The complement system is a tightly regulated enzymatic cascade of over 50 plasma and membrane proteins, whose activation leads to the killing and clearance of pathogens through the formation of opsonins, anaphylatoxins, and the membrane attack complex. Complement activation can occur via the classical, lectin, and alternative pathways (1, 2). The classical pathway is mainly activated by the binding of C1q to IgG- and IgM-containing immune complexes, or a range non-self (pathogen) and altered self (β-amyloid peptide, prion protein) ligands (3, 4); the lectin pathway is activated when mannan-binding lectin recognizes non-self-carbohydrate structures (5). The alternative pathway is activated when C3 spontaneously undergoes hydrolysis to generate C3b, which then interacts with various proteins, lipids, and carbohydrates on the pathogen surface (6, 7). Activation of the classical or lectin pathway leads to the breakdown of C4 and C2, resulting in the formation of C3 convertase (C4b2b), which cleaves C3 to produce C3b. In the alternative pathway, hydrolysis of the internal thioester bond in C3 produces C3(H₂O), which binds to factor B, leading to its cleavage by factor D into Bb. This generates C3(H₂O) Bb.

C1q is the target recognition subcomponent of the classical pathway and serves as a fundamental bridge between innate and adaptive immunity. Human C1q is a structurally complex hexameric glycoprotein (460 kDa), which associates with the Ca²⁺-dependent C1r₂–C1s₂ tetramer to form the pentameric C1, the first component of the complement classical pathway (8, 9). The basic subunit of C1q is composed of an N-terminal collagen-like region and a C-terminal globular domain (gC1q). The gC1q is the ligand recognition domain and has a heterotrimeric structure composed of the C-terminal halves of the A (ghA), B (ghB), and C (ghC) chains (10). Being a charge pattern recognition molecule, C1q can bind a diverse range of self and non-self ligands via its gC1q domain and then trigger the classical pathway following binding to antigen-antibody complex (11). In addition, C1q also binds to membrane blebs of apoptotic cells, thus promoting efferocytosis. Impaired clearance of apoptotic cells occurring due to C1q deficiencies can prompt autoantibody production, as in systemic lupus erythematosus (12, 13).

The C1Q gene cluster is located on chromosome 1 within a genomic region of ∼25 kb; it consists of three individual genes (i.e., C1QA, C1QC, and C1QB) in the same strand orientation (+ strand), being around 4.6, 8.9, and 5.0 kb long, respectively. Each gene is composed of three exons, two of which contain the sequence that is translated into protein. Phylogenetic analysis uncovered that C1QA, C1QB, and C1QC may derive from duplication of a single copy of a potential ancestor identified as C1QB; moreover, the C1q family seems closely related to the EMILIN family (11, 14, 15). The human C1q domain containing (C1qDC) proteins are a large group of proteins, whose members have been mainly categorized into two sub-families based on their sequence homology: the larger sub-family includes the C1q-like and cerebellin-like subgroups, while the smaller is composed of EMILINs and multimerins (11, 16). Interestingly, 32 open reading frames encoding C1qDC proteins have been identified within the human genome (17). C1qDC proteins are pivotal signaling molecules that control inflammation, adaptive immunity, and energy balance, suggesting their potential exploration as therapeutics or targets in drug development (17).

Although the production of complement proteins is mostly in the liver, C1q is extrahepatically synthesized by tissue macrophages, immature dendritic cells (DCs), and other myeloid-derived cells (18–20). The evolutionary advantages and transcriptional mechanisms underlying C1q production by these potent phagocytes and antigen-presenting cells are still poorly understood. The extrahepatic synthesis of C1q is also associated with its complement-activation independent functions. Alternative roles of C1q concern its capability to promote angiogenesis and placental development (21, 22). It has been reported that C1q may act as a proangiogenic factor in several contexts, such as wound healing (23), placentation (24), and tumor progression (25), in addition to neural development, ageing, and autoimmunity (26).

Studies investigating C1Q transcriptional regulation have identified specific transcription factors capable of activating C1Q promoters. Interestingly, the transcription of the three genes is synchronized, with a 53-bp element as a regulatory region on C1QB promoter and two transcription factors bound to this region, namely PU.1 and IRF8 (27). PU-1 was also demonstrated to regulate decidual C1Q expression in pregnancy (28). In addition, the transcription factor, MafB, specifically expressed by monocytes and macrophages, binds to and activates the transcription of each C1Q gene (29). However, little is known regarding the mechanisms of modulation of C1Q expression, in particular, at the level of the tumor microenvironment (25, 30, 31).

Over the past decade, multi-omic technologies have undergone significant advancements enabling comprehensive characterization of gene expression profiles in various cells and tissues, physio-pathological conditions. These methodologies have been instrumental in elucidating intricate regulatory mechanisms governing gene expression, including epigenetic variations and protein expression dynamics. Epigenetics plays a pivotal role in establishing and maintaining cellular identity, responding dynamically to developmental cues and environmental stimuli, thereby orchestrating precise gene expression patterns that are unique to distinct cellular states (32, 33). Broadly categorized, epigenetic modifications encompass post-transcriptional histone alterations, DNA methylation, and regulatory non-coding RNA molecules. The regulatory role of histone acetylation, exemplified by H3K27ac (acetylation of lysine 27 on histone H3 protein subunit), is closely linked to transcriptional activation, whereas histone methylation exhibits a nuanced impact, capable of both activating (e.g., H3K4me1 and H3K4me3) and repressing (e.g., H3K27me3) transcription, depending on the specific histone site and the extent of methylation (34). Additionally, DNA methylation predominantly occurs at CpG dinucleotides within gene promoters, exerting transcriptional repression by recruiting repressive complexes to methylated promoters (35).

In the current study, we aimed to examine the transcriptional regulation of C1QA, C1QB, and C1QC, by exploiting publicly available tools and datasets. We particularly focused on the epigenetic dynamics underlying C1Q expression, and providing critical insights into the intriguing contribution of C1Q in the tumor microenvironment.

The complement system plays a critical role in the immune response, bridging innate and adaptive immunity (1, 2). Among its components, C1q stands out as a key versatile molecule, initiating the classical pathway and participating in processes such as efferocytosis, differentiation, chemotaxis, aggregation, and adhesion (49). Understanding the regulation of C1q expression is crucial for elucidating its ambivalent roles in health and disease. Its expression at the tissue level plays an important role in physiological processes, such as trophoblast invasion during the first trimester of pregnancy (22) and synaptic pruning (50); at the same time, it plays pivotal roles in pathological processes, such as wound healing (23), tumor development (25, 48), autoimmune diseases, and endometriosis (51). Few studies on the transcriptional regulation of C1Q have been published: they identified specific transcription factors that can activate C1Q promoters such as PU.1. PU.1, an Ets-family transcription factor, is required for the development of hematopoietic myeloid lineage immune cells (52). The transcription factors PU.1 and IRF8 bind to a 53-bp element on C1QB promoter known to be a gene expression regulatory region (27). PU.1 influences C1Q expression in the decidua during placental development, and its presence is directly correlated with C1q synthesis (28). Additionally, the transcription factor, MafB, specifically expressed in monocytes and macrophages, binds to and activates the transcription of all three C1Q chain genes. C1Q is expressed at the microenvironment level in different types of gliomas with an ambivalent prognostic significance (30, 31, 53). Its crucial role has been widely demonstrated in mesothelioma, where in addition to directly promoting the proliferation and resistance of tumor cells (48), it modulates the metabolism of hyaluronic acid (54, 55). However, our understanding of how C1Q expression is modulated, particularly in the tumor microenvironment, remains limited.

Previous evidence revealed a synchronized transcription of C1Q genes (27). Our integrated analysis of TSS usage and expression patterns of the human C1Q genes (C1QA, C1QB, and C1QC) via Zenbu browser and FANTOM-CAT data highlighted a coordinated regulation and functional association across various cell types. This co-expression pattern, particularly prominent in immune-related cells (e.g., monocytes and macrophages), underscores the importance of C1q in immune regulation. Moreover, the functional annotation-based analysis confirmed C1q as one of the few non-liver-derived complement components whose major expression can be traced back to the bone marrow (56). Its expression in macrophages is also exemplified by recent identification of C1q+ macrophage populations in both healthy and tumor tissues (57). The evidence that, in certain cell populations, the expression of only one or two chains of C1q has been observed may not have a functional significance, but could be a spurious expression linked to the ancestral role of C1q as an acute phase molecule (24, 45).

Of interest is the association between C1QA, C1QB, and C1QC gene expression and CD14⁺ EPCs. We previously demonstrated the role of C1q in angiogenesis (21, 23), and its presence in several microenvironments characterized by active angiogenesis, such as decidua, endometriotic lesions, and tumors (22, 25, 51). These novel findings lead us to hypothesize that CD14⁺ EPCs could be the primary cells responsible for C1q synthesis in angiogenic regions.

Epigenetic modifications, including histone modifications and DNA methylation, play a central role in determining gene expression patterns. First, the analysis of histone modification patterns across different cell types revealed distinct epigenetic landscapes associated with C1Q expression. Specifically, permissive chromatin states characterized by activation histone marks are observed in cell types where C1Q is expressed (i.e., macrophages), while repressive marks dominate in non C1Q-expressing cell types (i.e., HUVEC cells). These findings suggest a direct link between chromatin state and transcriptional activity of C1Q genes. Moreover, examining DNA methylation profiles further elucidates the epigenetic regulation of C1Q expression. In fact, hypomethylation of the promoter regions in macrophages correlates with active transcription. The macrophage-specific activation signature further emphasizes the correlation between epigenetic marks and gene expression. In support of this evidence, the regulatory roles of histone and DNA modifications have recently emerged as crucial contributors to the monocyte and macrophage functions (58–60). The transition from monocyte to macrophage is marked by a specific gene expression profile, concomitant with extensive alterations in histone modification and chromatin accessibility (61). These findings underscore the indispensable involvement of epigenetic mechanisms in endowing macrophages with the capacity for phenotypic plasticity and functional versatility (62). Of interest, an epigenetic program is associated with monocyte-to-macrophage differentiation (61).

Previous reports have extensively emphasized a regulatory interdependence between C1q and microenvironment. Thus, C1q shapes the monocyte/macrophage/DC phenotype towards the development of a pro-efferocytic and anti-inflammatory phagocyte, creating an anti-inflammatory feed-forward loop (63), but also the microenvironment can influence C1q production. Unlike the monocyte-macrophage lineage, which constitutively produces C1q and modulates its expression in response to microenvironmental stimuli [e.g., the shift towards tumor-associated macrophages (TAMs)], some cells abruptly initiate or interrupt C1q production during their maturation process. Extravillous trophoblasts start expressing C1q during maturation and differentiation (24) via an active involvement of epigenetic dynamics in trophoblast biology (64). Conversely, the sustained production of active C1q by immature DCs is completely down-regulated upon DC maturation and differentiation in vitro (19). These findings strengthen our evidence of a fine regulation of C1Q expression in differentiation contexts.

We also aimed at shedding light on the ambivalent role of C1q in the tumor microenvironment. C1q has already been demonstrated to serve as a pro-tumorigenic factor in several tumor settings (25, 48, 54, 55, 65–67). In KIRC, infiltration by C1q-producing TAMs was associated with an immunosuppressed tumor microenvironment, characterized by high expression of immune-checkpoint molecules (PD-1, LAG-3, PD-L1, and PD-L2) (66, 68). Nevertheless, the prognostic impact of C1q is ambivalent and strictly dependent on the tumor type (30, 31, 51).

Interestingly, we found that tumor samples exhibit a distinct DNA methylation pattern compared to normal tissues, with decreased methylation associated with elevated C1Q expression, in accordance with previous studies (25, 65–67). Our observations suggest a potential role for epigenetic dysregulation in C1Q expression during tumorigenesis, and underscore the dynamic interconnection between DNA methylation and gene expression in C1QA, C1QB, and C1QC across various tumor types, shedding light on the intricate relationship between epigenetic modifications and the switching of transcriptional activation and deactivation processes. These findings not only substantiate the observed epigenetic patterns in macrophages but also emphasize the broader implications of DNA methylation in the transcriptional regulation of these immune response genes.

Overall, the comprehensive analysis of C1Q gene regulation sheds light on the intricate interplay between epigenetic mechanisms and transcriptional dynamics in modulating immune responses and disease processes. Further investigation into the epigenetic regulation of C1Q expression may offer new insights into the pathogenesis of immune-related disorders and cancer, providing potential therapeutic targets for intervention.