The Bintu lab at Stanford University uses systems and synthetic biology approaches to characterize the dynamics of gene and chromatin regulation in mammalian cells.
Our goal is to improve mammalian synthetic biology by building a quantitative, predictive framework of chromatin and gene regulation. This framework and the tools based on it are critical for dissecting the epigenetic mechanisms that rule development, cancer and the immune response, and for developing gene therapies that correct faulty expression states associated with disease.
To do this, we develop novel techniques and assays to manipulate gene expression by recruiting chromatin regulators, transcription factors, and their component effector domains to particular loci in cells. We use single-cell measurements such as time-lapse fluorescence microscopy and flow cytometry as well as high-throughput screens and next-generation sequencing to characterize the dynamics of chromatin-mediated gene regulation in mammalian cells.
Recently, we have developed new compact nanobodies for efficient CR recruitment, worked with the Bassik lab to identify hundreds of new transcriptional effectors in human cells, and characterized how quickly chromatin-mediated gene silencing spreads between nearby genes. Ongoing work in our lab includes systematically characterizing the dynamic capabilities of CRs and TFs alone and in combination, measuring how their effects spread on DNA in various cell types, and using this information to design more specific and efficient gene control tools.
We try to keep this page updated with brief descriptions of the stuff we’re excited to share — so if you’re curious, check out some of our recent work below!
High-throughput recruitment of chromatin regulators
Human gene expression is regulated by thousands of proteins that interact with chromatin to activate or repress transcription. While some of these have been characterized individually in a low-throughput manner, we lack a broad understanding of which protein domains can modulate gene expression, how strong their effects are, and the dynamics of transcriptional memory after their recruitment. To systematically discover and characterize these dynamics of transcriptional regulatory domains, we developed a high-throughput assay in which pooled libraries of protein domains are recruited to a reporter promoter and transcriptional effects are measured with a sequencing readout. This assay also allows us to perform deep mutational scanning of transcriptional regulatory domains, and expands the toolbox of proteins we can use to perturb and modify the epigenome. Learn more about HT-recruit here and here!
Temporal Signaling and Information Processing via Chromatin Regulation
Chromatin regulation is a key pathway cells use to regulate gene expression in response to temporal stimuli, and is becoming widely used as a platform for synthetic biology applications. Here, we build a mathematical framework for analyzing the response of genetic circuits containing chromatin regulators to temporal signals in mammalian cell populations. We demonstrate that repressive regulators without long-term epigenetic memory can filter out high frequency noise, and as part of an autoregulatory loop can precisely tune the fraction of cells in a population that expresses a gene of interest. Additionally, we find that repressive regulators with epigenetic memory can sum up and encode the total duration of their recruitment in the fraction of cells irreversibly silenced and, when included in a feed forward loop, enable perfect adaptation. Last, we use an information theoretic approach to show that all-or-none stochastic silencing can be used by populations to transmit information reliably and with high fidelity even in very simple genetic circuits. Altogether, we show that chromatin-mediated gene control enables a repertoire of complex cell population responses to temporal signals and can transmit higher information levels than previously measured in gene regulation. See here for more info!
Spreading of Transcriptional Repression and Insulators
In mammalian cells genes that are in close proximity are coupled transcriptionally: silencing or activating one gene can affect its neighbors. Understanding these dynamics is important for natural processes, such as heterochromatin spreading during development and aging, and when designing synthetic gene regulation. We built a dual-gene synthetic reporter system to systematically dissect this process in single cells by measuring how fast gene silencing and reactivation spread as a function of intergenic distance and insulator element configuration. We find KRAB-mediated silencing spreads between genes with a distance-dependent time delay, and is not sensitive to the presence of the classical cHS4 insulators. HDAC4-mediated silencing spreads more slowly than KRAB-mediated silencing, but does not feature a distance-dependent delay and can sometimes be stopped by insulators. We propose a new model of multi-gene regulation, where both gene silencing and gene reactivation can act at a distance, allowing for coordinated dynamics via chromatin regulator recruitment. Check it out here!
Nanobody-mediated control of gene expression
Epigenetic editing tools are often made by engineering synthetic enzymes that contain both DNA binding domains and chromatin-modifying domains, overexpressing those enzymes in cells, and recruiting those enzymes to a locus or gene of interest. However, this overexpression process can often lead to cell toxicity and inadvertent changes to cell phenotype and behavior. We have developed a new tool to avoid these problems by using nanobodies to recruit endogenous chromatin regulators to a specific genomic locus for gene expression control; fusing these nanobodies to other chromatin regulators has also allowed us to improve the silencing and memory of those chromatin regulators. This gives us a foundation to continue building a toolbox of more efficient and less invasive tools to manipulate the epigenome. Read more about it here!