Nuclear receptor SHP dampens transcription function and abrogates mitotic chromatin association of PXR and ERα via intermolecular interactions

Highlights

• Nuclear receptor SHP, though devoid of DBD, regulates target gene expression.

• Cell-based studies and in silico analyses reveal mechanism of gene regulation by SHP.

• SHP impedes gene bookmarking by directly interacting with other nuclear receptors.

• SHP, like RXR, regulates gene transcription function of selective nuclear receptors.

Abstract

Mitosis is a cellular process that produces two identical progenies. Genome-wide transcription is believed to be silenced during mitosis. However, some transcription factors have been reported to associate with the mitotic chromatin to uphold a role in ‘gene-bookmarking’. Here, we investigated the dynamic role of nuclear receptor SHP during cell cycle, and observed intermolecular interactions with PXR and ERα. This was reflected in altered subcellular localization, transcription function and mitotic chromatin behavior of these receptors. Subsequently, by in silico and live cell imaging approaches we identified the minimal domain(s) and crucial amino-acid residues required for such receptor-receptor interactions. It was apparent that both PXR/ERα interact with SHP to translocate cytoplasmic RFP-tagged SHP into the nucleus. In addition, during mitosis SHP interacted with some of the key nuclear receptors, altering partners, as well as, its own relationship with mitotic chromatin. SHP displaced a major fraction of PXR and ERα from the mitotic chromatin while promoted its own weak association reflected in its binding. Since SHP lacks DBD this association is attributed to receptor-receptor interactions rather than SHP-DNA interactions. The abrogation of PXR and ERα from the mitotic chromatin by SHP implies potential implications in regulation of gene bookmarking events in cellular development. Overall, it is concluded that intermolecular interactions between SHP and partner PXR/ERα result in attenuation of target promoter activities. It is proposed that SHP may act as an indirect physiological regulator and functions in a hog-tie manner by displacing the interacting transcription factor from gene regulatory sites.

Introduction

Maintenance of cell identity, proliferation and growth properties during clonal expansion must be maintained during genome replication and cell division [1,2]. Every cell accurately replicates its genome and transmits the information to its progeny [3]. It is a well-coordinated sequence of events for distribution of molecular belongings, ultimately poised for substantial dynamic changes in nuclear organization and differential gene expression of the cell [4]. The mitotic phase of the cell cycle is an indispensable event in the life of a cell, and it has attracted immense attention in recent years. During mitosis, the nuclear envelope is temporarily disassembled, which leads to massive release and mixing of soluble nuclear constituents within the cytoplasm [5]. At the same time, interphase chromatin begins to condense and progress towards the beginning of prophase [6]. It further leads to transient silencing of gene transcription, and dissociation of majority of transcription factors from chromatin [[7], [8], [9], [10]].

Recent observations suggest that during mitosis, some transcriptional regulators associate with mitotic chromatin and remain bound to their target gene promoters during the entire process [[11], [12], [13]]. A similar phenomenon of mitotic binding has also been reported for some nuclear receptors [2,8,[13], [14], [15]], coactivators [3], chromatin modifiers [16] and sequence-specific transcription factors such as RUNX2 [3], GATA1 [16], HNF1 [17] and FOXA1 [18]. This phenomenon appears to be appropriately referred to as ‘genomic or gene-bookmarking’. It has been proposed that mitotic gene-bookmarking may be coupled with the early-onset expression of essential regulatory genes, that help in the maintenance of specific cell proteome, cell identity and survival [8,19]. Recently, it has been postulated that functional impairment of specific bookmarking by transcription factors can result in delayed or impaired re-expression of target genes after a cell exits mitosis [20,21].

The initial finding that a nuclear receptor PXR remains associated with mitotic chromatin was novel and unexpected, and also a persuasive directive for further advances [13]. In that direction, our laboratory investigated the association of androgen receptor (AR) and estrogen receptor-α (ERα) with the mitotic chromatin [8,22]. However, unlike the PXR, mitotic chromatin association of AR and ERα was agonist-mediated. In this direction, a detailed study on AR conferred that only natural ligands and agonist provoke such an association. On the contrary, pure antagonist binds and translocates AR from the cytoplasm into the nucleus but prevents receptor binding to the mitotic chromatin [23]. The functional significance and molecular basis of such an association remain to be fully comprehended. However, as of current understand ding it has been contemplated that such an association may have a role in gene-bookmarking. Such a biological phenomenon is also believed to be similar to epigenetic mechanisms [24] involved in transmitting of interphase pattern of gene expression from progenitor to progeny cells via mitosis [7,13,19,21,25].

The nuclear receptor PXR acts as a xenobiotic-regulated transcription factor and forms a heterodimeric complex with retinoid X receptor (RXR) [26]. Investigations suggest that PXR constitutively associates with mitotic chromatin in a ligand-independent manner [13]. It is further reported that a specific region encompassing the NLS (Nuclear Localizing Signal) residing primarily in DBD (DNA binding domain) region of PXR is essential for mitotic chromatin binding [2,8]. Whereas, ERα, which functions as a homodimer associate with mitotic chromatin similar to AR in a ligand-dependent manner [8,27,28].

In view of the recent finding that PXR-DBD is central to the gene-bookmarking through interaction with chromatin [2], we decided to validate these findings further with atypical nuclear receptor SHP. Though SHP is devoid of DBD, it has been reported to interact with a few nuclear receptors and modulate their transcription function. This makes this receptor distinct from other conventional NRs. The functional cross-talk of SHP with other interacting partners is also compelling and may have importance in normal physiological controls and in the state of dysregulation. This warrants for extensive investigation not only on the unique regulatory mechanisms of SHP but also its distinctive relationship with other NRs and transcription factors. This may initiate ways to understand the SHP-mediated disease mechanisms (diabetes, cancer etc.) as well as its potential in therapeutic interventions [29]. From the simplistic point, it is apparent that SHP is an emerging atypical nuclear receptor and awaits an extensive investigation to assess its subcellular functional dynamics and molecular basis of transcriptional influence on its heterodimeric interacting partners. Also, a detailed study is warranted to unravel the emerging regulatory implications of gene-bookmarking events during mitosis which may affect the onset and progression of associated disorders.

Using live-cell fluorescence microscopy, we document that unlike some known nuclear receptors PXR, AR and ERα [15,30], SHP does not associate with mitotic chromatin by similar mechanisms. However, it plays a key role in influencing the mitotic chromatin association of its interacting partners and appears to be mutually responsive in influencing the gene-bookmarking events. Finally, we report that SHP interacts with some of the key nuclear receptors (like PXR and ERα) implying its modulatory role in execution of their cell regulatory functions. Altogether, we propose that SHP acts in a hog-tie manner to serve as an indirect physiological regulator.