Bat3 deficiency accelerates the degradation of Hsp70-2/HspA2 during spermatogenesis

Meiosis is critical for sexual reproduction. During meiosis, the dynamics and integrity of homologous chromosomes are tightly regulated. The genetic and molecular mechanisms governing these processes in vivo, however, remain largely unknown. In this study, we demonstrate that Bat3/Scythe is essential for survival and maintenance of male germ cells (GCs). Targeted inactivation of Bat3/Scythe in mice results in widespread apoptosis of meiotic male GCs and complete male infertility. Pachytene spermatocytes exhibit abnormal assembly and disassembly of synaptonemal complexes as demonstrated by abnormal SYCP3 staining and sustained γ-H2AX and Rad51/replication protein A foci. Further investigation revealed that a testis-specific protein, Hsp70-2/HspA2, is absent in Bat3-deficient male GCs at any stage of spermatogenesis; however, Hsp70-2 transcripts are expressed at normal levels. We found that Bat3 deficiency induces polyubiquitylation and subsequent degradation of Hsp70-2. Inhibition of proteasomal degradation restores Hsp70-2 protein levels. Our findings identify Bat3 as a critical regulator of Hsp70-2 in spermatogenesis, thereby providing a possible molecular target in idiopathic male infertility

RPRD1A and RPRD1B Are Human RNA Polymerase II C-Terminal Domain Scaffolds for Ser5 Dephosphorylation

The RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD) heptapeptide repeats (Y1-S2-P3-T4-S5-P6-S7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD interaction domains (CIDs) with CTD repeats phosphorylated at S2 and S7. Our high resolution crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an interesting example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2

Discovery of a chemical probe for the L3MBTL3 methyllysine reader domain

We describe the discovery of UNC1215, a potent and selective chemical probe for the methyl-lysine (Kme) reading function of L3MBTL3, a member of the malignant brain tumor (MBT) family of chromatin interacting transcriptional repressors. UNC1215 binds L3MBTL3 with a Kd of 120 nM, competitively displacing mono- or dimethyl-lysine containing peptides, and is greater than 50-fold selective versus other members of the MBT family while also demonstrating selectivity against more than 200 other reader domains examined. X-ray crystallography identified a novel 2:2 polyvalent mode of interaction. In cells, UNC1215 is non-toxic and binds directly to L3MBTL3 via the Kme-binding pocket of the MBT domains. UNC1215 increases the cellular mobility of GFP-L3MBTL3 fusion proteins and point mutants that disrupt the Kme binding function of GFP-L3MBTL3 phenocopy the effects of UNC1215. Finally, UNC1215 demonstrates a novel Kme-dependent interaction of L3MBTL3 with BCLAF1, a protein implicated in DNA damage repair and apoptosis

237 Empowering Youth in Mental Health Treatment: A Co-Designed Approach to Measurement-Based Care

OBJECTIVES/GOALS: This integrated knowledgetranslationproject aims to increase youth engagement in mental health services through enhancing shared decision-making processes. To do this, a knowledge product to support greater involvement in measurement-based care practices will be evaluated and co-developed with youth study participants. METHODS/STUDY POPULATION: This study population includes youth (aged 12-25) with lived/living experience of accessing mental health services through an integrated service delivery model in Ontario. Study methods focus on co-designing a knowledge product with youth partners guided by the Knowledge-to-Action (KTA) Framework. The prototype knowledge product addressing identified barriers to MBC understanding, will be quantitatively evaluated by study participants. Through semi-structured focus groups, qualitative perspectives of the intervention’s impact on shared decision-making and overall engagement in treatment will be collected. The study’s design ensures active collaboration with the study population and aims to enhance MBC understanding and engagement in mental health care among youth. RESULTS/ANTICIPATED RESULTS: We anticipate increased youth involvement within MBC practices, and overall increased engagement in shared-decision making in treatment. We anticipate the developed knowledge product will enhance youth’s understanding of MBC, foster dialogue between youth and clinicians, and promote active involvement and informed decision-making in their mental health treatment. By involving youth in the co-design process, our project is poised to foster a sense of ownership and relevance, ultimately improving youth engagement, decision-making, and mental health outcomes within integrated mental health services. DISCUSSION/SIGNIFICANCE: Youth deserve to have an active role in shaping treatment decisions. This tool may bridge a gap by equipping youth with the knowledge needed to engage in decisions that are often practitioner-led. This study will discuss the impact and strategies for increasing involvement in MBC practices on youth engagement within treatment

Collaborative Application of Resilience Education (CARE): Fostering Resilience to Combat Depression and Anxiety in Graduate Students

Background: Graduate students experience depression and anxiety at rates six times higher than the general population. A literature review of resilience education and an environmental scan of existing preventative resources found that current university-provided mental health interventions are primarily reactive, short-term, and crisis-oriented. There remains a need for preventative resources that prepare students to effectively face adversity and maintain wellbeing during graduate school.   Purpose: In collaboration with experts in mental health, psychology, pedagogy, resource development, and graduate students themselves, our team aims to develop and implement a proactive, skill-based resource that teaches strategies to foster resilience in graduate school while emphasizing the importance of self-care, self-awareness, and help-seeking.   Methods: Our new co-created resource is to be assessed in three phases: (1) Qualitative Needs Analysis, (2) Intervention Ideation, and (3) Resource Validation. All participants for this study will be graduate students in the first two years of their degree and recruited from the University of Toronto Temerty Faculty of Medicine (TFM). Phase One will be conducted through qualitative focus groups to explore student needs and experiences with mental health resources. Focus group data will be transcribed and analyzed using NVIVO to establish criteria for our resource prototype. In Phase Two, all data, in addition to input from a student advisory committee composed of TFM graduate students, will be compiled to design our prototype. In Phase Three, focus groups will assess the prototype and provide their feedback on the content and functionality of the resource, and where it might be improved. Anticipated Results and Potential Implications: Overall, we anticipate that this resource prototype will leave a positive impression on the Phase Two participants, helping them build their resilience-based skills and strategies for facing adverse events. Overall, given the correlation between increased resilience and improved mental health, we expect that this resource will reduce levels of depression and anxiety for students by preparing them for the stress they experience in graduate school. If successful, this resource may be adapted to address the needs of graduate students in different departments, universities, and institutions worldwide