Heller-Trulli D, Liu H, Mukherjee S
… +1 more, Moore CL
Mol Cell Biol
· 2022 Dec · PMID 36519931
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We have previously described the role of an essential Saccharomyces cerevisiae gene, important for cleavage and polyadenylation 1 (), in the regulation of gene expression through its interaction with Ysh1, the endonuclea...We have previously described the role of an essential Saccharomyces cerevisiae gene, important for cleavage and polyadenylation 1 (), in the regulation of gene expression through its interaction with Ysh1, the endonuclease subunit of the mRNA 3'-end processing complex. Through a similar mechanism, the mammalian homolog ubiquitin protein ligase E3D (UBE3D) promotes the migratory and invasive potential of breast cancer cells, but its role in the regulation of gene expression during normal cellular differentiation has not previously been described. In this study, we show that CRISPR/Cas9-mediated knockout of in 3T3-L1 cells blocks their ability to differentiate into mature adipocytes. Consistent with previous studies in other cell types, knockout leads to decreased levels of CPSF73 and global changes in cellular mRNAs indicative of a loss of 3'-end processing capacity. knockout cells also display decreased expression of known preadipogenic markers. Overexpression of either UBE3D or CPSF73 rescues the differentiation defect and partially restores protein levels of these markers. These results support a model in which UBE3D is necessary for the maintenance of the adipocyte-committed state via its regulation of the mRNA 3'-end processing machinery.
Ouvrard J, Muniz L, Nicolas E
… +1 more, Trouche D
Mol Cell Biol
· 2022 Dec · PMID 36445136
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Transcriptional gene silencing by small interfering RNAs (siRNAs) has been widely described in various species, including plants and yeast. In mammals, its extent remains somewhat debated. Previous studies showed that si...Transcriptional gene silencing by small interfering RNAs (siRNAs) has been widely described in various species, including plants and yeast. In mammals, its extent remains somewhat debated. Previous studies showed that siRNAs targeting gene promoters could induce the silencing of the targeted promoter, although the involvement of off-target mechanisms was also suggested. Here, by using nascent RNA capture and RNA polymerase II chromatin immunoprecipitation, we show that siRNAs targeting a chromatin-associated noncoding RNA induced its transcriptional silencing. Deletion of the sequence targeted by one of these siRNAs on the two alleles by genome editing further showed that this silencing was due to base-pairing of the siRNA to the target. Moreover, by using cells with heterozygous deletion of the target sequence, we showed that only the wild-type allele, but not the deleted allele, was silenced by the siRNA, indicating that transcriptional silencing occurred only in . Finally, we demonstrated that both Ago1 and Ago2 are involved in this transcriptional silencing. Altogether, our data demonstrate that siRNAs targeting a chromatin-associated RNA at a distance from its promoter induce its transcriptional silencing. Our results thus extend the possible repertoire of endogenous or exogenous interfering RNAs.
Snaurova R, Vdovin A, Durech M
… +5 more, Nezval J, Zihala D, Jelinek T, Hajek R, Simicek M
Mol Cell Biol
· 2022 Dec · PMID 36445135
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OTUD1 is a deubiquitinating enzyme involved in many cellular processes including cancer and innate, immune signaling pathways. Here, we perform a proximity labeling-based interactome study that identifies OTUD1 largely p...OTUD1 is a deubiquitinating enzyme involved in many cellular processes including cancer and innate, immune signaling pathways. Here, we perform a proximity labeling-based interactome study that identifies OTUD1 largely present in the translation and RNA metabolism protein complexes. Biochemical analysis validates OTUD1 association with ribosome subunits, elongation factors and the E3 ubiquitin ligase ZNF598 but not with the translation initiation machinery. OTUD1 catalytic activity suppresses polyA triggered ribosome stalling through inhibition of ZNF598-mediated RPS10 ubiquitination and stimulates formation of polysomes. Finally, analysis of gene expression suggests that OTUD1 regulates the stability of rare codon rich mRNAs by antagonizing ZNF598.
Suzuki C, Fujiwara T, Shima H
… +11 more, Ono K, Saito K, Kato H, Onodera K, Ichikawa S, Fukuhara N, Onishi Y, Yokoyama H, Nakamura Y, Igarashi K, Harigae H
Mol Cell Biol
· 2022 Dec · PMID 36374104
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Mitochondria play essential and specific roles during erythroid differentiation. Recently, , encoding a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1, as well as an erythropoietin-...Mitochondria play essential and specific roles during erythroid differentiation. Recently, , encoding a mitochondrial inner membrane protein, has been identified as a novel target of GATA-1, as well as an erythropoietin-inducible gene. While FAM210B protein is involved in regulate mitochondrial metabolism and heme biosynthesis, its detailed function remains unknown. Here, we generated both knockout and knockdown of endogenous FAM210B in human induced pluripotent stem-derived erythroid progenitor (HiDEP) cells using CRISPR/Cas9 methodology. Intriguingly, erythroid differentiation was more pronounced in the FAM210B-depleted cells, and this resulted in increased frequency of orthochromatic erythroblasts and decreased frequencies of basophilic/polychromatic erythroblasts. Comprehensive metabolite analysis and functional analysis indicated that oxygen consumption rates and the NAD (NAD)/NADH ratio were significantly decreased, while lactate production was significantly increased in FAM210B deletion HiDEP cells, indicating involvement of FAM210B in mitochondrial energy metabolism in erythroblasts. Finally, we purified FAM210B-interacting protein from K562 cells that stably expressed His/biotin-tagged FAM210B. Mass spectrometry analysis of the His/biotin-purified material indicated interactions with multiple subunits of mitochondrial ATP synthases, such as subunit alpha (ATP5A) and beta (ATP5B). Our results suggested that FAM210B contributes prominently to erythroid differentiation by regulating mitochondrial energy metabolism. Our results provide insights into the pathophysiology of dysregulated hematopoiesis.
Hartford CCR, Shrestha RL, Pongor L
… +16 more, Zhao Y, Chen X, Fromont C, Chaudhary R, Li XL, Pasterczyk KR, Kumar R, Muys BR, Tsitsipatis D, Chari R, Gorospe M, Aladjem MI, Khan J, Basrai MA, Grammatikakis I, Lal A
Mol Cell Biol
· 2022 Dec · PMID 36342127
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is a p53-induced lncRNA that suppresses basal p53 levels. Here, we investigated upon p53 activation in liver cancer cells, where it is expressed at significantly higher levels than other cell types. Using isoform sequen...is a p53-induced lncRNA that suppresses basal p53 levels. Here, we investigated upon p53 activation in liver cancer cells, where it is expressed at significantly higher levels than other cell types. Using isoform sequencing, we discovered novel transcripts that have a retained intron and/or previously unannotated exons. To determine function upon p53 activation, we performed transcriptome sequencing (RNA-Seq) after depleting using CRISPR interference (CRISPRi), followed by Nutlin treatment to induce p53. Strikingly, although loss of in untreated cells altered the expression of only 7 genes, loss of resulted in altered expression of ~800 genes upon p53 activation, revealing a context-dependent function of . Pathway analysis suggested that is important for fine-tuning the expression of specific genes required for mitosis. Consistent with these results, we observed a significant decrease in the percentage of mitotic cells upon depletion. Collectively, these data identify novel transcripts from the locus and suggest that delicately moderates the expression of mitotic genes in the context of p53 activation to control cell cycle arrest.
Kaida D, Satoh T, Ishida K
… +2 more, Yoshimoto R, Komori K
Mol Cell Biol
· 2022 Nov · PMID 36317925
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Pre-mRNA splicing is an indispensable mechanism for eukaryotic gene expression. Splicing inhibition causes cell cycle arrest at the G and G/M phases, and this is thought to be one of the reasons for the potent antitumor...Pre-mRNA splicing is an indispensable mechanism for eukaryotic gene expression. Splicing inhibition causes cell cycle arrest at the G and G/M phases, and this is thought to be one of the reasons for the potent antitumor activity of splicing inhibitors. However, the molecular mechanisms underlying the cell cycle arrest have many unknown aspects. In particular, the mechanism of G/M-phase arrest caused by splicing inhibition is completely unknown. Here, we found that lower and higher concentrations of pladienolide B caused M-phase and G-phase arrest, respectively. We analyzed protein levels of cell cycle regulators and found that a truncated form of the p27 cyclin-dependent kinase inhibitor, named p27*, accumulated in G-arrested cells. Overexpression of p27* caused partial G-phase arrest. Conversely, knockdown of p27* accelerated exit from G/M phase after washout of splicing inhibitor. These results suggest that p27* contributes to G/M-phase arrest caused by splicing inhibition. We also found that p27* bound to and inhibited M-phase cyclins, although it is well known that p27 regulates the G/S transition. Intriguingly, p27*, but not full-length p27, was resistant to proteasomal degradation and remained in G/M phase. These results suggest that p27*, which is a very stable truncated protein in G/M phase, contributes to G-phase arrest caused by splicing inhibition.
Lin YP, Scappini E, Landaverde C
… +4 more, Parekh-Glitsch F, Tucker CJ, Mirams GR, Parekh AB
Mol Cell Biol
· 2022 Nov · PMID 36317924
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A-kinase anchoring protein 79 (AKAP79) is a human scaffolding protein that organizes Ca/calmodulin-dependent protein phosphatase calcineurin, calmodulin, cAMP-dependent protein kinase, protein kinase C, and the transcrip...A-kinase anchoring protein 79 (AKAP79) is a human scaffolding protein that organizes Ca/calmodulin-dependent protein phosphatase calcineurin, calmodulin, cAMP-dependent protein kinase, protein kinase C, and the transcription factor nuclear factor of activated T cells (NFAT1) into a signalosome at the plasma membrane. Upon Ca store depletion, AKAP79 interacts with the N-terminus of STIM1-gated Orai1 Ca channels, enabling Ca nanodomains to stimulate calcineurin. Calcineurin then dephosphorylates and activates NFAT1, which then translocates to the nucleus. A fundamental question is how signalosomes maintain long-term signaling when key effectors are released and therefore removed beyond the reach of the activating signal. Here, we show that the AKAP79-Orai1 interaction is considerably more transient than that of STIM1-Orai1. Free AKAP79, with calcineurin and NFAT1 in tow, is able to replace rapidly AKAP79 devoid of NFAT1 on Orai1, in the presence of continuous Ca entry. We also show that Ca nanodomains near Orai1 channels activate almost the entire cytosolic pool of NFAT1. Recycling of inactive NFAT1 from the cytoplasm to AKAP79 in the plasma membrane, coupled with the relatively weak interaction between AKAP79 and Orai1, maintain excitation-transcription coupling. By measuring rates for AKAP79-NFAT interaction, we formulate a mathematical model that simulates NFAT dynamics at the plasma membrane.
Pal D, Dutta S, Iyer DP
… +3 more, Shriram D, Bhaduri U, Rao MRS
Mol Cell Biol
· 2022 Nov · PMID 36317923
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The long noncoding RNA (lncRNA) Mrhl has been shown to be involved in coordinating meiotic commitment of mouse spermatogonial progenitors and differentiation events in mouse embryonic stem cells. Here, we characterized t...The long noncoding RNA (lncRNA) Mrhl has been shown to be involved in coordinating meiotic commitment of mouse spermatogonial progenitors and differentiation events in mouse embryonic stem cells. Here, we characterized the interplay of Mrhl with lineage-specific transcription factors during mouse neuronal lineage development. Our results demonstrate that Mrhl is expressed in the neuronal progenitor populations in mouse embryonic brains and in retinoic acid-derived radial-glia-like neuronal progenitor cells. Depletion of Mrhl leads to early differentiation of neuronal progenitors to a more committed state. A master transcription factor, PAX6, directly binds to the Mrhl promoter at a major site in the distal promoter, located at 2.9 kb upstream of the transcription start site (TSS) of Mrhl. Furthermore, NFAT4 occupies the Mrhl-proximal promoter at two sites, at 437 base pairs (bp) and 143 bp upstream of the TSS. Independent knockdown studies for PAX6 and NFAT4 confirm that they regulate Mrhl expression in neuronal progenitors. We also show that PAX6 and NFAT4 associate with each other in the same chromatin complex. NFAT4 occupies the Mrhl promoter in PAX6-bound chromatin, implying possible coregulation of Mrhl. Our studies are crucial for understanding how lncRNAs are regulated by major lineage-specific transcription factors, in order to define specific development and differentiation events.
Mol Cell Biol
· 2022 Nov · PMID 36278823
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NAD synthesis is a fundamental process in living cells. The effects of local metabolite production on chromatin influence the epigenetic status of chromatin in DNA metabolism. We have previously shown that K5 acetylation...NAD synthesis is a fundamental process in living cells. The effects of local metabolite production on chromatin influence the epigenetic status of chromatin in DNA metabolism. We have previously shown that K5 acetylation of H2AX by TIP60 is required for the ADP ribosylation activity of PARP-1, for histone H2AX exchange at DNA damage sites. However, the detailed molecular mechanism has remained unclear. Here, we identified NAD synthetase 1 (NAD syn1) as a novel binding partner to H2AX. The enzymatic activity of NAD syn1 is crucial for the ADP ribosylation activity of PARP-1 for the H2AX dynamics at sites of DNA damage. Inhibition of the NAD synthetase activity in the cell nucleus decreased the overall cellular NAD concentration, leading to cellular senescence. Accordingly, the acetylation-dependent H2AX dynamics and homologous recombination repair were suppressed, leading to increased tumorigenesis. Our findings have revealed the importance of NAD production in the cell nucleus for protection against the decreased DNA repair capacity caused by cellular senescence and thus against tumorigenesis.
Mol Cell Biol
· 2022 Nov · PMID 36226970
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Protein arginylation mediated by arginyltransferase Ate1 is a posttranslational modification of emerging importance implicated in the regulation of mammalian embryogenesis, the cardiovascular system, tissue morphogenesis...Protein arginylation mediated by arginyltransferase Ate1 is a posttranslational modification of emerging importance implicated in the regulation of mammalian embryogenesis, the cardiovascular system, tissue morphogenesis, cell migration, neurodegeneration, cancer, and aging. deletion results in embryonic lethality in mice but does not affect yeast viability, making yeast an ideal system to study the molecular pathways regulated by arginylation. Here, we conducted a global analysis of cytoskeleton-related arginylation-dependent phenotypes in Schizosaccharomyces pombe, a fission yeast species that shares many fundamental features of higher eukaryotic cells. Our studies revealed roles of Ate1 in cell division, cell polarization, organelle transport, and interphase cytoskeleton organization and dynamics. We also found a role of Ate1 in mitochondria morphology and maintenance. Furthermore, targeted mass spectrometry analysis of the total Sc. pombe arginylome identified a number of arginylated proteins, including those that play direct roles in these processes; lack of their arginylation may be responsible for -knockout phenotypes. Our work outlines global biological processes potentially regulated by arginylation and paves the way to unraveling the functions of protein arginylation that are conserved at multiple levels of evolution and potentially constitute the primary role of this modification .
Dalwadi U, Corrado E, Fleming KD
… +4 more, Moeller BE, Nam SE, Burke JE, Yip CK
Mol Cell Biol
· 2022 Nov · PMID 36190236
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rimer ndependent of uA4 involved in ranscription nteractions with ucleosomes (TINTIN) is an integral module of the essential yeast lysine acetyltransferase complex NuA4 that plays key roles in transcription regulation an...rimer ndependent of uA4 involved in ranscription nteractions with ucleosomes (TINTIN) is an integral module of the essential yeast lysine acetyltransferase complex NuA4 that plays key roles in transcription regulation and DNA repair. Composed of Eaf3, Eaf5, and Eaf7, TINTIN mediates targeting of NuA4 to chromatin through the chromodomain-containing subunit Eaf3 that is shared with the Rpd3S histone deacetylase complex. How Eaf3 mediates chromatin interaction in the context of TINTIN and how is it different from what has been observed in Rpd3S is unclear. Here, we reconstituted recombinant TINTIN and its subassemblies and characterized their biochemical and structural properties. Our coimmunoprecipitation, AlphaFold2 modeling, and hydrogen deuterium exchange mass spectrometry (HDX-MS) analyses revealed that the Eaf3 MRG domain contacts Eaf7 and this binding induces conformational changes throughout Eaf3. Nucleosome-binding assays showed that Eaf3 and TINTIN interact non-specifically with the DNA on nucleosomes. Furthermore, integration into TINTIN enhances the affinity of Eaf3 toward nucleosomes and this improvement is a result of allosteric activation of the Eaf3 chromodomain. Negative stain electron microscopy (EM) analysis revealed that TINTIN binds to the edge of nucleosomes with increased specificity in the presence of H3K36me3. Collectively, our work provides insights into the dynamics of TINTIN and the mechanism by which its interactions with chromatin are regulated.
Onorati A, Havas AP, Lin B
… +4 more, Rajagopal J, Sen P, Adams PD, Dou Z
Mol Cell Biol
· 2022 Oct · PMID 36154662
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Cellular senescence is a stable form of cell cycle arrest associated with proinflammatory responses. Senescent cells can be cleared by the immune system as a part of normal tissue homeostasis. However, senescent cells ca...Cellular senescence is a stable form of cell cycle arrest associated with proinflammatory responses. Senescent cells can be cleared by the immune system as a part of normal tissue homeostasis. However, senescent cells can also accumulate in aged and diseased tissues, contributing to inflammation and disease progression. The mechanisms mediating the impaired immune-mediated clearance of senescent cells are poorly understood. Here, we report that senescent cells upregulate the immune checkpoint molecule PD-L1, the ligand for PD-1 on immune cells, which drives immune cell inactivation. The induction of PD-L1 in senescence is dependent on the proinflammatory program. Furthermore, the secreted factors released by senescent cells are sufficient to upregulate PD-L1 in nonsenescent control cells, mediated by the JAK-STAT pathway. In addition, we show that prolongevity intervention rapamycin downregulates PD-L1 in senescent cells. Last, we found that PD-L1 is upregulated in several tissues in naturally aged mice and in the lungs of idiopathic pulmonary fibrosis patients. Together, our results report that senescence and aging are associated with upregulation of a major immune checkpoint molecule, PD-L1. Targeting PD-L1 may offer new therapeutic opportunities in treating senescence and age-associated diseases.
Riehle C, Weatherford ET, McCarty NS
… +10 more, Seei A, Jaishy BP, Manivel R, Galuppo P, Allamargot C, Hameed T, Boudreau RL, Bauersachs J, Weiss RM, Abel ED
Mol Cell Biol
· 2022 Oct · PMID 36125265
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Insulin and insulin-like growth factor 1 (IGF1) signaling is transduced by insulin receptor substrate 1 (IRS1) and IRS2. To elucidate physiological and redundant roles of insulin and IGF1 signaling in adult hearts, we ge...Insulin and insulin-like growth factor 1 (IGF1) signaling is transduced by insulin receptor substrate 1 (IRS1) and IRS2. To elucidate physiological and redundant roles of insulin and IGF1 signaling in adult hearts, we generated mice with inducible cardiomyocyte-specific deletion of insulin and IGF1 receptors or IRS1 and IRS2. Both models developed dilated cardiomyopathy, and most mice died by 8 weeks post-gene deletion. Heart failure was characterized by cardiomyocyte loss and disarray, increased proapoptotic signaling, and increased autophagy. Suppression of autophagy by activating mTOR signaling did not prevent heart failure. Transcriptional profiling revealed reduced serum response factor (SRF) transcriptional activity and decreased mRNA levels of genes encoding sarcomere and gap junction proteins as early as 3 days post-gene deletion, in concert with ultrastructural evidence of sarcomere disruption and intercalated discs within 1 week after gene deletion. These data confirm conserved roles for constitutive insulin and IGF1 signaling in suppressing autophagic and apoptotic signaling in the adult heart. The present study also identifies an unexpected role for insulin and IGF1 signaling in regulating an SRF-mediated transcriptional program, which maintains expression of genes encoding proteins that support sarcomere integrity in the adult heart, reduction of which results in rapid development of heart failure.
Cugusi S, Bajpe PK, Mitter R
… +3 more, Patel H, Stewart A, Svejstrup JQ
Mol Cell Biol
· 2022 Oct · PMID 36121223
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During the heat shock response (HSR), heat shock factor (HSF1 in mammals) binds to target gene promoters, resulting in increased expression of heat shock proteins that help maintain protein homeostasis and ensure cell su...During the heat shock response (HSR), heat shock factor (HSF1 in mammals) binds to target gene promoters, resulting in increased expression of heat shock proteins that help maintain protein homeostasis and ensure cell survival. Besides HSF1, only a relatively few transcription factors with a specific role in ensuring correctly regulated gene expression during the HSR have been described. Here, we use proteomic and genomic (CRISPR) screening to identify a role for RPRD1B in the response to heat shock. Indeed, cells depleted for RPRD1B are heat shock sensitive and show decreased expression of key heat shock proteins (HSPs). These results add to our understanding of the connection between basic gene expression mechanisms and the HSR.
Mol Cell Biol
· 2022 Oct · PMID 36073913
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The cytosolic tryparedoxin peroxidase (cTXNPx) of Leishmania donovani is a defensive enzyme. Apart from the nonsecretory form, the cTXNPx is released in the spent media of cultures and also in the host cell cytosol. The...The cytosolic tryparedoxin peroxidase (cTXNPx) of Leishmania donovani is a defensive enzyme. Apart from the nonsecretory form, the cTXNPx is released in the spent media of cultures and also in the host cell cytosol. The secretory form of the enzyme from the parasite interacts with multiple proteins in the host cell cytosol, the apoptosis-inducing factor (AIF) being one of them. Immunoprecipitation with anti-cTXNPx and anti-AIF antibodies suggests a strong interaction between AIF and cTXNPx. Consequent to parasite invasion, the migration of AIF to the nucleus to precipitate apoptosis is inhibited in the presence of recombinant cTXNPx expressed in the host cell. This inhibition of AIF movement results in lesser host cell death, giving an advantage to the parasite for continued survival. Staurosporine-induced AIF migration to the nucleus was also inhibited in the presence of recombinant cTXNPx in the host cell. Therefore, this study demonstrates the ability of a parasite enzyme, cTXNPx, to interfere with the migration of the host AIF protein, providing a survival advantage to the parasite.
Reese RM, Helzer KT, Allen KO
… +3 more, Zheng C, Solodin N, Alarid ET
Mol Cell Biol
· 2022 Oct · PMID 36036613
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Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) is induced by estrogen and is the most abundant posttranslational mark associated with a transcriptionally active receptor. Cistromic analysis of pS118...Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) is induced by estrogen and is the most abundant posttranslational mark associated with a transcriptionally active receptor. Cistromic analysis of pS118-ER from our group revealed enrichment of the GRHL2 motif near pS118-ER binding sites. In this study, we used cistromic and transcriptomic analyses to interrogate the relationship between GRHL2 and pS118-ER. We found that GRHL2 is bound to chromatin at pS118-ER/GRHL2 co-occupancy sites prior to ligand treatment, and GRHL2 binding is required for maximal pS118-ER recruitment. pS118-ER/GRHL2 co-occupancy sites were enriched at active enhancers marked by H3K27ac and H3K4me1, along with FOXA1 and p300, compared to sites where each factor binds independently. Transcriptomic analysis yielded four subsets of ER/GRHL2-coregulated genes revealing that GRHL2 can both enhance and antagonize E2-mediated ER transcriptional activity. Gene ontology analysis indicated that coregulated genes are involved in cell migration. Accordingly, knockdown of GRHL2, combined with estrogen treatment, resulted in increased cell migration but no change in proliferation. These results support a model in which GRHL2 binds to selected enhancers and facilitates pS118-ER recruitment to chromatin, which then results in differential activation and repression of genes that control estrogen-regulated ER-positive breast cancer cell migration.
Basu S, Nandy A, Barad MK
… +2 more, Pal S, Biswas D
Mol Cell Biol
· 2022 Oct · PMID 36036574
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Although ELL-associated factors 1 and 2 (EAF1/2) have been shown to enhance RNA polymerase II-mediated transcription , their functional roles are poorly known. In this report, we show functions of these proteins in regu...Although ELL-associated factors 1 and 2 (EAF1/2) have been shown to enhance RNA polymerase II-mediated transcription , their functional roles are poorly known. In this report, we show functions of these proteins in regulating ELL stability through their competitive binding with HDAC3 at the N terminus of ELL. Reduced HDAC3 binding to ELL causes increased acetylation leading to reduced ubiquitylation-mediated degradation. Similar functional roles played by DBC1 in regulating ELL stability further prompted in-depth analyses that demonstrated presence of negative feedback loop mechanisms between DBC1 and EAF1/2 in maintaining overall ELL level. Mechanistically, increased DBC1 reduces EAF1/2 level through increased ubiquitylation involving E3 ubiquitin ligase TRIM28, whereas increased EAF1/2 reduces DBC1 level through reduced transcription. Physiologically, after a few passages, ELL levels in either DBC1 or EAF1 knockdown cells are restored through enhanced expression of EAF1 and DBC1, respectively. Interestingly, for maintenance of ELL level, mammalian cells prefer the EAF1-dependent pathway during exposure to genotoxic stress, and the DBC1-dependent pathway during exposure to growth factors. Thus, we describe coordinated functions of multiple factors, including EAF1/2, HDAC3, DBC1, and TRIM28 in regulating ELL protein level for optimal target gene expression in a context-dependent manner within mammalian cells.