The influence of tissue spatial geometry and functional organisation on liver regeneration.

The adult liver exerts crucial functions, including nutrient metabolism and storage, bile production and drug detoxification. These complex functions expose the liver to constant damage induced by toxins, metabolic intermediates and oxidative stress. However, the adult liver exhibits an exceptional regenerative potential, which allows fast and efficient restoration of tissue architecture and function both after tissue resection and toxic damage. To accomplish its vital role, the liver shows a peculiar tissue architecture into functional units, which follow the gradient of oxygen and nutrients within the parenchyma. Much less is known about the influence of tissue spatial geometry and functional organisation on adult liver regeneration. Here I examine the experimental evidence in mouse models showing that the spatial organisation of the epithelial and mesenchymal compartments plays a key role in liver regeneration and favours the establishment of regenerative adult liver progenitors following liver injury. I also discuss the advantages and limitations of human and mouse 3D hepatic organoid systems, which recapitulate key aspects of liver function and architecture, as models of liver regeneration and disease. Finally, I analyse the role of the YAP/TAZ transcriptional co-activators as a central hub sensing the extra-cellular matrix (ECM), metabolic and epigenetic remodelling that regulate liver regeneration and promote liver disease, such as fibrosis, chronic liver disease and liver cancer. Together, the findings summarised here demonstrate that local physical and functional cellular interactions determined by the liver peculiar spatial geometry, play a crucial role in liver regeneration, and that their alterations have important implications for human liver disease.

Epigenetic Regulation of Cell-Fate Changes That Determine Adult Liver Regeneration After Injury.

The adult liver has excellent regenerative potential following injury. In contrast to other organs of the body that have high cellular turnover during homeostasis (e.g., intestine, stomach, and skin), the adult liver is a slowly self-renewing organ and does not contain a defined stem-cell compartment that maintains homeostasis. However, tissue damage induces significant proliferation across the liver and can trigger cell-fate changes, such as trans-differentiation and de-differentiation into liver progenitors, which contribute to efficient tissue regeneration and restoration of liver functions. Epigenetic mechanisms have been shown to regulate cell-fate decisions in both embryonic and adult tissues in response to environmental cues. Underlying their relevance in liver biology, expression levels and epigenetic activity of chromatin modifiers are often altered in chronic liver disease and liver cancer. In this review, I examine the role of several chromatin modifiers in the regulation of cell-fate changes that determine efficient adult liver epithelial regeneration in response to tissue injury in mouse models. Specifically, I focus on epigenetic mechanisms such as chromatin remodelling, DNA methylation and hydroxymethylation, and histone methylation and deacetylation. Finally, I address how altered epigenetic mechanisms and the interplay between epigenetics and metabolism may contribute to the initiation and progression of liver disease and cancer.

Epigenetic remodelling licences adult cholangiocytes for organoid formation and liver regeneration.

Following severe or chronic liver injury, adult ductal cells (cholangiocytes) contribute to regeneration by restoring both hepatocytes and cholangiocytes. We recently showed that ductal cells clonally expand as self-renewing liver organoids that retain their differentiation capacity into both hepatocytes and ductal cells. However, the molecular mechanisms by which adult ductal-committed cells acquire cellular plasticity, initiate organoids and regenerate the damaged tissue remain largely unknown. Here, we describe that ductal cells undergo a transient, genome-wide, remodelling of their transcriptome and epigenome during organoid initiation and in vivo following tissue damage. TET1-mediated hydroxymethylation licences differentiated ductal cells to initiate organoids and activate the regenerative programme through the transcriptional regulation of stem-cell genes and regenerative pathways including the YAP-Hippo signalling. Our results argue in favour of the remodelling of genomic methylome/hydroxymethylome landscapes as a general mechanism by which differentiated cells exit a committed state in response to tissue damage.

Cellular plasticity in the adult liver and stomach.

Adult tissues maintain function and architecture through robust homeostatic mechanisms mediated by self-renewing cells capable of generating all resident cell types. However, severe injury can challenge the regeneration potential of such a stem/progenitor compartment. Indeed, upon injury adult tissues can exhibit massive cellular plasticity in order to achieve proper tissue regeneration, circumventing an impaired stem/progenitor compartment. Several examples of such plasticity have been reported in both rapidly and slowly self-renewing organs and follow conserved mechanisms. Upon loss of the cellular compartment responsible for maintaining homeostasis, quiescent or slowly proliferating stem/progenitor cells can acquire high proliferation potential and turn into active stem cells, or, alternatively, mature cells can de-differentiate into stem-like cells or re-enter the cell cycle to compensate for the tissue loss. This extensive cellular plasticity acts as a key mechanism to respond to multiple stimuli in a context-dependent manner, enabling tissue regeneration in a robust fashion. In this review cellular plasticity in the adult liver and stomach will be examined, highlighting the diverse cell populations capable of repairing the damaged tissue.

Polycomb Regulates Mesoderm Cell Fate-Specification in Embryonic Stem Cells through Activation and Repression Mechanisms.

Polycomb complexes (PRC1 and PRC2) are essential regulators of epigenetic gene silencing in embryonic and adult stem cells. Emerging evidence suggests that the core subunit composition regulates distinct biological processes, yet little is known about the mechanistic underpinnings of how differently composed Polycomb complexes instruct and maintain cell fate. Here we find that Mel18, also known as Pcgf2 and one of six Pcgf paralogs, uniquely regulates PRC1 to specify mesoderm cell fate in embryonic stem cells. Mechanistically, Mel18 functions as a classical Polycomb protein during early cardiac mesoderm differentiation by repressing pluripotency, lineage specification, late cardiac differentiation, and negative regulators of the BMP pathway. However, Mel18 also positively regulates expression of key mesoderm transcription factors, revealing an unexpected function of Mel18 in gene activation during cardiac differentiation. Taken together, our findings reveal that Mel18 is required to specify PRC1 function in both a context- and stage-specific manner.

ZRF1: a novel epigenetic regulator of stem cell identity and cancer.

The Zuotin-related factor 1, ZRF1, has recently been identified as an epigenetic regulator of gene transcription in stem cells and cancer. During differentiation of human teratocarcinoma cells, ZRF1 promotes transcriptional induction of developmental genes that are repressed by Polycomb complexes. Importantly, ZRF1 has recently been shown to be required for both neural differentiation of embryonic stem cells (ESCs) and for maintenance of neural progenitor cell (NPC) identity. Moreover, a dual role has now emerged for ZRF1 in cancer: on the one hand, ZRF1 plays a crucial role in oncogene-induced senescence (OIS) by activating the INK4/ARF locus, thus working as a tumor suppressor; on the other hand, ZRF1 promotes leukemogenesis in acute myeloid leukemia (AML) in a Polycomb-independent fashion. Therefore, increasing evidence points to ZRF1 as a novel target for therapy of neurodegenerative diseases and cancer.

Direct interaction between Id1 and Zrf1 controls neural differentiation of embryonic stem cells.

Id proteins are dominant-negative regulators within the HLH family of proteins. In embryonic stem cells (ESCs), Id1 and Id3 maintain the pluripotent state by preventing neural differentiation. The Id1-interacting protein Zrf1 plays a crucial role as a chromatin-bound factor in specification of the neural fate from ESCs. Here, we show that Id1 blocks Zrf1 recruitment to chromatin, thus preventing the activation of neural genes in ESCs. Upon differentiation, Id1 expression decreases thus inducing Zrf1 binding to neural genes. Importantly, depletion of Zrf1 rescues the expression of Polycomb targets involved in neural specification which are up-regulated in Id1 knock-out ESCs. We therefore identified Zrf1 as transcriptional regulator of neural fate downstream of Id1 in ESCs.

Zrf1 is required to establish and maintain neural progenitor identity.

The molecular mechanisms underlying specification from embryonic stem cells (ESCs) and maintenance of neural progenitor cells (NPCs) are largely unknown. Recently, we reported that the Zuotin-related factor 1 (Zrf1) is necessary for chromatin displacement of the Polycomb-repressive complex 1 (PRC1). We found that Zrf1 is required for NPC specification from ESCs and that it promotes the expression of NPC markers, including the key regulator Pax6. Moreover, Zrf1 is essential to establish and maintain Wnt ligand expression levels, which are necessary for NPC self-renewal. Reactivation of proper Wnt signaling in Zrf1-depleted NPCs restores Pax6 expression and the self-renewal capacity. ESC-derived NPCs in vitro resemble most of the characteristics of the self-renewing NPCs located in the developing embryonic cortex, which are termed radial glial cells (RGCs). Depletion of Zrf1 in vivo impairs the expression of key self-renewal regulators and Wnt ligand genes in RGCs. Thus, we demonstrate that Zrf1 plays an essential role in NPC generation and maintenance.

Polycomb complexes in stem cells and embryonic development.

Polycomb group (PcG) proteins are epigenetic modifiers involved in controlling gene repression. Organized within multiprotein complexes, they regulate developmental genes in multiple cell types and tissue contexts, including embryonic and adult stem cells, and are essential for cell fate transitions and proper development. Here, we summarize recent breakthroughs that have revealed the diversity of PcG complexes acting in different cell types and genomic contexts. Intriguingly, it appears that particular PcG proteins have specific functions in embryonic development, in pluripotent stem cells and in reprogramming somatic cells into a pluripotent-like state. Finally, we highlight recent results from analyzing PcG protein functions in multipotent stem cells, such as neural, hematopoietic and epidermal stem cells.

RYBP and Cbx7 define specific biological functions of polycomb complexes in mouse embryonic stem cells.

The Polycomb repressive complex 1 (PRC1) is required for decisions of stem cell fate. In mouse embryonic stem cells (ESCs), two major variations of PRC1 complex, defined by the mutually exclusive presence of Cbx7 or RYBP, have been identified. Here, we show that although the genomic localization of the Cbx7- and RYBP-containing PRC1 complexes overlaps in certain genes, it can also be mutually exclusive. At the molecular level, Cbx7 is necessary for recruitment of Ring1B to chromatin, whereas RYBP enhances the PRC1 enzymatic activity. Genes occupied by RYBP show lower levels of Ring1B and H2AK119ub and are consequently more highly transcribed than those bound by Cbx7. At the functional level, we show that genes occupied by RYBP are primarily involved in the regulation of metabolism and cell-cycle progression, whereas those bound by Cbx7 predominantly control early-lineage commitment of ESCs. Altogether, our results indicate that different PRC1 subtypes establish a complex pattern of gene regulation that regulates common and nonoverlapping aspects of ESC pluripotency and differentiation.

Direct targets of Klf5 transcription factor contribute to the maintenance of mouse embryonic stem cell undifferentiated state.

BACKGROUND: A growing body of evidence has shown that Krüppel-like transcription factors play a crucial role in maintaining embryonic stem cell (ESC) pluripotency and in governing ESC fate decisions. Krüppel-like factor 5 (Klf5) appears to play a critical role in these processes, but detailed knowledge of the molecular mechanisms of this function is still not completely addressed. RESULTS: By combining genome-wide chromatin immunoprecipitation and microarray analysis, we have identified 161 putative primary targets of Klf5 in ESCs. We address three main points: (1) the relevance of the pathways governed by Klf5, demonstrating that suppression or constitutive expression of single Klf5 targets robustly affect the ESC undifferentiated phenotype; (2) the specificity of Klf5 compared to factors belonging to the same family, demonstrating that many Klf5 targets are not regulated by Klf2 and Klf4; and (3) the specificity of Klf5 function in ESCs, demonstrated by the significant differences between Klf5 targets in ESCs compared to adult cells, such as keratinocytes. CONCLUSIONS: Taken together, these results, through the definition of a detailed list of Klf5 transcriptional targets in mouse ESCs, support the important and specific functional role of Klf5 in the maintenance of the undifferentiated ESC phenotype. See: http://www.biomedcental.com/1741-7007/8/125.

Differentiation of embryonic stem cells 1 (Dies1) is a component of bone morphogenetic protein 4 (BMP4) signaling pathway required for proper differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESCs) are pluripotent cells able to grow indefinitely in culture and to differentiate into all cell types of embryos upon specific stimuli. Molecular mechanisms controlling the unique characteristics of ESCs are still largely unknown. We identified Dies1 (Differentiation of ESCs 1), an unpublished gene, that encodes a type I membrane protein. ESCs stably transfected with Dies1 small hairpin RNAs failed to properly differentiate toward neural and cardiac cell fate upon appropriate stimuli and continued to express markers of undifferentiated cells, such as the membrane-associated alkaline phosphatase, and transcription factors, like Oct3/4 and Nanog, when grown under conditions promoting differentiation. Our results demonstrated that Dies1 is required for BMP4/Smad1 signaling cascade; in undifferentiated ESCs Dies1 knockdown did not affect the expression of leukemia inhibitory factor downstream targets, whereas it resulted in a strong decrease of BMP4 signaling, as demonstrated by the decrease of Id1, -2, and -3 mRNAs, the decreased activity of Id1 gene promoter, and the reduced phospho-Smad1 levels. Dies1 knockdown had no effect in murine ESCs when the expression of the BMP4 receptor Alk3 was suppressed. The phenotype induced by Dies1 suppression in ESCs is due to the indirect activation of the Nodal/Activin pathway, which is a consequence of the BMP4 pathway inhibition and is sufficient to support the mESC undifferentiated state in the absence of leukemia inhibitory factor.

Klf5 is involved in self-renewal of mouse embryonic stem cells.

Self-renewal of embryonic stem cells (ESCs) is maintained by a complex regulatory mechanism involving transcription factors Oct3/4 (Pou5f1), Nanog and Sox2. Here, we report that Klf5, a Zn-finger transcription factor of the Kruppel-like family, is involved in ESC self-renewal. Klf5 is expressed in mouse ESCs, blastocysts and primordial germ cells, and its knockdown by RNA interference alters the molecular phenotype of ESCs, thereby preventing their correct differentiation. The ability of Klf5 to maintain ESCs in the undifferentiated state is supported by the finding that differentiation of ESCs is prevented when Klf5 is constitutively expressed. Maintenance of the undifferentiated state by Klf5 is, at least in part, due to the control of Nanog and Oct3/4 transcription, because Klf5 directly binds to the promoters of these genes and regulates their transcription.

Receptor- and non-receptor tyrosine kinases induce processing of the amyloid precursor protein: role of the low-density lipoprotein receptor-related protein.

The Alzheimer's beta-amyloid peptides derive from the proteolytic processing of the beta-amyloid precursor protein, APP, by beta- and gamma-secretases. The regulation of this processing is not fully understood. Experimental evidence suggests that the activation of pathways involving protein tyrosine kinases, such as PDGFR and Src, could induce the cleavage of APP and in turn the generation of amyloid peptides. In this paper we addressed the effect of receptor and nonreceptor protein tyrosine kinases on the cleavage of APP and the mechanisms of their action. To this aim, we developed an in vitro system based on the APP-Gal4 fusion protein stably transfected in SHSY5Y neuroblastoma cell line. The cleavage of this molecule, induced by various stimuli, results in the activation of the transcription of the luciferase gene under the control of Gal4 cis-elements. By using this experimental system we demonstrated that, similarly to Src, three tyrosine kinases, TrkA, Ret and EGFR, induced the cleavage of APP-Gal4. We excluded that this effect was mediated by the activation of Ras-MAPK, PI3K-Akt and PLC-gamma pathways. Furthermore, the direct phosphorylation of the APP cytosolic domain does not affect Abeta peptide generation. On the contrary, experiments in cells lacking the LDL-receptor related protein LRP support the hypothesis that the interaction of APP with LRP is required for the induction of APP cleavage by tyrosine kinases.

Essential roles for Fe65, Alzheimer amyloid precursor-binding protein, in the cellular response to DNA damage.

Fe65 interacts with the cytosolic domain of the Alzheimer amyloid precursor protein (APP). The functions of the Fe65 are still unknown. To address this point we generated Fe65 knockout (KO) mice. These mice do not show any obvious phenotype; however, when fibroblasts (mouse embryonic fibroblasts), isolated from Fe65 KO embryos, were exposed to low doses of DNA damaging agents, such as etoposide or H2O2, an increased sensitivity to genotoxic stress, compared with wild type animals, clearly emerged. Accordingly, brain extracts from Fe65 KO mice, exposed to non-lethal doses of ionizing radiations, showed high levels of gamma-H2AX and p53, thus demonstrating a higher sensitivity to X-rays than wild type mice. Nuclear Fe65 is necessary to rescue the observed phenotype, and few minutes after the exposure of MEFs to DNA damaging agents, Fe65 undergoes phosphorylation in the nucleus. With a similar timing, the proteolytic processing of APP is rapidly affected by the genotoxic stress: in fact, the cleavage of the APP COOH-terminal fragments by gamma-secretase is induced soon after the exposure of cells to etoposide, in a Fe65-dependent manner. These results demonstrate that Fe65 plays an essential role in the response of the cells to DNA damage.