Scientists Study Dicer's Role in the Malignant Cerebellum

Dicer's Role in the Malignant Cerebellum

Vijay Swahari

In the early era of genomics, almost 98% of the human genome was considered noncoding. Recent advances in Encyclopedia of DNA Elements (ENCODE), a part of the Human Genome Project, have revealed that while only a small percentage of the genome is coding proteins, novel regulatory roles are also performed by the so called “noncoding” parts of DNA.¹Portions of noncoding DNA are transcribed into noncoding RNAs (ncRNAs) involved in an increasing number of cellular events, including the regulation of transcription through the process of RNA interference (RNAi).²

Dicer is an RNase enzyme that processes hairpin structures to yield small, double-stranded ncRNAs. These ncRNAs mediate RNAi during DNA damage response (DDR), specifically in the presence of exogenous DNA damage, as well as in cells undergoing oncogene-induced senescence.³The ncRNAs correspond to the sites of DNA double strand breaks and act as templates for efficient DNA repair.⁴

In a new study titled, Essential function of Dicer in resolving DNA damage in the rapidly dividing cells of the developing and malignant cerebellum, published inCell Reports, Swahari et al investigated the roles of Dicer in replication-associated DNA damage during development.⁵

The team, led by principal investigator Mohanish Deshmukh, PhD, professor of the Neuroscience Center, UNC Chapel Hill, chose a model of the developing cerebellum for the study. The cerebellum model was a good fit because it involved massive expansion of the cerebellar granule neuron precursors (CGNPs), a process associated with replicative stress.⁶

The hypothesis was based on correlations observed between Dicer expression and proliferation patterns. Dicer mRNA and protein levels were high in cerebellar lysates at P7, when proliferation is active and down regulated by P20 when the proliferation terminates.

Dicer roles were analyzed by generating mice lacking Dicer expression in specific domains of the cerebellum, because full knockout results in embryonic lethality. Mice lacking Dicer in CGNPs were generated by intercrossing Dicer floxed and Math1-Cre transgenic mice.⁷The Dicer Math1-Cre mice showed an extensive loss of CGNPs during cerebellar development. CGNP loss was detectable at P2 and resulted in near complete absence at P20. Analysis of P4 cerebellum with markers of proliferation and apoptosis revealed minimal differences in rates of proliferation, while a marked increase in expression of caspase-3, an apoptosis marker, was observed. Loss of CGNPs in the developing cerebellum where Dicer was deficient was attributed to increased apoptosis in CGNPs.

The team further examined the role of Dicer in replication-associated DNA damage in other proliferative regions of the brain by generating Dicer hGFAP-Cre in a mouse model where recombination occurs in primitive neural precursor, the dentate gurus of the hippocampus and cerebellum⁸. Dicerf/f; hGFAP-Cre (DicerhGFAP-Cre) mice also exhibited cerebellar progenitor degeneration. Dicer levels were also higher in mouse embryonic stem cells (mESCs) which are known to proliferate rapidly and undergo replicative stress.⁹

To determine whether or not Dicer was important for DDR in mESCs, the study’s authors examined the outcome of knock down Dicer in mESCs. A markedly increased cell death was observed. Importantly, cell death seen with Dicer inhibition in mESCs was p53 dependent, as knockdown of p53 reduced the Dicer-deficiency-induced mESC death.

Since replication-associated DNA damage is also associated with rapidly proliferating cancers, the team further decided to test the role of Dicer DDR in medulloblastoma tumor development and progression. To test this, the team used the SmoM2 medulloblastoma tumor model where a Smoothened mutation constitutively activates the Shh pathway in CGNPs, leading to aggressive tumor development in these mice by P20.¹⁰The team found that Dicer-deficient SmoM2 mice (Dicerf/f Math1-Cre; SmoM2) developed markedly smaller tumors compared with wild-type SmoM2 mice. The reduced tumor volume was not a consequence of reduced proliferation, but due to increased DNA damage and apoptosis. They also found that Dicer-deficient medulloblastoma tumors were more sensitive to chemotherapy.

Collectively, the study shows important roles of Dicer in DDR that resolves endogenous DNA damage in rapidly proliferating cells during development, a task that also is co-opted in tumors.

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