CITED2 IS A KEY REGULATOR OF PLACENTAL DEVELOPMENT AND PLASTICITY

Abstract

The biology of trophoblast cell lineage development and placentation is characterized by the involvement of several known transcription factors. Central to the action of a subset of these transcriptional regulators is CBP-p300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2). CITED2 acts as a co-regulator modulating transcription factor activities and affecting placental development and adaptations to physiological stressors. These actions of CITED2 on the trophoblast cell lineage and placentation are conserved across the mouse, rat, and human. Thus, aspects of CITED2 biology in hemochorial placentation can be effectively modeled in the mouse and rat. In this review, we present information on the conserved role of CITED2 in the biology of placentation and discuss the use of CITED2 as a tool to discover new insights into regulatory mechanisms controlling placental development.

Graphical Abstract

Hemochorial placentation in the human and rat is characterized by deep intrauterine trophoblast cell invasion and extensive trophoblast cell-guided spiral artery remodeling, which facilitates nutrient delivery to the fetus. CITED2 is a co-regulator modulating transcription factors central to hemochorial placentation, placental adaptations to environmental stressors, fetal development, and pregnancy outcome.

INTRODUCTION

CBP-p300 interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) is a transcriptional co-regulator^[1-8]. CITED2 was first identified in melanocytes and given the name melanocyte-specific gene related 1 (MRG1)^[1]. It was later shown, by Bhattacharya and coworkers that CITED2 acts a co-activator for the transcription factor AP-2 (TFAP2) family^[4]. CITED2 belongs to the CITED protein family. Other members of the CITED family in mammals include CITED1^[9-14] and CITED4^[15-18]. Members of the CITED protein family each share conserved regions, including a domain at their carboxy terminus, which interacts with CBP (also referred to as CREB-binding protein, CREBBP) and p300 (also referred to as EIA-binding protein p300; EP300)^[3,19]. The region of CREBBP and EP300 proteins that interacts with CITED2 also interacts with transcription factors.

CITED2 has been most extensively studied for its potential involvement in development, cancer, inflammation, metabolic, and congenital heart disease^[19-33]. Here, we present an assessment of CITED2 involvement in placental development.

OVERVIEW OF PLACENTATION

Our analysis focuses on the role of CITED2 in the process of hemochorial placentation, which is observed in the mouse, rat, and human^[34]. The hemochorial placenta exhibits extensive erosion of the uterine endometrium, placing maternal blood in direct contact with the extraembryonic epithelium of the placenta, which is referred to as trophoblast^[35,36]. The trophoblast cell lineage arises from the outer trophectoderm cell compartment of the blastocyst stage embryo^[37]. Trophoblast cells contribute to placental structures interfacing with the uterine vasculature and the fetal vasculature. These two main trophoblast cell populations are specialized to remodel the uterine vasculature, facilitating nutrient entry into the placenta, or alternatively, to act as a barrier, enabling or restricting the delivery of nutrients and harmful compounds, respectively, to the fetus^[35,36]. In the mouse and the rat, trophoblast cells engaged with the uterine vascular are referred to as invasive trophoblast cells and in the human, they are called extravillous trophoblast (EVT) cells^[35,36,38]. Invasive trophoblast cells arise from a structure termed the junctional zone in the mouse and rat, while EVT cells arise from cell columns in the human placenta^[35,36,38]. Trophoblast cell invasion in the mouse is shallow, whereas the rat and human exhibit deep intrauterine trophoblast cell invasion (Fig. 1)^[35,39]. The uterine-placental interface defines the site where invasive trophoblast cells are distributed within the uterus. The junctional zone also contains endocrine cells referred to as trophoblast giant cells and spongiotrophoblast cells and a population of cells that accumulate glycogen termed glycogen cells^[38]. Invasive/extravillous trophoblast cells come in two types based on their spatial location relative to the uterine vasculature: i) endovascular; ii) interstitial^[35,36]. Endovascular invasive trophoblast cells supplant the endothelium, whereas interstitial invasive trophoblast cells are positioned between blood vessels. The barrier component of the rodent placenta is referred to as the labyrinth zone, whereas in the human placenta it is termed the villous compartment^[35,36]. Syncytiotrophoblast (ST) contribute to the barrier of both rodent and human placentas and arise via fusion of progenitor trophoblast cell populations often referred to as cytotrophoblast, especially in human villous structures. Although there are prominent nomenclature differences, fundamental similarities exist in the structure and function of the mouse, rat, and human placentation sites. Thus, the mouse and rat can serve as effective models for investigating in vivo mechanisms regulating placental development, which have relevance to human placentation.

Fig. 1. Hemochorial placentation.

Schematic diagrams showing placentation sites of the mouse, rat, and human. The mouse exhibits shallow intrauterine trophoblast cell invasion, whereas the rat and human possess deep intrauterine trophoblast cell invasion. Invasive trophoblast (IT) and extravillous trophoblast (EVT) cells are functional equivalents, and the junctional zone (JZ) of the rodent placentation site is homologous to the EVT cell column (CC) of human placentation site. Uterine-placental interface (UPI), labyrinth zone (LZ), decidua (Dec), natural killer (NK) cells, villous core (VC), syncytiotrophoblast (STB), cytotrophoblast (CTB), basal CTB (bCTB), villous CTB (vCTB), spiral arteries (SpA), endothelial cells (EC), and red blood cells (RBC). Modified from Soares et al., 2017 with permission.

CITED2 AND PLACENTATION: ANIMAL MODELS

Roles for CITED2 in the biology of trophoblast cells and in placentation have been modeled in the mouse and rat (Table 1). In both species, CITED2 has been shown to possess a fundamental role in regulating placenta development and function.

Table 1.

CITED family and normal placentation.

Family member	Placental expression	Mutant rodent placenta phenotype	References
CITED1	X-linked gene, expression overlap with CITED2, low level expression in human trophoblast cells	Expanded junctional zone, enlarged maternal blood sinusoids, fetal growth restriction and embryonic death on the day of birth or shortly thereafter	[10,44]
CITED2	Highly expressed in both rodent and human placentas; expression increases with rat and human trophoblast stem cell differentiation	Placental and fetal growth restriction, abnormal trophoblast cell invasion, and prenatal (mouse) or immediate postnatal (rat) lethality, exaggerated responses to physiological stressors	[41,42,44,45,155]
CITED4	No reported placental expression	No reported involvement in placental biology	No reports on placental biology

Mouse

Global CITED2 deficiency mouse models have been generated^[4,21,40]. Disrupting CITED2 results in prenatal lethality, cardiac malformations, lung developmental anomalies, embryonic left-right patterning and neural crest defects, adrenal agenesis, exencephaly, and placental abnormalities^{[4,20-22,40,41]}. CITED2 deficiency leads to placental and fetal growth restriction^[41]. Both the junctional and labyrinth zones are affected by the absence of CITED2. Junctional zones lacking CITED2 possess fewer spongiotrophoblast, glycogen, and trophoblast giant cells, while labyrinth zones without CITED2 exhibit abnormal fetal vasculature characterized by stunted and irregular capillaries^[41]. Dunwoodie and coworkers specifically inactivated Cited2 in different cell types of the mouse placenta^[42]. They utilized a Tek-Cre recombinase mouse model to disrupt Cited2 in endothelial cells of the fetal vasculature and Tpbpa-Cre and Cyp19a1-Cre recombinase mouse models to disrupt Cited2 in subsets of trophoblast cells. Disrupting CITED2 in endothelial cells or subsets of trophoblast cells adversely affected placental and fetal growth, but in contrast to global CITED2 deficiency these conditionally mutant models were compatible with postnatal survival. Interestingly, CITED2 also participates in preparing the uterus for pregnancy^[43]. CITED2 is a progesterone responsive gene, and its uterine-specific ablation abrogates uterine stromal cell decidualization, which is essential for the establishment of pregnancy and the uterine-placental interface. Thus, CITED2 may directly contribute to both uterine and placental biology required for a successful pregnancy outcome.

Rat

A global CITED2 deficient rat model was generated utilizing CRISPR/Cas9 technology^[44]. Phenotypes for the Cited2 null rat and Cited2 null mouse were compared. Some similarities and differences in the rat and mouse Cited2 null phenotypes were observed. CITED2 deficient mice and rats shared heart, lung, placental abnormalities, and fetal growth restriction. However, deficits in adrenal gland morphogenesis, aberrations in neural tube development, and prenatal death were unique to the mouse. The explanation for the species differences is unknown, as is which species may best model CITED2 deficiencies in the human. In contrast, the placenta is a common site of action for CITED2 in the mouse, rat, and human^[41,42,44]. Furthermore, it is evident that placental and fetal growth restricted phenotypes are intrinsic to CITED2 actions within trophoblast cells^[44]. CITED2 is expressed prominently in cells of the rat junctional zone and in invasive trophoblast cells of the uterine-placental interface (Fig. 2A and B)^[44,45]. These tissue compartments also represent sites of deficits within the Cited2 null placentation site^[44]. CITED2 deficiency resulted in death within hours of delivery, most likely due to cardiac malformations, including ventral septal defects and double outlet right ventricle, and/or because of a delay in lung development. Within the placenta, CITED2 deficiency affected the junctional zone transcriptome, resulting in downregulation of transcripts such as matrix metallopeptidase 9 (Mmp9) and insulin like growth factor 2 (Igf2), and the upregulation of several interferon-responsive transcripts, including ISG15 ubiquitin like modifier (Isg15). MMP9 and IGF2 are established regulators of placental development^[46-49]. CITED2 deficiency also results in a delay in the onset of intrauterine trophoblast cell invasion. Furthermore, CITED2 deficient invasive trophoblast cells exhibit a distinct transcriptome, consistent with CITED2’s role as a co-regulator^[44]. Finally, CITED2 deficiency affects the ability of the placentation site to adapt to physiological stressors (see below). Thus, it is evident that the rat is a useful model for investigating conserved roles for CITED2 in placentation.

Fig. 2. CITED2 is expressed in both rat and human placentation sites.

A) Schematic showing the late gestation rat placentation site. Invasive trophoblast cells are depicted in green. B) In situ hybridization showing Cited2 transcript localization in a rat gestation day (gd) 18.5 placentation site (Scale bar, 500 μm). C) Schematic showing the late gestation human extravillous trophoblast (EVT) cell column (CC). Invasive EVT cells are depicted in green. D) In situ hybridization showing CITED2 transcript localization in first trimester (12 week) human placenta: CITED2, CDH1 (marker of basal cytotrophoblast, bCTB), NOTUM (marker of EVT cells) (Scale bar, 50 μm). Uterine-placental interface (UPI), junctional zone (JZ), labyrinth zone (LZ), decidua (Dec), natural killer (NK) cells, villous core (VC), syncytiotrophoblast (STB), cytotrophoblast (CTB), and villous CTB (vCTB). Modified from Kuna et al., 2023 with permission.

CITED2 AND PLACENTATION: HUMAN

CITED2 is prominently expressed in central and distal regions within the EVT cell column of first trimester human placenta (Fig. 2C and D)^[44]. Consistent with these observations, CITED2 exhibits an EVT cell differentiation-dependent increase in expression^[44]. In contrast, CITED2 shows limited expression in ST^[44]. Selective shRNA-mediated inhibition of CITED2 in human trophoblast stem (TS) cells interferes with normal EVT cell development, including their acquisition of invasive capabilities^[44]. Thus, CITED2 is poised to contribute to the regulation of trophoblast cell invasion and the actions of invasive trophoblast cells on the uterine vasculature.

CITED2 has also been implicated in disease states, such as diabetes^[50,51], congenital heart malformations^{[21,24,28-30,52-57]}, and cardiovascular dysfunction^[58-60]. Some of these disease states may be secondary to or exacerbated by CITED2 insufficiency leading to disruptions in placentation (see below). In fact, dysregulation of CITED2 expression has been associated with preeclampsia^[61]. CITED2 protein expression is decreased in the endoplasmic reticulum (ER) of stress-induced BeWo choriocarcinoma cells^[62]. ER stress has been implicated in preeclampsia^[63]. Whether non-transformed trophoblast cells exhibit a similar relationship of CITED2 protein expression and ER stress is unknown. Table 2 summarizes literature implicating CITED2 dysregulation in various disease states.

Table 2.

CITED2 involvement in disease states.

Disease states	CITED2 relevance	References
Glucose homeostasis and diabetes	Required for the regulation of hepatic gluconeogenesis	[50,156]
	Downregulation mediates high glucose-induced endoplasmic reticulum stress-dependent apoptosis	[51]
	Upregulated in vascular tissue of patients with obesity and type 2 diabetes	[157]
	Impairs insulin signaling in endothelial cells	[158]
	Decreased in heart tissue of maternal diabetic-exposed embryos	[58]
	Increased expression in adipose-derived stem cells from diabetic patients, which exhibit decreased proliferation and wound healing abilities	[159]
Inflammation	Potent repressor of macrophage proinflammatory activation	[23]
	Myeloid deficiency significantly increases macrophage and neutrophil recruitment	[32]
	Restrains signal transducer and activator of transcription 1 (STAT1) and interferon regulatory factor 1 (IRF1) signaling in macrophages and limits development of atherosclerotic plaques	[73]
	Deficiency results in enhanced placenta interferon-responsive gene expression	[44]
Pregnancy related disorders	Lower levels in cumulus cells are associated with higher rates of oocyte fertilization and embryo implantation	[161]
	Dysregulated in the placenta from mothers with hypertension	[160]
	Upregulated in placentas from women with severe preeclampsia	[61]
	Upregulated in placentas from growth restricted fetuses	[87]
	Decreased expression in endoplasmic-reticulum stress-induced BeWo choriocarcinoma cells (note: CITED2 may be restricted to the transformed character of the cells and not their trophoblast origin)	[62]
Cardiovascular disease	Essential for heart development	[20,21,26,27,44,162]
	Mutations contribute to congenital heart disease	[24,28-30,52-57]
Lung development	Dysregulation results in aberrations in lung development	[44,163,164]
Neurodevelopmental disorders	Deficiency results in an abnormal neural development (exencephaly) in the mouse, a phenotype reduced with folic acid treatment	[4,21,40]

EP300, an established partner in CITED2 action, has been implicated in the pathology of pregnancy disorders. The Rubinstein-Taybi syndrome is caused by disruptions in CREBBP or EP300. The subset of cases of the Rubinstein-Taybi syndrome affected by EP300 dysregulation are directly connected to pregnancy complications such as preeclampsia, HELLP syndrome, and fetal growth restriction^[64,65]. Meta-analysis of placental transcriptome data has also identified CREBBP/EP300 as a hub in preeclampsia-related pathways^[66]. Other anomalies in the EP300 gene have been linked to pregnancy miscarriages^[67]. These observations highlight the importance of a functional EP300 protein for the execution of key pregnancy-related events. Most interestingly, EP300 regulates human TS cell differentiation^[68]. Depletion of EP300 through short hairpin RNAs or CRISPR/Cas9 gene targeting inhibits TS cell differentiation and leads to retention of TS cells in a proliferative state. At least part of the mechanism of action of EP300 appears to be through inhibition of epidermal growth factor receptor (EGFR) signaling. Activation of EGFR is required for TS cell proliferation^[69]. Furthermore, EP300 is required for EVT and ST development^[68]. CITED2 shares involvement in regulating TS cell proliferation and in promoting EVT cell differentiation with EP300, but unlike EP300, is not involved in driving ST differentiation^[44]. Finally, actions on trophoblast cell development appear unique to EP300 and are not shared by the related protein, CREBBP^[68].

CITED2 AS A MODULATOR OF PLACENTAL PLASTICITY

A healthy placenta can adapt to environmental challenges affecting pregnancy^[35,70]. Stimuli driving adaptations, include nutrition status, oxygen tension, exposure to pathogens, inflammation, and many others^[38,71,72]. CITED2 has been implicated in cellular adaptations to hypoxia and to inflammation^{[23,31,32,73-79]}. CITED2 modulates placental adaptations to hypoxia and to a viral mimic, and thus is in an excellent position to affect placental health^[44]. In both cases, hypoxia and exposure to a viral mimic, the presence of CITED2 restrains trophoblast cell responses to the physiological stressor. Failure to attenuate these adaptive responses can lead to fetal growth restriction and fetal death^[44]. CITED2 may modulate trophoblast cell adaptations to other physiological stressors, and hence, potentially broadening the scope of its importance as a regulator of placentation.

INDIRECT ACTIONS OF PLACENTAL CITED2 ON FETAL DEVELOPMENT

There are many genes affecting placental development that also affect fetal heart and brain development^[80]. Some of these genes act within the placenta, heart, and brain, while it is apparent that the impact of other genes on the fetal heart and possibly brain is secondary to deficits in placental development and function^[80-82]. Cited2 represents a gene impacting the fetal heart and potentially the fetal brain both directly within each fetal tissue and indirectly through its actions on the placenta.

OTHER CITED FAMILY MEMBERS AND PLACENTATION

There are two other CITED family members: CITED1 and CITED4. CITED1 shows some overlap in its expression with CITED2, including expression in the embryo and the placenta, as well as the heart, mammary glands, and melanocytes^[10,83,84]. Dunwoodie and co-workers showed that disruption of the X-linked Cited1 gene in the mouse results in aberrant placental development^[11]. Cited1 mutants have an irregular and expanded junctional zone and enlarged maternal blood sinusoids within the labyrinth zone. The fetal vasculature component of the placenta is also disrupted due to junctional zone projections into the labyrinth zone. CITED1 deficiency also resulted in fetal growth restriction and fetal death on the day of birth or shortly thereafter^[11]. Little is known about a role for CITED1 in human placenta development. CITED1 does not appear relevant in human TS cells or their differentiation to ST or EVT cells^[44]. Some limited insight has emerged from CITED1 and placenta disease. CITED1 expression is increased in placenta of pregnant women with HELLP syndrome^[85]. CITED4 was identified as a co-activator for TFAP2 actions in heart development and function^[86]. CITED4 has not been directly implicated as a regulator of placentation but is downregulated in placentas from growth restricted fetuses^[87].

CITED2 MECHANISM OF ACTION - A MODULATOR OF TRANSCRIPTION FACTOR FUNCTION

The most well studied aspect of CITED2 action is its modulation of CREBBP and EP300 activities. CREBBP and EP300 are acetyltransferases that catalyze histone 3 lysine 27 acetylation at regulatory elements positively associated with transcriptional activation^[88]. All CITED family members possess three conserved regions (CR1, CR2, CR3) (Fig. 3A). CR2 is located at the carboxy terminus of each CITED family member and contains a potent transactivation domain (TAD) (Fig. 3B), which physically interacts with the Zn²⁺ binding cysteine/histidine-rich 1(CH1) domains of CREBBP and EP300 and is also called the transcription adaptor putative zinc finger 1 (TAZ1) domain^[3,9]. The CH1/TAZ1 domain is the region within CREBBP and EP300 that interacts with transcription factors such as hypoxia inducible factor 1 subunit alpha (HIF1A)^{[19,75,89-91]}, tumor protein p53 (TP53)^[92,93], RELA proto-oncogene, NF-kB subunit (RELA)^[94,95], and signal transducer and activator of transcription 2 (STAT2)^[96,97]. CITED2 can modulate the activity of transcription factors that interact with CH1/TAZ1. This interaction has been best demonstrated through the ability of CITED2 to effectively compete for HIF1A binding to CREBPP and EP300 resulting in the inhibition of HIF1A transcriptional activity^{[75,76,78,79,98-101]}. CITED2 blocks the HIF1A-EP300 interaction, most likely due to direct occupancy of the CH1/TAZ1 domain of EP300^[75]. These efforts have been extended to show that CITED2 controls hypoxia signaling by capturing EP300 from two distinct activation domains of HIF1A^[76]. CITED2 also has a higher affinity than HIF1A for EP300, and unidirectionally displaces HIF1A from its association with EP300^[102].

Fig. 3. The CITED protein family conserved domains.

A) Location of conserved regions (CR) within the CITED family. B) Amino acid sequence alignment of CR2 domains for each member of the CITED family.

CITED2 has also been shown to interact with other proteins, including LIM homeobox 2^[2], TFAP2 family transcription factors^[5], SMAD family members 2 and 3^[103], peroxisome proliferator-activated receptor gamma (PPARG)^[7], estrogen receptor^[104], MYC proto-oncogene, bHLH transcription factor^[105], and nucleolin^[106]. Several parameters of CITED family protein interactions with the TFAP2 family have been discerned^[86]. CITED2 exhibits stronger interactions with the TFAP2 family than does CITED1 or CITED4. Additionally, CITED protein interactions with TFAP2 proteins are within the region of TFAP2 proteins that binds to the CH1/TAZ1 domain of CREBBP and EP300. CITED2 may also interact with the dimerization domain of TFAP2C^[5]. In general, the mechanistic significance of CITED2 interactions with these other proteins is less than that currently understood about CITED2 interactions with CREBBP and EP300. Nonetheless, collectively, our knowledge of CITED2-protein interactions are all consistent with CITED2 functioning as a modulator of transcription.

In the following paragraphs, we discuss some examples of the potential involvement of CITED2 in modulating activities of specific transcription factors previously implicated in the regulation of placentation. CITED2 can act as either a co-activator or a co-repressor depending on the transcription factor.

CITED2 as a co-activator

TFAP2C.

TFAP2C is a member of the TFAP2 family of transcription factors. TFAP2C plays an important role in trophectoderm specification and TS cell self-renewal in the mouse^[107-111] and human^{[108,111-114]}. TFAP2C deficient mice fail to establish a proper maternal-fetal interface with malformations arising during the earliest stages of placentation^[115,116]. TFAP2C is also essential for junctional zone development^[117]. Potential TFAP2C gene targets have been identified in various trophoblast cell lineages^{[108,111,113,114,118]}. TFAP2C and CITED2 are co-expressed in the junctional zone and in invasive trophoblast cells situated within the rat uterine-placental interface^[44,119]. CITED2 may act as a rheostat to modulate TFAP2C transcriptional activities within the developing placenta. The negative aspects of CITED2 deficiency on placentation may be a consequence, at least in part, of less-than-optimal TFAP2C driven gene expression. TFAP2C dosage appears to be critical regarding its actions in trophoblast cells^[120]. As indicated above, CITED2 has been shown to physically and functionally interact with members of the TFAP2 family^[4,5]. More specifically, in the developing heart, CITED2 and TFAP2C cooperate in the transactivation of the paired-like homeodomain 2 gene^[121]. Target genes shared by TFAP2C and CITED2 in invasive trophoblast cells can be inferred from single cell RNA sequencing (scRNA-seq) and single nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) datasets^[119,122] but have not been empirically verified.

PPARG.

PPARG is a ligand activated nuclear receptor with a role in a variety of biological processes such as lipid and glucose homeostasis, metabolic disease, and inflammation^[123,124]. PPARG is expressed in both rodent and human placentas^[81,125-128]. Disruption of PPARG in the mouse results in prominent labyrinth zone dysfunction^[81]. The labyrinth zone does not represent a significant site of CITED2 expression and thus, CITED2 may not be a relevant co-regulator of PPARG action within the labyrinth zone. However, PPARG and CITED2 are co-expressed in invasive trophoblast cells, where their cooperativity may be more relevant^[119]. CITED2 is an established co-activator for PPARG^[7]. There is evidence that CITED2 partners with PPARG in other cell types, including PPARG’s involvement in regulating cortical neuron death associated with DNA damage^[129]. CITED2 is also required for optimal PPARG regulation of anti-inflammatory gene expression in macrophages^[23]. Target genes shared by PPARG and CITED2 in invasive trophoblast cells can be predicted from scRNA-seq and snATAC-seq datasets^[119,122].

CITED2 as a co-repressor

HIF1.

HIF1 is primarily a transcriptional activator and the main factor mediating adaptations to low oxygen tension in cells and tissues^[130]. HIF1 is composed of two subunits, an alpha or A subunit, which is regulated by oxygen tension, and a beta or B subunit, which is constitutively expressed^[131,132]. HIF1B is also called the aryl hydrocarbon receptor nuclear translocator (ARNT). In response to hypoxia, HIF1 is stabilized and promotes transcriptional changes in genes that are involved in angiogenesis, iron and glucose metabolism, cell proliferation, and cell survival. Early in gestation prior to uterine spiral artery remodeling intrauterine oxygen tensions are low and HIF1 is proposed to facilitate adaptations required to promote normal placenta development^[70,133-139].

Disruption of HIF signaling impairs TS cell responses to hypoxia and differentiation into the invasive trophoblast cell lineage^[140]. HIF signaling has also been identified as an important regulator of trophoblast cell plasticity and placental adaptations during low oxygen conditions^[141]. CITED2 is a negative regulator of HIF1. As described above, CITED2 prevents HIF1 from binding to CREBBP and EP300, and thus attenuates gene activation following exposure to hypoxic environments^{[3,76,79,98-100]}. CITED2 serves a critical role in modulating placental adaptations to hypoxia.

Nuclear factor kappa B (NFKB).

NFKB is a transcription factor involved in regulating innate immunity and inflammatory responses^[142,143]. The NFKB signaling pathway is involved in organizing cellular resistance to pathogens^[144-146]. NFKB possesses roles in trophoblast cell responses to pathogens^[147-151]. CITED2 has been identified as a repressor of NFKB^[152]. It prevents RELA from binding to its promoters and attenuates RELA acetylation by preventing its access to EP300. CITED2 has also been identified as a critical molecular switch that limits a broad proinflammatory gene network in macrophages by restraining inflammatory cytokine-induced NFKB activation^[32].

FINAL THOUGHTS

CITED2 has a pivotal and conserved role in regulating placenta development, especially at the uterine-placental interface, and in ensuring plasticity of the placentation site. These actions are accomplished through cooperation with other parts of the transcriptional regulatory apparatus. In general, CITED2 promotes placental growth and development through synergism with transcription factors such as TFAP2C and PPARG, while restraining responses to physiological stressors via antagonizing the actions of other transcription factors such as HIF1 and NFKB.

CITED2 can also be used as a tool to discover new transcription factor involvement in placental development. CITED2 has been shown to modulate the activity of several different transcription factors. As described above, a subset of these transcription factors has been implicated in the regulation of placentation (TFAP2C, PPARG) and placental plasticity (HIF1, NFKB). Other transcription factors are certainly involved in modulating aspects of placentation; however, the involvement of CITED2 as a modulator of any of these transcription factors is unknown. The existence of CITED2 deficient TS cells and animal models provides tools to discover new transcription factors targeting trophoblast cells and placental biology. Furthermore, examination of scRNA-seq and snATAC-seq in control versus CITED2 deficiency provides a strategy for identifying regulatory regions and transcription factor binding motifs specifically connected to CITED2. Such experimentation can be followed by chromatin immunoprecipitation, evaluation of the involvement of CREBBP and EP300, and assessment of direct physical interactions with CITED2. Any transcription factor utilizing CREBBP or EP300 is a potential target of CITED2 modulation. Such a strategy will expand our knowledge of the transcriptional control of trophoblast cell development, placentation, and adaptations of the placenta to physiological stressors.

The placenta is not the sole target of CITED2 actions. Other organs are dependent on CITED2 for orchestrating temporally and spatially appropriate morphogenesis. Several disease processes involve the dysregulation of CITED2. This represents a challenge that has experimentally been addressed using conditional mutagenesis^[42] and other methodologies to specifically manipulate the trophoblast cell lineage^[44]. Such approaches are dependent upon the availability of effective high-fidelity trophoblast driven Cre recombinase strains, which has represented a concern for in vivo manipulation of trophoblast cell lineages^[35,42,153], and/or the expertise in genetically manipulating and handling embryos for the creation of new trophoblast cell lineage- and placenta-specific mutant models. The precision of these approaches will improve and roles for CITED2 in specific trophoblast cell lineages will be addressed. The recent establishment of a highly specific invasive trophoblast cell-specific Cre recombinase rat model should facilitate understanding in vivo roles for CITED2 in the biology of invasive trophoblast cell-guided uterine transformation^[154].

It is apparent that there are species differences associated with the biology of CITED2. Genetic disruption of the Cited2 gene in the mouse and rat yield similarities but also pronounced phenotypic differences^[4,21,40,44]. The comparative biology of CITED2 reflects species-specific aspects of its expression pattern, the profile of other CITED family members, and undoubtedly the expression and activities of its many transcription factor targets. Making the leap from the mouse or rat to human CITED2 biology represents a challenge for understanding the biology of some organ systems; however, not for the placenta where CITED2 is a conserved core actor in the regulatory hierarchy directing trophoblast cell lineage development. This conservation likely emanates from the known actions of CITED2 modulated transcription factors in trophoblast cell development. We do not yet know the molecular targets of CITED2 action in mouse, rat, or human trophoblast cells. We expect to observe aspects of conservation and, also species specificity as we learn more about the involvement of CITED2 in trophoblast cell development and placentation.

Finally, it is evident that CITED2 dysregulation is linked to the etiology of an assortment of disease states not involving pregnancy or placentation. The involvement of CITED2 in both embryogenesis and disease should provide significant motivation for elucidating mechanisms underlying CITED2 action. Understanding the involvement of CITED2 in disease processes will provide insights into the role of CITED2 in placenta development. Similarly, knowledge of CITED2 in placentation should provide new directions for understanding the involvement of CITED2 in the etiology of diseases that impact human health. Not surprisingly, several pregnancy-related diseases involve the dysfunction of biological processes directly regulated by CITED2.

Fig. 4. Schematic of CITED2 positive and negative actions on gene regulation.

A) CITED2 facilitation of transcription factor (TF) interaction with CREBBP /EP300 promoting gene activation. B) CITED2 interference of TF interaction with CREBBP /EP300 preventing TF-mediated gene activation.

DATA AVAILABILITY

Data sharing is not applicable to this article as no new data were created or analyzed in this study.