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Metabolic dysfunction–associated steatotic liver disease (MASLD) is the most common chronic liver disease worldwide for which there is only one approved treatment. Adenosine monophosphate–activated protein kinase (AMPK) is an interesting therapeutic target since it acts as a central regulator of cellular metabolism. Despite efforts to target AMPK, no direct activators have yet been approved for treatment of this disease. This study investigated the effect of the AMPK activator ATX-304 in a preclinical mouse model of progressive fatty liver disease. The data demonstrated that ATX-304 diminishes body fat mass, lowers blood cholesterol levels, and mitigates general liver steatosis and the development of liver fibrosis, but with pronounced local heterogeneities. The beneficial effects of ATX-304 treatment were accompanied by a shift in the liver metabolic program, including increased fatty acid oxidation, reduced lipid synthesis, as well as remodeling of cholesterol and lipid transport. We also observed variations in lipid distribution among liver lobes in response to ATX-304, and a shift in the zonal distribution of lipid droplets upon treatment. Taken together, our data suggested that ATX-304 holds promise as a potential treatment for MASLD.

To facilitate the understanding of how a complex organ such as the pancreas is formed, this chapter illustrates the general anatomical dynamics of pancreas morphogenesis that occur during development in mice (and in humans where relevant). By applying recent advances in optical imaging techniques, including optical projection tomography and light sheet fluorescence microscopy (LSFM), this chapter presents a full image series demonstrating pancreatic bud formation and growth, as well as key morphological events that result in murine and human organs that are anatomically quite different. Further, it is now well established that pancreas development is governed by complex gene regulatory networks, where the timing and duration of gene expression, as well as the degree of molecular interactions are critical. Where appropriate, these key molecular determinants in inductive processes or other events are discussed in relation to pancreas organogenesis. Finally, this chapter describes the spatial and quantitative distribution of insulin as an example of pancreatic endocrine structure-function relationships, where lobular islet heterogeneity in the adult pancreata of mice and humans are evaluated and discussed.

The midbrain-hindbrain boundary (MHB), also known as the isthmic organizer (IsO), plays a critical role in the developmental patterning of the posterior midbrain and anterior hindbrain. Understanding the wiring of this organizer's deeply conserved gene regulatory network is of significant interest for both evolutionary and neurodevelopmental biology. Various secreted signalling molecules and transcription factors have been identified as being important components for the formation and function of the MHB. Among these, FGF8 is considered a primary mediator of IsO activity; it directs anterior-posterior patterning and promotes the specification and maintenance of the MHB. While the core gene regulatory network governing MHB development is well-characterized, the direct interactions between key regulatory genes and the cis-regulatory elements that control their spatiotemporal expression remain poorly understood. This review summarizes the current knowledge of the gene regulatory network underlying the formation of the vertebrate midbrain-hindbrain boundary. We focus in particular on Fgf8 and its regulatory landscape from an evolutionary perspective.

Several preclinical models have been recently developed for metabolic associated fatty liver disease (MAFLD) and associated hepatocellular carcinoma (HCC) but comprehensive analysis of the regulatory and transcriptional landscapes underlying disease in these models are still missing. We investigated the regulatory and transcriptional landscape in fatty livers and liver tumours from DIAMOND mice that faithfully mimic human HCC development in the context of MAFLD. RNA-sequencing and ChIP-sequencing revealed rewiring of the Wnt/β-catenin regulatory network in DIAMOND tumours, as manifested by chromatin remodelling and associated switching in the expression of the canonical TCF/LEF downstream effectors. We identified splicing as a major mechanism leading to constitutive oncogenic activation of β-catenin in a large subset of DIAMOND tumours, a mechanism that is independent on somatic mutations in the locus and that has not been previously shown. Similar splicing events were found in a fraction of human HCC and hepatoblastoma samples.

Background: Glioblastoma (GB) is the most aggressive of all primary brain tumours and due to its highly invasive nature, surgical resection is nearly impossible. Patients typically rely on radiotherapy with concurrent temozolomide (TMZ) treatment and face a median survival of ~ 14 months. Alterations in the Epidermal Growth Factor Receptor gene (EGFR) are common in GB tumours, but therapies targeting EGFR have not shown significant clinical efficacy.

Methods: Here, we investigated the influence of the EGFR regulatory genome on GB cells and identified novel EGFR enhancers located near the GB-associated SNP rs723527. We used CRISPR/Cas9-based approaches to target the EGFR enhancer regions, generating multiple modified GB cell lines, which enabled us to study the functional response to enhancer perturbation.

Results: Epigenomic perturbation of the EGFR regulatory region decreases EGFR expression and reduces the proliferative and invasive capacity of glioblastoma cells, which also undergo a metabolic reprogramming in favour of mitochondrial respiration and present increased apoptosis. Moreover, EGFR enhancer-perturbation increases the sensitivity of GB cells to TMZ, the first-choice chemotherapeutic agent to treat glioblastoma.

Conclusions: Our findings demonstrate how epigenomic perturbation of EGFR enhancers can ameliorate the aggressiveness of glioblastoma cells and enhance the efficacy of TMZ treatment. This study demonstrates how CRISPR/Cas9-based perturbation of enhancers can be used to modulate the expression of key cancer genes, which can help improve the effectiveness of existing cancer treatments and potentially the prognosis of difficult-to-treat cancers such as glioblastoma.

Chromatin organization controls transcription by modulating 3D-interactions between enhancers and promoters in the nucleus. Alterations in epigenetic states and 3D-chromatin organization result in gene expression changes contributing to cancer. Here, we map the promoter-enhancer interactome and regulatory landscape of glioblastoma, the most aggressive primary brain tumour. Our data reveals profound rewiring of promoter-enhancer interactions, chromatin accessibility and redistribution of histone marks in glioblastoma. This leads to loss of long-range regulatory interactions and overall activation of promoters, which orchestrate changes in the expression of genes associated to glutamatergic synapses, axon guidance, axonogenesis and chromatin remodelling. SMAD3 and PITX1 emerge as major transcription factors controlling genes related to synapse organization and axon guidance. Inhibition of SMAD3 and neuronal activity stimulation cooperate to promote proliferation of glioblastoma cells in co-culture with glutamatergic neurons, and in mice bearing patient-derived xenografts. Our findings provide mechanistic insight into the regulatory networks that mediate neurogliomal synaptic communication.

Genome architecture, epigenetics and enhancer function control the fate and identity of cells. Reprogramming to induced pluripotent stem cells (iPSCs) changes the transcriptional profile and chromatin landscape of the starting somatic cell to that of the pluripotent cell in a stepwise manner. Changes in the regulatory networks are tightly regulated during normal embryonic development to determine cell fate, and similarly need to function in cell fate control during reprogramming. Switching off the somatic program and turning on the pluripotent program involves a dynamic reorganization of the epigenetic landscape, enhancer function, chromatin accessibility and 3D chromatin topology. Within this context, we will review here the current knowledge on the processes that control the establishment and maintenance of pluripotency during somatic cell reprogramming.

Rhabdomyosarcoma is a soft tissue cancer that arises in skeletal muscle due to mutations in myogenic progenitors that lead to ineffective differentiation and malignant transformation. The transcription factors Pax3 and Pax7 and their downstream target genes are tightly linked with the fusion positive alveolar subtype, whereas the RAS pathway is usually involved in the embryonal, fusion negative variant. Here, we analyse the role of Pax3 in a fusion negative context, by linking alterations in gene expression in pax3a/pax3b double mutant zebrafish with tumour progression in kRAS-induced rhabdomyosarcoma tumours. Several genes in the RAS/MAPK signalling pathway were significantly down-regulated in pax3a/pax3b double mutant zebrafish. Progression of rhabdomyosarcoma tumours was also delayed in the pax3a/pax3b double mutant zebrafish indicating that Pax3 transcription factors have an unappreciated role in mediating malignancy in fusion negative rhabdomyosarcoma.

Developmental genes are often regulated by multiple elements with overlapping activity. Yet, in most cases, the relative function of those elements and their contribution to endogenous gene expression remain poorly characterized. An example of this phenomenon is that distinct sets of enhancers have been proposed to direct Fgf8 in the limb apical ectodermal ridge and the midbrain-hindbrain boundary. Using in vivo CRISPR/Cas9 genome engineering, we functionally dissect this complex regulatory ensemble and demonstrate two distinct regulatory logics. In the apical ectodermal ridge, the control of Fgf8 expression appears distributed between different enhancers. In contrast, we find that in the midbrain-hindbrain boundary, one of the three active enhancers is essential while the other two are dispensable. We further dissect the essential midbrain-hindbrain boundary enhancer to reveal that it is also composed by a mixture of essential and dispensable modules. Cross-species transgenic analysis of this enhancer suggests that its composition may have changed in the vertebrate lineage.

AMP-activated protein kinase (AMPK) has an important role in cellular energy homeostasis and has emerged as a promising target for treatment of Type 2 Diabetes (T2D) due to its beneficial effects on insulin sensitivity and glucose homeostasis. O304 is a pan-AMPK activator that has been shown to improve glucose homeostasis in both mouse models of diabetes and in human T2D subjects. Here, we describe the genome-wide transcriptional profile and chromatin landscape of pancreatic islets following O304 treatment of mice fed high-fat diet (HFD). O304 largely prevented genome-wide gene expression changes associated with HFD feeding in CBA mice and these changes were associated with remodelling of active and repressive chromatin marks. In particular, the increased expression of the β-cell stress marker Aldh1a3 in islets from HFD-mice is completely abrogated following O304 treatment, which is accompanied by loss of active chromatin marks in the promoter as well as distant non-coding regions upstream of the Aldh1a3 gene. Moreover, O304 treatment restored dysfunctional glucose homeostasis as well as expression of key markers associated with β-cell function in mice with already established obesity. Our findings provide preclinical evidence that O304 is a promising therapeutic compound not only for T2D remission but also for restoration of β-cell function following remission of T2D diabetes.