YAP controls transcriptional elongation through CKD9 recruitment for proximal pause release: “Hippo-thetical”, new therapeutic targets?

ABSTRACT

The Hippo-YAP signaling pathway has been established as a crucial regulator of tissue growth and tumorigenesis but the mechanism for transcriptional control remains unclear. In their study, Galli et al. show that YAP/TAZ binding is restricted to a small number of gene enhancers and that YAP/TAZ regulates transcriptional elongation by recruiting the Mediator complex.

KEYWORDS:

• Hippo/YAP pathway
• mediator complex
• tumorigenesis
• transcriptional elongation
• YAP-bound regulatory elements

Since the Hippo signaling pathway was initially discovered by Drosophila mosaic genetic screens in 2002, it has steadily gained recognition as an important step in tissue growth, stem cell activity and tumorigenesis.^1,2 As many cancers are marked by unchecked cell division, this signaling pathway has become an attractive target for cancer therapeutics. The way the Hippo/YAP pathway controls transcription is not yet clear, so additional data is required in order to fill the mechanistic gaps that currently exist.

In October 2015, Galli et al.³ reported in Molecular Cell the results of their study where, by using multiple genomic techniques, they attempted to answer the following questions: where does YAP, TAZ and TEA Domain transcription factors (TEADs) bind in the genome of cancer cells? And, which complexes are recruited to drive gene transcription? This study provided key points regarding YAP/TAZ-driven transcription that are discussed below.

In 2008, Zhao et al.,⁴ demonstrated that the YAP/TAZ activated complex is unable to bind to DNA by itself and requires binding with transcriptional factors like TEADs in order to translocate to the nucleus and allow expression of target genes. In this study, Galli et al. used ChIP-seq to check genome-wide occupancy for YAP, TAZ and members of the TEAD family (TEAD1, TEAD4) on 2 human cholangiocarcinoma line cells. They found that robust YAP/TAZ occupancy was associated with only a very small subset (about 7%) of the TEAD binding sites. In 2010 Lian et al.⁷ had used CHIP-seq on embryonic cells to explore the role of YAP transcription co-activator in regulating stem cell self-renewal and differentiation. Their study showed that YAP transcriptional functions were associated with binding of TEADs around the promoter of target genes. In the study by Galli et al., YAP was found to bind predominantly 20Kb away from the closest transcriptional start site (TSS), suggesting that the YAP/TAZ binding is restricted to distal regulator regions that have enhancer features.

The investigators looked into possible distinct differences between YAP-bound (YAP+) enhancer regions versus YAP- enhancers. They found that YAP+ enhancer regions had higher density of H3K27ac and H3K4me1 posttranscriptional modifications compared to the average signal at active YAP- enhancers. Higher density of such posttranscriptional modifications is typically associated with more robust enhancer activity. By assigning enhancers to their target genes and subsequently performing RNAseq, the authors were able to determine gene expression levels. Their results showed that genes associated with YAP+ enhancers were highly expressed compared to genes linked to active YAP- enhancers. Thus, their data indicated that these YAP+ enhancers are a defined subset of highly active enhancers that drive potent expression of their target genes, and were referred to as YAP-bound regulatory elements (YREs). These YREs demonstrated a higher transcription rate of target genes that appeared to be very similar to the activity of “super-enhancers” that have been previously described in other studies.⁵ For that reason, the investigators compiled a list of super-enhancers in the cholangiocarcinoma cells based on the presence of H3K27ac modification on the genome. They found that 25% of the YREs were listed as super-enhancers representing approximately 17% of all super-enhancers in the cell. In addition, they showed that YAP/TAZ plays a critical role in sustaining the high transcriptional output driven by the YREs. When silencing YAP/TAZ through YAP/TAZ knockdown (siYT) in cholangiocarcinoma cells, they noticed reductions of the H3K27ac and H3K4me1 presence at YREs. These reductions were directly correlated to loss of expression of the target genes while the gene expression levels of YAP- enhancers remained unchanged after siYT.

The results described above generated additional questions regarding the mechanism by which YAP/TAZ could regulate transcription from YREs. The investigators performed circular chromosome confirmation capture (4C-seq) using 21 different YREs as anchors in cholangiocarcinoma cells. Their results showed that YREs are sites of long-range chromatin interactions. When assigned to their target genes, YREs interacted with their respective TSS and other enhancer regions. Because there was minor or no difference in the chromatin interaction frequency upon siYT treatment, this led the investigators to conclude that YAP/TAZ is not required for normal chromatin looping. The authors next looked into a potential role of YAP/TAZ involving RNA polymerase II (Pol II) recruitment, pausing and/or transcription elongation. In 2012, Adelman and Lis,⁸ showed that proximal Pol II pausing is an important mechanism to regulate RNA transcription elongation in Drosophila S2 cells. The role of paused Pol II is to compete with nucleosomes for occupancy of highly regulated promoters in order to prevent the formation of repressive chromatin and therefore facilitate further or future gene activation. Recruitment of the P-TEFb complex is essential for catalyzing the Pol II Ser 2 phosphorylation and thus providing the elongating, productive Pol II.^8,9 Galli et al. noted that after siYT the YREs- associated genes had a dramatic decrease of Pol II presence at their gene bodies. After genomic data calculations, it was shown that there was a significant increase of the Pol II pausing index (PI), implying impaired transcription elongation. These results further supported the key role of YAP in elongation. The siYT cells were found to have a significant decrease in Ser 2-Pol II density at the YREs gene bodies, while no density changes were observed in the YAP- target genes. This suggested that when YAP/TAZ binds to YREs it results in high transcriptional rates of its target genes by promoting proximal pause release.

The interaction between YAP/TAZ and the Mediator complex (a protein complex that controls various aspects of transcriptional activation) on the chromatin has been established in previous studies and seems to be independent of TEAD binding.¹⁰ In order to further establish this functional interaction, the investigators performed ChIP-seq for the MED1 (Mediator complex subunit) and found that the co-occupancy with YAP across the genome exceeded 87%. After siYT, there was a dramatic decrease of MED1 signal around YREs, and the degree of decrease was directly correlated to the YAP binding density at the same sites while the occupancy of Cohesin (binding partner for the Mediator complex) remained unaffected. These results reinforced the impression that YAP is required for Mediator occupancy on the genome in a more selective way. It is important to mention that this functional interaction works both ways. In animal models, silencing of the Mediator subunits resulted in growth inhibition as well as weaker activation of the YREs associated genes.³

Previous studies have demonstrated that the Mediator complex is regulating transcription elongation through P-TEFb recruitment.^11,12 In this study, Galli et al. hypothesized that YAP/TAZ is promoting proximal pause release by recruiting P-TEFb through the Mediator complex. Their hypothesis was based on research data showing that there is higher CKD9 occupancy around the promoters of YREs-associated genes, and that YAP-bound genes display preferential CKD9 loss upon siYT. In order to explore the functional role of CKD9 downstream from YAP, 2 CKD inhibitors with CKD9 selectivity, Flavopiridol and NVP-2 were used. These inhibitors were used together in order to rescue, in a dose dependent manner, the activation of YAP +genes driven by silenced Mediator subunits (Dox-inducible YAPS127A transgenes). Transcriptional profiling in cells 3 hours post-treatment with the above mentioned compounds showed that the YERs-associated genes were very sensitive to CKD9 inhibition. The mechanism described above was corroborated in an animal model where the authors administered Flavopiridol (FP) to mice. One week of Flavopiridol administration resulted in full rescue of YAPS127A- driven increase in liver size and target gene expression.

During the past decade, the Hippo/YAP signaling pathway has emerged as a crucial regulator of tumorigenesis, and high YAP expression has been associated with several human cancers (liver, lung, breast, kidney, colon).⁶ The exact mechanism of how the Hippo-YAP pathway controls gene expression had not been elucidated but there was evidence indicating the Hippo/YAP signaling pathway functionally interacts with multiple other cellular pathways (e.g. Hedgehog, Wnt, or Notch) and serves as a central node in the regulation of cell division, especially in cancer cells. Galli et al. meticulously investigated certain aspects of the downstream signaling of the Hippo pathway and demonstrated the mechanisms that YAP utilizes to exert its oncogenic functions, while clarifying misconceptions of the past. For example, previous studies have shown widespread YAP-TEAD co-binding across the genome⁴ that takes place over the promoter of the target genes.⁶ In this study, the authors showed that in human cells there is a limited number of YAP-binding sites that represent only a fraction of the TEAD binding regions, and that YAP binding takes place predominantly 20kB away from the promoter regions. This means that YAP/TAZ binding is restricted to a small number of the most potent enhancers on the genome. In previous studies, the role of YAP/TAZ in transcriptional regulation beyond TEAD co-binding has been extensively studied.⁸ There is a number of developmental transcription factors (e.g., SMAD proteins, AP-1 transcription factor, RUNX2) that were identified as binding partners of YAP and TAZ and the list most likely expand in the future. Galli et al. demonstrated that YAP regulates transcriptional elongation through a Pol-II pause release with the help of the Mediator complex. The fact that this study showed an interaction between YAP and the most important core promoter for gene expression in eukaryotes¹⁴ is significant and unprecedented.

In 2015, Ehmer and Sage¹³ published a review of data regarding the important role Hippo signaling pathway in the control of proliferation, including unrestricted proliferation found in cancer. They suggested that targeting the Hippo signaling pathway to control proliferation is a promising therapeutic approach in cancers that show increased activity of YAP/TAZ. Furthermore, the oncogenic and tumor-suppressive attributes of YAP/TAZ were reviewed, emphasizing the relevance of the Hippo pathway in cancer. Previous studies had focused on a wide range of upstream regulators of the Hippo pathway with more emphasis on the GPCR family of cell surface receptors.¹⁵ While pharmacologically targeting this large family of signal transduction receptors seemed promising, these highly “druggable” targets, have broad physiological functions and there are concerns that interfering with their signaling may cause adverse side effects. Recently, studies have focused on finding downstream specific inhibitors of YAP/TAZ-TEAD function.¹⁶ A screening of >3300 drugs led to the identification of the Porphyrin family, including verteporfin (VP), hematoporphyrin (HP), and protoporphyrin IX (PPIX), as YAP inhibitors.^15,16 Porphyrin abrogates the interaction between YAP and TEAD. Most importantly, VP abolishes liver overgrowth induced by YAP overexpression or by inactivation of Nf2 in vivo, which clearly demonstrated the therapeutic potential of disrupting YAP/TAZ-TEAD interaction.^15,16 In this article, Galli et al.³ bring forward another possible therapeutic target that had not been previously identified. Their results show YAP/TAZ binding is predominantly restricted to enhancers associated with only a few hundred genes, and thus its function is mainly to control transcriptional elongation. These results make development of elongation inhibitors an appealing therapeutic target for YAP-driven tumors.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

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