The sections below detail the project areas we pursue in the West group. In general, we are interested in how transcription factors bound at gene regulatory elements can influence epigenetic chromatin states to regulate gene transcription. We seek a deeper understanding of chromosomal organisation by enhancer and insulator elements. We also translate this understanding to solving long standing problems in the biotechnology and genetic therapy sectors.
Inevitably, enabling technologies play a key part in our research. We have therefore invested considerable effort in developing a powerful gene reporter system, protein labelling in living cells and exciting genome / epigenome editing approaches using CRISPR and TALE technologies. These will be fundamental to our work in the next few years.
Chromatin domains and their boundaries
Evidence from chromatin profiling, chromosomal interaction mapping and functional assays all support the hypothesis that genome organization and long range gene regulation in metazoa involves the partitioning of genomes into distinct chromatin states. There is also strong evidence that the transcriptional regulation of genes and gene clusters within these chromatin domains can be maintained independently of their surroundings through the establishment of chromatin boundaries. These boundaries sometimes vary in position as a result of a balance between countervailing chromatin opening and condensing processes. Alternatively, chromatin boundaries of fixed position can be established by specific DNA sequence elements and their associated binding proteins. Such elements, collectively called insulators, possess a common ability to protect genes from inappropriate signals emanating from their surrounding environment.
Lessons from studying a model chromatin boundary
The chicken β-globin gene locus is a model example of chromatin domain organisation. Upon their expression, the β-globin genes are clustered within a thirty kilobase domain of open chromatin flanked by condensed chromatin that is repressive to transcription. The 5´ boundary of the globin domain is marked by the remarkable HS4 insulator element, which harbours enhancer blocking and heterochromatin barrier activities. The protein CTCF directs the enhancer blocking of HS4 and thousands of insulators throughout vertebrate genomes. CTCF interacts with cohesin proteins and forms chromosomal loop interactions that are widely considered to contribute to genome organization. Only a small subset of CTCF sites are proximal to chromatin boundary features, so it remains likely that other insulator proteins, with heterochromatin barrier-specific functions perhaps, function to partition chromosomal domains.
HS4 became useful again, as we found that its heterochromatin barrier activity did not require the CTCF site. Instead, HS4 requires the activities of the USF1/2 and VEZF1 transcription factors, which direct different mechanisms to counter the propagation of chromatin silencing.
Mechanisms of chromatin boundary formation
The role of H2B ubiquitination in chromatin boundary formation
The USF transcription factors act to recruit several histone modifying enzymes to HS4, including P300, PRMT1 and SET1, that result in the acetylation of H3, H4 and H2A.Z and the methylation of H3K4 and H4R3. Recently, we also found that USF1 mediates H2B mono-ubiquitination (H2Bub1) at insulators in chicken cells. Our experiments revealed that H2Bub1 is required for the histone modification signature at chromatin boundary elements, consistent with this mark acting at the head of a histone modification cascade. Collectively, these so-called “active” histone modifications at boundary elements act as a chain terminator to the propagation of histone modifications associated with heterochromatin assembly. Depletion of H2Bub1 allows the spreading of heterochromatin beyond its normal limits, resulting in the progressive silencing of nearby gene transcription. We are currently studying this process across the human genome.
The many roles of the VEZF1 transcription factor
Our earlier studies of the HS4 insulator found that multiple sites for the transcription factor VEZF1 were required for heterochromatin barrier activity. These sites were all associated with HS4’s resistance to DNA methylation. We also found VEZF1 sites abrogated DNA methylation at the Hprt CpG island promoter. In pursuing these interesting results, we have new data that shows VEZF1 has much broader gene regulatory roles than first expected. This is now a major focus of the group.
Identification of novel chromatin opening elements that facilitate stable gene expression
The understanding of mammalian chromatin boundary elements has been limited by the lack of a gene reporter assay that can be scaled up to assay new candidate elements and allow for direct comparisons between assays in controlled chromosomal locations. We therefore invested considerable effort in developing such an assay system. We have teamed up with our collaborators at UCB Celltech to identify and validate new elements that enable stable transgene expression. Recombinant genes tend to be become silenced when integrated in the genome of host cells as most of the genome is repressive to transcription at any given time. This represents a major bottleneck to the manufacturers of biopharmaceuticals, who currently invest extensive time and effort to identify mammalian cell lines that express recombinant genes at a high level for long periods. Our combined skills in identifying candidate chromatin opening elements, the new assay system and expertise in recombinant protein production are being used to reduce the effort required to develop highly productive cell lines, ultimately bringing down the cost and developmental timelines for the latest targeted therapies.
Genome and epigenome modification
Gene modification strategies
Recent advances in synthetic biology allow molecular biologists to make alterations to the genome of pretty much any species. The new approaches use DNA endonucleases to cleave a chosen sequence, which becomes mutated by error-prone DNA repair mechanism in host cells. This is a rapid and convenient way to mutate genes for functional studies. The West group has established both multiplex CRISPR/Cas9 and optimised TALEN platforms, each suited for different applications. Despite the ease and power of these new technologies for gene disruption, making accurate genetic modifications, particularly gene insertions, is challenging. The group are developing new strategies to address this challenge, which should impact the genetic therapy and biotechnology fields.
Transcription Activator Like Effector (TALE) proteins are transcription factors with a unique modular DNA-recognition mechanism. TALE proteins can be readily engineered to bind to any DNA sequence of choice. Likewise, dCas9, a deactivated version of the CRISPR enzyme, can be used as an RNA-guided DNA-binding protein. The fusion of effector domains onto TALE or dCas9 proteins creates new transcription factors that can alter gene expression programmes or interrogate candidate gene regulatory elements. The West group is using both platforms to activate, repress or label genetic elements to study gene regulation. Epigenetic engineering not only complements genome modification, but is also reversible and offers insight into the specific contributions of histone and DNA modifications.