Medical Education From a Theory-Practice-Philosophy Perspective.

Medical schooling, at least as structured in the United States and Canada, is commonly assembled intuitively or empirically to meet concrete goals. Despite a long history of scholarship in educational theory to address how people learn, this is rarely examined during medical curriculum design. We provide a historical perspective on educational theory-practice-philosophy and a tool to aid faculty in learning how to identify and use theory-practice-philosophy for the design of curriculum and instruction.

A WW-like module in the RAG1 N-terminal domain contributes to previously unidentified protein-protein interactions.

More than one-third of the RAG1 protein can be truncated from the N-terminus with only subtle effects on the products of V(D)J recombination in vitro or in a mouse. What, then, is the function of the N-terminal domain? We believe it to be regulatory. We determined, several years ago, that an included RING motif could function as an ubiquitin E3 ligase. Whether this activity is limited to automodification, or may alter other proteins in the cell, remains an open question. We revisited the issue of additional protein-protein interactions between RAG1 and other proteins by means of the yeast two-hybrid assay. We confirmed the interaction already described with KPNA2/RCH1/SRP1alpha and found two others--to the transcription factor GMEB1/PIF p96 and the splicing factor SF3A2/SF3a66. A luciferase reporter assay demonstrates that a protein complex containing RAG proteins and the transcription factor can assemble in cells. Further mapping identified a region within the N-terminal domain resembling a WW motif. Point mutation directed at residues conserved in WW motifs eliminated binding to one of the partners. Phylogenetic analysis shows the WW-like module to be highly conserved. The module contributes to protein-protein interactions that may also influence how RAG1 binds DNA targets.

Ku70 is stabilized by increased cellular SUMO.

Ku70 is a protein that finds itself at the heart of several important cellular processes. It is essential to the non-homologous end joining pathway as a part of the DNA-end binding complex, required for proper maintenance of telomeres and contributes to DNA damage recognition and regulation of apoptosis. Forces that regulate Ku70 are therefore likely to have large consequences on the physiologic state of the cell. We report here that transient expression of the small protein SUMO resulted in a dramatic increase in the abundance of Ku70. Surprisingly, the direct SUMOylation of Ku70 does not appear to be required for this effect. Rather, Ku70 appears to be stabilized through indirect effects on the rate of degradation. The same outcome was obtained by raising the expression of enzymes that promote SUMOylation. It is likely that many other proteins will be similarly regulated, providing a general control of cellular state.

PHD domain-mediated E3 ligase activity directs intramolecular sumoylation of an adjacent bromodomain required for gene silencing.

Tandem PHD and bromodomains are often found in chromatin-associated proteins and have been shown to cooperate in gene silencing. Each domain can bind specifically modified histones: the mechanisms of cooperation between these domains are unknown. We show that the PHD domain of the KAP1 corepressor functions as an intramolecular E3 ligase for sumoylation of the adjacent bromodomain. The RING finger-like structure of the PHD domain is required for both Ubc9 binding and sumoylation and directs modification to specific lysine residues in the bromodomain. Sumoylation is required for KAP1-mediated gene silencing and functions by directly recruiting the SETDB1 histone methyltransferase and the CHD3/Mi2 component of the NuRD complex via SUMO-interacting motifs. Sumoylated KAP1 stimulates the histone methyltransferase activity of SETDB1. These data provide a mechanistic explanation for the cooperation of PHD and bromodomains in gene regulation and describe a function of the PHD domain as an intramolecular E3 SUMO ligase.

SUMO modification of human XRCC4 regulates its localization and function in DNA double-strand break repair.

The nonhomologous end-joining (NHEJ) pathway is responsible for rejoining the majority of double-strand breaks in mammalian cells, including the programmed breaks introduced by V(D)J recombination. The regulation of the enzymatic activities associated with this recombination pathway is still largely unknown. Here we report that human XRCC4 (for X-ray cross-complementation group 4), a protein essential for NHEJ, is subject to posttranslational protein modification. The modifier peptide, SUMO, can be added to XRCC4 both in vitro and in vivo. The site of modification is mapped to lysine 210 by using specific mutagenesis. A protein mutated such that it cannot be SUMOylated remains localized in the cytoplasm rather than accumulating in the nucleus. Cells expressing only the mutated protein are radiation sensitive and fail to complete V(D)J recombination. Genetic fusion of the SUMO sequence to the C terminus of the mutant restores nuclear localization and radiation resistance. The modification may serve a regulatory role. Our finding fits with an emerging literature associating SUMO modification with the control of the repair and recombination associated with DNA breaks.

Recombination-activating gene proteins: more regulation, please.

Developing B and T cells assemble gene segments in order to create the variable regions of immunoglobulin and T-cell receptors required by our adaptive immune response. The chemistry of this recombination pathway requires a specific nuclease and a more general repair pathway for double-strand breaks. A complex of the recombination-activating gene 1 (RAG1) and RAG2 proteins provides the nuclease activity. In fact, RAG1 and RAG2 probably coordinate many steps involving the coding and signaling DNA sequences. Studies using deletion and truncation mutants of the RAG proteins demonstrate that each of these contain a functional core region, representing about two-thirds of the polypeptides. While the core regions are sufficient to catalyze recombination in test systems, the full-length proteins seem to show more complicated behaviors in vivo. A plausible explanation is that regions outside the core help in the proper regulation of recombination. The non-core region of RAG1 has been found to contain a ubiquitin ligase. Regulatory functions may contribute to autoregulation of the proteins involved, fidelity of the reaction, protection of the cell from translocations, coordination of recombination with the cell cycle, and possibly modification of the chromatin structure of target DNA.

Impaired V(D)J recombination and lymphocyte development in core RAG1-expressing mice.

RAG1 and RAG2 are the lymphocyte-specific components of the V(D)J recombinase. In vitro analyses of RAG function have relied on soluble, highly truncated "core" RAG proteins. To identify potential functions for noncore regions and assess functionality of core RAG1 in vivo, we generated core RAG1 knockin (RAG1(c/c)) mice. Significant B and T cell numbers are generated in RAG1(c/c) mice, showing that core RAG1, despite missing approximately 40% of the RAG1 sequence, retains significant in vivo function. However, lymphocyte development and the overall level of V(D)J recombination are impaired at the progenitor stage in RAG1(c/c) mice. Correspondingly, there are reduced numbers of peripheral RAG1(c/c) B and T lymphocytes. Whereas normal B lymphocytes undergo rearrangement of both JH loci, substantial levels of germline JH loci persist in mature B cells of RAG1(c/c) mice, demonstrating that DJH rearrangement on both IgH alleles is not required for developmental progression to the stage of VH to DJH recombination. Whereas VH to DJH rearrangements occur, albeit at reduced levels, on the nonselected alleles of RAG1(c/c) B cells that have undergone D to JH rearrangements, we do not detect VH to DH rearrangements in RAG1(c/c) B cells that retain germline JH alleles. We discuss the potential implications of these findings for noncore RAG1 functions and for the ordered assembly of VH, DH, and JH segments.