serpent-expressing cells in parasegments 4-9. The observed effect of en on fat body development is seen not only on mesodermal overexpression but also when en is overexpressed in the ectoderm. Loss of wingless leads to an expansion of the dorsolateral fat body primordium. decapentaplegic and one or more unknown genes determine the dorsoventral extent of these regions. High levels of Dpp repress serpent, resulting in the formation of visceral musculature, an alternative cell fate (Reichmann, 1998).

In each of the parasegments from 10 to12, one of these primary dorsolateral regions generates somatic gonadal precursors instead of generating the fat body. The balance between fat body and somatic gonadal fate in these serially homologous cell clusters is controlled by at least five genes. A model is suggested in which tinman, engrailed and wingless are necessary to permit somatic gonadal develoment, while serpent counteracts the effects of these genes and promotes fat body development. In wg mutant embryos, all dorsolateral mesodermal cells, including those in parasegments 10-12, acquire fat body fate. This phenotype can be interpreted as the combined effects of two separate functions of wg: (1) wg is necessary to repress fat body development in the dorsolateral mesoderm underlying the wg domain in all parasegments; (2) wg is required in the primary cluster to permit somatic gonadal precursor instead of fat body development in parasegments 10-12. Loss of engrailed results in the absence of demonstrable somatic gonadal precursors, similar to the situation in tinman mutants. Ubiquitous mesodermal en expression leads to the formation of additional somatic gonadal precursor cells in parasegments 10-12. The homeotic gene abdominalA limits the region of serpent activity by interfering in a mutually repressive feed back loop between gonadal and fat body development. It is unlikely that abdA represses srp directly, since srp can be expressed in cells in which abdA is active. abdA might prevent srp from inhibition of a somatic gonadal precursor competence factor (Riechmann, 1998).

serpent is necessary for embryonic fat-cell differentiation in Drosophila and has been proposed to function in a cell-fate choice between fat cell and somatic gonadal precursors. Deregulated expression of serpent in the mesoderm induces the formation of ectopic fat cells and prevents the migration and coalescence of the somatic gonadal precursors. The ectopic fat cells do not arise from hyperproliferation of the primary fat-cell clusters but they do associate with the endogenous fat cells to form a fat body that is expanded in both the dorsal/ventral and anterior/posterior axes. Misexpression of serpent also affects the differentiation of muscle cells. Few body-wall muscle precursors are specified and there is a loss of most body-wall muscle fibers. The precursors of the visceral mesoderm are also absent and concomitantly the visceral muscle is absent. It is suggested that the ectopic fat cells might originate from cells that have the potential, but do not normally, differentiate into fat cells or from cells that have acquired a fat-cell fate (Hayes, 2001).

To test whether srp is sufficient to induce fat-cell development, the GAL4 targeted expression system was employed. twist-GAL4 and UAS-srp constructs were used to express srp throughout the mesoderm beginning at gastrulation. In stage 10-11 embryos, srp expression is readily detected in the primary fat-cell clusters in PS 4-9 and in the secondary fat-cell clusters in PS 10-12. In twist-GAL4;UAS-srp embryos, srp expression is expanded into a continuous band of cells throughout the mesoderm. Several molecular cell markers were used to monitor the effects of ectopic srp expression on the segregation of mesodermal derivatives. Two genes, alcohol dehydrogenase (Adh) and Drosophila collagen gene 1 (DCg1) were used to detect the fat body. Both Adh and DCg1 are embryonic terminal fat-cell differentiation markers. Adh expression is first detected during embryogenesis at stage 14 in the anterior wall of the midgut and the atrium of the posterior spiracle, and in the late precursor fat cells at stage 15. By stage 16, high levels of Adh transcripts are detected in the lateral fat body, and lower levels are found in the dorsal fat-cell projections. Within the developing fat cells, the temporal expression pattern of DCg1 is similar to Adh although DCg1 transcripts are easily detected at equivalent levels in both the dorsal fat-cell projections and the lateral fat body by stage 16. DCg1 is also expressed in the hemocytes. In twist-GAL4;UAS-srp embryos, the premature expression of srp throughout the mesoderm does not induce premature expression of Adh. As in wild-type embryos, Adh transcripts are first detected in the fat cells of experimental embryos at stage 15. The Adh-positive fat cells however, occupied most of the lateral mesoderm and formed an expanded lateral fat body (Hayes, 2001).

Because srp is a direct transcriptional activator of the Adh promoter in transient transfection assays, the increase in Adh-expressing cells might be the result of ectopic transactivation of Adh by srp rather than a reflection of the activation of the entire fat-cell genetic program. To test this possibility, the expression pattern of a second fat-cell marker, DCg1, was examined. In embryos carrying twist-GAL4;UAS-srp, the temporal pattern of DCg1 expression was also normal. DCg1 transcripts were first detected in hemocytes and then in fat cells at stage 15. The number of DCg1-expressing hemocytes was comparable to that of wild type. However, similar to the results obtained with Adh, there was an increase in the number of DCg1-expressing fat cells. The DCg1-expressing fat cells formed an expanded lateral fat body and dorsal fat-cell projections were expanded and fused with the lateral fat body in the posterior region of the embryo. An increase in lateral fat body cells was also detected in experimental embryos using a third fat-cell marker, the imaginal disc growth factor 3 gene, IDGF3. Similar results, albeit less extreme, were obtained using a second mesodermal GAL4 driver, 24B, that is first active in the presumptive mesoderm. These data demonstrate that ectopic expression of srp in the mesoderm leads to an increase in cells composing the lateral fat body and dorsal fat-cell projections (Hayes, 2001).

To determine whether mesoderm-specific components are necessary for srp to promote fat-cell development, srp was ectopically expressed in the ectoderm starting at stage 9 using the 68B-GAL4 driver. Ectopic expression of srp in the ectoderm is not sufficient to induce either Adh or DCg1 in the ectoderm, nor does it alter the normal development or morphology of the lateral fat body and dorsal fat-cell projections. These data suggest that srp requires mesodermal factors for induction of fat cells (Hayes, 2001).

At least two distinct mechanisms could account for the expansion of the fat body in the twist-GAL4;UAS-srp embryos: (1) the misexpression of srp could alter cell fates within the mesoderm by recruiting cells into the fat-cell developmental pathway; (2) premature and ectopic expression of srp in the mesoderm could cause hyperproliferation of endogenous precursor fat cells. To distinguish between these possibilities, the P-element enhancer-trap line, P[29D], was employed as a lacZ reporter gene for the primary fat-cell clusters in PS 4-9 and in the secondary fat-cell clusters in PS 10-12. Early expression of P[29D] is independent of srp. By marking precursor fat cell with the P[29D] lacZ reporter, a test could be made of whether expression of srp throughout the mesoderm causes hyperproliferation of endogenous fat cells. It was found that lacZ expression is not significantly altered in twist-GAL4;UAS-srp embryos. Specifically, the number and organization of the fat cells marked by P[29D] appear normal. Thus, the increase in fat cells is likely not to be due to hyperproliferation of endogenous fat-cell lineage although the possibility that the ectopic fat cells originate from the secondary fat-cell clusters not marked by P[29D] could not be eliminated (Hayes, 2001).

The forced expression of srp in the mesoderm results in the production of ectopic fat cells. Because it is not likely that the endogenous fat-cell lineage undergoes hyperproliferation, it is suggested that srp might be capable of inducing fat-cell development in cells that normally would not contribute to the fat body. Such a capability has been proposed for srp in the developmental choice model between somatic gonadal precursors (SGPs) and precursor fat cells. If srp activity can direct a fat-cell fate upon a common precursor that otherwise would be specified as SGPs, then it follows that ectopic expression of srp should repress the specification of SGPs in PS 10-12 and these cells should be replaced by fat cells (Hayes, 2001).

To test for srp’s role in the repression of SGP specification, twist-GAL4;UAS-srp embryos were examined using 412 as a cell marker for the SGPs and the gonadal mesoderm. In wild-type stage-12 embryos, 412 is expressed in PS 10-12 in the SGPs and in cell clusters in PS 2-9 and 14. During germband retraction, expression of 412 declines but persists in the SGPs (PS 10-12). It is unclear which cell lineage(s) the PS 2-9 and PS 14 clusters of cells represent. However, by over-staining for 412 transcripts, late 412 expression can be detected in the dorsal-most cells of the lateral fat body. At stage 13, the SGPs migrate and coalesce to form the gonadal mesoderm cells that will eventually ensheath the germ cells. At stage 16, 412 transcripts are still detected in the gonadal mesoderm (Hayes, 2001).

In stage-12 twist-GAL4;UAS-srp embryos, the expression of 412 is similar to that observed in wild-type embryos. The SGPs are specified and initial association of pole cells with gonadal precursors appears normal. The first alteration in the development of the gonadal mesoderm is detected at stage 13 when the SGPs fail to migrate and coalesce. The number of 412-expressing cells gradually declines in the experimental embryos and the few remaining 412-expressing cells are dispersed in the posterior region of the embryo. Misexpression of srp in the mesoderm does not affect the formation of the SGPs, however it does disrupt the ability of the SGPs to migrate and coalesce into a gonad (Hayes, 2001).

To confirm the effects of srp on SGP and gonad formation, twist-GAL4;UAS-srp embryos were examined for expression of a second gonadal mesoderm cell maker, clift (cli). cli is expressed throughout the mesoderm but by early stage 11 is lost in most mesodermal cells. During late stage 11, cli expression is detected in SGPs and in lateral muscle precursors, as well as in the ectoderm. Based on mutational analysis of a transcript-producing cli mutant, cli^IIe, cli is necessary for maintenance of SGPs and their migration and coalescence into a gonad (Hayes, 2001).

In twist-GAL4;UAS-srp embryos, misexpression of srp does not affect early cli expression in the mesoderm. However, in older embryos, cli transcripts are not detected in either the SGPs or in the precursors of the lateral muscles although cli expression is still detected in the ectoderm. Because cli is necessary for the migration and coalescence of the SGPs, the inability of the SGPs to form a mature gonad is most likely due to the loss of cli expression in these cells. It is suggested that in the experimental embryos, srp does not prevent the specification of the SGPs but can block the differentiation of the SGPs by repressing cli expression in these cells (Hayes, 2001).

To test whether other lineages are affected by misexpression of srp, twist-GAL4;UAS-srp embryos were examined for heart and visceral muscle precursors. To mark heart precursors, the homeobox gene tinman (tin) was employed. tin is involved in the specification of the dorsal mesoderm and in the formation of heart muscle precursors. In embryos carrying twist-GAL4;UAS-srp, heart precursors develop normally to form the heart. Thus, ectopic expression of srp does not affect heart development (Hayes, 2001).

The bagpipe (bap) gene was used to mark the visceral mesoderm, which gives rise to visceral muscle precursors. The visceral mesoderm is made up of 10 metameric clusters of cells located in the dorsal mesoderm. bap is expressed in these cells and is necessary for the formation of the visceral muscle. twist-GAL4;UAS-srp embryos lack the bap-expressing cells and exhibit a bap-like phenotype, in which the midgut fails to undergo its normal constrictions. The loss of bap-expressing cells in the experimental embryos might reflect a cell-fate change that allows the replacement of visceral muscle precursors by fat-cell precursors (Hayes, 2001).

Finally, the effect of srp misexpression on somatic or body-wall muscle was examined. The absence of cli-expressing lateral (body wall) muscle precursors suggests that these muscles might be absent in the experimental embryos. The body-wall muscle is derived from the lateral region of the slp domain that exhibits the highest levels of twist expression. nautilus (nau) and S59 were used as cell markers for a subset of the founder or precursor cells for body-wall muscle and the tropomyosin I<.A> (Tm1) gene was used as a marker for body-wall muscle fibers. nau is first active at stage 10 and is expressed in a dynamic pattern. nau expression is detected in cells flanking the ventral midline and later in lateral and dorsolateral cell clusters. S59 expression is initially detected in a single, large mesodermal cell in a segmentally repeating pattern. At late stage 11, these cells give rise to two founder muscle cells and in each segment a second cluster of four S59-expressing cells appears that also contributes to precursor muscles. In embryos carrying twist-GAL4;UAS-srp, the early nau and S59 pattern of expression is disrupted and various nau- and S59-expressing cells are absent. To determine whether there is a general effect on body-wall muscle, twist-GAL4; UAS-srp embryos were stained for the body-wall muscle marker Tm1. Consistent with the loss of nau- and S59-expressing cells, a dramatic loss of muscle fibers was found. The few remaining muscle fibers were mono- and bi-nucleate and their number and location varied from embryo to embryo. These remaining muscle fibers might reflect an incomplete loss of the founder muscle cells and/or myoblasts. It is possible that the remaining fibers are due to loss of bap expression, which leads to visceral mesoderm precursors assuming a somatic muscle identity. It is concluded that the ectopic expression of srp in the mesoderm can disrupt body-wall muscle differentiation (Hayes, 2001).

Differential roles of the Drosophila EMT-inducing transcription factors Snail and Serpent in driving primary tumour growth

Several transcription factors have been identified that activate an epithelial-to-mesenchymal transition (EMT), which endows cells with the capacity to break through basement membranes and migrate away from their site of origin. A key program in development, in recent years it has been shown to be a crucial driver of tumour invasion and metastasis. However, several of these EMT-inducing transcription factors are often expressed long before the initiation of the invasion-metastasis cascade as well as in non-invasive tumours. Increasing evidence suggests that they may promote primary tumour growth, but their precise role in this process remains to be elucidated. To investigate this issue this study has focused studies on two Drosophila transcription factors, the classic EMT inducer Snail and the Drosophila orthologue of hGATAs4/6, Serpent, which drives an alternative mechanism of EMT; both Snail and GATA are specifically expressed in a number of human cancers, particularly at the invasive front and in metastasis. Thus, this study recreated conditions of Snail and of Serpent high expression in the fly imaginal wing disc and analysed their effect. While either Snail or Serpent induced a profound loss of epithelial polarity and tissue organisation, Serpent but not Snail also induced an increase in the size of wing discs. Furthermore, the Serpent-induced tumour-like tissues were able to grow extensively when transplanted into the abdomen of adult hosts. The differences between Snail and Serpent correlate with the genetic program they elicit; while activation of either results in an increase in the expression of Yorkie target genes, Serpent additionally activates the Ras signalling pathway. These results provide insight into how transcription factors that induce EMT can also promote primary tumour growth, and how in some cases such as GATA factors a 'multi hit' effect may be achieved through the aberrant activation of just a single gene (Campbell, 2018).

EMT transcription factors are often found upregulated in human tumours. Given their role in driving a transition from a polarised static epithelial cell to a migratory invasive cell state, much focus has been put on the pro-invasive and metastatic implications of their aberrant expression. However, tumour progression involves the progressive acquisition of many other biological capabilities including sustained proliferation, evasion of growth suppressors and resistance to cell death, and there is increasing evidence suggesting that EMT transcription factors contribute to these earlier stages of tumour progression. This study shows that the Drosophila EMT-inducers Sna and Srp drive not only EMT, but also over-proliferation in a well-established epithelial tumour model. Sna-driven proliferation is accompanied by extensive cell death and a decrease in cell size, and thus the overall effects of aberrant Sna expression on tissue size are negligible. In contrast, Srp drives an increase in cell size as well as cell proliferation, but not cell death, leading to a profound overall increase in the size of the tissue, which is particularly evident upon transplantation, when the tissue has more time to grow. This study finds that both Sna and Srp repress crb transcription, which has previously been shown to induce a repression of the Hippo pathway and thus drive Yki-dependent overproliferation. Indeed, this study shows that both Sna and Srp activate Yki activity, which has previously been shown to drive excess proliferation in the wing disc. However, in addition to this, Srp was also found to activate the mitogenic Ras pathway, which has recently been shown to act synergictically with Yki to promote hyperproliferation and tumour development in the Drosophila wing disc. Studies in breast cancer models and oesophageal epithelial cells have shown that the EMT transcription factors Twist and Zeb contribute to primary tumour growth through the activation of programs that prevent cells from undergoing oncogene-induced senescence and apoptosis. Taken together with the current results, this suggests that EMT transcription factors can contribute to the multistep process of tumour progression through the activation of different onco-promoting cell biological processes, and that this is both transcription factor and tumour dependent (Campbell, 2018).

EMT transcription factors drive a loss of epithelial cell polarity, which has been shown to activate cell death pathways in a number of contexts. For example, scribble (scrib) mutant clones are completely eliminated from wild type discs through programmed cell death pathways. Intriguingly, while overexpression of both Srp and Sna drives a loss of cell polarity, an increase in cell death is only seen with Sna, whereas Srp appears to correlate with an increase in cell survival. This is despite the fact thatan increase was found in the transcription of the key apoptosis inhibitor Diap1 in both scenarios. Diap1 functions as an E3-ubiquitin ligase that protects cells from unwanted death by blocking the activity of the caspase DRONC and the Drosophila apoptotic protease-activating factor-1 (Apaf-1) homolog, Dark, and the relative levels of Diap1, Dronc and Dark are important in determining the outcome ie. cell survival vs cell death. As a lot of cell death is seen in the wing disc upon overexpression of Sna, despite a clear increase in Diap1 expression, this suggests that the levels of Diap1 induced are not sufficient to block the level of cell death induced by Sna. While a comparable increase is seen in the levels of Diap1 upon Srp overexpression, Srp also activates Ras, which has been reported to protect cells with mutations in cell polarity genes from death. However, reducing Ras signalling does not lead to an increase in cell death when Srp is overexpressed. It has been previously seen that ectopic Srp also induces expression of Forkhead (Fkh), which has been reported to act as a survival factor in a number of Drosophila systems, including the midgut. These results therefore suggest that cells expressing ectopic Srp evade death through the upregulation of multiple cell survival factors (Campbell, 2018).

Intriguingly, so-called EMT transcription factors such as Sna, Srp, Twist and Zeb proteins often activate many developmental pathways and processes of which a loss of cell polarity and EMT is only a part. They are all expressed in multiple tissues during development and play pleotrophic roles, depending on the context and time window in which they are activated. The cell context dependence of the activity of these genes is emphasized, suggesting that other genes may collaborate to the Srp and Sna induced transformations. For example, in Drosophila Sna activates an EMT in mesoderm cells during early stages of embryonic development, but later on it plays distinct roles during central nervous system and peripheral eye development. Srp is required for EMT in the Drosophila midgut, but also for midgut cell specification, and additionally plays multiple roles during specification and maturation of the haemocytes. Hence, it is not surprising that activation of such transcription factors outside the normal controls imposed during development can impinge on multiple cell features and signalling programs in addition to EMT, and thus play key roles in the initiation and development of primary tumours, rather than being limited to the steps of cancer cell invasion and metastatic spread. Additionally, it is worth noting that the effect of Srp activity on tissue overgrowth in the wing disc is due, at least in part, to the ectopic triggering of effector genes normally elicited by Srp in the midgut, one of its regular domains of expression (Campbell, 2018).

A previously investigated the effects of triggering ectopic sna and srp in the Drosophila embryo, by driving their expression in ectodermal epithelial cells in which they are never normally expressed. While a previous study found that sna had no effect on ectoderm cell behaviour (Campbell, 2015), a more recent study showed that when sna was expressed at high levels using a maternal driver, it triggers adherens junction disassembly in ectodermal cells, and in rare cases, the movement of some cells to inside the embryo (Weng, 2016). Similarly, ectopic srp drives a loss of apicobasal polarity and junction disassembly, although with srp there is a profound migration of cells into the embryo. Remarkably, in the embryo no proliferation is seen in these circumstances. Conversely, overproliferation is seen in wing discs, but very little cell migration. Intriguingly, a 'Go or Grow' hypothesis has been proposed which postulates that cell division and cell migration are temporally exclusive events and that tumor cells defer migration to divide and vice versa. The results suggest that EMT transcription factors can drive migration or proliferation, but tend to favour one over the other at any given time. Given the fact that EMT transcription factors are increasingly associated with cancer stem cells, it will be important to unravel when and how EMT promotes one over the other (Campbell, 2018).

The transformation of a healthy cell into a cancerous one requires multiple mutations and cooperation between different oncogenic/tumor suppressor mutations. Not only can EMT transcription factors accelerate tumour progression by the activation of multiple biological processes, this can also be exacerbated through cooperative effects of the different pathways. For example, in breast cancer models cooperation between Twist and an active form of RAS is sufficient to trigger transformation of mammary epithelial cells into malignant cells exhibiting all the characteristic features of claudin-low tumors. Similarly the current results suggest that ectopic Sna in combination with situations where cells become resistant to cell death may have catastrophic effects. Remarkably, over-expression of Srp alone activates both the EGFR/Ras and Yki signalling pathways. Of note, over-activation of the Ras pathway in situations of compromised cell polarity often leads to dramatic tissue overgrowth, for example when oncogenic Ras is combined with a scribble mutation. Furthermore, it has previously been shown that loss of Drosophila cell polarity regulators such as Scribble promotes epithelial tissue overgrowth and cooperation with the Ras pathway through impaired Hippo pathway signaling. Thus the profound effects seen upon Srp activation are likely due to cooperation between these two pathways. GATA factors are increasingly found deregulated in human tumours, both at the invasive front and in primary lesions and are receiving increasing attention as onco-promoting genes. The current work suggests that GATA factors could be activating multiple tumour promoting pathways, that act cooperatively both in early stages of primary tumour growth and later in driving invasion and metastasis (Campbell, 2018).

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).

bfc, a novel Serpent co-factor for the expression of croquemort, regulates efferocytosis in Drosophila melanogaster

Efferocytosis is the process by which phagocytes recognize, engulf, and digest (or clear) apoptotic cells during development. Impaired efferocytosis is associated with developmental defects and autoimmune diseases. In Drosophila melanogaster, recognition of apoptotic cells requires phagocyte surface receptors, including the scavenger receptor CD36-related protein, Croquemort (Crq, encoded by crq). In fact, Crq expression is upregulated in the presence of apoptotic cells, as well as in response to excessive apoptosis. This study identified a novel gene bfc (booster for croquemort), which plays a role in efferocytosis, specifically the regulation of the crq expression. Bfc protein interacts with the zinc finger domain of the GATA transcription factor Serpent (Srp), to enhance its direct binding to the crq promoter; thus, they function together in regulating crq expression and efferocytosis. Overall, this study shows that Bfc serves as a Srp co-factor to upregulate the transcription of the crq encoded receptor, and consequently boosts macrophage efferocytosis in response to excessive apoptosis. Therefore, this study clarifies how phagocytes integrate apoptotic cell signals to mediate efferocytosis (Zheng, 2021).

Apoptosis is a developmentally programmed cell death process in multicellular organisms essential for the removal of excessive or harmful cells; whereby apoptotic cells (ACs) are swiftly removed by phagocytes to prevent the release of toxins and induction of inflammation, a process crucial for organ formation, tissue development, homeostasis, and normal immunoregulation. In fact, defects in AC clearance (efferocytosis) can lead to the development of various inflammatory and autoimmune diseases. During efferocytosis, the effective clearance of ACs is accomplished through the recognition and binding of engulfment receptors or bridging molecules on the surface of phagocytes to 'eat me' signals exposed on the surface of ACs. After receptor activation, downstream signals trigger actin cytoskeleton rearrangement and membrane extension around the ACs to form phagosomes. Finally, mature phagosomes fuse with lysosomes to form phagolysosomes, where the internalized ACs are ultimately digested and cleared (Zheng, 2021).

Since efferocytosis is conserved throughout evolution, it has been studied not only in mammals but also in Drosophila melanogaster. Of note, in D. melanogaster, ACs are removed by non-professional phagocytes, such as epithelial cells and professional phagocytes, such as macrophages and glial cells. Importantly, Drosophila macrophages perform similar functions to those of mammalian macrophages; they participate in both the phagocytosis of ACs and pathogens. Several engulfment receptors have been identified as key players in the recognition and removal of ACs in Drosophila. Franc and colleagues first characterized Croquemort (Crq), a Drosophila CD36-related receptor required by macrophages to engulf ACs. Additionally, Draper (Drpr, a homolog of CED-1/MEGF10) also mediates AC clearance in both glia and macrophages; JNK signaling plays a role in priming macrophages to rapidly respond to injury or microbial infections. Of note, Drpr and its adapter Dmel\Ced-6 (GULP homolog) also seemed important for axon pruning and the engulfment of degenerating neurons by glial cells. The Src tyrosine kinase Src42A (Frk homolog) promotes Drpr phosphorylation and its association with another soluble tyrosine kinase, Shark (ZAP70 homolog), which in turn activates the Drpr pathway. In addition to Drpr, Six-Microns-Under (SIMU) [10] and integrin αPS3 [21] contribute to efferocytosis. SIMU, a Nimrod family cell surface receptor, functions upstream of Drpr to mediate the recognition and clearance of ACs as well as of non-apoptotic cells at wound sites through the recognition of phosphatidylserine (PS). Importantly, the transcriptional factor Serpent (Srp), a GATA factor homolog, was recently found to be required for the efficient phagocytosis of ACs in the context of Drosophila embryonic macrophages and acted via the regulation of SIMU, Drpr, and Crq (Zheng, 2021).

Searching for other genes required for efferocytosis, this study performed transcriptomic analysis (RNA-seq) and RNAi screening, and discovered 12 genes required for AC clearance in Drosophila S2 cells. In particular, a novel gene, bfc (booster for croquemort), involved in efferocytosis that encodes a specific regulatory factor for the crq transcriptional expression, both in vitro and in vivo. Importantly, this study demonstrated that the GATA factor Srp directly binds to the crq promoter, while Bfc strengthens this binding by interacting with the Srp zinc finger domain. Therefore, a model is proposed in which Bfc cooperates with Srp to enhance crq expression and subsequently induce efferocytosis in D. melanogaster (Zheng, 2021).

In mammals, ACs are recognized by CD36, one of the several phagocyte cell surface receptors, with the AC surface molecules serving as cognate 'eat-me' signals/ligands. ACs also secrete molecules that attract distant phagocytes and modulate the immune response or phagocytic receptor activity. However, the mechanisms underlying this effect remain unclear. Crq is a CD36-related scavenger receptor in Drosophila and is expressed immediately after the onset of apoptosis in embryonic macrophages. The expression of Crq is regulated by the extent of apoptosis, although the regulatory mechanisms by which ACs control the expression of Crq and subsequently induce phagocytosis in embryonic macrophages have not been described (Zheng, 2021).

This study has revealed a novel protein, Bfc (Booster for Crq), that plays a key role in efferocytosis via specifically regulating the expression of crq in a manner dependent on the extent of apoptosis. Bfc interacts with the zinc finger domain of the transcription factor Srp as a cofactor to enhance the binding of Srp to the crq promoter, leading to the upregulation of crq expression and the consequent induction of efferocytosis in Drosophila melanogaster. Importantly, the data reveal the molecular mechanisms by which ACs affect Crq expression, as well as how the phagocytic ability of embryonic macrophages is boosted in the presence of excessive apoptosis (Zheng, 2021).

This study found that in S2 cells, the ACs induced the transcriptional upregulation of crq. In vivo, the macrophages developed as early as the first wave of developmentally programmed apoptosis began at embryogenic stage 11, when the expression of crq was activated and subsequently became widespread throughout the embryo. Importantly, these results are similar to the regulatory mechanisms associated with the expression of other phagocytic receptors, such as Drpr and integrin. For instance, studies showed that AC engulfment rapidly triggers an intracellular calcium burst followed by increased levels of drpr transcripts in Drosophila macrophages; similarly, Draper and integrins become apically enriched soon after the engulfment of apoptotic debris in epithelial follicle cells (Zheng, 2021).

That the expression of crq was elevated early after the co-culture of ACs and S2 cells, but gradually decreased to the basal levels as efferocytosis continued, suggesting that the regulation of AC clearance and crq expression follow a similar pattern. It was demonstrated that most AC samples added to live S2 cells were composed of apoptotic cells rather than necrotic cells. However, the upregulated expression of genes in response to the presence of a few necrotic cells cannot eliminated. Indeed, based on transcriptome analysis, 12 genes were identified that are required for AC clearance, which was confirmed by subsequent efferocytosis assays using their individual knockdown in S2 cells. Interestingly, among the 12 genes, two were related to innate immunity. CecA1, regulated at the transcriptional level encodes an antibacterial peptide, as well as a secreted protein that mediates the activation of the Toll pathway during bacterial infection. This result may contradict the discreet nature of the apoptotic process. However, ATPs released by bacteria are known to mediate inflammation, and the toll-like receptor 4 (TLR4) is activated by ACs to promote dendritic cell maturation and innate immunity in human monocyte-derived dendritic cells. These results indicate that innate immune pathways are activated in the presence of ACs, and may contribute to their recognition or clearance in Drosophila (Zheng, 2021).

Among these 12 genes, CG9129 (bfc) and CG30172 regulated the expression of crq and hence, efferocytosis. Further studies must be performed to elucidate the role of CG30172 in efferocytosis. On the other hand, The role played by bfc in efferocytosis as well as the underlying mechanism was clearly dissected. Using several different experimental approaches, this study demonstrated that bfc regulates crq expression in response to excessive apoptosis. First, bfc RNAi treatment decreased the crq expression levels in S2 cells exposed to ACs, but not in the absence of ACs. Second, the increase in crq transcription was proportional to the extent of apoptosis in embryos, which was blocked by the loss of bfc. Notably, other phagocytic receptors have been reported to be activated by dying cells. The integrin heterodimer αPS3/βPS can be enriched in epithelial follicle cells after the engulfment of dying germline cells. In addition, Drpr expression increases in follicle and glial cells, which activates the downstream JNK signaling during the clearance of apoptotic germline cells and neurons, respectively. Collectively, the available scientific literature suggests that the expression of phagocytic receptors can be stimulated by the presence of excessive ACs to improve the phagocytic activity of macrophages or epithelial cells in different tissues (Zheng, 2021).

Bioinformatics analysis of the conserved domains and gene structure indicated that Bfc does not likely function directly as a transcription factor. This study identified Srp as a Bfc interaction partner using yeast two-hybrid and Co-IP analyses. Shlyakhover (2018) reported that Srp is required for the expression of SIMU, Drpr, and Crq receptors in embryonic macrophages; however, the current results demonstrated that bfc only affects the expression of crq expression through interaction with Srp, with no impact on the expression of several other genes. A plausible hypothesis for this phenotype is that Bfc assistance for Srp binding to the promoters of simu and drpr, may have limited effects. Thus, the results suggest that Bfc may regulate the Crq expression levels in the first wave of AC recognition via binding to Srp, whereas other regulatory factors participate in the Srp-mediated regulation of Drpr and SIMU (Zheng, 2021).

Srp directly binds to the DNA consensus sequence GATA of the crq promoter via its highly conserved Cys-X2-Cys-X17-Cys-X2-Cys zinc finger binding domain (C4 motif). Meanwhile, Srp also interacts with Bfc through its zinc finger domain; curiously, while the mutation of the C4 motif did not affect the latter interaction, it completely blocked the former. Importantly, it was also shown that mutation in the GATA site abolished the expression of the crq in Drosophila embryo macrophages. As a potential Srp cofactor, Bfc increased the ability of Srp to bind to the crq promoter, while bfc knockdown inhibited the crq transcriptional activity. Ush (homolog of FOG-2 in Drosophila), a cofactor of GATA transcriptional factors, can bind Srp and limit crystal cell production during Drosophila blood cell development. Interestingly, genetic studies have demonstrated that Ush acts with Srp to maintain the pluripotency of hemocyte progenitors and suppresses their differentiation. Ush was reported to repress crq expression by interacting with the isoform of Srp, SrpNC (with two GATA zinc finger) while the other isoform of Srp, SrpC (with one GATA zinc finger) induced crq expression, which may indicate Bfc and Ush act on different isoforms of Srp to regulate crq expression by opposite mechanisms (Zheng, 2021).

Although the results elucidate several factors that contribute to efferocytosis in Drosophila embryos, some mechanistic details remain unresolved; for instance, how ACs induce Bfc-mediated regulation of crq expression in macrophages remains unclear. Bfc regulates Crq expression and efferocytosis, but not macrophage development. Moreover, this study found that Bfc-mediated activation of crq transcription and Crq accumulation leads to positive feedback to promote increased Bfc expression, which is required for engulfment. As expected, the upregulation of Bfc expression occurred earlier than that of crq in S2 cells after incubation with ACs. Therefore, further studies are required to elucidate the upstream signals in the context of the crq-mediated regulation of bfc expression. As previous studies have shown that Crq is required for phagosome maturation during the clearance of neuronal debris by epithelial cells and bacterial clearance, further studies should be conducted to determine whether Bfc is involved in the clearance of neuronal debris (Zheng, 2021).

This study is not without limitations. For instance, other potential regulators of efferocytosis, whose expression is not affected by ACs could not be detected in this study. In mammals, CD36 is involved in the clearance of ACs and regulates the host inflammatory response. As a CD36 family homolog, Crq promotes the clearance of ACs and bacterial uptake via efferocytosis. Researchers have reported that the GATA factor Srp is required for Crq expression; this study confirmed this finding and showed that Srp directly binds to the crq promoter via its GATA binding site, which is enhanced by Bfc. However, no apparent Bfc homologs exist in vertebrates, and whether GATA factors regulate the CD36 family in a mechanism similar to that in flies remains unclear. Nevertheless, it is predicted that one or more functional homologs of Bfc may exist in mammals and are likely involved in apoptotic cell clearance. Unraveling them as well as determining whether and how bfc participates in eliminating pathogens and innate immunity is essential (Zheng, 2021).

In summary, this study has shown that the expression of the engulfment receptor Crq is transcriptionally regulated by the presence of ACs, via Srp, and its newly identified cofactor, Bfc. Altogether, these findings imply that macrophages adopt a precise mechanism to increase the expression of engulfment receptors to boost their phagocytic activity, in the presence of excessive ACs. A similar role and mechanism is anticipated in the context of mammalian engulfment receptors in response to excessive ACs. Therefore, the findings of this study have significant implications for a wide range of human diseases, including those associated with aberrant apoptotic cell death and efferocytosis, such as tumor progression, neurodegenerative disorders, and other severe inflammatory conditions (Zheng, 2021).

GENE STRUCTURE

Bases in 5' UTR - 281

Exons - 6

Bases in 3' UTR - 581

PROTEIN STRUCTURE

Structural Domains

Serpent has a single Cys-4 zinc finger domain.

date revised: 5 August 2023 

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