Our laboratory is interested in deciphering the gene regulatory networks that control specification of cell lineages throughout human development. We use advanced molecular tools and human embryonic stem cell cultures to identify and study transcription and epigenetic factors that play critical roles in self-renewal, differentiation and reprogramming of human pluripotent cells. With this basic knowledge at hand, we hope to develop new ways to differentiate and reprogram cells, better understand and treat genetic diseases and reset cellular aging.
1. Global gene regulatory network for pluripotency maintenance in humans
While naïve pluripotency has been extensively studied in mice, primed pluripotency in humans remains poorly defined. We have begun systems level analyses of human ESC pluripotency by conducting whole-genome shRNA screens. Through these analyses we have compiled comprehensive lists of transcription and epigenetic factors whose depletion impairs pluripotency maintenance. We are employing perturbation analyses, genomics and biochemical approaches to gain a systems-level understanding of how a combined action of these factors creates a unique cellular phenotype.
1. Ivanova N, Dobrin R, Lu R, Kotenko I, Levorse J, DeCoste C, Schafer X, Lun Y, Lemischka IR. Dissecting self-renewal in stem cells with RNA interference. Nature 2006 Aug 3; 442: 533-8.
2. Wang Z., Oron E., Nelson B., Razis S., Ivanova N. Distinct lineage specification roles for NANOG, OCT4 and SOX2 in human embryonic stem cells. Cell Stem Cells 2012 10: 440-454.
3. Zhang Y, Schulz V, Reed B, Wang Z, Pan X, Mariani J, Euskirchen G, Snyder M, Vaccarino F, Ivanova N, Weissman S, Szekely A. Functional genomic screen of human stem cell differentiation reveals pathways involved in neurodevelopment and neurodegeneration. PNAS. 2013; 110(30):12361-6.
Funding: NIH R01 GM107092 (PI: Ivanova)
2. Mechanisms of targeting and repression by the non-canonical PRC1 complexes
Polycomb group proteins regulate self-renewal and differentiation in many stem cell systems. When assembled into two canonical complexes, (c)PRC1 and PRC2, they sequentially deposit H3K27me3 and H2AK119ub histone marks and establish repressive chromatin, known as Polycomb domains. Non-canonical (nc)PRC1 complexes which retain RNF2 E3-ubiquitin ligase but have unique sets of accessory subunits also play critical role in Polycomb silencing. How these non-canonical complexes recognize and regulate their target genes is an outstanding question in the field. We have recently shown that BCL6 co-repressor, BCOR, a ncPRC1.1 member, is critical for maintaining primed pluripotency in humans. BCOR depletion in hESCs leads to a loss of Polycomb domains at key developmental loci and initiation of differentiation. Our data suggest that BCOR-PRC1.1 is a novel subtype of the PRC1 complexes with distinct recruitment and repression mechanisms which are currently under investigation.
1. Wang Z., Gearhart MD, Lee YW, Kumar I, Ramazanov B, Zhang Y, Hernandez C, Lu AY, Neuenkirchen N, Deng J, Jin J, Kluger Y, Neubert TA, Bardwell VJ, Ivanova NB. BCOR, a component of the non-canonical Polycomb Repressive Complex PRC1.1, is required to repress critical developmental genes in human ESCs. Cell Stem Cell, 2018;22(2):235-51.
Funding: NIH R01 GM107092 (PI: Ivanova); State of Connecticut Regenerative Medicine Research Fund 16-RMB-YALE-07 (PI: Ivanova)
3. Chromatin remodeling during cellular reprogramming and differentiation
As somatic cells are converted into iPSCs, their chromatin is remodeled to a pluripotent configuration with unique euchromatin-to-heterochromatin ratio, DNA methylation patterns and enhancer/promoter status. The molecular machinery underlying this process is largely unknown. We have recently shown that ESC-specific factors Dppa2 and Dppa4 play a key role in resetting the epigenome to a pluripotent state. They are induced in reprogramming intermediates, function as a heterodimer and are required for efficient reprogramming of mouse and human cells. When co-expressed with OKSM factors, Dppa2/4 yield reprogramming efficiencies that exceed 80% and accelerate reprogramming kinetics, generating iPSCs in two to four days. When bound to chromatin, Dppa2/4 initiate global chromatin decompaction via the DNA damage response pathway, contribute to down-regulation of somatic genes and activation of ESC enhancers, all of which enables an efficient transition to pluripotency. Our new unpublished data indicate that in addition to their role in reprogramming, Dppa2/4 are crucial for the maintenance of developmental capacity of ESCs. Dppa2/4-depleted ESCs, both mouse and human, fail to form germ layer derivatives, while Dppa2/4 overexpression leads to a more efficient differentiation. Our ongoing studies will elucidate how Dppa2/4 and its associated chromatin remodeling factors maintain open chromatin at the regulatory elements of developmental genes to ensure correct execution of lineage-specific programs during differentiation.
1. Hernandez C, Wang Z, Ramazanov B, Tang Y, Mehta S, Dambrot C, Lee YW, Tessema K, Kumar I, Astudillo M, Neubert T, Guo S and Ivanova NB. Chromatin-associated factors Dppa2 and Dppa4 guide epigenetic remodeling during reprogramming to pluripotency. Cell Stem Cell.2018; 23 (3), 396–411.
Funding: NIH R01 GM105772 (PI: Ivanova)
4. Deciphering genetic and epigenetic regulatory logic of germ layer differentiation with single cell technologies
Of the 220 different cell types that exist in the human body, only a dozen or so have been recreated from hESCs in culture owing to a poor understanding of differentiation trajectories through which those cell types emerge during development. To decode the gene regulatory logic of germ layer differentiation in humans we have generated a scRNA-seq dataset of 30~,000 single hESCs grown as embryoid bodies and sampled continuously over a period of weeks. With our new computational method PHATE we generated a comprehensive and interpretable picture of differentiation. We captured all four branches of early development as they are being specified from hESCs. In collaboration with computational biologists at Yale we are conducting mechanistic studies to further unravel the molecular circuitry that controls differentiation of hESCs into specific cell types that are relevant to disease modeling.
1. Moon K, van Dijk D, Wang Z, Chen W, Hirn M, Coifman R, Ivanova N*#, Wolf G*, Krishnaswamy S* PHATE: Visualizing Trajectory Structures in High-Dimensional Biological Data. *Equal senior contributors, #Corresponding author for experiments, Nature Biotechnology – in press.
Funding: NIH R01 GM130847 (PI: Krishnaswamy; Co-I: Ivanova)
Derivation of human induced trophoblast stem cells
The trophectoderm (TE) is an extraembryonic tissue that supplies instructive signals required for embryo patterning during gastrulation and gives rise to the placenta, an organ that connects the developing fetus to the uterine wall to allow nutrient uptake, waste elimination, gas exchange and thermoregulation via the mother’s blood supply. In mice, trophoblast stem cells (mTSCs) derived from the preimplantation blastocyst or from fibroblasts via direct lineage conversion became an important in vitro tool for mechanistic dissection of the molecular pathways crucial for TE specification, maintenance and differentiation. However, the derivation of TSCs from early human embryos has proven surprisingly difficult. To better understand how TSCs form in the human embryo we have performed transcriptome profiling of single cells isolated directly from human blastocysts. From this data, we identified TE-specific transcription factors and signaling ligands expressed in the epiblast and/or in the TE itself that could be responsible for specification and/or maintenance of TSCs. We will use direct lineage conversion with a cocktail of TE-specific factors in the presence of maintenance cytokines to recreate human TSCs in vitro.
Funding: NIH R21 HD099694 (PI: Ivanova)