The role of CSB protein in Cockayne syndrome. Our major focus is to understand why defects in the human SWI/SNF chromatin remodeling protein known as CSB cause Cockayne syndrome (CS), a devastating childhood developmental disease characterized by neurological dysfunction, severe post-natal growth failure, and early death. The CSB protein is best understood as the central component of a DNA repair complex that performs transcription-coupled nucleotide excision repair (TC-NER), but despite continuing progress on the detailed biochemistry of TC-NER, a compelling connection between the DNA repair defect and CS disease has failed to emerge.
Our experimental evidence suggests instead that CS is not caused by a DNA repair defect (as generally assumed) but by a chromatin remodeling defect that deregulates the expression of many genes, somehow inducing a constitutive interferon-like response that resembles the early stages of Aicardi-Goutières syndrome (AGS). We are now collaborating with the Share and Care Network and the Luke O'Brien Foundation to determine whether CS newborns, infants, children, and young adults exhibit the same interferon-like response we observe in CS cell lines.
The CSB-PGBD3 fusion protein. While studying CSB, we accidentally discovered that the CSB gene actually generates two abundant proteins: intact CSB protein generated by default splicing of CSB exons 1-22, and a novel CSB-PGBD3 fusion protein in which CSB exons 1-5 are alternatively spliced in frame to a domesticated piggyBac transposase called PGBD3 which inserted into CSB intron 5 in marmosets over 43 million years ago. Conservation of the CSB-PGBD3 fusion protein for >43 My strongly suggests that it is advantageous in health, but the fusion protein may also contribute to the heterogeneous clinical presentation of CS because it continues to be expressed in some CS patients who lack functional CSB. We also found that the CSB-PGBD3 fusion protein binds to hundreds of specific genomic sites from which it regulates expression of nearby genes. Surprisingly, the fusion protein does not bind directly to DNA at these sites, but indirectly by protein/protein interactions ("tethering") with transcriptional activators (c-Jun and AP1), transcriptional repressors and silencers (CTCF), and RNA polymerase II itself.
PGBD5: a neural-specific piggyBac transposase domesticated >500 Mya and conserved from cephalochordates to humans. We are characterizing PGBD5, the only other domesticated piggyBac element in the human genome. We've shown that (1) PGBD5 was first domesticated in the common ancestor of the cephalochordate Branchiostoma (aka lancelet or amphioxus) and vertebrates, and is conserved in all vertebrates including lamprey, but cannot be found in more basal urochordates, hemichordates, or echinoderms; (2) the lancelet, lamprey, and human PGBD5 genes are syntenic and orthologous as expected for a single domestication at the base of the vertebrate lineage; (3) although derived from an IS4-related transposase, PGBD5 protein is unlikely to retain enzymatic activity because the catalytic DDD motif is not conserved; (4) PGBD5 is preferentially expressed in certain granule cell lineages of the mammalian brain and central nervous system based on available mouse and human in situ hybridization data; (5) the human PGBD5 promoter and gene region is rich in bound regulatory factors including the neuron-restrictive silencer factors NRSF/REST and CoREST, as well as SIN3, KAP1, STAT3, and CTCF; and (6) despite preferential localization within the nucleus, PGBD5 protein is unlikely to bind DNA or chromatin as neither DNase I digestion nor high salt extraction release PGBD5 from fractionated mouse brain nuclei. We speculate that the neural-specific PGBD5 transposase was domesticated >500 My after cephalochordates and vertebrates diverged from urocordates, and that PGBD5 may have played a role in the evolution of a primitive deuterosome neural network into a centralized nervous system (CNS).
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