Gene regulation during developmental transitions; from fertilization to genome activation to cell fate specification
Previous and Current Research
My lab studies how chromatin context influences the regulation of gene expression during vertebrate embryogenesis. We focus on how changes in chromatin structure orchestrate the developmental transitions from fertilization to genome activation to cell fate specification.
The early stages of animal development are characterized by dramatic changes in gene expression. After fertilization the embryonic genome is inactive until transcription is initiated during the maternal-zygotic transition. This transition coincides with the formation of pluripotent cells. Pluripotency refers to the ability of a cell to give rise to all cell lineages of the developing organism. Cells are only transiently pluripotent as cell fate specification signals initiate differentiation soon after the formation of pluripotent blastomeres. The regulation of transcription during the transitions from fertilization to genome activation to cell fate specification is a critical developmental process, yet it is poorly understood.
Chromatin influences transcription by restricting the accessibility of DNA binding proteins to the genome. Studies in cultured embryonic stem (ES) cells, and our analysis of zebrafish embryos have revealed that pluripotency is characterized by a unique chromatin signature. Genes essential for development are marked simultaneously by a repressive and an active chromatin mark (commonly referred to as a bivalent chromatin domain). These genes are thought to be transcriptionally inactive, yet poised for imminent activation. Our analysis has further shown that this profile is established during zygotic genome activation. While these studies have begun to clarify the in vivo relationship between chromatin structure, the onset of zygotic transcription, and pluripotency, they have raised several important questions that are the focus of my lab.
Function, function, function
The function of specific histone modifications in zygotic genome activation and embryonic pluripotency remains unknown. We interfere with histone modification marks in vivo, both genome-wide (mutant analysis) and gene-specifically (local disruption of chromatin structure), and analyze the effect on transcription and pluripotency. This will reveal the functional relationship between the appearance of specific histone modifications during embryogenesis, the activation of the genome, and embryonic pluripotency.
Preparing for genome activation and pluripotency
It is not known what directs establishment of the chromatin marks characteristic for pluripotency. We examine the establishment of pluripotent chromatin marks in a developmental context, by analyzing embryos from fertilization to pluripotency. We use a combined experimental and computational approach to predict whether sequence motifs and/or pre-existing histone modifications direct the recruitment of histone methyltransferases to their targets. We then test these predictions by transiently introducing recruitment sites or interfering with existing ones in vivo. These experiments will reveal how the embryo is prepared for zygotic genome activation and embryonic pluripotency.
Signaling to the genome
How developmental signaling pathways interact with chromatin to establish and maintain lineage-specific gene expression programs remains unclear. In order to address this question, we focus on the Nodal signaling pathway and analyze the chromatin profile of its target genes during the transition from pluripotency to fate specification. Ultimately, we will determine the molecular mechanism of these changes. This line of research will reveal how developmental signaling pathways interact with chromatin to establish specific gene expression programs.
We employ zebrafish for our studies because they provide an excellent model system to investigate the relationship between chromatin organization, gene regulation, and development in a dynamic environment in vivo. The broad applicability of many powerful tools in zebrafish, including genetics (generation of zebrafish mutants), molecular biology (chromatin IP, mRNA overexpression), and molecular and cellular embryology (in situ hybridization, transplantations, imaging), in combination with high throughput sequencing technologies (ChIP-Seq, RNA-Seq) and bio-informatics allow us to analyze all aspects of zygotic genome activation, pluripotency and cell fate specification in a single, developmentally relevant context.
Methodological and Technical Expertise
- Molecular biology such as cloning, (q)PCR, ChIP, and Western blotting
- Genomics approaches such as RNA-Seq, ChIP-Seq, and ATAC-Seq
- Zebrafish techniques such as transplantations, injections, in situs, and CRISPR mediated mutagenesis
Zhang Y, Vastenhouw NL, Feng J, Fu K, Wang C, Ge Y, Pauli A, van Hummelen P, Schier AF, Liu XS
Canonical nucleosome organization at promoters forms during genome activation.
Genome Res. 2014 Feb; 24 (2): 260–6. (3)
Vastenhouw NL, Schier AF
Bivalent histone modifications in early embryogenesis.
Current Opinion in Cell Biology 2012, Apr 16 (41)
Pauli A, Valen E, Lin MF, Garber M, Vastenhouw NL, Levin JZ, Fan L, Sandelin A, Rinn JL, Regev A, Schier AF
Systematic identification of long noncoding RNAs expressed during zebrafish embryogenesis.
Genome Research 2012 Mar; 22 (3): 577–91 (103)
Vastenhouw NL, Zhang Y, Woods IG, Imam F, Regev A, Liu XS, Rinn J, Schier AF
Chromatin signature of embryonic pluripotency is established during genome activation.
Nature 2010, Apr 8; 464 (7290): 922–6 (108)
Vastenhouw NL, Brunschwig K, Okihara KL, Müller F, Tijsterman M and Plasterk RH
Long-term gene silencing by RNA interference.
Nature 2006, Aug 24; 442 (7105): 882 (98)