Muktar A. Mahajan

A new family of nuclear receptor coregulators that integrate nuclear receptor signaling through CREB-binding protein.

ABSTRACT We describe the cloning and characterization of a new family of nuclear receptor coregulators (NRCs) which modulate the function of nuclear hormone receptors in a ligand-dependent manner. NRCs are expressed as alternatively spliced isoforms which may exhibit different intrinsic activities and receptor specificities. The NRCs are organized into several modular structures and contain a single functional LXXLL motif which associates with members of the steroid hormone and thyroid hormone/retinoid receptor subfamilies with high affinity. Human NRC (hNRC) harbors a potent N-terminal activation domain (AD1), which is as active as the herpesvirus VP16 activation domain, and a second activation domain (AD2) which overlaps with the receptor-interacting LXXLL region. The C-terminal region of hNRC appears to function as an inhibitory domain which influences the overall transcriptional activity of the protein. Our results suggest that NRC binds to liganded receptors as a dimer and this association leads to a structural change in NRC resulting in activation. hNRC binds CREB-binding protein (CBP) with high affinity in vivo, suggesting that hNRC may be an important functional component of a CBP complex involved in mediating the transcriptional effects of nuclear hormone receptors.

Microcephaly Gene Links Trithorax and REST/NRSF to Control Neural Stem Cell Proliferation and Differentiation

Microcephaly is a neurodevelopmental disorder causing significantly reduced cerebral cortex size. Many known microcephaly gene products localize to centrosomes, regulating cell fate and proliferation. Here, we identify and characterize a nuclear zinc finger protein, ZNF335/NIF-1, as a causative gene for severe microcephaly, small somatic size, and neonatal death. Znf335 null mice are embryonically lethal, and conditional knockout leads to severely reduced cortical size. RNA-interference and postmortem human studies show that ZNF335 is essential for neural progenitor self-renewal, neurogenesis, and neuronal differentiation. ZNF335 is a component of a vertebrate-specific, trithorax H3K4-methylation complex, directly regulating REST/NRSF, a master regulator of neural gene expression and cell fate, as well as other essential neural-specific genes. Our results reveal ZNF335 as an essential link between H3K4 complexes and REST/NRSF and provide the first direct genetic evidence that this pathway regulates human neurogenesis and neuronal differentiation.

Identification and Characterization of a Novel Nuclear Protein Complex Involved in Nuclear Hormone Receptor-mediated Gene Regulation

NRC/NCoA6 plays an important role in mediating the effects of ligand-bound nuclear hormone receptors as well as other transcription factors. NRC interacting factor 1 (NIF-1) was cloned as a novel factor that interacts in vivo with NRC. Although NIF-1 does not directly interact with nuclear hormone receptors, it enhances activation by nuclear hormone receptors presumably through its interaction with NRC. To further understand the cellular and biological function of NIF-1, we identified NIF-1-associated proteins by in-solution proteolysis followed by mass spectrometry. The identified components revealed factors involved in histone methylation and cell cycle control and include Ash2L, RbBP5, WDR5, HCF-1, DBC-1, and EMSY. Although the NIF-1 complex contains Ash2L, RbBP5, and WDR5, suggesting that the complex might methylate histone H3-Lys-4, we found that the complex contains a H3 methyltransferase activity that modifies a residue other than H3-Lys-4. The identified components form at least two distinctly sized NIF-1 complexes. DBC-1 and EMSY were identified as integral components of an NIF-1 complex of ∼1.5 MDa and were found to play an important role in the regulation of nuclear receptor-mediated transcription. Stimulation of the Sox9 and HoxA1 genes by retinoic acid receptor-α was found to require both DBC-1 and EMSY in addition to NIF-1 for maximal transcriptional activation. Interestingly, NRC was not identified as a component of the NIF-1 complex, suggesting that NIF-1 and NRC do not exist as stable in vitro purified complexes, although the separate NIF-1 and NRC complexes appear to functionally interact in the cell.

The Nuclear Hormone Receptor Coactivator NRC Is a Pleiotropic Modulator Affecting Growth, Development, Apoptosis, Reproduction, and Wound Repair

ABSTRACT Nuclear hormone receptor coregulator (NRC) is a 2,063-amino-acid coregulator of nuclear hormone receptors and other transcription factors (e.g., c-Fos, c-Jun, and NF-κB). We and others have generated C57BL/6-129S6 hybrid (C57/129) NRC+/− mice that appear outwardly normal and grow and reproduce. In contrast, homozygous deletion of the NRC gene is embryonic lethal. NRC−/− embryos are always smaller than NRC+/+ embryos, and NRC−/− embryos die between 8.5 and 12.5 days postcoitus (dpc), suggesting that NRC has a pleotrophic effect on growth. To study this, we derived mouse embryonic fibroblasts (MEFs) from 12.5-dpc embryos, which revealed that NRC−/− MEFs exhibit a high rate of apoptosis. Furthermore, a small interfering RNA that targets mouse NRC leads to enhanced apoptosis of wild-type MEFs. The finding that C57/129 NRC+/− mice exhibit no apparent phenotype prompted us to develop 129S6 NRC+/− mice, since the phenotype(s) of certain gene deletions may be strain dependent. In contrast with C57/129 NRC+/− females, 20% of 129S6 NRC+/− females are infertile while 80% are hypofertile. The 129S6 NRC+/− males produce offspring when crossed with wild-type 129S6 females, although fertility is reduced. The 129S6 NRC+/− mice tend to be stunted in their growth compared with their wild-type littermates and exhibit increased postnatal mortality. Lastly, both C57/129 NRC+/− and 129S6 NRC+/− mice exhibit a spontaneous wound healing defect, indicating that NRC plays an important role in that process. Our findings reveal that NRC is a coregulator that controls many cellular and physiologic processes ranging from growth and development to reproduction and wound repair.

NRC-Interacting Factor 1 Is a Novel Cotransducer That Interacts with and Regulates the Activity of the Nuclear Hormone Receptor Coactivator NRC

ABSTRACT We previously reported the cloning and characterization of a novel nuclear hormone receptor transcriptional coactivator, which we refer to as NRC. NRC is a 2,063-amino-acid nuclear protein which contains a potent N-terminal activation domain and several C-terminal modules which interact with CBP and ligand-bound nuclear hormone receptors as well as c-Fos and c-Jun. In this study we sought to clone and identify novel factors that interact with NRC to modulate its transcriptional activity. Here we describe the cloning and characterization of a novel protein we refer to as NIF-1 (NRC-interacting factor 1). NIF-1 was cloned from rat pituitary and human cell lines and was found to interact in vivo and in vitro with NRC. NIF-1 is a 1,342-amino-acid nuclear protein containing a number of conserved domains, including six Cys-2/His-2 zinc fingers, an N-terminal stretch of acidic amino acids, and a C-terminal leucine zipper-like motif. Zinc fingers 1 to 3 are potential DNA-binding BED finger domains recently proposed to play a role in altering local chromatin architecture. We mapped the interaction domains of NRC and NIF-1. Although NIF-1 does not directly interact with nuclear receptors, it markedly enhances ligand-dependent transcriptional activation by nuclear hormone receptors in vivo as well as activation by c-Fos and c-Jun. These results, and the finding that NIF-1 interacts with NRC in vivo, suggest that NIF-1 functions to regulate transcriptional activation through NRC. We suggest that NIF-1, and factors which associate with coactivators but not receptors, be referred to as cotransducers, which act in vivo either as part of a coactivator complex or downstream of a coactivator complex to modulate transcriptional activity. Our findings suggest that NIF-1 may be a functional component of an NRC complex and acts as a regulator or cotransducer of NRC function.

Components of the CCR4-NOT Complex Function as Nuclear Hormone Receptor Coactivators via Association with the NRC-interacting Factor NIF-1

CCR4-NOT is an evolutionarily conserved, multicomponent complex known to be involved in transcription as well as mRNA degradation. Various subunits (e.g. CNOT1 and CNOT7/CAF1) have been reported to be involved in influencing nuclear hormone receptor activities. Here, we show that CCR4/CNOT6 and RCD1/CNOT9, members of the CCR4-NOT complex, potentiate nuclear receptor activity. RCD1 interacts in vivo and in vitro with NIF-1 (NRC-interacting factor), a previously characterized nuclear receptor cotransducer that activates nuclear receptors via its interaction with NRC. As with NIF-1, RCD1 and CCR4 do not directly associate with nuclear receptors; however, they enhance ligand-dependent transcriptional activation by nuclear hormone receptors. CCR4 mediates its effect through the ligand binding domain of nuclear receptors and small interference RNA-mediated silencing of endogenous CCR4 results in a marked decrease in nuclear receptor activation. Furthermore, knockdown of CCR4 results in an attenuated stimulation of RARα target genes (e.g. Sox9 and HoxA1) as shown by quantitative PCR assays. The silencing of endogenous NIF-1 also resulted in a comparable decrease in the RAR-mediated induction of both Sox9 and HoxA1. Furthermore, CCR4 associates in vivo with NIF-1. In addition, the CCR4-enhanced transcriptional activation by nuclear receptors is dependent on NIF-1. The small interference RNA-mediated knockdown of NIF-1 blocks the ligand-dependent potentiating effect of CCR4. Our results suggest that CCR4 plays a role in the regulation of certain endogenous RARα target genes and that RCD1 and CCR4 might mediate their function through their interaction with NIF-1.

Nuclear receptor coactivator/coregulator NCoA6(NRC) is a pleiotropic coregulator involved in transcription, cell survival, growth and development.

NCoA6 (also referred to as NRC, ASC-2, TRBP, PRIP and RAP250) was originally isolated as a ligand-dependent nuclear receptor interacting protein. However, NCoA6 is a multifunctional coregulator or coactivator necessary for transcriptional activation of a wide spectrum of target genes. The NCoA6 gene is amplified and overexpressed in breast, colon and lung cancers. NCoA6 is a 250 kDa protein which harbors a potent N-terminal activation domain, AD1; and a second, centrally-located activation domain, AD2, which is necessary for nuclear receptor signaling. The intrinsic activation potential of NCoA6 is regulated by its C-terminal STL regulatory domain. Near AD2 is an LxxLL-1 motif which interacts with a wide spectrum of ligand-bound NRs with high-affinity. A second LxxLL motif (LxxLL-2) located towards the C-terminal region is more restricted in its NR specificity. The potential role of NCoA6 as a co-integrator is suggested by its ability to enhance transcriptional activation of a wide variety of transcription factors and from its in vivo association with a number of known cofactors including CBP/p300. NCoA6 has been shown to associate with at least three distinct coactivator complexes containing Set methyltransferases as core polypeptides. The composition of these complexes suggests that NCoA6 may play a fundamental role in transcriptional activation by modulating chromatin structure through histone methylation. Knockout studies in mice suggest that NCoA6 is an essential coactivator. NCoA6-/- embryos die between 8.5–12.5 dpc from general growth retardation coupled with developmental defects in the heart, liver, brain and placenta. NCoA6-/- MEFs grow at a reduced rate compared to WT MEFs and spontaneously undergo apoptosis, indicating the importance of NCoA6 as a prosurvival and anti-apoptotic gene. Studies with NCoA6+/- and conditional knockout mice suggest that NCoA6 is a pleiotropic coregulator involved in growth, development, wound healing and maintenance of energy homeostasis.

Fas Activated Serine-Threonine Kinase Domains 2 (FASTKD2) mediates apoptosis of breast and prostate cancer cells through its novel FAST2 domain

BackgroundExpression of NRIF3 (Nuclear Receptor Interacting Factor-3) rapidly and selectively leads to apoptosis of breast cancer cells. This occurs through binding of NRIF3 or its 30 amino acid Death Domain-1 (DD1) region to the transcriptional repressor, DIF-1 (DD1 Interacting Factor-1). DIF-1 acts in a wide variety of breast cancer cells but not other cell types to repress the pro-apoptotic gene, FASTKD2. Expression of NRIF3 or DD1 inactivates the DIF-1 repressor leading to rapid derepression of FASTKD2, which initiates apoptosis within 5–8 h of expression. Although FASTKD2 is an inner mitochondrial membrane protein, it does not require mitochondrial localization to initiate apoptosis.MethodsAndrogen dependent LNCaP cells as well as two androgen independent LNCaP cell lines (LNCaP-AI and LNCaP-abl) were studied and LNCaP-AI cells were engineered to conditionally express DD1 or the inactive DD1-S28A with 4-hydroxytamoxifen. Apoptosis was assessed by TUNEL assay. FASTKD2 is related to 4 other proteins encoded in the human genome (FASTKD1, 3, 4, 5). All contain a poorly conserved putative bipartite kinase domain designated as FAST1_FAST2. We examined whether expression of any of the other FASTKD isoforms leads to apoptosis and sought to identify the region of FASTKD2 necessary to initiate the apoptotic pathway.ResultsOf the FASTKD1-5 isoforms only expression of FASTKD2 leads to apoptosis. Although, the NRIF3/DD1/DIF-1 pathway does not mediate apoptosis of a wide variety of non-breast cancer cell lines, because of certain similarities and gene signatures between breast and prostate cancer we explored whether the NRIF3/DD1/DIF-1/FASTKD2 pathway mediates apoptosis of prostate cancer cells. We found that the pathway leads to apoptosis in LNCaP cells, including the two androgen-independent LNCaP cell lines that are generally resistant to apoptosis. Lastly, we identified that FASTKD2-mediated apoptosis is initiated by the 81 amino acid FAST2 region.ConclusionsThe NRIF3/DIF-1/FASTKD2 pathway acts as a “death switch” in breast and prostate cancer cells. Deciphering how this pathway is regulated and how FASTKD2 initiates the apoptotic response will allow for the development of therapeutic agents for the treatment of androgen-independent prostate cancer or Tamoxifen-unresponsive Estrogen Receptor negative tumors as well as metastatic breast or prostate cancer.

Insulin-activated Elk-1 recruits the TIP60/NuA4 complex to increase prolactin gene transcription

Insulin increases prolactin gene expression in GH4 cells through phosphorylation of Elk-1 (Jacob and Stanley, 2001). We preformed a reverse two-hybrid screen using Elk-1-B42 as bait to identify proteins from GH4 cells that might serve as co-activators or co-repressors for insulin-increased prolactin gene expression. A number of the components of the TIP60/NuA4 complex interacted with Elk-1 suggesting that Elk-1 might activate transcription by recruiting the TIP60 chromatin-remodeling complex to the prolactin promoter. Inhibition of insulin-increased prolactin-luciferase expression by wild type and mutant adenovirus E1A protein provided physiological context for these yeast studies. Inhibition of histone deacetylases dramatically increased both basal and insulin-increased prolactin gene transcription. Co-immune precipitation experiments demonstrated Elk-1 and TIP60 associate in vitro. Transient or stable expression of TIP60 activated insulin-increased prolactin gene expression while a mutated TIP60 blocked insulin-increased prolactin gene expression. Analysis of the prolactin mRNA by quantitative RT-PCR showed that insulin-increased prolactin mRNA accumulation and that this was inhibited in GH4 cells that stably expressed mutant TIP60. Finally, ChIP experiments demonstrate the insulin-dependent occupancy of the prolactin promoter by Elk-1 and TIP60. Our studies suggest that insulin activates prolactin gene transcription by activating Elk-1 that recruits the NuA4 complex to the promoter.

Functional consequences of interactions between thyroid hormone receptors and retinoid X receptor

Purpose of reviewThyroid hormone receptors mediate a wide variety of biologic processes in cells. They are members of the nuclear hormone receptor gene family, which includes the receptors that mediate the effects of steroid hormones, vitamin D, and the retinoids. These receptors are DNA binding proteins, which act as ligand-dependent transcription factors. One of the retinoid receptors, retinoid X receptor, acts as a dimerization partner for the thyroid hormone receptors and several other members of the nuclear receptor family. This review gives a historical perspective and overview of the thyroid hormone receptors and reviews the role of the retinoid X receptor in influencing the action of the thyroid hormones. Recent findingsRetinoid X receptor forms heterodimers with the thyroid hormone receptors and several other nuclear receptors and enhances the binding of these receptors to DNA. In addition, for thyroid hormone receptor/retinoid X receptor heterodimers, the general consensus has been that activation occurs through the thyroid hormone receptor component of the heterodimer and that retinoid X receptor does not bind its ligand or mediate cross-talk with the thyroid hormone receptor component of the heterodimer. Several recent studies, however, indicate that retinoid X receptor can bind its cognate ligand and modulate the activity of the thyroid hormone receptor/retinoid X receptor heterodimer through a variety of novel mechanisms. SummaryThe retinoid X receptors play an important role in mediating the action of the thyroid hormones and in part mediate these effects through cross-talk with thyroid hormone receptor in the heterodimer. Future studies that expand on recent findings in the field should help provide new insights into transcriptional regulation by thyroid hormone nuclear receptors.