INO80 requires a polycomb subunit to regulate the establishment of poised chromatin in murine spermatocytes

ABSTRACT INO80 is the catalytic subunit of the INO80-chromatin remodeling complex that is involved in DNA replication, repair and transcription regulation. Ino80 deficiency in murine spermatocytes (Ino80cKO) results in pachytene arrest of spermatocytes due to incomplete synapsis and aberrant DNA double-strand break repair, which leads to apoptosis. RNA-seq on Ino80cKO spermatocytes revealed major changes in transcription, indicating that an aberrant transcription program arises upon INO80 depletion. In Ino80WT spermatocytes, genome-wide analysis showed that INO80-binding sites were mostly promoter proximal and necessary for the regulation of spermatogenic gene expression, primarily of premeiotic and meiotic genes. Furthermore, most of the genes poised for activity, as well as those genes that are active, shared INO80 binding. In Ino80cKO spermatocytes, most poised genes demonstrated de-repression due to reduced H3K27me3 enrichment and, in turn, showed increased expression levels. INO80 interacts with the core PRC2 complex member SUZ12 and promotes its recruitment. Furthermore, INO80 mediates H2A.Z incorporation at the poised promoters, which was reduced in Ino80cKO spermatocytes. Taken together, INO80 is emerging as a major regulator of the meiotic transcription program by mediating poised chromatin establishment through SUZ12 binding.

eIF4A2 targets developmental potency and histone H3.3 transcripts for translational control of stem cell pluripotency

Translational control has emerged as a fundamental regulatory layer of proteome complexity that governs cellular identity and functions. As initiation is the rate-limiting step of translation, we carried out an RNAi screen for key translation initiation factors required to maintain embryonic stem cell (ESC) identity. We identified eIF4A2 and defined its mechanistic action through Rps26-independent and -dependent ribosomes in translation initiation activation of mRNAs encoding pluripotency factors and the histone variant H3.3 with demonstrated roles in maintaining stem cell pluripotency. eIF4A2 also mediates translation initiation activation of Ddx6, which acts together with eIF4A2 to restrict the totipotent 2-cell transcription program in ESCs through Zscan4 mRNA degradation and translation repression. Accordingly, knockdown of eIF4A2 disrupts ESC proteome causing the loss of ESC identity. Collectively, we establish a translational paradigm of the protein synthesis of pluripotency transcription factors and epigenetic regulators imposed on their established roles in controlling pluripotency.

IgA Plasma Cells Are Long-Lived Residents of Gut and Bone Marrow That Express Isotype- and Tissue-Specific Gene Expression Patterns

Antibody secreting plasma cells are made in response to a variety of pathogenic and commensal microbes. While all plasma cells express a core gene transcription program that allows them to secrete large quantities of immunoglobulin, unique transcriptional profiles are linked to plasma cells expressing different antibody isotypes. IgA expressing plasma cells are generally thought of as short-lived in mucosal tissues and they have been understudied in systemic sites like the bone marrow. We find that IgA+ plasma cells in both the small intestine lamina propria and the bone marrow are long-lived and transcriptionally related compared to IgG and IgM expressing bone marrow plasma cells. IgA+ plasma cells show signs of shared clonality between the gut and bone marrow, but they do not recirculate at a significant rate and are found within bone marrow plasma cells niches. These data suggest that systemic and mucosal IgA+ plasma cells are from a common source, but they do not migrate between tissues. However, comparison of the plasma cells from the small intestine lamina propria to the bone marrow demonstrate a tissue specific gene transcription program. Understanding how these tissue specific gene networks are regulated in plasma cells could lead to increased understanding of the induction of mucosal versus systemic antibody responses and improve vaccine design.

FOXO1 Dependent Transcription Network Is a Targetable Vulnerability of Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) often has an adverse prognosis and despite aggressive multimodal treatment with conventional and targeted therapies, the median survival of MCL patients remains approximately 4 years. Thus, there is a significant unmet need to find novel targets and rational combination treatments. Targeting lineage vulnerabilities driven by specific transcription factors has been broadly confirmed as an effective intervention in many human cancers. To identify the transcription program dependency of MCL cells, we conducted an unbiased domain-focused CRISPR-Cas9 screening against a library of 8,750 sgRNAs targeting 1,434 transcription factors in MCL cell lines (JEKO1, MAVER1, UPN1, and CCMCL1). We identified Forkhead Box O1 (FOXO1), EBF1, PAX5, and IRF4 as 4 transcription factors that are specifically required for MCL survival and growth. Chromatin-immunoprecipitation and sequencing (ChIP-Seq) analysis further revealed that the four transcription factors act together to orchestrate B cell lineage transcriptional program and MCL cell survival. Despite its well-recognized role as a tumor suppressor, FOXO1 has been implicated as a lineage specific transcription factor involved in mature B cell development. Genetic studies revealed a critical role of FOXO1 in germinal center dark zone formation and lymphomagenesis, raising the possibility that FOXO1 acts as a master transcription factor for lineage survival transcription program of MCL. Indeed, hierarchical interaction analysis revealed that FOXO1 functions as a pioneer factor that facilitate the chromatin access of other B cell lineage transcription factors. We demonstrated that interaction of FOXO1 to its cognate motif stabilizes B cell transcription factor complex and supports MCL progression. Along this line, we show that enforced expression of FOXO1 in myeloid leukemia cells induces transdifferentiation and B-cell specific gene expression. Mechanistically, we demonstrate through tiling CRISPR scanning screen that forkhead DNA binding and c-terminal transactivation domains of FOXO1 are specifically required for the viability of MCL cells. Given our finding of FOXO1 as a lineage-specific oncogene in MCL, we next explored the possibility of developing FOXO1-tageted inhibitors. We screened a library of potential small molecule inhibitors of FOXO1 (Forkhead BioTherapeutics) and identified cpd10 as one of the most potent and selective FOXO1 inhibitors (IC 50=76 ×/÷ 1.7. nM) Through the CCMCL1 MCL-NSG preclinical model, we found that cpd10 (100 mg/kg/daily) was well tolerated without overt toxicities. Importantly, prolonged treatment induced a robust cytotoxic response of MCL cells and suppressed MCL progression in vivo. Altogether, our findings identified FOXO1 as a MCL lineage-survival oncogene that can be exploited as a therapeutic target of future drug development. Disclosures Cantley: Petra Pharmaceuticals: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Elemento: Freenome: Consultancy, Other: Current equity holder in a privately-held company; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; Champions Oncology: Consultancy; Janssen: Research Funding; Owkin: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding. Baiocchi: Prelude Therapeutics: Consultancy; viracta: Consultancy, Current holder of stock options in a privately-held company; Codiak Biosciences: Research Funding; Atara Biotherapeutics: Consultancy. Belvedere: Forkhead BioTherapeutics: Current Employment. Paik: Forkhead BioTherapeutics: Research Funding.

Distinct requirements for the COMPASS core subunits Set1, Swd1, and Swd3 during meiosis in the budding yeast Saccharomyces cerevisiae

Meiosis-specific chromatin structures, guided by histone modifications, are critical mediators of a meiotic transient transcription program and progression through prophase I. Histone H3K4 can be methylated up to three times by the Set1-containing COMPASS complex and each methylation mark corresponds to a different chromatin conformation. The level of H3K4 modification is directed by the activity of additional COMPASS components. In this study, we characterized the role of the COMPASS subunits during meiosis in S. cerevisiae. In vegetative cells, previous studies revealed a role for subunits Swd2, Sdc1, and Bre2 for H3K4me2 while Spp1 supported trimethylation. However, we found that Bre2 and Sdc1 are required for H3K4me3 as yeast prepare to enter meiosis while Spp1 is not. Interestingly, we identified distinct meiotic functions for the core COMPASS complex members that required for all H3K4me, Set1, Swd1, and Swd3. While Set1 and Swd1 are required for progression through early meiosis, Swd3 is critical for late meiosis and spore morphogenesis. Furthermore, the meiotic requirement for Set1 is independent of H3K4 methylation, suggesting the presence of non-histone substrates. Finally, checkpoint suppression analyses indicate that Set1 and Swd1 are required for both homologous recombination and chromosome segregation. These data suggest that COMPASS has important new roles for meiosis that are independent of its well-characterized functions during mitotic divisions.

Systematic dissection of transcriptional regulatory networks by genome-scale and single-cell CRISPR screens

Millions of putative transcriptional regulatory elements (TREs) have been cataloged in the human genome, yet their functional relevance in specific pathophysiological settings remains to be determined. This is critical to understand how oncogenic transcription factors (TFs) engage specific TREs to impose transcriptional programs underlying malignant phenotypes. Here, we combine cutting edge CRISPR screens and epigenomic profiling to functionally survey ≈15,000 TREs engaged by estrogen receptor (ER). We show that ER exerts its oncogenic role in breast cancer by engaging TREs enriched in GATA3, TFAP2C, and H3K27Ac signal. These TREs control critical downstream TFs, among which TFAP2C plays an essential role in ER-driven cell proliferation. Together, our work reveals novel insights into a critical oncogenic transcription program and provides a framework to map regulatory networks, enabling to dissect the function of the noncoding genome of cancer cells.

Klf5 establishes bi-potential cell fate by dual regulation of ICM and TE specification genes

SummaryEarly blastomeres of mouse preimplantation embryos exhibit bi-potential cell fate, capable of generating both embryonic and extra-embryonic lineages in blastocysts. Here, we identified three major 2 cell (2C) specific endogenous retroviruses (ERVs) as the molecular hallmark of the bi-potential plasticity. Using the LTRs of all three 2C-ERVs, we identified Klf5 as their major upstream regulator. Klf5 is essential for bi-potential cell fate: a single Klf5-overexpressing ESC generated terminally differentiated embryonic and extra-embryonic lineages in chimeric embryos, and Klf5 directly induces both ICM and TE specification genes. Intriguingly, Klf5 and Klf4 act redundantly during ICM specification, whereas Klf5 deficiency alone impairs TE specification. Klf5 is regulated by multiple 2C-specific transcription factors, particularly Dux, and the Dux/Klf5 axis is evolutionarily conserved. Altogether, the 2C-specific transcription program converges on Klf5 to establish bi-potential cell fate, enabling a cell state with dual activation of ICM and TE genes.