Summary Understanding the topological configurations of chromatin may reveal valuable insights into how the genome and epigenome act in concert to control cell fate during development. Here we generate high-resolution architecture maps across seven genomic loci in embryonic stem cells and neural progenitor cells. We observe a hierarchy of 3-D interactions that undergo marked reorganization at the sub-Mb scale during differentiation. Distinct combinations of CTCF, Mediator, and cohesin show widespread enrichment in looping interactions at different length scales. CTCF/cohesin anchor long-range constitutive interactions that form the topological basis for invariant sub-domains. Conversely, Mediator/cohesin together with pioneer factors bridge shortrange enhancer-promoter interactions within and between larger sub-domains. Knockdown of Smc1 or Med12 in ES cells results in disruption of spatial architecture and down-regulation of genes found in cohesin-mediated interactions. We conclude that cell type-specific chromatin organization occurs at the sub-Mb scale and that architectural proteins shape the genome in hierarchical length scales.

SUMMARY A general mechanism for how intracellular signaling pathways in human pluripotent cells are co-ordinated and how they maintain self-renewal remain to be elucidated. In this report, we describe a novel signaling mechanism where PI3K/Akt activity maintains self-renewal by restraining pro-differentiation signaling through suppression of the Raf/Mek/Erk and canonical Wnt signaling pathways. When active, PI3K/Akt establishes conditions where Activin A/Smad2,3 performs a pro-self renewal function by activating target genes, including Nanog. When PI3K/Akt signaling is low, Wnt effectors are activated and function in conjunction with Smad2,3 to promote differentiation. The switch in Smad2,3 activity following inactivation of PI3K/Akt requires the activation of canonical Wnt signaling by Erk, which targets Gsk3β. In sum, we define a signaling framework that converges on Smad2,3 and determines its ability to regulate the balance between alternative cell states. This signaling paradigm has far-reaching implications for cell fate decisions during early embryonic development.

These authors contributed equally to this work.Keywords: AKT-MTOR pathway, autophagy, avibirnavirus, HSP90AA1, viral protein VP2Abbreviations: ANOVA, analysis of variance; ATG5, autophagy-related 5; BCA, bicinchoninic acid; BECN1, Beclin 1, autophagyrelated; cDNA, complementary DNA; CoIP, coimmunoprecipitation; DMEM, Dulbecco's modified Eagle's medium; dsRNA, double-stranded RNA; EBSS, Earle's balanced salt solution; EIF2AK2, eukaryotic translation initiation factor 2-alpha kinase 2; EIF2S1, eukaryotic translation initiation factor 2, subunit 1 alpha; eGFP, enhanced green fluorescent protein; ER, endoplasmic reticulum; Gg, Gallus gallus (chicken); GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GOPC, golgi-associated PDZ and coiled-coil motif containing; GST, glutathione S-transferase; HE-IBDV, heat-inactivated IBDV; hpi, hours post-infection; Hs, Homo sapiens (human); HSP90AA1, heat shock protein 90 kDa alpha (cytosolic), class A member 1; HSV-1, herpes simplex virus 1; IBDV, infectious bursal disease virus; IgG, immunoglobulin G; LPS, lipopolysaccharide; mAb, monoclonal antibody; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MOI, multiplicity of infection; MTOR, mechanistic target of rapamycin (serine/threonine kinase); Ni-NTA, nickel-nitrilotriacetic acid; PAMP, pathogen-associated molecular patterns; PBS, phosphate-buffered saline; PI3K, phosphoinositide 3-kinase; PRR, pattern recognition receptors; RNAi, RNA interference; SDS, sodium dodecyl sulfate; siRNA, small interfering RNA; shRNA, short hairpin RNA; SQSTM1, sequestosome 1; TCID 50 , 50% tissue culture infectious doses; TLR, tolllike receptors; TSC, tuberous sclerosis complex; VP, viral protein; SVP, subviral particle.Autophagy is an essential component of host innate and adaptive immunity. Viruses have developed diverse strategies for evading or utilizing autophagy for survival. The response of the autophagy pathways to virus invasion is poorly documented. Here, we report on the induction of autophagy initiated by the pathogen receptor HSP90AA1 (heat shock protein 90 kDa a [cytosolic], class A member 1) via the AKT-MTOR (mechanistic target of rapamycin)-dependent pathway. Transmission electron microscopy and confocal microscopy revealed that intracellular autolysosomes packaged avibirnavirus particles. Autophagy detection showed that early avibirnavirus infection not only increased the amount of light chain 3 (LC3)-II, but also upregulated AKT-MTOR dephosphorylation. HSP90AA1-AKT-MTOR knockdown by RNA interference resulted in inhibition of autophagy during avibirnavirus infection. Virus titer assays further verified that autophagy inhibition, but not induction, enhanced avibirnavirus replication. Subsequently, we found that HSP90AA1 binding to the viral protein VP2 resulted in induction of autophagy and AKT-MTOR pathway inactivation. Collectively, our findings suggest that the cell surface protein HSP90AA1, an avibirnavirus-binding receptor, induces autophagy through the HSP90AA1-AKT-MTOR pathway in early infection. We reveal that upon...

SummaryHere we show that bivalent domains and chromosome architecture for bivalent genes are dynamically regulated during the cell cycle in human pluripotent cells. Central to this is the transient increase in H3K4-trimethylation at developmental genes during G1, thereby creating a “window of opportunity” for cell-fate specification. This mechanism is controlled by CDK2-dependent phosphorylation of the MLL2 (KMT2B) histone methyl-transferase, which facilitates its recruitment to developmental genes in G1. MLL2 binding is required for changes in chromosome architecture around developmental genes and establishes promoter-enhancer looping interactions in a cell-cycle-dependent manner. These cell-cycle-regulated loops are shown to be essential for activation of bivalent genes and pluripotency exit. These findings demonstrate that bivalent domains are established to control the cell-cycle-dependent activation of developmental genes so that differentiation initiates from the G1 phase.