You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: The bHLH Factors Extramacrochaetae and Daughterless Control Cell Cycle in Drosophila Imaginal Discs through the Transcriptional Regulation of the cdc25 Phosphatase string}
Article:
Abstract:
One of the major issues in developmental biology is about having a better understanding of the mechanisms that regulate organ growth. Identifying these mechanisms is essential to understand the development processes that occur both in physiological and pathological conditions, such as cancer. The E protein family of basic helix-loop helix( bHLH) transcription factors, and their inhibitors the Id proteins, regulate cell proliferation in metazoans. This notion is further supported because the activity of these factors is frequently deregulated in cancerous cells. The E protein orthologue Daughterless( Da) and the Id orthologue Extramacrochaetae( Emc) are the only members of these classes of bHLH proteins in Drosophila. Although these factors are involved in controlling proliferation, the mechanism underlying this regulatory activity is poorly understood. Through a genetic analysis, we show that during the development of epithelial cells in the imaginal discs, the G2/M transition, and hence cell proliferation, is controlled by Emc via Da. In eukaryotic cells, the main activator of this transition is the Cdc25 phosphatase, string. Our genetic analyses reveal that the ectopic expression of string in cells with reduced levels of Emc or high levels of Da is sufficient to rescue the proliferative defects seen in these mutant cells. Moreover, we present evidence demonstrating a role of Da as a transcriptional repressor of string. Taken together, these findings define a mechanism through which Emc controls cell proliferation by regulating the activity of Da, which transcriptionally represses string.
Introduction:
Cell proliferation is a critical event in organ formation and it is regulated by multiple cellular signals. During development, individual cells interpret these signals to determine whether to continue proliferating or to induce cell cycle arrest and initiate differentiation. The way that cells integrate these different signals to control the cell cycle machinery is a crucial issue that has received a great deal of attention[1]–[2] although many aspects of this process have yet to be elucidated. The basic Helix-Loop-Helix( bHLH) family of transcription factors are key regulatory molecules that control multiple developmental processes in species from yeast to humans[3]. In metazoans, bHLH proteins orchestrate cell cycle control, cell lineage commitment and cell differentiation( reviewed in[4]). Dimerization of the HLH domain allows these proteins to form homo- or heterodimers, while their basic domain is responsible for their capacity to bind to DNA[5]–[6]. The bHLH proteins are classified into different categories according to their function, distribution and DNA-binding properties. Class II encompasses bHLH proteins that are expressed in tissue-specific patterns, including MyoD, Myogenin, NeuroD/BETA2, MASH, HAND and TAL. The activity of these factors is related to the acquisition of particular developmental fates or potentials. These tissue-specific HLH factors dimerize with ubiquitous bHLH proteins called E proteins, which are “class I” bHLH factors. Vertebrate class I bHLH proteins are encoded by differentially spliced transcripts from the E2A( E12, E47 and E2-5/ITF1 proteins), E2-2/ITF2 and HEB/HTF4 genes. The currently accepted model is that in these heteromers, the tissue-specific class II factors confer spatial and temporal specificity, whereas the ubiquitous class I factors drive DNA binding and transcriptional activation[7]–[9]. The basic domains of the dimers contact a DNA target site known as the E-box, which is responsible for transcriptional activation. As dimerization is essential for DNA binding and transcriptional activity in vivo[10],[4], proteins that prevent the formation of these complexes act as negative regulators. These negative regulators, which contain an HLH domain but lack a basic region, are members of the Inhibitor of Differentiation( Id) protein family, composed of Id1-4. These factors represent “class V” proteins and they bind to ubiquitous bHLH proteins to inhibit their activity[11]–[13]. Although Id proteins were discovered due to their ability to prevent cell differentiation, they play a broad range of biological roles during development and significantly contribute to tumour development[reviewed in 14]–[15]. In addition to the widely accepted function of E proteins as regulators of tissue specific gene expression, they are also believed to serve as cell cycle effectors. However, this latter function remains controversial due to the diversity of cell cycle-specific target genes identified for E proteins[16]–[19]. The functional redundancy between the members of these two families has also complicated the study of their influence on cell proliferation[20]–[21]. However, the problem of redundancy is eliminated in Drosophila, in which Daughterless( Da) and Extramacrochaetae( Emc) are the only representatives of classes I and V[22]–[24], thereby facilitating functional studies. The interaction between these proteins and with other bHLH proteins is a determinant of cell fate decisions in different developmental contexts[25]–[27]. Moreover, null mutations for emc cause severe defects in cell division, suggesting that emc may be necessary to maintain a proliferative state during organ development[28]–[29]. An evolutionary conserved regulatory network between Da and Emc, in which Da controls its own activity by enhancing emc expression, was described recently[30]. According to this model, Emc functions as a negative feedback regulator that prevents runaway self-stimulation of da expression. Thus, changes in the expression of emc by different extracellular signals modulate the levels of Da. Furthermore, because Emc can bind directly to Da, elimination of Emc promotes an increase in Da homodimers. Nevertheless, little is known about how Emc and Da control cell proliferation. In vertebrates, Id2 is thought to bind to the retinoblastoma tumour suppressor protein( pRB), abolishing its growth-suppressor activity[31]–[32], although no physical association between pRb and Emc has been detected in Drosophila[33]. To date, the role of da in regulating proliferation has not been studied. In the present study, we investigated the control of cell proliferation by Da and Emc, and we found that both emc and da were required for the G2/M transition in the cell cycle. In eukaryotic cells, the main activator of mitosis is the( Cdc25) phosphatase String, which triggers the G2/M transition. We observed that defects in cell proliferation seen in emc mutant cells and da overexpressing cells were a result of reduced string expression. Thus, our results indicate that Da functions as a transcriptional repressor of string, thereby regulating G2/M transition. The role of bHLHs in integrating signals and permitting cell fate acquisition has been studied previously, yet these proteins are also essential for cell proliferation and tissue growth. It is well established that Id factors regulate G1-S transition in vertebrate cells, and our data indicate that at least in Drosophila, E proteins can also control the G2/M transition in conjunction with Id factors. Thus, here we have described the first mechanism through which interacting class I and class V bHLHs intercede in this cell cycle transition.
Discussion:
It is widely accepted that basic helix-loop helix( bHLH) transcription factors, E proteins and their inhibitors, the Id factors, play important roles in controlling the balance between proliferation and differentiation that determines the correct proportion of differentiated cell types in the adult nervous system. In addition to their influence on nervous system precursors, these factors act as essential proliferative factors for a large variety of cell types in which they are expressed[16]–[18],[20],[49],[50]. In Drosophila, most epithelial cells express the sole representatives of class I and V bHLH factors found in this organism, Daughterless and Extramacrochaetae, respectively. In the present and previous studies, overexpression of da or the absence of emc in different tissues has been shown to block cell proliferation. These findings are consistent with the hypothesis that the expression of these factors defines thresholds for the differentiation of most cells, controlling the timing of differentiation and exit from the cell cycle[51]–[52]. In Drosophila, an evolutionarily conserved cross-interacting regulatory network that links E proteins and class V gene expression was recently identified[30]. Accordingly, changes in Emc levels alter da expression, and moreover, the effects of emc depletion on the control of cell proliferation are mediated by the upregulation of da expression. The present findings confirm that the elimination of da in emc mutant cells suppresses the growth defects caused by emc. Understanding how E proteins control cell proliferation is crucial to identify the mechanism by which emc regulates this process. Very little is known about the role of Da in controlling cell proliferation. Our results indicate that the elimination of emc or the overexpression of da results in a greater accumulation of cells in the G2 phase. Strikingly, the growth defects observed in these mutants were almost abolished by ectopic expression of string. Moreover, we present different results that indicate that Da transcriptionally regulates string expression. Our data are consistent with a model in which da binds to the stg promoter to regulate its expression. We have defined a regulatory region upstream of the transcriptional initiation site of stg that drives the expression of this gene in the wing discs and that is transcriptionally regulated by Da. In this region as well as throughout stg promoter we have identified multiple putative Da binding sites. The results obtained with our ChIP experiments, as well as the data from the modENCODE annotation project, suggest that Da binds in vivo to different regions of the stg promoter, including the regulatory region that we have identified as required for the regulation of the stg expression in the discs. Considered together, these results suggest that Da exerts this function binding to different regions of string promoter to repress its expression. Based on these findings, we propose that the main mitotic defect provoked by the overexpression of da or the absence of emc is the transcriptional downregulation of string. Given the regulatory network that exists between Emc and Da[30], changes in Emc expression can modulate the levels of Da. Thus, when the levels of Emc were reduced, the levels of Da would increase. When the levels of Da rise above a certain threshold, the expression of string would be reduced, and then the cells will be retained in the G2 phase. Therefore, Da levels must be kept below this threshold in order for cells to remain in the proliferative cycle. Although our data indicate that the main mitotic defect caused by the overexpression of da, is the transcriptional down-regulation of string, some of our observations also suggest that da might be also affecting other factors that regulate cell cycle. Thus, whereas emc1 mutant cells exhibited a prolonged cell doubling time compared with control cells, alterations in other G/M regulators did not change the total duration of cell cycle[53]. A plausible explanation for this discrepancy is that the expression of different target genes involved in the control of cell cycle might be regulated by Da. According to our model, we would expect the loss of da to increase string expression. However, neither the ectopic expression of emc nor the elimination of da induced the ectopic expression of stg. Although we do not fully understand the reasons for this, one possibility is that the system ensures its robustness through the existence of genetic redundancy. This redundancy may occur with other bHLH genes or another transcriptional repressor, and could ensure that cell proliferation will be precisely regulated even in the absence of one or more genes. Alternatively, it has previously been shown that different antagonistic transcriptional regulators control the expression of stg. For example, during eye development, it has been proposed that Pointed, an activator, and Tramtrack69, a repressor, directly regulate the transcription of string. The absence of the repressor was not sufficient to promote the transcription of stg, as a positive signal is necessary to activate its transcription[54]. The absence of emc blocks cell proliferation in different tissues. However, the proliferation of cells completely null for emc function can be recovered using the Minute technique[55], suggesting that while emc is not absolutely required for cell division, its activity is necessary for the competitive success of cells. We have shown that string downregulation is the main mitotic defect caused by the absence of emc or the overexpression of da. Cells homozygous for a null stg allele divide only once, generating clones of two cells that are eliminated by cell competition[56],[37]. Interestingly, very large clones of string mutant cells can be generated when the function of string is not completely eliminated and clones are induced using the Minute technique[37]. Hence, the longer cell cycle of cells in which string is depleted would appear to result in slower growth than that which is seen in wild-type neighbours, which subsequently outcompete the string-deficient cells. Accordingly, the downregulation of string caused by the elimination of emc or overexpression of da can produce a similar growth defect. A large body of evidence demonstrates the involvement of Id proteins in the control of cell proliferation. This family of proteins has been extensively linked to cancer in humans and it mediates several processes that are regarded as hallmarks of cancer[57]. Id proteins trigger entry into S-phase, relieving E2F transcription from the inhibitory influence of pRB. Moreover, Ids interfere with the transcriptional activation by bHLH proteins of the cyclin-dependent kinase inhibitor p21/WAF1/CIP1. While much less is known about the role of E proteins as cell cycle regulators, our data from Drosophila define a new mechanism through which the E protein Da and its inhibitor, the Id orthologue emc, regulate cell cycle progression. In our model, emc is required to downregulate da, which in turn triggers mitosis via the transcriptional activation of a universally conserved cell cycle component, Cdc25. Since the mechanisms controlling cell cycle progression are evolutionarily conserved, it seems possible that the mechanism described here will be conserved in organisms other than Drosophila, and that mammalian E and Id proteins may also regulate cell proliferation. It will be important to ascertain whether E proteins repress mitosis by downregulating cdc25 expression. Although the role of Id factors in regulating G1-S transition is well documented, our data suggest that E proteins and class V factors also influence the G2 phase of the cell cycle. If the only role of Id factors were to promote the G1-S transition, they might simply arrest cells in the next phase of the cell cycle, the G2-M transition. However, the fact that Id proteins are associated with the development of tumours suggests that these factors drive cells through the different phases of the cell cycle.
Precise control of cell proliferation is critical for normal development and tissue homeostasis . Members of the inhibitor of differentiation ( Id ) family of helix-loop-helix ( HLH ) proteins are key regulators that coordinate the balance between cell division and differentiation . These proteins exert this function in part by combining with ubiquitously expressed bHLH transcription factors ( E proteins ) , preventing these transcription factors from forming functional hetero- or homodimeric DNA binding complexes . Deregulation of the activity of Id proteins frequently leads to tumour formation . The Daughterless ( Da ) and Extramacrochaetae ( Emc ) proteins are the only members of the E and Id families in Drosophila , yet their role in the control of cell proliferation has not been determined . In this study , we show that the elimination of emc or the ectopic expression of da arrests cells in the G2 phase of the cell cycle . Moreover , we demonstrate that emc controls cell proliferation via Da , which acts as a transcriptional repressor of the Cdc25 phosphatase string . These results provide an important insight into the mechanisms through which Id and E protein interactions control cell cycle progression and therefore how the disruption of the function of Id proteins can induce oncogenic transformation .