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Resistance to DNA-damaging agents is a major unsolved challenge for breast cancer patients undergoing chemotherapy. Here, we show that elevated expression of transcriptional repressor GATA binding 1 (TRPS1) is associated with lower drug sensitivity, reduced response rate, and poor prognosis in chemotherapy-treated breast cancer patients. Mechanistically, elevated TRPS1 expression promotes hyperactivity of DNA damage repair (DDR) in breast cancer cells. We provide evidence that TRPS1 dynamically localizes to DNA breaks in a Ku70-and Ku80-dependent manner and that TRPS1 is a new member of the DDR protein family. We also discover that the dynamics of TRPS1 assembly at DNA breaks is regulated by its reversible PARylation in the DDR, and that mutations of the PARylation sites on TRPS1 lead to increased sensitivity to chemotherapeutic drugs. Taken together, our findings provide new mechanistic insights into the DDR and chemoresistance in breast cancer patients and identify TRPS1 as a critical DDR protein. TRPS1 may also be considered as a target to improve chemo-sensitization strategies and, consequently, clinical outcomes for breast cancer patients., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Triple-negative breast cancer (TNBC) is a prevalent malignancy in women, casting a formidable shadow on their well-being. Positioned within the nucleolus, SUMO-specific protease 3 (SENP3) assumes a pivotal role in the realms of development and tumorigenesis. However, the participation of SENP3 in TNBC remains a mystery. Here, we elucidate that SENP3 exerts inhibitory effects on migration and invasion capacities, as well as on the stem cell-like phenotype, within TNBC cells. Further experiments showed that YAP1 is the downstream target of SENP3, and SENP3 regulates tumorigenesis in a YAP1-dependent manner. YAP1 is found to be SUMOylated and SENP3 deconjugates SUMOylated YAP1 and promotes degradation mediated by the ubiquitin-proteasome system. More importantly, YAP1 with a mutation at the SUMOylation site impedes the capacity of WT YAP1 in TNBC tumorigenesis. Taken together, our findings firmly establish the pivotal role of SENP3 in the modulation of YAP1 deSUMOylation, unveiling novel mechanistic insight into the important role of SENP3 in the regulation of TNBC tumorigenesis in a YAP1-dependent manner., Competing Interests: Conflict of interest The authors declare that they have no conflict of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The tricarboxylic acid (TCA) cycle plays a crucial role in mitochondrial ATP production in the healthy heart. However, in heart failure, the TCA cycle becomes dysregulated. Understanding the mechanism by which TCA cycle genes are transcribed in the healthy heart is an important prerequisite to understanding how these genes become dysregulated in the failing heart. PPARγ coactivator 1α (PGC-1α) is a transcriptional coactivator that broadly induces genes involved in mitochondrial ATP production. PGC-1α potentiates its effects through the coactivation of coupled transcription factors, such as estrogen-related receptor (ERR), nuclear respiratory factor 1 (Nrf1), GA-binding protein-a (Gabpa), and Yin Yang 1 (YY1). We hypothesized that PGC-1α plays an essential role in the transcription of TCA cycle genes. Thus, utilizing localization peaks of PGC-1α to TCA cycle gene promoters would allow the identification of coupled transcription factors. PGC-1α potentiated the transcription of 13 out of 14 TCA cycle genes, partly through ERR, Nrf1, Gabpa, and YY1. ChIP-sequencing showed PGC-1α localization peaks in TCA cycle gene promoters. Transcription factors with binding elements that were found proximal to PGC-1α peak localization were generally essential for the transcription of the gene. These transcription factor binding elements were well conserved between mice and humans. Among the four transcription factors, ERR and Gabpa played a major role in potentiating transcription when compared to Nrf1 and YY1. These transcription factor-dependent PGC-1α recruitment was verified with Idh3a, Idh3g, and Sdha promoters with DNA binding assay. Taken together, this study clarifies the mechanism by which TCA cycle genes are transcribed, which could be useful in understanding how those genes are dysregulated in pathological conditions., Competing Interests: Conflict of interest The authors declare that they have no competing interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Our previous studies determined that elevating SOX2 in a wide range of tumor cells leads to a reversible state of tumor growth arrest. Efforts to understand how tumor cell growth is inhibited led to the discovery of a SOX2:MYC axis that is responsible for downregulating c-MYC (MYC) when SOX2 is elevated. Although we had determined that elevating SOX2 downregulates MYC transcription, the mechanism responsible was not determined. Given the challenges of targeting MYC clinically, we set out to identify how elevating SOX2 downregulates MYC transcription. In this study, we focused on the MYC promoter region and an upstream region of the MYC locus that contains a MYC super-enhancer encompassing five MYC enhancers and which is associated with several cancers. Here we report that BRD4 and p300 associate with each of the MYC enhancers in the upstream MYC super-enhancer as well as the MYC promoter region and that elevating SOX2 decreases the recruitment of BRD4 and p300 to these sites. Additionally, we determined that elevating SOX2 leads to increases in the association of SOX2 and H3K27me3 within the MYC super-enhancer and the promoter region of MYC. Importantly, we conclude that the increases in SOX2 within the MYC super-enhancer precipitate a cascade of events that culminates in the repression of MYC transcription. Together, our studies identify a novel molecular mechanism able to regulate MYC transcription in two distinctly different tumor types and provide new mechanistic insights into the molecular interrelationships between two master regulators, SOX2 and MYC, widely involved in multiple cancers., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The HIRA histone chaperone complex is comprised of four protein subunits: HIRA, UBN1, CABIN1, and transiently associated ASF1a. All four subunits have been demonstrated to play a role in the deposition of the histone variant H3.3 onto areas of actively transcribed euchromatin in cells. The mechanism by which these subunits function together to drive histone deposition has remained poorly understood. Here we present biochemical and biophysical data supporting a model whereby ASF1a delivers histone H3.3/H4 dimers to the HIRA complex, H3.3/H4 tetramerization drives the association of two HIRA/UBN1 complexes, and the affinity of the histones for DNA drives release of ASF1a and subsequent histone deposition. These findings have implications for understanding how other histone chaperone complexes may mediate histone deposition., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Diamond Blackfan Anemia (DBA) is a rare macrocytic red blood cell aplasia that usually presents within the first year of life. The vast majority of patients carry a mutation in one of approximately 20 genes that results in ribosomal insufficiency with the most significant clinical manifestations being anemia and a predisposition to cancers. Nemo-like Kinase (NLK) is hyperactivated in the erythroid progenitors of DBA patients and inhibition of this kinase improves erythropoiesis, but how NLK contributes to the pathogenesis of the disease is unknown. Here we report that activated NLK suppresses the critical upregulation of mitochondrial biogenesis required in early erythropoiesis. During normal erythropoiesis, mTORC1 facilitates the translational upregulation of Transcription factor A, mitochondrial (TFAM), and Prohibin 2 (PHB2) to increase mitochondrial biogenesis. In our models of DBA, active NLK phosphorylates the regulatory component of mTORC1, thereby suppressing mTORC1 activity and preventing mTORC1-mediated TFAM and PHB2 upregulation and subsequent mitochondrial biogenesis. Improvement of erythropoiesis that accompanies NLK inhibition is negated when TFAM and PHB2 upregulation is prevented. These data demonstrate that a significant contribution of NLK on the pathogenesis of DBA is through loss of mitochondrial biogenesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In the Neurospora circadian system, the White Collar Complex (WCC) formed by WC-1 and WC-2 drives expression of the frequency (frq) gene whose product FRQ feedbacks to inhibit transcriptional activity of WCC. Phosphorylation of WCC has been extensively studied, but the extent and significance of other post-translational modifications (PTM) have been poorly studied. To this end, we used mass-spectrometry to study alkylation sites on WCC, resulting in discovery of nine acetylation sites. Mutagenesis analysis showed most of the acetylation events individually do not play important roles in period determination. Moreover, mutating all the lysines falling in either half of WC-1 or all the lysine residues in WC-2 to arginines did not abolish circadian rhythms. In addition, we also found nine mono-methylation sites on WC-1, but like acetylation, individual ablation of most of the mono-methylation events did not result in a significant period change. Taken together, the data here suggest that acetylation or mono-methylation on WCC is not a determinant of the pace of the circadian feedback loop. The finding is consistent with a model in which repression of WCC's circadian activity is mainly controlled by phosphorylation. Interestingly, light-induced expression of some light-responsive genes has been modulated in certain wc-1 acetylation mutants, suggesting that WC-1 acetylation events differentially regulate light responses., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite the ever-growing research interest in polyhydroxyalkanoates (PHAs) as green plastic alternatives, our understanding of the regulatory mechanisms governing PHA synthesis, storage, and degradation in the model organism Ralstonia eutropha remains limited. Given its importance for central carbon metabolism, PHA homeostasis is probably controlled by a complex network of transcriptional regulators. Understanding this fine-tuning is the key for developing improved PHA production strains thereby boosting the application of PHAs. We conducted promoter pull-down assays with crude protein extracts from R. eutropha Re2058/pCB113, followed by liquid chromatography with tandem mass spectrometry, to identify putative transcriptional regulators involved in the expression control of PHA metabolism, specifically targeting phasin phaP1 and depolymerase phaZ3 and phaZ5 genes. The impact on promoter activity was studied in vivo using β-galactosidase assays and the most promising candidates were heterologously produced in Escherichia coli, and their interaction with the promoters investigated in vitro by electrophoretic mobility shift assays. We could show that R. eutropha DNA-binding xenobiotic response element-family-like protein H16_B1672, specifically binds the phaP1 promoter in vitro with a K D of 175 nM and represses gene expression from this promoter in vivo. Protein H16_B1672 also showed interaction with both depolymerase promoters in vivo and in vitro suggesting a broader role in the regulation of PHA metabolism. Furthermore, in vivo assays revealed that the H-NS-like DNA-binding protein H16_B0227 and the peptidyl-prolyl cis-trans isomerase PpiB, strongly repress gene expression from PphaP1 and PphaZ3, respectively. In summary, this study provides new insights into the regulation of PHA metabolism in R. eutropha, uncovering specific interactions of novel transcriptional regulators., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The Hippo-YAP signaling pathway plays a central role in many biological processes such as regulating cell fate, organ size, and tissue growth, and its key components are spatiotemporally expressed and posttranslationally modified during these processes. Neddylation is a posttranslational modification that involves the covalent attachment of NEDD8 to target proteins by NEDD8-specific E1-E2-E3 enzymes. Whether neddylation is involved in Hippo-YAP signaling remains poorly understood. Here, we provide evidence supporting the critical role of NEDD8 in facilitating the Hippo-YAP signaling pathway by mediating neddylation of the transcriptional coactivator yes-associated protein 1 (YAP1). Overexpression of NEDD8 induces YAP1 neddylation and enhances YAP1 transactivity, but inhibition of neddylation suppresses YAP1 transactivity and attenuates YAP1 nuclear accumulation. Furthermore, inhibition of YAP1 signaling promotes MLN4924-induced ovarian granulosa cells apoptosis and disruption of nedd8 in zebrafish results in downregulation of yap1-activated genes and upregulation of yap1-repressed genes. Further assays show that the xiap ligase promotes nedd8 conjugates to yap1 and that yap1 neddylation. In addition, we identify lysine 159 as a major neddylation site on YAP1. These findings reveal a novel mechanism for neddylation in the regulation of Hippo-YAP signaling., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Menstrual toxic shock syndrome (mTSS) is a rare but severe disorder associated with the use of menstrual products such as high-absorbency tampons and is caused by Staphylococcus aureus strains that produce the toxic shock syndrome toxin-1 (TSST-1) superantigen. Herein, we screened a library of 3920 small bioactive molecules for the ability to inhibit transcription of the TSST-1 gene without inhibiting the growth of S. aureus. The dominant positive regulator of TSST-1 is the SaeRS two-component system (TCS), and we identified phenazopyridine hydrochloride (PP-HCl) that repressed the production of TSST-1 by inhibiting the kinase function of SaeS. PP-HCl competed with ATP for binding of the kinase SaeS leading to decreased phosphorylation of SaeR and reduced expression of TSST-1 as well as several other secreted virulence factors known to be regulated by SaeRS. PP-HCl targets the virulence of S. aureus, and it also decreases the impact of TSST-1 on human lymphocytes without affecting the healthy vaginal microbiota. Our findings demonstrate the promising potential of PP-HCl as a therapeutic strategy against mTSS., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Progesterone (P 4 ), acting via its nuclear receptor (PR), is critical for pregnancy maintenance by suppressing proinflammatory and contraction-associated protein (CAP)/contractile genes in the myometrium. P 4 /PR partially exerts these effects by tethering to NF-κB bound to their promot-ers, thereby decreasing NF-κB transcriptional activity. However, the underlying mechanisms whereby P 4 /PR interaction blocks proinflammatory and CAP gene expression are not fully understood. Herein, we characterized CCR-NOT transcription complex subunit 1 (CNOT1) as a corepressor that also interacts within the same chromatin complex as PR-B. In mouse myome-trium increased expression of CAP genes Oxtr and Cx43 at term coincided with a marked decline in expression and binding of CNOT1 to NF-κB-response elements within the Oxtr and Cx43 promoters. Increased CAP gene expression was accompanied by a pronounced decrease in enrichment of repressive histone marks and increase in enrichment of active histone marks to this genomic region. These changes in histone modification were associated with changes in expression of corresponding histone modifying enzymes. Myometrial tissues from P 4 -treated 18.5 dpc pregnant mice manifested increased Cnot1 expression at 18.5 dpc, compared to vehicle-treated controls. P 4 treatment of PR-expressing hTERT-HM cells enhanced CNOT1 expression and its recruitment to PR bound NF-κB-response elements within the CX43 and OXTR promoters. Furthermore, knockdown of CNOT1 significantly increased expression of contractile genes. These novel findings suggest that decreased expression and DNA-binding of the P 4 /PR-regulated transcriptional corepressor CNOT1 near term and associated changes in histone modifications at the OXTR and CX43 promoters contribute to the induction of myometrial contractility leading to parturition., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

AT-rich interacting domain (ARID)-containing proteins, Arids, are a heterogeneous DNA-binding protein family involved in transcription regulation and chromatin processing. For the member Arid5a, no exact DNA-binding preference has been experimentally defined so far. Additionally, the protein binds to mRNA motifs for transcript stabilization, supposedly through the DNA-binding ARID domain. To date, however, no unbiased RNA motif definition and clear dissection of nucleic acid-binding through the ARID domain have been undertaken. Using NMR-centered biochemistry, we here define the Arid5a DNA preference. Further, high-throughput in vitro binding reveals a consensus RNA-binding motif engaged by the core ARID domain. Finally, transcriptome-wide binding (iCLIP2) reveals that Arid5a has a weak preference for (A)U-rich regions in pre-mRNA transcripts of factors related to RNA processing. We find that the intrinsically disordered regions flanking the ARID domain modulate the specificity and affinity of DNA binding, while they appear crucial for RNA interactions. Ultimately, our data suggest that Arid5a uses its extended ARID domain for bifunctional gene regulation and that the involvement of IDR extensions is a more general feature of Arids in interacting with different nucleic acids at the chromatin-mRNA interface., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Novel components in the noncanonical Hippo pathway that mediate the growth, metastasis, and drug resistance of breast cancer (BC) cells need to be identified. Here, we showed that expression of SAM and SH3 domain-containing protein 1 (SASH1) is negatively correlated with expression of mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) in a subpopulation of patients with luminal-subtype BC. Downregulated SASH1 and upregulated MAP4K4 synergistically regulated the proliferation, migration, and invasion of luminal-subtype BC cells. The expression of LATS2, SASH1, and YAP1 and the phosphorylation of YAP1 were negatively regulated by MAP4K4, and LATS2 then phosphorylated SASH1 to form a novel MAP4K4-LATS2-SASH1-YAP1 cascade. Dephosphorylation of Yes1 associated transcriptional regulator (YAP1), YAP1/TAZ nuclear translocation, and downstream transcriptional regulation of YAP1 were promoted by the combined effects of ectopic MAP4K4 expression and SASH1 silencing. Targeted inhibition of MAP4K4 blocked proliferation, cell migration, and ER signaling both in vitro and in vivo. Our findings reveal a novel MAP4K4-LATS2-SASH1-YAP1 phosphorylation cascade, a noncanonical Hippo pathway that mediates ER signaling, tumorigenesis, and metastasis in breast cancer. Targeted intervention with this noncanonical Hippo pathway may constitute a novel alternative therapeutic approach for endocrine-resistant BC., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest related to the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The stability of ribosomal DNA (rDNA) is maintained through transcriptional silencing by the NAD + -dependent histone deacetylase Sir2 in Saccharomyces cerevisiae. Alongside proteostasis, rDNA stability is a crucial factor regulating the replicative lifespan of S. cerevisiae. The unfolded protein response (UPR) is induced by misfolding of proteins or an imbalance of membrane lipid composition and is responsible for degrading misfolded proteins and restoring endoplasmic reticulum (ER) membrane homeostasis. Recent investigations have suggested that the UPR can extend the replicative lifespan of yeast by enhancing protein quality control mechanisms, but the relationship between the UPR and rDNA stability remains unknown. In this study, we found that the deletion of ARV1, which encodes an ER protein of unknown molecular function, activates the UPR by inducing lipid bilayer stress. In arv1Δ cells, the UPR and the cell wall integrity pathway are activated independently of each other, and the high osmolarity glycerol (HOG) pathway is activated in a manner dependent on Ire1, which mediates the UPR. Activated Hog1 translocates the stress response transcription factor Msn2 to the nucleus, where it promotes the expression of nicotinamidase Pnc1, a well-known Sir2 activator. Following Sir2 activation, rDNA silencing and rDNA stability are promoted. Furthermore, the loss of other ER proteins, such as Pmt1 or Bst1, and ER stress induced by tunicamycin or inositol depletion also enhance rDNA stability in a Hog1-dependent manner. Collectively, these findings suggest that the induction of the UPR enhances rDNA stability in S. cerevisiae by promoting the Msn2-Pnc1-Sir2 pathway in a Hog1-dependent manner., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Thoracic aortic dissection (TAD) is a highly dangerous cardiovascular disorder caused by weakening of the aortic wall, resulting in a sudden tear of the internal face. Progressive loss of the contractile apparatus in vascular smooth muscle cells (VSMCs) is a major event in TAD. Exploring the endogenous regulators essential for the contractile phenotype of VSMCs may aid the development of strategies to prevent TAD. Krüppel-like factor 15 (KLF15) overexpression was reported to inhibit TAD formation; however, the mechanisms by which KLF15 prevents TAD formation and whether KLF15 regulates the contractile phenotype of VSMCs in TAD are not well understood. Therefore, we investigated these unknown aspects of KLF15 function. We found that KLF15 expression was reduced in human TAD samples and β-aminopropionitrile monofumarate-induced TAD mouse model. Klf15KO mice are susceptible to both β-aminopropionitrile monofumarate- and angiotensin II-induced TAD. KLF15 deficiency results in reduced VSMC contractility and exacerbated vascular inflammation and extracellular matrix degradation. Mechanistically, KLF15 interacts with myocardin-related transcription factor B (MRTFB), a potent serum response factor coactivator that drives contractile gene expression. KLF15 silencing represses the MRTFB-induced activation of contractile genes in VSMCs. Thus, KLF15 cooperates with MRTFB to promote the expression of contractile genes in VSMCs, and its dysfunction may exacerbate TAD. These findings indicate that KLF15 may be a novel therapeutic target for the treatment of TAD., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The polybromo, brahma-related gene 1-associated factors (PBAF) chromatin remodeling complex subunit polybromo-1 (PBRM1) contains six bromodomains that recognize and bind acetylated lysine residues on histone tails and other nuclear proteins. PBRM1 bromodomains thus provide a link between epigenetic posttranslational modifications and PBAF modulation of chromatin accessibility and transcription. As a putative tumor suppressor in several cancers, PBRM1 protein expression is often abrogated by truncations and deletions. However, ∼33% of PBRM1 mutations in cancer are missense and cluster within its bromodomains. Such mutations may generate full-length PBRM1 variant proteins with undetermined structural and functional characteristics. Here, we employed computational, biophysical, and cellular assays to interrogate the effects of PBRM1 bromodomain missense variants on bromodomain stability and function. Since mutations in the fourth bromodomain of PBRM1 (PBRM1-BD4) comprise nearly 20% of all cancer-associated PBRM1 missense mutations, we focused our analysis on PBRM1-BD4 missense protein variants. Selecting 16 potentially deleterious PBRM1-BD4 missense protein variants for further study based on high residue mutational frequency and/or conservation, we show that cancer-associated PBRM1-BD4 missense variants exhibit varied bromodomain stability and ability to bind acetylated histones. Our results demonstrate the effectiveness of identifying the unique impacts of individual PBRM1-BD4 missense variants on protein structure and function, based on affected residue location within the bromodomain. This knowledge provides a foundation for drawing correlations between specific cancer-associated PBRM1 missense variants and distinct alterations in PBRM1 function, informing future cancer personalized medicine approaches., Competing Interests: Conflict of interest The authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Inhibition of protein kinase C (PKC) efficiently promoted the self-renewal of embryonic stem cells (ESCs). However, information about the function of PKC inhibition remains lacking. Here, RNA-sequencing showed that the addition of Go6983 significantly inhibited the expression of de novo methyltransferases (Dnmt3a and Dnmt3b) and their regulator Dnmt3l, resulting in global hypomethylation of DNA in mouse ESCs. Mechanistically, PR domain-containing 14 (Prdm14), a site-specific transcriptional activator, partially contributed to Go6983-mediated repression of Dnmt3 genes. Administration of Go6983 increased Prdm14 expression mainly through the inhibition of PKCδ. High constitutive expression of Prdm14 phenocopied the ability of Go6983 to maintain` mouse ESC stemness in the absence of self-renewal-promoting cytokines. In contrast, the knockdown of Prdm14 eliminated the response to PKC inhibition and substantially impaired the Go6983-induced resistance of mouse ESCs to differentiation. Furthermore, liquid chromatography-mass spectrometry profiling and Western blotting revealed low levels of Suv39h1 and Suv39h2 in Go6983-treated mouse ESCs. Suv39h enzymes are histone methyltransferases that recognize dimethylated and trimethylated histone H3K9 specifically and usually function as transcriptional repressors. Consistently, the inhibition of Suv39h1 by RNA interference or the addition of the selective inhibitor chaetocin increased Prdm14 expression. Moreover, chromatin immunoprecipitation assay showed that Go6983 treatment led to decreased enrichment of dimethylation and trimethylation of H3K9 at the Prdm14 promoter but increased RNA polymerase Ⅱ binding affinity. Together, our results provide novel insights into the pivotal association between PKC inhibition-mediated self-renewal and epigenetic changes, which will help us better understand the regulatory network of stem cell pluripotency., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The nucleolus, a membrane-less organelle, is responsible for ribosomal RNA transcription, ribosomal RNA processing, and ribosome assembly. Nucleolar size and number are indicative of a cell's protein synthesis rate and proliferative capacity, and abnormalities in the nucleolus have been linked to neurodegenerative diseases and cancer. In this study, we demonstrated that the nucleolar protein ZNF692 directly interacts with nucleophosmin 1 (NPM1). Knocking down ZNF692 resulted in the nucleolar redistribution of NPM1 in ring-like structures and reduced protein synthesis. Purified NPM1 forms spherical condensates in vitro but mixing it with ZNF692 produces irregular condensates more closely resembling living cell nucleoli. Our findings indicate that ZNF692, by interacting with NPM1, plays a critical role in regulating nucleolar architecture and function in living cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Starvation of Schizosaccharomyces pombe for inorganic phosphate elicits adaptive transcriptome changes in which mRNAs driving ribosome biogenesis, tRNA biogenesis, and translation are globally downregulated, while those for autophagy and phosphate mobilization are upregulated. Here, we interrogated three components of the starvation response: upregulated autophagy; the role of transcription factor Pho7 (an activator of the PHO regulon); and upregulated expression of ecl3, one of three paralogous genes (ecl1, ecl2, and ecl3) collectively implicated in cell survival during other nutrient stresses. Ablation of autophagy factor Atg1 resulted in early demise of phosphate-starved fission yeast, as did ablation of Pho7. Transcriptome profiling of phosphate-starved pho7Δ cells highlighted Pho7 as an activator of genes involved in phosphate acquisition and mobilization, not limited to the original three-gene PHO regulon, and additional starvation-induced genes (including ecl3) not connected to phosphate dynamics. Pho7-dependent gene induction during phosphate starvation tracked with the presence of Pho7 DNA-binding elements in the gene promoter regions. Fewer ribosome protein genes were downregulated in phosphate-starved pho7Δ cells versus WT, which might contribute to their shortened lifespan. An ecl3Δ mutant elicited no gene expression changes in phosphate-replete cells and had no impact on survival during phosphate starvation. By contrast, pan-ecl deletion (ecl123Δ) curtailed lifespan during chronic phosphate starvation. Phosphate-starved ecl123Δ cells experienced a more widespread downregulation of mRNAs encoding aminoacyl tRNA synthetases vis-à-vis WT or pho7Δ cells. Collectively, these results enhance our understanding of fission yeast phosphate homeostasis and survival during nutrient deprivation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

PKC is a multifunctional family of Ser-Thr kinases widely implicated in the regulation of fundamental cellular functions, including proliferation, polarity, motility, and differentiation. Notwithstanding their primary cytoplasmic localization and stringent activation by cell surface receptors, PKC isozymes impel prominent nuclear signaling ultimately impacting gene expression. While transcriptional regulation may be wielded by nuclear PKCs, it most often relies on cytoplasmic phosphorylation events that result in nuclear shuttling of PKC downstream effectors, including transcription factors. As expected from the unique coupling of PKC isozymes to signaling effector pathways, glaring disparities in gene activation/repression are observed upon targeting individual PKC family members. Notably, specific PKCs control the expression and activation of transcription factors implicated in cell cycle/mitogenesis, epithelial-to-mesenchymal transition and immune function. Additionally, PKCs isozymes tightly regulate transcription factors involved in stepwise differentiation of pluripotent stem cells toward specific epithelial, mesenchymal, and hematopoietic cell lineages. Aberrant PKC expression and/or activation in pathological conditions, such as in cancer, leads to profound alterations in gene expression, leading to an extensive rewiring of transcriptional networks associated with mitogenesis, invasiveness, stemness, and tumor microenvironment dysregulation. In this review, we outline the current understanding of PKC signaling "in" and "to" the nucleus, with significant focus on established paradigms of PKC-mediated transcriptional control. Dissecting these complexities would allow the identification of relevant molecular targets implicated in a wide spectrum of diseases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The protein kinase RNA-like endoplasmic reticulum kinase (PERK)-eukaryotic translation initiation factor 2 subunit α (eIF2α) pathway plays an essential role in endoplasmic reticulum (ER) stress. When the PERK-eIF2α pathway is activated, PERK phosphorylates eIF2α (p-eIF2α) at Ser51 and quenches global protein synthesis. In this study, we verified eIF2α as a bona fide substrate of the E3 ubiquitin ligase carboxyl terminus of the HSC70-interaction protein (CHIP) both in vitro and in cells. CHIP mediated the ubiquitination and degradation of nonphosphorylated eIF2α in a chaperone-independent manner and promoted the upregulation of the cyclic AMP-dependent transcription factor under endoplasmic reticulum stress conditions. Cyclic AMP-dependent transcription factor induced the transcriptional enhancement of the tumor suppressor genes PTEN and RBM5. Although transcription was enhanced, the PTEN protein was subsequently degraded by CHIP, but the expression of the RBM5 protein was upregulated, thereby suppressing the proliferation and migration of A549 cells. Overall, our study established a new mechanism that deepened the understanding of the PERK-eIF2α pathway through the ubiquitination and degradation of eIF2α. The crosstalk between the phosphorylation and ubiquitination of eIF2α shed light on a new perspective for tumor progression., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The CCAAT/enhancer-binding proteins (C/EBPs) constitute a family of pivotal transcription factors involved in tissue development, cellular function, proliferation, and differentiation. NFIL3, as one of them, plays an important role in regulating immune cell differentiation, circadian clock system, and neural regeneration, yet its specific DNA recognition mechanism remains enigmatic. In this study, we showed by the ITC binding experiments that NFIL3 prefers to bind to the TTACGTAA DNA motif. Our structural studies revealed that the α-helical NFIL3 bZIP domain dimerizes through its leucine zipper region, and binds to DNA via its basic region. The two basic regions of the NFIL3 bZIP dimer were pushed apart upon binding to DNA, facilitating the snug accommodation of the two basic regions within the major grooves of the DNA. Remarkably, our binding and structural data also revealed that both NFIL3 and C/EBPα/β demonstrate a shared preference for the TTACGTAA sequence. Furthermore, our study revealed that disease-associated mutations within the NFIL3 bZIP domain result in either reduction or complete disruption of its DNA binding ability. These discoveries not only provide valuable insights into the DNA binding mechanisms of NFIL3 but also elucidate the causal role of NFIL3 mutations in disease pathogenesis., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Liver cancer is notoriously refractory to conventional therapeutics. Tumor progression is governed by the interplay between tumor-promoting genes and tumor-suppressor genes. BRD4, an acetyl lysine-binding protein, is overexpressed in many cancer types, which promotes activation of a pro-tumor gene network. But the underlying mechanism for BRD4 overexpression remains incompletely understood. In addition, understanding the regulatory mechanism of BRD4 protein level will shed insight into BRD4-targeting therapeutics. In this study, we investigated the potential relation between BRD4 protein level and P53, the most frequently dysregulated tumor suppressor. By analyzing the TCGA datasets, we first identify a strong negative correlation between protein levels of P53 and BRD4 in liver cancer. Further investigation shows that P53 promotes BRD4 protein degradation. Mechanistically, P53 indirectly represses the transcription of USP1, a deubiquitinase, through the P21-RB1 axis. USP1 itself is also overexpressed in liver cancer and we show USP1 deubiquitinates BRD4 in vivo and in vitro, which increases BRD4 stability. With cell proliferation assays and xenograft model, we show the pro-tumor role of USP1 is partially mediated by BRD4. With functional transcriptomic analysis, we find the USP1-BRD4 axis upholds expression of a group of cancer-related genes. In summary, we identify a functional P53-P21-RB1-USP1-BRD4 axis in liver cancer., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Prolidase (PEPD) is the only hydrolase that cleaves the dipeptides containing C-terminal proline or hydroxyproline-the rate-limiting step in collagen biosynthesis. However, the molecular regulation of prolidase expression remains largely unknown. In this study, we have identified overlapping binding sites for the transcription factors Krüppel-like factor 6 (KLF6) and Specificity protein 1 (Sp1) in the PEPD promoter and demonstrate that KLF6/Sp1 transcriptionally regulate prolidase expression. By cloning the PEPD promoter into a luciferase reporter and through site-directed deletion, we pinpointed the minimal sequences required for KLF6 and Sp1-mediated PEPD promoter-driven transcription. Interestingly, Sp1 inhibition abrogated KLF6-mediated PEPD promoter activity, suggesting that Sp1 is required for the basal expression of prolidase. We further studied the regulation of PEPD by KLF6 and Sp1 during transforming growth factor β 1 (TGF-β 1 ) signaling, since both KLF6 and Sp1 are key players in TGF-β1 mediated collagen biosynthesis. Mouse and human fibroblasts exposed to TGF-β1 resulted in the induction of PEPD transcription and prolidase expression. Inhibition of TGF-β1 signaling abrogated PEPD promoter-driven transcriptional activity of KLF6 and Sp1. Knock-down of KLF6 as well as Sp1 inhibition also reduced prolidase expression. Chromatin immunoprecipitation assay supported direct binding of KLF6 and Sp1 to the PEPD promoter and this binding was enriched by TGF-β1 treatment. Finally, immunofluorescence studies showed that KLF6 co-operates with Sp1 in the nucleus to activate prolidase expression and enhance collagen biosynthesis. Collectively, our results identify functional elements of the PEPD promoter for KLF6 and Sp1-mediated transcriptional activation and describe the molecular mechanism of prolidase expression., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In contrast to stage-specific transcription factors, the role of ubiquitous transcription factors in neuronal development remains a matter of scrutiny. Here, we demonstrated that a ubiquitous factor NF-Y is essential for neural progenitor maintenance during brain morphogenesis. Deletion of the NF-YA subunit in neural progenitors by using nestin-cre transgene in mice resulted in significant abnormalities in brain morphology, including a thinner cerebral cortex and loss of striatum during embryogenesis. Detailed analyses revealed a progressive decline in multiple neural progenitors in the cerebral cortex and ganglionic eminences, accompanied by induced apoptotic cell death and reduced cell proliferation. In neural progenitors, the NF-YA short isoform lacking exon 3 is dominant and co-expressed with cell cycle genes. ChIP-seq analysis from the cortex during early corticogenesis revealed preferential binding of NF-Y to the cell cycle genes, some of which were confirmed to be downregulated following NF-YA deletion. Notably, the NF-YA short isoform disappears and is replaced by its long isoform during neuronal differentiation. Forced expression of the NF-YA long isoform in neural progenitors resulted in a significant decline in neuronal count, possibly due to the suppression of cell proliferation. Collectively, we elucidated a critical role of the NF-YA short isoform in maintaining neural progenitors, possibly by regulating cell proliferation and apoptosis. Moreover, we identified an isoform switch in NF-YA within the neuronal lineage in vivo, which may explain the stage-specific role of NF-Y during neuronal development., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interests with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Upon Mg 2+ starvation, a condition often associated with virulence, enterobacteria inhibit the ClpXP-dependent proteolysis of the master transcriptional regulator, σ s , via IraM, a poorly understood antiadaptor that prevents RssB-dependent loading of σ s onto ClpXP. This inhibition results in σ s accumulation and expression of stress resistance genes. Here, we report on the structural analysis of RssB bound to IraM, which reveals that IraM induces two folding transitions within RssB, amplified via a segmented helical linker. These conformational changes result in an open, yet inhibited RssB structure in which IraM associates with both the C-terminal and N-terminal domains of RssB and prevents binding of σ s to the 4-5-5 face of the N-terminal receiver domain. This work highlights the remarkable structural plasticity of RssB and reveals how a stress-specific RssB antagonist modulates a core stress response pathway that could be leveraged to control biofilm formation, virulence, and the development of antibiotic resistance., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A renewable source of porcine macrophages derived from pluripotent stem cells (PSCs) would be a valuable alternative to primary porcine alveolar macrophages (PAMs) in the research of host-pathogen interaction mechanisms. We developed an efficient and rapid protocol, within 11 days, to derive macrophages from porcine PSCs (pPSCs). The pPSC-derived macrophages (pPSCdMs) exhibited molecular and functional characteristics of primary macrophages. The pPSCdMs showed macrophage-specific surface protein expression and macrophage-specific transcription factors, similar to PAMs. The pPSCdMs also exhibited the functional characteristics of macrophages, such as endocytosis, phagocytosis, porcine respiratory and reproductive syndrome virus infection and the response to lipopolysaccharide stimulation. Furthermore, we performed transcriptome sequencing of the whole differentiation process to track the fate transitions of porcine PSCs involved in the signaling pathway. The activation of transforming growth factor beta signaling was required for the formation of mesoderm and the inhibition of the transforming growth factor beta signaling pathway at the hematopoietic endothelium stage could enhance the fate transformation of hematopoiesis. In summary, we developed an efficient and rapid protocol to generate pPSCdMs that showed aspects of functional maturity comparable with PAMs. pPSCdMs could provide a broad prospect for the platforms of host-pathogen interaction mechanisms., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Bromodomains (BDs) regulate gene expression by recognizing protein motifs containing acetyllysine. Although originally characterized as histone-binding proteins, it has since become clear that these domains interact with other acetylated proteins, perhaps most prominently transcription factors. The likely transient nature and low stoichiometry of such modifications, however, has made it challenging to fully define the interactome of any given BD. To begin to address this knowledge gap in an unbiased manner, we carried out mRNA display screens against a BD-the N-terminal BD of BRD3-using peptide libraries that contained either one or two acetyllysine residues. We discovered peptides with very strong consensus sequences and with affinities that are significantly higher than typical BD-peptide interactions. X-ray crystal structures also revealed modes of binding that have not been seen with natural ligands. Intriguingly, however, our sequences are not found in the human proteome, perhaps suggesting that strong binders to BDs might have been selected against during evolution., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The bacteriophage capsid protein, Psu (polarity suppression), inhibits the bacterial transcription terminator, Rho. In an effort to find nontraditional antibacterial agents, we previously designed peptides from the Psu C terminus that function as inhibitors of Rho. Here, we demonstrated that these peptides have positive surface-charge densities, and they downregulate many genes in Escherichia coli. We hypothesized that these peptides could bind to nucleic acids and repress gene expression. One of these peptides, peptide 33, represses in vitro transcription from the T7A1 and P lac promoters efficiently by blocking the access of RNA polymerase to the promoter, a mode of transcription repression akin to many bacterial repressors. In vivo, expressions of the peptides reduce the total RNA level as well as transcription from P lac and P osm promoters significantly. However, they are less efficient in repressing transcription from the rRNA promoters with a very high turnover of RNA polymerase. The peptide 33 binds to both single and dsDNA as well as to RNA with dissociation constants ranging from 1 to 5 μM exhibiting preferences for the single-stranded DNA and RNAs. These interactions are salt-resistant and not sequence-specific. Interactions with dsDNA are entropy-driven, while it is enthalpy-driven for the ssDNA. This mode of interaction with nucleic acids is similar to many nonspecific ssDNA-binding proteins. Expression of peptide 33 induces cell elongation and impaired cell division, possibly due to the dislodging of the DNA-binding proteins. Overall, we surmised that these synthetic transcription repressors would function like bacterial nucleoid-associated proteins., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen-induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Rax flox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokinesignaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and are heavily influenced by genetic factors. Genome-wide association studies have mapped >90% of CVD-associated variants within the noncoding genome, which can alter the function of regulatory proteins, such as transcription factors (TFs). However, due to the overwhelming number of single-nucleotide polymorphisms (SNPs) (>500,000) in genome-wide association studies, prioritizing variants for in vitro analysis remains challenging. In this work, we implemented a computational approach that considers support vector machine (SVM)-based TF binding site classification and cardiac expression quantitative trait loci (eQTL) analysis to identify and prioritize potential CVD-causing SNPs. We identified 1535 CVD-associated SNPs within TF footprints and putative cardiac enhancers plus 14,218 variants in linkage disequilibrium with genotype-dependent gene expression in cardiac tissues. Using ChIP-seq data from two cardiac TFs (NKX2-5 and TBX5) in human-induced pluripotent stem cell-derived cardiomyocytes, we trained a large-scale gapped k-mer SVM model to identify CVD-associated SNPs that altered NKX2-5 and TBX5 binding. The model was tested by scoring human heart TF genomic footprints within putative enhancers and measuring in vitro binding through electrophoretic mobility shift assay. Five variants predicted to alter NKX2-5 (rs59310144, rs6715570, and rs61872084) and TBX5 (rs7612445 and rs7790964) binding were prioritized for in vitro validation based on the magnitude of the predicted change in binding and are in cardiac tissue eQTLs. All five variants altered NKX2-5 and TBX5 DNA binding. We present a bioinformatic approach that considers tissue-specific eQTL analysis and SVM-based TF binding site classification to prioritize CVD-associated variants for in vitro analysis., Competing Interests: Conflict of interest The authors declare no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In enterobacteria such as Escherichia coli, the general stress response is mediated by σ s , the stationary phase dissociable promoter specificity subunit of RNA polymerase. σ s is degraded by ClpXP during active growth in a process dependent on the RssB adaptor, which is thought to be stimulated by the phosphorylation of a conserved aspartate in its N-terminal receiver domain. Here we present the crystal structure of full-length RssB bound to a beryllofluoride phosphomimic. Compared to the structure of RssB bound to the IraD anti-adaptor, our new RssB structure with bound beryllofluoride reveals conformational differences and coil-to-helix transitions in the C-terminal region of the RssB receiver domain and in the interdomain segmented helical linker. These are accompanied by masking of the α4-β5-α5 (4-5-5) "signaling" face of the RssB receiver domain by its C-terminal domain. Critically, using hydrogen-deuterium exchange mass spectrometry, we identify σ s -binding determinants on the 4-5-5 face, implying that this surface needs to be unmasked to effect an interdomain interface switch and enable full σ s engagement and hand-off to ClpXP. In activated receiver domains, the 4-5-5 face is often the locus of intermolecular interactions, but its masking by intramolecular contacts upon phosphorylation is unusual, emphasizing that RssB is a response regulator that undergoes atypical regulation., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Yeast mRNAs are polyadenylated at multiple sites in their 3' untranslated regions (3' UTRs), and poly(A) site usage is regulated by the rate of transcriptional elongation by RNA polymerase II (Pol II). Slow Pol II derivatives favor upstream poly(A) sites, and fast Pol II derivatives favor downstream poly(A) sites. Transcriptional elongation and polyadenylation are linked at the nucleotide level, presumably reflecting Pol II dwell time at each residue that influences the level of polyadenylation. Here, we investigate the effect of Pol II elongation rate on pausing patterns and the relationship between Pol II pause sites and poly(A) sites within 3' UTRs. Mutations that affect Pol II elongation rate alter sequence preferences at pause sites within 3' UTRs, and pausing preferences differ between 3' UTRs and coding regions. In addition, sequences immediately flanking the pause sites show preferences that are largely independent of Pol II speed. In wild-type cells, poly(A) sites are preferentially located < 50 nucleotides upstream from Pol II pause sites, but this spatial relationship is diminished in cells harboring Pol II speed mutants. Based on a random forest classifier, Pol II pause sites are modestly predicted by the distance to poly(A) sites but are better predicted by the chromatin landscape in Pol II speed derivatives. Transcriptional regulatory proteins can influence the relationship between Pol II pausing and polyadenylation but in a manner distinct from Pol II elongation rate derivatives. These results indicate a complex relationship between Pol II pausing and polyadenylation., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A substantial body of evidence has established the contributions of both mitochondrial dynamics and lipid metabolism to the pathogenesis of diabetic kidney disease (DKD). However, the precise interplay between these two key metabolic regulators of DKD is not fully understood. Here, we uncover a link between mitochondrial dynamics and lipid metabolism by investigating the role of carbohydrate-response element-binding protein (ChREBP), a glucose-responsive transcription factor and a master regulator of lipogenesis, in kidney podocytes. We find that inducible podocyte-specific knockdown of ChREBP in diabetic db/db mice improves key biochemical and histological features of DKD in addition to significantly reducing mitochondrial fragmentation. Because of the critical role of ChREBP in lipid metabolism, we interrogated whether and how mitochondrial lipidomes play a role in ChREBP-mediated mitochondrial fission. Our findings suggest a key role for a family of ether phospholipids in ChREBP-induced mitochondrial remodeling. We find that overexpression of glyceronephosphate O-acyltransferase, a critical enzyme in the biosynthesis of plasmalogens, reverses the protective phenotype of ChREBP deficiency on mitochondrial fragmentation. Finally, our data also points to Gnpat as a direct transcriptional target of ChREBP. Taken together, our results uncover a distinct mitochondrial lipid signature as the link between ChREBP-induced mitochondrial dynamics and progression of DKD., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Coactivator-associated arginine methyltransferase 1 (CARM1) is an arginine methyltransferase that posttranslationally modifies proteins that regulate multiple levels of RNA production and processing. Its substrates include histones, transcription factors, coregulators of transcription, and splicing factors. CARM1 is overexpressed in many different cancer types, and often promotes transcription factor programs that are co-opted as drivers of the transformed cell state, a process known as transcription factor addiction. Targeting these oncogenic transcription factor pathways is difficult but could be addressed by removing the activity of the key coactivators on which they rely. CARM1 is ubiquitously expressed, and its KO is less detrimental in embryonic development than deletion of the arginine methyltransferases protein arginine methyltransferase 1 and protein arginine methyltransferase 5, suggesting that therapeutic targeting of CARM1 may be well tolerated. Here, we will summarize the normal in vivo functions of CARM1 that have been gleaned from mouse studies, expand on the transcriptional pathways that are regulated by CARM1, and finally highlight recent studies that have identified oncogenic properties of CARM1 in different biological settings. This review is meant to kindle an interest in the development of human drug therapies targeting CARM1, as there are currently no CARM1 inhibitors available for use in clinical trials., Competing Interests: Conflict of interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Mark T. Bedford is the co-founder of EpiCypher., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The application of genetic and biochemical techniques in yeast has informed our knowledge of transcription in mammalian cells. Such systems have allowed investigators to determine whether a gene was essential and to determine its function in rDNA transcription. However, there are significant differences in the nature of the transcription factors essential for transcription by Pol I in yeast and mammalian cells, and yeast RNA polymerase I contains 14 subunits while mammalian polymerase contains 13 subunits. We previously reported the adaptation of the auxin-dependent degron that enabled a combination of a "genetics-like" approach and biochemistry to study mammalian rDNA transcription. Using this system, we studied the mammalian orthologue of yeast RPA34.5, PAF49, and found that it is essential for rDNA transcription and cell division. The auxin-induced degradation of PAF49 induced nucleolar stress and the accumulation of P53. Interestingly, the auxin-induced degradation of AID-tagged PAF49 led to the degradation of its binding partner, PAF53, but not vice versa. A similar pattern of co-dependent expression was also found when we studied the non-essential, yeast orthologues. An analysis of the domains of PAF49 that are essential for rDNA transcription demonstrated a requirement for both the dimerization domain and an "arm" of PAF49 that interacts with PolR1B. Further, we demonstrate this interaction can be disrupted to inhibit Pol I transcription in normal and cancer cells which leads to the arrest of normal cells and cancer cell death. In summary, we have shown that both PAF53 and PAF49 are necessary for rDNA transcription and cell growth., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Mn 2+ is an essential nutrient whose concentration is tightly controlled in bacteria. In Bacillus subtilis, the Mn 2+ -activated transcription factor MntR controls Mn 2+ transporter genes. However, factors regulating intracellular Mn 2+ concentration are incompletely understood. Here, we found that glucose addition induces an increase in intracellular Mn 2+ concentration. We determined this upshift was mediated by glucose induction of the major Mn 2+ importer gene mntH by the transcription factor AhrC, which is known to be involved in arginine metabolism and to be indirectly induced by glucose. In addition, we identified novel AhrC-regulated genes encoding the Mn 2+ importer YcsG and the ABC-type exporter YknUV. We found the expression of these genes was also regulated by glucose and contributes to the glucose induction of Mn 2+ concentrations. ycsG expression is regulated by MntR as well. Furthermore, we analyzed the interaction of AhrC and MntR with the promoter driving ycsG expression and examined the Mn 2+ -dependent induction of this promoter to identify the transcription factors responsible for the Mn 2+ induction. RNA-Seq revealed that disruption of ahrC and mntR affected the expression of 502 and 478 genes, respectively (false discovery rate, <0.001, log 2 [fold change] ≥ |2|. The AhrC- and/or MntR-dependent expression of twenty promoters was confirmed by LacZ analysis, and AhrC or MntR binding to some of these promoters was observed via EMSA. The finding that glucose promotes an increase in intracellular Mn 2+ levels without changes in extracellular Mn 2+ concentrations is reasonable for the bacterium, as intracellular Mn 2+ is required for enzymes and pathways mediating glucose metabolism., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The epithelium of the pulmonary airway is composed of several distinct cell types that differentiate from common progenitor cells to provide defense against environmental insults. Epigenetic mechanisms regulating lineage differentiation of airway epithelial progenitors remain poorly understood. Protein arginine methyltransferase 5 (Prmt5) is a predominant type II arginine methyltransferase that methylates >85% of symmetric arginine residues. Here, we provide evidence for the function of Prmt5 in promoting ciliated cell fate specification of airway epithelial progenitors. We show that lung epithelial-specific deletion of Prmt5 resulted in a complete loss of ciliated cells, an increased number of basal cells, and ecotopic-expressed Tp63 - Krt5 + putative cells in the proximal airway. We further identified that transcription factor Tp63 is a direct target of Prmt5, and Prmt5 inhibited Tp63 transcription expression through H4R3 symmetric dimethylation (H4R3sme2). Moreover, inhibition of Tp63 expression in Prmt5-deficient tracheal progenitors could partially restore the ciliated cell deficient phenotype. Together, our data support a model where Prmt5-mediated H4R3sme2 represses Tp63 expression to promote ciliated cell fate specification of airway progenitors., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lung branching morphogenesis relies on a complex coordination of multiple signaling pathways and transcription factors. Here, we found that ablation of the LIM homeodomain transcription factor Islet1 (Isl1) in lung epithelium resulted in defective branching morphogenesis and incomplete formation of five lobes. A reduction in mesenchymal cell proliferation was observed in Isl1 ShhCre lungs. There was no difference in apoptosis between the wild-type (Shh Cre ) and Isl1 ShhCre embryos. RNA-Seq and in situ hybridization analysis showed that Shh, Ptch1, Sox9, Irx1, Irx2, Tbx2, and Tbx3 were downregulated in the lungs of Isl1 ShhCre embryos. ChIP assay implied the Shh gene served as a direct target of ISL1, since the transcription factor ISL1 could bind to the Shh epithelial enhancer sequence (MACS1). Also, activation of the Hedgehog pathway via ectopic gene expression rescued the defects caused by Isl1 ablation, confirming the genetic integration of Hedgehog signaling. In conclusion, our works suggest that epithelial Isl1 regulates lung branching morphogenesis through administrating the Shh signaling mediated epithelial-mesenchymal communications., Competing Interests: Conflict of interest The authors have declared that no competing interests exist., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The Gram-positive bacterium Bacillus subtilis can utilize several proteinogenic and non-proteinogenic amino acids as sources of carbon, nitrogen, and energy. The utilization of the amino acids arginine, citrulline, and ornithine is catalyzed by enzymes encoded in the rocABC and rocDEF operons and by the rocG gene. The expression of these genes is controlled by the alternative sigma factor SigL. RNA polymerase associated with this sigma factor depends on ATP-hydrolyzing transcription activators to initiate transcription. The RocR protein acts as a transcription activator for the roc genes. However, the details of amino acid metabolism via this pathway are unknown. Here, we investigated the contributions of all enzymes of the Roc pathway to the degradation of arginine, citrulline, and ornithine. We identified the previously uncharacterized RocB protein as responsible for the conversion of citrulline to ornithine. In vitro assays with the purified enzyme suggest that RocB acts as a manganese-dependent N-carbamoyl-L-ornithine hydrolase that cleaves citrulline to form ornithine and carbamate. Moreover, the molecular effector that triggers transcription activation by RocR has not been unequivocally identified. Using a combination of transcription reporter assays and biochemical experiments, we demonstrate that ornithine is the molecular inducer of RocR activity. Taken together, our work suggests that binding of ATP to RocR triggers its hexamerization, and binding of ornithine then allows ATP hydrolysis and activation of roc gene transcription. Thus, ornithine is the central molecule of the roc degradative pathway as it is the common intermediate of arginine and citrulline degradation and the molecular effector of RocR., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A nucleosome is typically positioned with its proximal edge (NPE) ∼50 bp downstream from the transcription start site of metazoan RNA polymerase II promoters. This +1 nucleosome has distinctive characteristics, including the presence of variant histone types and trimethylation of histone H3 at lysine 4. To address the role of these features in transcription complex assembly, we generated templates with four different promoters and nucleosomes located at a variety of downstream positions, which were transcribed in vitro using HeLa nuclear extracts. Two promoters lacked TATA elements, but all supported strong initiation from a single transcription start site. In contrast to results with minimal in vitro systems based on the TATA-binding protein (TBP), TATA promoter templates with a +51 NPE were transcriptionally inhibited in extracts; activity continuously increased as the nucleosome was moved downstream to +100. Inhibition was much more pronounced for the TATA-less promoters: +51 NPE templates were inactive, and substantial activity was only seen with the +100 NPE templates. Substituting the histone variants H2A.Z, H3.3, or both did not eliminate the inhibition. However, addition of excess TBP restored activity on nucleosomal templates with TATA promoters, even with an NPE at +20. Remarkably, nucleosomal templates with histone H3 trimethylated at lysine 4 are active with an NPE at +51 for both TATA and TATA-less promoters. Our results strongly suggest that the +1 nucleosome interferes with promoter recognition by TFIID. This inhibition can be overcome with TBP alone at TATA promoters or through positive interactions with histone modifications and TFIID., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Neuroblastoma (NB) is one of the most common extracranial solid tumors in children. MYCN gene amplification is highly associated with poor prognosis in high-risk NB patients. In non-MYCN-amplified high-risk NB patients, the expression of c-MYC (MYCC) and its target genes is highly elevated. USP28 as a deubiquitinase is known to regulate the stability of MYCC. We show here USP28 also regulates the stability of MYCN. Genetic depletion or pharmacologic inhibition of the deubiquitinase strongly destabilizes MYCN and stops the growth of NB cells that overexpress MYCN. In addition, MYCC could be similarly destabilized in non-MYCN NB cells by compromising USP28 function. Our results strongly suggest USP28 as a therapeutic target for NB with or without MYCN amplification/overexpression., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In the negative feedback loop composing the Neurospora circadian clock, the core element, FREQUENCY (FRQ), binds with FRQ-interacting RNA helicase (FRH) and casein kinase 1 to form the FRQ-FRH complex (FFC) which represses its own expression by interacting with and promoting phosphorylation of its transcriptional activators White Collar-1 (WC-1) and WC-2 (together forming the White Collar complex, WCC). Physical interaction between FFC and WCC is a prerequisite for the repressive phosphorylations, and although the motif on WCC needed for this interaction is known, the reciprocal recognition motif(s) on FRQ remains poorly defined. To address this, we assessed FFC-WCC in a series of frq segmental-deletion mutants, confirming that multiple dispersed regions on FRQ are necessary for its interaction with WCC. Biochemical analysis shows that interaction between FFC and WCC but not within FFC or WCC can be disrupted by high salt, suggesting that electrostatic forces drive the association of the two complexes. As a basic sequence on WC-1 was previously identified as a key motif for WCC-FFC assembly, our mutagenetic analysis targeted negatively charged residues of FRQ, leading to identification of three Asp/Glu clusters in FRQ that are indispensable for FFC-WCC formation. Surprisingly, in several frq Asp/Glu-to-Ala mutants that vastly diminish FFC-WCC interaction, the core clock still oscillates robustly with an essentially wildtype period, indicating that the interaction between the positive and negative elements in the feedback loop is required for the operation of the circadian clock but is not a determinant of the period length., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

The BarA/UvrY two-component signal transduction system mediates adaptive responses of Escherichia coli to changes in growth stage. At late exponential growth phase, the BarA sensor kinase autophosphorylates and transphosphorylates UvrY, which activates transcription of the CsrB and CsrC noncoding RNAs. CsrB and CsrC, in turn, sequester and antagonize the RNA binding protein CsrA, which posttranscriptionally regulates translation and/or stability of its target mRNAs. Here, we provide evidence that during stationary phase of growth, the HflKC complex recruits BarA to the poles of the cells and silences its kinase activity. Moreover, we show that during the exponential phase of growth, CsrA inhibits hflK and hflC expression, thereby enabling BarA activation upon encountering its stimulus. Thus, in addition to temporal control of BarA activity, spatial regulation is demonstrated., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Transcription regulation is a critical means by which microorganisms sense and adapt to their environments. Bacteria contain a wide range of highly conserved families of transcription factors that have evolved to regulate diverse sets of genes. It is increasingly apparent that structural similarities between transcription factors do not always equate to analogous transcription regulatory networks. For example, transcription factors within the copper-sensing operon repressor (CsoR)-resistance to cobalt and nickel repressor family have been found to repress a wide range of gene targets, including various metal efflux genes, as well as genes involved in sulfide and formaldehyde detoxification machinery. In this study, we identify the preferred DNA-binding sequence for the CsoR-like protein, TTHA1953, from the model extremophile Thermus thermophilus HB8 using the iterative selection approach, restriction endonuclease, protection, selection, and amplification. By mapping significant DNA motifs to the T. thermophilus HB8 genome, we identify potentially regulated genes that we validate with in vitro and in vivo methodologies. We establish TTHA1953 as a master regulator of the sulfur oxidation pathway, providing the first link between CsoR-like proteins and Sox regulation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondria are organelles known primarily for generating ATP via the oxidative phosphorylation process. Environmental signals are sensed by whole organisms or cells and markedly affect this process, leading to alterations in gene transcription and, consequently, changes in mitochondrial function and biogenesis. The expression of mitochondrial genes is finely regulated by nuclear transcription factors, including nuclear receptors and their coregulators. Among the best-known coregulators is the nuclear receptor corepressor 1 (NCoR1). Muscle-specific knockout of NCoR1 in mice induces an oxidative phenotype, improving glucose and fatty acid metabolism. However, the mechanism by which NCoR1 is regulated remains elusive. In this work, we identified the poly(A)-binding protein 4 (PABPC4) as a new NCoR1 interactor. Unexpectedly, we found that silencing of PABPC4 induced an oxidative phenotype in both C2C12 and MEF cells, as indicated by increased oxygen consumption, mitochondria content, and reduced lactate production. Mechanistically, we demonstrated that PABPC4 silencing increased the ubiquitination and consequent degradation of NCoR1, leading to the derepression of PPAR-regulated genes. As a consequence, cells with PABPC4 silencing had a greater capacity to metabolize lipids, reduced intracellular lipid droplets, and reduced cell death. Interestingly, in conditions known to induce mitochondrial function and biogenesis, both mRNA expression and PABPC4 protein content were markedly reduced. Our study, therefore, suggests that the lowering of PABPC4 expression may represent an adaptive event required to induce mitochondrial activity in response to metabolic stress in skeletal muscle cells. As such, the NCoR1-PABPC4 interface might be a new road to the treatment of metabolic diseases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Heparan sulfate (HS) is a long, linear polysaccharide that is ubiquitously expressed in all animal cells and plays a key role in many cellular processes, including cell signaling and development. Dysregulation of HS assembly has been implicated in pathophysiological conditions, such as tumorigenesis and rare genetic disorders. HS biosynthesis occurs in a non-template-driven manner in the endoplasmic reticulum and Golgi through the activity of a large group of biosynthetic enzymes. While much is known about its biosynthesis, little is understood about the regulation of HS assembly across diverse tissue types and disease states. To address this gap in knowledge, we recently performed genome-wide CRISPR/Cas9 screens to identify novel regulatory factors of HS biosynthesis. From these screens, we identified the alpha globin transcription factor, TFCP2, as a top hit. To investigate the role of TFCP2 in HS assembly, we targeted TFCP2 expression in human melanoma cells using the CRISPR/Cas9 system. TFCP2 knockout cells exhibited decreased fibroblast growth factor binding to cell surface HS, alterations in HS composition, and slowed cell growth compared to wild-type cells. Additionally, RNA sequencing revealed that TFCP2 regulates the expression of multiple enzymes involved in HS assembly, including the secreted endosulfatase, SULF1. Pharmacological targeting of TFCP2 activity similarly reduced growth factor binding and increased SULF1 expression, and the knockdown of SULF1 expression in TFCP2 mutant cells restored melanoma cell growth. Overall, these studies identify TFCP2 as a novel transcriptional regulator of HS and highlight HS-protein interactions as a possible target to slow melanoma growth., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Hypoxia-inducible factor-1 (HIF-1) is a critical transcription factor that regulates the expression of genes involved in cellular adaptation to low oxygen levels. Aberrant regulation of the HIF-1 signaling pathway is linked to various human diseases. Previous studies have established that HIF-1α is rapidly degraded in a von Hippel-Lindau protein (pVHL)-dependent manner under normoxic conditions. In this study, we find that pVHL binding protein 1 (VBP1) is a negative regulator of HIF-1α but not HIF-2α using zebrafish as an in vivo model and in vitro cell culture models. Deletion of vbp1 in zebrafish caused Hif-1α accumulation and upregulation of Hif target genes. Moreover, vbp1 was involved in the induction of hematopoietic stem cells (HSCs) under hypoxic conditions. However, VBP1 interacted with and promoted the degradation of HIF-1α in a pVHL-independent manner. Mechanistically, we identify the ubiquitin ligase CHIP and HSP70 as new VBP1 binding partners and demonstrate that VBP1 negatively regulated CHIP and facilitated CHIP-mediated degradation of HIF-1α. In patients with clear cell renal cell carcinoma (ccRCC), lower VBP1 expression was associated with worse survival outcomes. In conclusion, our results link VBP1 with CHIP stability and provide insights into underlying molecular mechanisms of HIF-1α-driven pathological processes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

In recent years, lactate has been recognized as an important circulating energy substrate rather than only a dead-end metabolic waste product generated during glucose oxidation at low levels of oxygen. The term "aerobic glycolysis" has been coined to denote increased glucose uptake and lactate production despite normal oxygen levels and functional mitochondria. Hence, in "aerobic glycolysis," lactate production is a metabolic choice, whereas in "anaerobic glycolysis," it is a metabolic necessity based on inadequate levels of oxygen. Interestingly, lactate can be taken up by cells and oxidized to pyruvate and thus constitutes a source of pyruvate that is independent of insulin. Here, we show that the transcription factor Foxp1 regulates glucose uptake and lactate production in adipocytes and myocytes. Overexpression of Foxp1 leads to increased glucose uptake and lactate production. In addition, protein levels of several enzymes in the glycolytic pathway are upregulated, such as hexokinase 2, phosphofructokinase, aldolase, and lactate dehydrogenase. Using chromatin immunoprecipitation and real-time quantitative PCR assays, we demonstrate that Foxp1 directly interacts with promoter consensus cis-elements that regulate expression of several of these target genes. Conversely, knockdown of Foxp1 suppresses these enzyme levels and lowers glucose uptake and lactate production. Moreover, mice with a targeted deletion of Foxp1 in muscle display systemic glucose intolerance with decreased muscle glucose uptake. In primary human adipocytes with induced expression of Foxp1, we find increased glycolysis and glycolytic capacity. Our results indicate Foxp1 may play an important role as a regulator of aerobic glycolysis in adipose tissue and muscle., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Bacterial pathogens like Mycobacterium tuberculosis (Mtb) employ transcription factors to adapt their physiology to the diverse environments within their host. CarD is a conserved bacterial transcription factor that is essential for viability in Mtb. Unlike classical transcription factors that recognize promoters by binding to specific DNA sequence motifs, CarD binds directly to the RNA polymerase to stabilize the open complex intermediate (RP o ) during transcription initiation. We previously showed using RNA-sequencing that CarD is capable of both activating and repressing transcription in vivo. However, it is unknown how CarD achieves promoter-specific regulatory outcomes in Mtb despite binding indiscriminate of DNA sequence. We propose a model where CarD's regulatory outcome depends on the promoter's basal RP o stability and test this model using in vitro transcription from a panel of promoters with varying levels of RP o stability. We show that CarD directly activates full-length transcript production from the Mtb ribosomal RNA promoter rrnAP3 (AP3) and that the degree of transcription activation by CarD is negatively correlated with RP o stability. Using targeted mutations in the extended -10 and discriminator region of AP3, we show that CarD directly represses transcription from promoters that form relatively stable RP o . DNA supercoiling also influenced RP o stability and affected the direction of CarD regulation, indicating that the outcome of CarD activity can be regulated by factors beyond promoter sequence. Our results provide experimental evidence for how RNA polymerase-binding transcription factors like CarD can exert specific regulatory outcomes based on the kinetic properties of a promoter., Competing Interests: Conflict of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)