@article {163840, title = {The assembly and functions of multiway enhancer-promoter hubs.}, journal = {Curr Opin Genet Dev}, volume = {97}, year = {2026}, month = {2026 Feb 19}, pages = {102446}, abstract = {

Single enhancer-promoter communication, once conceptualized through a simple {\textquoteright}bridge model{\textquoteright} of direct molecular contact, is now redefined by the concept of hubs. The term {\textquoteright}hub{\textquoteright} is often used interchangeably with other nuclear structures and concepts; however, in this review, we define a hub as a spatial cluster of multiple cis-regulatory elements (CREs), including enhancers and promoters, originating from the same or different chromosomes. These hubs represent dynamic and transient assemblies that coordinate regulatory interactions within the three-dimensional nuclear landscape. A variety of partially overlapping mechanisms, including phase-separation, loop extrusion, protein scaffolding, and actin myosin-driven chromatin mobility, have been proposed to facilitate their formation. However, the organizational principles governing these multi-CREs interacting hubs and their roles in orchestrating gene expression programs remain only partially understood. In this review, we synthesize emerging insights into the formation, function, and regulatory architecture of these transcriptional hubs and propose that they confer robustness, coordination, and flexibility to gene regulation across diverse cellular and developmental contexts.

}, issn = {1879-0380}, doi = {10.1016/j.gde.2026.102446}, url = {https://www.sciencedirect.com/science/article/pii/S0959437X26000134?ref=pdf_download\&fr=RR-2\&rr=9d0a84d9df331473}, author = {Islam, Zubairul and Shigvan, Sakshi and Singh, Amanjot and Notani, Dimple} } @article {156309, title = {Androgen receptor activation for breast cancer therapy.}, journal = {Nat Rev Cancer}, year = {2025}, month = {2025 Apr 24}, abstract = {

Breast cancer overwhelmingly affects women (approximately 1000:1 ratio of women to men), with around 80\% of cases driven by oncogenic oestrogen receptor-α (ERα) activity. Endocrine therapy, including surgical removal of ovaries (oophorectomy), aromatase inhibitors or pharmaceutical ERα ablation, has long been the cornerstone of treatment in ER+\ breast cancer. Although effective in localized disease, it fails against metastatic endocrine-resistant breast cancer, the primary cause of mortality. This underscores the need for new treatment strategies beyond targeting oestrogen and ERα. In this context, the androgen receptor (AR), a key player in breast cancer biology, has long been overlooked. In the normal breast, oestrogen drives mammary gland growth, while androgens counterbalance it. The majority of ER+\ breast cancers also express AR, and treatment strategies adapted from prostate cancer using AR antagonists have failed in clinical trials. Considering the role of AR in normal breast biology, this might have been the wrong approach. Instead of blocking AR, does it need to be activated?

Hickey et al. tackled this question, conducting a large-scale preclinical study using cell lines and patient-derived breast cancer models, including endocrine-resistant ones with\ ESR1\ and\ CCND1\ amplification or mutations. The authors used ChIP{\textendash}seq (chromatin immunoprecipitation followed by sequencing) to map genome-wide interactions of AR and ERα with chromatin under different hormonal conditions. When AR was activated with dihydrotestosterone (DHT) and ERα with oestradiol, AR hijacked the transcriptional program of ERα by sequestering shared transcriptional coactivators such as SRC3 and p300 from ERα genomic sites. In doing so, AR effectively displaced ERα from target chromatin, shutting down ERα-driven tumour growth.

This explained why past AR antagonists such as enzalutamide had failed; blocking AR removed a crucial tumour suppressor. However, AR agonists such as DHT or the selective, non-virilizing agonist enobosarm suppressed tumour growth in ER+\ breast cancer models.

Encouragingly, a phase II clinical trial (NCT02463032) confirmed that enobosarm was not only effective but also well tolerated in women who had exhausted standard endocrine therapies.

The study by Hickey et al. represents a paradigm shift, highlighting how the intricate cross-talk between steroid receptors, beyond a singular focus on ERα, can be strategically leveraged for breast cancer therapy. It also underscores the need for further research into the dynamic hormonal landscape in women, particularly the substantial hormonal shifts post-menopause, which may alter transcriptional programs and influence responses to endocrine therapy.

{\textquotedblleft}blocking AR removed a crucial tumour suppressor{\textquotedblright}

My lab explores transcriptional mechanisms of nuclear receptors and, inspired by the study discussed here, we are examining the ERα{\textendash}AR interplay across a range of oestradiol-to-DHT ratios to mimic shifts from pre- to post-menopause. Our initial findings suggest that ligand balance shapes receptor interactions, highlighting the need to consider ligand ratios for effective therapy.

}, issn = {1474-1768}, doi = {10.1038/s41568-025-00821-y}, url = {https://www.nature.com/articles/s41568-025-00821-y}, author = {Notani, Dimple} } @article {162687, title = {Fibrillarin regulates epithelial integrity via EZH2-mediated modulation of scribble expression.}, journal = {Cell Rep}, volume = {44}, year = {2025}, month = {2025 Nov 25}, pages = {116608}, abstract = {

Fibrillarin (FBL) is a nucleolar protein critical for rRNA biogenesis. We show that FBL is essential for the maintenance of epithelial integrity through its regulation of cell-cell adhesion proteins. RNA-seq analyses upon FBL depletion revealed deregulation of adhesion- and apical membrane organization-associated pathways. FBL loss deregulates cell polarity via increased deposition of H3K27me3 on the SCRIB promoter, driven by the localization of a subpopulation of EZH2 from the nucleolus to the nucleoplasm. Disrupting the FBL-EZH2 interaction increases cell migration, underscoring a requirement for retaining EZH2 also in the nucleolus. Furthermore, FBL depletion induces EMT in breast epithelial cells, owing to increased levels of mesenchymal factors (Snail1, Twist1, and Zeb1) and activation of Akt. Since Scribble tethers PHLPP1 and PTEN to antagonize Akt, repression of Scribble induces EMT. In summary, FBL safeguards epithelial integrity, by regulating the expression of Scribble, uncovering an FBL-EZH2 axis in EMT and metastasis.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2025.116608}, author = {Saha, Santam and A K, Balaji and Meel, Sweety and Notani, Dimple and Sengupta, Kundan} } @article {154146, title = {Hypoxia-induced CTCF mediates alternative splicing via coupling chromatin looping and RNA Pol II pause to promote EMT in breast cancer.}, journal = {Cell Rep}, volume = {44}, year = {2025}, month = {2025 Feb 04}, pages = {115267}, abstract = {

Hypoxia influences the epithelial-mesenchymal transition (EMT) through the remodeling of the chromatin structure, epigenetics, and alternative splicing. Hypoxia drives CCCTC-binding factor (CTCF) induction through hypoxia-inducible factor 1-alpha (HIF1α), which promotes EMT, although the underlying mechanisms remain unclear. We find that hypoxia significantly increases CTCF occupancy at various EMT-related genes. We present a CTCF-mediated intricate mechanism promoting EMT wherein CTCF binding at the collagen type V alpha 1 chain (COL5A1) promoter is crucial for COL5A1 upregulation under hypoxia. Additionally, hypoxia drives exon64A inclusion in a mutually exclusive alternative splicing event of COL5A1exon64 (exon64A/64B). Notably, CTCF mediates COL5A1 promoter-alternatively spliced exon upstream looping that regulates DNA demethylation at distal exon64A. This further regulates the CTCF-mediated RNA polymerase II pause at COL5A1exon64A, leading to its inclusion in promoting the EMT under hypoxia. Genome-wide study indicates the association of gained CTCF occupancy with the alternative splicing of many cancer-related genes, similar to the proposed model. Specifically, disrupting the HIF1α-CTCF-COL5A1exon64A axis through the dCas9-DNMT3A system alleviates the EMT in hypoxic cancer cells and may represent a novel therapeutic target in breast cancer.

}, keywords = {COL5A1; CP: Cancer; CP: Molecular biology; CRISPR-dCas9-mediated editing; CTCF; EMT; alternative splicing; breast cancer; epigenetics; hypoxia; promoter-exon upstream looping.}, issn = {2211-1247}, doi = {10.1016/j.celrep.2025.115267}, url = {https://pubmed.ncbi.nlm.nih.gov/39913285/}, author = {Kakani, Parik and Dhamdhere, Shruti Ganesh and Pant, Deepak and Joshi, Rushikesh and Mishra, Sachin and Pandey, Anchala and Notani, Dimple and Shukla, Sanjeev} } @article {162678, title = {Organization principles of dynamic three-dimensional genome architecture associated with centromere clustering states.}, journal = {Proc Natl Acad Sci U S A}, volume = {122}, year = {2025}, month = {2025 Dec 16}, pages = {e2520310122}, abstract = {

Fungal centromeres are clustered near microtubule organizing centers to help adopt the Rabl chromosomal organization. The role of centromere clustering in driving large-scale changes in structural and functional chromatin assembly remains unclear. Here, using Hi-C and superresolution microscopy, we show that cell cycle-dependent centromere declustering and clustering states in drive global changes in the 3D genome architecture. Centromeres and telomeres are scattered around the nuclear periphery at interphase, and this arrangement constrains the interarm interactions within a chromosome, providing a unique interphase chromosome organization. Moreover, centromeres and telomeres are organized as separate compartments, segregating them from active euchromatic regions. Polymer modeling reveals that the transition from the unclustered to clustered centromere state during the cell cycle involves changes from a globular to an elongated chromosome architecture. Strikingly, while clustered centromeric regions replicate early in most yeasts, centromeres replicate late in S-phase, hinting at a possible link between centromere clustering dynamics and DNA replication timing. Overall, our study uncovers several unique organizational principles governing the dynamic genome architecture in an evolutionarily diverged basidiomycete yeast.

}, keywords = {Cell Cycle, Centromere, Chromosomes, Fungal, Cryptococcus neoformans, Genome, Fungal, Telomere}, issn = {1091-6490}, doi = {10.1073/pnas.2520310122}, author = {Polisetty, Satya Dev and Dutta, Shuvadip and Vadnala, Rakesh Netha and Notani, Dimple and Padinhateeri, Ranjith and Sanyal, Kaustuv} } @article {161318, title = {Paused RNA polymerase primes promoters via RNA-mediated stabilisation of transcription factor ERα.}, journal = {Nat Commun}, volume = {16}, year = {2025}, month = {2025 Oct 29}, pages = {9553}, abstract = {

Regulated pausing of RNA polymerase II (Pol II) is essential for enabling rapid and coordinated transcriptional responses to signalling cues.\ Pausing also contributes to the formation of nucleosome-free regions with the help of chromatin remodellers. However, if these nucleosome-free regions engage with transcription factors to stimulate the transcription potential of paused promoters is not known. In this study, we demonstrate that ligand-induced estrogen receptor-alpha (ERα) binding is stabilized at Pol II-paused sites. This stabilization results from an increased dwell time of ERα on chromatin, as revealed by single molecule tracking (SMT) experiments. Notably, short chromatin-associated RNAs generated by the paused Pol II contribute to enhancing ERα binding at paused promoters. We also observe that pausing increases H3K27ac levels, which primes paused promoters for robust transcriptional activation upon release. Collectively, these findings suggest that paused Pol II plays a central role in enhancing transcription factor binding through an RNA-dependent mechanism. This, in turn, results in a more vigorous transcriptional response following pausing release, thus contributing to the fine-tuning of ERα-mediated gene regulation.

}, keywords = {Chromatin, Estrogen Receptor alpha, Histones, Humans, Nucleosomes, Promoter Regions, Genetic, Protein Binding, RNA, RNA Polymerase II, Transcription, Genetic, Transcriptional Activation}, issn = {2041-1723}, doi = {10.1038/s41467-025-64569-7}, author = {Mann, Rajat and Soota, Deepanshu and Das, Ayan and Podh, Nitesh Kumar and Mehta, Gunjan and Notani, Dimple} } @article {159721, title = {upstream deletion leads to dysregulation of and autosomal gene expression.}, journal = {Genome Res}, year = {2025}, month = {2025 Aug 06}, abstract = {

long noncoding RNA is the master regulator of the X-Chromosome inactivation (XCI) process. is expressed from the inactive X and coats the inactive X to facilitate XCI. -regulation of expression remains poorly understood in the context of maintenance of XCI. Here, we have explored the role of the upstream sequences (\~{}6 kb) lying between and in the regulation of and XCI in mouse extra-embryonic endoderm stem cells (XEN), which represent the maintenance phase of imprinted XCI. Here, we show that the deletion of this upstream sequence in the inactive X leads to the upregulation of expression accompanied by the dispersal of the cloud. Notably, we find the loss of enrichment of repressive marks such as H3K27me3, H4K20me1, and MacroH2A, except that of H2AK119ub, in dispersed nuclei. However, X-linked genes remain silent despite dispersal and loss of enrichment of repressive marks. Notably, we find that many autosomal genes, including cohesin , are dysregulated in -upstream-deleted cells. Additionally, we demonstrate that -upstream deletion leads to alterations of topological contacts of the locus with its upstream positive regulator and across the inactive X and autosomes. Finally, we show genome-wide alterations of the occupancy of architectural proteins CTCF/RAD21, including at many loci of the inactive X such as the upstream regions and the locus, which is critical for maintaining inactive X conformation. Taken together, we demonstrate that the upstream sequence imparts a multifaceted role in genome regulation beyond the XCI.

}, issn = {1549-5469}, doi = {10.1101/gr.279822.124}, author = {Majumdar, Sudeshna and Bammidi, Lakshmi Sowjanya and Naik, Hemant C and Manhas, Avinchal and Baro, Runumi and Kalita, Akash and Naskar, Amlan Jyoti and Nidharshan, Sundarraj and Bariha, Girija S and Notani, Dimple and Gayen, Srimonta} } @article {150019, title = {Acute Activation of Genes Through Transcriptional Condensates Impact Non-target Genes in a Chromatin Domain}, journal = {eLife}, year = {2024}, month = {11/2024}, abstract = {

Transcription activation of genes by estrogens is driven by enhancers, which are often located within the same Topologically Associating Domain (TAD) as non-targeted promoters. We investigated how acute enhancer-driven activation affects neighbouring non-target genes within the same TAD. Using single-molecule RNA FISH (smFISH), we tracked the transcription of TFF1 (enhancer-targeted) and TFF3 (non-targeted) during estrogen stimulation. We observed mutually exclusive expression patterns: TFF1 expression peaked at 1 hour, while TFF3 reached its peak at 3 hours, after TFF1{\textquoteright}s activation had diminished. Chromatin looping data indicated that the enhancer loops with TFF1 but not TFF3, suggesting that TFF3 upregulation is not due to direct enhancer-promoter interactions. CRISPR deletion of the TFF1 enhancer and 1,6-hexanediol (HD) exposure revealed that the TFF1 enhancer:promoter undergo Liquid-Liquid Phase Separation (LLPS), which sequesters the transcriptional machinery and inhibits TFF3 expression. As estrogen signalling wanes or LLPS is disrupted, TFF1 expression declines while TFF3 expression increases. Our findings reveal that enhancer-driven activation can indirectly influence neighbouring genes, highlighting a dynamic shift in gene expression as signalling progresses.

}, doi = {10.7554/eLife.102417.1}, author = {Bohra, Darshika and Islam, Zubairul and Nidharshan, Sundarraj and Mazumder, Aprotim and Notani, Dimple} } @article {152142, title = {Non-coding genetic variants underlying higher prostate cancer risk in men of African ancestry.}, journal = {Res Sq}, year = {2024}, month = {2024 Dec 05}, abstract = {

Incidence and severity of prostate cancer (PrCa) substantially varies across ancestries. American men of African ancestry (AA) are more likely to be diagnosed with and die from PrCa than the those of European ancestry (EA). Published polygenic risk scores for developing prostate cancer, even those based on multi-ancestry genome-wide association studies, do not address population-specific genetic mechanisms underlying PrCa risk in men of African ancestry. Specifically, the role of non-coding regulatory polymorphisms in driving inter-ancestry variation in PrCa has not been suficiently explored. Here, by employing a sequence-based deep learning model of prostate regulatory enhancers, we identified ~ 2,000 SNPs with higher alternate allele frequency in AA men that potentially affect enhancer function associated with PrCa susceptibility, as supported by our experimental validation. The identified enhancer SNPs (eSNPs) may influence PrCa development through two complementary mechanisms: 1) the alternate allele that increase enhancer activity result in immune suppression and telomere elongation, and 2) the alternate alleles that decrease enhancer activity, lead to de-differentiation and inhibition of apoptosis. Notably, the eSNPs tend to disrupt the binding of known prostate transcription factors including FOX, AR and HOX families. Lastly, the identified eSNPs can be combined into a polygenic risk score that adds value to current GWAS-based risk variants in assessing PrCa risk in independent cohorts.

}, issn = {2693-5015}, doi = {10.21203/rs.3.rs-5485172/v1}, author = {Hannenhalli, Sridhar and Li, Shan and Fatema, Kaniz and Nidharshan, Sundarraj and Singh, Arashdeep and Rajagopal, Padma Sheila and Notani, Dimple and Takeda, David} } @article {148261, title = {RNA fine-tunes estrogen receptor-alpha binding on low-affinity DNA motifs for transcriptional regulation.}, journal = {EMBO J}, year = {2024}, month = {2024 Sep 16}, abstract = {

Transcription factors (TFs) regulate gene expression by binding with varying strengths to DNA via their DNA-binding domain. Additionally, some TFs also interact with RNA, which modulates transcription factor binding to chromatin. However, whether RNA-mediated TF binding results in differential transcriptional outcomes remains unknown. In this study, we demonstrate that estrogen receptor α (ERα), a ligand-activated TF, interacts with RNA in a ligand-dependent manner. Defects in RNA binding lead to genome-wide loss of ERα recruitment, particularly at weaker ERα-motifs. Furthermore, ERα mobility in the nucleus increases in the absence of its RNA-binding capacity. Unexpectedly, this increased mobility coincides with robust polymerase loading and transcription of ERα-regulated genes that harbor low-strength motifs. However, highly stable binding of ERα on chromatin negatively impacts ligand-dependent transcription. Collectively, our results suggest that RNA interactions spatially confine ERα on low-affinity sites to fine-tune gene transcription.

}, issn = {1460-2075}, doi = {10.1038/s44318-024-00225-y}, author = {Soota, Deepanshu and Saravanan, Bharath and Mann, Rajat and Kharbanda, Tripti and Notani, Dimple} } @article {145426, title = {UBR7 in concert with EZH2 inhibits the TGF-β signaling leading to extracellular matrix remodeling.}, journal = {Cell Rep}, volume = {43}, year = {2024}, month = {2024 Jun 25}, pages = {114394}, abstract = {

The intricate interplay between resident cells and the extracellular matrix (ECM) profoundly influences cancer progression. In triple-negative breast cancer (TNBC), ECM architecture evolves due to the enrichment of lysyl oxidase, fibronectin, and collagen, promoting distant metastasis. Here we uncover a pivotal transcription regulatory mechanism involving the epigenetic regulator UBR7 and histone methyltransferase EZH2 in regulating transforming growth factor (TGF)-β/Smad signaling, affecting the expression of ECM genes. UBR7 loss leads to a dramatic reduction in facultative heterochromatin mark H3K27me3, activating ECM genes. UBR7 plays a crucial role in matrix deposition in adherent cancer cells and spheroids, altering collagen content and lysyl oxidase activity, directly affecting matrix stiffness and invasiveness. These findings are further validated in\ vivo in mice models and TNBC patients, where reduced UBR7 levels are accompanied by increased ECM component expression and activity, leading to fibrosis-mediated matrix stiffness. Thus, UBR7 is a master regulator of matrix stiffening, influencing the metastatic potential of TNBC.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2024.114394}, author = {Adhikari, Swagata and Singh, Vipin and Nandi, Sandhik and Ghosal, Manorama and Raj, Nidharshan Sundar and Khanna, Jayati and Bhattacharya, Apoorva and Kabiraj, Aindrila and Mondal, Atanu and Vasudevan, Madavan and Senapati, Dulal and Roy, Himansu and Sengupta, Kundan and Notani, Dimple and Das, Chandrima} } @article {123130, title = {Active enhancers strengthen insulation by RNA-mediated CTCF binding at chromatin domain boundaries.}, journal = {Genome Res}, year = {2023}, month = {2023 Jan 17}, abstract = {

Vertebrate genomes are partitioned into chromatin domains or topologically associating domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at domain boundaries correlates with better insulation; however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant and TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer-promoter interactions, and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters, we show that active TAD enhancers, but not promoters, induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.

}, issn = {1549-5469}, doi = {10.1101/gr.276643.122}, author = {Islam, Zubairul and Saravanan, Bharath and Walavalkar, Kaivalya and Farooq, Umer and Singh, Anurag Kumar and Sabarinathan, Radhakrishnan and Thakur, Jitendra and Pandit, Awadhesh and Henikoff, Steven and Notani, Dimple} } @article {136529, title = {Cis-regulatory effect of HPV integration is constrained by host chromatin architecture in cervical cancers.}, journal = {Mol Oncol}, year = {2023}, month = {2023 Nov 27}, abstract = {

HPV infections are the primary drivers of cervical cancers, and often HPV DNA gets integrated into the host genome. Although the oncogenic impact of HPV encoded genes is relatively well known, the cis-regulatory effect of integrated HPV DNA on host chromatin structure and gene regulation remains less understood. We investigated genome-wide patterns of HPV integrations and associated host gene expression changes in the context of host chromatin states and TADs. HPV integrations were significantly enriched in active chromatin regions and depleted in inactive ones. Interestingly, regardless of chromatin state, genomic regions flanking HPV integrations showed transcriptional upregulation. Nevertheless, upregulation (both local and long-range) was mostly confined to TADs with integration, but not affecting adjacent TADs. Few TADs showed recurrent integrations associated with overexpression of oncogenes within them (e.g. MYC, PVT1, TP63 and ERBB2) regardless of proximity. Hi-C and 4C-seq analyses in cervical cancer cell line (HeLa) demonstrated chromatin looping interactions between integrated HPV and MYC/PVT1 regions (~500kb apart), leading to allele-specific overexpression. Based on these, we propose HPV integrations can trigger multimodal oncogenic activation to promote cancer progression.

}, issn = {1878-0261}, doi = {10.1002/1878-0261.13559}, author = {Singh, Anurag Kumar and Walavalkar, Kaivalya and Tavernari, Daniele and Ciriello, Giovanni and Notani, Dimple and Sabarinathan, Radhakrishnan} } @article {129160, title = {Editing the core region in HPFH deletions alters fetal and adult globin expression for treatment of β-hemoglobinopathies.}, journal = {Mol Ther Nucleic Acids}, volume = {32}, year = {2023}, month = {2023 Jun 13}, pages = {671-688}, abstract = {

Reactivation of fetal hemoglobin (HbF) is a commonly adapted strategy to ameliorate β-hemoglobinopathies. However, the continued production of defective adult hemoglobin (HbA) limits HbF tetramer production affecting the therapeutic benefits. Here, we evaluated deletional hereditary persistence of fetal hemoglobin (HPFH) mutations and identified an 11-kb sequence, encompassing putative repressor region (PRR) to β-globin exon-1 (βE1), as the core deletion that ablates HbA and exhibits superior HbF production compared with HPFH or other well-established targets. PRR-βE1-edited hematopoietic stem and progenitor cells (HSPCs) retained their genome integrity and their engraftment potential to repopulate for long-term hematopoiesis in immunocompromised mice producing HbF positive cells . Furthermore, PRR-βE1 gene editing is feasible without HSPC culture. Importantly, the editing induced therapeutically significant levels of HbF to reverse the phenotypes of both sickle cell disease and β-thalassemia major. These findings imply that PRR-βE1 gene editing of patient HSPCs could lead to improved therapeutic outcomes for β-hemoglobinopathy gene therapy.

}, issn = {2162-2531}, doi = {10.1016/j.omtn.2023.04.024}, author = {Venkatesan, Vigneshwaran and Christopher, Abisha Crystal and Rhiel, Manuel and Azhagiri, Manoj Kumar K and Babu, Prathibha and Walavalkar, Kaivalya and Saravanan, Bharath and Andrieux, Geoffroy and Rangaraj, Sumathi and Srinivasan, Saranya and Karuppusamy, Karthik V and Jacob, Annlin and Bagchi, Abhirup and Pai, Aswin Anand and Nakamura, Yukio and Kurita, Ryo and Balasubramanian, Poonkuzhali and Pai, Rekha and Marepally, Srujan Kumar and Mohankumar, Kumarasamypet Murugesan and Velayudhan, Shaji R and Boerries, Melanie and Notani, Dimple and Cathomen, Toni and Srivastava, Alok and Thangavel, Saravanabhavan} } @article {128080, title = {Transcription factor condensates and signaling driven transcription.}, journal = {Nucleus}, volume = {14}, year = {2023}, month = {2023 Dec}, pages = {2205758}, abstract = {

Transcription Factor (TF) condensates are a heterogenous mix of RNA, DNA, and multiple co-factor proteins capable of modulating the transcriptional response of the cell. The dynamic nature and the spatial location of TF-condensates in the 3D nuclear space is believed to provide a fast response, which is on the same pace as the signaling cascade and yet ever-so-specific in the crowded environment of the nucleus. However, the current understanding of how TF-condensates can achieve these feet so quickly and efficiently is still unclear. In this review, we draw parallels with other protein condensates and share our speculations on how the nucleus uses these TF-condensates to achieve high transcriptional specificity and fidelity. We discuss the various constituents of TF-condensates, their properties, and the known and unknown functions of TF-condensates with a particular focus on steroid signaling-induced transcriptional programs.

}, keywords = {Cell Nucleus, Chromatin, DNA, Signal Transduction, Transcription Factors}, issn = {1949-1042}, doi = {10.1080/19491034.2023.2205758}, author = {Mann, Rajat and Notani, Dimple} } @article {109940, title = {First glimpse of enhancers in gene regulation.}, journal = {Nat Rev Genet}, year = {2022}, month = {2022 Apr 27}, issn = {1471-0064}, doi = {10.1038/s41576-022-00492-7}, author = {Notani, Dimple} } @article {117967, title = {Transcriptional regulation of INK4/ARF locus by cis and trans mechanisms.}, journal = {Front Cell Dev Biol}, volume = {10}, year = {2022}, month = {2022}, pages = {948351}, abstract = {

9p21 locus is one of the most reproducible regions in genome-wide association studies (GWAS). The region harbors genes that code for p16, p15, and p14 proteins, and it also harbors a long gene desert adjacent to these genes. The polymorphisms that are associated with several diseases and cancers are present in these genes and the gene desert region. These proteins are critical cell cycle regulators whose transcriptional dysregulation is strongly linked with cellular regeneration, stemness, aging, and cancers. Given the importance of this locus, intense scientific efforts on understanding the regulation of these genes via promoter-driven mechanisms and recently, via the distal regulatory mechanism have provided major insights. In this review, we describe these mechanisms and propose the ways by which this locus can be targeted in pathologies and aging.

}, issn = {2296-634X}, doi = {10.3389/fcell.2022.948351}, author = {Farooq, Umer and Notani, Dimple} } @article {91470, title = {An interdependent network of functional enhancers regulates transcription and EZH2 loading at the INK4a/ARF locus.}, journal = {Cell Rep}, volume = {34}, year = {2021}, month = {2021 Mar 23}, pages = {108898}, abstract = {

The INK4a/ARF locus encodes important cell-cycle regulators p14, p15, and p16. The neighboring gene desert to this locus is the most reproducible GWAS hotspot that harbors one of the densest enhancer clusters in the genome. However, how multiple enhancers that overlap with GWAS variants regulate the INK4a/ARF locus is unknown, which is an important step in linking genetic variation with associated diseases. Here, we show that INK4a/ARF promoters interact with a subset of enhancers in the cluster, independent of their H3K27ac and eRNA levels. Interacting enhancers transcriptionally control each other and INK4a/ARF promoters over long distances as an interdependent single unit. The deletion of even a single interacting enhancer results in an unexpected collapse of the entire enhancer cluster and leads to EZH2 enrichment on promoters in an ANRIL-independent manner. Dysregulated genes genome-wide mimic 9p21-associated diseases under these scenarios. Our results highlight intricate dependencies of promoter-interacting enhancers on each other.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2021.108898}, author = {Farooq, Umer and Saravanan, Bharath and Islam, Zubairul and Walavalkar, Kaivalya and Singh, Anurag Kumar and Jayani, Ranveer Singh and Meel, Sweety and Swaminathan, Sudha and Notani, Dimple} } @article {94543, title = {IP3-mediated Ca2+ signals regulate larval to pupal transition under nutrient stress through the H3K36 methyltransferase Set2.}, journal = {Development}, volume = {148}, year = {2021}, month = {2021 Jun 01}, abstract = {

Persistent loss of dietary protein usually signals a shutdown of key metabolic pathways. In Drosophila larvae that have reached a {\textquoteright}critical weight{\textquoteright} and can pupariate to form viable adults, such a metabolic shutdown would needlessly lead to death. Inositol 1,4,5-trisphosphate-mediated calcium (IP3/Ca2+) release in some interneurons (vGlutVGN6341) allows Drosophila larvae to pupariate on a protein-deficient diet by partially circumventing this shutdown through upregulation of neuropeptide signaling and the expression of ecdysone synthesis genes. Here, we show that IP3/Ca2+ signals in vGlutVGN6341 neurons drive expression of Set2, a gene encoding Drosophila Histone 3 Lysine 36 methyltransferase. Furthermore, Set2 expression is required for larvae to pupariate in the absence of dietary protein. IP3/Ca2+ signal-driven Set2 expression upregulates key Ca2+-signaling genes through a novel positive-feedback loop. Transcriptomic studies, coupled with analysis of existing ChIP-seq datasets, identified genes from larval and pupal stages that normally exhibit robust H3K36 trimethyl marks on their gene bodies and concomitantly undergo stronger downregulation by knockdown of either the intracellular Ca2+ release channel IP3R or Set2. IP3/Ca2+ signals thus regulate gene expression through Set2-mediated H3K36 marks on select neuronal genes for the larval to pupal transition.

}, issn = {1477-9129}, doi = {10.1242/dev.199018}, author = {Mitra, Rishav and Richhariya, Shlesha and Jayakumar, Siddharth and Notani, Dimple and Hasan, Gaiti} } @article {102257, title = {Optimized protocol to create deletion in adherent cell lines using CRISPR/Cas9 system.}, journal = {STAR Protoc}, volume = {2}, year = {2021}, month = {2021 Dec 17}, pages = {100857}, abstract = {

The CRISPR/Cas9 system is a powerful tool for genome editing and is adaptable for a wide range of applications. Here, we have put together a step-by-step protocol for generating knockout cell lines (coding or non-coding region) using CRISPR/Cas9 tool. The protocol below has been tested on adherent cell lines such as HeLa and MCF7. However, it may easily be adapted to other adherent cell lines with minor variations. For complete details on the use and execution of this protocol, please refer to Farooq et\ al. (2021).

}, issn = {2666-1667}, doi = {10.1016/j.xpro.2021.100857}, author = {Farooq, Umer and Notani, Dimple} } @article {99109, title = {Testing the super-enhancer concept.}, journal = {Nat Rev Genet}, year = {2021}, month = {2021 Sep 03}, issn = {1471-0064}, doi = {10.1038/s41576-021-00398-w}, author = {Blobel, Gerd A and Higgs, Douglas R and Mitchell, Jennifer A and Notani, Dimple and Young, Richard A} } @article {78108, title = {Beyond the coding genome: non-coding mutations and cancer.}, journal = {Front Biosci (Landmark Ed)}, volume = {25}, year = {2020}, month = {2020 Jun 01}, pages = {1828-1838}, abstract = {

Latest advancements in genomics involving individuals from different races and geographical locations has led to the identification of thousands of common as well as rare genetic variants and copy number variations (CNVs). These studies have surprisingly revealed that the majority of genetic variation is not present within the coding region but rather in the non-coding region of the genome, which is also termed as "Medical Genome". This short review describes how mutations/variations within; regulatory sequences, architectural proteins and transcriptional regulators give rise to the aberrant gene expression profiles that drives cellular transformations and malignancies.

}, issn = {1093-4715}, author = {Walavalkar, Kaivalya and Notani, Dimple} } @article {71098, title = {Ligand dependent gene regulation by transient ERα clustered enhancers.}, journal = {PLoS Genet}, volume = {16}, year = {2020}, month = {2020 Jan 06}, pages = {e1008516}, abstract = {

Unliganded Estrogen receptor alpha (ERα) has been implicated in ligand-dependent gene regulation. Upon ligand exposure, ERα binds to several EREs relatively proximal to the pre-marked, or persistent, ERα-bound sites and affects transient but robust gene expression. However, the underlying mechanisms are not fully understood. Here we demonstrate that upon ligand stimulation, persistent sites interact extensively, via chromatin looping, with the proximal transiently ERα-bound sites, forming Ligand Dependent ERα Enhancer Cluster in 3D (LDEC). The E2-target genes are regulated by these clustered enhancers but not by the H3K27Ac super-enhancers. Further, CRISPR-based deletion of TFF1 persistent site disrupts the formation of its LDEC resulting in the loss of E2-dependent expression of TFF1 and its neighboring genes within the same TAD. The LDEC overlap with nuclear ERα condensates that coalesce in a ligand and persistent site dependent manner. Furthermore, formation of clustered enhancers, as well as condensates, coincide with the active phase of signaling and their later disappearance results in the loss of gene expression even though persistent sites remain bound by ERα. Our results establish, at TFF1 and NRIP1 locus, a direct link between ERα condensates, ERα enhancer clusters, and transient, but robust, gene expression in a ligand-dependent fashion.

}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1008516}, author = {Saravanan, Bharath and Soota, Deepanshu and Islam, Zubairul and Majumdar, Sudeshna and Mann, Rajat and Meel, Sweety and Farooq, Umer and Walavalkar, Kaivalya and Gayen, Srimonta and Singh, Anurag Kumar and Hannenhalli, Sridhar and Notani, Dimple} } @article {79800, title = {A rare variant of African ancestry activates 8q24 lncRNA hub by modulating cancer associated enhancer.}, journal = {Nat Commun}, volume = {11}, year = {2020}, month = {2020 Jul 17}, pages = {3598}, abstract = {

Genetic variation at the 8q24 locus is linked with the greater susceptibility to prostate cancer in men of African ancestry. One such African ancestry specific rare variant, rs72725854 (A\>G/T) (~6\% allele frequency) has been associated with a ~2-fold increase in prostate cancer risk. However, the functional relevance of this variant is unknown. Here we show that the variant rs72725854 is present in a prostate cancer-specific enhancer at 8q24 locus. Chromatin-conformation capture and dCas9 mediated enhancer blocking establish a direct regulatory link between this enhancer and lncRNAs PCAT1, PRNCR1 and PVT1. The risk allele ({\textquoteright}T{\textquoteright}) is associated with higher expression of PCAT1, PVT1 and c-myc in prostate tumors. Further, enhancer with the risk allele gains response to androgen stimulation by recruiting the transcription factor SPDEF whereas, non-risk alleles remain non-responsive. Elevated expression of these lncRNAs and c-myc in risk allele carriers may explain their greater susceptibility to prostate cancer.

}, issn = {2041-1723}, doi = {10.1038/s41467-020-17325-y}, author = {Walavalkar, Kaivalya and Saravanan, Bharath and Singh, Anurag Kumar and Jayani, Ranveer Singh and Nair, Ashwin and Farooq, Umer and Islam, Zubairul and Soota, Deepanshu and Mann, Rajat and Shivaprasad, Padubidri V and Freedman, Matthew L and Sabarinathan, Radhakrishnan and Haiman, Christopher A and Notani, Dimple} } @article {53190, title = {A SUV39H1-low chromatin state characterises and promotes migratory properties of cervical cancer cells.}, journal = {Exp Cell Res}, year = {2019}, month = {2019 Feb 14}, abstract = {

Metastatic progression is a major cause of mortality in cervical cancers, but factors regulating migratory and pre-metastatic cell populations remain poorly understood. Here, we sought to assess whether a SUV39H1-low chromatin state promotes migratory cell populations in cervical cancers, using meta-analysis of data from The Cancer Genome Atlas (TCGA), immunohistochemistry, genomics and functional assays. Cervical cancer cells sorted based on migratory ability in vitro have low levels of SUV39H1 protein, and SUV39H1 knockdown in vitro enhanced cervical cancer cell migration. Further, TCGA SUV39H1-low tumours correlated with poor clinical outcomes and showed gene expression signatures of cell migration. SUV39H1 expression was examined within biopsies, and SUV39H1 cells within tumours also demonstrated migratory features. Next, to understand genome scale transcriptional and chromatin changes in migratory populations, cell populations sorted based on migration in vitro were examined using RNA-Seq, along with ChIP-Seq for H3K9me3, the histone mark associated with SUV39H1. Migrated populations showed SUV39H1-linked migratory gene expression signatures, along with broad depletion of H3K9me3 across gene promoters. We show for the first time that a SUV39H1-low chromatin state associates with, and promotes, migratory populations in cervical cancers. Our results posit SUV39H1-low cells as key populations for prognosis estimation and as targets for novel therapies.

}, issn = {1090-2422}, doi = {10.1016/j.yexcr.2019.02.010}, author = {Rodrigues, Calvin and Pattabiraman, Chitra and Vijaykumar, Anjali and Arora, Reety and Narayana, Suma Mysore and Kumar, Rekha V and Notani, Dimple and Varga-Weisz, Patrick and Krishna, Sudhir} }