Enhancers and long noncoding RNAs (lncRNAs) are key determinants of lineage specification during development. Here, we evaluate remodeling of the enhancer landscape and modulation of the lncRNA transcriptome during mesendoderm specification. We sort mesendodermal progenitors from differentiating embryonic stem cells (ESCs) according to Eomes expression, and find that enhancer usage is coordinated with mesendoderm-specific expression of key lineage-determining transcription factors. Many of these enhancers are associated with the expression of lncRNAs. Examination of ESC-specific enhancers interacting in three-dimensional space with mesendoderm-specifying transcription factor loci identifies MesEndoderm Transcriptional Enhancer Organizing Region (Meteor). Genetic and epigenetic manipulation of the Meteor enhancer reveal its indispensable role during mesendoderm specification and subsequent cardiogenic differentiation via transcription-independent and -dependent mechanisms. Interestingly, Meteor-deleted ESCs are epigenetically redirected towards neuroectodermal lineages. Loci, topologically associating a transcribed enhancer and its cognate protein coding gene, appear to represent therefore a class of genomic elements controlling developmental competence in pluripotency.

Early in female mammalian embryonic development, cells randomly inactivate one of the two X chromosomes to achieve overall equal inactivation of parental X-linked alleles. Hcfc1 is a highly conserved X-linked mouse gene that encodes HCF-1 - a transcriptional co-regulator implicated in cell proliferation in tissue culture cells. By generating a Cre-recombinase inducible Hcfc1 knock-out (Hcfc1(lox)) allele in mice, we have probed the role of HCF-1 in actively proliferating embryonic cells and in cell-cycle re-entry of resting differentiated adult cells using a liver regeneration model. HCF-1 function is required for both extraembryonic and embryonic development. In heterozygous Hcfc1(lox/+) female embryos, however, embryonic epiblast-specific Cre-induced Hcfc1 deletion (creating an Hcfc1(epiKO) allele) around E5.5 is well tolerated; it leads to a mixture of HCF-1-positive and -negative epiblast cells owing to random X-chromosome inactivation of the wild-type or Hcfc1(epiKO) mutant allele. At E6.5 and E7.5, both HCF-1-positive and -negative epiblast cells proliferate, but gradually by E8.5, HCF-1-negative cells disappear owing to cell-cycle exit and apoptosis. Although generating a temporary developmental retardation, the loss of HCF-1-negative cells is tolerated, leading to viable heterozygous offspring with 100% skewed inactivation of the X-linked Hcfc1(epiKO) allele. In resting adult liver cells, the requirement for HCF-1 in cell proliferation was more evident as hepatocytes lacking HCF-1 fail to re-enter the cell cycle and thus to proliferate during liver regeneration. The survival of the heterozygous Hcfc1(epiKO/+) female embryos, even with half the cells genetically compromised, illustrates the developmental plasticity of the post-implantation mouse embryo - in this instance, permitting survival of females heterozygous for an X-linked embryonic lethal allele.

Host cell factor 1 (HCF-1), encoded by the ubiquitously expressed X-linked gene Hcfc1, is an epigenetic coregulator important for mouse development and cell proliferation, including during liver regeneration. We used a hepatocyte-specific inducible Hcfc1 knockout allele (called Hcfc1hepKO) to induce HCF-1 loss in hepatocytes of hemizygous Hcfc1hepKO/Y males by 4 days. In heterozygous Hcfc1hepKO/+ females, owing to random X-chromosome inactivation, upon Hcfc1hepKO allele induction, a 50/50 mix of HCF-1-positive and -negative hepatocyte clusters is engineered. The livers with Hcfc1hepKO/Y hepatocytes displayed a 21- to 24-day terminal nonalcoholic fatty liver (NAFL), followed by nonalcoholic steatohepatitis (NASH) disease progression typical of severe NAFL disease (NAFLD). In contrast, in livers with heterozygous Hcfc1hepKO/+ hepatocytes, HCF-1-positive hepatocytes replaced HCF-1-negative hepatocytes and revealed only mild NAFL development. Loss of HCF-1 led to loss of PGC1α protein, probably owing to its destabilization, and deregulation of gene expression, particularly of genes involved in mitochondrial structure and function, likely explaining the severe Hcfc1hepKO/Y liver pathology. Thus, HCF-1 is essential for hepatocyte function, likely playing both transcriptional and nontranscriptional roles. These genetically engineered loss-of-HCF-1 mice can be used to study NASH as well as NAFLD resolution.

Mouse liver regeneration after partial hepatectomy involves cells in the remaining tissue synchronously entering the cell division cycle. We have used this system and H3K4me3, Pol II and Pol III profiling to characterize adaptations in Pol III transcription. Our results broadly define a class of genes close to H3K4me3 and Pol II peaks, whose Pol III occupancy is high and stable, and another class, distant from Pol II peaks, whose Pol III occupancy strongly increases after partial hepatectomy. Pol III regulation in the liver thus entails both highly expressed housekeeping genes and genes whose expression can adapt to increased demand.

Mammalian partial hepatectomy (PH) induces an orchestrated compensatory hyperplasia, or regeneration, in remaining tissue to restore liver mass; during this process, liver functions are maintained. We probed this process in mice with feeding‐ and light/dark‐entrained animals subjected to sham or PH surgery. Early on (i.e., 10 hours), irrespective of sham or PH surgery, hepatocytes equidistant from the portal and central veins (i.e., midlobular) accumulated the G1‐phase cell‐division‐cycle marker cyclin D1. By 24 hours, however, cyclin D1 disappeared absent PH but was reinforced in midlobular hepatocytes after PH. At 48 hours after PH and 2 hours fasting, synchronously mitotic hepatocytes possessed less glycogen than surrounding nonproliferating hepatocytes. The differential glycogen content generated a conspicuous entangled pattern of proliferating midlobular and nonproliferating periportal and pericentral hepatocytes. The nonproliferating hepatocytes maintained aspects of normal liver properties. Conclusion: In the post‐PH regenerating mouse liver, a binary switch segregates midlobular cells to proliferate side‐by‐side with nonproliferating periportal and pericentral cells, which maintain metabolic functions. Our results also indicate that mechanisms of liver regeneration display evolutionary flexibility. (Hepatology Communications 2017;1:871–885)

BackgroundCompensatory liver hyperplasia—or regeneration—induced by two-thirds partial hepatectomy (PH) permits the study of synchronized activation of mammalian gene expression, particularly in relation to cell proliferation. Here, we measured genomic transcriptional responses and mRNA accumulation changes after PH and sham surgeries.ResultsDuring the first 10–20 h, the PH- and sham-surgery responses were very similar, including parallel early activation of cell-division-cycle genes. After 20 h, however, whereas post-PH livers continued with a robust and coordinate cell-division-cycle gene-expression response before returning to the resting state by 1 week, sham-surgery livers returned directly to a resting gene-expression state. Localization of RNA polymerase II (Pol II), and trimethylated histone H3 lysine 4 (H3K4me3) and 36 (H3K36me3) on genes dormant in the resting liver and activated during the PH response revealed a general de novo promoter Pol II recruitment and H3K4me3 increase during the early 10–20 h phase followed by Pol II elongation and H3K36me3 accumulation in gene bodies during the later proliferation phase. H3K36me3, generally appearing at the first internal exon, was preceded 5′ by H3K36me2; 3′ of the first internal exon, in about half of genes H3K36me3 predominated and in the other half H3K36me2 and H3K36me3 co-existed. Further, we observed some unusual gene profiles with abundant Pol II but little evident H3K4me3 or H3K36me3 modification, indicating that these modifications are neither universal nor essential partners to Pol II transcription.ConclusionsPH and sham surgical procedures on mice reveal striking early post-operatory gene expression similarities followed by synchronized mRNA accumulation and epigenetic histone mark changes specific to PH.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0222-0) contains supplementary material, which is available to authorized users.

Background: Compensatory liver hyperplasia -or regeneration -induced by two-thirds partial hepatectomy (PH) permits the study of synchronized activation of mammalian gene expression, particularly in relation to cell proliferation. Here, we measured genomic transcriptional responses and mRNA accumulation changes after PH and sham surgeries. Results:During the first 10-20 hours, the PH-and sham-surgery responses were very similar, including parallel early activation of cell-division-cycle genes. After 20 hours, however, whereas post-PH livers continued with a robust and coordinate cell-division-cycle gene-expression response before returning to the resting state by one week, sham-surgery livers returned directly to a resting gene-expression state. Localization of RNA polymerase II (Pol II), and trimethylated histone H3 lysine 4 (H3K4me3) and 36 (H3K36me3) on genes dormant in the resting liver and activated during the PH response revealed a general de novo promoter Pol II recruitment and H3K4me3 increase during the early 10-20 hour phase followed by Pol II elongation and H3K36me3 accumulation in gene bodies during the later proliferation phase. H3K36me3, generally appearing at the first-internal exon, was preceded 5' by H3K36me2; 3' of the first-internal exon, in about half of genes H3K36me3 predominated and in the other half H3K36me2 and H3K36me3 co-existed. Further, we observed some unusual gene profiles with abundant Pol II but little evident H3K4me3 or H3K36me3 modification, indicating that these modifications are neither universal nor essential partners to Pol II transcription.Conclusions: PH and sham surgical procedures on mice reveal striking early post-operatory gene expression similarities followed by synchronized mRNA accumulation and epigenetic histone mark changes specific to PH.

The following paragraph should be added at the end of Materials and Methods. "Accession number(s). The GEO number for the data sets corresponding to the expression and genome binding/occupancy profilings by high-throughput sequencing is GSE115768." Supplemental material: The files originally posted inadvertently were missing the final page (Supplemental file 1) and the figures (Supplemental file 2). Revised supplemental material is posted at