Mediator's Kinase Module: A Modular Regulator of Cell Fate

Abstract

Selective gene expression is crucial in maintaining the self-renewing and multipotent properties of stem cells. Mediator is a large, evolutionarily conserved, multi-subunit protein complex that modulates gene expression by relaying signals from cell type-specific transcription factors to RNA polymerase II. In humans, this complex consists of 30 subunits arranged in four modules. One critical module of the Mediator complex is the kinase module consisting of four subunits: MED12, MED13, CDK8, and CCNC. The kinase module exists in variable association with the 26-subunit Mediator core and affects transcription through phosphorylation of transcription factors and by controlling Mediator structure and function. Many studies have shown the kinase module to be a key player in the maintenance of stem cells that is distinct from a general role in transcription. Genetic studies have revealed that dysregulation of this kinase subunit contributes to the development of many human diseases. In this review, we discuss the importance of the Mediator kinase module by examining how this module functions with the more recently identified transcriptional super-enhancers, how changes in the kinase module and its activity can lead to the development of human disease, and the role of this unique module in directing and maintaining cell state. As we look to use stem cells to understand human development and treat human disease through both cell-based therapies and tissue engineering, we need to remain aware of the on-going research and address critical gaps in knowledge related to the molecular mechanisms that control cell fate.

Introduction

Our understanding of genomics and transcriptomics has advanced considerably in the past 30 years. The Human Genome Project, exponential increases in computing power, and the advent of next-generation sequencing have formed a solid foundation upon which to study the transcriptome: the gateway between the potentiality of genomic information and the actuality of cellular protein expression. Regulation of cell type-specific transcription is the key to that gateway, and the keeper of the key is the Mediator complex. The Mediator complex is a highly conserved transcriptional regulatory complex composed of many subunits divided into four modules, and it facilitates the convergence of environment, signaling cascades, cell type-specific transcription factors, and core transcriptional machinery to control cell state [1 –3]. Mediator's activity is associated with recently characterized super-enhancers and their interaction with Mediator's four modules.

Perhaps the most poorly understood aspect of Mediator is its kinase module composed of the subunit 12 or 12-like (MED12/L), subunit 13 or 13-like (MED13/L), cyclin-dependent kinase 8 or 19 (CDK8/19), and Cyclin C (CCNC). The final step in deciphering Mediator's role in cell fate determination may lie in the greater understanding of this enigmatic module. Stem cells offer a unique opportunity to delve deeper into the role of Mediator and the kinase domain, as these cells are normal, self-renew, and differentiate [4,5]. Despite their incredible clinical relevance, particularly in the fields of regenerative therapy and tissue engineering, there are still too many unanswered questions that hinder their widespread clinical deployment, including questions about how the environment, signaling pathways, transcription factors, and Mediator work together to drive self-renewal and differentiation toward specific lineages.

In this review, we will describe the Mediator complex, its association with super-enhancers, its kinase module, and the kinase module's role in controlling cell state and lineage commitment. The discussion of the kinase module will include its structure, function, association with disease, and role in regulating cell type-specific transcription. Finally, we identify gaps that remain in our current understanding of the kinase module and point to those as opportunities for continued exploration.

The Mediator Complex

In 1990, around the time that the Human Genome Project was beginning in earnest, Roger Kornberg's laboratory at Stanford University stumbled upon a phenomenon while studying transcription activation in Saccharomyces cerevisiae. The work involved the potent activator GAL4-VP16 (a hybrid protein composed of GAL4's DNA-binding domain and the activator portion of the herpes simplex VP-16 protein) and its in vitro inhibition of T-rich binding factor. GAL4-VP16 was responsible for activating genes with GAL4 binding sites while T-rich binding factor activated genes possessing thymine-rich regions. Reasonably, neither activator should have interfered with the activity of the other unless an unknown coactivator was also required to complete the activation, and this coactivator was acting as a limiting reagent owing to its low availability. This squelching between the two activators was relieved when S. cerevisiae protein fractions were added, indicating that there was indeed a novel coactivator at work. Neither RNA polymerase II (RNAP II) nor the general transcription factor (GTF) TFIID proved to be this coactivator, and so the mysterious factor was at once named the Mediator of RNAP II Transcription, describing its observed function in the transcriptional process [6,7] (for a more extensive history of Mediator's discovery, see review by Kornberg) [8].

Over the next two decades, the Mediator complex was found to be universally conserved throughout all eukaryotic organisms while completely absent in prokaryotes. Eukaryotes, being far more complex than prokaryotes, owe their incredible complexity to the regulation of gene expression provided, in large part, by the Mediator complex [8]. Mediator is a large, 1 million Dalton [8] protein complex composed of between 20 and 30 subunits depending on species [3], and is arranged in four distinct modules: head, middle, tail, and kinase [9]. Bioinformatic and biochemical analyses have identified most of the Homo sapiens Mediator subunits as similar in sequence, structure, and function to those found in S. cerevisiae and D. melanogaster, indicating a high degree of sequence conservation across eukaryotic species [10] (Fig. 1).

FIG. 1.

Structural differences between Saccharomyces cerevisiae (yeast) and Homo sapiens (human) Mediator complex. Shared and highly conserved subunits are shown in blue, whereas additional subunits that exist in humans but not yeast are highlighted in orange. This includes subunits that can substitute for each other in the kinase domain, including CDK8/CDK19, MED12/MED12L, and MED13/MED13L.

Mediator functions as a bridge-like adaptor to facilitate the interaction between activators and RNAP II during activator-dependent transcription [1]. Each module—and in some cases individual subunits—of the H. sapiens Mediator complex has a unique role in the regulation of transcription and interactions with the transcriptional machinery [2,3]. Recent studies have indicated that Mediator is a key regulator and functional unit of super-enhancers that are responsible for properly regulating cell type-specific gene expression profiles [11,12]. The roles of the head, middle, and tail modules of the Mediator complex have been largely defined within the context of directly regulating RNAP II activity [1,3]. The head module, together with the middle module, plays an essential role in stabilizing the interaction of transcription factors and RNAP II during pre-initiation complex (PIC) assembly on a cell type-specific promoter. The head module changes conformation to accommodate the subunit organization that controls the Mediator-RNAP II and Mediator-promoter interactions [13,14]. Whereas the head and middle modules bind RNAP II and the PIC, the tail module interacts with transcription factors bound to peripheral gene regulatory elements [1,3]. In addition, the tail module along with the kinase module regulates the head and middle modules, whereas the core complex (head, middle, and tail modules) represents the essential function of the Mediator complex [15]. The kinase module of the Mediator complex, composed of subunits MED12/L, MED13/L, CDK8/19, and CCNC, is reversibly associated with the core complex [16 –18] and is perhaps the most versatile and mysterious of all of Mediator modules.

Mediator and Super-Enhancers

In the past 10 years, super-enhancers have been characterized as clustered enhancers whose combined influence over the cellular transcription program is greater than other known gene regulatory elements. The complexity and specificity of super-enhancers make them critical components of identifying and characterizing cell type-specific genes. Super-enhancers were initially defined in embryonic stem cells (ESCs) by Whyte et al. in 2013 as genomic elements occupied by (1) a significant number of cell type-specific transcription factors such as OCT4, SOX2, and NANOG; (2) an abundance of the histone mark H3K27Ac, and perhaps most importantly; (3) the Mediator complex [11]. Traditionally, enhancers have been defined as regions of DNA spanning only a few hundred base pairs to which transcription factors bind. These elements occur with a frequency of one every 3,000–30,000 bases and are responsible for regulating the expression of a corresponding downstream gene by helping to recruit RNAP II to the gene's promoter [19].

The unique feature of super-enhancers is the high level of Mediator occupancy along with cell type-specific transcription factors compared with typical enhancers. Because of this, the identification of super-enhancers hinges primarily on the presence of MED1, the largest and most highly conserved of the Mediator core subunits [11]. Using chromatin immunoprecipitation sequencing (ChIP-seq), enhancers are ranked by their MED1 enrichment and are plotted against the MED1 signal. All enhancers beyond the inflection point—where the slope of the generated line graph equals 1—are deemed super-enhancers and further investigated for co-occupancy by other identifiers [20]. Given the role of Mediator as a bridge between cell type-specific transcription factors and the transcriptional machinery, the detection of Mediator at the enhancer and promoter of cell type-specific genes confirms DNA looping [21]. In addition, it further identifies the promoter of a specific super-enhancer target gene (Fig. 2), making Mediator a significant factor in identifying cell type-specific gene profiles and understanding transcriptional control that defines cell state [11,22 –27].

FIG. 2.

Top: linear representation of a super-enhancer. Grey ovals represent DNA-bound transcription factors and orange circles represent the histone mark H3K27Ac with the gene promoter potentially located over 10 kilobases downstream of the super-enhancer. Bottom: DNA looping mediated by the Mediator complex and Cohesin leading to the expression of the downstream gene [21].

Understanding Mediator's role in occupying enhancers and super-enhancers began as early as 2010 when Kagey et al. used shRNAs to reduce expression of Mediator and Cohesin—a protein complex involved in looping chromatin. The results of this study indicated a loss of pluripotency in murine ESCs (mESCs) following the knockdown of several Mediator subunits, most of the Cohesin subunits, and the Cohesin loading complex subunit Nipbl. ChIP-seq analysis revealed that Mediator and Cohesin co-occupied both the enhancers and promoters of key mESC regulators pou5f1 (the gene encoding OCT4) and nanog, indicating that Mediator was interacting with distant enhancers and promoters simultaneously. Chromatin Conformation Capture assays further confirmed DNA looping between these enhancers and promoters, supporting a model for Mediator in which the complex reaches across a three-dimensional chromatin landscape to activate the expression of stem cell transcription factors and cell type-specific genes [21]. This work was later confirmed and expanded on in a series of publications from other laboratories [28 –32].

In 2013, Whyte et al. performed ChIP-seq for OCT4, SOX2, and NANOG in mESCs and observed yet another novel pattern in genome occupancy. Of the 8,794 sites that were co-occupied by all three transcription factors, 231 of those sites were also occupied by MED1 at levels at least 10 times higher than typical enhancers. In addition to patterns of transcription factor and co-factor occupancy, super-enhancer-associated genes have higher rates of expression than genes associated with typical enhancers. To demonstrate this, a luciferase assay was performed where pou5f1 was incorporated into either a cloned region from a typical enhancer or a super-enhancer. This study revealed at least a threefold increase in pou5f1 expression when coupled with the super-enhancer region compared with a typical enhancer region. Conversely, expression of genes associated with super-enhancers was found to be reduced by 0.5-fold in the absence of OCT4 or MED12, showing that super-enhancer regulation is indeed dependent on the presence of key transcription factors and Mediator [11].

Work in the same year—also performed in the laboratory of Dr. Richard Young at the Whitehead Institute—expanded the search for super-enhancers beyond mESCs. Hnisz et al. performed ChIP-seq for RNAP II and RNA-seq to monitor all transcripts, and their data revealed that super-enhancers are enriched for both RNAP II and transcript with signals that are at least 24 times greater than those observed for typical enhancers. These data support the evidence initially provided by Whyte et al. that super-enhancer-associated genes are transcribed at higher levels. Finally, H3K27Ac histone marks were measured using ChIP-seq to identify super-enhancers in 86 human cells and tissue samples ranging from adipose and aorta tissue to small intestine and thymus tissue [25].

This line of exploration revealed that super-enhancers are not a unique feature of mouse and human ESCs but are indeed found in a host of cell types where they are associated with master transcription factors such as MYOD1 (myoblasts) and PPARG (adipocytes). Genome-wide association studies revealed that noncoding single nucleotide polymorphisms (SNPs) that are associated with phenotypic traits also occur disproportionately in super-enhancers. Some noncoding SNPs identified in super-enhancers are linked with human diseases such as Alzheimer's disease and type 1 diabetes, and cancers like pancreatic cancer and colorectal cancer. Such research has further elucidated the importance of super-enhancers not only in the regulation of cell type specificity, but also in the onset and progression of disease and oncogenesis [25].

Given the interaction of Mediator with transcription factors, its regulation of cell type-specific gene expression, and its intimate relationship with super-enhancers, Aranda-Orgilles et al. sought to better understand the role of Mediator's transient kinase module in the regulation of cell state, namely MED12's role in the maintenance of hematopoietic stem cells (HSCs). First, MED12 knockouts were induced in mouse embryos, resulting in mortality of all knockout mice within a month. The knockout of MED12 also reduced the growth of bone marrow and thymus tissue in these mice compared with the untreated control. Considering the interdependence of the kinase module subunits, separate knockouts of MED13, CDK8, and CCNC (each discussed in further detail later) were performed in murine HSCs but none could replicate the severity of the MED12 knockout. This suggested MED12's critical importance in maintaining HSC viability. The expression of HSC-specific genes decreased in the absence of MED12, which indicated a role in regulating cell type specificity. MED12 ChIP-seq was performed in human HSCs, and 84% of the sites found to be occupied by MED12 were located more than 10 kilobases from promoters. MED12 co-occupied genomic regions along with the enhancer-associated histone mark H3K27ac and the transcription factors RUNX1, FLI1, GATA2, and ERG. MED12 also occupied super-enhancers associated with hematopoietic-specific genes [33]. This line of research revealed that beyond the interaction of Mediator's core with super-enhancers and promoters, Mediator's four-subunit kinase module is also quite active on its own in genome-wide transcription regulation and so deserving more in-depth study.

Despite all the previous findings, the exact mechanism for how signaling factors efficiently interact with transcription factors scattered across the enhancers and super-enhancers of the genome remained poorly understood. More recently, Zamudio et al. initiated studies to determine how signaling factors, namely β-catenin, interacted with chromatin, transcription factors, and Mediator to form condensates: three-dimensional pockets of enhancers and super-enhancers all interacting together to drive cell type-specific gene transcription. RNA-FISH was performed to monitor the location of nanog transcripts and IF staining was used to confirm that the signaling factors β-catenin, STAT3, SMAD3, and coactivator MED1 all localize to regions of nanog involved in activating transcription. ChIP-seq analysis confirmed that all four factors interact with the nanog-associated super-enhancer [34]. The model for β-catenin's interaction with the nanog-associated super-enhancer established by this research serves as an explanation for how signaling factors can simultaneously influence the expression of several genes important for the maintenance and development of stem cells.

Taken together, these studies over the past 10 years indicate a cooperativity between chromatin structure in the form of super-enhancers, the Mediator complex, and Mediator's kinase module that is integral for the direction of cell type-specific gene transcription. Now that Mediator and super-enhancers form part of the foundation of transcription regulation, the outstanding question from our existing information involves the extent to which Mediator's kinase module plays a role in the overall process of cell type determination, either in regulating Mediator itself, or as independent coactivating subunits, or both.

The Mediator Kinase Module

The Mediator complex exists in two forms: The Mediator complex with the kinase module (30 subunits) and the core Mediator (26 subunits), which lacks the four-subunit kinase module. The 600 kDa kinase module is stable and able to exist and function independently of the core Mediator complex and can transiently associate with the core complex through MED13 [8,35]. The structural and functional aspects of the kinase module, including information on paralogs of the kinase module subunits and the importance of kinase activity in transcriptional regulation, is described hereunder.

Structure and function

A tightly organized network of both physical and functional subunit interactions maintains the structural integrity of the kinase module. Detailed studies of this complex structure have revealed the subunit organization within the kinase module, adding critical insight into the mechanism of CDK8's enzymatic activity. Specifically, early electron microscopy analysis in S. cerevisiae showed the kinase module structure having two bent protruding ends, identified as CDK8-CCNC, MED13, and a central globular protein, MED12. This model correlated with the previously observed human kinase module structure, with both models confirming the connection of the kinase module to the Mediator core through MED13 (Fig. 1) [36].

The association of the kinase module through MED13/MED13L allows for the adoption of different conformations and reversible interactions not just with the core Mediator but with other factors involved in transcription and cell state regulation [37]. In addition, physical interactions between kinase module subunits and the core Mediator leads to the adoption of different complex confirmations, which also plays a role in activating CDK8 kinase activity. Compiling data from different studies, it has been shown that CDK8 kinase activity is activated through a series of sequential steps: the first is the binding of CDK8 to its cyclin partner, CCNC, resulting in partial activation of CDK8. This is then followed by the binding of MED12, which results in the final necessary conformational change [35,38]. Biochemical studies revealed that CCNC possesses a surface groove that acts as a MED12 docking site. Mutations in either CCNC or the MED12 interface located at the N-terminal of MED12 leads to the dissociation of CCNC-CDK8 from the core Mediator complex followed by impaired kinase activity of CDK8 [39]. These findings suggest that the MED12-CCNC binding interface serves as an anchor that also activates CCNC-CDK8.

Kinase module paralogs

Although highly conserved as a complete complex, the kinase module in vertebrates is unique and more complex compared with the core complex as three of the four subunits have paralogs. CDK8 can be replaced by CDK19, MED12 can be replaced by MED12L, and MED13 can be replaced by MED13L [40,41]. The replacement of these subunits by their paralogs is mutually exclusive, meaning that the kinase module can possess either MED12 or MED12L but not both subunits simultaneously, leading to eight different forms of the kinase module subunits (Fig. 3) [42].

FIG. 3.

Mutually exclusive paralogs of subunits from the Mediator kinase domain. These paralogs, CDK19, MED12L, and MED13L, are replaced by CDK8, MED12, and MED13, respectively, under specific conditions, although the full details of how and why remain unknown.

CDK19, the homolog of CDK8, shares a 91% sequence homology with CDK8. There is an especially high degree of sequence conservation in the kinase activity domain and cyclin binding domain, with more divergence in the C-terminal sequence. The high degree of similarity suggests that CDK8 and CDK19 have overlapping functions and that one may be able to compensate for loss or absence of the other as it relates to kinase activity. For example, isolation and purification of CDK8/CDK19 interacting proteins demonstrates that both CDK19 and CDK8 interact with PRMT5 during repression of transcription in HeLa cells [43]. However, given that the tail portion of CDK8/CDK19 interacts with transcription factors and cofactors, the difference in the C-terminal sequence suggests that CDK8 and CDK19 more likely regulate different transcriptional programs [40,44]. Supporting this is the fact that CDK8 and CDK19 are differentially expressed across tissues, with CDK19 expression restricted to prostate, testis, thymus, and salivary glands, whereas CDK8 is expressed more ubiquitously across tissues [43].

Homology studies reveal that MED12 and MED12L share a 67% sequence identity, sharing two of four protein domains: PQL (proline/glutamine/leucine-rich) and OPA (C-terminal opposite paired domain). Of interest, according to the human protein atlas, after analyzing ∼37 human tissue samples, MED12L is found to be most highly expressed in the brain, whereas MED12 is expressed ubiquitously in all tissues [45,46] (for more information about the human protein atlas visit proteinatlas.org). Although they only share similarity in two of the domains, there does appear to be functional redundancy between MED12 and MED12L. For example, a study by Vogl et al. suggests MED12 and MED12L have the same binding sites for SOX10, which is an essential component of the transcriptional network that regulates the development and terminal differentiation of myelinating glia. From a series of pull-down experiments of MED12/MED12L and SOX10 proteins, it is evident that the C-terminal region of both MED12 and MED12L interact with SOX10. This discovery leads to the assumption that both proteins have the same function in the regulation of transcription during the differentiation of myelinating glia [47]. However, there is currently little research in this area, making it difficult to evaluate the complete functional redundancy between MED12 and MED12L.

Finally, MED13L shares a 51% sequence similarity to MED13 [48] and they both serve to link the kinase module to core Mediator by associating with middle module subunits, MED14 and MED19 [49]. Of note, this interaction of MED13/MED13L with core Mediator is regulated by SCF-FBW7 ubiquitin ligase through the proteasomal degradation of MED13/MED13L. Immunoprecipitation and in vitro ubiquitylation assays reveal that MED13/MED13L is directly ubiquitylated by SCF-FBW7 in vitro [16]. This degradation of MED13/MED13L prevents the kinase module from associating with the core complex suggesting that MED13/MED13L serves as an anchor for the kinase module. Of interest, MED13L, but not MED13, associates with MED26, indicating there may be unique functions for each subunit. When co-purified, MED13L is accompanied by a high abundance of MED26; however, MED13L is not present in MED26 isolations. This association is found to be specific to MED13L, as the same set of pull-down assays does not show a robust association between MED13 and MED26 [42]. According to the human protein atlas, MED13L is primarily expressed in heart and brain, whereas MED13 is expressed in all human tissues. More recent evidence supports previous studies indicating that mutations in med13l are associated with neurodevelopmental defects and heart diseases [50,51]. Knockdown and conditional knockout studies of MED13 in murine zygotes suggest that MED13L can partially substitute for MED13 and function during the development of embryo [52]. This study demonstrates that MED13 is required for both transcriptional activation and repression during zygote genome activation, as demonstrated through the up- and downregulated transcripts observed in MED13-knockdown embryos. Furthermore, med13 knockout embryos showed arrested development postimplantation. MED13L compensated for MED13 function during the OET (oocyte to embryo transition) enough to support embryo development to the blastocyst stage during preimplantation in med13 knockout murine zygotes. Clearly, the functional compensation of MED13 by MED13L is limited in context, as evident by its lack of compensation during postimplantation development and other studies showing mutations of MED13/MED13L that lead to disruptions of cellular functions resulting in respective disorders [53,54].

Together, studies surrounding the kinase module and each of the seven subunits indicate that the kinase module of the Mediator complex can present itself in many forms by changing its subunit configuration to diversify its function. Although it appears that there is some amount of functional overlap between paralogs, there is no complete functional redundancy for any of these kinase module subunits. Some of these paralogs may in fact play different roles in development and cell-type-specific transcription programs. In addition, individual genetic mutations of kinase subunits leads to embryonic lethality in mice and many human disorders with clear developmental disruptions [33,52,55,56]. In addition, research to date excludes the possibility of complete functional redundancy among the paralogs as they failed to substitute for their paralogous subunits (except for the partial functional compensation of MED13 by MED13L in murine zygote development as described in MED13 paralog section) when the other is genetically disrupted. The mutually exclusive nature, the unique expression profiles, and the relative functional contribution of each kinase module paralog still requires more thorough investigation to understand the structural interactions and functional relationships within the kinase module and what role each subunit plays in human development and disease.

Kinase module in transcriptional control

The kinase module functions as both an activator and repressor of gene transcription, making the study of the module that much more complicated. Initial functional studies revealed a repressive function for the kinase module in S. cerevisiae where the Mediator core, together with the kinase module, repressed transcription and the Mediator core alone enhanced activator-dependent transcription [57]. Early electron microscopy and subsequent functional studies in human HeLa cells showed that the kinase module repressed transcription by preventing the binding of RNAP II to Mediator thereby blocking the formation of the PIC-scaffold complex. This inhibition was achieved using multiple mechanisms, including kinase-independent regulation of Mediator-RNAP II interaction [37], kinase-dependent inactivation of TFIIH by phosphorylation [57], and gene silencing through the recruitment of histone methyl-transferases [36]. Later, biochemical and molecular studies supported the view that the kinase module has a context-dependent role in both gene repression and activation [11,58]. It now appears that when the Mediator core is absent, the kinase module acts to inhibit core Mediator function within the PIC [36,58]. Furthermore, the kinase module appears to mediate transcriptional activation through ncRNA-a (noncoding RNA-a, a class of ncRNAs that activate neighboring genes) by interacting with MED12 and chromatin [59]. Finally, a new role for the kinase module in the regulation of transcription elongation has been reported where the kinase module appears to coordinate with positive transcription elongation factor b (P-TEFb) by regulating its kinase activity. ChIP analysis and genome occupancy profiles of elongation factors in human cells indicate that CDK8 orchestrates key events in the formation of a functional elongation complex. CDK8 is required for RNAP II-dependent elongation by phosphorylating the C-terminal domain of RNAP II, as they demonstrate that RNAP II elongation is impaired upon knockdown of CDK8 [60]. This study also suggests that the kinase module may facilitate the interaction of P-TEFb with core Mediator to regulate RNAP II phosphorylation and transcription elongation. These studies represent a marked advance in our understanding of how the kinase module acts to both repress and promote gene expression while also revealing additional questions about the role the kinase module has as part of the core Mediator complex in regulating transcription.

Although the precise mechanism that regulates the reversible association of the kinase module with core Mediator is not clearly understood, a few studies offer some insight into why the regulatory association of the kinase module with core Mediator occurs. Initial clues were provided by studies in yeast suggesting that the reversible association between the kinase module and core Mediator existed as way of regulating the output of signaling-dependent transcription. Similarly, later studies in mammals suggest that specific cellular signals and signaling pathways regulate Mediator-kinase module association. For example, an in vivo study by Mo et al. in human HeLa cells showed that in response to Ras signaling, the repressed promoter of the C/EBP-β is kinase module bound, whereas upon activation, the kinase module is lost [17]. Another study by Pavri et al. demonstrated the switch from inactive to active Mediator executed by PARP-1 during retinoic acid (RA)-induced gene expression in vivo. ChIP analysis of promoter occupancy in PARP-1 present and absent cells shows that in the absence of PARP-1, Mediator did not attain its active conformation (accompanied by loss of kinase module) upon RA induction as evidenced by the retention of CDK8 after RA treatment [18].

Although these studies do not prove the exact mechanism for what triggers the kinase module to dissociate from the core, they do suggest that the kinase-containing Mediator complex requires an interaction with other factors to detach from the kinase module and adopt the conformation required to facilitate transcriptional activation. Indeed, Davis et al. demonstrated mechanistic evidence of kinase module dissociation involving SCF-FBW7 ubiquitin ligase-mediated proteasomal degradation of MED13/MED13L, which anchors the kinase module to core Mediator [16]. A recent study by Youn et al. also demonstrated that the Mediator complex in mouse liver undergoes dynamic physiologic regulation through nutrient signaling-dependent downregulation of the kinase module to core Mediator. This dissociation and degradation of the kinase module is induced by SCF-FBW7 E3 ligase [61].

Finally, the Mediator kinase module has been implicated as a gene regulator in physiological processes from development and differentiation to the maintenance of cell fate and function. Several genetic studies have revealed critical roles for kinase subunits in regulating signal-dependent gene expression during development [55,62,63]. In mice, kinase module subunits are found to be critical in early development, as evident by embryonic lethality resulting from mutations in kinase subunits. For example, embryo implantation failure is observed when CDK8 is inactivated through gene trap insertions, suggesting that CDK8 is necessary for preimplantation of mouse embryos [56]. CCNC knockout murine embryos failed to survive past implantation owing to severe developmental retardation and an underdeveloped placental layer [64]. MED12 mutant and knockout embryos failed to survive later embryonic stages as they suffered from acute defects in developmental processes including neural tube closure and heart formation [55].

Several studies show that these developmental disruptions stem from defects in key developmental signaling pathways, including Wnt, Notch, mTORC1, and TGF-β. The kinase module subunits are found to be involved in the expression of signaling pathway target genes and when mutated, lose their ability to activate or repress expression of required target genes for respective signaling pathways, leading to impaired responses of signaling pathways as illustrated by a growing number of developmental disorders. For example, loss of CDK8 in murine embryos is shown to disrupt Wnt target gene expression [62]. Mutant MED12 murine embryos showed aberrant Wnt/β-catenin target gene expression, indicating that MED12 is essential for Wnt signaling during embryogenesis where MED12 mutant embryos recapitulated phenotypes similar to those observed in the absence of β-catenin [55]. Recently, CDK8/CDK19 has been shown to have a role in negatively regulating Notch 1 signaling, a developmental pathway that regulates self-renewal and differentiation in several cell types including stem cells [64]. Kinase subunits have also been linked to TGF-β, a developmental signaling pathway that regulates cell proliferation, differentiation, cell fate, and apoptosis. CDK8-CCNC plays a critical role in regulation of SMADS in TGF-β-driven transcriptional responses by limiting the SMAD2/3-dependent induction of mesodermal cell fate in response to TGF-β signaling [65]. Recently, a study by Youn et al. demonstrated that when mice were fasted and refed, the kinase module dissociated and degraded upon nutrient activation of mTORC1 in mouse livers. This dissociation and degradation of the kinase module is necessary for the induction of lipogenic gene expression because genetic/pharmacological inhibition of mTORC1 in the fed state restores the kinase module suggesting that the kinase module plays a role in repressing lipogenic gene expression. In addition, genetically insulin-resistant and obese mice in the fasted state showed elevated levels of lipogenic gene expression and loss of the kinase module was reversed after mTORC1 inhibition [61]. In agreement with these studies, the kinase module has been involved as the terminal factor of cell signaling pathways owing to its representation as a final and functional target for transcription factors. Together, these studies imply a highly targeted role of individual Mediator subunits in the regulation of cell state and lineage commitment through the regulation of developmental signaling pathways.

Kinase activity

Finally, one of the most important and highly conserved functions of the kinase module is its kinase activity. The best characterized of these activities is the kinase module's phosphorylation of the C-terminal domain of RNAP II during transcription initiation, elongation, and RNA processing [3]. Human CDK8 appears to negatively regulate transcription by phosphorylating TFIIH [57], whereas inhibition of CDK8 kinase activity suppresses the RNAP II CTD phosphorylation thereby preventing elongation of transcription [66]. Despite the requirement for CDK8 and CCNC to interact and bring the kinase module together, the CDK8-CCNC interaction is not sufficient for CDK8 kinase activity. In the past 2 years, a series of studies by two different laboratories have provided evidence that MED12 is required for CDK8 kinase activity [67,68]. With a combination of Hi-C and cryo-EM studies along with knockdown studies in mESCs, MED12 knockdown cells showed acute depletion of Mediator and RNAP II, indicating that MED12 is required for proper phosphorylation of RNAP II. Another recent study by Klatt et al. described the binding location of MED12 and CDK8-CCNC dimer providing insight into activation of CDK8 by MED12. In vitro biochemical and in vivo studies together with cross-linking coupled with mass spectroscopy, demonstrated that the N-terminal of MED12 wraps around CDK8 at its T-loop to form an activation helix, which activates the enzymatic activity of CDK8 [68]. With MED12 now known to activate CDK8 as a kinase, the association of MED12 with the CDK8-CCNC dimer provides a critical element of regulation and prevents uncontrolled and inappropriate substrate targeting.

Overall, the genetic and biochemical analysis of the kinase module is consistent with the functional studies indicating a role for this unique module in both gene activation and repression. The kinase activity of CDK8 is required for activator-dependent transcription and has been shown to direct multiple steps in transcription including initiation, elongation, and re-initiation. Although several studies have begun to shed light on the signaling and mechanistic role for the kinase module and Mediator core, there is still much to be explored. Involvement of kinase subunits in many physiological processes and pathways proves the complexity and the functional implications of the kinase module subunits either individually or as a complete structure. The significant functional involvement of the Mediator kinase module in regulating aspects of transcription has a significant impact on human development as illustrated by an increasing number of diseases and developmental disorders that have been associated Mediator subunit mutations (Table 1).

Table 1.

Currently Known Roles for Mediator Kinase Subunits in Development or Disease

Subunit	Role
CCNC	Multiple deletions associated with T cell acute lymphoblastic leukemia; function as a tumor suppressor [64,73]
	Deletion associated with osteosarcoma; function as an inhibitor of cell growth [74]
	Regulation of adipogenesis [75]
	G₀ to G₁ transition in CD34⁺ cord blood cells [76]
CDK8	Phosphorylation of TFIIH to activate transcription [57]
	Phosphorylation of E2F to activate transcription [77]
	Phosphorylation of RNA Pol II C-terminal domain to repress transcription [78,79]
	Maintenance of ESC pluripotency mediated by MYC protein [80]
	Colorectal cancer oncogenesis [62,81,82]
	Melanoma oncogenesis [83]
	Breast tumorigenesis [84]
	Endometrial cancer tumor suppression [85]
	Alzheimer's disease [86]
CDK19	Upregulated in prostate cancer [87]
	Intellectual disability [88]
	Epileptic encephalopathy [87]
MED12	Designated a cancer driver gene [89,90]
	Uterine leiomyoma oncogenesis [71,91 –104]
	Breast fibroadenoma oncogenesis and phyllodes tumorigenesis [105 –110]
	Prostate cancer oncogenesis [111 –113]
	Interaction with NANOG and SOX2 C-terminals to regulate ESC pluripotency [114]
	Interaction with Wnt/β-catenin to recruit core Mediator to target genes [55]
	Interaction with PRC1 to repress differentiation genes during pluripotency [115]
	Super-enhancer-associated co-activator [33]
	Maintenance of HSC viability [33]
	Neural development in zebrafish [116 –118]
	Interaction with SOX10 in myelinating glia [47]
	Development of epithalamus in zebrafish [119]
	FG syndrome [120]
	Lujan syndrome [121]
	Ohdo syndrome [122]
MED12L	Designated a cancer driver gene [123]
MED12L	Interaction with SOX10 in myelinating glia [47]
MED13	Neurodevelopmental disease [124]
	Breast cancer oncogenesis [125]
	Regulation of early embryogenesis [52]
	Interaction with Smad7 to regulate myogenesis [126]
MED13L	Congenital heart defects [48,127]

Most cancers noted in this table were previously compiled and reviewed by Clark, Oldenbroek, & Boyer [72].

Kinase Module and Human Diseases

The Mediator kinase module is a critical component of the transcriptional machinery required for proper regulation of gene expression and lineage commitment of cells during development and tissue differentiation [1,6,8]. Kinase module subunits, as part of the Mediator complex, have been linked to many key developmental and oncogenic signaling pathways including Wnt, mTORC1, EGF, SHH, and Notch [10,12,55,69]. Alterations of individual subunits of the kinase module have been associated with developmental defects and diseases including Lujan syndrome, schizophrenia, breast and uterine cancers, and cardiovascular diseases [70]. Studies have found that the kinase module subunits are direct targets of genetic alteration in human tumors and specifically, MED12, the largest subunit of the kinase module, has been consistently implicated in many female cancers and cognitive developmental conditions. Research indicates that it is the role of MED12 in regulating the kinase activity of the module that leads to a number of these outcomes [71]. Specific roles for each of the kinase module subunits in development and disease is given in Table 1.

Kinase and Stem Cells

All cells in an adult human have the same 3 billion base pair genome, but it is how that genome is expressed that determines whether a cell becomes a myocyte, an osteocyte, a neuron, and so on. Stem cells are both able to clone themselves and able to become specialized cell types by pursuing one of a number of possible cellular lineages. Pluripotent stem cells, such as ESCs in the blastocyst stage of embryonic development, are ultimately committed to cells of either the endoderm, mesoderm, or ectoderm lineage [4]. Gene regulation is tightly controlled to ensure proper transcription for healthy organismal development. Aberrant gene expression is implicated in a multitude of developmental defects and disease and would be more rampant if not for the highly specialized, complex method for cell type-specific transcriptional control [128,129]. The unique properties of stem cells to both self-renew and differentiate under controlled laboratory conditions allows for the effects and functional characterization of deliberate perturbations in the regulation of gene expression [4]. This includes altering the function and expression of the GTFs that assemble the transcription PIC on gene promoters, activators, and repressors that bind to gene regulatory elements located upstream or downstream of promoters, and the essential co-activator of cell type-specific genes like the Mediator complex. The fate of any stem cell is ultimately determined by regulating the transcription of specific genes, a feature largely facilitated by the Mediator complex.

Stem cells are categorized based on their differentiation potential. Totipotent stem cells have the potential to become any cell type in the body including extra-embryonic tissue, an example of which is the zygote [4,130]. Pluripotent stem cells, such as ESCs in the blastocyst stage of embryonic development, are committed to cells of either the endoderm, mesoderm, or the ectoderm lineage [131]. Adult stem cells are a multipotent cell type that can be found in umbilical cord blood and tissue, bone marrow, adipose tissue, and several other fully developed organs [132,133]. These cells have a more restricted developmental pathway and, in the body, will only become cells of that tissue or organ. Of course, the potential for stem cells to differentiate allows them to change their transcription program in response to the environment. In this way, a stem cell is a progenitor possessing a certain identity, and by changing its transcription program, it can assume a new identity as a differentiated somatic cell. Despite different levels of differentiation potential possessed by different classes of stem cells, all stem cells share the ability to self-renew [4]. We will begin this section by exploring stem cell self-renewal and the role of the Mediator complex kinase module in this process, followed by the kinase module regulation of differentiation down the various possible lineages.

Kinase module and self-renewal

Self-renewal is the process by which stem cells replicate themselves to maintain a stable population of undifferentiated clones. For individual stem cells, self-renewal and differentiation are mutually exclusive in that lineage commitment down a differentiation pathway alters a stem cell's gene expression profile away from self-renewal permanently. If a stem cell is not self-renewing, it is differentiating, and vice versa [134]—until the stem cell reaches quiescence, temporarily halting further self-renewal [135].

Work by Miyata et al. revealed that, when exposed to cytokines, human cord blood CD34⁺ cells in the quiescent G₀ phase of the cell cycle expressed CCNC at a higher level compared with cells in the more growth-oriented G₁ phase. This led them to perform an shRNA-mediated knockdown of CCNC in the cord blood cells, which resulted in an increase in G₀ cells in their culture population as determined by Hoechst and Pyronin Y staining. It must be noted that the knockdown of CCNC did not increase differentiation. Of interest, in the absence of CCNC, cord blood cells were able to maintain expression of CD34 at higher levels than control cells through up to 4 weeks of cell culture, possibly because of the induced quiescence keeping the cells in a protracted immature state [76].

Like CCNC, CDK8 has also been shown to play a role in stem cell self-renewal. Murine ESCs showed a significant reduction in both transcript and protein expression of CDK8 postdifferentiation. After this, an shRNA knockdown of CDK8 was performed in murine ESCs that resulted in a loss of ESC pluripotency. This was determined by the reduced expression of NANOG and OCT4, reduced alkaline phosphatase staining, and reduced colony formation in these cells. Further study revealed that CDK8 regulation of pluripotency was at least partially mediated by MYC protein [80].

Co-IPs were used to determine the interaction between MED12 and the C-terminal domains of NANOG and SOX2 in ESCs. Furthermore, an siRNA-mediated knockdown of MED12 was performed in murine ESCs. NANOG transcript and protein levels fell by as much as 65% in the absence of MED12 in addition to the onset of spontaneous differentiation. This loss of pluripotency during MED12 knockdown could not be rescued by a NANOG-expressing doxycycline system, indicating the requirement for MED12 for proper differentiation. In ESCs undergoing differentiation, although the expression of NANOG, OCT4, and SOX2 decreased, MED12 expression increased 3.5-fold. NANOG and MED12 co-occupy NANOG target genes during pluripotency, but this co-occupancy ends during differentiation [114]. This work was later challenged by data collected in a recombinant murine ES cell line expressing a hypomorphic MED12 mRNA at low levels. Despite this, the absence of MED12 was embryonic lethal in mice, and this was attributed to MED12 interaction with Wnt/β-catenin signaling pathway for recruiting core Mediator to target genes [55].

ChIP-seq analysis conducted in murine ESCs revealed a small degree of overlapping genome occupancy between CDK8, MED12, and the polycomb repressor complex 1 (PRC1) subunit RING1B. PRC proteins are known regulators of ESC state. Of the identified MED12 targets, 21% were co-occupied by RING1B. Furthermore, 80% of these regions occupied by MED12 and RING1B were also occupied by MED1, indicating that the entire core Mediator was present at these sites. Gene ontology identified these sites as genes necessary for differentiation and development. An shRNA-mediated knockdown of RING1B in murine ESCs saw a marked decrease in MED12 chromatin interaction. The inverse was not true in a MED12 knockdown condition where MED12 recruitment to core Mediator was reduced in the absence of RING1B, and Co-IP confirmed that MED12 and RING1B interact directly. Quantitative PCR found that, in pluripotent murine ESCs, almost 600 genes were more highly expressed in the absence of either MED12 or RING1B, showing a role for MED12 and RING1B in development-associated gene repression during pluripotency; however, further research indicated that MED12—not RING1B—was responsible for activating development genes during murine ESC differentiation [115].

As discussed previously, MED12 is critical for maintaining HSC viability. After performing MED12 knockouts in mice using a Cre recombinase system, a 75% decrease in cell numbers was observed in the bone marrow and thymus of the MED12 knockout mice compared with a control. A CFU assay revealed that HSCs lacking MED12 were unable to form colonies, highlighting a failure in HSC self-renewal. Of interest, the researchers knocked out MED12 in an immortalized mouse embryonic fibroblast cell line and observed no significant changes in cell growth compared with a control. A similar experiment in murine ESCs were able to maintain pluripotency markers and self-renewal capability despite the absence of MED12 [33]. These findings suggest that, although different stem cells have similar properties such as self-renewal and differentiation, potency and lineage commitment of different stem cell classes is governed by highly specific regulators.

Finally, zygotes and other totipotent stem cells also offer an opportunity to broaden our understanding of self-renewal. Microarray analysis revealed that, of all Mediator subunits, MED13 was the most highly translated Mediator subunit during oocyte maturation with MED13L and MED12L ranking at second and third, respectively. This high level of MED13 translation continues into the two-cell (2C) stage of embryo development. Morpholino oligonucleotides were used to block MED13 translation at the single-cell zygote stage 4 h after fertilization, and whereas the zygotes underwent cleavage into the 2C stage, only 40% of tested embryos were able to advance to the 4C stage. Those embryos that reached 4C halted thereafter. Performing the same experiment 6 h after fertilization allowed 78% to advance to the morula stage. After ruling out transcription and DNA replication failures as reasons for the MED13 knockdown effects, RNA-seq analysis revealed 1201 upregulated and 2203 downregulated genes in the absence of MED13. RNA processing, cell cycle, transcription, protein catabolism, and chromatin modification were among the categories of the downregulated genes discovered during gene ontology (GO) analysis [52]. Given that these sets of genes are important for determining cell state, these results point toward the Mediator kinase module as an integral cell state regulator.

Kinase module and differentiation

Differentiation is the process by which a stem cell alters its transcription program in response to the external environment and internal regulation to commit to one of its many potential lineages. In normal, healthy stem cells, differentiation is a permanent epigenetic transformation that, once started, causes a stem cell to lose its potency and self-renewal capacity while gaining specialized forms and functions important for the operation of tissues and organs. Here we review some of the lineage commitments where the kinase module has been shown to play a significant role to further support the critical role of Mediator in directing cell fate and need for continued research in this area.

Adipogenesis

Work by Song et al. involved screening adipocytes with shRNA libraries to identify genes whose expression was altered during adipogenesis. CCNC and CDK19 were found to be downregulated, whereas CDK8 was upregulated during adipogenesis. CCNC and CDK19 expression in brown adipose tissue of 2-year-old mice was down to 25% of the expression levels present in 3-month-old mice. Conversely, CDK8 expression was twice as high in the brown adipose tissue of older mice compared with the younger mice. Because brown adipose tissue is responsible for producing heat, groups of mice were exposed to either 4°C or 22°C conditions. The transcription levels of ccnc, cdk8, and cdk19 mRNA were determined to be unaffected by these differences in temperature; however, CCNC and CDK19 protein expression fell slightly, whereas CDK8 rose in the 4°C group compared with 22°C. After performing siRNA knockdown of ccnc, the expression of adipogenesis markers including PPARG, FABP4, and CEBPA fell during the first 2 days of adipogenesis, but expression of these genes increased after 5 days to the levels in the control group. After switching to an inducible knockout system for CCNC, cells undergoing CCNC knockout did not undergo adipogenesis and did not express the previously tested adipogenic markers and a host of other genes associated with brown adipose tissue, mitochondria, and lipogenesis. Retroviruses expressing CCNC only partly rescued adipogenesis in CCNC knockout cells but did not improve adipogenesis in cells unaffected by CCNC knockout. The most downregulated pathway in the absence of CCNC was the PPAR pathway, a master regulator of adipogenesis. PPARG-2 overexpression rescued both lipid vesicle formation and adipogenic marker expression in CCNC knockout cells, showing that PPARG's activity does not depend on CCNC. C/EBP-α, a coregulator of adipogenesis along with PPARG, could not rescue adipogenesis upon overexpression in CCNC knockout cells. Owing to C/EBPα being an important regulator of white adipose tissue, siRNA knockdown of CCNC was also performed in 3T3-L1 cells, revealing a similar decrease in lipid accumulation and adipogenic marker expression as was seen in brown adipose tissue [75]. In summary, CCNC regulates adipogenesis by interacting with C/EBPα.

Myogenesis

Like adipogenesis, the process of differentiating into skeletal muscle tissue involves interactions between kinase module subunits and transcription factors to achieve proper regulation. MED12 and β-catenin are known to interact [136], and β-catenin is active during myogenesis [137 –139]. Further research found that not only does Smad7 interact with β-catenin to regulate myogenesis, but Smad7 also interacts directly with MED13. The significance of this finding is that the Smad7:β-catenin complex could be responsible for recruiting core Mediator to myogenesis-specific promoters by kinase module association through MED12 and MED13 [126].

Osteogenesis

Bone tissue is maintained by the actions of osteoblasts and osteoclasts. Osteoblasts perform mineralization that increases bone density, whereas osteoclasts resorb bone matrix that decreases bone density. Initial research revealed that the inhibition of CDK8/19 could interfere with the self-renewal capacity of bone progenitor stem cells by virtue of the Wnt signaling pathway. Although bone formation was disrupted by CDK8/19 inhibition, it was through a mechanism independent of Wnt [140]. Later work tested the effects of CDK8/19 inhibition on murine bone marrow macrophages, osteoblasts, and osteoclasts. Although inhibition did not affect macrophage and osteoblast self-renewal, it did reduce the osteoclast differentiation of the macrophages and lead to significantly decreased bone matrix resorption. Both effects were reversed by withdrawing the CDK8/19 inhibitors. Expression of osteoclastogenic genes were downregulated in macrophages exposed to CDK8/19 inhibition. Meanwhile, CDK8/19 inhibition increased ALP activity in murine osteoblasts and boosted calcium deposition [141]. This research demonstrates the role of CDK8 and 19 in maintaining bone tissue by playing on the side of osteoblast and osteoclast activity simultaneously. It is possible that all the kinase module subunits are responsible for acting as both drivers and repressors to a certain degree, especially given their relationships to oncogenesis.

Neurogenesis

Owing to the difficulty of studying mammalian neurogenesis directly, murine or human stem cells and tissue, along with zebrafish models represent the primary means of exploring this area of development and differentiation [119]. Research in this area began by identifying mutations of MED12 (at that time referred to as TRAP230, or thyroid hormone receptor-associated protein) that produced truncated MED12 protein, resulting in zebrafish brain tissue that developed all the correct regions but failed to expand anteriorly and posteriorly while also failing to form the forebrain and midbrain ventricles [116]. Another mutation of MED12 was found to disrupt neural tissue development in zebrafish, and the mutant embryos were rescued by the introduction of wild-type med12 mRNA. In situ hybridization was used to determine that MED12 is most active in the part of the zebrafish brain forming the ventricle lining. Overexpression of MED12 lead to premature neuronal development and increased differentiation of monoaminergic neurons but did not increase neuron differentiation overall [117]. MED12 was later found to interact with the intracellular domain of amyloid precursor protein (AICD), a protein essential for brain development and function [118]. MED12 coactivates tbx2b transcription that guides neural progenitor cells toward correct epithalamic differentiation at a critical moment in brain development in zebrafish [119]. Beyond neurons, MED12 and MED12L were also discovered to interact with SOX10 to direct the development of Schwann cells and oligodendrocytes, two types of glial cells that are responsible for the formation of myelin sheaths that insulate neuron axons and accelerate action potentials [47].

Conclusion

Nearly 30 years ago, a question about in vitro gene activation led to the discovery of the Mediator complex. At once, this expanded our understanding of how complex life can be achieved with relatively few genes. Since then, each answered question has bred a multitude of further questions. Because of that, we now know how near (or far) gene regulatory elements are from gene promotors, how Mediator reaches across vast linear distances through the three-dimensional folding of chromatin to reach those elements, and that super-enhancers work with Mediator to regulate cell type-specific gene transcription. The kinase module has expanded the complexity of this regulatory relationship given its transient nature, and we are certain of the kinase module's subunit and paralog composition with CDK8/19 possessing kinase activity. It is understood that each kinase module subunit plays an integral role in the module's overall stability, thereby enhancing its regulatory and kinase activity. Unfortunately, mutations in these subunits have been implicated in a host of developmental disorders and diseases, including many cancers, wherein lies the importance of proper kinase module functioning to maintain healthy development and cell fate determination. Research is now branching into the kinase module's activity in the context of stem cell self-renewal and differentiation.

Despite what we have learnt, we still do not have a complete understanding of how, precisely, the kinase module interacts with nuclear machinery and signaling molecules to drive cell state into a particular direction: either to maintain homeostasis or to differentiate down the epigenetic landscape toward any particular lineage while avoiding oncogenesis. Although we know several targets of kinase module interaction in this context, we are far from knowing them all. A large focus has been placed on MED12 (warranted considering its importance in oncogenesis and development), and this has elevated the profile of the individual subunit and that of the entire kinase module. Although this is bearing good fruit in terms of thorough research (ChIP-seq and genome-interactions), more abundant fruit would be reaped by similarly thorough elucidation of the other subunits and their interactions with each other. It must be stressed that there is much about the paralogs (MED12L, MED13L, CDK19) that we have yet to explore. What regulatory advantages do the paralogs offer the kinase module? How much functional overlap is there between the paralogs, and what is the relative expression between them? Does their expression change depending on cell state, and how would their relative expression levels affect cell state? And then, there is the added dimension of cell signaling factors that may or may not influence Mediator and its kinase module, or vice versa.

The great web of interactions between Mediator, the kinase module, the various transcription factors, and the multitude of signaling molecules is falling into place piece-by-piece, but work is far from finished. The benefits of this knowledge extend beyond molecular biology, stem cell biology, and biochemistry and into the realm of clinical application. Given a more complete map of cell-state regulation, we may one day see the efforts of this research pay off in the form of regenerative medicine involving stem cell therapies that are tailorable to individual patients, therefore maximizing patient benefit and minimizing patient harm. We may discover ways to manipulate stem cells that we previously could have only imagined, and then we would have uncovered the elusive fountain of youth. These possibilities are beyond our grasp now, but no great height was ever reached without a solid foundation, and it is this foundation that current research must continue to build.

Ultimately, the study of Mediator and its kinase module will necessitate increased collaboration between the fields of stem cell biology, cell signaling, and transcriptomics. That level of research is a difficult undertaking given the shear complexity of gene expression regulation. One of the joys of biology is that despite how complex it all may seem, there is a hidden elegance to all the intertwined systems that make cells and organisms function, and function well. Unfortunately, the nature of foundational research tends to render an individual researcher unable to see the forest for the trees, but as scientists, we have the pleasure of knowing that the forest is there, waiting to be fully explored.

Footnotes

Acknowledgments

The authors thank Tom Futrell, Associate Professor of Graphic Design at Louisiana Tech University, for creating the figures in this review. The authors also thank Foram Patel, Onyekachi Idigo, Caroline Rinderle, and John Bradley Cart at Louisiana Tech University for their proofreading and constructive feedback.

Author Disclosure Statement

The authors do not have any conflicts of interest to disclose.

Funding Information

No funding was received for this article.

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