Transcription Factor CP2-like 1 Research Articles

Fluorescence-activated nuclear sorting (FANS) of nuclei from in vitro-generated syncytiotrophoblast

Large, multinucleated cells, like syncytiotrophoblasts (STB), are not readily analyzed by standard methods used for single cells, such as single-cell RNA-sequencing and fluorescence-activated cellular sorting (FACS). Here we have demonstrated that fluorescence-activated nuclear sorting (FANS) is suitable to analyze nuclei from STB. Human pluripotent stem cells (PSCs) can be differentiated into a mixed trophoblast populations comprising approximately 20 % STB by treatment with BMP4 (Bone Morphogenetic Protein-4), plus A83-01 and PD173074, inhibitors of activin and FGF2 signaling, respectively (the BAP model) in about a week. Here we demonstrate that FANS can be used to separate two types of STB nuclei from the nine different clusters of trophoblast nuclei previously identified in the BAP model by single nucleus RNA sequencing (snRNAseq). Rather than using cell surface markers, as in FACS, transcription factors in various combinations were employed to target specific nuclear types. Nuclei were isolated at d 8 of BAP differentiation of H1 human embryonic stem cells and fixed in 4% paraformaldehyde. After permeabilization in 0.1 % triton X-100, nuclei were incubated for 3 and 1 h at 4 °C with primary and secondary antibodies respectively and nuclear samples were then subjected to FANS. By using markers identified by snRNA and immunohistochemistry, nuclei were first sorted into a topoisomerase, or TOP1 (Topoisomerase-1), bright population and then into the two STB subpopulations by using antibodies to JUNB (Jun B Proto-Oncogene) and TFCP2L1 (Transcription Factor CP2 Like 1). The protocol established here is simple, straightforward, and efficient and can be used on a relatively large scale to sort individual subtypes of nuclei from mixed populations of trophoblasts for further analysis.

Development of stemness-related signature to optimize prognosis prediction and identify XMD8-85 as a novel therapeutic compound for glioma

Glioma is a highly invasive and aggressive type of brain cancer with poor treatment response. Stemness-related transcription factors form a regulatory network that sustains the malignant phenotype of gliomas. We conducted an integrated analysis of stemness-related transcription factors using The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) datasets, established the characteristics of stemness-related transcription factors, including Octamer-Binding Protein 4 (OCT4), Meis Homeobox 1 (MEIS1), E2F Transcription Factor 1 (E2F1), Transcription Factor CP2 Like 1 (TFCP2L1), and RUNX Family Transcription Factor 1 (RUNX1). The characteristic of stemness-related transcription factors was identified as an independent prognostic factor for glioma patients. Patients in the high-risk group have a worse prognosis than those in the low-risk group. The glioma microenvironment in the high-risk group exhibited a more active immune status. Single-cell level analysis revealed that stem cell-like cells exhibited stronger intercellular communication than glioma cells. Meanwhile, patients in different risk stratification exhibited varying sensitivities to immunotherapy and small molecule drug therapy. XMD8-85 was more effective in the high-risk group, and its antitumor effects were validated both in vivo and in vitro. Our results indicate that this prognostic feature will assist clinicians in predicting the prognosis of glioma patients, guiding immunotherapy and personalized treatment, as well as the potential clinical application of XMD8-85 in glioma treatment, and helping to develop effective treatment strategies.

Mechanism of tumor‑derived extracellular vesicles in regulating renal cell carcinoma progression by the delivery of MALAT1.

Renal cell carcinoma (RCC) is a major healthcare burden globally. Tumor-derived extracellular vesicles (EVs) contribute to the formation of a pro-metastatic microenvironment. In the present study, we explored the role and mechanism of RCC cell 786-O-derived EVs (786-O-EVs) in RCC. First, 786-O-EVs were extracted and identified, and EV internalization of RCC cells was observed. RCC cell malignant behaviors and long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) expression patterns were detected before and after 786-O-EV treatment. MALAT1 was intervened to evaluate RCC cell behaviors. The downstream mechanism involving MALAT1 was predicted. In addition, the relationship among MALAT1, transcription factor CP2 like 1 (TFCP2L1) and ETS proto-oncogene 1, transcription factor (ETS1) was analyzed. TFCP2L1 expression patterns were measured after 786-O-EV exposure. Tumor xenograft formation assay and lung metastasis model were adopted to verify the role of 786-O-EVs in vivo in RCC. It was found that 786-O-EVs could be internalized by RCC cells. 786-O-EVs promoted RCC cell malignant behaviors, accompanied by elevated MALAT1 expression levels. The 786-O-EVs with MALAT1 knockdown attenuated the promotive effect of sole 786-O-EVs on RCC cells. MALAT1 located ETS1 in the TFCP2L1 promoter and negatively regulated TFCP2L1, and ETS1 protein could specifically bind to MALAT1. 786-O-EVs enhanced the binding of ETS1 and the TFCP2L1 promoter and decreased TFCP2L1 expression. In vivo, 786-O-EVs promoted tumor growth and RCC lung metastasis, which was suppressed following inhibition of MALAT1. Our findings indicated that 786-O-EVs promoted RCC invasion and metastasis by transporting MALAT1 to promote the binding of transcription factor ETS1 and TFCP2L1 promoter.

Gene manipulation in liver ductal organoids by optimized recombinant adeno-associated virus vectors

Understanding the mechanism of how liver ductal cells (cholangiocytes) differentiate into hepatocytes would permit liver-regenerative medicine. Emerging liver ductal organoids provide an ex vivo system to investigate cholangiocyte-to-hepatocyte differentiation. However, as current gene manipulation methods require organoid dissociation into single cells and have only low efficiency, it is difficult to dissect specific gene functions in these organoids. Here we developed the adeno-associated virus (AAV) vector AAV-DJ as a powerful tool to transduce mouse and human liver ductal organoids. Via AAV-DJ-mediated up- or down-regulation of target genes, we successfully manipulated cholangiocyte-to-hepatocyte differentiation. We induced differentiation by overexpressing the hepatocyte-specifying regulator hepatocyte nuclear factor 4α (HNF4α) and blocked differentiation by stimulating Notch signaling or interfering with Smad signaling. Further screening for transcriptional factors critical for cholangiocyte-to-hepatocyte differentiation identified HOP homeobox (HOPX), T-box 15 (TBX15), and transcription factor CP2-like 1 (TFCP2L1) as master regulators. We conclude that this highly efficient and convenient gene manipulation system we developed could facilitate investigation into genes involved in cell lineage transitions and enable application of engineered organoids in regenerative medicine.

The transcription factor TFCP2L1 induces expression of distinct target genes and promotes self-renewal of mouse and human embryonic stem cells

TFCP2L1 (transcription factor CP2-like 1) is a transcriptional regulator critical for maintaining mouse and human embryonic stem cell (ESC) pluripotency. However, the direct TFCP2L1 target genes are uncharacterized. Here, using gene overexpression, immunoblotting, quantitative real-time PCR, ChIP, and reporter gene assays, we show that TFCP2L1 primarily induces estrogen-related receptor β (Esrrb) expression that supports mouse ESC identity and also selectively enhances Kruppel-like factor 4 (Klf4) expression and thereby promotes human ESC self-renewal. Specifically, we found that in mouse ESCs, TFCP2L1 binds directly to the Esrrb gene promoter and regulates its transcription. Esrrb knockdown impaired Tfcp2l1's ability to induce interleukin 6 family cytokine (leukemia inhibitory factor)-independent ESC self-renewal and to reprogram epiblast stem cells to naïve pluripotency. Conversely, Esrrb overexpression blocked differentiation induced by Tfcp2l1 down-regulation. Moreover, we identified Klf4 as a direct TFCP2L1 target in human ESCs, bypassing the requirement for activin A and basic fibroblast growth factor in short-term human ESC self-renewal. Enforced Klf4 expression recapitulated the self-renewal-promoting effect of Tfcp2l1, whereas Klf4 knockdown eliminated these effects and caused loss of colony-forming capability. These findings indicate that TFCP2L1 functions differently in naïve and primed pluripotency, insights that may help elucidate the different states of pluripotency.

Regulatory gene network from a genome-wide association study for sow lifetime productivity traits.

Among swine reproductive traits, sow lifetime productivity (SLP) is considered a profitable trait in commercial pig farming. Notably, longevity and efficiency in SLP can be adopted as the key phenotype representing SLP. In this study, we conducted a co-association network analysis using results from a genome-wide association study for SLP-related traits. A total of 656 purebred Landrace female pigs were genotyped using a 60K SNP array. Significantly associated SNPs identified from the GWAS were annotated for the specific genes. Then, we constructed an association weight matrix to build a network based on the co-associations between the genes and 10 SLP traits. The entire network consisted of 495 nodes and 37755 significant edges. We identified three key regulatory transcription factors: STAT2 (signal transducer and activator of transcription 2), MYF6 (myogenic factor 6) and TFCP2L1 (transcription factor CP2 like 1). The network revealed that the STAT2 and MYF6 regulatory modules cooperate with each other and specifically influence the longevity and efficiency of sows, whereas the TFCP2L1 family specifically affects the improvement of litter size.

SILAC-Based Quantitative Proteomics Approach to Identify Transcription Factors Interacting with a Novel Cis-Regulatory Element

The motif TMTCGCGANR (M being C or A, R being A or G, and N any nucleotide) called M8 was discovered as a putative cis-regulatory element present in 368 human gene promoters. Of these, 236 (64%) are conserved within promoter sequences of four related organisms: human, mouse, rat and dog. However, transcription factors (TFs) interacting with the M8 motif has not yet been described. We previously reported the use of quantitative proteomics coupled to one-step DNA affinity purification as a means of screening for TFs associated with given functional DNA elements. The procedure is performed in-vitro employing SILAC-labeled nuclear extracts and making use of a well-characterized cis-regulatory motifs. Building on that, in this study we have combined our method with statistical analysis to filter out false positive hits from the one-step DNA affinity pull-down experiments. This resulted in the identification of zinc finger BED domain-containing protein 1 (ZBED1), alpha globin transcription factor CP2 (TFCP2), upstream binding protein 1 (UBP) and transcription factor CP2 like 1(TFCP2L1), as specific M8 interacting factors. We validated our screen demonstrating the in vivo binding of alpha globin transcription factor TFCP2 to selected genes harboring M8-containing promoters using ChIP (chromatin immuno-precipitation) assays. This not only implicates a functional role of the above proteins in regulating M8 motif containing genes, but also suggests the potential use of our approach to decipher protein-DNA interactions occurring in living cells.