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DNA fragmentation factor-related protein 4: Biological Overview | References

Phagocytic macrophages are crucial for innate immunity and tissue homeostasis. Most tissue-resident macrophages develop from embryonic precursors that populate every organ before birth to lifelong self-renew. However, the mechanisms for versatile macrophage differentiation remain unknown. This study used in vivo genetic and cell biological analysis of the Drosophila larval hematopoietic organ, the lymph gland that produces macrophages. The developmentally regulated transient activation of DNA fragmentation factor-related protein 4-mediated DNA strand breaks in intermediate progenitors is essential for macrophage differentiation. Insulin receptor-mediated PI3K/Akt signaling regulates the apoptosis signal-regulating kinase 1 (Ask1)/c-Jun kinase (JNK) axis to control sublethal levels of caspase activation, causing DNA strand breaks during macrophage development. Furthermore, caspase activity is also required for embryonic-origin macrophage development and efficient phagocytosis. This study provides insights into developmental signaling and CAD-mediated DNA strand breaks associated with multifunctional and heterogeneous macrophage differentiation (Maurya, 2024).

Multifunctional phagocytic macrophages populate most tissues during fetal development and can self-renew. However, the macrophage differentiation mechanisms remain unknown. This study shows that during the normal development of Drosophila macrophages in the larval lymph gland, apoptotic caspases are activated in the differentiating cells. Sublethal executioner caspase activation induces CAD, triggering DNA strand breaks in differentiating macrophages. InR/PI3K/Akt-mediated signaling induces a transient caspase cascade through Ask1/JNK signaling in differentiating macrophages. Furthermore, for efficient phagocytic activity, caspase activation is required in embryonic-origin macrophage development. Therefore, this research using in vivo genetic analysis revealed that developmental signal-mediated caspase activation and DNA damage response (DDR) signals play a role in determining macrophage differentiation during normal development (Maurya, 2024).

In several types of cell differentiation, programmed DNA breaks are reported to coordinate gene expression changes without causing cell death. However, the signals that cause DNA damage in these cases were not addressed. Single-cell transcriptomics on lymph glands revealed a group of cells (1.2%) called cluster X, or glutathione S-transferase (GST)-rich, with unique genetics and enrichment of DDR, Myb, and cell cycle genes. These cells are most likely the CAD-mediated DNA-damaged cells that are reported in this study, as their location and numbers in the lymph gland are comparable. This study revealed that caspase-mediated Drosophila CAD causes DNA breaks, which is essential for macrophage differentiation, as depletion of CAD/ICAD in the lymph gland causes loss of phagocytic markers and DNA damage, but caspase activity is still seen (Maurya, 2024).

Many Drosophila cells show caspase activation to have non-lethal roles in development and differentiation. Studies showed that lymph gland progenitors must balance ROS-mediated JNK signaling to maintain and differentiate. ROS in lymph gland progenitors might induce caspase activation in differentiating macrophages, and by the time DDR is seen, ROS becomes lower. Monocyte-to-macrophage differentiation requires CSF1-Akt-mediated caspase activation. InR-mediated PI3K-Akt signaling has a role in autonomous apoptotic activation and caspase activity control. However, a partially redundant role of other signaling (e.g., Pvr, EGFR, GABA-calcium) cannot be ruled out at present. Further, the differentiated macrophages require both initiator and effector caspases for Draper expression and phagocytic efficiency. The current data support previous research showing that loss of RHG genes causes low levels of Draper expression in embryonic macrophages. Lineage trace experiments for caspase-positive cells confirm that differentiated macrophages undergo caspase activation (Maurya, 2024).

How the executioner caspase levels and dynamics predict cell survival vs. cell death remains unclear. A cancer cell line model this study showed that high caspase activity kills all cells but low levels allow survival. In the differentiating cells, PI3K/Akt signaling through the Ask1/JNK axis regulates caspase and CAD activity at a sublethal level. Also, CAD depletion rescues the PI3K active phenotypes except for caspase activity, suggesting that macrophage differentiation requires InR/PI3K-mediated CAD activation. Other mechanisms might also help survival after caspase activation. For example, caspase-mediated skeletal muscle cell differentiation studies reported that nuclear pore complex trimming alters the intracellular environment, and CAD-mediated DNA damage is repaired by base excision repair protein XRCC1, resulting in gene expression changes.Differential accessibility of transient CAD for DNA fragmentations helps cells survive due to their chromatin architecture (Maurya, 2024).

Caspase/CAD-mediated DNA breaks for macrophage differentiation may modulate chromatin organization to control macrophage-specific gene expression. CAD-mediated DNA breaks around chromatin modifying CTCT binding factor sites (chromatin insulators) induce chromatin landscape change by directly acting on promoter or altering promoter-enhancer interaction, which regulates gene expression. A Drosophila study showed that DNA damage increases chromatin insulator enrichment at insulator sites by regulating the γH2Av. Interestingly, previous research found that mammalian macrophage functions require a set of transcriptional regulators accomplished by the tissue-specific macrophage chromatin landscape. Together, it is hypothesized that caspase/CAD-mediated DNA breaks in differentiating macrophages may influence the specification of macrophage fate, possibly by regulating the chromatin landscape and the gene expression that prepares the macrophages for trained immunity and efficient tissue-specific functions. Further research will determine how caspase/CAD-mediated DNA breaks cause macrophage-specific gene expression in Drosophila and whether these are also relevant to macrophages in higher organisms (Maurya, 2024).

This genetic analysis showed that InR/PI3K/Akt signaling through the Ask1/JNK axis activates sublethal caspase and CAD, causing DNA strand breaks during macrophage differentiation. However, present studies do not rule out other redundant signalings. Due to technical and biological difficulties, it was not possible to determine how Ask1 controls transient caspase activity and the exact levels of caspase activity that cause DNA damage without cell death. The CAD-mediated DNA damage locations in the developing macrophage genome and DNA repair mechanisms are not known. This DNA breakage could be site specific, which needs to be identified, and may involve the altered chromatin landscape and macrophage-specific gene expression (Maurya, 2024).

Interaction mode of CIDE family proteins in fly: DREP1 and DREP3 acidic surfaces interact with DREP2 and DREP4 basic surfaces

Cell death-inducing DNA fragmentation factor 45 (DFF45)-like effector (CIDE) domains were initially identified as protein interaction modules in apoptotic nucleases and are now known to form a highly conserved family with diverse functions that range from cell death to lipid homeostasis. In the fly, four CIDE domain-containing proteins (DFF-related protein [DREP]-1-4) and their functions, including interaction relationships, have been identified. This study introduced and investigated acidic side-disrupted mutants of DREP1, DREP2, and DREP3. The acidic surface patches of DREP1 and DREP3 are critical for the homo-dimerization. In addition, the acidic surface sides of DREP1 and DREP3 interact with the basic surface sides of DREP2 and DREP4. This study provides clear evidence demonstrating the mechanism of the interactions between four DREP proteins in the fly (Kim, 2017).

CIDE domains form functionally important higher-order assemblies for DNA fragmentation

Cell death-inducing DFF45-like effector (CIDE) domains, initially identified in apoptotic nucleases, form a family with diverse functions ranging from cell death to lipid homeostasis. This study shows that the CIDE domains of Drosophila and human apoptotic nucleases Drep2, Drep4, and DFF40 all form head-to-tail helical filaments. Opposing positively and negatively charged interfaces mediate the helical structures, and mutations on these surfaces abolish nuclease activation for apoptotic DNA fragmentation. Conserved filamentous structures are observed in CIDE family members involved in lipid homeostasis, and mutations on the charged interfaces compromise lipid droplet fusion, suggesting that CIDE domains represent a scaffold for higher-order assembly in DNA fragmentation and other biological processes such as lipid homeostasis (Choi, 2017).

Why does the lack of oligomerization compromise the DNA fragmentation activity of apoptotic nucleases? As demonstrated by the crystal structure, the nuclease domain of CAD can only function as a dimer, yet its dimer interface is loosely packed with poorly defined electron densities. It is suggested that the intrinsically weak nuclease dimers require that the nuclease domains be brought into proximity by the CIDE domains. This idea is supported by the earlier in vitro observation that on treatment of the DFF40/DFF45 complex by caspase-3, only the oligomeric fraction of DFF40 was associated with catalytic activity, whereas the monomeric fraction was inactive. Although the size distribution of DFF40 helical oligomers may differ in cells, the mechanism of oligomerization-driven activation remains the same. Thus, release from their inhibitors is not sufficient for activation of apoptotic nucleases. Instead, helical oligomerization of apoptotic nucleases is a missing step in their activation, which promotes nuclease dimerization for DNA fragmentation (Choi, 2017).

Therefore, caspase-mediated activation of apoptotic nucleases may proceed in several steps. In a basal state, a nuclease binds its inhibitor using both the CIDE and inhibitor/chaperone domains to form an inactive heterodimer. On induction of apoptosis, the caspase cascade cleaves the inhibitor, releasing the nuclease for nuclear translocation. The monomeric nuclease then oligomerizes via its CIDE domain, which efficiently brings the nuclease domain into proximity for dimerization and thus catalytic activation, in a manner analogous to many caspase-activating complexes. The dimeric nuclease domain exhibits the shape of molecular scissors, with the distance between the blades compatible with a dsDNA strand. The deep active-site crevice may distinguish internucleosomal DNA from nucleosomal DNA to generate apoptotic DNA ladders (Choi, 2017).

In recent years, higher-order assemblies have been identified as mechanisms of signaling in cell death and immunity. In particular, the death domain (DD) fold superfamily members, including DD, CARD, PYD, and DED, ubiquitously form helical assemblies to mediate signal transduction, signal amplification, and proximity-induced enzyme activation. The present study demonstrates that the CIDE domain family proteins form kinds of higher-order structures, which provide insight into CIDE domain-mediated processes ranging from DNA fragmentation to lipid droplet exchange and fusion. It is not yet known how higher-order structures facilitate lipid droplet homeostasis; however, in analogy to apoptotic nucleases and DD-mediated caspase and kinase activation, it is suspected that the increased local concentration by oligomerization also may promote the lipid-binding activity of CIDEA, CIDEB, and FSP27 (Choi, 2017).

Unlike the more stable cooperatively formed DD filaments, the head-to-tail oligomerization observed for CIDE domains may be more dynamic in assembly and disassembly and is reminiscent of the head-to-tail oligomerization by PB1, DIX, and SAM domains. In general, open-ended oligomers pose challenges to structural biology; they are often ignored because of their apparent size heterogeneity, and their structural mechanisms of assembly may be more difficult to elucidate. When the oligomerization leads to ordered assemblies such as the CIDE domain filaments, structure determination may be achieved by cryo-EM and sometimes by crystallography. When the oligomerization is less ordered and more dynamic, investigation with multiple approaches may be required to tease out the mechanisms. Perhaps because of these challenges, it is likely that many more higher-order structures exist in diverse biological functions that have escaped attention so far (Choi, 2017).

Dual apoptotic DNA fragmentation system in the fly: Drep2 is a novel nuclease of which activity is inhibited by Drep3

DNA fragmentation is the hallmark of apoptotic cells and mainly mediated by the DNA fragmentation factor DFF40(CAD)/DFF45(ICAD). DFF40 is a novel nuclease, whereas DFF45 is an inhibitor that can suppress the nuclease activity. Apoptotic DNA fragmentation in the fly is controlled by four DFF-related proteins, known as Drep1, 2, 3 and 4. However, the functions of Drep2 and Drep3 are totally unknown. This study found that Drep2 is a novel nuclease whose activity is inhibited by Drep3 through a tight interaction with the CIDE domain. The results suggest that the fly has dual apoptotic DNA fragmentation systems: Drep1: Drep4 and Drep2: Drep3 complexes (Park, 2012).

Functions of Drep4 orthologs in other species]

Molecular basis of apoptotic DNA fragmentation by DFF40

Although the functions of CIDE domain-containing proteins, including DFF40, DFF45, CIDE-A, CIDE-B, and FSP27, in apoptotic DNA fragmentation and lipid homeostasis have been studied extensively in mammals, the functions of four CIDE domain-containing proteins identified in the fly, namely DREP1, 2, 3, and 4, have not been explored much. Recent structural study of DREP4, a fly orthologue of mammalian DFF40 (an endonuclease involved in apoptotic DNA fragmentation), showed that the CIDE domain of DREP4 (and DFF40) forms filament-like assembly, which is critical for the corresponding function. The current study aimed to investigate the mechanism of filament formation of DREP4 CIDE and to characterize the same. DREP4 CIDE was shown to specifically bind to histones H1 and H2, an event important for the nuclease activity of DREP4. Based on the current experimental results, the mechanism underlying the process of apoptotic DNA fragmentation is proposed (Ha, 2022).

DFF40 deficiency in cancerous T cells is implicated in chemotherapy drug sensitivity and resistance through the regulation of the apoptotic pathway

The regulation of the apoptotic pathway is one of the most studied mechanisms regarding cancer cell resistance. Many mutations have been linked to drug resistance. The DNA fragmentation factor 40 (DFF40) has been gaining interest regarding cancer cell response to chemotherapy and patient outcomes. Glioblastomas and uterine leiomyosarcomas have been shown to have a downregulation of DFF40 expression, conferring a poor patient prognosis. In concordance with these observations, this study showed that DFF40 gene is also downregulated in breast, endocervical, ovarian, lung, pancreas and glioblastomas. DFF40 is the endonuclease responsible of DNA fragmentation during apoptosis. This study sought to determine if a DFF40 deficiency in Jurkat T cells could impact the sensitivity to conventional chemotherapy drugs. CRISPR-cas9 generated DFF40 knockout (DFF40 KO) stable Jurkat cells and wild-type (DFF40 WT) cells were treated with different antimetabolites and topoisomerase II (TOP2) inhibitors, and cell viability was subsequently assessed. DFF40 deficient cells show chemoresistance to antimetabolites (e.g. methotrexate, 6-mercaptopurine and cytarabine) and surprisingly, they are more sensitive to TOP2 inhibitors (e.g. etoposide and teniposide). DFF40 deficient cells exposed to cytarabine present lower phosphatidylserine translocation levels to the outer cell membrane layer. Etoposide exposure in DFF40 deficient cells induces higher mortality levels and downregulation of Bcl-xL cells compared to DFF40 expressing T cells. The abolition of DFF40 expression in Jurkat cells significantly impairs histone H2AX phosphorylation following etoposide and cytarabine treatments. These findings suggest that DFF40 is a novel key target in cancer cell resistance that potentially regulates genomic stability (Kulbay, 2021).

Crystal structure and mutation analysis revealed that DREP2 CIDE forms a filament-like structure with features differing from those of DREP4 CIDE

Cell death-inducing DFF45-like effect (CIDE) domain-containing proteins, DFF40, DFF45, CIDE-A, CIDE-B, and FSP27, play important roles in apoptotic DNA fragmentation and lipid homeostasis. The function of DFF40/45 in apoptotic DNA fragmentation is mediated by CIDE domain filament formation. Although recent structural study of DREP4 CIDE revealed the first filament-like structure of the CIDE domain and its functional importance, the filament structure of DREP2 CIDE is unclear because this structure was not helical in the asymmetric unit. This study presents the crystal structure and mutagenesis analysis of the DREP2 CIDE mutant, which confirmed that DREP2 CIDE also forms a filament-like structure with features differing from those of DREP4 CIDE (Ha, 2018).

Interaction mode of CIDE family proteins in fly: DREP1 and DREP3 acidic surfaces interact with DREP2 and DREP4 basic surfaces

Cell death-inducing DNA fragmentation factor 45 (DFF45)-like effector (CIDE) domains were initially identified as protein interaction modules in apoptotic nucleases and are now known to form a highly conserved family with diverse functions that range from cell death to lipid homeostasis. In the fly, four CIDE domain-containing proteins (DFF-related protein [DREP]-1-4) and their functions, including interaction relationships, have been identified. This study introduced and investigated acidic side-disrupted mutants of DREP1, DREP2, and DREP3. The acidic surface patches of DREP1 and DREP3 are critical for the homo-dimerization. In addition the acidic surface sides of DREP1 and DREP3 interact with the basic surface sides of DREP2 and DREP4. This study provides clear evidence demonstrating the mechanism of the interactions between four DREP proteins in the fly (Kim, 2017).

Filament-like DREP4 CIDE domain: characterization and preliminary X-ray crystallographic studies

DREP4 is a nuclease from fruit fly that is involved in apoptotic DNA fragmentation. DREP4 contains a conserved CIDE domain that acts as a protein-interaction module and is critical for its function. This study found that DREP4 CIDE domains form filament-like structures in solution. The length of the highly ordered filament-like structure is dependent on the salt concentration. By adjusting the salt concentration the DREP4 CIDE domain could be crystallized, and X-ray diffraction data were collected to a resolution of 1.9 A. The crystals were found to belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 53.08, b = 76.58, c = 174.59 A (Park, 2017).

In vitro analysis of the complete CIDE domain interactions of the Drep system in fly

CIDE domain containing proteins are involved in apoptosis and lipid metabolism, and four CIDE containing proteins, Drep1, Drep2, Drep3, and Drep4, have been identified in fly. This study found that Drep3 interacts with Drep4 via the CIDE domain specifically, which completes the interaction map of Drep system in fly, cyclic interactions: Drep1-Drep2-Drep3-Drep4-Drep1. In addition, the dynamic stoichiometry changes of Drep proteins upon binding to their binding partners was analyzed. The current studies will help in understanding the Drep system in fly as well as CIDE domain for protein-protein interactions (Lee, 2014).

A putative role of Drep1 in apoptotic DNA fragmentation system in fly is mediated by direct interaction with Drep2 and Drep4

DNA fragmentation is common phenomenon for apoptotic cell death. DNA fragmentation factor, called DFF40 (CAD: mouse homologue), is a main nuclease for apoptotic DNA fragmentation. Nuclease activity of DFF40 is normally inhibited by DFF45 by tight interaction via CIDE domain without apoptotic stimuli. Once effector caspase is activated during apoptosis signaling, it cleaves DFF45, allowing DFF40 to enter the nucleus and cleave chromosomal DNA. Unlike mammalian system, apoptotic DNA fragmentation in the fly might be controlled by four DFF-related proteins, known as Drep1, Drep2, Drep3 and Drep4. Although the function of Drep1 and Drep4 is well known as DFF45 and DFF40 homologues, respectively, the function of Drep2 and Drep3 is still unclear. DFF-related proteins contain a conserved CIDE domain of ~90 amino acid residues that is involved in protein-protein interaction. This study showed that Drep1 directly bind to Drep2 as well as Drep4 via CIDE domain. In addition, the interaction of Drep2 and Drep4 to Drep1 was not competitive indicating that Drep2 and Drep4 bind different place of Drep1. All together, it is suggested that Drep1 might be involved in apoptotic DNA fragmentation of fly system by direct interaction with Drep2 as well as Drep4 (Park, 2013).

Structural mechanism for inactivation and activation of CAD/DFF40 in the apoptotic pathway

CAD/DFF40 is responsible for the degradation of chromosomal DNA into nucleosomal fragments and subsequent chromatin condensation during apoptosis. It exists as an inactive complex with its inhibitor ICAD/DFF45 in proliferating cells but becomes activated upon cleavage of ICAD/DFF45 into three domains by caspases in dying cells. The molecular mechanism underlying the control and activation of CAD/DFF40 was unknown. In this study, the crystal structure of activated CAD/DFF40 reveals that it is a pair of molecular scissors with a deep active-site crevice that appears ideal for distinguishing internucleosomal DNA from nucleosomal DNA. Ensuing studies show that ICAD/DFF45 sequesters the nonfunctional CAD/DFF40 monomer and is also able to disassemble the functional CAD/DFF40 dimer. This capacity requires the involvement of the middle domain of ICAD/DFF45, which by itself cannot remain bound to CAD/DFF40 due to low binding affinity for the enzyme. Thus, the consequence of the caspase-cleavage of ICAD/DFF45 is a self-assembly of CAD/DFF40 into the active dimer (Woo, 2004).

Search PubMed for articles about Drosophila Drep4

Choi, J. Y., Qiao, Q., Hong, S. H., Kim, C. M., Jeong, J. H., Kim, Y. G., Jung, Y. K., Wu, H., Park, H. H. (2017). CIDE domains form functionally important higher-order assemblies for DNA fragmentation. Proc Natl Acad Sci U S A, 114(28):7361-7366 PubMed ID: 28652364

Ha, H. J., Park, H. H. (2018). Crystal structure and mutation analysis revealed that DREP2 CIDE forms a filament-like structure with features differing from those of DREP4 CIDE. Sci Rep, 8(1):17810 PubMed ID: 30546036

Ha, H. J., Park, H. H. (2022). Molecular basis of apoptotic DNA fragmentation by DFF40. Cell Death Dis, 13(3):198 PubMed ID: 35236824

Kim, C. M., Jeon, S. H., Choi, J. H., Lee, J. H., Park, H. H. (2017). Interaction mode of CIDE family proteins in fly: DREP1 and DREP3 acidic surfaces interact with DREP2 and DREP4 basic surfaces. PLoS One, 12(12):e0189819 PubMed ID: 29240809

Kulbay, M., Johnson, B., Fiola, S., Diaz, R. J., Bernier, J. (2021). DFF40 deficiency in cancerous T cells is implicated in chemotherapy drug sensitivity and resistance through the regulation of the apoptotic pathway. Biochem Pharmacol, 194:114801 PubMed ID: 34678222

Lee, S. M., Park, H. H. (2014). In vitro analysis of the complete CIDE domain interactions of the Drep system in fly. Apoptosis, 19(3):428-435 PubMed ID: 24233238

Maurya, D., Rai, G., Mandal, D., Mondal, B. C. (2024). Transient caspase-mediated activation of caspase-activated DNase causes DNA damage required for phagocytic macrophage differentiation. Cell Rep, 43(5):114251 PubMed ID: 38761374

Park, O. K., Park, H. H. (2012). Dual apoptotic DNA fragmentation system in the fly: Drep2 is a novel nuclease of which activity is inhibited by Drep3. FEBS Lett, 586(19):3085-3089 PubMed ID: 22850116

Park, O. K., Park, H. H. (2013). A putative role of Drep1 in apoptotic DNA fragmentation system in fly is mediated by direct interaction with Drep2 and Drep4. Apoptosis, 18(4):385-392 PubMed ID: 23417746

Park, H. H. (2017). Filament-like DREP4 CIDE domain: characterization and preliminary X-ray crystallographic studies. Acta Crystallogr F Struct Biol Commun, 73(Pt 8):481-485 PubMed ID: 28777092

Woo, E. J., Kim, Y. G., Kim, M. S., Han, W. D., Shin, S., Robinson, H., Park, S. Y., Oh, B. H. (2004). Structural mechanism for inactivation and activation of CAD/DFF40 in the apoptotic pathway. Mol Cell, 14(4):531-539 PubMed ID: 15149602

date revised: 5 April 2026

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