A Stringent Systems Approach Uncovers Gene-Specific Mechanisms Regulating Inflammation.

Much has been learned about transcriptional cascades and networks from large-scale systems analyses of high-throughput datasets. However, analysis methods that optimize statistical power through simultaneous evaluation of thousands of ChIP-seq peaks or differentially expressed genes possess substantial limitations in their ability to uncover mechanistic principles of transcriptional control. By examining nascent transcript RNA-seq, ChIP-seq, and binding motif datasets from lipid A-stimulated macrophages with increased attention to the quantitative distribution of signals, we identified unexpected relationships between the in vivo binding properties of inducible transcription factors, motif strength, and transcription. Furthermore, rather than emphasizing common features of large clusters of co-regulated genes, our results highlight the extent to which unique mechanisms regulate individual genes with key biological functions. Our findings demonstrate the mechanistic value of stringent interrogation of well-defined sets of genes as a complement to broader systems analyses of transcriptional cascades and networks.

Identification and functional characterization of transcriptional activators in human cells.

Transcription is orchestrated by thousands of transcription factors (TFs) and chromatin-associated proteins, but how these are causally connected to transcriptional activation is poorly understood. Here, we conduct an unbiased proteome-scale screen to systematically uncover human proteins that activate transcription in a natural chromatin context. By combining interaction proteomics and chemical inhibitors, we delineate the preference of these transcriptional activators for specific co-activators, highlighting how even closely related TFs can function via distinct cofactors. We also identify potent transactivation domains among the hits and use AlphaFold2 to predict and experimentally validate interaction interfaces of two activation domains with BRD4. Finally, we show that many novel activators are partners in fusion events in tumors and functionally characterize a myofibroma-associated fusion between SRF and C3orf62, a potent p300-dependent activator. Our work provides a functional catalog of potent transactivators in the human proteome and a platform for discovering transcriptional regulators at genome scale.

Redox modification of nuclear actin by MICAL-2 regulates SRF signaling.

The serum response factor (SRF) binds to coactivators, such as myocardin-related transcription factor-A (MRTF-A), and mediates gene transcription elicited by diverse signaling pathways. SRF/MRTF-A-dependent gene transcription is activated when nuclear MRTF-A levels increase, enabling the formation of transcriptionally active SRF/MRTF-A complexes. The level of nuclear MRTF-A is regulated by nuclear G-actin, which binds to MRTF-A and promotes its nuclear export. However, pathways that regulate nuclear actin levels are poorly understood. Here, we show that MICAL-2, an atypical actin-regulatory protein, mediates SRF/MRTF-A-dependent gene transcription elicited by nerve growth factor and serum. MICAL-2 induces redox-dependent depolymerization of nuclear actin, which decreases nuclear G-actin and increases MRTF-A in the nucleus. Furthermore, we show that MICAL-2 is a target of CCG-1423, a small molecule inhibitor of SRF/MRTF-A-dependent transcription that exhibits efficacy in various preclinical disease models. These data identify redox modification of nuclear actin as a regulatory switch that mediates SRF/MRTF-A-dependent gene transcription.

A myocardin-adjacent lncRNA balances SRF-dependent gene transcription in the heart.

Myocardin, a potent coactivator of serum response factor (SRF), competes with ternary complex factor (TCF) proteins for SRF binding to balance opposing mitogenic and myogenic gene programs in cardiac and smooth muscle. Here we identify a cardiac lncRNA transcribed adjacent to myocardin, named CARDINAL, which antagonizes SRF-dependent mitogenic gene transcription in the heart. CARDINAL-deficient mice show ectopic TCF/SRF-dependent mitogenic gene expression and decreased cardiac contractility in response to age and ischemic stress. CARDINAL forms a nuclear complex with SRF and inhibits TCF-mediated transactivation of the promitogenic gene c-fos, suggesting CARDINAL functions as an RNA cofactor for SRF in the heart.

Blood-borne circadian signal stimulates daily oscillations in actin dynamics and SRF activity.

In peripheral tissues circadian gene expression can be driven either by local oscillators or by cyclic systemic cues controlled by the master clock in the brain's suprachiasmatic nucleus. In the latter case, systemic signals can activate immediate early transcription factors (IETFs) and thereby control rhythmic transcription. In order to identify IETFs induced by diurnal blood-borne signals, we developed an unbiased experimental strategy, dubbed Synthetic TAndem Repeat PROMoter (STAR-PROM) screening. This technique relies on the observation that most transcription factor binding sites exist at a relatively high frequency in random DNA sequences. Using STAR-PROM we identified serum response factor (SRF) as an IETF responding to oscillating signaling proteins present in human and rodent sera. Our data suggest that in mouse liver SRF is regulated via dramatic diurnal changes of actin dynamics, leading to the rhythmic translocation of the SRF coactivator Myocardin-related transcription factor-B (MRTF-B) into the nucleus.

SRF transcriptionally regulates the oligodendrocyte cytoskeleton during CNS myelination.

Myelination of neuronal axons is essential for nervous system development. Myelination requires dramatic cytoskeletal dynamics in oligodendrocytes, but how actin is regulated during myelination is poorly understood. We recently identified serum response factor (SRF)-a transcription factor known to regulate expression of actin and actin regulators in other cell types-as a critical driver of myelination in the aged brain. Yet, a major gap remains in understanding the mechanistic role of SRF in oligodendrocyte lineage cells. Here, we show that SRF is required cell autonomously in oligodendrocytes for myelination during development. Combining ChIP-seq with RNA-seq identifies SRF-target genes in oligodendrocyte precursor cells and oligodendrocytes that include actin and other key cytoskeletal genes. Accordingly, SRF knockout oligodendrocytes exhibit dramatically reduced actin filament levels early in differentiation, consistent with its role in actin-dependent myelin sheath initiation. Surprisingly, oligodendrocyte-restricted loss of SRF results in upregulation of gene signatures associated with aging and neurodegenerative diseases. Together, our findings identify SRF as a transcriptional regulator that controls the expression of cytoskeletal genes required in oligodendrocytes for myelination. This study identifies an essential pathway regulating oligodendrocyte biology with high relevance to brain development, aging, and disease.

CCG-1423-derived compounds reduce global RNA synthesis and inhibit transcriptional responses.

Myocardin-related transcription factors (MRTFs) are coactivators of serum response factor (SRF), and thereby regulate cytoskeletal gene expression in response to actin dynamics. MRTFs have also been implicated in transcription of heat shock protein (HSP)-encoding genes in fly ovaries, but the mechanisms remain unclear. Here, we demonstrate that, in mammalian cells, MRTFs are dispensable for gene induction of HSP-encoding genes. However, the widely used small-molecule inhibitors of the MRTF-SRF transcription pathway, derived from CCG-1423, also efficiently inhibit gene transcription of HSP-encoding genes in both fly and mammalian cells in the absence of MRTFs. Quantifying RNA synthesis and RNA polymerase distribution demonstrates that CCG-1423-derived compounds have a genome-wide effect on transcription. Indeed, tracking nascent transcription at nucleotide resolution reveals that CCG-1423-derived compounds reduce RNA polymerase II elongation, and severely dampen the transcriptional response to heat shock. The effects of CCG-1423-derived compounds therefore extend beyond the MRTF-SRF pathway into nascent transcription, opening novel opportunities for their use in transcription research.

Sall1 and Sall4 cooperatively interact with Myocd and SRF to promote cardiomyocyte proliferation by regulating CDK and cyclin genes.

Sall1 and Sall4 (Sall1/4), zinc-finger transcription factors, are expressed in the progenitors of the second heart field (SHF) and in cardiomyocytes during the early stages of mouse development. To understand the function of Sall1/4 in heart development, we generated heart-specific Sall1/4 functionally inhibited mice by forced expression of the truncated form of Sall4 (ΔSall4) in the heart. The ΔSall4-overexpression mice exhibited a hypoplastic right ventricle and outflow tract, both of which were derived from the SHF, and a thinner ventricular wall. We found that the numbers of proliferative SHF progenitors and cardiomyocytes were reduced in ΔSall4-overexpression mice. RNA-sequencing data showed that Sall1/4 act upstream of the cyclin-dependent kinase (CDK) and cyclin genes, and of key transcription factor genes for the development of compact cardiomyocytes, including myocardin (Myocd) and serum response factor (Srf). In addition, ChIP-sequencing and co-immunoprecipitation analyses revealed that Sall4 and Myocd form a transcriptional complex with SRF, and directly bind to the upstream regulatory regions of the CDK and cyclin genes (Cdk1 and Ccnb1). These results suggest that Sall1/4 are critical for the proliferation of cardiac cells via regulation of CDK and cyclin genes that interact with Myocd and SRF.

Comparative epigenomic analysis of murine and human adipogenesis.

We report the generation and comparative analysis of genome-wide chromatin state maps, PPARγ and CTCF localization maps, and gene expression profiles from murine and human models of adipogenesis. The data provide high-resolution views of chromatin remodeling during cellular differentiation and allow identification of thousands of putative preadipocyte- and adipocyte-specific cis-regulatory elements based on dynamic chromatin signatures. We find that the specific locations of most such elements differ between the two models, including at orthologous loci with similar expression patterns. Based on sequence analysis and reporter assays, we show that these differences are determined, in part, by evolutionary turnover of transcription factor motifs in the genome sequences and that this turnover may be facilitated by the presence of multiple distal regulatory elements at adipogenesis-dependent loci. We also utilize the close relationship between open chromatin marks and transcription factor motifs to identify and validate PLZF and SRF as regulators of adipogenesis.

The benzoylphenylurea derivative BPU17 acts as an inhibitor of prohibitin and exhibits antifibrotic activity.

Inflammation-induced choroidal neovascularization followed by the epithelial-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPEs) is a cause of neovascular age-related macular degeneration (nAMD). RPE-derived myofibroblasts overproduce extracellular matrix, leading to subretinal fibrosis. We already have demonstrated that benzylphenylurea (BPU) derivatives inhibit the function of cancer-associated fibroblasts. Here, we investigated the anti-myofibroblast effects of BPU derivatives and examined such BPU activity on subretinal fibrosis. A BPU derivative, BPU17, exhibits the most potent anti-myofibroblast activity among dozens of BPU derivatives and inhibits subretinal fibrosis in a mouse model of retinal degeneration. Investigations with primary cultured RPEs reveal that BPU17 suppresses cell motility and collagen synthesis in RPE-derived myofibroblasts. These effects depend on repressing the serum response factor (SRF)/CArG-box-dependent transcription. BPU17 inhibits the expression of SRF cofactor, cysteine and glycine-rich protein 2 (CRP2), which activates the SRF function. Proteomics analysis reveals that BPU17 binds to prohibitin 1 (PHB1) and inhibits the PHB1-PHB2 interaction, resulting in mild defects in mitochondrial function. This impairment causes a decrease in the expression of CRP2 and suppresses collagen synthesis. Our findings suggest that BPU17 is a promising agent against nAMD and the close relationship between PHB function and EMT.

ERK-Induced Activation of TCF Family of SRF Cofactors Initiates a Chromatin Modification Cascade Associated with Transcription.

We investigated the relationship among ERK signaling, histone modifications, and transcription factor activity, focusing on the ERK-regulated ternary complex factor family of SRF partner proteins. In MEFs, activation of ERK by TPA stimulation induced a common pattern of H3K9acS10ph, H4K16ac, H3K27ac, H3K9acK14ac, and H3K4me3 at hundreds of transcription start site (TSS) regions and remote regulatory sites. The magnitude of the increase in histone modification correlated well with changes in transcription. H3K9acS10ph preceded the other modifications. Most induced changes were TCF dependent, but TCF-independent TSSs exhibited the same hierarchy, indicating that it reflects gene activation per se. Studies with TCF Elk-1 mutants showed that TCF-dependent ERK-induced histone modifications required Elk-1 to be phosphorylated and competent to activate transcription. Analysis of direct TCF-SRF target genes and chromatin modifiers confirmed this and showed that H3S10ph required only Elk-1 phosphorylation. Induction of histone modifications following ERK stimulation is thus directed by transcription factor activation and transcription.

Vascular injury activates the ELK1/SND1/SRF pathway to promote vascular smooth muscle cell proliferative phenotype and neointimal hyperplasia.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation is the leading cause of vascular stenosis or restenosis. Therefore, investigating the molecular mechanisms and pivotal regulators of the proliferative VSMC phenotype is imperative for precisely preventing neointimal hyperplasia in vascular disease. METHODS: Wire-induced vascular injury and aortic culture models were used to detect the expression of staphylococcal nuclease domain-containing protein 1 (SND1). SMC-specific Snd1 knockout mice were used to assess the potential roles of SND1 after vascular injury. Primary VSMCs were cultured to evaluate SND1 function on VSMC phenotype switching, as well as to investigate the mechanism by which SND1 regulates the VSMC proliferative phenotype. RESULTS: Phenotype-switched proliferative VSMCs exhibited higher SND1 protein expression compared to the differentiated VSMCs. This result was replicated in primary VSMCs treated with platelet-derived growth factor (PDGF). In the injury model, specific knockout of Snd1 in mouse VSMCs reduced neointimal hyperplasia. We then revealed that ETS transcription factor ELK1 (ELK1) exhibited upregulation and activation in proliferative VSMCs, and acted as a novel transcription factor to induce the gene transcriptional activation of Snd1. Subsequently, the upregulated SND1 is associated with serum response factor (SRF) by competing with myocardin (MYOCD). As a co-activator of SRF, SND1 recruited the lysine acetyltransferase 2B (KAT2B) to the promoter regions leading to the histone acetylation, consequently promoted SRF to recognize the specific CArG motif, and enhanced the proliferation- and migration-related gene transcriptional activation. CONCLUSIONS: The present study identifies ELK1/SND1/SRF as a novel pathway in promoting the proliferative VSMC phenotype and neointimal hyperplasia in vascular injury, predisposing the vessels to pathological remodeling. This provides a potential therapeutic target for vascular stenosis.

Direct GPCR-EGFR interaction enables synergistic membrane-to-nucleus information transfer.

We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or -mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.

Serum Response Factor Expression in Excess Permits a Dual Contractile-Proliferative Phenotype of Airway Smooth Muscle.

The transcription factors (TFs) MyoCD (myocardin) and Elk-1 (ETS Like-1 protein) competitively bind to SRF (serum response factor) and control myogenic- and mitogenic-related gene expression in smooth muscle, respectively. Their functions are therefore mutually inhibitory, which results in a contractile-versus-proliferative phenotype dichotomy. Airway smooth muscle cell (ASMC) phenotype alterations occur in various inflammatory airway diseases, promoting pathological remodeling and contributing to airflow obstruction. We characterized MyoCD and Elk-1 interactions and their roles in phenotype determination in human ASMCs. MyoCD overexpression in ASMCs increased smooth muscle gene expression, force generation, and partially restored the loss of smooth muscle protein associated with prolonged culturing while inhibiting Elk-1 transcriptional activities and proliferation induced by EGF (epidermal growth factor). However, MyoCD overexpression failed to suppress these responses induced by FBS, as FBS also upregulated SRF expression to a degree that allowed unopposed function of both TFs. Inhibition of the RhoA pathway reversed said SRF changes, allowing inhibition of Elk-1 by MyoCD overexpression and suppressing FBS-mediated contractile protein gene upregulation. Our study confirmed that MyoCD in increased abundance can competitively inhibit Elk-1 function. However, SRF upregulation permits a dual contractile-proliferative ASMC phenotype that is anticipated to exacerbate pathological alterations, whereas therapies targeting SRF may inhibit pathological ASMC proliferation and contractile protein gene expression.

Myocardin regulates fibronectin expression and secretion from human pleural mesothelial cells.

During the progression of pleural fibrosis, pleural mesothelial cells (PMCs) undergo a phenotype switching process known as mesothelial-mesenchymal transition (MesoMT). During MesoMT, transformed PMCs become myofibroblasts that produce increased extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1) that is critical to develop fibrosis. Here, we studied the mechanism that regulates FN1 expression in myofibroblasts derived from human pleural mesothelial cells (HPMCs). We found that myocardin (Myocd), a transcriptional coactivator of serum response factor (SRF) and a master regulator of smooth muscle and cardiac muscle differentiation, strongly controls FN1 gene expression. Myocd gene silencing markedly inhibited FN1 expression. FN1 promoter analysis revealed that deletion of the Smad3-binding element diminished FN1 promoter activity, whereas deletion of the putative SRF-binding element increased FN1 promoter activity. Smad3 gene silencing decreased FN1 expression, whereas SRF gene silencing increased FN1 expression. Moreover, SRF competes with Smad3 for binding to Myocd. These results indicate that Myocd activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression in HPMCs. In HPMCs, TGF-ß induced Smad3 nuclear localization, and the proximity ligation signal between Myocd and Smad3 was markedly increased after TGF-ß stimulation at nucleus, suggesting that TGF-ß facilitates nuclear translocation of Smad3 and interaction between Smad3 and Myocd. Moreover, Myocd and Smad3 were coimmunoprecipitated and isolated Myocd and Smad3 proteins directly bound each other. Chromatin immunoprecipitation assays revealed that Myocd interacts with the FN1 promoter at the Smad3-binding consensus sequence. The results indicate that Myocd regulates FN1 gene activation through interaction and activation of the Smad3 transcription factor.NEW & NOTEWORTHY During phenotype switching from mesothelial to mesenchymal, pleural mesothelial cells (PMCs) produce extracellular matrix (ECM) proteins, including collagen and fibronectin (FN1), critical components in the development of fibrosis. Here, we found that myocardin, a transcriptional coactivator of serum response factor (SRF), strongly activates FN1 expression through Smad3, whereas SRF inhibits FN1 expression. This study provides insights about the regulation of FN1 that could lead to the development of novel interventional approaches to prevent pleural fibrosis.

TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity.

The transcription factor TRPS1 is a context-dependent oncogene in breast cancer. In the mammary gland, TRPS1 activity is restricted to the luminal population and is critical during puberty and pregnancy. Its function in the resting state remains however unclear. To evaluate whether it could be a target for cancer therapy, we investigated TRPS1 function in the healthy adult mammary gland using a conditional ubiquitous depletion mouse model where long-term depletion does not affect fitness. Using transcriptomic approaches, flow cytometry and functional assays, we show that TRPS1 activity is essential to maintain a functional luminal progenitor compartment. This requires the repression of both YAP/TAZ and SRF/MRTF activities. TRPS1 represses SRF/MRTF activity indirectly by modulating RhoA activity. Our work uncovers a hitherto undisclosed function of TRPS1 in luminal progenitors intrinsically linked to mechanotransduction in the mammary gland. It may also provide new insights into the oncogenic functions of TRPS1 as luminal progenitors are likely the cells of origin of many breast cancers.

The actin cytoskeleton-MRTF/SRF cascade transduces cellular physical niche cues to entrain the circadian clock.

The circadian clock is entrained to daily environmental cues. Integrin-linked signaling via actin cytoskeleton dynamics transduces physical niche cues from the extracellular matrix to myocardin-related transcription factor (MRTF)/serum response factor (SRF)-mediated transcription. The actin cytoskeleton organization and SRF-MRTF activity display diurnal oscillations. By interrogating disparate upstream events in the actin cytoskeleton-MRTF-A/SRF signaling cascade, we show that this pathway transduces extracellular niche cues to modulate circadian clock function. Pharmacological inhibition of MRTF-A/SRF by disrupting actin polymerization or blocking the ROCK kinase induced period lengthening with augmented clock amplitude, and genetic loss of function of Srf or Mrtfa mimicked the effects of treatment with actin-depolymerizing agents. In contrast, actin polymerization shortened circadian clock period and attenuated clock amplitude. Moreover, interfering with the cell-matrix interaction through blockade of integrin, inhibition of focal adhesion kinase (FAK, encoded by Ptk2) or attenuating matrix rigidity reduced the period length while enhancing amplitude. Mechanistically, we identified that the core clock repressors Per2, Nr1d1 and Nfil3 are direct transcriptional targets of MRTF-A/SRF in mediating actin dynamics-induced clock response. Collectively, our findings defined an integrin-actin cytoskeleton-MRTF/SRF pathway in linking clock entrainment with extracellular cues that might facilitate cellular adaptation to the physical niche environment.

Lysine acetyltransferase 14 mediates TGF-ß-induced fibrosis in ovarian endometrioma via co-operation with serum response factor.

BACKGROUND: Fibrogenesis within ovarian endometrioma (endometrioma), mainly induced by transforming growth factor-ß (TGF-ß), is characterized by myofibroblast over-activation and excessive extracellular matrix (ECM) deposition, contributing to endometrioma-associated symptoms such as infertility by impairing ovarian reserve and oocyte quality. However, the precise molecular mechanisms that underpin the endometrioma- associated fibrosis progression induced by TGF-ß remain poorly understood. METHODS: The expression level of lysine acetyltransferase 14 (KAT14) was validated in endometrium biopsies from patients with endometrioma and healthy controls, and the transcription level of KAT14 was further confirmed by analyzing a published single-cell transcriptome (scRNA-seq) dataset of endometriosis. We used overexpression, knockout, and knockdown approaches in immortalized human endometrial stromal cells (HESCs) or human primary ectopic endometrial stromal cells (EcESCs) to determine the role of KAT14 in TGF-ß-induced fibrosis. Furthermore, an adeno-associated virus (AAV) carrying KAT14-shRNA was used in an endometriosis mice model to assess the role of KAT14 in vivo. RESULTS: KAT14 was upregulated in ectopic lesions from endometrioma patients and predominantly expressed in activated fibroblasts. In vitro studies showed that KAT14 overexpression significantly promoted a TGF-ß-induced profibrotic response in endometrial stromal cells, while KAT14 silencing showed adverse effects that could be rescued by KAT14 re-enhancement. In vivo, Kat14 knockdown ameliorated fibrosis in the ectopic lesions of the endometriosis mouse model. Mechanistically, we showed that KAT14 directly interacted with serum response factor (SRF) to promote the expression of α-smooth muscle actin (α-SMA) by increasing histone H4 acetylation at promoter regions; this is necessary for TGF-ß-induced ECM production and myofibroblast differentiation. In addition, the knockdown or pharmacological inhibition of SRF significantly attenuated KAT14-mediating profibrotic effects under TGF-ß treatment. Notably, the KAT14/SRF complex was abundant in endometrioma samples and positively correlated with α-SMA expression, further supporting the key role of KAT14/SRF complex in the progression of endometrioma-associated fibrogenesis. CONCLUSION: Our results shed light on KAT14 as a key effector of TGF-ß-induced ECM production and myofibroblast differentiation in EcESCs by promoting histone H4 acetylation via co-operating with SRF, representing a potential therapeutic target for endometrioma-associated fibrosis.

Serum Response Factor Reduces Gene Expression Noise and Confers Cell State Stability.

The role of serum response factor (Srf), a central mediator of actin dynamics and mechanical signaling, in cell identity regulation is debated to be either a stabilizer or a destabilizer. We investigated the role of Srf in cell fate stability using mouse pluripotent stem cells. Despite the fact that serum-containing cultures yield heterogeneous gene expression, deletion of Srf in mouse pluripotent stem cells leads to further exacerbated cell state heterogeneity. The exaggerated heterogeneity is detectible not only as increased lineage priming but also as the developmentally earlier 2C-like cell state. Thus, pluripotent cells explore more variety of cellular states in both directions of development surrounding naïve pluripotency, a behavior that is constrained by Srf. These results support that Srf functions as a cell state stabilizer, providing rationale for its functional modulation in cell fate intervention and engineering.

Altered extracellular matrix-related pathways accelerate the transition from normal to prefibroid myometrium in Black women.

BACKGROUND: Black women experience a disproportionate impact of uterine fibroids compared to White women, including earlier diagnosis, higher frequency, and more severe symptoms. The etiology underlying this racial disparity remains elusive. OBJECTIVE: The aim of this study was to evaluate the molecular differences in normal myometrium (fibroid-free uteri) and at-risk myometrium (fibroid-containing uteri) tissues in Black and White women. STUDY DESIGN: We conducted whole-genome RNA-seq on normal and at-risk myometrium tissues obtained from both self-identified Black and White women (not Hispanic or Latino) to determine global gene expression profiles and to conduct enriched pathway analyses (n=3 per group). We initially assessed the differences within the same type of tissue (normal or at-risk myometrium) between races. Subsequently, we analyzed the transcriptome of normal myometrium compared to at-risk myometrium in each race and determined the differences between them. We validated our findings through real-time PCR (sample size range=5-12), western blot (sample size range=5-6), and immunohistochemistry techniques (sample size range=9-16). RESULTS: The transcriptomic analysis revealed distinct profiles between Black and White women in normal and at-risk myometrium tissues. Interestingly, genes and pathways related to extracellular matrix and mechanosensing were more enriched in normal myometrium from Black than White women. Transcription factor enrichment analysis detected greater activity of the serum response transcription factor positional motif in normal myometrium from Black compared to White women. Furthermore, we observed increased expression levels of myocardin-related transcription factor-serum response factor and the serum response factor in the same comparison. In addition, we noted increased expression of both mRNA and protein levels of vinculin, a target gene of the serum response factor, in normal myometrium tissues from Black women as compared to White women. Importantly, the transcriptomic profile of normal to at-risk myometrium conversion differs between Black and White women. Specifically, we observed that extracellular matrix-related pathways are involved in the transition from normal to at-risk myometrium and that these processes are exacerbated in Black women. We found increased levels of Tenascin C, type I collagen alpha 1 chain, fibronectin, and phospho-p38 MAPK (Thr180/Tyr182, active) protein levels in at-risk over normal myometrium tissues from Black women, whereas such differences were not observed in samples from White women. CONCLUSION: These findings indicate that the racial disparities in uterine fibroids may be attributed to heightened production of extracellular matrix in the myometrium in Black women, even before the tumors appear. Future research is needed to understand early life determinants of the observed racial differences.