The Why of YY1: Mechanisms of Transcriptional Regulation by Yin Yang 1

1Yin Yang 1 (YY1) is a transcription factor (TF) that was first described in 1991 by three independent groups, who all named the protein differently based on the molecular mechanisms they associated it with. It was called NF-E1, as it binds the μE1 intron enhancer at the immunoglobulin heavy chain (IgH) locus or δ, as it binds the delta motif in the promoter of ribosomal protein genes. The name Yin Yang 1 was eventually broadly adopted, as it captures the dual activity of YY1 as both a transcriptional activator and repressor. Similarly, the YY1 family member YY2 has this dual activity.

2YY1 is ubiquitously expressed in mammalian cells. It forms homodimers that seem to be stabilized by low specificity RNA binding. YY1 homodimers bind a relatively small sequence motif (5′-CCGCCATNTT-3′), often found in enhancers and promoters, via the four C2H2 zinc fingers in its C-terminal domain (;; Figure 1). YY1 and its paralog, YY2, share overall 56% homology at the aminoacid sequence, reaching 86% of conservation in the zinc-fingers region. This high degree of homology might result in YY2 binding to the same YY1 DNA binding motif. The DNA binding zinc fingers partially overlap with sequences involved in transcriptional repression. In the N-terminal region a transcriptional activator domain is located. YY1 influences transcription by the recruitment of cofactors. For example, the REPO domain interacts with polycomb group proteins, to recruit repressive cofactors to specific genes. Cohesin and condensin, key factors of 3D chromatin organization, also interact with the REPO domain of YY1. Another YY1 domain contains a his tract consisting of eleven consecutive histidine residues, and is proposed to stimulate YY1 accumulation in nuclear speckles, that play an important role in RNA metabolism. Interestingly, YY1 itself plays a role in pre-mRNA splicing. Binding intronic enhancer motives allows YY1 to activate gene expression and promote splicing.

3YY1 is a crucial factor regulating cell proliferation and apoptosis and it has been implicated in many regulatory mechanisms in different tissues. In the brain, YY1 plays a well-established role in neuronal development. In mice, a homozygous Yy1 knock-out results in peri-implantation lethality while heterozygous mutations cause growth retardation and neurulation defects. In humans, YY1 haploinsufficiency causes Gabriele-de Vries syndrome (OMIM #617557), characterized by psychomotor delay and intellectual disability alongside many comorbidities, including craniofacial dysmorphisms, intra-uterine growth restriction and behavioral alterations.

4Recent findings concerning YY1 activity in chromatin rearrangement and its importance in neuronal development have caused re-evaluation of YY1 function. This review focusses on basic transcriptional mechanisms that YY1 has been implicated in and considers its function in context of neuronal development and disease.

5A key question in the YY1 field is how a DNA-binding TF can act as both a transcriptional activator and repressor. Multiple mechanisms have been suggested, including post-translational modifications of YY1 and co-factor dependency. Acetylation of the C-terminal domain of YY1, for example, reduces DNA-binding capacity in vitro, whereas acetylation of the central domain is required for YY1 to act as a full repressor. In mouse anterior neuroectoderm development, the acetylation of YY1 is required for Otx2 activation. Only acetylated YY1 binds an essential enhancer 5kb upstream of the homeobox gene Otx2 to prompt its expression. Post-translational modifications of YY1, thus, seem to influence whether YY1 can activate or repress transcription.

6Another explanation for YY1s context-dependent transcriptional activation or repression is the interplay between YY1 and its cofactors. Protein-protein interactions exist between YY1 and DNA-binding cofactors such as SpI and c-Myc. Bound to DNA, YY1 tethers cofactors directly to promoters, such as YY1 associated factor 1 (YY1AP), that boosts transcription. YARP, a structural homolog of YY1AP, is expressed in brain, heart and placenta tissue suggesting it acts as another co-activator of YY1. Additionally, YY1 also recruits co-repressors to the DNA, such as Smad family members repressing TGF-β signaling and cell differentiation. In vitro, YY1 is able to bind promoter sequences and recruit polymerase by interacting with general TFs. Overlapping DNA binding sites of YY1 and transcriptional activators provide another explanation for YY1-mediated repression. Since DNA-binding of YY1 and an activator at a given locus can be mutually exclusive, YY1 binding can block the activating factor. For example, in mammary epithelial cells, YY1 represses β-casein competing with the latent-state mammary gland specific factor (MGF) of the STAT family, STAT5A. Upon lactation, MGF is activated, increasing its DNA-binding affinity and enabling it to replace YY1 and de-repress β-casein. Independent of binding the DNA, YY1 can regulate transcription by protein-protein interactions with other regulators. In mouse neurons, Yy1 and its interacting partner Brd4 activate Senp1, an upstream regulator of glutamate signaling, which plays a pivotal role in neuronal plasticity. Dephosphorylation of Yy1 upon membrane depolarization depletes the Senp1 promoter of Yy1-Brd4, consequently repressing Senp1 expression. This example illustrates the mechanisms of both post-translational YY1 modifications and co-factor dependency that partly explain how a ubiquitously expressed TF can be an activator or repressor depending on the cellular context.

7Among YY1 interactors, are multiple chromatin modifiers, suggesting chromatin modifications might explain YY1 functioning as a transcriptional repressor or activator. YY1 can direct the Polycomb complex to specific DNA loci, initiating methylation of histone 3 lysine 27 (H3K27me3). Furthermore, YY1 interacts with histone deacetylases (HDACs) associated with gene silencing. Several members of this family, such as HDAC1, HDAC2 and HDAC3, interact with YY1 both in vitro and in vivo. Interestingly, YY1 also interacts with histone acetyltransferases (HATs) like p300 and CREB binding protein (CBP), activating transcription. Besides acetylating or de-acetylating histones, HATs and HDACs modify YY1 itself, regulating its DNA-binding affinity and activity as a transcriptional regulator. In addition to histone 3 modifications, YY1 promotes transcription by the recruitment of the methyltransferase PRMT1, which methylates histone 4 arginine 3.

8Moreover, YY1 has been shown to activate transcription by interacting with chromatin remodeling complexes involved in the shifting and repositioning of nucleosomes, such as the INO80 complex and, more recently, the BAF complex. The interaction with the INO80 complex is also thought to play a role in facilitating the access of YY1 to its target genes. Hence, through a plethora of molecular co-factor interactions, YY1 influences chromatin modifications and ultimately gene expression.

9At first glance, YY1 does not seem essential for 3D chromatin organization, as the majority of its binding sites are close to transcription start sites (TSSs) and only a minority is located distal from regulated genes. However, in YY1-haploinsufficient lymphoblastoid cell lines, the most differentially expressed genes were controlled by those distal YY1 binding sites. Likewise, in T-helper cells, YY1 seems to mainly influence gene expression through long-distance DNA interactions. Interactions between YY1 and proteins involved in chromatin organization, such as CTCF and cohesin, further implicate a role in 3D chromatin organization.

10Loop extrusion allows cohesin to actively form DNA loops. CTCF, a DNA-binding protein, delimits these DNA loops, demarcating isolated chromatin structures that can be as large as 1 Megabase, called topologically associating domains (TADs). Within TADs, long-distance DNA interaction is facilitated between enhancers and promoters by chromatin structuring proteins. CTCF itself is, however, not crucial for the majority of enhancer-promoter interactions. Recently, YY1 was identified as the structural factor that regulates the formation of DNA loops within the larger CTCF-CTCF domains in a wide variety of mammalian cell types, indicating that YY1-mediated long-distance promoter-enhancer interactions are a general mechanism in mammalian cells. Like CTCF, YY1 forms homodimers and binds hypomethylated DNA to facilitate long-distance DNA interactions. In contrast to CTCF, however, YY1 binds to a consensus sequence mainly present in enhancers and promoters and is only scarcely associates with insulators.

11In neuronal differentiation specifically, TAD organization was found to be less conserved between cell types and differentiation stages than initially thought. These findings triggered research into the possible role of dynamic chromatin organization during differentiation of neural progenitor cells (NPCs). Surprisingly, YY1 appeared to instigate DNA loop formation and NPC-specific promoter-enhancer interactions. These YY1-mediated DNA loop formations occur within CTCF-CTCF DNA loops. These findings introduce a new identity of YY1 as a structural protein in addition to its role as a traditional TF, providing an even broader understanding of the multitude of cellular mechanisms that employ the ubiquitously expressed YY1 protein to regulate transcription.

12Several studies show that YY1 plays a role in neurodevelopmental disorders, but the mechanisms behind this are still incompletely understood. In mouse embryos, YY1 has proven essential for neurodevelopment. In murine models, Yy1 conditional knock-out (cKO) induced at an early stage of cortical development, caused an increased apoptosis rate and cell cycle arrest in neuroepithelium and NPCs. This effect on NPCs however decreased markedly when cKO was induced at later stages. Accordingly, another study by Varum and colleagues showed that Yy1 cKO in mice affects neural crest (NC) development in a strict stage dependent manner. Early KO caused a reduction of multiple NC-derived lineages, whereas late KO (after embryonic day 11.5) resulted in no clear phenotypic difference compared to control, showing a decreased dependency on YY1 regulated processes at later stages of neuronal development.

13Surprisingly, early and late Yy1 cKO, caused similar changes in gene expression, which indicates that the decreased importance of YY1 during neuronal development does not coincide with a shift in YY1 target genes. As YY1-regulated genes seemed to be mainly implicated in metabolic pathways and protein translation, influencing the cell cycle machinery indirectly in NPCs and NC, Zurkirchen and colleagues hypothesized that a decreasing dependency on YY1 during cortical development is due to a decreased biosynthetic demand and decreased proliferation rate of cells at later stages of corticogenesis, making these cells less vulnerable to defects in these pathways.

14The importance of YY1 in early development is also attributed to apoptosis inhibition. In mice, cKO of Yy1 at an early embryonic stage caused an accumulation of p53 protein and increased apoptosis. This effect could be partially reversed in Yy1/Trp53 double cKO mice, indicating that YY1 is important in the downregulation of p53, an important apoptosis regulator, to facilitate NPC survival. Additionally, YY1 inhibits apoptosis by regulation of the Planar cell polarity effector gene FUZ, an important apoptosis factor in neuronal development. Alterations of FUZ/Fuz expression cause neural tube defects in humans and are associated with an increased number and disorganization of NC cells in mice. YY1 can induce hypermethylation of the FUZ promoter, resulting in FUZ downregulation and inhibition of its apoptotic signal. A reduction in soluble YY1 protein reverses this hypermethylation at the FUZ promoter and is associated with increased apoptosis.

15Recently, YY1 was also linked to NPC differentiation by downregulation of Sox2 expression in mice. SOX2 is a known pluripotency factor and is also involved in the maintenance of the undifferentiated state of NPCs. YY1 was implicated in Sox2 downregulation in mouse brain cortexes during neuronal development by binding the Sox2 locus and physically halting transcription. These results accentuate a pro-differentiation role for YY1 that contradicts previously described work, which shows that YY1 is vital for NPCs maintenance and proliferation in early development.

16Given its complex function, it is not surprising that the role of YY1 in neurodevelopment remains a topic of active discussion. It remains important to also consider YY1 function in a context dependent manner. Indeed, YY1 has been shown to exert context dependent functions in several cell types, like B-cells and heart muscle. For neurodevelopment specifically, context dependent YY1 functionality is still incompletely understood and will be an interesting topic to further explore in future studies.

17In humans, YY1 haploinsufficiency causes Gabriele−de Vries Syndrome, which is characterized by cognitive impairment, behavioral alterations, intrauterine growth restriction, feeding problems and sometimes congenital malformations. One of the consequences of YY1 haploinsufficiency in humans is a loss of H3K27 acetylation at enhancers bound by YY1. H3K27 acetylation is tightly associated with active promoters and distal enhancers which indicates that downregulation of YY1 affects chromatin regulation and gene transcription. Additionally, mutations in YY1 binding sites in specific non-coding regulatory regions cause neurodevelopmental disorders with a milder phenotype, since the effect of such mutations is limited to the expression of the gene associated with this regulatory region. For example, a disrupted YY1-binding site in a brain-specific enhancer of ADGRL3 creates a predisposition to attention-deficit/hyperactivity disorder (ADHD).

18Throughout life, YY1 regulates various neuroprotective pathways, playing a central role in ischemic damage, Parkinson’s and Alzheimer’s disease. For example, YY1 activates NRF2, which in turn initiates an antioxidant response to protect brain cells against ischemic damage following cerebrovascular accidents. In Parkinson’s Disease, YY1 is downregulated in microglia, along with other neuroprotective pathways like mTOR and TGF-β. YY1 regulates the expression of NRF2-mediated anti-oxidant response and the transmembrane transporter SVCT2-dependent import of the protective drug ascorbate, which are key targets for Parkinson’s disease treatment developments. A bioinformatics approach to uncover regulators of Alzheimer’s Disease appointed YY1 as one of the master regulators. Interestingly, in contrast to Parkinson’s, in Alzheimer disease higher levels of YY1 mRNA were detected in human autopsy-derived whole brain samples and isolated neuron samples compared to controls. It would be tempting to speculate that the protective function requires tightly regulated levels of YY1, while aberrant levels contribute to the onset and progression of neurodegenerative diseases.

19Analogous to its role in healthy state, YY1 has a highly context-dependent function also in cancer. YY1 can act as a tumor suppressor or stimulator. For example, YY1 inhibits proliferation in pancreatic cancer through downregulation of SOX2, while in melanoma, YY1 promotes tumor growth, and YY1 cKO prevents tumorigenesis in a melanoma murine model. Interestingly, both early NC development and melanoma tumorigenesis seem to be particularly sensitive to alterations in the same metabolic processes that are regulated by YY1. Of note, next to YY1, YY2 can also be upregulated in cancer cells. Possibly, due to the high degree of homology, some YY1 antibodies might not discriminate between the two paralogs, which can be relevant for the interpretation of studies which only employ immunohistochemistry.

20In gliomas, YY1 is overexpressed and contributes to tumor progression. YY1 has been implicated in glioma tumor cell proliferation due to its inhibitory effect on p53 protein levels. In glioma cell lines, YY1 has been shown to be regulated by the MicroRNA miR-218. Normally, miR-218 is downregulated in glioma. When miR-218 is overexpressed, YY1 is downregulated and the inhibition of the tumor suppressor p53 is nullified. Additionally, YY1 was shown to upregulate transcription of the long non-coding RNA SNHG17, which activates the Wnt/β-catenin signaling pathway and thus contributes to the proliferation of glioma cells and inhibition of apoptosis.

21Proteomics identified YY1 as a regulatory factor involved in cancer stem cell (CSC) maintenance across 17 different cancer types. YY1 expression was associated with that of CSC regulators SOX2, BMI1, and OCT4. In glioma stem cells (GSCs) Notch signaling was shown to direct YY1 to repress gene transcription involved in cell differentiation by causing locus specific methylation of histone H3K27. Notch signaling is known to inhibit differentiation and its regulatory mechanisms seem to be vital in neuronal development as well as in GSC maintenance. Together, the ability of YY1 to inhibit apoptosis and its association with CSC maintenance could indicate a broader role for YY1 in tumor biology.

22YY1 activates or represses genes depending on the cellular context. These seemingly contradictory functions led to the name Yin Yang 1 and sparked investigations into YY1 on a mechanistic level. Here, we briefly discussed the roles that YY1 plays at different levels of transcriptional regulation. First, as a (traditional) DNA-binding TF, YY1 interacts with an extensive list of general and context-dependent cofactors. Recruitment of these cofactors by YY1 is important for direct repression or activation of genes and was shown to play a role in pre-mRNA splicing. Second, YY1 interacts with chromatin remodeling complexes to regulate transcription through chromatin accessibility. Third, by controlling chromatin looping, YY1 can enable and stabilize enhancer-promoter interactions to activate expression (Figure 2).

23For the human brain, YY1 is essential for proper development, maintenance, and protection from degeneration. It remains difficult to speculate which of the described roles of YY1 is most important in the brain. As a structural factor, YY1 was shown to facilitate NPC-specific long-distance promoter-enhancer DNA interactions. A developmental stage-specific dependency on YY1 in neuronal development, stresses YY1s role in metabolic pathways and protein translation. Further research is needed to elucidate the interplay between YY1 as a regulator of chromatin looping and its role as a traditional TF. Another topic for further research would be the RNA-binding capability of YY1 in relation to transcriptional regulation, as it was shown that RNA binding stabilizes YY1 homodimers and that, compared to DNA binding, the RNA binding occurs with low sequence specificity. In light of the extensive list of YY1 cofactors, it is important to consider the recently proposed phase-separation mechanism, where DNA bound TFs drive the formation of droplets with high concentrations of other TFs and cofactors necessary for transcription (; Figure 2D). For example, the TFs OCT4 and GCN4 can form such phase-separated droplets with a high concentration of many cofactors upon enhancer binding. The high concentration of factors in phase-separated droplets would allow rapid interactions of many factors that during in vitro assays would seem too weak. In highly concentrated phase-separated droplets YY1 might be able to interact with even more cofactors than those that have been identified in in vitro assays. The ambivalence of YY1 being a repressing and activating TF fits the proposition of the studies on phase-separation that transcriptional regulation is a dynamic biochemical equilibrium.

24Being a ubiquitously expressed transcriptional regulator, it comes as no surprise that YY1 plays a role in multiple regulatory pathways that work in concert to execute the correct transcriptional program in different cell types and at different stages of development. Reviewing the body of literature on YY1, the dualistic reference in its name still fits well. The mechanisms behind seemingly opposite forces of repression and activation are complementary and interconnected. The molecular mechanisms of YY1 also teach us about transcriptional regulation in general. Similar to YY1, other TFs might depend on the cellular context to play different roles with different transcriptional outcomes. GATA1 and co-factor FOG1 for example, activate or repress their target genes in a context-depended manner, which is mediated by the Nucleosome Remodeling and Deacetylase (NuRD) complex. Further research into the different roles of YY1 is warranted. In addition to answering fundamental questions on transcriptional regulation, future studies on YY1 could have clinical implications for neurodevelopmental and neurodegenerative disorders as well as cancer.

25TV, LH, and EP performed the literature research and wrote sections of the manuscript. TB conceived the manuscript, wrote sections, supervised the work, and obtained funding. All authors approved the final version of the manuscript.

26The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.