A PWWP Domain of Histone-Lysine N-Methyltransferase NSD2 Binds to Dimethylated Lys-36 of Histone H3 and Regulates NSD2 Function at Chromatin.

The readout of histone modifications plays a critical role in chromatin-regulated processes. Dimethylation at Lys-36 on histone H3 (H3K36me2) is associated with actively transcribed genes, and global up-regulation of this modification is associated with several cancers. However, the molecular mechanism by which H3K36me2 is sensed and transduced to downstream biological outcomes remains unclear. Here we identify a PWWP domain within the histone lysine methyltransferase and oncoprotein NSD2 that preferentially binds to nucleosomes containing H3K36me2. In cells, the NSD2 PWWP domain interaction with H3K36me2 plays a role in stabilizing NSD2 at chromatin. Furthermore, NSD2's ability to induce global increases in H3K36me2 via its enzymatic activity, and consequently promote cellular proliferation, is compromised by mutations within the PWWP domain that specifically abrogate H3K36me2-recognition. Together, our results identify a pivotal role for NSD2 binding to its catalytic product in regulating its cellular functions, and suggest a model for how this interaction may facilitate epigenetic spreading and propagation of H3K36me2.

A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by the SETMAR Enzyme.

The lysine methyltransferase (KMT) SETMAR is implicated in the response to and repair of DNA damage, but its molecular function is not clear. SETMAR has been associated with dimethylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates, we utilized a strategy on the basis of quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 of the mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates monomethylation at this position. Furthermore, we show that SETMAR methylates snRNP70 Lys-130 in cells. Because snRNP70 is a key early regulator of 5' splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for large-scale mapping of KMT substrates.

Residency Program Directors' Views on Research Conducted During Medical School: A National Survey.

PURPOSE: With the United States Medical Licensing Examination Step 1 transition to pass/fail in 2022, uncertainty exists regarding how other residency application components, including research conducted during medical school, will inform interview and ranking decisions. The authors explore program director (PD) views on medical student research, the importance of disseminating that work, and the translatable skill set of research participation. METHOD: Surveys were distributed to all U.S. residency PDs and remained open from August to November 2021 to query the importance of research participation in assessing applicants, whether certain types of research were more valued, productivity measures that reflect meaningful research participation, and traits for which research serves as a proxy. The survey also queried whether research would be more important without a numeric Step 1 score and the importance of research vs other application components. RESULTS: A total of 885 responses from 393 institutions were received. Ten PDs indicated that research is not considered when reviewing applicants, leaving 875 responses for analysis. Among 873 PDs (2 nonrespondents), 358 (41.0%) replied that meaningful research participation will be more important in offering interviews. A total of 164 of 304 most competitive specialties (53.9%) reported increased research importance compared with 99 of 282 competitive (35.1%) and 95 of 287 least competitive (33.1%) specialties. PDs reported that meaningful research participation demonstrated intellectual curiosity (545 [62.3%]), critical and analytical thinking skills (482 [55.1%]), and self-directed learning skills (455 [52.0%]). PDs from the most competitive specialties were significantly more likely to indicate that they value basic science research vs PDs from the least competitive specialties. CONCLUSIONS: This study demonstrates how PDs value research in their review of applicants, what they perceive research represents in an applicant, and how these views are shifting as the Step 1 exam transitions to pass/fail.

CRISPR-Cas9 Gene Editing in Yeast: A Molecular Biology and Bioinformatics Laboratory Module for Undergraduate and High School Students.

CRISPR-Cas9 genome editing technology is widely used in scientific research and biotechnology. As this technology becomes a staple tool in life sciences research, it is increasingly important to incorporate it into biology curricula to train future scientists. To demonstrate the molecular underpinnings and some limitations of CRISPR-based gene editing, we designed a laboratory module to accompany a discussion-based course on genome editing for college and advanced high school biology students. The laboratory module uses CRISPR-Cas9 to target and inactivate the ADE2 gene in Saccharomyces cerevisiae so as to give red colonies, employing an inexpensive yeast model system with a phenotypic readout that is easily detectable without specialized equipment. Students begin by accessing the yeast ADE2 sequence in a genome database, applying their understanding of Cas9 activity to design guide RNA (gRNA) sequences, using a CRISPR analysis tool to compare predicted on- and off-target effects of various gRNAs, and presenting and explaining their choice of an optimal gRNA to disrupt the ADE2 gene. They then conduct yeast transformations using Cas9 and preselected gRNA plasmids with or without donor templates to explore the importance of DNA repair pathways in genome editing. Lastly, they analyze the observed editing rates across different gRNAs targeting ADE2, leading to a discussion of editing efficiency. This module engages students in experimental design, provides hands-on experience with CRISPR-Cas9 gene editing and collaborative data analysis, and stimulates discussion on the uses and limitations of CRISPR-based gene editing technology.

Characterization of H3.3K36M as a tool to study H3K36 methylation in cancer cells.

Recurrent mutations at key lysine residues in the histone variant H3.3 are thought to play an etiologic role in the development of distinct subsets of pediatric gliomas and bone and cartilage cancers. H3.3K36M is one such mutation that was originally identified in chondroblastomas, and its expression in these tumors contributes to oncogenic reprogramming by triggering global depletion of dimethylation and trimethylation at H3K36 with a concomitant increase in the levels of H3K27 trimethylation. H3.3K36M expression can also cause epigenomic changes in cell types beyond chondrocytic cells. Here we show that expression of H3.3K36M in HT1080 fibrosarcoma cancer cells severely impairs cellular proliferation, which contrasts its role in promoting transformation of chondrocytic cells. H3.3K36M-associated cellular toxicity phenocopies the specific depletion of H3K36me2, but not loss of H3K36me3. We further find that the H3K36me2-associated toxicity is largely independent of changes in H3K27me3. Together, our findings lend support to the argument that H3K36me2 has distinct roles in cancer cells independent of H3K36me3 and H3K27me3, and highlight the use of H3.3K36M as an epigenetic tool to study H3K36 and H3K27 methylation dynamics in diverse cell types.

Implementation of a project-based molecular biology laboratory emphasizing protein structure-function relationships in a large introductory biology laboratory course.

In introductory laboratory courses, many universities are turning from traditional laboratories with predictable outcomes to inquiry-inspired, project-based laboratory curricula. In these labs, students are allowed to design at least some portion of their own experiment and interpret new, undiscovered data. We have redesigned the introductory biology laboratory course at Brandeis University into a semester-long project-based laboratory that emphasizes concepts and contains an element of scientific inquiry. In this laboratory, students perform a site-directed mutagenesis experiment on the gene encoding human γD crystallin, a human eye lens protein implicated in cataracts, and assess the stability of their newly created protein with respect to wild-type crystallin. This laboratory utilizes basic techniques in molecular biology to emphasize the importance of connections between DNA and protein. This project lab has helped engage students in their own learning, has improved students' skills in critical thinking and analysis, and has promoted interest in basic research in biology.