Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma. The oncogene product Tax of HTLV-I is thought to play crucial roles in leukemogenesis by promoting proliferation of the virus-infected cells through activation of growth-promoting genes. These genes code for growth factors and their receptors, cytokines, cell adhesion molecules, growth signal transducers, transcription factors and cell cycle regulators. We show here that Tax activates the gene coding for coactivator-associated arginine methyltransferase 1 (CARM1), which epigenetically enhances gene expression through methylation of histones. Tax activated the Carm1 gene and increased protein expression, not only in human T-cell lines but also in normal peripheral blood lymphocytes (PHA-PBLs). Tax increased R17-methylated histone H3 on the target gene IL-2Rα, concomitant with increased expression of CARM1. Short hairpin RNA (shRNA)-mediated knockdown of CARM1 decreased Tax-mediated induction of IL-2Rα and Cyclin D2 gene expression, reduced E2F activation and inhibited cell cycle progression. Tax acted via response elements in intron 1 of the Carm1 gene, through the NF-κB pathway. These results suggest that Tax-mediated activation of the Carm1 gene contributes to leukemogenic target-gene expression and cell cycle progression, identifying the first epigenetic target gene for Tax-mediated trans-activation in cell growth promotion.

Zygotic genome activation (ZGA) is a pivotal event in mammalian embryogenesis, marking the transition from maternal to zygotic control of development. During the ZGA process that is characterized by the intricate cascade of gene expression, who tipped the first domino in a meticulously arranged sequence is a subject of paramount interest. Recently, Dux, Obox and Nr5a2 were identified as pioneer transcription factors that reside at the top of transcriptional hierarchy. Through co-option of retrotransposon elements as hubs for transcriptional activation, these pioneer transcription factors rewire the gene regulatory network, thus initiating ZGA. In this review, we provide a snapshot of the mechanisms underlying the functions of these pioneer transcription factors. We propose that ZGA is the starting point where the embryo\'s own genome begins to influence development trajectory, therefore in-depth dissecting the functions of pioneer transcription factors during ZGA will form a cornerstone of our understanding for early embryonic development, which will pave the way for advancing our grasp of mammalian developmental biology and optimizing in vitro production (IVP) techniques.

Pertuzumab (Perjeta®), a humanized antibody binding to the dimerization arm of HER2 (Human epidermal growth factor receptor-2), has failed as a monotherapy agent in HER2 overexpressing malignancies. Since the molecular interaction of HER2 with ligand-bound EGFR (epidermal growth factor receptor) has been implied in mitogenic signaling and malignant proliferation, we hypothesized that this interaction, rather than HER2 expression and oligomerization alone, could be a potential molecular target and predictor of the efficacy of pertuzumab treatment. Therefore, we investigated static and dynamic interactions between HER2 and EGFR molecules upon EGF stimulus in the presence and absence of pertuzumab in HER2+ EGFR+ SK-BR-3 breast tumor cells using Förster resonance energy transfer (FRET) microscopy and fluorescence correlation and cross-correlation spectroscopy (FCS/FCCS). The consequential activation of signaling and changes in cell proliferation were measured by Western blotting and MTT assay. The autocorrelation functions of HER2 diffusion were best fitted by a three-component model corrected for triplet formation, and among these components the slowly diffusing membrane component revealed aggregation induced by EGFR ligand binding, as evidenced by photon-counting histograms and co-diffusing fractions. This aggregation has efficiently been prevented by pertuzumab treatment, which also inhibited the post-stimulus interaction of EGFR and HER2, as monitored by changes in FRET efficiency. Overall, the data demonstrated that pertuzumab, by hindering post-stimulus interaction between EGFR and HER2, inhibits EGFR-evoked HER2 aggregation and phosphorylation and leads to a dose-dependent decrease in cell proliferation, particularly when higher amounts of EGF are present. Consequently, we propose that EGFR expression on HER2-positive tumors could be taken into consideration as a potential biomarker when predicting the outcome of pertuzumab treatment.

Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a protein arginine methyltransferase that deposits asymmetrical dimethylation marks on both histone and nonhistone substrates. The regulatory role of CARM1 in transcription was first identified in estrogen receptor positive (ER+) breast cancer. Since then, the mechanism of CARM1 in activating ER-target genes has been further interrogated. CARM1 is expressed at the highest level in ER negative (ER-) breast cancer and higher expression correlates with poor prognosis, suggesting an oncogenic role of CARM1. Indeed, in ER- breast cancer, CARM1 can promote proliferation and metastasis at least partly through methylation of proteins and activation of oncogenes. In this review, we summarize the mechanisms of transcriptional activation by CARM1 in breast cancer. The methyltransferase activity of CARM1 is important for many of its functions; here, we also highlight the nonenzymatic roles of CARM1. We also cover the biological processes regulated by CARM1 that are often deregulated in cancer and the ways to harness CARM1 in cancer treatment.

Gene Transfer Agents (GTAs) are phage-like particles that cannot self-multiply and be infectious. Caulobacter crescentus, a bacterium best known as a model organism to study bacterial cell biology and cell cycle regulation, has recently been demonstrated to produce bona fide GTA particles (CcGTA). Since C. crescentus ultimately die to release GTA particles, the production of GTA particles must be tightly regulated and integrated with the host physiology to prevent a collapse in cell population. Two direct activators of the CcGTA biosynthetic gene cluster, GafY and GafZ, have been identified, however, it is unknown how GafYZ controls transcription or how they coordinate gene expression of the CcGTA gene cluster with other accessory genes elsewhere on the genome for complete CcGTA production. Here, we show that the CcGTA gene cluster is transcriptionally co-activated by GafY, integration host factor (IHF), and by GafZ-mediated transcription anti-termination. We present evidence that GafZ is a transcription anti-terminator that likely forms an anti-termination complex with RNA polymerase, NusA, NusG, and NusE to bypass transcription terminators within the 14 kb CcGTA cluster. Overall, we reveal a two-tier regulation that coordinates the synthesis of GTA particles in C. crescentus.

Triple-negative breast cancer (TNBC) is more aggressive and has a higher metastasis rate compared with other subtypes of breast cancer. Due to the lack of drug-targetable receptors, chemotherapy is now the only available systemic treatment for TNBC. However, some patients might still develop drug resistance and have poor prognosis. Therefore, novel molecular biomarkers and new treatment targets are urgently needed for patients with TNBC. To provide molecular insights into TNBC progression, we investigated the function and the underlying mechanism of Defective in cullin neddylation 1 domain containing 5 (DCUN1D5) in the regulation of TNBC. By TCGA dataset and surgical specimens with immunohistochemical (IHC) staining method, DCUN1D5 was identified to be significantly upregulated in TNBC tumor tissues and negatively associated with prognosis. A series of in vitro and in vivo experiments were performed to confirm the oncogenic role of DCUN1D5 in TNBC. Overexpression of FN1 or PI3K/AKT activator IGF-1 could restore the proliferative and invasive ability induced by DCUN1D5 knockdown and DCUN1D5 could act as a novel transcriptional target of transcription factor Yin Yang 1 (YY1). In conclusion, YY1-enhanced DCUN1D5 expression could promote TNBC progression by FN1/PI3K/AKT pathway and DCUN1D5 might be a potential prognostic biomarker and therapeutic target for TNBC treatment.

The mammalian SWI/SNF-like BAF complexes play critical roles during animal development and pathological conditions. Previous gene deletion studies and characterization of human gene mutations implicate that the complexes both repress and activate a large number of genes. However, the direct function of the complexes in cells remains largely unclear due to the relatively long-term nature of gene deletion or natural mutation. Here we generate a mouse line by knocking in the auxin-inducible degron tag (AID) to the Smarca4 gene, which encodes BRG1, the essential ATPase subunit of the BAF complexes. We show that the tagged BRG1 can be efficiently depleted by osTIR1 expression and auxin treatment for 6 to 10 h in CD4 + T cells, hepatocytes, and fibroblasts isolated from the knock-in mice. The acute depletion of BRG1 leads to decreases in nascent RNAs and RNA polymerase II binding at a large number of genes, which are positively correlated with the loss of BRG1. Further, these changes are correlated with diminished accessibility at DNase I Hypersensitive Sites (DHSs) and p300 binding. The acute BRG1 depletion results in three major patterns of nucleosome shifts leading to narrower nucleosome spacing surrounding transcription factor motifs and at enhancers and transcription start sites (TSSs), which are correlated with loss of BRG1, decreased chromatin accessibility and decreased nascent RNAs. Acute depletion of BRG1 severely compromises the Trichostatin A (TSA) -induced histone acetylation, suggesting a substantial interplay between the chromatin remodeling activity of BRG1 and histone acetylation. Our data suggest BRG1 mainly plays a direct positive role in chromatin accessibility, RNAPII binding, and nascent RNA production by regulating nucleosome positioning and facilitating transcription factor binding to their target sites.

CRISPR-based gene therapy offers precise targeting and specific editing of disease-related gene sequences, potentially yielding long-lasting treatment effects. However, efficient delivery remains a significant challenge for its widespread application. In this study, we design a novel short peptide-conjugated bioreducible polymer named TSPscp as a safe and effective delivery vector for the CRISPR system. Our results show that TSPscp markedly boosts transcriptional activation and genome editing activities of multiple CRISPR systems as confirmed by decomposition-seq and Deep-seq, which is resulted from its capability in facilitating delivery of plasmid DNA by promoting cellular uptake and lysosomal escape. Additionally, TSPscp further enhances genome editing of CRISPR by delivery of minicircle DNA, a condensed form of regular plasmid DNA. More importantly, TSPscp significantly improves delivery and genome editing of CRISPR system in vivo. In summary, our study highlights TSPscp as a promising delivery tool for CRISPR applications in vivo.

How enhancers regulate their target genes in the context of 3D chromatin organization is extensively studied and models which do not require direct enhancer-promoter contact have recently emerged. Here, we use the activation of estrogen receptor-dependent enhancers in a breast cancer cell line to study enhancer-promoter communication at two loci. This allows high temporal resolution tracking of molecular events from hormone stimulation to efficient gene activation. We examine how both enhancer-promoter spatial proximity assayed by DNA fluorescence in situ hybridization, and contact frequencies resulting from chromatin in situ fragmentation and proximity ligation, change dynamically during enhancer-driven gene activation. These orthogonal methods produce seemingly paradoxical results: upon enhancer activation enhancer-promoter contact frequencies increase while spatial proximity decreases. We explore this apparent discrepancy using different estrogen receptor ligands and transcription inhibitors. Our data demonstrate that enhancer-promoter contact frequencies are transcription independent whereas altered enhancer-promoter proximity depends on transcription. Our results emphasize that the relationship between contact frequencies and physical distance in the nucleus, especially over short genomic distances, is not always a simple one.