Rolf Ohlsson lab
The laboratory of nuclear architecture and genome organization
The chromatin platform and its folding in 3D interdigitates the many different pathways converging on the regulation of gene transcription. These principles enable enhancers and promoters to find each other in the nuclear space as well as providing physical obstacles to attenuate such interactions to influence the transcriptional repertoire. To achieve this, the higher order chromatin structures both contribute to and are guided by hallmarks of the nuclear architecture, such as the nuclear periphery. These perspectives have long been in the focus, but has recently been revived by the advent of high throughput sequencing. These include a detailed understanding of the partitioning of the genome in topologically associated domains (TADs) that encompass up to a few million bps to facilitate local regulation of gene expression. However, current chromatin technologies have severe limitations in either resolution or sensitivity, as well as in their abilities to correctly quantify key features of chromatin biology. This two-tier progress of the chromatin field, i.e. high throughput sequencing versus single cell dynamics, is further compounded by the fact that regulatory chromatin features are very dynamic and are governed by stochastic principles.
- Discovery of genomic imprinting in humans (Nature genetics, 1993)
- Discovery of the role of CTCF in genomic imprinting (Curr Biol 2000, Curr Biol 2003)
- Discovery of the role of CTCF in regulating higher order chromatin conformations (PNAS 2006, Nature genetics 2006)
- Discovery of a new role for super-enhancers: To gate target genes to the nuclear pores (under review)
- Innovation of the following techniques: In situ detection of RNA expression (Cell 1984, Cell 1985); Allele-specific in situ hybridization (Development 2005, Cancer Res 2006)); Circular Chromosome Conformation Capture (4C) technique (Nature genetics, 2006); Chromatin in situ Proximity Assay (ChrISP) (Biotechniques 2014; Epigenetics 2014); Nodewalk to examine chromatin structures in less than 10 cells (BioRxiv, under review).
Our lab thus has a long track record in generating techniques dedicated to the high-sensitivity analyses of both gene expression and higher order chromatin conformations. Below we list current projects in which these and other techniques are being employed to explore the symbiosis between the nuclear architecture and genome organization with a special emphasis on understanding how these features are dysregulated in cancer.
Project 1: Development of a novel assay to visualize chromosome conformations in single cells
Next to nothing is known about how entire chromosomes are folded in single cells. This is an important drawback, as dynamic changes in chromosome 3D structures and how these relate to the nuclear architecture can be expected to provide key information of pivotal events in response to environmental cues both in normal development and in complex diseases, such as cancer. We have earlier innovated the ChrISP technique to examine the degree of compaction and/or presence of chromatin marks in either whole chromosomes or at individual loci. Although this technique has been a key to uncover important principles, such as gene gating events (see below), it is based on a fixed distance (<162Å in all three dimensions). In this project we want to visualise a gradient of chromatin compaction and examine how this relates to structural hallmarks of the nucleus, such as nucleolar and peripheral heterochromatin. Moreover, we plan to refine the analyses of these features and examine their dynamic presentations during normal differentiation and in cancer stem-like (CSCs) cell states as well as derived, more mature tumor cells. We will also examine how these features relate to specific hallmarks within normal and tumor tissues, such as angiogenesis, presence of immune cells and inflammation, and build 3D models from the results.
Project 2: Gene gating: An old enigma in new clothes
Although the nuclear periphery is in general linked to repressive micro-domains, both active and inactive transcriptional states are associated with nuclear pores. Moreover, certain nucleoporins can directly interact with active regions including enhancers, although their prominent presence in the nucleoplasm has blurred the discovery of their specific functions at the nuclear pores and the potential existence of gene gating also in higher animals. Using beyond the state of the art techniques, such as the ChrISP technique to analyze opportunities for enhancer-promoter proximities in relation to the nuclear architecture, we have documented that the oncogenic colorectal super-enhancer recruits the transcriptionally active MYC gene to the nuclear pores, thereby facilitating the export of nuclear MYC transcripts. This principle is facilitated by that the potential for interactions between the colorectal super-enhancer and MYC increases in a manner directly proportional to their proximity to the nuclear pores, showing a previously unrecognized connection between enhancer-gene communications and hallmarks of the nuclear architecture (Fig. 1). As the MYC mRNA decay rate is several-fold higher in the nucleus than in the cytoplasm, the net result is increased MYC expression driving the neoplastic process. In this project, we now seek to understand how this version of gene gating is initiated and to examine the role of nucleoporins and other factors that recruit nucleoporins to chromatin in the oncogenic process.
Fig. 1. The influence of the nuclear architecture on a specific set of enhancer-promoter interactions. The dynamic juxtaposition of enhancers and genes to the nuclear periphery/nuclear pore is accompanied by increased opportunities for their interactions. The Lamina-Associated Domains (LADs) and H3K9me2 LOCKs represent robustly inactivated compartments that are perceived to increase the physical constraints on the mobility of chromatin fibres.
Project 3: Cancer stem-like cell states: Dysregulated peripheral chromatin structures and gene gating
The CSC concept, well established in breast cancer, posits that immature cells with self-renewal capacity drive tumorigenesis and metastasis due to their multi-lineage potential. While such cells make up only a minority of the tumor mass, the continuous maturation process will, according to the model, produce tumors with a hierarchical organization. Moreover, the different cell states within a tumor can undergo stochastic transitions to increase cellular heterogeneity. As also more mature cells can de-differentiate into CSC cell states, cancer cells are able to re-establish the immature/mature mix when cultured individually with continuous de novo production of CSC cell states (reviewed in Feinberg et al 2016). In this project, we explore the possibility that epimutations at LADs/LOCKs pave the way for gene gating of pluripotency genes. As described above for project 2, this principle might increase the expression of pluripotency genes to perturb the differentiation pathway to promote CSC cell states. This project will benefit from the availability of purified breast CSCs from patient material within the KA Wallenberg consortium, as well as from the repertoire of single cell techniques that our lab has innovated. Our aim is moreover to identify compounds that specifically interfere with nuclear pore-gene interactions to enforce the specific loss of CSC cell states.
- Knut och Alice Wallenbergs Foundation
- Science Research Council (VR)
- Cancer Research Society