Research conducted in the Knott laboratory is aimed at understanding the epigenetic basis for cellular differentiation in the mammalian embryo and stem cell. The model systems we are implementing include mouse preimplantation embryos, embryonic stem (ES) cells, and trophoblast Stem (TS) cells. Presently, we are investigating the role of Brahma related gene 1 (BRG1)-dependent chromatin remodeling complexes in blastocyst development and ES cell pluripotency. These epigenetic modifiers represent a subclass of SWItch/Sucrose NonFermentable (SWI/SNF) ATP-dependent enzymes that play key roles in embryo development, differentiation, and cell-cycle regulation. In the nucleus BRG1 functions as either an activator or repressor of transcription. BRG1 accomplishes this via remodeling of nucleosomes at core regulatory elements and recruiting various co-activators and co-repressors to target gene promoters. Current projects are elucidating the biological function of BRG1 in blastocyst development and stem cell pluripotency. Specifically, we are establishing the underlying mechanism by which BRG1 regulates transcription of embryonic genes in the first cellular lineages (i.e., inner cell mass and trophoblast). To test this we are utilizing a number of molecular tools including RNA interference (RNAi), gene expression analysis, proteomics, chromatin-remodeling assays and Chromatin-immunoprecipitation (ChIP) analysis. So far, we have identified a subset of BRG1 target genes that are essential for normal embryo development and stem cell pluripotency. Moreover, we have established that BRG1 cooperates with core transcription factors and co-repressors to regulate these genes. This work has implications in assisted reproductive technologies (ART) and cellular reprogramming for cell replacement therapies as well as broader implications in cancer. These studies are funded through a grant from the National Institutes of Health (NIH).
Role of Brg1 in cell-fate decisions and repression of pluripotency
The first cell-fate decision in the preimplantation embryo, the differentiation of the ICM and trophoblast, is regulated by the transcription factors OCT4 and CDX2. Initially, both Oct4 and Cdx2 are widely expressed. However, during blastocyst formation Oct4 expression is restricted to the ICM and Cdx2 expression is confined to the trophoblast. Evidence indicates that suppression of Oct4 expression in the trophectoderm lineage is mediated by CDX2. Nonetheless, the underlying epigenetic modifiers required for CDX2-dependent repression of the Oct4 gene are largely unknown. We have identified an important function for BRG1 in CDX2-mediated repression of Oct4 expression in the developing trophectoderm. We found that: (1) combined knockdown (KD) of BRG1 and CDX2 levels in blastocysts resulted in increased levels of Oct4 transcripts compared to KD of BRG1 or CDX2 alone, (2) endogenous BRG1 coimmunoprecipitated with CDX2 in TS cell extracts, (3) in blastocysts BRG1 and CDX2 co-localize in trophectoderm nuclei and (4) in Cdx2-induced ES cells BRG1 and CDX2 are recruited to the Oct4 promoter. To determine how BRG1 may induce epigenetic silencing of theOct4 gene, we evaluated CpG methylation at the Oct4 promoter in the trophectoderm of BRG1 KD blastocysts. This analysis revealed that BRG1 dependent repression of Oct4 expression is independent of DNA methylation at the blastocyst stage. Ongoing studies are establishing the underlying epigenetic mechanism(s) by which BRG1 facilitates Oct4 silencing in the trophectoderm. This work is done in collaboration with Bill Henry in the Department of BMB.
Manipulation of Brg1 target genes/interacting proteins in the developing mouse embryo
Brg1 knock-out/down embryos exhibit perturbations in gene expression and arrest around the blastocyst stage. To better understand the biological role of BRG1 in embryo development we are manipulating the levels of candidate BRG1 interacting proteins and BRG1 target genes in wild-type preimplantation embryos. These candidates were selected based on our microarray, co-immunoprecipitation, and ChIP studies. Particularly, we are utilizing siRNA and/or lentiviral expression vectors to knockdown or ectopically express specific proteins in the trophectoderm. Through this analysis we have begun to establish the BRG1-based mechanisms that regulate normal embryo development.
Research Assistant II
Catherine received her BS degree in biology from Drexel University. She manages the Knott laboratory and provides cell and tissue culture support for ongoing projects.
Tim received his BS from Washington State University. His research focuses on the role of BRG1 in transcriptional regulation of blastocyst development and stem cell pluripotency.
Inchul received his PhD from the University of Nottingham. His research focuses on the role of BRG1 and the transcription factor TCFAP2C in preimplantation development and ES cell pluripotency.
Ragina N.P., Schlosser K, Knott J.G., Senagore P.K., Swiatek P.J., Chang E.A., Fakhouri W.D., Schutte B.G., Kiupel M, Cibelli J.B. Down-regulation of H19 improves the differentiation potential of mouse parthenogenetic embryonic stem cells. Stem Cells Dev. 2011; Jul 27, Epub.
Luo J, Suhr S, Chang E.A., Wang K, Ross P.J., Nelson L, Venta P, Knott J.G., Jose J.B. Generation of LIF and bFGF-Dependent Induced Pluripotent Stem Cells from Canine Adult Somatic Cells. Stem Cells Dev. 2011; Jun 15, Epub.
Wang K, Sengupta S, Magnani L, Wilson C.A., Henry R.W., Knott J.G. Brg1 is required for Cdx2-mediated repression of Oct4 expression in mouse blastocysts. PloS One 2010 5(5): e10622.
Wang K, Chen Y, Chang E.A., Knott J.G., Cibelli J.B. Dynamic epigenetic regulation of the Oct4 and Nanog regulatory regions during neural differentiation in rhesus nuclear transfer embryonic stem cells. Cloning and Stem Cells 2009; 4: 483-96.
Kidder B.L., Palmer S, Knott J.G. SWI/SNF-Brg1 regulates self-renewal and occupies core pluripotency-related genes in embryonic stem cells. Stem Cells 2009; 27: 317-328.