Osf2/Cbfa1, hereafter called Osf2, is a member of the Runt-related family of transcription factors that plays a critical role during osteoblast differentiation. Like all Runt-related proteins, it contains a runt domain, which is the DNA-binding domain, and a C-terminal proline-serine-threonine-rich (PST) domain thought to be the transcription activation domain. Additionally, Osf2 has two amino-terminal domains distinct from any other Runt-related protein. To understand the mechanisms of osteoblast gene regulation by Osf2, we performed an extensive structure-function analysis. After defining a short Myc-related nuclear localization signal, a deletion analysis revealed the existence of three transcription activation domains and one repression domain. AD1 (for activation domain 1) comprises the first 19 amino acids of the molecule, which form the first domain unique to Osf2, AD2 is formed by the glutamine-alanine (QA) domain, the second domain unique to Osf2, and AD3 is located in the N-terminal half of the PST domain and also contains sequences unique to Osf2. The transcription repression domain comprises the C-terminal 154 amino acids of Osf2. DNA-binding, domainswapping, and protein interaction experiments demonstrated that full-length Osf2 does not interact with Cbf␤, a known partner of Runt-related proteins, whereas a deletion mutant of Osf2 containing only the runt and PST domains does. The QA domain appears to be responsible for preventing this heterodimerization. Thus, our results uncover the unique functional organization of Osf2 by identifying functional domains not shared with other Runt-related proteins that largely control its transactivation and heterodimerization abilities.

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 receptorinteracting 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.Nuclear hormone receptors comprise a superfamily of ligand-dependent transcription factors involved in controlling diverse cellular processes, including growth, differentiation, development, and homeostasis (37). The nuclear hormone receptor superfamily includes type I receptors which mediate the effects of glucocorticoids (glucocorticoid receptor , and the PPARs. These receptors share a similar modular structure consisting of an Nterminal A/B domain, a DNA-binding C domain, and a D, E, and F ligand-binding domain (LBD) (4, 37). The DNA-binding C domain is highly conserved among members of type I and type II nuclear hormone receptors. Although the LBDs of nuclear hormone receptors (ϳ300 amino acids) are diverse in sequence, accounting for ligand specificity, they exhibit certain similarities in their overall structure (4, 13). Thus, the LBDs of all nuclear receptors are organized into 12 helical regions which play an important role in determining the conformation of the LBD in the presence and absence of ligand (59) and in mediating heterodimerization of type II receptors with the RXRs (2, 13).[Ligand-dependent conformational changes in the LBD are thought to recruit coactivators or coregulators to the DNAbound receptor, which leads to transcriptional activation (37). The activation function mediated through the LBD has been referred to as activation function 2 (AF2) (38, 54). The majority of the coactivators, identified in a yeast two-hybrid screen, fall into two main groups: the p160/SRC family (SRC-1 (5,20,28). Coactivators which fall outside these groups include PGC-1 (44), ARA70 (62), p/CAF (3, 60), and NRIF3, which exhibits specificity for only the TRs and the RXRs (33). Using a biochemical approach, another class of factors have been identified...

SUMMARY 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.

How predictable are life trajectories? We investigated this question with a scientific mass collaboration using the common task method; 160 teams built predictive models for six life outcomes using data from the Fragile Families and Child Wellbeing Study, a high-quality birth cohort study. Despite using a rich dataset and applying machine-learning methods optimized for prediction, the best predictions were not very accurate and were only slightly better than those from a simple benchmark model. Within each outcome, prediction error was strongly associated with the family being predicted and weakly associated with the technique used to generate the prediction. Overall, these results suggest practical limits to the predictability of life outcomes in some settings and illustrate the value of mass collaborations in the social sciences.

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-B). 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.

Nuclear hormone receptor coregulator (NRC) (also referred to as activating signal cointegrator-2, thyroid hormone receptor-binding protein, peroxisome proliferator activating receptor-interacting protein, and 250-kDa receptor associated protein) belongs to a growing class of nuclear cofactors widely known as coregulators or coactivators that are necessary for transcriptional activation of target genes. The NRC gene is also amplified and overexpressed in breast, colon, and lung cancers. NRC is a 2063-amino acid protein that harbors a potent N-terminal activation domain (AD1) and a second more centrally located activation domain (AD2) that is rich in Glu and Pro. Near AD2 is a receptor-interacting domain containing an LxxLL motif (LxxLL-1), which interacts with a wide variety of ligand-bound nuclear hormone receptors with high affinity. A second LxxLL motif (LxxLL-2) located in the C-terminal region of NRC is more restricted in its nuclear hormone receptor specificity. The intrinsic activation potential of NRC is regulated by a C-terminal serine, threonine, leucine-regulatory domain. The potential role of NRC as a cointegrator 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 transcriptional regulators including CBP/p300. Recent studies in mice indicate that deletion of both NRC alleles leads to embryonic lethality resulting from general growth retardation coupled with developmental defects in the heart, liver, brain, and placenta. NRC(-/-) mouse embryo fibroblasts spontaneously undergo apoptosis, indicating the importance of NRC as a prosurvival and antiapoptotic gene. Studies with 129S6 NRC(+/-) mice indicate that NRC is a pleiotropic regulator that is involved in growth, development, reproduction, metabolism, and wound healing.

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 ϳ1.5 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-␣ 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.A number of crucial insights into the multilayered regulation of transcription have been uncovered through study of nuclear hormone receptor-mediated gene expression. Nuclear hormone receptors are hormone-and ligand-dependent transcription factors that control the coordinated expression of gene networks in numerous physiological, developmental, and metabolic processes (1). Dysfunction of nuclear receptor signaling leads to a number of proliferative, reproductive, and metabolic diseases such as cancer, infertility, obesity, and diabetes (2). The biological functions of nuclear hormone receptors rely on coactivators that represent a diverse group of proteins that enhance nuclear receptor-mediated transcription (3). Extensive studies on the in vivo functions of nuclear receptors have led to the identification and characterization of ϳ200 coactivators, all of which have been catalogued on-line at the Nuclear Receptor Signaling Atlas. Coactivators, in general, are known to act by: (i) bridging factors to recruit additional cofactors to DNA-bound nuclear receptors, e.g. p160/SRC proteins (4, 5); (ii) exhibiting various enzymatic activities such as methylation, acetylation, and others to modulate chromatin (6 -8); and (iii) interfacing between DNA-bound nuclear receptors and the basal transcriptional machinery, e.g. TRAP/DRIP complex (9).Nuclear receptor coregulator (NRC) 3 also referred to as ASC-2, TRBP...

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.Nuclear hormone receptors comprise a family of liganddependent transcription factors that have a broad effect on gene expression, growth, and development (2, 38, 39). These include the thyroid hormone receptors (TRs) for thyroid hormone (T3), the retinoic acid (RA) receptors (RARs) for all trans RA, the RARs and the retinoid X receptors (RXRs) for 9-cis RA, vitamin D receptor (VDR) for 1,25-(OH) 2 vitamin D3, glucocorticoid receptor (GR), progesterone receptor, estrogen receptors (ERs), and the peroxisome proliferator-activated receptors (PPARs), which are regulated by a variety of lipophilic compounds. These receptors share a similar modular structure consisting of an N-terminal A/B domain, a DNAbinding C domain, and a D, E, and F ligand binding domain (LBD) (7, 38). The LBDs of nuclear receptors are organized into 12 helical regions, and the binding of ligand to the LBD of a DNA-bound receptor mediates a conformational change which recruits coactivators or coregulators, leading to transcriptional activation (38,49).Coactivators which have been identified include members of the p160 family (SRC-1/NCoA-1 [27, 42], TIF-2/GRIP-1/ NCoA-2 [23, 50, 52] and AIB1/p/CIP/A...