Multidimensional chromatin profiling of zebrafish pancreas to uncover and investigate disease-relevant enhancers

Most alleles uncovered by genome-wide association studies of pancreatic dysfunctions overlap with non-coding sequences, many of them, containing epigenetic marks of cis-regulatory elements (CREs) active in pancreatic cells, suggesting that alterations in these sequences contribute to pancreatic diseases. Animal models greatly help to understand the role of non-coding alterations in disease. However, interspecies identification of equivalent CREs faces fundamental challenges, including lack of sequence conservation. A team led by José Bessa from at the i3S, combined epigenetic assays with reporter assays in zebrafish and human pancreatic cells to identify interspecies functionally equivalent CREs, regardless of sequence conservation. Among other potential disease-relevant enhancers, they identified a zebrafish ptf1a distal-enhancer whose deletion causes pancreatic agenesis, a phenotype previously described in humans. This approach helps to uncover interspecies functionally equivalent CREs and their potential role in human disease. The paper entitled "Multidimensional chromatin profiling of zebrafish pancreas to uncover and investigate disease-relevant enhancers" was published in Nature Communications.


Transcription in waves

During mitosis, chromatin condensation is accompanied by a global arrest of transcription.  Recent studies suggest transcriptional reactivation upon mitotic exit occurs in temporally coordinated waves, but the underlying regulatory principles have yet to be elucidated.  Work by Mário Soares and collaborators, led by Diogo S. Castro at the i3S Institute of Research and Innovation in Health, revealed how differential binding of transcription factors to mitotic chromosomes, governed by their electrostatic properties, plays an important role in determining the timing of transcriptional reactivation during Mitosis-to-G1 transition.  The team reached this conclusion by focusing on Brn2 and Ascl1, two important regulators of neural stem cell biology.  The article entitled “Hierarchical reactivation of transcription during mitosis-to-G1 transition by Brn2 and AScl1 in neural stem cells” was published in Genes and Development.

Targeting senescent cells improves functional recovery after spinal cord injury

Persistent senescence was shown to be deleterious for tissue repair. A team led by Leonor Saude from iMM in collaboration with Antonio Jacinto lab from CEDOC demonstrated that upon a spinal cord injury, senescent cells start to accumulate at the lesion periphery. Targeting senescent cells through the administration of senolytic drugs improved functional recovery after injury as it promoted myelin sparing, reduced fibrotic scar and attenuated inflammation, which correlated with decreased levels of pro-fibrotic and pro-inflammatory factors in the microenvironment. The paper entitled "Targeting senescent cells improves functional recovery after spinal cord injury" was published in Cell Reports.

A new role for foxm1 in zebrafish muscle homeostasis and consequences of Cas9 overexpression in vivo

foxm1is a master regulator of the cell cycle but recent data have suggested that this transcription factor also modulates gene networks associated with other cellular mechanisms. In their recent work, a team led by Elsa Logarinho and José Bessa from i3S used CRISPR/Cas9 to disrupt foxm1 in the zebrafish terminally differentiated fast-twitching muscle cells. They observed that Cas9 expression alone was strongly deleterious to muscle cells, inducing apoptosis. Interestingly, disruption of foxm1 increased myofiber death and contributed to non-autonomous satellite cell activation and proliferation. The report was published on Cells with the title “foxm1 Modulates Cell Non-Autonomous Response in Zebrafish Skeletal Muscle Homeostasis” and suggests a new function for foxm1 in muscle non-autonomous response to myofiber death, in zebrafish.

CRISPR/Cas9 mediated genomic deletions protocol in zebrafish

Since its first application for site-directed mutagenesis in 2013, the CRISPR/Cas9 system revolutionized genome engineering. In this protocol, João Amorim, Chiara Perrod and collaborators, lead by José Bessa from i3S, present a validated workflow for the generation of targeted genomic deletions in zebrafish. Here, step-by-step details can be found from design, cloning and synthesis of single-guide RNAs and Cas9 mRNA, followed by microinjection in zebrafish embryos and subsequent genotyping screening for the establishment of a mutant line. The versatility and efficiency of this pipeline makes the generation of zebrafish models a widely-used approach in functional genetics.

The article entitled CRISPR-Cas9-Mediated Genomic Deletions Protocol in Zebrafish was published in STAR protocols.