Substrate Specificities and Identification of Putative Substrates of ATM Kinase Family Members

https://www.cell.com/cell/pdf/0092-8674(95)90512-X.pdf

Histone H3 and H4 N-termini interact with SIR3 and SIR4 proteins: A molecular model for the formation of heterochromatin in yeast

Fibroblast growth factor 4 (FGF-4) has been shown to be a signaling molecule whose expression is essential for postimplantation mouse development and, at later embryonic stages, for limb patterning and growth. The FGF-4 gene is expressed in the blastocyst inner cell mass and later in distinct embryonic tissues but is transcriptionally silent in the adult. In tissue culture FGF-4 expression is restricted to undifferentia ted embryonic stem (ES) cells and embryonal carcinoma (EC) cell lines. Previously, we determined that EC cell-specific transcriptional activation of the FGF-4 gene depends on a synergistic interaction between octamer-binding proteins and an EC-specific factor, Fx, that bind adjacent sites on the FGF-4 enhancer. Through the cloning and characterization of an F9 cell cDNA we now show that the latter activity is Sox2, a member of the Sry-related Sox factors family. Sox2 can form a ternary complex with either the ubiquitous Oct-1 or the embryonic-specific Oct-3 protein on FGF-4 enhancer DNA sequences. However, only the Sox2/Oct-3 complex is able to promote transcriptional activation. These findings identify FGF-4 as the first known embryonic target gene for Oct-3 and for any of the Sox factors, and offer insights into the mechanisms of selective gene activation by Sox and octamer-binding proteins during embryogenesis.

/pdf/developmental-specific-activity-of-the-fgf-4-enhancer-lua3s2r7nz.pdf

Developmental-specific activity of the FGF-4 enhancer requires the synergistic action of Sox2 and Oct-3.

Organization of chromatin by epigenetic mechanisms is essential for establishing and maintaining cellular identity in developing and adult organisms. A key question that remains unresolved about this process is how epigenetic marks are transmitted to the next cell generation during cell division. Here we provide a model to explain how trimethylated Lys 27 of histone 3 (H3K27me3), which is catalysed by the EZH2-containing Polycomb Repressive Complex 2 (PRC2), is maintained in proliferating cells. We show that the PRC2 complex binds to the H3K27me3 mark and colocalizes with this mark in G1 phase and with sites of ongoing DNA replication. Efficient binding requires an intact trimeric PRC2 complex containing EZH2, EED and SUZ12, but is independent of the catalytic SET domain of EZH2. Using a heterologous reporter system, we show that transient recruitment of the PRC2 complex to chromatin, upstream of the transcriptional start site, is sufficient to maintain repression through endogenous PRC2 during subsequent cell divisions. Thus, we suggest that once the H3K27me3 is established, it recruits the PRC2 complex to maintain the mark at sites of DNA replication, leading to methylation of H3K27 on the daughter strands during incorporation of newly synthesized histones. This mechanism ensures maintenance of the H3K27me3 epigenetic mark in proliferating cells, not only during DNA replication when histones synthesized de novo are incorporated, but also outside S phase, thereby preserving chromatin structure and transcriptional programs.

A model for transmission of the H3K27me3 epigenetic mark

Nucleosome assembly by a complex of CAF-1 and acetylated histones H3/H4.

DNA-dependent and DNA-independent associations of DNA-binding proteins are important in transcriptional regulation. The analysis of DNA-independent associations frequently relies on assaying protein interaction in the absence of target DNA sequences. We have found that contaminating DNA in protein preparations can stabilize DNA-dependent associations that may appear DNA-independent. Three cellular proteins of 70, 85, and 110 kDa coimmunoprecipitated with the octamer motif-binding protein Oct-2 because of the presence of contaminating DNA in the cell extracts. In addition, heterodimer formation between Oct-1 (or Oct-2) and Pit-1 during protein-affinity chromatography was stabilized by the contaminating DNA. In both instances, these DNA-dependent protein associations were selectively inhibited by ethidium bromide in the precipitation reaction without any evident effect on DNA-independent protein associations. Thus, ethidium bromide may serve as a simple and general indicator of DNA-dependent and DNA-independent protein associations.

https://www.pnas.org/content/pnas/89/15/6958.full.pdf

Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations

Promoter-selective activation domains in Oct-1 and Oct-2 direct differential activation of an snRNA and mRNA promoter

The selective association of the herpesvirus trans-activator VP16 with the human Oct-1 homeo domain is a model for differential positive transcriptional control by homeo domains. VP16 discriminates between the closely related homeo domains of Oct-1 and Oct-2 by distinguishing among their seven amino-acid differences; these differences lie on the surface that is thought to be accessible when the homeo domain is bound to DNA. Only two of these seven differences are recognized by VP16, one in each of the first two alpha-helices of the tri-alpha-helical homeo domain. The major determinant for selective association with VP16 in vitro and VP16-induced positive control in vivo is a single glutamic acid residue at position 22 in the first alpha-helix of the Oct-1 homeo domain, but the acidic properties of this residue are not critical for association with VP16 in vitro or in vivo, because it can be replaced by glutamine with little or no deleterious effect. Mere replacement of the single corresponding alanine residue in the Oct-2 homeo domain with the key glutamic acid residue is sufficient to confer on the Oct-2 homeo domain the ability to associate with VP16 in vitro and respond to VP16-induced positive control in vivo. Thus, the specificity of homeo domain positive control can be conferred by a single amino acid difference.

/pdf/a-single-amino-acid-exchange-transfers-vp16-induced-positive-38aemcw4vw.pdf

A single amino acid exchange transfers VP16-induced positive control from the Oct-1 to the Oct-2 homeo domain.

Upon infection, the herpes simplex virus (HSV) activator of immediate-early (IE) gene transcription VP16 forms a multiprotein-DNA complex with two cellular proteins, Oct-1 and HCF. First, VP16 associates with HCF independently of DNA, and this association stimulates subsequent association with Oct-1 on the DNA target of VP16 activation, the TAATGARAT motif found in HSV IE promoters. We have analyzed the involvement of VP16 residues lying near the carboxy-terminal transcriptional activation domain of VP16 in associating with HCF, Oct-1, and DNA. To assay VP16 association with HCF, we developed an electrophoretic mobility retardation assay in which HCF is used to retard the mobility of a hybrid VP16-GAL4 DNA-binding domain fusion protein bound to a GAL4 DNA-binding site. Analysis of an extensive set of individual and combined alanine substitutions over a 61-amino-acid region of VP16 shows that, even within a region as small as 13 amino acids, there are separate residues involved in association with either HCF, DNA, or Oct-1 bound to DNA; indeed, of two immediately adjacent amino acids in VP16, one is important for DNA binding and the other is important for HCF binding. These results suggest that a small region in VP16 is important for linking in close juxtaposition the four components of the VP16-induced complex and support the hypothesis that the structure of the Oct-1-VP16 interaction in this complex is similar to that formed by the yeast transcriptional regulatory proteins MATa1 and MAT alpha2. We propose that HCF stabilizes this Oct-1-VP16 interaction.

Interdigitated residues within a small region of VP16 interact with Oct-1, HCF, and DNA.

Background CD19-targeted chimeric antigen receptor therapies (CAR19) have driven a paradigm shift in the treatment of relapsed/refractory B-cell malignancies. However, >50% of CAR19-treated patients experienced progressive disease mainly due to antigen escape and low persistence. Clinical prognosis is heavily influenced by CAR-T cell function and systemic cytokine toxicities. Furthermore, it remains a challenge to efficiently, cost-effectively, and consistently manufacture clinically relevant number of virally engineered CAR-T cells. Methods Using a highly efficient piggyBac transposon-based vector, Quantum pBac, we developed a virus-free cell engineering system, Quantum CART (qCART™), for development and production of multiplex CAR-T therapies. Results Here, we demonstrated in vitro and in vivo that consistent, robust, and functional CD20/CD19 dual-targeted CAR-T stem cell memory (TSCM) cells can be efficiently manufactured using the qCART™ system for clinical application. qCART™-manufactured CAR-T cells from cancer patients expanded efficiently, rapidly eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug. Conclusions The qCART™ system is an elegant system for the manufacturing of CAR-T products having all the desired CAR-T therapy attributes. We believe that the simplicity of manufacturing multiplex CAR-T cells using the qCART™ system will not only significantly enhance the accessibility of CAR-T therapy but also unlock the full potential of armored CAR-T therapy for the treatment of solid tumors in the future. What is already known on this topic Despite the considerable success which has been achieved with CD19-targeted chimeric antigen receptor therapies (CAR19), >50% of CAR19-treated patients still experienced progressive disease. Therefore, there is a need to further improve CAR19 therapies. Current CAR19 therapies commonly utilize virus-based cell engineering methods. CAR-T production using these methods face multiple hurdles, including difficulties to efficiently, cost-effectively, and consistently manufacture clinically relevant number of CAR-T cells. We have previously used a highly efficient piggyBac transposon-based vector, Quantum pBac, to establish Quantum CART (qCART™) which is a virus-free cell engineering system for development and production of multiplex CAR-T therapies. What this study adds In this report, we further demonstrate in vitro and in vivo that consistent, robust, and functional iCasp9-regulatable, CD20/CD19 dual-targeted CAR-T stem cell memory (TSCM) cells can be efficiently manufactured using the qCART™ system for clinical application. These cells possess all the desired attributes for ensuring therapeutic efficacy in CAR-T therapy, including high CAR-TSCM, balanced CD8/CD4 ratio, low exhaustion and senescence marker expressions, and high ex vivo and in vivo expansion capacity. Importantly, we show that qCART™-manufactured CAR-T cells from hematological cancer patients expanded efficiently, effectively eradicated tumors, and can be safely controlled via an iCasp9 suicide gene-inducing drug. We believe that the simplicity of manufacturing multiplex CAR-T cells using the qCART™ system will not only significantly enhance the accessibility of CAR-T therapy but also unlock the full potential of armored CAR-T therapy for the treatment of solid tumors in the future. How this study might affect research, practice or policy Our findings demonstrate that qCART™ is a virus-free CAR-T engineering system for manufacturing CAR-TSCM cells from either healthy donors or hematological cancer patients, that possess all the desired attributes for a successful CAR-T therapy. These cells expanded efficiently, rapidly eradicated tumors, and can be safely controlled via activation of iCasp9. We expect that this simple yet robust system for manufacturing multiplex CAR-T cells will advance the CAR-T field.

/pdf/manufacturing-cd20-cd19-targeted-icasp9-regulatable-car-tscm-3eels7l8.pdf

Jiann-Shiun Lai

Papers

Ethidium bromide provides a simple tool for identifying genuine DNA-independent protein associations

Promoter-selective activation domains in Oct-1 and Oct-2 direct differential activation of an snRNA and mRNA promoter

A single amino acid exchange transfers VP16-induced positive control from the Oct-1 to the Oct-2 homeo domain.

Interdigitated residues within a small region of VP16 interact with Oct-1, HCF, and DNA.

Manufacturing CD20/CD19-targeted iCasp9 regulatable CAR-TSCM cells using qCART, the Quantum pBac-based CAR-T system