Skeletal muscle differentiation induces wide-ranging nucleosome repositioning in muscle gene promoters.

In a previous report, we demonstrated that Cbx1, PurB and Sp3 inhibited cardiac muscle differentiation by increasing nucleosome density around cardiac muscle gene promoters. Since cardiac and skeletal muscle express many of the same proteins, we asked if Cbx1, PurB and Sp3 similarly regulated skeletal muscle differentiation. In a C2C12 model of skeletal muscle differentiation, Cbx1 and PurB knockdown increased myotube formation. In contrast, Sp3 knockdown inhibited myotube formation, suggesting that Sp3 played opposing roles in cardiac muscle and skeletal muscle differentiation. Consistent with this finding, Sp3 knockdown also inhibited various muscle-specific genes. The Cbx1, PurB and Sp3 proteins are believed to influence gene-expression in part by altering nucleosome position. Importantly, we developed a statistical approach to determine if changes in nucleosome positioning were significant and applied it to understanding the architecture of muscle-specific genes. Through this novel statistical approach, we found that during myogenic differentiation, skeletal muscle-specific genes undergo a set of unique nucleosome changes which differ significantly from those shown in commonly expressed muscle genes. While Sp3 binding was associated with nucleosome loss, there appeared no correlation with the aforementioned nucleosome changes. In summary, we have identified a novel role for Sp3 in skeletal muscle differentiation and through the application of quantifiable MNase-seq have discovered unique fingerprints of nucleosome changes for various classes of muscle genes during myogenic differentiation.

Specificity Proteins (Sp) and Cancer.

The specificity protein (Sp) transcription factors (TFs) Sp1, Sp2, Sp3 and Sp4 exhibit structural and functional similarities in cancer cells and extensive studies of Sp1 show that it is a negative prognostic factor for patients with multiple tumor types. In this review, the role of Sp1, Sp3 and Sp4 in the development of cancer and their regulation of pro-oncogenic factors and pathways is reviewed. In addition, interactions with non-coding RNAs and the development of agents that target Sp transcription factors are also discussed. Studies on normal cell transformation into cancer cell lines show that this transformation process is accompanied by increased levels of Sp1 in most cell models, and in the transformation of muscle cells into rhabdomyosarcoma, both Sp1 and Sp3, but not Sp4, are increased. The pro-oncogenic functions of Sp1, Sp3 and Sp4 in cancer cell lines were studied in knockdown studies where silencing of each individual Sp TF decreased cancer growth, invasion and induced apoptosis. Silencing of an individual Sp TF was not compensated for by the other two and it was concluded that Sp1, Sp3 and Sp4 are examples of non-oncogene addicted genes. This conclusion was strengthened by the results of Sp TF interactions with non-coding microRNAs and long non-coding RNAs where Sp1 contributed to pro-oncogenic functions of Sp/non-coding RNAs. There are now many examples of anticancer agents and pharmaceuticals that induce downregulation/degradation of Sp1, Sp3 and Sp4, yet clinical applications of drugs specifically targeting Sp TFs are not being used. The application of agents targeting Sp TFs in combination therapies should be considered for their potential to enhance treatment efficacy and decrease toxic side effects.

A novel Cbx1, PurB, and Sp3 complex mediates long-term silencing of tissue- and lineage-specific genes.

miRNA-based cellular fate reprogramming offers an opportunity to investigate the mechanisms of long-term gene silencing. To further understand how genes are silenced in a tissue-specific manner, we leveraged our miRNA-based method of reprogramming fibroblasts into cardiomyocytes. Through screening approaches, we identified three proteins that were downregulated during reprogramming of fibroblasts into cardiomyocytes: heterochromatin protein Cbx1, transcriptional activator protein PurB, and transcription factor Sp3. We show that knockdown of Cbx1, PurB, and Sp3 was sufficient to induce cardiomyocyte gene expression in fibroblasts. Similarly, gene editing to ablate Cbx1, PurB, and Sp3 expression induced fibroblasts to convert into cardiomyocytes in vivo. Furthermore, high-throughput DNA sequencing and coimmunoprecipitation experiments indicated that Cbx1, PurB, and Sp3 also bound together as a complex and were necessary to localize nucleosomes to cardiomyocyte genes on the chromosome. Finally, we found that the expression of these genes led to nucleosome modification via H3K27me3 (trimethylated histone-H3 lysine-27) deposition through an interaction with the polycomb repressive PRC2 complex. In summary, we conclude that Cbx1, PurB, and Sp3 control cell fate by actively repressing lineage-specific genes.

Caspase-Mediated Cleavage of the Transcription Factor Sp3: Possible Relevance to Cancer and the Lytic Cycle of Kaposi's Sarcoma-Associated Herpesvirus.

The open reading frame 50 (ORF50) protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is the master regulator essential for initiating the viral lytic cycle. Previously, we have demonstrated that the ORF50 protein can cooperate with Sp3 to synergistically activate a set of viral and cellular gene promoters through highly conserved ORF50-responsive elements that harbor a Sp3-binding motif. Herein, we show that Sp3 undergoes proteolytic cleavage during the viral lytic cycle, and the cleavage of Sp3 is dependent on caspase activation. Since similar cleavage patterns of Sp3 could be detected in both KSHV-positive and KSHV-negative lymphoma cells undergoing apoptosis, the proteolytic cleavage of Sp3 could be a common event during apoptosis. Mutational analysis identifies 12 caspase cleavage sites in Sp3, which are situated at the aspartate (D) positions D17, D19, D180, D273, D275, D293, D304 (or D307), D326, D344, D530, D543, and D565. Importantly, we noticed that three stable Sp3 C-terminal fragments generated through cleavage at D530, D543, or D565 encompass an intact DNA-binding domain. Like the full-length Sp3, the C-terminal fragments of Sp3 could still retain the ability to cooperate with ORF50 protein to activate specific viral and cellular gene promoters synergistically. Collectively, our findings suggest that despite the proteolytic cleavage of Sp3 under apoptotic conditions, the resultant Sp3 fragments may retain biological activities important for the viral lytic cycle or for cellular apoptosis. IMPORTANCE The ORF50 protein of Kaposi's sarcoma-associated herpesvirus (KSHV) is the key viral protein that controls the switch from latency to lytic reactivation. It is a potent transactivator that can activate target gene promoters via interacting with other cellular DNA-binding transcription factors, such as Sp3. In this report, we show that Sp3 is proteolytically cleaved during the viral lytic cycle, and up to 12 caspase cleavage sites are identified in Sp3. Despite the proteolytic cleavage of Sp3, several resulting C-terminal fragments that have intact zinc-finger DNA-binding domains still retain substantial influence in the synergy with ORF50 to activate specific gene promoters. Overall, our studies elucidate the caspase-mediated cleavage of Sp3 and uncover how ORF50 utilizes the cleavage fragments of Sp3 to transactivate specific viral and cellular gene promoters.

The androgen receptor inhibits transcription of GPER1 by preventing Sp1 and Sp3 from binding to the promoters in prostate cancer cells.

G-1, a GPER1 agonist, was shown to inhibit the growth of castration-resistant mouse xenografts but not their parental androgen-dependent tumors. It is currently unknown how the androgen receptor (AR) represses GPER1 expression. Here, we found that two GPER1 mRNA variants (GPER1v2 and GPER1v4) were transcriptionally repressed, not via transcript destabilization, by the androgen-activated AR. Although no AR binding was found in all active promoters near GPER1, data from promoter assays suggested that both variants' promoters were inhibited by androgen treatment. Site-directed mutagenesis on Sp1/Sp3 binding sites revealed their role in supporting the basal expression of GPER1. Knockdown of Sp1 and Sp3 together but not separately repressed GPER1 expression whereas overexpression of both Sp1 and Sp3 together was required to alleviate AR repression of GPER1. Based on the chromatin immunoprecipitation data, Sp3 was found to bind to the promoters prior to the binding of Sp1 and RNA polymerase II. However, the binding of all three transcription factors was inhibited by DHT treatment. Concordantly, DHT treatment induced nuclear interactions between AR and Sp1 or Sp3. Taken together, these results indicate that AR represses transcription of GPER1 by binding to Sp1 and Sp3 independently to prevent their transactivation of the GPER1 promoters.

Contribution of the Transcription Factors Sp1/Sp3 and AP-1 to Clusterin Gene Expression during Corneal Wound Healing of Tissue-Engineered Human Corneas.

In order to reduce the need for donor corneas, understanding of corneal wound healing and development of an entirely tissue-engineered human cornea (hTECs) is of prime importance. In this study, we exploited the hTEC to determine how deep wound healing affects the transcriptional pattern of corneal epithelial cells through microarray analyses. We demonstrated that the gene encoding clusterin (CLU) has its expression dramatically repressed during closure of hTEC wounds. Western blot analyses confirmed a strong reduction in the expression of the clusterin isoforms after corneal damage and suggest that repression of CLU gene expression might be a prerequisite to hTEC wound closure. Transfection with segments from the human CLU gene promoter revealed the presence of three regulatory regions: a basal promoter and two more distal negative regulatory regions. The basal promoter bears DNA binding sites for very potent transcription factors (TFs): Activator Protein-1 (AP-1) and Specificity protein-1 and 3 (Sp1/Sp3). By exploiting electrophoretic mobility shift assays (EMSA), we demonstrated that AP-1 and Sp1/Sp3 have their DNA binding site overlapping with one another in the basal promoter of the CLU gene in hCECs. Interestingly, expression of both these TFs is reduced (at the protein level) during hTEC wound healing, thereby contributing to the extinction of CLU gene expression during that process. The results of this study contribute to a better understanding of the molecular mechanisms accounting for the repression of CLU gene expression during corneal wound healing.

Editing of DNA methylation using CRISPR/Cas9 and a ssDNA template in human cells.

Programmable DNA methylation is required for understanding of transcriptional regulation and elucidating gene functions. We previously reported that MMEJ-based promoter replacement enabled targeted DNA methylation in human cells. ssDNA-mediated knock-in has recently been reported to completely reduce random integrations. We speculated that by changing MMEJ-to ssDNA-based knock-in, targeted DNA methylation may be achieved through a hemimethylation-symmetric methylation pathway. We herein successfully developed a new system that enables the replacement of an unmethylated promoter with a methylated ssDNA promoter through ssDNA-based knock-in. A DNA methylation ratio of approximately 100% was achieved at the cancer-associated gene SP3 in HEK293 cells. The present results provide a promising framework for artificial epigenetic modifications.

Dietary biotin supplementation increases proliferation pathways in mice testes without affecting serum follicle-stimulating hormone levels and stem cell factor expression.

Supplements containing pharmacological concentrations of biotin are commercially available. The mechanisms by which biotin at pharmacological concentrations exerts its action have been the subject of multiple investigations, particularly for biotin's medicinal potential and wide use for cosmetic purposes. Several studies have reported that biotin supplementation increases cell proliferation; however, the mechanisms involved in this effect have not yet been characterized. In a previous study, we found that a biotin-supplemented diet increased spermatogonia proliferation. The present study was focused on investigating the molecular mechanisms involved in biotin-induced testis cell proliferation. Male BALB/cAnNHsd mice were fed a control or a biotin-supplemented diet (1.76 or 97.7 mg biotin/kg diet) for eight weeks. Compared with the control group, the biotin-supplemented mice presented augmented protein abundance of the c-kit-receptor and pERK1/2Tyr204 and pAKTSer473, the active forms of ERK/AKT proliferation signaling pathways. No changes were observed in the testis expression of the stem cell factor and in the serum levels of the follicle-stimulating hormone. Analysis of mRNA abundance found an increase in cyclins Ccnd3, Ccne1, Ccna2; Kinases Cdk4, Cdk2; and E2F; and Sp1 & Sp3 transcription factors. Decreased expression of cyclin-dependent kinase inhibitor 1a (p21) was observed but not of Cdkn2a inhibitor (p16). The results of the present study identifies, for the first time, the mechanisms associated with biotin supplementation-induced cell proliferation, which raises concerns about the effects of biotin on male reproductive health because of its capacity to cause hyperplasia, especially because this vitamin is available in large amounts without regulation.

lncRNA UCA1 induced by SP1 and SP3 forms a positive feedback loop to facilitate malignant phenotypes of colorectal cancer via targeting miR-495.

AIMS: Long noncoding RNA (LncRNA) urothelial cancer associated 1 (UCA1) was dysregulated in colorectal cancers (CRC) and promoted tumor progression of CRC. The aims of this study are to further investigate the underlying mechanism. MAIN METHODS: Short hairpin RNAs (shRNAs) were applied for gene knockdown. microRNA mimic and pcDNA-UCA1 plasmids were transfected for miR-495 and UCA1 overexpression, respectively. MTT was applied to determine cell viability and sensitivity of 5-fluorouracil (FU). Transwell assays were performed to evaluate cell migration/invasion. Angiogenesis was evaluated by tube formation. Western blotting and quantitative PCR were utilized for protein and mRNA detection, respectively. The interaction of UCA1, miR-495 and SP1/SP3 were explored by dual-luciferase assay. RNA pulldown was adopted to determine the UCA1/miR-495 interaction. KEY FINDINGS: UCA1 was significantly upregulated in CRC tissues. UCA1 enhanced cell proliferation, migration/invasion, angiogenesis, epithelial-mesenchymal transition, and resistance to 5-FU in CRC cell lines. MiR-495 was inversely correlated to the expression of UCA1. The results indicated that UCA1 sponged miR-495, leading to the disinhibition of SP1/SP3 expression. SP1/SP3 induced the expression of DNA methyltransferases and, in turn, contributed to UCA1 mediated tumor-promoting actions. Reduction of SP1/SP3 exerted anti-cancer effects, which can be reversed by forced expression of UCA1. SIGNIFICANCE: UCA1-miR-495-SP1/SP3 axis is dysregulated in CRC and contributed to malignant phenotypes of CRC. UCA1-SP1/SP3 may form a positive feedback loop in CRC.

A novel Lnc408 maintains breast cancer stem cell stemness by recruiting SP3 to suppress CBY1 transcription and increasing nuclear ß-catenin levels.

Tumor initiation, development, and relapse may be closely associated with cancer stem cells (CSCs). The complicated mechanisms underlying the maintenance of CSCs are keeping in illustration. Long noncoding RNAs (lncRNAs), due to their multifunction in various biological processes, have been indicated to play a crucial role in CSC renewal and stemness maintenance. Using lncRNA array, we identified a novel lncRNA (named lnc408) in epithelial-mesenchymal transition-related breast CSCs (BCSCs). The lnc408 is high expressed in BCSCs in vitro and in vivo. The enhanced lnc408 is critical to BCSC characteristics and tumorigenesis. Lnc408 can recruit transcript factor SP3 to CBY1 promoter to serve as an inhibitor in CBY1 transcription in BCSCs. The high expressed CBY1 in non-BCSC interacts with 14-3-3 and ß-catenin to form a ternary complex, which leads a translocation of the ternary complex into cytoplasm from nucleus and degradation of ß-catenin in phosphorylation-dependent pattern. The lnc408-mediated decrease of CBY1 in BCSCs impairs the formation of 14-3-3/ß-catenin/CBY1 complex, and keeps ß-catenin in nucleus to promote CSC-associated CD44, SOX2, Nanog, Klf4, and c-Myc expressions and contributes to mammosphere formation; however, restoration of CBY1 expression in tumor cells reduces BCSC and its enrichment, thus lnc408 plays an essential role in maintenance of BCSC stemness. In shortly, these findings highlight that the novel lnc408 functions as an oncogenic factor by recruiting SP3 to inhibit CBY1 expression and ß-catenin accumulation in nucleus to maintain stemness properties of BCSCs. Lnc408-CBY1-ß-catenin signaling axis might serve as a new diagnostic and therapeutic target for breast cancer.

Phosphorylation of Specificity Protein 3 Is Critical for Activation of ß4-Galactosyltransferase 3 Gene Promoter in SH-SY5Y Human Neuroblastoma Cell Line.

Elevated expression of ß4-galactosyltransferase (ß4GalT) 3 is correlated with poor clinical outcome of neuroblastoma patients. Our recent study has revealed that the transcription of the ß4GalT3 gene is activated by Specificity protein (Sp) 3 in SH-SY5Y human neuroblastoma cell line. Here we report the biological significance of the Sp3 phosphorylation in the transcriptional activation of the ß4GalT3 gene. The treatment of SH-SY5Y cells with 10% fetal bovine serum (FBS) increased the mitogen-activated protein kinase (MAPK) signaling and the promoter activity of the ß4GalT3 gene. Meanwhile, the treatment with U0126, an inhibitor for MAPK kinase, decreased the MAPK signaling and the promoter activity. These findings indicate that the transcriptional activation of the ß4GalT3 gene is mediated by the MAPK signaling. In SH-SY5Y cells cultured in the medium containing 10% FBS, the serine (Ser) residues in Sp3 were phosphorylated. Human Sp3 contains four Ser residues, Ser73, Ser563, Ser566, and Ser646, as the putative phosphorylation sites. Sp3 mutant with the mutation of Ser73 did not decrease the promoter activation of the ß4GalT3 gene, indicating that Ser73 is uninvolved in the promoter activation of the ß4GalT3 gene by Sp3. In contrast, Sp3 mutants with the mutations of Ser563, Ser566, and Ser646 significantly reduced the promoter activation by Sp3. The results suggest that the phosphorylation of these Ser residues is implicated in the promoter activation by Sp3. This study demonstrates that the phosphorylation of Sp3 plays important roles in the transcriptional activation of the ß4GalT3 gene in human neuroblastoma.

SP3 is associated with migration, invasion, and Akt/PKB signalling in MDA-MB-231 breast cancer cells.

Specificity proteins (SPs) have pro-oncogenic functions in cancer cells, ranging from cancer cell proliferation, migration, invasion, and angiogenesis. There is strong evidence that several antineoplastic drugs target depletion of SP proteins via different pathways. However, the mode of action of SP3 and the underlying consequences of its depletion are not well understood. Here, we demonstrate that SP3 is overexpressed in invasive breast cancer cells vs normal counterparts. The gene expression analysis from The Cancer Genome Atlas datasets indicated that SP3 is strongly correlated with Akt signalling-related proteins, G protein subunit alpha 13, and RAB33B (RAB33B, member RAS oncogene family). RNA interference of SP3 decreased active phosphorylation of Akt at serine and threonine sites. These findings indicate that SP3 exhibits a pro-oncogenic function, which clearly fits the description of an nononcogene addiction gene. Future analyses are prompted to uncover the SP3 gene regulation function and to reveal downstream targets of SP3 in breast cancer.

Estrogen-regulated expression of SK3 channel in rat colonic smooth muscle contraction.

AIMS: Estrogen can induce inhibition of colonic smooth muscle contraction in male and female mice, which may lead to constipation; however, the mechanisms of inhibition are poorly understood. Hence, this study investigated the effect of estrogen on rat colonic smooth muscle contraction and role of small-conductance Ca2+-activated K+ 3 (SK3) and transcription factors (Sp1 and Sp3) in the underlying mechanisms. MAIN METHODS: The experiment included 24 female Sprague-Dawley (SD) rats divided into 4 groups. The rats were oophorectomized surgically, and a silicone tube containing blank solvent, 0.3 mg/mL estrogen (E2), equal-concentration of estrogen and estrogen receptor antagonist (EI), and bovine serum albumin-E2 (BSA-E2) was implanted. The rats were sacrificed on day 14. The molecular insights were confirmed using real-time quantitative reverse transcription PCR (qRT-PCR) and western blot analyses to determine the effect of estrogenic stimulation on gene and protein expression analyses, respectively. KEY FINDINGS: The E2 group showed significantly greater SK3 expression (P < .005) compared with other groups and significantly lowers smooth muscle cell (SMC) contractility (P < .005). Estrogen stimulation and SK3 overexpression resulted in a significant decrease (P < .05) in Ca2+ mobilization in the E2 group versus the control group. Further, the E2 group showed significantly higher Sp1 mRNA (P < .05) but lower Sp3 mRNA expression (P < .05) and protein expression (P < .001) compared with other groups. SIGNIFICANCE: E2 may promote SK3 expression by its genomic effect and inhibit colonic contraction by affecting SK3 expression via an interaction between Sp1 and Sp3.

GR/Sp3/HDAC1/UGDH signaling participated in the maternal dexamethasone-induced dysplasia of the rat fetal growth plate.

Maternal dexamethasone decreases the body length of the newborn. However, whether dexamethasone inhibits the development of the growth plate of the fetal long bone is still unknown. Here, we found that lengths of fetal femur and growth plate were both shorter in the fetuses with maternal dexamethasone (0.2 mg/kg.d from gestation day 9 to 20), with a decreased proteoglycan content of the growth plate in the fetal rat. Notable decreases in both the gene expression and H3K9 acetylation of UDP-glucose dehydrogenase (Ugdh) gene, which codes a key enzyme in the proteoglycan biosynthesis in the chondrocyte, were also observed. Meanwhile, up-regulation of glucocorticoid receptor (GR), specific protein 3 (Sp3), and histone deacetylase 1 (Hdac1) gene expression were detected in the fetal growth plate. Similar changes were also observed in the chondrogenic rat bone marrow stromal cells (BMSCs) with excessive exogenous dexamethasone. However, antagonizing GR with RU486 and silencing Hdac1 or Sp3 with specific siRNAs could all stimulate the H3K9 acetylation and gene expression of Ugdh previously inhibited by dexamethasone. Meanwhile, dexamethasone also induced the nuclear translocation of GR, which further directly bound to the Ugdh promoter and interacted with HDAC1 and Sp3, respectively. Collectively, our study revealed that maternal dexamethasone induced the direct binding of GR to the Ugdh promoter of the chondrocyte in the rat fetal growth plate, which recruited HDAC1 and Sp3, induced deacetylation of the H3K9, and subsequently inhibited Ugdh gene expression. Such changes further led to attenuated proteoglycan synthesis in the developing chondrocyte and therefore disrupted the development of growth plate and fetal long bone.

Sp3 Transcription Factor Cooperates with the Kaposi's Sarcoma-Associated Herpesvirus ORF50 Protein To Synergistically Activate Specific Viral and Cellular Gene Promoters.

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded open reading frame 50 (ORF50) protein is the key transactivator responsible for the latent-to-lytic switch. Here, we investigated the transcriptional activation of the ORF56 gene (encoding a primase protein) by ORF50 and successfully identified an ORF50-responsive element located in the promoter region between positions -97 and -44 (designated 56p-RE). This 56p-RE element contains a noncanonical RBP-Jκ-binding sequence and a nonconsensus Sp1/Sp3-binding sequence. Electrophoretic mobility shift assays revealed that RBP-Jκ, Sp3, and ORF50 could form stable complexes on the 56p-RE element. Importantly, transient-reporter analysis showed that Sp3, but not RBP-Jκ or Sp1, acts in synergy with ORF50 to activate the 56p-RE-containing reporter construct, and the synergy mainly depends on the Sp1/Sp3-binding region of the 56p-RE element. Sequence similarity searches revealed that the promoters for ORF21 (thymidine kinase), ORF60 (ribonucleotide reductase, small subunit), and cellular interleukin-10 (IL-10) contain a sequence motif similar to the Sp1/Sp3-binding region of the 56p-RE element, and we found that these promoters could also be synergistically activated by ORF50 and Sp3 via the conserved motifs. Noteworthily, the conversion of the Sp1/Sp3-binding sequence of the 56p-RE element into a consensus high-affinity Sp-binding sequence completely lost the synergistic response to ORF50 and Sp3. Moreover, transcriptional synergy could not be detected through other ORF50-responsive elements from the viral PAN, K12, ORF57, and K6 promoters. Collectively, the results of our study demonstrate that ORF50 and Sp3 can act in synergy on the transcription of specific gene promoters, and we find a novel conserved cis-acting motif in these promoters essential for transcriptional synergy.IMPORTANCE Despite the critical role of ORF50 in the KSHV latent-to-lytic switch, the molecular mechanism by which ORF50 activates its downstream target genes, especially those that encode the viral DNA replication enzymes, is not yet fully understood. Here, we find that ORF50 can cooperate with Sp3 to synergistically activate promoters of the viral ORF56 (primase), ORF21 (thymidine kinase), and ORF60 (ribonucleotide reductase) genes via similar Sp1/Sp3-binding motifs. Additionally, the same synergistic effect can be seen on the promoter of the cellular IL-10 gene. Overall, our data reveal an important role for Sp3 in ORF50-mediated transactivation, and we propose a new subclass of ORF50-responsive elements in viral and cellular promoters.

Transcriptional regulatory network controlling the ontogeny of hematopoietic stem cells.

Hematopoietic stem cell (HSC) ontogeny is accompanied by dynamic changes in gene regulatory networks. We performed RNA-seq and histone mark ChIP-seq to define the transcriptomes and epigenomes of cells representing key developmental stages of HSC ontogeny in mice. The five populations analyzed were embryonic day 10.5 (E10.5) endothelium and hemogenic endothelium from the major arteries, an enriched population of prehematopoietic stem cells (pre-HSCs), fetal liver HSCs, and adult bone marrow HSCs. Using epigenetic signatures, we identified enhancers for each developmental stage. Only 12% of enhancers are primed, and 78% are active, suggesting the vast majority of enhancers are established de novo without prior priming in earlier stages. We constructed developmental stage-specific transcriptional regulatory networks by linking enhancers and predicted bound transcription factors to their target promoters using a novel computational algorithm, target inference via physical connection (TIPC). TIPC predicted known transcriptional regulators for the endothelial-to-hematopoietic transition, validating our overall approach, and identified putative novel transcription factors, including the broadly expressed transcription factors SP3 and MAZ. Finally, we validated a role for SP3 and MAZ in the formation of hemogenic endothelium. Our data and computational analyses provide a useful resource for uncovering regulators of HSC formation.

Robust hematopoietic specification requires the ubiquitous Sp1 and Sp3 transcription factors.

BACKGROUND: Both tissue-specific and ubiquitously expressed transcription factors, such as Sp-family members, are required for correct development. However, the molecular details of how ubiquitous factors are involved in programming tissue-specific chromatin and thus participate in developmental processes are still unclear. We previously showed that embryonic stem cells lacking Sp1 DNA-binding activity (Sp1ΔDBD/ΔDBD cells) are able to differentiate into early blood progenitors despite the inability of Sp1 to bind chromatin without its DNA-binding domain. However, gene expression during differentiation becomes progressively deregulated, and terminal differentiation is severely compromised. RESULTS: Here, we studied the cooperation of Sp1 with its closest paralogue Sp3 in hematopoietic development and demonstrate that Sp1 and Sp3 binding sites largely overlap. The complete absence of either Sp1 or Sp3 or the presence of the Sp1 DNA-binding mutant has only a minor effect on the pattern of distal accessible chromatin sites and their transcription factor binding motif content, suggesting that these mutations do not affect tissue-specific chromatin programming. Sp3 cooperates with Sp1ΔDBD/ΔDBD to enable hematopoiesis, but is unable to do so in the complete absence of Sp1. Using single-cell gene expression analysis, we show that the lack of Sp1 DNA binding leads to a distortion of cell fate decision timing, indicating that stable chromatin binding of Sp1 is required to maintain robust differentiation trajectories. CONCLUSIONS: Our findings highlight the essential contribution of ubiquitous factors such as Sp1 to blood cell development. In contrast to tissue-specific transcription factors which are required to direct specific cell fates, loss of Sp1 leads to a widespread deregulation in timing and coordination of differentiation trajectories during hematopoietic specification.

lncRNA TINCR participates in ALA-PDT-induced apoptosis and autophagy in cutaneous squamous cell carcinoma.

This study aims to investigate whether terminal differentiation-induced ncRNA (TINCR) has an effect on apoptosis and autophagy induced by ALA-PDT in cutaneous squamous cell carcinoma (CSCC). A431 cells were treated with 5-aminolevulinic acid (ALA) solution at different concentrations and for different duration time. A431 cell viability was detected by Cell Counting Kit-8 (CCK-8) assay, relative TINCR messenger RNA expression was detected by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). A431 cell apoptosis was examined by flow cytometry. Relative apoptosis/autophagy-related protein expression was analyzed by Western blot analysis. The effect of TINCR on cell autophagy was detected by RFP-LC3 immunofluorescence assay. Reactive oxygen species concentration was detected by 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. Relative expressions of ERK1/2 and specificity protein 3 (Sp3) in A43 cells were detected by Western blot analysis and qRT-PCR. Sp3 binding sites were analyzed by ChIP-qPCR. The relative transcription activity was measured with luciferase reporter assay. ALA-PDT treatment at 3.2 mmol/L for 120 minutes significantly promoted TINCR expression in CSCC A431 cells, and TINCR promoted ALA-PDT-induced apoptosis and cell autophagy. Furthermore, ALA-PDT promoted TINCR expression through ERK1/2-SP3 pathway. Sp3 promoted TINCR transcription by binding TINCR promoters. Our data indicated that TINCR involves in ALA-PDT-induced apoptosis and autophagy in CSCC.

The transcription factor SP3 drives TNF-α expression in response to Smac mimetics.

The controlled production and downstream signaling of the inflammatory cytokine tumor necrosis factor-α (TNF-α) are important for immunity and its anticancer effects. Although chronic stimulation with TNF-α is detrimental to the health of the host in several autoimmune and inflammatory disorders, TNF-α-contrary to what its name implies-leads to cancer formation by promoting cell proliferation and survival. Smac mimetic compounds (SMCs), small-molecule antagonists of inhibitor of apoptosis proteins (IAPs), switch the TNF-α signal from promoting survival to promoting death in cancer cells. Using a genome-wide siRNA screen to identify factors required for SMC-to-TNF-α-mediated cancer cell death, we identified the transcription factor SP3 as a critical molecule in both basal and SMC-induced production of TNF-α by engaging the nuclear factor κB (NF-κB) transcriptional pathway. Moreover, the promotion of TNF-α expression by SP3 activity confers differential sensitivity of cancer versus normal cells to SMC treatment. The key role of SP3 in TNF-α production and signaling will help us further understand TNF-α biology and provide insight into mechanisms relevant to cancer and inflammatory disease.

Fibronectin fragment inhibits xylosyltransferase-1 expression by regulating Sp1/Sp3- dependent transcription in articular chondrocytes.

OBJECTIVE: We investigated the effects of 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) on xylosyltransferase-1 (XT-1), an essential anabolic enzyme that catalyzes the initial and rate-determining step in glycosaminoglycan chain synthesis, in human primary chondrocytes. METHODS: Proteoglycan and XT-1 expression in cartilage tissue was analyzed using safranin O staining and immunohistochemistry. The effects of 29-kDa FN-f on XT-1 expression and its relevant signaling pathway were analyzed by quantitative real-time-PCR, immunoblotting, chromatin immunoprecipitation, and immunoprecipitation assays. The receptors for 29-kDa FN-f were investigated using small interference RNA and blocking antibodies. RESULTS: The expression of XT-1 was significantly lower in human osteoarthritis cartilage than in normal cartilage. Intra-articular injection of 29-kDa FN-f reduced proteoglycan levels and XT-1 expression in murine cartilage. In addition, in 29-kDa FN-f-treated cells, XT-1 expression was significantly suppressed at both the mRNA and protein levels, modulated by the transcription factors specificity protein 1 (Sp1), Sp3, and activator protein 1 (AP-1). The 29-kDa FN-f suppressed the binding of Sp1 to the promoter region of XT-1 and enhanced the binding of Sp3 and AP-1. Inhibition of mitogen-activated protein kinase and nuclear factor kappa B signaling pathways restored the 29-kDa FN-f-inhibited XT-1 expression, along with the altered expression of Sp1 and Sp3. Blockading toll-like receptor 2 (TLR-2) and integrin α5ß1 via siRNA and blocking antibodies revealed that the effects of 29-kDa FN-f on XT-1 expression were mediated through the TLR-2 and integrin α5ß1 signaling pathways. CONCLUSION: These results demonstrate that 29-kDa FN-f negatively affects cartilage anabolism by regulating glycosaminoglycan formation through XT-1.