Research Publications

Research – ACS Biochemistry – Design, construction, and validation of histone-binding effectors in vitro and in cells

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Design, construction, and validation of histone-binding effectors in vitro and in cells
Tekel SJ, Barrett CM, Vargas DA, Haynes KA. (2018) ACS Biochemistry. https://pubs.acs.org/doi/10.1021/acs.biochem.8b00327 (Just Accepted manuscript)

In a special topic issue from ACS Biochemistry: From the Bench, we describe our workflow for quick screening and validation of customized histone-binding fusion proteins. Since our previous report where we enhanced the activity of one such fusion called PcTF, we modified our procedure to circumvent the need to generate large quantities of fusion proteins in bacterial cultures. Instead, we use cell-free transcription-translation (TXTL) to generate small batches of variant proteins, quantify the product with ELISA, and determine relative avidities using immobilized histone peptides in an ELISA format. We demonstrate that relative binding determined by ELISA is consistent with the strength of gene-regulation activity at a target gene in HEK293 cells. Our ongoing work aims to miniaturize this technique even further for rapid exploration of the vast design space for synthetic epigenetic effectors.

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Research – bioRxiv Pre-print – Characterization of Diverse Homoserine Lactone Synthases in Escherichia coli

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Characterization of Diverse Homoserine Lactone Synthases in Escherichia coli
Daer R, Barrett CM, Melendez EL, Wu J, Tekel SJ, Xu J, Dennison B, Muller R, Haynes KA. (2018) bioRxiv. https://www.biorxiv.org/content/early/2018/03/09/279349

The Haynes lab focuses on advanced chromosome engineering in human cells, but also provides opportunities for undergraduates to learn synthetic biology using simpler organisms like bacteria (E. coli). In this paper, the 2016 ASU International Genetically Engineered Machines (iGEM) Competition team and their graduate advisors report their work to identify useful, new cell-cell communication components to use in engineered systems. Homoserine lactone (HSL) synthases appear as a wide variety of different forms in the bacterial kingdom, and produce various chemical signals that regulate genes in neighboring bacteria. When these are combined to build synthetic circuits in a common lab strain (E. coli), the signals are sometimes not produced as expected. Therefore, it is important to systematically characterize HSL synthases in context. The team also used experiments to identify the most effective way to neutralize unused HSLs in biological waste. The ten HSL synthases characterized in this paper were contributed to public collections for use by the scientific community.

Research – bioRxiv Pre-print – Histone modifications and active gene expression are associated with enhanced CRISPR activity in de-silenced chromatin

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Histone modifications and active gene expression are associated with enhanced CRISPR activity in de-silenced chromatin
Daer R, Barrett C, Haynes KA. (2017) bioRxiv. https://www.biorxiv.org/content/early/2018/03/11/228601

CRISPR is a powerful and popular tool for editing DNA in living cells. Scientists are becoming more interested in using CRISPR to correct mistakes in DNA that lead to diseases, to artificially generate mutations to research the origins of diseases, and for other important applications. However, CRISPR originated in bacteria and has probably not evolved to function very well in genomes that are packed in configurations (open and closed chromatin) as complex as those found in human cells. In a recent report (Daer et al. 2017), we demonstrated that CRISPR activity was inhibited at a DNA sequence that became artificially condensed into closed chromatin. Our new study shows that targeted re-opening of closed chromatin leads to enhanced CRISPR activity in the same region. The epigenetic drug we tested (UNC1999) was not sufficient to generate a transcriptionally active or CRISPR-accessible state. In contrast, strong direct activation with a DNA-binding p65 protein did enhance CRISPR accessibility. Importantly, we learned that a recovery period (following treatment with p65) is needed to generate the CRISPR-accessible state.

Research – bioRxiv Pre-print – The synthetic histone-binding regulator protein PcTF activates interferon genes in breast cancer cells

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The synthetic histone-binding regulator protein PcTF activates interferon genes in breast cancer cells
Olney KC, Nyer DB, Wilson Sayres MA, Haynes KA. (2017) bioRxiv. http://www.biorxiv.org/content/early/2017/09/07/186056

Certain types of breast cancer can be difficult to treat because cancer cells in different patients may not be completely identical. Here, we investigated and artificially manipulated the expression states of genes in drug responsive and non-responsive (triple-negative) breast cancer cell lines. Similar to findings from other researchers, we observed that certain groups of genes are commonly or differentially expressed. A large group of genes is epigenetically silenced in breast cancer cells compared to non-cancer cells. We used a synthetic fusion protein called PcTF to physically bridge histone methylation at silenced genes with proteins that drive gene activation. This experiment revealed that nineteen common PcTF-upregulated genes (PUGs) from the interferon pathway as well as other tumor suppressors became activated in all three cell types, including the triple negative cells. PcTF has the potential to act as a powerful therapeutic protein (biologic) that activates multiple anti-cancer genes at once.

Research – ACS Synthetic Biology – Tandem histone binding domains enhance the activity of a synthetic chromatin effector

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Tekel2017_biorxivTandem histone-binding domains enhance the activity of a synthetic chromatin effector
Tekel SJ, Vargas DA, Song L, LaBaer J, Caplan M, Haynes KA. (2017) ACS Synthetic Biol. 7:842-852. https://pubs.acs.org/doi/pdf/10.1021/acssynbio.7b00281

Here, we report the behavior of a re-engineered PcTF,  a gene-regulating fusion protein that is designed to activate genes that have been suppressed by chromatin condensation in cancer cells. We added an extra histone-binding domain to create Pc2TF and observed 2- to 4-fold enhancement of target binding and target gene activation. The new design was inspired by natural proteins that also have double-motifs that contribute to target affinity. The specific combination of motifs in Pc2TF does not exist in nature. By using design rules inferred from pre-existing motif patterns, we have improved the performance a novel synthetic chromatin effector. This improved activity advances PcTF towards clinical translation for anti-cancer therapy.

Related resources:

  1. Pre-print: Tandem Histone-Binding Domains Enhance the Activity of a Synthetic Chromatin Effector. bioRxiv. http://biorxiv.org/content/early/2017/06/03/145730
  2. Conference Proceeding: In Vitro Development of Synthetic Chromatin Proteins That Function in Live Cells. FASEB. http://www.fasebj.org/content/31/1_Supplement/922.8.short

Research – ACS Synthetic Biology – The impact of chromatin dynamics on Cas9-mediated genome editing in human cells

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BioRxiv_Daer2016The impact of chromatin dynamics on Cas9-mediated genome editing in human cells
Daer RM, Cutts JP, Brafman DA, Haynes KA (2016) ACS Synthetic Biology. doi: 10.1021/acssynbio.5b00299

We used a chromatin switch system to compare the efficiency of human gene editing (via CRISPR/Cas9) before and after DNA had become packaged with nuclear proteins. The DNA-nuclear protein complex (called chromatin) ‘turns the dials’ of gene expression. Here, we discovered that this dialing mechanism can also disrupt artificial genome editing. We also found that readjusting chromatin could restore gene editing, which has implications for improving CRISPR for use in stem cell genomes, where key genes are often tightly packaged in chromatin.

Related resources:

Research – Nature – Regulation of cancer epigenomes with a histone-binding synthetic transcription factor

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Figure6_finalRegulation of cancer epigenomes with a histone-binding synthetic transcription factor.
Nyer DB, Daer R, Vargas D, Hom C, Haynes KA. (2017) Nature Genomic Medicine. http://rdcu.be/oqv7

This work expands our 2011 report in many important ways. We studied the behavior of a synthetic chromatin protein that we designed called PcTF in bone, blood, and brain cancer-derived cells. We expected to see PcTF bind to methylated histones, but instead saw strong signals closer to histone-free gene promoters. However, PcTF activity still required the methyl-histone binding domain to interact with its targets. It appears that PcTF bridges methylated histone signals with the transcription complex. We also discovered that PcTF activates a key tumor suppressor, CASZ1 as well as other silenced genes in all three cancer cell types. This new information has advanced our understanding of how a potentially therapeutic histone-binding protein behaves in cancer cells.

Related resources: