The β-Cell Response to the Challenge of Weaning

July 24, 2017

Embryonic development of the pancreas depends heavily on the Pdx1 transcription factor, which contributes to the differentiation and function of every cell type, including the insulin-secreting β-cells of the Islets of Langerhans. Expression of the gene encoding Pdx1 is controlled by four 5′-flanking enhancer-like domains designated Areas I, II, III, and IV. A variety of transcription factors bind to these areas with changes in identity and quantity occurring throughout development and after birth. Prior work revealed that Area II is necessary for development of pancreatic progenitor cell formation, leading Vanderbilt Basic Sciences investigators Roland Stein, Christopher Wright, and Mark Magnuson to investigate the role of Area IV. To accomplish their goal, they created a mouse bearing a Pdx1 gene lacking area IV. Mice homozygous for this gene exhibited no significant abnormalities, suggesting that the other regulatory areas could compensate for the Area IV deficit. However, heterozygous mice bearing one area IV knockout gene and one Pdx1-null gene exhibited a small but consistent reduction in the number of insulin-producing cells during embryogenesis. These mice appeared normal at birth and through the first several weeks of life, but at the time of weaning (>week 3), hyperglycemia appeared in the males. Further studies in these male mice demonstrated a reduction in β-cell area and proliferation by weeks 4-5. Consistently, a decrease in expression of β-cell-associated genes, and genes regulating endoplasmic reticulum function and the cell cycle was also evident. Because these abnormalities only occurred in mice expressing low levels of Pdx1 due to knockout of one allele, the investigators hypothesized that binding of Pdx1 itself to Area IV must occur at the time of weaning. Experimental data supported this idea. Weaning is associated with a marked change in diet that leads to differential expression of 1600 β-cell genes, over 500 of which are regulated by Pdx1. Together, the findings support a role for Pdx1 in the β-cell response to weaning. The work is published in the journal Diabetes [J.M. Spaeth, et al., (2017) Diabetes, published online July 13, DOI: 10.2337/db16-1516]. 

RADX: A New DNA Replication and Repair Protein

July 20, 2017

Single-stranded DNA (ssDNA) binding proteins protect and promote the processing of ssDNA that is formed during replication and repair. Among these are RPA (replication protein A), which associates with ssDNA at the replication fork to facilitate lagging-strand synthesis and prevent fork collapse, and RAD51, which forms filaments on ssDNA at resected double strand breaks (DSBs) to promote homology-directed repair (HDR). Now, Vanderbilt Basic Sciences investigator David Cortez and his laboratory describe a new ssDNA binding protein that they discovered using iPOND (identification of Proteins on Nascent DNA), a method developed in their laboratory to identify proteins associated with DNA replication forks. The new protein, which they named RADX, accumulated in large quantities at replication forks that had stalled as a result of exposure to hydroxyurea. Use of siRNA or CRISPR-Cas9 to reduce expression of RADX in cells resulted in increased DNA damage. Structural studies revealed the presence of multiple ssDNA binding domains similar to those in RPA, suggesting that RADX is an ssDNA-binding protein. Further work showed that RAD51 accumulates at DNA replication forks when RADX expression is blocked and that reduction of RAD51 expression reduces the DNA damage associated with RADX depletion. This led the investigators to hypothesize that RADX’s function is to antagonize the action of RAD51. An important mutation found in some common cancers is in the gene that encodes BRCA2, a protein required for RAD51 binding to ssDNA. These cancers are usually highly susceptible to DNA damaging chemotherapeutic agents but often acquire resistance to the drugs. The Cortez lab hypothesized that a reduction in RADX expression to match the decreased RAD51 function might be a mechanism of resistance in BRCA2-deficient cells, and reported experimental data to support this idea. Their findings provide new insight into a key protein involved in DNA replication and repair and the role it might play in cancer drug resistance. The work is published in the journal Molecular Cell [H. Dungrawala, K. Bhat, et al., (2017) Mol. Cell, published online July 20, DOI: 10.1016/j.molcel.2017.06.023

Attribution would be: Figure kindly provided by Bianca Sirbu of the Cortez lab. Copyright 2013.

Understanding Myelination in the Peripheral Nervous System

July 19, 2017

The ability of the nervous system to transmit impulses rapidly depends on the presence of myelin, a complex membranous structure that provides insulation to neuronal axons. In the peripheral nervous system (PNS), myelin is formed by multiple Schwann cells, each of which wraps its membrane in a spiral fashion around a section of the axon. The composition of myelin membrane, which is distinct from that of other cellular membranes, is notable for its high cholesterol and sphingolipid content and the presence of specialized proteins, including peripheral myelin protein 22 (PMP22). PMP22 is an integral membrane protein that accounts for 2 – 5% of the total protein in PNS myelin. A number of inherited neurological diseases result from failure of proper PNS myelination due to insufficient or excessive expression of PMP22; however, the mechanism by which PMP22 promotes myelin formation is not fully understood. Now, Basic Sciences investigators Melanie Ohi and Chuck Sanders, along with their collaborator Jun Li (Department of Neurology) show that PMP22 reconstituted in lipid vesicles forms myelin-like assemblies (MLAs). These MLAs comprised multiple compressed vesicles, stacked and wrapped around each other in a horseshoe shape. As in the case of myelination, MLA formation required the right proportion of PMP22 to lipid. Studies using competing peptides demonstrated a role for the two extracellular loops of PMP22 in MLA formation, suggesting that these loops enable PMP22 on opposing membrane leaflets to interact, sealing the membranes together. A disease-associated mutation of PMP22 resulted in failure of MLA formation in this model system. These findings provide key insights into the role of PMP22 in myelin formation and help to explain why mutations affecting myelin quantity or structure lead to diseases of the PNS.  The work is published in the journal Science Advances [K. F. Mittendorf, J. T. Marinko, et al., (2017), Sci. Adv., 3, e1700220].