Chromatin proteins have expanded the mammalian synthetic biology toolbox by enabling control of active and silenced states at endogenous genes. Others have reported synthetic proteins that bind DNA and regulate genes by altering chromatin marks, such as histone modifications. Previously, we reported the first synthetic transcriptional activator, the “Polycomb-based transcription factor” (PcTF) that reads histone modifications through a protein–protein interaction between the polycomb chromodomain motif and trimethylated lysine 27 of histone H3 (H3K27me3). Here, we describe the genome-wide behavior of the polycomb-based transcription factor fusion protein. Transcriptome and chromatin profiling revealed several polycomb-based transcription factor-sensitive promoter regions marked by distal H3K27me3 and proximal fusion protein binding. These results illuminate a mechanism in which polycomb-based transcription factor interactions bridge epigenomic marks with the transcription initiation complex at target genes. In three cancer-derived human cell lines tested here, some target genes encode developmental regulators and tumor suppressors. Thus, the polycomb-based transcription factor represents a powerful new fusion protein-based method for cancer research and treatment where silencing marks are translated into direct gene activation.

Fusion proteins that specifically interact with biochemical marks on chromosomes represent a new class of synthetic transcriptional regulators that decode cell state information rather than DNA sequences. In multicellular organisms, information relevant to cell state, tissue identity, and oncogenesis is often encoded as biochemical modifications of histones, which are bound to DNA in eukaryotic nuclei and regulate gene expression states. We have previously reported the development and validation of the "polycomb-based transcription factor" (PcTF), a fusion protein that recognizes histone modifications through a protein-protein interaction between its polycomb chromodomain (PCD) motif and trimethylated lysine 27 of histone H3 (H3K27me3) at genomic sites. We demonstrated that PcTF activates genes at methyl-histone-enriched loci in cancer-derived cell lines. However, PcTF induces modest activation of a methyl-histone associated reporter compared to a DNA-binding activator. Therefore, we modified PcTF to enhance its binding avidity. Here, we demonstrate the activity of a modified regulator called PcTF, which has two tandem copies of the H3K27me3-binding PCD at the N-terminus. PcTF has a smaller apparent dissociation constant value in vitro and shows enhanced gene activation in HEK293 cells compared to PcTF. These results provide compelling evidence that the intrinsic histone-binding activity of the PCD motif can be used to tune the activity of synthetic histone-binding transcriptional regulators.

The aim of this study was to determine if aging or dietary restriction (DR) alters activation-induced cell death, which is known to regulate cell proliferation and eliminate the high number of activated cells during an immune response. Splenic T cells were isolated from young (4^6 months) and old (25^26 months) Fischer 344 rats that had free access to food, ad libitum (AL), and from dietary-restricted (DR) old (25^26 months) rats that beginning at 6 weeks of age were fed 60% (40% food-restricted) of the diet consume by the AL rats. T cells were incubated with anti-CD3 antibody, or staphylococcal enterotoxin B (primary stimulus) for 72^96 h, followed by restimulation with anti-CD3 (secondary stimulus) for 72 h. Activation-induced apoptosis was assessed by DNA fragmentation and the expression of Fas/CD95 receptor and Fas ligand (Fas-L) was measured by flow cytometry. We found that the amount of DNA fragmentation was significantly (P 6 0.05) higher in the stimulated and restimulated T cells from AL old rats and DR old rats compared to young rats. The increase in DNA fragmentation with age was paralleled by an increase in the proportion of the cells expressing Fas and Fas-L. However, DR had no significant effect on the age-related increase in DNA fragmentation or the expression of Fas or Fas-L. We also measured the levels of Bcl-2 and Bax protein and found that the level of Bcl-2 decreased and Bax increased with age and that DR had no effect on the agerelated changes in the level of Bcl-2 or Bax protein. These results demonstrate that aging but not DR alters activation-induced apoptosis in rat T cells. ß

Chromatin is a system of nuclear proteins and nucleic acids that plays a pivotal role in gene expression and cell behavior and is therefore the subject of intense study for cell development and cancer research. Biochemistry, crystallography, and reverse genetics have elucidated the macromolecular interactions that drive chromatin regulation. One of the central mechanisms is the recognition of post-translational modifications (PTMs) on histone proteins by a family of nuclear proteins known as "readers". This knowledge has launched a wave of activity around the rational design of proteins that interact with histone PTMs. Useful molecular tools have emerged from this work, enabling researchers to probe and manipulate chromatin states in live cells. Chromatin-based proteins represent a vast design space that remains underexplored. Therefore, we have developed a rapid prototyping platform to identify engineered fusion proteins that bind histone PTMs in vitro and regulate genes near the same histone PTMs in living cells. We have used our system to build gene activators with strong avidity for the gene silencing-associated histone PTM H3K27me3. Here, we describe procedures and data for cell-free production of fluorescently tagged fusion proteins, enzyme-linked immunosorbent assay-based measurement of histone PTM binding, and a live cell assay to demonstrate that the fusion proteins modulate transcriptional activation at a site that carries the target histone PTM. This pipeline will be useful for synthetic biologists who are interested in designing novel histone PTM-binding actuators and probes.

The aim of this study was to determine if long-term treatment with melatonin (MEL), a purported anti-aging agent, was as effective as calorie restriction (CR) in modulating immune parameters in aging Fischer 344 male rats. Splenic lymphocytes were isolated from 17-month-old rats that, beginning at 6 weeks of age, were treated with MEL (4 or 16 μg/ml in drinking water) and from 17-month-old rats fed ad libitum (AL) or rats fed a CR diet (55% of AL intake). The number of splenic T cell populations and T cell subsets was measured by flow cytometry, the proliferative response of splenocytes to Concanavalin A (Con A) and lipopolysaccharide (LPS) was measured by [3H]thymidine incorporation, and the induction of cytokine production (IL-2 and IFN-γ) was measured by ELISA assay. In addition, the level of the natural killer (NK) cell activity was assessed by fluorimetric assay. CR rats had a higher number of lymphocytes expressing the naïve T cell marker (CD3 OX22) than AL rats (P < 0.05). CR rats also showed greater induction of proliferative response, IL-2 and IFN-γ levels following Con A simulation, and NK cell activity than AL rats (P < 0.05). MEL-treated rats did not differ from AL rats in any of these parameters or in any other measurement. These results indicate that MEL treatment is unable to modulate immune function in a manner comparable with that of CR.