Program in Cellular and Molecular Medicine (PCMM)

The Alt lab previously made transformative leaps in our mechanistic understanding of two distinct programmed rearrangement mechanisms in lymphocytes by discovering that cohesin-mediated loop extrusion plays fundamental roles in immunoglobulin heavy chain locus (Igh) V(D)J recombination and IgH class switch recombination (CSR). Thus, these very different recombination processes, which occur at different stages of B lymphocyte development, both use cohesin-mediated chromatin loop extrusion to reel long loops of chromatin past recombination centers. For each, loop extrusion juxtaposes cis-regulatory elements, substrate DNA sequences, and initiating enzymes.

The lab discovered that, during Igh variable region assembly in early B cell development, loop extrusion-mediated V(D)J recombination is regulated via down-regulation of the WAPL cohesin-complex factor, leading to substantial neutralization of chromatin impediments to loop extrusion. Impediment neutralization allows the V(D)J recombination initiating enzyme (RAG endonuclease) bound to the fixed Igh recombination center (RC) location to scan megabases (Mb) of chromatin linearly presented to it by loop extrusion. This active loop extrusion-based process, as opposed to diffusion, is the underlying mechanism by which the many diverse Igh VH gene segments are able to be located and to contribute to diverse antibody repertoires.

After solving the long-range Igh V(D)J recombination mechanism, which only joins V segments when they are directly juxtaposed and are oriented for deletional joining, the Alt lab addressed the quite different mechanism by which the Igκ light chain locus is able to employ V segments oriented for both deletional and inversional joining. The mechanism by which the Igκ locus joins V segments in both orientations was a long-standing, and much discussed, mystery for the field.

In a paper published June 6, 2024 in Nature, the Alt lab reported that, in contrast to Igh long-range V(D)J recombination that directly aligns gene segments in the RC for joining, the Igκ locus extrudes gene segments to positions within short-range diffusional distance from the RAG-bound Igκ RC, with the diffusion process allowing transient alignment for joining regardless of orientation. The lab further reported that the mechanism by which Igκ can use such a diffusion-based joining mechanism is based on the Igκ RAG endonuclease target sequences (“RSSs") being much stronger than those of the Igh locus.

Overall, the study revealed that the Igκ locus evolved remarkably strong RSSs that function robustly in the context of more transient RC interactions that occur during diffusion-mediated primary Vκ-to-Jκ joining. In contrast, the Igh locus employs relatively weak RSSs to facilitate mediation of VH utilization by WAPL down-regulated modulation of scanning impediments during long-range linear RAG scanning. Thus, rather than previously speculated differences in chromatin folding principles, the key difference between the long-range V(D)J recombination mechanisms of the Igh and Igκ loci appears to be RSS strength. Indeed, this study also showed that Igκ RSS substitutions for Igh RSSs could program the Igh locus to undergo diffusional V(D)J recombination.

The findings from this study should also have broader implications regarding how loop-extrusion-mediated V(D)J recombination could be differentially regulated in the various T Cell Receptor loci, for the evolution of the V(D)J recombination process, and for regulation of loop extrusion mediated control of functional interactions between elements located in large adjacent chromatin loop domains more generally.

Aritra Bhattacherjee, PhD, a Research Fellow in Yi Zhang’s laboratory, was awarded a Harvard Brain Science Initiative Postdoc Pioneers Grant. Emerging evidence indicates that chronic pain can worsen opioid addiction. Mechanistically, the prefrontal cortex (PFC) is thought to be instructive in the pain/addiction interaction, but its cellular heterogeneity and functional complexity presented a challenge in understanding the mechanisms of such interaction. Using single cell genomics techniques, Aritra recently identified a distinct spatially and molecularly-defined neuron subtype, the activation of which can aggravate pain and addiction. In his project, he will use a genetic mouse model that he created to study how chronic pain facilitates addiction liability through these neurons, which could reveal molecular mechanisms that can promote better understanding for potential therapeutic interventions.

Paolo Cadinu, PhD, a Research Fellow in Jeffrey Moffitt’s laboratory, was awarded a  Charles A. King Trust Postdoctoral Research Fellowship. In addition to providing tissue structure, fibroblasts have been recently shown to affect the immune response and become inflammation-associated fibroblast (IAFs). By using spatial transcriptomics, Paolo has recently shown an unprecedented IAF diversity within the gastrointestinal (GI) tract of a colitis mouse model. However, it still remains unknown how these IAFs contribute to tissue repair, interact with other cell types, and the extent to which they retain memory of the insult. To address this question, he will create a comprehensive atlas characterizing these heterogenous fibroblast populations further, to potentially uncover mechanisms of tissue repair that can be targeted in treatment of gut-related diseases.

Uriel López-Sánchez, PhD, a Research Fellow in Timothy Springer’s laboratory, was awarded a Charles A. King Trust Postdoctoral Research Fellowship. Integrins α4β1 and α4β7 are cell surface receptors that play essential roles in cell adhesion and migration and are involved in processes such as circulation of immune cells during inflammation. However, the mechanism of how these integrins affect cell migration remains not fully understood. Using biochemical, structural, and cellular approaches, Uriel’s research aims to elucidate the principles underlying the conformational changes in α4β1 and α4β7 and to clarify how these changes influence their ability to control immune cell migration. Insights gained from this study may contribute to accelerating the development of new drugs and improvement of therapeutics for conditions such as multiple myeloma and inflammatory bowel diseases.

Yingying Zhang, PhD, a Postdoctoral Fellow in Michael Carroll’s laboratory, was awarded a Brain and Behavior Research Foundation Young Investigator Grant. Genetic studies have implicated an increase of an innate immune system protein C4A as a major risk factor in schizophrenia. Yingying has established a mouse model of C4A overexpression, in which complement-mediated microglia excessively prune synapses. Additionally, other schizophrenia-like phenotypes were observed: increased monocyte infiltration into the choroid plexus (ChP), hydrocephalus, dramatically enlarged cerebral ventricles (ventriculomegaly), and subsequent behavioral anomalies. In this study, Yingying will probe the complement overactivation-mediated mechanisms underlying the observed pathology and test for a causal relationship between ChP inflammation and schizophrenia-like neuropsychiatric manifestations.

Congratulations to Aritra, Paolo, Uriel, and Yingying!

The overall goal of Li Laboratory is to uncover the missing link between repeat DNA, genomic instability, and viral infection. Half of our genome contains repeat sequences that resemble viral DNA, yet their functional significance remains a mystery. As abnormal DNA repeat sequences are found at unstable genomic regions, and DNA viruses—like Epstein Barr Virus (EBV)— are also detected in cancer, these DNA elements have been prime suspects in promoting genomic instability in cancer. For decades, these seemingly separate but connected observations pointed towards a missing link.

Dr. Li’s recent discovery of a cluster of EBV-like repeats provides the missing piece to the puzzle, functionally linking repeat DNA, genomic instability, and viruses. She showed that these virus-like repeats can break and trigger chromosomal abnormalities when bound by EBV nuclear antigen 1 (EBNA1) in cells infected with EBV. Going forward, the lab will investigate how virus-like repeat sequences in our genome pose a threat to genome stability, yet contribute to normal genome structure and function. Specifically, the lab will leverage the cluster of breakage-prone EBV-like repeat sequences to determine how virus-like DNA repeat sequences undergo breakage, the role of such breakage in shaping abnormal genomes that underlie cancer and genetic diseases, the pathological conditions in viral infection that trigger breakage, and finally, the functional significance of these DNA repeat sequences in normal genome structure and function. The lab’s research is relevant for understanding the role of virus-like repeat sequences in the development of viral infection-associated cancer and genetic diseases. In the long run, Dr. Li is excited about potentially uncovering a class of viral proteins that bind virus-like repeats in our genome and the role they play in health and disease.

Pietro Fontana, PhD, an instructor in Hao Wu’s laboratory, was awarded a 2024 Boston Children’s Hospital OFD/BTREC/CTREC Faculty Career Development Fellowship. He will continue his research on the mechanistic study of innate immune pathways, with a particular focus on immunogenic cell death and the role of gasdermin D (GSDMD) in this process. While GSDMD has been recognized for its involvement in pyroptosis, its broader functions and potential roles in immune regulation have not been fully explored. He will use small molecule agonists to induce cancer cell death in a GSDMD-dependent manner and investigate combinatorial therapy with existing cancer therapies. Overall, the project contributes to filling gaps in knowledge about immunogenic cell death, the potential of agonizing GSDMD for anti-tumor immunity, the development of targeted immunotherapies, and mechanistic understanding of GSDMD regulation.

Ibraheem Alshareedah, PhD, a postdoctoral fellow in TJ Ha’s laboratory, has been named a Jane Coffin Childs-HHMI Fellow. In the area of DNA break repair, it is known that BRCA2 loads RAD51 onto single stranded DNA (ssDNA), yet homologous recombination still occurs in BRCA2-mutant cancers, suggesting that there is redundancy in this pathway. Hypothesizing that RAD52 nanoclusters in cells recruit RAD51 and load it onto ssDNA, even in the absence of functional BRCA2, he will examine if RAD51, ssDNA, and other DNA-break repair proteins are recruited to RAD52 nanoclusters in cells, and will then determine which RAD52 protein features are required for cluster formation. By understanding the partial redundancies in the DNA repair pathway, his research may reveal novel targets for treating BRCA2-mutant cancers.

Man Wu, PhD, a postdoctoral fellow in Hao Wu’s laboratory, has received an Irvington Postdoctoral Fellowship from the Cancer Research Institute. She will focus on the mechanism for LPS-rendered NEK7-independent NLRP3 inflammasome activation in human macrophages. NEK7 was previously found as a scaffolding protein mediating NLRP3 cage structure opening to further assemble into supracomplexes together with ASC and caspase-1, which is the hallmark of NLRP3 inflammasome activation. Using biochemical, structural, and cellular imaging approaches, she aims to interrogate whether and what condition of NLRP3 activation requires NEK7 and the mechanism of NEK7-indenpendent NLRP3 activation, which will provide a comprehensive understanding of NLRP3 activity to guide future therapeutic intervention.

Congratulations to Pietro, Ibraheem, and Man!

He was recognized “For revolutionizing the understanding of how genomic rearrangements occur, elucidating their role in cancer development and progression, and for discovering dihydrofolate reductase gene amplification in methotrexate-resistant cancer cells and MYCN amplification in neuroblastoma, as well as discovering the mechanisms regulating V(D)J recombination to form exons that encode antibody variable regions.”  Margaret Foti, PhD, MD (hc), chief executive officer of the AACR, said “These scientific pioneers from around the globe have fundamentally shaped cancer research through their respective scientific accomplishments.  The 2024 class of Fellows includes leaders from many scientific disciplines and areas of study who have collectively contributed to the improvement of the understanding and treatment of cancer. We are thrilled and honored to have them join our 312 existing Fellows and look forward to celebrating their enormous scientific achievements at our upcoming Annual Meeting in April.”  Congratulations, Fred!

During Brandeis University’s 75th anniversary weekend held October 13-15, Fred was presented with an Alumni Achievement Award from the Brandeis Alumni Association, that organization’s highest honor.  He was an undergraduate researcher with Prof. Jerome Schiff before earning his Ph.D. in Biology at Stanford, and he was the second alumnus to receive Brandeis’ Rosenstiel Award for Distinguished Work in Biomedical Science in 2014 for pioneering research exploring the mechanisms of genomic instability and its implications for the immune system and cancer cells.  He has championed undergraduate research opportunities at Brandeis, formalizing his long-standing funding of summer research programs by endowing the Dr. Frederick W. Alt ’71 Summer Biology Research Fellowship earlier this year.  Fred was also named the winner of the 2023 William E. Paul Memorial Award for Excellence in Immunology and Cell Biology by the Foundation for Primary Immunodeficiency Diseases.  The award was established to honor and celebrate the contributions of Dr. William E. Paul, a pioneer in cytokine research known for his discovery of interleukin-4, who served the NIH as Chief of the Laboratory of Immunology at NIAID and Director of the Office of the AIDS Research.  Fred will be honored at the 18th International Symposium on Inborn Errors of Immunity on November 16-18 in Newport Beach, California, where he will present the William E. Paul Memorial Lecture.   PCMM congratulates Fred on his two latest honors!