Biology subjects

Shilatifard, A.

Publications and source records attributed to Shilatifard, A..

13 recordsLinked to original sources

bioRxiv2026

POU2AF2/OCA-T1 coactivates POU2F2 and defines a lineage-specific dependency in diffuse large B-cell lymphoma

Lineage-restricted transcriptional programs establish cell identity and can create selective dependencies in cancer. Here, we identify POU2AF2, encoding the transcriptional co-activator OCA-T1, as a critical lineage-specific dependency in a subset of diffuse large B-cell lymphoma (DLBCL). Pan-cancer dependency analyses and patient cohorts reveal elevated POU2AF2 expression in genetically aggressive DLBCL, where its depletion markedly suppresses tumor growth in vitro and in vivo. Mechanistically, POU2AF2 cooperates with the B-cell lineage-defining transcription factor POU2F2 (OCT2) to activate lymphocyte activation gene programs through direct chromatin engagement, thereby sustaining malignant transcriptional networks. We further identified a key epigenetic regulatory axis composed of the lineage-specific transcription factor TCF3 and the histone methyltransferase SET1A-COMPASS that drives POU2AF2 expression downstream of B-cell receptor signaling. Single-cell transcriptomic analysis reveals that POU2AF2 marks and sustains an innate-like B1 B-cell population in vivo, a candidate cell of origin for lymphoma. Together, these findings define a lineage-restricted POU2AF2/POU2F2 transcriptional module, controlled by a TCF3/SET1A epigenetic network, that sustains both innate-like B-cell identity and malignant fitness in DLBCL. Our study uncovers a previously unrecognized lineage-specific transcriptional dependency and highlights POU2AF2 and its associated regulatory circuitry as potential therapeutic targets in aggressive B-cell malignancies.

cell biology↗

bioRxiv2026

FCD-11: A First-in-Class COMPASS Inhibitor in Cancer Therapeutics

COMPASS (Complex of proteins associated with Set1) are highly conserved chromatin regulatory complexes responsible for all methylation marks on histone H3 lysine 4 (H3K4). The COMPASS protein SET1A is upregulated in metastatic breast cancer, and its H3K4 methyltransferase activity promotes metastasis in a palmitic acid diet setting. We identify and characterize FCD-11, a first-in-class small molecule inhibitor of COMPASS activity designed to disrupt the termolecular interface between COMPASS SET domains, ASH2L, and RBBP5. FCD-11 significantly inhibited the H3K4me3 methyltransferase activity of SET1A/COMPASS and MLL1/COMPASS in vitro. ChIP-seq and CETSA indicated that FCD-11 selectively inhibits SET1A/COMPASS activity in mouse embryonic stem cells and breast cancer cell lines. FCD-11 significantly reduced tumor size and extended survival in mouse models of breast cancer. Our findings establish FCD-11 as a potent, COMPASS-specific inhibitor lead compound with preclinical efficacy in breast cancer models and therapeutic potential for cancers with abnormal dependence on SET1A/COMPASS activity.

biochemistry↗

bioRxiv2025

BRD2 bridges TFIID and histone acetylation to promote transcriptional initiation

Members of the bromodomain and extraterminal domain (BET) protein family play a central role in transcription by RNA Polymerase II (Pol II). Small-molecule inhibitors that block interaction between BET bromodomains and acetylated histones have been developed to achieve therapeutic benefit. However, the BET protein BRD4 does not require bromodomains to perform its major transcriptional elongation function, and the mechanisms by which other BET proteins regulate transcription remain incompletely understood. Addressing the disparity between pan-BET degraders and BRD4-specific depletion, we report that the BET protein BRD2 generally functions to promote transcriptional initiation in a bromodomain-dependent manner at both promoters and enhancers in human cells. We demonstrate that BRD2 bromodomains preferentially bind to tetra-acetylated histones harboring MOF-mediated H4K16ac, while the BRD2 C terminal domain facilitates recruitment of TFIID. Our studies provide mechanistic insight into the distinct roles of BRD2 in transcriptional initiation through the recruitment of TFIID and BRD4 in transcriptional elongation through the recruitment of CDK9 and controlling proper regulation of gene expression.

biochemistry↗

bioRxiv2025

Transcriptional Elongation-Associated RNA Processing Errors in Induced Cellular Growth Arrest

Summary ParagraphTranscription elongation factors control post-initiation steps of gene expression by RNA polymerase II (RNAPII). We have established distinct mechanistic roles for the essential elongation factors PAF1, NELF, SPT5, SPT6, and the Super Elongaiton Complex (SEC) via acute depletion of each individually in auxin-inducible degron lines. Here, we leverage these degron lines to explore the regulatory intersection of transcription elongation control and pre-mRNA processing. Integrating long- and short-read RNA-seq data to quantify transcript isoform usage at single-molecule resolution, we identify elongation factor-specific RNA processing regulons including a cellular senescence-enriched regulon shared by NELF and SPT6. We then show that long-term depletion of NELF or SPT6 results in reversible growth arrest following early upregulation of a small group of genes, which include the senescence-associated genes CDKN1A (p21) and CCN2. We perform genetic suppressor screens that implicate the elongation factor Elongin A (ELOA) in NELF or SPT6 depletion-induced growth arrest. ELOA loss suppresses NELF depletion-induced pre-mRNA processing defects and the 3 extension of RNAPII occupancy past transcription end sites (TES) at genes induced by NELF depletion. ELOA also occupies TES-proximal regions under normal conditions, and acute ELOA depletion results in a loss of RNAPII processivity at the 3 end of genes, opposing the effects of NELF or SPT6 depletion. Finally, we demonstrate that genetic loss of ELOA confers a growth advantage to aging human primary dermal fibroblasts. These findings establish the existence of novel ELOA-dependent mechanisms regulating transcription maturation, and links these mechanisms to the complex phenomena of cellular senescence and aging.

molecular biology↗

bioRxiv2024

MeCP2 Interacts with the Super Elongation Complex to Regulate Transcription

Loss-of-function mutations in methyl-CpG binding protein 2 (MECP2) cause Rett syndrome, a postnatal neurodevelopmental disorder that occurs in [~]1/10,000 live female births. MeCP2 binds to methylated cytosines across genomic DNA and recruits various partners to regulate gene expression. MeCP2 has been shown to repress transcription in vitro and interacts with co-repressors such as the Sin3A and NCoR complexes. Based on these observations, MeCP2 has been largely considered as a repressor of transcription. However, a mouse model of RTT displays many down-regulated genes, and those same genes are up-regulated in a MECP2 duplication mouse model. Furthermore, TCF20, which has been associated with transcriptional activation, have recently been identified as a protein interactor of MeCP2. These data broaden the potential functions of MeCP2 as a regulator of gene expression. Yet, the molecular mechanisms underlying MeCP2-dependent gene regulation remain largely unknown. Here, using a human MECP2 gain-of-function Drosophila model, we screened for genetic modifiers of MECP2-induced phenotypes. Our approach identified several subunits of the Drosophila super elongation complex, a P-TEFb containing RNA polymerase II (RNA pol II) elongation factor required for the release of promoter-proximally paused RNA pol II, as genetic interactors of MECP2. We discovered that MeCP2 physically interacts with the SEC in human cells and in the mouse brain. Furthermore, we found that MeCP2 directly binds AFF4, the scaffold of the SEC, via the transcriptional repression domain. Finally, loss of MeCP2 in the mouse cortex caused reduced binding of AFF4 specifically on a subset of genes involved in the regulation of synaptic function, which also displayed the strongest decrease in RNA pol II binding in the genebody. Taken together, our study reveals a previously unrecognized mechanism through which MeCP2 regulates transcription, providing a new dimension to its regulatory role in gene expression.

molecular biology↗

bioRxiv2024

Release of P-TEFb from the Super Elongation Complex promotes HIV-1 latency reversal

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state, and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes, such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs), which play broad regulatory roles in host gene expression. Still, it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here, we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is, at least in part, dependent on the viral Tat protein. Finally, we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV on suppressive ART, most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together, these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation. AUTHOR SUMMARYSince the start of the HIV pandemic, it is estimated that nearly 86 million people have been infected with the virus, and about 40 million people have died. Modern antiretroviral therapies potently restrict viral replication and prevent the onset of AIDS, saving millions of lives. However, these therapies are not curative due to the persistence of the virus in a silenced or latent state in long-lived cells of the body. One proposed strategy to clear this latent reservoir, termed "shock and kill", is to activate these silenced viruses such that the infected cells can be cleared from the body by the immune system. While several drugs have been developed that can activate latent viruses, none have proven effective at reducing the size of the latent reservoir in patients in clinical trials. Here, we describe a new method for latency reactivation using a small molecule inhibitor of a human protein complex called the Super Elongation Complex (SEC). Inhibiting the SEC enhances viral transcription during active infection and triggers the reactivation of latent viruses, especially when in combination with other latency reversing agents. These results pave the way for developing more effective strategies to reactivate latent viruses towards a functional cure.

microbiology↗

bioRxiv2024

NELF coordinates Pol II transcription termination and DNA replication initiation

Regulation of RNA polymerase II (Pol II) transcription is closely associated with cell proliferation. However, it remains unclear how the Pol II transcription program is altered in cancer to favour cell growth. Here, we find that gene expression of NELFCD, a known negative elongation factor, is up-regulated in colorectal tumours. To dissect the direct role of NELF-C on Pol II transcription in such cancer, we employed an auxin-dependent protein degradation system for NELF-C in combination with nascent transcript sequencing technologies. Strikingly, we demonstrated that the acute loss of NELF-C protein globally perturbs Pol II transcription termination and also increases transcription elongation rate, independently of promoter-proximal Pol II pausing. This results in Pol II transcription into DNA replication initiation zones, and may link to failure of the cell cycle transition into S phase. We anticipate that NELF will be a potential therapeutic target to restrict colorectal cancers by promoting transcription-replication conflict. HIGHLIGHTSO_LIExpression of NELFCD transcript is up-regulated in colorectal tumors C_LIO_LINELF-C protein is mandatory for the transition between G1-S phases during cell cycle C_LIO_LINELF-C loss impairs transcription termination independently of Pol II promoter-proximal pausing C_LIO_LINELF-C loss leads Pol II to invade DNA replication initiation zones C_LI

molecular biology↗

bioRxiv2023

Expansion of profibrotic monocyte-derived alveolar macrophages in patients with persistent respiratory symptoms and radiographic abnormalities after COVID-19

As many as 10-30% of the over 760 million survivors of COVID-19 develop persistent symptoms, of which respiratory symptoms are among the most common. To understand the cellular and molecular basis for respiratory PASC, we combined a machine learning based analysis of lung computed tomography (CT) with flow cytometry, single-cell RNA-sequencing analysis of bronchoalveolar lavage fluid and nasal curettage samples, and alveolar cytokine profiling in a cohort of thirty-five patients with respiratory symptoms and radiographic abnormalities more than 90 days after infection with COVID-19. CT images from patients with PASC revealed abnormalities involving 73% of the lung, which improved on subsequent imaging. Interstitial abnormalities suggestive of fibrosis on CT were associated with the increased numbers of neutrophils and presence of profibrotic monocyte-derived alveolar macrophages in BAL fluid, reflecting unresolved epithelial injury. Persistent infection with SARS-CoV-2 was identified in six patients and secondary bacterial or viral infections in two others. These findings suggest that despite its heterogenous clinical presentations, respiratory PASC with radiographic abnormalities results from a common pathobiology characterized by the ongoing recruitment of neutrophils and profibrotic monocyte-derived alveolar macrophages driving lung fibrosis with implications for diagnosis and therapy.

immunology↗

bioRxiv2020

Decoding the Protein Composition of Whole Nucleosomes with Nuc-MS

Nuc-MS characterizes histone modifications and variants directly from intact endogenous nucleosomes. Preserving whole nucleosome particles enables precise interrogation of their protein content, as for H3.3-containing nucleosomes which had 6-fold co-enrichment of variant H2A.Z over bulk chromatin. Nuc-MS, validated by ChIP-seq, showed co-occurrence of oncogenic H3.3K27M with euchromatic marks (e.g., H4K16ac and >15-fold enrichment of H3K79me2). By capturing the entire epigenetic landscape, Nuc-MS provides a new, quantitative readout of nucleosome-level biology.

biochemistry↗

bioRxiv2020

Alveolitis in severe SARS-CoV-2 pneumonia is driven by self-sustaining circuits between infected alveolar macrophages and T cells

Some patients infected with Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) develop severe pneumonia and the acute respiratory distress syndrome (ARDS) [1]. Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from other types of pneumonia [2]. We collected bronchoalveolar lavage fluid samples from 86 patients with SARS-CoV-2-induced respiratory failure and 252 patients with known or suspected pneumonia from other pathogens and subjected them to flow cytometry and bulk transcriptomic profiling. We performed single cell RNA-Seq in 5 bronchoalveolar lavage fluid samples collected from patients with severe COVID-19 within 48 hours of intubation. In the majority of patients with SARS-CoV-2 infection at the onset of mechanical ventilation, the alveolar space is persistently enriched in alveolar macrophages and T cells without neutrophilia. Bulk and single cell transcriptomic profiling suggest SARS-CoV-2 infects alveolar macrophages that respond by recruiting T cells. These T cells release interferon-gamma to induce inflammatory cytokine release from alveolar macrophages and further promote T cell recruitment. Our results suggest SARS-CoV-2 causes a slowly unfolding, spatially-limited alveolitis in which alveolar macrophages harboring SARS-CoV-2 transcripts and T cells form a positive feedback loop that drives progressive alveolar inflammation. This manuscript is accompanied by an online resource: https://www.nupulmonary.org/covid-19/ One sentence summarySARS-CoV-2-infected alveolar macrophages form positive feedback loops with T cells in patients with severe COVID-19.

immunology↗

bioRxiv2020

Screening of PCRP transcription factor antibodies in biochemical assays

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability are problematic and epitope tagging can be impractical in many cases. In an effort to improve this situation, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human-presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. 887 unique antibodies against 681 unique human transcription factors (TFs), were assayed by ultra-high resolution ChIP-exo/seq, primarily in a single pass in one cell type (K562). Deep systematic analyses of the resulting [~]1,200 ChIP-exo datasets can be found at www.PCRPvalidation.org. Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that was distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We demonstrate and discuss the metrics and challenges to antibody validation in chromatin-based assays.

biochemistry↗

bioRxiv2018

LEDGF and HDGF2 relieve the nucleosome-induced barrier to transcription

FACT (facilitates chromatin transcription) is a protein complex that allows RNAPII to overcome the nucleosome-induced barrier to transcription. While abundant in undifferentiated cells and many cancers, FACT is not abundant or is absent in most tissues. Therefore, we screened for additional proteins that might replace FACT upon differentiation. Here we report the identification of two such proteins, LEDGF and HDGF2, each containing two HMGA-like AT-hooks and a PWWP methyl-lysine reading domain known to bind to H3K36me2 and H3K36me3. LEDGF and HDGF2 localize with H3K36me2/3 at genomic regions containing active genes, usually adjacent to H3K27me3 domains. In myoblasts, where FACT expression is low, LEDGF and HDGF2 are enriched on most active genes. Upon differentiation to myotubes, LEDGF levels decrease across the genome while HDGF2 levels are maintained. Moreover, HDGF2 is recruited to the majority of myotube up-regulated genes and is required for their proper expression. HDGF2 knockout myoblasts exhibit an accumulation of paused RNAPII proximal to the first nucleosome within the transcribed region of many HDGF2 target genes, indicating a defect in early elongation. We propose that LEDGF and HDGF2 substitute for FACT in differentiated tissues and that their distribution on the genome helps maintain transcriptional programs unique to particular cell types.\n\nOne Sentence SummaryChromatin bound LEDGF and HDGF2 proteins allow RNAPII to overcome the nucleosome-induced block to transcription.

molecular biology↗

bioRxiv2018

Single-Cell Transcriptomic Analysis of Human Lung Reveals Complex Multicellular Changes During Pulmonary Fibrosis

Pulmonary fibrosis is a devastating disorder that results in the progressive replacement of normal lung tissue with fibrotic scar. Available therapies slow disease progression, but most patients go on to die or require lung transplantation. Single-cell RNA-seq is a powerful tool that can reveal cellular identity via analysis of the transcriptome, but its ability to provide biologically or clinically meaningful insights in a disease context is largely unexplored. Accordingly, we performed single-cell RNA-seq on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and one bronchoscopic cryobiospy sample. Integrated single-cell transcriptomic analysis of donors and patients with pulmonary fibrosis identified the emergence of distinct populations of epithelial cells and macrophages that were common to all patients with lung fibrosis. Analysis of transcripts in the Wnt pathway suggested that within the same cell type, Wnt secretion and response are restricted to distinct non-overlapping cells, which was confirmed using in situ RNA hybridization. Single-cell RNA-seq revealed heterogeneity within alveolar macrophages from individual patients, which was confirmed by immunohistochemistry. These results support the feasibility of discovery-based approaches applying next generation sequencing technologies to clinically obtained samples with a goal of developing personalized therapies.\n\nOne Sentence SummarySingle-cell RNA-seq applied to tissue from diseased and donor lungs and a living patient with pulmonary fibrosis identifies cell type-specific disease-associated molecular pathways.

systems biology↗