Histone-fold domain protein NF-Y promotes chromatin accessibility for cell type-specific master transcription factors.

Cell type-specific master transcription factors (TFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that the ubiquitous CCAAT-binding NF-Y complex is required for the maintenance of embryonic stem cell (ESC) identity and is an essential component of the core pluripotency network. Genome-wide studies in ESCs and neurons reveal that NF-Y regulates not only genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with master TFs. Mechanistically, NF-Y's distinct DNA-binding mode promotes master/pioneer TF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a conceptually unique function for histone-fold domain (HFD) protein NF-Y in promoting chromatin accessibility and suggest that other HFD proteins with analogous structural and DNA-binding properties may function in similar ways.

CBFß stabilizes HIV Vif to counteract APOBEC3 at the expense of RUNX1 target gene expression.

The HIV-1 accessory protein Vif hijacks a cellular Cullin-RING ubiquitin ligase, CRL5, to promote degradation of the APOBEC3 (A3) family of restriction factors. Recently, the cellular transcription cofactor CBFß was shown to form a complex with CRL5-Vif and to be essential for A3 degradation and viral infectivity. We now demonstrate that CBFß is required for assembling a well-ordered CRL5-Vif complex by inhibiting Vif oligomerization and by activating CRL5-Vif via direct interaction. The CRL5-Vif-CBFß holoenzyme forms a well-defined heterohexamer, indicating that Vif simultaneously hijacks CRL5 and CBFß. Heterodimers of CBFß and RUNX transcription factors contribute toward the regulation of genes, including those with immune system functions. We show that binding of Vif to CBFß is mutually exclusive with RUNX heterodimerization and impacts the expression of genes whose regulatory domains are associated with RUNX1. Our results provide a mechanism by which a pathogen with limited coding capacity uses one factor to hijack multiple host pathways.

Epigenetic role of the nuclear factor NF-Y on ID gene family in endometrial tissues of women with endometriosis: a case control study.

BACKGROUND: A predominant difference between endometrial and normal cells is higher proliferation rate in the former cells which is benign. The genes of inhibitor of differentiation (ID) family play a major role in cell proliferation regulation which might be targeted by the nuclear transcription factor Y (NF-Y) for subsequent epigenetic modifications through the CCAAT box regulatory region. The present study was designed to investigate the epigenetic role of NF-Y on ID gene family in endometrial tissue of patients with endometriosis. MATERIALS & METHODS: In this case-control study, 20 patients with endometriosis and 20 normal women were examined for the relative expression of the NF-YA, NF-YB, NF-YC and ID genes by real-time PCR during the proliferative phase. The occupancy of NF-Y on CCAAT box region of ID genes was investigated using chromatin immunoprecipitation (ChIP) followed by real-time PCR. RESULTS: The NF-YA was over-expressed in eutopic endometrium during the proliferative phase. Although the expression level of NF-YB and NF-YC were unchanged in eutopic samples, they were remarkably higher in ectopic group (P<0.05). The ID2 and ID3 genes were up-regulated in ectopic and eutopic tissues, however ID1 and ID4 genes were down-regulated in these samples (P<0.05). The ChIP analysis revealed significant enrichment of NF-Y on regulatory regions of ID2,3 genes in eutopic group, but reduced binding level of NF-Y to the ID1,3 promoters in ectopic specimens (P<0.05). CONCLUSION: The ability of NF-Y to regulate ID genes via CCAAT box region suggests the possible role of NF-Y transcription factor in epigenetic changes in endometrial tissues which may open novel avenues in finding new therapeutic strategies.

Gene structure, expression pattern and interaction of Nuclear Factor-Y family in castor bean (Ricinus communis).

MAIN CONCLUSION: Nuclear Factor-Y transcription factors, which function in regulating seed development (including storage reservoir accumulation) and responding to abiotic stresses, were identified and characterized in castor bean. Nuclear Factor-Y (NF-Y) transcription factors in plants contain three subunits (NF-YA, NF-YB and NF-YC), and function as a heterodimer or heterotrimer complex in regulating plant growth, development and response to stresses. Castor bean (Ricinus communis, Euphorbiaceae) one of the most economically important non-edible oilseed crops, able to grow in diverse soil conditions and displays high tolerance to abiotic stresses. Due to increasing demands for its seed oils, it is necessary to elucidate the molecular mechanism underlying the regulation of growth and development. Based on the available genome data, we identified 25 RcNF-Y members including six RcNF-YAs, 12 RcNF-YBs and seven RcNF-YCs, and characterized their gene structures. Yeast two-hybrid assays confirmed the protein-protein interactions among three subunits. Using transcriptomic data from different tissues, we found that six members were highly or specifically expressed in endosperms (in particular, two LEC1-type members RcNF-YB2 and RcNF-YB12), implying their involvement in regulating seed development and storage reservoir accumulation. Further, we investigated the expression changes of RcNF-Y members in two-week-old seedlings under drought, cold, hot and salt stresses. We found that the expression levels of 20 RcNF-Y members tested were changed and three RcNF-Y members might function in response to abiotic stresses. This study is the first reported on genomic characterization of NF-Y transcription factors in the family Euphorbiaceae. Our results provide the basis for improved understanding of how NF-Y genes function in the regulation of seed development and responses to abiotic stresses in both castor bean and other plants in this family.

Ambient temperature regulates the expression of a small set of sRNAs influencing plant development through NF-YA2 and YUC2.

Plants substantially alter their developmental programme upon changes in the ambient temperature. The 21-24 nt small RNAs (sRNAs) are important gene expression regulators, which play a major role in development and adaptation. However, little is known about how the different sRNA classes respond to changes in the ambient temperature. We profiled the sRNA populations in four different tissues of Arabidopsis thaliana plants grown at 15°C, 21°C, and 27°C. We found that only a small fraction (0.6%) of the sRNA loci are ambient temperature-controlled. We identified thermoresponsive microRNAs and identified their target genes using degradome libraries. We verified that the target of the thermoregulated miR169, NF-YA2, is also ambient temperature-regulated. NF-YA2, as the component of the conserved transcriptional regulator NF-Y complex, binds the promoter of the flowering time regulator FT and the auxin biosynthesis gene YUC2. Other differentially expressed loci include thermoresponsive phased siRNA loci that target various auxin pathway genes and tRNA fragments. Furthermore, a temperature-dependent 24-nt heterochromatic siRNA locus in the promoter of YUC2 may contribute to the epigenetic regulation of auxin homeostasis. This holistic approach facilitated a better understanding of the role of different sRNA classes in ambient temperature adaptation of plants.

Direct non transcriptional role of NF-Y in DNA replication.

NF-Y is a heterotrimeric transcription factor, which plays a pioneer role in the transcriptional control of promoters containing the CCAAT-box, among which genes involved in cell cycle regulation, apoptosis and DNA damage response. The knock-down of the sequence-specific subunit NF-YA triggers defects in S-phase progression, which lead to apoptotic cell death. Here, we report that NF-Y has a critical function in DNA replication progression, independent from its transcriptional activity. NF-YA colocalizes with early DNA replication factories, its depletion affects the loading of replisome proteins to DNA, among which Cdc45, and delays the passage from early to middle-late S phase. Molecular combing experiments are consistent with a role for NF-Y in the control of fork progression. Finally, we unambiguously demonstrate a direct non-transcriptional role of NF-Y in the overall efficiency of DNA replication, specifically in the DNA elongation process, using a Xenopus cell-free system. Our findings broaden the activity of NF-Y on a DNA metabolism other than transcription, supporting the existence of specific TFs required for proper and efficient DNA replication.

The gene pair PRR11 and SKA2 shares a NF-Y-regulated bidirectional promoter and contributes to lung cancer development.

Head-to-head gene pairs represent a unique feature of gene organization in eukaryotes, accounting for >10% of genes in the human genome. Identification and functional analysis of such gene pairs is only in its infancy. Recently, we identified PRR11 as a novel cancer-related gene that is implicated in cell cycle and lung cancer. Here we demonstrate that PRR11 is oriented in a head-to-head configuration with its neighboring gene, SKA2. 5'-RACE assay revealed that the intergenic spacer region between the two genes is <500 bp. Serial luciferase reporter assays demonstrated that a minimal 80-bp intergenic region functions as a core bidirectional promoter to drive basal transcription in both the PRR11 and SKA2 orientations. EMSA and ChIP assays demonstrated that NF-Y binds to and directly transactivates the PRR11-SKA2 bidirectional promoter. SiRNA-mediated NF-Y depletion significantly downregulated PRR11 and SKA2 expression. Expression of both PRR11 and SKA2 was significantly upregulated in lung cancer. Expression of the two genes was highly correlated with each other and with NF-Y expression. Remarkably, high expression of both PRR11 and SKA2 was associated with poorer prognosis in lung cancer patients compared with high expression of one gene or low expression of both genes. Knockdown of PRR11 and/or SKA2 remarkably reduced cell proliferation, migration, and invasion in lung cancer cells. Thus, the PRR11-SKA2 bidirectional transcription unit, which is a novel direct target of NF-Y, is essential for the accelerated proliferation and motility of lung cancer cells and may represent a potential target in the diagnosis and/or treatment of human lung cancer.

Transcriptional activation of PRMT5 by NF-Y is required for cell growth and negatively regulated by the PKC/c-Fos signaling in prostate cancer cells.

Protein arginine methyltransferase 5 (PRMT5) symmetrically methylates arginine residues of histones and non-histone protein substrates and regulates a variety of cellular processes through epigenetic control of target gene expression or post-translational modification of signaling molecules. Recent evidence suggests that PRMT5 may function as an oncogene and its overexpression contributes to the development and progression of several human cancers. However, the mechanism underlying the regulation of PRMT5 expression in cancer cells remains largely unknown. In the present study, we have mapped the proximal promoter of PRMT5 to the -240bp region and identified nuclear transcription factor Y (NF-Y) as a critical transcription factor that binds to the two inverted CCAAT boxes and regulates PRMT5 expression in multiple cancer cell lines. Further, we present evidence that loss of PRMT5 is responsible for cell growth inhibition induced by knockdown of NF-YA, a subunit of NF-Y that forms a heterotrimeric complex with NF-YB and NF-YC for function. Significantly, we have found that activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) in LNCaP prostate cancer cells down-regulates the expression of NF-YA and PRMT5 at the transcription level in a c-Fos-dependent manner. Given that down-regulation of several PKC isozymes is implicated in the development and progression of several human cancers, our findings suggest that the PKC-c-Fos-NF-Y signaling pathway may be responsible for PRMT5 overexpression in a subset of human cancer patients.

Epigenetic role of CCAAT box-binding transcription factor NF-Y on ID gene family in human embryonic carcinoma cells.

Nuclear factor Y (NF-Y) is a histone substitute protein that specifically binds to the CCAAT box of the target genes and thereby promotes their regulation. NF-Y transcription factor, with defined CCAAT element-binding activities, target a gene family that encodes a group of basic helix-loop-helix ID factors (ID1-ID4), with or without CCAAT box at their promoter region. In this study, the expressions of NF-Y in mRNA and protein level were evaluated in a human embryonic carcinoma cell line, named NTera2, before and after 7 days induction of differentiation. We also looked into expression levels of ID genes in NTera2 cells during differentiation because of their critical role in development. By using chromatin immunoprecipitation coupled with real-time polymerase chain reaction, NF-Y incorporation and acetylation/dimethylation of histone H3 at lysine 9 (H3K9ac/me2) was quantitatively evaluated on the regulatory regions of considered genes to monitor the changes in epigenetic markers at ID gene promoters throughout differentiation. The results demonstrated a marked down-regulation of ID1, ID2, and ID3 genes, parallel to a loss of NF-Y binding to the promoters of these genes. The data show that although the genes encoding NF-Y complex remained expressed at mRNA level, NF-YC is lost at the protein level onset of differentiation. Additionally, the epigenetic marks of H3K9ac and H3K9me2 at the target gene promoters decreased and increased, respectively, after 1 day of differentiation. It is suggested that, in the absence of NF-Y binding, the corresponding regions adopt a heterochromatic nature, whereas when NF-Y comes back after 7 days of differentiation, the ID1-3 promoters become again converted into active chromatin. The ID4 gene, lacking a CCAAT box, behaves differently and does not show any incorporation. This experiment implies for the first time that the presence of NF-Y transcription factor plays a pivotal role in transcriptional regulation of ID genes in development.

Transcription factor NF-Y inhibits cell growth and decreases SOX2 expression in human embryonal carcinoma cell line NT2/D1.

Transcription factor NF-Y belongs to the embryonic stem cell transcription factor circuitry due to its role in the regulation of cell proliferation. We investigated the role of NF-Y in pluripotency maintenance using NT2/D1 cells as one of the best-characterized human embryonal carcinoma cell line. We investigated the efficiency of protein transduction and analyzed the effects of forced expression of short isoform of NF-Y A-subunit (NF-YAs) on NT2/D1 cell growth and expression of SOX2. We found that protein transduction is an efficient method for NF-Y overexpression in NT2/D1 cells. Next, we analyzed the effect of NF-YAs overexpression on NT2/D1 cell viability and detected significant reduction in cell growth. The negative effect of NF-YAs overexpression on NT2/D1 cell pluripotency maintenance was confirmed by the decrease in the level of the pluripotency marker SOX2. Finally, we checked the p53 status and determined that the NF-Y-induced inhibition of NT2/D1 cell growth is p53-independent.

Inactivation of nuclear factor-Y inhibits vascular smooth muscle cell proliferation and neointima formation.

OBJECTIVE: Atherosclerosis and restenosis are multifactorial diseases associated with abnormal vascular smooth muscle cell (VSMC) proliferation. Nuclear factor-Y (NF-Y) plays a major role in transcriptional activation of the CYCLIN B1 gene (CCNB1), a key positive regulator of cell proliferation and neointimal thickening. Here, we investigated the role of NF-Y in occlusive vascular disease. APPROACH AND RESULTS: We performed molecular and expression studies in cultured cells, animal models, and human tissues. We find upregulation of NF-Y and cyclin B1 expression in proliferative regions of murine atherosclerotic plaques and mechanically induced lesions, which correlates with higher binding of NF-Y to target sequences in the CCNB1 promoter. NF-YA expression in neointimal lesions is detected in VSMCs, macrophages, and endothelial cells. Platelet-derived growth factor-BB, a main inductor of VSMC growth and neointima development, induces the recruitment of NF-Y to the CCNB1 promoter and augments both CCNB1 mRNA expression and cell proliferation through extracellular signal-regulated kinase 1/2 and Akt activation in rat and human VSMCs. Moreover, adenovirus-mediated overexpression of a NF-YA-dominant negative mutant inhibits platelet-derived growth factor-BB-induced CCNB1 expression and VSMC proliferation in vitro and neointimal lesion formation in a mouse model of femoral artery injury. We also detect NF-Y expression and DNA-binding activity in human neointimal lesions. CONCLUSIONS: Our results identify NF-Y as a key downstream effector of the platelet-derived growth factor-BB-dependent mitogenic pathway that is activated in experimental and human vasculoproliferative diseases. They also identify NF-Y inhibition as a novel and attractive strategy for the local treatment of neointimal formation induced by vessel denudation.

A C subunit of the plant nuclear factor NF-Y required for rhizobial infection and nodule development affects partner selection in the common bean-Rhizobium etli symbiosis.

Legume plants are able to interact symbiotically with soil bacteria to form nitrogen-fixing root nodules. Although specific recognition between rhizobia and legume species has been extensively characterized, plant molecular determinants that govern the preferential colonization by different strains within a single rhizobium species have received little attention. We found that the C subunit of the heterotrimeric nuclear factor NF-Y from common bean (Phaseolus vulgaris) NF-YC1 plays a key role in the improved nodulation seen by more efficient strains of rhizobia. Reduction of NF-YC1 transcript levels by RNA interference (RNAi) in Agrobacterium rhizogenes-induced hairy roots leads to the arrest of nodule development and defects in the infection process with either high or low efficiency strains. Induction of three G2/M transition cell cycle genes in response to rhizobia was impaired or attenuated in NF-YC1 RNAi roots, suggesting that this transcription factor might promote nodule development by activating cortical cell divisions. Furthermore, overexpression of this gene has a positive impact on nodulation efficiency and selection of Rhizobium etli strains that are naturally less efficient and bad competitors. Our findings suggest that this transcription factor might be part of a mechanism that links nodule organogenesis with an early molecular dialogue that selectively discriminates between high- and low-quality symbiotic partners, which holds important implications for optimizing legume performance.

An RNA interference-based screen of transcription factor genes identifies pathways necessary for sensory regeneration in the avian inner ear.

Sensory hair cells of the inner ear are the mechanoelectric transducers of sound and head motion. In mammals, damage to sensory hair cells leads to hearing or balance deficits. Nonmammalian vertebrates such as birds can regenerate hair cells after injury. In a previous study, we characterized transcription factor gene expression during chicken hair cell regeneration. In those studies, a laser microbeam or ototoxic antibiotics were used to damage the sensory epithelia (SE). The current study focused on 27 genes that were upregulated in regenerating SEs compared to untreated SEs in the previous study. Those genes were knocked down by siRNA to determine their requirement for supporting cell proliferation and to measure resulting changes in the larger network of gene expression. We identified 11 genes necessary for proliferation and also identified novel interactive relationships between many of them. Defined components of the WNT, PAX, and AP1 pathways were shown to be required for supporting cell proliferation. These pathways intersect on WNT4, which is also necessary for proliferation. Among the required genes, the CCAAT enhancer binding protein, CEBPG, acts downstream of Jun Kinase and JUND in the AP1 pathway. The WNT coreceptor LRP5 acts downstream of CEBPG, as does the transcription factor BTAF1. Both of these genes are also necessary for supporting cell proliferation. This is the first large-scale screen of its type and suggests an important intersection between the AP1 pathway, the PAX pathway, and WNT signaling in the regulation of supporting cell proliferation during inner ear hair cell regeneration.

Expression and functional analysis of NUCLEAR FACTOR-Y, subunit B genes in barley.

NUCLEAR FACTOR-Y, subunit B (NF-YB) comprises a multigene family in plants and has been shown to play important roles in growth, development, and response to environmental stress. In this study, five NF-YBs containing the full-length coding region were obtained from barley (Hordeum vulgare) through database sequence analysis, cloning, and sequencing. Sequence alignment and phylogenetic analysis showed that HvNF-YB3 and HvNF-YB1 were clustered with NF-YB2 and NF-YB3 in Arabidopsis, suggesting these NF-YBs are evolutionary and functionally related. To test this hypothesis, HvNF-YB3 and HvNF-YB1 were overexpressed in Arabidopsis. Overexpression of HvNF-YB1 greatly promoted early flowering in Arabidopsis, supporting that HvNF-YB1may have conserved gene function in flowering time control as NF-YB2 and NF-YB3 in Arabidopsis. Overexpression of HvNF-YB3 in Arabidopsis had no effect on flowering time. An analysis of barley single-nucleotide polymorphism (SNP) data, however, revealed a significant association between an HvNF-YB3 SNP and heading date. While it is unknown whether HvNF-YB3 directly contributes to heading date regulation, the results implied that HvNF-YB3 may also have conserved function in flowering time (heading date in barley) control. Further studies are needed to directly verify these gene functions in barley. Barley NF-YBs showed different expression patterns associated with tissue types, developmental stages, and response to different stress treatments, suggesting that barley NF-YBs may be involved in other physiological processes.

Role of nuclear factor Y in stress-induced activation of the herpes simplex virus type 1 ICP0 promoter.

Herpesviruses are characterized by the ability to establish lifelong latent infections and to reactivate periodically, leading to recurrent disease. The herpes simplex virus type 1 (HSV-1) genome is maintained in a quiescent state in sensory neurons during latency, which is characterized by the absence of detectable viral protein synthesis. Cellular factors induced by stress may act directly on promoters within the latent viral genome to induce the transcription of viral genes and trigger reactivation. In order to identify which viral promoters are induced by stress and elucidate the cellular mechanism responsible for the induction, we generated a panel of HSV-1 promoter-luciferase constructs and measured their response to heat shock. Of the promoters tested, those of ICP0 and ICP22 were the most strongly upregulated after heat shock. Microarray analysis of lytically infected cells supported the upregulation of ICP0 and ICP22 promoters by heat shock. Mutagenic analysis of the ICP0 promoter identified two regions necessary for efficient heat-induced promoter activity, both containing predicted nuclear factor Y (NF-Y) sites, at bases -708 and -75 upstream of the transcriptional start site. While gel shift analysis confirmed NF-Y binding to both sites, only the site at -708 was important for efficient heat-induced activity. Reverse transcription-PCR analysis of selected viral transcripts in the presence of dominant-negative NF-Y confirmed the requirement for NF-Y in the induction of the ICP0 but not the ICP22 promoter by heat shock in lytically infected cells. These findings suggest that the immediate-early ICP0 gene may be among the first genes to be induced during the early events in HSV-1 reactivation, that NF-Y is important for this induction, and that other factors induce the ICP22 promoter.

NF-YC complexity is generated by dual promoters and alternative splicing.

The CCAAT box is a DNA element present in the majority of human promoters, bound by the trimeric NF-Y, composed of NF-YA, NF-YB, and NF-YC subunits. We describe and characterize novel isoforms of one of the two histone-like subunits, NF-YC. The locus generates a minimum of four splicing products, mainly located within the Q-rich activation domain. The abundance of each isoform is cell-dependent; only one major NF-YC isoform is present in a given cell type. The 37- and 50-kDa isoforms are mutually exclusive, and preferential pairings with NF-YA isoforms possess different transcriptional activities, with specific combinations being more active on selected promoters. The transcriptional regulation of the NF-YC locus is also complex, and mRNAs arise from the two promoters P1 and P2. Transient transfections, chromatin immunoprecipitations, and reverse transcription-PCRs indicate that P1 has a robust housekeeping activity; P2 possesses a lower basal activity, but it is induced in response to DNA damage in a p53-dependent way. Alternative promoter usage directly affects NF-YC splicing, with the 50-kDa transcript being excluded from P2. Specific functional inactivation of the 37-kDa isoform affects the basal levels of G(1)/S blocking and pro-apoptotic genes but not G(2)/M promoters. In summary, our data highlight an unexpected degree of complexity and regulation of the NF-YC gene, demonstrating the existence of a discrete cohort of NF-Y trimer subtypes resulting from the functional diversification of Q-rich transactivating subunits and a specific role of the 37-kDa isoform in suppression of the DNA damage-response under growing conditions.

Expression levels of histone deacetylases determine the cell fate of hematopoietic progenitors.

Histone deacetylases (HDACs) are globally implicated in the growth and differentiation of mammalian cells; however, relatively little is known about their specific roles in hematopoiesis. In this study, we investigated the expression of HDACs in human hematopoietic cells and their functions during hematopoiesis. The expression of HDACs was very low in hematopoietic progenitor cells, which was accompanied by histone hyperacetylation. HDACs were detectable in more differentiated progenitors and erythroid precursors but down-regulated in mature myeloid cells especially granulocytes. In contrast, acute myeloid leukemias showed HDAC overexpression and histone hypoacetylation. Transcription of the HDAC1 gene was repressed by CCAAT/enhancer binding proteins during myeloid differentiation, and activated by GATA-1 during erythro-megakaryocytic differentiation. Small interfering RNA-mediated knockdown of HDAC1 enhanced myeloid differentiation in immature hematopoietic cell lines and perturbed erythroid differentiation in progenitor cells. Myeloid but not erythro-megakaryocytic differentiation was blocked in mice transplanted with HDAC1-overexpressing hematopoietic progenitor cells. These findings suggest that HDAC is not merely an auxiliary factor of genetic elements but plays a direct role in the cell fate decision of hematopoietic progenitors.

Transcriptional activators HAP/NF-Y rescue a cytochrome c oxidase defect in yeast and human cells.

Cell survival and energy production requires a functional mitochondrial respiratory chain. Biogenesis of cytochrome c oxidase (COX), the last enzyme of the mitochondrial respiratory chain, is a very complicated process and requires the assistance of a large number of accessory factors. Defects in COX assembly alter cellular respiration and produce severe human encephalomyopathies. Mutations in SURF1, a COX assembly factor of exact unknown function, produce Leigh's syndrome (LS), the most frequent cause of COX deficiency in infants. In the yeast Saccharomyces cerevisiae, deletion of the SURF1 homologue SHY1 results in a similar COX deficiency. In order to identify genetic modifiers of the shy1 mutant phenotype, we have explored for genetic interactions involving SHY1. Here we report that overexpression of Hap4p, the catalytic subunit of the CCAAT binding transcriptional activator Hap2/3/4/5p complex, suppresses the respiratory defect of yeast shy1 mutants by increasing the expression of nuclear-encoded COX subunits that interact with the mitochondrially encoded Cox1p. Analogously, overexpression of the Hap complex human homologue NF-YA/B/C transcription complex in SURF1-deficient fibroblasts from an LS patient efficiently rescues their COX deficiency.

Systematic identification of cis-regulatory sequences active in mouse and human embryonic stem cells.

Understanding the transcriptional regulation of pluripotent cells is of fundamental interest and will greatly inform efforts aimed at directing differentiation of embryonic stem (ES) cells or reprogramming somatic cells. We first analyzed the transcriptional profiles of mouse ES cells and primordial germ cells and identified genes upregulated in pluripotent cells both in vitro and in vivo. These genes are enriched for roles in transcription, chromatin remodeling, cell cycle, and DNA repair. We developed a novel computational algorithm, CompMoby, which combines analyses of sequences both aligned and non-aligned between different genomes with a probabilistic segmentation model to systematically predict short DNA motifs that regulate gene expression. CompMoby was used to identify conserved overrepresented motifs in genes upregulated in pluripotent cells. We show that the motifs are preferentially active in undifferentiated mouse ES and embryonic germ cells in a sequence-specific manner, and that they can act as enhancers in the context of an endogenous promoter. Importantly, the activity of the motifs is conserved in human ES cells. We further show that the transcription factor NF-Y specifically binds to one of the motifs, is differentially expressed during ES cell differentiation, and is required for ES cell proliferation. This study provides novel insights into the transcriptional regulatory networks of pluripotent cells. Our results suggest that this systematic approach can be broadly applied to understanding transcriptional networks in mammalian species.

The Sp1 and CBF/NF-Y transcription factors cooperatively regulate the mouse pro-alpha3(V) collagen gene (Col5a3) in osteoblastic cells.

The purpose of this study was to clarify the mechanism responsible for the transcriptional regulation of the mouse Col5a3 gene in osteoblastic cells. Transient transfection into rat osteosarcoma ROS17/2.8 cells demonstrated that a region from nucleotides 337 to 1 was involved in the transcriptional activity of the Col5a3 gene. An electrophoretic mobility shift assay showed that Sp1/Sp3 and CBF/NF-Y bound to a GC-rich domain (194/186) and a CCAAT box (134/130) in the Col5a3 gene, respectively. Introduction of mutations or deletion into a GC-rich domain, the CCAAT box, or both elements decreased the transcription activity. Overexpression of Sp1 increases the transcription activity and interferes with Sp family binding to the GC-rich domain to decrease promoter activity. Therefore, the transcription of the mouse Col5a3 gene is cooperatively regulated by Sp1 and CBF/NF-Y in osteoblastic cells.